itaconate production by ustilago maydis
TRANSCRIPT
"Itaconate production by Ustilago maydis;
the influence of genes and cultivation conditions"
Von der Fakultät für Mathematik, Informatik und
Naturwissenschaften der RWTH Aachen University
zur Erlangung des akademisches Grades einer
Doktorin der Naturwissenschaften
genehmigte Dissertation vorgelegt von
M.Sc.
Monika Maasem Panáková
aus
Šumperk - Tschechische Republik
Berichter: Universitätsprofessor Dr. em. Ulrich Klinner
Universitätsprofessor Dr. Michael Bölker
Universitätsprofessor Dr. Jan Schirawski
Tag der mündlichen Prüfung: 10.10.2013
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar.
Summary
Due to the increasing CO2 emissions and the necessity for substitutions to the petrochemical
fuels, the interest in biofuels originating from alternative renewable resources is on the rise.
Itaconate represents a newly identified platform molecule, which can be used for the produc-
tion of various polymers and can also act as an intermediate for biofuel production. It can
be produced by microorganisms from hydrolyzed biomass, without these resources being
in competition with common sources of human diet. Ustilago maydis MB215 represents
an alternative itaconate producer; it exhibits an unicellular yeast-like form of growth and
possesses the ability to produce itaconate naturally. However, its itaconate yields do not
compare to those from the currently most favored producer, Aspergillus terreus. Therefore,
the aim of this work was to investigate and understand the itaconate synthesis pathway in
U . maydis and determine the factors influencing the production process in order to enhance
the efficiency of the itaconate production.
Several genes putatively involved in the itaconate synthesis pathway were determined and
deleted and/or overexpressed in order to elucidate the biosynthetic pathway. Deletion of
the UM06344 gene, presumably encoding a cis-aconitate decarboxylase, did not decrease
the itaconate production. Although overexpression of this gene might indirectly improve
the itaconate production, it is not directly involved. Nevertheless, three mutants with sig-
nificantly increased itaconate generation were obtained during the phenotypical screening
of the overexpression mutants. These mutants were subjected to further characterization
under selected cultivation conditions. On the other hand, disruption of the UM11778 gene,
encoding an enzyme similar to 3-methylitaconate-∆-isomerase (Mii), drastically decreased
itaconate production. The Mii enzyme can catalyze the isomerization between citraconate
and itaconate, and based on this finding and physiological characterization of itaconate
production by U . maydis, possible itaconate synthesis pathways were proposed.
Biotechnological production of chemicals is strongly influenced by the cultivation conditions.
In order to establish an optimal production process, factors having an impact on the cells and
consequently on the itaconate yields were determined and studied. The most influencing
factor proved to be the oxygen supply. Cultures suffering from oxygen limitation failed to
efficiently synthesize itaconate, whereas more aerated cultures were capable of significantly
enhanced itaconate production. The necessity for sufficient aeration originates from the
indispensability of cofactor regeneration during itaconic acid production. The respiratory
chain represents the first option after the cessation of the biomass formation, which is the
main route for the cofactor regeneration. However, during the oxygen limitation, these
cofactors are regenerated via by-product synthesis. The two most distinct by-products are
glycolipids and malate. A mutant with blocked glycolipid production exhibited increased
itaconate production with regard to the wt strain.
The initial glucose concentration also represents an important factor and therefore vari-
ous glucose amounts and their impact on the cells as well as on the itaconate yields were
studied. Even though calculations revealed that the maximum theoretical itaconate yield
increases with increasing initial glucose concentrations, the cultivation experiments showed
that the cells undergo a substantial osmotic stress while being cultivated in high-glucose-
concentration solutions. Understanding these relations led to the determination of the
optimal initial glucose concentration for the itaconate production in U . maydis, which under
the conditions tested is 65 g l−1.
Not only the depletion of the nitrogen (N) functions as a trigger for the itaconate synthesis but
its concentration influences the itaconate yield and production rate; in theory, more N means
high rates but low yields and vice versa. However, the biomass formation increases with the
increasing N concentration and soon the culture reaches oxygen limitation and the itaconate
production decreases. Taken all these facts into consideration, the optimal N concentration
for the itaconate production by U . maydis was determined to be 0.8 g l−1 NH4Cl.
In order to decrease the overall fermentation costs, alternative carbon sources for the it-
aconate production were tested. U . maydis is capable of utilization of xylose, arabinose and
sucrose as substrates for itaconate production. However, itaconate production from these
sugars was very low, and further optimization is required.
Based on the findings presented within this work, U . maydis proved to be a suitable alternat-
ive itaconate producer. Although its itaconate yields do not yet reach those from A. terreus,
there are several advantages U . maydis possesses in comparison to the Aspergillus and with
suggested further improvements the itaconate yields will improve.
Zusammenfassung
Aufgrund der Zunahme an CO2-Emissionen und der Notwendigkeit einen Ersatz für petro-
chemische Treibstoffe zu finden, zeigt sich ein steigendes Interesse an Biokraftstoffen aus
regenerativen Ausgangsstoffen.
Hierzu gehört Itaconat als ein neu identifiziertes Plattformmolekül, das sowohl für die Produk-
tion verschiedener Polymere als auch als Intermediär für die Biokraftstoff-Produktion ver-
wendet werden kann. Mikroorganismen können Itaconat aus hydrolysierter Biomasse bilden,
so dass die verwendeten Ressourcen nicht mit menschlichen Nahrungsquellen konkurrieren.
Bei Ustilago maydis MB215 handelt es sich um einen alternativen Itaconat-Produzenten, der
ein einzelliges, hefe-ähnliches Wachstum aufweist und die Fähigkeit besitzt Itaconat auf
natürliche Weise zu generieren. Die Ausbeute ist jedoch nicht mit der des zurzeit favorisier-
ten Itaconat-Produzenten Aspergillus terreus vergleichbar. Deshalb lag das Ziel dieser Arbeit
in der Untersuchung und dem Verständnis des Itaconat- Syntheseweges in U . maydis und
der Bestimmung der Einflussfaktoren im Produktionsprozess, um dessen Effizienz bei der
Itaconat-Produktion zu steigern.
Einige Gene, die mutmaßlich in den Syntheseweg von Itaconat eingebunden sind, kon-
nten bestimmt, deletiert und/oder überexpressioniert werden, um den tatsächlichen Bio-
syntheseweg aufzudecken. Das Entfernen des UM06344-Gens, das wahrscheinlich eine
cis-Aconitat-Decarboxylase kodiert, verringerte den Itaconat-Ausstoß nicht. Auch wenn
die Überexpression dieses Gens die Itaconat möglicherweise indirekt verbessert, ist es je-
doch nicht direkt beteiligt. Bei der phänotypischen Überprüfung der überexpressionier-
ten Mutanten wurden jedoch drei Mutanten gewonnen werden, die die Itaconat-Ausbeute
signifikant steigerten. Diese Mutanten wurden daraufhin unter ausgewählten Kultivier-
ungsbedingungen genauer charakterisiert. Die Spaltung des Gens UM11778, das ein 3-
Methylitaconat-∆-Isomerase (Mii) ähnliches Enzym kodiert, führte zu einer drastischen
Abnahme der Itaconat-Produktion. Das Mii Enzym kann die Isomerisierung zwischen Cit-
raconat und Itaconat katalysieren. Auf dieser Erkenntnis und der physiologischen Charakter-
isierung der Itaconat-Produktion von U . maydis aufbauend, werden mögliche Synthesewege
der Itaconat aufgezeigt.
Die biotechnologische Produktion der Chemikalien hängt stark von den genutzten Kultivier-
ungsbedingungen ab. Um einen optimalen Produktionsprozess zu gewährleisten, wurden
die Einflussfaktoren auf die Zellen und folglich auf die Itaconat-Ausbeute ermittelt und näher
untersucht. Als Faktor mit dem meisten Einfluss stellte sich hier die Sauerstoffzufuhr heraus.
Kulturen, die nur eine limitierte Sauerstoffzufuhr erhielten, konnten Itaconat nicht effizient
synthetisieren, wohingegen besser mit Sauerstoff versorgte Kulturen eine deutliche Produk-
tionssteigerung aufwiesen. Die Notwendigkeit ausreichender Sauerstoffzufuhr liegt in der
unverzichtbaren Kofaktor-Regeneration während der Itaconsäure-Produktion begründet.
Die Atmungskette ist die erste Regenerationsmöglichkeit nach Einstellung der Biomasse-
bildung, die den Hauptpfad der Kofaktor-Regeneration darstellt. Es ist anzumerken, dass
die Kofaktoren bei verringerter Sauerstoffzufuhr über Nebenprodukt-Synthese regeneriert
werden. Die beiden am häufigsten ausgeprägten Nebenprodukte sind Glykolipide und Malat.
Ein Mutant mit blockierter Glykolipid-Produktion zeigte eine gesteigerte Itaconat-Produktion
bezüglich des Wildtyp Stamms.
Die anfängliche Glukose-Konzentration stellt einen bedeutenden Faktor dar, so dass ver-
schiedene Glukosekonzentration eingestellt und die jeweiligen Auswirkungen auf die Zellen
als auch die sich jeweils ergebenden Itaconat-Ausbeuten untersucht wurden. Auch wenn die
Berechnungen ergeben, dass die maximale Itaconat-Ausbeute mit zunehmender initialer
Glukosekonzentration ansteigt, zeigen die durchgeführten Kultivierungsexperimente jedoch
dass die Zellen einem nicht zu vernachlässigbaren osmotischen Schock ausgesetzt werden,
wenn sie sich in hohen Glukosekonzentrationen befinden. Das Verständnis dieser Wirkungs-
beziehung führte zur Bestimmung der optimalen initialen Glukose-Konzentration für die
Itaconat-Produktion in U . maydis, die unter den getesteten Bedingungen einen Wert von 65
g l−1 ergab.
Die Itaconat-Ausbeute und Produktionsrate werden jedoch nicht nur durch die Erschöp-
fung des Stickstoffs (N), als Auslöser der Itaconat-Synthese, sondern auch durch die Stick-
stoffkonzentration beeinflusst. In der Theorie geht ein mehr an Stickstoff mit höheren Raten
aber geringerer Ausbeute einher und umgekehrt. Die Bildung der Biomasse nimmt hingegen
mit zunehmender Stickstoffkonzentration zu, so dass die Kultur bald darauf das eingestellte
Sauerstofflimit erreicht und sich die Itaconat-Produktion wieder verringert. Unter Berück-
sichtigung aller Fakten ergab sich eine optimale N-Konzentration von 0.8 g l−1 NH4Cl für die
Itaconat-Produktion von U . maydis.
Zur Reduktion der Fermentationskosten wurden alternative Kohlenstoffquellen für die Gewin-
nung der Itaconat getestet. U . maydis kann hierbei Xylose, Arabinose und Saccharose als
Substrate für die Itaconat-Produktion nutzen. Die Ausbeute mit diesen Zuckern war jedoch
sehr gering, so dass eine weitere Optimierung notwendig ist.
Basierend auf den Ergebnissen der vorliegenden Arbeit konnte U . maydis als passender
alternativer Itaconat-Produzent ermittelt werden. Obwohl die produzierte Menge an It-
aconat bisher nicht mit der durch A. terreus zu erreichenden Ausbeute mithalten kann, weist
U . maydis doch einige Vorteile auf, die im Vergleich mit Aspergillus und mit den vorgeschla-
genen Verbesserungen die Ausbeute noch weiter steigern werden können.
Acknowledgements
This thesis represents the final work of my Ph.D. study at the Institute of Applied Microbiology
at the RWTH Aachen University.
I would like to thank several individuals for their support and encouragement during the last
almost five years; Dr. Nicole Maaβen for her guidance throughout the practical part of the
work, Dr. Nick Wierckx for his much welcomed insights, ideas and patience during the writing
of the thesis. I would also like to thank him for the countless hours he spent proofreading
the manuscript. I would like to thank Prof. Dr. em. Ulrich Klinner, who has given me the
great opportunity to perform this challenging work. I would also like to express my gratitude
and respect to Prof. Dr. Michael Bölker for his much appreciated ideas, encouragement and
for being a member of the dissertation committee. I am also very grateful for his technical
support concerning cell strains, plasmids and primers. Moreover, I am very thankful for the
possibility he gave me to spend much valued time within his research team where I have
learnt methods I have used throughout this work. I would also like to thank Prof. Dr. Jan
Schirawski that he was so kind and agreed to be a member of the dissertation committee and
to write the report.
Last but not least, I would like to thank to Chris for his endless moral support, patience and
love, which have given me the strength to complete this thesis. A lot of thanks to Susan for
lifting up my spirits during dark times.
This work was performed as part of the Cluster of Excellence “Tailor-Made Fuels from Bio-
mass“, which is funded by the Excellence Initiative by the German federal and state govern-
ments to promote science and research at German universities.
Contents
1 Introduction 1
1.1 Biofuels for a sustainable society . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Alternative carbon sources for biofuel production . . . . . . . . . . . . . . . . . . 3
1.3 Itaconic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.4 Industrial bioproduction of itaconic acid . . . . . . . . . . . . . . . . . . . . . . . 6
1.5 Ustilago maydis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.6 Aim of this study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2 Materials and methods 11
2.1 Strains, plasmids and primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3 Cultivation methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.4 Analytical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.4.1 Fermentation broth analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.4.2 Off-gas analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.5 Molecular biological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.5.1 Genomic DNA isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.5.2 Plasmid DNA isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.5.3 Gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.5.4 In vitro DNA methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.5.5 Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.5.6 Southern blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3 Results 25
3.1 Selection of the gene of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2 Disruption of UM06344 in U. maydis . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.3 Overexpression of UM06344 in U. maydis . . . . . . . . . . . . . . . . . . . . . . . 27
3.3.1 Screening of transformants for increased itaconate production . . . . . . 28
3.3.2 Southern blot analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.4 Itaconate production kinetics under various cultivation conditions . . . . . . . 33
3.4.1 Influence of glucose concentration . . . . . . . . . . . . . . . . . . . . . . 34
3.4.2 Influence of culture aeration . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.4.3 Influence of nitrogen concentration . . . . . . . . . . . . . . . . . . . . . . 42
3.4.4 Selected optimal conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.5 Itaconate production during fermenter batch cultivation . . . . . . . . . . . . . 47
3.6 Biosurfactant production and its influence on the itaconate production . . . . 49
3.7 Alternative carbon sources for the itaconate production . . . . . . . . . . . . . . 51
3.8 Deletion of UM11778 in U. maydis . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4 Discussion 57
4.1 Itaconate synthesis pathway in U. maydis . . . . . . . . . . . . . . . . . . . . . . 57
4.2 Factors influencing the itaconate production . . . . . . . . . . . . . . . . . . . . 63
4.2.1 Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
4.2.2 Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4.2.3 Oxygen supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.2.4 Growth-limiting factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
4.3 Alternative carbon sources for itaconate production . . . . . . . . . . . . . . . . 70
4.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.5 Further recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
5 Supplementary data 75
List of Figures III
List of Tables V
Bibliography VII
CHAPTER 1
Introduction
1.1 Biofuels for a sustainable society
Increasing carbon dioxide emissions, a rising energy demand and limited availability of fossil
energy resources are leading to an ever growing necessity for a more sustainable society.
Especially transportation fuels, which are 98 % derived from petroleum, are highly depend-
ent on fossil resources. The use of these resources leads to high net CO2 emissions, and
the number of (often politically unstable) oil producing countries is small, leading to large
fluctuations in supply and price. Biofuels are considered a promising sustainable alternative
to petrochemical fuels. Their main advantage is that they are produced from biomass, which
means there is little net CO2 emission, and the biomass can be produced virtually everywhere
[1].
Up to now, research into biological transportation fuel substitutes has focused mainly on
ethanol. Saccharomyces cerevisiae is by far the most commonly used organism for ethanol
production, and this organism is used at a large scale for fuel production today. There are also
other ethanol fermenting organisms, such as Zymomonas mobilis [2]and Escherichia coli [3],
which have been engineered to produce ethanol at a high rate, titer and yield.
Alternatively, some molecules present in gasoline can also be produced by microorganisms,
both natural and engineered. The advantages of these molecules with regard to ethanol
are higher energy content, lower hygroscopicity and corrosivity. For instance, straight- and
branched-chain alkanes and alkenes can be produced through isoprenoid pathways, and
various alcohols and esters can be derived from fatty acid biosynthesis. Moreover, itaconic
acid, the newly identified key molecule for biofuel production [4], can be produced from
hydrolyzed biomass using engineered microorganisms (Figure 1.1).
1
Chapter 1: Introduction
Arabinose
Ribulose
R5P
Xylose
Xylulose X5P 6PG G6P
Glucose
G1P G´1P
Galactose
F6P
FBP
G3P
DHAP
M6P
Mannose
E4P
S7P
PEP
Pyruvate Formate Hydrogen
CO2
Acetaldehyd
Ethanol
CO2
Acetyl-CoA
CIT
ICT
OGA
SUC SUC-CoA
Glyoxylate
FUM
MAL
OAA
Propyonyl-CoA Propanol
Butyryl-CoA Butanol
Valeryl-CoA Isopentanol
Even-chain fatty acids
Alcohols
Odd-chain fatty acids
Even-chain fatty alcohol
Various esters
Odd-chain fatty alcohol
Even-chain alkane
Odd-chain alkane
IPP DMAPP
GPP
FPP
GGPP Geranylgeraniol
Diterpene
Farnesol
Sesquiterpene
Geraniol
Monoterpene
Isopentenol Isopentanol
Acetoacetyl-CoA
Cis-Ac
CO2
ITACONIC ACID
Figure 1.1: Overview of potential fuel precursors derived from various products of
hydrolyzed lignocellulose. Blue box: isoprenoid pathways and isoprenoid-
derived molecules. Yellow box: fatty acid pathways and fatty acid-derived
molecules. Green box: products of hydrolyzed lignocellulose. In green font
are shown short-chain alcohols. Abbreviations: 6PG, 6-phosphogluconate;
Cis-Ac, cis-aconitate; CIT, citrate; DHAP, dihydroxyacetone phosphate;
DMAPP, dimethylallyl pyrophosphate; E4P, erythrose-4-phosphate; F6P,
fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; FPP, farnesyl pyro-
phosphate; FUM, fumarate; G’1P, galactose-1-phosphate; G1P, glucose-1-
phosphate; G3P, glyceraldehyde-3-phosphate; G6P, glucose-6-phosphate;
GGPP, geranylgeranyl pyrophosphate; GPP, geranyl pyrophosphate; ICT,
isocitrate; IPP, isopentenyl pyrophosphate; M6P, mannose-6-phosphate;
MAL, malate; OAA, oxaloacetate; OGA, 2-oxoglutarate; PEP, phosphoen-
olpyruvate; R5P, ribose-5-phosphate; S7P, sedoheptulose-7-phosphate;
SUC, succinate; SUC-CoA, succinyl coenzyme A; X5P, xylulose-5-phosphate
(figure adapted from [5]).
2
1.2 Alternative carbon sources for biofuel production
High-yield production of these molecules using microbial systems could function as a re-
placement of some or all of the supply currently derived from petroleum [5].
Currently, there are two main biomass substrates for biofuel production. Cane juice is favored
in Brazil, while starch-crops like corn are used in the USA [6]. Corn is used as human food,
and the production of sugar cane competes with food production for arable land. Using some
of human’s main diet sources for ethanol production has raised an intense discussion about
its effect on food production and costs. These concerns were the base and the driving force
in development of new ways of biofuels production based on biomass that does not compete
with human food production [5].
1.2 Alternative carbon sources for biofuel production
The next generation of biofuels will most likely be developed from abundantly present ligno-
cellulosic biomass due to the political pressure and physical limitations on tillable land [5].
Moreover lignocellulosic biomass such as straw or wood is not used as human nutrient.
Therefore there is no competition between human food and biofuel production.
Lignocellulose consists of cellulose and hemicellulose, which are tightly bound to lignin [7].
Cellulose is the major structural component of cell wall of plants. This polysaccharide is
organized in microfibrils stabilized by hydrogen bonds [8] and consists of D-glucose units
linked together via β-1,4 glycosidic bonds into linear chains. The length of these chains varies
with the origin and treatment of the biomass [9]. The cellulose chains bind together through
hydrogen bonds to form microfibrils, which are in turn interconnected by hemicelluloses
and polymers of various other molecules (sugars, pectin). This structure is then covered by
lignin. Hemicellulose consists mainly of pentoses (xylose, arabinose) and hexoses (glucose,
mannose, galactose). It has a lower molecular weight than cellulose, and its polymer chains
are considerably more branched [8]. The composition of hemicelluloses varies depending on
the biomass source [7]. Lignin is an amorphous polymer which functions mainly as physical
support of the plant cell wall, giving it its firmness. It also functions as a barrier against
microbial attack [8].
