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Enhanced S-adenosylmethionine production by increasing ATP levels in baker´s yeast (Saccharomyces cerevisiae) yawei chen, and Tianwei Tan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00819 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018

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Journal of Agricultural and Food Chemistry

Enhanced S-adenosylmethionine production by increasing ATP levels in baker´s yeast (Saccharomyces cerevisiae) Yawei Chen1* and Tianwei Tan2 1. College of Chemical and Pharmaceutical Engineering, Henan University of Science and Technology, Luoyang 471023, PR China 2. National Energy R&D Center for Biorefinery, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China *

Corresponding Author: Yawei Chen

Email: [email protected] Tel: +8637964231914

1

ACS Paragon Plus Environment


1

ABSTRACT: In the biosynthesis of S-adenosylmethionine (SAM) in baker´s yeast

2

(Saccharomyces cerevisiae), ATP functions both as a precursor and a driving force.

3

However, few published reports have dealt with the control of ATP concentration

4

using genetic design. In this study we have adopted a new ATP regulation strategy in

5

yeast for enhancing SAM biosynthesis, including altering NADH availability and

6

regulating the oxygen supply. Different ATP regulation systems were designed based

7

on the introduction of water-forming NADH oxidase, Vitreoscilla hemoglobin and

8

phosphite dehydrogenase in combination with overexpression of the gene SAM2.

9

Applying this strategy, after 28 h cultivation, the SAM titer in the yeast strain

10

ABYSM-2 reached a maximum level of close to 55 mg/L, an increase of 67%

11

compared to the control strain. The results show that the ATP regulation strategy is a

12

valuable tool for SAM production and might further enhance the synthesis of other

13

ATP-driven metabolites in yeast.

14

KEYWORDS: ATP; S-adenosylmethionine (SAM); Saccharomyces cerevisiae;

15

Vitreoscilla hemoglobin (VHb)

16 17 18 19 20 21 22 2


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INTRODUCTION

24

S-Adenosylmethionine (SAM) is a key component in various biological reactions. Its

25

major function is to serve as a methyl group donor for the modification of

26

biomolecules such as DNA, RNA, proteins and various small molecules1, 2. It is

27

widely used in the therapy of various diseases, such as osteoarthritis, affective

28

disorders and liver disease3-6. Therefore, SAM is of commercial interest as a research

29

reagent, a pharmaceutical and as a dietary supplement. In recent years, several studies

30

of the pharmacological effects of SAM have been reported7-9. To this end, attempts

31

have been made to achieve SAM production by fermentation utilizing various types of

32

microorganisms10-12.

33

In living organisms, SAM is synthesized from L-methionine and ATP with

34

methionine adenosyltransferase (MAT) as catalyst13. Thus the addition of

35

L-methionine is required for enhanced SAM production as L-methionine is the

36

limiting component for biosynthesis of SAM in yeast10, 12, 14. Strains of S. cerevisiae

37

have been genetically engineered using a variety of approaches to overproduce SAM,

38

as this yeast is recognized as GRAS (generally recognized as safe) by FDA15, 16. SAM

39

has also been produced on a large scale after optimizing the fermentation conditions,

40

including the supply level of L-methionine. However, ATP, which not only provides

41

the adenosyl moiety but also acts as an energy carrier, also becomes a limiting factor

42

for SAM production when L-methionine is in excess. To this end, chemicals such as

43

citrate (auxiliary energy substrate) and n-hexadecane (oxygen vector)17,

44

supplied as well. Attempts were made to control the intracellular ATP level by 3


18

were


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45

suppressing the metabolic pathways of by-products using a synthetic sRNA-based

46

regulation strategy, as reported in a previous study 19. However, this approach failed to

47

supply enough ATP to satisfy high SAM production.

48

Traditional metabolic engineering studies have focused on controlling enzyme

49

levels through the addition, overexpression, or deletion of enzymes in the particular

50

pathway. An alternative approach, cofactor regulation, has the potential to become a

51

powerful tool for metabolic engineering. Cofactors such as NADPH/NADP+,

52

ATP/ADP and NADH/NAD+ are the most powerful metabolites in metabolic

53

networks in all living organisms. They provide redox carriers for biosynthetic

54

reactions and act as principal agents in the energy transformation inside the cell. It has

55

been reported that control of the concentration and forms of these cofactors could

56

regulate the expression level of many genes and redirect the flux of the central carbon

57

metabolism20, 21. Expression of the gene adk1 (encoding adenylate kinase) led to

58

increased supply of ATP and enhanced SAM production with higher L-methionine

59

conversion efficiency in Pichia pastoris

60

vhb (encoding Vitreoscilla hemoglobin, VHb) could increase the synthesis rate of ATP

61

and thus enhance cell growth and SAM production in recombinant P. pastoris

62

has also been reported that increasing the availability of methionine and ATP by

63

co-expression of SAM2 and MET6, and the addition of sodium citrate, led to a 2.3-fold

64

increase in SAM accumulation in S. cerevisiae CGMCC 2842 (WT) 24. Although it is

65

generally known that cofactors play a major role in the production of metabolites, the

66

application of a genetically based ATP regulation strategy to promote SAM

22

. Some reports have shown that the gene

4


23

. It

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production in S. cerevisiae has seldom been considered.

