studies on breeding of yeast saccharomyces cerevisiae for

TitleStudies on breeding of yeast Saccharomyces cerevisiae foreffective macroalgae utilization based on the metabolism ofmarine bacterium( Dissertation_全文 )

Author(s) Takagi, Toshiyuki

Citation 京都大学

Issue Date 2017-03-23

URL https://doi.org/10.14989/doctor.k20440

Right学位規則第9条第2項により要約公開; 許諾条件により本文は2019-06-20に公開; 許諾条件により要約は2018-03-22に公開

Type Thesis or Dissertation

Textversion ETD

Kyoto University

Studies on breeding of yeast Saccharomyces cerevisiae for effective macroalgae utilization based on the metabolism of marine bacterium

Toshiyuki TAKAGI

2017

Contents

Introduction

Chapter I Elucidation of alginate assimilation mechanism of the

marine bacterium Saccharophagus degradans 2-40 based

on quantitative proteomic analysis

Chapter II Engineered yeast whole-cell biocatalyst for degradation of

alginate and production of non-commercialized useful

monosaccharide 4-deoxy-L-erythro-5-hexoseulose uronic

acid

Chapter III Generation of Saccharomyces cerevisiae for direct ethanol

production from alginate of brown macroalgae by

synthetic biology

Conclusions

Acknowledgements

Publications

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Abbreviations

PolyM polymannuronic acid

PolyG polyguluronic acid

PL polysaccharide lyase

CAZy carbohydrate-active enzymes

DEH 4-deoxy-L-erythro-5-hexoseulose uronic acid

KDG 2-keto-3-deoxy-D-gluconic acid

DehR DEH reductase

KdgK KDG kinase

KdgpA 2-keto-3-deoxy-6-phosphogluconate aldolase

GAP glyceraldehyde-3-phosphate

GPI glycosylphosphatidylinositol

ER endoplasmic reticulum

TMT tandem mass tag

FDR false discovery rate

PCA principal component analysis

SDR short-chain dehydrogenase/reductase

MFS major facilitator superfamily

TonB phage T-one

2OG oxygenase 2-oxoglutarate/Fe2+-dependent oxygenase

GntR transcriptional regulator gluconate repressor

YPD yeast extract-peptone-dextrose

SD synthetic dextrose

MES 2-morpholinoethanesulfonic acid

PBS phosphate-buffered saline

TIM triose phosphate isomerase

F16BPase fructose-1,6-bisphosphatase

DNS 3,5-dinitrosalicylic acid

TLC thin layer chromatography

YPA yeast extract-peptone-alginate

YPM yeast extract-peptone-mannitol

SM synthetic mannitol

YPAM yeast extract-peptone-alginate-mannitol

HPLC high-performance liquid chromatography

1

Introduction

Over the past hundred years, heavy consumption of fossil fuels has increased

the carbon dioxide level in the atmosphere, contributing to environmental problems such

as climate change and ocean acidification. Furthermore, in accordance with the

population growth, the world energy demand is continuously increasing. Thus, the

development of a renewable and clean source of bioenergy is essential for a sustainable

energy future. Bioethanol has been produced from agricultural biomass such as corn,

sugar cane, wheat, and sugar beet (Quintero et al. 2008; Wang et al. 2002; Ogbonna et al.

2001). Relative to fossil fuels, bioethanol is less toxic and its utilization produces fewer

harmful environmental consequences (John et al. 2011). However, the use of biomass for

energy production competes with its value as a foodstuff for humans and livestock.

Although lignocellulosic biomass such as sugar cane bagasse, rice straw, and wheat straw

has also been considered as an alternative feedstock for bioethanol production

(Velmurugan et al. 2011; Okamoto et al. 2011; Shinozaki et al. 2011), the presence of

lignin in lignocellulose biomass complicates the release of the polysaccharide in the

conversion to bioethanol and results in inefficient bioethanol production. Therefore, using

biomass with low lignin content is effective for the low cost refining of bioethanol.

Potential of brown macroalgae as feedstock for biofuel production

Macroalgae are a promising alternative biomass feedstock for bioethanol

production because they contain little or no lignin; thus simple biorefinery processes can

be used to produce sugars (Wargacki et al. 2012). Macroalgae are plentiful in ocean

ecosystems and can grow at rates that far exceed those of terrestrial plants, mainly

because the ocean is not a water-limited environment (Ross et al. 2008). Commercial

scale cultivation of macroalgae for both food product and their biochemical constituents

is already being carried out in many countries. Aquaculture-based world production of

macroalgae was estimated to be approximately 15.1 million wet metric tons annually,

based on 2006 data (Table 1) (Roesijadi et al. 2010). Macroalgae production from

aquaculture is concentrated in Asian countries and is mainly practiced by China, which

accounted for 73% of the total value of macroalgae farming, based on the 2006 data.

Macroalgae farming does not require arable land for cultivation and grows in salt water,

avoiding competition for the fresh water resources required for field crop production.

2

With these considerations, macroalgae appear well suited to use in biofuel production,

circumventing adverse impacts on food supplies.

Macroalgae are classified according to their photosynthetic pigments, color

schemes (e.g., green, red, and brown) (Fig. 1) and habitat (Ross et al. 2008; Demirbas et

al. 2010). The exclusive economic zone of Japan is 4,479,358 km², the sixth-largest in

scale in the world (Fig. 2). Macroalgae present in Japan constitute more than 1,000

species. Among them, brown macroalgae are characterized by large and containing useful

metabolite phlorotannins, which exhibit anti-allergy and inflammatory activities. For

instance, Undaria pinnatifida, a kelp speacies native to temperate shores of Japan, grow

to 3 m long (Akiyama and Kurogi 1982). Brown macroalgae contain cellulose,

hemicellulose, mannitol, laminarin, and alginate as major carbohydrates. Among the

carbohydrates, brown macroalgae are characterized by the high content of mannitol,

laminarin, and alginate. Laminarin is a β-1,3-linked glucose polymer with β-1,6 cross-

link branches and contains 30 glucose residues on average (Fig. 3). The higher degree of

β-1,6 branching makes laminarin more soluble. It is used as a long-term storage

compound produced in brown macroalgae and has seasonal variation ranging from 0 to

35% of the dry weight (Kadam et al. 2015). Mannitol is also the carbon storage compound

of brown macroalgae and represents up to 20–30% of the dry weight depending on the

species, harvesting season, and habitat (Reed et al. 1985) (Fig. 3). This polyol is produced

from fructose-6-phosphate via the enzymatic reaction of a mannitol-1-phosphate

dehydrogenase and mannitol-1-phosphatase (Groisillier et al. 2014). Mannitol not only

acts as carbon storage but also acts as an osmoprotectant and antioxidant in brown

macroalgae. Although several key studies have demonstrated ways to utilize the

carbohydrates, complete utilization of brown macroalgae cannot be realized without the

development of alginate (Ota et al. 2013a; Motone et al. 2016). In the current study, in

order to complete the process of brown macroalgae utilization, I developed a method for

efficient ethanol production from alginate.

Fig. 1 Green, red, and brown macroalgae. (http://chibadai.flier.jp/algae/algae/) Ulva linza (Green) . (Green)

Costaria costata (Brown) Meristotheca papulose (Red)

. (Red)

3

Table 1 Top ten producers of cultured macroalgae and world total, plus monetary

value of cultures (USD) in 2006 (Roesijadi et al. 2010).

Fig. 2 Exclusive economic zones of Japan.

(http://www.kaiho.mlit.go.jp/e/index_e.htm)

Fig. 3 Chemical structures of laminarin (left) and mannitol (right).

Source (country) Production

(metric ton) % of total

Value

US$1,000s $/metric ton

World total 15,075,612 100.00 7,187,125 476.74

China 10,867,410 72.09 5,240,819 482.25

Philippines 1,468,905 9.74 173,963 118.43

Indonesia 910,636 6.04 127,489 140.00

Republic of Korea 765,595 5.08 269,657 352.22

Japan 490,062 3.25 1,051,361 2,145.36

Korea DPRp 444,300 2.95 244,365 550.00

Chile 33,586 0.22 52,394 1,560.00

Malaysia 30,000 0.20 4,500 150.00

Vietnam 30,000 0.20 15,000 500.00

Cambodia 16,000 0.11 4,000 250.00

4

Properties of alginate and its applications

Alginate, a major component of brown macroalgae, is a linear polysaccharide

composed of α-L-guluronic acid and its C5-epimer β-D-mannuronic acid (Fig. 4A).

Alginate has 1,4-glycosidic bonds between the pyranose rings of the two monosaccharide

components (Fig. 4B). In the cases of Ascophyllum nodosum and Laminaria digitata, the

alginate contents are 22–30% and 25–44% on a dry weight basis, respectively (Qin 2008).

The biological function and physiological properties of alginate in brown macroalgae is

thought to be similar to those of cellulose in terrestrial plants (Draget et al. 2005).

The sequence of alginate is variable but consists of three distinct regions: a

polymannuronic acid (polyM), a polyguluronic acid (polyG), and a random region. A

higher ratio of mannuronic acid increases the viscosity and gel strength of the polymer,

and O-acetylation of mannuronic acid decreases the efficiency of degradation within the

cell (Wong et al. 2000). The relation between structure and function varies according to

species and tissues from the same algae. For instance, in Laminaria hyperborean, the

stipe and holdfast have a very high content of guluronic acid, providing high mechanical

rigidity. In contrast, the leaves of the same macroalgae have a low content of guluronic

acid, providing a flexible texture (Draget et al. 2005). Annual industrial production of

alginate is estimated to be approximately 30,000 metric tons, mainly from the genera

Laminaria and Macrocystis of brown macroalgae (Pawar et al. 2012). Alginate is widely

used across a variety of industries in food, textiles, cosmetics, and bioplastics (Hay et al.

2013). More recently, alginate has been utilized in the area of high-value biomedical

applications including wound management, anti-adhesion, drug delivery systems, and

tissue encapsulation for regenerative therapy (Lee et al. 2012). However, alginate is not only of interest as a polymer; the degradation products

of alginate are useful for various applications. The oligosaccharides are known to have

various physiological properties such as radical scavenging activity toward superoxide

radicals, enhancing the cytokine secretion from human macrophages, and growth of

human keratinocytes and endothelial cells (Iwamoto et al. 2005; Iwamoto et al. 2003;

Kawada et al. 1999; Courtois 2009; Falkeborg et al. 2014; Yamamoto et al. 2007). The

monosaccharides are useful for biofuel production using a metabolically engineered

recombinant microorganism (Enquist-Newman et al. 2014). Therefore, a stable source of

alginate lyase is essential for obtaining alginate-derived natural antioxidants and biofuels.

In alginate-assimilating bacterium, an endolytic alginate lyase first degrades

5

alginate into smaller oligosaccharides. These oligosaccharides are further broken down

into monosaccharides by an exolytic alginate lyase, and the monosaccharides are then

non-enzymatically converted into 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH)

(Takase et al. 2010). Polysaccharide lyases (PL) are categorized into 21 families in the

carbohydrate-active enzymes (CAZy) database. Among these, the families containing

alginate lyases are PL-5, 6, 7, 14, 15, 17, and 18 (Garron and Cygler 2010). Various

alginate lyases have been discovered in marine and soil bacteria. For examples, FlAlyA

from Flavobacterium sp. strain UMI-01 (Inoue et al. 2014), A1-IV from Sphingomonas

sp. A1 (Miyake et al. 2003), AlgMsp from Microbulbifer sp. 6532A (Swift et al. 2014),

Atu3025 from Agrobacterium tumefaciens (Ochiai et al. 2010), Vibrio sp. QY101 (Han

et al. 2004), Vibrio splendidus 12B01 (Jagtap et al. 2014), Corynebacterium strain ALY-

1 (Matsubara et al. 2000), Pseudoalteromonas elyakovii IAM (Sawabe et al. 2001), and

Streptomyces sp. ALG-5 (Kim et al. 2009), have been successfully produced in

Escherichia coli cells and characterized.