Pretreatment of lignocellulose is a necessary step, its main function being to make the cellu-
lose and hemicelluloses available for cellulolytic enzymes. Several kinds of pretreatment are
used. These can be divided into physical (e.g. milling, irradiation), physico-chemical and
chemical (e.g. several types of explosion, usage of oxidizing agents) or biological approaches
(treatment with enzymes of fungi and Actinomycetes) [10]. Most of the techniques require
high temperatures, long run time, extreme pH levels and are energy intensive. Moreover,
the target of each of these methods is different and therefore also the quantitative inter-
action between the pretreatment parameters must still be clarified [11]. Hydrolysis of the
3
Chapter 1: Introduction
pretreated lignocellulose is performed mainly by highly specific enzymes (endoglucanases,
exoglucanases, β-glucosidases) [12] or by using diluted acids (sulfuric, phosphoric or nitric
acid) [13]. The most important factors influencing hydrolysis include porosity, cellulose fiber
crystallinity and lignin and cellulose content [12].
The lignocellulose hydrolysates resulting from the pretreatment contain a highly diverse
mixture of sugars, lignin and their degradation products. The main components are usually
glucose and xylose, although arabinose, galacturonic acid and rhamnose can also be found in
small concentrations. Lignin and its residues also represent a large part of most hydrolysates
[5]. In order to reach economically efficient bioconversion of this feedstock to bulk and
commodity chemicals, a high percentage of the available monosaccharides must be used
effectively.
Glucose utilization is common among microorganisms. Even though exceptions can be found
in E. coli mutant [14] or Pichia stipitis wt [15], which can utilize also C5 sugars, the ability to
metabolize other sugars, such as xylose and arabinose is rather rare. This work shows, that
U . maydis possesses such ability of utilization the above mentioned sugars.
The interest in engineering of strains capable of utilizing other monosaccharides rises.
S. cerevisiae [16], [17], [18] and Z . mobilis [2] have been engineered with heterologous path-
ways for xylose and arabinose utilization. Although these steps were made in order to optimize
ethanol production, these advances are also applicable for the production of other molecules,
which could be used for biofuel production [5].
1.3 Itaconic acid
Itaconic acid (ITA, CAS number: 97-65-4) is a promising new platform compound for produc-
tion of alternative biofuels and chemicals. Based on its industrial potential, it was selected by
US Department of Energy as one of the top 12 building block chemicals to be produced from
biomass [19].
Itaconic acid is a white crystalline powder soluble in water. More than 80 000 tons are pro-
duced per year, mostly for the synthesis of polymers, but also for bioactive compounds in
the fields of medicine and agriculture [20]. Several starting molecules for various purposes
originating from itaconic acid have been already identified (Figure 1.2).
4
1.3 Itaconic acid
Itaconic acid
3-Methyl THF
2-Methyl-1,4 BDO
Poly(itaconic acid)
Biofuels
Medical polymers
n
m n o
SBR Latex Technical polymers
Figure 1.2: Examples of possible itaconic acid application. Abbreviations: 3-MTHF -
3-methyltetrahydrofuran, MBD - 2-methyl-1,4-butanediol, SBR - styrene-
butadiene resin, [19], [21], [22].
There are chemical [23] and biological ways to produce itaconic acid. The biological way is
mostly studied in Aspergillus terreus, which produces itaconate via the Embden-Meyerhof-
-Parnas (EMP) pathway and the tricarboxylic acid (TCA) cycle. The enzymes involved in
the last part of the pathway are citrate synthase (EC: 2.3.3.1), pyruvate dehydrogenase (EC:
1.2.4.1), aconitase (EC: 4.2.1.3), malate dehydrogenase (EC: 1.1.1.37), pyruvate carboxylase
(EC: 6.4.1.1) and cis-aconitic acid decarboxylase (CAD; EC: 4.1.1.6). The first three enzymes
are localized in the mitochondrion, the last two enzymes in cytosol and malate dehydro-
genase can be found in both environments. The transport of the substrates between the
mitochondrion and the cytosol might be carried out via citrate/malate antiport (Figure 1.3)
[24].
The CAD activity was first identified by Bentley and Thiessen in A. terreus crude cell-free
extracts. This cytoplasmic enzyme [25] is implicated in the itaconate formation from the TCA
cycle intermediate cis-aconitate [26]. The sequence of the CAD gene of A. terreus has been
published quite recently [27]. Although this is the best characterized biochemical pathway
5
Chapter 1: Introduction
for the itaconate production, alternative routes may exist.
Figure 1.3: Proposed subcellular organization of the itaconate pathway in A. terreus.
Figure adapted from [24].
1.4 Industrial bioproduction of itaconic acid
Currently, almost all itaconic acid is produced using filamentous Aspergilli. Industrial itaconic
acid production by submerged fermentation of A. terreus was started by Pfizer Co. Inc. in
1955 in their plant in Brooklyn. This process was stopped in 1995. Nowadays, most of the
main itaconic acid suppliers come from China, some of them also from Japan, France and
the USA [20].
The optimal conditions for itaconate production by A. terreus are under phosphate limita-
tion with sugar concentrations between 100 and 150 g l−1 usually at 37 °C. Molasses is the
substrate of choice, but trace elements such as N, P, Fe, Mn, Mg, Cu and Zn significantly
influence the itaconate production, necessitating pre-purification of this substrate [28]. In
the initial growth phase the pH of the medium drops from 5 to less than 3. When phosphate
is depleted and sufficient biomass has been formed, the second (production) phase starts,
6
1.4 Industrial bioproduction of itaconic acid
where itaconate is produced, substantially free from other organic acids. In this phase, further
growth is prevented by keeping the phosphate level low.
It was previously shown that higher concentration of itaconate in culture broth inhibits its
production by A. terreus. Therefore the improvement of the production strain is focused on
engineering itaconate resistance [29]. However, depending on the cultivation conditions and
strain, A. terreus is able to produce 30 - 90 g l−1 itaconate in 3 - 25 days with a production rate
of 0.13 - 0.98 g l−1 h−1 and 0.34 - 0.62 g g−1 yield [30].
The process for the industrial purification of itaconic acid from the culture broth consists
of several steps; filtration in order to remove mycelia and other suspended solids, filtrate
concentration and itaconic acid crystallization (twice), decolorization of crystals with active
carbon, evaporation of the decolorized broth and recrystallization. Subsequently the itaconic
acid crystals are dried and packaged. The total itaconic acid recovery yield throughout the
whole process is approximately 80 % [20].
Filamentous fungi are known to be particularly difficult to handle in bioreactors [31]. Moreover,
certain fermentation conditions, such as substrate impurities, influence fermentations using
Aspergillus [32]. Also commercial fermentations must be cost-effective and therefore min-
imum of nutrients should be used. Furthermore utilization of both C5 and C6 sugars would
be a great advantage [19].
Therefore a search for alternative itaconate producers without disadvantages which are con-
nected with large scale cultivation of filamentous fungi is important. Many strains have
already been screened, both wild type and engineered (Table 1.1).
Among these, Ustilago is able to metabolize both C5 (arabinose, xylose) and C6 sugars (gluc-
ose, fructose) [this thesis, [33]]. This ability makes it an ideal microbial system for production
of biofuels and chemicals from hydrolyzed biomass.
Strain Itaconate concentration and time it was reached Reference
Pseudozyma antarctica 30 g l−1 [34]
Rhodotorula sp. 15 g l−1, 7 days [32]
Candida sp. 35 g l−1, 5 days [32]
Candida mutant 42 g l−1, 5 days [32]
Ustilago zeae * 15 g l−1, 5 days [35]
Ustilago maydis 53 g l−1, 5 days [32]
Table 1.1: Selection of alternative itaconate production hosts. * U . zeae is the former
designation for U . maydis.
7
Chapter 1: Introduction
1.5 Ustilago maydis
Ustilago maydis, a phytopathogenic basidiomycetous fungus, is the causative agent of smut
disease in corn (Figure 1.4A). U . maydis represents a microorganism with dimorphic life style.
The haploid form divides by budding, has a yeast-like appearance (sporidia) (Figure 1.4B)
and can be easily propagated on artificial media. The pathogenic dikaryotic filamentous
stage is formed by fusion of two sporidia that carry different alleles of a and b mating types
and needs its plant host to propagate [36].
Figure 1.4: (A) Smut disease on corn caused by dikaryotic form of U . maydis and (B)
the unicellular haploid form of U . maydis with buds (arrow), 630 ×.
U . maydis is a genetically well-characterized model organism and in 2006 its genome se-
quence has been published [37]. The genes are organized in highly compact manner and
many of them lack introns. The genome consists of about 6900 protein encoding genes and
many of them code for secreted proteins. The availability of genome sequence made identify-
ing of genes and metabolic pathways as well as developing U . maydis for biotechnological
purposes not only easier, but also quicker and more effective [38].
U . maydis is known to produce large concentrations of surface-active compounds. They can
be divided into two groups; mannosylerythritol lipids (MEL, ustilipids) and ustilagic acid.
MEL are found in the culture broth as extracellular oil droplets with higher density than water.
They consist of disaccharide units containing mannose and erythritol and a fatty acid of
8
1.6 Aim of this study
variable length. Ustilagic acid is a cellobiose lipid with antibiotic activity forming needle-like
crystals [38]. Both of these types of biosurfactants are produced under nitrogen (N) starvation
and their concentrations can reach up to 23 g l−1 [39]. The type of available carbon source is
the most important factor influencing the yield and ratio of both glycolipids [40].
The first large screening of Ustilago for metabolite products was performed in 1955 by Haskins
et al. [35]. 180 isolates underwent a screening for ustilipids and ustilagic acid production.
Several other metabolic products were identified, among them also the itaconate. In the
majority of the samples, more itaconate was produced in presence of CaCO3 than without.
This study represents the starting point of understanding Ustilago as a successful itaconate
producer.
The greatest advantage of industrial itaconic acid production using U . maydis is the fact, that
itaconate is secreted to the medium during cultivation by the yeast-like form of Ustilago and
therefore handling of the biomass in large scale fermenters even in high concentrations does
not bring the difficulties that are observed while fermenting filamentous fungi. Moreover
utilization of both C5 and C6 sugars by U . maydis is a fundamental industrial advantage.
Nevertheless as yet there is not much known about U . maydis and itaconate production. How-
ever, the preliminary study of Chandragiri and Sastry [41] and Rafi et al. [42] and the more
detailed study of Klement et al. [33] suggest that there is an increasing interest in this topic.
The goal of this work is to deliver more insights into this interesting but poorly studied subject.
1.6 Aim of this study
This study focuses on U . maydis as an alternative itaconate producer. The metabolic basis of
the itaconate production, as well as various aspects influencing the production process, was
investigated.
In order to find genes, which might be involved in the itaconate production pathway, mutants
with deletion and overexpression of genes possibly involved in the itaconate synthesis path-
way were created. These mutants were phenotypically characterized with regard to high-yield
itaconate production conditions. The influence of aeration of the culture, N concentration
as well as glucose concentration in the medium was studied. Moreover, various carbon
sources, which can be found in hydrolyzed biomass, were tested as potential substrates for
the itaconate production by U . maydis. Finally, the production of glycolipids as side products
of the itaconate synthesis was described.
Ultimately, the aim of this study is to optimize the itaconate production in U . maydis to such
extent, that it can be applied in sustainable production process.
9
Chapter 1: Introduction
10
CHAPTER 2
Materials and methods
2.1 Strains, plasmids and primers
Strain Description Origin
U . maydis MB215 wt DSM 17144, DSMZ Braun-
schweig, Germany *
U . maydis MB215 6_15 Overexpressed UM06344 This work
U . maydis MB215 6_17 Overexpressed UM06344 This work
U . maydis MB215 2_29 Overexpressed UM06344 This work
U . maydis MB215 ∆UM06344 ∆UM06344 *
U . maydis ∆cyp1∆emt1 ∆cyp1∆emt1, deleted cyp1 and
emt1 are responsible for glycol-
ipid production
[39] *
U . maydis MB215 ∆UM11778 ∆UM11778 *
E. coli DH5α ampR, fhuA2,
∆(argF − lacZ)U169,
phoA, glnV44, theta80,
delta(lacZ)M15, gyrA96, recA1,
relA1, endA1, thi−1, hsdR17
Strain no. 795, Cell collec-
tion of Institute of Applied
Microbiology, RWTH
Aachen University
S. cerevisiae DF5his1-1 his1−1, leu2−3, 2−112,
lys2−801, trp1−1, ura3−52
Strain was modified by H.
Ullrich from strain DH5
[43] *
Table 2.1: Strains used in this work. * Strains kindly provided by Prof. Michael Bölker.
11
Chapter 2: Materials and methods
Plasmid Description Origin
pMF1h ampR , hygR , oriC, lacZ , MCS in lacZ [44] *
pRS426 ori(f1), lacZ, T7 promoter, MCS (KpnI −SacI), T3
promoter, lacI , ori(pMB1), ampR , ori (2 micron),
URA3
*
pSMUT ampR , hygR [45] *
pMF2-3h hygR , Po2te f [44] *
UM06344 disruption vec-
tor
Integrated disruption cassette for UM06344, hygro-
mycin marker; derived from pRS426
This work
UM06344 overexpression
vector
Overexpressed UM06344; derived from pRS426 This work
Table 2.2: Plasmids used in this work. * Plasmids kindly provided by Prof. Michael
Bölker.
Primer Sequence 5’- 3’ Purpose
LF for * gtaacgccagggttttcccagtcacgac
gaatattcgcagcctgtcacgcggccca
acttg
Amplification of left flanking region
for UM06344 disruption vector
LF rev * attgtcacgccatggtggccatctaggc
cctggatacgaacgaaaacgagagatg
agc
Amplification of left flanking region
for UM06344 disruption vector
RF for * gtgcggccgcattaataggcctgagtg
gccactgggcagcagctagatcggcg
gacgc
Amplification of right flanking re-
gion for UM06344 disruption vec-
tor
RF rev * gcggattaacaatttcacacaggaaac
agcaatattgaaaagtcagatgggtaag
gagcg
Amplification of right flanking re-
gion for UM06344 disruption vec-
tor
Hyg pMF 2-3 h for ctgtcctgatttccaccctc Hygromycin probe for Southern
blot
Hyg pMF 2-3 h rev cagctatttacccgcaggac Hygromycin probe for Southern
blot
Continues on next page
12
2.1 Strains, plasmids and primers
Primer Sequence 5’- 3’ Purpose
pRS426+Hyg for * actcactatagggcgaattgggtacaata
ttggccgcactcgagtggcggcagatg
Amplification of hygromycin and
Po2te f for UM06344 overexpression
vector
Prom+CAD rev * tggattgggaaacgagtcgaggcatccgtg
gatgatgttgtctgtgtatg
Amplification of hygromycin and
Po2te f for UM06344 overexpression
vector
Prom+CAD for * catacacagacaacatcatccacggat
gcctcgactcgtttcccaatcca
Amplification of UM06344 gene for
UM06344 overexpression vector
CAD+pRS426 rev * ctcactaaagggaacaaaagctggagcaa
atattctactggtcgcgaacccag aggtca
Amplification of UM06344 gene for
UM06344 overexpression vector
Po2te f end for gctgtctcggcactatctttctttc Sequencing
UM06344 for agcagggctacaactcggta UM06344 probe for Southern blot
UM06344 rev tcttgatggtgacgctcttg UM06344 probe for Southern blot
UM06344 ORF rev taccgaattccgagttgcaagagcgtccg Amplification of UM06344 ORF
UM06344 ORF rev accggaattctccagatgcctcgactcg Amplification of UM06344 ORF
pUM UM06344 for ttaagttgggtaacgccagg Amplification of UM06344 overex-
pression cassette
pUM UM06344 rev ttccggctcctatgttgtgt Amplification of UM06344 overex-
pression cassette
Disruption cassette
for
aaagtgccatccatcctgga Amplification of integrated disrup-
tion cassette
Disruption cassette
rev
cgaaagcgtcgaagtggaag Amplification of integrated disrup-
tion cassette
Actin for cttcctgacggacaggtgat Amplification of part of actin gene
Actin rev tcctttcggatgtccaagtc Amplification of part of actin gene
pRS426-LF for acgacgttgtaaaacgacgg Sequencing
DownUM06344-LF
rev
agaggtctgcaatgcttggt Sequencing
DownUM06344-LF
for
agctctgcacagctctgttg Sequencing
Hyg-LF rev cgcgtgtcgattcacgtttt Sequencing
Hyg-RF r for ctgcaggcatgcaagcttca Sequencing
Hyg-RF for aacccagcatcaccaactgt Sequencing
Hyg-DownUM06344
rev
tgggcgagctcgaattcatc Sequencing
Continues on next page
13
Chapter 2: Materials and methods
Primer Sequence 5’- 3’ Purpose
pRS426-RF rev cgcaattaaccctcactaaaggg Sequencing
Primer 2 rev * cgataagccaggttaacctgtcttgttg
agacggatgagc
Screening
Table 2.3: List of used primers. * Primers were designed following the in vivo homo-
logous recombination [46].
2.2 Media
If not mentioned otherwise, all chemicals were supplied by Roth, Fluka or Merck.
YPD
10 g yeast extract
20 g pepton
20 g glucose
20 g agar for solid media
were dissolved in 1000 ml deionized water and sterilized in autoclave.
YPD-Hyg
YPD medium containing 400 µg ml−1 hygromycin (sterile filtered).
LB
10 g pepton
5 g NaCl
5 g yeast extract
20 g agar for solid media
were dissolved in 1000 ml deionized water and sterilized in autoclave.
LB-A
LB medium containing 100 mg l−1 ampicillin.
Modified Tabuchi medium (MTM; [47])
65, 110 or 250 g glucose, xylose, arabinose or sucrose
0.8, 1.6 or 4 g NH4Cl
0.5 g KH2PO4
0.2 g MgSO4
14
2.2 Media
0.14 g FeSO4 × 7H2O
1 g yeast extract
33 g CaCO3
was dissolved in such volume of water so that after sterilization in autoclave and subsequent
addition of appropriate volume of 50 % (w/w) sterile carbon source solution and 30 % (w/v)
sterile CaCO3 suspension total volume of 1 l medium was achieved.
SC-Ura
2.4 g (NH4)2SO4
1.7 g Difco Yeast Nitrogen Base w/o Amino Acids (BD)
12 g glucose
0.5 g Yeast Synthetic Drop-out Medium Supplement (Sigma)
10 g agar for solid media
were dissolved in 1000 ml deionized water and pH of 5.6 was adjusted using 10 M NaOH.
Consequently the mixture was sterilized in autoclave.
Regeneration agar
10 g yeast extract
20 g pepton
20 g sucrose
182.2 g sorbitol
15 g agar for solid media
were dissolved in 1000 ml deionized water and sterilized in autoclave. When medium cooled
down sufficiently, 400 µg ml−1 hygromycin (sterile filtered) was added. Agar plates were
poured according to Figure 2.1.
Figure 2.1: Outline of regeneration agar plate.
15
Chapter 2: Materials and methods
2.3 Cultivation methods
Cultivation on the solid media
All strains were transferred every two weeks on appropriate fresh plates and incubated for 24 -
48 h in 28 °C prior further use.
Cultivation in the liquid media
All strains were cultivated in 28 °C at 100 rpm on side-to-side motion agitator. If not men-
tioned otherwise, the cells were inoculated at starting OD O.5 AU (600 nm).
Precultures
If not mentioned otherwise, all precultures were cultivated in YPD and cells were washed
with 0.9 % NaCl solution before inoculation into main culture.
Precultures were carried out in 100 ml Erlenmeyer flasks containing 20 ml medium. The
incubation was performed at 28 °C and 100 rpm for 20 h.
Precultures for fermenter cultivation were carried out in 1 l Fernbach flasks containing 300
ml MTM supplemented with 30 g l−1 glucose, 1.6 g l−1 NH4Cl, w/o CaCO3. Cultivations were
carried out for 16 - 18 h in 28 °C at 100 rpm.
Cultivation for the screening tests
In order to screen overexpression mutants for increased itaconate production cells were
cultivated in 6-well plates (BD) containing 2.5 ml MTM (250 g l−1 glucose, 1.6 g l−1 NH4Cl).
Cells were cultivated in 28 °C at 100 rpm for 24, 48, 72 or 96 h, resp. Cultures were inoculated
with cells at OD 0.5 AU (600 nm).
To find out whether the disruption cassette was integrated into the right position in U . maydis
genome, DNA isolations were performed. For these purposes cells were cultivated in 10 ml
YPD in 100 ml Erlenmeyer flasks in 28 °C at 100 rpm for 24 h. Cultures were inoculated with
cells at OD 0.5 AU (600 nm).
Cultivation for the off-gas analysis
Cultivations in airtight DURAN bottles (Schott) were performed according to Robertz et al.
(1999) [48] with various filling volumes (10, 50 or 100 ml l−1, resp.) in MTM containing 65, 110
or 250 g l−1 glucose. In each case MTM was supplemented with 1.6 g l−1 NH4Cl. Bottles were
shaken in 28 °C at 100 rpm for about 144 h. Cells were inoculated at OD of 0.5 AU (600 nm).
Cultivation in the fermenter
Fermenter batch cultivations were performed in 2 l fermenter model Biostat A Plus (Sar-
torius).
