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A synthetic consortium of Escherichia coli for n-butanol production by fermentation of the glucose-xylose mixture Mukesh Saini, Li-Jen Lin, Chung-Jen Chiang, and Yun-Peng Chao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04275 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on October 30, 2017

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Journal of Agricultural and Food Chemistry

A synthetic consortium of Escherichia coli for n-butanol production by fermentation of the glucose-xylose mixture Mukesh Saini,1 Li-Jen Lin,2 Chung-Jen Chiang,3* and Yun-Peng Chao,1,4,5*

1

Department of Chemical Engineering, Feng Chia University

100 Wenhwa Road, Taichung 40724, Taiwan 2

School of Chinese Medicine, College of Chinese Medicine, China Medical

University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan 3

Department of Medical Laboratory Science and Biotechnology, China Medical

University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan 4

Department of Medical Research, China Medical University Hospital, Taichung

40447, Taiwan 5

Department of Health and Nutrition Biotechnology, Asia University, Taichung

41354, Taiwan

*Correspondence should be addressed to: Dr. Chung-Jen Chiang E-mail:[email protected] Phone:886-4-22053366 ext. 7227; Fax: 886-4-22057414

Dr. Yun-Peng Chao E-mail: [email protected] Phone:886-4-24517250 ext. 3677; Fax: 886-4-24510890

1

ACS Paragon Plus Environment


1

ABSTRACT

2

The microbial production of n-butanol using glucose and xylose, the major

3

components of plant biomass, can provide a sustainable and renewable fuel as crude

4

oil replacement. However, Escherichia coli prefers glucose to xylose as

5

programmed by carbohydrate catabolite repression (CCR). In this study, a synthetic

6

consortium consisting of two strains was developed by transforming the

7

CCR-insensitive strain into a glucose-selective strain and a xylose-selective strain.

8

Furthermore, the dual culture was reshaped by distribution of the synthetic pathway

9

of n-butanol into two strains. Consequently, the co-culture system enabled effective

10

co-utilization of both sugars and production of 5.2 g/L n-butanol at 30 h. The result

11

leads to the conversion yield and productivity accounting for 63% of the theoretical

12

yield and 0.17 g/L/h, respectively. Overall, the technology platform as proposed is

13

useful for production of other value-added chemicals which require complicated

14

pathways for their synthesis by microbial fermentation of a sugar mixture.

15 16 17

Keywords: Metabolic engineering; n-butanol; sugar mixture; sustainable fuel

18 19 20 21 22 23 24 25 26 27 28 2


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29

INTRODUCTION

30

Compatible with gasoline, n-butanol possesses a superior feature of energy

31

density, volatility, and hygroscopicity and is conceived as one of the most potential

32

alternative fuels.1 The industrial production of n-butanol has long been performed

33

by the acetone-butanol-ethanol (ABE) fermentation process with Clostridium

34

species.2 However, continued improvement of this production scheme still remains

35

technically challenging.3 Thanks to the advance of genetic engineering, many

36

surrogate microbes have been equipped with the clostridial CoA-dependent

37

synthetic pathway for n-butanol.4-6 Although feasible, these research efforts result in

38

a low production titer of n-butanol with glucose and are generally less promising.

39

The implementation of Escherichia coli for n-butanol production is perhaps the

40

most encouraging approach so far. A high level of n-butanol can be obtained in E.

41

coli by generation of more NADH output from glucose catabolism.7-9 However, all

42

these studies were illustrated with super rich TB medium, which makes them

43

industrially impractical. To address this issue, we designed a dual culture system

44

which carries a redox-balanced synthetic pathway of n-butanol.10 Moreover, a single

45

strain with a high level of NADH was developed by rewiring the fueling pathways

46

including glycolysis, the pentose phosphate (PP) pathway, and the tricarboxylic acid

47

(TCA).11 Consequently, these two approaches lead genetically-modified E. coli

48

strains to high production of n-butanol by effective fermentation of glucose on the

49

cost-effective M9Y (M9 mineral salt plus yeast extract) medium.

50

The concern over the insecure supply of fossil fuels and the greenhouse effect

51

has called on the pressing need for the sustainable and environment-friendly energy

52

source.12 It seems appealing to address this issue by microbial fermentation of

53

renewable feedstock for production of n-butanol as crude oil replacement.

