Efficient gene editing system of pichia pastoris, construction method of efficient gene editing system and application of efficient gene editing system in high-yield cordycepin engineering strains

Abstract

The invention relates to the fields of gene editing technology, bioengineering and applied microbial fermentation, and discloses a pichia pastoris efficient gene editing system, a construction method thereof and application of the pichia pastoris efficient gene editing system in high-yield cordycepin engineering strains. The construction of the pichia pastoris CRISPR-Cas9 gene editing system disclosed by the invention mainly comprises the following steps: refilling histidine HISS4 auxotrophy to realize nonreactive assembly of KhCas9; the sgRNA-tRNA array and the IIS type restriction enzyme are combined to simplify the gene editing operation so as to realize simultaneous efficient editing of multiple genes; and fusion of the Brex27 structural domain and KhCas9 enhances the homologous recombination efficiency. By utilizing the gene editing system and optimizing methanol assimilation, 3 '-AMP and adenosine precursor supply, ATP / NADPH energy supply and the like, the construction of a cordycepin engineering strain without resistance gene residue is realized for the first time, so that the gene editing efficiency of pichia pastoris is greatly improved, and a powerful tool is provided for industrial production of cordycepin.

Description

Technical Field

[0001] The present invention relates to the fields of gene editing technology, bioengineering, and applied microbial fermentation. Specifically, it relates to a high-efficiency gene editing system for Pichia pastoris, a method for constructing the same, and its application in high-yield cordycepin engineering strains. Background Art

[0002] Pichia pastoris is a generally recognized as safe (GRAS) methylotrophic yeast, widely used in the agriculture, food, pharmaceutical, and feed industries. Pichia pastoris promotes sustainable chemical production by efficiently utilizing methanol, which can promote its high-density growth and strong induced expression. In fed-batch fermentation, the temporal separation of the growth phase (glycerol feeding) and the production phase (methanol induction) makes Pichia pastoris an effective vector for synthesizing antibacterial compounds such as cordycepin, thus overcoming the growth inhibition caused by product accumulation. However, the current gene editing strategies for Pichia pastoris mainly rely on plasmid-based single crossover integration technology, which depends on antibiotic tags, inevitably introducing yeast-sensitive resistance genes such as bleomycin, nourseothricin, hygromycin, and G418 into the genome. These residual resistance genes will lead to an increase in the cost of industrial waste treatment and the risk of environmental drift of resistance genes. Therefore, the development of a CRISPR-Cas9 system without introducing resistance gene tags into the yeast genome is of great significance for the green production of Pichia pastoris cell factories.

[0003] The plasmid construction efficiency and multiplex genome editing ability are the key bottlenecks in the development of Pichia pastoris cell factories. Currently, the CRISPR-Cas9 system in Pichia pastoris relies on HH and HDV ribozymes to express sgRNA. However, the variable 6bp sequence in the HH ribozyme needs to be replaced according to different gRNA sequences, which complicates vector construction and significantly prolongs the working cycle during target gene replacement or multiplex editing. In contrast, the ability of RNase P and RNase Z to cleave tRNA without discrimination helps to achieve sequence-independent sgRNA-tRNA fusion cleavage, thus providing a rapid sgRNA assembly strategy. Precise genome editing depends on homologous directed repair (HDR). However, in Pichia pastoris, non-homologous end joining (NHEJ) dominates double-strand break (DSB) repair, while HDR itself is extremely inefficient. Overexpressing HDR-related proteins can improve HDR efficiency without disrupting NHEJ, which is beneficial to maintaining genetic stability. Notably, local enrichment of HDR proteins near DSBs has been shown to play an important role in HDR repair. RAD51 is involved in all stages of HDR. Studies have shown that the Brex27 domain of humans can recruit RAD51 to the DSB site, effectively improving HDR efficiency. Combining the sgRNA-tRNA array with Brex27-mediated RAD51 recruitment will establish a streamlined and efficient CRISPR-Cas9 system, significantly optimizing genome editing in Pichia pastoris.

[0004] Cordycepin is a naturally occurring nucleoside analogue, mainly present in ascomycetes such as Cordyceps militaris, Cordyceps cicadae, Ophiocordyceps sinensis, and Cordyceps kyushuensis. Cordycepin has powerful biological activities, including antibacterial, antiviral, antioxidant, immunomodulatory, and neuromodulatory properties. Its anticancer potential has recently been verified in a phase I clinical trial of NUC-7738. Currently, the chemical synthesis methods of cordycepin face many challenges such as low yield, difficult chiral control, high prices of substrates and catalysts, high tin residue, and serious organic solvent pollution. Biosynthesis remains the main production route of cordycepin. However, the cordycepin yields of cell factories such as Cordyceps militaris, Saccharomyces cerevisiae, Yarrowia lipolytica, and Aspergillus oryzae are low, and they all rely heavily on food-derived raw materials such as wheat, rice, and glucose. With the CRISPR-Cas9 technology provided by the present invention, Pichia pastoris can be transformed into a high-yield cordycepin cell factory. At the same time, using methanol as a carbon source can effectively avoid the phenomenon of competing with the people for food in cordycepin production, which is an important method for realizing the green, efficient, and low-cost synthesis of cordycepin. Summary of the Invention

[0005] The object of the present invention is to provide a high-efficiency gene editing system for Pichia pastoris, its construction method, and application.

[0006] Another object of the present invention is to provide a high-yield cordycepin engineering strain, its construction method and application.

[0007] To achieve the object of the present invention, in a first aspect, the present invention provides a KhCas9-Brex27 expression cassette, which comprises the following elements connected in sequence: PpHIS4-1 fragment - GAP promoter - Kozak sequence - hCas9 protein - Linker - Brex27 motif - SV40 nuclear localization signal (NLS) - AOX1 terminator - DAS1 terminator - PpHIS4-2 fragment; Among them, the PpHIS4-1 fragment and the PpHIS4-2 fragment are from plasmid pAO815 (purchased from Invitrogen); the primers for amplifying the PpHIS4-1 fragment are F-PpHIS4-1 and R-PpHIS4-1, and the primers for amplifying the PpHIS4-2 fragment are F-PpHIS4-2 and R-PpHIS4-2; The primer sequences are as follows (5'-3'): F-PpHIS4-1: ATGACATTTCCCTTGCTACCTGCAT R-PpHIS4-1: CTTGTGAGGAGGCCTTAGAAACGTC F-PpHIS4-2: AAATTGAAGATGCCCTTCGTCGTCCT R-PpHIS4-2: TTAAATAAGTCCCAGTTTCTCCATACGAACC The Brex27 motif is from humans, and its amino acid sequence is ALDFLSRLPLPPPVSPICTFVSPAAQKAFQPPRSCG (SEQ ID NO: 211); the DNA sequence encoding the above Brex27 motif after codon optimization for Pichia pastoris is as shown in SEQ ID NO: 5; The hCas9 protein is from Streptococcus pyogenes ( Streptococcus pyogenes ), and its sequence has been optimized according to human codon preference, and the reference sequence number in NCBI is 69900935; The GAP promoter (pGAP), AOX1 terminator (tAOX1) and DAS1 terminator (tDAS1) are all from Pichia pastoris ( Pichia pastoris ) GS115.

[0008] The Kozak sequence is CGGACC.

[0009] Preferably, the amino acid sequence of the Linker is (GGGGS)3 (SEQ ID NO: 212).

[0010] More preferably, the sequence of the KhCas9-Brex27 expression cassette optimized by Pichia pastoris codons is as shown in SEQ ID NO:10.

[0011] In a second aspect, the present invention provides an sgRNA expression plasmid for use in conjunction with the KhCas9-Brex27 expression cassette, comprising at least the following elements: a gRNA expression cassette, a Zeocin resistance marker, a yeast replication origin (panARS), and a bacterial replication origin (ori); Among them, the gRNA expression cassette contains a TEF1 promoter, an AOX1 terminator, Pichia pastoris endogenous tRNA1, tRNA3, tRNA4, and tRNA5, type IIS restriction endonuclease sites BsaI, BspQI, and BbsI, and three gRNA scaffolds; The TEF1 promoter (pTEF1) is from Pichia pastoris GS115.

[0012] The nucleotide sequences of Pichia pastoris endogenous tRNA1, tRNA3, tRNA4, and tRNA5 are as shown in SEQ ID NO:1-4, respectively.

[0013] Furthermore, the gRNA expression cassette includes the following elements connected in sequence: TEF1 promoter - tRNA① - endonuclease site① - gRNA scaffold - tRNA② - endonuclease site② - gRNA scaffold - tRNA③ - endonuclease site③ - gRNA scaffold - tRNA④ - AOX1 terminator; Among them, tRNA①, tRNA②, tRNA③, and tRNA④ are different from each other and respectively correspond to four Pichia pastoris endogenous tRNAs; Endonuclease site①, endonuclease site②, and endonuclease site③ are different from each other and respectively correspond to three type IIS restriction endonuclease sites.

[0014] Preferably, the gRNA expression cassette includes the following elements connected in sequence: TEF1 promoter - tRNA1 - endonuclease site BsaI - gRNA scaffold - tRNA3 - endonuclease site BspQI - gRNA scaffold - tRNA4 - endonuclease site BbsI - gRNA scaffold - tRNA5 - AOX1 terminator.

[0015] More preferably, the sgRNA expression plasmid of the present invention is pZBJ, and the full sequence of plasmid pZBJ is as shown in SEQ ID NO:11.

[0016] In a third aspect, the present invention provides a method for constructing a highly efficient gene editing system for Pichia pastoris. The KhCas9-Brex27 expression cassette is integrated into the genome of Pichia pastoris GS115 to obtain the Pichia pastoris strain GS115-KhCas9-Brex27 that highly expresses the hCas9 protein. Then, the sgRNA expression plasmid and the donor DNA fragment are introduced into Pichia pastoris GS115-KhCas9-Brex27, and the Pichia pastoris CRISPR-Cas9 gene editing system is obtained.

[0017] Furthermore, the donor DNA may contain elements such as a Kanamycin resistance tag, a bacterial replication origin ori, homologous arms (about 0.5-1 kb in size) on both sides of the Cas9 cleavage site, a promoter, a terminator, and a target gene.

[0018] In a fourth aspect, the present invention provides a highly efficient gene editing system for Pichia pastoris constructed according to the described method.

[0019] In a fifth aspect, the present invention provides the application of the highly efficient gene editing system for Pichia pastoris in constructing a high-yield cordycepin engineering strain.

