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Bioprocess Technology
(Industrial biotechnology)
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Biological ScienceNon-Biological Science
&
Engineering
Biotechnology
&
Biochemical engineering
Bioprocess Technology
Product
Biotransformation /
Fermentation
by
Bio-agent
(enzymes/organisms)
Biomaterial /
biomass
Renewable / non-renewable
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Bioprocess Technology
Bioreactor/ fermentation
Food processing
Immobilized enzymes
Detoxification of wastes
Bye product utilization
Biosensor
Several others
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Upstream processing:
Selection and preparation of the suitable biosystem
[enzyme/organism(microbe)]
Selection and preparation of the substrate (for enzyme) / rawmaterial (for proper growth of the organism/microbe) for
product formation
Fermentation and Biotransformation:
Immobilization of the catalytic enzyme or growth of thecandidate microbe in a large bioreactor (usually > 100 lit) and
the target product formation by the single enzyme /pathway
enzymes
Design of the bioreactor, monitoring and controlling the
fermentation /biotransformation is very critical for yield of thetarget product
Downstream processing:
Purification of the target metabolite or molecule from either
the immobilized material/ the cell mass/ the culture medium
Bioprocess Technology/ Industrial biotechnology
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Microbial Cell Factories (MCFs)
for chemicals & fuels
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Microorganisms have the diverse metabolic pathways and pathwayenzymes to produce value-added chemicals, fuels and other bulk
products from simple, readily available and inexpensive starting
material.
Currently, these fuels and chemical products are derived from the
non-renewable resources, like petroleum or other fossil reserves.
However, the lignocellulosic biomass-derived sugars are renewable
resources.
Microbes have the capacity to utilize these biomass-derived sugars
to convert them into varieties of chemicals, drugs and fuels.
What is MCF?
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Glucose
Transformation of renewable biomass-derived sugars
to chemicals by MCF
Ref: Science 2010, 330: 1355-1358
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Critical considerations for development of a successful MCFs(i) Cost and availability of starting materials (e.g., carbon substrates);
(ii) Metabolic route and corresponding genes encoding the enzymes in the
pathway to produce the desired product;[Lack of well characterized pathway & enzymes, poor activity of the selected pathway
enzymes, metabolic burden, unfavorable cofactor balance. Thus designing and
engineering the pathway and the pathway enzymes followed by experimental
validation]
(i) Most appropriate microbial host;
(ii) Robust and responsive genetic control system for the desired pathways
and chosen host;
(iii) Methods for debugging and debottlenecking the constructed/designed
pathway; and
(iv) Bioprocess optimization: ways to maximize yields, titers, and
productivities
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Lecture # 1, delivered up to this slide on 23.07.2013
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1) Improvement of the upstream processes
i) Development and Improvement of the microbial strainincluding the pathwayand the enzymes involved for the
target metabolite/product
ii), iii), iv) etc. for other considerations in upstream
processes
2) Improvement in fermentation/ biotransformation
processes; designing, monitoring and controlling the
bioreactor.
3) Improvement of the downstream processes
Optimization of the bioprocess technology
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Strain development and improvement
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Biotransformation in large-scale by natural microbes is less than optimum
and not economic.Thus, strain improvement is essential and vital
1) Screening & selection from naturally occurring diversity and/
artificially induced genetic mutation:
2) Genetic or metabolic engineering: up-regulation of the desired
pathway or down-regulation of the competitive pathway to increase
the metabolic flux towards a desirable/targeted metabolite or
product by gene manipulation.
3) Rational design & engineering of the metabolic pathway: Recent
development in the re-design and model-based engineering of the
naturally exiting pathway and the pathway enzymes in one specific
host, and combination of enzymes and pathways from different hosts.
Optimization of the bioprocess technology:
Naturally occurring vs. engineered MCF
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Improved or engineered microbial strains coupled with the
optimization of the upstream and downstream processes have
enabled successful production of the desired metabolites (natural or
novel)
Optimization of the bioprocess technology:
Naturally occurring vs. engineered MCF
Thus, MCFs:
now, become a complementary and
in future, may be rival to the synthetic organic chemistry
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Design & engineering of the pathway for microbial cell
factories (MCFs): Conceptual strategies
a) Selection of specific production pathway
(Analogy: one can reach the destination by different routes)
b) Selection of suitable enzyme(s) for the pathway
(Analogy: each route has set of stations with bypasses)
c) Pathway optimization to enhance the product yield/titer
(Analogy: One combination of route, station and bypass for cost-
effective way/better comfort of journey/specific purpose)
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Design & engineering of the pathway for microbial cell
factories (MCFs): Conceptual strategies
a) Selection of specific production pathway:
Identification and selection of the target
metabolite, the suitable pathway and the
desired substrate/feedstock/biomass from several
possibilities at each level.
