Engineering Saccharomyces cerevisiae for Enhanced Production of

Subscriber access provided by University of Sunderland

Biotechnology and Biological Transformations

Engineering Saccharomyces cerevisiae for enhanced production of protopanaxadiol with cofermentation of glucose and xylose Xiao Gao, Qinggele Caiyin, Fanglong Zhao, Yufen Wu, and Wenyu Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04916 • Publication Date (Web): 23 Oct 2018 Downloaded from http://pubs.acs.org on October 24, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

Engineering Saccharomyces cerevisiae for enhanced production of protopanaxadiol with cofermentation of glucose and xylose Xiao Gao a, Qinggele Caiyin a, Fanglong Zhao a, Yufen Wu a, Wenyu Lu a,b,c* (a) School of Chemical Engineering and Technology, Tianjin University, Tianjin, People’s Republic of China (b) Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, People’s Republic of China (c) SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, People’s Republic of China *Corresponding

author: Wenyu Lu

Tel: +86-022-27892132; Fax: +86-022-27892132. E-mails: [email protected]

1

ACS Paragon Plus Environment


1

Abstract

2

Protopanaxadiol (PPD), an active triterpene compound, is the precursor of high-value

3

ginsenosides. In this study, we report a strategy for the enhancement of PPD production in

4

Saccharomyces cerevisiae by cofermentation of glucose and xylose. In mixed sugar

5

fermentation, strain GW6 showed higher PPD titer and yield than that obtained from

6

glucose cultivation. Then, engineering strategies were implemented on GW6 to enhance

7

the PPD yields, such as adjustment of the central carbon metabolism, optimization of the

8

mevalonate pathway, reinforcement of the xylose assimilation pathway and regulation of

9

cofactor balance, namely overexpression of xPK/PTA, ERG10/ERG12/ERG13,

10

XYL1/XYL2/TAL1 and POS5, respectively. In particular, the final obtained strain GW10,

11

harboring overexpressed POS5, exhibited the highest PPD yield, which was 2.06 mg PPD/g

12

mixed sugar. In a 5-L fermenter, PPD titer reached to 152.37mg/L. These promising results

13

demonstrate the great advantages of mixed sugar over glucose for high yield production of

14

PPD.

15 16

Keywords:

17

Protopanaxadiol; Cofermentation; Xylose; Saccharomyces cerevisiae; POS5

2


Page 2 of 38

Page 3 of 38


18

1. Introduction

19

With the increasing demand for energy and environmental protection, efficient

20

utilization of lignocellulose is essential for replacing conventional fossil fuels with

21

renewable biomass to some extent. The amount of lignocellulosic biomass produced

22

annually by photosynthesis is more than 100 billion tons, making this biomass the most

23

abundant renewable resource on Earth1. Monosaccharides such as glucose and xylose can

24

be obtained from the hydrolysates of lignocellulosic feedstock. The xylose content in the

25

hydrolysates is inferior to only that of glucose2; therefore, there has been extensive research

26

on the microbial conversion of xylose into biofuels and chemicals3, 4, such as in the

27

production of xylitol5, ethanol6, butanol7, acetoin8, 2,3-butanediol9, pullulan10, PHA11.

28

To date, many microbes that are capable of metabolizing xylose have been discovered

29

by researchers, including bacteria, yeast and filamentous fungi. With the emergence of the

30

ethanol industry, Saccharomyces cerevisiae has become the preferred microbe used for

31

ethanol production due to the superior abilities of this organism, such as tolerance to

32

ethanol and its byproducts, high ethanol productivity and robustness in industrial

33

environments12, 13. However, wild-type S. cerevisiae is generally regarded as being unable

34

to utilize xylose. Thus, intensive efforts have been made to develop xylose-fermenting S.

35

cerevisiae strains14, 15. Via the introduction of the xylose assimilation pathway genes XYL1

36

and XYL2, encoding NADPH-dependent xylose reductase (XR) and NAD+-dependent

37

xylitol dehydrogenase (XDH), respectively, in Scheffersomyces stipitis, xylose can be 3



38

converted into xylulose by budding yeast16, 17. However, the different cofactor preferences

39

of XR and XDH lead to an intracellular redox imbalance, which is likely to result in

40

challenges such as inefficient xylose utilization, low production yields and byproduct

41

accumulation14, 18. To avoid these problems, xylose isomerase (XI) has been introduced in

42

S. cerevisiae to convert xylose into xylulose directly without the involvement of cofactors19,

43

20.

44

(XK) and then metabolized via the pentose phosphate pathway (PPP) and glycolytic

45

pathway. Improvement of the activity of XK can moderately accelerate cell growth21, 22.

46

Furthermore, overexpression of the nonoxidative PPP genes TAL1/TKL1/RPE1/RKI1 from

47

S. cerevisiae, encoding transaldolase, transketolase, ribulose-phosphate 3-epimerase and

48

ribose-5-phosphate isomerase, respectively, can also significantly improve the growth rate

49

and xylose consumption23,

50

capability to produce fuels and chemicals derived from acetyl-CoA, such as isoprenoids,

51

due to a rigid flux toward ethanol25. Accordingly, xylose and glucose were used as sole

52

carbon sources to produce squalene and amorphadiene, respectively. The results showed

53

that the squalene titer of the squalene-producing strain grown on xylose was 8-fold higher

54

than that on glucose (18.7 vs. 150 mg/L) and that the amorphadiene yield of the

55

amorphadiene-producing strain grown on xylose was 2-fold higher than that on glucose

56

despite the similar titers. The above is a promising strategy for the enhancement of

Xylulose can be phosphorylated into xylulose-5-phosphate (X-5-P) by xylulokinase

24.

During glucose fermentation, S. cerevisiae has limited

4


Page 4 of 38

Page 5 of 38


57

isoprenoid production by using xylose instead of glucose as the sole carbon source, which

58

occurs due to reduction of the flux partition toward ethanol production in yeast.

