Systematic Characterization of the Metabolism of Acetoin and Its

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Food and Beverage Chemistry/Biochemistry

Systematic Characterization of the Metabolism of Acetoin and Its Derivative Ligustrazine in Bacillus subtilis under Micro-Oxygen Conditions Youqiang Xu, Yuefeng Jiang, Xiuting Li, Baoguo Sun, Chao Teng, Ran Yang, ke xiong, Guangsen Fan, and Wenhua Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00113 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Journal of Agricultural and Food Chemistry

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Systematic Characterization of the Metabolism of Acetoin and Its

2

Derivative Ligustrazine in Bacillus subtilis under Micro-Oxygen

3

Conditions

4 5

Youqiang Xu1,2, Yuefeng Jiang1,2, Xiuting Li1,2,3,*, Baoguo Sun1,4, Chao Teng2,4, Ran

6

Yang2,4, Ke Xiong1,2, Guangsen Fan1,3, Wenhua Wang1,2

7 8

1

9

Technology and Business University, Beijing 100048, China.

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

10

2

11

Technology and Business University, Beijing 100048, China.

12

3

13

University, Beijing 100048, China.

14

4

15

University, Beijing 100048, China.

Beijing Engineering and Technology Research Center of Food Additives, Beijing

School of Food and Chemical Engineering, Beijing Technology and Business

Beijing Key Laboratory of Flavor Chemistry, Beijing Technology and Business

16 17

* Correspondence to: X. Li, School of Food and Chemical Engineering, Beijing

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Technology and Business University. No. 11, Fucheng Road, Haidian District, Beijing

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100048, China, E-mail: [email protected] (Li Xiuting)

20 21

ACS Paragon Plus Environment


Bacillus subtilis is an important microorganism for brewing of

22

ABSTRACT:

23

Chinese Baijiu, which contributes to the formation of flavour chemicals including

24

acetoin and its derivative ligustrazine. The first stage of Baijiu brewing process is

25

under the micro-oxygen condition; however, there are few studies about B. subtilis

26

metabolism under this condition. Effects of various factors on acetoin and ligustrazine

27

metabolism were investigated under this condition including key genes and

28

fermentation conditions. Mutation of bdhA (encoding acetoin reductase) or

29

overexpression of glcU (encoding glucose uptake protein) increased acetoin

30

concentration. Addition of Vigna angularis powder to the culture medium also

31

promoted acetoin production. The optimal culture conditions for ligustrazine synthesis

32

were pH 6.0 and 42°C. Ammonium phosphate was shown to promote ligustrazine

33

synthesis in situ. This is the first report of acetoin and ligustrazine metabolism in B.

34

subtilis under micro-oxygen conditions, which will ultimately promote the application

35

of B. subtilis for maintaining Baijiu quality.

36

KEYWORDS:

37

Chinese Baijiu

Bacillus subtilis, acetoin, ligustrazine, micro-oxygen conditions,

38


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INTRODUCTION

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Chinese Baijiu (also named Chinese liquor or Chinese spirit) is one kind of the oldest

41

distillates in the world, and is renowned overseas.1 Numerous microorganisms exist in

42

the Baijiu brewing process, and their propagations and metabolisms greatly affect the

43

quality of Baijiu.2 B. subtilis is one of the most dominant microorganisms found

44

during the brewing process of Chinese Baijiu.3,4 The Baijiu brewing process is

45

relatively special with constantly changing parameters, such as pH, moisture,

46

dissolved oxygen, temperature and microbial flora,5 thus making it challenging for the

47

adapting and propagating representative mixed cultures. B. subtilis demonstrates good

48

resistance to extreme environments,6 and thus, exists in the brewing process of

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Chinese Baijiu. In addition, B. subtilis can produce protease, amylase and lipase,7,8

50

which play important roles for the hydrolysis of proteins, starches and lipids in the

51

raw materials used to produce Chinese Baijiu.7 B. subtilis can also be used to control

52

the formation of off-odor chemicals such as geosmin in Chinese Baijius.9 The

53

chemicals produced by carbon metabolism of B. subtilis significantly contribute to the

54

formation of flavour compounds during Baijiu brewing process.10 Therefore, B.

55

subtilis was recognized as an important bacteria for the brewing of Chinese

56

Baijiu.3,4,6-11 Among all the flavour chemicals, acetoin and ligustrazine produced by B.

57

subtilis have important impact on the flavour and quality of Chinese Baijiu.10,12

58

Acetoin (also named 3-hydroxy-2-butanone or acetyl methyl carbinol, AC) is a

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popular food flavour additive.13 It is generally recognized as safe (GRAS) by the

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Flavor and Extract Manufactures Association of the United States (FEMA) with No.



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2008, and widely used to enhance the flavour of food products.13 AC is an important

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substance in Baijiu industry, by its significance in the fruity aroma of the liquor. 14 Its

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derivate ligustrazine (also named 2,3,5,6-tetramethylpyrazine, TMP) is a food flavour

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additive with FEMA No. 3237.15 TMP is found in a variety of fermented foods such

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as cheese,16 whiskey,17 vinegar18 and also Chinese Baijiu.12 TMP is recognized as a

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health factor in Chinese Baijiu, since studies find that this monomer can be used as a

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promising remedy for cardiovascular diseases.12,19 The possible molecular

68

pharmacological

69

proliferation and migration of vascular smooth muscle cell, regulating inflammation

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and apoptosis, and preventing aggregation of platelet.19 The pharmacological value of

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TMP endows the alcoholic beverage with certain health promoting factors. Some

72

studies have concluded that moderate drinking of alcoholic drinks could reduce the

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mortality of patients with cardiovascular diseases.20,21

mechanisms

include

modulating

ion

channels,

inhibiting

74

Due to the flavour and health promoting factors, AC and TMP metabolism have

75

drawn much attention. TMP is one of the derivatives of AC produced by condensing

76

AC with NH4+.13,22 Although studies find that many species are capable of producing

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AC and TMP, B. subtilis has been confirmed as the key strain for the synthesis of AC

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and TMP during the Chinese Baijiu brewing process.11,23 For the brewing of Chinese

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Baijiu, the mixed grains are fermented in the “mud pit” covered by the “cellar mud”

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for 2 months or more.25 During the first two weeks, the aerobic and facultative

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anaerobic microorganisms grow rapidly, including fungi and bacteria such as Bacillus

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species.2 At this stage, the limited oxygen in the “mud pit” is gradually consumed,


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which can be recognized as a micro-oxygen environment in the “mud pit”.2,26 This

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growth stage is crucial for Baijiu brewing, since with microbial growth, a series of

85

enzymes are produced that hydrolyze starches, proteins or lipids of the grains into

86

small molecules, which then greatly influence the following microbial growth and

87

metabolism. Oxygen is a very important factor affecting the growth and metabolism

88

of microbes, including B. subtilis. However, most studies on B. subtilis metabolism

89

have been carried out under aerobic or anaerobic conditions,24 there has been no

90

detailed investigation of B. subtilis metabolism under micro-oxygen conditions until

91

now.