54

Lignocellulose derived from plant cell walls appears to be the most abundant

55

resource in nature. It is mainly composed of cellulose and hemicelluloses and

56

decomposed

to

glucose

and

xylose

after

hydrolysis.13

3


Like

most

of


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57

naturally-occurring microbes, E. coli is unable to co-utilize these two sugars as

58

programmed by carbohydrate catabolite repression (CCR).14 Glucose is taken up by

59

permease

60

(PEP):carbohydrate phosphotransferase system (PTS) and subsequently converted to

61

glucose-6-phosphate by EIIAGlc (encoded by crr). EIIAGlc then becomes

62

dephosporylated and prevents activation of adenylate cyclase. The result gives rise

63

to the limited availability of the catabolite activator protein (CAP) and cAMP

64

complex. Without the threshold level of cAMP-CAP, the genes involved in xylose

65

catabolism are not expressed. Accordingly, E. coli utilizes xylose after glucose is

66

depleted, which prolongs the fermentation and reduces productivity.15

EIICBglc

(encoded

by

ptsG)

of

the

phosphoenolpyruvate

67

There are several strategies proposed to reshape E. coli for co-utilization of the

68

glucose-xylose mixture. Such a mutant strain was created by deletion of ptsG or

69

mgsA. The former mutant displays a high level of cAMP-CAP while the expression

70

of ptsG is lowered in the latter.16 After the removal of ptsHI and crr genes, the

71

PTS-negative mutant was evolved to restore its growth on glucose as a result of

72

exhibiting high GalP and Glk activities.17 Alternatively, the strain devoid of CCR

73

was constructed by generation of the CRP* or mlc* mutation. Without the need for

74

cAMP, CRP* functions as CRP-cAMP.18 Meanwhile, the strain with the mlc*

75

mutation shows a marginal level of ptsG.19 Moreover, the CCR-insensitive strain

76

could be developed by elimination of multiple pathway genes. This was carried out

77

by reducing the functional space of the central metabolism involving zwf, ndh, sfcA,

78

maeB, ldhA, frdA, poxB, and pta genes.20 Another study reported the mutation

79

mapped in araC, xylA, pyrE, araE, and ybjG genes.21 Nevertheless, most of these

80

approaches are afflicted with a low utilization rate of glucose.

81

In this study, we found that the glucose-xylose catabolism interfered with the

82

n-butanol synthesis. Therefore, the issue was addressed by implementing a

83

co-culture

84

glucose-selective strain and a xylose-selective strain. Each equipped with the

system.

The

bacterial

consortium

was

4


constructed

with

a

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85

synthetic pathway of n-butanol, both strains were co-cultured to produce n-butanol

86

by co-fermentation of the sugar mixture. Furthermore, the synthetic pathway of

87

n-butanol was distributed into the glucose-selecting strain and the xylose-selecting

88

strain. As a result, a high level of n-butanol was obtained by the dual culture which

89

enabled efficient co-utilization of glucose and xylose.

90 91

MATERIALS AND METHODS

92

Bacteria Culturing. The method for the microaerobic production of n-butanol

93

essentially followed our previous report.10 The seeding culture was prepared with

94

the strains grown on Luria-Bertani medium overnight. The cell density was

95

measured using a spectrophotometer with the wave length of 550 nm (OD550). The

96

fermentation was then carried out by the inoculum culture with initial OD550 at 0.2

97

in a capped Erlenmeyer flask (125 mL) containing the culture medium of 50 mL.

98

For the co-culture system, the cell density ratio of two strains was adjusted to reach

99

initial OD550 at 0.2 for the experiments. Unless stated otherwise, the shake-flask

100

cultures were grown on M9Y medium (6 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L

101

NaCl, 1 g/L NH4Cl, 1 mM MgSO4, 0.1 mM CaCl2, and 5 g/L yeast extract)

102

containing 13.4 g/L glucose and 6.7 g/L xylose.