16
2.3 Cultivation methods
Cells were collected from preculture by centrifugation (5 000 rpm, 5 min) and resuspended in
0.1 l MTM w/o CaCO3. The suspension was then transferred into the fermenter containing
1.4 l MTM w/o CaCO3.
Fermenter batch cultivation conditions were:
Working volume: 1.5 l
Temperature: 28 °C
Dissolved oxygen tension (DOT): 80 or 15 %
pH 6.
pH value was controlled automatically by adding 10 M NaOH. Sterile silicone antifoaming
emulsion was automatically added, when foam occurred.
An easyferm Plus K8 200 electrode (Hamilton) measured the pH level while an Oxyferm FDA
225 electrode (Hamilton) determined the DOT level.
If necessary, fermenters were cooled by Frigomix 1000 cooling system (Sartorius). Data were
collected using BioPAT MFCS/DA 2.1 software (Sartorius).
Cell growth quantification
Determination of optical density
The optical density was determined using Pharmacia LKB Ultrospec III (Pharmacia) at 600
nm through 1 cm thick cell layer.
In order to avoid the interference of the CaCO3 with the OD determination, 36% HCl was
used to dissolve the particles. In all cases 500 µl sample and 50 µl HCl (1/10 of the sample
volume) were used. However, the OD determination represents only an indication of the
biomass production, because the HCl used for the OD determination could precipitate some
of the cultivation products (i.e. organic acids or glycolipids).
The cells themselves were not influenced by the addition of HCl, which was demonstrated in
a separate experiment (data not shown). Briefly, the cells of U . maydis wt were cultivated for
144 h in YPD medium. Since there was no itaconate produced in this medium, the presence
of the buffer, CaCO3, which disturbs the OD determination, was not necessary. Each sample
was divided into two aliquots and only one was treated with HCl. Subsequently the OD was
measured in both aliquots.
Determination of the dry cell biomass from the fermenter batch cultivations
The samples of cell cultures of 5 ml were filtered through filter paper type MN 218 B (Macherey-
Nagel). These were washed with 5 ml deionized water, dried for 72 hours at 65 °C and
consequently weighted.
17
Chapter 2: Materials and methods
2.4 Analytical methods
2.4.1 Fermentation broth analysis
Samples were taken from a homogenous culture using a sterile pipet. A syringe was filled with
the content of the pipet and sterile filtered. The filtrate was used for organic acids, carbon
sources and TCA cycle intermediates determination.
Determination of the itaconate content
In order to verify the occurance of the itaconate in the culture broth, a fraction containing
the itaconate peak was isolated and its presence was confirmed by means of NMR (data not
shown; personal communication with Dr. Nicolas Lassauque).
Itaconate concentration determination was performed using Beckman Coulter System Gold
series HPLC (Beckman Coulter) consisting of an autosampler, binary pump, thermostatted
compartment for column, injection system and UV detector. The separation of cultivation
products was accomplished with LiChrospher 100 RP-18 column (5 µm particle size, Merck).
Chromatographic conditions were: mobile phase, 0.1 % phosphoric acid; flow-rate, 2 ml
min−1; injection volume, 30 µl; column temperature, 60 °C; UV detection, 210 nm; run time,
8 min, autosampler temperature: 4 °C. Data were recorded and processed using 32 Karat
Software (Beckman Coulter).
Determination of the carbon sources content
Glucose, xylose, arabinose and sucrose concentration was detected using HPLC based on
measurement of refraction index.
Determination was carried out using a set containing SDS 9414 Solvent Delivery System
(Schambeck SFD), thermostatted compartment for column type SFD 12560 (Schambeck SFD)
and refractometric detector RI 2000 (Schambeck SFD). Samples were injected to the system
manually using Exmire Microsyringe. Data were collected using PeakSimple Chromatography
Data System SRI Model 333 (SRI Instruments) and processed with PeakSimple 3.21 software
(Schambeck SFD).
Glucose, xylose and arabinose were separated using column filled with organic-acid resins
(300 × 0.8 mm) and fitting pre-column (CS-Chromatographie Service). Deionized water was
used as mobile phase.
Sucrose was separated using column filled with carbohydrate Ca2+ (300 × 0.8 mm) and fitting
pre-column (CS-Chromatographie Service). 0.016 M H2SO4 was used as mobile phase.
In both cases the chromatographic conditions were: column temperature, 60 °C; injection
volume, 20 µl; run time, 10 min (sucrose: 15 min); flow-rate, 0.8 ml min−1.
18
2.4 Analytical methods
Determination of TCA cycle intermediates
Fumarate, succinate, malate and pyruvate titers were determined using UltiMate 3000 HPLC
system (Dionex). The system consisted of a degasser, isocratic pump, autosampler, ther-
mostatted compartment for column and variable wavelength UV detector. The TCA cycle
intermediates separation was based on the ion exchange mehanism using Trentec 308R-
Gel.H column (300 × 0.8 mm, Trentec Anylysentechnik). The determination parameters
were: mobile phase, 5 mM H2SO4; flow-rate, 0.6 ml min−1; injection volume, 5 µl; column
temperature, 25 °C; UV detection, 210 nm; run time, 30 min, autosampler temperature: 4 °C.
Data were recorded and processed using Chromeleon Software (Dionex).
Determination of the ammonium content
The consumption or release of ammonium was determined by means of Kjeldahl method
[49]. Briefly, the sample was distilled together with NaOH and back titrated using Na2CO3
solution.
Surface tension determination
The surface tension of fermentation broth was measured using table-top tensiometer (Krüss),
using the du Noüy ring method.
Lipid-specific staining
The samples containing lipids underwent the lipid-specific staining according to Burdon
(1946) [50]. Solutions of Sudan black B and Safranin were used.
Cell photographs
Photographs of microscopical view of cells were obtained using Leica DM750 microscope
containing built-in camera type Leica ICC50 Digital Camera Module (Leica Microsystems).
Photographs were processed using either Debut Video Capture Software or LAS EZ software
(Leica Microsystems).
2.4.2 Off-gas analysis
The biochemical oxygen demand (BOD), CO2 production (CBP) and respiratory quotient
(RQ) was followed using OxiTop system [48].
Each sample was set in 2 parallels, one for BOD (w/o NaOH) and the other for CBP (with
NaOH) determination. The flask for CBP determination contained filter paper soaked with
50% (w/v) NaOH solution for CO2 absorption. The other flask (for BOD determination) was
set without NaOH. While BOD was measured automatically by a sensor attached to the flask,
19
Chapter 2: Materials and methods
the CBP was calculated from BOD.
In order to find out whether the cells in the poorly aerated culture were cultivated under
oxygen limited conditions, calculations were performed based on the formula:
6 O2 + C6H12O6 → 6 CO2 + 6 H2O.
Results from the fermenter batch cultivation showed, that one half of utilized glucose was
used for biomass production (27 g/l utilized glucose/14 g/l biomass/first 24 h) and the other
half for energy generation. The amount of oxygen needed by the cells in the first 24 h of cul-
tivation to convert half of the utilized glucose into energy was calculated from the following
formula:
n (O2) = c(g l ucose)M(g l ucose) ∗ 0.5 ∗ 6
V q ;
where n is the amount of substance [mol], c is concentration [g/l], M is molecular weight
[g/mol], 0.5 represents the half of the utilized glucose used to generate energy, 6 is molar
coefficient of oxygen (from formula 1) and Vq is the quotient between 1 l to which all the
calculations were applied and the culture volume [l].
Consequently the amount of oxygen available to the cells at the beginning of the experiment
was calculated from the formula:
n (O2) = V22.4 ∗ 0.21;
where n is the amount of substance [mol], V is the volume of the gas phase present in the flask
[l], 22.4 is the volume of 1 mol of gas [l] and 0.21 represents the relative amount of oxygen in
air. The values used for the calculations (concentration of utilized glucose) were obtained
from the experiment dealing with the influence of the culture aeration on the itaconate
production. While in the poorly aerated cultures, the cells utilized 25 g glucose in the first 24
h, the cells in the highly aerated culture utilized 37 g glucose.
The RQ values were calculated based of the following formula:
RQ = |BODC BP |;
where the BOD and the CBP are first recalculated from g/l to mol.
20
2.5 Molecular biological methods
2.5 Molecular biological methods
2.5.1 Genomic DNA isolation
The isolation of U . maydis genomic DNA was performed as described by Hoffman and Win-
ston (1987) [51] with some modifications. The final protocol was as follows:
• Cultivate the cells in 10 ml culture
• Collect the cells by centrifugation (5 000 rpm, 5 min) and resuspend the pellet in 0.5 ml
water
• Spin down the cell suspension (5 000 rpm, 1 min), discard the supernatant and resus-
pend the cells in remaining water
• Add 0.4 ml of buffer A (2 % Triton X-100, 1 % SDS, 100 mM NaCl, 10 mM Tris-HCl pH
8.0, 1 mM EDTA) and transfer the suspension to glass tube
• Add 0.4 ml phenol-chloroform-isoamyl alcohol (25:24:1) and 0.3 g acid-washed glass
beads (0.75 - 1 mm)
• Vortex for 2 × 5 min
• Add 0.2 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and spin down (12 000 rpm,
5 min)
• Transfer the aqueous layer to a fresh tube, add 1 ml 100 % ethanol and mix by inverting
• Spin it down (12 000 rpm, 2 min), decant the supernatant and add 0.4 ml TE buffer and
5 µl RNAse (10 mg ml−1, Roche)
• Incubate the mixture for 1 h in 37 °C
• Add 0.5 ml phenol-chloroform-isoamyl alcohol (25:24:1) and vortex strongly for 20 sec
• Spin it down (12 000 rpm, 10 min), transfer the aqueous layer to a fresh tube, add 0.5
ml chloroform-isoamyl alcohol (24:1) - repeat this step 3 ×
• Add 0.3 M ammonium acetate-ethanol solution - 2.5 fold sample volume, mix by
inverting and spin it down (12 000 rpm, 2 min)
• Discard the supernatant and dry the pellet
• Resuspend the pellet in 50 µl nuclease-free water and store it in 4 °C.
21
Chapter 2: Materials and methods
2.5.2 Plasmid DNA isolation
Plasmid DNA was isolated using either the QIAprep Spin Microprep Kit (Qiagen) or the In-
visorb Spin Plasmid Mini Two (stratec molecular).
2.5.3 Gel electrophoresis
The DNA fragments were separated using 1 % agarose gel in TAE buffer (4.84 g l−1 Tris, 0.372
g l−1 Na2EDTA, pH 8.0). The separation was performed in 75 V environment.
Consequently, the gel was stained with ethidium bromide solution and evaluated in UV light
(265 nm).
Fermentas GeneRuler 1 kb Ladder or Fermentas GeneRuler 100 bp Ladder (Fermentas) was
used as DNA fragment size standard.
For the extraction of the DNA fragments from gel, Biozym LE GP Agarose (Biozym) and Min-
iElute Gel Extraction Kit (Qiagen) were used. The gel concentration was chosen appropriately
to the DNA fragment size.
2.5.4 In vitro DNA methods
Purification of DNA
The restricted or amplified DNA was purified using High Pure PCR Product Purification Kit
(Roche) or MSB Spin PCRapace (Invitek).
DNA concentration determination
The concentration of the isolated genomic DNA was determined using UV-VIS Spectropho-
tometer UV-1650PC (Schimadzu) and UVProbe 2.31 software (Schimadzu) at 260 nm and
concentration was calculated using the following formula:
(dsDNA) = A260 nm ∗ 50 µl ml−1 ∗ dilution factor.
The concentration of plasmid DNA was estimated from gel of appropriate concentration
using DNA frangment size standard (Fermentas, see Gel electrophoresis).
Polymerase chain reaction (PCR)
All reactions were performed as described by Newton and Graham (1994) [52] using GoTaq
DNA Polymerase (Promega).
For the amplification of the ORFs and the DNA fragments for sequencing, Phusion High-
Fidelity DNA Polymerase (Biozym) was used.
All reactions were performed using FlexCycler (analytik Jena).
22
2.5 Molecular biological methods
DNA sequencing
For the sequencing of the DNA fragments services of either 4base lab or Eurofins MWG
Operon were used.
2.5.5 Transformation
The transformation of the E. coli DH5α followed the heat shock method by Sambrook et. al
(1989) [53].
The yeast cells were transformed according to Gietz and Woods (2002) [54] using the lithium
acetate/ss DNA/PEG method. The in vivo homologous recombination was used for vector
construction [46].
The transformation of U . maydis cells was performed using protoplasts. The protocol fol-
lowed the method by Tsukuda et al. (1988) [55] using STC/PEG and sorbitol solution.
2.5.6 Southern blot
The Southern blot assays were performed as described by Ausubel et. al (1991) [56].
For the transfer of the DNA fragments pressure was applied evenly on the gel and the mem-
brane. For this purpose a stack of paper towels and weight was used.
The DIG Easy Hyb solution, Blocking reagent, Anti-Dioxigenin-AP Fab fragments, CDP Star
solution, DIG DNA Labeling mix for probe labeling, DNA Molecular Weight Marker VII, DIG-
labeled (all Roche) and Biodyne B 0.45 µm membrane (Pall Corporation) were used.
23
Chapter 2: Materials and methods
24
CHAPTER 3
Results
3.1 Selection of the gene of interest
In A. terreus, the cis-aconitate decarboxylase (CAD), an enzyme that converts cis-aconitate
into itaconate, plays a crucial role in the itaconate production [27]. A sequence of the gene
coding for CAD in A. terreus, was recently published by Kanamasa et al. [27]. The biosynthetic
pathway of the itaconate in U . maydis is not known, but the gene UM06344 encodes a protein
with 23% identity to the CAD gene of A. terreus (ATEG 09971). Although the similarity between
the A. terreus and U . maydis proteins is very low, UM06433 was investigated in more detail in
order to learn whether it plays a role in the itaconate production.
3.2 Disruption of UM06344 in U. maydis
In order to knockout the UM06344 gene a disruption cassette was constructed according to
Mézard et al. [46] and Jansen et al. [57]. This method is based on the homologous recom-
bination system of S. cerevisiae. The vector consisted of carrier plasmid pRS426, containing
the ampicillin selection marker and the ura3 gene, left (LF) and right (RF) flanking regions of
UM06344 and a hygromycin resistance marker (Figure 3.1).
The individual cassette parts were amplified using PCR and chimeric primers (LF for, LF
rev, RF for and RF rev; see Materials and methods, Table 2.3). In order to verify, whether
the constructs were correct, restriction analysis with SspI restriction enzyme (Roche) and
sequencing were performed. Four out of twelve tested constructs were correct.
Selected correct constructs were restricted with SspI and transformed into U . maydis proto-
plasts. About 5 µg DNA was transformed, resulting in about 150 colonies per plate after 120 -
25
Chapter 3: Results
168 h of cultivation. Some of the colonies were cultivated further in shaken flasks and those
with sufficient growth (about 80 transformants) were submitted to screening.
Figure 3.1: Map of UM06344 disruption vector (A), outline of the region around the
UM06344 gene in U . maydis genome (B) and the cassette integrated into
U . maydis genome (C). (A) HygR : hygromycin resistance marker, RF: right
flank, AmpR : ampicillin resistance marker, LF: left flank.
Genomic DNA from transformants was isolated and PCRs with three different sets of primers
were performed (disruption cassette for and rev, primer 2 rev and ORF RF rev; see Materi-
als and methods, Table 2.3). The aim was to find an UM06344 deletion mutant. Although
the cultivation experiments in YPD containing hygromycin suggested that the cassette was
successfully integrated into the genome, no bands of the correct size were found in the PCR
analyses. Thus, obtaining the mutants with deleted UM06344 gene was not successful.
A mutant with deleted UM06344 gene was kindly provided later by Prof. M. Bölker. A com-
parison of the itaconate production between U . maydis wt and ∆UM06344 mutant strain is
shown in Figure 3.2.
Substantially different results from cultivation experiments were obtained. If UM06344 in-
deed encodes a CAD enzyme, the deletion of this gene would likely lead to reduced itaconate
production. Figure 3.2A shows significantly increased itaconate titer from the ∆UM06344
mutant in comparison to the wt strain. However, the results from further cultivations were
inconsistent and did not confirm previous findings (Figure 3.2B). Nevertheless, none of the
cultures exhibited decreased itaconate production by the ∆UM06344 mutant with regard to
the wt, indicating that the UM06344 gene does not encode a CAD enzyme.
26
3.3 Overexpression of UM06344 in U. maydis
Figure 3.2: Production kinetics of the itaconate under various conditions. Cells were
cultivated in (A) 10 ml MTM containing 65 g l−1 glucose, 0.8 g l−1 NH4Cl
and CaCO3 (n = 3; the values are mean of three analytical determinations
± standard deviation) and (B) 50 ml MTM containing 65 g l−1 glucose,
1.6 g l−1 NH4Cl and CaCO3 (n = 1). Green line: U . maydis wt, black line:
U . maydis ∆UM06344 mutant. 5.1
3.3 Overexpression of UM06344 in U. maydis
Although the results from the experiments with the ∆UM06344 mutant were inconsistent,
certain cultures did show that this gene might be otherwise related to the itaconate produc-
tion. Therefore, in order to obtain additional information, an overexpression construct of this
gene was created.
The UM06344 overexpression vector construction was also based on the protocols of Mézard
et al. [46] and Jansen et al. [57]. The vector consisted of hygromycin marker, Po2tef promoter
and UM06344 ORF. Plasmid pRS426 containing an ampicillin resistance marker and ura3
gene was used as a carrier plasmid. The map of this construct is shown in Figure 3.3. Each part
of the construct was amplified via PCR using chimeric primers (pRS426+Hyg for, Prom+CAD
for and rev and CAD+pRS426 rev; see Materials and methods, Table 2.3). The constructs were
tested via restriction with NdeI (Fermentas). Ten of twelve tested constructs proved to be
correct, which was later confirmed by sequencing.
Prior the transformation, the vector was restricted using SspI and the mixture was heat in-
activated. About 5 µg of the DNA was transformed into the Ustilago maydis wt protoplasts.
Transformation resulted in 0 - 80 colonies per plate after 120 - 168 h of cultivation.
27
Chapter 3: Results
Figure 3.3: Outline of the UM06344 overexpression vector. HygR : hygromycin resist-
ance marker, AmpR : ampicillin resistance marker.
3.3.1 Screening of transformants for increased itaconate production
One hundred thirty eight transformants were submitted to screening for increased itaconate
production. The cells were cultivated in 6-well plates, where each well contained 2.5 ml MTM
with 250 g l−1 glucose, 1.6 g l−1 NH4Cl and CaCO3. The plates were cultivated in 28 °C at
100 rpm for 24-96 hours. The culture broth was filtered and the itaconate production was
analyzed by HPLC.
The UM06344 overexpression cassette contained four different DNA regions originating from
U . maydis genome. Three integration events could occur (Figure 3.5). (A) Random integra-
tion of single or multiple copies of the linearized cassette into the genome [58]. In this case
random gene-knockout takes place. (B) Integration of the cassette based on the homologous
recombination at a non-random site (in this case probably an exchange of the hsp70 gene).
As a result, the Po2tef promoter and the UM06344 ORF would get lost. (C) Integration based
on a single cross-over between any of the homologous cassette parts of the circular vector
and the genome. Although this last event is improbable since the vector was digested with
SspI before transformation, it is still possible that a trace of undigested vector remained.
In order to find a mutant where integration of the construct led to a gene knockout which
is beneficial for the itaconate production, 138 individual transformants were screened for
increased the itaconate production (Figure 3.4).
28
3.3 Overexpression of UM06344 in U. maydis
Figure 3.4: Itaconate production by UM06344 overexpression cassette transformants
compared to the U . maydis wt. The wt itaconate titers were used as 100 %.
In average, the mutants produced 1.2-fold more itaconate with regard to the wt. About half of
the mutants produced itaconate in a standard deviation range. However, the graph (Figure 3.4)
is clearly asymmetrical towards the improved production (42 mutants produced significantly
more itaconate and 27 significantly less itaconate with regard to the wt), indicating that even
though the UM06344 gene is unlikely to encode a CAD enzyme, its overexpression or random
integration of the construct does have a certain positive effect on the itaconate production.
Transformants with significantly higher itaconate titer were present. The effect of UM06344
overexpression or random integration of the construct is considered to be small. The increase
in the itaconate production was most likely due to a positional effect of a random insertion
of the overexpression construct into the genome (Figure 3.5A). The three mutants with the
highest itaconate production (U . maydis 6_15, 6_17 and 2_29) were submitted to further
characterization.
29
Chapter 3: Results
3.3.2 Southern blot analyses
As discussed previously, there are three different ways of integration of the UM06344 over-
expression cassette. To find out whether and how UM06344 overexpression construct was
integrated into the genome of the selected mutants, Southern blot analyses were performed.
Genomic DNA was digested with BamHI (Roche) and probes for the hygromycin resistance
marker and the UM06344 gene were used (Figure 3.5A,B).