92

The flavour substances in the liquors determine the taste and quality of Chinese

93

Baijiu.27 However, the formation of these substances are not clear by microorganisms

94

during the brewing process, and the flavour and quality of Chinese Baijius are

95

unstable

96

characteristics of microorganisms is an effective way to scientifically study the

97

formation of various compounds in Baijiu, and ultimately allow the process control

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for maintaining a high consistent quality in Chinese Baijiu.11 Considering the

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importance of AC and TMP in maintaining the flavour and high quality of Chinese

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Baijiu and the micro-oxygen conditions on Baijiu brewing, this study provided

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detailed insights into the effects of key genes on the metabolism of AC and TMP and

102

also the culture conditions for B. subtilis under micro-oxygen conditions (i.e., glucose

103

concentration, nitrogen source and concentrations, initial pH and temperature).

among

batches.11

A comprehensive

understanding

104


the

metabolic


105 106

MATERIALS AND METHODS Bacterial Strains, Media and Chemical Reagents.

Strains used in this study are

107

listed in Table 1. E. coli DH5α was used for vector propagation and purchased from

108

TIANGEN Biotech. Co., Ltd. (Beijing, China). Bacillus subtilis 16828 was purchased

109

from American type culture collection (ATCC) with No. ATCC 23857, and was used

110

as the target strain for investigating AC and TMP metabolism. The genomic DNA of B.

111

subtilis 168 was extracted and used as the template for amplification of AC synthetic

112

gene cluster alsSD through polymerase chain reaction (PCR). The FastDigest

113

restriction enzymes were obtained from Takara (Dalian, China). T4 DNA ligase and

114

Q5 DNA polymerase were purchased from New England Biolabs (Beijing, China).

115

Ampicillin, erythromycin and tetracycline were purchased from Amresco (Solon, OH,

116

USA). AC (99.0%) and TMP (98.0%) standards were obtained from Sigma-Aldrich

117

(St Louis, MO, USA). All other chemicals were analytical-grade reagents and

118

commercially available. LB broth (5.0 g/L yeast extract, 10.0 g/L tryptone and 10.0

119

g/L NaCl) was used as the culture medium during the construction of the recombinant

120

strains. LB broth with glucose added was used as the culture medium for fermentation

121

studies. NB/glucose medium (8 g/L nutrient broth supplemented with 50 mM glucose)

122

was used for cell culture during gene mutation. The antibiotic was added into the

123

culture media for cultivating the recombinant E. coli or B. subtilis to a final

124

concentration of 15 µg/mL for tetracycline, 100 µg/mL for ampicillin, and 5 µg/mL

125

for erythromycin, respectively.

126


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{Please insert Table 1 about here}

128 129

DNA Manipulation and Vector Construction.

The primer pair PpHY.f(XhoI)/

130

PpHY.r(BglII) was used to amplify the vector pHY300PLK. The primers P43.f(XhoI),

131

P43r.2, P43f.3, P43r.4, P43f.5, P43r.6, P43f.7, P43r.8, P43f.9 and P43.r were designed

132

according to the sequence of promoter P43,29 and ligated together by gene splicing

133

through overlap extension. Thereafter the promoter was confirmed by gene

134

sequencing. The promoter P43 was further amplified using the primer pair

135

P43.f(XhoI)/P43.r, and spliced together with the primer pair Bs.f(OV)/Bs.r(BglII)

136

amplified AC synthetic gene cluster alsSD including genes alsS and alsD from B.

137

subtilis 168. The DNA fragment P43-alsSD was treated with the restriction enzymes

138

XhoI and BglII, and ligated with the same restriction enzymes treated pHY300PLK to

139

generate the vector designated pHYP43-alsSD. The promoter P43 was further

140

amplified by the primer pair P43.f(XhoI)/ P43-glcU.r, and spliced with the primer pair

141

PglcU.f(OV)/PglcU.r(BglII) amplified glucose uptake encoding gene glcU using the

142

genomic DNA of B. subtilis 168 as the template, and generated the DNA fragment

143

P43-glcU. The DNA fragments P43-pfkA and P43-pyK were produced using the same

144

protocol. Gene pfkA encoded 6-phosphofructokinase and gene pyK encoded pyruvate

145

kinase. The DNA fragments P43-glcU, P43-pfkA and P43-pyK were treated with the

146

restriction enzymes XhoI and BglII, and ligated with the same restriction enzymes

147

treated pHY300PLK to generate the vectors designated pHYP43-glcU, pHYP43-pfkA

148

and pHYP43-pyK, respectively. The homologous repair template flanking the



149

designated deletion region was obtained through overlap extension PCR of the DNA

150

fragments upstream and downstream of the target genomic DNA locus. The resulting

151

DNA fragment ∆bdhA or ∆acoABC was treated with the restriction enzymes BglII and

152

SalI, and inserted into the BglII and SalI sites of pKVM1 to generate the vector

153

pKVM-∆bdhA or pKVM-∆acoABC, respectively. All the sequences of the primers

154

were listed in Table 2. The above expression vectors were transferred into B. subtilis

155

168 to produce the respective recombinant strains as listed in Table 1. The gene

156

mutation vector pKVM-∆bdhA or pKVM-∆acoABC was first constructed and

157

transferred into E. coli S17-1 λpair.30 All the PCR conditions for synthesis of

158

promoter P43, gene cloning and splicing could be seen in the supporting information.

159


160 161 162

Gene Mutation.

The gene mutation vector pKVM-∆bdhA or pKVM-∆acoABC

163

was extracted from E. coli S17-1 λpair and transferred into B. subtilis 168 by

164

electroporation. The electroporation condition was as follows: 2000 V, 25 µF and 200

165

Ω using a 0.1 cm cuvette. A three-step gene mutation procedure was carried out

166

according to the previous study.31 B. subtilis 168 with the vector pKVM-∆bdhA or

167

pKVM-∆acoABC was first plated onto NB/glucose medium with erythromycin (5

168

µg/mL) and X-gal for 12 – 24 hours. The blue colonies were inoculated into

169

NB/glucose medium with erythromycin (5 µg/mL) and incubated at 30°C for vector

170

replication with the temperature sensitive origin for 12 hours. Thereafter, the culture


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was serial diluted and plated onto NB/glucose agar added with erythromycin (5

172

µg/mL) and X-gal and incubated at 42°C for 12 – 24 hours. The temperature sensitive

173

origin prevented vector replication, thus vector integration occurred by homologous

174

recombination within one of the regions flanking the deletion target site, which could

175

be detected due to the β-galactosidase gene on pKVM1. The blue colonies were

176

further inoculated into NB/glucose medium without an antibiotic and cultivated at

177

30°C for 3 – 4 passages. At this temperature, the replication origin of the integrated

178

pKVM derivative was functional, which resulted in counter selection against the

179

integrated vector. White colonies were selected, which had lost the β-galactosidase

180

gene along with the integrated vector due to the second recombination. Some of the

181

white colonies were wild type strains and others were mutant ones. The white

182

colonies were subjected to further analysis by colony PCR to select the target mutants.

183

The colony PCR conditions could be seen in the supporting information.

184

AC and TMP Metabolism in B. subtilis under Different Fermentation LB broth supplemented with glucose and adjusted to pH 7.0 before

185

Conditions.