59

Protopanaxadiol (PPD), a 30-carbon isoprenoid (tetracyclic triterpene), exhibits

60

promising anticancer activity with little toxicity toward normal cells26, 27, 28. Although PPD

61

can be derived from plant extracts, this method is time-consuming and cannot meet

62

consumer’s demands. To overcome the limitations of traditional extraction processes, a

63

strategy involving the engineering of the PPD biosynthesis pathway into S. cerevisiae to

64

produce PPD has emerged29, 30. In previous studies, to enhance isoprenoid production in

65

budding yeast, metabolic engineering approaches mainly focused on the following aspects:

66

increasing the cytosolic acetyl-CoA supply31,

67

pathway33, 34, balancing redox cofactors35, 36, engineering subcellular compartments and

68

modifying the central carbon metabolism of yeast37, 38, 39.

32,

optimizing the mevalonate (MVA)

69

To circumvent the limitations of glucose fermentation and enhance PPD production,

70

in this study, the xylose assimilation pathway genes XYL1/XYL2 from S. stipites and

71

XKS1/TAL1/TKL1 from S. cerevisiae were overexpressed along with genes involved in the

72

PPD synthesis pathway in budding yeast. Additionally, certain approaches were adopted

73

to improve PPD production, including adjustment of the central carbon metabolism,

74

optimization of the mevalonate pathway, reinforcement of the xylose assimilation pathway,

75

and regulation of cofactor balance. Subsequently, fermentation was conducted with mixed

76

sugar or glucose to evaluate the performance of different carbon sources. 5



77

2. Materials and Methods

78

2.1 Strains and plasmids

79

S. cerevisiae W303-1a was used as a parent strain for all the engineered strains, which

80

were used to produce PPD from mixed sugar. Engineered yeasts were grown in SD medium

81

(Synthetic Dropout Medium, containing 0.17% Yeast Nitrogen Base, 0.5% ammonia

82

sulfate and 0.2% amino acid mixture, supplemented with glucose or mixed sugar) lacking

83

adenine, uracil, histidine, leucine and tryptophan where appropriate. The strains used in

84

this study are listed in Table 1. The plasmids pXP218, pXP320, pRS405, and pSH47 were

85

obtained from ATCC (American Type Culture Collection, America).

86

2.2 Genetic manipulation

87

Genes encoding dammarenediol-II synthase (DS), protopanaxadiol synthase (PPDS)

88

and NADPH-cytochrome P450 reductase (ATR1) were synthesized by GENEWIZ

89

(Suzhou, China) with codon optimization for S. cerevisiae. Genes encoding xylulose-5-

90

phosphate specific phosphoketolase (xPK), phosphotransacetylase (PTA) and NADH-

91

specific HMG-CoA reductase (NADH-HMGr) were synthesized by Genecreate (Wuhan,

92

China) with codon optimization for S. cerevisiae. The S. stipites genes XYL1 and XYL2

93

were generously donated by Prof. Yingjin Yuan at Tianjin University. The synthesized

94

genes were cloned into the pUC57 vector to generate the plasmids p-DS, p-PPDS, p-ATR1,

95

p-xPK, p-PTA and p-NADH-HMGr.

6


Page 6 of 38

Page 7 of 38


96

Based on previous reports25, 40, a set of strong constitutive promoters that exhibited

97

similar expression levels for both glucose and xylose were selected for overexpression of

98

genes involved in the xylose assimilation pathway and PPD biosynthetic pathway. These

99

promoters and the genes TAL1, TKL1, ERG1, ERG9, ERG20, ERG10, ERG12, ERG13, and

100

POS5 as well as the corresponding terminators were amplified from the genomic DNA of

101

S. cerevisiae W303-1a. PPDS-ATR1 was amplified from the genomic DNA of W3a41. DS,

102

xPK, PTA, NADH-HMGr were amplified from the plasmids p-DS, p-PPDS, p-ATR1, p-

103

xPK, p-PTA and p-NADH-HMGr, respectively. XYL1 and XYL2 were amplified from

104

SyBE_Sc1700442. PCCW12-XYL1-THSP26, PHXT7-XYL2-THXT7, XKS1(R)-HIS3-PFBA1-XKS1(L),

105

PCCW12-TKL1-THSP26, PHXT7-TAL1-THXT7, PPGK1-DS-TCYC1, PTEF1-ERG1-TADH1, PTPI1-ERG9-

106

TADH2, PTPI1-ERG20 (including the native terminator), PPGK1-NADH-HMGr-TCYC1, PTDH3-

107

PPDS-ATR1-TADH1, PHXT7-ERG10 (including the native terminator), PPGK1-ERG12

108

(including the native terminator), PTEF1-ERG13 (including the native terminator), PFBA1-

109

TAL1 (including the native terminator), PTDH3-xPK-TCYC1, PFBA1-PTA-TTDH1, and PTDH3-

110

POS5-TTDH1 expression cassettes were constructed by fusion PCR. All primers used in this

111

study are listed in Supplementary Table SI.

112

2.3 Yeast transformation

113

Transformation of yeast was conducted by the LiAc/ssDNA/PEG method43. The

114

cassettes were integrated into chromosomes of S. cerevisiae using DNA assembler44. Yeast

115

nitrogen base (YNB) lacking amino acids and supplemented with 20 g/L glucose and 7



116

necessary nutrients was used to screen transformants. 5-Fluoroorotic acid (5-FOA) was

117

added when required.

118

2.4 Fermentation for PPD production at the flask level

119

All the engineered strains were stored at -80 °C in 25% glycerol. For the seed culture,

120

the engineered yeasts were inoculated into tubes containing 5 mL of SD medium lacking

121

adenine, uracil, histidine, leucine and tryptophan where appropriate and cultivated at 30 ○C

122

and 220 rpm for 24 h. Then, the seed cultures were inoculated into flasks containing 30 mL

123

of the corresponding SD medium (supplemented with glucose or mixed sugar) at an initial

124

cell OD600 of 0.1 and cultivated at 30 ○C and 220 rpm under aerobic conditions for 96 h.