[0020] In a sixth aspect, the present invention provides a high-yield cordycepin Pichia pastoris engineering bacterium, and its construction method includes the following steps: (1) The KhCas9-Brex27 expression cassette is integrated into the genome of Pichia pastoris GS115 to obtain the strain GS115-KhCas9-Brex27. Then, the expression plasmid containing sgRNA1 and the donor DNA1 fragment are introduced into the strain GS115-KhCas9-Brex27 to construct the engineering bacterium 1; Among them, the nucleotide sequence of sgRNA1 targeting Pichia pastoris chromosome Chr2-5 is 5’-AACTTTGAAACAAAAGAAGG -3’; The target gene carried by the donor DNA1 fragment is the gene from Cordyceps militaris ( Cordyceps militaris ) CP1 and optimized by Pichia pastoris codons Ppcns1 and Ppcns2 ; among them, the gene Ppcns1 is driven by the promoter pAOX1, and the gene Ppcns2 is driven by the promoter pFLD1; The gene Ppcns1 and Ppcns2 The nucleotide sequences of are shown in SEQ ID NO:6 and 7 respectively; The promoters pAOX1 and pFLD1 are from Pichia pastoris GS115; (2) The expression plasmid containing sgRNA2 and the donor DNA2 fragment are introduced into the engineering bacterium 1 to construct the engineering bacterium 2; Among them, the nucleotide sequence of sgRNA2 targeting the Pichia pastoris chromosome Chr2-4 is 5'-CCTAAATACTACCTAAACAG -3'; The target genes carried by the donor DNA2 fragment are genes from Cordyceps militaris CP1 and optimized by Pichia pastoris codons Ppcns3 / NK , and genes from Escherichia coli ( Escherichia coli ) and optimized by Pichia pastoris codons PpcpdB ; among them, gene Ppcns3 / NK is driven by the promoter pAOX1, and gene PpcpdB is driven by the promoter pFLD1; The nucleotide sequences of gene Ppcns3 / NK and PpcpdB are shown in SEQ ID NO:8 and 9 respectively; (3) An expression plasmid containing sgRNA3 and a donor DNA3 fragment are introduced into engineering bacteria 2 to construct engineering bacteria 3; Among them, the nucleotide sequence of sgRNA3 targeting the Pichia pastoris chromosome Chr1-2 is 5'-GGTTGGTACTATGTCCAACA -3'; The target genes carried by the donor DNA3 fragment are Pichia pastoris endogenous genes das1 and pex8 ; among them, gene das1 is driven by the promoter pAOX1, and gene pex8 is driven by the promoter pFLD1; The reference sequence numbers of gene das1 and pex8 in NCBI are PAS_chr3_0832 and PAS_chr1-4_0349 respectively; (4) An expression plasmid containing sgRNA4 and a donor DNA4 fragment are introduced into engineering bacteria 3 to construct engineering bacteria 4; Among them, the nucleotide sequence of sgRNA4 targeting the Pichia pastoris chromosome Chr1-5 is 5'-CACGAGCCGAGTAATAACCG -3'; The target genes carried by the donor DNA4 fragment are Pichia pastoris endogenous genes purF and adss ; among them, gene purF is driven by the promoter pCAT1, and gene adss is driven by the promoter pFDH1; The reference sequence numbers of gene purF and adss in NCBI are PAS_chr1-1_0430 and PAS_chr4_0613 respectively; The promoters pCAT1 and pFDH1 are from Pichia pastoris GS115; (5)The expression plasmid containing sgRNA5 and the donor DNA5 fragment were introduced into the engineering bacterium 4 to construct the engineering bacterium 5; Among them, the nucleotide sequence of sgRNA5 targeting the chromosome Chr3-5 of Pichia pastoris is 5'-ATACTAGTTAACAAACTGGG -3'; The target gene carried by the donor DNA5 fragment is an endogenous gene of Pichia pastoris zwf1 and pgk ; Among them, the gene zwf1 is driven by the promoter pTEF1, and the gene pgk is driven by the promoter pPGI1; The gene zwf1 and pgk The reference sequence numbers in NCBI are PAS_chr2-1_0308 and PAS_chr1-4_0292 respectively; The promoters pTEF1 and pPGI1 are from Pichia pastoris GS115; (6)The expression plasmid containing sgRNA6 and the donor DNA6 fragment were introduced into the engineering bacterium 5 to knockout the hCas9 protein, and the high-yield cordycepin Pichia pastoris engineering bacterium was obtained.

[0021] Among them, the nucleotide sequence of sgRNA6 targeting hCas9 in the chromosome of the engineering bacterium 5 is 5'-TACGCCGGATACATTGACGG-3'; The construction method of the donor DNA6 fragment includes: amplifying the ΔCas9-UP-OE fragment from the genome of Pichia pastoris GS115 using the primers F-ΔCas9-UP-OE and R-ΔCas9-UP-OE; amplifying the ΔCas9-DW-OE fragment from the genome of Pichia pastoris GS115 using the primers F-ΔCas9-DW-OE and R-ΔCas9-DW-OE; the ΔCas9-UP-OE fragment and the ΔCas9-DW-OE fragment were subjected to overlap extension PCR to obtain the ΔCas9-UP-DW donor DNA fragment (SEQ ID NO:12); The primer sequences are as follows (5'-3'): F-ΔCas9-UP-OE: TTTTTGTAGAAATGTCTTGGTGTCCTCGTCC R-ΔCas9-UP-OE: CTTCTGCTCATTATCTTCCCGAAATTTGATCATATGCGCCAGC F-ΔCas9-DW-OE: CATATGATCAAATTTCGGGAAGATAATGAGCAGAAGCAGCTGTTCG R-ΔCas9-DW-OE: ACGGGAAGTCTTTACAGTTTTAGTTAGGAGCC The expression plasmid containing sgRNA is the same as the aforementioned sgRNA expression plasmid.

[0022] In the seventh aspect, the present invention provides a high-yield cordycepin Pichia pastoris engineering bacterium constructed by the above method.

[0023] In the eighth aspect, the present invention provides the application of the engineering bacterium in the production of cordycepin, including: culturing the engineering bacterium by a fed-batch fermentation process to obtain a culture, and collecting the produced cordycepin from the culture.

[0024] Among them, the fed-batch fermentation process includes: S1. Activation of the seed liquid; S2. Fermentation tank culture: Inoculate the seed liquid with an OD 600 value of 4.0 - 5.0 into the fermentation tank for fermentation tank culture at a volume ratio of 5% - 10%; The culture conditions are as follows: The medium filling volume of the fermentation tank is 30% - 40% of the fermentation tank volume; the temperature is controlled at 28 - 30 °C throughout the process, the pH value is 4.0 - 5.0, the stirring speed is 200 - 800 rpm, the aeration rate is maintained at 2 - 4 vvm, and the dissolved oxygen DO value of the fermentation broth is 20% - 60%; The formula of the medium is: 0.8 - 1.2 g / L CaSO4, 15 - 20 g / L K2SO4, 12 - 18 g / L MgSO4·7H2O, 2 - 5 g / L KOH, 30 - 50 g / L glycerol, 25 - 30 mL / L of 85% concentrated phosphoric acid; Preferably, the formula of the medium is: 0.93 g / L CaSO4, 18.2 g / L K2SO4, 14.9 g / L MgSO4·7H2O, 4.13 g / L KOH, 40.0 g / L glycerol, 26.7 mL / L of 85% concentrated phosphoric acid; S3. Glycerol feeding: When the dissolved oxygen DO value shows the first rebound increase, it indicates that the glycerol in the fermentation broth has been exhausted, and the glycerol feeding medium is added dropwise at a uniform speed of 15 - 25 mL / L / h until the OD 600 increases to 300 - 350, and the glycerol feeding stage ends; The culture conditions are as follows: The fermentation temperature is 28 - 30 °C, the pH value is 4.0 - 5.0, the stirring speed is 500 - 800 rpm, and the aeration rate is maintained at 4 - 8 vvm; The formula of the glycerol feeding medium is: a 40 - 60% m / v glycerol solution containing 10 - 15 mL / L of PTM1; Preferably, the formula of the glycerol feeding medium is: a 50% m / v glycerol solution containing 12 mL / L of PTM1; S4. Methanol induction and cordycepin synthesis: After stopping the supplementation of glycerol, continue culturing for 2 - 3 h to deplete the residual glycerol in the cells. Record the time as t0, and then start methanol induction; Add the methanol feeding medium at the following gradient increasing speeds: 0.5 mL / L / h, 1 mL / L / h, 2 mL / L / h, 3 mL / L / h, 4 mL / L / h, 5 mL / L / h. Starting from t0 + 7 h, maintain a methanol feeding rate of 5 - 10 mL / L / h to keep the methanol concentration in the fermenter at 0.5 - 1.0%; The culture conditions are as follows: fermentation temperature 28 - 30 °C, pH value controlled at 5.1 - 6.0, stirring speed 500 - 800 rpm, aeration rate maintained at 4 - 8 vvm, control the dissolved oxygen DO value of the fermentation broth at 15 - 75%. After 6 - 9 days of fermentation, end the fermentation and discharge the fermenter.

[0025] The formula of the methanol feeding medium is: 100% methanol containing 10 - 15 mL / L of PTM1; Preferably, the formula of the methanol feeding medium is: 100% methanol containing 12 mL / L of PTM1.

[0026] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects: (1) The present invention provides a method for editing the CRISPR - Cas9 gene of Pichia pastoris without residual resistance genes and its application in the construction of high - yield cordycepin engineering strains, especially involving simplifying gene editing operations and enhancing homologous recombination efficiency through sgRNA - tRNA arrays, type IIS restriction endonucleases, and Brex27 domains to achieve the construction of cordycepin engineering strains without residual resistance genes.

[0027] (2) The construction and application process of the CRISPR - Cas9 gene editing system in the present invention mainly includes the realization of the antibiotic - free assembly of KhCas9 through the complementation of the histidine HIS4 nutritional defect; the combination of sgRNA - tRNA arrays and type IIS restriction endonucleases simplifies gene editing operations to achieve simultaneous and efficient editing of multiple genes; the fusion of the Brex27 domain with KhCas9 enhances homologous recombination efficiency and improves gene editing efficiency; the knockout of the Cas9 protein realizes scarless gene editing.

[0028] (3) With the gene editing system constructed by the present invention, by optimizing methanol assimilation, 3'-AMP and adenosine precursor supply, ATP / NADPH energy supply, etc., the construction of a cordycepin engineering strain without residual resistance genes was achieved. Through the staged fermentation method, the cordycepin yield reached 18.3 g / L after 144 h of fermentation and 27.2 g / L after 216 h of fermentation. The CRISPR-Cas9 gene editing method provided by the present invention greatly improved the gene editing efficiency of Pichia pastoris, and for the first time realized the construction of a high-yield cordycepin engineering strain without relying on resistance gene tags, providing a powerful tool for the industrial production of cordycepin. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the integration of hCas9 without resistance in Pichia pastoris GS115 by the present invention using histidine auxotrophy.

[0030] Figure 2 This is the construction of the sgRNA plasmid by the present invention using the sgRNA-tRNA array and type IIS restriction endonuclease.

[0031] Figure 3 This is the glycerol kinase gene in the preferred embodiment of the present invention gut1 as the target to verify the gene editing ability of the CRISPR-Cas9 system. (A) Growth defect plate diagram of the glycerol kinase gene gut1 knockout strain; (B) NHEJ knockout efficiency of the glycerol kinase gene gut1 using the BsaI, BspQI and BbsI sites on the sgRNA plasmid respectively; (C) Three mutation types were generated at the gut1 locus by NHEJ-mediated DSB repair.

[0032] Figure 4 This is that the Brex27 domain of the present invention enhances the HDR ability by attracting PpRAD51 and strengthens the multi-gene editing ability.

[0033] Figure 5 This is the glycerol kinase in the preferred embodiment of the present invention gut1 , alcohol oxidase aox1 , histidine multifunctional enzyme his4 as the target to verify the effect of the Brex27 domain on the multi-gene editing ability of the CRISPR-Cas9 system.

[0034] Figure 6Enhancing multiplex genome editing ability by improving HDR efficiency in the preferred embodiment of the present invention. (A) Targeting glycerol kinase, alcohol oxidase, and histidine multifunctional enzyme to verify the improvement of multiplex genome editing ability; (B) The effect of Brex27 domain on single-gene editing efficiency; (C) The effect of Brex27 domain on double-gene editing efficiency; (D) The effect of Brex27 domain on triple-gene editing efficiency.