186

sterilization was used as the culture medium for B. subtilis under aerobic and

187

micro-oxygen conditions, respectively. Glucose solution (600 g/L) was sterilized

188

separately and added to the medium to a final concentration of about 60 g/L. For

189

aerobic fermentation, the seed culture was inoculated (1%, v/v) into fresh medium

190

using 500 mL flasks containing 100 mL of medium and shaken at 200 r/min and 37°C

191

for 36 h. For micro-oxygen fermentations, the seed culture was inoculated (1%, v/v)

192

into fresh medium with 250 mL flasks containing 200 mL medium and cultivated at



193

37°C for 6 d under static conditions. The final glucose concentration was adjusted to

194

20, 40, 60, 80, 100 and 120 g/L to verify its effect on B. subtilis metabolism of AC

195

and TMP. When investigating nitrogen sources on B. subtilis metabolism, different

196

bean powders [soya bean (Glycine max L.), peas (Pisum sativum L.), black soya bean

197

(Dumasia truncata), small red bean (Vigna angularis) and mung bean (Vigna radiata

198

L.)] were added to different flasks containing the modified LB broth to give a final

199

concentration of 20 g/L. Beans were used in this study because they had served as

200

substrates for brewing of Chinese Baijiu.2 In addition, beans are rich in proteins which

201

have been shown to promote the formation of free ammonia during microbial protein

202

degradation, and therefore might contribute to the production of TMP. Since previous

203

work found (NH4)2HPO4 a preferred ammonium salt for TMP production.32 It was

204

used in this study as a model nitrogen source using the same concentration and

205

medium as above for different bean substrates. Thereafter, the preferred bean powder

206

was added into the culture broth at different concentrations to verify its effect on AC

207

and TMP metabolism in B. subtilis under the micro-oxygen conditions at pH 7.0 and

208

37°C. For pH investigation, the pH of medium was adjusted before sterilization to 4.0,

209

5.0, 6.0, 7.0 and 8.0, respectively. For temperature analysis, the inoculated media

210

were kept at 20°C, 25°C, 30°C, 34°C, 37°C, 42°C and 45°C, respectively with the

211

medium pH adjusted to 7.0. In addition, a long-term study of AC and TMP

212

metabolism was carried out under micro-oxygen conditions.

213 214

Analytical Methods.

Cell density was measured by recording the optical density

at 600 nm after an appropriate dilution using a spectrophotometer (PERSEE TU-1901,


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Jiangsu, China). The culture medium was centrifuged at 13,000 × g for 10 min, and

216

the supernatant was used for further analysis. Glucose concentration was quantified

217

by a bio-analyzer (SBA-40D, Shandong Academy of Sciences, China). The

218

concentrations of AC, TMP and 2,3-butanediol were analyzed simultaneously by gas

219

chromatography (GC) equipped with an Agilent DB-WAX-123-7032 column (30 m ×

220

0.32 mm × 0.25 µm). After centrifugation, the supernatant of culture suspension was

221

extracted by equal volume of ethyl acetate, and filtered through a 0.22 µm filter

222

before injection. Isoamyl alcohol was used as the internal standard. Quantitative

223

analysis was carried out using GC with an oven temperature program of 40°C for 3

224

min, and gradiently increased to 240°C with a rate of 15°C/min and maintained at

225

240°C for 1 min. The carrier gas was nitrogen with a flow-rate of 1.0 mL/min. The

226

instrument was equipped with an autosampler. The injection volume was 1.0 µL with

227

a split ratio of 1:5. Chemicals were quantified by the external standard method. AC,

228

TMP or 2,3-butanediol standard solution was serial diluted by 50% for five rounds

229

and samples from each round were measured in triplicate to generate the standard

230

curve. The R value of the standard curve was above 0.9999 for each chemical (data

231

not shown). The concentrations of byproducts were detected by high performance

232

liquid chromatography (HPLC) as described in the previous report.32 The HPLC

233

analysis was carried out with a Bio-Rad Aminex HPX-87H column (300 × 7.8 mm)

234

using a refractive index detector. A mobile phase of 5 mM H2SO4 solution was used at

235

55°C with a flow rate of 0.5 mL/min. The injection volume was 10 µL.

236



237 238

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RESULTS AND DISCUSSION Effects of bdhA and acoABC Mutation and alsSD Expression on B. subtilis AC is a flavour chemical widely used in food industry, and serves also

239

Metabolism.

240

as a platform chemical for the production of many other important compounds.13

241

Microbial AC metabolism has drawn considerable attention in recent years. Its direct

242

substrate is pyruvate, which originates from glycolysis, the standard metabolic

243

pathway used by most microorganisms. There are three rate-limiting enzymes in this

244

pathway, hexokinase (HK, EC 2.7.1.1), 6-phosphofructokinase (PFKA, EC 2.7.1.11)

245

and pyruvate kinase (PYK, EC 2.7.1.40) (Figure 1). HK catalyzes the irreversible

246

phosphorylation

247

fructose-6-phosphate to fructose-1,6-diphosphate; and PYK catalyzes the formation of

248

pyruvate from phosphoenolpyruvate. The AC synthetic gene cluster includes two

249

genes encoding α-acetolactate synthetase (AlsS) and α-acetolactate decarboxylase

250

(AlsD), respectively.22 During AC production from glucose, several byproducts are

251

formed such as 2,3-butanediol, succinate, lactate and acetate, and the main byproduct

252

is 2,3-butanediol, produced by acetoin reductase (encoded by gene bdhA) on its

253

substrate, acetoin22 (Figure 1). When the carbon source such as glucose is completely

254

consumed, AC degradation is activated and catalyzed by the acetoin dehydrogenase

255

enzyme system (AoDH ES),33 which consists of acetoin dehydrogenase E1

256

component (TPP-dependent alpha subunit) encoded by gene acoA, acetoin

257

dehydrogenase E1 component (TPP-dependent beta subunit) encoded by gene acoB,

258

acetoin dehydrogenase E2 component (dihydrolipoamide acetyltransferase) encoded

of

glucose;

PFKA

catalyzes


the

phosphorylation

of

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259

by gene acoC, and acetoin dehydrogenase E3 component (dihydrolipoamide

260

dehydrogenase) encoded by gene acoL in B. subtilis.

261 262

{Please insert Figures 1 and 2 about here}

263 264

Gene bdhA was first knocked out and generated the strain B. subtilis 1 (Figure 2A).

265

Mutation of gene bdhA inhibited the production of 2,3-butanediol, with a

266

concentration of only 27.9% compared with that of B. subtilis 168 (Figure 2F). On the

267

contrary, AC concentration increased from 0.84 g/L to 2.41 g/L (Figure 2E). With a

268

further deletion of gene cluster acoABC (B. subtilis 2) (Figure 2B), no obvious

269

metabolic divergence was observed between strains B. subtilis 1 and B. subtilis 2. The

270

culture broth was analyzed and showed it contained glucose. Results from a previous

271

study suggested that after the exhaustion of carbon in the culture media, the

272

transcription and translation of gene cluster acoABC would begin and encode the

273

enzymes that catalyzed AC breakdown.33 Due to the presence of glucose in the culture

274

media, the effect of acoABC mutation was not significant for AC metabolism in B.

275

subtilis (Figure 2E). Thereafter, gene cluster alsSD was cloned and overexpressed

276

using promoter P43 (Figure 2C and 2D) but AC production only slightly increased

277

with strain B. subtilis 4 compared with B. subtilis 2 and B. subtilis 3 (B. subtilis

278

168∆bdhA∆acoABC harboring pHY300PLK) (Figure 2E). The main reason for this

279

observation might be due to poor glycolysis efficiency of the B. subtilis strains under

280

the micro-oxygen conditions, as only 7.5 g/L to 9.5 g/L glucose was consumed within



281

6 d (Figure 2E). Due to the low rate of glucose consumption, the concentration of

282

pyruvate was maintained at a low level and AC production was not significant among

283

the engineered B. subtilis strains.

284

Effects of Glycolysis Pathway Gene Over-Expression on B. subtilis Metabolism.