68

The aim of this study is to increase the availability of ATP during SAM production

69

by introducing three heterologous pathways coupled to the ATP synthesis (Fig.1). The

70

effects of ATP on production rate and energy metabolism during SAM biosynthesis

71

have been investigated as well. Differential metabolomics analysis by LC-MS

72

combined with transcriptional analysis have been applied to explore the overall

73

metabolic effects of the different strategies in S. cerevisiae.

74

MATERIALS AND METHODS

75

Strains, media and cultivation conditions

76

All strains used in this study are listed in Table 1. E. coli Trans 10 was used for

77

propagating the plasmids before transformation into the yeast strains. The transformed

78

E. coli was screened on LB (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 20 g/L

79

agar) plates supplemented with 100 µg/mL Ampicillin at 37℃. The recombinant

80

yeasts were selected in synthetic media containing 6.7 g/L yeast nitrogen base with

81

ammonium sulfate and without amino acids, 20 g/L glucose, and 20 g/L agar.

82

Modified S. cerevisiae BY4741 (BY4741-MH, MATa; leu2∆0; ura3∆0) was used as

83

the parent strain for the SAM production. The yeast strains were pre-cultured in 50

84

mL synthetic complete (SC) medium (6.7 g/L yeast nitrogen base with ammonium

85

sulfate and without amino acids, 1.4 g/L amino acids dropout mixture lacking leucine

86

and uracil) supplemented with amino acids as required and 20 g/L glucose at 30℃,

87

220 rpm for 24 h. The main cultures were inoculated to an initial OD600 of 0.5 in 100

88

mL of SC medium or YPD medium (10 g/L yeast extract, 20 g/L peptone, 20 g/L 5



89

glucose) supplemented with 2 g/L L-methionine in 250 mL flasks and grown at 30℃,

90

220 rpm for 28 h. For strain ABYSM-3, 15 mM phosphite was added to the SC

91

medium or YPD medium in the flask. All flask fermentations were carried out in

92

triplicates of both the recombinant and the control strain.

93

Table 1

94

Plasmid construction

95

All plasmids used and constructed in this study are listed in Table 1. Primers for PCR

96

are listed in Table 2. Standard genetic techniques were used for DNA manipulation.

97

Cloning procedures were carried out in E.coli Trans10. Phusion High-Fidelity DNA

98

polymerase, restriction enzymes, T4 DNA ligase and other enzymes were all

99

purchased from New England Biolabs (USA), following the manufacturer’s

100 101

instructions. To complement the auxotrophic markers (met15∆0, his3∆1) in strain BY4741, the

102

expression cassettes of MET15 and HIS3 were both amplified from S. cerevisiae

103

S288c genomic DNA25 and strain BY4741-MH was produced. Two rounds of overlap

104

PCR were applied to efficiently construct the recombinant plasmids. The overlap PCR

105

primers for promoters, genes of interest and terminators were all designed according

106

to the NEB Assembly Builder online tools (http://nebuilder.neb.com/). The SAM2

107

gene10 from S. cerevisiae BY4741was inserted into pRS425 using TEF1 promoter and

108

PGI1 terminator to yield pRS425-PTEF1-SAM2-TPGI1. The genes of noxE (from

109

Lactococcus lactis) 26, vhb (encoding Vitreoscilla hemoglobin) 35 and ptxD (S.

110

cerevisiae codon-optimized from Pseudomonas stutzeri) 27 were assembled with 6


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HXT7 promoter and HXT7 terminator into YCPlac33 and yield the recombinant

112

plasmids of YCplac33-PHXT7-noxE-THXT7, YCplac33-PHXT7-vhb-THXT7 and

113

YCplac33-PHXT7-ptxD-THXT7, respectively (Fig.2). The recombinant vectors were

114

transformed into E. coli Trans 10 by a heat shock at 42℃ for 30 s. The expression

115

vector was transformed into yeast S.cerevisiae BY4741-MH using the LiAc/SS carrier

116

DNA/PEG method.

117

Table 2

118

SAM quantitation

119

SAM was extracted from the fermentation broth after treatment with 10% (w/v)

120

perchloric acid by shaking (220 rpm) at 30℃ for 1 h. After centrifugation at 12000×g

121

for 10 min at 4℃, the supernatant was collected and filtered through a 0.22 µm

122

membrane. Subsequent analysis of the SAM concentration was carried out by HPLC

123

(Shimadzu, Japan) equipped with a C18 column (Agela Technologies, China) and a

124

UV detector at 260 nm. The mobile phase was 0.01 mol/L ammonium formate at pH

125

3.5 operated at a flow rate of 1.0 mL/min28.