(A)

β-D-Mannuronic acid α-L-Guluronic acid

(B)

Fig. 4 Structural characteristics of alginates: (A) alginate monomers, (B) chain

conformation.

Alginate assimilation by microbes and strategies for alginate utilization

Cellulose and starch are linear polysaccharides of glucose and bioconversion

of their degradation products can be easily processed (Gray et al. 2006). In contrast,

alginate is a linear polysaccharide composed of mixed mannuronic and guluronic acids.

The use of technologies previously developed for cellulose and starch processing would

6

result in difficulties in the bioconversion of alginate.

Although several alginate-assimilating bacteria were isolated from the marine

environment, a Gram-negative alginate-assimilating bacterium Sphingomonas sp. Strain

A1 isolated from soil is the only microorganism for which the complete alginate-

assimilating processes has been investigated in detail (Hayashi et al. 2014).

Sphingomonas sp. strain A1 cells possess a unique alginate assimilating process; in

particular, the process of alginate uptake (Fig. 5). When grown on alginate,

Sphingomonas sp. strain A1 cells form a mouth-like pit on the cell surface for

reorganization and transporting-alginate into the periplasm directly. After alginate is

degraded into DEH with various endolytic and exolytic alginate lyases in cytoplasmic

space, DEH is converted to 2-keto-3-deoxy- D-gluconic acid (KDG) by DEH reductase

(DehR). The KDG can then be metabolized to pyruvate and glyceraldehyde-3-phosphate

(GAP) by KDG kinase (KdgK) and 2-keto-3-deoxy-6-phosphogluconate aldolase

(KdgpA), and fed into central metabolic pathways (Takase et al. 2010).

In contrast, although the alginate derived from brown macroalgae is important

for marine ecosystems as an essential nutrient, the detailed mechanisms behind alginate

assimilation remain largely unknown in marine bacteria. Saccharophagus degradans 2-

40 is one of the most powerful biomass degrading microbes with a salt requirement

typical of marine bacteria (Andrykovitch and Marx 1988). S. degradans is a gamma-

subgroup proteobacterium of the Alteromonadales group. This bacterium was isolated

from decaying saltwater marsh grass in a marine estuary (Andrykovitch and Marx 1988).

The unique character of this bacterium was further revealed by complete genome

sequencing in 2008 (Weiner et al. 2008). The genome sequencing characterization

revealed that a large portion of predicted genes were involved in the assimilation of the

complex polysaccharides. The current study is focused on S. degradans, a microbe that

can metabolize whole plant material in monocultures. S. degradans expresses genes

encoding multi-enzyme complexes that can degrade at least 10 different carbon

polysaccharides, including cellulose, hemicellulose, xylan, agar, pectin, and alginate

(Hutcheson et al. 2011).

Although a subset of the alginate utilization mechanisms has been examined,

including alginate degradation, the detailed mechanism(s) of alginate utilization is not

well known (Kim et al. 2012; Kim et al. 2012; Wang et al. 2014; Hutcheson et al. 2011).

7

Fig. 5 Alginate uptake and assimilation system in Sphingomonas sp. strain A1.

Yeast cell surface engineering technology

Yeast Saccharomyces cerevisiae is the most widely used yeast for industrial

ethanol fermentation. Although it has been used for bioethanol fermentation from brown

macroalgae in previous studies (Enquist-Newman et al. 2014), S. cerevisiae lacks the

ability to utilize polymers, in this case, alginate. Among the alginate assimilation

processes, alginate degradation into DEH by alginate lyases is first essential step for direct

ethanol fermentation from alginate.

The yeast cell surface display system should be applied to develop the alginate

degradation process using yeast. The yeast cell surface display system is an innovative

method for the construction of whole-cell biocatalysts (Kuroda and Ueda 2013) (Fig. 6A).

Although intracellular expression and extracellular secretion have been employed in

conventional strategies for molecular breeding of yeast, this system enables the

immobilization of functional heterologous proteins and displays one million molecules

of enzymes on the yeast cell surface (Kondo and Ueda 2004). Cell surface-engineered

yeast have been introduced in “Chemical & Engineering News” and were termed “arming

yeasts” (Ueda and Tanaka 2000; Ueda and Tanaka 2000).

8

(A) (B)

Fig. 6 (A) Mechanism of cell surface display of proteins by cell surface display

system; (B) molecular design of cell-surface displayed enzyme.

The yeast has a rigid cell wall, about 200 nm thick, mainly composed of an

outer layer of mannoproteins and an internal skeletal layer of β-linked glucans, and lies

outside of the plasma membrane. The bilayered structure of the cell wall consists of a

fibrillary-like outer layer, which is composed of glycosylated proteins covalently linked

to the cell wall glucan, and an internal skeletal layer of glucan, which is composed of β-

1, 3- and β-1, 6- linked glucose.

Among the glycosylated proteins on the cell surface of yeast, the mating-type-

specific agglutinins, which mediate the cell to cell adhesion between cells of the opposite

mating type during mating, are representatives of cell wall-anchoring proteins used for

yeast cell surface display system. In particular, α-agglutinin is the most widely utilized

approach because of the N-terminus of displayed proteins freely on the cell surface. α-

agglutinin is composed of an N-terminal secretion signal peptide for transportation to the

cell surface, an active domain, and C-terminal glycosylphosphatidylinositol (GPI) anchor

attachment signal. Molecular information on α-agglutinin is used to display the target

heterologous protein on the yeast cell surface. The target protein fused with the N-

terminal of secretion signal sequence and the C-terminal half of α-agglutinin, which is

the cell wall binding domain containing GPI anchor attachment signal at the C-terminal

end (Fig. 6B). After transcription, the fusion proteins are translocated into the lumen of

the endoplasmic reticulum (ER), and further transported from the ER onto the plasma

membrane via Golgi apparatus and the secretory vesicle. The GPI anchored proteins are

9

released from the plasma membrane via the cleavage by phosphatidylinositol-specific

phospholipase, and bind to the outer most surface of the yeast cell walls with a covalent

bond by transglycosylation.

Although some purified alginate lyases have been used to produce the

oligosaccharides and monosaccharides from alginate (Falkeborg et al. 2014; Enquist-

Newman et al. 2014), it is costly to produce purified enzyme on a commercial scale. To

solve commercial problems, I employed the yeast cell surface engineering system to

utilize alginate lyases. This technology does not require a separation step as the active

enzymes are produced on the yeast cell surface, and enables: (1) an easy display of 1 ×

104 to 1×105 enzymes on the yeast cell surface; (2) the enzyme-displaying yeast to be

treated as a whole-cell biocatalyst; (3) utilization without purification; and (4) reusability

(Nakanishi et al. 2012; Ota et al. 2013b; Inokuma et al. 2014; Bae et al. 2015). In addition,

although some alginate lyase-producing bacteria are pathogens of seaweed and plants,

using yeast displaying alginate lyase avoids the risks of using pathogenic bacteria (Ochiai

et al. 2010; Han et al. 2004).

Moreover, as a result of the lack of ability of DEH assimilation, further

molecular breeding of yeast is required for direct ethanol production from alginate of

brown macroalgae. Therefore, a DEH metabolic pathway derived from alginate

assimilating microorganisms will be introduced into alginate lyase-displaying yeast using

synthetic biological techniques.

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of AlgMsp, an alginate lyase from Microbulbifer sp. 6532A. PloS ONE 9:e112939

Takase R, Ochiai A, Mikami B, Hashimoto W, Murata K (2010) Molecular identification

of unsaturated uronate reductase prerequisite for alginate metabolism in Sphingomonas

sp. A1. Biochim Biophys Acta 1804:1925-1936

Ueda M, Tanaka A (2000) Cell surface engineering of yeast: construction of arming yeast

with biocatalyst. J Biosci Bioeng 90:125-136

Ueda M, Tanaka A (2000) Genetic immobilization of proteins on the yeast cell surface.

Biotechnol Adv 18:121-140

Velmurugan R, Muthukumar K (2011) Utilization of sugarcane bagasse for bioethanol

production: sono-assisted acid hydrolysis approach. Bioresour Technol 102:7119-7123

Wang da M, Kim HT, Yun EJ, Kim do H, Park YC, Woo HC, Kim KH (2014) Optimal

production of 4-deoxy-L-erythro-5-hexoseulose uronic acid from alginate for brown

macro algae saccharification by combining endo- and exo-type alginate lyases.

Bioprocess Biosyst Eng 37:2105-2111

Wang R, Dominguez-Espinosa RM, Leonard K, Konutinas A, Webb C (2002) The

application of a generic feedstock from wheat for microbial fermentation. Biotechnol

Prog 18:1033-1038

Wargacki AJ, Leonard E, Win MN, Regitsky DD, Santos CN, Kim PB, Cooper SR,

Raisner RM, Herman A, Sivitz AB, Lakshmanaswamy A, Kashiyama Y, Baker D,

14

Yoshikuni Y (2012) An engineered microbial platform for direct biofuel production from

brown macroalgae. Science 335:308-313

Weiner RM, Taylor LE, Henrissat B, Hauser L, Land M, Coutinho PM, Rancurel C,

Saunders EH, Longmire AG, Zhang HT, Bayer EA, Gilbert HJ, Larimer F, Zhulin IB,

Ekborg NA, Lamed R, Richardson PM, Borovok I, Hutcheson S (2008) Complete

genome sequence of the complex carbohydrate-degrading marine bacterium,

Saccharophagus degradans strain 2-40T. PloS Genet 4:e1000087

Wong TY, Preston LA, Schiller NL (2000) ALGINATE LYASE: review of major sources

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Yamamoto Y, Kurachi M, Yamaguchi K, Oda T (2007) Stimulation of multiple cytokine

production in mice by alginate oligosaccharides following intraperitoneal administration.

Carbohydr Res 342:1133-1137

15

Chapter I

Elucidation of alginate assimilation mechanism of the marine bacterium

Saccharophagus degradans 2-40 based on quantitative proteomic analysis

Alginate is major carbohydrate of brown macroalgae. In order to utilize brown

macroalgae completely, elucidation of alginate assimilation process of the marine

bacterium is essential step. Marin bacterium S. degradans, one of the most versatile

polysaccharide-degrading microbes, capable of assimilating whole plant material by

releasing sugars through the coordinated production of carbohydrase (Hutcheson et al.

2011). Based on genome sequencing of S. degradans, existence of 13 genes encoding

alginate lyase were predicted (Weiner et al. 2008). However, detailed mechanisms for

alginate degradation, transport, and metabolism have not been determined.

In this chapter, I quantitatively analyzed the proteome using tandem mass tag

(TMT)-labeled samples with unique long monolithic silica capillary column in order to

characterize the abundance of glucose-, pectin-, and alginate-metabolism-associated

proteins in S. degradans. Proteins associated with the specific carbon sources were

successfully identified and quantified, including membrane transporter, enzymes relating

to degradation and metabolism of alginate by S. degradans. Furthermore, I elucidated

alginate assimilation mechanisms of S. degradans based on the results of quantitative

proteomic analysis.

Materials and methods

Strains, media, and culture conditions

S. degradans 2-40 (ATCC43961) was pre-cultured in minimal media [2.3%

(w/v) Instant Ocean (Aquarium Systems, Mentor, Ohio), 0.2% (w/v) glucose, 0.5% (w/v)

ammonium chloride, and 50 mM Tris-HCl, pH 7.4] at 30 °C for 12 h. Cultured cells were

harvested by centrifugation at 6,000×g for 10 min at 25 °C and resuspended in fresh

minimal media containing 0.2% (w/v) glucose (Nacalai Tesque, Kyoto, Japan), highly

methylated (70-75%) pectin from apple (P8471; Sigma, MO, USA), or alginate from

brown algae (A2033; Sigma) at 30 °C, and cell growth was monitored by measuring the

OD600 in triplicate.

