CN109486871B

CN109486871B - Method for producing acetoin by fermentation of bacillus licheniformis engineering strain

Info

Publication number: CN109486871B
Application number: CN201811497781.4A
Authority: CN
Inventors: 高超; 马翠卿; 严金鑫; 刘秋媛; 张一鹏
Original assignee: Shenzhen Research Institute Of Shandong University; Shandong University
Current assignee: Shenzhen Research Institute Of Shandong University; Shandong University
Priority date: 2018-12-07
Filing date: 2018-12-07
Publication date: 2021-10-26
Anticipated expiration: 2038-12-07
Also published as: CN109486871A

Abstract

The invention discloses a method for producing acetoin by fermentation of a Bacillus licheniformis engineering strain, which comprises the steps of taking Bacillus licheniformis (Bacillus licheniformis)10-1-A as a starting strain to knock out a glycerol dehydrogenase gdh gene and a 2, 3-butanediol dehydrogenase gene budC of the Bacillus licheniformis, replacing a lactate dehydrogenase gene ldh with an NADH oxidase gene nox from Thermococcus profundus DT5432 to construct the Bacillus licheniformis engineering strain capable of producing acetoin, then taking glucose as a substrate, biologically fermenting the Bacillus licheniformis strain, and obtaining the acetoin from a fermentation liquid. Experiments prove that the engineering bacteria can produce acetoin of 82.14 g/L, the production efficiency reaches 2.28 g/L/h, and the production process has low cost, high yield, good application value and considerable economic benefit.

Description

Method for producing acetoin by fermentation of bacillus licheniformis engineering strain

Technical Field

The invention relates to a method for producing acetoin by fermentation, in particular to a method for producing acetoin by fermentation of a bacillus licheniformis engineering strain.

Background

Acetoin (3-hydroxy-2-butanone) is a volatile compound, and is widely applied to food addition, plant growth promotion, biological pest control and the like. In addition, acetoin can also be used as a precursor for chemical synthesis of a series of compounds, such as diacetyl and alkyl-containing pyrazines, wherein the latter comprises 2,3,5, 6-tetramethylpyrazine. In view of the wide application and potential for industrial production of acetoin, acetoin is listed as one of 30 platform compounds with priority development by the U.S. department of energy.

At present, the main source of commercially available acetoin is a chemical synthesis mode based on non-renewable resources, the process steps are complex, the reaction conditions are harsh, the pollution is serious, and the application of the chemically synthesized acetoin the fields of food, medicine, daily chemicals and the like is limited. Many researchers are dedicated to the industrial production of acetoin by microbial fermentation, but the cost for producing the acetoin by microbial fermentation is still higher compared with that of a chemical process method. Therefore, the method reduces the dependence of the acetoin industry on non-renewable petroleum resources, realizes the green production of the acetoin, improves the economic competitiveness of the microbial acetoin fermentation process, and has important economic benefits and social significance.

And (3) searching and finding: a plurality of microorganisms are reported to be used for acetoin fermentation production, and a strain of bacillus pumilus is obtained by high-sugar-resistant screening and NTG mutagenesis in the U.S. Pat. No.4, 0815840, wherein the yield of the acetoin reaches 63 g/L. Sun Jianan et al enhanced intracellular NAD by exogenous expression of NADH oxidase in Serratia marcescens⁺And the concentration is that glucose is used as a carbon source, and the final yield of the acetoin reaches 75.2g/L, which is the highest value of the yield of the acetoin reported at present. However, no report has been found on a method for constructing a bacillus licheniformis engineering strain by knocking out key enzyme genes (gdh and budC) of a byproduct 2, 3-butanediol synthesis pathway and replacing a bacillus licheniformis lactate dehydrogenase gene (ldh) with an NADH oxidase gene (nox) from a thermochcus profundus DT5432 strain to reduce the yield of a byproduct lactic acid.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for producing acetoin by using a constructed bacillus licheniformis engineering strain through fermentation.