126

ATP and ADP determination

127

For ATP and ADP quantitation, the harvested cells (in SC medium) were

128

re-suspended in 0.4 mol/L perchloric acid and treated by ultra-sonication in an

129

ice-water bath. After centrifugation at 12000×g (4℃) for 10 min, the supernatant was

130

neutralized with saturated potassium carbonate. The concentrations of ATP and ADP

131

were detected by HPLC equipped with a C18 column (Agela Technologies, China)

132

and a UV detector at 254 nm. The mobile phase used was phosphate buffer consisting 7



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of 0.06 M K2HPO4 and 0.04 M KH2PO4 at pH 7.0 with a flow rate of 1.0 mL/min.

134

LC-MS analysis of intracellular metabolites

135

An amount of 10 mL S. cerevisiae culture was collected and immediately quenched

136

with 20 mL methanol (HPLC grade, precooled to -40℃) at -40℃ The samples of the

137

intracellular metabolites were prepared as previously reported29. Before detection an

138

amount of 150 µL acetonitrile-water (v/v: 1/1) mixture was added to the dried samples

139

to dissolve the metabolites. The concentrations of the metabolites were measured by

140

LC-MS in the supernatants obtained after centrifugation.

141

All measurements were performed on a LC-20AD HPLC system (Shimadzu, Japan)

142

equipped with a QTRAP5500 mass spectrometer (AB SCIEX, USA). The HPLC

143

system was equipped with a BEHAmide column (Waters, USA) (oven temperature

144

40℃). The source was operated in ESI+ mode (CUR 40 psi, CXP 10, GS1 50 psi,

145

GS260 psi, IS 1500 V, CAD Medium, TEM 600℃, DP 40 and EP 10). The pump

146

supplied a gradient with the following settings: 0 min, 10% mobile phase A (0.1%

147

formic acid, 99.9% H2O), 90% mobile phase B (95% acetonitrile, 5% mixture of 1

148

mmoL ammonium formate and 0.01% formic acid), and maintained for 5 min. Then

149

the 20% mobile phase A and 80% mobile phase B were introduced and maintained for

150

3 min. Subsequently, mobile phase A was increased to 60% after 8 min and held

151

constant for 6 min. Then mobile phase B was increased to 90% after 6 s and held

152

constant for 5 min. The flow rate was set to 0.3 mL/min. All data obtained from the

153

LC-MS were calculated using the software Analyst1.6.1 (AB SCIEX, USA) and

154

further

analyzed

using

the

web-based

8


MetaboAnalyst

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(http://www.metaboanalyst.ca/)30 .

156

Quantitative real-time PCR

157

All primers used for the qPCR are listed in Table 3. An amount of 1 mL S. cerevisiae

158

culture after 16 h cultivation was collected in order to extract the total RNA using

159

Yeast RNAiso kit (Takara, China). The concentration of the RNA samples was

160

measured using the NanoDrop K5500 (Beijing Kaiao Technology Development

161

Company, China). The extracted RNA was converted into cDNA using the Trans

162

Script First Strand cDNA Synthesis Supermix (Beijing TransGen Biotech Company,

163

China). Quantitative real-time PCR (qPCR) was performed on a Rotor-Gene Q system

164

(Analytikjena, Germany) using the Trans Start Top Green qPCR Supermix (Beijing

165

TransGen Biotech Company, China). Each qPCR reaction was performed with three

166

replicates.

167

Table 3

168

RESULTS AND DISCUSSION

169

Effect of the ATP regulation strategy on SAM production

170

Methionine adenosyltransferase (MAT) catalyzes the reaction that combines

171

L-methionine, ATP and water to produce the target metabolite SAM, pyrophosphate

172

and inorganic phosphate. Many experiments have shown that overexpression of MAT

173

greatly increases SAM accumulation. Among the different kinds of MAT, SAM2

174

encoding MAT from S. cerevisiae is not repressed in the presence of L-methionine 31,

175

32

176

production SAM is the first choice. The constitutive promoters TEF1 and truncated

. Therefore, overexpressing SAM2 in S. cerevisiae BY4741-MH to facilitate the

9



177

HXT7 from S. cerevisiae show strong transcription ability when glucose or ethanol

178

serves as the carbon source, and were thus selected as the promoters for the

179

expression of SAM2 and the genes involved in the ATP synthesis, respectively33.