103

Strain Construction. E. coli strains applied in this study were obtained in

104

several steps (Table 1). The DNA carrying the fusion of lpdA* with the λPL

105

promoter (PλPL) was integrated into strain BuT-8 by using plasmid pLam-LpdA*

106

according to the reported method.11 Moreover, the aceEF operon of strain BuT-8

107

was fused with PλPL by λ Red-mediated homologous recombination of the

108

passenger DNA which was amplified by PCR from plasmid pPR-aceE with

109

RC12060-RC12086.11 The antibiotic marker associated with each genomic

110

integration event was subsequently removed to give strain BuT8-PDH.

111

The glk and xylA genes of strain BuT8-PDH were deleted as follows. The

112

passenger DNAs containing the FRT site-flanked kan (FRT-kan-FRT) cassette with 5



113

two extensions of the genes were amplified from strains CGSC9905 (△glk-726::kan)

114

and CGSC10610 ( △ xylA-748::kan)22 using primers Glk1-Glk2 and Xyl1-Xyl2,

115

respectively. With the aid of λ Red, the target genes were knocked out after

116

electroporation of the passengers DNAs into the host strain. The same approach was

117

employed to eliminate xylA of strain BuT-8L-Z and glk of strain BuT-3E-Z.

118

Similarly, zwf of strains BuT-3E and BuT-8L-ato was removed using the passenger

119

DNA which was obtained by PCR from strain CGSC9537 (△zwf-777::kan)22 with

120

primer Zwf1-Zwf2.

121

Analytical Methods. The microbial fermentation was carried out and sampled

122

for determination of products along the time course. The production of n-butanol in

123

the cell culture was analyzed by Gas Chromatograph (GC) Trace 1300 (Thermo

124

Scientific, USA). The concentration of sugars was measured using High

125

Performance Liquid Chromatography (HPLC) equipped with Reflective Index

126

RID-10A (Shimadzu, Japan). The conditions for GC and HPLC analyses were based

127

on the previous report.10 The theoretical yield of n-butanol based on glucose and

128

xylose was calculated according to the stoichiometric equations for conversion of

129

glucose and xylose to n-butanol (Table 2).

130 131

RESULTS

132

Production of n-Butanol by a Single Strain on Mixed Sugars. To produce

133

n-butanol, strain BuT-8 was previously constructed with recruitment of the

134

clostridial CoA-dependent pathway for n-butanol and deletion of adhE, frdA, ldhA,

135

poxB, and pta genes to conserve NADH and curtail the carbon waste.10 This strain is

136

deficient in ptsG and relies on Zymomonas mobilis glf (encoding glucose facilitator)

137

for glucose uptake (Fig 1A). The glucose-mediated repression program in the

138

ptsG-null E. coli strain is accordingly absent, which results in the strain’s phenotype

139

of co-utilizing glucose and non-PTS sugars.23 To increase the intracellular NADH

140

level, the pyruvate dehydrogenase complex of strain BuT-8 was enhanced by fusion 6


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141

of PλPL with aceEF and lpdA* (the NADH-insensitive lpdA mutant), respectively.11

142

This gene construction gave rise to strain BuT-8-PDH. The n-butanol fermentation

143

for strain BuT-8-PDH was then carried out with the weight ratio of glucose to

144

xylose (G/X ratio) at 2:1. As depicted in Fig. 1B, the strain co-utilized both sugars

145

but was unable to consume them at the end of the fermentation. Consequently, the

146

result gave the n-butanol titer of 2.6 g/L and the conversion yield reaching 32% of

147

the theoretical yield.

148

Production of n-Butanol by a Co-culture System. The synthetic microbial

149

consortium appears to be the most common strategy employed for bacterial

150

co-utilization of mixed sugars.24 Therefore, the co-culture system consisting of the

151

glucose- and xylose-utilizing strains was developed as shown in Fig. 2A. Strain

152

BuT-8-Glu and BuT-8-Xyl were obtained from strain BuT-8-PDH deprived of xylA

153

and glk, respectively. The phenotypes of both strains were characterized with the

154

M9Y medium containing the glucose-xylose mixture. As a result, strain BuT-8-Glu

155

without xylA selectively metabolized glucose while strain BuT-8-Xyl deficient in

156

glk selectively utilized xylose (Figs. 2B and 2C).

