Search

CN-122012539-A - Fatty acid beta-oxidation pathway coding gene from aspergillus nidulans and application thereof

CN122012539ACN 122012539 ACN122012539 ACN 122012539ACN-122012539-A

Abstract

The invention discloses a fatty acid beta-oxidation pathway coding gene from Aspergillus nidulans and application thereof, wherein the coding gene is selected from the group consisting of (a) Aspergillus0G048390 gene with a nucleotide sequence shown as SEQ ID NO. 1, (b) Aspergillus0G044330 gene with a nucleotide sequence shown as SEQ ID NO. 3, and/or (c) Aspergillus0G004350 gene with a nucleotide sequence shown as SEQ ID NO. 5. The engineering strain constructed by the invention has remarkable echinocandin B yield, and in shake flask fermentation, the over-expression strain yield is improved by 33.55%, 25.14% and 25.62% compared with the original strain, so that important technical support is provided for the industrialized high-efficiency production of the echinocandin B, and the engineering strain has remarkable economic value and application prospect.

Inventors

  • LIU ZHIQIANG
  • Kou tong
  • PANG AIPING
  • HUANG LIANGGANG
  • YANG XIAOZHANG
  • ZHENG YUGUO

Assignees

  • 浙江工业大学

Dates

Publication Date
20260512
Application Date
20260211

Claims (10)

  1. 1. A fatty acid beta-oxidation pathway coding gene derived from Aspergillus nidulans is characterized in that the coding gene is selected from the group consisting of (a) Aspergillus0G048390 gene with a nucleotide sequence shown as SEQ ID NO. 1, (b) Aspergillus0G044330 gene with a nucleotide sequence shown as SEQ ID NO. 3, and/or (c) Aspergillus0G004350 gene with a nucleotide sequence shown as SEQ ID NO. 5.
  2. 2. The protein encoded by the gene encoding the beta-oxidation pathway of fatty acids derived from Aspergillus nidulans as claimed in claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 2, SEQ ID NO. 4 and/or SEQ ID NO. 6, wherein SEQ ID NO. 2 corresponds to the protein encoded by the Aspergillus0G048390 gene having the nucleotide sequence shown as SEQ ID NO. 1, SEQ ID NO. 4 corresponds to the protein encoded by the Aspergillus0G044330 gene having the nucleotide sequence shown as SEQ ID NO. 3, and SEQ ID NO. 6 corresponds to the protein encoded by the Aspergillus0G004350 gene having the nucleotide sequence shown as SEQ ID NO. 5.
  3. 3. A recombinant overexpression vector comprising a gene encoding the β -oxidation pathway of fatty acids from aspergillus nidulans as claimed in claim 1, and a promoter and a terminator operably linked.
  4. 4. The recombinant overexpression vector according to claim 3, characterized in that it is obtained by inserting a fatty acid β -oxidation pathway coding gene derived from aspergillus nidulans, based on plasmid pDht-sk, and said coding gene is driven by the constitutive promoter gpdA, terminated by terminator TtrpC.
  5. 5. A genetically engineered bacterium comprising the coding gene of claim 1 or the recombinant overexpression vector of claim 3.
  6. 6. The genetically engineered bacterium of claim 5, wherein the genetically engineered bacterium is an engineered strain that overexpresses Aspergillus0G048390 gene, aspergillus0G044330 gene, and/or Aspergillus0G004350 gene by transforming the recombinant overexpression vector into a host bacterium with Aspergillus nidulans producing echinocandin B as the host bacterium.
  7. 7. The method for constructing genetically engineered bacteria of claim 5, comprising the steps of: (1) The encoding genes of the fatty acid beta-oxidation pathway are obtained by screening from the genome of Aspergillus nidulans ZJB16068, wherein the encoding genes are Aspergillus0G048390 gene, aspergillus0G044330 gene and/or Aspergillus0G004350 gene; (2) Constructing recombinant overexpression vectors containing Aspergillus0G048390 gene, aspergillus0G044330 gene and Aspergillus0G004350 gene respectively by taking the Escherichia coli-Agrobacterium shuttle plasmid pDht-sk as a basic vector; (3) And transforming the constructed recombinant overexpression vector into Aspergillus nidulans ZJB16068 by adopting an agrobacterium AGL 1-mediated transformation method to obtain engineering strains over-expressing Aspergillus0G048390 gene, aspergillus0G044330 gene or Aspergillus0G004350 gene.
  8. 8. Use of the coding gene of claim 1, the protein of claim 2, the recombinant overexpression vector of claim 3 or 4, or the genetically engineered bacterium of claim 5 or 6 in the fermentative production of echinocandin B.
  9. 9. A production method of echinocandin B yield, characterized in that the genetic engineering bacteria of claim 5 or 6 are inoculated into a fermentation medium for fermentation culture, and echinocandin B is obtained.
  10. 10. The production method according to claim 8, wherein the fermentation culture conditions are a temperature of 25 ℃, a rotation speed of 200r/min and a fermentation period of 10 hours, and the fermentation culture medium is 70% methyl oleate 90, tween 80.98, sucrose 9.44, peanut oil 20, glycerol 10, soybean meal 40, sucrose 9.44, tryptone 8.6, threonine 3.1 and ornithine hydrochloride 6.1,K 2 HPO 4 6.1,MgSO 4 ·7H 2 O 0.5,MnSO 4 ·H 2 O 0.2,FeSO 4 ·7H 2 O 0.05,CaCl 2 0.3,CuSO 4 ·5H 2 O 0.6,pH.

