A pichia pastoris high-efficiency gene editing system, a construction method thereof and application in high-yield cordycepin engineering strain

Abstract

The present application relates to the field of gene editing technology, bioengineering and application of microbial fermentation, and discloses a Pichia pastoris high-efficiency gene editing system, a construction method thereof and application in a high-yield cordycepin engineering strain.The construction of the Pichia pastoris CRISPR-Cas9 gene editing system mainly includes the following steps: the histidine HIS4 nutritional deficiency back complementation is used to realize the resistance-free assembly of KhCas9; the sgRNA-tRNA array and the IIS type restriction endonuclease combination are used to simplify the gene editing operation and realize the simultaneous and high-efficiency editing of multiple genes; and the Brex27 domain is fused with KhCas9 to strengthen the homologous recombination efficiency. By using the gene editing system, through the optimization of 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 residues is realized for the first time, the gene editing efficiency of Pichia pastoris is greatly improved, and a powerful tool is provided for the industrialized production of cordycepin.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene editing technology, bioengineering and application of microbial fermentation, in particular, to a Pichia pastoris high-efficiency gene editing system, a construction method thereof and application in high-yield cordycepin engineering strain. BACKGROUND

[0002] Pichia pastoris is a recognized safe (GRAS) methylotrophic yeast, which is widely used in 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 separation of growth phase (glycerol feeding) and production phase (methanol induction) in time makes Pichia pastoris an effective carrier for synthesizing antibacterial compounds such as cordycepin, thereby overcoming the growth inhibition caused by product accumulation. However, the current gene editing strategy of Pichia pastoris is mainly based on the single cross integration technology of plasmid, which relies on antibiotic markers, which inevitably introduces yeast sensitive resistance genes such as bleomycin, nourseothricin, hygromycin and G418 into the genome. These residual resistance genes will lead to increased industrial waste disposal costs and the risk of environmental drift of resistance genes. Therefore, developing a CRISPR-Cas9 system without the need to introduce resistance gene markers into the yeast genome is of great significance for realizing green production of Pichia pastoris cell factories.

[0003] Plasmid construction efficiency and multiplex genome editing capabilities are key bottlenecks in the development of Pichia pastoris cell factories. The current CRISPR-Cas9 system in Pichia pastoris relies on the HH and HDV ribozymes to express sgRNAs. However, the variable 6-bp sequence in the HH ribozyme requires replacement according to the gRNA sequence, complicating vector construction and significantly increasing the work-cycle during target gene replacement or multiple editing. In contrast, the indiscriminate cleavage of tRNA by RNase P and RNase Z facilitates sequence-independent sgRNA-tRNA fusion cleavage, providing a rapid sgRNA assembly strategy. Precise genome editing relies on homology-directed repair (HDR). However, in Pichia pastoris, double-strand break (DSB) repair is dominated by non-homologous end joining (NHEJ), and HDR itself is extremely inefficient. Overexpression of HDR-related proteins can improve HDR efficiency without impairing NHEJ, thereby maintaining genetic stability. Notably, localized enrichment of HDR proteins near DSBs has been shown to be crucial for HDR repair. RAD51 is involved in all stages of HDR. Studies have shown that the human Brex27 domain can recruit RAD51 to DSB sites, effectively improving HDR efficiency. Combining sgRNA-tRNA arrays 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 analog found primarily in ascomycete fungi such as Cordyceps militaris, Cicadae, Cordyceps sinensis, and Kyushu Cordyceps. Cordycepin exhibits potent biological activities, including antibacterial, antiviral, antioxidant, immunomodulatory, and neuromodulatory properties. Its anticancer potential has recently been demonstrated in a Phase I clinical trial of NUC-7738. Current chemical synthesis methods for cordycepin face numerous challenges, including low yields, difficulty in controlling chirality, expensive substrates and catalysts, high levels of residual tin, and severe organic solvent contamination. Biosynthesis remains the primary production pathway for cordycepin. However, cell factories produced by methods such as Cordyceps militaris, Saccharomyces cerevisiae, Yarrowia lipolytica, and Aspergillus oryzae produce low cordycepin yields and rely heavily on food-derived raw materials such as wheat, rice, and glucose. The CRISPR-Cas9 technology provided by the present invention can transform Pichia pastoris into a high-yield cordycepin cell factory. Using methanol as a carbon source effectively avoids the competition for food with the public during cordycepin production, making it an important approach for achieving green, efficient, and low-cost cordycepin synthesis. Summary of the Invention

[0005] The purpose of the present invention is to provide a Pichia pastoris efficient gene editing system and its construction method and application.

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

[0007] To achieve the purpose 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;

[0008] The PpHIS4-1 and PpHIS4-2 fragments were derived from plasmid pAO815 (purchased from Invitrogen). 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.

[0009] The primer sequences are as follows (5'-3'):

[0010] F-PpHIS4-1:ATGACATTTCCCTTGCTACCTGCAT

[0011] R-PpHIS4-1:CTTGTGAGGAGGCCTTAGAAACGTC

[0012] F-PpHIS4-2: AAATTGAAGATGCCCTTCGTCGTCCT

[0013] R-PpHIS4-2:TTAAATAAGTCCCAGTTTCTCCATACGAACC

[0014] The Brex27 motif is derived from humans and has an amino acid sequence of ALDFLSRLPLPPPVSPICTFVSPAAQKAFQPPRSCG (SEQ ID NO: 211). The Pichia pastoris codon-optimized DNA sequence encoding the Brex27 motif is shown in SEQ ID NO: 5.

[0015] hCas9 protein from Streptococcus pyogenes ( Streptococcus pyogenes ), which has been sequence-optimized according to human codon preference, and the reference sequence number in NCBI is 69900935;

[0016] The GAP promoter (pGAP), AOX1 terminator (tAOX1), and DAS1 terminator (tDAS1) were all from Pichia pastoris ( Pichia pastoris )GS115.

[0017] The Kozak sequence is CGGACC.

[0018] Preferably, the amino acid sequence of the linker is (GGGGS)3 (SEQ ID NO: 212).

[0019] More preferably, the sequence of the KhCas9-Brex27 expression cassette after Pichia codon optimization is shown in SEQ ID NO: 10.

[0020] In a second aspect, the present invention provides an sgRNA expression plasmid for use 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);

[0021] The gRNA expression cassette contains the TEF1 promoter, the AOX1 terminator, Pichia pastoris endogenous tRNA1, tRNA3, tRNA4, and tRNA5, type IIS restriction endonuclease sites BsaI, BspQI, and BbsI, and three gRNA scaffolds (gRNAscaffold);

[0022] TEF1 promoter (pTEF1) was from Pichia pastoris GS115.

[0023] The nucleotide sequences of endogenous tRNA1, tRNA3, tRNA4 and tRNA5 of Pichia pastoris are shown in SEQ ID NOs: 1-4, respectively.

[0024] 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;

[0025] Among them, tRNA①, tRNA②, tRNA③, and tRNA④ are different and correspond to four endogenous tRNAs of Pichia pastoris;

[0026] Endonuclease site ①, endonuclease site ②, and endonuclease site ③ are different and correspond to three types of IIS restriction endonuclease sites respectively.

[0027] 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.

[0028] More preferably, the sgRNA expression plasmid of the present application is pZBJ, and the full sequence of the plasmid pZBJ is shown in SEQ ID NO: 11.

[0029] In a third aspect, the present application provides a method for constructing a Pichia pastoris high-efficiency gene editing system, wherein the KhCas9-Brex27 expression cassette is integrated into the genome of Pichia pastoris GS115 to obtain a Pichia pastoris strain GS115-KhCas9-Brex27 that highly expresses hCas9 protein, and then the sgRNA expression plasmid and the donor DNA fragment are introduced into the Pichia pastoris GS115-KhCas9-Brex27 to obtain the Pichia pastoris CRISPR-Cas9 gene editing system.

[0030] Further, the donor DNA can comprise a Kanamycin resistance marker, a bacterial replication origin ori, a homologous arm (about 0.5-1 kb in size) flanking the Cas9 enzyme cutting site, a promoter, a terminator, and a target gene, etc.

[0031] In a fourth aspect, the present application provides a Pichia pastoris high-efficiency gene editing system constructed according to the method.

[0032] In a fifth aspect, the present application provides an application of the Pichia pastoris high-efficiency gene editing system in constructing an engineered strain for high-yield cordycepin.