The invention discloses a method for producing acetoin by fermentation of a bacillus licheniformis engineering strain, which comprises the following steps:

(1) using Bacillus licheniformis (Bacillus licheniformis)10-1-A as a starting bacterium, knocking out a glycerol dehydrogenase gdh gene and a 2, 3-butanediol dehydrogenase gene budC of the Bacillus licheniformis (Bacillus licheniformis)10-1-A and replacing a lactate dehydrogenase gene ldh by an NADH oxidase gene nox from Thermococcus profundus DT5432 to construct a Bacillus licheniformis engineering strain capable of producing acetoin, wherein the engineering strain is named as Bacillus licheniformis Δ gdh Δ budC Δ ldh:: nox;

wherein the starting strain Bacillus licheniformis (Bacillus licheniformis)10-1-A is preserved in China general microbiological culture Collection center at 11/14/2011 with the preservation number as follows: CGMCC NO. 5461;

(2) biologically fermenting Bacillus licheniformis delta gdh delta budC delta ldh with glucose as a substrate, wherein nox is obtained from fermentation liquor;

wherein the fermentation conditions are: the culture temperature is 50 +/-1 ℃, the culture mode is stirring culture, the stirring rotation speed is 500 +/-50 revolutions per minute, the rotation radius is 33 +/-1 mm, the ventilation volume is 1.0 +/-0.1 vvm, and the culture time is 18-36 hours;

the fermentation medium comprises the following components: 50-80 g/L of glucose, 12 g/L of yeast powder, 6.5 g/L of anhydrous sodium acetate, 1 g/L of ammonium citrate, 2g/L of dipotassium hydrogen phosphate, 0.25 g/L of magnesium sulfate heptahydrate, 10 ml/L of metal ion mother liquor and the balance of water; the pH was 7.0; the formula of the metal ion mother liquor is as follows: ferrous sulfate 2.25 g/L, zinc sulfate 0.75 g/L, manganese sulfate 0.38 g/L.

In the method for producing acetoin by fermenting the engineering strain of Bacillus licheniformis, the engineering strain Bacillus licheniformis delta gdh delta budC delta ldh in the step (1) is as follows:

taking a bacillus licheniformis10-1-A genome as a template, obtaining a recombinant fragment containing upstream and downstream homologous sequences of a gdh gene by PCR, connecting the recombinant fragment to pKMM 1, transferring the recombinant fragment into the bacillus licheniformis10-1-A through parental hybridization, and screening a single-exchange transformant through resistance; after double exchange is generated by temperature induction, a strain with a gdh gene successfully knocked out is obtained by PCR screening and named as Bacillus licheniformis delta gdh;

taking a bacillus licheniformis10-1-A genome as a template, obtaining a recombinant fragment containing upstream and downstream homologous sequences of a budC gene by PCR, connecting the recombinant fragment to pKMM 1, transferring the recombinant fragment into bacillus licheniformis delta gdh through parental hybridization, and performing resistance screening on a single-exchange transformant; after double exchange is carried out by temperature induction, a strain with successfully knocked-out budC gene is obtained by PCR screening, and the strain is named as Bacillus licheniformis delta gdh delta budC.

Taking a bacillus licheniformis10-1-A genome as a template, and obtaining a sequence containing upstream and downstream homologous arms of an ldh gene by PCR; using plasmid pUC57-nox carrying NADH oxidase synthesized by whole gene as a template, obtaining a nox sequence by PCR, recombining an upstream and downstream homologous arm sequence of an ldh gene and the nox sequence by PCR to obtain a gene replacement recombinant fragment, connecting the gene replacement recombinant fragment to pKMV 1, transferring the gene replacement recombinant fragment into Bacillus licheniformis delta gdh delta budC by parental hybridization, and screening a single-exchange transformant by resistance; after double exchange is carried out by temperature induction, an engineering strain with the nox gene successfully replacing ldh gene is obtained by PCR screening, and the strain is named as Bacillus licheniformis delta gdh delta budC delta ldh:: nox.

The Bacillus licheniformis delta gdh delta budC delta ldh is that nox is gram-positive bacteria, grows aerobically or facultative anaerobically, has strong protein secretion capacity and fast growth rate, and is a recognized biosafety bacterium. The bacillus subtilis is rod-shaped, has the length of 1.5-3.0 mu m and the diameter of 0.6-0.7 mu m, has red or white colony color, produces spores, has positive VP reaction, can produce acid by using glucose, sucrose and fructose, can hydrolyze casein, gelatin and Tween 80, can grow in a culture medium containing 100g/L NaCl by using citrate, and can grow at the temperature of 42-60 ℃.

In the method for producing acetoin by fermentation of the bacillus licheniformis engineering strain, the fermentation conditions in the step (2) are preferably as follows: the culture temperature is 50 ℃, the culture mode is stirring culture, the stirring rotation speed is 500 r/min, the rotation radius is 33 mm, the ventilation volume is 1.0vvm, and the culture time is 25-36 hours.

