Method for producing acetoin by efficient bioconversion of 2,3-butanediol by using Bacillus subtilis nicotinamide adenine dinucleotide (NAD)<+> regeneration system

A technology of acetoin and butanediol, applied in the fields of genetic engineering and bioengineering, can solve the problems of low accumulation concentration, difficult separation in downstream engineering, inability to obtain pure acetoin, etc., and achieves efficient production, cost reduction, and simplified separation Effect

Inactive Publication Date: 2014-09-03
JIANGNAN UNIV
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Problems solved by technology

At present, the research on the production of acetoin by fermentation is mostly focused on screening wild strains and optimizing the fermentation medium or process, while there are relatively few reports on genetically engineered strains, and in the metabolic process of most strains, acetoin is It exists as a by-product of the metabolism of 2,3-butanediol and diacetyl, and the accumulation concentration is low. In addition, many by-products are produced during the fermentation process, such as acetic acid, lactic acid, ethanol and 2,3-butanediol. Thereby directly cause the downstream project to be difficult to separate, finally can't obtain the higher purity acetoin

Method used

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  • Method for producing acetoin by efficient bioconversion of 2,3-butanediol by using Bacillus subtilis nicotinamide adenine dinucleotide (NAD)&lt;+&gt; regeneration system
  • Method for producing acetoin by efficient bioconversion of 2,3-butanediol by using Bacillus subtilis nicotinamide adenine dinucleotide (NAD)&lt;+&gt; regeneration system
  • Method for producing acetoin by efficient bioconversion of 2,3-butanediol by using Bacillus subtilis nicotinamide adenine dinucleotide (NAD)&lt;+&gt; regeneration system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1: Amplification of target gene and construction of recombinant Bacillus subtilis

[0015] The schematic diagram of the construction of plasmid pMA5-bdhA-yodC is as follows figure 1 As shown, the specific process is as follows:

[0016] First, using the chromosomal DNA of the bacterial strain B.subtilis JNA as a template, using primers P1 and P2, a nucleotide sequence of 1041 bp is amplified by PCR to obtain a bdhA gene as shown in SEQ ID NO: 1, and the purified After the bdhA gene was digested by restriction endonucleases MluI and BamHI, it was connected with the plasmid pMA5 digested by the above two restriction endonucleases to construct the recombinant plasmid pMA5-bdhA. Plasmid construction was successful. Subsequently, using primers P3 and P4 to obtain the gene yodC whose nucleotide sequence is shown in SEQ ID NO: 2 by PCR amplification, after the purified yodC gene was digested by restriction endonucleases MluI and BamHI, the same process as describe...

Embodiment 2

[0025] Embodiment 2: Determination of recombinant strain enzyme activity

[0026] (1) Enzyme activity assay buffer system

[0027] Acetoin reductase: 0.05M acetoin, 50mM sodium phosphate buffer pH6.5, 5mM NAD + ;

[0028] 2,3-butanediol dehydrogenase: 0.1M 2,3-butanediol, 50mM sodium phosphate buffer pH8.0, 5mM NADH;

[0029] NADH oxidase: 50mM potassium phosphate buffer pH7.0, 0.3mM EDTA, 50μM FAD, 0.3mMβ-NADH;

[0030] (2) Determination of enzyme activity

[0031] The recombinant bacteria B.subtilis JNA / pMA5-bdhA, B.subtilis JNA / pMA5-yodC and B.subtilis JNA / pMA5-bdhA-yodC constructed in Example 1 were inoculated with the starting strain B.subtilis JNA in 10 mL of card-containing In the LB medium of namycin, shake culture overnight at 37°C, transfer to LB medium the next day at 4% inoculum size, culture at 37°C for 24 hours, take the fermentation broth at 4°C, centrifuge at 10000r / min for 10min, pH7 .0 sodium phosphate buffer, washed three times, suspended in pH 7.0 sodi...

Embodiment 3

[0032] Example 3: Determination of intracellular cofactor levels and whole cell viability of recombinant bacterial strains

[0033] The intracellular cofactor levels of the recombinant strains were detected using the AAT Bioquest reagent kit. Detect NADH, NAD+ concentration and NADH / NAD at Ex / Em=540 / 590nm by fluorescent microplate reader + Proportion.

[0034] The acetoin generated by the reaction system was used to detect the transformation activity of the whole cells. The recombinant cells were suspended in 50 mM phosphate buffer (pH8.0) containing 40 g / L 2,3-butanediol, and reacted on a shaking table at 37 ° C for 20 minutes. Reaction solution is centrifuged 1min with the rotating speed of 12000rpm, measures the content of acetoin in the supernatant, and measuring system is as follows: 0.5mL creatine (0.5%w / v), 0.5mLα-naphthol (5%w / v is dissolved in 95% ethanol), 0.5mL potassium hydroxide (10%w / v), 0.01mL centrifuged supernatant and 3.5mL deionized water, put the measurem...

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Abstract

The invention discloses a method for producing acetoin by efficient bioconversion of 2,3-butanediol by using a Bacillus subtilis nicotinamide adenine dinucleotide (NAD)<+> regeneration system, and belongs to the field of genetic engineering. Acetoin reductase and educed form of nicotinamide-adenine dinucleotide (NADH) oxidase in a high-yield acetoin strain B.subtilis JNA with independent intellectual property rights, which are autonomously screened from a laboratory are cloned by the method disclosed by the invention, and excessive coexpression of the acetoin reductase and NADH oxidase in B.subtilis JNA is carried out, so that the production of acetoin by effective conversion of the 2,3-butanediol in the wild-type high-yield acetoin bacillus subtilis by virtue of the NAD<+> regeneration system is realized for the first time at home and abroad. The enzyme activity determination and intracellular coenzyme level research on the built gene engineering strain prove that the 2,3-butanediol can be lastingly and effectively converted by B.subtilis JNA/pMA5-bdhA-yodC to produce the acetoin. 120g/L of 2,3-butanediol can be finally converted into about 92.5g/L of acetoin by the B.subtilis JNA/pMA5-bdhA-yodC when the temperature is 40 DEG C and the pH is 8.0 under the optimal whole-cell conversion condition of adding 5mM of MnC12, the acetoin yield can be up to 2.31g/(L.h), and is the highest lever for producing the acetoin from the bacillus subtilis in the current report, and a foundation is provided for industrial production of the acetoin from microorganisms.

Description

technical field [0001] Utilize NAD + The invention relates to a method for efficiently biotransforming 2,3-butanediol to produce acetoin in a regeneration system, and the invention belongs to the fields of genetic engineering and bioengineering. Specifically, it relates to the construction of a genetically engineered bacterial strain co-expressed with acetoin reductase and NADH oxidase and a method for using the bacterial strain to transform 2,3-butanediol into whole cells to produce acetoin. technical background [0002] Acetoin has a wide range of applications in food flavoring, biochemistry and pharmacology. At present, there are relatively in-depth researches on its synthesis and production at home and abroad. In recent years, the production of acetoin by microbial fermentation has received more and more attention, and scientists from various countries have carried out work in this area one after another. At present, the research on the production of acetoin by ferment...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12P7/26C12R1/125
Inventor 包腾饶志明张显赵晓静杨套伟徐美娟
Owner JIANGNAN UNIV
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