180

To alleviate the redox imbalance and improve the fermentation performance of

181

SAM production in S. cerevisiae, the three genes noxE, vhb and ptxD were tested

182

separately. The noxE gene from Lactococcus lactis was used as control to examine the

183

effect of the NAD (H) on ATP and SAM synthesis. The noxE gene encoding

184

water-forming NADH oxidase provides an extra route for NAD+ regeneration. It

185

regenerates NAD+ from NADH using molecular oxygen 26. Vitreoscilla hemoglobin

186

as a membrane protein could enhance the respiration and energy metabolism by

187

promoting oxygen transfer to the intracellular terminal oxidases. The oxygen

188

dissociation rate constant of VHb is hundreds of times higher than that of other

189

hemoglobins34, 35. However, it has been shown that VHb is predominantly localized in

190

the cytoplasm when expressed in S. cerevisiae36. In order to regenerate NADH, a

191

codon-optimized phosphite dehydrogenase PtxD (encoded by ptxD) from

192

Pseudomonas stutzeri, was selected because of its ability to catalyze the nearly

193

irreversible oxidation of phosphite to phosphate, with the concomitant reduction of

194

NAD+ to NADH37. VHb, PtxD and NoxE have been widely used in recombinant

195

strains to improve the production of target compounds21, 34. However, no published

196

reports have so far been found regarding the application of these enzymes for SAM

197

production in S. cerevisiae.

198

The biomass and glucose consumption were shown in Fig.3A-Fig.3D. The biomass 10


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of recombinant strains in YPD medium was significantly higher than that in SC

200

medium. The biomass of the recombinant strain ABYSM-1 in either medium was

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lower than other recombinant strains, indicating that noxE might cause growth

202

retardation of the strain. In addition, the SAM titer of the strain ABYSM-1 was lower

203

at most of the time points tested than that of recombinant strains ABYSM-2 and

204

ABYSM-3 (Fig.3E and Fig.3F). Previous studies have demonstrated that NoxE is

205

mainly localized in the cytosol of S. cerevisiae and has a high affinity for NADH26, 38.

206

The extra intracellular NADH cannot flow to the oxidative phosphorylation pathway

207

in strain ABYSM-1, thereby reducing the synthesis of intracellular ATP and disrupting

208

the cellular redox state, resulting in inhibition of cell growth.

209

The SAM titers obtained in flask experiments using recombinant strains in YPD

210

medium and SC medium are shown in Fig.3E and Fig.3F. The titer gradually

211

increases during the fermentation process, and in the strain PBYSM-1 increases 2- to

212

3-fold compared to the control, the highest titer reaching 32.80 mg/L. In addition, the

213

transcription level of SAM2 in the strain PBYSM-1 was twice that of the strain

214

PBYSM-0 (Fig.S1). These results proved that the activity of MAT in a wild S.

215

cerevisiae strain is the limiting factor for the synthesis of SAM, and thus, promotion

216

of the SAM production could benefit a lot from the enhanced activity of MAT10, 24.

217

The intracellular SAM titers of strain ABYSM-2 and ABYSM-3 increased by 37%

218

(42.58 mg/L) and 24% (40.06 mg/L) after 28 h, compared to that of PBYSM-1 in SC

219

medium. The L-methionine conversion efficiency and the SAM productivity of the

220

strain ABYSM-2 was higher than other recombinant strains either in SC medium or in 11



221

YPD medium (Table S1). The results obtained above imply that the expression of the

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vhb gene would enhance oxygen supply and that introduction of the ptxD gene would

223

increase NADH availability, leading to promotion of the synthesis of SAM.

224

Regulating the oxidative phosphorylation seems to be a more efficient way to control

225

the intracellular ATP concentration, as most ATP production comes from an oxidative

226

phosphorylation pathway under aerobic conditions. Intracellular NADH, produced

227

from the central carbon metabolism and other metabolic pathways, can be converted

228

to NAD+ in different ways. Under aerobic growth, NADH oxidation occurs through

229

ETC (electron transfer chain), in which oxygen is used as the final electron acceptor,

230

producing a large amount of ATP. In addition, the transcription level of SAM2 in the

231

strain ABYSM-2 and ABYSM-3 was higher than that of the strain PBYSM-1 (Fig.S1).

232

The reason could be that the cofactor regulation strategies promoted the

233

transcriptional expression of SAM2. Overall, the results obtained showed that the

234

strategies of controlling NADH availability and regulation of oxygen supply could be

235

potential tools for enhancing SAM production.

236

Higher SAM titers were observed in all recombinant strains after 24 h fermentation,

237

indicating that the SAM concentration is correlated to cell growth. The SAM titer of

238

strain ABYSM-2 with the vhb gene increased by 19% in the SC medium and by 22%

239

in the YPD medium during the fermentation period 24 to 28 h. The possible reason

240

may be that the dissolved oxygen in the medium decreased in the late stages of the

241

fermentation process, enhancing the VHb’s ability to bind to oxygen to increase the

242

intracellular levels of ATP, and thus significantly increasing the production of 12


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intracellular SAM.