In the case of the hygromycin probe, there was a band visible in the wt lane as well as in
the transformants lanes. Since this band was present in the wt sample, it had to be un-
specific. This may be explained by the position of the hygromycin probe in the cassette,
where the probe consisted partly of the hygromycin ORF and partly of the Hsp70 promoter
(Figure 3.5A,B). The presence of this promoter, originating from the U . maydis genome, de-
creased the specificity of the probe.
Each of the transformants exhibited a 4.9 kb band which corresponds to the original UM06344
gene (Figure 3.6). If the overexpression cassette would integrate into the genome based on
the single cross-over (Figure 3.5C), the bands on the Southern blot film would be as follows:
UM06344: 4946 and 10141 bp; Po2tef : 18403 bp; Hsp70 promotor: 12346 bp and in case of
Hsp70 terminator: 16077 bp. None of these bands are present on the film (Figure 3.6). This
indicates that the overexpression cassette did not integrate into the genomic DNA based
on single cross-over. Moreover, the transformant 6_15 exhibited a clearly brighter band
(Figure 3.6A1, B), which is most likely a result of multiple tandem integrations of the linear
construct. The integration numbers are summarized in Table 3.1.
The results from the Southern blot analyses revealed, that each of the selected mutants con-
tained at least one extra copy of the UM06344 gene in its genome.
Moreover, the UM06344 overexpression cassette integrated into the genome of each strain
based on the scenario in Figure 3.5A and therefore a random gene-knockout can be expected.
30
3.3 Overexpression of UM06344 in U. maydis
gDNAPo2
tef
C
UM06344 ORF
pBR322 originAmpR
2μ origin
ura3
HygR
Figure 3.5: Outline of the three possibilities of the integration of the UM06344 over-
expression cassette into the gDNA of U . maydis. (A) Random insertion in
the genome, (B) double cross-over between the Hsp70 promotor and the
terminator resulting in the loss of the Po2tef and UM06344 ORF parts and
(C) single cross-over of the circular plasmid with any of the homologous
regions in the cassette, here with the Po2tef promoter as an example. AmpR :
ampicillin resistance marker, HygR : hygromycin resistance marker.
31
Chapter 3: Results
Figure 3.6: Southern blot analysis of the genomic DNA of U . maydis wt and the trans-
formants with integrated UM06344 overexpression cassette (6_15, 6_17,
2_29). (A1, A2) UM06344 probe and (B) hygromycin probe was used. The
black arrows show the original UM06344 gene (A1, A2) or the unspecific
annealing position for hygromycin gene (B), the red arrows show the integ-
rated UM06344 gene (A1, A2) or gene coding for hygromycin originating
from the overexpression cassette (B). Abbreviations: M - marker (DNA
fragment sizes are shown in bp).
Transformant No. of UM06344 genes No. of hygromycin genes
U . maydis 6_15 3 + 1 original 3
U . maydis 6_17 1 + 1 original 1
U . maydis 2_29 1 + 1 original 2
Table 3.1: Summary of integration numbers of UM06344 overexpression cassette
parts.
32
3.4 Itaconate production kinetics under various cultivation conditions
3.4 Itaconate production kinetics under various
cultivation conditions
After the production mutants were selected, the aim of the further investigation focused on
selection of optimal cultivation conditions with regard to high-yield itaconate production.
A certain pattern in the distribution of the lines representing each determined parameter
was visible among all the strains and all studied conditions. Figure 3.7 shows an example of a
cultivation chart. The U . maydis wt cells were cultivated in 50 ml MTM containing 250 g l−1
glucose, 1.6 g l−1 NH4Cl and CaCO3 (basic cultivation conditions, Table 3.2). This chart rep-
resents a typical cultivation graph, which can be divided into two distinct phases: 1) A growth
phase, with a high rate of biomass growth and no itaconate production. 2) A production
phase, where the itaconate is produced while the biomass growth rate is greatly decreased.
The trigger for the transition from phase 1 to phase 2 was the ammonium depletion, which
usually occurred within the first 24 - 48 h.
Figure 3.7: A typical cultivation chart of U . maydis wt. Green line: glucose utilization,
blue line: OD, red line: ammonium consumption, yellow line: itaconate
production. Cells were cultivated under the basic cultivation conditions.
The MTM contained CaCO3. The values are mean of three analytical
determinations ± standard deviation. 5.2
33
Chapter 3: Results
Aside of the itaconate, a high concentration of malate is produced. This phenomenon is
known from the literature [47] and is strain-dependent.
In order to produce the itaconate with the highest possible titer and in the most efficient way,
several cultivation conditions which may influence the itaconate concentration were tested
in further experiments.
Briefly, three parameters were studied; glucose and ammonium concentration and culture
aeration. Two of these parameters were always kept constant and the third one varied (Table
3.2).
Condition 1 Condition 2 Condition 3
Setup 1 1.6 g l−1 NH4Cl 50 ml culture
65 g l−1 glucose
110 g l−1 glucose
250 g l−1 glucose
Setup 2 250 g l−1 glucose 1.6 g l−1 NH4Cl
10 ml culture
50 ml culture
100 ml culture
Setup 3 50 ml culture 250 g l−1 glucose
0.8 g l−1 NH4Cl
1.6 g l−1 NH4Cl
4 g l−1 NH4Cl
Table 3.2: Experimental design of optimization of the itaconate production paramet-
ers.
3.4.1 Influence of glucose concentration
Since the carbon source concentration is known to affect the biomass production and the
itaconate yield and titer in A. terreus [59], cultivation experiments with varying initial gluc-
ose concentrations were performed in order to find the optimal glucose concentration for
U . maydis.
Three different glucose concentrations were applied, 65, 110 and 250 g l−1. The cells were
cultivated in 50 ml MTM per 1 l flask in medium containing 1.6 g l−1 NH4Cl and CaCO3. As
can be seen in Figure 3.8, the high concentrations of available initial glucose seemed to have a
negative effect on the itaconate production. In the experiments with 65 and 250 g l−1 glucose,
the highest itaconate production rate was observed between 72 and 96 h while with 110 g l−1
glucose it was between 120 and 144 h of cultivation. In the case of 65 g l−1 glucose the average
itaconate production rate was 0.109 ± 0.051 g l−1 h−1, with 250 g l−1 glucose it was 0.027 ±0.017 g l−1 h−1 and with 110 g l−1 glucose 0.06 ± 0.016 g l−1 h−1.
34
3.4 Itaconate production kinetics under various cultivation conditions
Figure 3.8: The ammonium utilization (dashed lines) and the itaconate production
(full lines) in MTM containing 250 (A) and 65 (B) g l−1 glucose. Green
lines: U . maydis wt, red lines: U . maydis 6_15, blue lines: U . maydis 6_17
and yellow lines: U . maydis 2_29. The values are mean of three analytical
determinations ± standard deviation. 5.3
In the cultures with the high glucose concentration, the substrate was not yet depleted and the
itaconate titer was still increasing, suggesting that the itaconate production was not finished
after 144 h. Therefore, the final itaconate titer could be much higher. The mutant strains
exhausted the 65 g l−1 glucose already within 120 h. The end-point glucose concentrations as
well as the final OD and itaconate yield values are summarized in Table 3.3.
After 144 h, significantly more itaconate was produced from the medium containing 65 g l−1
glucose. The high glucose concentration seemed to have a negative effect on the itaconate
production rate and yield. The reason might be the osmotic stress caused by such high
glucose concentration. Indeed, the ammonium was depleted at a lower rate in the culture
with 250 g l−1 glucose, indicating that the growth rate was decreased under this condition. In
the experiments with only 110 g l−1 glucose the itaconate production did not improve much
compared to the 250 g l−1 culture.
An overview of the influence of the glucose concentration on the final itaconate titer is
summarized in Figure 3.9. While the itaconate titers obtained from the two higher glucose
concentrations (250 and 110 g l−1) were in average very similar, the final itaconate titers
obtained with the 65 g l−1 initial glucose concentration were significantly higher. Moreover,
all the three selected mutants produced substantially more itaconate with regard to the wt,
where the effect of the glucose concentration was the least visible.
35
Chapter 3: Results
Initial glucose
concentration
U . maydis
wt
U . maydis
6_15
U . maydis
6_17
U . maydis
2_29
OD (AU)
65 g l−1 17.0 ± 0.4 17.8 ± 1.4 15.3 ± 1.7 13.1 ± 1.5
110 g l−1 22.1 ± 0.7 25.9 ± 2.0 29.1 ± 0.6 19.5 ± 1.8
250 g l−1 17.9 ± 0.8 24.3 ± 1.2 25.5 ± 1.2 19.7 ± 1.4
Glucose (g l−1)
65 g l−1 2.0 ± 0.8 0 0 0
110 g l−1 64.0 ± 5.0 64.5 ± 15.6 67.8 ± 7.0 56.4 ± 11.5
250 g l−1 152.4 ± 6.1 145.8 ± 1.7 160.0 ± 3.5 169.7 ± 11.8
Yield (g g−1)
65 g l−1 0.05 0.121 0.074 0.161
110 g l−1 0.041 0.065 0.053 0.106
250 g l−1 0.032 0.017 0.025 0.051
Table 3.3: The summary of the end-point values of OD (AU), glucose concentration (g
l−1) and yield (itaconate/utilized glucose, g g−1). The values (mean of three
analytical determinations ± standard deviation) were obtained after 144 h
of cultivation.
Figure 3.9: An overview of the influence of the initial glucose concentration on the
itaconate titer. Initial glucose concentration: 250 g l−1 (red columns), 110
g l−1 (green columns) and 65 g l−1 (orange columns). The values (mean
of three analytical determinations ± standard deviation) were obtained
after 144 h of cultivation. 5.4
36
3.4 Itaconate production kinetics under various cultivation conditions
In order to verify the hypothesis concerning the influence of the osmotic stress caused by
the high glucose concentration, a cultivation experiment in air-tight flasks with medium
containing the high and low glucose concentrations was performed. In these flasks, the
biological oxygen demand (BOD) and the biological production of CO2 (CBP) were studied.
The results are shown in Figure 3.10. Since there were no differences among the four strains,
the chart of the 2_29 mutant was used as an example.
Figure 3.10: Influence of various glucose concentration in MTM on BOD and CBP in
the U . maydis 2_29 strain. The cells were cultivated in 50 ml MTM in 1 l
flask containing 1.6 g l−1 NH4Cl and CaCO3. Blue line: BOD (250 g l−1
glucose), red line: BOD (65 g l−1), orange line: CBP (250 g l−1) and green
line: CBP (65 g l−1 glucose). The values in are mean of three analytical
determinations.
The most apparent differences in the BOD and the CBP were present between 8 and 36 h of
cultivation. Then the values reached stationary phase and the previous differences caused by
the glucose concentration vanished.
The presence of the high glucose concentration and consequently also the high osmotic
pressure led to slower O2 consumption and CO2 production compared to the low glucose
concentration. This indicates decreased growth rate and therefore overall lower metabolism
level (e.g. N utilization in Figure 3.8A).
37
Chapter 3: Results
The respiratory quotient (RQ) was among all the four strains the same - slightly above 1 (data
not shown). The reason, why the RQ is above 1 could be the extra CO2 released from the
reaction between CaCO3 present in the culture and the acids produced by the cells.
3.4.2 Influence of culture aeration
The aeration of the culture, as one of the putative factors influencing the itaconate production,
was studied.
The cells of U . maydis wt as well as the 6_15, 6_17 and 2_29 mutants were cultivated in 1 l
flasks with 10 ml (highly aerated), 50 ml or 100 ml (poorly aerated) MTM containing 250 g l−1
glucose, 1.6 g l−1 NH4Cl and CaCO3. The flasks were agitated at 100 rpm.
The findings revealed that the aeration of the culture has a strong influence on the itaconate
titer. Substantially more itaconate (in average 10-fold more) was produced in the 10 ml culture
(Figure 3.11. Moreover, the mutants produced significantly more itaconate in comparison to
the wt regardless of the culture aeration. Interestingly, the itaconate production rate of strain
2_29 was approximately 6-fold higher than the other strains under low aeration, indicating
that this mutant is capable of relatively efficient itaconate production even at low aeration
levels.
Figure 3.11: The kinetics of ammonium utilization (dashed lines) and the itaconate
production (full lines) in 100 ml culture (A) and 10 ml culture (B). Green
lines: U . maydis wt, red lines: U . maydis 6_15, blue lines: U . maydis
6_17 and yellow lines: U . maydis 2_29. The values are mean of three
analytical determinations ± standard deviation. 5.5
38
3.4 Itaconate production kinetics under various cultivation conditions
Interestingly, while in the 10 ml culture the N exhaustion was the trigger for the itaconate pro-
duction as expected, in the 100 ml culture the itaconate production started even though the
N was not depleted. The severe oxygen limitation might in this case have induced itaconate
production, although the exact mechanism is not clear.
In the poorly aerated culture, the final biomass concentration and the N consumption rate
were greatly reduced, indicating that the cultures were severely oxygen limited. As a result,
the cells in this culture consumed 4-fold less glucose in comparison to the highly aerated
culture. The end-point OD values as well as the final glucose concentration and the itaconate
yield values are summarized in Table 3.4.
Culture
volume
U . maydis
wt
U . maydis
6_15
U . maydis
6_17
U . maydis
2_29
OD (AU)
10 ml 45.3 ± 2.4 33.7 ± 2.9 42.3 ± 1.8 27.2 ± 1.3
50 ml 17.9 ± 0.8 24.3 ± 1.2 25.5 ± 1.2 19.7 ± 1.4
100 ml 10.1 ± 0.9 10.7 ± 0.7 11.9 ± 1.2 11.3 ± 0.7
Glucose (g l−1)
10 ml 102.7 ± 16.2 100.7 ± 4.9 88.7 ± 10.2 131.2 ± 18.4
50 ml 166.3 ± 8.6 151.4 ± 9.2 149.8 ± 4.7 165.6 ± 5.0
100 ml 179.1 ± 2.9 195.8 ± 6.9 194.0 ± 3.0 217.6 ± 4.0
Yield (g g−1)
10 ml 0.022 0.076 0.095 0.189
50 ml 0.002 0.003 0.005 0.012
100 ml 0.008 0.013 0.022 0.107
Table 3.4: The summary of the end-point values of OD (AU), glucose concentration (g
l−1) and yield (itaconate/utilized glucose, g g−1). The values (mean of three
analytical determinations ± standard deviation) were obtained after 144 h
of cultivation.
As shown in the Figure 3.12, the aeration of the culture plays an important role in the op-
timization of the high-yield itaconate production conditions. The final itaconate titers were
increased manifold in the highly aerated cultures in comparison to the poorly aerated ones
(100 and 50 ml). There were no big differences in the itaconate titers obtained from the latter
two cultures by any of the three mutants. The only difference was observed at the wt strain.
Moreover, the selected mutants were able to produce significantly more itaconate with regard
to the wt in the 10 ml cultures (6_15: 3.6-fold more, 6_17: 4.8-fold more and 2_29: 7-fold more).
39
Chapter 3: Results
Figure 3.12: An overview of the influence of the culture aeration on the itaconate titer.
Culture volumes: 100 ml (red columns), 50 ml (green columns) and 10 ml
(orange columns). The values (mean of three analytical determinations
± standard deviation) were obtained after 144 h of cultivation. 5.6
In order to verify the reason for the low cell performance in the poorly aerated culture in
comparison to the highly aerated one, cultivation experiments in 1 l air-tight flasks were
performed. The BOD and the CBP were determined in MTM with 250 g l−1 glucose, 1.6 g l−1
NH4Cl and CaCO3 and flasks were filled with either 10 or 100 ml of medium.
Each strain used in this experiment demonstrated the same pattern in O2 consumption and
CO2 production. Therefore, results from strain 2_29 are given as an example (Figure 3.13).
The highly aerated cultures used oxygen at an almost linear rate, which was 3-fold higher than
the initial rate in the poorly aerated culture. In the case of 100 ml culture the BOD reached
stationary phase after the first 30 h of cultivation, indicating that oxygen was depleted at this
point in the closed flasks.
In order to find out whether the cells in the poorly aerated cultures were cultivated under
oxygen limited conditions, calculations were performed (for the calculations see Material
and methods, section 2.4.2).
40
3.4 Itaconate production kinetics under various cultivation conditions
Figure 3.13: Overview of the results from the off-gas analysis. The BOD values are
located in the positive and the CBP values in the negative areas of the
charts. The U . maydis 2_29 mutant was used as an example. The results
were obtained from the cultivation in MTM containing 250 g l−1 glucose,
1.6 g l−1 NH4Cl and CaCO3. Blue line: BOD in 10 ml, green line: BOD
in 100 ml, orange line: CBP in 100 ml and red line: CBP in 10 ml. The
values are mean of three analytical determinations.
Since the experiments were performed in closed flasks, only a fixed amount of oxygen is
available to the cells. In the 100 ml culture (900 ml air) 8 mmol oxygen was available at the
beginning of the experiment. Assuming that half of the glucose consumed during growth is
used to generate energy according to formula above, the cells would need 42 mmol oxygen.
Therefore, the cells likely reached the oxygen limitation after few hours of cultivation.
In the case of the 10 ml culture (990 ml air), there was 9 mmol oxygen available. Although the
biomass concentration was higher under these conditions, the 10-fold lower culture volume
means that the cells would need only 6.3 mmol oxygen. These cells therefore were cultivated
under non-limiting oxygen conditions.
These calculations clarified why the cells performed so poorly in the 100 ml culture, and also
indicate that the 10 ml culture was not limited by the total available oxygen amount.
41
Chapter 3: Results
3.4.3 Influence of nitrogen concentration
Previous experiments showed that in some cases the itaconate production is triggered while
N is still present in the medium. Therefore cultivations in MTM containing excess as well as
limiting concentration of N were performed in order to test whether other growth limitations
can also trigger the itaconate production.
U . maydis wt and mutants 6_15, 6_17 and 2_29 were cultivated for 96 h in 50 ml MTM per 1 l
flask in medium containing 250 g l−1 glucose, CaCO3 and either 0.8, 1.6 or 4 g l−1 NH4Cl.
The N/P molar ratio in MTM containing 0.8 g l−1 NH4Cl is 4.54 and with 1.6 g l−1 NH4Cl
8.65. In MTM with 4 g l−1 NH4Cl it is already 20.8. Assuming that the cellular composition
of U . maydis is similar to yeast, (N/P ratio of 14.7; [60]), the presence of 0.8 and 1.6 g l−1
NH4Cl in medium will lead to N-limitation, while 4 g l−1 NH4Cl will cause a P-limitation. In
all calculations it was assumed that 0.1 g l−1 N was added with the yeast extract.
In the case of 4 g l−1 NH4Cl, the N was not depleted and yet the itaconate production started
(Figure 3.14A). The sudden decrease in the N utilization likely means that the phosphate was
exhausted in the first 24 h, indicating that under these conditions the limiting factor and
therefore the itaconate production trigger was the phosphorus.
Figure 3.14: The kinetics of ammonium utilization (dashed lines) and the itaconate
production (full lines) in MTM containing 4 g l−1 NH4Cl (A) and 0.8 g
l−1 NH4Cl (B). Green lines: U . maydis wt, red lines: U . maydis 6_15, blue
lines: U . maydis 6_17 and yellow lines: U . maydis 2_29. The values are
mean of three analytical determinations ± standard deviation. 5.7
42
3.4 Itaconate production kinetics under various cultivation conditions
Nevertheless the limiting concentration of N led to higher titers of the itaconate with regard
to the medium with limiting concentration of phosphorus (Figure 3.14B). This suggests that
an N-limitation is a better trigger for the high-yield itaconate production than P-limitation,
although there were no substantial differences in the itaconate titers with 4 and 1.6 g l−1
NH4Cl among the strains (Figure 3.15). The only exception represented the strain 2_29, which
produced also significantly more itaconate with 4 g l−1 NH4Cl (44-fold with regard to the
wt). This indicates that this mutant is capable of relatively efficient itaconate production
regardless of the available N concentration.
Figure 3.15: An overview of the influence of the concentration of the N supply on the
itaconate titer. NH4Cl concentrations: 4 g l−1 (red columns), 1.6 g l−1
(green columns) and 0.8 g l−1 (orange columns). The values (mean of
three analytical determinations ± standard deviation) were obtained
after 96 h of cultivation
The excess of N led to 1.6-fold more biomass even though the glucose consumption was
comparable among the conditions. The end-point glucose concentration, OD values and
itaconate yields are listed in Table 3.5.
As shown in the Figure 3.15, all the selected mutants produced significantly more itaconate
with regard to the wt under the highly N limited conditions (6_15: 6.6-fold more, 6_17: 10.5-
fold more and 2_29: 9-fold more with 0.8 g l−1 NH4Cl).