[0033] In a sixth aspect, the present application provides a Pichia pastoris engineered strain for high-yield cordycepin, and a method for constructing the same comprises the following steps:

[0034] (1) the KhCas9-Brex27 expression cassette is integrated into the genome of Pichia pastoris GS115 to obtain a strain GS115-KhCas9-Brex27, and then the sgRNA1-containing expression plasmid and the donor DNA1 fragment are introduced into the strain GS115-KhCas9-Brex27 to construct an engineered strain 1;

[0035] wherein the nucleotide sequence targeted by sgRNA1 on the chromosome Chr2-5 of Pichia pastoris is 5'-AACTTTGAAACAAAAGAAGG-3';

[0036] The donor DNA1 fragment carries a target gene from Cordyceps militaris (Cp1) that is optimized for Pichia pastoris codons Cordyceps militaris Ppcns1 and Ppcns2 ; wherein the gene Ppcns1 is driven by the promoter pAOX1, and the gene Ppcns2 is driven by the promoter pFLD1;

[0037] the gene Ppcns1 ​and Ppcns2 The nucleotide sequences are shown in SEQ ID NOs: 6 and 7, respectively;

[0038] Promoters pAOX1 and pFLD1 were from Pichia pastoris GS115;

[0039] (2) The expression plasmid containing sgRNA2 and the donor DNA2 fragment were introduced into the engineered bacteria 1 to construct the engineered bacteria 2;

[0040] Among them, the nucleotide sequence of sgRNA2 targeting Pichia chromosome Chr2-4 is 5'-CCTAAATACTACCTAAACAG -3';

[0041] The target gene carried by the donor DNA2 fragment is a gene from Cordyceps militaris CP1 and has been codon-optimized in Pichia pastoris. Ppcns3 / NK , and from Escherichia coli ( Escherichia coli ), and the gene after Pichia pastoris codon optimization PpcpdB Among them, genes Ppcns3 / NK Driven by the promoter pAOX1, the gene PpcpdB Driven by promoter pFLD1;

[0042] Gene Ppcns3 / NK and PpcpdB The nucleotide sequences are shown in SEQ ID NOs: 8 and 9, respectively;

[0043] (3) The expression plasmid containing sgRNA3 and the donor DNA3 fragment were introduced into the engineered bacteria 2 to construct the engineered bacteria 3;

[0044] Among them, the nucleotide sequence of sgRNA3 targeting Pichia chromosome Chr1-2 is 5'-GGTTGGTACTATGTCCAACA -3';

[0045] The target gene carried by the donor DNA3 fragment is an endogenous gene of Pichia pastoris das1 and pex8 Among them, genes das1 Driven by the promoter pAOX1, the gene pex8 Driven by promoter pFLD1;

[0046] Gene das1 and pex8 The reference sequence numbers in NCBI are PAS_chr3_0832 and PAS_chr1-4_0349;

[0047] (4) The expression plasmid containing sgRNA4 and the donor DNA4 fragment were introduced into the engineered bacteria 3 to construct the engineered bacteria 4;

[0048] Among them, the nucleotide sequence of sgRNA4 targeting Pichia chromosome Chr1-5 is 5'-CACGAGCCGAGTAATAACCG -3';

[0049] The target gene carried by the donor DNA4 fragment is an endogenous gene of Pichia pastoris purF and ads Among them, genes purF Driven by the promoter pCAT1, the gene ads Driven by promoter pFDH1;

[0050] Gene purF and ads The reference sequence numbers in NCBI are PAS_chr1-1_0430 and PAS_chr4_0613;

[0051] Promoters pCAT1 and pFDH1 were from Pichia pastoris GS115;

[0052] (5) The expression plasmid containing sgRNA5 and the donor DNA5 fragment were introduced into the engineered bacteria 4 to construct the engineered bacteria 5;

[0053] Among them, the nucleotide sequence of sgRNA5 targeting Pichia chromosome Chr3-5 is 5'-ATACTAGTTAACAAACTGGG -3';

[0054] The target gene carried by the donor DNA5 fragment is an endogenous gene of Pichia pastoris zwf1 and pgk Among them, genes zwf1 Driven by the promoter pTEF1, the gene pgk Driven by promoter pPGI1;

[0055] Gene zwf1 and pgk The reference sequence numbers in NCBI are PAS_chr2-1_0308 and PAS_chr1-4_0292;

[0056] Promoters pTEF1 and pPGI1 were from Pichia pastoris GS115;

[0057] (6) The expression plasmid containing sgRNA6 and the donor DNA6 fragment were introduced into the engineered bacteria 5, and the hCas9 protein was knocked out to obtain the high-yield cordycepin Pichia pastoris engineered bacteria.

[0058] Among them, the nucleotide sequence of hCas9 targeted by sgRNA6 in chromosome 5 of the engineered bacteria is 5'-TACGCCGGATACATTGACGG-3';

[0059] The method for constructing the donor DNA6 fragment includes: using primers F-ΔCas9-UP-OE and R-ΔCas9-UP-OE to amplify the ΔCas9-UP-OE fragment from the Pichia pastoris GS115 genome; using primers F-ΔCas9-DW-OE and R-ΔCas9-DW-OE to amplify the ΔCas9-DW-OE fragment from the Pichia pastoris GS115 genome; and performing overlap extension PCR on the ΔCas9-UP-OE fragment and the ΔCas9-DW-OE fragment to obtain a ΔCas9-UP-DW donor DNA fragment (SEQ ID NO: 12).

[0060] The primer sequences are as follows (5'-3'):

[0061] F-ΔCas9-UP-OE:TTTTTGTAGAAATGTCTTGGTGTCCTCGTCC

[0062] R-ΔCas9-UP-OE: CTTCTGCTCATTATCTTCCCGAAATTTGATCATATGCGCCAGC

[0063] F-ΔCas9-DW-OE:CATATGATCAAATTTCGGGAAGATAATGAGCAGAAGCAGCTGTTCG

[0064] R-ΔCas9-DW-OE: ACGGGAAGTCTTTACAGTTTTAGTTAGGAGCC

[0065] The above-mentioned expression plasmid containing sgRNA is the same as the aforementioned sgRNA expression plasmid.

[0066] In a seventh aspect, the present invention provides an engineered strain of Pichia pastoris with high cordycepin production constructed according to the above method.

[0067] In an eighth aspect, the present invention provides a use of the engineered bacteria in the production of cordycepin, comprising: culturing the engineered bacteria using a fed-batch fermentation process to obtain a culture, and collecting the produced cordycepin from the culture.

[0068] Among them, the fed-batch fermentation process includes:

[0069] S1, seed liquid activation;

[0070] S2, fermentation tank culture: OD 600 The seed solution with a value of 4.0-5.0 is inoculated into a fermentation tank at a volume ratio of 5%-10% for fermentation tank culture;

[0071] The culture conditions are as follows: the volume of the culture medium in the fermentation tank is 30%-40% of the volume of the fermentation tank; the temperature is controlled at 28-30°C throughout the fermentation process, the pH value is 4.0-5.0, the stirring speed is 200-800 rpm, the ventilation volume is maintained at 2-4 vvm, and the dissolved oxygen DO value of the fermentation liquid is 20-60%;

[0072] The culture medium formula 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;

[0073] Preferably, the formula of the culture 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 85% concentrated phosphoric acid;

[0074] S3, glycerol feeding: When the dissolved oxygen DO value rebounds for the first time, it indicates that the glycerol in the fermentation broth has been exhausted. Glycerol feeding medium is added at a constant rate of 15-25 mL / L / h until the OD 600 Increase to 300-350, the glycerol feeding stage ends;

[0075] The culture conditions were as follows: fermentation temperature 28-30°C, pH 4.0-5.0, stirring speed 500-800 rpm, and ventilation maintained at 4-8 vvm;

[0076] The formula of glycerol feed medium is: 40-60% m / v glycerol solution containing 10-15 mL / L PTM1;

[0077] Preferably, the formula of the glycerol feed medium is: 50% m / v glycerol solution containing 12 mL / L PTM1;

[0078] S4, methanol induction and cordycepin synthesis: After stopping the glycerol supplementation, continue the culture for 2-3 hours to deplete the residual glycerol in the bacteria. The time is recorded as t0, and methanol induction begins;

[0079] Add methanol feed medium at the following 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. From t0+7h, maintain a methanol feed rate of 5-10 mL / L / h to maintain the methanol concentration in the fermenter at 0.5-1.0%.

[0080] The culture conditions are as follows: fermentation temperature 28-30℃, pH value controlled at 5.1-6.0, stirring speed 500-800 rpm, ventilation volume maintained at 4-8 vvm, dissolved oxygen DO value of the fermentation liquid controlled at 15-75%, and fermentation ended after 6-9 days, and the tank was released.

[0081] The formula of methanol feed medium is: 100% methanol containing 10-15 mL / L PTM1;

[0082] Preferably, the formula of methanol feed medium is: 100% methanol containing 12 mL / L PTM1.

[0083] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0084] (1) The present invention provides a CRISPR-Cas9 gene editing method for Pichia pastoris without residual resistance genes and its application in the construction of a high-yield cordycepin engineered strain, particularly involving simplifying gene editing operations and enhancing homologous recombination efficiency by using sgRNA-tRNA arrays, type IIS restriction endonucleases, and Brex27 domains, thereby achieving the construction of a cordycepin engineered strain without residual resistance genes.

[0085] (2) The construction and application process of the CRISPR-Cas9 gene editing system in the present invention mainly includes complementation of the histidine HIS4 auxotrophy to achieve the antibody-free assembly of KhCas9; the combination of sgRNA-tRNA array and type IIS restriction endonuclease simplifies the gene editing operation to achieve efficient simultaneous editing of multiple genes; the fusion of Brex27 domain and KhCas9 enhances the efficiency of homologous recombination and improves the efficiency of gene editing; and the knockout of Cas9 protein achieves scarless gene editing.

[0086] (3) With the help of 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 engineered strain without resistance gene residues was achieved. Through a staged fermentation method, the cordycepin production reached 18.3g / L after 144h of fermentation, and the cordycepin production reached 27.2g / L after 216h of fermentation. The CRISPR-Cas9 gene editing method provided by the present invention has greatly improved the gene editing efficiency of Pichia pastoris and, for the first time, achieved the construction of a high-yield cordycepin engineered strain that does not rely on resistance gene tags, providing a powerful tool for the industrial production of cordycepin. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 The present invention utilizes histidine auxotrophy to achieve resistance-free integration of hCas9 in Pichia pastoris GS115.

[0088] Figure 2The present invention utilizes sgRNA-tRNA array and type IIS restriction endonuclease to construct sgRNA plasmid.

[0089] Figure 3 In the preferred embodiment of the present invention, the glycerol kinase gene gut1 Validation of the gene editing capability of the CRISPR-Cas9 system for target (A) Glycerol kinase gene gut1 Growth defect plate image of knockout strain; (B) Glycerol kinase gene was knocked out using BsaI, BspQI and BbsI sites on sgRNA plasmid respectively. gut1 NHEJ knockout efficiency; (C) NHEJ-mediated DSB repair in gut1 Three types of mutations occurred at the locus.