16

E. coli strain DH5α (F−, ϕ80dlacZΔM15, Δ (lacZYA-argF)U169, deoR, recA1,

endA1, hsdR17(rK-, mK

+), phoA, supE44, λ−, thi-1, gyrA96, relA1) (Toyobo, Osaka,

Japan) and BL21 (DE3) (BioDynamics Laboratory Inc., Tokyo, Japan) were used as the

host for recombinant DNA manipulation and recombinant protein expression,

respectively. Each E. coli transformant was grown in Luria-Bertani media (1% tryptone,

0.5% yeast extract, 0.5% sodium chloride) containing 100 µg/mL ampicillin.

Sample preparation for proteomic analysis

Samples were prepared as described previously with modifications as indicated

(Aoki et al. 2013). After incubation, each cell culture was instantly cooled in ice-cold

water and centrifuged at 6,000×g for 10 min at 4 °C. Harvested cells were resuspended

in 700 µL lysis buffer [2% (w/v) 3-(3-cholamidepropyl) dimethylammonio-1-

propanesulfonate, 10 mM dithiothreitol, 1% (v/v) protease inhibitor cocktail (Sigma-

Aldrich, MO, USA), 7 M urea, 2 M thiourea, and 50 mM Tris-HCl pH 7.5]. The cells

were then disrupted by sonication using Bioruptor UCD-250T (Cosmo Bio, Tokyo,

Japan) (250 W, 15 s on and 15 s off/ 15 cycles, on ice). The crude solution was centrifuged

at 12,000×g for 10 min, and the supernatant was harvested. Each supernatant was

subjected to reduction, alkylation, and digestion by trypsin (Esaka et al. 2015). The tryptic

digests were labeled using a TMT 6-plex labeling kit (Thermo Fisher Scientific, Waltham,

MA, USA) with reporters at m/z = 126, 127, 128, 129, 130, and 131, respectively, in 41

µL acetonitrile. After 60 min at room temperature, 8µL of 5% (w/v) hydroxylamine was

added to each tube and further incubated for 15 min. Aliquots were pooled and evaporated

under vacuum, then dissolved in 60 µL of formic acid (0.1%) and subjected to LC-MS/MS

analysis.

Quantitative proteomic analyses of S. degradans cultured in the presence of different

carbon sources

Proteomic analyses were performed using an LC (Ultimate 3000; Thermo

Fisher Scientific)-MS/MS (LTQ Orbitrap Velos Mass Spectrometer; Thermo Fisher

Scientific) system equipped with a long monolithic column as described previously

(Morisaka et al. 2012). The monolithic silica column is a unique separation medium of

liquid chromatography that exhibits lower column back pressure owning by its high

permeability. This property enables the utilization of the long column that is impossible

17

by conventional particle-packed columns and shows higher performance in comparison

to conventional columns (Morisaka et al. 2012). Tryptic digests were separated by

reversed-phase chromatography using a monolithic silica capillary column (500 cm × 0.1

mm ID) at a flow rate of 500 nL/min. The gradient was achieved by changing the mixing

ratio of the two eluents from 5% to 45% B for 600 min (A, 0.1% (v/v) formic acid and B,

80% acetonitrile containing 0.1% (v/v) formic acid). The separated analytes were

detected using a mass spectrometer with a full scan range of 350–1500 m/z (resolution

60,000) followed by 10 data-dependent HCD MS/MS scans acquired for the TMT

reporter ions. A normalized collision energy of 40% for HCD with a 0.1 ms activation

time was used. An ESI voltage of 2.4 kV was applied directly to the LC buffer distal to

the chromatography column. The temperature of the ion transfer tube on the LTQ Velos

ion trap was set at 300 °C.

The mass spectrometry data files were used for protein identification and

quantification by using Protein Discoverer 1.4 (Thermo Fisher Scientific). Protein

identification was performed with a Mascot algorithm against the amino acid sequence

data of S. degradans 2-40, including the 4007 sequences from NCBI

(http://www.ncbi.nlm.nih.gov/) with a precursor mass tolerance of 30 ppm and a fragment

ion mass tolerance of 30 mmu. Carbamidomethylation of cysteine, oxidation of

methionine, and TMT 6-plex of the N-term were set as translational modifications.

Protein quantification was performed by Reporter Ions Quantifier using the TMT 6-plex

method. The data were then filtered at a q-value of ≤ 0.01 corresponding to 1% false

discovery rate (FDR) on the spectral level. Proteins with no missing values were accepted

as “identified proteins.” A global median normalization was carried out to normalize the

data to the amount of tryptic digests assessed by mass spectrometry.

Construction of plasmid for the overexpression of short-chain

dehydrogenase/reductase Sde_3281

The short-chain dehydrogenase/reductase (SDR) Sde_3281 gene was amplified

from S. degradans isolated genomic DNA by PCR using the following primers: 5’-

AGAAGGAGATATACAATGAAACTTGAAAATAAAGTATGTG-3’ and 5’-

GGTGGTGGTGCTCGATTATGAGAAGTACAAGCCGCCGTTT-3’. The PCR

products were cloned into the pET-21a expression vector (Novagen, Madison, WI) using

the In-Fusion HD cloning kit (Takara Bio Inc., Shiga, Japan).

18

SDR Sde_3281 expression and activity assay

The E. coli strain BL21 (DE3) strain harboring the SDR Sde_3281 expression

plasmid was grown in Luria-Bertani medium containing 100 µg/mL ampicillin at 30 oC

to OD600 of 0.5. Protein expression was induced by the addition of IPTG (0.1 mM), and

culturing was continued for an additional 12 h. The pellet was washed and resuspended

with HEPES buffer (50 mM, pH 7.4). The samples were sonicated using Bioruptor UCD-

250T (Cosmo Bio) (250 W, 15 s on and 15 s off/ 15 cycles, on ice), and the supernatant

obtained after centrifugation at 15,000×g for 20 min at 4 °C were used for enzyme assays.

A spectrophotometric assay modified from a previous report to identify DehR

activity in the E. coli strain BL21 (DE3) lysates was developed (Enquist-Newman et al.

2014). The DEH reaction mixture contained 50 mM HEPES (pH 7.4), 100 mM NaCl, 50

mM KCl, 5 mM MgCl2, 10 mM DEH, and 2.0 mM NADPH in a final volume of 100 µL.

The reaction was initiated by adding 5 µL of E. coli BL21 lysate at 25 oC. The activity

was determined by continuously monitoring the decrease in absorbance at 340 nm, which

corresponds to the oxidation of NADPH.

Results

Growth confirmation of S. degradans

The cells of S. degradans reached early stationary phase at 9 h in minimal

medium with glucose, pectin, or alginate, respectively (Fig. 1). Although the cells reached

stationary phase at 9 h, glucose, pectin, or alginate were not completely exhausted in the

medium at 12 h (data not shown). From these observations, I selected a culture time of

12 h for proteomic analysis.

19

Fig. 1 Growth confirmation of S. degradans cultured with three different substrates. Growth of S. degradans with glucose (●), pectin (■), or alginate (▲) were monitored

by measuring the OD600. Error bars indicated SEM (n = 3).

Quantitative proteomic analysis of S. degradans cultured in the presence of different

carbon sources

Quantitative proteomic analyses, based on an isobaric tag method, were

performed to assess the assimilation processes of S. degradans when cultured in the

presence of different carbon sources (Fig. 2). The mass spectrometry data obtained were

used for protein identification and quantification by comparison with the sequence data

of S. degradans from NCBI (http://www.ncbi.nlm.nih.gov/). In total, with the cut-off

criteria, I identified 987 proteins in the samples from four biological replicates of each

carbon source.

Statistical analysis for the selection of carbon source-specific proteins

Principal component analysis (PCA) was performed to examine the similarity

between the protein profiles of the four biological replicate proteomic samples obtained

from S. degradans grown on glucose, pectin, or, alginate and the dataset was analyzed

using R. A score plot of the PCA (Fig. 3) showed significant similarities between each of

00.20.40.60.8

11.21.41.61.8

2

0 5 10 15

Cel

lgro

wth

(OD

600)

Culture time (h)

Glucose Pectin Alginate

20

the biological replicates. The plots derived from each carbon source formed individual

groups indicating that each had a distinct protein profile.

In an attempt to select the proteins that show significant fold-changes

dependent upon the different carbon sources, an empirical Bayes moderated t-test was

performed (Matsui et al. 2013). P-values were adjusted using the Benjamini-Hochberg

method to avoid the problem of multiple testing. Volcano plots of each carbon source

were used to select statistically differentially expressed proteins (Fig. 4). Proteins with

FDR-adjusted P-values of <0.01 and fold changes of the protein ratio of >2 for both

glucose and pectin were accepted as alginate-specific proteins, and are listed in Table 1.

Fig. 2 Schematic representation of the techniques used to quantify proteins of S.

degradans.

The proteins extracted from S. degradans, grown in the presence of carbon sources

(glucose, pectin, or alginate) were individually subjected to reduction, alkylation, and

digestion by trypsin. Each peptide sample was labeled with tandem mass tags (TMTs)

using various reporters at m/z 126, 127, 128, 129, 130, and 131. After labeling, each

sample was simultaneously injected onto a nano LC-MS/MS with long monolithic

column (Morisaka et al. 2012) to identify and quantify proteins. Statistical analyses of the

data were performed to determine the quantitative changes in the proteomic profile for

each of the three different carbon sources.

21

Fig. 3 Score plot of the principal component analysis (PCA) of 4 biological replicates

proteomic samples obtained from S. degradans grown on glucose, pectin, or alginate.

The cumulative contribution rate for principal component PC1 to PC2 was 89%. A score

plot of the PCA showed significant similarities between each of the biological replicates.

Fig. 4 Volcano plots of (A) pectin/alginate and (B) glucose/alginate.

Volcano plots reveal significantly produced proteins in response to alginate compared to

the control substrate pectin and glucose, respectively. Plots showed an FDR-adjusted P-

value of <0.01 and fold change of protein ratio of >2, to compare the specific-proteins

expressed in S. degradans grown on different carbon sources (blue dots). Non-specific

proteins are shown as red dots.

22

Table 1 Alginate-specific proteins selected by volcano plot.

Discussion

The alginate-specific proteins including alginate lyases were identified (Table

1). The 12 genes encoding the alginate lyases annotated in CAZy database

(http://www.cazy.org) are categorized into the PL family (4 of PL6, 5 of PL7, 1 of PL14,

1 of PL17, and 1 of PL18) (Cantarel et al. 2009; Garron and Cygler 2010). Based on the

quantitative proteomic analysis, Alg17C (Sde_3284), Alg6B (Sde_3285), Alg6F

(Sde_2873), Alg6H (Sde_3275), Alg7E (Sde_2478), Alg7K (Sde_2839), and Alg6I

(Sde_3274) were selected (Table 1). Alg6F, Alg7E, and Alg7K were previously annotated

as putative alginate lyases, but for this analysis, they were selected as alginate-specific

proteins. However, the genes related to alginate utilization were not confirmed near the

genes for Alg6F, Alg7E, or Alg7K. On the other hand, the genes for Alg17C, Alg6B,

Alg6H, and Alg6I are located in a large cluster of 16 genes (2.4 kbp, Fig. 5). Most of

23

these genes encoded proteins that have a predicted function related to carbohydrate

utilization: 2-oxoglutarate/Fe2+-dependent oxygenase (Sde_3273), triose phosphate

isomerase (Sde_3277), fructose-1,6-bisphosphatase (Sde_3278), fructose-bisphosphate

aldolase (Sde_3279), KdgK (Sde_3280), SDR (Sde_3281), major facilitator superfamily

transporter (MFS transporter) (Sde_3282), and transcriptional regulator gluconate

repressor (GntR) family (Sde_3287). Therefore, I hypothesized that S. degradans has an

alginate-specific gene cluster for efficient alginate utilization.