244

The ATP regulation strategy disturbed the ATP pool

245

The intracellular ATP concentration, ADP concentration and ATP/ADP ratio of the

246

different strains in the SC medium were determined. As shown in Fig.4, the

247

intracellular ATP concentrations of ABYSM-2 and ABYSM-3 were higher than that

248

of the control strain PBYSM-1 at 28 h. And the ratio of ATP to ADP of ABYSM-2

249

and ABYSM-3 were higher than that of the control strain PBYSM-1 at 24 h. The

250

ATP/ADP ratio of strains ABYSM-2 and ABYSM-3 increased by 28% and 18%,

251

respectively, at 24 h. The absolute concentration of ATP in the ABYSM-3 strain with

252

the ptxD gene was higher than that of ABYSM-2. However, the SAM titer of the

253

ABYSM-3 strain was less than that of ABYSM-2. The possible reason could be that

254

the ATP concentration of ABYSM-3 strain was presumably improved by the change

255

of intracellular NADH. Also NADH, as an important cofactor, is not only involved in

256

the synthesis of ATP but also in the maintenance of the intracellular redox balance.

257

The cofactor NAD+ is utilized to produce the reducing equivalent in the form of

258

NADH. Meanwhile, the cell regenerates NAD+ from NADH to achieve a redox

259

balance. While the enhancement of NADH could improve the intracellular ATP level,

260

it might also disturb the redox micro-environment, changing the distribution of carbon,

261

and ultimately affect the synthesis of the target product39. Therefore, alterations in the

262

availability of NADH are expected to have a profound effect in the whole metabolic

263

network during the fermentation process.

264

Effects of ATP regulation on the distribution of intracellular metabolites 13



265

The metabolomes of the strains ABYSM-2

266

(BY4741-MH/pRS425-SAM2/YCplac33-vhb) and ABYSM-3

267

(BY4741-MH/pRS425-SAM2/YCplac33-ptxD) which had a higher SAM titer were

268

chosen and tested by LC-MS. The data were imported and analyzed using the

269

MetaboAnalyst software according to the published protocol30, 40. Strain PBYSM-1

270

(BY4741-MH/pRS425-SAM2/ YCplac33) was used as the control. The results of the

271

statistical analysis and the metabolic pathway analysis are shown in Fig.5 and

272

Fig.S2-S3. There were 87 valid metabolites detected by LC-MS in all of the three

273

strains. The metabolic data were normalized before statistical analysis (Fig.S2). The

274

heatmap analysis showed that the quantitative results of the determined metabolites in

275

each strain were consistent between replicates and could be used for further analysis

276

(Fig.5A). The heatmap analysis and the principal component analysis (PCA) showed

277

that there were significant variations in the relative contents of metabolites in the

278

different strains, which indicates that the three strains have their own distinct

279

character after genetic manipulation (Fig.5A, Fig.S3-A and Fig.S3-B). However, no

280

separate metabolite could be identified that would individually account for the

281

differences among the three strains (Fig.S3-C). An over-representation analysis plot

282

reveals the most relevant pathways that are affected by the genetic changes (Fig.5B).

283

Several amino acid pathways, i.e. the arginine and proline metabolisms, methionine

284

metabolism and glycine metabolism and so on, were considered to be highly affected

285

which was confirmed by further amino acid analysis. The amino acid analysis (Table

286

S2) showed that the concentration of several amino acids in the recombinant strains 14


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increased to different degree compared to the control strain. In particular, the

288

concentrations of glycine, glutamic acid, serine and valine increased by more than

289

50%. It has been reported that the synthesis of these amino acids is positively

290

correlated with the synthesis of SAM41. Changes in the concentrations of several

291

amino acids indicate that the levels of ATP and NADH in vivo were involved in the

292

changes of the intracellular metabolic network.

293

Analysis of transcriptional levels of key genes in the central carbon metabolism

294

In an attempt to shed light upon the mechanism of the ATP promotion of SAM

295

biosynthesis, qPCR was used to analyze the transcription level of the key genes in the

296

central carbon metabolism of strains PBYSM-1(control strain), ABYSM-3 and

297

ABYSM-2 (Fig.6). The gene S. cerevisiae act1 (encoding actin) was chosen as

298

reference for normalizing the cDNA samples during analysis. The normalized mRNA

299

level in the control was assumed to be 1.0. The qPCR primers for genes hxk1

300

(encoding hexokinase), tdh1 (encoding glyceraldehyde-3-phosphate dehydrogenase),

301

tdh3 (encoding glyceraldehyde-3-dehydrogenase), pyk2 (encoding pyruvate kinase) in

302

the glycolytic pathway, cit1 (encoding citrate synthase), idh1 (encoding isocitrate

303

dehydrogenase) and mdh1 (encoding malate dehydrogenase) in the TCA cycle, and

304

zwf1 (encoding glucose-6-phosphate dehydrogenase) in the pentose phosphate

305

pathway (PPP) were designed and listed in Table 3.