In the method for producing acetoin by fermentation of the bacillus licheniformis engineering strain, the detection method of the substrate glucose comprises the following steps: after appropriate dilution of the sample, it was measured using a biosensor analyzer SBA-40D (institute of biological research, academy of sciences, Shandong province). The determination principle is that the immobilized glucose oxidase membrane is used for specifically determining the glucose content.

In the method for producing acetoin by fermentation of the bacillus licheniformis engineering strain, the method for detecting the acetoin as the fermentation product comprises the following steps:

adding 0.7 ml of isoamyl alcohol into every 500 ml of ethyl acetate to serve as an extracting agent; the extractant is used for performing isovolumetric extraction on a fermentation product sample in fermentation liquor, a vortex oscillator is used for oscillating the extracted sample for 30 seconds, then standing is performed, and an upper layer sample is taken for gas phase detection. The specific gas phase detection conditions were as follows:

the model of the gas chromatograph is Agilent 6820, nitrogen is used as carrier gas, and the temperatures of the sample injector and the detector are both set to be 280 ℃; the capillary column is Supelcobeta-DexTM120 (inner diameter 0.25 mm, length 30 m); the column temperature during the detection was set as: the temperature was maintained at 40 ℃ for 3 minutes, then increased to 80 ℃ at a rate of 1.5 ℃ per minute, to 86 ℃ at a rate of 0.5 ℃ per minute, and to 200 ℃ at a rate of 30 ℃ per minute. The sample size was 1.0. mu.l, and gas phase detection was carried out.

The invention has the characteristics and the outstanding effects that:

(1) nox is a biologically safe strain with high yield of acetoin, key enzyme genes (gdh and budC) of a byproduct 2, 3-butanediol synthesis pathway are knocked out, so that metabolic flow of the acetoin converted into the 2, 3-butanediol is blocked, in addition, NADH oxidase gene (nox) in a Thermococcus profundus DT5432 strain is used for replacing a Bacillus licheniformis lactate dehydrogenase gene (ldh) so as to reduce the yield of the byproduct lactic acid, and the NADH dependent byproduct synthesis pathway is reduced so as to improve the yield of the acetoin.

(2) The engineering strain provided by the invention takes glucose as a substrate, the maximum concentration of acetoin obtained under the fermentation condition provided by the invention can reach 82.14 g/L, and the production efficiency can reach 2.28 g/L/h;

(3) the method for producing acetoin provided by the invention fully utilizes the advantages of a high-temperature fermentation process, has low probability of contaminating infectious microbes, and simultaneously realizes the aims of high yield and high production strength.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the present invention in any way, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention fall within the scope of the technical solution of the present invention.

Example 1: constructing engineering bacteria Bacillus licheniformis delta gdh delta budC delta ldh for producing acetoin

The starting strain is Bacillus licheniformis10-1-A, which has been preserved in China general microbiological culture Collection center at 11/14/2011 with the preservation number as follows: CGMCC NO. 5461.

(1) Knockout of the gdh Gene of Glycerol dehydrogenase

The length of the gdh sequence of the glycerol dehydrogenase gene is 1104 basic groups, and the nucleotide sequence is shown in SEQ ID NO. 1.

Preparing genomic DNA of B.licheniformis 10-1-A by a conventional method, wherein the genomic DNA of Bacillus licheniformis10-1-A is extracted by a method for preparing a bacterial genome in a small amount according to 'a guide to molecular biology finely compiled by scientific publishers'; the upstream and downstream homology arms of the gdh-encoding gene were PCR amplified using primers "gdh 1-f and gdh 1-r" and "gdh 2-f and gdh 2-r". Then, recombinant PCR was performed using the obtained upstream and downstream homology arms as templates using primers "gdh 1-f and gdh 2-r" to obtain a gdh truncated fragment containing BglII and BamHI cleavage sites at both ends.

Carrying out double enzyme digestion on the gdh truncated fragment and the suicide plasmid pKMG 1 by BglII and BamHI respectively, recovering the enzyme digestion product by glue, and using T₄DNA ligase ligation to obtain the knock-out plasmid pKMM 1-delta gdh.