2-Oxoglutarate/Fe2+-dependent oxygenase (2OG oxygenase) uses an Fe2+

cofactor, with 2OG and molecular oxygen as co-substrates. 2OG oxygenases are widely

distributed, evolutionarily conserved enzymes involved in many biologically important

processes such as protein modification, lipid metabolism, and secondary metabolite

production in microbes. 2OG oxygenases catalyze a diverse array of oxidative reactions,

epimerizations, and carbon-carbon bond cleavage (van Staalduinen and Jia 2014). Triose

phosphate isomerase (TIM), fructose-1,6-bisphosphatase (F16BPase), and fructose-

bisphosphate aldolase are related to carbohydrate metabolism pathways. TIM is a

ubiquitous enzyme found in glycolysis, gluconeogenesis, and the oxidative pentose

phosphate pathways (Schurig et al. 1995). F16BPase catalyzes the hydrolysis of D-

fructose-1,6-bisphosphate to D-fructose-6-phosphate and inorganic phosphate and is a

key enzyme in gluconeogenesis (Sato et al. 2004). SDR is an enzyme of great functional

diversity, which catalyzes NAD(P)(H)-dependent oxidation/reduction reactions

(Kavanagh et al. 2008). The MFS transporter is a member of one of the two largest

families of membrane transporters. It is a secondary carrier specific for

hexuronate/hexarate, and transports DEH from the periplasmic space into the cytoplasm

(Kabisch et al. 2014). GntR is a transcription factor that negatively regulates the gluconate

operon involved in the uptake and catabolism of gluconate in E. coli (Izu et al., 1997).

Thus, the alginate-specific gene cluster of S. degradans was comprised of alginate lyases,

enzymes related to carbohydrate metabolism, membrane transporters, and transcriptional

factors.

24

Fig. 5 Predicted alginate-specific gene cluster of S. degradans based on quantitative

proteomic analysis of S. degradans.

The functions of the proteins are color-coded: red, alginate lyase; blue, enzymes related

to carbohydrate utilization; orange, membrane transporter; green, transcriptional

regulator; and grey, other. Sde_3274 (Alg6I), Sde_3275 (Alg6H), Sde_3276 (hypothetical

protein), Sde_3279 (Fructose-bisphosphate aldolase), Sde_3280 (KdgK), Sde_3281

(SDR), Sde_3282 (MFS transporter), Sde_3283 (Cupin 2, conserved barrel), Sde_3284

(Alg17C), Sde_3285 (Alg6B), and Sde_3287 (transcriptional regulator, GntR family)

were selected as alginate-specific proteins (Table 1).

Kabisch et al. (2014) reported that marine microorganisms have proteins on the

cell surface, which degrade polysaccharides into smaller components and uptake

degradation products into the cell. Such a strategy would prevent a loss of these proteins

by diffusion and increase the efficiency of degradation and uptake. ‘Lipobox’ motifs

designate the attachment of host proteins to the outer face of the cell membrane

(Hutcheson et al. 2010). Four alginate lyases (Alg6H, Alg7E, Alg7K, and Alg6I) are

included in a lipobox, suggesting their localization to the cell surface (Table 1). Although

the secretion-type alginate lyases Alg17C and Alg7D have been characterized in previous

studies (Kim et al. 2012; Kim et al. 2012; Wang et al. 2014), the characterization of

alginate lyases included in a lipobox will be important for clarification of the alginate

degradation mechanisms of S. degradans. Notably, Alg7K has been shown to be an

exolytic alginate lyase, mainly releasing unsaturated monosaccharides, which are

spontaneously converted into DEH (data shown in chapter II). Thus, characterization of

alginate lyases including a lipobox would lead to elucidate detailed alginate degradation

mechanism of S. degradans.

The current results indicate that S. degradans produces a specific Phage T-one

(TonB)-dependent receptor (Koebnik 2005; Taylor and Trotter 1967). Among 47 genes

25

encoding TonB-dependent receptor-related proteins in S. degradans, 3 TonB-dependent

receptors (Sde_1508, Ade_1386, and Sde_2991) were categorized as alginate-specific

proteins. The TonB-dependent receptors localize to the outer membrane of Gram-

negative bacteria and interact with the TonB protein in the cytoplasmic membrane. The

receptors recognize specific substrates and are important for the detection of

environmental signals (Braun 1995; Kabisch et al. 2014; Koebnik 2005). Additionally,

Tang et al. (2012) reported that TonB-dependent receptors are important for the uptake of

glycans by marine Gram-negative bacteria. Among the group of cytoplasmic membrane

transporters, the MFS transporter (Sde_3282) located in the alginate-specific gene cluster

was identified as an alginate-specific protein by the selection criteria. As mentioned

above, MFS transporter incorporates DEH from periplasmic space into cytoplasm. The

TonB-dependent receptors (Sde_1508, Sde_1386, and Sde_2991) and the MFS

transporter (Sde_3282) may be specific for the incorporation of alginate degradation

products.

Based on the results of our quantitative proteomic analysis, I suggest that the

sugars released from alginate degradation, mannuronic and guluronic acids, are converted

to DEH. DehR, key enzyme to metabolize DEH, reduces DEH into KDG, a common

metabolite that is fed into the Entner-Doudoroff pathway (Wargacki et al. 2012).

Although the gene encoding DehR is not annotated in the genome of S. degradans, I

considered the possibility that SDR, especially Sde_3281 in the alginate-specific gene

cluster, might be an active DehR. To demonstrate this proposal, I constructed an

overexpression system for SDR Sde_3281 in E. coli cells. The resulting protein of 27 kDa

was confirmed by SDS-PAGE (Fig. 6). As it is difficult to obtain commercially available

DEH, the activity of SDR Sde_3281 was assayed by monitoring the oxidation of NADPH

in the presence of DEH released by Alg7K. A decrease in absorbance at 340 nm was

observed in the presence of Sde_3281 (Fig. 7). This result showed that SDR Sde_3281

converted DEH to KDG with the oxidation of NADPH or NADH. SDR Sde_3281

exhibits 33% and 19% sequence identity on amino acids with DehR of Sphingomonas sp.

A1 and V. splendidus 12B01, respectively (Takase et al. 2010; Wargacki et al. 2012).

Therefore, SDR Sde_3281 is considered to be a homologous protein to DehR.

26

Fig. 6 SDS-PAGE analysis of recombinant SDR Sde_3281.

SDS-PAGE was performed in 5-20% polyacrylamide gradient gel staining with CBB: M,

molecular weight standards; lane 1, lysate of BL21 cells transformed with empty vector;

lane 2, lysate of BL21 cells overexpressing recombinant SDR Sde_3281. The band in

lane 2 is SDR Sde_3281.

Fig. 7 Spectrophotometric assay modified from a previous report to identify DehR

activity in the lysate of E. coli strain BL21 cells overexpressing recombinant SDR

Sde_3281 was developed (Enquist-Newman et al. 2014).

The reaction was initiated by adding 5 µL of E. coli strain BL21 lysate at 25 oC into DEH

reaction mixture. The activity was determined by continuously monitoring the decrease

in absorbance at 340 nm. All the data are given as measns±SEM, n=3.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 20 40 60 80 100

Abs

340

nm

Time (s)

Empty vector Sde_3281

27

KDG is metabolized to pyruvate and GAP by KdgK and KdgpA, respectively.

KdgK was selected as an alginate-specific protein (Table 1). In contrast, KdgpA was not

a carbon source-specific protein, but was detected as a constitutively produced protein in

all samples. S. degradans has 4 genes encoding KdgK (Sde_0939, Sde_1269, Sde_2656,

and Sde_3280), which produces enzymes catalyze the phosphorylation of KDG to 2-keto-

3-deoxy-6-phosphogluconate. S. degradans used Sde_3280 to generate the KDG derived

from alginate (Table 1) and Sde_0939 to generate the KDG derived from pectin. The 4-

deoxy-L-threo-5-hexoseulose uronic acid, which is a monosaccharide released from

pectin, is an isomer of DEH. Hence, these different selections of KdgK by S. degradans

might be interestingly attributed to the structural distinction of KDGs derived from

alginate and pectin. Furthermore, genes encoding enzymes related to pectin metabolism

such as glucuronate isomerase (Sde_0940), pectate lyase (Sde_0943), and 4-deoxy-L-

threo-5-hexoseulose-uronate ketol-isomerase (Sde_0950) were found near KdgK

(Sde_0939). Therefore, different selections of KdgK by S. degradans may result from the

polycistronic expression system for efficient transcription of alginate or pectin-specific

gene clusters.

Summary

I elucidated alginate assimilation mechanism of the marine bacterium S.

degradans based on quantitative proteomic analysis. From the quantitative proteomic

analysis, I postulate that S. degradans has an alginate-specific gene cluster for efficient

alginate utilization. The SDR Sde_3281, one of the key enzymes in the alginate-specific

gene cluster, showed DehR enzymatic activity. Furthermore, I suggest that different

selections of KdgK play an important role in how S. degradans utilizes alginate and pectin.

The predicted alginate assimilation mechanism of S. degradans was illustrated

in Fig. 8. Alginate degraded into monosaccharides by freely secreted alginate lyases and

lipobox containing cell surface-attached alginate lyases. The monosaccharides are non-

enzymatically converted into DEH during transportation into cytoplasm by TonB-

dependent receptors (Sde_1508, Sde_1386, and Sde_2991) and MFS transporter

(Sde_3282). DEH metabolizes into pyruvate and GAP by DehR (Sde_3281), KdgK

(Sde_3280), and KdgpA (Sde_1382).

28

Fig. 8 Predicted alginate assimilation mechanism of S. degradans.

29

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33

Chapter II

Engineered yeast whole-cell biocatalyst for degradation of alginate and production

of non-commercialized useful monosaccharide 4-deoxy-L-erythro-5-hexoseulose

uronic acid

Yeast S. cerevisiae is widely used to produce bioethanol in industrial scale.

Among the processes, alginate degradation into DEH by alginate lyases is indispensable

for direct ethanol fermentation (Takeda et al. 2011; Wargacki et al. 2012; Enquist-

Newman et al. 2014). Since S. cerevisiae lacks the ability to degrade alginate, molecular

breeding is required for alginate degradation into DEH.

In chapter I, I suggested alginate lyases including lipobox motif will be

important for clarification of the alginate degradation mechanisms of S. degradans.

Lipobox destine the attachment of host proteins to the outer face of the cell membrane

(Pugsley et al. 1986; Pugsley 1993). The localization of these alginate lyases on the cell

surface prevents loss of the enzymes through diffusion, and increases the efficiency of

alginate degradation and uptake (Kabisch et al. 2014). Alg7A, Alg7E, Alg6H, Alg6I, and

Alg7K of S. degradans also include lipobox, suggesting their localization on the cell

surface (Weiner et al. 2008; Hutcheson et al. 2011).

In this chapter, I focused on both freely secreted alginate lyases (Alg7D and

Alg18J) and lipobox containing cell surface-attached alginate lyases (Alg7A and Alg7K)

of S. degradans. In order to breed alginate degrading-yeast, alginate lyases from S.

degradans were produced on S. cerevisiae W303-1A with yeast cell surface display

system (Kuroda and Ueda 2013).



E. coli strain DH5α (Toyobo) was used as the host for recombinant DNA

manipulation as described in Chapter I. S. cerevisiae strain W303-1A (MATa, leu2-3/112,

ura3-1, trp1-1, his3-11/15, ade2-1, can1-100) was used for the cell- surface display of

alginate lyases. Yeast host cells were grown in yeast extract-peptone-dextrose (YPD)

medium [1% (w/v) yeast extract, 2% (w/v) glucose, 2% (w/v) peptone] for transformation.