[0090] Figure 4 The Brex27 domain of the present invention enhances HDR ability by attracting PpRAD51, thereby strengthening the multi-gene editing ability.

[0091] Figure 5 In the preferred embodiment of the present invention, glycerol kinase gut1 , alcohol oxidase aox1 , histidine multifunctional enzyme his4 Three reporter genes were used as targets to verify the effect of the Brex27 domain on the multi-gene editing ability of the CRISPR-Cas9 system.

[0092] Figure 6 Figure 1 shows the enhanced multiplex genome editing capability achieved by improving HDR efficiency in a preferred embodiment of the present invention. (A) Targeting glycerol kinase, alcohol oxidase, and histidine multifunctional enzyme to verify the enhanced multiplex genome editing capability; (B) Effect of the Brex27 domain on single-gene editing efficiency; (C) Effect of the Brex27 domain on dual-gene editing efficiency; (D) Effect of the Brex27 domain on triple-gene editing efficiency.

[0093] Figure 7 Figures 1 and 2 show PCR amplification bands after single, double, and triple knockout in preferred embodiments of the present invention. (A) Banding pattern for single ΔGUT1 knockout in KhCas9 and khCas9-Brex27; (B) Banding pattern for double ΔGUT1-ΔAOX1 knockout in KhCas9 and khCas9-Brex27; (C) Banding pattern for triple ΔGUT1, ΔAOX1, and ΔHIS4 knockout in KhCas9 and khCas9-Brex27.

[0094] Figure 8Figure 2 shows the sequencing results of NHEJ knockouts in multiplex editing of ΔGUT1, ΔAOX1, and ΔHIS4 in a preferred embodiment of the present invention. (A) NHEJ sequencing of ΔGUT1; (B) NHEJ sequencing of ΔAOX1; (C) NHEJ sequencing of ΔHIS4.

[0095] Figure 9 In a preferred embodiment of the present invention, yeast two-hybrid was used to verify the protein interaction between the Brex27 domain and PpRAD51.

[0096] Figure 10 Figure 1 illustrates the construction and optimization of the cordycepin biosynthesis pathway in Pichia pastoris using the CRISPR-Cas9 system in a preferred embodiment of the present invention. (A) Pichia pastoris was modified to achieve cordycepin biosynthesis in the cytoplasm; (B) Chromatograms and mass spectra of cordycepin in standard solution and fermentation broth; (C) Effects of promoter combinations of varying strength and copy number on cordycepin production and cell density; (D) Effects of increasing 3'-AMP supply on cordycepin production and cell density.

[0097] Figure 11 The present invention shows a preferred embodiment of a multi-pathway modification strategy for improving cordycepin production. (A) Mechanism of multi-pathway modification using the CRISPR-Cas9 system to improve cordycepin production; (B) 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) Effect of purine pathway enhancement on cordycepin production and cell density; (E) Effect of pathway modification on adenosine content; (F) Effect of enhanced cofactor synthesis on cordycepin production and cell density; (G) Effect of enhanced cofactor supply on ATP content and NADPH / NADP in PC16 and PC19. + The impact of the ratio.

[0098] Figure 12 OD is the cell density of the cordycepin engineered strain PC19 in a preferred embodiment of the present invention. 600 Changes with fermentation time.

[0099] Figure 13 The cordycepin production of the cordycepin engineered strain PC19 in a preferred embodiment of the present invention changes with fermentation time. DETAILED DESCRIPTION

[0100] To address the current difficulties in gene editing in Pichia pastoris strains, the present invention provides a Pichia pastoris CRISPR-Cas9 gene editing system without any resistance gene residues, and applies it to the construction of a high-yield cordycepin engineered strain to achieve low-carbon and efficient synthesis of cordycepin.

[0101] The application adopts the technical scheme as follows:

[0102] In the first aspect, the codon-optimized hCas9 protein is fused with a K7 sequence (i.e., a Kozak sequence CGGACC) to construct a KhCas9, and the KhCas9 is integrated into the genome of Pichia pastoris GS115 by means of a histidine nutritional deficiency tag HIS4 to obtain a strain GS115-KhCas9 that efficiently expresses the Cas9 protein.

[0103] Further, the sgRNA-tRNA array is constructed by using the endogenous tRNA1, tRNA3, tRNA4 and tRNA5 of Pichia pastoris and a gRNA scaffold, and the sgRNA plasmid is constructed by combining the three type-IIS restriction endonucleases BsaI / BspQI / BbsI. gut1 Further, the glycerol kinase gene

[0104] It should be noted that one of the technical advantages of the application is that the type-IIS restriction endonucleases BsaI / BspQI / BbsI are used to quickly replace different genomic target gRNAs, which avoids the complex homologous sequence replacement and multiple rounds of PCR of the traditional HH and HDV ribozyme, and has extremely high convenience.

[0105] Further, the codon-optimized Brex27 domain is fused with the KhCas9 to construct the GS115-KhCas9-Brex27 strain, which significantly strengthens the aggregation of the DNA repair protein PpRAD51 at the genomic fracture, and can simultaneously achieve efficient seamless knockout of the glycerol kinase gut1 , alcohol oxidase aox1 , and histidine multifunctional enzyme his4 The knockout efficiency is significantly better than that of another improved strain GS115-KhCas9-PpRAD52.

[0106] The construction method of the strain GS115-KhCas9-PpRAD52 is as follows:

[0107] The GAP promoter-PpRad52 gene-AOX1 terminator expression cassette is integrated into the Chr1-2 site of the chromosome of the GS115-KhCas9 strain to obtain the GS115-KhCas9-PpRAD52 strain. The PpRad52 gene (NCBI accession number PAS_chr2-1_0153) is cloned from the genome of Pichia pastoris GS115.

[0108] 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. The local spatial concentration of the recruited DNA repair proteins is high, and there is no need to occupy a new genomic integration site, which saves space for subsequent cordycepin pathway modification.

[0109] In the second aspect, the present invention uses Cordyceps militaris Cordyceps militaris CP1 nucleoside / nucleotide kinase gene Cmcns3 / NK , metal-dependent phosphohydrolase genes Cmcns2 and oxidoreductase / dehydrogenase genes Cmcns1 According to the codon preference of Pichia pastoris, the unique Ppcns3 / NK 、 Ppcns2 and Ppcns1 Coding sequence. Cmcns3 / NK 、 Cmcns2 、 Cmcns1 Compared to the sequence published by NCBI, the CP1 strain has one, two, and eight optimized amino acid sites, respectively: Cns1 (p.F12C, p.P30S, p.E425D, p.N440G, p.S599A, p.A611V, p.S635P, p.D770E), Cns2 (p.K130E, p.P314Q), and Cns3 / NK (p.N154S). These amino acid modifications promote cordycepin synthesis. The CP1 strain of Cordyceps militaris is currently deposited with the China Center for Type Culture Collection (CCTCC M2019671).

[0110] Furthermore, the present invention uses pAOX1-pFLD1, pAOX1-pCAT1, and pAOX1-pDAS2 three strong promoter combinations to control Ppcns1 and Ppcns2 Efficient extracellular synthesis of cordycepin was achieved, among which the pAOX1-pFLD1 combination was the best, and further increasing the copy number did not effectively accelerate the synthesis of cordycepin.

[0111] Furthermore, the present invention uses pAOX1 and pFLD1 promoters to overexpress Ppcns3 、 Ppcns3 / NK 、 PpcpdB To strengthen the synthesis of cordycepin's direct precursor 3'-AMP, the cordycepin production is significantly improved.

[0112] Thirdly, the present invention utilizes combined metabolic engineering of enhanced methanol assimilation, enhanced purine pathway, and enhanced cofactor NADPH / ATP supply to achieve a significant increase in cordycepin production.

[0113] Preferably, the present invention uses pAOX1 and pFLD1 promoters to overexpress dihydroxyacetone synthase DAS1 and peroxisome biogenesis factor 8 PEX8, thereby enhancing the transfer of methanol assimilation enzymes from the cytoplasm to peroxisomes and accelerating the conversion of formaldehyde, thereby providing sufficient carbon metabolic flow for cordycepin synthesis.

[0114] Preferably, the present invention uses pCAT1 and pFDH1 promoters to overexpress phosphoribosylpyrophosphate amidotransferase PurF and adenylate succinate synthase AdSS, thereby strengthening the transfer of purine pathway metabolic flux to the cordycepin synthesis pathway, mainly involving the strengthening of the supply of cordycepin precursor adenosine.

[0115] Preferably, the present invention uses pTEF1 and pPGI1 promoters to overexpress glucose-6-phosphate dehydrogenase ZWF1 and phosphoglycerate kinase PGK, thereby enhancing the supply of cofactor NADPH and energy carrier ATP, and providing sufficient reducing power and energy for the synthesis of cordycepin.

[0116] In a fourth aspect, the present invention provides a high-density fermentation method for cordycepin-engineered Pichia pastoris based on a fed-batch strategy. 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 hours, at which time the OD 600 When the total fermentation time reaches 45-55h, OD 600 After a 2-3h starvation period, the PC19 strain entered the methanol induction period. 1.9 g / L of cordycepin was detected 60h after the start of methanol induction. The fermentation was terminated after 144h. At this time, the yield and production rate of cordycepin were 18.3 g / L and 3.05 g / L / d, respectively. The conversion rate of methanol to cordycepin was 50.2 mg / g, and the OD 600 The concentration of glutathione reached 368.3, the dry weight of the fungus (DCW) reached 149.6 g / L, the fermentation time was extended to 216 h, and the cordycepin yield reached 27.2 g / L.