125

2.5 Batch fermentation for PPD production in a 5-L fermenter

126

For the seed culture, the engineered strain GW10 was cultured in 5 mL of SD medium

127

and cultivated at 30 ○C and 220 rpm for 24 h. Preculture was performed by inoculating the

128

seed culture in 100 mL of SD medium in flasks and cultivating at 30 ○C and 220 rpm for

129

24 h. For PPD production, the cells were then transferred to a 5-L bioreactor (BLBIO-5GL,

130

ShangHai Bailun Biotechnology CO., LTD, China) at an initial OD600 of 0.5, with a

131

working volume of 2.5-L of medium containing 20 g/L peptone, 10 g/L yeast extract and

132

supplemented with 80 g/L glucose or 80 g/L mixed sugar (20 g/L glucose and 60 g/L

133

xylose). Fermentation was carried out at 30 °C with an agitation speed of 400 rpm and an

134

air flow rate of 3 L/min. The pH was automatically maintained at 5.8 with HCl (2 M) and

135

NaOH (2 M), and the dissolved oxygen (DO) level was maintained above 30%. 8


Page 8 of 38

Page 9 of 38

136


2.6 Extraction and analysis of PPD in fermentation broth

137

PPD exists in the intracellular and extracellular space during fermentation. Four

138

hundred microliters of n-butanol and 0.2 g of glass beads (0.5 mm diameter) were then

139

added into 1 mL of culture broth, and the mixture was agitated by vortexing for 20 min,

140

followed by ultrasonic extraction for 20 min. Then, the mixture was centrifuged at

141

12,000×g for 5 min, and the n-butanol phase was collected for HPLC analysis.

142

HPLC analysis was carried out on an Elite (Dalian, China) HPLC system equipped

143

with an Elite P230II pump and an UV230II detector at 203 nm. PPD was separated by a

144

Hypersil C18 column (4.6 mm × 250 mm, 5 μm; Elite Analytical Instruments Co., Ltd.,

145

Dalian, China) with a methanol-acetonitrile (4:6, v/v) mobile phase at a flow rate of 1

146

mL/min.

147

2.7 Analytical methods

148

Cell growth was monitored by measuring the OD600 with a UV-Vis spectrophotometer

149

(Oppler, 752N, China). The concentrations of glucose, xylose, ethanol, xylitol, glycerol

150

and acetate were detected by an Elite HPLC system equipped with an Elite P230II pump,

151

a refractive-index detector (Shodex RI-201H) and an Aminex HPX-87H column (Bio-Rad,

152

USA). H2SO4 (5 mM) was used as the mobile phase at a flow rate of 0.6 mL/min, and the

153

column and detection temperatures were 40 ○C and 65 ○C, respectively.

154

9



155

3. Results and Discussion

156

3.1 Construction of a xylose-fermenting strain with enhanced metabolic flux in the

157

PPP

158

Since the introduction of the xylose assimilation pathway genes XYL1/XYL2, encoding

159

XR and XDH from S. stipitis enables S. cerevisiae to metabolize xylose, the XYL1/XYL2

160

genes from S. stipitis were overexpressed in the S. cerevisiae W303-1a strain for xylose

161

utilization, which was essential for PPD production from xylose (Figure 1). The yeast

162

transformant that exhibited the best performance in terms of xylose consumption was

163

screened out, and strain GW1 was obtained. Subsequently, the endogenous xylulokinase

164

gene XKS1 was overexpressed in GW1 by replacement of the native promoter PXKS1 with

165

the strong promoter PFBA1 to generate strain GW2. Then, the engineered strain GW2 was

166

cultured in 30 mL of SD medium supplemented with 50 g/L xylose as the sole carbon

167

source at 30 ○C and 220 rpm for 96 h. During this period, the OD600 and xylose

168

concentration were monitored. The results (Supplementary Figure S1) showed that GW2

169

could metabolize xylose and grow at a low rate. Only approximately 10 g of xylose was

170

consumed during fermentation, which was likely due to the limited metabolic flux of the

171

PPP. To overcome this problem, multiple copies of the endogenous transaldolase gene

172

TAL1 and transketolase gene TKL1 were integrated at the rDNA locus to generate the

173

efficient xylose-fermenting strain GW3. Then, the engineered strain GW3 was cultured in

174

a 250-mL flask under the same conditions as those used for GW2 fermentation. The GW3 10


Page 10 of 38

Page 11 of 38


175

strain exhibited significantly enhanced performance and could utilize approximately 44 g/L

176

xylose in 96 h (Figure 2). In addition, the production levels of byproducts such as xylitol,

177

glycerol, acetate and ethanol remained very low, and among these byproducts, the

178

maximum concentrations of ethanol and xylitol reached 6.5 g/L and 0.7 g/L, respectively,

179

with little impact on cell growth and xylose consumption. Considering the results of flask

180

fermentation, overexpression of the TAL1/TKL1 genes greatly improved xylose

181

assimilation and made strain GW3 eligible for xylose assimilation and as a chassis for

182

construction of a PPD-producing strain.

183

3.2 Construction of a PPD-producing S. cerevisiae strain

184

In strain GW3, URA3 and HIS3 were employed as selection markers flanked by two

185

loxP sites. To recycle these genes, the Cre-expressing plasmid pSH47 was used, resulting

186

in strain GW445. Biosynthesis of PPD begins with the cyclization of 2, 3-oxidosqualene

187

into dammarenediol-II (DMD) by DS, then DMD is hydroxylated at the C-12 position to

188

yield PPD by the cytochrome P450-type protopanaxadiol synthase (PPDS) 46. To construct

189

a strain for PPD production from mixed sugar, DS/ERG1/ERG9 expression cassettes were

190

integrated into the rDNA locus of strain GW4, resulting in strain GW5, which could

191

produce DMD from mixed sugar. Subsequently, ERG20/NADH-HMGr/PPDS-ATR1

192

expression cassettes were integrated into the δ locus of strain GW5, generating strain GW6,

193

which could produce PPD from mixed sugar. The engineered strains GW5 and GW6 were

194

cultured in 30 mL of SD medium supplemented with 40 g/L glucose or 40 g/L mixed sugar 11



195

(10 g/L glucose and 30 g/L xylose) as a carbon source at 30 ○C and 220 rpm for 96 h.