157

The n-butanol fermentation was carried out with the culture medium containing

158

the G/X ratio at 2:1. Since more glucose was present, the cell inoculum was

159

composed of the glucose-utilizing and xylose-utilizing strains with the cell density

160

ratio at 2:1. As indicated in Fig. 2D, the co-culture system simultaneously utilized

161

both sugars and consumed almost all sugars at 36 h. The final production titer of

162

n-butanol was around 4 g/L, roughly accounting for 50% of the theoretical yield.

163

This result indicates that the co-culture system is superior to the single strain in

164

terms of production titer and conversion yield.

165

Production of n-Butanol by a Co-culture with the Redox-balanced Pathway.

166

The synthetic pathway of n-butanol entails many NADH-dependent genes. The

167

NADH output from glucose or xylose catabolism is not sufficient for the synthesis

168

of n-butanol, consequently lowering the production titer.11 This issue was addressed 7



169

by our recent approach to distribute the synthetic pathway into two strains (e.g.,

170

strain BuT-3E and BuT-8L-ato), which is simple and straightforward.10 Strain

171

BuT-3E mainly carries Clostridium adhE2 and enhanced atoDA, and strain

172

BuT-8L-ato resembling strain BuT-8 is equipped with enhanced atoDA but without

173

Clostridium adhE2. The former strain enables conversion of n-butyrate to n-butanol

174

associated with the production of acetate while the latter strain produces n-butyrate

175

at the expense of acetate (refer to Fig. 3A). Like strain BuT-8, both strains are

176

deficient in ptsG and able to co-utilize mixed sugars. As revealed in Figs. 1B and 2B,

177

the n-butanol production in the engineered strain is likely disturbed by the

178

concurrent catabolism of glucose and xylose. To moderate the interference of two

179

glycolytic flux, the Zwf-mediated reaction step that connects the glycolysis with the

180

oxidative PP pathway was blocked in strains BuT-3E and BuT-8L-ato to give strains

181

BuT-3EZ and BuT-8LZ, respectively. The n-butanol fermentation was then carried

182

out with the cell density ratio of strain BuT-8LZ to strain BuT-3EZ at 2:1. This

183

bacterial consortium consumed almost all sugars and produced n-butanol of 4.4 g/L

184

at 36 h (Fig. 3B).

185

Improved Production of n-Butanol by the Dual Culture. Finally, the dual

186

culture as illustrated in Fig. 3A was modified to contain a glucose-utilizing strain

187

and a xylose-utilizing strain (Fig. 4A). Strain BuT-LZ-Glu and BuT-EZ-Xyl were

188

obtained by deleting either xylA of strain BuT-8LZ or glk of strain BuT-3EZ,

189

respectively. Subsequently, the phenotype of both resulting strains was

190

characterized in the way as described earlier. The cell density ratio of strain

191

BuT-LZ-Glu to strain BuT-EZ-Xyl at 2:1 was employed for the n-butanol

192

fermentation with the medium containing the G/X ratio at 2:1. As shown in Fig. 4B,

193

this dual culture utilized all sugars and produced 5.2 g/L n-butanol at 30 h. The

194

conversion yield accounts for around 63% of the theoretical yield.

195

It was also intriguing to investigate the effect of the cell ratio on the production

196

of n-butanol. Therefore, the n-butanol fermentation was performed with various cell 8


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197

density ratios and G/X ratios. In general, the production titer decreased with a lower

198

ratio of strain BuT-LZ-Glu to strain BuT-EZ-Xyl in the case with the G/X ratio at

199

2:1 (Fig. 4C). Similarly, the maximal production of n-butanol was obtained with the

200

cell density ratio of strain BuT-LZ-Glu to strain BuT-EZ-Xyl at 2:1 when the G/X

201

ratio was set at 1:1 (Fig. 4C). The result suggests that glucose is more favorable than

202

xylose for production of n-butanol by this co-culture system.