285

The key genes affecting glycolysis included hK (encoding hexokinase), pfkA

286

(encoding 6-phosphofructokinase) and pyK (encoding pyruvate kinase). Unfortunately,

287

the gene encoding hexanoate kinase was still not annotated in B. subtilis strains.

288

Studies found that overexpression of genes pfkA or pyK could increase glycolysis

289

efficiency in Clostridium acetobutylicum and Corynebacterium glutamicum.34,35

290

Therefore, genes pfkA and pyK were overexpressed in this study, respectively (Figure

291

3A and 3B), and the respective effects were verified on B. subtilis metabolism. In

292

addition, glucose utilization rate might not only be affected by the enzymes during

293

glycolysis. Glucose transfer rate was also a crucial factor influencing glycolysis in B.

294

subtilis, but no study focused on this so far. The effects of glucose transfer rate on B.

295

subtilis metabolism were examined here by overexpressing another gene, glcU

296

(encoding glucose uptake protein) (Figure 3C). The overexpression of genes pfkA and

297

pyK had almost no positive effects on AC production (Figure 3D), which could

298

similarly be due to the low efficiency of glucose consumption. On the contrary, the

299

overexpression of glcU increased glucose utilization rate by 26.0% (Figure 3E), and

300

the concomitant concentration of AC increased by 23.8% compared with that of B.

301

subtilis 2 (Figures 2E and 3D), this indicated that the glucose uptake rate was

302

important for glycolysis and AC metabolism in B. subtilis under the micro-oxygen


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303

conditions.

304 305

{Please insert Figure 3 about here}

306 307

The above strains were also cultivated under aerobic conditions using the identical

308

culture media as used with micro-oxygen conditions, in order to better understand the

309

metabolic characteristics of B. subtilis (Table 3). Under aerobic conditions, the

310

glucose uptake and AC production rates in Bacillus strains greatly increased

311

compared to when cultivated under micro-oxygen conditions. Glucose uptake rate

312

increased by 11.93 folds, and AC productivity enhanced by 27.38 folds. This shows

313

the importance of oxygen on B. subtilis metabolism. The main metabolic byproducts

314

were 2,3-butanediol, lactate and acetate. No succinate was detected during aerobic

315

fermentation. For B. subtilis 168, 2,3-butanediol accounted for 89.3% (g/g) of the

316

total byproducts, while B. subtilis 1 produced 2,3-butanediol with a concentration of

317

0.51 ± 0.07 g/L (18.6% of that of B. subtilis 168). AC concentration increased with B.

318

subtilis 1 to 8.53 ± 0.04 g/L, which was an additional 43.0% more than that of B.

319

subtilis 168 (5.96 ± 0.18 g/L). The overexpression of gene cluster alsSD, genes pfkA

320

and pyK each could enhance AC production, which was consistent with previous

321

studies.34–36 In addition, overexpression of gene glcU also showed a positive effect on

322

glucose consumption (Table 3), which indicated that glucose uptake rate was a key

323

factor for glucose utilization in B. subtilis. To our knowledge, this was the first report

324

that overexpression of gene glcU promoted glucose consumption and AC metabolism



325

in B. subtilis under either aerobic or micro-oxygen conditions.

326 327


328 329

B. subtilis was generally recognized as safe, and a common host strain for the

330

production of many enzymes.37 However, there were not many studies on using B.

331

subtilis for production of chemicals. One of the reasons was the inefficiency of

332

glycolysis in B. subtilis.24 The results of this study suggested that overexpression of

333

gene glcU helped to increase the efficiency of glycolysis and this might lead to further

334

improving glycolysis efficiency through a systematic metabolic engineering strategy.

335 336


337 338

Glucose metabolic flux distribution was investigated with the B. subtilis strains

339

under both aerobic and micro-oxygen conditions to interpret and examine the

340

metabolic behaviors (Table 4). For the micro-oxygen conditions, AC yields decreased

341

with every B. subtilis strain tested. One of the reason was the poor efficiency of

342

glycolysis (Figures 2 and 3), and another reason would be the in vivo cofactor

343

imbalance.32 AC production from pyruvate did not consume NADH, but glycolysis

344

generated 2 mols of NADH during consumption of 1 mol glucose. The production of

345

lactate, acetate and succinate coupled with the conversion of NADH to NAD+ could

346

help to rebalance in vivo cofactors. On the contrary, under aerobic fermentation


Page 16 of 37

Page 17 of 37


347

conditions, the oxygen level was enough to spontaneously react with NADH to form

348

NAD+. This would be consistent with the observation that low concentrations of

349

byproducts 2,3-butanediol, lactate and acetate and no succinate were produced under

350

the aerobic conditions.

351

Effects of Different Fermentation Conditions on B. subtilis Metabolism.

B.

352

subtilis 2 (B. subtilis ∆bdhA∆acoABC), a markerless deletion mutant, was used in the

353

following study. It was known that the composition of the culture medium and

354

fermentation conditions had significant influence on metabolism in microorganisms.

355

Glucose concentration was examined and found to have a slight effect on AC

356

metabolism in B. subtilis. The source of nitrogen showed an obvious influence on AC

357

production, especially in the case of where the presence of small red bean (Vigna

358

angularis) into culture media increased AC production by 108% (Figure 4B).

359

Thereafter, the influence of the amount of small red bean was determined in AC

360

fermentation. Figure 4C indicated that the optimal dosage was 40 g/L, and AC

361

concentration was 5.93 ± 0.34 g/L after cultivation for 6 d under the micro-oxygen

362

conditions. No TMP was produced during the above cultivation, but TMP was

363

detected in the fermentation broth after addition of (NH4)2HPO4 (Figure 4B). These

364

results indicated that B. subtilis metabolism could not produce enough NH4+, which

365

was a precursor for production of TMP. They also support the observation that B.

366

subtilis inoculated into fermenting grains does not, by itself, significantly promote the

367

accumulation of TMP in the Baijiu brewing process. Isolation or breeding of a strain

368

with efficient amino acid metabolic capability and co-culture with B. subtilis would



369

help to produce TMP in situ during Baijiu brewing process, since amino acid

370

metabolism could produce free ammonia, and was the main origin for ammonia

371

formation during Baijiu brewing.23

372 373

{Please insert Figures 4 and 5 about here}

374 375

The initial pH of culture media and temperature were investigated thereafter, and

376

Figure 5 illustrated that the optimal pH was 6.0 (Figure 5A), and 42°C was most

377

suitable for AC accumulation (Figure 5B). A long-term cultivation was carried out

378

using B. subtilis 2 under the optimized culture conditions to further verify B. subtilis

379

metabolic characteristics (Figure 6). AC reached a concentration of 9.44 ± 0.56 g/L

380

after 17 d, but no TMP was produced (Figure 6A). (NH4)2HPO4 could promote the

381

synthesis of TMP in situ, and when added 2.37 ± 0.20 g/L TMP was achieved,

382

coupled with 4.96 ± 0.58 g/L AC within 15 d (Figure 6B). However, the addition of

383

(NH4)2HPO4 to culture media delayed the growth of B. subtilis, which was in

384

accordance with previous studies.15,38

385 386

{Please insert Figure 6 about here}

387 388

In this study, AC and TMP metabolism were investigated using B. subtilis under the

389

various conditions of aeration, composition of culture media and fermentation

390

conditions. Mutation of gene bdhA or overexpression of gene glcU contributed to


Page 18 of 37

Page 19 of 37


391

accumulation of AC. Small red bean was found to promote AC synthesis when added

392

to culture broth. The optimum pH and temperature of the fermentation were

393

determined to be pH 6.0 and 42°C, respectively, for AC production under

394

micro-oxygen conditions. No TMP was detected during cultivation without the

395

addition of (NH4)2HPO4 into the culture media, indicating that B. subtilis metabolism

396

could not produce enough NH4+, which was a required precursor for synthesis of TMP.