196

During this period, the OD600 and xylose concentration were monitored. During glucose

197

fermentation, the glucose was depleted in 24 h, and then ethanol was reutilized as a carbon

198

source. For the mixed sugar fermentation, xylose could not be utilized completely in 96 h,

199

which was likely due to inefficient xylose assimilation and lack of NADPH (Figure 3A and

200

4B). However, xylitol and ethanol remained at low levels.

201

The DMD and PPD titers and yields were measured at the end of fermentation at 96

202

h. Strain GW5 produced 9.66 mg/L and 10.69 mg/L DMD under glucose and mixed sugar

203

culture conditions, respectively (Figure 4A). Although the titers of DMD were similar, the

204

yield under mixed sugar fermentation conditions reached 0.57 mg DMD/g mixed sugar,

205

which was 2.38-fold higher than that observed with glucose fermentation (0.24 mg DMD/g

206

glucose) (Figure 4B). Strain GW6 produced 8.75 mg/L and 11.16 mg/L PPD under glucose

207

and mixed sugar culture conditions, respectively (Figure 4A). In addition, the yield under

208

mixed sugar fermentation conditions reached 0.56 mg PPD/g mixed sugar, which was 2.55-

209

fold higher than that observed with glucose fermentation (0.22 mg PPD/g glucose) (Figure

210

4B). As demonstrated by the results, mixed sugar fermentation had a distinct advantage

211

over glucose fermentation, but there remained many aspects that required improvement,

212

such as the xylose consumption rate and PPD yield.

213

3.3 Introduction of the xPK/PTA genes for enhancement of the acetyl-CoA supply

12


Page 12 of 38

Page 13 of 38


214

Acetyl-CoA, the initial compound of the mevalonate pathway, is a key precursor in

215

the biosynthesis of PPD. In S. cerevisiae, cytosolic acetyl-CoA is mainly derived from the

216

PDH bypass pathway, which is composed of pyruvate decarboxylase (PDC), acetaldehyde

217

dehydrogenase (ACDH) and acetyl-CoA synthetase (ACS). Pyruvate, the starting material

218

of the PDH bypass, is derived from glucose or xylose via EMP and then converted into

219

acetyl-CoA via the PDH bypass. However, there are many steps from glucose to acetyl-

220

CoA and many more from xylose to acetyl-CoA (Figure 1). In this study, strain GW7 was

221

generated to increase the acetyl-CoA supply of strain GW6 by introducing a new pathway,

222

composed of a xylulose-5-phosphate specific phosphoketolase (xPK) from Leuconostoc

223

mesenteroides and a phosphotransacetylase (PTA) from Clostridium kluyveri. This

224

pathway can convert xylose-5-phosphate into acetyl-CoA in two steps, greatly truncating

225

the route from xylose to acetyl-CoA (Figure 1). Meanwhile, to some extent, the xylose

226

consumption rate was also improved in GW7 compared to GW6 (Figure 3C). The titers of

227

PPD reached 19.66 mg/L and 15.36 mg/L under glucose and mixed sugar culture

228

conditions, respectively (Figure 4A). In contrast, the yield of PPD in mixed sugar

229

fermentation was 0.70 mg PPD/g mixed sugar, which was 1.43-fold higher than that in

230

glucose fermentation (0.49 mg PPD/g glucose) (Figure 4B). Compared to the values for

231

strain GW6, the titer and yield in mixed sugar medium increased by 38% and 25%,

232

respectively. These results indicated that introduction of the xPK/PTA pathway led to

233

enhancement of the acetyl-CoA supply, which contributed to xylose assimilation and PPD 13



234

production. Furthermore, the xPK/PTA pathway bypassed the PPP and EMP and

235

uncoupled xylose metabolism from central carbon metabolism (CCM) to a large extent,

236

making this a promising strategy for the coutilization of glucose and xylose. Meanwhile,

237

the dependence of the PPD biosynthetic pathway on CCM decreased, facilitating the

238

coordination of cell growth and product synthesis.

239

3.4 Overexpression of ERG10/ERG12/ERG13 for enhanced PPD production

240

To improve the production of PPD, the mevalonate pathway (MVA) in strain GW6

241

was upregulated by overexpression of the acetyl-CoA C-acetyltransferase (ERG10),

242

mevalonate kinase (ERG12) and hydroxymethylglutaryl-CoA synthase (ERG13) genes;

243

therefore, strain GW8 was obtained. In both glucose and mixed sugar fermentation, strain

244

GW8 exhibited a higher growth rate than GW6, but the xylose consumption rate decreased

245

slightly (Figure 3D). The titers of PPD under glucose and mixed sugar culture conditions

246

reached 17.91 mg/L and 14.48 mg/L, respectively (Figure 4A). In contrast, the yield of

247

PPD in mixed sugar fermentation reached 0.89 mg PPD/g mixed sugar, which was 1.97-

248

fold higher than that in glucose fermentation (0.45 mg PPD/g glucose) (Figure 4B). The

249

PPD titers and yields of strain GW8 were higher than those of strain GW6, especially in

250

the glucose medium. These improvements were caused by the upregulation of the MVA

251

pathway, which led to enhancement of the acetyl-CoA metabolic flux toward the PPD

252

biosynthesis pathway.

14


Page 14 of 38

Page 15 of 38


253

However, when the expression of ERG10/ERG12/ERG13 improved, the growth rates

254

of strain GW8 increased, whereas the xylose consumption rate decreased in mixed sugar

255

fermentation. These results showed that overexpression of ERG10/ERG12/ERG13 did not

256

exert growth stress on the cells. The slight decrease in the xylose assimilation rate under

257

high cell density was likely due to the competition for NADPH between XR (in the xylose

258

assimilation pathway) and HMG-CoA reductase, as well as PPD synthase (in the PPD

259

biosynthetic pathway). Overall, this genetic perturbation was beneficial for PPD

260

production, resulting in a highly increased PPD yield under mixed sugar culture conditions.

