Pichia pastoris chassis with high efficiency of supplying terpenoid precursor IPP and construction method thereof

Abstract

This invention relates to a Pichia pastoris chassis for efficiently supplying the terpene precursor IPP and its construction method, belonging to the field of molecular biology. The invention involves screening three highly expressed HMGR genes from the latex tissue of the Brazilian rubber tree using transcriptome sequencing. After codon optimization in Pichia pastoris, these genes were assembled into an expression cassette with a screening marker, a bleomycin resistance gene, expression elements, and a weak promoter. Using two fragments from different upstream regions of the CDS of the endogenous EGR9 gene in Pichia pastoris as homologous arms, the expression cassette was integrated into the Pichia pastoris genome through homologous recombination, replacing the original ERG9 gene promoter to weaken the competitive consumption of IPP by the squalene synthesis pathway. The constructed Pichia pastoris mutant strain showed a 430% higher IPP content than the wild type, with no significant difference in growth rate. It efficiently supplies IPP, providing a sufficient precursor for the heterologous synthesis of terpenoids, and has significant industrial application value.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more particularly to Pichia pastoris, specifically a Pichia pastoris disc substrate for efficiently supplying the terpene precursor IPP and its construction method. Background Technology

[0002] Terpenes (including terpenes and isoprene-like compounds) are the largest family of natural products synthesized by plants, with over 55,000 known members. Their core structure consists of one or more isoprene (C5H8) units. The synthetic pathway of terpenes involves three key stages: the formation of the precursor IPP and its isomer dimethylallyl pyrophosphate (DMIPP), chain elongation and cyclization of the terpenoid skeleton, and further modification of the skeleton. Studies have confirmed that increasing the supply of IPP in the synthetic biology chassis host is crucial for significantly increasing terpenoid yields.

[0003] Yeast expression systems, serving as eukaryotic chassis, are widely used in synthetic biology. Their endogenous methylvaleric acid pathway (MVA pathway) can produce IPP, providing C5 monomers for terpenoids. While *Saccharomyces cerevisiae* possesses the advantage of a redox system supporting cytochrome P450 modification of the terpene skeleton, it suffers from low production capacity. The unconventional yeast *Pichia pastoris* can proliferate to extremely high cell densities in simple media, resists phage contamination, requires simple fermentation conditions, and can perform green fermentation using methanol as the sole carbon source. It has been proven to be a highly efficient chassis for producing high-value chemicals, including polyketides and terpenoids.

[0004] The Brazilian rubber tree can synthesize high-polymerization-degree polyisoprene due to its strong ability to synthesize terpenoid precursors. Transcriptome sequencing identified three highly expressed rate-limiting enzymes in the MVA pathway from the latex tissue of the Brazilian rubber tree: 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase, HMGR) gene. Therefore, this invention aims to heterologously express these HMGR genes in Pichia pastoris, screen for functional genes that efficiently enhance IPP supply, construct a high-IPP-producing Pichia pastoris chassis strain, and thus efficiently synthesize terpenoid compounds. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a Pichia pastoris chassis for efficiently supplying the terpene precursor IPP and its construction method, thereby achieving efficient synthesis of terpene compounds.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solution: 1. Screening, acquisition, and optimization of the HMGR gene: (1) Gene screening: Three highly expressed HMGR genes were screened from the latex tissue of Brazilian rubber tree by transcriptome sequencing and named EVM0006956, EVM0017893 and EVM0023638, respectively. (2) Gene acquisition: Attempts were made to amplify the above three genes from the latex cDNA of Brazilian rubber tree. Only the original fragments of EVM0017893 and EVM0023638 were successfully obtained, and EVM0006956 was not amplified. (3) Codon optimization: Due to the difference in codon usage preferences between the Brazilian rubber tree and Pichia pastoris, direct heterologous expression of plant-derived HMGR genes may result in low translation efficiency. Therefore, Pichia pastoris codon optimization was performed on the three HMGR genes: For the unamplified EVM0006956, the optimized coding sequence EVM0006956*p was artificially synthesized based on its sequence information. For the amplified EVM0017893 and EVM0023638, rare codons were removed and the mRNA secondary structure was optimized through codon optimization to obtain EVM0017893*p and EVM0023638*p, so as to improve the stability of the mRNA secondary structure and enhance its translation efficiency in Pichia pastoris.

[0007] 2. Bioinformatics characteristics of the HMGR gene: (1) Sequence alignment: Analysis using NCBI BLAST tool showed that EVM0017893 is Brazilian rubber tree HMGR1, sequence ID: NP_001392267.1, length 575 amino acids. EVM0023638 is highly similar to Brazilian rubber tree HMGR2. The sequence ID of Brazilian rubber tree HMGR2 is XP_057987249.1, length 686 amino acids. EVM0006956 is highly similar to HMGR1, with only 33 amino acid differences between amino acids 35-67. (2) Functional domain and structure prediction: SMART analysis showed that all three genes possessed an HMG-CoA reductase functional domain at their C-terminus and a transmembrane domain of [missing information]. The helix is ​​used to anchor HMGR to a specific region of the cell membrane; the N-terminus of EVM0023638 has 100 more amino acids than the other two genes, forming a cavity-like morphology; the 3D structure predicted by the Robbetta tool shows that EVM0023638 contains three N-terminal transmembrane domains (the other two genes contain only two), and there is a unique pouch-like structure near the first transmembrane domain, which can improve membrane anchoring, enzyme stability or substrate transport.