[0117] The following examples are used to illustrate the present invention but are not intended to 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.

[0118] Related Materials and Methods

[0119] 1. Main Reagents

[0120] 10×YNB amino-free yeast nitrogen source:

[0121] 13.4 g YNB dissolved in 100 mL of deionized water, 0.22 μm filter sterilized.

[0122] 500x biotin (500x B):

[0123] 20 mg biotin dissolved in 100 mL of deionized water, 0.22 μm filter sterilized.

[0124] 10% glycerol:

[0125] 100 mL glycerol mixed with 900 mL of deionized water, sterilized at 121 °C for 20 min.

[0126] 1 M phosphate buffer PBS (pH 6.0):

[0127] 132 mL of 1 M K2HPO4and 868 mL of 1 M KH2PO4mixed, pH adjusted with KOH, sterilized at 121 °C for 20 min.

[0128] 1 M sorbitol solution:

[0129] 18.2 g sorbitol dissolved in 100 mL of deionized water, 0.22 μm filter sterilized.

[0130] PTM1 trace element solution:

[0131] 6.0 g / L CuSO4·5H2O, 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, 0.2 g / L biotin, 5.0 mL / L concentrated sulfuric acid.

[0132] 2. Main medium

[0133] LB liquid / solid medium:

[0134] Yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, agar 15 g / L added for solid.

[0135] Low salt LB liquid / solid medium:

[0136] Yeast extract 5 g / L, tryptone 10 g / L, NaCl 5 g / L, agar 15 g / L added for solid.

[0137] YPD liquid / solid medium:

[0138] Yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, solidified with agar 20 g / L.

[0139] BMD solid medium:

[0140] Glucose 20 g / L, YNB 13.4 g / L, Biotin 0.4 mg / L, agar 20 g / L.

[0141] BMG solid medium:

[0142] Glycerol 10 g / L, YNB 13.4 g / L, Biotin 0.4 mg / L, agar 20 g / L.

[0143] BMM solid medium:

[0144] Methanol 10 mL / L, YNB 13.4 g / L, Biotin 0.4 mg / L, agar 20 g / L.

[0145] BMGY medium:

[0146] Yeast extract 10 g / L, peptone 20 g / L, sterile water 700 mL / L, sterilized at 115°C for 15 min, cooled to room temperature; add 10x YNB 100 mL / L, 500x Biotin 2 mL / L, 10x glycerol 100 mL / L, 1 M PBS (pH=6.0) 100 mL / L.

[0147] BMMY medium:

[0148] 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 10x YNB 100 mL / L, 500x Biotin 2 mL / L, 1 M PBS (pH=6.0) 100 mL / L; 100% methanol 10 mL / L.

[0149] Basic salt BSM medium:

[0150] 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%).

[0151] 50% (m / v) glycerol feed medium:

[0152] Weigh glycerol to prepare a 50% glycerol solution, sterilize it at 115°C for 20 min, and add 12 mL / L PTM1 before use.

[0153] 100% methanol feed medium:

[0154] Measure 100% methanol and add 12 mL / L PTM1 before use.

[0155] 3. Yeast genome extraction method

[0156] Aspirate the YPD bacterial suspension and centrifuge at 12,000 rpm for 2 minutes to retain the bacterial cells. After grinding with liquid nitrogen, the yeast cells were disrupted and the yeast genome was extracted using the Tiangen Yeast Genomic DNA Extraction Kit DP307. Refer to the kit instructions for related procedures.

[0157] 4. PCR amplification of target gene fragments

[0158] Prepare a 50 μL PCR system with 2 μL each of upstream and downstream primers, 25 μL of 2× PCR mix, and 1-2 μL of template. The reaction program is: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 56-72°C annealing for 15 s, 72°C extension for 15-60 s / kb, 25-35 cycles, and 72°C full extension for 5 min.

[0159] 5. E. coli Competent Cell Transformation Method

[0160] Mix 5-20 μg of recombinant plasmid with Bio-Tech DH5α competent cells and place on ice for 20-30 min. Heat shock the cells in a 42°C water bath for 40-60 min, then place them on ice for 3-5 min. Resuspend the cells in 500-700 μL of LB and incubate them at 37°C for 45 min. Spread the cells on LB plates containing 70 mg / L bleomycin or kanamycin and incubate them upside down at 37°C in the dark for 10-15 h.

[0161] 6. E. coli Plasmid Extraction Method

[0162] Take 1-5 ml of the overnight shake culture and centrifuge at 12,000 rpm for 1 min to retain the bacteria. Use the Novozymes Plasmid Extraction Kit DC201 to extract the plasmid from E. coli. Refer to the kit instructions for related procedures.

[0163] 7. Plasmid Enzyme Digestion System

[0164] Taking 100 μL of enzyme digestion system as an example, add 5.0 μL of restriction endonuclease, 10.0 μL of 10× Buffer, and 3-5 μg of plasmid; add dd H2O to 100 μL; and digest in a 37°C water bath for 2-3 hours.

[0165] 8. PCR product purification and recovery method

[0166] Cut the target DNA fragment under UV light. Purify and recover the product using the Novozymes PCR Product Recovery and Gel Extraction Kit DC301. Refer to the kit instructions for related procedures.

[0167] 9. Gibson Assembly Homologous Recombination System

[0168] Prepare 10 μL of homologous recombination system and 5 μL of recombinase at a molar ratio of cloning vector to insert of 1:1 to 1:2. Perform single-fragment recombination reactions at 50°C for 5 min; multi-fragment recombination reactions at 50°C for 15 min. Transfer to an ice bath. Homologous assembly was performed using Norvegase C115. Refer to the kit instructions for related procedures.

[0169] 10. Preparation of Pichia pastoris Competent Cells

[0170] YPD was shaken for about 10-14 h, and OD 600 The pH was 1.2-1.5, centrifuged at 5000 rpm at 4°C for 5 min, and resuspended the cells in 40 mL of sterile water at 4°C; centrifuged at 5000 rpm at 4°C for 5 min, and resuspended the cells in 20 mL of sterile water at 4°C; centrifuged at 5000 rpm at 4°C for 5 min, and resuspended the cells in 10 mL of 1 M sorbitol at 4°C; centrifuged at 5000 rpm at 4°C for 5 min, retained the cells, added 300-500 μL of 1 M sorbitol at 4°C to resuspend the cells, and placed in an ice bath for later use.

[0171] 11. Pichia pastoris electroporation transformation method

[0172] Mix 80 μL of competent cells and 5-20 μg of linearized plasmid by pipetting into a 0.2 cm electroporation cuvette; voltage 1500-2000 V, capacitance 25 μF, resistance 200-400 Ω, and electroporation time 6.0-8.0 ms. After the electroporation, add 1 mL of 4°C 1 M sorbitol solution. Incubate at 30°C for 2-4 h. Spread 200 μL of the bacterial solution on a YPDZ plate and culture inverted at 28-30°C for 3-4 days.

[0173] 12. Pichia pastoris Induced Expression

[0174] Pick up yeast strains with an inoculation loop and place them in YPD, shake and incubate at 30°C and 200 rpm for 18-22 h; inoculate 1% of the yeast strains into BMGY and wait until the OD 600 The cell count was about 12.0-13.0, and the cells were collected by centrifugation and resuspended in BMMY. The cells were shaken at 30°C and 200 rpm for 7 days, and 100% methanol was added at 1% / day.

[0175] 13. Synthesis of Cordycepin by High-Density Fermentation of Pichia pastoris

[0176] (1) Seed liquid activation stage

[0177] Store the strain at -80℃ and inoculate into 20mL YPD medium. Shake and incubate at 28-30℃, 150-200 rpm for 20-24 hours until the OD reaches 0. 600 About 2.0-4.0. 1mL of bacterial solution was inoculated into 100mL of YPD medium, and cultured at 28-30℃, 150-200 rpm, for 18-24h until the OD 600 Then, inoculate the fermenter at a volume ratio of 5%-10%.

[0178] (2) Fermentation tank preparation stage

[0179] Culture medium: Fill the fermenter at a volume of 30%-40% of the fermentor volume and sterilize at 115-121°C for 15-20 minutes. Since fermentation is conducted in fed-batch mode, the final fermentation volume will be approximately 1.5-2 times the initial fermentation volume. Note: PTM1 trace element solution should not be autoclaved in the fermenter. It should be filtered and sterilized before addition (bring the fermenter medium to 30°C, 4.4 mL / L) to prevent oxidation and inactivation of some trace element components.

[0180] (3) Segmented fermentation and efficient synthesis of cordycepin

[0181] Rapid strain growth: The glycerol batch phase lasts for 20-24 hours. When the dissolved oxygen (DO) value shows its first rebound, it indicates that the 4% glycerol initially added to the fermentation broth has been consumed and the fermentation broth is now carbon-deficient, marking the end of the first glycerol batch phase. During the initial 20-24 hours of biomass accumulation, the fermentation temperature is maintained at 28-30°C, the pH at 4.0-5.0, the agitation speed at 200-800 rpm, the aeration rate at 2-4 vvm, and the dissolved oxygen (DO) value of the fermentation broth at 15-75%.

[0182] Glycerol fermentation: During the glycerol feeding phase, 40-60% (w / v) glycerol culture medium (supplemented with 10-15 mL / L PTM1 trace element solution) was added at a constant rate of 15-25 mL / L / h for a total of 20-30 hours. OD was measured every 6 hours. 600 Value, up to OD 600 The second stage of glycerol fermentation ends when the pH increases to 300-350. The glycerol feeding phase, which lasts 20-30 hours, is maintained at a temperature of 28-30°C, a pH of 4.0-5.0, a stirring speed of 500-800 rpm, and a ventilation volume of 4-8 vvm. The bacteria grow rapidly and achieve high-density growth.