261

3.5 Reintroduction of xylose assimilation pathway for improvement of xylose

262

consumption rate and PPD production

263

Strain GW6 was constructed to produce PPD from mixed sugar, but the low rate of

264

xylose assimilation was a limitation for efficient PPD production. To facilitate xylose

265

metabolism, the xylose assimilation pathway genes XYL1/XYL2/TAL1 were reintroduced

266

into strain GW6 to obtain GW9. The engineered strain GW9 exhibited an improved cell

267

growth rate and xylose consumption rate. Approximately 15 g/L xylose was utilized in 96

268

h, which was greatly improved compared with the value observed for the GW6 strain

269

(Figure 3E). The titers of PPD under glucose and mixed sugar culture conditions reached

270

26.42 mg/L and 18.69 mg/L, respectively (Figure 4A). The PPD yield under mixed sugar

271

culture conditions was 0.77 mg PPD/g mixed sugar, which was 1.16-fold higher than that

272

under glucose fermentation (0.66 mg PPD/g glucose) (Figure 4B). Compared to the values 15



273

obtained for strain GW6, the PPD titer and yield of GW9 were enhanced under both culture

274

conditions, which was likely due to the increased NADPH supply caused by the increase

275

in PPP flux. However, the improvement under glucose culture conditions was more

276

obvious than observed in mixed sugar fermentation, indicating competition for NADPH

277

between XR and PPD synthase. Therefore, increasing the NADPH supply was essential for

278

high-level production of PPD under mixed sugar culture conditions, which could benefit

279

xylose assimilation and PPD biosynthesis.

280

3.6 Enhancement of the NADPH supply via overexpression of the NADH kinase

281

POS5

282

In this study, it was found that NADPH is vital for xylose metabolism and PPD

283

production. In the xylose assimilation pathway, XR is NADPH dependent. Meanwhile, in

284

the PPD biosynthetic pathway, reactions catalyzed by HMG-CoA reductase

285

(HMG1/HMG2), squalene synthase (ERG9), and protopanaxadiol synthase (PPDS) all

286

require NADPH, especially HMG1/HMG2. To produce 1 molecule of PPD, at least 14

287

molecules of NADPH are required. To reduce the need for NADPH, NADH-dependent

288

HMGr (NADH-HMGr) from Silicibacter pomeroyi was induced as an alternative to HMG-

289

CoA reductase in strain GW6. However, the demand for NADPH was still not met in the

290

engineered strains. Consequently, the NADH kinase encoded by POS5, which

291

phosphorylates NADH into NADPH, was overexpressed in strain GW6, and strain GW10

292

was generated. Strain GW10 exhibited PPD titers of 26.52 mg/L and 50.78 mg/L in glucose 16


Page 16 of 38

Page 17 of 38


293

and mixed sugar fermentation, respectively, which were 3.03-fold and 4.55-fold higher,

294

respectively, than those in strain GW6 (Figure 4A). Furthermore, the yield of PPD under

295

mixed sugar culture conditions reached 2.06 mg PPD/g mixed sugar, which was 3.12-fold

296

higher than that under glucose fermentation (0.66 mg PPD/g glucose) (Figure 4B). In

297

contrast to other engineered strains, the improvement of the PPD titer of strain GW10 in

298

mixed sugar fermentation exceeded that in glucose fermentation, indicating that the

299

NADPH supply was a key factor for PPD production from xylose. Meanwhile, the increase

300

in the PPD titer and yield under glucose culture conditions suggested that the NADPH

301

supply was also insufficient in the glucose medium. These results showed that the redox

302

balance is essential for xylose metabolism and PPD production and that increasing the

303

NADPH supply great benefited isoprenoid production because of the NADPH-consuming

304

MVA pathway.

305

3.7 Production of PPD in shake flask cultivation

306

In this study, to evaluate the PPD production ability of the engineered strains, these

307

strains were fermented in shake flasks. When comparing the PPD titer of engineered strains

308

in glucose and mixed sugar fermentation, we found that only GW6 and GW10 exhibited

309

high PPD titers under mixed sugar culture conditions, not the other strains. The titers of

310

PPD under mixed sugar cultivation for engineered strains (GW6, GW7, GW8, GW9 and

311

GW10) were 1.28, 0.78, 0.81, 0.71 and 1.91 times of those acquired from glucose

312

fermentation. When the PPD yields under both culture conditions were compared, all the 17



313

engineered strains exhibited high PPD yields in mixed sugar cultivation (Figure 4). In

314

mixed sugar fermentation, the PPD yields of engineered strains with different strategies,

315

namely GW6, GW7, GW8, GW9 and GW10, were 2.55-, 1.43-, 1.97-, 1.16- and 3.12-fold

316

higher than those obtained from glucose fermentation, respectively. Among these strains,

317

the best performing strain, namely, GW10, produced the highest titer and yield of PPD

318

compared to the other strains, with values of 50.78 mg/L and 2.06 mg PPD/g mixed sugar,

319

respectively. Furthermore, the cell growth rate and xylose consumption rate of strain GW10

320

were also the highest among all the engineered strains (Figure 3).

321

3.8 Batch fermentation of GW10 for PPD production in a 5-L bioreactor

322

To assess the potential of strain GW10 as a PPD producer, scale-up experiments were

323

implemented in a 5-L fermenter with a working volume of 2.5-L of medium for cultivating

324

strain GW10. Because a low concentration of glucose could enhance the xylose uptake rate

325

and provide cofactors in mixed substrate fermentation47, glucose and mixed sugar batch

326

fermentation was carried out (Figure 5). After 144 h of cultivation, the strain GW10

327

produced 152.37 mg/L PPD under mixed sugar culture conditions, which was 2.58-fold

328

higher than the value of 59.04 mg/L PPD obtained with glucose fermentation. The low

329

PPD production in glucose medium was likely due to the Crabtree effect. High

330

concentrations of glucose accelerate glycolysis, which results in the production of

331

appreciable amounts of ATP via substrate-level phosphorylation48. This effect reduces the

332

need for oxidative phosphorylation by the TCA cycle via the electron transport chain; 18


Page 18 of 38

Page 19 of 38


333

therefore, respiratory metabolism is strongly repressed by fermentative metabolism, even

334

in the presence of oxygen. Subsequently, pyruvate cannot enter the TCA cycle to be

335

converted into considerable amounts of ethanol, which would be detrimental for cell

336

growth and PPD production. However, the repression of respiratory metabolism was

337

alleviated during xylose metabolism because hardly any ethanol accumulated during mixed

338

sugar fermentation, indicating that the carbon flux toward ethanol was greatly reduced.