Bioinformatics approach- Several computational
tools are available and
Experimental validationin small-scale is crucial
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Design & engineering of the pathway for microbial cell
factories (MCFs): Conceptual strategiesb) Selection of suitable
enzyme(s) for the pathway using
various enzyme information
databases.
Selected Enzymes:
1) clearly known activityfor thesubstrate to specific
metabolite conversion
2) promiscuous activity for
structurally and chemically
similar substrates (or similarcatalytic reactions with
different substrate)
Bioinformatics approach &
Experimental verification
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Design & engineering of the pathway for microbial cell
factories (MCFs): Conceptual strategies
c) Pathway optimization to enhance the
product yield/titer:
1) Genetic/Metabolic engineering by up-
regulation or down-regulation of the
desired enzyme(s) by over-expression
or gene-knockout, respectively.2) Protein engineering to enhance the
activity (catalytic turn over) and
specificity (substrate binding) of the
pathway enzyme.
3) Cofactor balancing by effectiverecycling of suitable cofactor involved
in the target metabolite production.
Bioinformatics approach &
Experimental verification
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Design & engineering of the pathway for microbial cell
factories (MCFs): Conceptual strategies
Ref: Current Opinion of Structural Biology 2011, 21: 1-7
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Lecture # 2, delivered up to this slide on 30.07.2013
i & i i f h h f i bi l ll
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Design & engineering of the pathway for microbial cell
factories (MCFs): Operational strategies
a) Recruitment of partial pathways from independent sources
b) Using engineered or promiscuous enzymes,
c) de novo design or retro-biosynthetic approach
D i & i i f h h f i bi l ll
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Design & engineering of the pathway for microbial cell
factories (MCFs): Operational strategies
Ref: Current Opinion of Biotechnology 2008, 19:468-474
a) Recruitment of partial pathways fromindependent sources and co-localization
in a single host to produce a known target
product
D i & i i f h h f i bi l ll
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Design & engineering of the pathway for microbial cell
factories (MCFs): Operational strategies
Ref: Current Opinion of Biotechnology 2008, 19:468-474
b) Using engineered orpromiscuous enzymes,
new pathways can be
constructed for the
production of novel,non-natural products
M1
M2
D i & i i f th th f i bi l ll
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Design & engineering of the pathway for microbial cell
factories (MCFs): Operational strategies
Ref: Current Opinion of Biotechnology 2008, 19:468-474
c) de novo design or retro-biosynthetic
approach considers the biotransformation ofthe functional group rather than entire
structure, exploiting the tremendous natural
diversity of enzyme-catalyzed reactions
occurring across many living systems.
This can result into enhanced yield/titer of
already known product/metabolite
This can also lead to the production of novel
metabolites for which natural pathways have
not been elucidated.This strategy just started to develop, but the
situation is analogous to the retro-synthesis
scheme widely practiced by organic chemists.
D i & i i f th th f i bi l ll
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Design & engineering of the pathway for microbial cell
factories (MCFs): Operational strategies
Ref: Current Opinion of Biotechnology 2008, 19:468-474
D i & i i f th th f i bi l ll
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1) Combinations of existing pathways from different
organisms/strains into a single host
2) Engineering of existing pathway with naturally occurring
promiscuous enzymes or engineered enzymes or in combination
3) de novopathway design using synthetic & systems biology
Design & engineering of the pathway for microbial cell
factories (MCFs): Objectives/outcomes/examples
D i & i i f th th f i bi l ll
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1) Combinations of existing pathways from different
organisms/strains having individual native enzyme activities into asingle host:
Design & engineering of the pathway for microbial cell
factories (MCFs): Objectives/outcomes/examples
Outcome: new/hybrid pathway
to produce the same naturalproduct/metabolite
Example: 4-step pathway to produce isopropanol from acetyl-CoA,
was re-constructed in E. coli using enzyme encoding genes from E.
coli, Clostridia acetobutylicum and C. beijerinckii (Ref: Appl Environ
Micrbiol 2007, 73:7814-7818).
Desi n & en ineerin of the path a for mi robial ell
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2) Engineering of existing pathway with naturally occurring
promiscuous enzymes or engineered enzymes or in combination
Design & engineering of the pathway for microbial cell
factories (MCFs): Objectives/outcomes/examples
Outcome:new pathwayand novel product/metabolite
Example:Synthesis of novel 1,2,4-butanetriol from xylose in E. coli
using combination of promiscuous and engineered enzymes from
Pseudomonous fragi, E. coliand P. putida(Ref: J Am Chem Soc 2003,
125:12998-12999).
