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CN-121975833-A - Small molecular compound for improving homologous recombination efficiency of pichia pastoris and method thereof

CN121975833ACN 121975833 ACN121975833 ACN 121975833ACN-121975833-A

Abstract

The invention belongs to the technical field of biology, and discloses a small molecular compound for improving homologous recombination efficiency of pichia pastoris and a method thereof, aiming at the problem of low homologous recombination efficiency of pichia pastoris, wherein the small molecular compound comprises at least one of compounds SCR7, EPZ5676 and nocodazole. The method for improving the homologous recombination efficiency of the pichia pastoris comprises the steps of mixing a constructed recombinant vector targeting the OPI1 gene with a purified OPI1 knockout donor DNA fragment, adding the mixture into the prepared pichia pastoris competent cells, performing electric shock transformation, adding a small molecular compound, standing for resuscitation and culturing to obtain the recombinant pichia pastoris strain. According to the invention, the micromolecular compounds SCR7, EPZ5676 and nocodazole are applied to the Pichia pastoris CRISPR/Cas9 system, and the homologous recombination efficiency of the Pichia pastoris is remarkably improved by regulating and controlling the repair route and the cell cycle.

Inventors

  • LIU HUAN
  • XIONG XIAOBO
  • AN YAJUN
  • LUO LIANG

Assignees

  • 深圳普罗吉医药科技有限公司

Dates

Publication Date
20260505
Application Date
20260126

Claims (8)

  1. 1. A small molecular compound for improving the homologous recombination efficiency of pichia pastoris, which is characterized by comprising at least one of the compounds SCR7, EPZ5676 and nocodazole.
  2. 2. A method for improving the homologous recombination efficiency of pichia pastoris, comprising the steps of: S1, constructing a recombinant vector of a targeted OPI1 gene; S2, constructing a purified OPI1 knockout donor DNA fragment; S3, preparing pichia pastoris competent cells; S4, mixing the recombinant vector of the targeted OPI1 gene with the purified OPI1 knockout donor DNA fragment, adding the mixture into pichia pastoris competent cells, carrying out ice bath 5 min, and then carrying out electric shock transformation to obtain electric shock transformed pichia pastoris cells; S5, resuspending the electric shock transformed pichia pastoris cells, adding the small molecular compound of claim 1, standing for resuscitation and culturing to obtain the recombinant pichia pastoris strain.
  3. 3. A method for improving the efficiency of homologous recombination in pichia pastoris according to claim 2, wherein S1 comprises the steps of: S11, amplifying a Cas9 plasmid by using an sgRNA oligonucleotide chain containing an OPI1 target sequence; S12, treating the amplified Cas9 plasmid for 2 hours by NEB restriction endonuclease DpnI at 37 ℃ to recover fragments; s13, recombining and cloning the recovered fragments into pPICZC-A-Cas9 vector to obtain a connection product, transforming E.coli DH5 alpha competent cells by using the connection product, and verifying positive cloning by colony PCR and sequencing to obtain the recombinant vector of the target OPI1 gene.
  4. 4. A method for improving the efficiency of homologous recombination in pichia pastoris according to claim 2, wherein S2 comprises the steps of: S21, respectively taking KO OPI1-5F-F/KO OPI1-5F-R and KO OPI1-3F-F/KO OPI1-3F-R as primers, and taking the genome of pichia pastoris as a template for amplification to obtain an upstream 5F fragment and a downstream 5F fragment of a homologous arm of OPI1 gene knockout and a 3F fragment; S22, after fusion PCR amplification, fusing the upstream and downstream 5F fragments and the 3F fragment together to obtain a purified OPI1 knockout donor DNA fragment.
  5. 5. A method for improving the efficiency of homologous recombination in pichia pastoris according to claim 2, wherein S3 comprises the steps of: S31, picking a single colony of a pichia pastoris strain containing human serum albumin, inoculating the single colony into a YPD culture medium, and culturing overnight at 30 ℃ and 250 rpm; S32, transferring the cultured colonies into a fresh YPD culture medium according to the proportion of 1:100, and culturing until OD 600 = 1.3-1.5; S33, collecting thalli, cleaning with a precooled 1M sorbitol solution, and re-suspending in 100 mu L of 1M sorbitol to prepare pichia pastoris competent cells.
  6. 6. The method for improving the homologous recombination efficiency of Pichia pastoris according to claim 2, wherein the ratio of the mixed mass of the recombinant vector targeting the OPI1 gene to the purified OPI1 knockout donor DNA fragment in S4 is 1:2.
  7. 7. The method for improving the homologous recombination efficiency of Pichia pastoris according to claim 2, wherein the parameters for shock transformation in S4 are 600V,4 ms.
  8. 8. A method for improving the efficiency of homologous recombination in pichia pastoris according to claim 2, wherein S5 comprises the steps of: s51, resuspending the electric shock transformed pichia pastoris cells, adding the small molecular compound of claim 1, standing and resuscitating for 4-6 hours at 30 ℃; S52, coating the Pichia pastoris cells subjected to standing recovery on a YPD plate containing the nocardiacin resistance, and inversely culturing for 3-5 days at the temperature of 30 ℃ until single colonies appear, so as to obtain the recombinant Pichia pastoris strain.