Wherein, the primers for amplifying the recombinant fragments are designed as follows:

gdh1-f:5’-ATTTAGATCTAACAAGCCGCGTCATTCAAG-3’(BglII)

gdh1-r:5’-ACTTGGCGCCATTCTTCTTCGACACATCGCAAATGATA-3’

gdh2-f:5’-TATCATTTGCGATGTGTCGAAGAAGAATGGCGCCAAGT-3’

gdh2-r:5’-TACCGTGGATCCGCTTTAAG-3’(BamHI)

1) inoculation of E.coli S17-1 lambda harboring a knock-out plasmidpir and Bacillus licheniformis10-1-A, culturing the strain to OD at 37 deg.C₆₀₀1.2. 4-7 ml of escherichia coli liquid and 1 ml of bacillus licheniformis are centrifuged at 6000 rpm for 5 minutes, washed twice with 50mM phosphate buffer with pH 7.5, mixed, resuspended and centrifuged, then resuspended in LB medium and dropped on an LB plate, and cultured overnight at 30 ℃. After collecting cells with LB liquid medium preheated at 30 ℃ and diluting 10^-4And 10^-5Coating on LB solid plate added with erythromycin and polymyxin B, and culturing at 30 ℃ for 48-72 hours.

2) Selecting a transformant to an LB culture medium added with erythromycin resistance, culturing for 6-12 hours at 37 ℃, washing with the LB culture medium once, transferring to a fresh LB culture medium for culturing for 6-12 hours at 42 ℃, then diluting to an erythromycin LB plate in a gradient manner, culturing overnight at 42 ℃, selecting the transformant for culturing at 42 ℃, selecting a growing single colony, and carrying out PCR verification by using upstream and downstream primers to obtain a bacterium which can simultaneously carry out PCR on long fragments and short fragments and has a correct single exchange purpose.

3) Selecting transformant to LB culture medium, culturing at 30 deg.C without resistance, transferring for two generations, diluting 10^-5And 10^-6Coating a plate to LB, culturing for 12 hours at 37 ℃, selecting a white transformant to 50 ℃, culturing and carrying out molecular verification, carrying out PCR verification by using an upstream primer and a downstream primer, carrying out PCR verification on a double-exchange target strain to obtain a short fragment (constructed upstream and downstream homologous arms) and selecting a correct double-exchange strain, and carrying out phenotype verification to obtain a correct engineering strain Bacillus licheniformis delta gdh for knocking out the gdh gene of the Bacillus licheniformis10-1-A glycerol dehydrogenase.

(2) Knock-out of 2, 3-butanediol dehydrogenase budC Gene

The length of the sequence of the 2, 3-butanediol dehydrogenase gene budC is 783 bases, and the nucleotide sequence is shown as SEQ ID NO. 2.

Knockout of Gene budC referring to the knockout of gdh in step (1) of this example, the primer sequences were as follows:

budC1-f:5’-AAACCATGGAATAAACGAGTTGACGGAAA-3’(NcoI)

budC1-r:5’-GCAAAGCAATTGCGGTTAAATTGCATTAAAACGCTTATCC-3’

budC2-f:5’-GGATAAGCGTTTTAATGCAATTTAACCGCAATTGCTTTGC-3’

budC2-r:5’-TTTGGATCCTATGCTCGCGGTGTTCTAT-3’(BamHI)

the engineering strain which correctly knocks out the glycerol dehydrogenase gdh gene and the 2, 3-butanediol dehydrogenase budC gene in the Bacillus licheniformis is named as Bacillus licheniformis delta gdh delta budC.

(3) Replacement of lactate dehydrogenase gene ldh in Bacillus licheniformis delta gdh delta budC strain by NADH oxidase gene nox in Thermococcus profundus DT5432

The length of the sequence of the NADH oxidase gene nox is 1320 basic groups, and the nucleotide sequence is shown as SEQ ID NO. 3. The length of the lactate dehydrogenase ldh sequence is 960 bases, and the nucleotide sequence is shown as SEQ ID NO. 4.

Preparing genomic DNA of B.licheniformis 10-1-A by a conventional method, wherein the genomic DNA of Bacillus licheniformis10-1-A is extracted by a method for preparing a bacterial genome in a small amount according to 'a guide to molecular biology finely compiled by scientific publishers'; the genomic DNA of Bacillus licheniformis10-1-A is used as a template, and primers are used for PCR amplification to replace the upstream and downstream homologous arms of the gene nox, wherein the primers comprise nox1-f and ldh, the nox1-r and the primers comprise nox3-f and ldh, and the nox 3-r. The middle-segment replacement gene nox is amplified by using a gene of NADH oxidase nox in a Thermococcus profundus DT5432 synthesized by a whole gene as a template and using primers 'ldh:: nox2-f and ldh:: nox 2-r'. Then, recombinant PCR was carried out using the primers "gdh 1-f and gdh 2-r" using the obtained upstream homology arm and the middle nox gene as templates to obtain 1-2 fragments. Finally, recombinant PCR was performed using the primers "gdh 1-f and gdh 3-r" and the obtained 1-2 fragment and the downstream homology arm fragment as templates to obtain a gene replacement fragment of Δ ldh:: nox, both ends of which contain BamHI and SmaI cleavage sites.

