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CN-121991994-A - Gene-deficient amycolatopsis mediterranean and construction method and application thereof

CN121991994ACN 121991994 ACN121991994 ACN 121991994ACN-121991994-A

Abstract

The invention provides a gene-defective amycolatopsis mediterranean and a construction method and application thereof. The first aspect of the invention provides a construction method of amycolatopsis mediterranei with a gene defect, which comprises the steps of knocking out a rifamycin biosynthesis gene cluster and an integration plasmid pDZLCas a containing FnCas a coding genes in a genome of a receptor amycolatopsis mediterranei strain U32 (Cas 12 a) to obtain the amycolatopsis mediterranei with the gene defect. The invention constructs the amycolatopsis mediterranean with gene defect by knocking out the rifamycin biosynthesis gene cluster in the genome of amycolatopsis mediterranean strain U32 (Cas 12 a) and the integration plasmid pDZLCas a containing FnCas a coding gene, which can be used as a chassis strain for excavating identified or unidentified biosynthesis gene cluster in actinomycetes.

Inventors

  • YUAN HUA
  • WANG WEI
  • WANG XINYUN

Assignees

  • 上海师范大学

Dates

Publication Date
20260508
Application Date
20241108

Claims (10)

  1. 1. The construction method of the amycolatopsis mediterranean with gene defects is characterized by comprising the following steps: Knocking out a rifamycin biosynthesis gene cluster in a receptor amycolatopsis mediterranei U32 (Cas 12 a) genome and an integration plasmid pDZLCas a containing FnCas a coding genes to obtain the gene-defective amycolatopsis mediterranei.
  2. 2. The method of claim 1, wherein the rifamycin biosynthesis gene cluster comprises rifS、rifT、rif35、rif0、rifA、rifB、rifC、rifD、rifE、rifF、rif1、rifG、rifH、rifI、rifK、rifL、rifM、rifN、rifO、rif2、rifP、rifQ、rif3、rif4、rif5、rif6、rif7、rif8、rif9、rif10、rif11、rif17、rif18、rif19、rif20、rifR、rif13、rif14、rif15A、rif15B、rif16、rifJ、rif36 total 43 genes, wherein: The nucleotide sequence of rifS is a sequence numbered AMED-0613 in Genebank No. CP 002000; the nucleotide sequence of rifT is a sequence numbered AMED-0614 in Genebank No. CP 002000; the nucleotide sequence of the rib 35 is a sequence numbered AMED-0615 in Genebank No. CP 002000; The nucleotide sequence of the rib 0 is a sequence numbered AMED-0616 in Genebank No. CP 002000; The nucleotide sequence of rifA is the sequence numbered AMED-0617 in Genebank No. CP 002000; The nucleotide sequence of rifB is a sequence numbered AMED-0618 in Genebank No. CP 002000; the nucleotide sequence of rifC is the sequence numbered AMED-0619 in Genebank No. CP 002000; the nucleotide sequence of rifD is a sequence numbered AMED _0620 in Genebank number CP 002000; the nucleotide sequence of rifE is a sequence numbered AMED _0621 in Genebank No. CP 002000; the nucleotide sequence of rifF is a sequence numbered AMED _0622 in Genebank number CP 002000; The nucleotide sequence of the rib 1 is a sequence numbered AMED-0623 in Genebank No. CP 002000; the nucleotide sequence of rifG is the sequence numbered AMED _0624 in Genebank number CP 002000; the nucleotide sequence of rifH is the sequence numbered AMED _0625 in Genebank number CP 002000; the nucleotide sequence of rifI is the sequence numbered AMED _0626 in Genebank number CP 002000; The nucleotide sequence of rifK is the sequence numbered AMED _0627 in Genebank number CP 002000; The nucleotide sequence of rifL is the sequence numbered AMED _0628 in Genebank number CP 002000; the nucleotide sequence of rifM is the sequence numbered AMED _0629 in Genebank number CP 002000; the nucleotide sequence of rifN is the sequence numbered AMED _0630 in Genebank number CP 002000; the nucleotide sequence of rifO is the sequence numbered AMED _0631 in Genebank number CP 002000; the nucleotide sequence of the rib 2 is a sequence numbered AMED-0632 in Genebank number CP 002000; the nucleotide sequence of rifP is the sequence numbered AMED _0633 in Genebank number CP 002000; the nucleotide sequence of rifQ is the sequence numbered AMED _0634 in Genebank number CP 002000; The nucleotide sequence of the