34

For the activity assay of alginate lyase, cells were cultivated in synthetic dextrose (SD)

medium [0.67% (w/v) yeast nitrogen base without amino acids, 2% (w/v) glucose, 2%

(w/v) casamino acids, 0.002% (w/v) L-tryptophan, and 0.002% (w/v) adenine] buffered

at pH 6.0 with 50 mM 2-morpholinoethanesulfonic acid (MES). For the co-display of

endolytic and exolytic alginate lyases, cells were cultivated in SD medium [0.67% (w/v)

yeast nitrogen base without amino acids, 2% (w/v) glucose, 2% (w/v) casamino acids,

and 0.002% (w/v) adenine] buffered at pH 6.0 with MES.

Construction of plasmids

All the primers used for constructing plasmids are listed in Table 1. The alginate

lyase-encoding genes were amplified from S. degradans 2-40 (ATCC 43961) genomic

DNA by polymerase chain reaction using KOD-Plus-Neo DNA polymerase (Toyobo).

The amplified fragments were cloned into vectors using the In-Fusion HD cloning kit

(Takara Bio Inc.).

The Alg7A (Sde_3286) gene was PCR-amplified using in-fusion Alg7A-F and

in-fusion Alg7A-R primers. The Alg7K (Sde_2839) gene was PCR-amplified using in-

fusion Alg7K-F and in-fusion Alg7K-R primers. Each of the amplified fragments was

inserted into the vector pULD1 (Ota et al. 2013), digested with BglII and XhoI. The

resulting plasmids were named pALG7A and pALG7K (Fig. 1A). The Alg7D (Sde_2547)

gene was PCR-amplified using in-fusion Alg7D-F and in-fusion Alg7D-R primers. The

Alg18J (Sde_3272) gene was PCR-amplified using in-fusion Alg18J-F and in-fusion

Alg18J-R primers. Each of the amplified fragments was inserted into pULD1, which was

digested with BglII. The resulting plasmids were named pALG7D and pALG18J (Fig.

1A).

The plasmids pALG7A, pALG7D, and pALG18J, except the URA3sequence,

were PCR-amplified using pULD1-PCR-F and pULD1-PCR-R primers and used as

vectors. The TRP1 gene was PCR-amplified using pRS404-TRP1-F and pRS404-TRP1-

R primers from pRS404. The amplified TRP1 fragments were cloned into vectors, and

the resulting plasmids were named pALG7A-TRP, pALG7D-TRP, and pALG18J-TRP

(Fig. 1B).

The strep-tagged plasmid for displaying Alg7K was constructed as follows: the

Alg7K (Sde_2839) gene was PCR-amplified using in-fusion Alg7K-S-F and in-fusion

Alg7K-S-R primers. The amplified fragment was inserted into pULD1-s (Ota et al. 2013),

35

which was digested with BglII. The resulting plasmid was named pALG7K-s (Fig. 1C).

Table 1 Plasmids and primers used in chapter II.

Fig. 1 Constructed plasmids for yeast cell

surface expression of alginate lyases.

(A) FLAG-tagged pALG for cell surface

display of alginate lyases (B) FLAG-tagged

pALG-TRP for cell surface display of

endolytic alginate lyases (Alg: Alg7A,

Alg7D, and Alg18J) (C) Strep-tagged

pALG7K-s for cell surface display of

exolytic alginate lyase (Alg7K)

36

Yeast transformation

Constructed plasmids were introduced into yeast using the lithium acetate

method (Ito et al. 1983), with the EZ-Yeast transformation kit (BIO 101, CA, USA).

Transformants were selected on SD plate medium containing the appropriate amino acids.

Immunofluorescence microscopy

To confirm the display of alginate lyases on the yeast cell surface, cells were

immunofluorescently labeled using FLAG epitope tag as follows: cells were incubated in

phosphate-buffered saline (PBS; pH 7.4) containing 1% (w/v) bovine serum albumin for

30 min prior to immunostaining. A mouse monoclonal anti-FLAG M2 antibody (1:300

dilution; Sigma-Aldrich) was used as the primary antibody. A mixture of cells and

antibody were incubated for 1.5 h at room temperature on a rotator, and the cells were

washed with PBS (pH 7.4). As secondary antibody, Alexa Fluor 488-conjugated goat anti-

mouse Ig G antibody (1:300 dilution; Invitrogen, CA, USA) was incubated with cells for

1.5 h at room temperature on a rotator. After washing with PBS (pH 7.4), the cells were

suspended in 30 µL of PBS (pH 7.4) and observed by fluorescence microscopy.

Fluorescence was detected using an inverted microscope IX71 (Olympus, Tokyo, Japan)

through a U-MNIBA2 mirror unit with a BP470-490 excitation filter, DM505 dichronic

mirror, and BA510-550 emission filter (Olympus). Live images were obtained using the

Aqua Cosmos 2.0 software (Hamamatsu Photonics, Shizuoka, Japan) to control a digital

change-coupled device camera (Hamamatsu Photonics).

Measurement of alginate lyase activity on yeast cell surface by 3,5-dinitrosalicylic

acid assay

After precultivation in buffered SD medium at 30 oC for 24 h, the alginate lyase

co-displaying yeast was cultivated in buffered SD medium at 30 oC for 24 h, and collected

by centrifugation for 5 min at 6,000×g and 4 oC. After the cells (OD600 = 10 for Alg7A

and Alg18J, OD600 = 50 for Alg7D and Alg7K) were washed with PBS (pH 7.4), 0.5 mL

of the cell suspension was mixed with 0.5 mL of 1.0% (w/v) sodium alginate solution

(dissolved in PBS containing 10 mM magnesium chloride) and the mixture was incubated

at 40 oC for 30 min (Alg7A and Alg18J) or 3 h (Alg7D and Alg7K), on a rotator. After

this, the mixture was centrifuged at 6,000×g for 5 min at 4 oC and the supernatant was

collected. The reaction was stopped by boiling the supernatant at 100 oC for 2 min. After

37

cooling, the supernatant was mixed with 3,5-dinitrosalicylic acid (DNS) solution (NaOH,

16 g/L; potassium sodium tartrate, 300 g/L; DNS, 5 g/L) in 1:2 ration to quantify the

reduced ends of degradation products. The mixed solutions were incubated at 100 oC for

5 min, and after cooling them to room temperature, 200µL of each solution was used for

measuring absorbance at 530 nm with D-glucose as the standard.

Thin layer chromatography analysis of alginate degradation

The changes in the carbohydrate profile after degradation by displayed alginate

lyases were examined using thin layer chromatography (TLC). Reaction products were

developed with the solvent system of 1-butanol/acetic acid/water (3:2:2 by volume), and

visualized using 20% (v/v) sulfuric acid solution in ethanol containing 0.2% (w/v) 1, 3-

naphthalenediol (Nacalai Tesque), followed by heating the TLC plate at 80 oC for 5 min.

Effects of pH and temperature on alginate lyase activity

The effect of pH on the alginate lyase activity was determined by incubating

the alginate lyase co-displaying yeast cells over pH range 4.0-11.0, using standard assay

conditions. The buffers used were 50 mM acetate buffer (pH 4.0-5.0), 50 mM potassium

phosphate buffer (pH 5.0-7.5), 50 mM tricine-KOH buffer (pH 7.5-9.0), and glycine-

KOH buffer (pH 9.0-11.0). The optimal temperature for activity of the displayed alginate

lyases was determined over a temperature range of 20-60 oC in 50 mM tricine-KOH

buffer (pH 8.5).

Activity measurement of co-displayed alginate lyases as a whole-cell biocatalyst on

yeast cell surface

Cultivation of alginate lyase co-displaying yeast cells was carried out as

described above. After the cells (OD600 = 10) were washed with tricine-KOH (pH 8.5),

0.5 mL of the cell suspension was mixed with 0.5 mL of 1.0% (w/v) sodium alginate

solution (dissolved in tricine-KOH (pH 8.5) containing 10 mM magnesium chloride) and

the mixture was incubated at 40 oC for 1 h on a rotator. The amount of alginate

degradation products was determined by the DNS assay.

38

Results

Construction of yeasts displaying alginate lyases from S. degradans

To display alginate lyases from S. degradans on the yeast cell surface, a

multicopy expression plasmid pALG was constructed and introduced into the S.

cerevisiae W303-1A strain (Fig. 1A).

The display of alginate lyases on yeast cell surface was confirmed by

immunofluorescence labeling with mouse monoclonal anti-FLAG M2 antibody. Yeast

harboring pULD1-s, which contains a strep-tag instead of a FLAG-tag, was used as a

negative control. These results indicated that Alg7A (Sde_3286), Alg7D (Sde_2547),

Alg7K (Sde_2839), and Alg18J (Sde_3272) were displayed on yeast cell surface (Fig. 2).

Fig. 2 Fluorescence observed of yeast cells after immunofluorescence labeling using

an anti-FLAG antibody and an Alexa Fluor 488-conjugated goat anti-mouse Ig G

antibody.

S. cerevisiae displaying: (A) Alg7A, (B) Alg7D, (C) Alg7K, (D) Alg18J, and (E) control

cells harboring pULD1-s. The scale bar is 5 µm.

(A) (B)

(C) (D)

(E)

39

Activity of yeast cell surface-displaying alginate lyases derived from S. degradans

The degradation products of alginate degraded by displayed alginate lyases on

yeast cell surface were analyzed by TLC. Alginate could be converted into saccharides of

varying lengths (Fig. 3). Alg7A, Alg7K, and Alg18J previously annotated as putative

alginate lyases, were confirmed to show alginate lyase activity in this assay.

Alginate lyase S (Nagase Enzymes, Kyoto, Japan) served as positive control as

it has been shown to possess endolytic activity and produce oligosaccharide from alginate

(Fig. 3) (Falkeborg et al. 2014). Degradation products of Alg7A, Alg7D, and Alg18J

mainly consisted of oligosaccharides (Fig. 3). These results indicate that Alg7A, Alg7D,

and Alg18J on yeast cell surface possess the typical mode of endolytic alginate lyase

activity. It is interesting that I observed the migration distance of degradation products of

Alg7K was greater than other degradation products. Additionally, by mass spectrometric

analysis, the peak of 175 m/z [M-H]- was specifically detected in degradation products of

Alg7K (Fig. 4). These results indicated that Alg7K displayed on yeast cell surface had

exolytic activity. This is the first demonstration that alginate lyase of S. degradans

included in a lipobox-type enzyme, showed exolytic activity.

Fig. 3 TLC analysis of degradation products from alginate substrate.

The reactions were performed by alginate lyases displayed on yeast cell surface: Alg7A

(A), Alg7D (D), Alg7K (K), and Alg18J (J), and alginate lyase S (S), at 40 oC in 50 mM

tricine-KOH (pH 8.5) containing 10 mM magnesium chloride for 24 h.

40

Fig. 4 Total ion chromatogram (top) and extracted ion chromatogram for 175 m/z

[M-H]- (bottom) of degradation product of Alg7K.

The direct infusion MS technique was carried out on the LTQ Velos Mass spectrometer

(Thermo Fisher Scientific). The sample was injected into the LC–MS mobile phase (50

µl/min of 80% (v/v) acetonitrile containing 0.1% (v/v) formic acid) and then directly into

the ESI source. ESI-MS spectra (negative mode) were acquired from m/z 150–800. The

following parameters were used: capillary temperature: 275 oC, spray voltage: 5 kV,

sheath gas: 8 arbitrary units. Extracted ion chromatogram for 175 m/z [M-H]- show the

production of the monomeric sugar acid from the reaction by Alg7K.

41

Optimal pH and temperature of the alginate lyases displayed on the yeast cells

To investigate the optimal pH and temperature of alginate lyases displayed on

yeast cell surface, its degradation products of alginate were measured at different pHs and

temperatures. The maximum activity of Alg7A, Alg7D, Alg7K, and Alg18J were at pH

8.5 in 50 mM tricine-NaOH and at 40 oC, pH 8.5 in 50 mM tricine-NaOH and at 30 oC,

pH 7.5 in 50 mM potassium phosphate and at 40 oC, and pH 7.5 in 50 mM potassium

phosphate and at 50 oC, respectively (Figs. 5 and 6).