Design & engineering of the pathway for microbial cell
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3) de novo pathway design
Design & engineering of the pathway for microbial cell
factories (MCFs): Objectives/outcomes/examples
Outcome:new pathwayand novel product/metabolite or/and
Increasing yield of naturally-existing (already known) product/metabolite
Previously described two operational strategies are based upon the naturally
occurring known or promiscuous enzymes of the pathways.
Most recent approaches, such as BNICE (Biochemical Network Integrated
Computational Explorer*) and others go beyond the current knowledge
boundaries to explore the entire space of possible metabolic pathways
generated based upon in silicodesign using the enzyme reaction rules and
starting or target metabolites.
*Trends in Biotechnology (2010) 28: 501508
Examples: Production of artemisinic acid (precursor of anti-malerial drug)
and taxadiene (precursor of anticancer drug) in S. cerevisiae; Production of
polylactic acid(PLA, biodegradable thermoplastic) and taxadiene in E. coli.
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Lecture # 3, delivered up to this slide on 06.08.2013
Design & engineering of the pathway for microbial cell
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Design & engineering of the pathway for microbial cell
factories (MCFs): de novopathway design
Trends in Biotechnology (2010) 28: 501508
Design & engineering of the pathway for microbial cell
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Design & engineering of the pathway for microbial cell
factories (MCFs): de novopathway design
Trends in Biotechnology (2010) 28: 501508
Design & engineering of the pathway for microbial cell
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Design & engineering of the pathway for microbial cell
factories (MCFs): de novopathway design
Trends in Biotechnology (2010) 28: 501508
Design & engineering of the pathway for microbial cell
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Design & engineering of the pathway for microbial cell
factories (MCFs): de novopathway design
Trends in Biotechnology (2010) 28: 501508
Design & engineering of the pathway for microbial cell
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Design & engineering of the pathway for microbial cell
factories (MCFs): de novopathway design
Trends in Biotechnology (2010) 28: 501508
DREAMS of metabolism = Discovery, Retrosynthesis, Evolution, Aalysis of the pathways,
Mining of omics , Selection of targets for enzyme engg.
Overview of various high-throughput databases available for
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Overview of various high-throughput databases available for
metabolic pathway engineering
FEBS Letters (2010) 584: 2556-2564
Overview of various high-throughput databases available for
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Overview of various high throughput databases available for
metabolic pathway engineering
FEBS Letters (2010) 584: 2556-2564
Overview of various high-throughput databases available for
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Overview of various high throughput databases available for
metabolic pathway engineering
FEBS Letters (2010) 584: 2556-2564
Overview of various high-throughput databases available for
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Overview of various high throughput databases available for
metabolic pathway engineering
FEBS Letters (2010) 584: 2556-2564
Overview of various high-throughput databases available for
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Overview of various high throughput databases available for
metabolic pathway engineering
FEBS Letters (2010) 584: 2556-2564
Overview of various high-throughput databases available for
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Overview of various high throughput databases available for
metabolic pathway engineering
FEBS Letters (2010) 584: 2556-2564
Overview of various high-throughput databases available for
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Overview of various high throughput databases available for
metabolic pathway engineering
FEBS Letters (2010) 584: 2556-2564
Overview of various high-throughput databases available for
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Overview of various high throughput databases available for
metabolic pathway engineering
FEBS Letters (2010) 584: 2556-2564
Integration of system biology, synthetic biology and
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Integration of system biology, synthetic biology and
evolutionary engineering into metabolic engineering
Trends in Biotechnology August 2011, Vol. 29, No. 8 (pp 370-378)
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Systems metabolic engineering resulted:
Successful development of several designer pathways for
fine/value-added chemicals and biofuels production in MCFs
Development of a few synthetic microbes with minimumgenome for basic and applied research
Systems biology is an integrative theoretical and experimental
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Systems biology is an integrative theoretical and experimentaldiscipline that studies biological system at a holistic level .
Experimental systems biology utilize high through-put and genome-
wide tools at whole-cell or sub-cellular levels, and include methods to
profile genome, transcriptome, proteome, metabolome,and fluxome,
which are the hierarchical components for the flow of information for
life from genotype to phenotype.
Theoretical systems biology allows mathematical description of the
biological network that can be computationally simulated to predict
systematically the phenotypesof an organism under various conditions
of interest for several possible applications, including systemsmetabolic engineering.
Trends in Biotechnology August 2011, Vol. 29, No. 8 (pp 370-378)
Synthetic biology aims at creating novel functional parts modules
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Synthetic biologyaims at creating novel functional parts, modules,circuits, and/or organisms using synthetic DNAs (bio-bricks) and
mathematical/ logical methodologies.