306

The glycolysis ability of recombinant strain ABYSM-3 might have been suppressed,

307

when the transcriptional levels of hxk1, tdh1 and pyk2 were reduced by 62%, 87% and

308

85% respectively. The transcription levels of some key enzymes in the TCA cycle 15



309

were also severely inhibited, and the transcription levels of cit1 and idh1 decreased by

310

58% and 86%, respectively. This might be due to the presumably higher level of

311

NADH in strain ABYSM-3 that disturbed the TCA cycle and glycolysis, which is

312

consistent with literature reports26. The key enzyme in the PPP, glucose-6-phosphate

313

dehydrogenase encoded by gene zwf1, was also partially inhibited, indicating that the

314

glycolysis metabolism in PPP was suppressed in strains where intracellular NADH

315

was enhanced. The transcription level of the TCA cycle mitochondrial malate

316

dehydrogenase in strains ABYSM-2 and ABYSM-3 was increased by nearly 50%.

317

Regarding the key glycolysis enzymes of strain ABYSM-2, only the transcription

318

level of hexokinase was reduced by 48%. The levels of the other transcripts tested

319

were not significantly changed.

320

The central carbon flux is selectively regulated by the intracellular levels of NAD+,

321

NADH and ATP. In particular, these cofactors play an important role in glucose

322

metabolism through regulation of both the expression and activities of various key

323

enzymes in glycolysis and the TCA cycle20, 26, 39. Thus, re-distribution of the central

324

carbon flux potentially contributed to the increase in the SAM titer of S. cerevisiae.

325

However, the higher ATP concentration in this yeast could also cause allosteric

326

inhibition of the glycolytic pathway, which would eventually lead to a reduction of

327

SAM productivity. Although it is difficult to control the concentration of ATP in

328

order to achieve maximum metabolite production, it was still possible to increase the

329

synthesis of ATP-driven metabolites by applying optimized ATP regulation strategies.

330

ATP is an essential cofactor in many metabolic pathways, and is also an important 16


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precursor as well as the driving force for SAM biosynthesis in the yeast S. cerevisiae.

332

In this work, different ATP regulation systems were designed on the basis of

333

overexpression of SAM2 to study the effect of intracellular ATP on SAM synthesis in

334

S. cerevisiae. The results showed that the activity of MAT is a major factor in SAM

335

biosynthesis, and the intracellular SAM titer doubled in strains where SAM2 was

336

overexpressed. The genes vhb and ptxD have a positive effect on promoting SAM

337

production. The intracellular SAM titer in the strain ABYSM-2 reached maximum

338

level at 28 h, a 67% (54.92 mg/L) increase compared to the control strain. The results

339

showed that the strategy of controlling NADH availability and regulation of oxygen

340

supply contributed to the enhancement of ATP and SAM synthesis in S. cerevisiae. In

341

particular the introduction of VHb can boost energy metabolism, resulting in

342

increased biomass and SAM titer. The metabolites were determined by LC-MS

343

combined with metabolomics analysis. The concentration variation of certain

344

metabolites and amino acids were significant between strains, which correlated with

345

the variation in SAM production. In addition, it was found that genetic perturbations

346

significantly inhibited transcription of key enzymes in glycolysis and the TCA cycle.

347

As a further development, these efficient ATP regulation systems and strategies will

348

be combined and applied to build high yield genetic engineering strain.

349

ASSOCIATED CONTENT

350

Supporting Information

351

Data normalization view; Principal component analysis; List of concentrations of

352

amino acids in S. cerevisiae 17



353

AUTHOR INFORMATION

354

Corresponding Author

355

Tel.: +8637964231914. E-mail:[email protected]

356

Funding

357

This work was supported by the National Nature Science Foundation of China

358

(21606073, 21390202, 21436002), the National Basic Research Program of China

359

(973 program) (2013CB733600, 2012CB725200) and the National Key Scientific

360

Instruments and Equipment Development Special Fund (2012YQ0401400302).

361

Notes

362

The authors declare no competing financial interest.

363

ACKNOWLEDGMENTS

364

The authors thank Prof. Jan-Christer Janson (Uppsala University) for revising the

365

language of this manuscript.

366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382

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and glutathione by an ATP-oriented amino acid addition strategy. Bioresource Technology 2012, 107, 19-24. (42) Li, Y. J.; Wang, M. M.; Chen, Y. W.; Wang, M.; Fan, L. H.; Tan, T. W., Engineered yeast with a

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21



Figure captions Fig. 1 Schematic diagram of ATP regulation strategies in S. cerevisiae MAT: methionine adenosyltransferase; NoxE: water-forming NADH oxidase; VHb: Vitreoscilla hemoglobin; PtxD: phosphite dehydrogenase Fig. 2 Schematic overview of the plasmid construction in yeast Fig. 3 Flask fermentation results of recombinant strains. (A) biomass in SC medium; (B) biomass in YPD medium; (C) glucose consumption in SC medium; (D) glucose consumption in YPD medium; (E) SAM titer in SC medium; (F) SAM titer in YPD medium 482