rib 3 is a sequence numbered AMED-0635 in Genebank No. CP 002000; the nucleotide sequence of the rib 4 is a sequence numbered AMED-0636 in Genebank No. CP 002000; the nucleotide sequence of the rib 5 is a sequence numbered AMED-0637 in Genebank No. CP 002000; The nucleotide sequence of the rib 6 is a sequence numbered AMED-0638 in Genebank No. CP 002000; the nucleotide sequence of the rib 7 is a sequence numbered AMED-0639 in Genebank No. CP 002000; the nucleotide sequence of the rib 8 is a sequence numbered AMED-0640 in Genebank number CP 002000; the nucleotide sequence of the rib 9 is a sequence numbered AMED-0641 in Genebank number CP 002000; the nucleotide sequence of the rib 10 is a sequence numbered AMED-0642 in Genebank number CP 002000; the nucleotide sequence of the rib 11 is a sequence numbered AMED-0643 in Genebank number CP 002000; the nucleotide sequence of the rib 17 is a sequence numbered AMED-0644 in Genebank number CP 002000; The nucleotide sequence of the rib 18 is a sequence numbered AMED-0645 in Genebank number CP 002000; the nucleotide sequence of the rib 19 is a sequence numbered AMED-0646 in Genebank number CP 002000; The nucleotide sequence of the rib 20 is a sequence numbered AMED-0647 in Genebank number CP 002000; The nucleotide sequence of rifR is the sequence numbered AMED _0648 in Genebank number CP 002000; The nucleotide sequence of the rib 13 is a sequence numbered AMED-0649 in Genebank number CP 002000; The nucleotide sequence of the rib 14 is a sequence numbered AMED-0650 in Genebank number CP 002000; The nucleotide sequence of the rib 15A is a sequence numbered AMED-0651 in Genebank No. CP 002000; The nucleotide sequence of the rib 15B is a sequence numbered AMED-0652 in Genebank No. CP 002000; The nucleotide sequence of the rib 16 is a sequence numbered AMED-0653 in Genebank number CP 002000; the nucleotide sequence of rifJ is the sequence numbered AMED _0654 in Genebank number CP 002000; the nucleotide sequence of the rib 36 is a sequence numbered AMED-0655 in Genebank No. CP 002000.
  3. 3. The method of claim 1, wherein the FnCas a coding gene is the Cpf1 gene sequence in Genbank number MF193599, the protein sequence is the sequence in Genbank number ASK09413.1, and the integration site of FnCas a coding gene is located within the gene numbered AMED _0846 in Genbank number CP 002000.
  4. 4. A genetically deficient amycolatopsis mediterranean bacterium constructed according to the method of any one of claims 1-3.
  5. 5. Use of a genetically deficient amycolatopsis mediterranean as defined in claim 4 as a chassis strain for the identification of actinomycete biosynthetic gene clusters and/or biosynthesis of a compound of interest.
  6. 6. A method for identifying a cluster of actinomycete biosynthetic genes, comprising the steps of: Introducing a target biosynthesis gene cluster derived from a target actinomycete into the amycolatopsis mediterranei with the gene defect as set forth in claim 4, performing heterologous expression in the amycolatopsis mediterranei with the gene defect, and analyzing and structure measuring a product of the heterologous expression to complete identification of the biosynthesis gene cluster of the actinomycete.
  7. 7. The method according to claim 6, wherein the objective actinomycetes is at least one of amycolatopsis, saccharopolyspora, micromonospora, and Streptomyces.
  8. 8. A method for the heterologous synthesis of a compound of interest derived from actinomycetes, comprising: Introducing a target compound biosynthesis gene cluster derived from actinomycetes into the gene-deficient amycolatopsis mediterranei of claim 4, performing heterologous expression in the gene-deficient amycolatopsis mediterranei, and isolating the target compound from the culture product of the gene-deficient amycolatopsis mediterranei.
  9. 9. A knockout vector for knocking out a rifamycin biosynthesis gene cluster in the genome of receptor amycolatopsis mediterranei U32 (Cas 12 a).
  10. 10. A knockout vector for knockout of integrative plasmid pDZLCas a containing FnCas a coding gene in the genome of receptor amycolatopsis mediterranei U32 (Cas 12 a).

