Method for producing acetoin through high-efficiency fermentation by appropriately expressing novel bacillus subtilis NADH oxidizing enzyme

A technology of Bacillus subtilis and oxidase, which is applied in the fields of genetic engineering and fermentation engineering to achieve the effect of shortening the fermentation period

Inactive Publication Date: 2014-09-03
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Method for producing acetoin through high-efficiency fermentation by appropriately expressing novel bacillus subtilis NADH oxidizing enzyme
  • Method for producing acetoin through high-efficiency fermentation by appropriately expressing novel bacillus subtilis NADH oxidizing enzyme
  • Method for producing acetoin through high-efficiency fermentation by appropriately expressing novel bacillus subtilis NADH oxidizing enzyme

Examples

Experimental program
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Embodiment 1

[0016] Example 1: Construction of bdhA gene-deleted strains by means of genetic engineering

[0017] Construct the bdhA gene fragment of the reporter gene insertion inactivation, use primers P1 and P2 to clone the complete expression gene fragment of the cat gene from the pBGSC6 plasmid, then use EcoR47III to treat the fragment recovered from the gel, and finally treat the gene fragment with alkaline phosphatase and remove Connect with T-bdhA after the same treatment, transform Escherichia coli JM109, and screen positive clones on ampicillin and chloramphenicol double resistance plates. The constructed T-bdhA::cat was transformed into B. subtilis JNA, and the bdhA gene-deleted recombinant strain was screened on a chloramphenicol plate. Colony PCR and enzyme activity verification experiments were performed to confirm whether the bdhA gene was successfully inserted and inactivated.

[0018] P1: 5'-CGCAGCGCTAAAAAAGGATTGATTCTAATG-3' (Eco47III)

[0019] P2: 5'-CGCAGCGCTTAGTGACATTA...

Embodiment 2

[0021] Example 2: NADH oxidase primer design

[0022] First, using the chromosomal DNA of the bacterial strain B. subtilis JNA as a template, using primers P3, P4, P5, P6, P7, P8, P9 and P10, a section of ydfN, ydgI, yodC and yfhC genes were respectively amplified by PCR technology, After the purified ydfN, ydgI, yodC and yfhC genes were digested by restriction endonucleases MluI and BamHI, they were connected with the plasmid pMA5 digested by the above two restriction endonucleases to construct the recombinant plasmid pMA5-bdhA. 4 Under the action of DNA ligase, connect overnight at 16°C, transform the ligation liquid into E. coli competent E.coli JM109, pick positive transformants, extract the plasmids in the transformants, and confirm the recombinant plasmid pMA5-ydfN by enzyme digestion , pMA5-ydgI, pMA5-yodC and pMA5-yfhC were constructed successfully. After verification by double enzyme digestion, it indicated that the recombinant plasmid was constructed successfully. ...

Embodiment 3

[0032] Embodiment 3: NADH oxidase activity assay

[0033] The recombinant bacteria B.subtilis JNA / pMA5-ydfN constructed in Example 4, B.subtilis JNA / pMA5-ydgI, B.subtilis JNA / pMA5-yodC and B.subtilis JNA / pMA5-yfhC, and the starting strain B.subtilis JNA were respectively inoculated in 10 mL of LB medium containing kanamycin, cultured with shaking at 37°C overnight, transferred to LB medium at 4% inoculum the next day, cultured at 37°C for 24 hours, and the fermentation broth was taken at 4°C. Centrifuge at 10000r / min for 10min, wash with pH7.0 sodium phosphate buffer for 3 times, resuspend the cells in pH6.5 sodium phosphate buffer, then place the liquid in an ultrasonic breaker to treat the cells for 20min, 15000r·min -1 Centrifuge for 30 minutes, and the supernatant is the crude enzyme solution. Add 20 μl of crude enzyme solution to the enzyme activity assay buffer system to detect A immediately 340 Changes in absorbance.

[0034] Determination of NADH oxidase activity: T...

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Abstract

The invention relates to a method for producing acetoin through high-efficiency fermentation by appropriately expressing a novel bacillus subtilis NADH oxidizing enzyme and belongs to the fields of genetic engineering and fermentation engineering. According to the method, the noval bacillus subtilis NADH oxidizing enzyme is found for the first time in genome of a high-yield acetoin bacterial strain B.subtilis JNA which is autonomously screened by a laboratory and has independent intellectual property, the enzymatic property of the noval bacillus subtilis NADH oxidizing enzyme is determined while an NADH oxidizing enzyme is expressed in bdhA gene deleted bacillus subtilis by appropriately expressing a carrier pMA5-PbdhA, finally the acetoin is produced through fermentation by utilizing recombinant bacteria modified by metabolic engineering, about 56.7g/L acetoin and 1.2g/L of 2,3-butanediol are obtained, the contents of 2,3-butanediol, lactic acid and acetic acid are respectively reduced by about 92.3%, 70.1% and 75.0%, the production efficiency of the acetoin is increased to 0.68g/(L.h), and a fermentation period is shortened for 1.7 times compared with the original bacterial strain; NADH regulation in bacillus subtilis is utilized for producing the acetoin for the first time at home and abroad, and the aims of shortening the fermentation period and reducing a NADH-dependent by-product are realized.

Description

technical field [0001] The method utilizes moderately expressed novel NADH oxidase to reduce NADH-dependent by-products and increase the yield of acetoin. The invention belongs to the fields of genetic engineering and fermentation engineering. It specifically relates to the screening of a novel NADH oxidase, the construction of genetically engineered bacteria and its fermentation production. technical background [0002] Acetoin occurs naturally in corn, grapes, cocoa, apples, bananas, cheese, meat, and many other foods. It is a widely used and lovable food spice, with a strong creamy, fat, butter-like aroma, and a pleasant milky aroma after being highly diluted, so acetoin is mainly used to prepare milk-flavored, meat-flavored type, strawberry-flavored flavor, or directly used in dairy products. my country's national standard GB2760-86 clearly stipulates that it is a food-grade spice that is allowed to be used, and the safety number of the American Food and Extraction Ass...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P7/26C12N9/02C12R1/125
Inventor 饶志明张显包腾赵晓静杨套伟徐美娟
Owner JIANGNAN UNIV
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