It has been shown to be practical and useful in various biotechnological
applications, e.g. production of various chemicals and materials that are
heterologous to the original host strain.
More sophisticated and optimal design of such smart biologicalsystemswill be a continued challenge of synthetic biology for metabolic
engineering.
Trends in Biotechnology August 2011, Vol. 29, No. 8 (pp 370-378)
Evolutionary engineering is defined as a technique to select or
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Evolutionary engineering is defined as a technique to select orscreen cells that have desired phenotypes from variant cell libraries
that are created either adaptively or randomly.
To optimize metabolic pathways by evolutionary engineering,
mutagenesis followed by selective breeding, i.e. selection based on the
desired phenotype are required.
Some objectivesof evolution can includebetter cell growth,
utilization of desired carbon sources,
higher yield of product,
good tolerance to the product or inhibitory intermediate metabolite,elimination of byproduct formation.
Trends in Biotechnology August 2011, Vol. 29, No. 8 (pp 370-378)
Advantages of strain development by systems metabolic engineering
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Complementarily and synergistically overcomes the weaknesses of each technology.
Allows the creation of novel enzymes and metabolic pathways and gene regulatory circuits.
Fine-tuning and optimization of the metabolic fluxes that lead to increased yield andconcentration of a desired product.
Increased tolerance of cells to the product, harmful medium components or inhibitory
intermediate metabolite.
Formation of fewer or no byproducts.
Enhanced utilization of desired carbon substrates (biomass).
Development of cost-effective fermentation and downstream processes.
The starting point of systems metabolic engineering can be decided based on several criteria
such as the availability of native producer, biosynthetic pathways, enzymes, and others.
Several cycles of systems metabolic engineering can be performed until the efficiencies of
production of the desired chemicals and materials become satisfactory.
Trends in Biotechnology August 2011, Vol. 29, No. 8 (pp 370-378)
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Trendsin
BiotechnologyAugust2011,Vol.29,No.8
(pp
37
0-378)
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Lecture # 4, delivered up to this slide on 06.08.2013
This is Prof Bahadurs class, he was absent & told me to take this class.
What is the need for universal platform microbe for MCF ?
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pNo straightforward wayto extract the natural products (desired metabolite) from the native
producerat economic level
Most of the native producers are not cultivable in laboratory/ fermentor [Only 1% of bacteria
and 5% of fungi are cultivable in lab conditions]
Even if some are cultivable in laboratory, extensive optimization procedures are required to
standardize their growth conditions
Many organisms grow slowly and low-yielderof the desired product/metabolite
Lack of genetic and physiological information in these native producers and suitable
engineering tools to genetically manipulatethese organisms to improve the product yield and
productivity
Some platform microbes are:
Escherichia coliSaccharomyces cerevisiae
Bacillus subtilis
Pseudomonas putida
Streptomyces spp.
Certain problems of over-production of secondary metabolites in plants, but advantageous in
microbes
Example of systems metabolic engineering approach: Saccharomyces
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cerevisiaeplatform for production of value-added compounds
Ref: J Ind Microbiol Biotechnol 2011
Native pathway in yeast
Anti-malerial drug
Anti-cancer drug
IPP = isopentenyl pyrophosphate;
DMAPP = dimethylallyl pyrophosphate;
GPP = geranyl pyrophospahe;
GGPP = geranylgeranyl pyrophosphate
FPP = farnesyl pyrophosphate;
ADS = amorphadiene synthase
P450 = cytochrome P450 monooxygenase;CPR = cytochrome P450 reductase
Titer: From 115 mg/L to 1g/L
Titer: From 204 g/L to 8.7 mg/L
Engineered yeast to
produce
increased level of FPP and
reduced level of sterols
Twomoregenesaddedf
romA
rtemisiaannua
E.g., terpenoids
Two genes added from Taxus chinensis
Example of systems metabolic engineering approach: Saccharomyces
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cerevisiaeplatform for production of value-added compounds
Ref: J Ind Microbiol Biotechnol 2011
Native pathway in yeast
Anti-malerial drug
Anti-cancer drug
IPP = isopentenyl pyrophosphate;
DMAPP = dimethylallyl pyrophosphate;
GPP = geranyl pyrophospahe;
GGPP = geranylgeranyl pyrophosphate
FPP = farnesyl pyrophosphate;
ADS = amorphadiene synthase
P450 = cytochrome P450 monooxygenase;CPR = cytochrome P450 reductase
Titer: From 115 mg/L to 1g/L
Titer: From 204 g/L to 8.7 mg/L
Engineered yeast to
produce
increased level of FPP and
reduced level of sterols
TwomoregenesaddedfromA
rtemisiaannua
E.g., terpenoids
Two genes added from Taxus chinensis
Example of systems metabolic engineering approach: Escherichia coli
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platform for production of value-added compounds
Production of taxadiene 5 -ol, the precursor of taxol
Multivariate modular pathway
engineering, metabolomics and
synthetic biology approach.