[0035] Figure 7 PCR amplification bands after single knockout, double knockout, and triple knockout in the preferred embodiment of the present invention. (A) Band diagram of single knockout of ΔGUT1 in KhCas9 and khCas9-Brex27; (B) Band diagram of double knockout of ΔGUT1-ΔAOX1 in KhCas9 and khCas9-Brex27; (C) Band diagram of triple knockout of ΔGUT1, ΔAOX1, and ΔHIS4 in KhCas9 and khCas9-Brex27.

[0036] Figure 8 Sequencing results of NHEJ knockout in multiplex editing of ΔGUT1, ΔAOX1, and ΔHIS4 in the preferred embodiment of the present invention. (A) NHEJ sequencing of ΔGUT1; (B) NHEJ sequencing of ΔAOX1; (C) NHEJ sequencing of ΔHIS4.

[0037] Figure 9 Verifying the protein interaction between Brex27 domain and PpRAD51 using yeast two-hybrid in the preferred embodiment of the present invention.

[0038] Figure 10 Constructing and optimizing the cordycepin biosynthetic pathway in Pichia pastoris using the CRISPR-Cas9 system in the preferred embodiment of the present invention. (A) Engineering Pichia pastoris to achieve cordycepin biosynthesis in the cytoplasm; (B) Chromatogram and mass spectrum of cordycepin in the standard solution and fermentation broth; (C) Effects of promoter combinations with different strengths and copy numbers on cordycepin production and cell density; (D) Effects of increasing 3'-AMP supply on cordycepin production and cell density.

[0039] Figure 11In a preferred embodiment of the present invention, a multi - pathway modification strategy is used to increase cordycepin production. (A) The mechanism of using the CRISPR - Cas9 system for multi - pathway modification to increase cordycepin production; (B) The effect of enhanced methanol assimilation on cordycepin production and cell density; (C) Cordycepin production and cell density of PC10 and PC13 under 0.5 - 3% methanol conditions; (D) The effect of enhanced purine pathway on cordycepin production and cell density; (E) The effect of pathway modification on adenosine content; (F) The effect of enhanced cofactor synthesis on cordycepin production and cell density; (G) The effect of enhanced cofactor supply on ATP content and NADPH / NADP + ratio.

[0040] Figure 12 In a preferred embodiment of the present invention, the cell density OD of the cordycepin engineering strain PC19 600 changes with fermentation time.

[0041] Figure 13 In a preferred embodiment of the present invention, the cordycepin production of the cordycepin engineering strain PC19 changes with fermentation time. Detailed implementation manners

[0042] Aiming at the problem of difficult gene editing in current Pichia pastoris strains, the present invention provides a Pichia pastoris CRISPR - Cas9 gene editing system without residual resistance genes and applies it to the construction of high - yield cordycepin engineering strains to achieve low - carbon and high - efficiency synthesis of cordycepin.

[0043] The present invention adopts the following technical solutions: In the first aspect, the present invention fuses the codon - optimized hCas9 protein with the K7 sequence (i.e., the Kozak sequence CGGACC) to construct KhCas9, and realizes the antibiotic - free integration of KhCas9 into the genome of Pichia pastoris GS115 with the help of the histidine auxotrophic tag HIS4, obtaining the strain GS115 - KhCas9 that highly expresses the Cas9 protein.

[0044] Furthermore, the present invention uses Pichia pastoris endogenous tRNA1, tRNA3, tRNA4, tRNA5 and the gRNA scaffold to construct the sgRNA - tRNA array, and combines three IIS - type restriction enzymes, BsaI / BspQI / BbsI, to jointly construct the sgRNA plasmid. Furthermore, using the glycerol kinase gene gut1 as the target, it is verified that all three BsaI / BspQI / BbsI sites can achieve efficient gene editing.

[0045] It should be noted that one of the technical advantages of the present invention is to utilize IIS-type restriction endonucleases BsaI / BspQI / BbsI to achieve rapid replacement of different genomic target gRNAs, avoiding the complex homologous sequence replacement and multiple rounds of PCR of traditional HH and HDV ribozymes, and having extremely high convenience.

[0046] Furthermore, the present invention constructs the GS115-KhCas9-Brex27 strain by fusing the codon-optimized Brex27 domain with KhCas9, significantly enhancing the aggregation of the DNA repair protein PpRAD51 at genomic breaks, and simultaneously enabling the gut1 efficient seamless knockout of the glycerol kinase aox1 and alcohol oxidase his4 and histidine multifunctional enzyme

[0047] The construction method of the GS115-KhCas9-PpRAD52 strain is as follows: Construct a GAP promoter-PpRad52 gene-AOX1 terminator expression cassette and integrate it into the Chr1-2 site of the chromosome of the GS115-KhCas9 strain to obtain the GS115-KhCas9-PpRAD52 strain. Among them, the PpRad52 gene (NCBI accession number: PAS_chr2-1_0153) is cloned from the genome of Pichia pastoris GS115.

[0048] It should be noted that the second technical advantage of the present invention is that the Brex27 domain is directly fused to the C-terminus of KhCas9, with a high local spatial concentration of the recruited DNA repair protein and without occupying a new genomic integration site, saving space for subsequent cordycepin pathway modification.

[0049] In the second aspect, the present invention optimizes the nucleoside / nucleotide kinase gene Cordyceps militaris of Cordyceps militaris Cmcns3 / NK CP1, the metal-dependent phosphohydrolase gene Cmcns2 and the oxidoreductase / dehydrogenase gene Cmcns1 according to the codon preference of Pichia pastoris to obtain the unique Ppcns3 / NK , Ppcns2 and Ppcns1 coding sequences of the present invention. Their Cmcns3 / NK , Cmcns2 , Cmcns1They have 1, 2, and 8 amino acid optimization sites respectively compared with the sequences published by NCBI, specifically: Cns1 (p.F12C, p.P30S, p.E425D, p.N440G, p.S599A, p.A611V, p.S635P, p.D770E), Cns2 (p.K130E, p.P314Q), Cns3 / NK (p.N154S). These amino acid optimizations promoted the synthesis of cordycepin. The Cordyceps militaris CP1 strain is currently deposited in the China Center for Type Culture Collection (CCTCC M2019671).

[0050] Furthermore, the present invention uses a combination of three promoter strengths, pAOX1-pFLD1, pAOX1-pCAT1, and pAOX1-pDAS2, to control Ppcns1 and Ppcns2 achieve efficient extracellular synthesis of cordycepin. Among them, the pAOX1-pFLD1 combination is the best, and further increasing the copy number cannot effectively accelerate the synthesis of cordycepin.

[0051] Furthermore, the present invention uses the pAOX1 and pFLD1 promoters to overexpress Ppcns3 , Ppcns3 / NK , PpcpdB to strengthen the synthesis of 3'-AMP, the direct precursor of cordycepin, and the yield of cordycepin is significantly improved.

[0052] Thirdly, the present invention uses a combined metabolic engineering of methanol assimilation enhancement, purine pathway enhancement, and cofactor NADPH / ATP supply enhancement to achieve a significant increase in the yield of cordycepin.

[0053] Preferably, the present invention uses the pAOX1 and pFLD1 promoters to overexpress dihydroxyacetone synthase DAS1 and peroxisome biogenesis factor 8 PEX8, strengthen the transfer of methanol assimilating enzymes from the cytoplasm to peroxisomes, and accelerate the conversion of formaldehyde to provide sufficient carbon metabolic flux for cordycepin synthesis.

[0054] Preferably, the present invention uses the pCAT1 and pFDH1 promoters to overexpress phosphoribosyl pyrophosphate amidotransferase PurF and adenylosuccinate synthase AdSS, strengthen the transfer of the purine pathway metabolic flow to the cordycepin synthesis pathway, mainly involving the enhancement of the supply of adenosine, the precursor of cordycepin.

[0055] Preferably, the present invention uses the pTEF1 and pPGI1 promoters to overexpress glucose-6-phosphate dehydrogenase ZWF1 and phosphoglycerate kinase PGK, strengthen the supply of cofactor NADPH and energy carrier ATP, and provide sufficient reducing power and energy for cordycepin synthesis.

[0056] Fourthly, the present invention provides a high-density fermentation method for cordycepin engineering Pichia pastoris based on a fed-batch strategy. The fed-batch fermentation is divided into three stages: Batch, Fed Batch, and methanol induction. The pH values of the Batch and Fed Batch stages are set to 4.0 - 5.0, while the pH value of the methanol induction stage is 5.1 - 6.0. The Batch stage lasts for 20 - 25 h, and at this time, OD 600 reaches 30 - 35. When the total fermentation duration reaches 45 - 55 h, OD 600 reaches 290 - 320. The PC19 strain enters the methanol induction period after a starvation period of 2 - 3 h. Cordycepin at a concentration of 1.9 g / L is detected 60 h after the start of methanol induction. Fermentation is terminated after 144 h of fermentation. At this time, the yield and productivity of cordycepin are 18.3 g / L and 3.05 g / L / d, respectively. The conversion rate of methanol to cordycepin is 50.2 mg / g, OD 600 reaches 368.3, and the dry cell weight (DCW) reaches 149.6 g / L. When the fermentation duration is extended to 216 h, the cordycepin yield reaches 27.2 g / L.

[0057] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art, and the raw materials used are all commercially available products.

[0058] Related materials and methods 1. Main reagents 10×YNB yeast nitrogen base without amino acids: 13.4 g of YNB is dissolved in 100 mL of deionized water and filtered through a 0.22 μm filter membrane for sterilization.

[0059] 500×Biotin (500×B): 20 mg of biotin is dissolved in 100 mL of deionized water and filtered through a 0.22 μm filter membrane for sterilization.

[0060] 10% Glycerol: 100 mL of glycerol is mixed with 900 mL of deionized water and sterilized at 121 °C for 20 min.

[0061] 1M Phosphate buffer PBS (pH 6.0): 132 mL of 1M K2HPO4 and 868 mL of 1M KH2PO4 are mixed, and the pH is adjusted with KOH and sterilized at 121 °C for 20 min.

[0062] 1M Sorbitol solution: 18.2 g of sorbitol is dissolved in 100 mL of deionized water and filtered through a 0.22 μm filter membrane for sterilization.

[0063] PTM1 Trace Element Solution: 6.0 g / LCuSO4·5H2O, 0.08 g / L NaI, 3.0 g / L MnSO4·H2O, 0.2 g / L MoNa2O4·2H2O, 0.02 g / L H3BO3, 0.5 g / L CoCl2, 20.0 g / L ZnCl2, 65.0 g / L FeSO4·7H2O, 0.2g / L Biotin, 5.0 mL / L Concentrated Sulfuric Acid.

[0064] 2. Main Culture Medium LB Liquid / Solid Medium: Yeast Extract 5 g / L, Tryptone 10 g / L, NaCl 10 g / L, add 15 g / L Agar for solid medium.

[0065] Low-Salt LB Liquid / Solid Medium: Yeast Extract 5 g / L, Tryptone 10 g / L, NaCl 5 g / L, add 15 g / L Agar for solid medium.

[0066] YPD Liquid / Solid Medium: Yeast Extract 10 g / L, Peptone 20 g / L, Glucose 20 g / L, add 20 g / L Agar for solid medium.

[0067] BMD Solid Medium: Glucose 20 g / L, YNB 13.4 g / L, Biotin 0.4 mg / L, Agar 20 g / L.