203 204

DISCUSSION

205

Glucose and xylose are two main carbohydrates present in lingocellulose. It

206

would be advantageous to produce n-butanol by microorganisms which assimilate

207

both

208

co-utilization of the two sugars. In E. coli, the genes which engage in xylose

209

catabolism are subject to catabolite repression as exerted by glucose.14 Accordingly,

210

the regulation program dictates E. coli to preferentially metabolize glucose. The

211

glucose-mediated control circuit can be decoupled when the function of ptsG is

212

nullified and in turn increases the cAMP-CAP level in E. coli.16 This strategy has

213

been commonly employed for co-utilization of the sugar mixture in E. coli.23 In this

214

study, strain BuT-8-PDH without ptsG enabled co-utilization of glucose and xylose

215

but inefficiently (Fig. 1B), which is likely due to the limited availability of

216

intracellular NADH. It is possible to increase the NADH level by considerably

217

rewiring the fueling pathways of E. coli, involving redirection of glycolysis,

218

enhancement of the PP pathway, and downregulation of the TCA cycle.11 However,

219

this work is complicated to perform. In addition to ptsG, manXYZ, mglABC, and

220

galP in E. coli share a similar function for transport of glucose and are not essential

221

for cell growth on glucose as reported previously.25 Apparently, the ptsG-null strain

222

relies on Z. mobilis glf for glucose uptake and utilizes glucose more efficiently than

223

xylose as previously recognized.26

sugars.

However,

naturally-occurring

microbes

9


display

inefficient


224

The co-culture system consisting of two distinct microbes has been extensively

225

applied for ethanol production by fermentation of the glucose-xylose mixture.27

226

Nevertheless, this system is not limited to the same strain species. One typical

227

example is the development of E. coli for specific selection of either glucose or

228

xylose.24 In this study, strain BuT-8-PDH was genetically modified to the

229

glucose-selective strain (BuT-8-Glu) and the xylose-selective strain (BuT-8-Xyl).

230

The synthesis of n-butanol from glucose is a more energy-efficient route than from

231

xylose whereas both routes are limited by NADH on the basis of the stoichiometric

232

equations shown in Table 2. As depicted in Figs. 2B and 2C, strain BuT-8-Glu and

233

BuT-8-Xyl produced n-butanol by selective utilization of glucose and xylose,

234

respectively. Their combined production of n-butanol was equal to the yield by the

235

bacterial consortium consisting of strain BuT-8-Glu and BuT-8-Xyl (Fig. 2D). It

236

indicates that the n-butanol fermentation of the co-culture remains unaffected in the

237

presence of two sugars. Consequently, the n-butanol production by the co-culture

238

showed a 54% increase in the production titer as compared to strain BuT-8-PDH.

239

This result is attributed to the dual culture with a superior ability of co-utilizing

240

glucose and xylose (Figs. 1B and 2D). Note that the strains in the co-culture system

241

are in general isogenic to the single strain BuT-8-PDH. It implies the mutual

242

interference between the glucose-xylose catabolism and the n-butanol anabolism

243

(see below).

244

A novel production platform of n-butanol has been recently proposed based on

245

the distribution of the synthetic pathway into two E. coli strains.10 This approach

246

results in a co-culture system consisting of the n-butyrate-conversion strain BuT-3E

247

and the n-butyrate-producing strain BuT-8L-ato, which establishes a redox-balanced

248

synthetic pathway favorable for n-butanol production by fermentation of glucose.

249

However, the n-butanol yield of 2.3 g/L was obtained by application of this dual

250

culture in the presence of the glucose-xylose mixture (data not shown). The

251

production yield in the co-culture system was improved to 4.4 g/L after removal of 10


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zwf in strains BuT-3E and BuT-8L-ato (Fig. 3B). Without zwf, the glycolytic flux of

253

glucose is prevented from entry into the oxidative PP pathway, which reduces the

254

production of NADPH but increases the glucose uptake rate and the carbon flux in

255

the TCA cycle.28 Moreover, the n-butyrate-conversion and the n-butyrate-producing

256

strains

257

glucose-selective strains, respectively. Consequently, the modified co-culture

258

system showed 1.3-fold improvement in the n-butanol titer (e.g., 5.2 g/L). The result

259

implies that the catabolic route of glucose interfere with that of xylose, which

260

negates the n-butanol production. Note that this co-culture system on the mixed

261

sugars produces a comparable yield of n-butanol as the counterpart on glucose alone.

262

In the case of glucose fermentation, both strains in the synthetic ecosystem compete

263

for the substrate and the cell composition is required to adjust during the time course

264

of fermentation.10 In contrast, the two strains in the bacterial consortium for

265

fermentation of the glucose-xylose mixture need not to compete for the substrates

266

and grow in concert to provide the mutual need (e.g. acetate and n-butyrate in Fig.

