CN-120591181-B - Genetically engineered strain for high-yield acetoin and 2, 3-butanediol, construction method and application

CN120591181BCN 120591181 BCN120591181 BCN 120591181BCN-120591181-B

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a genetic engineering strain for high-yield acetoin and 2, 3-butanediol, a construction method and application thereof. According to the invention, through knocking out the regulatory gene acoR of an acetoin dehydrogenase enzyme system, a acoR deletion mutant MW 03-delta A is obtained, the degradation of AoDHES to acetoin is blocked, the generation of byproducts is effectively reduced, so that more acetoins can be accumulated, a strong promoter expression vector is constructed in the MW 03-delta A strain, an alpha-acetolactate decarboxylase encoding gene alsD is connected, and the alpha-acetolactate decarboxylase encoding gene alsD is introduced into a Bacillus licheniformis acoR deletion mutant MW 03-delta A, so that the alpha-acetolactate decarboxylase is overexpressed, and a genetically engineered strain MW-BL4 for high yield of acetoin and 2, 3-butanediol is obtained. Compared with engineering strains with single products, the invention can realize the production of various products such as acetoin, 2, 3-butanediol and the like by using one strain, has considerable yield, can obviously reduce the strain cost in industrial production and improves the production benefit.

Inventors

MENG WU
WANG LIQIAN
GAO YINHAO
QIN WEISHUAI
ZHANG NA

Assignees

齐鲁工业大学(山东省科学院)

Dates

Publication Date: 20260508
Application Date: 20250425

Claims (3)

1. The genetically engineered strain is characterized in that the genetically engineered strain is preserved in China center for type culture collection, the preservation address is China university of Wuhan, the preservation date is 2025, the date is 03 months and 31 days, the preservation number is CCTCC NO: M2025653, and the genetically engineered strain is classified and named as bacillus licheniformis MW-BL4 Bacillus licheniformis MW-BL4.
2. The use of the genetically engineered strain of claim 1 for the production of 2, 3-butanediol and acetoin.
3. The use according to claim 2, wherein the fermentation conditions for producing 2, 3-butanediol and acetoin by the genetically engineered strain are as follows: The fermentation temperature is 35-38 ℃, the pH is 6.2-7.8, the inoculum size of the seed liquid is 8% -12%, the constant temperature culture is carried out at the rotating speed of 180-250 r/min, the sterile glucose solution is added from the 12 th h during the fermentation period, then 10 mL of 70g/L of the sterile glucose solution is added every 12h, and the fermentation period is 3-5 days.

Description

Genetically engineered strain for high-yield acetoin and 2, 3-butanediol, construction method and application Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to a genetic engineering strain for high-yield acetoin and 2, 3-butanediol, a construction method and application thereof. Background With the increasing severity of global energy crisis and the gradual exhaustion of petroleum resources, the search for alternative chemical production methods is an important topic in the current chemical industry field. The traditional chemical production depends on petroleum-based raw materials, and the production mode not only generates huge pressure on the environment, but also aggravates the consumption of petroleum resources, so that development of sustainable production technology independent of petroleum resources is of great significance for realizing environmental friendliness and efficient utilization of resources. 2, 3-Butanediol (2, 3-BD) and acetoin are two chemicals with wide application prospects, and have wide application in the fields of foods, medicines, agriculture, chemical industry and the like. However, the traditional production methods of 2, 3-butanediol and acetoin mainly depend on fossil resources, so that the energy consumption is high, the pollution is serious, and the risk of resource exhaustion is also faced. Microbial fermentation is becoming an important way to produce these high-value chemicals by virtue of its low cost, low energy consumption, environmental friendliness, etc. as an alternative production process. 2, 3-butanediol, acetoin and other high-value chemicals are produced by a microbial fermentation method, so that dependence on petroleum resources can be effectively reduced, powerful support can be provided for environmental protection and efficient resource utilization, and important economic value and social significance are achieved. Disclosure of Invention In order to solve the technical problems, the invention provides a genetic engineering strain for high-yield acetoin and 2, 3-butanediol, a construction method and application thereof. According to the invention, the acoR deletion mutant MW 03-delta A is obtained by knocking out the positive regulation Gene acoR of the acetoin dehydrogenase system (AoDHES) operon aco, so that the degradation of AoDHES to acetoin is blocked, the generation of byproducts is effectively reduced, more acetoin can be accumulated, a strong promoter expression vector is further constructed in the MW 03-delta A strain, and the Gene encoding alpha-acetolactate decarboxylase alsD (Gene ID: 936857) is connected and then introduced into the Bacillus licheniformis acoR deletion mutant MW 03-delta A, so that the alpha-acetolactate decarboxylase (ALDC) is overexpressed, and a genetically engineered strain MW-BL4 for high yield of acetoin and 2, 3-butanediol is obtained. The first aspect of the invention provides a genetically engineered strain for high-yield of acetoin and 2, 3-butanediol, wherein the genetically engineered strain is preserved in China center for type culture collection, the preservation address is China university of Wuhan, the preservation date is 2025 and 31 days 03, the preservation number is CCTCC NO: M2025653, and the genetically engineered strain is classified and named as bacillus licheniformis MW-BL4 Bacillus licheniformis MW-BL4. The second aspect of the invention provides a construction method of the genetic engineering strain, which comprises the following steps: S1 acoR deletion mutant strain MW 03-delta A is constructed by taking Bacillus licheniformis B.lichenifermis MW03 (source: microbial enzyme technology laboratory of the university of Qilu university student engineering department) as an original strain, knocking out acetoin dehydrogenase regulatory gene acoR in the Bacillus licheniformis to obtain a acoR deletion mutant strain which is named MW 03-delta A; S2, constructing a genetic engineering strain MW-BL4, namely connecting a target gene alpha-acetolactate decarboxylase encoding gene alsD to an expression vector to obtain a recombinant plasmid, and introducing the recombinant plasmid into acoR deletion mutant strain MW 03-delta A constructed in S1 to obtain the genetic engineering strain named MW-BL4. Preferably, in S2, the recombinant plasmid is prepared by synthesizing a promoter Plaps fragment and a target gene alsD, obtaining a target gene fragment Plaps +alsD by using a primer alsD-F/alsD-F, and connecting the gene fragment to an expression vector pHY300PLK to obtain a recombinant plasmid pHY300PLK-Plaps-alsD over-expressing the gene alsD. The third aspect of the invention provides application of the genetically engineered strain in the production of 2, 3-butanediol and acetoin. Preferably, the fermentation conditions for producing 2, 3-butanediol and acetoin by the genetically engineered strain are as follows: The fermentation temperature is 35-