Relative expression levels of two
modules are optimized to balance the
overall flux distribution for enhanced
production of the desired metabolitealong with the synthetic taxadiene 5-
hydroxylase with evolved N-terminal
transmembrane domain.
Metabolomics identified that indole isthe inhibitorof E. colicell growth and
taxadiene synthesis.
Titer: About 1g/L of taxadiene
Taxa-4,11-dieneGGPP
GLC = glucose
Trends in Biotechnology August 2011, Vol. 29, No. 8 (pp 370-378)
Example of systems metabolic engineering approach: Escherichia coli
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platform for production of value-added compounds
Production of polylactic acid (PLA) The artificially evolved heterologous
enzymes- propionate Coenzyme A (CoA)
transferase can produce lactyl-CoA from
lactate; and polyhydroxyalkanoate (PHA)synthase produces polylactic acid (PLA)
from lactyl-CoA.
Besides PLA homopolymer, the engineered
PHA synthase can also make copolymer
poly(3-hydroxybutyrate-co-lactic acid) orP(3HB-co-LA).
To increase the flux towards the precursors
of these biopolymers, the central carbon
metabolic pathways have been engineered
based upon genome-scale simulation by
eliminating acetate kinase (ackA), PEP
carboxylase (ppc) and alcohol
dehydrogenase (adhE) along with the
overexpression of lactate dehydrogenase
(ldhA) and acetyl-CoA synthetase (acs).
Yield: PLA 11 wt% ; P(3HB-co-LA) 56 wt% of dry cell wtTrends in Biotechnology August 2011,Vol. 29, No. 8 (pp 370-378)
The key issue: Genetic regulation of the pathway enzymes
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Successful development and operation of MCFs depend upon the
devices or machines made up of constituent parts- genes,
enzymes, reactions and metabolites.
Pathway enzymes are the important machine parts of the MCFs.
Suitable genetic transformation technique is required to introduce
the foreign or modified genes encoding the enzymes of themetabolic pathway.
Control over their expression is important to maximize yields and
titers.
All these genes need not to be highly expressed, but must be
produced in catalytic amounts sufficient to adequately transform the
metabolic intermediates into the desired products at a sufficient rate.
The key issue: Genetic regulation of the pathway enzymes (Contd..)
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High level expression of one gene dose not necessarily increase the
pathway flux.
However, coordinated and balanced expressions of all the genes
involved in the particular metabolic pathway are crucial for
minimizing the accumulation of the toxic metabolite and maximizing
the product formation.
Whether transgene(s) integrated in the genome or not
Copy number of the plasmidbearing the genes of interest
Promoterstrength & types (constitutive or inducible)
Presence of terminator Proper uses of activatorand repressor
Specificity/efficiency of ribosome binding site (RBS)
Codonusage
mRNAstability
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Lecture # 5, delivered up to this slide on 20.08.2013
The key issue: Genetic regulation of the pathway enzymes (Contd..)
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Over-expression or underexpression of the genes may be problem:
Expression of the desired genes at too high a level will rob the cell of
metabolites that might otherwise be used to produce the desiredmolecule of interest, particularly important for production of low-
margin chemicals, while under-expressed genes will create pathway
bottlenecks.
When intermediate metabolite is toxic/inhibitory:
Furthermore, because intermediates of a foreign metabolic pathway can
be toxic to the heterologous host, which results in decreased production
of the desired final compound, it is essential that the relative levels of
the enzymes be coordinated.
Use of regulatory RNA (encoded by gene R) or protein
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Use of regulatory RNA (encoded by gene R) or protein
(encoded by gene P) to modulate the metabolic pathway
that has a toxic/inhibitory metabolite
Ref: Science 2010, 330: 1355-1358
Use of regulatory RNA (encoded by gene R) or protein
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g y ( y g ) p
(encoded by gene P) to modulate the metabolic pathway
that has a toxic/inhibitory metabolite
Ref: Science 2010, 330: 1355-1358
Use of regulatory RNA (encoded by gene R) or protein
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g y ( y g ) p
(encoded by gene P) to modulate the metabolic pathway
that has a toxic/inhibitory metabolite
Binding of the regulatory RNA and protein to the toxic metabolite
down-regulate the toxic metabolite synthesis and up-regulate the
product formation Ref: Science 2010, 330: 1355-1358
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MCFs for production of building block
chemicals & biopolymers
MCFs for production of building block chemicals & biopolymersMicroorganisms are endowed with capabilities to synthesize a wide range of building
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Microorganisms are endowed with capabilities to synthesize a wide range of building
block chemicals, i.e. monomers and polymers/co-polymers from varieties of carbon
sources.