[0183] Methanol Induction and Cordycepin Synthesis: After stopping glycerol supplementation, continue the culture for 2-3 hours to deplete the residual glycerol in the cells. The third methanol induction phase begins. Methanol is added at a gradual rate of 0.5 mL / L / h, 1 mL / L / h, 2 mL / L / h, 3 mL / L / h, 4 mL / L / h, and finally 5 mL / L / h. Starting at hour 7, maintain a methanol feed rate of 5-10 mL / L / h to maintain a methanol concentration in the fermenter between 0.5-1.0%. The methanol induction phase lasts for 144-216 hours of fermentation. The fermentation temperature is 28-30°C, the pH of the fermentation broth is controlled between 5.1-6.0, the agitation speed is 500-800 rpm, the aeration rate is maintained at 4-8 vvm, and the dissolved oxygen (DO) value in the fermentation broth is controlled between 20-60% to achieve cordycepin synthesis and accumulation.

[0184] 14. High Performance Liquid Chromatography and Mass Spectrometry Detection Methods

[0185] Cordycepin standards were purchased from Solebol. Analysis was performed on a Thermo Fisher Ultimate 3000 HPLC system using an Agilent Zorbax SB-C18 column. Mobile phase A consisted of 10 mmol / L acetic acid in water, and mobile phase B was a 1:1 (volume ratio) mixture of methanol and acetonitrile. The injection volume was 10 μL, the column temperature was 40°C, and the detection wavelength was 260 nm. LC-MS detection was performed with a spray voltage of 3200 V, a capillary temperature of 300°C, a sheath gas of 40 Arb, an auxiliary gas of 8 Arb, a maximum spray current of 100 μA, and a probe temperature of 300°C.

[0186] 15. Yeast Two-Hybrid

[0187] Protein-protein interactions were 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 (provided with the kit) and fused to the GAL4 DNA activation domain (DNA-AD) and GAL4 DNA binding domain (DNA-BD) as prey and bait constructs, respectively. After co-transformation into Y2HGold competent cells, yeast two-hybrid analysis was performed using SC-Leu-Trp, SC-Leu-Trp-His-Ade, and SC-Leu-Trp-His-Ade-containing defective plates.

[0188] 16. Gene Sequence

[0189] Cordyceps militaris Cordyceps militaris Oxidoreductase / dehydrogenase gene of CP1 (CCTCC M2019671) Cmcns1 ( CCM_04436 ), metal-dependent phosphohydrolase genes Cmcns2 ( CCM_04437 ), nucleoside / nucleotide kinase genes Cmcns3 / NK ( CCM_04438 ), Escherichia coli 2',3'-cyclic nucleotide-2'-phosphodiesterase gene cpdB ( b4213 ), the human Brex27 domain gene was optimized according to the codon preference of Pichia pastoris. 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).

[0190] The genomic DNA of Pichia pastoris GS115 (ATCC 20864 purchased from Invitrogen) was used as a template to amplify the following endogenous genes: Pichia pastoris endogenous tRNA, dihydroxyacetone synthase ( das1 , PAS_chr3_0832 ), peroxisome biogenesis factor 8 ( pex8,PAS_chr1-4_0349 ), phosphoribosylpyrophosphate amidotransferase ( purF, PAS_chr1- 1_0430 ), adenylate succinate synthase ( adss, PAS_chr4_0613 ), glucose-6-phosphate dehydrogenase ( zwf1, PAS_ chr2-1_0308 ) and phosphoglycerate kinase ( pgk, PAS_chr1-4_0292 ).

[0191] The gRNA sequences involved in the present invention are shown in Table 1:

[0192] Table 1 gRNA sequences involved in the present invention (SEQ ID NO: 13-25)

[0193]

[0194] Reference: 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.

[0195] 17. Plasmids and primers

[0196] The plasmids and corresponding primers used in the present application are shown in Tables 2-6 (SEQ ID NOs: 26-210):

[0197] Table 2 Plasmids and primers involved in the present application

[0198]

[0199] Table 3 Plasmids and primers involved in the present application

[0200]

[0201] Table 4 Plasmids and primers involved in the present application

[0202]

[0203] Table 5 Plasmids and primers involved in the present application

[0204]

[0205] Table 6 Plasmids and primers involved in the present application

[0206]

[0207] Note: The plasmids involved in Tables 2-6 are all constructed according to the three-step basic construction process of single fragment amplification, multi-fragment overlap extension, and multi-fragment Gibson assembly into a circular plasmid, and then screened by Zeocin and kanamycin resistance tags to obtain positive clones and correct plasmids.

[0208] Example 1 Construction and optimization of CRISPR-Cas9 system in Pichia pastoris

[0209] 1. Genome integration of Cas9 protein in Pichia pastoris

[0210] The hCas9 gene was fused with Kozak sequence (CGGACC), GAP promoter (pGAP), AOX1 terminator (tAOX1), DAS1 terminator (tDAS1), and SV40 nuclear localization signal (NLS) to construct the KhCas9 expression cassette (pPICZK-hCas9) (Figure 1A). Figure 1). The PpHIS4-1 and PpHIS4-2 fragments amplified from plasmid pAO815 were flanked with the KhCas9 expression cassette to form the KhCas9 genomic recombination fragment Figure 1 ). The recombination fragment was inserted into the Pichia pastoris GS115 genome (His4 - , p.C557R), to construct the strain GS115-KhCas9 (His4 + , p.557C) ( Figure 1 ). The complementation of the histidine auxotrophy can strengthen the growth of Pichia pastoris and the synthesis of target products.

[0211] The primers used for amplifying the PpHIS4-1 fragment are F-PpHIS4-1 and R-PpHIS4-1, and the primers used for amplifying the PpHIS4-2 fragment are F-PpHIS4-2 and R-PpHIS4-2.

[0212] 2. Construction of sgRNA plasmid

[0213] The sgRNA plasmid is composed 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 a TEF1 promoter (PpTEF1), tAOX1, Pichia pastoris endogenous tRNA, 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 Pichia pastoris endogenous tRNA1 (72bp, SEQ ID NO: 1), tRNA3 (80bp, SEQ ID NO: 2), tRNA4 (85bp, SEQ ID NO: 3), and tRNA5 (72bp, SEQ ID NO: 4) for constructing the sgRNA-tRNA array ( Figure 2 ). The 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 linearized pZBJ sgRNA plasmid can realize the rapid replacement of the gRNA sequence ( Figure 2 ).

[0214] 3. Construction of donor DNA plasmid

[0215] The donor plasmid backbone includes a kanamycin resistance tag and a bacterial origin of replication (ori). Homology arms (approximately 1 kb) flanking the Cas9 restriction site (i.e., the sgRNA targeting site) were amplified from Pichia pastoris GS115 genomic DNA (gDNA). The Kan-GJ fragment, upstream and downstream homology arms, promoter, terminator, and target gene were integrated into the donor plasmid using OE-PCR (overlap extension PCR) and Gibson assembly. The donor plasmid was selected for kanamycin resistance and confirmed by Sanger sequencing. Transformations were performed by electroporation using a GenePulser Xcell electroporation system using 500 ng of sgRNA plasmid and 1 μg of donor DNA fragment. The parameters were set to 1.5 kV, 25 μF, and 200 Ω. Transformants were cultured on YPDZ plates for 3 days. Twenty colonies were randomly selected from each transformant for genomic extraction and PCR verification. To eliminate the sgRNA plasmid, culture the positive strain in 20 mL of YPD medium for 24 hours and then streak onto YPD plates. Confirm successful plasmid elimination by repeating the plasmid on YPDZ plates.

[0216] 4. Preliminary verification of the gene editing capability of the Pichia pastoris CRISPR-Cas9 system

[0217] Glycerol kinase-deficient strains have limited growth on BMG plates, and the glycerol kinase gene gut1 Evaluate the NHEJ targeting efficiency of each of the BsaI / BspQI / BbsI sites for the target ( Figure 3 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 The differences in ΔGUT1 targeting efficiency at the three sites may be related to the differences in the self-cleavage efficiency of the four tRNAs. The NHEJ repair mechanism randomly generates insertions and deletions of individual bases at genomic DSBs. ΔGUT1 has three types of NHEJ mutations: single-base deletion, two-base deletion, and single-base insertion. Figure 3 Middle C).

[0218] 5. Fusion of Brex27 and Cas9 enhances homologous recombination ability

[0219] In order to enhance homologous recombination ability, the codon-optimized Brex27 motif (ALDFLSRLPLPPPVSPICTFVSPAAQKAFQPPRSCG) was genetically fused to the C-terminus of KhCas9 via a (GGGGS)3 flexible linker peptide to form a KhCas9-Brex27 expression cassette (the sequence of the KhCas9-Brex27 expression cassette after Pichia codon optimization is shown in SEQ ID NO: 10). The expression cassette was integrated into the Pichia pastoris GS115 genome (His4 - , p.C557R), and constructed strain GS115-KhCas9-Brex27 (His4 + , p.557C) ( Figure 4 We tested the multiple 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 rates 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%, which were also higher than those of the GS115-KhCas9-PpRAD52 strain (85.7%, 57.1%, and 50.8%). Figure 6 The reason why HDR efficiency is lower than the total targeting efficiency is that some auxotrophic positive clones are NHEJ-type knockouts. This conclusion was verified by simulation analysis, PCR amplification, and Sanger sequencing ( Figure 7 , Figure 8 ). Saccharomyces cerevisiae ScRAD51 and hRAD51 have an amino acid identity of 50.12%, while Pichia pastoris PpRAD51 and hRAD51 have an amino acid identity of 56.47%. Yeast two-hybrid assay results showed that Brex27 can recruit PpRAD51 and interact with it, so we speculate that KhCas9-Brex27 can also enrich PpRAD51 around DSBs and improve HDR efficiency ( Figure 9 ).