Description

Small molecular compound for improving homologous recombination efficiency of pichia pastoris and method thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a small molecular compound for improving homologous recombination efficiency of pichia pastoris and a method thereof. Background Pichia pastoris (Pichia pastoris) has become one of the preferred hosts for efficient protein expression and metabolic engineering as a methylotrophic yeast. This has the advantage of enabling high density fermentation, possessing a strong and tightly regulated promoter (e.g. pAOX 1) and post-translational modification capability. In eukaryotic cells, DNA Double Strand Breaks (DSB) are repaired primarily by two pathways, error-prone non-homologous end joining (NHEJ) and precise homologous recombination repair (HDR). Pichia pastoris faces a major bottleneck in metabolic engineering application, namely low homologous recombination efficiency, which severely limits the construction and optimization of the polygenic biosynthetic pathway. Pichia pastoris typically requires 500-1000 bp long homology arms to achieve efficient genome integration compared to traditional Saccharomyces cerevisiae, which makes the procedure time consuming and inefficient. Recent studies have shown that significant progress has been made in improving the efficiency of homologous recombination by genetic engineering techniques. For example, overexpression of homologous recombination-related genes from Saccharomyces cerevisiae, such as RAD52, RAD59, MRE11 and SAE2 genes, can increase the efficiency of multiple genome integration of short homology arms (40 bp) to over 80%. The research team of the institute of Dalian chemical and physical of China academy of sciences also found that the deletion of the MPH1 gene increased the efficiency of multi-fragment recombination by 13.5 times, while the high expression of the RAD52 gene increased the editing efficiency of single gene to 90%. Although these genetic modification methods are effective, they involve permanent modification of the genome, which may lead to reduced cell fitness or defective growth. For example, studies have shown that over-expression of the SAE2 gene, while improving recombination efficiency, causes significant growth defects. The advent of CRISPR/Cas9 gene editing technology has revolutionized microbial genetic engineering. It directs Cas9 nucleases through guide RNAs (sgrnas) to generate precise DSBs at specific genomic sites. To increase HDR efficiency of CRISPR/Cas9, researchers have tried a variety of strategies including optimizing sgRNA design, modulating cell cycle, and interfering with DNA repair pathways using small molecule compounds. The small molecular compound has simple operation, reversible action and low cost, and becomes a hot spot direction for improving editing efficiency. Although some small molecule compounds that increase HDR efficiency have been found to be useful in mammalian cell studies, their use presents significant species-specific and effectiveness challenges. For Pichia pastoris, an important industrial microorganism, a targeted and systematic small molecular compound screening and effect verification system is lacking. This makes it difficult for researchers to select the appropriate compound and determine its optimal use concentration. Because of the uncertainty, an optimized standardized small molecule treatment scheme suitable for pichia pastoris is lacking, which brings a barrier to large-scale and high-repeatability pichia pastoris metabolic expression practice. Disclosure of Invention The invention aims to overcome the defects of the prior art and provide a small molecular compound for improving the homologous recombination efficiency of pichia pastoris and a method thereof. The first object of the invention is to provide a small molecular compound for improving the homologous recombination efficiency of pichia pastoris, which comprises at least one of the compounds SCR7, EPZ5676 and nocodazole. The second object of the present invention is to provide a method for improving the homologous recombination efficiency of Pichia pastoris, comprising the steps of: S1, constructing a recombinant vector of a targeted OPI1 gene; S2, constructing a purified OPI1 knockout donor DNA fragment; S3, preparing pichia pastoris competent cells; S4, mixing the recombinant vector of the targeted OPI1 gene with the purified OPI1 knockout donor DNA fragment, adding the mixture into pichia pastoris competent cells, carrying out ice bath 5 min, and then carrying out electric shock transformation to obtain electric shock transformed pichia pastoris cells; S5, resuspending the electric shock transformed pichia pastoris cells, adding the small molecular compound of claim 1, standing for resuscitation and culturing to obtain the recombinant pichia pastoris strain. Preferably, S1 comprises the steps of: S11, amplifying a Cas9 pl