Fig. 5 Effect of different pHs on activity of the alginate lyases displayed on the yeast

cells.

(A) Alg7A (B) Alg7D (C) Alg7K (D) Alg18J. All the data are given as measns±SEM, n=3. The buffers used were 50 mM acetate buffer (●), 50 mM potassium phosphate

buffer (■), 50 mM tricine-KOH buffer (▲), and glycine-KOH buffer (◆).

42

Fig. 6 Effect of different temperatures on activity of the alginate lyases displayed on

the yeast cells.

(A) Alg7A (B) Alg7D (C) Alg7K (D) Alg18J. All the data are given as measns±SEM,

n=3. The optimal temperature for activity of the displayed alginate lyases was determined

over a temperature range of 20-60 oC in 50 mM tricine-KOH buffer (pH 8.5).

43

Substrate specificity of the alginate lyases displayed on the yeast cells

Based on the substrate specificity, alginate lyases are classified into

polymannuronate lyase (EC 4.2.2.3) and polyguluronate lyase (EC 4.2.2.11). As expected,

polyM and polyG were degraded by our alginate lyase-displaying yeasts. The polyM and

polyG were prepared from sodium alginate by the partial acid hydrolysis method (Haug

et al. 1966; Gacesa and Wusteman 1990). The results showed that all alginate lyases

displayed on yeast cell surface could degrade polyM and polyG (Fig. 7). These results

indicated that Alg7A, Alg7D, Alg7K, and Alg18J possessed the activities of both

polyguluronate lyase and polymannuronate lyase. Additionally, polyG can be much easily

degraded by Alg7A, Alg7K, and Alg7D than polyM. In contrast, polyM can be much

easily degraded by Alg18J than polyG.

Fig. 7 Degradation of polyM, and polyG.

Reducing sugars generated by S. cerevisiae displaying (A) Alg7A, (B) Alg7D, (C)

Alg7K, and (D) Alg18J. All the data are given as mean±SEM, n=3.

44

Degradation of alginate by endolytic and exolytic alginate lyase co-displaying yeast

In order to degrade alginate more efficiently, I constructed endolytic (Alg7A,

Alg7D, and Alg18J) and exolytic (Alg7K) alginate lyase co-displaying yeasts (Fig. 8 and

10). The multi-copy plasmids for co-displaying endolytic (Alg7A, Alg7D, and Alg18J)

and exolytic (Alg7K) alginate lyases were fused to FLAG and strep tags, respectively,

and introduced into the yeast W303-1A strain. The display of alginate lyases on yeast cell

surface was confirmed by immunofluorescence labeling with mouse monoclonal anti-

FLAG M2 antibody and strepMAB-classic antibody. The green fluorescence of Alexa

Fluor 488 for each primary antibody was observed on the cell surface of each

transformant (Fig. 8). The results indicated that endolytic (Alg7A, Alg7D, and Alg18J)

and exolytic (Alg7K) alginate lyase were co-displayed on the surface of single yeast cells.

Alg7A, Alg7D, Alg7K, and Alg18J-displaying yeast produced 1.58, 0.07, 0.06,

and 1.12 g/L of reducing sugar, respectively, in a 60 min reaction. In comparison,

Alg7K/Alg7A, Alg7K/Alg7D, and Alg7K/Alg18J co-displaying yeast produced

significantly higher amounts of reducing sugar: 1.98, 0.26, and 1.90 g/L, respectively, in

a 60 min reaction (Fig. 9). Degradation products of alginate lyase co-displaying yeasts

were significantly more than those of single alginate lyase-displaying yeasts. These

results indicated that alginate lyase co-displaying yeast could degrade alginate more

effectively than individual single alginate lyase-displaying yeast.

45

Fig. 8 Fluorescence observation of the cells after immunofluorescence labeling.

Cells were labeled with primary and secondary antibodies. The primary antibodies were

anti-strep IgG for exolytic alginate lyase (Alg7K) detection (A) Alg7K/Alg7A co-

displaying yeast, (C) Alg7K/Alg7D co-displaying yeast, (E) Alg7K/Alg18J co-displaying

yeast, (G) S. cerevisiae harboring pULD1; anti-FLAG IgG for endolytic alginate lyases

(Alg7A, Alg7D, and Alg18J) detection (B) Alg7K/Alg7A co-displaying yeast, (D)

Alg7K/Alg7D co-displaying yeast, (F) Alg7K/Alg18J co-displaying yeast, (H) S.

cerevisiae harboring pULD1-s. All antibodies were derived from mice, and the secondary

antibody was Alexa Fluor 488 anti-mouse IgG. The scale bar is 5 µm.

46

Fig. 9 Degradation of alginate with endolytic alginate lyase displaying yeasts,

exolytic alginate lyase displaying yeast, or endolytic and exolytic alginate lyase co-

displaying yeasts.

S. cerevisiae co-displaying: (A) Alg7K/Alg7A, (B) Alg7K/Alg7D, and (C)

Alg7K/Alg18J. The experimental conditions were: amount of yeast tested, OD600 = 10;

pH, 8.5 (tricine-KOH); reaction temperature, 40 °C; reaction time, 1 h. All the data are

given as mean±SEM, n=3. *P < 0.05 was determined by one-way analysis of variance

followed by Turkey’s test for multiple comparison.

47

Discussion

Alg7A, Alg7D, and Alg18J displayed on yeast cell surface showed endolytic

alginate lyase activity. Among endolytic alginate lyases, Alg7A, included in lipobox

containing enzymes, showed maximum alginate degradation activity. Moreover, Alg7K

included in lipobox containing enzymes, when displayed on yeast cell surface, showed

exolytic alginate lyase activity. These results suggested that alginate lyases belonging to

the lipobox containing enzymes, may be very useful in elucidating the alginate

degradation mechanism of S. degradans. In a previous study, although a cell surface

display system of Yarrowia lipolica produced only oligosaccharides (more than

pentasaccharide chains) (Liu et al. 2009), I succeeded in producing monosaccharides

from alginate with exolytic Alg7K-displaying yeast. Based on measurement of the

substrate specificity, Alg7A, Alg7D, Alg7K, and Alg18J possessed the activities of both

polyguluronate lyase and polymannuronate lyase. Alg7A, Alg7K, and Alg7D showed

higher activity towards polyG than towards polyM. In contrast, Alg18J showed higher

activity towards polyM than towards polyG.

Furthermore, I successfully constructed endolytic (Alg7A, Alg7D, and Alg18J)

and exolytic (Alg7K) alginate lyase co-displaying yeasts (Fig. 8 and 10). Alginate lyase

activities of Alg7K/Alg7A, Alg7K/Alg7D, and Alg7K/Alg18J co-displaying yeasts were

significantly higher than individual single alginate lyase-displaying yeasts. The

degradation products of Alg7K/Alg7A, Alg7K/Alg7D, and Alg7K/Alg18J co-displaying

yeasts were 1.25-, 3.71-, 1.70-fold higher than the degradation products of single Alg7A,

Alg7D, and Alg18J-displaying yeasts at 60 min, respectively. Such an enhanced

degradation efficiency can be attributed to two major factors: (i) proximity effect (Bae et

al. 2015) among endolytic and exolytic alginate lyase displayed on yeast cell surface; and

(ii) sequential alginate degradation by endolytic and exolytic alginate lyase on yeast cell

surface (Fig. 10). Bae et al. (2015) reported the proximity effect among enzymes on the

yeast cell surface. The proximity effect among endolytic and exolytic alginate lyase on

yeast cell surface is considered to provide efficient alginate degradation. Additionally,

oligosaccharides produced by endolytic alginate lyase were sequentially degraded by

exolytic alginate lyase displayed on yeast cell surface (Fig. 10). Consequently, the

endolytic and exolytic alginate lyase co-displaying system is highly efficient in alginate

degradation (Fig. 9). However, since degradation activity of Alg7A on yeast cell surface

48

is much higher than Alg7K on yeast cell surface in the initial 30 min (Fig. 9), it seems

that saturation of oligosaccharides occurred at that time. Hence, co-display exolytic

alginate lyase stronger than Alg7K with the endolytic alginate lyase Alg7A will further

enhance effective degradation of alginate.

Wang et al. (2014) reported an optimized saccharification process of alginate

using purified Alg7D and Alg17C expressed in E. coli (Kim et al. 2012; Kim et al. 2012).

However, use of purified enzymes is unsuitable for industrial scale application as it is

highly expensive. In contrast, the yeast cell surface display method has major advantages:

(i) no enzyme immobilization step is needed; (ii), the separation of the biocatalyst from

the products is easy; and (iii) enables reutilization of the active enzymes on the yeast cell

surface without any special separation step (Nakanishi et al. 2012; Ota et al. 2013b;

Inokuma et al. 2014; Bae et al. 2015).

Although DEH is useful monosaccharide for use as a biomaterial, it is not

commercialized. However, DEH can be produced on a very large scale by the alginate

lyase co-displaying system described here, at substantially low cost. Especially efficient

is the Alg7K/Alg7A co-displaying yeast, which produces 1.98 g/L of reducing sugar from

alginate in a 60 min reaction, which represents a conversion efficiency (reducing sugar

produced/ initial alginate, in a 60 min reaction (OD600 = 10)) of 36.8%.

49

Summary

I have successfully constructed endolytic (Alg7A-, Alg7D-, and Alg18J-) and

exolytic (Alg7K-) alginate lyase displaying yeasts. After investigating substrate

specificity, optimal pH, and temperature of displayed alginate lyases, I have constructed

yeasts co-displaying endolytic and exolytic alginate lyases (Fig. 10). These alginate lyase

co-displaying yeasts enable sequential degradation of alginate to the monosaccharide,

DEH (Fig. 10). Additionally, I have compared alginate degradation activity between

individual yeast displaying a single alginate lyase and yeast co-displaying alginate lyases.

I have demonstrated that alginate lyase co-displaying yeasts degraded alginate more

effectively than single alginate lyase-displaying yeast. Our system and findings will

contribute to the efficient utilization of alginate by S. cerevisiae and large-scale

production of DEH, useful non-commercialized monosaccharide.

Fig. 10 Direct alginate degradation by endolytic and exolytic alginate lyase co-

displaying yeast.

50

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53

Chapter III

Generation of Saccharomyces cerevisiae for direct ethanol production from alginate

of brown macroalgae by synthetic biology

The alginate-assimilating bacterium, Sphingomonas sp. strain A1, degrades

alginate into DEH by the endolytic and exolytic alginate lyases. DEH is converted to

KDG by DehR, which is metabolized to pyruvate and GAP by KdgK and KdgpA and

channelized into the central metabolic pathways (Takase et al. 2010). In chapter I, I

showed that S. degradans also metabolized DEH to pyruvate and GAP by DehR, KdgK,

and KdgpA. Several studies showed that the genetically engineered Sphingomonas sp.

A1 and E. coli, and Defluviitalea phaphyphila are capable of directly producing ethanol

from alginate (Takeda et al. 2011; Wargacki et al. 2012; Ji et al. 2016). However, to the

best of our knowledge, direct production of ethanol from alginate by yeast has not yet

been reported. In chapter II, I showed that the yeast co-displaying the endolytic and

exolytic alginate lyases could degrade alginate and produce DEH effectively. Since the

yeast S. cerevisiae lacks the ability to metabolize DEH, the genes encoding the DEH

transporter and components of the metabolic pathway must also be introduced into S.

cerevisiae to produce ethanol from alginate. Therefore, I used synthetic biological

techniques to introduce the genes encoding the DEH transporter and metabolic pathway

components from alginate-assimilating microorganisms in S. cerevisiae that expressed

alginate lyases.



E. coli strain DH5α (Toyobo) was used for recombinant DNA manipulation

described in chapter I. S. cerevisiae strain SEY6210 (MATα, leu2-3/112, ura3-52, trp1-

Δ901, his3-Δ200, lys2-801, suc2-Δ9) was used for the generation of the alginate-

assimilating yeast. Yeast cells were grown in YPD medium for transformation. For the

activity assay of alginate lyase and alginate degradation, cells were cultured in the YPD

medium buffered at pH 6.0 with 50 mM MES.