These monomers and biopolymers/bio-co-polymers serve diverse biological
functions in natural hosts.
They have varying chemical and material propertiessuitable for numerous industrial
(food/feed & non-food/feed) and medical applications.
Due to increasing concerns on the environmental pollution (because of chemical
industries), adverse climate change (affecting plants as bioreactors) and depletion offossil-fuel (and stored chemicals), MCFs are considered as the suitable platform for
production of these chemicals from renewable biomass.
Enhanced production and isolation of the particular monomeric chemicals that may
be used for synthesis of biopolymer outside the host cells or
direct synthesis of tailor-made biopolymers with highly applicable material
properties has been possible by superior strain or MCFs coupled with modern
fermentation technologyand advanced downstream processes.
Recent advances in systems metabolic engineering helped us to develop superior
strains or MCFs.
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MCFs for production of building block chemicals & biopolymers
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(A) Production of building block chemicals: A few examples
1) Succinic acid producers:
Natural strains are mostly rumen bacteria.
Natural isolates of Actinobacillus succinogenes, Anaerobiospirillum
succiniciproducens, Mannheimia succiniciproducens, Corynebacterium
glutamicumand their engineered derivativescan produce 52106 g/L
of succinic acid.
Metabolically engineered, acid-tolerant (cells grow at low pH, which is
required for industrial production and purification), osmo-tolerant yeast
Yarrowia lipolytica can produce a titer of 45.5 g/L succinic acid
Systems metabolic engineeredE. colican produce 87 g/Lsuccinic acid
Succinic acid is commonly used as surfactant, precursor of other
chemicals (e.g. 1,4-butanediol = BDO) and poly(butylene succinate) =
PBS polymer
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(A) Production of building block chemicals: A few successful examples
2) Lactic acid producers:
Natural isolates of Lactobacillus plantarum, Lactococcus lactis,
Lactobacillus delbrueckii, and Lactobacillus caseiand their engineered
derivativescan produce 30135 g/Lof lactic acid.
Metabolically engineeredE. colican produce 138 g/Llactic acid
3) Itaconic acid producers:
Natural isolates of Aspergillus terreus after strain improvement by
random mutagenesis produce 82 g/Lof itaconic acid.Systems metabolic engineeredE. colican produce 4g/Litaconic acid
Lactic acid is the precursorof polylactic acid (PLA) homopolymer andpoly(3-hydroxybutyrate-co-lactic acid) or P(3HB-co-LA) copolymer
Itaconic acid is the precursor of polyitaconate homopolymer and
poly(acrylate-co-itaconate)copolymer
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(A) Production of building block chemicals: A few successful examples
4) Glucaric acid producers:
Naturally present in fruits, vegetables and mammals, but not produced
by natural microbes.
Systems metabolic engineered E. coli with a synthetic polypeptide
scaffolds composed of protein-protein interaction domains can produce
2.37 g/Lglucaric acid
Glucaric acid is the precursorof poly(hexamethylene glucaramide) and
poly(glucaramide), a type of water-resistant hydroxylated nylon
PtsG= PEP-dependent glucose phosphotransferase; Ino1= myo-inositol-1-phosphate
synthase; SuhB= inositol monophosphatase; MIOX= myo-inositol oxygenase; Udh=
uronate dehydrogenase
MCFs for production of building block chemicals & biopolymers
( ) d f b ld bl k h l f f l l
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(A) Production of building block chemicals: A few successful examples
5) Adipic acid producers:
It is the most important dicarboxylic acid for industrial uses. Naturally
rarely present (in some plants and historically first isolated from
oxidation of fats?), but not produced by natural microbes.
Systems metabolic engineered E. coli can produce 36.8 g/L muconic
acid, which after chemical hydrogenation produces adipic acid.
Adipic acid is the precursor of nylon-4,6 and nylon-6,6 polymer and
polyurethane
DHS= 3-dehydroshikimic acid; PCA= protocatechuic acid; CTL= catechol; MCA= cis,cis-
muconic acid; AroZ= DHS dehydratase; AroY= PCA decarboxylase; CatA= CTL 1,2-
dioxygenase
MCFs for production of building block chemicals & biopolymers
( ) d i f b ildi bl k h i l f f l l
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(A) Production of building block chemicals: A few successful examples
6) Isoprene producers:
It is a 5-carbon diene known as 2-methyl-1,3-butadiene. Naturally present in
all classes of living organisms as different modified forms or polymers.Systems metabolic engineeredE. colican produce 60 g/Lisoprene.