[0068] BMG Solid Medium: Glycerol 10 g / L, YNB 13.4 g / L, Biotin 0.4 mg / L, Agar 20 g / L.

[0069] BMM Solid Medium: Methanol 10mL / L, YNB 13.4 g / L, Biotin 0.4 mg / L, Agar 20 g / L.

[0070] BMGY Medium: Yeast Extract 10 g / L, Peptone 20 g / L, Sterile Water 700 mL / L, sterilize at 115℃ for 15 min, cool to room temperature; add 10×YNB 100 mL / L, 500×Biotin 2 mL / L, 10×Glycerol 100 mL / L, 1 M PBS (pH = 6.0) 100 mL / L.

[0071] BMMY medium: Yeast extract 10 g / L, peptone 20 g / L, sterile water 800 mL / L, sterilized at 115 °C for 15 min, cooled to room temperature; add 10×YNB 100 mL / L, 500×Biotin 2 mL / L, 1 M PBS (pH = 6.0) 100 mL / L; 100% methanol 10 mL / L.

[0072] Basic salt BSM medium: 0.93 g / L CaSO4, 18.2 g / L K2SO4, 14.9 g / L MgSO4·7H2O, 4.13 g / L KOH, 40.0 g / L glycerol, 26.7 mL / L concentrated phosphoric acid (85%).

[0073] 50% (m / v) glycerol feeding medium: Weigh glycerol to prepare 50% glycerol solution, sterilized at 115 °C for 20 min, add 12 mL / L PTM1 before use.

[0074] 100% methanol feeding medium: Measure 100% methanol, add 12 mL / L PTM1 before use.

[0075] 3. Yeast genomic DNA extraction method Absorb YPD bacterial liquid, centrifuge at 12000 rpm for 2 min, and retain the cells. After grinding and breaking yeast cells with liquid nitrogen, use Tiangen yeast genomic DNA extraction kit DP307 to extract yeast genomic DNA, and the related operations refer to the kit instruction manual.

[0076] 4. PCR amplification of target gene fragment Configure according to a 50 μL PCR system, 2 μL of each upstream and downstream primer, 25 μL of 2×PCR mix, and 1 - 2 μL of template. The reaction program is set as follows: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 15 s, annealing at 56 - 72 °C for 15 s, extension at 72 °C for 15 - 60 s / kb, 25 - 35 cycles, and final extension at 72 °C for 5 min.

[0077] 5. Transformation method of Escherichia coli competent cells Take Shenggong DH5α competent cells and mix with 5 - 20 μg of recombinant plasmid, incubate on ice for 20 - 30 min; heat shock in a 42 °C water bath for 40 - 60 s and then incubate on ice for 3 - 5 min; resuspend with 500 - 700 μL of LB, incubate at 37 °C for 45 min; spread on an LB plate containing 70 mg / L bleomycin or kanamycin, and incubate in the dark at 37 °C for 10 - 15 h.

[0078] 6. Escherichia coli plasmid extraction method Take 1 - 5 ml of overnight cultured bacterial solution, centrifuge at 12,000 rpm for 1 min, and retain the bacterial cells. Use the Novoprotein Plasmid Extraction Kit DC201 to extract plasmids from Escherichia coli, and refer to the kit instruction manual for relevant operations.

[0079] 7. Plasmid digestion system Taking a 100 μL digestion system as an example, add 5.0 μL of restriction endonuclease, 10.0 μL of 10× Buffer, and 3 - 5 μg of plasmid; make up to 100 μL with dd H2O; incubate at 37℃ in a water bath for 2 - 3 h.

[0080] 8. PCR product purification and recovery method Cut the target DNA fragment under ultraviolet light. Use the Novoprotein PCR Product Recovery and Gel Extraction Kit DC301 for product purification and recovery, and refer to the kit instruction manual for relevant operations.

[0081] 9. Gibson Assembly homologous recombination system For a 10 μL homologous recombination system, add 5 μL of recombinase, and the molar ratio of the cloning vector to the insert fragment is 1:1 to 1:2. For single - fragment recombination reaction, incubate at 50℃ for 5 min; for multi - fragment recombination reaction, incubate at 50℃ for 15 min; place on ice for transformation. Use the Novoprotein homologous recombinase C115 for homologous assembly, and refer to the kit instruction manual for relevant operations.

[0082] 10. Preparation of Pichia pastoris competent cells Culture in YPD shaker for about 10 - 14 h until OD 600 is 1.2 - 1.5, centrifuge at 5000 rpm at 4℃ for 5 min, resuspend the bacterial cells with 40 mL of 4℃ sterile water; centrifuge at 5000 rpm at 4℃ for 5 min, resuspend the bacterial cells with 20 mL of 4℃ sterile water; centrifuge at 5000 rpm at 4℃ for 5 min, resuspend the bacterial cells with 10 mL of 4℃ 1 M sorbitol; centrifuge at 5000 rpm at 4℃ for 5 min, retain the bacterial cells, add 300 - 500 μL of 4℃ 1 M sorbitol to resuspend the bacterial cells, and keep on ice for use.

[0083] 11. Pichia pastoris electrotransformation method Mix 80 μL of competent cells with 5 - 20 μg of linear plasmid by pipetting, use a 0.2 cm electroporation cuvette; voltage 1500 - 2000 V; capacitance 25 μF; resistance 200 - 400 Ω; electroporation time is 6.0 - 8.0 ms; after electroporation, add 1 mL of 4℃ 1 M sorbitol solution; incubate at 30℃ for 2 - 4 h, spread 200 μL of the bacterial solution on the YPDZ plate, and incubate upside - down at 28 - 30℃ for 3 - 4 days.

[0084] 12. Pichia pastoris Induced Expression Pick up yeast strains with an inoculation loop and transfer them to YPD. Incubate at 30 °C and 200 rpm for 18 - 22 h; inoculate into BMGY at 1%, and wait until the OD 600 is approximately 12.0 - 13.0. Centrifuge to collect the cells and resuspend them in BMMY. Incubate at 30 °C and 200 rpm for 7 days, and supplement 100% methanol at 1% per day.

[0085] 13. High - density Fermentation of Pichia pastoris for Cordycepin Synthesis (1) Seed Liquid Activation Stage Inoculate the strains stored at - 80 °C into 20 mL of YPD medium. Incubate at 28 - 30 °C and 150 - 200 rpm for 20 - 24 h until the OD 600 is approximately 2.0 - 4.0. Inoculate 1 mL of the bacterial solution into 100 mL of YPD medium. Incubate at 28 - 30 °C and 150 - 200 rpm for 18 - 24 h until the OD 600 is approximately 4.0 - 5.0. Then, inoculate into the fermenter according to a volume ratio of 5% - 10%.

[0086] (2) Fermenter Preparation Stage Medium: The filling volume of the medium is 30% - 40% of the fermenter volume. Sterilize at 115 - 121 °C for 15 - 20 min. Since the fermentation is carried out in a fed - batch mode, the final fermentation volume will be approximately 1.5 - 2 times the initial fermentation volume. Note that the PTM1 trace element solution should not be sterilized at high temperature in the fermenter. It needs to be filter - sterilized and then added (when the fermenter medium cools to 30 °C, 4.4 mL / L) to prevent the oxidation and inactivation of some trace element components.

[0087] (3) Step - wise Fermentation and Efficient Cordycepin Synthesis Rapid Strain Growth: The glycerol batch stage is maintained for 20 - 24 h. When the dissolved oxygen (DO) value shows the first rebound increase, it indicates that the initially added 4% glycerol in the fermentation broth has been exhausted. At this time, there is a lack of carbon source in the fermentation broth, which represents the end of the first - stage glycerol batch stage. During the initial accumulation stage of cell biomass in the first 20 - 24 h, the fermentation temperature is controlled at 28 - 30 °C throughout, the pH value is 4.0 - 5.0, the stirring speed is 200 - 800 rpm, the aeration rate is maintained at 2 - 4 vvm, and the dissolved oxygen (DO) value of the fermentation broth is 15 - 75%.

[0088] Glycerol Fermentation: During the glycerol feeding stage, a 40 - 60% (w / v) glycerol medium (supplemented with 10 - 15 mL / L of PTM1 trace element solution) is continuously added at a constant speed of 15 - 25 mL / L / h for a total of 20 - 30 h. Measure the OD every 6 h600 numerical value until OD 600 It is increased to 300 - 350, and the second-stage glycerol fermentation ends. When entering the glycerol feeding stage lasting for 20 - 30 h, the fermentation temperature is 28 - 30 °C, the pH value is 4.0 - 5.0, the stirring speed is 500 - 800 rpm, the aeration rate is maintained at 4 - 8 vvm, and the cells grow rapidly to achieve high cell density growth.

[0089] Methanol induction and cordycepin synthesis: After stopping the glycerol supplementation and continuing the cultivation for 2 - 3 h, after depleting the residual glycerol in the cells, it enters the third-stage methanol induction period. Methanol is added at a gradient increasing rate, 0.5 mL / L / h, 1 mL / L / h, 2 mL / L / h, 3 mL / L / h, 4 mL / L / h, 5 mL / L / h gradually increasing, and starting from the 7th h, the methanol feeding rate of 5 - 10 mL / L / h is maintained to keep the methanol concentration in the fermenter at 0.5 - 1.0 %. The methanol induction period lasts until 144 - 216 h of the total fermentation duration, the fermentation temperature is 28 - 30 °C, the pH value of the fermentation broth is controlled at 5.1 - 6.0, the stirring speed is 500 - 800 rpm, the aeration rate is maintained at 4 - 8 vvm, and the dissolved oxygen DO value of the fermentation broth is controlled at 20 - 60 % to achieve the synthesis and accumulation of cordycepin.

[0090] 14. High-performance liquid chromatography and mass spectrometry detection methods The cordycepin standard product is purchased from Solarbio. Detection and analysis are carried out using the Thermo Fisher Ultimate 3000 HPLC system and the Agilent ZorbaxSB-C18 column. Mobile phase A is 10 mmol / L acetic acid aqueous solution, and mobile phase B is a mixed solution of methanol and acetonitrile at a volume ratio of 1:1. The injection volume is 10 μL, the column temperature is 40 °C, and the detection wavelength is 260 nm. LCMS mass spectrometry detection, spray voltage 3200 V, capillary temperature 300 °C, sheath gas 40 Arb, auxiliary gas 8 Arb, maximum spray current 100 μA, probe temperature 300 °C.

[0091] 15. Yeast two-hybrid The protein-protein interaction was verified using the Clontech Matchmaker Gold Yeast Two-Hybrid System Kit (No. 630489, TaKaRa, Tokyo, Japan). PpRad51 and Brex27 were integrated into the plasmids pGADT7 and pGBKT7 (supplied with the kit) and fused with the GAL4 DNA activation domain (DNA-AD) and GAL4 DNA binding domain (DNA-BD), respectively, as prey and bait constructs. After co-transformation into Y2HGold competent cells, the yeast two-hybrid results were analyzed using SC-Leu-Trp, SC-Leu-Trp-His-Ade, and SC-Leu-Trp-His-Ade defective plates containing X-α-Gal.