54

Construction of plasmids

All the primers used for constructing plasmids are listed in Table 1. PCR was

performed using the KOD-Plus-Neo DNA polymerase (Toyobo). The genes encoding the

DEH transporter, DHT1, from Asteromyces cruciatus, dehR from V. harveyi, kdgK from

E. coli, and kdgpA from V. splendidus were codon-optimized and chemically synthesized

(GeneArt, Thermo Fisher Scientific, Braunschweig, Germany) for expression in S.

cerevisiae. The fragments were cloned into vectors using the In-Fusion HD cloning kit

(Takara Bio Inc.).

DHT1 was amplified using the infusion DHT1-F and infusion DHT1-R primers.

The fragment was inserted into the vector pGK426, which was pre-digested with EcoRI

and BamHI. The resulting plasmid was named pGK426-DHT1. dehR was amplified using

the infusion DehR-F and infusion DehR-R primers. The fragment was inserted into the

vector p413GPD (Mumberg et al. 1995), which was pre-digested with BamHI and SalI

and the resulting plasmid was named p413GPD-DehR. kdgK was amplified using the

infusion KdgK-F and infusion KdgK-R primers. kdgpA was amplified using the infusion

KdgpA-F and infusion KdgpA-R primers. Each of the amplified fragments was inserted

into the vector p413TEF (Mumberg et al. 1995), pre-digested with BamHI and SalI and

the resulting plasmids were named p413TEF-KdgK and p413TEF-KdgpA, respectively.

The gene cassette used for producing DHT1 (PGK1 promoter, DHT1, and the

PGK1 terminator) was amplified using the infusion PGK-promoter-F and infusion PGK-

promoter-R primers. The fragment was inserted into the vector pRS404, pre-digested with

XhoI and NotI and the resulting plasmid was named pRS404-DHT1. The gene cassette

used for DehR production (GAPDH promoter, dehR, and the CYC1 terminator) was

amplified with the infusion GAPDH-promoter-F and infusion CYC-terminator-R primers.

The fragment was inserted into the vector pRS307, pre-digested with XbaI and EcoRI

and the resulting plasmid was named pRS307-DehR. The gene cassettes used for KdgK

and KdgpA production (TEF2 promoter, kdgK or kdgpA, and the CYC1 terminator) were

amplified using the infusion TEF-promoter-F and CYC-terminator-R2 primers. The

fragment was inserted into the vector pRS405 and pRS406, pre-digested with XhoI and

NotI and the resulting plasmids were named pRS405-KdgK and pRS406-KdgpA,

respectively.

Furthermore, the gene cassettes used for the cell surface display of Alg7A and

Alg7K (GAPDH promoter, genes/elements encoding the secretion signal of glucoamylase,

55

alginate lyase, and the C-terminal region of α-agglutinin) were amplified using the

infusion GAPDH-promoter-F2 and infusion GAPDH-terminator-R primers from

pALG7A and pALG7K-S, respectively. The fragments were inserted into the vector

pRS405-KdgK and pRS406-KdgpA, which had been digested with SacI and the resulting

plasmids were named pRS405-KdgK-Alg7A and pRS406-KdgpA-Alg7K, respectively.

Thus, I constructed 10 plasmids that were used for the generation of the alginate-

assimilating yeast.

Table 1 Plasmids and primers used in chapter III.

56

Yeast transformation and construction of the alginate-assimilating yeast strain

The constructed plasmids were introduced into yeast using the lithium acetate

method (Ito et al. 1983) and the EZ-yeast transformation kit (BIO 101). Transformants

were selected on plates containing the synthetic dropout medium with the appropriate

amino acids.

The plasmids pRS307-DehR, pRS404-DHT1, pRS405-KdgpA-Alg7K, and

pRS406-KdgK-Alg7A were integrated into genome of S. cerevisiae SEY6210. The

integration of DHT1, dehR, kdgK, and kdgpA into the SEY6210 genome were confirmed

by colony PCR using appropriate primers and the KOD FX neo (Toyobo) (Table 2). The

resulting strain was named Alg1. Alg1 was sub-cultured at 29 °C in the yeast extract-

peptone-alginate (YPA) medium [1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v)

sodium alginate, and 10 mM magnesium chloride] buffered at pH 6.0 with 50 mM MES

for enhancing alginate assimilation, and cell growth was monitored by measuring the

OD600 in triplicates. The YPA medium after degradation with Alg1 was used for sub-

culture. After pre-cultivation in the buffered YPD medium at 30 °C for 24 h, Alg1 was

cultivated in the buffered YPD medium at 30 °C for 24 h, and collected by centrifugation

for 5 min at 6,000×g and 4 °C. Afterwards, the cells (OD600 = 10) were washed with PBS

(pH 7.4) and mixed with the buffered YPA medium, and the mixture was incubated at

40 °C for 24 h on a rotator. After this, the mixture was centrifuged at 6,000×g for 5 min

at 4 °C and the supernatant was collected. The medium was filter sterilized by

a Nalgene sterilization unit (pore size 0.2 µm; Nalgene, Rochester, NY). The empty

vector pRS307 was integrated into the genome of SEY6210 as a negative control.

Table 2 Primers used for colony PCR amplification.

TLC analysis of alginate degradation

The changes in the carbohydrate profile after degradation by Alg1 were

57

examined using TLC. Reaction products were developed with the solvent system of 1-

butanol/acetic acid/water (3:2:2 by volume), and visualized using 20% (v/v) sulfuric acid

solution in ethanol containing 0.2% (w/v) 1, 3-naphthalenediol (Nacalai Tesque),

followed by heating the TLC plate at 80 oC for 5 min.

Construction of the alginate and mannitol assimilating yeast strain

Alg1 was cultured in the synthetic mannitol (SM) medium [0.67% (w/v) yeast

nitrogen base without amino acids, 2% (w/v) mannitol, 0.079% (w/v)

complete supplement mixture (Formedium, Norwich, UK)]. Alg1 grown in the liquid SM

medium was plated on plates containing the SM medium and incubated at 30 °C for 3

days. Single colonies obtained on the SM plate medium were named AM1 and were used

for growth confirmation and ethanol production experiments. Cell growth was monitored

by measuring the OD600 in triplicate.

Ethanol production

The strain AM1 was cultured in the YPD medium, buffered at pH 6.0 with 50

mM MES for 24 h at 30 °C, and the cells (OD600 = 50) were centrifuged at 6,000×g for 5

min at 25 °C and washed with PBS (pH 6.7). The cell pellets were inoculated in the YPA

medium, yeast extract-peptone-mannitol (YPM) medium [1% (w/v) yeast extract, 2%

(w/v) peptone, 2% (w/v) mannitol, and 10 mM magnesium chloride], and yeast extract-

peptone-alginate-mannitol (YPAM) medium [1% (w/v) yeast extract, 2% (w/v) peptone,

2% (w/v) sodium alginate and 2% (w/v) mannitol, and 10 mM magnesium chloride]

buffered at pH 6.0 with 50 mM MES, respectively. The fermentations were performed in

closed tubes at 37 °C on a rotator.

Measurement of sugar and ethanol levels

After filtering through an ultrafree-MC 0.45 mm centrifugal filter device

(Millipore, MA, USA), mannitol and ethanol in the fermentation medium were analyzed

by high-performance liquid chromatography (HPLC; Shimadzu, Kyoto, Japan) coupled

with a RID-10A refractive index detector (Shimadzu) and an Aminex HPX-87H column

(Bio-Rad, Hercules, CA, USA). The HPLC system was operated at 60 °C with 5 mM

H2SO4 (flow rate, 0.75 mL/min) as the mobile phase.

58

Results

Confirmation of the genomic integration of DHT1 and the genes encoding the

components of the DEH metabolic pathway in SEY6210

To confirm the occurrence of the genomic integrations of DHT1, dehR, kdgK,

and kdgpA in SEY6210, colony PCR was performed with the appropriate primers in Alg1

(Table 2).

As shown in Fig. 1, DHT1, dehR, kdgK, and kdgpA were successfully amplified

from the genome of Alg1. These results indicated that DHT1, dehR, kdgK, and kdgpA

were successfully integrated in the genome of SEY6210.

Fig. 1 Colony PCR amplification of gene encoding DHT1 and DEH metabolic

pathway.

Lane1: 2-log ladder. Lane 2: DHT1 (1947 bp) and native 3-phosphoglycerate kinase

(1593 bp) were PCR-amplified using ColonyP-PGK-F and ColonyP-PGK-R primers.

Lane 3: DehR (925bp) was PCR-amplified using ColonyP-GAPDH-F and ColonyP-

CYC-R primers. Lane4: KdgK (1066 bp), KdgpA (763 bp) were PCR-amplified using

ColonyP-TEF-F and ColonyP-CYC-R primers.

59

Confirmation of the alginate degradation ability of Alg1

To confirm the alginate degrading activity of Alg1, the degradation products of

alginate were analyzed by TLC. The degradation products of alginate predominantly

contained DEH, with certain amount of oligosaccharides (Fig. 2). These results indicated

that Alg7K and Alg7A had been successfully integrated in the genome of SEY6210.

Fig. 2 TLC analysis of the alginate degradation products of Alg1.

The reactions were performed by Alg1 at 40 °C in the YPA medium buffered at pH 6.0

with 50 mM MES for 24 h.

Growth characteristics of Alg1 in the YPA medium

Following the confirmation of the genomic integration of the genes required for

alginate assimilation, the growth of Alg1 in the YPA medium was examined (Fig. 3). Alg1

showed better growth in the YPA medium compared to that of the negative control, with

OD600 consistently exceeding 1.0 when cultured for 3-4 days. However, since the negative

control strain exhibited slower growth, the cultures were examined microscopically.

Typical yeast cell morphology was observed for Alg1. In contrast, an abnormal

enlargement of vacuoles was observed in the negative control strain that lacked dehR,

suggesting that the intracellular accumulation of DEH was a physiological load on the

yeast cells (Fig. 4).

60

Fig. 3 Growth characteristics of Alg1 and the negative control strain in the YPA

medium.

Growth analysis of Alg1 and the negative control strain lacking dehR were performed. Alg1 (●) and the negative control (▲) strain were sub-cultured in the YPA medium

obtained after degradation. Alg1 showed better growth in the YPA medium compared to

the negative control.

Fig. 4 Changes in vacuole morphology in Alg1 and the negative control strain.

The strains were grown at 29 °C for 96 h in the YPA medium. The scale bar is 5 µm.

(A) Alg1 (B) Negative control strain without dehR.

00.10.20.30.40.50.60.70.80.9

11.11.21.31.4

0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450

Yea

st c

ell g

row

th (O

D60

0)

Time (h)

Alg1 Negative control

61

Mannitol assimilation by Alg1

DEH metabolism into KDG by dehR produces redox imbalance in yeast cells.

In fact, although the strain Alg1 was able to grow in the YPA medium under aerobic

conditions (Fig. 3), its growth under anaerobic conditions was not determined. The

oxidation of mannitol into fructose is catalyzed by mannitol-2-dehydrogenase, which

produces excessive reducing power. Additionally, wild-type S. cerevisiae is known to

acquire the ability to assimilate mannitol during prolonged culturing in medium

containing mannitol as the sole carbon source (Chujo et al. 2015). Therefore, to regulate

the redox balance in the presence of the co-metabolites alginate and mannitol, Alg1 was

cultured in the SM medium. After 4-6 days, the growth of the cultures reached saturation.