Description

Gene-deficient amycolatopsis mediterranean and construction method and application thereof Technical Field The invention relates to a gene-defective amycolatopsis mediterranei and a construction method and application thereof, and relates to the technical field of microorganisms. Background Microbial genomes contain a vast array of natural product biosynthetic gene clusters, but most of these functions have not been identified, in a "silent" state, and their encoded products are known as microbial "life-dark matter". The complex and intricate biosynthesis of microbial natural products, greatly affected by itself and environmental factors, and how to effectively activate and mine these microorganisms for "life-dark substances" has been a bottleneck limiting the discovery of new natural products and is a challenge in the art. Actinomycetes have been the main source of microbial drugs. Among them, amycolatopsis (Amycolatopsis) belongs to rare actinomycetes, and from the initial discovery, it belongs to Streptomyces (Sreptomyces), then to Nocardia (Nocarpia), and finally it is divided into a new genus (Amycolatopsis), which means that Amycolatopsis has a unique "position" in the history of evolution. The species that are currently published are less than a hundred, but the natural products produced are very high in drug-forming and play a critical role clinically, such as the glycopeptides vancomycin, the oricomycin (derivative of the vancomycin analogue chloroeremomycin), and the ansamycin. In addition to these three clinical antibiotics, the genus strain synthesizes other natural products with more than 160 activities of antibacterial, cytotoxic, antioxidant, etc. In addition, large-scale genome sequencing and functional annotation analysis indicated that the genus strain encoded a vast number and variety of gene clusters, and that the homology was lower than that identified in the database, suggesting that the encoded products were mostly novel unidentified compounds. Therefore, gene cluster functional identification analysis of amycolatopsis strains is one of the hot spots of interest in the art. Various methods of activating gene clusters have been reported, mainly including two strategies based on natural hosts and heterologous hosts. The development of a highly efficient gene operating system in a natural host is advantageous for remodeling the transcriptional regulatory loop of a target gene cluster for activation purposes, but given that gene operating system development is strain-specific, time-consuming and often futile, natural host-based activation strategies are generally not suitable for exploiting the natural product production potential of most microorganisms, and are more unsuitable for the mining of gene clusters in the genome of uncultured microorganisms. Cloning and heterologous expression of gene clusters is regarded as a common strategy for activating silent gene clusters, which is more flexible based on heterologous hosts, and can be applied to gene cluster resources from culturable microorganisms as well as microorganisms not yet cultivated in the laboratory. The strategy mainly comprises the steps of 1) cloning (or reconstructing) target gene clusters from microbial genome DNA, 2) carrying out heterologous expression in host/chassis cells (such as Streptomyces coelicolor, escherichia coli and the like) which are relatively mature in an operating system, and 3) carrying out analysis and structural determination on chemical components of products of the heterologous expression. Wherein, the heterologous host/chassis cell is crucial for the activation of the silencing gene cluster, and the good chassis cell can provide a clean metabolic background, thereby facilitating the separation and purification of the expressed compound. Streptomyces chassis cells, such as Streptomyces coelicolor derived strains (CH 999, M1154, etc.), streptomyces albus (J1074, del14, etc.), streptomyces avermitilis derived strains (SUKA, SUKA, etc.), have generally been given priority in mining the gene cluster resources of actinomycetes, and many successful cases of expressing known or unknown gene clusters have been presented. Based on the unique "position" of amycolatopsis in its evolutionary history, common Streptomyces chassis cells are not necessarily suitable for the excavation of biosynthetic gene clusters derived from amycolatopsis. How to provide a chassis strain suitable for digging a biosynthesis gene cluster of amycolatopsis is a technical problem to be solved in the field. Disclosure of Invention The invention provides a amycolatopsis mediterranei with gene defects and a construction method thereof, which are used for providing a chassis strain suitable for digging biosynthesis gene clusters of amycolatopsis mediterranei. The invention also provides application of the amycolatopsis mediterranean with the gene defect in digging actinomycete biosynthesis gene clusters and synthesizing natural products.