339

During mixed sugar fermentation, the PPD concentration in the broth reached a peak at 36

340

h, with a value of 101.02 mg/L. Then, the PPD concentration decreased and remained

341

constant at approximately 65 mg/L. However, as the fermentation proceeded, red

342

substances were produced and accumulated on the tank walls. After 144 h of fermentation,

343

these substances were collected and analyzed for PPD content, which reached 86.32 mg/L.

344

These findings demonstrated that mixed sugar fermentation was more suitable for the

345

production of PPD than glucose fermentation and had the potential to circumvent the

346

limitations associated with glucose fermentation. Mixed sugar fermentation is a promising

347

strategy for the production of specific acetyl-CoA-derived chemicals at a high conversion

348

rate and to overcome the bottleneck associated with glucose fermentation.

349

Conflicts of interest

350 351

The authors declare no conflicts of interest. Acknowledgment

19



352

This work was financially supported by the National Natural Science Foundation of

353

China (no.21878220) and the Major Research Plan of Tianjin (no.16YFXTSF00460).

354

Supporting information

355

Table SI. All the primers used in this study.

356

Table SII. Titers and yields of PPD in the engineered strains under different culture

357

conditions.

358

Figure S1. Technical route for strain construction.

359

Figure S2. Comparison of fermentation performance between strains GW2 and GW3 in

360

xylose fermentation.

361

Figure S3. Growth status of the engineered strains under mixed sugar culture conditions

362

in flasks.

363

Figure S4. Xylose consumption of engineered strains under mixed sugar culture

364

conditions in flasks.

365

Figure S5. Fermentation performance of strain GW10 under glucose culture conditions in

366

a 5-L fermenter.

367

20


Page 20 of 38

Page 21 of 38


References 1.

Lin, H., Research progress in biorefinery of lignocellulosic biomass. Chinese Journal of Bioprocess Engineering 2017, 15, 44-54. (In Chinese).

2.

Jeffries, T. W., Emerging technology for fermenting d-xylose. Trends in Biotechnology 1985, 3, 208-212.

3.

Stephanopoulos, G., Challenges in engineering microbes for biofuels production. Science 2007, 315, 801-4.

4.

Kwak, S.; Jin, Y. S., Production of fuels and chemicals from xylose by engineered Saccharomyces cerevisiae: a review and perspective. Microbial Cell Factories 2017, 16, 82.

5.

Cirino, P. C.; Chin, J. W.; Ingram, L. O., Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. Biotechnology & Bioengineering 2006, 95, 1167.

6.

Cao, L.; Tang, X.; Zhang, X.; Zhang, J.; Tian, X.; Wang, J.; Xiong, M.; Wei, X., Twostage transcriptional reprogramming in Saccharomyces cerevisiae for optimizing ethanol production from xylose. Metabolic Engineering 2014, 24, 150-159.

7.

Yu, M.; Zhang, Y.; Tang, I. C.; Yang, S. T., Metabolic engineering of Clostridium tyrobutyricum for n-butanol production. Metabolic Engineering 2011, 13, 373-382.

21



8.

Chen, T.; Liu, W. X.; Fu, J.; Zhang, B.; Tang, Y. J., Engineering Bacillus subtilis for acetoin production from glucose and xylose mixtures. Journal of Biotechnology 2013, 168, 499-505.

9.

Kim, S. J.; Sim, H. J.; Kim, J. W.; Lee, Y. G.; Park, Y. C.; Seo, J. H., Enhanced production of 2,3-butanediol from xylose by combinatorial engineering of xylose metabolic pathway and cofactor regeneration in pyruvate decarboxylase-deficient Saccharomyces cerevisiae. Bioresource Technology 2017.

10. Chen, Y.; Guo, J.; Li, F.; Liu, M.; Zhang, X.; Guo, X.; Xiao, D., Production of pullulan from xylose and hemicellulose hydrolysate by Aureobasidium pullulans AY82 with pH control and DL-dithiothreitol addition. Biotechnology & Bioprocess Engineering 2014, 19, 282-288. 11. Le, M. S.; Manfred, Z.; Thomas, E.; Linda, T. M.; Ren, Q., Production of mediumchain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440. Bmc Biotechnology 2012, 12, 53. 12. Amorim-Costa, C.; Gonçalves, H.; Bernardes, J.; Ayres-De-Campos, D., Genomewide identification of genes involved in tolerance to various environmental stresses in Saccharomyces cerevisiae. Journal of Applied Genetics 2009, 50, 301-310. 13. Almeida, J. R.; Modig, T.; Petersson, A.; Hähn-Hägerdal, B.; Lidén, G.; GorwaGrauslund, M. F., Increased tolerance and conversion of inhibitors in lignocellulosic 22


Page 22 of 38

Page 23 of 38


hydrolysates by Saccharomyces cerevisiae. Journal of Chemical Technology & Biotechnology Biotechnology 2007, 82, 340–349. 14. Matsushika, A.; Inoue, H.; Kodaki, T.; Sawayama, S., Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives. Applied Microbiology & Biotechnology 2009, 84, 37-53. 15. Moysés, D. N.; Reis, V. C. B.; Almeida, J. R. M. D.; Moraes, L. M. P. D., Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects. International Journal of Molecular Sciences 2016, 17. 16. Walfridsson, M.; Anderlund, M.; Bao, X.; Hahn-Hägerdal, B., Expression of different levels of enzymes from the Pichia stipitis XYL1 and XYL2 genes in Saccharomyces cerevisiae and its effects on product formation during xylose utilisation. Applied Microbiology & Biotechnology 1997, 48, 218. 17. Zha, J.; Hu, M. L.; Shen, M. H.; Li, B. Z.; Wang, J. Y.; Yuan, Y. J., Balance of XYL1 and XYL2 expression in different yeast chassis for improved xylose fermentation. Frontiers in Microbiology 2012, 3, 355. 18. Choi, Y., Cofactor engineering in cyanobacteria to overcome imbalance between NADPH and NADH: A mini review. Frontiers of Chemical Science & Engineering 2017, 11(1), 1-6.