397

TMP was produced at a concentration of 2.37 ± 0.20 g/L after 15 d under the optimal

398

fermentation conditions with added (NH4)2HPO4 using B. subtilis 2. This study

399

further contributed to further understanding the synthesis of other flavour chemicals

400

during the production of Chinese Baijius and improvements to process control.

401

Nucleotide Sequence Accession Numbers: bdhA (BSU06240), acoA (BSU08060),

402

acoB (BSU08070), acoC (BSU08080), alsS (BSU36010), alsD (BSU36000), pfkA

403

(BSU29190), pyK (BSU29180), glcU (BSU03920).

404 405

AUTHOR INFORMATION

406

Corresponding Author

407

*Telephone: +86-10-68985342; fax: +86-10-68985342; e-mail: [email protected]

408

Funding

409

This research was supported by the National Natural Science Foundation of China

410

(No. 31671798), Beijing Postdoctoral Research Foundation (Type A), China

411

Postdoctoral Science Foundation (2016M600019), the Foundation of Beijing

412

Technology and Business University (No. LKJJ2017-10), and the Project of 2017



413

Graduate Scientific Research Ability Enhancement Program of Beijing Technology

414

and Business University.

415

Notes

416

The authors declare no competing financial interest.

417

We give our great thanks to Prof. Dr. Sharon P. Shoemaker from California Institute of

418

Food and Agricultural Research and Ph.D Yan Jiang from Tsinghua University for

419

their helps in manuscript language improvement.

420 421

Supporting Information Available: The PCR conditions for the synthesis of promoter

422

P43, gene cloning, gene splicing and colony PCR.

423 424


Page 20 of 37

Page 21 of 37


425

REFERENCES

426

(1) Wu, J.; Zheng, Y.; Sun, B.; Sun, X.; Sun, J.; Zheng, F.; Huang, M. The

427

occurrence of propyl lactate in Chinese Baijius (Chinese liquors) detected by direct

428

injection coupled with gas chromatography-mass spectrometry. Molecules 2015, 20,

429

19002–19013.

430

(2) Xu, Y.; Sun, B.; Fan, G.; Teng, C.; Xiong, K.; Zhu, Y.; Li, J.; Li, X. The brewing

431

process and microbial diversity of strong flavour Chinese spirits: a review. J. Inst.

432

Brew. 2017, 123, 5–12.

433

(3) Yan, Z.; Zheng, X. W.; Chen, J. Y.; Han, J. S.; Han, B. Z. Effect of different

434

Bacillus strains on the profile of organic acids in a liquid culture of Daqu. J. Inst.

435

Brew. 2013, 119, 78–83.

436 437

(4) Zheng, X. W.; Tabrizi, M. R.; Nout, M. J.; Han, B. Z. Daqu—a traditional Chinese liquor fermentation starter. J. Inst. Brew. 2011, 117, 82−90.

438

(5) Liu, Z. H.; Zhang, W. X.; Zhang, Q. S.; Yue, Y.; Hu, C.; Wang, Z. Y. Research

439

on the change rules of environmental factors during the fermentation in simulant pit.

440

Liquor Making Sci. Technol. 2006, 17−20. (In Chinese)

441

(6) Nicholson, W. L.; Munakata, N.; Horneck, G.; Melosh, H. J.; Setlow, P.

442

Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial

443

environments. Microbiol. Mol. Biol. Rev. 2000, 64, 548−572.

444

(7) Liang, H.; Li, W.; Luo, Q.; Liu, C.; Wu, Z.; Zhang, W. Analysis of the bacterial

445

community in aged and aging pit mud of Chinese Luzhou-flavour liquor by combined

446

PCR-DGGE and quantitative PCR assay. J. Sci. Food Agri. 2015, 95, 2729−2735.

447

(8) Ramos, C. L.; de Almeida, E. G.; Freire, A. L.; Schwan, R. F. Diversity of

448

bacteria and yeast in the naturally fermented cotton seed and rice beverage produced

449

by Brazilian Amerindians. Food Microbiol. 2011, 28, 1380−1386.

450

(9) Zhi, Y.; Wu, Q.; Du, H.; Xu, Y. Biocontrol of geosmin-producing Streptomyces

451

spp. by two Bacillus strains from Chinese liquor. Int. J. Food Microbiol. 2016, 231,

452

1−9.

453

(10) Lin, Q.; Dong, S.; Fu, Q.; Hu, C.; Pan, Q.; Zhou, G. Isolation of



454

aroma-producing Bacillus subtilis and analysis of its fermentation metabolites. Liquor

455

Making Sci. Technol. 2013, 30−32. (In Chinese)

456

(11) Xu, Y. Study on liquor-making microbes and the regulation & control of their

457

metabolism based on flavor-oriented technology. Liquor-Making Sci. Technol. 2015,

458

1−11,16. (In Chinese)

459

(12) Zhu, B. F.; Xu, Y.; Fan, W. L. High-yield fermentative preparation of

460

tetramethylpyrazine by Bacillus sp. using an endogenous precursor approach. J. Ind.

461

Microbiol. Biotechnol. 2010, 37, 179−186.

462 463 464 465

(13) Xiao, Z.; Lu, J. R. Generation of acetoin and its derivatives in foods. J. Agri. Food Chem. 2014, 62, 6487−6497. (14) Lytra, G.; Tempere, S.; Revel, G. D.; Barbe, J. C. Impact of perceptive interactions on red wine fruity aroma. J. Agri. Food Chem. 2012, 60, 12260−12269.

466

(15) Xiao, Z.; Hou, X.; Lyu, X.; Xi, L.; Zhao, J. Y. Accelerated green process of

467

tetramethylpyrazine production from glucose and diammonium phosphate. Biotechnol.

468

Biofuels 2014, 7, 106.

469 470 471 472

(16) Sloot, D.; Hofman, H. J. Alkylpyrazines in emmental cheese. J. Agri. Food Chem. 1975, 23, 358−358. (17) Boothroyd, E.; Linforth, R. S.; Jack, F.; Cook, D. J. Origins of the perceived nutty character of new-make malt whisky spirit. J. Inst. Brew. 2014, 120, 16−22.

473

(18) Liang, J.; Xie, J.; Hou, L.; Zhao, M.; Zhao, J.; Cheng, J.; Wang, S.; Sun, B. G.

474

Aroma constituents in Shanxi aged vinegar before and after aging. J. Agri. Food

475

Chem. 2016, 64, 7597−7605.

476 477

(19) Sinclair, S. Chinese herbs: a clinical review of astragalus, ligusticum, and schizandrae. Altern. Med. Rev. 1998, 3, 338−344.

478

(20) Arranz, S.; Chiva-Blanch, G.; Valderas-Martínez, P.; Medina-Remón, A.;

479

Lamuela-Raventós, R. M.; Estruch, R. Wine, beer, alcohol and polyphenols on

480

cardiovascular disease and cancer. Nutrients 2012, 4, 759−781.

