CN-121991858-A - Bacillus amyloliquefaciens for producing Fengyuan by using corn steep liquor and application thereof

Abstract

Bacillus amyloliquefaciens for producing Fengyuan by using corn steep liquor and application thereof belong to the technical fields of microbial fermentation and genetic engineering. In order to solve the technical problems that the existing strain for producing the Fengyin by fermentation has too high cost of a culture medium and can not utilize corn steep liquor to efficiently synthesize Feng Yuan of the Fengyin, bacillus amyloliquefaciens TF28 is subjected to random mutation, a bacillus amyloliquefaciens mutant strain with strong growth capacity in corn steep liquor is obtained by screening, and an original promoter is replaced by a strong promoter in series connection on the basis of the mutant strain, so that the expression of a Fengyin synthesis operon is directionally enhanced. The transformed bacillus amyloliquefaciens engineering bacteria can effectively utilize the corn steep liquor to grow and synthesize high-level Fengyuan, and are expected to be used for industrial production of the Fengyuan.

Inventors

• Yan Gengxuan
• ZHOU LU
• XU YAN
• SUN MENG
• WANG ZIXUAN
• TIAN YUAN
• YU CHONG
• XIA HAIHUA

Assignees

• 黑龙江省科学院微生物研究所

Dates

Publication Date

20260508

Application Date

20260325

Claims (10)

1. 1. The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) TF29 is characterized in that the preservation number is CGMCC No.37503.
2. 2. Use of bacillus amyloliquefaciens TF29 according to claim 1 for the production of Feng Yuan th element using corn steep liquor.
3. 3. The use according to claim 2, wherein the corn steep liquor is prepared by concentrating wastewater from a corn steep liquor process, and comprises the main components of 15% nitrogen compounds, 10% reducing sugars, 7% lactic acid, 4% lactose and 5% ash.
4. 4. The method according to claim 3, wherein the fermentation is carried out using a fermentation medium containing corn steep liquor, the fermentation medium containing corn steep liquor comprises, by mass, 4% glucose, 2% fructose, 8% corn steep liquor, 2.21% magnesium sulfate, 0.1% calcium chloride, 0.1% manganese sulfate, 1.5% potassium dihydrogen phosphate, 3% dipotassium hydrogen phosphate, and the balance water, and the pH is adjusted to 7.5.
5. 5. The method according to claim 4, wherein the seed solution is prepared by inoculating a seed culture medium with a bacterial solution of Bacillus amyloliquefaciens TF29, and culturing the seed solution, and further inoculating the seed solution into a fermentation culture medium containing corn steep liquor for fermentation culture.
6. 6. The use according to claim 5, wherein the seed medium has a composition of 2% tryptone, 0.5% yeast extract, 10 mM sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium chloride, 10 mM magnesium sulfate, 20 mM dextrose, the balance being water.
7. 7. The use according to claim 5, wherein the seed culture conditions are 37 ℃ and 200 r/min culture 16-24 h.
8. 8. The use according to claim 5, wherein the seed culture conditions are 37 ℃ and 200 r/min culture 16 h.
9. 9. The use according to claim 5, wherein the conditions of the fermentation culture are 37 ℃ and 200 r/min culture 48 h.
10. 10. The use according to claim 5, wherein the seed liquid is inoculated in an amount of 10% in the fermentation culture.

Description

Bacillus amyloliquefaciens for producing Fengyuan by using corn steep liquor and application thereof Technical Field The invention belongs to the technical field of microbial fermentation and genetic engineering, and particularly relates to bacillus amyloliquefaciens for producing Fengyuan by using corn steep liquor and application thereof. Background Fengycin (English name Fengycin) is a lipopeptide with strong antifungal activity, is formed by combining lipid molecules with amino acids through chemical bonds, and bacillus is the most common synthetic source of Fengycin. At present, the Fengyuan can only be synthesized by a biological fermentation method, depends on carbon and nitrogen sources such as glucose, peptone and the like, has the yield limited by the levels of thallus fatty acid and amino acid, has quite high fermentation cost and has quite high production difficulty. The corn steep liquor is the waste remained after corn starch is produced by corn, has low price, contains a large amount of nutrient substances such as short peptide, amino acid and the like and rich nitrogen sources, and can meet the requirement of bacterial growth and metabolism. However, due to the limitation of the characteristics of the strain, the conventional bacillus can only utilize a few corn steep liquor nutrient components for synthesizing the Fengyuan, the prior art does not systematically analyze the transportation and synthesis paths of the strain for producing the Fengyuan by utilizing the nutrient substances in the corn steep liquor, and a targeted strain transformation strategy is also lacking. Therefore, the construction of the bacillus engineering bacteria capable of producing the Fender through the corn steep liquor has important significance for realizing the industrial production of the Fender. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) TF28 is a bacillus capable of synthesizing Fengyuan at a high level, and the inventor modifies the bacillus in the earlier research to remarkably improve the yield of Feng Yuan element produced by fermentation (see patent number ZL202210288093.7, and the invention name is a Bacillus amyloliquefaciens engineering bacterium for producing Fengyuan as well as a construction method and an applied Chinese invention patent). However, the Bacillus amyloliquefaciens engineering bacteria obtained in the above patent promote the synthesis of all types of non-ribosomal synthetic peptides by strengthening the expression of global regulatory factors so as to indirectly improve the yield of Feng Yuan elements and not specifically improve the yield of Feng Yuan elements, and meanwhile, when the Bacillus amyloliquefaciens engineering bacteria is used for fermenting and producing the Fengyuan element, yeast extract, beef extract, ammonium sulfate and the like are still used as composite nitrogen sources in the early stage, so that excessive waste of raw material resources is caused. At present, no technology improvement method for effectively utilizing a strain for synthesizing the Fengyuan through corn steep liquor is reported. Disclosure of Invention In order to solve the technical problems that the existing strain for producing the Fengyin by fermentation has too high cost of a culture medium and can not utilize corn steep liquor to efficiently synthesize Feng Yuan of the Fengyin, bacillus amyloliquefaciens TF28 is subjected to random mutation, a bacillus amyloliquefaciens mutant strain with strong growth capacity in corn steep liquor is obtained by screening, and an original promoter is replaced by a strong promoter in series connection on the basis of the mutant strain, so that the expression of a Fengyin synthesis operon is directionally enhanced. The transformed bacillus amyloliquefaciens engineering bacteria can effectively utilize the corn steep liquor to grow and synthesize high-level Fengyuan. In order to solve the technical problems and realize the corresponding technical effects, the invention provides the following technical scheme: The first object of the present invention is to provide a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) TF29 which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.37503, the preservation date of 2026, 01 month and 22 days, and the preservation unit address of North Star Xiyang area of Beijing, 1 st West, 3 rd, and the institute of microbiology of China academy of sciences. A second object of the present invention is to provide the use of the above Bacillus amyloliquefaciens TF29 for producing Feng Yuan elements using corn steep liquor. In one embodiment of the invention, the corn steep liquor is prepared by concentrating wastewater generated in a corn soaking process, and the main components of the corn steep liquor comprise 15% of nitrogen-containing compounds, 10% of reducing sugar, 7% of lactic acid, 4% of lactose and 5% of ash. In one embodiment of the invention