43
Chapter 3: Results
Initial NH4Cl
concentration
U . maydis
wt
U . maydis
6_15
U . maydis
6_17
U . maydis
2_29
OD (AU)
0.8 g l−1 19 ± 1 19 ± 2 21 ± 1 18 ± 2
1.6 g l−1 17 ± 1 20 ± 1 22 ± 1 14 ± 1
4 g l−1 31 ± 2 30 ± 4 31 ± 6 30 ± 3
Glucose (g l−1)
0.8 g l−1 192 ± 4 186 ± 6 185 ± 5 186 ± 5
1.6 g l−1 179 ± 10 162 ± 8 157 ± 4 171 ± 5
4 g l−1 192 ± 10 199 ± 6 209 ± 7 214 ± 11
Yield (g g−1)
0.8 g l−1 0.0074 0.0449 0.0696 0.0604
1.6 g l−1 0.0012 0.0024 0.0033 0.0109
4 g l−1 0.0008 0.004 0.0115 0.0553
Table 3.5: The summary of the end-point values of OD (AU), glucose concentration (g
l−1) and yield (itaconate/utilized glucose, g g−1). The values (mean of three
analytical determinations ± standard deviation) were obtained after 96 h
of cultivation.
3.4.4 Selected optimal conditions
Based on the results from the previous experiments, optimal cultivation conditions for high-
yield itaconate production from U . maydis were selected and tested.
The cells were cultivated in MTM containing CaCO3 and the selected conditions were: high
culture aeration (10 ml culture per 1 l flask), low initial concentration of carbon source (65 g
l−1 glucose) and low N concentration (0.8 g l−1 NH4Cl). Four strains of U . maydis were com-
pared; wt and the three selected mutants 6_15, 6_17 and 2_29. The kinetics of the itaconate
production and yield, ammonium utilization, malate production, cell growth, and glucose
utilization were studied (Figure 3.16).
Itaconate production started, when the N, which was present in the medium in growth-
limiting concentration, was depleted (Figure 3.16A), which is in correlation with the previous
experiments. All the four strains exhausted the N within the first 24 h of cultivation, but the
itaconate production in the wt strain started only after 72 h.
The highest rate in the itaconate production was observed between 24 and 48 h by the strain
2_29 (0.29 ± 0.029 g l−1 h−1). Also the most itaconate was produced by this strain, 18.8 g
l−1 after 144 h, which is 9.8-fold more itaconate than wt produced (1.9 g l−1 after 144 h).
Generally, all three selected mutants produced significantly more itaconate than the wt.
44
3.4 Itaconate production kinetics under various cultivation conditions
Figure 3.16: The kinetics of the ammonium utilization and the itaconate production
(A, the dashed lines - ammonium concentration, the full lines - itaconate
production), malate production (B), cell growth (C) and glucose util-
ization (D) under selected optimal cultivation conditions. Green lines:
U . maydis wt, red lines: U . maydis 6_15, blue lines: U . maydis 6_17,
yellow lines: U . maydis 2_29. The values are mean of three analytical
determinations ± standard deviation. 5.9
Although the cells were cultivated under selected optimal conditions, the itaconate titer ob-
tained in this experiment by the strain 2_29 did not improve when compared to the previous
experiments. There was a comparable itaconate concentration reached in the cultivation of
the 10 ml culture in the experiments with various aeration levels (22.6 g l−1 itaconate after
144 h, strain 2_29, Figure 3.16B). This may suggest that the most influencing condition for the
itaconate production from those studied is the aeration of the culture.
Aside of the itaconate, malate was produced in high titers. Any side product which accom-
panies the itaconate production process potentially decreases the efficiency of the itaconate
production by the cells. Figure 3.16B shows production kinetics of the malate. In contrast
to the itaconate, the malate was produced directly from the beginning of the cultivation. It
45
Chapter 3: Results
appears that the malate yield decreases when the itaconate yield is increased. The ratio values
between the itaconate and the malate are summarized in Table 3.6. While the highest ratio
was obtained from the 2_29 strain, the lowest one belonged to the wt strain.
U . maydis wt U . maydis 6_15 U . maydis 6_17 U . maydis 2_29
Yield (g g−1) 0.03 0.24 0.22 0.3
Itaconate (g l−1) 2 ± 0.1 15 ± 0.2 14 ± 1 19 ± 0.2
Itaconate/malate 0.3 4.1 4.0 8.9
Table 3.6: Summary of values of the itaconate yield (itaconate/utilized glucose), titer
and itaconate/malate ratio. The values were calculated from samples ob-
tained after 144 h of cultivation. The itaconate titer values are mean of
three analytical determinations ± standard deviation.
A new pattern in the cell growth was observed during this experiment (Figure 3.16C). This
rapid increase in the OD in the first 24 h of cultivation and a decline in the OD after 48 - 72 h
was not present in any of the previous experiments. Nevertheless, a decrease in the OD was
visible among all strains after about 96 h.
On the contrary, the glucose was utilized throughout the whole experiment and with no signi-
ficant differences among the strains (Figure 3.16D), as was already observed in the previous
experiments. After 144 h, there was between 1.5 g l−1 (2_29 strain) and 5 g l−1 (wt) glucose left
in the culture.
A strong positive influence of the integration of the UM06344 overexpression cassette on the
itaconate titer was observed. Comparison between the results obtained from this experiment
revealed that the condition optimization had significantly higher effect on the mutants than
on the wt strain. The difference in the itaconate titer obtained from the mutants based on
the cultivation conditions was however huge. While the strain 2_29 produced only about 4 g
l−1 itaconate from 0.8 g l−1 NH4Cl in the N concentration optimization experiment, about 11
g l−1 itaconate from 65 g l−1 glucose in the glucose concentration optimization experiment
and about 22 g l−1 itaconate in the 10 ml culture in the experiment focusing on the culture
aeration, the same strain produced almost 19 g l−1 itaconate under the selected conditions.
Therefore, not only an improvement in the itaconate production was reached by the culture
conditions optimization but moreover, further optimization should be conducted with main
focus on the high-yield strain.
46
3.5 Itaconate production during fermenter batch cultivation
3.5 Itaconate production during fermenter batch
cultivation
The results from the shaken flasks experiments revealed, that the most important factor for
the itaconate production is the aeration of the culture. Since the conditions in the fermenter
are highly controlled, the oxygen limitation can be excluded. Moreover, cultivation in the
fermenter is closer to the industrial production. Therefore, fermenter batch cultivations were
performed.
U . maydis 2_29 strain was cultivated alongside the wt strain in 1.5 l MTM containing 208 or
200 g l−1 glucose and 1.6 g l−1 NH4Cl. The dissolved oxygen tension (DOT) was 80 %. The pH
level was kept at 6 by automatic addition of NaOH, and thus no CaCO3 was present, enabling
the accurate determination of dry biomass weight.
Even though the U . maydis wt cells in the fermenter were cultivated in the same medium as
those in the shaken flask experiments, the discrepancy between the itaconate titer obtained
from the fermenter batch cultivation and the shaken flask experiments was large. The highest
itaconate titer produced by wt strain was obtained in the experiment dealing with the culture
aeration (10 ml culture, 3.2 g l−1 itaconate after 144 h). This is manifold less in comparison to
the fermenter batch cultivation. The reason could be the culture aeration, since it appears to
be the most influencing factor for the itaconate production. Apparently, the oxygen transfer
rate in the 10 ml culture is still much lower with regard to the transfer rate in the fermenter
and therefore only such low itaconate titer was obtained.
Big differences in the itaconate titers between the wt and the 2_29 strain were observed
(Figure 3.17A). Although the wt strain produced 37 g l−1 itaconate after 116 h, a decrease in
the production rate is obvious. While at the beginning the itaconate production rate was 0.8 g
l−1 h−1, at the end of the cultivation it dropped to 0.095 g l−1 h−1. The 2_29 mutant produced
29 g l−1 itaconate after 160 h, but with a rather stable production rate of about 0.21 g l−1 h−1.
Nevertheless, the wt strain reached a higher final itaconate titer and yield than the 2_29 strain.
There can be several reasons for that, e.g. the higher amount of biomass in the 2_29 strain cul-
ture (Figure 3.17B). Such a high quantity of biomass requires certain amount of maintenance
energy, which would negatively affect the itaconate production. Another reason could be that
the 2_29 strain was selected under conditions, which were at least to a certain extend oxygen
limited. If the mutation in strain 2_29 specifically affected the itaconate production under
low oxygen conditions, the improved production may be lost in a highly aerated fermenter.
In order to obtain further information about the influence of the culture aeration on the it-
aconate production under highly controlled conditions, fermenter batch cultivation with low
DOT (15 %) was performed. Even though this cultivation was only a preliminary experiment
and needs to be repeated in order to confirm the findings, some very interesting results were
47
Chapter 3: Results
obtained.
The cells of U . maydis wt and the 2_29 strain were cultivated under the same conditions as
above (1.5 l MTM containing 270 g l−1 glucose, 1.6 g l−1 NH4Cl, pH 6 and no CaCO3). As
mentioned previously, the only difference was the DOT, which was kept at about 15 %.
The difference in the itaconate production between the wt and the 2_29 strain was vast
(Figure 3.17C). While the 2_29 strain produced almost 20 g l−1 itaconate after 115 h, the
itaconate titer obtained from wt cells after the same cultivation time period was only 0.3 g l−1.
It appears, that the wt cells did not even start the itaconate production.
0
50
100
150
200
250
0
5
10
15
20
25
30
35
40
0 20 40 60 80 100 120 140 160
Glu
cose
(g
/l)
Dry
bio
mas
s w
eigh
t (g
/l)
Time (h)
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0 20 40 60 80 100 120 140 160
0
5
10
15
20
25
30
35
40
Am
mo
niu
m (
g/l
)
Time (h)
ITA
(g
/l)
0
5
10
15
20
25
0
1
2
3
4
0 24 48 72 96 120
ITA
(g
/l)
Mal
ic a
cid
(g
/l)
Time (h)
0
50
100
150
200
250
300
0
5
10
15
20
25
30
35
0 24 48 72 96 120
Glu
cose
(g
/l)
Dry
bio
mas
s w
eigh
t (g
/l)
Time (h)
A B
D C
Figure 3.17: Fermenter batch cultivations with 80 % (A, B; n = 2) and 15 % DOT (C,
D; n = 1). (A) Ammonium consumption (dashed lines) and itaconate
production (full lines), (B) dry biomass weight (dashed lines) and glucose
utilization (full lines), (C) malate (dashed lines) and itaconate produc-
tion (full lines) and (D) dry biomass weight (dashed lines) and glucose
utilization (full lines). Green lines: U . maydis wt, blue lines: U . maydis
2_29. 5.10
48
3.6 Biosurfactant production and its influence on the itaconate production
A relatively high concentration of malate was produced alongside the itaconate. Even though
the titers were comparable among the both strains (about 3 g l−1), when compared to the
itaconate concentrations, the differences became apparent. The ratio itaconate/malate in
the case of the wt strain is 0.14, for the mutant 5.5. Furthermore, in contrary to the previous
findings, the malate production started later, parallel to the itaconate production.
While there were differences in the glucose utilization among the strains especially in the
second half of the cultivation, the biomass production from both the strains was comparable
(Figure 3.17D).
The results from these two fermenter batch cultivations confirmed the necessity of the proper
culture aeration for high-yield itaconate production as well as the fact, that also in the case of
fermenter batch experiments, the condition optimization should be performed with regard
to the mutant strain.
3.6 Biosurfactant production and its influence on the
itaconate production
The production of surface active compounds by the cells of U . maydis is well known [38].
The determination of the surface tension of the culture broth from cultivation experiments
revealed that all three selected mutants produced glycolipids under the conditions men-
tioned above (data not shown). Production of oil droplets was also observed by microscopy
(Figure 3.18). Under optimal conditions, up to about 23 g l−1 of these surface active com-
pounds can be produced [39], indicating that the synthesis of glycolipids as side products can
represent a major obstacle for the itaconate production, because it is energy and resources
demanding.
In order to investigate the extent of influence of the glycolipid synthesis on the itaconate pro-
duction, cultivation experiments with the wt and the U . maydis ∆cyp1 ∆emt1 strain (kindly
provided by Prof. Michael Bölker, Phillips University Marburg; see Materials and methods,
Table 2.1) were conducted. The cyp1 and emt1 are genes responsible for the production of
ustilagic acid and MEL respectively in U . maydis [39].
The cells were cultivated in 10 ml MTM containing CaCO3, 65 g l−1 glucose and 0.8 g l−1
NH4Cl. The kinetics of the itaconate production, ammonium utilization, cell growth, and
glucose utilization were studied (Figure 3.19).
The itaconate production was significantly increased in the ∆cyp1 ∆emt1 strain with regard
to the wt (about 2-fold, Figure ??A). Moreover, the production started after the N depletion,
which occurred within the first 24 h, only in the case of ∆cyp1 ∆emt1 strain.
49
Chapter 3: Results
Figure 3.18: Microscopic view of U . maydis 6_17 mutant without staining (A, 630 x)
and after the lipid-specific staining with Sudan black B and Safranin (B,
1000 x). Some lipid droplets are marked with arrows.
Figure 3.19: The kinetics of the ammonium utilization and the itaconate production
(A, dashed lines - ammonium concentration, full lines - itaconate produc-
tion) and cell growth and glucose utilization (B, dashed lines - biomass
production, full lines - glucose utilization) under selected optimal cul-
tivation conditions. Green lines: U . maydis wt, purple lines: U . maydis
∆cyp1 ∆emt1. The values are mean of three analytical determinations ±standard deviation. 5.11
The ∆cyp1 ∆emt1 strain grew much better in comparison to the wt, probably because this
mutant did not need to invest the gained energy into the glycolipid production (Figure ??B).
The deletion of the glycolipid production represents a great advantage for the itaconate
50
3.7 Alternative carbon sources for the itaconate production
production. The cells can use their energy for the itaconate production rather than for
biosurfactant synthesis. Moreover, the absence of the glycolipids in the culture broth may sig-
nificantly simplify the downstream processing, i.e. the membrane filtration or centrifugation
and thus reduce the overall production costs.
3.7 Alternative carbon sources for the itaconate
production
The ability to utilize the products of hydrolyzed biomass would be a great advantage in order
to establish U . maydis as a successful industrial itaconate producer. Therefore utilization of
L-arabinose, xylose and sucrose as well as itaconate production from them by various strains
of U . maydis was studied.
The cells of U . maydis wt and the 6_15, 6_17 and 2_29 mutants were cultivated in 50 ml
medium in 1 l flasks. The MTM contained 1.6 g l−1 NH4Cl, CaCO3 and 240, 260 or 240 g l−1
L-arabinose, xylose or sucrose.
The itaconate titers obtained from these alternative carbon sources were significantly lower
in comparison to the glucose (Table 3.7). One reason could be the cultivation conditions,
which may have not been optimal. Fermenter batch cultivation experiments performed by
other members of our research group revealed that the use of mixed carbon sources leads to
efficient production of itaconate (Dr. Nicole Maaßen, personal communication).
The cell growth on the different carbon sources was comparable. With regard to glucose, the
cells were able to produce even more biomass from these alternative carbon sources. The
end-point OD values obtained after 144 h are summarized in Table 3.7. While about 60 g l−1
arabinose and 28 g l−1 xylose was utilized by the cells over the 144-h cultivation, the cells used
about 241 g l−1 sucrose.
The U . maydis cells were able to utilize L-arabinose, xylose and sucrose, the two first being
products of biomass hydrolysis. Although the culture conditions clearly need to be optimized,
the use of these alternative carbon sources is possible.
51
Chapter 3: Results
Carbon
source
U . maydis
wt
U . maydis
6_15
U . maydis
6_17
U . maydis
2_29
OD (AU)
Arabinose 15 ± 2 26 ± 1 27 ± 2 23 ± 2
Xylose nd 18 ± 2 18 ± 2 15 ± 0.4
Sucrose 21 ± 2 24 ± 1 24 ± 1 17 ± 1
ITA (g l−1)
Arabinose 0.016 ± 0.002 0.017 ± 0.002 0.013 ± 0.0003 0.015 ± 0.001
Xylose 0.023 ± 0.0007 0.103 ± 0.019 0.176 ± 0.025 0.054 ± 0.012
Sucrose 0.091 ± 0.01 0.148 ± 0.021 0.212 ± 0.015 0.123 ± 0.013
Table 3.7: The summary of the end-point values of OD (AU) and itaconate (g l−1) from
various carbon sources. The values (mean of three analytical determina-
tions ± standard deviation) were obtained after 144 h of cultivation; ITA -
itaconate, nd - not determined.
3.8 Deletion of UM11778 in U. maydis
Results obtained from previous experiments revealed that the UM06344 gene does not func-
tion as a cis-aconitate decarboxylase, as was originally assumed.
Therefore, another gene putatively involved in the itaconate synthesis pathway, UM11778,
was selected by Prof. Michael Bölker and an U . maydis UM11778 knockout was constructed
by his work group and kindly provided for further analyses.
The basic local alignment search tool analysis (BLAST; [61]) of the protein encoded by
this gene revealed that it belongs to the prpF superfamily and it has 33% identity to a 3-
methylitaconate-∆-isomerase (Mii) from Eubacterium barkeri. This enzyme, among other
functions, catalyzes the isomerization of itaconate to citraconate [62].
The strains of U . maydis wt and the U . maydis ∆UM11778 were cultivated in 50 ml medium
in 1 l flasks. The MTM contained 65 g l−1 glucose, 1.6 g l−1 NH4Cl and CaCO3.
Fumarate, itaconate, glucose, succinate, malate and pyruvate were detected in the culture
broth. The production kinetics of these substances are shown in Figure 3.20.
There were great production differences between the∆UM11778 strain and the wt strain. The
deletion of the UM11778 gene led to a significant decrease in the itaconate production (0.014
g l−1 after 192 h), indicating that this gene plays a crucial role in the itaconate biosynthesis.
Still, a clearly defined peak of the itaconate was visible in the chromatogram (Figure 3.21).
The itaconate titer obtained from the wt strain was about 330-fold higher than the itaconate
titer from the ∆UM11778 strain.
52
3.8 Deletion of UM11778 in U. maydis
Figure 3.20: The production kinetics of various substances putatively involved in the
itaconate synthesis pathway. Green lines - U . maydis wt, brown lines -
U . maydis ∆UM11778; n = 1. 5.12
The Figure 3.20 shows, that all the TCA cycle intermediates were produced in significantly
lower concentration by the ∆UM11778 strain with regard to the wt. The pyruvate concentra-
tion, conversely, was higher in the ∆UM11778 strain after 24 hours.
Apart from the peaks of the identified compounds, a peak of unknown substance eluting at
53
Chapter 3: Results
13.4 min was present in the culture broth of ∆UM11778 strain during the whole experiment
(Figure 3.22). A similarity with the terreic acid obtained by Bonnarme et al. [25] is possible.
The cultivation experiments with the ∆UM11778 strain revealed, that this gene is indeed
strongly involved in the itaconate synthesis pathway of U . maydis. This finding represent a
significant step forward in understanding the process of the itaconate synthesis.
Figure 3.21: HPLC profile of culture broth of U . maydis ∆UM11778 strain obtained
after 192 h of cultivation. A clearly defined itaconate peak is visible at
14.4 min.
54
3.8 Deletion of UM11778 in U. maydis
Figure 3.22: HPLC profile of supernatant from 2-day-old culture of U . maydis
∆UM11778 strain. A peak of unidentified compound is visible at 13.4
min.
55
Chapter 3: Results
56
CHAPTER 4
Discussion
The aim of this work was to characterize the itaconate production in U . maydis, understand
the itaconate synthesis pathway and describe conditions and aspects which may influence
the itaconate yields.
U . maydis strain MB215 is presented as an alternative itaconate producer and compared
to the currently most used one, A. terreus. The great advantage of U . maydis with regard
to A. terreus is its unicellular growth. However, the production characteristics are for the
time being much lower than those from A. terreus. Therefore, various aspects putatively
influencing the itaconate production were studied within this work in order to improve them.
Genes which might be involved in the synthesis pathway of itaconate were either deleted
and/or overexpressed and the consequent cellular phenotype was examined. Various initial
carbon and nitrogen source concentrations as well as oxygen supply were investigated in
order to understand their impact on the itaconate yields. Moreover, generation of various
side products and their influence on the itaconate yields was also analyzed.
4.1 Itaconate synthesis pathway in U. maydis
In A. terreus the itaconate is synthesized via Embden-Meyerhof-Parnas (EMP) pathway fol-
lowed by the initial steps of the tricarboxylic acid (TCA) cycle. The last step of the pathway
involves the conversion of cis-aconitate to itaconate (Figure 4.1, cis-aconitate route). This
reaction is catalyzed by cis-aconitate decarboxylase (EC: 4.1.1.6). Bonnarme et al. [25] proved
by employing 14C-labelled TCA cycle intermediates, that A. terreus uses this pathway to syn-
thesize the itaconate. The detected radioactivity levels corroborate the cis-aconitate route
but exclude the citraconate route (Figure 4.1). These experiments also excluded the pentose
57
Chapter 4: Discussion
phosphate pathway, leaving the EMP route the only one used for processing the glucose
during itaconate synthesis in A. terreus. These findings were supported by detection of most
of the TCA cycle intermediates in the culture broth.
The itaconate synthesis pathway in U . maydis remains unclear. There are three possible
routes for itaconate production in U . maydis.