267

4A).

were

genetically

transformed

to

the

xylose-selective

and

the

268

In conclusion, we developed the co-culture system equipped with a

269

redox-balanced pathway and a substrate-selective trait. The system enables effective

270

co-fermentation of the glucose-xylose mixture, thus leading to high production of

271

n-butanol (associated with CO2) which accounts for 75% carbon recovery of the

272

consumed sugars (Table 2). This technology platform is simple and useful to

273

implement for production of a chemical which requires a complicated synthesis

274

pathway. It can be also extended for production of other value-added chemicals by

275

microbial fermentation of the sugar mixture.29, 30

276 277 278

COMPETING INTERESTS The authors declare that they have no competing interests.

279 11



280 281 282

ACKNOWLEDGEMENTS This work is supported by Ministry of Science and Technology (MOST 105-2221-E-035-085-MY3), Taiwan.

283 284

REFERENCES

285

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products. Biotechnol. Bioeng. 2003, 83, 687-694.

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15



Page 16 of 31

FIGURE LEGEND Figure 1. (A) The central metabolic pathways leading to n-butanol in strain BuT-8-PDH. The PP pathway was marked. The genes involved in the metabolic pathways: aceEF-lpdA*: pyruvate dehydrogenase complex; adhE, aldehyde-alcohol dehydrogenase; adhE2, butyraldehyde-butanol dehydrogenase; crt, crotonse; hbd, 3-hydroxybutyryl-CoA dehydrogenase; ldhA, lactate dehydrogenase; frdA, subunit of fumarate reductase; glf, glucose facilitator; glk, glucokinase; pflB, pyruvate-formate lyase; phaA, acetoacetyl-CoA thiolase; pta, phosphate acetyltransferase; poxB, pyruvate oxidase; ter, trans-enoyl-CoA reductase; zwf, glucose-6-phosphate dehydrogenase; xylA, xylose isomerase; xylB, xylulokinase. The deleted genes are indicated by “X”. Abbreviations: EtOH, ethanol; F6P, fructose-6-phosphate; Lac, lactate; G6P, glucose-6-phosphate; Glc, glucose; G6P, glucose-6-phosphate; PEP, phosphoenolpyruvate;

3PGA,

3-phosphoglyceraldehyde;

Pyr,

pyruvate;

Suc,

succinate; Xyl, xylose; X5P, xylulose-5-phosphate. (B) The time course of n-butanol production for the single strain. The n-butanol production was carried out with strain BuT-8-PDH by fermentation of the glucose-xylose mixture. The experiment was conducted in triplicate. Symbols: glucose (solid circle); xylose (open circle); cell density (open square); n-butanol (solid square).

Figure 2. (A) The schematic illustration of the co-culture system involving strains BuT-8-Glu and BuT-8-Xyl. Refer to the legend of Fig. 1A for detailed information. (B) The time course of n-butanol production for strain BuT-8-Glu. The n-butanol production based on the G/X ratio at 2:1 was carried out with the initial cell density at OD550 of 0.1. (C) The time course of n-butanol production for strain BuT-8-Xyl. The n-butanol production based on the G/X ratio at 2:1 was carried out with the initial cell density at OD550 of 0.1. (D) The time course of n-butanol production for the co-culture system. The n-butanol production based on the G/X ratio at 2:1 was carried 16


Page 17 of 31


out using strains BuT-8-Glu and BuT-8-Xyl (at 2:1) with the initial cell density at OD550 of 0.2. The experiment was conducted in triplicate. Symbols: glucose (solid circle); xylose (open circle); cell density (open square); n-butanol (solid square).

Figure 3. (A) The schematic illustration of the co-culture system involving strains BuT-3EZ and BuT-8LZ. Refer to the legend of Fig. 1A for detailed information. Butyrate and acetate are shuttled as a result of the reaction mediated by atoDA (encoding acetoacetyl-CoA transferase). (B) The time course of n-butanol production for the co-culture system. The n-butanol production based on the G/X ratio at 2:1 was carried out using strains BuT-8LZ and BuT-3EZ (at 2:1) with the initial cell density at OD550 of 0.2. The experiment was conducted in triplicate. Symbols: glucose (solid circle); xylose (open circle); cell density (open square); n-butanol (solid square).