To obtain a single colony of Alg1, cells from the stationary phase in the liquid SM

medium were spread onto the SM medium on plates. After 3 days of incubation, several

visible colonies appeared on the plates. A single colony of Alg1 on the SM plate medium

was named as AM1 and used for ethanol production experiments. To confirm the ability

to assimilate mannitol, the adapted strain AM1 was cultured in the SM medium. The

culture reached saturation after 24 h (Fig. 5). These results indicated that AM1 was able

to metabolize alginate and mannitol.

Fig. 5 Growth characteristics of AM1 in the SM medium. The adapted strain AM1 (●) was grown in medium containing mannitol as the sole

carbon source (SM medium). In the case of flocculated cells, growth was measured after the culture was mixed with 0.1 volumes of 50 mM EDTA. Wild type SEY6210 (▲) was

used as a negative control. Results are shown as means of triplicate experiments, and error

bars represent the standard errors.

0123456789

0 10 20 30 40

Yea

st c

ell g

row

th (O

D60

0)

Time (h)

Strain AM1

SEY6210

62

Ethanol production in media containing alginate and mannitol

To analyze the ethanol production by AM1 using alginate and mannitol, ethanol

fermentation experiments were performed with the YPA, YPM, and YPAM media. AM1

successfully produced ethanol from the YPM and YPAM media, whereas it produced

negligible amounts of ethanol from the YPA medium. The amount of ethanol produced

with the YPAM medium was 4.8 g/L at 44 h, which was more than those obtained with

the YPM (2.2 g/L) and YPA (0.2 g/L) media (Fig. 6A). By contrast, the negative control

strain lacking dehR had low ethanol productivity in the YPAM medium (0.9 g/L). These

results indicated that co-metabolism of alginate and mannitol enabled regulation of the

redox balance. Furthermore, the mannitol concentration in the YPAM medium at 72 h

was 1.7 g/L, which was considerably lower than that in the YPM (6.6 g/L) and YPAM

media with the negative control strain (17.6 g/L) (Fig. 6B).

Fig. 6 Ethanol production in the YPAM medium by AM1.

(A) Graph depicting ethanol production in each medium (B) Graph depicting mannitol concentration in each medium. Strain AM1 (OD600 = 50) was inoculated in the YPA (■),

YPM (▲), and YPAM (●) media buffered at pH 6.0 with 50 mM MES. Empty vector

pRS307 (instead of pRS307-dehR) was integrated in SEY6210 in the negative control strain (◆). Results are shown as means of triplicate experiments and error bars represent

the standard errors.

63

Discussion

In this chapter, I have discussed the construction and characterization of S.

cerevisiae strains co-expressing alginate lyases, DHT1, and the genes encoding the

components of the DEH pathway to produce ethanol directly from alginate. The genes

encoding the endolytic alginate lyase alg7A and exolytic alginate lyase alg7K from S.

degradans, DHT1 from A. cruciatus, dehR from V. harveyi, kdgK from E. coli, and kdgpA

from V. splendidus were integrated in the genome of the S. cerevisiae strain SEY6210,

and the resulting strain was named Alg1. After confirmation of the genomic integration

of the above genes by colony PCR and TLC, Alg1 was sub-cultured in the YPA medium

for improving the alginate utilization ability. Furthermore, the strain AM1 was

constructed for direct ethanol production from alginate and mannitol by prolonged

culturing in the medium containing mannitol as the sole carbon source.

The ethanol concentration produced by AM1 in the YPAM medium at 44 h was

2.2- and 24.0-fold higher than those in the YPM and YPA media, respectively.

Additionally, the assimilated percentage of mannitol in the YPAM medium at 72 h by

AM1 was 91.9%, whereas those in the YPM and YPAM media by the negative control

strain (lacking dehR) were 66.4% and 12.6%, respectively. These results indicated that

the co-metabolism of alginate and mannitol improved ethanol productivity and the rate

of mannitol utilization.

Although previous studies showed that the genetically engineered

Sphingomonas sp. A1 and E. coli, and D. phaphyphila were able to produce ethanol from

alginate directly (Takeda et al. 2011; Wargacki et al. 2012; Ji et al. 2016), they are

unsuitable for industrial-scale use as these bacteria have low ethanol tolerance. In contrast,

S. cerevisiae is an ideal host for industrial-scale bioethanol production and is the standard

microbe used in the industry. Recently, the yeast platform has been constructed to enable

the production of ethanol from DEH and mannitol, albeit without the alginate degradation

capacity (Enquist-Newman et al. 2014). Purification of alginate lyases on a commercial

scale requires a separate process for ethanol fermentation, which results in high

production cost. In order to solve this problem, I used synthetic biology techniques to

construct a yeast strain that can directly produce ethanol from alginate. Furthermore,

the released sugar DEH is utilized rapidly by the cells in this system, which reduces

contamination risks by other microorganisms (Apiwatanapiwat et al. 2011).

64

I have successfully constructed the first S. cerevisiae strain by synthetic

biological techniques that enabled the assimilation of alginate and mannitol for direct

ethanol fermentation from brown macroalgae. The results of this chapter are important as

S. cerevisiae is the standard microbe for industrial ethanol production. Therefore, this

system will contribute to the efficient utilization of brown macroalgae for ethanol

production by S. cerevisiae.

Summary

I have constructed an alginate-assimilating yeast strain, Alg1, by introducing

alginate lyases, the DEH transporter, and the components of the DEH metabolic pathway

into the S. cerevisiae strain SEY6210. Although Alg1 was able to assimilate alginate

under aerobic conditions, its growth under anaerobic conditions was not confirmed.

Therefore, the mannitol metabolizing capacity of Alg1 was enhanced by controlling the

redox balance upon prolonged culturing in medium containing mannitol as the sole

carbon source. The adapted strain AM1 enabled co-metabolism of alginate and mannitol.

Finally, I demonstrated that ethanol can be produced directly from alginate and mannitol

with an engineered yeast strain (Fig. 7).

Fig. 7 Illustration of the direct ethanol production from alginate by a recombinant

yeast.

65

References

Apiwatanapiwat W, Murata Y, Kosugi A, Yamada R, Kondo A, Arai T, Rugthaworn P,

Mori Y (2011) Direct ethanol production from cassava pulp using a surface-engineered

yeast strain co-displaying two amylases, two cellulases, and beta-glucosidase. Appl

Microbiol Biotechnol 90:377-384

Chujo M, Yoshida S, Ota A, Murata K, Kawai S (2015) Acquisition of the ability to

assimilate mannitol by Saccharomyces cerevisiae through dysfunction of the general

corepressor Tup1-Cyc8. Appl Environ Microbiol 81:9-16







Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated

with alkali cations. J Bacteriol 153:163-168

Ji SQ, Wang B, Lu M, Li FL (2016) Direct bioconversion of brown algae into ethanol by

thermophilic bacterium Defluviitalea phaphyphila. Biotechnol Biofuels 9:81

Mumberg D, Muller R, Funk M (1995) Yeast vectors for the controlled expression of

heterologous proteins in different genetic backgrounds. Gene 156:119-122




Takeda H, Yoneyama F, Kawai S, Hashimoto W, Murata K (2011) Bioethanol production

from marine biomass alginate by metabolically engineered bacteria. Energ Environ Sci

4:2575-2581





66

Conclusions

The present study has been carried out to understand alginate assimilation

mechanism of marine bacterium S. degradans toward the development of method for

direct ethanol production from alginate of brown macroalgae.

In chapter I, I conducted to assess the assimilation processes of S. degradans

cultured with three different substrates using quantitative proteomics analysis. I suggested

alginate assimilation mechanism of the marine bacterium S. degradans based on the

analysis. S. degradans degrades alginate into monosaccharides by both freely secreted

alginate lyases and lipobox-containing cell surface-attached alginate lyases. The

monosaccharides are non-enzymatically converted into DEH during transportation into

cytoplasm by TonB-dependent receptors and MFS transporter. DEH is metabolized into

pyruvate and GAP by DehR, KdgK, and KdgpA.

In chapter II, alginate lyase-displaying yeasts were constructed using the yeast

cell surface display system. Alg7A-, Alg7D-, and Alg18J-displaying yeasts showed

endolytic alginate lyase activity. On the other hand, Alg7K-displaying yeast showed

exolytic alginate lyase activity. In order to produce DEH from alginate effectively, I

further constructed yeasts co-displaying endolytic and exolytic alginate lyases.

Degradation efficiency by the co-displaying yeasts was significantly higher than single

alginate lyase-displaying yeasts. Especially, Alg7K/Alg7A co-displaying yeast had

maximum alginate degrading activity.

In chapter III, in order to produce ethanol from alginate directly, I further bred

yeast by using synthetic biological techniques. The genes encoding endolytic alginate

lyase Alg7A from S. degradans, exolytic alginate lyase Alg7K from S. degradans, DHT1

from A. cruciatus, DehR from V. harveyi, KdgK from E. coli, and KdgpA from V.

splendidus were integrated into the genome of S. cerevisiae SEY6210. The resulting

strain, Alg1, was sub-cultured in medium with alginate as the only carbon source for

enhancing the alginate assimilating ability. Moreover, the mannitol metabolizing capacity

of Alg1 was also enhanced for controlling the redox balance. Finally, I have successfully

produced ethanol from alginate and mannitol directly with an engineered yeast strain.

67

Acknowledgments

This thesis is submitted by the author to Kyoto University for the Doctor degree

of Agriculture. The studies presented here have been carried out under the direction of

Professor Mitsuyoshi Ueda in the Laboratory of Biomacromolecular Chemistry, Division

of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, during

2013-2017.

I would like to express my sincere gratitude to Professor Mitsuyoshi Ueda for

his continuous guidance and helpful discussions throughout the course of this study. I

deeply thank Associate Professor Kouichi Kuroda for his critical comments throughout

my study. I am also deeply grateful to Assistant Professor Wataru Aoki for his valuable

comments and discussion. I express my appreciation to Secretary Fukuko Suzuki for her

support of my laboratory life. My deepest appreciation goes to all the members in this

laboratory for their constant encouragement and support throughout this work. I am

deeply grateful to the late ex-Assistant Professor Hironobu Morisaka for his guidance,

encouragement, understanding, experimental suggestion, and skilled technical supports.

I would like to gratefully thank Dr. Naoto Urano, Professor of Tokyo University

of Marine Science and Technology, for his continuous warm encouragements and

thoughtful discussions on this work. I would like to sincerely thank Dr. Masami Ishida,

Professor of Tokyo University of Marine Science and Technology, for his continuous

warm encouragements and thoughtful suggestions.

I am grateful to Research Fellowship of the Japan Society for the Promotion of

Science for Young Scientists and Shoshisha scholarship foundation for financial support.

Lastly, I would like to express my great gratitude to my parents Mr. Katsuhiko Takagi and

Ms. Kunie Takagi for their kind understanding of my career decisions, moral supports,

and warm encouragements.

Toshiyuki TAKAGI

Laboratory of Biomacromolecular Chemistry

Division of Applied Life Sciences

Graduate School of Agriculture

Kyoto University

68

Publications

Chapter I

Takagi T, Morisaka H, Aburaya S, Tatsukami Y, Kuroda K, Ueda M (2016) Putative

alginate assimilation process of the marine bacterium Saccharophagus

degradans 2-40 based on quantitative proteomic analysis. Mar Biotechnol 18, 15-

23

Chapter II

Takagi T, Yokoi T, Shibata T, Morisaka H, Kuroda K, Ueda M (2016) Engineered

yeast whole-cell biocatalyst for direct degradation of alginate from macroalgae and

production of non-commercialized useful monosaccharide from alginate. Appl

Microbiol Biotechnol 100, 1723-1732

Chapter III

Takagi T, Kuroda K, Ueda M, Direct ethanol production from alginate of brown

macroalgae with engineered yeasts. submitted.

Other publication

Motone K, Takagi T, Sasaki Y, Kuroda K, Ueda M (2016) Direct ethanol

fermentation of the algal storage polysaccharide laminarin with an optimized

combination of engineered yeasts. J Biotechnol 231, 129-135

studies on breeding of yeast saccharomyces cerevisiae for

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