The first route includes the condensation of two acetyl-CoA molecules and further production
of 1,2,3-tricarboxypropanoate from succinate [63]. This pathway was excluded for A. terreus
by Bonnarme et al. [25] based on the disproportion of the itaconate yields. Although this
route is considered highly unlikely due to the inefficiency of the pathway and the fact that
there has not been any further literature dealing with this route published, it can not be
completely excluded for the itaconate production of U . maydis. The theoretical itaconate
yield of this pathway is 56 Cmol %. This value was calculated based on the stoichiometry of
the following reaction:
1.5 glucose → 3 CO2 + 3 acetyl-CoA → 4 CO2 + 1 itaconate.
The second possible pathway (Figure 4.1, cis-aconitate route) is similar to the pathway in
A. terreus, via the conversion of cis-aconitate to itaconate, which is catalyzed by cis-aconitate
decarboxylase (CAD). Based on a blast analysis between the protein sequence of the CAD
from A. terreus [27] and the U . maydis genome, the UM06344 gene was selected as a putative
CAD in U . maydis. Both of these genes contain similar conserved domains.
If the UM06344 gene does indeed encode a CAD enzyme, the deletion of this gene should have
led to decreased itaconate production, which was not the case. Moreover, the itaconate titers
from the deletion mutant were in some cases even higher than the those from the wt strain.
Thus, although a certain influence of this gene on the itaconate production was observed,
the findings suggest that this gene is not the cis-aconitic acid decarboxylase responsible for
itaconate production.
The overexpression of UM06344 or random integration of the overexpression construct led
on average to 1.2-fold higher itaconate titers with regard to the wt, although a wide range of
titers was observed. This suggests that the UM06344 overexpression construct influences the
itaconate production only to a small extent.
Three consequences of the overexpression of the UM06344 gene or random integration of the
overexpression construct simultaneously affect the final cell phenotype;
• An increase in the itaconate production due to the overexpression of the UM06344 gene
itself, as originally expected. However, since the deletion of UM06344 gene did not result in
decreased itaconate production, this gene is not directly involved in the itaconate production.
58
4.1 Itaconate synthesis pathway in U. maydis
Glucose
Acetyl-CoA Pyruvate
CO2
Oxaloacetate
CO2
R-citramalate
Itaconate (Citraconate route)
Cis-aconitate
Itaconate (Cis-aconitate route)
CO2
Cis-aconitic acid decarboxylase
Citraconate
Mii (UM11778)
R-citramalate synthase
R-citramalate dehydratase
EMP pathway
Figure 4.1: Putative itaconate synthesis routes in U . maydis and A. terreus (figure was
adapted from [25]); Mii - 3-methylitaconate-∆-isomerase, EMP - Embden-
Meyerhof-Parnas pathway.
59
Chapter 4: Discussion
• As no terminator for the UM06344 ORF was present in the overexpression construct, the
termination was not efficient and therefore the gene expression did not reach its putative
maximum. However, if the overexpression cassette integrated into the U . maydis genome
in front of a terminator, the expression of the UM06344 gene would be greatly enhanced.
Such arrangement would contribute to the variation of the itaconate titers obtained from the
transformant screening.
• A gene knock-out as a result of the overexpression construct integration into the U . maydis
genome. If a gene involved in the itaconate production pathway would be disrupted, the
itaconate production would be either decreased or obstructed. However, a disruption of
another gene involved in a competing route might as well lead to an increase of the metabolic
flux through the itaconate synthesis pathway.
Therefore, it is difficult to distinguish between the outcome resulting from the UM06344
overexpression and the random integration of this construct and the consequent mutation.
The latter two points can have either negative or positive effect due to the integration locus.
Moreover, the second point strongly influences the first one.
A further BLAST analysis of the protein encoded by the UM06344 gene revealed a 96%
identity to 2-methylcitrate dehydratase (PrpD, EC: 4.2.1.79) from Sporisorium reilianum.
Furthermore, an identity to the 2-methylcitrate dehydratase higher that 60 % was found
among wide range of microorganisms, e.g. Puccinia graminis, several strains of Burkholderia,
Salmonella enterica and E. coli. The PrpD of E. coli showed the highest affinity to 2-methylcit-
rate and citrate and no affinity to cis- or trans-aconitate [64]. Thus, overexpression of
UM06344 or random integration of the overexpression construct could increase the rate
of the dehydration of the citrate to form the cis-aconitate, and therefore the concentration
of the substrate for the next step in the itaconate synthesis pathway, the conversion of the
cis-aconitate into itaconate [65]. This may explain the slight overall improvement of itaconate
production resulting from introduction of the UM06344 overexpression construct. However,
this applies only if U . maydis employs the cis-aconitate route (Figure 4.1) for the itaconate
production.
Nowakowska-Waszczuk proposed yet another possible itaconate production route for A.
terreus, which involves the production of citramalate from pyruvate and acetyl-CoA (Fig-
ure 4.1, citraconate route; [66]). This assumption originates from the experiment with isolated
mitochondria from cell extracts from A. terreus. These preparations were unable to oxidize
the TCA cycle intermediates and therefore Nowakowska-Waszczuk concluded, that the it-
aconate synthesis pathway most likely does not involve the TCA cycle, but rather occurs
via dehydration of citramalate. However, this pathway for itaconate production in A. terreus
was excluded by the findings by Bonnarme et al. [25] based on the 13C and 14C labeling
experiments. Nevertheless, for U . maydis this route should still be taken into consideration.
Velarde et al. [62] described an enzyme catalyzing the isomerization between 3-methylitacona-
60
4.1 Itaconate synthesis pathway in U. maydis
te and 2,3-dimethylmaleate in Eubacterium barkerii. This enzyme, 3-methylitaconate-∆-
isomerase (Mii, EC 5.3.3.6), also catalyzes the reversible conversion between itaconate and
citraconate. The BLAST analysis between the protein sequence of Mii and the U . maydis
genome resulted in three putative 3-methylitaconate-∆-isomerases (UM11778, UM06058 and
UM02807). Although the sequence similarities are very low (33 - 35 %), a conserved domain
search classifies them into the same PrpF superfamily as the Mii enzyme. These three genes
were deleted in the U . maydis MB215 wt strain by Prof. Michael Bölker and kindly provided
for phenotypic characterization.
Since the deletion of the UM11778 gene almost completely disrupted the itaconate produc-
tion, the respective enzyme plays a crucial role in the itaconate synthesis pathway. This
finding could indicate that the citraconate route is in U . maydis active. However, a clearly
defined peak was visible in the HPLC profile of the culture broth from this strain (Figure 3.21).
Although the deletion of the other two genes, UM06058 and UM02807, did not result in
any detectable decrease in itaconate production, the residual itaconate production in the
UM11778 knockout could be explained by the activity of the enzymes encoded by either one
of these two genes or even the UM06344 mentioned above.
As described previously, Mii catalyzes the isomerization between itaconate and citracon-
ate. In theory, citraconate can be produced by dehydration of R-citramalate by the enzyme
R-citramalate dehydratase (EC: 4.2.1.35). This R-citramalate in turn can be formed from pyr-
uvate and acetyl-CoA, catalyzed by R-citramalate synthase (EC: 2.3.1.182). This citraconate
route is summarized in Figure 4.1.
A BLAST analysis of a well characterized R-citramalate dehydratase from Methanocaldococcus
jannaschii revealed a putative R-citramalate dehydratase in the U . maydis genome (UM06010).
However, this gene is annotated as 3-isopropylmalate dehydratase (EC: 4.2.1.33). The max-
imal identity between these two proteins is only 35 %. Rubin et al. [67] characterized the
UM06010 gene as a LEU1 encoding the isopropylmalate isomerase. Therefore, this gene can
be excluded from the genes participating in the citraconate pathway.
The presence of the R-citramalate synthase gene in the genome of U . maydis was not con-
firmed since the BLAST analysis of this protein from Sulfobacillus acidophilus resulted in two
hypothetical proteins; UM04115 and UM04156. The BLAST analysis of the protein encoded
by the first gene resulted in 97% identity with a LYS20-homocitrate synthase (EC: 2.3.3.14) of
Sporisorium reilianum and 93% identity with the same enzyme from Ustilago hordei. Since
this enzyme is involved in the lysine biosynthesis, the role of the UM04115 gene in the itacon-
ate synthesis pathway is not likely. The BLAST analysis of the protein encoded by the second
gene showed a 89% and 88% identity to the LEU4-2-isopropylmalate synthase (EC: 2.3.3.13)
from S. reilianum and U . hordei, resp. The LEU4-2-isopropylmalate synthase is a part of the
biosynthesis route of leucine and similarly to the LYS20-homocitrate synthase, it is also not
likely to be involved in the itaconate synthesis route.
61
Chapter 4: Discussion
Since the itaconate is not essential for the viability of U . maydis, it is considered a secondary
metabolite. The secondary metabolites are usually produced under specific cultivation con-
ditions and support the cells in adjusting to the environmental fluctuations or in competition
with other microorganisms [68]. Secondary metabolism genes are often arranged together in
clusters and coregulated [69].
A gene cluster for another secondary metabolite of U . maydis, the ustilagic acid, has been
identified and described [68]. The expression of this gene cluster is induced under N starva-
tion, leading to extensive production of ustilagic acid. Since itaconate synthesis is triggered
among other by N starvation, it might be possible, that a similar gene cluster responsible for
itaconate synthesis and involving the UM11778 gene is present in the genome of U . maydis.
The R-citramalate dehydratase and the R-citramalate synthase genes might be located in
such cluster. However, further investigations are necessary.
To summarize the findings and the assumptions about the itaconate synthesis pathway, it is
very difficult to identify which particular pathway U . maydis uses for the itaconate synthesis.
The UM11778 gene has a significant influence on the itaconate production, but putative
genes encoding the remaining enzymes in the citraconate route could not be identified.
Thus, the presence of the remaining enzymes, R-citramalate dehydratase and R-citramalate
synthase, in U . maydis is so far only speculative. Although the citraconate pathway may
exist, due to the insufficient identification of the genes involved in it, further investigation is
needed.
The cis-aconitate pathway can also be neither dismissed nor confirmed. Although several
of its intermediates were detected in the culture broth of U . maydis cells, the activity and
consequently the involvement of the key enzyme of this pathway, the putative U . maydis
CAD encoded by the UM06344 gene, remains unclear.
The theoretical itaconate yields from both of these pathways are 83 Cmol %.The calculation
is based on the theoretical itaconate yield and the stoichiometry of the following reaction,
which summarizes both of the itaconate synthesis pathways (Figure 4.1):
1 glucose → 1 CO2 + 1 itaconate.
Since the highest itaconate yield obtained during the cultivation experiments was only 38
Cmol %, both these routes can be taken into consideration.
However, without further investigation, no unequivocal conclusion concerning the itaconate
synthesis pathway in U . maydis can be drawn.
62
4.2 Factors influencing the itaconate production
4.2 Factors influencing the itaconate production
The biotechnological production process of itaconate is greatly influenced by the cultivation
conditions. Therefore, their optimization may lead to an increase in the itaconate yields.
Hence, U . maydis wt and the three selected high-yield itaconate mutants of U . maydis (strains
6_15, 6_17 and 2_29) were phenotypically characterized under various cultivation conditions.
The most promising parameters for optimization in order to obtain an economically feasible
production process were chosen to be the initial glucose and ammonium concentration
and the oxygen supply. The carbon source usually represents the highest expense within
the fermentation process, while as mentioned above, N depletion can act as an itaconate
synthesis trigger. However, the most important factor influencing the itaconate synthesis
might be the oxygen supply as it is known for example for the citric acid production [70]. The
influence of these factors is interconnected.
Theoretical maximum yields and production rates of itaconate depend on the initial glucose
and ammonium concentrations as shown in Figure 4.2. Numbers were estimated based on
the experiment dealing with various initial glucose concentrations (Figure 3.8). The theoret-
ical maximum itaconate yields were calculated according to the following formula:
ycomp = ymax∗ (g l ucosei ni t − g l ucosebi om)
(g l ucosei ni t )= ymax∗ (g l ucosei ni t − ( f ∗ammoni umi ni t ))
g l ucosei ni t,
where ycomp (Cmol %) is the theoretical maximum yield (%) compensated for biomass pro-
duction, ymax is theoretical maximum yield (83 %) based on the stoichiometry of the reaction
from glucose to itaconate and CO2, glucosei ni t is the initial glucose concentration (g l−1),
glucosebi om is the glucose concentration used for biomass production (g l−1), f is a factor
related to the C/N ratio of the biomass estimated from Figure 3.8 (utilized glucose (g) per
utilized ammonium (g) in the first 24 h of cultivation) and ammoniumi ni t is the initial am-
monium concentration (g l−1).
The itaconate production rates were calculated from formula below:
rp,max = rp,spec ∗ NH4+
i ni t ,
where rp,max is the theoretical itaconate production rate (g l−1 h−1) at a specific nitrogen
concentration, rp,spec is the specific itaconate production rate estimated from Figure 3.8 (g
ITA l−1 h−1 g−1 NH4+) and NH4
+i ni t is the initial ammonium concentration (g l−1). Great
disproportion can be observed in the production rates between the strains and therefore two
63
Chapter 4: Discussion
different rp,spec were used. The itaconate production rate of U . maydis wt (Figure 4.2, black
full line) is almost 2-fold lower than that of the 2_29 mutant (Figure 4.2, black dashed line).
Figure 4.2: Theoretical yields and production rates of itaconate vs. ammonium con-
centration by U . maydis wt (black full lines) and 2_29 mutant (black
dashed lines) strains. Yields are based on the initial glucose concentration;
green - 65 g l−1, red - 110 g l−1, yellow - 250 g l−1 glucose. Arrows indicate
the NH4+ concentrations tested (0.25, 0.52 and 1.35 g l−1).
4.2.1 Nitrogen
The concentration of the N source represents an important factor influencing the itaconate
production. N is essential for the cells, amino acids and consequently proteins, as well as
polyamines, nucleic acids and vitamins being the products of its utilization. Moreover, nitro-
gen source depletion represents a common trigger for production of various biotechnological
products (citric acid by A. niger [71], [72] or lipids by oleaginous yeast [73]).
There are several substrates used in order to supplement the cells with N. The most common
ones are nitrate, nitrite, ammonium and urea. While not all microorganisms are capable of
nitrate utilization, U . maydis possess this ability, although the presence of ammonium ions
drastically decreases the activity of the nitrate reductase [74].
The cells in the cultivation experiments throughout this work were supplemented with NH4Cl.
However, NH4NO3 is used in most cases for the itaconate production by A. terreus [75], [76],
64
4.2 Factors influencing the itaconate production
[29], [26] due to its less significant effect on the culture pH [47].
The optimal N initial concentration should be selected based on the specific preferences
for the production process. In theory, lower initial N concentrations lead to high yields but
very low production rates, while high N concentrations lead to low yields and high produc-
tion rates (Figure 4.2). However, with increasing initial N concentration, the culture quickly
approaches oxygen limitation due to the high biomass production. Therefore, an optimal
combination of glucose and N concentrations should be selected. As an example, low glucose
concentration would not be suitable in combination with high concentration of ammonium,
because all the glucose would be utilized for biomass production.
In the cultivation experiments, the highest itaconate yields were achieved with low N con-
centration, which is in contradiction to the theoretical expectations (Figure 4.2). Under the
conditions used, higher N concentration likely led to a high biomass formation and con-
sequently to oxygen limitation, which prevented the cells from efficient itaconate production
(Figure 3.11).
4.2.2 Glucose
As shown in Figure 4.2, high glucose concentrations are more favorable, since higher itaconate
yields can be achieved. However, high glucose concentrations can have negative effects of the
cells. Moreover, the highest expense within the itaconate fermentation process represents
the carbon source [77]. Therefore, optimization of the glucose concentration is necessary.
High glucose concentrations (about 250 g l−1) significantly decreased the level of cell meta-
bolism, e.g. the ammonium depletion occurred at a lower rate, growth rate decreased and
hence also the itaconate production was reduced. Most likely the reason for this is the high
osmotic pressure caused by the presence of the glucose and the high energy demand re-
quired to sustain the cell integrity in such environment. The hyperosmotic environment
itself represents a stress and nutrient shortage just adds to that [78]. Such finding is not
completely unexpected, because the cellular adaptations (e.g. up- or down-regulations of
metabolic pathways, switching on and off sensing systems) to an environment containing
nutrients in growth-limiting concentrations are closely related to responses to stresses such
as a hyperosmotic environment. As an example, Diano et al. [79] reported a slower growth
of A. niger when a high carbon source concentration was used (100 g l−1 glucose or 90 g
l−1 xylose). During the cultivation experiments performed within the present work, large
differences in the metabolic rate based on the various initial glucose concentrations (65 or
250 g l−1, respectively) were observed (Figure 3.10).
A significantly higher itaconate yield was obtained in the low-glucose-concentration environ-
ment (65 g l−1). Since the osmotic pressure was much lower, also the level of cell metabolism
65
Chapter 4: Discussion
was substantially increased. This finding is in contrast to the theoretical yield estimations
(Figure 4.2), where a higher glucose concentration would be more favorable for itaconate
production. In the presence of the high glucose concentration, the osmotic pressure in
addition to the oxygen limitation cause the decrease in the itaconate production. Until this
issue has been resolved, lower glucose concentrations should be applied for itaconate pro-
duction with U . maydis. However, rather low titers can be reached with these concentrations.
Such difficulty can be overcome by alternative fermentation strategies such as fed-batch or
continuous cultures.
While the glucose concentration in the production medium for itaconate by A. terreus is usu-
ally between 130 and 160 g l−1 [80], [20], [81], [29], [75], [27], for U . maydis and the itaconate
production, the determined optimal sugar concentration was about 2-fold lower (65 g l−1).
This may suggest, that either A. terreus possesses better mechanisms for coping with osmotic
stress caused by the presence of high glucose concentrations than U . maydis, or that the
oxygen limitation itself prevents U . maydis from reaching high itaconate yields.
4.2.3 Oxygen supply
The importance of sufficient oxygen supply for production of organic acids generally [79] and
itaconate in particular is indisputable. The role of the oxygen in the process of the itacon-
ate synthesis is very well shown in the experiment with A. terreus performed by Gyamerah
[82], where a 5 min interruption of the oxygen supply during fermentation led to complete
cessation of the itaconate production. The process was only slowly renewed after 24 h. The
same results were obtained by Guevarra et al. [83] with Ustilago cynodontis. The findings
from the experiments concerning the culture aeration within this work with U . maydis are
in agreement with the literature. Cells of U . maydis cultivated in higher aerated shake flasks
(10 ml culture/1 l flask) produced up to 6.5-fold more itaconate than those in poorly aerated
ones (100 ml culture/1 l flask). A DOT of 20% in the cultivation experiments with A. niger
is considered to be a critical value, under which the culture is oxygen-limited [79]. Such
situation can be recognized by decreased oxygen uptake rate and consequently also lowered
CO2 production rate. As a result, the glucose utilization rate diminished as well as the biomass
yield. A very similar situation was observed during the cultivation of the U . maydis cells in the
poorly aerated shaken flask cultures within this work. Further experiments and calculations
revealed that the cells cultivated in the poorly aerated cultures depleted the total available
oxygen within the first few hours and during the rest of the several-day experiment the cells
suffered from lack of oxygen. On the other hand, the cells cultivated in the higher aerated
media had more than enough available oxygen and did not experience the stress of oxygen
depletion.
66
4.2 Factors influencing the itaconate production
Nevertheless, a limitation in the oxygen transfer rate was most likely present, because Kle-
ment et al. [33] obtained approximately 20 g l−1 itaconate with the wt strain of U . maydis.
This is substantially more than the itaconate titers obtained with the U . maydis wt strain
within this work (0.2 g l−1 itaconate) under otherwise comparable conditions. This 20 g l−1
itaconate was reached although the ratio of flask volume to culture volume was relatively
low (12.5). However, these cultures were agitated at a speed of 300 rpm, which significantly
increased the oxygen transfer rate, while all the cultivation experiments within this work were
performed at a speed of only 100 rpm. Therefore, it is likely that even the ‘high’ aeration
shaken flasks used in this study were to some extent limited in the oxygen transfer rate.
During glycolysis and the consequent itaconate or citrate synthesis routes (the synthesis and
the production conditions are very similar for both of these organic acids), NADH and ATP
are produced in excess (Figure 4.3; [70]). A high internal concentration of ATP may have
an inhibitory effect on the enzymes involved in glycolysis [28], and an excess of NADH can
cause metabolic cessation [84]. Since ATP and NADH are no longer used for cell growth
during itaconate production (in the case of U . maydis after the N or P exhaustion), the it-
aconate production leads to excess of these cofactors, which must be regenerated (Figure 4.3).
Figure 4.3: Putative possibilities of cofactor regeneration in U . maydis.
During the citric acid production process in A. niger one of the two major cofactor regenera-
tion routes includes an alternative (ubiquinol) oxidase (AOX; [28]). The main ability of this
terminal oxidase is to bypass the proton-pumping cytochrome pathway. Consequently, the
final ATP yield is lower with regard to the full length pathway, while the NADH regeneration
remains preserved. This is advantageous mainly when ATP is already in excess (during the
organic acids production).