PBYSM-0: control strain; PBYSM-1: PBYSM-0 strain with SAM2 overexpressed;

483

ABYSM-1: PBYSM-1strain with noxE expressed; ABYSM-2: PBYSM-1strain with

484

vhb expressed; ABYSM-3: PBYSM-1strain with ptxD expressed

485

Fig. 4 Intracellular ATP/ADP concentrations and the ratio of ATP to ADP in the

486

control and the recombinant S. cerevisiae

487

PBYSM-0: control strain; PBYSM-1: PBYSM-0 strain with SAM2 overexpressed;

488

ABYSM-1: PBYSM-1 strain with noxE expressed; ABYSM-2: PBYSM-1 strain with

489

vhb expressed; ABYSM-3: PBYSM-1strain with ptxD expressed

490

Fig. 5 The metabolic analysis. (A) Clustering result shown as heatmap; (B) Overview

491

of over representation analysis plot for metabolomic data, The P-value indicates the

492

probability of observing at least a particular number of metabolites from a certain

493

metabolite set in a given compound list.

494

PBYSM-1: control strain with SAM2 overexpressed; ABYSM-1: PBYSM-1 strain 22


Page 22 of 34

Page 23 of 34


495

with noxE expressed; ABYSM-2: PBYSM-1 strain with vhb expressed

496

Fig. 6 Relative transcriptional levels of key genes in the central carbon metabolic

497

pathways

498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 23



Page 24 of 34

517

Table 1 Strains and plasmids in this study

518

strains

description

source

E. coli Tans 10

cloning host

Beijing TransGen Biotech Company

BY4741

MATa his3∆1 leu2∆0 met15∆0 ura3∆0

10

BY4741-MH

BY4741 his3::HIS3, met15::MET15

this study

PBYSM-0

BY4741-MH/pRS425/YCplac33

this study

PBYSM-1

BY4741-MH/pRS425-SAM2/YCplac33

this study

ABYSM-1

BY4741-MH/pRS425-SAM2/YCplac33-noxE

this study

ABYSM-2

BY4741-MH/pRS425-SAM2/YCplac33-vhb

this study

ABYSM-3

BY4741-MH/pRS425-SAM2/YCplac33-ptxD

this study

URA3，CEN/ARS origin

42

Plamids YCplac33 pETDuet-SAM2-doc

Amp ，harboring SAM2 gene

10

pUC19-PTEF1-TPGI1

harboring PTEF1 promoter and TPGI1 terminator

43

pRS424-PHXT7-mcherry

harboring PHXT7 promoter and THXT7 terminator

43

R

-THXT7 pUC57-noxE

AmpR，synthetic gene noxE, optimized in S. cerevisiae codon

Institute

pRS425-SAM2

PTEF1-SAM2-TPGI1

this study

YCplac33-noxE

PHXT7-noxE-THXT7

this study

YCplac33-vhb

PHXT7-vhb-THXT7

this study

YCplac33-ptxD

PHXT7-ptxD-THXT7

this study

519 520 521 522 523 524 525 526

24


The Beijing Genomics

Page 25 of 34


527

Table 2 Primers used for PCR

528 constructs

oligo name

PTEF1(SAM2)

Pst I-PTEF1-F

oligonucleotides(5’-3) AACTGCAGAACCAATGCATTGGATAGCTTCAAA ATGTTTCTACTC

PTEF1(SAM2)-overlap-R

TCTTGGACATTTTGTAATTAAAACTTAGATTAGA TTGC

SAM2

(PTEF1)SAM2-overlap-F

TTAATTACAAAATGTCCAAGAGCAAAACTTTC

SAM2(TPGI1)-overlap-R

GCGATTTGTTTTAAAATTCCAATTTCTTTGGTTTT TC

TPGI1

(SAM2)TPGI1-overlap-F

GGAATTTTAAAACAAATCGCTCTTAAATATATA CC

Pst I-TPGI1-overlap-R

AACTGCAGAACCAATGCATTGGGGTATACTGGA GGCTTCATG

(YCplac33)-Sac PHXT7(vhb)

GGATCCCCGGGTACCGAGCTACCGAGCTCACTTC

I-PHXT7-F

TCGT

PHXT7-(vhb)-R

GGTCTAACATTTTTTGATTAAAATTAAAAAAACTT TTTGTTTTTGTG

vhb THXT7

(PHXT7)-vhb-F

TTAATCAAAAAATGTTAGACCAGCAAACC

vhb-(THXT7)-R

TGTTCGCAAATTATTCAACCGCTTGAGC

THXT7-(vhb)-F

GGTTGAATAATTTGCGAACACTTTTATTAATTCAT

Spe I-THXT7(YCplac33)-R

TATTAGGACTTCCACACCAAGGACTAGTATAACT

G GACTCATTAG PHXT7(ptxD)