[0092] 16. Gene sequences The oxidoreductase / dehydrogenase gene Cordyceps militaris from Cordyceps militaris Cmcns1 ( CCM_04436 ) and the metal-dependent phosphohydrolase gene Cmcns2 ( CCM_04437 ), the nucleoside / nucleotide kinase gene Cmcns3 / NK ( CCM_04438 ), the 2',3'-cyclic nucleotide-2'-phosphodiesterase gene of Escherichia coli cpdB ( b4213 ) and the Brex27 domain gene of humans were optimized according to the codon preference of Pichia pastoris to Ppcns1 (SEQ ID NO:6), Ppcns2 (SEQ ID NO:7), Ppcns3 / NK (SEQ ID NO:8), PpcpdB (SEQ ID NO:9) , PpBrex27 (SEQ ID NO:5).

[0093] Using the genomic DNA of Pichia pastoris GS115 (purchased from Invitrogen, ATCC 20864) as a template, the following endogenous genes were amplified: the endogenous tRNA of Pichia pastoris, dihydroxyacetone synthase ( das1 , PAS_chr3_0832 ), peroxisome biogenesis factor 8 ( pex8, PAS_chr1-4_0349 ), phosphoribosyl pyrophosphate amidotransferase ( purF, PAS_chr1- 1_0430 ), adenylosuccinate synthase ( adss, PAS_chr4_0613 ), glucose-6-phosphate dehydrogenase ( zwf1, PAS_ chr2-1_0308 ) and phosphoglycerate kinase ( pgk, PAS_chr1-4_0292)。

[0094] The gRNA sequences involved in the present invention are shown in Table 1: Table 1 gRNA sequences involved in the present invention (SEQ ID NO: 13-25)

[0095] References: Cai, P., Duan, X., Wu, X., Gao, L., Ye, M., Zhou, Y., 2021. Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichiapastoris . Nucleic Acids Res. 49(13), 7791-7805. https: / / doi.org / 10.1093 / nar / gkab535. 17. Plasmids and Primers The plasmids and corresponding primers used in the present invention are shown in Tables 2-6 (SEQ ID NO: 26-210): Table 2 Plasmids and Primers Involved in the Present Invention

[0096] Table 3 Plasmids and Primers Involved in the Present Invention

[0097] Table 4 Plasmids and Primers Involved in the Present Invention

[0098] Table 5 Plasmids and Primers Involved in the Present Invention

[0099] Table 6 Plasmids and Primers Involved in the Present Invention

[0100] Note: For the self-constructed plasmids involved in Tables 2-6, they are all constructed according to the three basic construction processes of single-fragment amplification, multi-fragment overlap extension, and multi-fragment Gibson assembly into circular plasmids, and then screened by resistance tags such as Zeocin and kanamycin to obtain positive clones and correct plasmids.

[0101] Example 1 Construction and Optimization of the CRISPR-Cas9 System in Pichia pastoris 1. Genomic Integration of Cas9 Protein in Pichia pastoris The hCas9 gene was fused with the Kozak sequence (CGGACC), the GAP promoter (pGAP), the AOX1 terminator (tAOX1), the DAS1 terminator (tDAS1), and the SV40 nuclear localization signal (NLS) to construct the KhCas9 expression cassette ( Figure 1 ). The PpHIS4-1 and PpHIS4-2 fragments amplified from the plasmid pAO815 were ligated to the flanks of the KhCas9 expression cassette to form the KhCas9 genomic recombination fragment ( Figure 1 ). This recombination fragment was inserted into the Pichia pastoris GS115 genome (His4 - , p.C557R) by single-crossover recombination to construct the strain GS115-KhCas9 (His4 + , p.557C) ( Figure 1 ). The complementation of histidine auxotrophy can enhance the growth of Pichia pastoris and the synthesis of target products.

[0102] Among them, the primers used to amplify the PpHIS4-1 fragment were F-PpHIS4-1 and R-PpHIS4-1, and the primers used to amplify the PpHIS4-2 fragment were F-PpHIS4-2 and R-PpHIS4-2.

[0103] 2. Construction of the sgRNA plasmid The sgRNA plasmid consists of a gRNA expression cassette, a Zeocin resistance marker, a yeast replication origin (panARS), and a bacterial replication origin (ori) ( Figure 2 ). The gRNA expression cassette contains the TEF1 promoter (PpTEF1), tAOX1, an endogenous tRNA of Pichia pastoris, three type IIS restriction enzyme sites (BsaI / BspQI / BbsI), and a gRNA scaffold ( Figure 2 ). All gRNA target sequences were designed using CHOPCHOP. The sgRNA-tRNA array can effectively improve the construction efficiency of the sgRNA vector. Therefore, we selected the endogenous tRNA1 (72bp, SEQ ID NO:1), tRNA3 (80bp, SEQ ID NO:2), tRNA4 (85bp, SEQ IDNO:3), and tRNA5 (72bp, SEQ ID NO:4) of Pichia pastoris to construct the sgRNA-tRNA array ( Figure 2 ). Three type IIs restriction enzyme sites were placed between the tRNA and the gRNA scaffold to form three gRNA integration sites of BsaI / BspQI / BbsI, and the enzyme-linearized pZBJ sgRNA plasmid can achieve rapid replacement of the gRNA sequence ( Figure 2 ).

[0104] 3. Construction of Donor DNA Plasmid The donor plasmid backbone includes a Kanamycin resistance tag and a bacterial replication origin ori. Homologous arms (about 1 kb) on both sides of the Cas9 cleavage site (i.e., the sgRNA targeting site) were amplified from the genomic DNA (gDNA) of Pichia pastoris GS115. Through OE-PCR (Overlap Extension PCR) and Gibson assembly, the Kan-GJ fragment, upstream and downstream homologous arms, promoter, terminator, and target gene were integrated into the donor plasmid. The donor plasmid was screened by Kanamycin resistance and then confirmed by Sanger sequencing. Using 500 ng of sgRNA plasmid and 1 μg of donor DNA fragment, electrotransformation was carried out with the GenePulser Xcell electroporation system. The parameters were set as voltage 1.5 kV, capacitance 25 μF, and resistance 200 Ω. The transformed strains were cultured on YPDZ plates for 3 days. 20 colonies were randomly selected from each transformant for genomic DNA extraction and PCR verification. To eliminate the sgRNA plasmid, the confirmed positive strains were cultured in 20 mL of YPD medium for 24 hours and then streaked onto YPD plates. The successful removal of the plasmid was confirmed by repeated plating on YPDZ plates.

[0105] 4. Preliminary Verification of the Gene Editing Ability of the Pichia pastoris CRISPR-Cas9 System The glycerol kinase-deficient strain grows restricted on BMG plates. Using the glycerol kinase gene gut1 as the target, the NHEJ targeting efficiency of each of the BsaI / BspQI / BbsI three sites ( Figure 3 A in it) was evaluated. The results showed that the NHEJ targeting efficiency of ΔGUT1 at the BspQI site (98.4%) was significantly higher than that at the BsaI site (84.1%) and the BbsI site (71.4%) ( Figure 3 B in it). The difference in the targeting efficiency of the three sites of ΔGUT1 may be related to the difference in the self-cleavage efficiency of four tRNAs. The NHEJ repair mechanism randomly generates insertions and deletions of individual bases at genomic DSBs. ΔGUT1 shows three NHEJ mutation types: single-base deletion, two-base deletion, and single-base insertion ( Figure 3 C in it).

[0106] 5. Fusion of Brex27 and Cas9 to Enhance Homologous Recombination Ability To enhance homologous recombination ability, the codon-optimized Brex27 motif (ALDFLSRLPLPPPVSPICTFVSPAAQKAFQPPRSCG) was gene-fused to the C-terminus of KhCas9 via a (GGGGS)3 flexible linker peptide to form the KhCas9-Brex27 expression cassette (the sequence of the KhCas9-Brex27 expression cassette optimized for Pichia pastoris codons is shown in SEQ ID NO:10). This expression cassette was integrated into the Pichia pastoris GS115 genome (His4 - , p.C557R) by single-crossover recombination to construct the strain GS115-KhCas9-Brex27(His4 + , p.557C) ( Figure 4 ). We tested the multiplex editing effects of three reporter genes, glycerol kinase, alcohol oxidase, and histidine multifunctional enzyme, on BMG, BMM, and BMD plates respectively ( Figure 5 ). The results showed that the HDR knockout ratios of the GS115-KhCas9-Brex27 strain in ΔGUT1, ΔGUT1-ΔAOX1, and ΔGUT1-ΔAOX1-ΔHIS4 were 93.7%, 87.3%, and 81.0% respectively, while those of the GS115-KhCas9 strain were only 77.8%, 31.7%, and 20.6%, and were also higher than those of the GS115-KhCas9-PpRAD52 strain, which were 85.7%, 57.1%, and 50.8% ( Figure 6 ). The reason for the lower HDR efficiency than the total targeting efficiency was that some positive clones of auxotrophs were NHEJ-type knockouts, and this conclusion was verified by simulation analysis, PCR amplification, and Sanger sequencing ( Figure 7 , Figure 8 ). The amino acid identity rate between ScRAD51 of Saccharomyces cerevisiae and hRAD51 is 50.12%, while the amino acid identity rate between PpRAD51 of Pichia pastoris and hRAD51 is 56.47%. The results of yeast two-hybrid experiments showed that Brex27 could recruit PpRAD51 and interact with it protein-wise. Therefore, we speculated that KhCas9-Brex27 could also enrich PpRAD51 around DSBs and improve HDR efficiency ( Figure 9 ).

[0107] The construction method of the strain GS115-KhCas9-PpRAD52 is as follows: The GAP promoter-PpRad52 gene-AOX1 terminator expression cassette was constructed and integrated into the Chr1-2 site of the chromosome of the GS115-KhCas9 strain to obtain the GS115-KhCas9-PpRAD52 strain. Among them, the PpRad52 gene (NCBI accession number PAS_chr2-1_0153) was cloned from the Pichia pastoris GS115 genome.

[0108] Example 2 Construction and multi-pathway optimization of the biosynthetic pathway of cordycepin in Pichia pastoris 1. Construction and optimization of the direct synthesis pathway of cordycepin in Pichia pastoris The synthesis of cordycepin is affected by cns1 and cns2 Its direct precursor 3'-AMP comes from the mRNA degradation pathway and adenosine conversion pathway ( Figure 10 The retention time of cordycepin in the standard and fermentation broth was 10.9 min, and the recovery rate of the standard was 98.8% ~ 100.7% ( Figure 10 The standard molecular weight of cordycepin is 251.24, and the mass-to-charge ratios of cordycepin in the standard and sample are 252.10873 and 252.10890, respectively, which confirms that the Pichia pastoris engineered strain synthesizes cordycepin molecules ( Figure 10 In B). Among the methanol-inducible promoters of Pichia pastoris, pAOX1 and pDAS2 were classified as strong methanol-inducible promoters (denoted as Q), pCAT1 (~50% pAOX1) was classified as a moderate methanol-inducible promoter (denoted as Z), and pFLD1 (~30% pAOX1) was classified as a weak methanol-inducible promoter (denoted as R). The results showed that Ppcns1 - Ppcns2 The cordycepin production controlled by the three promoter combinations of pAOX1-pFLD1 (denoted as QR), pAOX1-pCAT1 (denoted as QZ), and pAOX1-pDAS2 (denoted as QQ) were 1709.7 mg / L, 1483.3 mg / L, and 1388.9 mg / L, respectively. Figure 10 Increasing the gene copy number is a common method to increase the yield of the target product, but the cordycepin yield of strains PC04 and PC05 was not significantly different from that of strain PC01 ( Figure 10 This indicates that strain PC01 Ppcns1 and Ppcns2 The expression level of AMP is sufficient, and the rate-limiting step of cordycepin synthesis is located upstream of 3'-AMP. The results of fed-batch fermentation showed that the cordycepin production reached 1999.2 mg / L after the addition of 350 mg / L 3'-AMP, and the conversion rate of 3'-AMP to cordycepin reached 82.7% ( Figure 10In C). PpCns3 / NK can phosphorylate adenosine to 3'-AMP, and PpCpdB can further enhance the supply of 3'-AMP in the mRNA degradation pathway. Figure 10 In A). After overexpressing PpCns3, PpCns3 / NK, PpCpdB, PpCns3-PpCpdB, and PpCns3 / NK-PpCpdB, the cordycepin yields of PC06, PC07, PC08, PC09, and PC10 were significantly increased by 10.6%, 9.6%, 6.0%, 13.6%, and 15.7% respectively compared with PC01. Figure 10 In D). 3'-AMP is a more direct cordycepin precursor than adenosine, and the limited availability of 3'-AMP in the cells is the bottleneck for high-yield cordycepin production.