Carrying out double enzyme digestion on the gene replacement fragment of the Deltaldh: nox and the suicide plasmid pKMG 1 by using BamHI and SmaI respectively, recovering the enzyme digestion product by using glue, and using T₄DNA ligase is connected to obtain a gene replacement plasmid pKMM 1-delta ldh:: nox.

ldh::nox1-f:5’-AATGGATCCTAGCGTAGCAAGCAGTGTTCCGCTGTT-3’(BamHI)

ldh::nox1-r:5’-TGACGACTCTTTTGCGTTCCATGACTCATCATTCCTTTGCCG-3’

ldh::nox2-f:5’-CGGCAAAGGAATGATGAGTCATGGAACGCAAAAGAGTCGTCA-3’

ldh::nox2-r:5’-AGTATCTTCATGGTGTTCAGTTAAAATTTCAGGACGCGCG-3’

ldh::nox3-f:5’-CGCGCGTCCTGAAATTTTAACTGAACACCATGAAGATACT-3’

ldh::nox3-r:5’-AATCCCGGGCGGTGACCAAGCGTTCATAAT-3’(SmaI)

gene replacement procedure for replacement of lactate dehydrogenase ldh in Bacillus licheniformis. DELTA. gdh. DELTA. budC Strain with NADH oxidase Gene nox referring to the gdh knockout procedure in step (1) of this example, recombinant Bacillus licheniformis. DELTA. gdh. DELTA. budC. DELTA. ldh: nox was finally obtained.

The experiment proves that: the Bacillus licheniformis delta gdh delta budC delta ldh is that the nox genetic engineering bacteria are gram-positive bacteria, and the optimal culture temperature is

Furthermore, the Bacillus licheniformis delta gdh delta budC delta ldh shows that the nox grows aerobically or facultative anaerobically, the protein secretion capability is strong, the growth rate is high, and the Bacillus licheniformis delta gdh is a recognized biosafety bacterium. The bacillus subtilis is rod-shaped, has the length of 1.5-3.0 mu m and the diameter of 0.6-0.7 mu m, has red or white colony color, produces spores, has positive VP reaction, can produce acid by using glucose, sucrose and fructose, can hydrolyze casein, gelatin and Tween 80, can grow in a culture medium containing 100g/L NaCl by using citrate, and can grow at the temperature of 42-60 ℃.

Example 2: rotation speed optimization of fermentation production of acetoin by a nox strain in a 1L fermentation tank

Referring to example 1, a wild type Bacillus licheniformis10-1-A strain is used as a starting strain, a glycerol dehydrogenase gdh gene and a 2, 3-butanediol dehydrogenase gene budC are knocked out, an NADH oxidase gene nox in Thermococcus profundus DT5432 is expressed to replace lactate dehydrogenase ldh, and the strain is named as Bacillus licheniformis delta gdh delta budC delta ldh:: nox.

(1) Drawing a line of nox on an LB culture medium plate containing agar with the mass-volume ratio of 1.5-1.8%, and performing shaking culture on the line at 50 +/-1 ℃ for 12 +/-1 hours;

(2) under the aseptic condition, picking a single colony on the plate in the step (1) by using an aseptic toothpick, then inoculating the single colony into 5mL of LB culture medium, and carrying out shake culture for 12 +/-1 hours at the temperature of 50 +/-1 ℃ in a shaking table to obtain a first-level seed of the recombinant bacillus licheniformis;

(3) under the aseptic condition, inoculating the primary seed bacterial liquid obtained in the step (2) into two bottles of 100mL LB culture medium in an inoculation amount of 2-4% in volume ratio, and performing shaking culture for 12 +/-1 hours at 50 +/-1 ℃ in a shaking table to obtain a recombinant bacillus licheniformis secondary seed bacterial liquid;

(4) and (3) under the aseptic condition, inoculating the secondary seed bacterial liquid obtained in the step (3) into three 1-liter fermentation tanks filled with sterilized 0.8-liter fermentation culture medium in an inoculation amount of 5% by volume, wherein the ventilation amount is 1.0vvm under the condition of 50 ℃, the stirring rotation speed of the fermentation tanks is 400 r/min, 500 r/min and 600 r/min respectively, and the fermentation is carried out for 18-36 hours.