67
Chapter 4: Discussion
U . maydis possess a gene encoding such alternative oxidase (UM02774) [85]. Thus, under
sufficiently aerobic conditions, the cofactors are regenerated via the respiratory chain, mak-
ing a sufficient oxygen supply in U . maydis cultures an important factor in the process of
itaconate production. Therefore this oxidase activity represents a very important factor in the
maintenance of the cofactor balance, and can be considered as the physiological basis for the
strong relation between oxygen supply and itaconate production.
Under anaerobic or oxygen limited conditions, the cofactors can be regenerated via various
routes. For S. cerevisiae it is the production of ethanol and glycerol [84], for E. coli ethanol
and a mixture of organic acids (e.g. lactate, acetate, formate [86]) and for A. niger mannitol or
other polyols [79]. While S. cerevisiae does not use malate production in order to regenerate
the NADH [84], for example Corynebacterium glutamicum does [87]. The main NADH regen-
erating reaction is catalyzed by a cytoplasmic malate dehydrogenase (EC: 1.1.1.37). However,
such regeneration is efficient only when malate is produced in cytosol from oxaloacetate [24]
since the malate production via the TCA cycle leads to further cofactor imbalance.
U . maydis possesses two genes encoding probable malate dehydrogenase (UM00403 and
UM04919). Since the shaken flask experiments were conducted under certain oxygen limita-
tion and large concentrations of malate were detected in the cultures, it is quite likely that
also U . maydis is capable of the cofactor regeneration via malate production [88]. Moreover,
this finding suggests, that the cis-aconitate route for the itaconate synthesis (Figure 4.1) might
be active in U . maydis, since malate is one of the key intermediates in the proposed pathway
[24]. Therefore, malate production can be used as an indicator for cofactor imbalance or
oxygen limitation during the production of itaconate by U . maydis.
Alternatively, lipid production represents yet another possibility to regenerate ATP and NADH
[89]. In comparison to U . maydis, A. terreus is not capable of lipid synthesis. The acetyl-CoA
used for fatty acid synthesis is transported from mitochondria, where it is produced, to the
cytosol in the form of citrate. Consequently, the citrate is cleaved back to oxaloacetate and
acetyl-CoA (citrate/malate cycle). This reaction is catalyzed by ATP-citrate lyase (EC: 2.3.3.8),
an enzyme present in oleaginous microorganisms. NADH aside of ATP is being recycled
in a cytosolic pyruvate regeneration cycle consisting of pyruvate, oxaloacetate and malate,
which essentially produces NADPH at a cost of NADH and ATP [89]. Acetyl-CoA from the
citrate/malate cycle together with the NADPH enables the consequent lipid biosynthesis.
The BLAST analysis between the protein sequence of ATP-citrate lyase from Aspergillus oryzae,
a typical oleaginous fungus, and the U . maydis genome resulted in a gene encoding a hypo-
thetical protein (UM01005). Consequent protein BLAST analysis of this putative U . maydis
ATP-citrate lyase revealed up to 59% identity to lyases of various microorganisms. Based
on all the previously listed findings, it is very likely that U . maydis regenerates the cofactors
also via the lipid production. However, the first choice for the cofactor regeneration during
the itaconate production is the respiratory chain, followed by both the malate and the lipid
68
4.2 Factors influencing the itaconate production
synthesis.
The production of surface active compounds by fungi and namely by Ustilago is known for a
long time and very well described [90], [39]. The cultivation experiments performed within
this work with U . maydis MB215 wt and ∆cyp1∆emt1 (U . maydis MB215 ∆cyp1 crossed with
U . maydis FB1 ∆emt1) revealed that disabling the glycolipid production has a positive effect
on the itaconate production. When the genes responsible for the glycolipid production in
U . maydis (cyp1 and emt1, [39]) were deleted, the itaconate titers as well as the biomass
production significantly increased in comparison to the parental strain. The inability of the
U . maydis cells to produce the lipids comes as an advantage in the process of the itaconate
production not only from these biosurfactants is not only favorable from an energetic point
of view, but also for the downstream processing.
As described previously the aeration of the culture is the most important factor influencing
the itaconate production. However, U . maydis mutant 2_29 is capable of significantly in-
creased itaconate production with regard to the other mutants as well as to the wt, even in the
poorly aerated cultures, whether it was the shaken flask culture experiments or the fermenter
batch cultivations with only 15 % DOT. Interestingly, fermenter batch cultivation with 80 %
DOT resulted in substantially higher itaconate titers from U . maydis wt strain in comparison
to the 2_29 mutant. Since this mutant was selected under oxygen limited conditions, it might
be capable of higher itaconate production selectively under oxygen limitation. However,
these fermentation experiments were preliminary and need to be repeated in order to exclude
any errors. The lower malate production as well as the ability of the 2_29 strain to sustain
its metabolism even under oxygen-limited conditions may imply that this strain possesses
alternative ways for NADH and ATP regeneration. This feature of the 2_29 strain might be
very important considering the putative application of this strain in the industrial itaconate
production, because it represents a decrease of the production costs due to the lower energy
demand required for aeration of the culture.
One of the most profound advantages of U . maydis with regard to A. terreus is its unicellu-
lar yeast-like form of growth. Cultivation experiments performed by Park et al. [81] with
A. terreus showed that since Aspergillus forms hyphae, it is very difficult to supply the culture
with sufficient oxygen. The filamentous growth of Aspergillus increases the viscosity of the
culture, which greatly influences the oxygen transfer into the medium and subsequently the
respiration rate of the cells. Moreover, a too high impeller tip speed in stirred batch cultures
leads to a shear stress and hyphal breaks from mechanical agitation. Consequently, the condi-
tions switch from aerobic to oxygen-limited, resulting in production of large concentrations
of various polyols, causing problems in industrial process [79]. Furthermore, A. terreus forms
pellets during its growth, and cells in the center of such pellet are exposed to conditions of
substantial oxygen limitation.
Thus, the hyphal growth pattern of Aspergillus leads to severe limitations in oxygen transfer,
69
Chapter 4: Discussion
a factor which is critical for itaconate production. Therefore, alternative ways to increase the
oxygen supply, such as decreased biomass concentrations, enriched oxygen supply [91] or
alternative reactor designs [92] need to be employed. However, all of these measures either
decrease the efficiency of itaconate production or add to the total operational cost.
Herein lies the main advantage of U . maydis as an alternative itaconate production host.
Since its cells exhibit a yeast-like unicellular growth pattern, the above mentioned difficulties
can be avoided, leading to an overall more efficient and reliable process.
4.2.4 Growth-limiting factors
For most microorganisms, the production of chemicals is coupled to growth. The most com-
mon example is S. cerevisiae and ethanol production. Therefore, in order to obtain high titers
of desired product, large concentrations of biomass, essentially an undesired by-product, are
produced as well [93]. Such phenomenon is not observed in U . maydis cultivations. After
exhaustion of N source, which occurs usually within the first 24 hours, the growth rate sig-
nificantly decreases and itaconate production starts (Figure 3.7). This represents rather an
exception among microorganisms, but also a great advantage, because after the first 24 hours
of cultivation, the utilization of the carbon source can be directed mostly to the production of
chemicals, without being used for unnecessary further biomass production.
In the cases of A. terreus [94], [59] and Pseudozyma antarctica [34], it is also N and P which
can trigger itaconate production. Interestingly, P is the more efficient trigger for the itaconate
production in A. terreus. In the cultivation experiments with A. terreus performed by Riscald-
ati et al. [95] the critical P concentration is 10 mg l−1. Consequently, the primary mycelia
growth is terminated and itaconate excretion starts as well as the secondary growth [59].
Secondary growth can be also observed in the U . maydis cultures [33]. After N depletion from
the medium, such growth is possible most likely due to the utilization of N stored within the
cells. However, secondary growth and consequent extra N utilization may lead to a reduced
concentration of proteins necessary for the itaconate production. This can be the reason for
the use of a P-limitation in the Aspergillus process.
4.3 Alternative carbon sources for itaconate production
Since the highest itaconate titers can be reached by utilizing glucose, it is currently the most
suitable carbon source for itaconate production. However, utilizing solely glucose is not
economically admissible due to its high price, and in bulk scale the use of edible substrates is
undesirable from a societal perspective. In order to find a suitable substituent, several other
70
4.4 Conclusions
carbon sources were investigated as putative substrates for the itaconate production.
One of the best substrates would be corn starch [20]. However, despite its low price, high
purity and stability in mass supply, its usage is rather limited. The reason is its gelatinization
during heat sterilization. This obstacle can be overcome by starch hydrolysis, although this
process is accompanied with several further difficulties [76], [20]. Other substrates commonly
used for the itaconate synthesis are sucrose, xylose [96] or glycerol [97]. Newly, also citric
acid has been tested as a putative substrate for the itaconate synthesis [92]. Citric acid with
its price being only 1/10 of the price of itaconate could prove itself to be an economically
interesting carbon source. However, its price still exceeds the price of glucose. Nonetheless,
the most frequently used substrates are beet and sugarcane molasses [32].
The most interesting substrates for the purposes of this work are those, which can be found
in hydrolyzed biomass. Not only are these carbon sources cheap, but most importantly, they
do not compete with common human diet sources. Aside of glucose, it is mainly xylose and
to a certain extent also arabinose, which are present in the hydrolysates in the highest con-
centrations [5]. However, the utilization of a single carbon source at a time (xylose, sucrose or
arabinose) by U . maydis resulted in significantly lower itaconate titers compared to glucose.
Begum et al. [98] showed, that combining two substrates in ratio 1:1 significantly increases the
citric acid titers by A. niger. Fermenter batch cultivations with mixed substrates conducted
within our institute with U . maydis cells resulted in similar findings. Solely used substrates
gave substantially lower itaconate titers, while mixtures of carbon sources led to much higher
itaconate concentrations (Maaßen et al., unpublished results).
Therefore, in order to employ these alternative carbon sources in successful high-yield it-
aconate production, they should be used in mixtures. This finding is also favorable from an
economical aspect, because separation and further purification of the substrates from the
hydrolyzed biomass is therefore not necessary.
4.4 Conclusions
This work describes U . maydis as an alternative itaconate producer to the current indus-
trial workhorse A. terreus. The itaconate titers from U . maydis do not yet reach those from
A. terreus. However, genes putatively involved in the itaconate synthesis pathway were se-
lected and respective U . maydis mutants created. The UM06344 gene, initially considered
as a putative cis-aconitate decarboxylase, is not essential for the itaconate production. On
the other hand, the UM11778 gene, putatively encoding 3-methylitaconate-∆-isomerase,
is crucial for itaconate production. This enzyme is thought to catalyze the last step in the
putative itaconate synthesis pathway, the conversion of citraconate to itaconate, and its
deletion greatly reduced the itaconate production. However, until the enzyme activity of
71
Chapter 4: Discussion
the enzyme encoded by UM11778 can be determined, a clear conclusion concerning the
itaconate synthesis pathway can not yet be drawn.
Nevertheless, high-yield itaconate mutants were obtained and the itaconate production
from these mutants was significantly improved with regard to the parental strain. These
mutants were selected under oxygen limited conditions, which is reflected by the ability of
mutant 2_29 to cope with low oxygen environments and the production. Even though some
of these improvements need to be further investigated and explained, the findings described
above brought certain insight into the itaconate production pathway. However, the route still
remains just putative.
Substantial improvements to the cultivation medium were employed. Contrary to theoretical
estimations, the most favorable glucose and NH4Cl concentrations were the lowest ones
tested. This is most likely due to oxygen limiting conditions in the shake flask experiments
used in this study.
Therefore, the influence of the various levels of oxygen supply on the itaconate production
as well as on the overall cell metabolism was investigated. As expected, culture aeration
represents a crucial factor in the final itaconate titers and the cell viability. From the three
aeration levels tested, the highest level proved to be the best. However, even the highly
aerated cultures in the shaken flask experiment are likely oxygen limited to a certain extend
and therefore further increase in the culture aeration could be beneficial.
Various carbon sources were tested as putative substrates for itaconate production, most
of them to be found in hydrolyzed biomass. Not only is U . maydis capable of utilization
of these sugars, moreover, it possesses the ability to form itaconate from these substrates.
However, further optimization is clearly required since the itaconate titers reached in these
experiments were very low.
Based on the findings presented within this work, U . maydis appears to be a suitable itacon-
ate producer due to its morphological qualities and the ability to produce itaconate naturally.
Nevertheless, the efficiency levels of the itaconate production of U . maydis do not meet those
of A. terreus. However, the previously stated results show, that improvement of the production
conditions and the host itself is possible and therefore, the U . maydis strain should ultimately
reach the efficiency levels of A. terreus.
4.5 Further recommendations
Although substantial improvements in itaconate production were achieved within this work,
there are still many aspects of both the process and the organism itself, which should be
further investigated and eventually employed.
Mutants with significantly improved itaconate production were obtained and several at-
72
4.5 Further recommendations
tempts with various methods (LILIS PCR - Ligated Linker Supported PCR or the recovery of
the integrated plasmid using PCR) were conducted in order to determine the integration loci
of the overexpression cassette without success. Clarification of the genetic background of
improved production in these mutants will be beneficial for consequent strain enhancement
and therefore new approaches, such as the whole genome sequencing of pooled mutants,
should be employed.
Although the phenotypic characterization of the mutants provided certain insight into the
itaconate synthesis pathway, it remains unclear via which biosynthetic route itaconate is
produced and whether there is no other pathway involved. The labeling of putative precurs-
ors of itaconate as described by Bonnarme et al. [25] would enable a definite conclusion in
this respect. Furthermore, several enzymatic steps such as R-citramalate and citraconate
production are thus far only theoretical, and therefore the identification of the genes and
enzymes responsible for these steps would be necessary.
For further U . maydis strain improvement, cell enhancements performed within this work
should be combined in one strain. The 2_29 mutant strain would be a good choice for a
parental strain, since its itaconate production is substantially increased and malate synthesis
reduced. In addition, the genes responsible for the glycolipid production in U . maydis (cyp1
and emt1) should be deleted in order to facilitate the downstream process and increase
carbon efficiency. If no lipids were produced, more glucose can be utilized for itaconate
production. Since the UM11778 gene plays a crucial role in the itaconate synthesis pathway,
its overexpression would likely also be highly beneficial as would also be the identification of
the other genes involved in the citraconate pathway. Further improvement of the itaconate
titers could also be obtained by identification of the itaconate transporter through the cell
wall and consequent overexpression of the gene encoding this protein.
As findings within this work revealed, the aeration of the culture represents a key factor
in the cell metabolism and consequently also in the itaconate yields. Nevertheless, even
in the highly aerated cultures within the shaken flask experiments, the oxygen supply was
to a certain extent limited. Therefore, further increase of the aeration of the cultures may
increase the itaconate yields. There are multiple ways to enhance the culture aeration; in
the fermenter, adding oxygen either directly into the culture or to the air stream is possible
as well as simple stirring with high speed. Since U . maydis exhibits an unicellular form of
growth, the negative effect of the shear stress does not need to be taken into consideration.
To ensure optimal oxygen supply in shaken flask experiment, increasing the agitation rate
should be sufficient.
Aside of the depletion of the N being the trigger for the itaconate synthesis, P may represent
another interesting growth limiting factor. However, experiments with low P and constant N
concentration would be necessary in order to verify this hypothesis as well as to pose a valid
comparison between N and P as the triggers for the itaconate production for U . maydis.
73
Chapter 4: Discussion
In order to establish a sustainable production process, alternative carbon sources should
be employed. As described above, sugars from hydrolyzed biomass should be utilized in
mixtures in order to reach high itaconate yields. However, both the optimal composition and
percentage of such mixture is yet to be determined.
Identification of the peak of the unknown substance visible in the HPLC profile of the
∆UM11778 strain might reveal further information about the itaconate production pathway
in U . maydis.
As mentioned above, current itaconate yields from U . maydis do not reach the same level as
those from A. terreus. However, applying these recommendations and further understanding
of the itaconate synthesis pathway will most certainly lead to the optimal production process
and consequently also to the desired itaconate titers and yields.
74
CHAPTER 5
Supplementary data
75
Chapter 5: Supplementary data
0h
24h
48h
72h
96h
120
h14
4h
Fig.
Aw
t0.
00.
02±
0.0
0.17
±0.
040.
2±
0.01
1.40
±0.
231.
75±
0.19
1.92
±0.
13
∆U
M06
344
0.0
1.23
±0.
076.
08±
0.25
9.85
±0.
3712
.38±
0.97
13.8
6±
1.03
13.8
3±
0.70
Fig.
Bw
tn
d0.
009
0.09
50.
345
0.40
20.
465
4.2
∆U
M06
344
nd
0.00
80.
20.
358
0.37
70.
483
5.05
Tab
le5.
1:C
omp
aris
onof
ITA
pro
du
ctio
n(g
l−1
)by
U.m
ayd
isw
tan
d∆
UM
0634
4st
rain
s;n
d-
not
det
erm
ined
(Fig
ure
3.2)
.
0h
24h
48h
72h
96h
120
h14
4h
ITA
(gl−
1)
0.0
0.01
3±
0.00
10.
02±
0.00
10.
05±
0.02
0.08
±0.
040.
13±
0.04
0.18
±0.
07
NH
4+
(gl−
1)
0.52
±0.
00.
06±
0.03
0.0
0.0
0.0
0.0
0.0
OD
(AU
)0.
4±
0.01
9.6±
0.44
16.9
±0.
818
.5±
1.05
16.5
±0.
9715
.5±
0.45
17.9
±0.
78
Glu
cose
(gl−
1)
251±
4.0
206±
5.1
196±
7.3
183±
4.7
170±
4.4
161±
6.1
152±
6.1
Tab
le5.
2:A
typ
ical
cult
ivat
ion
char
tofU
.may
dis
wt(
Figu
re3.
7).
76
Stra
in0
h24
h48
h72
h96
h12
0h
144
h
Fig.
A
ITA
(gl−
1)
wt
0.0
0.01
6±
0.00
20.
19±
0.04
60.
5±
0.11
1.17
±0.
212.
25±
0.38
3.1±
0.38
6_15
0.0
0.01
4±
0.00
10.
14±
0.03
0.35
±0.
090.
75±
0.22
1.22
±0.
361.
74±
0.55
6_17
0.0
0.02
6±
0.00
80.
56±
0.11
1.08
±0.
103
1.4±
0.06
1.76
±0.
032.
3±
0.13
2_29
0.0
0.01
2±
0.00
20.
76±
0.1
1.85
±0.
43.
08±
0.3
3.2±
0.3
4.18
±0.
34
NH
4+
(gl−
1)
wt
0.52
±0.
00.
14±
0.02
0.0
0.0
0.0
0.0
0.0
6_15
0.52
±0.
00.
1±
0.03
0.0
0.0
0.0
0.0
0.0
6_17
0.52
±0.
00.
11±
0.04
0.0
0.0
0.0
0.0
0.0
2_29
0.52
±0.
00.
08±
0.01
0.0
0.0
0.0
0.0
0.0
Fig.
B
ITA
(gl−
1)
wt
0.0
0.01
2±
0.00
20.
082±
0.01
40.
53±
0.17
2.0±
0.35
2.8±
0.29
3.15
±0.
13
6_15
0.0
0.01
±0.
0006
0.14
±0.
021
1.5±
0.00
64.
6±
0.09
7.9±
0.17
7.9±
0.29
6_17
0.0
0.01
1±
0.00
030.
15±
0.03
61.
0±
0.07
82.
75±
0.3
4.54
±0.
544.
8±
0.53
2_29
0.0
0.02
8±
0.00
31.
25±
0.29
3.9±
0.64
8.0±
0.8
10.0
4±
0.82
10.5
±0.
52
NH
4+
(gl−
1)
wt
0.52
±0.
00.
00.
00.
00.
00.
00.
0
6_15
0.52
±0.
00.
2±
0.0
0.0
0.0
0.0
0.0
0.0
6_17
0.52
±0.
00.
2±
0.01
0.0
0.0
0.0
0.0
0.0
2_29
0.52
±0.
00.
1±
0.0
0.0
0.0
0.0
0.0
0.0
Table 5.3: Influence of initial glucose amount on ITA production and ammonium
utilization by U . maydis wt and mutants (Figure 3.8).
77
Chapter 5: Supplementary data
Initial glucose amount (g l−1) Strain ITA (g l−1)
250
wt 3.1 ± 0.38
6_15 1.74 ± 0.55
6_17 2.27 ± 0.13
2_29 4.18 ± 0.34
110
wt 1.9 ± 0.32
6_15 2.9 ± 0.49
6_17 2.23 ± 0.32
2_29 5.67 ± 0.95
65
wt 3.15 ± 0.13
6_15 7.86 ± 0.29
6_17 4.8 ± 0.53
2_29 10.45 ± 0.52
Table 5.4: An overview of the influence of the initial glucose amount on ITA titers by
U . maydis wt and mutants (Figure 3.9).
78
Stra
in0
h24
h48
h72
h96
h12
0h
144
h
Fig.
A
ITA
(gl−
1)
wt
0.0
0.01
2±
0.00
040.