[0109] The construction method of strain PC01 is as follows: Using GS115-KhCas9-Brex27 as the starting strain, with the help of pZBJ-Chr2-5(BspQI) plasmid and Chr2-5-COR-QR donor DNA plasmid, strain PC01 was constructed.

[0110] Among them, the construction process of pZBJ-Chr2-5(BspQI) plasmid is as follows: pZBJ plasmid was digested with BspQI, and the linearized pZBJ plasmid was recovered by gel extraction; then the gRNA sequence 5'-AACTTTGAAACAAAAGAAGG-3' at the Chr2-5 locus of the Pichia pastoris genome was integrated into the BspQI cleavage site. After heat shock transformation into Escherichia coli DH5α, pZBJ-Chr2-5(BspQI) plasmid was obtained by Zeocin screening. This plasmid can efficiently express sgRNA targeting the Chr2-5 locus in Pichia pastoris cells.

[0111] Among them, the construction process of the Chr2-5-COR-QR donor DNA plasmid is as follows: construct a target gene expression cassette of AOX1 promoter - Kozak sequence (CGGACC) - Ppcns1 - CYC1 terminator (from Saccharomyces cerevisiae S288C) - FLD1 promoter - Kozak sequence (CGGACC) - Ppcns2 - AOX1 terminator; use primers F-Chr2-5-UP-OE and R-Chr2-5-UP-OE to amplify the Chr2-5-UP-OE fragment from the genome of Pichia pastoris GS115; use primers F-Chr2-5-DW-OE and R-Chr2-5-DW-OE to amplify the Chr2-5-DW-OE fragment from the genome of Pichia pastoris GS115; the plasmid screening marker is kanamycin; the plasmid replication origin is ori; the above five fragments are obtained by Gibson assembly to obtain the Chr2-5-COR-QR donor DNA plasmid. Using the Chr2-5-COR-QR donor DNA plasmid as a template and F-Chr2-5-UP and R-Chr2-5-DW as primers, the Chr2-5-COR-QR donor DNA fragment is amplified.

[0112] Finally, 500 ng of pZBJ-Chr2-5(BspQI) plasmid and 1000 ng of Chr2-5-COR-QR donor DNA fragment are co-electroporated into the GS115-KhCas9-Brex27 strain, and the strain PC01 is obtained by Zeocin screening.

[0113] The construction method of strain PC10 is as follows: Using PC01 as the starting strain, with the help of pZBJ-Chr2-4(BspQI) plasmid and Chr2-4-Ppcns3NK-PpcpdB donor DNA plasmid, strain PC10 is constructed.

[0114] Among them, the construction process of the pZBJ-Chr2-4(BspQI) plasmid is as follows: use BspQI to digest the pZBJ plasmid and recover the linearized pZBJ plasmid by gel extraction; then integrate the gRNA sequence 5’-CCTAAATACTACCTAAACAG-3’ at the Chr2-4 locus of the Pichia pastoris genome into the BspQI cleavage site, transform it into Escherichia coli DH5α by heat shock, and obtain the pZBJ-Chr2-4(BspQI) plasmid by Zeocin screening. This plasmid can efficiently express the sgRNA targeting the Chr2-4 locus in Pichia pastoris cells.

[0115] Among them, the construction process of the Chr2-4-Ppcns3NK-PpcpdB donor DNA plasmid is as follows: construct a target gene expression cassette of AOX1 promoter - Kozak sequence (CGGACC) - Ppcns3 / NK - CYC1 terminator - FLD1 promoter - Kozak sequence (CGGACC) - PpcpdB - AOX1 terminator; use primers F-Chr2-4-UP-OE and R-Chr2-4-UP-OE to amplify the Chr2-4-UP-OE fragment from the genome of Pichia pastoris GS115; use primers F-Chr2-4-DW-OE and R-Chr2-4-DW-OE to amplify the Chr2-4-DW-OE fragment from the genome of Pichia pastoris GS115; the plasmid screening marker is kanamycin; the plasmid replication origin is ori; assemble the above five fragments by Gibson assembly to obtain the Chr2-4-Ppcns3NK-PpcpdB donor DNA plasmid. Using the Chr2-4-Ppcns3NK-PpcpdB donor DNA plasmid as a template, amplify the Chr2-4-Ppcns3NK-PpcpdB donor DNA fragment with primers F-Chr2-4-UP and R-Chr2-4-DW.

[0116] Finally, 500 ng of pZBJ-Chr2-4(BspQI) plasmid and 1000 ng of Chr2-4-Ppcns3NK-PpcpdB donor DNA fragment were co-electroporated into strain PC01, and strain PC10 was obtained through Zeocin screening.

[0117] 2. Strengthen the methanol assimilation pathway and enzyme transmembrane transport to promote the synthesis of cordycepin Methanol is oxidized to formaldehyde by AOX, and DAS can rapidly convert formaldehyde into glyceraldehyde 3'-phosphate (G3P) through the Xu5P cycle, thereby alleviating the cytotoxicity of methanol metabolism ( Figure 11 in A). After overexpression of DAS1 by pAOX1, the cordycepin yield increased by 2.8% ( Figure 11 in B). Peroxisome is a functional sub-organelle responsible for methanol assimilation and dissimilation. The peroxisome import protein PEX8 is responsible for transmembrane transport of methanol metabolism pathway enzymes from the cytoplasm into the peroxisome. After overexpression of PEX8 by pFLD1, the cordycepin yield increased significantly by 3.6%. After co-overexpression of DAS1-PEX8, the cordycepin yield increased significantly by 6.8%, while the cell density did not change significantly ( Figure 11 in B). In this study, a methanol concentration gradient of 0.5% - 3% was further used to test the methanol tolerance and the changes in cordycepin yield of strains PC10 and PC13. Figure 11C). When the methanol concentration was increased from 0.5% to 1%, the cordycepin yields of strains PC10 and PC13 were significantly increased by 13.7% and 20.8% respectively, but the highest cell densities of strains PC10 and PC13 both appeared in the 0.75% methanol treatment group( Figure 11 C). However, during the process of increasing the methanol concentration from 1% to 3%, there was no continuous increase in the cordycepin yield, and the toxicity of high-concentration methanol led to a gradual decrease in cell density( Figure 11 C). The results showed that 1% methanol was sufficient to supply the carbon metabolic flux required for cell growth and cordycepin synthesis, and the limiting site of cordycepin synthesis was located downstream of the methanol assimilation pathway.

[0118] The construction method of strain PC13 is as follows: Using PC10 as the starting strain, with the help of pZBJ-Chr1-2(BsaI) plasmid and Chr1-2-PpDAS1-PpPEX8 donor DNA plasmid, strain PC13 was constructed.

[0119] Among them, the construction process of pZBJ-Chr1-2(BsaI) plasmid is as follows: pZBJ plasmid was digested with BsaI, and the linearized pZBJ plasmid was recovered by gel extraction; subsequently, the gRNA sequence 5'-GGTTGGTACTATGTCCAACA-3' at the Chr1-2 locus of the Pichia pastoris genome was integrated into the BsaI cleavage site, and after heat shock transformation into Escherichia coli DH5α, the pZBJ-Chr1-2(BsaI) plasmid was obtained through Zeocin screening. This plasmid can highly express sgRNA targeting the Chr1-2 locus in Pichia pastoris cells.

[0120] Among them, the construction process of the Chr1-2-PpDAS1-PpPEX8 donor DNA plasmid is as follows: construct a target gene expression cassette of AOX1 promoter - Kozak sequence (CGGACC) - PpDAS1 - CYC1 terminator - FLD1 promoter - Kozak sequence (CGGACC) - PpPEX8 - AOX1 terminator; amplify the Chr1-2-UP-OE fragment from the genome of Pichia pastoris GS115 using primers F-Chr1-2-UP-OE and R-Chr1-2-UP-OE; amplify the Chr1-2-DW-OE fragment from the genome of Pichia pastoris GS115 using primers F-Chr1-2-DW-OE and R-Chr1-2-DW-OE; the plasmid screening marker is kanamycin; the plasmid replication origin is ori; assemble the above five fragments by Gibson assembly to obtain the Chr1-2-PpDAS1-PpPEX8 donor DNA plasmid. Using the Chr1-2-PpDAS1-PpPEX8 donor DNA plasmid as a template, amplify the Chr1-2-PpDAS1-PpPEX8 donor DNA fragment using primers F-Chr1-2-UP and R-Chr1-2-DW.

[0121] Finally, 500 ng of pZBJ-Chr1-2(BsaI) plasmid and 1000 ng of Chr1-2-PpDAS1-PpPEX8 donor DNA fragment were co-electroporated into strain PC10, and strain PC13 was obtained by screening with Zeocin.

[0122] 3. Strengthen the supply of precursor substances in the purine synthesis pathway to promote the synthesis of cordycepin The de novo purine synthesis pathway is the key metabolic flux source for the synthesis of nucleosides and nucleoside analogs. 5-Phosphoribosyl-1-pyrophosphate (PRPP) is converted into inosine monophosphate (IMP) through 10 consecutive catalytic reactions, and IMP is further converted into adenosine monophosphate (AMP). PRPP is the link connecting the pentose phosphate pathway and the de novo purine synthesis pathway, and PurF is the key rate-limiting enzyme that catalyzes the initial reaction in the de novo purine synthesis pathway ( Figure 11 in A). After catalysis by AdSS, IMP will accelerate the conversion to AMP, and the adenosine generated by the dephosphorylation of AMP supplies the substrate for the efficient synthesis of cordycepin ( Figure 11 in A). After overexpressing PurF and AdSS, the cordycepin yields of strains PC14, PC15, and PC16 reached 2256.8 mg / L, 2136.9 mg / L, and 2362.6 mg / L respectively, which were significantly increased by 9.4%, 3.6%, and 14.5% compared with strain PC13 respectively ( Figure 11D). The results of the adenosine content change showed that the adenosine synthesis in the engineered strain was affected by multiple factors, including the cordycepin biosynthesis pathway, methanol assimilation pathway, and de novo purine synthesis pathway. Figure 11 E). The adenosine content of strains PC16 and PC13 was analyzed every 24 hours. The adenosine content of PC16 was significantly increased by 7.5% - 16.9% compared to PC13, indicating that sufficient precursor supply could accelerate the synthesis of cordycepin. Figure 11 E). Adenosine, adenine, inosine, and hypoxanthine are often used as precursors for cordycepin biosynthesis. However, the use of precursors will significantly increase production costs, and strengthening the endogenous supply of precursors is a more cost - effective production strategy.