481 482 483

(21) Klatsky, A. L. Moderate drinking and reduced risk of heart disease. Alcohol Res. Health. 1999, 23, 15−24. (22) Xu, Y.; Chu, H.; Gao, C.; Tao, F.; Zhou, Z.; Li, K.; Li, L.; Ma, C.; Xu, P. ACS Paragon Plus Environment

Page 22 of 37

Page 23 of 37


484

Systematic metabolic engineering of Escherichia coli for high-yield production of

485

fuel bio-chemical 2,3-butanediol. Metab. Eng. 2014, 23, 22−33.

486

(23) Xu, Y.; Wu, Q.; Fan, W. L.; Zhu, B. The discovery & verification of the

487

production pathway of tetramethylpyrazine (TTMP) in Chiese liquor. Liquor-Making

488

Sci. Technol. 2011, 37–40. (In Chinese)

489

(24) Nakano, M. M.; Dailly, Y. P.; Zuber, P.; Clark, D. P. Characterization of

490

anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end

491

products and genes required for growth. J. Bacteriol. 1997, 179, 6749−6755.

492

(25) Li, K.; Zhang, Q.; Zhong, X. T.; Jia, B. H.; Yuan, C. H.; Liu, S.; Che, Z. M.;

493

Xiang, W. L. Microbial diversity and succession in the Chinese Luzhou-flavor liquor

494

fermenting cover lees as evaluated by SSU rRNA profiles. Indian J. Microbiol. 2013,

495

53, 425–431.

496

(26) Xiang, S.; Chen, J.; Zhang, Z. The relationship between the fermentation

497

temperature curve and solid state brewing between pre control conditions. Liquor

498

Making. 2015, 42, 28–31. (In Chinese)

499 500 501 502

(27) Fan, W.; Xu, Y. The review of the research of aroma compounds in Chinese liquors. Liquor Making 2007, 34, 31−37. (In Chinese) (28) Kunst, F.; Vassarotti, A.; Danchin, A. Organization of the European Bacillus subtilis genome sequencing project. Microbiol. 1995, 389, 84–87.

503

(29) Wang, P. Z.; Doi, R. H. Overlapping promoters transcribed by Bacillus subtilis

504

sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary

505

phases. J. Biol. Chem. 1984, 259, 8619−8625.

506

(30) Simon, R.; Priefer, U.; Pühler, A. A broad host range mobilization system for

507

in vivo genetic engineering: transposon mutagenesis in gramnegative bacteria. Nat.

508

Biotechnol. 1983, 1, 784−791.

509

(31) Rachinger, M.; Bauch, M.; Strittmatter, A.; Bongaerts, J.; Evers, S.; Maurer, K.

510

H.; Daniel, R.; Liebl, W.; Liesegang, H.; Ehrenreich, A. Size unlimited markerless

511

deletions by a transconjugative plasmid-system in Bacillus licheniformis. J.

512

Biotechnol. 2013, 167, 365−369.

513

(32) Xu, Y.; Xu, C.; Li, X.; Sun, B.; Eldin, A. A.; Jia, Y. A combinational ACS Paragon Plus Environment


Page 24 of 37

514

optimization method for efficient synthesis of tetramethylpyrazine by the recombinant

515

Escherichia coli. Biochem. Eng. J. 2018, 129, 33−43.

516

(33) Huang, M.; Oppermann-Sanio, F. B.; Steinbüchel, A. Biochemical and

517

molecular characterization of the Bacillus subtilis acetoin catabolic pathway. J.

518

Bacteriol. 1999, 181, 3837−3841.

519

(34) Ventura, J. R. S.; Hu, H.; Jahng, D. Enhanced butanol production in

520

Clostridium

521

6-phosphofructokinase and pyruvate kinase genes. Appl. Microbiol. Biotechnol. 2013,

522

97, 7505−7516.

523

acetobutylicum

ATCC

824

by

double

overexpression

of

(35) Yamamoto, S.; Gunji, W.; Suzuki, H.; Toda, H.; Suda, M.; Jojima, T.; Inui, M.;

524

Yukawa,

525

Corynebacterium glutamicum enhances glucose metabolism and alanine production

526

under oxygen deprivation conditions. Appl. Environ. Microbiol. 2012, 78, 4447−4457.

527

(36) Wang, M.; Fu, J.; Zhang, X.; Chen, T. Metabolic engineering of Bacillus

528 529 530

H.

Overexpression

of

genes

encoding

glycolytic

enzymes

in

subtilis for enhanced production of acetoin. Biotechnol. Lett. 2012, 34, 1877−1885. (37) van Dijl, J.; Hecker, M. Bacillus subtilis: from soil bacterium to super-secreting cell factory. Microb. Cell Fact. 2013, 12, 3.

531

(38) Hao, F.; Wu, Q.; Xu, Y. Precursor supply strategy for tetramethylpyrazine

532

production by Bacillus subtilis on solid-state fermentation of wheat bran. Appl.

533

Biochem. Biotechnol. 2013, 169, 1346–1352.

534


Page 25 of 37


535

FIGURE LEGENDS

536

The acetoin and ligustrazine metabolic network of B. subtilis. GlcU,

537

Figure 1.

538

glucose uptake protein; LDH, lactate dehydrogenase; PDC, pyruvate dehydrogenase

539

complex; PTA, phospho-transacetylase; AckA, acetate kinase; AldH, acetaldehyde

540

dehydrogenase; AlsS, α-acetolactate synthase; AlsD, α-acetolactate decarboxylase;

541

BdhA, acetoin reductase; EDH, ethanol dehydrogenase; AoDH ES, acetoin

542

dehydrogenase enzyme system; TCA, tricarboxylic acid; SES, secretion system; HK,

543

hexokinase; PFKA, 6-phosphofructokinase; PYK, pyruvate kinase; GPI, glucose

544

phosphate isomerase

545

Mutation of bdhA (A), acoABC (B) and overexpression of gene cluster

546

Figure 2.

547

alsSD (C) by promoter P43 (D) in B. subtilis and the fermentation analysis (E and F).

548

(A) M, marker; 1, PCR of bdhA using the genomic DNA of B. subtilis 168 as template;

549

2, PCR of bdhA using the genomic DNA of B. subtilis 1 as template. (B) M, marker; 1,

550

PCR of bdhA (left) and acoABC (right) using the genomic DNA of B. subtilis 168 as

551

template; 2, PCR of bdhA (left) and acoABC (right) using the genomic DNA of B.

552

subtilis 2 as template. (C) M, marker; 1, PCR of gene cluster alsSD using the genomic

553

DNA of B. subtilis 168 as template. (D) M, marker; 1, synthesis of promote P43 using

554

designed primers through overlap extension.

555 556

Figure 3.

Cloning of genes pfkA (A), pyK (B), glcU (C) and the effects of their



557

respective overexpression on B. subtilis metabolism (D and E). (A) M, marker; 1,

558

PCR of pfkA using the genomic DNA of B. subtilis 168 as template. (B) M, marker; 1,

559

PCR of pyK using the genomic DNA of B. subtilis 168 as template. (C) M, marker; 1,

560

PCR of glcU using the genomic DNA of B. subtilis 168 as template.

561

Acetoin and ligustrazine metabolism by B. subtilis 2 under different

562

Figure 4.

563

glucose concentrations (A), nitrogen sources (B) and small red bean concentrations

564

(C).

565

Optimization of initial pH of culture media (A) and temperature (B) using

566

Figure 5.

567

B. subtilis 2.

568

The long-term cultivation of B. subtilis 2 with or without (NH4)2HPO4 in

569

Figure 6.