Description

Fatty acid beta-oxidation pathway coding gene from aspergillus nidulans and application thereof Technical Field The application belongs to the field of genetic engineering, and relates to a fatty acid beta-oxidation pathway coding gene derived from aspergillus nidulans and application thereof, in particular to application in industrial efficient fermentation production of echinocandin B. Background Invasive fungal infection has become a major challenge in the field of global public health, and the existing antifungal medicines have the problems of strong toxic and side effects, frequent drug resistance, limited curative effect and the like. Echinocandin B (ECB) is used as a novel antifungal drug precursor, plays a role by inhibiting fungal cell wall beta-1, 3-glucan synthase, has the advantages of broad-spectrum antibacterial activity, low toxic and side effects and the like, and is a core raw material for synthesizing clinical drugs such as anidulafungin and the like, so that the market demand is urgent. At present, ECB is mainly produced by aspergillus nidulans (Aspergillus nidulans) fermentation, but the yield of a natural strain is low (the shake flask fermentation yield of an original strain is usually lower than 1500 mg/L), and the industrial production requirement is difficult to meet. The biosynthesis of ECB belongs to a non-ribosomal peptide synthesis pathway, the construction of which requires acetyl-CoA as a key precursor, whereas the fatty acid beta-oxidation pathway is one of the important supply pathways of intracellular acetyl-CoA of Aspergillus nidulans. The method is characterized in that fatty acid is degraded into acetyl-CoA through the stepwise catalysis of key enzymes such as acyl-CoA oxidase (FOX 1), 3-hydroxy acyl-CoA dehydrogenase (FOX 2), thiolase (POT 1) and the like, and each catalytic reaction is a potential regulation node of the pathway flux, so that the precursor supply efficiency of ECB synthesis is directly influenced. In the prior art, strategies for improving ECB yield are mainly focused on electronic transfer system optimization (such as cytochrome P450 reductase gene overexpression), fermentation condition regulation and control and the like, but the application of single key enzyme genes in the fatty acid beta-oxidation pathway in excavation, identification and independent overexpression has not been reported. Therefore, screening the core functional genes in the Aspergillus nidulans fatty acid beta-oxidation pathway strengthens the efficiency of a single catalytic step through single overexpression, further improves the acetyl-CoA supply flux, is an effective strategy for directionally improving the ECB yield, and has important significance for promoting the industrialized production of the ECB. Therefore, development of an engineering bacterium for producing echinocandin B, which has high ECB yield, simple strain construction and strong genetic stability, is needed. Disclosure of Invention The invention aims to overcome the defects of the prior art, provides a fatty acid beta-oxidation pathway coding gene derived from aspergillus nidulans and application thereof, and aims to solve the technical problem of low ECB yield of the existing aspergillus nidulans strain and realize the efficient biosynthesis of ECB. In order to achieve the above object and other related objects, the following technical solutions are adopted: A first object of the present invention is to provide a fatty acid beta-oxidation pathway encoding gene derived from Aspergillus nidulans, which is selected from the group consisting of (a) Aspergillus0G048390 gene having a nucleotide sequence shown as SEQ ID NO. 1, (b) Aspergillus0G044330 gene having a nucleotide sequence shown as SEQ ID NO. 3, and/or (c) Aspergillus0G004350 gene having a nucleotide sequence shown as SEQ ID NO. 5. Because of the specificity of the nucleotide sequence, any variant of the polynucleotides shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, as long as it has more than 90% homology with the polynucleotide, falls within the scope of the present invention. A mutant of the polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes. Mutants of this polynucleotide may be either naturally occurring allelic variants or non-naturally occurring variants, including substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded amino acid. The second object of the present invention is to provide a protein encoded by the fatty acid beta-oxidation pathway encoding gene derived from Aspergillus nidulans, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 2, SEQ ID NO.4 and/or SEQ ID NO. 6. As a preference of the application, SEQ ID NO. 2 corresponds to the protein encoded by th