Isoprene is the precursor of the largest class of naturally occurring isoprenoids or terpenoids
molecules having medicinal and industrial values. Its polymer, i.e. poly(isoprene) mainly
extracted from rubber tree.
MvaE= AACoA thiolase/HMGCoA reductase; MvaS= Mev synthase; HMG-CoA= 3-hydroxy-3-methyl-glutaryl-
CoA; MVK= Mev kinase; PMK= phosphomevalonate kinase; MVD= diphosphomevalonate decarboxylase; Idi=
isopentenyl diphosphate isomerase; IspS= isoprene synthase
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Lecture # 6, delivered up to this slide on 27.08.2013
MCFs for production of building block chemicals & biopolymers(B) Production of biopolymers: A few successful examples
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(B) Production of biopolymers: A few successful examples
Some microbes naturallyproduce biopolymers or can be engineeredto produce the
biopolymers directlyby fermentation/transformation without the chemical catalytic
process.One step microbial biopolymer formation has several advantages including the
control on composition of polymer, particularly the heteropolymer by balancing the
ratio of various monomers, and circumvent the costly and environmentally harmful
chemical catalytic process
1. Polysaccharides: Intracellular- Glycogen
Extracellular- Alginate, Xanthan , Dextran, Curdlan,
Gellan, Cellulose, Hyaluronic acid
2. Polyamides: Intracellular- Cyanophycin
Extracellular- Poly- -glutamate, -poly-L-lysine
3. Polyesters: Intracellular- Polyhydroxyalkanoate (PHA), Polylactic acid
(PLA, not natural in microbes)
4. Polyanhydrides: Intracellular- Polyphosphate
Four major classes of naturally occurring microbial biopolymers & a few examples:
Bacterial biopolymer synthesis pathways from intermediates ofcentral metabolism
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central metabolism
Nature Review Microbiology (2010) 8: 578- 592
l l d d l
Examples of bacterial polymers, features and their applications
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Polymer Primary structure Main component Natural producer Industrial
applications
Glycogen -(1,6)-branched-(1,4 )- linked
homopolymer
Glucose Bacteria and
archaea
----------
Alginate -(1,4)-linked non-repeating
heteropolymer
Mannuronic acid
and guluronic acid
Pseudomonas spp.
andAzotobacter spp.
Biomaterial (for example, as a
tissue scaffold or for drug delivery)
Xanthan -(1,4)-linked repeating
heteropolymer consisting of
pentasaccharide units
Glucose, mannose
and glucuronate
Xanthomonasspp. Food additive (for example, as a
thickener or an emulsifier)
Dextran -(1,2)/-(1,3)/ -(1,4)-branched
-(1,6)-linked homopolymer
Glucose Leuconostocspp. and
Streptococcus spp.
Blood plasma extender and
chromatography media
Curdlan -(1,3)-linked
homopolymer
Glucose Agrobacterium spp.,
Rhizobium spp. etc
Food additive (for example, as a
thickener or a gelling agent)
Gellan -(1,3)-linked repeating
heteropolymer consisting of
tetrasaccharide units
Glucose, rhamnose
and glucuronate
Sphingomonas spp. Culture media additive, food
additive (for example, as a gelling
agent) or for encapsulation
Cellulose -(1,4)-linked homopolymer Glucose Alpha-, Beta- and
Gamma- proteobacteria,
Gram-positive bacteria
Food, diaphragms of acoustic
transducers and wound dressing
Hyaluronicacid
-(1,4)-linked repeatingheteropolymer consisting of
disaccharide units
Glucuronate and N-acetyl glucosamine
Streptococcusspp. andPasteurella multocida
Cosmetics, viscosupplementation,tissue repair and drug delivery
Cyanophycin
granule
Repeating heteropolymer
consisting of dipeptide units
Aspartate and
arginine
Cyanobacteria,
Acinetobacterspp. , etc.
Dispersant and water softener
(after removal of arginyl residues)
Poly-
-glutamate
Homopolymer d-glutamate and/or
L-glutamate
Few Gram +, Gram -
bacteria, & few archaea
Replacement of polyacrylate,
thickener, humectant, drug
delivery and cosmetics
Nature Review Microbiology (2010) 8: 578- 592
P l P i M i N l d I d i l
Examples of bacterial polymers, features and their applications
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Polymer Primary structure Main component Natural producer Industrial
applications
Glycogen -(1,6)-branched-(1,4 )- linked
homopolymer
Glucose Bacteria and
archaea
----------
Alginate -(1,4)-linked non-repeating
heteropolymer
Mannuronic acid
and guluronic acid
Pseudomonas spp.
andAzotobacter spp.