Sampling every 3 hours during fermentation, and detecting OD_600nm. Centrifuging the sample at 8,000 Xg for 10 minutes, detecting the concentrations of acetoin and glucose in the supernatant, and supplementing dry glucose powder according to the glucose concentration to maintain the glucose concentration at 20-50 g/L; and (4) performing gas chromatography analysis on the supernatant to determine the concentration of the acetoin in the fermentation liquor, and stopping fermentation when the concentration of the acetoin is not increased any more.

The detection result after 36 hours shows that Bacillus licheniformis delta gdh delta budC delta ldh is that the optimal rotating speed condition for producing acetoin by nox fermentation is 500 r/min.

The fermentation medium comprises the following components: 50-80 g/L of glucose, 12 g/L of yeast powder, 6.5 g/L of anhydrous sodium acetate, 1 g/L of ammonium citrate, 2g/L of dipotassium hydrogen phosphate, 0.25 g/L of magnesium sulfate heptahydrate, 10 ml/L of metal ion mother liquor and the balance of water; the pH value is 6-7.

Wherein, the formula of the metal ion mother liquor is as follows: ferrous sulfate 2.25 g/L, zinc sulfate 0.75 g/L, manganese sulfate 0.38 g/L.

Example 3: bacillus licheniformis delta gdh delta budC delta ldh-beta-delta-beta

(4) and (3) under the aseptic condition, inoculating the secondary seed bacterial liquid obtained in the step (3) into three 1-liter fermentation tanks filled with sterilized 0.8 liter of fermentation culture medium in an inoculation amount of 5% by volume, and fermenting for 36 hours at the temperature of 50 ℃ at the fermentation tank stirring speed of 500 r/min and the ventilation amounts of 0.5vvm, 1.0vvm and 1.5vvm respectively.

Sampling every 3 hours during fermentation, and detecting OD_600nm. The samples were centrifuged at 8,000 Xg for 10 minutes and the supernatant assayed for acetoin and glucose concentrationAdding glucose dry powder according to the glucose concentration to maintain the glucose concentration at 20-50 g/L; and (4) performing gas chromatography analysis on the supernatant to determine the concentration of the acetoin in the fermentation liquor, and stopping fermentation when the concentration of the acetoin is not increased any more.

The detection result after 36 hours shows that Bacillus licheniformis delta gdh delta budC delta ldh of the acetoin fermentation of nox has the optimal ventilation condition of 1.0 vvm.

Example 4: production of acetoin by fermentation of a Bacillus licheniformis strain delta gdh delta budC delta ldh in a fermentation medium in a 5L fermenter using a Bacillus licheniformis strain delta gdh delta budC delta ldh

(4) and (3) under the aseptic condition, inoculating the secondary seed bacterial liquid obtained in the step (3) into a 5-liter fermentation tank filled with 4 liters of sterilized fermentation culture medium according to the inoculation amount of 5 percent by volume, stirring the fermentation tank at the rotation speed of 500 r/min at the temperature of 50 ℃, and fermenting for 36 hours with the ventilation quantity of 1.0 vvm.

After 36 hours, the detection result shows that: the concentration of acetoin reaches 57.98 g/l, and the production efficiency is 1.61 g/l/h.

Example 5: production of acetoin by fermentation of Bacillus licheniformis delta gdh delta budC delta ldh with a fermentation medium using a nox strain in a 50L fermenter

Referring to example 1, a wild type Bacillus licheniformis10-1-A strain is used as a starting strain, a glycerol dehydrogenase gdh gene and a 2, 3-butanediol dehydrogenase gene budC are knocked out, a NADH oxidase gene nox in a thermophilic coccus profundus DT5432 is expressed to replace a lactate dehydrogenase ldh, and the strain is named as Bacillus licheniformis delta gdh delta budC delta ldh:: nox.