Figure 4. (A) The schematic illustration of the co-culture system involving strains BuT-EZ-Xyl and BuT-LZ-Glu. Refer to the legend of Fig. 1A for detailed information. Butyrate and acetate are shuttled as a result of the reaction mediated by atoDA (encoding acetoacetyl-CoA transferase). (B) The time course of n-butanol production for the co-culture system. The n-butanol production based on the G/X ratio at 2:1 was carried out using strains BuT-LZ-Glu and BuT-EZ-Xyl (at 2:1) with the initial cell density at OD550 of 0.2. The experiment was conducted in triplicate. Symbols: glucose (solid circle); xylose (open circle); cell density (open square); n-butanol (solid square). (C) The n-butanol production by the co-culture system at various cell ratios. The n-butanol fermentation was carried out with the cell ratio of strain BuT-LZ-Glu to BuT-EZ-Xyl at 2:1, 1:1, and 1:2 for 36 h. Symbols: the G/X ratio at 2:1 (solid bar); the G/X ratio at 1:1 (open bar).

17



Page 18 of 31

Table 1. E. coli strains and primers applied in this study. Characteristic

Source

Strain BuT-8

∆ptsG ∆pgi ∆frdA ∆poxB HK022:: PλPL-glf ɸ80attB:: PλPL-ter λattB:: PλPL-crt ∆adhE::ɸ80attB::PλPL-pha-hbd ∆ldhA::λattB::PλPL-adhE2

10

BuT-8-PDH

as BuT-8 λattB::PλPL-lpdA* PλPL-aceEF

This study

BuT-8-Glu

as BuT-8-PDH ∆xylA

This study

BuT-8-Xyl

as BuT-8-PDH ∆glk

This study

BuT-3E

∆ptsG ∆pgi ∆frdA ∆poxB HK022:: PλPL-glf ∆adhE PλPL-atoDABE ∆ldhA::λattB::PλPL-adhE2

BuT-8L-ato

10

∆ptsG ∆pgi ∆frdA ∆poxB HK022:: PλPL-glf ɸ80attB:: PλPL-ter λattB:: PλPL-crt ∆adhE::ɸ80attB::PλPL-pha-hbd ∆ldhA PλPL-atoDABE

10

BuT-3EZ

as BuT-3E ∆zwf

This study

BuT-8LZ

as BuT-8L-ato ∆zwf

This study

BuT-EZ-Xyl

as BuT-3EZ ∆glk

This study

BuT-LZ-Glu

as BuT-8LZ ∆xylA

This study

Primer Glk1(gcccagcttgcaaaaaggc)-Glk2 (cgtgcaaaacaaatcgccg) Xyl1(ccaagatctatcccgatatac)-Xyl2 (gcgcacacttgtgaattatc)

Zwf1(cgcaagctcgtaaaagcag)-Zwf 2 (acaatctgcgcaagatcatg)

18


Page 19 of 31


Table 2. Carbon recovery of fermentation products for single and co-culture strains. EtOH

Lac

Ace

But

BuOH

CO2

Total (%)

33.7

0.3

1.3

0.2

1.2

33.2

21.8

91.7

－

4.7

Strain

Pyr

BuT-8 BuT-LZ-Glu + BuT-EZ-Xyl

0.1

4.7

5.7

41.9

33.2

90.3

Carbon recovery was calculated as the molar percent of carbon in products per carbon in consumed glucose and xylose. The microbial biomass was not included for calculation. The carbon recovery of CO2 was calculated according to the following stoichiometric equations for the theoretical conversion of glucose or xylose to n-butanol, which considers CO2 formation from the decarboxylation of pyruvate. For the single strain, the CO2 production was deducted from accumulated pyruvate. Abbreviations: Ace, acetate; But, n-butyric acid; BuOH, n-butanol. Refer to Fig. 1 legend for other abbreviations. Glucose + 2 NADH → n-Butanol + 2 CO2 + 2 ATP Xylose + 1.67 NADH → 0.83 n-Butanol + 1.67 CO2 + 0.67 ATP

19



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