Biomaterial (for example, as a
tissue scaffold or for drug delivery)
Xanthan -(1,4)-linked repeating
heteropolymer consisting of
pentasaccharide units
Glucose, mannose
and glucuronate
Xanthomonasspp. Food additive (for example, as a
thickener or an emulsifier)
Dextran -(1,2)/-(1,3)/ -(1,4)-branched
-(1,6)-linked homopolymer
Glucose Leuconostocspp. and
Streptococcus spp.
Blood plasma extender and
chromatography media
Curdlan -(1,3)-linked
homopolymer
Glucose Agrobacterium spp.,
Rhizobium spp. etc
Food additive (for example, as a
thickener or a gelling agent)
Gellan -(1,3)-linked repeating
heteropolymer consisting of
tetrasaccharide units
Glucose, rhamnose
and glucuronate
Sphingomonas spp. Culture media additive, food
additive (for example, as a gelling
agent) or for encapsulation
Cellulose -(1,4)-linked homopolymer Glucose Alpha-, Beta- and
Gamma- proteobacteria,
Gram-positive bacteria
Food, diaphragms of acoustic
transducers and wound dressing
Hyaluronicacid
-(1,4)-linked repeatingheteropolymer consisting of
disaccharide units
Glucuronate and N-acetyl glucosamine
Streptococcusspp. andPasteurella multocida
Cosmetics, viscosupplementation,tissue repair and drug delivery
Cyanophycin
granule
Repeating heteropolymer
consisting of dipeptide units
Aspartate and
arginine
Cyanobacteria,
Acinetobacterspp. , etc.
Dispersant and water softener
(after removal of arginyl residues)
Poly-
-glutamate
Homopolymer D-glutamate and/or
L-glutamate
Few Gram +, Gram -
bacteria, & few archaea
Replacement of polyacrylate,
thickener, humectant, drug
delivery and cosmetics
Nature Review Microbiology (2010) 8: 578- 592
Gl l h t li t i d t i l t b d t
Example of waste by-product utilization to make biopolymer
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Glycerol, whey, corn steep liquor etc. are industrial waste by-products
How the whey, a waste by-product of cheese- and yogurt-making
industries is converted into economically valuable biopolymer?
Whey contains ~94% water, ~ 5% lactose, rest small amount of
proteins, minerals etc.
Enormous quantities are generated by diary industry
Disposal is a problem and cause ground-water contamination &
environmental pollution
Releasing into rivers and lakes cause available oxygen depletion,
killing aquatic organisms
When used in processed food product, may be a problem to lactoseintolerant people
Recovering the solid component from whey is cost-expensive
Thus, effective way of whey utilization was sought for
How E. coli lactose catabolism was introduced into other bacteriacapable of synthesizing biopolymer?
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Xanthomonas campestrisis a natural producer of xanthan gum. The wild type
X. campestrisutilizes glucose, sucrose and starch, but not lactose as carbon.
The genes encoding for lactose catabolism enzymes from E. coli were cloned
onto a broad-host range plasmid and under the transcriptional control of X.
campestrisbacteriophage promoter.
lacZ encodes -galactosidase enzyme that cleaves the disaccharide lactose
into glucose and galactose
lacY encodes -galactoside permease, a membrane-bound transport protein
that pumps lactose into the cell
This recombinant plasmid was then introduced into X. campestris by
triparental mating.
capable of synthesizing biopolymer?
Mol Biotech book Glick et al.
How E. coli lactose catabolism & biopolymer synthesizing genes ofother bacteria were put together in E. coli?
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other bacteria were put together in E. coli?
Several bacteria like Alcaligenes eutrophus, Ralstonia eutropha, Pesudomonassp.,
Azotobacter sp. naturally synthesize polyhydroxybutyrate (PBA) , a type of
polyhydroxylalkanote (PHA),but not E. coli.
The PBA synthesizing genes from Azotobacter sp. were cloned onto the plasmid
under the transcriptional control of lacpromoter.
This recombinant plasmid was then introduced into an E. coli strain that has the
genes for lactose uptake and assimilation, but not the lactose repressor gene. Thus,
this transformed E. coli cells express the lactose catabolism and PBA biosynthesizingconstitutively.
phaA for 3-ketothiolase, phaB for acetoacetyl-CoA reductase, phaC for
polyhydroxylalkanoate synthase
The engineered E. colican grow on whey or corn steep liquor(a by-product of corn
processing, and source of nitrogen, amino acids, vitamins and other nutrients) to
produce PBA upto 73% of dry cell weightin fed-batch culture.
Mol Biotech book Glick et al.
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Lecture # 7, delivered up to this slide on 10.09.2013
On 03.09.2013, Prof. D. Das took my class. Hence, I took two classes
(lecture # 7 & 8) on 10.09.2013