23



19. Kuyper, M.; Hartog, M. M. P.; Toirkens, M. J.; Almering, M. J. H.; Winkler, A. A.; Dijken, J. P. V.; Pronk, J. T., Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. Fems Yeast Research 2005, 5, 399. 20. Takahiro, B.; Tomohisa, H.; Akihiko, K., Disruption of PHO13 improves ethanol production via the xylose isomerase pathway. Amb Express 2016, 6, 1-10. 21. Jin, Y. S.; Ni, H. J.; Jeffries, T. W., Optimal growth and ethanol production from xylose by recombinant Saccharomyces cerevisiae require moderate D-xylulokinase activity. Appl Environ Microbiol 2003, 69, 495-503. 22. Richard, P.; Toivari, M. H.; Penttilä, M., The role of xylulokinase in Saccharomyces cerevisiae xylulose catabolism. Fems Microbiology Letters 2000, 190, 39-43. 23. Walfridsson, M.; Hallborn, J.; Penttilä, M.; Keränen, S.; Hahnhägerdal, B., Xylosemetabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase. Applied & Environmental Microbiology 1995, 61, 4184-90. 24. Karhumaa, K.; Hahnhägerdal, B.; Gorwagrauslund, M. F., Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering. Yeast 2005, 22, 359-368.

24


Page 24 of 38

Page 25 of 38


25. Kwak, S.; Kim, S. R.; Xu, H.; Zhang, G. C.; Lane, S.; Kim, H.; Jin, Y. S., Enhanced isoprenoid production from xylose by engineered Saccharomyces cerevisiae. Biotechnology & Bioengineering 2017, 114, 176-185. 26. Sun, L.; Wang, Q.; Liu, X.; Brons, N. H. C.; Wang, N.; Steinmetz, A.; Lv, Y.; Liao, Y.; Zheng, H., Anti-cancer effects of 20(S)-protopanoxadiol on human acute lymphoblastic leukemia cell lines Reh and RS4;11. Medical Oncology 2011, 28, 813821. 27. Popovich, D. G.; Kitts, D. D., Ginsenosides 20(S)-protopanaxadiol and Rh2 reduce cell proliferation and increase sub-G1 cells in two cultured intestinal cell lines, Int-407 and Caco-2. Can J Physiol Pharmacol 2004, 82, 183-190. 28. Gao, J. L.; Lv, G. Y.; He, B. C.; Zhang, B. Q.; Zhang, H.; Wang, N.; Wang, C. Z.; Du, W.; Yuan, C. S.; He, T. C., Ginseng saponin metabolite 20(S)-protopanaxadiol inhibits tumor growth by targeting multiple cancer signaling pathways. Oncology Reports 2013, 30, 292-298. 29. Dai, Z.; Liu, Y.; Zhang, X.; Shi, M.; Wang, B.; Wang, D.; Huang, L.; Zhang, X., Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. Metabolic Engineering 2013, 20, 146-156.

25



30. Zhao, F.; Du, Y.; Bai, P.; Liu, J.; Lu, W.; Yuan, Y., Enhancing Saccharomyces cerevisiae reactive oxygen species and ethanol stress tolerance for high-level production of protopanoxadiol. Bioresource Technology 2017, 227, 308-316. 31. Shiba, Y.; Paradise, E. M.; Kirby, J.; Ro, D. K.; Keasling, J. D., Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids. Metabolic Engineering 2007, 9, 160-168. 32. Chen, Y.; Daviet, L.; Schalk, M.; Siewers, V.; Nielsen, J., Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism. Metabolic Engineering 2013, 15, 48-54. 33. Yuan, J.; Ching, C. B., Combinatorial engineering of mevalonate pathway for improved amorpha-4, 11-diene production in budding yeast. Biotechnology & Bioengineering 2014, 111, 608. 34. Westfall, P. J.; Pitera, D. J.; Lenihan, J. R.; Eng, D.; Woolard, F. X.; Regentin, R.; Horning, T.; Tsuruta, H.; Melis, D. J.; Owens, A., Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proceedings of the National Academy of Sciences of the United States of America 2012, 109, 111-8.

26


Page 26 of 38

Page 27 of 38


35. Zhao, X.; Shi, F.; Zhan, W., Overexpression of ZWF1 and POS5 improves carotenoid biosynthesis in recombinant Saccharomyces cerevisiae. Letters in Applied Microbiology 2015, 61, 354-360. 36. Paramasivan, K.; Mutturi, S., Regeneration of NADPH coupled with HMG-CoA reductase activity increases squalene synthesis in Saccharomyces cerevisiae. Journal of Agricultural & Food Chemistry 2017, 65, 8162. 37. Farhi, M.; Marhevka, E.; Masci, T.; Marcos, E.; Eyal, Y.; Ovadis, M.; Abeliovich, H.; Vainstein, A., Harnessing yeast subcellular compartments for the production of plant terpenoids. Metabolic Engineering 2011, 13, 474-481. 38. Lv, X.; Wang, F.; Zhou, P.; Ye, L.; Xie, W.; Xu, H.; Yu, H., Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae. Nature Communications 2016, 7, 12851. 39. Meadows, A. L.; Hawkins, K. M.; Tsegaye, Y.; Antipov, E.; Kim, Y.; Raetz, L.; Dahl, R. H.; Tai, A.; Mahatdejkulmeadows, T.; Xu, L., Rewriting yeast central carbon metabolism for industrial isoprenoid production. Nature 2016, 537, 694. 40. Nambu-Nishida, Y.; Sakihama, Y.; Ishii, J.; Hasunuma, T.; Kondo, A., Selection of yeast Saccharomyces cerevisiae promoters available for xylose cultivation and fermentation. Journal of Bioscience & Bioengineering 2017, 125.