(3) under the aseptic condition, inoculating the primary seed bacterial liquid obtained in the step (2) into 50mL of LB culture medium in an inoculation amount of 2-4% in volume ratio, and performing shaking culture for 12 +/-1 hours at 50 +/-1 ℃ in a shaking table to obtain a recombinant bacillus licheniformis secondary seed bacterial liquid;

(4) under the aseptic condition, inoculating the secondary seed bacterial liquid obtained in the step (3) into two bottles of 1L LB culture medium in an inoculation amount of 2-4% in volume ratio, and performing shaking culture for 12 +/-1 hours at 50 +/-1 ℃ in a shaking table to obtain a recombinant bacillus licheniformis tertiary seed bacterial liquid;

(5) and (3) under the aseptic condition, inoculating the third-level seed bacterial liquid obtained in the step (4) into a 50-liter fermentation tank filled with 40 liters of sterilized fermentation culture medium in an inoculation amount of 5% by volume, and fermenting for 36 hours at the temperature of 50 ℃ at the stirring rotating speed of 500 r/min and the ventilation quantity of 1.0 vvm.

After 36 hours, the detection result shows that: the concentration of acetoin reaches 82.14 g/l, and the production efficiency is 2.28 g/l/h.

Sequence listing

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ttcgctgttc gcgggattac ccaaactgcg gcaaaagatc tagctgaatt aggtattact 540

