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CN-122012361-A - Penicillium beliae bacillus resistant genetically engineered bacterium, and preparation method and application thereof

CN122012361ACN 122012361 ACN122012361 ACN 122012361ACN-122012361-A

Abstract

The invention belongs to the technical field of genetically engineered bacteria, and particularly discloses a genetically engineered bacteria resistant to bacillus penicillium and preparation method and application thereof. The genetically engineered bacterium is a cluster of polyketide synthase gene clusters in the genome of bacillus beleiensis, wherein the gene clusters comprise one cluster of PKS5, PKS6 and PKS10 gene clusters. Through specific knocking out polyketide synthase gene clusters competing with target products, metabolic flows are redistributed while the excellent growth performance of the strain is maintained, the yield of anti-penicillium active substances is remarkably improved, and a new technical scheme is provided for solving the bottleneck of low strain titer in the existing biological control technology. Compared with the original strain, the method strengthens the utilization efficiency of nutrient substances and the adaptability to fermentation environment in industrial production, improves the inhibition effect on penicillium by 10.4 percent, and has good application prospect in the aspect of improving the yield of industrial anti-penicillium active substances.

Inventors

  • ZHANG HUITU
  • LU FUPING
  • GUO QINGPING
  • WANG HAIKUAN

Assignees

  • 天津科技大学

Dates

Publication Date
20260512
Application Date
20260213

Claims (10)

  1. 1. A genetically engineered strain against bacillus penicillium, wherein a cluster of polyketide synthase gene clusters in the genome of bacillus penicillium is knocked out or inactivated, said gene clusters comprising one of PKS5, PKS6, PKS10 gene clusters.
  2. 2. The genetically engineered strain of bacillus penicillium resistance according to claim 1, wherein the knocked out or inactivated gene cluster is PKS6.
  3. 3. The genetically engineered strain of bacillus penicillium resistance as claimed in claim 1, wherein the bacillus penicillium has a preservation number of CGMCC No.36999.
  4. 4. The genetically engineered strain of anti-Bacillus beleiensis according to claim 3, wherein the whole genome sequence position code of Bacillus beleiensis having a accession number of CGMCC No.36999 is registered in GenBank, the pks5 gene cluster is located at 1426080-1514310 th position of the whole genome sequence, the pks6 gene cluster is located at 1735334-1845433 th position of the whole genome sequence, and the pks10 gene is located at 2475831-2496721 th position of the whole genome sequence.
  5. 5. The method for constructing genetically engineered bacteria against bacillus penicillium as claimed in claim 4, comprising the steps of: Providing bacillus belgium according to claim 3; designing a gene manipulation element for achieving knocking out or inactivation of a polyketide synthase gene cluster in the bacillus bailii genome; Introducing the gene manipulation element into the bacillus beljalis such that the polyketide synthase gene cluster is knocked out or inactivated; obtaining the bacillus bailii genetically engineered bacterium in which the polyketide synthase gene cluster is knocked out or inactivated.
  6. 6. The method of construction of claim 5, wherein the gene operational element comprises a homologous recombination fragment, a CRISPR/Cas gene editing system, a transposon element, or a combination thereof.
  7. 7. The method of claim 5, wherein the polyketide synthase gene cluster is one of PKS5, PKS6, and PKS10 gene clusters.
  8. 8. A fermentation method of the genetically engineered bacteria resistant to bacillus penicillium as claimed in any one of claims 1 to 4, which is characterized by comprising the steps of inoculating the genetically engineered bacteria to a culture medium for fermentation culture to obtain a target fermentation product, wherein the culture medium comprises 8-12 g/L of corn starch, 20-30 g/L of yeast extract, 0.001-0.01 g/L of manganese sulfate, 0.3-0.7 g/L of monopotassium phosphate and pH 7.0-8.0.
  9. 9. The fermentation method according to claim 8, wherein the amount of inoculation is 2% -5% of the culture medium, and fermentation is performed at 30 ℃ -37 ℃ for 48-72 hours.
  10. 10. Use of a genetically engineered bacillus belicus as defined in any one of claims 1 to 4 or a fermentation product obtained by a fermentation process as defined in any one of claims 8 to 9 for the preparation of a microbial preparation for inhibiting the growth of penicillium.