[0123] The construction method of strain PC16 is as follows: Using PC13 as the starting strain, with the help of pZBJ - Chr1 - 5(BspQI) plasmid and Chr1 - 5 - purF - AdSS donor DNA plasmid, strain PC16 was constructed.

[0124] Among them, the construction process of pZBJ - Chr1 - 5(BspQI) plasmid is as follows: pZBJ plasmid was digested with BspQI, and the linearized pZBJ plasmid was recovered by gel extraction; subsequently, the gRNA sequence 5’ - CACGAGCCGAGTAATAACCG - 3’ at the Chr1 - 5 locus of Pichia pastoris genome was integrated into the BspQI cleavage site. After heat - shock transformation into Escherichia coli DH5α and screening with Zeocin, pZBJ - Chr1 - 5(BspQI) plasmid was obtained, which could efficiently express sgRNA targeting the Chr1 - 5 locus in Pichia pastoris cells.

[0125] Among them, the construction process of Chr1 - 5 - purF - AdSS donor DNA plasmid is as follows: construct the target gene expression cassette of CAT1 promoter - purF - DAS1 terminator - FDH1 promoter - AdSS - AOX1 terminator; amplify the Chr1 - 5 - UP - OE fragment from Pichia pastoris GS115 genome using primers F - Chr1 - 5 - UP - OE and R - Chr1 - 5 - UP - OE; amplify the Chr1 - 5 - DW - OE fragment from Pichia pastoris GS115 genome using primers F - Chr1 - 5 - DW - OE and R - Chr1 - 5 - DW - OE; the plasmid selection marker is kanamycin; the plasmid replication origin is ori; the above five fragments were assembled by Gibson assembly to obtain Chr1 - 5 - purF - AdSS donor DNA plasmid. Using Chr1 - 5 - purF - AdSS donor DNA plasmid as the template and F - Chr1 - 5 - UP and R - Chr1 - 5 - DW as primers, the Chr1 - 5 - purF - AdSS donor DNA fragment was amplified.

[0126] Finally, 500 ng of pZBJ-Chr1-5 (BspQI) plasmid and 1000 ng of Chr1-5-purF-AdSS donor DNA fragment were co-electroporated into strain PC13, and strain PC16 was obtained through Zeocin screening.

[0127] 4. Strengthen the supply of cofactors NADPH and ATP to promote cordycepin synthesis Adequate ATP supply and a stable intracellular reducing environment provide the necessary energy, phosphate groups, and reducing power for cordycepin synthesis. In the metabolic pathway of adenosine synthesizing cordycepin, the process of synthesizing 1 molecule of adenosine from 1 molecule of PRPP and then converting it into 1 molecule of cordycepin requires the consumption of 8 molecules of ATP. Insufficient ATP supply may be the rate-limiting step in cordycepin synthesis. Research shows that the strong hydrogen-donating ability of NADPH plays an important role in the process of reducing the carbonyl group to a hydroxyl group. Yeast mainly synthesizes NADPH through the pentose phosphate pathway (PPP), and NADPH may be an important hydrogen donor for the conversion of 2′-C-3′-dA to cordycepin ( Figure 11 in A). After overexpressing ZWF1 and PGK, the cordycepin yields of strains PC17, PC18, and PC19 reached 2436.4 mg / L, 2409.2 mg / L, and 2509.7 mg / L respectively, which were significantly increased by 4.0%, 2.8%, and 7.1% compared with PC16 ( Figure 11 in F). The NADPH / NADP+ ratio and ATP content of strains PC19 and PC16 were compared and analyzed every 24 hours ( Figure 11 in G). The results showed that the ATP content of strain PC19 was significantly increased by 7.5% - 16.9% compared with PC16, and at the same time, the NADPH / NADP+ ratio of PC19 was also always higher than that of PC16 ( Figure 11 in G). By strengthening the regeneration and supply capacity of ATP and NADPH, the synthesis environment of cordycepin in Pichia pastoris was significantly optimized, which was beneficial to the efficient accumulation of cordycepin. Finally, the hCas9 protein in strain PC19 was knocked out to avoid the phenomenon of spontaneous gene editing in the strain. The construction method of strain PC19 is as follows: Using PC16 as the starting strain, with the help of pZBJ-Chr3-5 (BspQI) plasmid and Chr3-5-ZWF1-PGK donor DNA plasmid, strain PC19 was constructed.

[0128] Among them, the construction process of the pZBJ-Chr3-5(BspQI) plasmid is as follows: Use BspQI to digest the pZBJ plasmid, and recover the linearized pZBJ plasmid by gel extraction; Subsequently, integrate the gRNA sequence 5’-ATACTAGTTAACAAACTGGG-3’ at the Chr3-5 locus of the Pichia pastoris genome into the BspQI cleavage site. After heat shock transformation into Escherichia coli DH5α, the pZBJ-Chr3-5(BspQI) plasmid is obtained by Zeocin screening. This plasmid can highly express the sgRNA targeting the Chr3-5 locus in Pichia pastoris cells.

[0129] Among them, the construction process of the Chr3-5-ZWF1-PGK donor DNA plasmid is as follows: Construct the target gene expression cassette of TEF1 promoter-ZWF1-DAS1 terminator-PGI1 promoter-PGK-AOX1 terminator; Use primers F-Chr3-5-UP-OE and R-Chr3-5-UP-OE to amplify the Chr3-5-UP-OE fragment from the Pichia pastoris GS115 genome; Use primers F-Chr3-5-DW-OE and R-Chr3-5-DW-OE to amplify the Chr3-5-DW-OE fragment from the Pichia pastoris GS115 genome; The plasmid screening marker is kanamycin; The plasmid replication origin is ori; The above five fragments are obtained by Gibson assembly to get the Chr3-5-ZWF1-PGK donor DNA plasmid. Using the Chr3-5-ZWF1-PGK donor DNA plasmid as a template, and F-Chr3-5-UP and R-Chr3-5-DW as primers to amplify the Chr3-5-ZWF1-PGK donor DNA fragment.

[0130] Finally, 500 ng of the pZBJ-Chr3-5(BspQI) plasmid and 1000 ng of the Chr3-5-ZWF1-PGK donor DNA fragment are co-electroporated into the PC16 strain, and the strain PC19 is obtained by Zeocin screening.

[0131] The method for knocking out the hCas9 protein in the strain PC19 is as follows: To remove the hCas9 protein from the PC19 genome, with the help of the pZBJ-ΔhCas9(BspQI) plasmid and the ΔCas9-UP-DW donor DNA fragment, the knockout of the hCas9 protein in the PC19 strain is achieved.

[0132] Among them, the construction process of the pZBJ-ΔhCas9(BspQI) plasmid is as follows: pZBJ plasmid is digested with BspQI, and the linearized pZBJ plasmid is recovered by gel extraction; subsequently, the gRNA sequence 5’-TACGCCGGATACATTGACGG-3’ of the hCas9 gene in the Pichia pastoris genome is integrated into the BspQI cleavage site, and after heat shock transformation into Escherichia coli DH5α, the pZBJ-ΔhCas9(BspQI) plasmid is obtained by screening with Zeocin. This plasmid can efficiently express the sgRNA targeting the hCas9 site in Pichia pastoris cells.

[0133] Among them, the construction process of the ΔCas9-UP-DW donor DNA fragment is as follows: the ΔCas9-UP-OE fragment is amplified from the Pichia pastoris GS115 genome using the primers F-ΔCas9-UP-OE and R-ΔCas9-UP-OE; the ΔCas9-DW-OE fragment is amplified from the Pichia pastoris GS115 genome using the primers F-ΔCas9-DW-OE and R-ΔCas9-DW-OE; the ΔCas9-UP-DW donor DNA fragment is obtained after overlap PCR extension of the ΔCas9-UP-OE fragment and the ΔCas9-DW-OE fragment.

[0134] Finally, 500 ng of the pZBJ-ΔhCas9(BspQI) plasmid and 1000 ng of the ΔCas9-UP-DW donor DNA fragment are co-electroporated into the PC19 strain, and the strain PC19 with the hCas9 protein knocked out is obtained by screening with Zeocin.

[0135] Example 3 Low-carbon and high-efficiency synthesis of cordycepin by segmented high-density fermentation High-density fermentation of the Pichia pastoris PC19 strain is carried out in a 10 L fermenter using a fed-batch strategy. The fed-batch fermentation process is divided into three stages: Batch (fermentation in the fermenter, consuming the original glycerol in the medium), Fed Batch (glycerol feeding), and methanol induction. The staged feeding of glycerol and methanol effectively separates the biomass accumulation stage and the cordycepin synthesis stage of Pichia pastoris. The stirring speed of the fermenter is cascaded with the DO level. When the dissolved oxygen (DO) value is lower than 20%, the stirring speed is actively increased to ensure that the DO is not lower than the threshold. Research shows that a weakly acidic environment is beneficial to the synthesis and accumulation of cordycepin. Therefore, the pH values of the Batch and Fed Batch stages are set to 5.0, while the pH value of the methanol induction stage is 5.5. The Batch stage lasts for 22 h in total. The end of the Batch stage is marked by the DO rising back above 60%, at which time the OD 600 reaches 32.8( Figure 12 ). When the total fermentation time reaches 50 h, the OD 600 reaches 311.8(Figure 12 ), at this time, the DO rising above 75% marks the end of the Fed Batch stage. After experiencing a 3-hour starvation period, the PC19 strain enters the methanol induction period. Cordycepin at 1.9 g / L was detected 60 h after the start of methanol induction ( Figure 13 ). Fermentation was terminated after a total fermentation duration of 144 h. At this time, the cordycepin yield and productivity were 18.3 g / L and 3.05 g / L / d, respectively, the conversion rate of methanol to cordycepin was 50.2 mg / g, OD 600 reached 368.3, and the dry cell weight (DCW) of the cells reached 149.6 g / L ( Figure 13 ). When the total fermentation duration was extended to 216 h, the cordycepin yield reached 27.2 g / L ( Figure 13 ).

[0136] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. The KhCas9-Brex27 expression cassette, characterized in that, The expression cassette comprises the following elements connected in sequence: PpHIS4-1 fragment - GAP promoter - Kozak sequence - hCas9 protein - Linker - Brex27 motif - SV40 nuclear localization signal - AOX1 terminator - DAS1 terminator - PpHIS4-2 fragment; Among them, the PpHIS4-1 fragment and PpHIS4-2 fragment are from plasmid pAO815; the primers for amplifying the PpHIS4-1 fragment are F-PpHIS4-1 and R-PpHIS4-1, and the primers for amplifying the PpHIS4-2 fragment are F-PpHIS4-2 and R-PpHIS4-2; The primer sequences are as follows (5'-3'): F-PpHIS4-1: ATGACATTTCCCTTGCTACCTGCAT R-PpHIS4-1: CTTGTGAGGAGGCCTTAGAAACGTC F-PpHIS4-2: AAATTGAAGATGCCCTTCGTCGTCCT R-PpHIS4-2: TTAAATAAGTCCCAGTTTCTCCATACGAACC The Brex27 motif is from humans, and the amino acid sequence is ALDFLSRLPLPPPVSPICTFVSPAAQKAFQPPRSCG; The hCas9 protein is derived from Streptococcus pyogenes ( Streptococcus pyogenes ), and its reference sequence number in NCBI is 69900935; The GAP promoter, AOX1 terminator, and DAS1 terminator are all from Pichia pastoris ( Pichia pastoris ), GS115.