03±
0.00
730.
13±
0.03
80.
25±
0.08
0.4±
0.08
0.6±
0.15
6_15
0.0
0.01
1±
0.00
080.
016±
0.00
50.
08±
0.02
0.25
±0.
050.
47±
0.04
0.71
±0.
007
6_17
0.0
0.01
3±
0.00
20.
033±
0.01
80.
18±
0.06
70.
45±
0.22
0.83
±0.
441.
28±
0.49
2_29
0.0
0.01
1±
0.00
070.
25±
0.07
71.
52±
0.15
2.52
±0.
363.
07±
0.43
3.52
±0.
53
NH
4+
(gl−
1)
wt
0.52
±0.
00.
27±
0.01
0.2±
0.01
0.09
±0.
030.
02±
0.01
0.0
0.0
6_15
0.52
±0.
00.
32±
0.02
0.21
±0.
020.
08±
0.04
0.05
±0.
030.
03±
0.01
0.01
±0.
0
6_17
0.52
±0.
00.
31±
0.02
0.2±
0.05
0.09
±0.
050.
05±
0.02
0.03
±0.
010.
01±
0.0
2_29
0.52
±0.
00.
32±
0.02
0.17
±0.
010.
13±
0.05
0.09
±0.
040.
05±
0.02
0.05
±0.
02
Fig.
B
ITA
(gl−
1)
wt
0.0
0.04
6±
0.00
40.
22±
0.11
1.19
±0.
322.
13±
0.36
2.56
±0.
083.
23±
0.07
6_15
0.0
0.05
5±
0.00
52.
16±
0.82
6.18
±1.
779.
52±
1.68
10.8
1±
1.33
11.4
8±
1.37
6_17
0.0
0.05
8±
0.00
72.
42±
0.53
7.69
±1.
6411
.56±
1.45
14.0
±0.
5415
.5±
1.0
2_29
0.0
0.06
1±
0.00
013.
12±
0.35
10.3
8±
1.82
16.2
6±
1.42
20.4
1±
0.85
22.6
±1.
43
NH
4+
(gl−
1)
wt
0.52
±0.
00.
07±
0.03
0.0
0.0
0.0
0.0
0.0
6_15
0.52
±0.
00.
00.
00.
00.
00.
00.
0
6_17
0.52
±0.
00.
00.
00.
00.
00.
00.
0
2_29
0.52
±0.
00.
7±
0.02
0.0
0.0
0.0
0.0
0.0
Table 5.5: Influence of culture aeration on ITA production and ammonium utilization
by U . maydis wt and mutants (Figure 3.11). 79
Chapter 5: Supplementary data
Culture volume (ml) Strain ITA (g l−1)
100
wt 0.6 ± 0.15
6_15 0.71 ± 0.007
6_17 1.28 ± 0.5
2_29 3.52 ± 0.53
50
wt 3.1 ± 0.38
6_15 1.74 ± 0.55
6_17 2.3 ± 0.13
2_29 4.18 ± 0.34
10
wt 3.23 ± 0.07
6_15 11.5 ± 1.4
6_17 15.5 ± 1.0
2_29 22.6 ± 1.4
Table 5.6: An overview of the influence of the culture aeration on ITA titers by
U . maydis wt and mutants (Figure 3.12).
80
Stra
in0
h24
h48
h72
h96
h
Fig.
A
ITA
(gl−
1)
wt
0.0
0.01
4±
0.00
30.
006±
0.00
10.
016±
0.00
40.
046±
0.02
3
6_15
0.0
0.00
8±
0.00
020.
019±
0.01
20.
12±
0.05
0.21
±0.
08
6_17
0.0
0.00
8±
0.00
040.
0103
±0.
009
0.12
4±
0.09
0.48
±0.
2
2_29
0.0
0.00
5±
0.00
010.
028±
0.01
90.
73±
0.1
2.04
±0.
32
NH
4+
(gl−
1)
wt
1.35
±0.
00.
83±
0.09
0.81
±0.
050.
76±
0.11
0.6±
0.11
6_15
1.35
±0.
00.
91±
0.12
0.86
±0.
130.
71±
0.07
0.83
±0.
08
6_17
1.35
±0.
00.
89±
0.05
0.86
±0.
120.
79±
0.1
0.67
±0.
12
2_29
1.35
±0.
00.
89±
0.02
0.83
±0.
110.
71±
0.08
0.61
±0.
11
Fig.
B
ITA
(gl−
1)
wt
0.0
0.01
7±
0.00
10.
027±
0.00
40.
23±
0.04
0.44
±0.
05
6_15
0.0
0.03
8±
0.02
60.
57±
0.32
1.66
±0.
442.
9±
0.35
6_17
0.0
0.02
9±
0.01
30.
095±
0.06
52.
2±
0.6
4.6±
0.16
2_29
0.0
0.02
9±
0.01
70.
65±
0.21
2.0±
0.44
3.95
±0.
16
NH
4+
(gl−
1)
wt
0.25
0.0
0.0
0.0
0.0
6_15
0.25
0.0
0.0
0.0
0.0
6_17
0.25
0.0
0.0
0.0
0.0
2_29
0.25
0.0
0.0
0.0
0.0
Table 5.7: Influence of initial amount of nitrogen supply on ITA production and
ammonium utilization by U . maydis wt and mutants (Figure 3.14).
81
Chapter 5: Supplementary data
Initial NH4+ amount (g l−1) Strain ITA (g l−1)
4
wt 0.046 ± 0.023
6_15 0.21 ± 0.12
6_17 0.48 ± 0.2
2_29 2.04 ± 0.32
1.6
wt 0.18 ± 0.07
6_15 0.29 ± 0.07
6_17 0.54 ± 0.04
2_29 1.04 ± 0.12
8
wt 0.44 ± 0.05
6_15 2.9 ± 0.35
6_17 4.6 ± 0.16
2_29 4.0 ± 0.16
Table 5.8: An overview of the influence of the initial nitrogen supply on ITA titers by
U . maydis wt and mutants (Figure 3.12).
82
Stra
in0
h24
h48
h72
h96
h12
0h
144
h
Fig.
A
ITA
(gl−
1)
wt
0.0
0.02
±0.
00.
2±
0.04
0.2±
0.01
1.4±
0.2
1.8±
0.2
1.9±
0.13
6_15
0.0
1.5±
0.3
5.8±
0.9
10.7
±0.
113
.6±
0.2
14.2
±1.
014
.8±
0.2
6_17
0.0
1.4±
0.3
5.3±
0.1
9.6±
1.1
13.0
±1.
313
.7±
1.0
14.0
±1.
0
2_29
0.0
1.1±
0.03
8.0±
0.7
14.4
±1.
018
.5±
0.14
18.7
±0.
318
.8±
0.2
NH
4+
(gl−
1)
wt
0.25
±0.
00.
00.
00.
00.
00.
00.
0
6_15
0.25
±0.
00.
00.
00.
00.
00.
00.
0
6_17
0.25
±0.
00.
00.
00.
00.
00.
00.
0
2_29
0.25
±0.
00.
00.
00.
00.
00.
00.
0
Fig.
BM
alat
e(g
l−1
)
wt
nd
1.7±
0.6
3.8±
0.8
5.5±
1.1
6.3±
0.5
6.4±
0.6
6.4±
0.6
6_15
nd
1.0±
0.2
2.4±
0.3
2.8±
0.07
3.2±
0.5
3.6±
0.5
3.6±
0.2
6_17
nd
1.1±
0.1
2.2±
0.15
3.4±
0.6
4.1±
0.3
3.9±
0.5
3.5±
0.6
2_29
nd
0.28
±0.
031.
2±
0.11
1.7±
0.13
2.3±
0.09
2.2±
0.3
2.1±
0.3
Fig.
CO
D(A
U)
wt
0.5
18.3
±1.
422
.4±
0.8
21.4
±1.
121
.6±
0.4
20.7
±1.
417
.5±
1.0
6_15
0.5
20.6
±0.
421
.6±
1.1
18.1
±1.
617
.5±
0.7
16.1
±1.
115
.6±
0.4
6_17
0.5
19.0
±0.
619
.7±
1.0
18.2
±1.
813
.2±
1.6
9.8±
1.8
7.4±
1.7
2_29
0.5
16.0
±1.
119
.2±
1.1
19.1
±1.
39.
5±
1.1
7.8±
0.3
7.1±
0.6
Fig.
DG
luco
se(g
l−1
)
wt
65.0
39.7
±3.
724
.4±
3.5
15.6
±4.
318.
11±
3.2
4.4±
0.3
4.8±
0.4
6_15
65.0
38.8
±2.
325
.8±
1.5
15.2
±2.
28.
4±
3.5
4.4±
1.8
2.7±
1.5
6_17
65.0
38.0
±1.
924
.0±
4.9
12.6
±1.
82.
7±
0.9
2.0±
0.5
2.1±
0.4
2_29
65.0
39.1
±3.
625
.0±
2.7
12.7
±3.
23.
6±
2.1
1.6±
0.4
1.6±
0.2
Table 5.9: Cultivation of U . maydis wt and mutants under selected optimal condi-
tions; nd - not determined (Figure 3.16).
83
Chapter 5: Supplementary data
DO
Tan
d
stra
in0
h4
h20
h26
h50
h76
h92
h98
h11
6h
80%
,wt
ITA
(gl−
1)
0.0
0.0
0.19
0.77
20.6
32.2
34.6
35.2
36.9
NH
4+
(gl−
1)
0.51
0.48
0.00
90.
00.
00.
00.
00.
00.
0
DW
(gl−
1)
11.9
12.9
27.1
28.6
28.9
30.3
31.5
30.9
31.1
Glu
cose
(gl−
1)
199.
619
7.1
163.
615
0.8
104.
174
.753
.044
.024
.3
DO
Tan
d
stra
in2
h7
h21
h27
h32
h56
h62
h85
h11
0h
128
h13
4h
138
h16
0h
80%
,2_2
9
ITA
(gl−
1)
0.0
0.0
0.03
0.65
2.63
9.4
12.8
619
.56
23.9
525
.71
25.9
126
.52
28.9
9
NH
4+
(gl−
1)
0.61
0.57
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
DW
(gl−
1)
21.7
21.6
35.3
37.4
nd
34.8
34.5
35.3
32.0
30.7
30.4
3231
.6
Glu
cose
(gl−
1)
209.
020
2.7
182.
316
2.6
147.
611
9.7
110.
078
.354
.038
.235
.432
.925
.2
DO
TSt
rain
1h
20h
28h
51h
76h
94h
100
h11
6h
15%
wt
ITA
(gl−
1)
0.02
0.00
60.
038
0.23
0.24
0.26
0.36
0.34
Mal
ate
(gl−
1)
0.0
0.07
2.0
2.4
2.5
2.2
2.6
2.5
DW
(gl−
1)
12.1
23.7
25.2
26.6
24.8
25.0
23.8
25.0
Glu
cose
(gl−
1)
267.
123
0.0
215.
117
0.0
150.
013
9.0
147.
413
7.7
2_29
ITA
(gl−
1)
0.02
0.02
1.04
8.9
16.1
17.2
18.2
19.3
Mal
ate
(gl−
1)
0.0
0.0
0.5
2.2
3.0
3.1
nd
3.5
DW
(gl−
1)
16.1
27.4
31.7
31.2
28.1
29.8
30.1
29.5
Glu
cose
(gl−
1)
277.
724
7.7
225.
716
0.2
115.
197
.396
.289
.8
Tab
le5.
10:F
erm
ente
rb
atc
hcu
ltiv
ati
ons
ofU
.may
dis
wt
an
d2_
29st
rain
s;D
W-
dry
bio
ma
ssw
eigh
t,n
d-
not
det
erm
ined
(Fig
-
ure
3.17
).
84
Stra
in0
h24
h48
h72
h96
h12
0h
144
h
Fig.
A
ITA
(gl−
1)
wt
0.0
0.02
±0.
00.
17±
0.04
0.2±
0.01
1.4±
0.23
1.75
±0.
191.
92±
0.13
∆cy
p1∆
emt1
0.0
0.06
±0.
011.
36±
0.12
3.0±
0.65
3.96
±0.
764.
19±
0.99
4.1±
0.53
NH
4+
(gl−
1)
wt
0.25
±0.
00.
00.
00.
00.
00.
00.
0
∆cy
p1∆
emt1
0.25
±0.
00.
00.
00.
00.
00.
00.
0
Fig.
B
OD
(AU
)w
t0.
518
.3±
1.4
22.4
±0.
821
.4±
1.1
21.6
±0.
420
.7±
1.4
17.5
±1.
0
∆cy
p1∆
emt1
0.5
22.4
±0.
936
.3±
1.3
41.1
±1.
044
.6±
2.4
41.4
±2.
540
.2±
2.0
Glu
cose
(gl−
1)
wt
65.0
39.7
±3.
724
.4±
3.5
15.6
±4.
38.
1±
3.2
4.4±
0.3
4.8±
0.4
∆cy
p1∆
emt1
65.0
36.9
±0.
520
.0±
2.0
6.3±
1.0
1.0±
0.4
1.1±
0.5
1.1±
0.5
Table 5.11: Overview of the influence of the biosurfactant production on ITA titers
using U . maydis wt and ∆cyp1 ∆emt1 strains (Figure ??).
I
Chapter 5: Supplementary data
Strain 24 h 48 h 72 h 96 h 120 h 192 h
ITA (g l−1)wt 0.009 0.1 0.35 0.4 0.47 4.2
∆UM11778 0.0 0.004 0.004 0.01 0.012 0.014
Fumarate (g l−1)wt 0.01 0.06 0.14 0.35 0.44 0.56
∆UM11778 0.02 0.02 0.05 0.12 0.22 0.32
Glucose (g l−1)wt 60.6 55.3 50.1 37.1 28.5 0.0
∆UM11778 61.0 54.9 43.0 32.1 21.9 0.0
Succinate (g l−1)wt 0.25 1.0 2.8 3.0 2.8 3.2
∆UM11778 0.0 0.0 0.0 0.0 0.0 0.7
Malate (g l−1)wt 0.83 2.28 4.6 6.2 6.7 9.2
∆UM11778 0.0 0.0 0.12 0.95 2.0 5.0
Pyruvate (g l−1)wt 0.1 0.15 0.3 0.1 0.08 0.0
∆UM11778 0.6 0.23 0.16 0.14 0.16 0.0
Table 5.12: The production kinetics of various substances putatively involved in ITA
synthesis pathway. U . maydis wt and ∆UM11778 strains were used (Fig-
ure 3.20).
II
List of Figures
1.1 Overview of potential fuel precursors derived from various products of hydro-
lyzed lignocellulose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 Examples of possible itaconic acid application. . . . . . . . . . . . . . . . . . . . 5
1.3 Proposed subcellular organization of the itaconate pathway in A. terreus. . . . . 6
1.4 Smut disease on corn caused by dikaryotic form of U . maydis and the unicellu-
lar haploid form of U . maydis with buds. . . . . . . . . . . . . . . . . . . . . . . . 8
2.1 Outline of regeneration agar plate . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.1 Integrated disruption cassette. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2 Comparison of production of the itaconate by U . maydis wt and ∆UM06344
strains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.3 Outline of the UM06344 overexpression vector. . . . . . . . . . . . . . . . . . . . 28
3.4 Itaconate production by all the UM06344 overexpression transformants. . . . . 29
3.5 Outline of the integrated UM06344 overexpression vector. . . . . . . . . . . . . . 31
3.6 Southern blot analyses of U . maydis wt and UM06344 overexpression trans-
formants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.7 A typical cultivation chart of U . maydis wt . . . . . . . . . . . . . . . . . . . . . . 33
3.8 Influence of glucose concentration on the itaconate production and ammonium
utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.9 An overview of the influence of the initial glucose concentration on the itacon-
ate titer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.10 Influence of various glucose concentration in MTM on BOD and CBP in the
U . maydis 2_29 strain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.11 Influence of the culture aeration on the itaconate titer . . . . . . . . . . . . . . . 38
3.12 An overview of the influence of the culture aeration on the itaconate titer . . . . 40
3.13 Overview of the results from the off-gas analysis. . . . . . . . . . . . . . . . . . . 41
3.14 Influence of the initial NH4Cl concentration on the itaconate titer . . . . . . . . 42
III
List of Figures
3.15 An overview of the influence of the initial NH4Cl concentration on the itaconate
titer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.16 Kinetics of the itaconate production under selected optimal conditions . . . . . 45
3.17 Fermenter batch cultivation charts . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.18 Microscopic view of biosurfactants produced by U . maydis cells . . . . . . . . . 50
3.19 The itaconate production by U . maydis wt and ∆cyp1 ∆emt1 strains . . . . . . . 50
3.20 The production kinetics of various substances putatively involved in the itacon-
ate synthesis pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.21 HPLC profile of supernatant from U . maydis ∆UM11778 strain with clearly
visible itaconate peak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.22 HPLC profile of supernatant from U . maydis ∆UM11778 strain containing a
large peak of undefined compound . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.1 Putative itaconate synthesis routes in U . maydis and A. terreus . . . . . . . . . . 59
4.2 Theoretical yields and production rates of itaconate vs. ammonium concentra-
tion by U . maydis wt and 2_29 mutant strains . . . . . . . . . . . . . . . . . . . . 64
4.3 Putative possibilities of cofactor regeneration in U . maydis. . . . . . . . . . . . . 67
IV
List of Tables
1.1 Selection of alternative itaconate production hosts . . . . . . . . . . . . . . . . . 7
2.1 List of used strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2 List of used plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.3 List of used primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.1 Summary of integration numbers of UM06344 overexpression cassette parts . . 32
3.2 Experimental design of optimization of the itaconate production parameters . 34
3.3 The summary of the end-point values of OD, glucose concentration and yield
based on various initial glucose concentration . . . . . . . . . . . . . . . . . . . . 36
3.4 The summary of the end-point values of OD, glucose concentration and yield
based on various culture aeration . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.5 The summary of the end-point values of OD, glucose concentration and yield
based on various initial NH4Cl concentration . . . . . . . . . . . . . . . . . . . . 44
3.6 Selection of final values obtained from the optimal cultivation conditions ex-
periment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.7 The summary of the end-point values of OD, glucose concentration and yield
based on various carbon sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
5.1 Comparison of ITA production by U . maydis wt and ∆UM06344 strains . . . . . 76
5.2 A typical cultivation chart of U . maydis wt . . . . . . . . . . . . . . . . . . . . . . 76
5.3 Influence of initial glucose amount on ITA production and ammonium utilization 77
5.4 An overview of the influence of the initial glucose amount on ITA titers by
U . maydis wt and mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
5.5 Influence of culture aeration on ITA production and ammonium utilization . . 79
5.6 An overview of the influence of the culture aeration on ITA titer . . . . . . . . . 80
5.7 Influence of initial amount of nitrogen supply on ITA production and am-
monium utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
V
List of Tables
5.8 An overview of the influence of the initial nitrogen supply on ITA titer . . . . . . 82
5.9 Cultivation of U . maydis wt and mutants under selected optimal conditions . . 83
5.10 Fermenter batch cultivations of U . maydis wt and 2_29 strains . . . . . . . . . . 84
5.11 Overview of the influence of the biosurfactant production on ITA titers . . . . . I
5.12 The production kinetics of various substances putatively involved in ITA syn-
thesis pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II
VI
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XVI
Curriculum Vitae
Personal detailsName: Monika Maasem Panáková
Date of birth: 7.1.1982
Place of birth: Šumperk, Czech Republic
Nationality: Czech
Education9/1988 - 8/1990 5th Elementary School, Šumperk, Czech Republic
9/1990 - 8/1996 3r d Elementary School with Extended Foreign Language Education,
Šumperk, Czech Republic
9/1996 - 6/2001 Grammar School in Šumperk, Šumperk, Czech Republic; A-levels with
final "excellent" grade
9/2001 - 8/2004 University of Pardubice, Czech Republic, Faculty of Chemical Techno-
logy; Bachelor’s degree in Clinical Biology and Chemistry with final
"excellent" grade
9/2004 - 6/2006 University of Pardubice, Czech Republic, Faculty of Chemical Techno-
logy; Master’s degree in Analysis of Biological Materials with final "ex-
cellent" grade; Master thesis "The Influence of Low Molecular Weight
Hyaluronic Acid to Human Chondrocytes”
4/2008 - 10/2013 RWTH Aachen University, Aachen, Germany, Institute of Applied Mi-
crobiology; Ph.D. thesis "Itaconate production by Ustilago maydis; the
influence of genes and cultivation conditions"
Professional experiences2/2005 - 3/2006 Research assistant at Contipro Group, Dolní Dobrouc, Czech Republic
7/2006 - 8/2006 Research assistant at Department of Biochemistry, Faculty of Medicine
of Charles University in Prague, Hradec Králové, Czech Republic
10/2006 - 8/2007 Research assistant at Institute of Molecular Biophysics, CNRS Orleans,
France
Declaration of independent work
I declare that I have independently written the work presented here, and I have not used any
help other than from the stated sources and resources.
Aachen, January 29, 2013