570

culture media. (A) LB added with 40 g/L small red bean and 40 g/L glucose, (B) LB

571

added with 40 g/L small red bean, 40 g/L glucose and 20 g/L (NH4)2HPO4. ■, glucose;

572

●, acetoin; ▲, cell density (OD600 nm); ◆, ligustrazine

573


Page 26 of 37

Page 27 of 37


574

Table 1.

Strains and Vectors Used in This Study

Name

Characteriatic

Reference

Strain Escherichia coli

F-, φ80d lacZ∆M15, ∆(lacZYA-argF)U169, recA1, endA1, hsdR17(rk-, +

-

Novagen

-1

DH5α

mk ), phoA, supE44λ , thi , gyrA96, relA1

E. coli S17-1 λpir

Tpr Smr recA thi pro hsd(r- m+)RP4-2-Tc::Mu::Km Tn7 λpir

30

Bacillus subtilis 168

Type strain, used as host strain

28

B. subtilis 1

B. subtilis 168∆bdhA

This work

B. subtilis 2

B. subtilis 168∆bdhA∆acoABC

This work

B. subtilis 3

B. subtilis 168∆bdhA∆acoABC harboring pHY300PLK

This work

B. subtilis 4

B. subtilis 168∆bdhA∆acoABC harboring pHYP43-alsSD

This work

B. subtilis 5

B. subtilis 168∆bdhA∆acoABC harboring pHYP43-glcU

This work

B. subtilis 6

B. subtilis 168∆bdhA∆acoABC harboring pHYP43-pfkA

This work

B. subtilis 7

B. subtilis 168∆bdhA∆acoABC harboring pHYP43-pyK

This work

ori-pAMα1, ori-177, Apr, Tcr

Takara

Vector pHY300PLK

r

r

pKVM1

oriR-pBR322, oriT, oriR-pE194ts, bgaB, Ap , Ery

31

pKVM-∆bdhA

pKVM1 carrying the mutation gene fragment of bdhA

This work

pKVM-∆acoABC

pKVM1 carrying the mutation gene fragment of acoABC

This work

pHYP43-alsSD

pHY300PLK carrying P43 promoter and acetoin synthetic gene cluster

This work

pHYP43-glcU

pHY300PLK carrying P43 promoter and glucose uptake gene glcU from

alsSD originated from B. subtilis 168 This work

B. subtilis 168 pHYP43-pfkA

pHY300PLK carrying P43 promoter and 6-phosphofructokinase gene

This work

pfkA from B. subtilis 168 pHYP43-pyK

pHY300PLK carrying P43 promoter and pyruvate kinase gene pyK from B. subtilis 168

575 576


This work


577

Table 2.

Primers Used in This Study

Primer namea

Sequence (5’ – 3’)b

PpHY.f(XhoI)

CCGCTCGAGCTGTTATAAAAAAAGGATC

PpHY.r(BglII)

GAAGATCTGCAAAGCGTTTTTCCATAG

P43.f(XhoI)

CCGCTCGAGGAGCTCAGCATTATTGAG

P43r.2

GCGAAAACATACCACCTATCAAAAGGAATATAATCATCCACTCAATAATGCTGAG

P43f.3

GGTGGTATGTTTTCGCTTGAACTTTTAAATACAGCCATTGAACATACGGTTGATTTAAT

P43r.4

TCCTTGGCCGCTTTAGCAAGAGGGTGATGTTTGTCAGTTATTAAATCAACCGTATG

P43f.5

CTAAAGCGGCCAAGGACGCTGCCGCCGGGGCTGTTTGCGTTTTTGCCGTGATTTCGTG

P43r.6

GCCATTACAGCTTTGGCAAAAAAATAAGTAAACCAATGATACACGAAATCACGGCA

P43f.7

CAAAGCTGTAATGGCTGAAAATTCTTACATTTATTTTACATTTTTAGAAATGGGCGTG

P43r.8

GTACCGCTATCACTTTATATTTTACATAATCGCGCGCTTTTTTTCACGCCCATTTCTA

P43f.9

AGTGATAGCGGTACCATTATAGGTAAGAGAGGAAAAAAA

P43.r

TTTTTTTCCTCTCTTACC

P43-glcU.r

GAGCCAATAATAAATCCATTTTTTTTCCTCTCTTACC

P43-pfkA.r

TACCCCTATTCGTTTCATTTTTTTTCCTCTCTTACC

P43-pyK.r

CAATTTTAGTTTTTCTCATTTTTTTTCCTCTCTTACC

Bs.f(OV)

GGTAAGAGAGGAAAAAAAGTGAGGGTGTTGACAAAAG

Bs.r(BglII)

GAAGATCTTTATTCAGGGCTTCCTTC

PglcU.f(OV)

GGTAAGAGAGGAAAAAAAATGGATTTATTATTGGCTC

PglcU.r(BglII)

GAAGATCTTTATGAATTTGTTTTGGCG

PpfkA.f(ov)

GGTAAGAGAGGAAAAAAAATGAAACGAATAGGGGTA

PpfkA.r(BglII)

GAAGATCTTTAGATAGACAGTTCTTTTG

PpyK.f(OV)

GGTAAGAGAGGAAAAAAAATGAGAAAAACTAAAATTG

PpyK.r(BglII)

GAAGATCTTTAAAGAACGCTCGCACG

BdhA-UP.f(BglII)

GGCAGATCTCCGTTTCTAGGAACATCA

BdhA-UP.r(OV)

CCTAGCCTCGAGGTTTTTGGCTCTTCGAT

BdhA-DN.f(OV)

CCTCGAGGCTAGGGGAAAAGTAAAGATCA

BdhA-DN.r(SalI)

GAAGTCGACCTGCAATCTCAGCGACTAC

acoABC-UP.f(BamHI)

CGGGATCCGATTTCCAAGGAAATAAA

acoABC-UP.r(OV)

GCTTTCATCGTGATTCCATTTGCGCCTAA

acoABC-DN.f(OV)

CAAATGGAATCACGATGAAAGCTGATATC

acoABC-DN.r(BglII)

GAAGATCTCTATAAAATTAATGCTGC

578 579 580

a

“.f” in the primer name means that this is the sense primer; “.r” in the primer name means that this is

the antisense primer. b The restriction site is underlined.

581


Page 28 of 37

Page 29 of 37


582

Table 3.

583

Using Shake Flasks within 36 h under the Aerobic Condition

Strainsa

Cell Density, Glucose Consumption, Acetoin and the Main Byproducts Production by the B. subtilis Recombinant Strains

P valueb

Cell density

Glucose consumption

Acetoin

2,3-Butanediol

Lactate concentration

Acetate concentration

Glucose uptake

(OD600nm)

(g/L)

concentration (g/L)

concentration (g/L)

(g/L)

(g/L)

rate (g/[L h])