27



41. Zhao, F.; Bai, P.; Liu, T.; Li, D.; Zhang, X.; Lu, W.; Yuan, Y., Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae. Biotechnology & Bioengineering 2016, 113, 1787-1795. 42. Shen, M.; Song, H.; Li, B.; Yuan, Y., Deletion of D-ribulose-5-phosphate 3-epimerase (RPE1) induces simultaneous utilization of xylose and glucose in xylose-utilizing Saccharomyces cerevisiae. Biotechnology Letters 2015, 37, 1031-1036. 43. Gietz, R. D.; Woods, R. A., 4 Transformation of Yeast by the Lithium Acetate/SingleStranded Carrier DNA/PEG Method. Methods in Microbiology 1998, 26, 53-66. 44. Shao, Z.; Zhao, H.; Zhao, H., DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Research 2009, 37, e16. 45. Gueldener, U.; Heinisch, J.; Koehler, G. J.; Voss, D.; Hegemann, J. H., A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Research 2002, 30, e23. 46. Han, J. Y.; Kim, H. J.; Kwon, Y. S.; Choi, Y. E., The Cyt P450 enzyme CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediol-II during ginsenoside biosynthesis in Panax ginseng. Plant & Cell Physiology 2011, 52, 2062-73. 47. Krahulec, S.; Petschacher, B.; Wallner, M.; Longus, K.; Klimacek, M.; Nidetzky, B., Fermentation of mixed glucose-xylose substrates by engineered strains of

28


Page 28 of 38

Page 29 of 38


Saccharomyces cerevisiae: role of the coenzyme specificity of xylose reductase, and effect of glucose on xylose utilization. Microbial Cell Factories 2010, 9, 16. 48. Thomson, J.; Gaucher, E.; Burgan, M. K., Dw; Li, T.; Aris, J.; Benner, S., Resurrecting ancestral alcohol dehydrogenases from yeast. Nature Genetics 2005, 37, 630-5.

29



Page 30 of 38

Figure 1 Overview of protopanaxadiol synthesis from xylose in engineered Saccharomyces cerevisiae strains. Purple words represent the xylose assimilation pathway; blue words represent the pentose phosphate pathway; black words represent the PDH bypass; green words represent the protopanaxadiol biosynthetic pathway; and red words represent genes upregulated in this study. XR, xylose reductase; XDH, xylitol dehydrogenase; XI, xylose isomerase; XK, xylulokinase; TKL, transketolase; TAL, transaldolase; PDC1/5/6, pyruvate decarboxylase; ALD6, aldehyde dehydrogenase; ACS2, acetyl-CoA synthetase; ERG10, acetyl-CoA Cacetyltransferase; ERG13, hydroxymethylglutaryl-CoA synthase; NADH-HMGr, NADHdependent hydroxymethylglutaryl-CoA reductase; ERG12, mevalonate kinase; ERG8, phosphomevalonate

kinase;

ERG19,

diphosphomevalonate

decarboxylase;

IDI1,

isopentenyl-diphosphate delta-isomerase; ERG20,farnesyl diphosphate synthase; ERG9, farnesyl-diphosphate

farnesyltransferase;

ERG1,

squalene

monooxygenase;

DS,

dammarenediol-II synthase; PPDS, protopanaxadiol synthase; ATR1, Arabidopsis thaliana NADPH-cytochrome P450 reductase. PPP, pentose phosphate pathway; F6P, fructose 6-phosphate; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; HMG-CoA, hydroxymethylglutaryl-CoA; MVA, mevalonate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; DMD,

30


Page 31 of 38


dammarenediol-II;

PPD,

protopanaxadiol.

xPK,

xylulose-5-phosphate

specific

phosphoketolase; PTA, phosphotransacetylase. Figure 2 Fermentation profiles of the engineered GW3 strain under mixed sugar culture conditions. Figure 3 Growth status and xylose consumption of the engineered strains during mixed sugar fermentation in flasks. (A) GW5, (B) GW6, (C) GW7, (D) GW8, (E) GW9, and (F) GW10 in SD medium with 10 g/L glucose and 30 g/L xylose. Symbols: upward-facing triangles, OD600; circles, xylose. Figure 4 Product titers and yields of the engineered strains in mixed sugar and glucose fermentation in flasks. The product of strain GW5 is DMD, and that of the other strains is PPD. Figure 5 Fermentation performance of strain GW10 under mixed sugar conditions in a 5L fermenter. Symbols: filled circles, OD600; filled downward-facing triangles, xylose; filled squares, PPD; open squares, DMD.

31



Page 32 of 38

Table 1 Strains used in this study Strains

Description

Source

W303-1a

{MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100} XYL1 and XYL2 expression cassettes were integrated into the δ site of W303-1a Native promoter PXKS1 of XKS1 was replaced with strong the promoter PFBA1 in GW1 TAL1 and TAKL1 expression cassettes were integrated into the rDNA site of GW2 HIS3 and URA3 markers were deleted in GW3 by the Cre-expressing plasmid pSH47 DS, ERG1 and ERG9 expression cassettes were integrated into the rDNA site of GW4 ERG20, NADH-HMGr and PPDS-ATR1 expression cassettes were integrated into the rDNA site of GW5 xPK and PTA expression cassettes were integrated into the trp1 site of GW6 ERG10, ERG12 and ERG13 expression cassettes were integrated into the δ site of GW6 XYL1, XYL2 and TAL1 expression cassettes were integrated into the δ site of GW6 POS5 expression cassette was integrated into the trp1 site of GW6

Laboratory stock This study

GW1 GW2 GW3 GW4 GW5 GW6 GW7 GW8 GW9 GW10

32


This study This study This study This study This study This study This study This study This study

Page 33 of 38


Figure 1

33



Figure 2

34


Page 34 of 38

Page 35 of 38


Figure 3

35



Figure 4

36


Page 36 of 38

Page 37 of 38


Figure 5

37



Table of Contents

38


Page 38 of 38

Engineering Saccharomyces cerevisiae for Enhanced Production of

Recommend Documents