PHXT7-(ptxD)-R

TAGGCAGCATTTTTTGATTAAAATTAAAAAAACT TTTTGTTTTTGTG

(PHXT7)-ptxD-F ptxD

TTAATCAAAAAATGCTGCCTAAACTGGTCATC

ptxD-(THXT7)-R

TGTTCGCAAATTAGCACGCCGCAGGTTC

THXT7-(ptxD)-F

GGCGTGCTAATTTGCGAACACTTTTATTAATTCAT G

PHXT7(noxE)

PHXT7-(noxE)-R

GATCTTCATTTTTTGATTAAAATTAAAAAAACTTT TTGTTTTTGTG

noxE

(PHXT7)-noxE-F

TTAATCAAAAAATGAAGATCGTTGTCATCG

noxE-(THXT7)-R

GTTCGCAAATCACTTGGCGTTCAAAGC

THXT7-(noxE)-F

CCAAGTGATTTGCGAACACTTTTATTAATTCATG

529 530 531 532 533 534 535 25



536 537 538

Table 3 Primers used for qPCR oligo name

oligonucleotides(5’-3)

0-act1-F

TGACCAAACTACTTACAACTCC

0-act1-R

AGAAGCCAAGATAGAACCA

1-hxk1-F

CAAAGATGGACCAAGGG

1-hxk1-R

CGCTACAATTTCAATAGGC

2-tdh1-F

TGAAGGTCCAATGAAGGGTGT

2-tdh1-R

CTCTGGCGGAGTAACCGTATT

3-pyk2-F

GAAGTATCGGATGTGGGTAA

3-pyk2-R

AGTGGAAGTAGGTTTGGGAG

4-mdh1-F

ATTAGAGCCGCCAGATTCAT

4-mdh1-R

CGACTTCGTCACCACCAAAC

5-cit1-F

GGTATTAGATTTAGGGGTCG

5-cit1R

TAACTTGAGCGTCAGTAGGT

6-idh1-F

AAGAAGTATGGCGGTCGTT

6-idh1-R

TGTTTGGTCAGCAGGAGTG

7-tdh3-F

TTTTGGGTTACACCGAAGACG

7-tdh3-R

ATTGGATACCAGCGGAAGCA

8-zwf1-F

CCCTGGTCTGTCAAATGCTAC

8-zwf1-R

CACCTCGTAAGCCTCTGGAA

539 540 541 542 543 544 545 546 547 548 549 26


Page 26 of 34

Page 27 of 34


550

Fig. 1

551 552 553 554 555 556 557 558 559 560 27



561

Fig. 2

562 563 564 565 566 567 568 569 570 571 572 28


Page 28 of 34

Page 29 of 34


573

12 8

A

PBYSM-0 PBYSM-1 ABYSM-1 ABYSM-2 ABYSM-3

7 6

9 8

4

B

7

OD600

OD600


11 10

5

6 5

3

4

2

3 2

1

1 0

0 0

10

20

30

40

0

50

10

20

30


C

15

20

Glucose concentration(g/L)

Glucose concentration(g/L)

20

40

50

Time(h)

Time(h)

10

5


D

15

10

0

5

0 0

10

20

30

0

10

20

Time(h)

30

Time(h)

60

60

E


50 45 40


55 50 45

SAM titer(mg/L)

55

SAM titer(mg/L)

574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615

Fig. 3

35 30 25

40

F

35 30 25 20

20

15

15

10

10

5 0

5 16

18

20

22

24

26

28

16

18

20

22

Time(h)

Time(h)

29


24

26

28


Page 30 of 34

616

1.4 1.2

0.30

1.0

0.25 0.8 0.20 0.6 0.15 0.4

0.10

0.2

0.05 0.00

0.0

PBYSM-0

ATP/ADP concentration(µg/mL/OD)

ATP ADP

0.35

0.40

24h 0.35

1.4

ATP ADP

28h 1.2

0.30

1.0

0.25 0.8 0.20 0.6 0.15 0.4

0.10

0.2

0.05 0.00

PBYSM-1 ABYSM-1 ABYSM-2 ABYSM-3 Strains

0.0

PBYSM-0

PBYSM-1 ABYSM-1 ABYSM-2 ABYSM-3 Strains

634 635 636 637 638 639 640 641 642 643 644 645 646 30


Ratio of ATP to ADP

0.40

Ratio of ATP to ADP

618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633

Fig. 4

ATP/ADP concentration(µg/mL/OD)

617

Page 31 of 34


647

Fig. 5

648 649 1：PBYSM-1；2：ABYSM-2；3：ABYSM-3 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664

A 665 666 667

31



668 669 670 671 672 673 674 675 676 677 678 32


Page 32 of 34

Page 33 of 34


679

Fig. 6

680

681 682 683 684 685 686 687 688 33



689 690 691

graphical abstract

692 693 694 695 696 697 698 699

34


Page 34 of 34

Saccharomyces cerevisiae

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