B. subtilis 168

5.26 ± 0.49

24.25 ± 1.71

5.96 ± 0.18

3.01 ± 0.21

0.18 ± 0.014

0.18 ± 0.032

0.674

-

B. subtilis 1

5.33 ± 0.96

24.50 ± 0.58

8.53 ± 0.04

0.56 ± 0.012

0.24 ± 0.024

0.19 ± 0.025

0.681

-

B. subtilis 2

5.51 ± 1.29

24.40 ± 1.14

8.76 ± 0.24

0.62 ± 0.081

0.27 ± 0.045

0.12 ± 0.064

0.678

-

B. subtilis 3

5.12 ± 0.26

22.33 ± 2.52

8.58 ± 0.09

0.49 ± 0.085

0.29 ± 0.013

0.26 ± 0.033

0.620

-

B. subtilis 4

5.40 ± 0.20

25.17 ± 1.04

9.45 ± 0.34

0.50 ± 0.014

0.30 ± 0.011

0.21 ± 0.015

0.699

0.0042

B. subtilis 5

5.20 ± 0.29

26.67 ± 0.58

9.65 ± 0.04

0.67 ± 0.027

0.32 ± 0.041

0.15 ± 0.033

0.741

0.000046

B. subtilis 6

5.06 ± 0.11

25.67 ± 0.58

9.14 ± 0.01

0.59 ± 0.017

0.41 ± 0.016

0.22 ± 0.007

0.713

0.00043

B. subtilis 7

4.60 ± 0.13

25.33 ± 2.08

9.18 ± 0.30

0.48 ± 0.020

0.30 ± 0.041

0.23 ± 0.047

0.704

0.029

584 585 586

a

Data are the means ± standard deviations (SDs) from three parallel experiments.

b

P values are the acetoin data of the groups B. subtilis 4, 5, 6, 7 compared with B. subtilis 3 (B. subtilis 168∆bdhA∆acoABC harboring pHY300PLK), respectively, as all the

groups with significant differences compared with B. subtilis 168 (p < 0.01).

587



588

Table 4.

589

(Unit: Mol/Mol Glucose)

Namea

Page 30 of 37

Glucose Metabolic Flux Distribution of Different B. subtilis Strains

Acetoin

2,3-Butanediol

Lactate

Acetate

Succinate

P valueb

Micro-oxygen conditions B. subtilis 168

0.229 ± 0.009

0.488 ± 0.016

0.077 ± 0.002

0.084 ± 0.004

0.016 ± 0.001

-

B. subtilis 1

0.616 ± 0.111

0.127 ± 0.017

0.100 ± 0.030

0.028 ± 0.005

0.018 ± 0.002

-

B. subtilis 2

0.618 ± 0.083

0.132 ± 0.005

0.102 ± 0.031

0.025 ± 0.003

0.017 ± 0.002

-

B. subtilis 3

0.559 ± 0.045

0.096 ± 0.000

0.100 ± 0.000

0.050 ± 0.003

0.016 ± 0.000

-

B. subtilis 4

0.620 ± 0.006

0.078 ± 0.001

0.091 ± 0.003

0.054 ± 0.005

0.015 ± 0.001

0.029

B. subtilis 5

0.609 ± 0.003

0.087 ± 0.008

0.072 ± 0.001

0.026 ± 0.002

0.014 ± 0.000

0.049

B. subtilis 6

0.641 ± 0.016

0.101 ± 0.010

0.106 ± 0.010

0.040 ± 0.006

0.016 ± 0.001

0.016

B. subtilis 7

0.674 ± 0.013

0.091 ± 0.006

0.099 ± 0.003

0.047 ± 0.004

0.019 ± 0.001

0.0042

Aerobic conditions B. subtilis 168

0.503 ± 0.015

0.248 ± 0.017

0.015 ± 0.001

0.022 ± 0.004

-

-

B. subtilis 1

0.712 ± 0.003

0.046 ± 0.001

0.020 ± 0.002

0.023 ± 0.003

-

-

B. subtilis 2

0.734 ± 0.020

0.051 ± 0.007

0.022 ± 0.004

0.015 ± 0.008

-

-

B. subtilis 3

0.786 ± 0.008

0.044 ± 0.008

0.026 ± 0.001

0.035 ± 0.005

-

-

B. subtilis 4

0.768 ± 0.028

0.040 ± 0.001

0.024 ± 0.001

0.025 ± 0.002

-

0.048

B. subtilis 5

0.740 ± 0.003

0.050 ± 0.002

0.024 ± 0.003

0.017 ± 0.004

-

0.00084

B. subtilis 6

0.728 ± 0.001

0.046 ± 0.001

0.032 ± 0.001

0.026 ± 0.001

-

0.00026

B. subtilis 7

0.741 ± 0.024

0.038 ± 0.002

0.024 ± 0.003

0.027 ± 0.006

-

0.039

590 591 592 593

a

Data are the means ± standard deviations (SDs) from three parallel experiments.

b

P values are the acetoin data of the groups B. subtilis 4, 5, 6, 7 compared with B. subtilis 3 (B. subtilis

168∆bdhA∆acoABC harboring pHY300PLK), respectively, as all the groups with significant differences compared with B. subtilis 168 (p < 0.01).

594


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Figure 1. The acetoin and ligustrazine metabolic network of B. subtilis. GlcU, glucose uptake protein; LDH, lactate dehydrogenase; PDC, pyruvate dehydrogenase complex; PTA, phospho-transacetylase; AckA, acetate kinase; AldH, acetaldehyde dehydrogenase; AlsS, α-acetolactate synthase; AlsD, α-acetolactate decarboxylase; BdhA, acetoin reductase; EDH, ethanol dehydrogenase; AoDH ES, acetoin dehydrogenase enzyme system; TCA, tricarboxylic acid; SES, secretion system; HK, hexokinase; PFKA, 6phosphofructokinase; PYK, pyruvate kinase; GPI, glucose phosphate isomerase 86x86mm (300 x 300 DPI)



Figure 2. Mutation of bdhA (A), acoABC (B) and overexpression of gene cluster alsSD (C) by promoter P43 (D) in B. subtilis and the fermentation analysis (E and F). (A) M, marker; 1, PCR of bdhA using the genomic DNA of B. subtilis 168 as template; 2, PCR of bdhA using the genomic DNA of B. subtilis 1 as template. (B) M, marker; 1, PCR of bdhA (left) and acoABC (right) using the genomic DNA of B. subtilis 168 as template; 2, PCR of bdhA (left) and acoABC (right) using the genomic DNA of B. subtilis 2 as template. (C) M, marker; 1, PCR of gene cluster alsSD using the genomic DNA of B. subtilis 168 as template. (D) M, marker; 1, Synthesis of promote P43 using designed primers through overlap extension. 126x192mm (300 x 300 DPI)


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Figure 3. Cloning of genes pfkA (A), pyK (B), glcU (C) and the effects of their respective overexpression on B. subtilis metabolism (D and E). (A) M, marker; 1, PCR of pfkA using the genomic DNA of B. subtilis 168 as template. (B) M, marker; 1, PCR of pyK using the genomic DNA of B. subtilis 168 as template. (C) M, marker; 1, PCR of glcU using the genomic DNA of B. subtilis 168 as template. 123x187mm (300 x 300 DPI)



Figure 4. Acetoin and ligustrazine metabolism by B. subtilis 2 under different glucose concentrations (A), nitrogen sources (B) and small red bean concentrations (C). 86x216mm (300 x 300 DPI)


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Figure 5. Optimization of initial pH of culture media (A) and temperature (B) using B. subtilis 2. 160x70mm (300 x 300 DPI)



Figure 6. The long-term cultivation of B. subtilis 2 with or without (NH4)2HPO4 in culture media. (A) LB added with 40 g/L small red bean and 40 g/L glucose, (B) LB added with 40 g/L small red bean, 40 g/L glucose and 20 g/L (NH4)2HPO4. ■, glucose; ●, acetoin; ▲, cell density (OD600 nm); ◆, ligustrazine 86x124mm (300 x 300 DPI)


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TOC Graphic 42x22mm (600 x 600 DPI)


Systematic Characterization of the Metabolism of Acetoin and Its

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