[0220] The strain GS115-KhCas9-PpRAD52 was constructed as follows:

[0221] A GAP promoter-PpRad52 gene-AOX1 terminator expression cassette was constructed and integrated into the Chr1-2 locus of the chromosome of the GS115-KhCas9 strain to generate the GS115-KhCas9-PpRAD52 strain. The PpRad52 gene (NCBI accession number PAS_chr2-1_0153) was cloned from the Pichia pastoris GS115 genome.

[0222] Example 2 Construction and multi-pathway optimization of the biosynthetic pathway of cordycepin in Pichia pastoris

[0223] 1. Construction and optimization of the direct synthesis pathway of cordycepin in Pichia pastoris

[0224] 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 both the standard and fermentation broth was 10.9 min, and the recovery rate of the standard was 98.8% to 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 the cordycepin molecule ( Figure 10 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 ( Figure 10 Increasing gene copy number is a common method to increase the yield of target products, but the cordycepin yields of strains PC04 and PC05 were not significantly different from those of strain PC01 ( Figure 10 C). This indicates that strain PC01 Ppcns1 and Ppcns2The expression of PpCns3 / NK is sufficient, and the rate-limiting step of cordycepin synthesis is located upstream of 3'-AMP. The results of fed-batch fermentation show that the cordycepin yield reaches 1999.2 mg / L after adding 350 mg / L of 3'-AMP, and the conversion rate of 3'-AMP to cordycepin reaches 82.7%( Figure 10 PpCns3 / NK can phosphorylate adenosine to 3'-AMP, and PpCpdB can further strengthen the supply of 3'-AMP in the mRNA degradation pathway Figure 10 After overexpressing PpCns3, PpCns3 / NK, PpCpdB, PpCns3-PpCpdB, and PpCns3 / NK-PpCpdB, the cordycepin yield of PC06, PC07, PC08, PC09, and PC10 is significantly increased by 10.6%, 9.6%, 6.0%, 13.6%, and 15.7% respectively compared with PC01 Figure 10 3'-AMP is a more direct cordycepin precursor than adenosine, and the limited availability of 3'-AMP in the cell is the bottleneck of high cordycepin production.

[0225] The construction method of strain PC01 is as follows:

[0226] 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, the construction of strain PC01 is realized.

[0227] Among them, the construction process of pZBJ-Chr2-5(BspQI) plasmid is as follows: BspQI is used to cut pZBJ plasmid, and the linearized pZBJ plasmid is recovered by gel; then the gRNA sequence 5'-AACTTTGAAACAAAAGAAGG-3' of Pichia pastoris genome Chr2-5 site is integrated into the BspQI cutting site, and after heat shock transformation into E. coli DH5α, pZBJ-Chr2-5(BspQI) plasmid is obtained by Zeocin screening, which can efficiently express sgRNA targeting Chr2-5 site in Pichia pastoris cells.

[0228] The construction process of the Chr2-5-COR-QR donor DNA plasmid is as follows: construct the 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 Pichia pastoris GS115 genome; use primers F-Chr2-5-DW-OE and R-Chr2-5-DW-OE to amplify the Chr2-5-DW-OE fragment from the Pichia pastoris GS115 genome; the plasmid selection marker is kanamycin; the plasmid replication origin is ori; the above five fragments are assembled by Gibson to obtain the Chr2-5-COR-QR donor DNA plasmid. The Chr2-5-COR-QR donor DNA fragment was obtained by amplification using the Chr2-5-COR-QR donor DNA plasmid as a template and F-Chr2-5-UP and R-Chr2-5-DW as primers.

[0229] Finally, 500 ng of pZBJ-Chr2-5 (BspQI) plasmid and 1000 ng of Chr2-5-COR-QR donor DNA fragment were co-electroporated into the GS115-KhCas9-Brex27 strain, and strain PC01 was obtained after Zeocin selection.

[0230] The strain PC10 was constructed as follows:

[0231] Using PC01 as the starting strain, strain PC10 was constructed with the help of pZBJ-Chr2-4 (BspQI) plasmid and Chr2-4-Ppcns3NK-PpcpdB donor DNA plasmid.

[0232] Among them, the construction process of the pZBJ-Chr2-4 (BspQI) plasmid is as follows: the pZBJ plasmid is digested with BspQI, and the linearized pZBJ plasmid is recovered by gel extraction; then the gRNA sequence 5'-CCTAAATACTACCTAAACAG-3' of the Chr2-4 site of the Pichia pastoris genome is integrated into the BspQI cleavage site, and after heat shock transformation into Escherichia coli DH5α, the pZBJ-Chr2-4 (BspQI) plasmid is obtained by Zeocin screening. This plasmid can efficiently express the sgRNA targeting the Chr2-4 site in Pichia pastoris.

[0233] The construction process of the Chr2-4-Ppcns3NK-PpcpdB donor DNA plasmid is as follows: construct the 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 obtain the target gene expression cassette from P. The Chr2-4-UP-OE fragment was amplified from the yeast GS115 genome. The Chr2-4-DW-OE fragment was amplified from the Pichia pastoris GS115 genome using primers F-Chr2-4-DW-OE and R-Chr2-4-DW-OE. The plasmid selection marker was kanamycin, and the plasmid replication origin was ori. The five fragments were assembled by Gibson assembly to generate the Chr2-4-Ppcns3NK-PpcpdB donor DNA plasmid. The Chr2-4-Ppcns3NK-PpcpdB donor DNA fragment was amplified using the Chr2-4-UP and R-Chr2-4-DW primers, using the Chr2-4-UP and R-Chr2-4-DW donor DNA plasmids as templates.

[0234] Finally, 500 ng of pZBJ-Chr2-4(BspQI) plasmid and 1000 ng of Chr2-4-Ppcns3NK-PpcpdB donor DNA fragment were co-electroporated into PC01 strain, and strain PC10 was obtained after Zeocin selection.

[0235] 2. Strengthen the methanol assimilation pathway and enzyme transmembrane transport to promote the synthesis of cordycepin

[0236] Methanol is oxidized to formaldehyde by AOX, and DAS can rapidly convert formaldehyde into 3'-phosphoglyceraldehyde (G3P) through the Xu5P cycle, thereby alleviating the cytotoxicity of methanol metabolism ( Figure 11 Middle A). After DAS1 was overexpressed in pAOX1, the cordycepin production increased by 2.8% ( Figure 11 Peroxisomes are functional subcellular organelles responsible for methanol assimilation and dissimilation. The peroxisomal import protein PEX8 is responsible for transporting methanol metabolic enzymes from the cytoplasm across the membrane into the peroxisome. Overexpression of PEX8 via pFLD1 significantly increased cordycepin production by 3.6%, while combined overexpression of DAS1 and PEX8 significantly increased cordycepin production by 6.8%, without significantly changing cell density. Figure 11 In this study, a 0.5%-3% methanol concentration gradient was used to test the methanol tolerance and cordycepin production of strains PC10 and PC13 ( Figure 11When the methanol concentration increased from 0.5% to 1%, the cordycepin production of strains PC10 and PC13 increased significantly by 13.7% and 20.8%, respectively. However, the highest cell density of strains PC10 and PC13 appeared in the 0.75% methanol treatment group ( Figure 11 However, increasing the methanol concentration from 1% to 3% did not result in a sustained increase in cordycepin production, and the toxicity of high methanol concentrations led to a gradual decrease in cell density ( Figure 11 The results indicate that 1% methanol is sufficient to supply the carbon metabolic flux required for bacterial growth and cordycepin synthesis, and the limiting site of cordycepin synthesis is located downstream of the methanol assimilation pathway.

[0237] The strain PC13 was constructed as follows:

[0238] PC10 was used as the starting strain, and the strain PC13 was constructed with the help of pZBJ-Chr1-2(BsaI) plasmid and Chr1-2-PpDAS1-PpPEX8 donor DNA plasmid.

[0239] Among them, the construction process of the pZBJ-Chr1-2 (BsaI) plasmid is as follows: the pZBJ plasmid is digested with BsaI, and the linearized pZBJ plasmid is recovered by gel extraction; then the gRNA sequence 5'-GGTTGGTACTATGTCCAACA-3' of the Chr1-2 site of the Pichia pastoris genome is integrated into the BsaI cutting site, and after heat shock transformation into Escherichia coli DH5α, the pZBJ-Chr1-2 (BsaI) plasmid is obtained by Zeocin screening. This plasmid can efficiently express the sgRNA targeting the Chr1-2 site in Pichia pastoris.

[0240] The construction process of the Chr1-2-PpDAS1-PpPEX8 donor DNA plasmid is as follows: construct the target gene expression cassette of AOX1 promoter-Kozak sequence (CGGACC)-PpDAS1-CYC1 terminator-FLD1 promoter-Kozak sequence (CGGACC)-PpPEX8-AOX1 terminator; use primers F-Chr1-2-UP-OE and R-Chr1-2-UP-OE to obtain the target gene expression cassette from Pichia pastoris. The Chr1-2-UP-OE fragment was amplified from the GS115 genome. The Chr1-2-DW-OE fragment was amplified from the Pichia pastoris GS115 genome using primers F-Chr1-2-DW-OE and R-Chr1-2-DW-OE. The plasmid selection marker was kanamycin, and the plasmid replication origin was ori. The five fragments were assembled by Gibson assembly to generate the Chr1-2-PpDAS1-PpPEX8 donor DNA plasmid. The Chr1-2-PpDAS1-PpPEX8 donor DNA fragment was amplified using the Chr1-2-UP and R-Chr1-2-DW primers, using the Chr1-2-UP and R-Chr1-2-DW donor DNA plasmids as templates.

