Systematic Characterization of the Metabolism of Acetoin and Its

Bacillus subtilis is an important microorganism for brewing of Chinese Baijiu, which contributes to the ... (1) Numerous microorganisms exist in the B...
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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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Systematic Characterization of the Metabolism of Acetoin and Its

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Derivative Ligustrazine in Bacillus subtilis under Micro-Oxygen

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Conditions

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

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

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1

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Technology and Business University, Beijing 100048, China.

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

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2

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Technology and Business University, Beijing 100048, China.

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3

13

University, Beijing 100048, China.

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4

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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

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* 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)

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Bacillus subtilis is an important microorganism for brewing of

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ABSTRACT:

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

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

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

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

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metabolism were investigated under this condition including key genes and

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

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overexpression of glcU (encoding glucose uptake protein) increased acetoin

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

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promoted acetoin production. The optimal culture conditions for ligustrazine synthesis

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

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

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

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of B. subtilis for maintaining Baijiu quality.

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KEYWORDS:

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Chinese Baijiu

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

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INTRODUCTION

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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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

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pharmacological

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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

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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

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

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

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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

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

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

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

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

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

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

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now.

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

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

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

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unstable

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characteristics of microorganisms is an effective way to scientifically study the

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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

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

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

among

batches.11

A comprehensive

understanding

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MATERIALS AND METHODS Bacterial Strains, Media and Chemical Reagents.

Strains used in this study are

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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for erythromycin, respectively.

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{Please insert Table 1 about here}

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DNA Manipulation and Vector Construction.

The primer pair PpHY.f(XhoI)/

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

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

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

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through overlap extension. Thereafter the promoter was confirmed by gene

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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{Please insert Table 2 about here}

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Gene Mutation.

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

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

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

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

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

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

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

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

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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

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

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origin prevented vector replication, thus vector integration occurred by homologous

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

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

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

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

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

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

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

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

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

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

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

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Conditions.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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metabolism was carried out under micro-oxygen conditions.

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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Metabolism.

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

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

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

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

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

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

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phosphorylation

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

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pyruvate from phosphoenolpyruvate. The AC synthetic gene cluster includes two

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

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

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

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

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

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

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

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

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

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acetoin dehydrogenase E2 component (dihydrolipoamide acetyltransferase) encoded

of

glucose;

PFKA

catalyzes

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by gene acoC, and acetoin dehydrogenase E3 component (dihydrolipoamide

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dehydrogenase) encoded by gene acoL in B. subtilis.

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{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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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the engineered B. subtilis strains.

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

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

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

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

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

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efficiency in Clostridium acetobutylicum and Corynebacterium glutamicum.34,35

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

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

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

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

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

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subtilis metabolism were examined here by overexpressing another gene, glcU

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

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

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

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

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

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

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

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conditions.

304 305

{Please insert Figure 3 about here}

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

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

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

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glucose uptake and AC production rates in Bacillus strains greatly increased

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

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

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

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

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

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

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

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

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

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

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

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

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

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that overexpression of gene glcU promoted glucose consumption and AC metabolism

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

326 327

{Please insert Table 3 about here}

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

{Please insert Table 4 about here}

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

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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

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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

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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

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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

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19002–19013.

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

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

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

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

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

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

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anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end

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

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

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

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

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

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

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(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.

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

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

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

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Biotechnol. 1983, 1, 784−791.

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

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

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optimization method for efficient synthesis of tetramethylpyrazine by the recombinant

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

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

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

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Bacteriol. 1999, 181, 3837−3841.

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

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

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acetobutylicum

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by

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Yukawa,

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Corynebacterium glutamicum enhances glucose metabolism and alanine production

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

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

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Overexpression

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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.

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

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

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

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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

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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

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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

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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.

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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).

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588

Table 4.

589

(Unit: Mol/Mol Glucose)

Namea

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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).

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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)

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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)

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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)

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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)

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