Description

Penicillium beliae bacillus resistant genetically engineered bacterium, and preparation method and application thereof Technical Field The invention relates to the technical field of genetically engineered bacteria, in particular to genetically engineered bacteria resistant to bacillus penicillium and preparation method and application thereof. Background Mold growth is one of the core causes of spoilage and deterioration of fruits, vegetables, grains and various foods, and is directly related to daily life and diet safety of people. The mould has extremely strong reproductive capacity, the reproductive process is mainly completed through diversified asexual spores or sexual spores, the spore quantity is huge, the method is a key factor for realizing quick propagation and large-scale propagation, and more importantly, part of mould can generate mycotoxins with carcinogenicity or other toxicity in the growth process, and once the mycotoxins enter a human body along with food, the body health of people is seriously threatened. In recent years, problems of fungal contamination of agricultural products and foods, particularly, contamination with mold and mycotoxin have been receiving a great deal of attention worldwide, and mold control has become one of the key research subjects in the fields of food hygiene and feed hygiene. According to the review of Li Can in research progress of mycotoxin degradation method in feed, the current methods for controlling mold and toxin mainly comprise physical method, chemical method and biological method. Limitations of the prior art: 1. physical methods (e.g., adsorbents, heat treatments) are widely used, but often involve non-specific loss of feed nutrients (e.g., vitamins, minerals) and adsorption efficiency often fluctuates with the type of toxin. 2. Chemical methods (such as ammoniation and oxidation) have quick effect, but are extremely easy to destroy the palatability of the feed, and residual chemical reagents can bring potential food safety hazards and cause secondary pollution. 3. Biological methods (using microorganisms or enzymes) are considered to be the most promising because of their strong specificity, safety and no residues. However, most of the biological agents currently used are wild-type strains (e.g., wild-type bacillus, yeast, etc.), and a large number of unnecessary secondary metabolic gene clusters exist in the genome. The redundant gene clusters not only do not generate target antibacterial substances, but also competitively consume substrate and cell energy, so that the synthesis efficiency of the target anti-mildew active substances is low, the yield is unstable, and the industrial low-cost and high-efficiency application requirements are difficult to meet. Disclosure of Invention The invention aims at overcoming the defects of the prior art, and provides a genetically engineered strain for resisting penicillium bacillus, a preparation method and application thereof, wherein cell resources are redistributed by knocking out redundant polyketide synthase (PKS) gene clusters in genome, and the synthesis capability of the strain for resisting penicillium active substances is obviously improved by combining with an optimized fermentation process. In order to achieve the above purpose, the present invention adopts the following technical scheme: In a first aspect, the present invention provides a genetically engineered bacterium against bacillus penicillium in which a cluster of polyketide synthase gene clusters in the genome of bacillus penicillium is knocked out or inactivated, the gene clusters comprising one of the PKS5, PKS6, PKS10 gene clusters. Further, the gene cluster that is knocked out or inactivated is PKS6. Further, the preservation number of the bacillus belicus is CGMCC No.36999. Further, the bacillus belicus is preserved in China general microbiological culture collection center (CGMCC) for 12 months and 10 days in 2025, the address of a preservation unit is North Star Xiyu No. 1 and No. 3 in the Korean area of Beijing city, and the bacillus belicus is classified and named as bacillus belicus Bacillus velezensis, and the preservation number is CGMCC No. 36999. Further, the whole genome sequence position of the bacillus beleiensis is encoded, the pks5 gene cluster is positioned at 1426080-1514310 th position of the whole genome sequence, the pks6 gene cluster is positioned at 1735334-1845433 th position of the whole genome sequence, and the pks10 gene is positioned at 2475831-2496721 th position of the whole genome sequence. The second aspect of the invention provides a construction method of genetically engineered bacteria resistant to bacillus penicillium, comprising the following steps: Providing the bacillus beleiensis; designing a gene manipulation element for achieving knocking out or inactivation of a polyketide synthase gene cluster in the bacillus bailii genome; Introducing the gene manipulation element into the bacillus beljalis