[0241] Finally, 500 ng of pZBJ-Chr1-2(BsaI) plasmid and 1000 ng of Chr1-2-PpDAS1-PpPEX8 donor DNA fragment were co-electroporated into PC10 strain, and strain PC13 was obtained after Zeocin selection.

[0242] 3. Strengthen the purine synthesis pathway to supply precursor substances to promote the synthesis of cordycepin

[0243] The de novo purine synthesis pathway is a key metabolic flow 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 between the pentose phosphate pathway and the de novo purine synthesis pathway, while PurF is the key rate-limiting enzyme that catalyzes the initiation reaction in the de novo purine synthesis pathway ( Figure 11 A). IMP is converted to AMP after being catalyzed by AdSS. The adenosine generated by AMP dephosphorylation provides a substrate for the efficient synthesis of cordycepin ( Figure 11 After overexpression of PurF and AdSS, the cordycepin production 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 ( Figure 11The results of the changes in adenosine content showed that the synthesis of adenosine in the engineered strain was affected by multiple pathways including the cordycepin biosynthesis pathway, the methanol assimilation pathway, and the purine de novo synthesis pathway ( Figure 11 E). Comparative analysis of the adenosine content of strains PC16 and PC13 was performed every 24 hours. The adenosine content of PC16 was significantly increased by 7.5%-16.9% compared with PC13, indicating that sufficient precursor supply can accelerate the synthesis of cordycepin ( Figure 11 Adenosine, adenine, inosine, and hypoxanthine are commonly used as precursors for cordycepin biosynthesis. However, the use of these precursors significantly increases production costs, and enhancing the endogenous supply of these precursors is a more cost-effective production strategy.

[0244] The strain PC16 was constructed as follows:

[0245] PC13 was used as the starting strain, and the strain PC16 was constructed with the help of pZBJ-Chr1-5 (BspQI) plasmid and Chr1-5-purF-AdSS donor DNA plasmid.

[0246] The construction process of the pZBJ-Chr1-5 (BspQI) plasmid is as follows: the pZBJ plasmid is digested with BspQI, and the linearized pZBJ plasmid is recovered by gel extraction; then the gRNA sequence 5'-CACGAGCCGAGTAATAACCG-3' of the Chr1-5 site of the Pichia pastoris genome is integrated into the BspQI cleavage site, and after heat shock transformation into Escherichia coli DH5α, the pZBJ-Chr1-5 (BspQI) plasmid is obtained by Zeocin screening. This plasmid can efficiently express the sgRNA targeting the Chr1-5 site in Pichia pastoris.

[0247] Among them, the construction process of Chr1-5-purF-AdSS donor DNA plasmid is as follows: construct the target gene expression frame of CAT1 promoter-purF-DAS1 terminator-FDH1 promoter-AdSS-AOX1 terminator; use primers F-Chr1-5-UP-OE and R-Chr1-5-UP-OE to amplify the Chr1-5-UP-OE fragment from the Pichia pastoris GS115 genome; use primers F-Chr1-5-DW-OE and R-Chr1-5-DW-OE to amplify the Chr1-5-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 assembled by Gibson to obtain the Chr1-5-purF-AdSS donor DNA plasmid. The Chr1-5-purF-AdSS donor DNA plasmid was used as a template and F-Chr1-5-UP and R-Chr1-5-DW were used as primers to amplify the Chr1-5-purF-AdSS donor DNA fragment.

[0248] Finally, 500 ng of pZBJ-Chr1-5(BspQI) plasmid and 1000 ng of Chr1-5-purF-AdSS donor DNA fragment were co-electroporated into PC13 strain, and strain PC16 was obtained after Zeocin selection.

[0249] 4. Strengthen the supply of cofactors NADPH and ATP to promote the synthesis of cordycepin

[0250] Adequate ATP supply and a stable intracellular reducing environment provide the necessary energy, phosphate groups and reducing power for the synthesis of cordycepin. In the metabolic pathway of adenosine synthesis of cordycepin, the process of synthesizing one adenosine molecule from one molecule of PRPP and then converting it into one molecule of cordycepin requires the consumption of 8 molecules of ATP. Insufficient ATP supply may be the rate-limiting step in the synthesis of cordycepin. Studies have shown that the strong hydrogen-donating ability of NADPH plays an important role in the reduction of carbonyl groups to hydroxyl groups. 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 After overexpression of ZWF1 and PGK, the cordycepin production 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 F). The NADPH / NADP+ ratio and ATP content of strains PC19 and PC16 were compared and analyzed every 24 hours ( Figure 11The results showed that the ATP content of strain PC19 was significantly increased by 7.5%-16.9% compared with PC16, and the NADPH / NADP+ ratio of PC19 was also consistently higher than that of PC16 ( Figure 11 By enhancing the regeneration and supply of ATP and NADPH, the synthetic environment of cordycepin in Pichia pastoris was significantly optimized, facilitating its efficient accumulation. Finally, the hCas9 protein was knocked out in the PC19 strain to prevent spontaneous gene editing. The construction method for strain PC19 is as follows:

[0251] PC16 was used as the starting strain, and the strain PC19 was constructed with the help of pZBJ-Chr3-5 (BspQI) plasmid and Chr3-5-ZWF1-PGK donor DNA plasmid.

[0252] Among them, the construction process of the pZBJ-Chr3-5 (BspQI) plasmid is as follows: the pZBJ plasmid is digested with BspQI, and the linearized pZBJ plasmid is recovered by gel extraction; then the gRNA sequence 5'-ATACTAGTTAACAAACTGGG-3' of the Chr3-5 site of the Pichia pastoris genome is integrated into the BspQI cleavage site, and after heat shock transformation into Escherichia coli DH5α, the pZBJ-Chr3-5 (BspQI) plasmid is obtained by Zeocin screening. This plasmid can efficiently express the sgRNA targeting the Chr3-5 site in Pichia pastoris.

[0253] Among them, the construction process of the Chr3-5-ZWF1-PGK donor DNA plasmid is as follows: construct the target gene expression frame 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 assembled by Gibson to obtain the Chr3-5-ZWF1-PGK donor DNA plasmid. The Chr3-5-ZWF1-PGK donor DNA fragment was obtained by amplification using the Chr3-5-ZWF1-PGK donor DNA plasmid as a template and F-Chr3-5-UP and R-Chr3-5-DW as primers.

[0254] Finally, 500 ng of pZBJ-Chr3-5(BspQI) plasmid and 1000 ng of Chr3-5-ZWF1-PGK donor DNA fragment were co-electroporated into PC16 strain, and strain PC19 was obtained after Zeocin selection.

[0255] The hCas9 protein knockout method for strain PC19 is as follows:

[0256] In order to remove the hCas9 protein from the PC19 genome, the hCas9 protein was knocked out in the PC19 strain with the help of the pZBJ-ΔhCas9(BspQI) plasmid and the ΔCas9-UP-DW donor DNA fragment.

[0257] The construction process of the pZBJ-ΔhCas9 (BspQI) plasmid is as follows: the pZBJ plasmid is digested with BspQI, and the linearized pZBJ plasmid is recovered by gel electrophoresis; the gRNA sequence 5'-TACGCCGGATACATTGACGG-3' of hCas9 in the Pichia pastoris genome is then integrated into the BspQI cleavage site, and after heat shock transformation into Escherichia coli DH5α, the pZBJ-ΔhCas9 (BspQI) plasmid is obtained by Zeocin screening. This plasmid can efficiently express sgRNA targeting the hCas9 site in Pichia pastoris.

[0258] 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 primers F-ΔCas9-UP-OE and R-ΔCas9-UP-OE; the ΔCas9-DW-OE fragment is amplified from the Pichia pastoris GS115 genome using primers F-ΔCas9-DW-OE and R-ΔCas9-DW-OE; the ΔCas9-UP-OE fragment and the ΔCas9-DW-OE fragment are extended by overlapping PCR to obtain the ΔCas9-UP-DW donor DNA fragment.

[0259] Finally, 500 ng of pZBJ-ΔhCas9(BspQI) plasmid and 1000 ng of ΔCas9-UP-DW donor DNA fragment were co-electroporated into the PC19 strain, and the hCas9 protein knockout strain PC19 was obtained by Zeocin screening.

[0260] Example 3: Segmented high-density fermentation to achieve low-carbon and high-efficiency synthesis of cordycepin

[0261] A fed-batch strategy was used to carry out high-density fermentation of Pichia pastoris PC19 strain in a 10 L fermentor. The fed-batch fermentation process is divided into three stages: Batch (fermentation in the fermentor, consuming the original glycerol in the culture medium), Fed Batch (glycerol feeding) and methanol induction. The staged addition of glycerol and methanol effectively separates the biomass accumulation stage and cordycepin synthesis stage of Pichia pastoris. The stirring speed of the fermentor is cascaded with DO. When the dissolved oxygen DO value is lower than 20%, the stirring speed is actively increased to ensure that DO is not lower than the threshold. Studies have shown that a weakly acidic environment is conducive to the synthesis and accumulation of cordycepin. Therefore, the pH value of the Batch and Fed Batch stages was set to 5.0, and the pH value of the methanol induction stage was 5.5. The Batch stage lasted for a total of 22 hours. The rise of DO to above 60% marked the end of the Batch stage. At this time, OD 600 Reached 32.8 ( Figure 12 ). When the total fermentation time reaches 50h, OD 600 Reached 311.8 ( Figure 12 ), at which point the DO returned to above 75%, marking the end of the Fed Batch phase. After a 3-hour starvation period, the PC19 strain entered the methanol induction phase. 1.9 g / L cordycepin ( Figure 13 The fermentation was terminated after the total fermentation time reached 144 h. At this time, the yield and production rate of cordycepin were 18.3 g / L and 3.05 g / L / d, respectively. The conversion rate of methanol to cordycepin was 50.2 mg / g, and the OD 600 reached 368.3, and the dry weight of the bacteria (DCW) reached 149.6 g / L ( Figure 13 When the total fermentation time was extended to 216 h, the cordycepin yield reached 27.2 g / L ( Figure 13 ).