gtaaacgcct tttgtccggg tatcgttaaa actcctatga tgatggggat tgcacagcaa 600

accgctgatg aagcaggcaa gccgtttgaa tggggcatgg aacaattcgc taaaaatatt 660

gcattaaaac gcttatccga gccggaagat gtagcagcat gcgtttctta ccttgcaggg 720

ccagattcag attatatgac tggtcaagct cttatcattg atggcggaat ggtatttaat 780

taa 783

<210> 3

<211> 1320

<212> DNA

<213> Thermococcus profundus DT5432

<221> NADH oxidase gene nox sequence

<400> 3

caagtccgtg gagagaaacg aacccttcca cgtccggttt gagtccaaac tcctccggcg 60

agtgcctcag gatataggcc atcagcttgc ttaccctcgt cctctttgat cccatagtaa 120

aaagttgggg agaaaagcta aaaacctcac ggaagctcca ccagagccca caccggcctg 180

tggtctgaaa cctccacgtc acaaaggcag ccgtagtctt taatttcagc cggccagttc 240

ttcttaagca ggatgtagtc tatgttctct ttgtccctga cgccgttcct ctcccagagg 300

aaggtgtaag gcggcctctg ctcaaatgca tccctgtact cgcgggtgag tatctctatc 360

gctttctcgt ccggctcggc gttcgtgtcg ccgagtatta tctccgctat gggtccgctc 420

tccgcgaact tgaggagctc ctcggcctgc atcgccctct cctcttcgct caaacccata 480

tggacgttca cgagggtgag acccaactcc tcgaagctaa ccttctgggc tggcctggcc 540

tgaccgacgc tcttgaggtt gagctcgccc tccgtcttca tgtgccagtg ggagaagacc 600

gctattccgt aggtgccctc cactgcaggc ttgtactcgt aaacgtagcc gaggtaggcc 660

gagagcatga gcgggacgtc ctggtagccg ttgcctatca tcccaccgac gacctcctgg 720

gacgcccaga tgtcaggttt ctgttctttt aggagattaa caagctcgta gccgttgaac 780

ttcccgtcgt aaggcccgaa accctggtgg acgttgtagg tccagatgag aacctctttt 840

ttggccggtt cgtagaccgg agaggcgttg aagatggcta ggacgattat agaggccagg 900

aggaggcccc cgagggaagc cgctatctcc cttacgcttg gaaccctgat ctccaccgac 960

tttccatagg cgctcatggc gtagactacg gatgccgcaa ggatgagggc ctcaagcctg 1020

tcctccatga aagctaaacc gatgtccctg cccacatagg ccccgagggc aagggtcgcg 1080

accaggaaga gatagaccgc tccgatgact cctcccctgc tgcccttgga gctttcgacg 1140

agagctattg aagatgccag cgcaagcggg aggccgatta aagcggccgg ccttacgaag 1200

agcgccgcag agccaaggat cagcaggagt gcggctatgc caggcttttt gcccagatat 1260

gggccgagga ggatagccag tgcgacgacg aacgagaagc cgacgaactg tgggaggtag 1320

<210> 4

<211> 960

<212> DNA

<213> Bacillus licheniformis (Bacillus licheniformis) Δ gdh Δ budC

<221> lactate dehydrogenase Gene ldh sequence

<400> 4

gtgacgcacg gttatttcta tcaaagggcg cgggaaagcg aaaggcgttt ggcacgcagg 60

gcggtcaacg gggagcggct ttggccggca aagttcagtt tgtggtattt cagaccggcg 120

ggggcgtgtc ccgcacagtg gtacaatcag ccgcatgtcg gccgctttaa atcgcattgt 180

ttttacgagc cgacagcgga agagtgtgag aatgtctata atacgttcta acaaaaacat 240

cccttttcaa cggtttttga aaagggatgt ttttcgttca aaataattct gccagtcttt 300

cgtaagactg cttcttatct tcaaaccggt agacgttagt taaaatcata aactcatctg 360

tttgatatcg ttcagaaagc tcgagcagtt ttcttttgac tgtttgagga gacccgatca 420

ccatacggct gcggttttgg gcaagcttct ttttatcggc agctgtgtaa gcggccgatt 480

tcgcctcttc aatgcttggg acaaggctgt ccagcccttt ttcaacccgg agaagccata 540

aatcttggct caagcagagc tcctcggctt tttcatcgga ctccgcacaa acgacgaaaa 600

cggcggccaa tgattggggg cgtttaaaga aaggggaagg ccgaaagctt tcacggtatg 660

attgaaaggc gttctttcct ctcgcgggag agatgaaatg gccgaatacg tatcccgctc 720

ccatgctacc ggccaatttt gcgctgtttt ctcccaggcc taaaagccac acctccgggg 780

gcgaatccgt caaaggggag gctttaattc cgaagtaatc atgtccggaa ggcacggaat 840

cagtcagaaa gctaaccaag tcttgtaact gtctcgggaa ttcgtgcagg cttttatgaa 900

ccccgtctgt cagcgccagc ctcgttttag ttgtgcctcc cggagagcgg ccgatcccca 960

Claims

1. A method for producing acetoin by fermentation of a bacillus licheniformis engineering strain comprises the following steps:

the engineering strain Bacillus licheniformis delta gdh delta budC delta ldh comprises the following steps:

taking a bacillus licheniformis10-1-A genome as a template, obtaining a recombinant fragment containing upstream and downstream homologous sequences of a budC gene by PCR, connecting the recombinant fragment to pKMM 1, transferring the recombinant fragment into bacillus licheniformis delta gdh through parental hybridization, and performing resistance screening on a single-exchange transformant; after double exchange is carried out by temperature induction, a strain with successfully knocked-out budC gene is obtained by PCR screening, and the strain is named as Bacillus licheniformis delta gdh delta budC;

taking a bacillus licheniformis10-1-A genome as a template, and obtaining a sequence containing upstream and downstream homologous arms of an ldh gene by PCR; using plasmid pUC57-nox carrying NADH oxidase synthesized by whole gene as a template, obtaining a nox sequence by PCR, recombining an upstream and downstream homologous arm sequence of an ldh gene and the nox sequence by PCR to obtain a gene replacement recombinant fragment, connecting the gene replacement recombinant fragment to pKMV 1, transferring the gene replacement recombinant fragment into Bacillus licheniformis delta gdh delta budC by parental hybridization, and screening a single-exchange transformant by resistance; after double exchange is carried out by temperature induction, an engineering strain with a nox gene successfully replacing an ldh gene is obtained by PCR screening, and the engineering strain is named as Bacillus licheniformis delta gdh delta budC delta ldh as shown in the specification, nox;

the method is characterized in that:

the fermentation conditions of the step (2) are as follows: the culture temperature is 50 +/-1 ℃, the culture mode is stirring culture, the stirring rotation speed is 500 +/-50 revolutions per minute, the rotation radius is 33 +/-1 mm, the ventilation volume is 1.0 +/-0.1 vvm, and the culture time is 18-36 hours; the fermentation medium comprises the following components: 50-80 g/L of glucose, 12 g/L of yeast powder, 6.5 g/L of anhydrous sodium acetate, 1 g/L of ammonium citrate, 2g/L of dipotassium hydrogen phosphate, 0.25 g/L of magnesium sulfate heptahydrate, 10 ml/L of metal ion mother liquor and the balance of water; the pH was 7.0; the formula of the metal ion mother liquor is as follows: ferrous sulfate 2.25 g/L, zinc sulfate 0.75 g/L, manganese sulfate 0.38 g/L.

2. The method for producing acetoin by fermentation of the engineered strain of bacillus licheniformis according to claim 1, wherein the fermentation conditions in the step (2) are: the culture temperature is 50 ℃, the culture mode is stirring culture, the stirring rotation speed is 500 r/min, the rotation radius is 33 mm, the ventilation volume is 1.0vvm, and the culture time is 25-36 hours.