2. The expression cassette according to claim 1, characterized in that The amino acid sequence of the Linker is (GGGGS)3; The sequence of the KhCas9-Brex27 expression cassette optimized by Pichia pastoris codons is as shown in SEQ ID NO:

10.

3. An sgRNA expression plasmid for use in combination with the expression cassette according to claim 1 or 2, characterized in that, It includes at least the following elements: gRNA expression cassette, Zeocin resistance marker, yeast replication origin panARS, and bacterial replication origin ori; Among them, the gRNA expression cassette contains the TEF1 promoter, AOX1 terminator, endogenous tRNA1, tRNA3, tRNA4, and tRNA5 of Pichia pastoris, type IIS restriction endonuclease sites BsaI, BspQI, and BbsI, and three gRNA scaffolds; The TEF1 promoter is from Pichia pastoris GS115; The nucleotide sequences of the endogenous tRNA1, tRNA3, tRNA4, and tRNA5 of Pichia pastoris are as shown in SEQ ID NO:1-4 respectively.

4. The sgRNA expression plasmid according to claim 3, wherein The gRNA expression cassette comprises the following elements connected in sequence: TEF1 promoter - tRNA① - endonuclease site① - gRNA scaffold - tRNA② - endonuclease site② - gRNA scaffold - tRNA③ - endonuclease site③ - gRNA scaffold - tRNA④ - AOX1 terminator; Among them, tRNA①, tRNA②, tRNA③, and tRNA④ are different from each other and correspond to four endogenous tRNAs of Pichia pastoris respectively; Endonuclease site①, endonuclease site②, and endonuclease site③ are different from each other and correspond to three type IIS restriction endonuclease sites respectively.

5. The sgRNA expression plasmid according to claim 4, characterized in that, The gRNA expression cassette comprises the following elements connected in sequence: TEF1 promoter - tRNA1 - endonuclease site BsaI - gRNA scaffold - tRNA3 - endonuclease site BspQI - gRNA scaffold - tRNA4 - endonuclease site BbsI - gRNA scaffold - tRNA5 - AOX1 terminator; The complete sequence of the sgRNA expression plasmid is shown in SEQ ID NO:

11.

6. Method for constructing a high-efficiency gene editing system for Pichia pastoris, characterized in that, Integrate the expression cassette described in claim 1 or 2 into the genome of Pichia pastoris GS115 to obtain the Pichia pastoris strain GS115 - KhCas9 - Brex27 that highly expresses the hCas9 protein, and then introduce the sgRNA expression plasmid described in any one of claims 3 - 5 and the donor DNA fragment into Pichia pastoris GS115 - KhCas9 - Brex27, thus obtaining the Pichia pastoris CRISPR - Cas9 gene editing system.

7. The highly efficient Pichia pastoris gene editing system constructed by the method according to claim 6.

8. Use of the highly efficient Pichia pastoris gene editing system described in claim 7 in constructing a high - yield cordycepin engineering strain.

9. A Pichia pastoris engineering strain with high cordycepin production, characterized in that, Its construction method comprises the following steps: (1) Integrate the expression cassette described in claim 1 or 2 into the genome of Pichia pastoris GS115 to obtain the strain GS115 - KhCas9 - Brex27, and then introduce the expression plasmid containing sgRNA1 and the donor DNA1 fragment into the strain GS115 - KhCas9 - Brex27 to construct engineering bacterium 1; Among them, the nucleotide sequence of sgRNA1 targeting the chromosome Chr2 - 5 of Pichia pastoris is 5’ - AACTTTGAAACAAAAGAAGG - 3’; The target gene carried by the donor DNA1 fragment is a gene from Cordyceps militaris ( Cordyceps militaris ), namely CP1, and the gene optimized by Pichia pastoris codons Ppcns1 and Ppcns2 ; among them, the gene Ppcns1 is driven by the promoter pAOX1, and the gene Ppcns2 is driven by the promoter pFLD1; Gene Ppcns1 and Ppcns2 The nucleotide sequences are shown in SEQ ID NO:6 and 7 respectively; The promoters pAOX1 and pFLD1 are from Pichia pastoris GS115; (2) Introduce the expression plasmid containing sgRNA2 and the donor DNA2 fragment into engineering bacterium 1 to construct engineering bacterium 2; Among them, the nucleotide sequence of sgRNA2 targeting the chromosome Chr2 - 4 of Pichia pastoris is 5’ - CCTAAATACTACCTAAACAG - 3’; The target gene carried by the donor DNA2 fragment is the gene from Cordyceps militaris CP1 and optimized by Pichia pastoris codons. Ppcns3 / NK , and the gene from Escherichia coli ( Escherichia coli ), and optimized by Pichia pastoris codons PpcpdB ; among them, the gene Ppcns3 / NK is driven by the promoter pAOX1, and the gene PpcpdB is driven by the promoter pFLD1; Gene Ppcns3 / NK and PpcpdB The nucleotide sequences are shown in SEQ ID NO:8 and 9, respectively; (3) Introduce the expression plasmid containing sgRNA3 and the donor DNA3 fragment into engineering bacterium 2 to construct engineering bacterium 3; Among them, the nucleotide sequence of sgRNA3 targeting the chromosome Chr1 - 2 of Pichia pastoris is 5’ - GGTTGGTACTATGTCCAACA - 3’; The target gene carried by the donor DNA3 fragment is an endogenous gene of Pichia pastoris das1 and pex8 ; among them, gene das1 is driven by the promoter pAOX1, and gene pex8 is driven by the promoter pFLD1; Gene das1 and pex8 The reference sequence numbers in NCBI are PAS_chr3_0832 and PAS_chr1-4_0349, respectively; (4) Introduce the expression plasmid containing sgRNA4 and the donor DNA4 fragment into engineering bacterium 3 to construct engineering bacterium 4; Among them, the nucleotide sequence targeted by sgRNA4 on the chromosome Chr1-5 of Pichia pastoris is 5’-CACGAGCCGAGTAATAACCG -3’; The target gene carried by the donor DNA4 fragment is an endogenous gene of Pichia pastoris purF and adss ; among them, the gene purF is driven by the promoter pCAT1, and the gene adss is driven by the promoter pFDH1; Gene purF and adss The reference sequence numbers in NCBI are PAS_chr1-1_0430 and PAS_chr4_0613 respectively; The promoters pCAT1 and pFDH1 are from Pichia pastoris GS115; (5) An expression plasmid containing sgRNA5 and a donor DNA5 fragment are introduced into engineering bacterium 4 to construct engineering bacterium 5; Among them, the nucleotide sequence targeted by sgRNA5 on the chromosome Chr3-5 of Pichia pastoris is 5’-ATACTAGTTAACAAACTGGG -3’; The target gene carried by the donor DNA5 fragment is an endogenous gene of Pichia pastoris zwf1 and pgk ; among them, gene zwf1 is driven by the promoter pTEF1, and gene pgk is driven by the promoter pPGI1; Gene zwf1 and pgk The reference sequence numbers in NCBI are PAS_chr2-1_0308 and PAS_chr1-4_0292 respectively; The promoters pTEF1 and pPGI1 are from Pichia pastoris GS115; (6) An expression plasmid containing sgRNA6 and a donor DNA6 fragment are introduced into engineering bacterium 5, and the hCas9 protein is knocked out to obtain the Pichia pastoris engineering bacterium with high-yield cordycepin; Among them, the nucleotide sequence targeted by sgRNA6 on the chromosome of engineering bacterium 5 for hCas9 is 5’-TACGCCGGATACATTGACGG-3’; The construction method of the donor DNA6 fragment includes: amplifying the ΔCas9-UP-OE fragment from the genome of Pichia pastoris GS115 using primers F-ΔCas9-UP-OE and R-ΔCas9-UP-OE; amplifying the ΔCas9-DW-OE fragment from the genome of Pichia pastoris GS115 using primers F-ΔCas9-DW-OE and R-ΔCas9-DW-OE; the ΔCas9-UP-OE fragment and the ΔCas9-DW-OE fragment are subjected to overlap extension PCR to obtain the ΔCas9-UP-DW donor DNA fragment; The primer sequences are as follows (5'-3'): F-ΔCas9-UP-OE: TTTTTGTAGAAATGTCTTGGTGTCCTCGTCC R-ΔCas9-UP-OE: CTTCTGCTCATTATCTTCCCGAAATTTGATCATATGCGCCAGC F-ΔCas9-DW-OE: CATATGATCAAATTTCGGGAAGATAATGAGCAGAAGCAGCTGTTCG R-ΔCas9-DW-OE: ACGGGAAGTCTTTACAGTTTTAGTTAGGAGCC The above-mentioned expression plasmid containing sgRNA is the same as the sgRNA expression plasmid described in any one of claims 3-5.

10. Use of the engineering bacteria according to claim 9 in cordycepin production, characterized in that, Including: The engineering bacterium is cultured by a fed-batch fermentation process to obtain a culture, and cordycepin produced is collected from the culture; Among them, the fed-batch fermentation process includes: S1. Activation of the seed solution; S2. Fermenter culture: Inoculate the seed liquid with an OD 600 value of 4.0 - 5.0 into the fermenter for fermenter culture at a volume ratio of 5% - 10%; The culture conditions are as follows: the filling volume of the medium is 30%-40% of the volume of the fermenter; the temperature is controlled at 28-30 °C, the pH value is 4.0-5.0, the stirring speed is 200-800 rpm, the aeration rate is maintained at 2-4 vvm, and the dissolved oxygen (DO) value of the fermentation broth is 20-60%; The formula of the medium is: 0.8-1.2 g / L CaSO4, 15-20 g / L K2SO4, 12-18 g / L MgSO4·7H2O, 2-5 g / L KOH, 30-50 g / L glycerol, 25-30 mL / L of 85% concentrated phosphoric acid; S3, Glycerol feeding: When the dissolved oxygen (DO) value shows the first rebound increase, it indicates that the glycerol in the fermentation broth has been exhausted. Then, the glycerol feeding medium is added at a constant rate of 15 - 25 mL / L / h until the OD 600 increases to 300 - 350, and the glycerol feeding stage ends; The culture conditions are as follows: the fermentation temperature is 28-30 °C, the pH value is 4.0-5.0, the stirring speed is 500-800 rpm, and the aeration rate is maintained at 4-8 vvm; The formula of the glycerol feeding medium is: a 40-60% m / v glycerol solution containing 10-15 mL / L of PTM1; S4. Methanol induction and cordycepin synthesis: After stopping the addition of glycerol, continue to culture for 2-3 h to deplete the residual glycerol in the cells. Record the time as t0, and then start methanol induction; Add the methanol feeding medium at the following gradient increasing speeds: 0.5 mL / L / h, 1 mL / L / h, 2 mL / L / h, 3 mL / L / h, 4 mL / L / h, 5 mL / L / h. Starting from t0 + 7 h, maintain a methanol feeding rate of 5-10 mL / L / h to keep the methanol concentration in the fermenter at 0.5-1.0%; The culture conditions are as follows: the fermentation temperature is 28-30 °C, the pH value is controlled at 5.1-6.0, the stirring speed is 500-800 rpm, the aeration rate is maintained at 4-8 vvm, and the dissolved oxygen (DO) value of the fermentation broth is controlled at 15-75%. After 6-9 days of fermentation, end the fermentation and empty the fermenter; The formula of the methanol feeding medium is: 100% methanol containing 10-15 mL / L of PTM1.

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