[0262] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. KhCas9-Brex27 expression cassette, characterized in that The expression cassette includes 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 the PpHIS4-2 fragment were from plasmid pAO815; 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; 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 derived from humans, with the amino acid sequence ALDFLSRLPLPPPVSPICTFVSPAAQKAFQPPRSCG; hCas9 protein from Streptococcus pyogenes ( Streptococcus pyogenes ), the reference sequence number in NCBI is 69900935; The GAP promoter, AOX1 terminator, and DAS1 terminator were all from Pichia pastoris ( Pichia pastoris )GS115; The amino acid sequence of Linker is (GGGGS)3; The sequence of the KhCas9-Brex27 expression cassette after Pichia codon optimization is shown in SEQ ID NO:

10.

2. An sgRNA expression plasmid for use with the expression cassette according to claim 1, characterized in that: Include 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, the AOX1 terminator, Pichia pastoris endogenous tRNA1, tRNA3, tRNA4 and tRNA5, type IIS restriction endonuclease sites BsaI, BspQI and BbsI, and three gRNA scaffolds; TEF1 promoter from Pichia pastoris GS115; The nucleotide sequences of Pichia pastoris endogenous tRNA1, tRNA3, tRNA4, and tRNA5 are shown in SEQ ID NOs: 1-4, respectively; 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 and correspond to four endogenous tRNAs of Pichia pastoris; Endonuclease site ①, endonuclease site ②, and endonuclease site ③ are different and correspond to three types of IIS restriction endonuclease sites respectively; 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; The full sequence of the sgRNA expression plasmid is shown in SEQ ID NO:

11.

3. A method for constructing an efficient gene editing system for Pichia pastoris, characterized in that: The expression cassette of claim 1 is integrated into the genome of Pichia pastoris GS115 to obtain the Pichia pastoris strain GS115-KhCas9-Brex27 that efficiently expresses the hCas9 protein, and then the sgRNA expression plasmid and the donor DNA fragment of claim 2 are introduced into Pichia pastoris GS115-KhCas9-Brex27 to obtain the Pichia pastoris CRISPR-Cas9 gene editing system.

4. The efficient gene editing system of Pichia pastoris constructed according to the method of claim 3.

5. Use of the efficient Pichia pastoris gene editing system according to claim 4 in constructing a high-yield cordycepin engineered strain.

6. An engineered strain of Pichia pastoris with high cordycepin production, characterized in that: The construction method includes the following steps: (1) Integrating the expression cassette described in claim 1 into the genome of Pichia pastoris GS115 to obtain strain GS115-KhCas9-Brex27, and then introducing the expression plasmid containing sgRNA1 and the donor DNA1 fragment into the strain GS115-KhCas9-Brex27 to construct an engineered strain 1; Among them, the nucleotide sequence of sgRNA1 targeting Pichia chromosome Chr2-5 is 5'-AACTTTGAAACAAAAGAAGG -3'; The target gene carried by the donor DNA1 fragment is from Cordyceps militaris ( Cordyceps militaris ) CP1, and the gene after Pichia pastoris codon optimization Ppcns1 and Ppcns2 Among them, genes Ppcns1 Driven by the promoter pAOX1, the gene Ppcns2 Driven by promoter pFLD1; Gene Ppcns1 and Ppcns2 The nucleotide sequences are shown in SEQ ID NOs: 6 and 7, respectively; Promoters pAOX1 and pFLD1 were from Pichia pastoris GS115; (2) The expression plasmid containing sgRNA2 and the donor DNA2 fragment were introduced into the engineered bacteria 1 to construct the engineered bacteria 2; Among them, the nucleotide sequence of sgRNA2 targeting Pichia chromosome Chr2-4 is 5'-CCTAAATACTACCTAAACAG -3'; The target gene carried by the donor DNA2 fragment is a gene from Cordyceps militaris CP1 and has been codon-optimized in Pichia pastoris. Ppcns3 / NK , and from Escherichia coli ( Escherichia coli ), and the gene after Pichia pastoris codon optimization PpcpdB Among them, genes Ppcns3 / NK Driven by the promoter pAOX1, the gene PpcpdB Driven by promoter pFLD1; Gene Ppcns3 / NK and PpcpdB The nucleotide sequences are shown in SEQ ID NOs: 8 and 9, respectively; (3) The expression plasmid containing sgRNA3 and the donor DNA3 fragment were introduced into the engineered bacteria 2 to construct the engineered bacteria 3; Among them, the nucleotide sequence of sgRNA3 targeting Pichia chromosome Chr1-2 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, genes das1 Driven by the promoter pAOX1, the gene pex8 Driven by promoter pFLD1; Gene das1 and pex8 The reference sequence numbers in NCBI are PAS_chr3_0832 and PAS_chr1-4_0349; (4) The expression plasmid containing sgRNA4 and the donor DNA4 fragment were introduced into the engineered bacteria 3 to construct the engineered bacteria 4; Among them, the nucleotide sequence of sgRNA4 targeting Pichia chromosome Chr1-5 is 5'-CACGAGCCGAGTAATAACCG -3'; The target gene carried by the donor DNA4 fragment is an endogenous gene of Pichia pastoris purF and ads Among them, genes purF Driven by promoter pCAT1, gene ads Driven by promoter pFDH1; Gene purF and ads The reference sequence numbers in NCBI are PAS_chr1-1_0430 and PAS_chr4_0613; Promoters pCAT1 and pFDH1 were from Pichia pastoris GS115; (5) The expression plasmid containing sgRNA5 and the donor DNA5 fragment were introduced into the engineered bacteria 4 to construct the engineered bacteria 5; Among them, the nucleotide sequence of sgRNA5 targeting Pichia chromosome Chr3-5 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, genes zwf1 Driven by the promoter pTEF1, the gene pgk Driven by promoter pPGI1; Gene zwf1 and pgk The reference sequence numbers in NCBI are PAS_chr2-1_0308 and PAS_chr1-4_0292; Promoters pTEF1 and pPGI1 were from Pichia pastoris GS115; (6) The expression plasmid containing sgRNA6 and the donor DNA6 fragment were introduced into the engineered bacteria 5 to knock out the hCas9 protein, thereby obtaining the high-yield cordycepin Pichia pastoris engineered bacteria; Among them, the nucleotide sequence of hCas9 targeted by sgRNA6 in chromosome 5 of the engineered bacteria is 5'-TACGCCGGATACATTGACGG-3'; The method for constructing the donor DNA6 fragment includes: using primers F-ΔCas9-UP-OE and R-ΔCas9-UP-OE to amplify the ΔCas9-UP-OE fragment from the Pichia pastoris GS115 genome; using primers F-ΔCas9-DW-OE and R-ΔCas9-DW-OE to amplify the ΔCas9-DW-OE fragment from the Pichia pastoris GS115 genome; and performing overlap extension PCR on the ΔCas9-UP-OE fragment and the ΔCas9-DW-OE fragment 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 according to any one of claims 3 to 5.

7. The use of the engineered bacteria according to claim 6 in the production of cordycepin, characterized in that: include: Cultivating the engineered bacteria using a fed-batch fermentation process to obtain a culture, and collecting the produced cordycepin from the culture; Among them, the fed-batch fermentation process includes: S1, seed liquid activation; S2, fermentation tank culture: OD 600 The seed solution with a value of 4.0-5.0 is inoculated into a fermentation tank at a volume ratio of 5%-10% for fermentation tank culture; The culture conditions are as follows: the volume of the culture medium in the tank 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 ventilation volume is maintained at 2-4 vvm, and the dissolved oxygen DO value of the fermentation liquid is 20-60%; The culture medium formula 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 85% concentrated phosphoric acid; S3, glycerol feeding: When the dissolved oxygen DO value rebounds for the first time, it indicates that the glycerol in the fermentation broth has been exhausted. Glycerol feeding medium is added at a constant rate of 15-25 mL / L / h until the OD 600 Increase to 300-350, the glycerol feeding stage ends; The culture conditions were as follows: fermentation temperature 28-30°C, pH 4.0-5.0, stirring speed 500-800 rpm, and ventilation maintained at 4-8 vvm; The formula of glycerol feed medium is: 40-60% m / v glycerol solution containing 10-15 mL / L PTM1; S4, methanol induction and cordycepin synthesis: After stopping the glycerol supplementation, continue the culture for 2-3 hours to deplete the residual glycerol in the bacteria. The time is recorded as t0, and methanol induction begins; Add methanol feed medium at the following 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. From t0+7h, maintain a methanol feed rate of 5-10 mL / L / h to maintain 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, ventilation volume maintained at 4-8 vvm, dissolved oxygen DO value of the fermentation liquid controlled at 15-75%, and fermentation ended after 6-9 days, and the tank was released; The formula of methanol feed medium is: 100% methanol containing 10-15 mL / L PTM1.

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