[0008] 3. Construction of expression cassettes and mutant strains: (1) Homologous arm design and acquisition: The endogenous ERG9 gene of Pichia pastoris was selected as the integration target. ERG9 is a key gene in the squalene synthesis pathway of Pichia pastoris. Its encoded squalene synthase catalyzes the conversion of FPP to squalene, which is the main consumption pathway of IPP. Two homologous arm fragments were amplified from the genomic DNA of wild-type Pichia pastoris: ① The first homologous arm: a 500 bp fragment distal to the upstream end of ERG9 CDS, located upstream of the original ERG9 promoter, denoted as ERG9 500F; ② The second homologous arm: a 500 bp fragment adjacent to the start end of ERG9 CDS, located downstream of the original ERG9 promoter. The two homologous arms together define the original ERG9 promoter region, providing an anchor for the directional integration of exogenous fragments. (2) Expression cassette assembly: Using two homologous arms as boundaries, expression elements, HMGR genes (EVM0006956*p / EVM0017893*p / EVM0023638*p / EVM0017893 original fragment / EVM0023638 original fragment), selection marker bleomycin resistance gene (BleoR) and if promoter pSer1 were inserted in sequence to construct 5 expression cassettes targeting different HMGR genes. The expression elements are assembled according to the classic expression unit of "promoter-target gene-terminator": the promoters selected are the endogenous constitutive promoters PRAP (glyceraldehyde-3-phosphate dehydrogenase promoter, which continuously drives gene expression) and pHis4 (histidine synthase 4 gene promoter, which provides medium and low intensity expression to meet the needs of different genes) from Pichia pastoris; the terminators selected are the commonly used Pichia pastoris tAOX1 (alcohol oxidase 1 gene terminator) and tCyc1 (yeast Yc1 gene terminator), which ensure precise termination of transcription and improve mRNA stability; the selection marker BleoR is used to screen positive clones, so that yeast strains that successfully integrate the expression cassette acquire bleomycin resistance and can be produced on bleomycin-containing media; the weak promoter pSer1 is used to replace the original strong promoter of ERG9, reduce the expression level of ERG9 gene, and thus reduce the consumption of IPP by squalene synthesis; (3) Homologous recombination and mutant construction: Five expression cassettes were introduced into Pichia pastoris competent cells, and the expression cassettes were integrated into the ERG9 locus of the Pichia pastoris genome through homologous recombination, replacing the original ERG9 promoter, and five mutant strains were constructed: P1 (containing EVM0006956*p), P2 (containing EVM0017893*p), P3 (containing EVM0023638*p), P4 (containing the original fragment of EVM0017893), and P5 (containing the original fragment of EVM0023638).

[0009] 4. Identification of positive mutant strains: (1) Resistance screening: Transformed yeast cells were spread on YPD screening plates containing bleomycin and cultured at 30℃ for 48-72 h to select resistant single colonies; (2) PCR identification: Genomic DNA was extracted from resistant single colonies, and eight pairs of specific primers (Primer 8F / R, Primer 9F / R, Primer 10F / R, Primer 11F / R, Primer 12F / R, Primer 13F / R, Primer 14F / R, Primer 15F / R) were designed for PCR amplification at key junctions: "first homologous arm-promoter", "promoter-HMGR gene", "HMGR gene-terminator", "terminator-promoter", "promoter-BleoR", "BleoR-terminator", "terminator-pSer1", and "pSer1-second homologous arm". The complete insertion of the fragment was verified by gel electrophoresis. The results showed that all 10 constructed mutant strains (each expression cassette corresponding to 2 duplicate clones) were positive clones, and the gene insertion sites were correct. (3) Strain screening: Biomass and IPP quantification were performed on positive mutant strains to screen for Pichia pastoris chassis strains with growth rates not significantly different from wild-type and significantly increased IPP content. For biomass detection, sterile YPD was used as a blank control, and the absorbance of the yeast culture was measured at 600 nm using a spectrophotometer. For IPP quantification, an isopentenyl pyrophosphate (IPP) ELISA kit was used, a standard curve and a blank control were set, and the absorbance values ​​were read using an ELISA reader. The IPP content in the sample was calculated based on the standard curve. All tests were repeated three times, and the average value was taken.

[0010] Compared with the prior art, the present invention has the following advantages: (1) This application significantly increases the endogenous IPP content of Pichia pastoris through dual metabolic modification that enhances synthesis and weakens consumption. The IPP content of all mutant strains is higher than that of wild type, and the IPP content of P5 strain is even higher, which is attributed to the highly efficient catalytic activity brought about by the unique protein structure of EVM0023638. (2) The mutant strain of this application has good growth stability. The integration of exogenous genes and the replacement of ERG9 promoter did not have an adverse effect on the normal growth of yeast, and it has the basis for industrial fermentation application. (3) The expression cassette design of this application is scientific and efficient. It improves the translation efficiency of heterologous genes through codon optimization, selects Pichia pastoris endogenous adapted expression elements to ensure stable gene expression, uses ERG9 locus as integration target to achieve targeted modification, and obtains positive clones quickly through BleoR resistance screening. The construction process is standardized and reproducible. (4) The chassis of this application has wide applicability and can efficiently supply IPP precursors. It is suitable for heterogeneous synthesis of various terpenoids such as monoterpenes, sesquiterpenes, and diterpenes, providing an efficient and stable host platform for the industrial production of high-value terpenoids. It has important economic value and application prospects. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the de novo synthesis pathway and precursor metabolism modification of terpenoids in yeast according to this application (the ERG9-mediated squalene synthesis pathway is marked as an IPP competitive consumption pathway). Figure 2 The sequence alignment of three highly expressed HMGR genes screened from the latex tissue of the Brazilian rubber tree in this application in the Blast database is shown. A is EVM0006956, B is EVM0017893, and C is EVM0023638. Figure 3 Analysis of the structure and functional domains of the EVM0006956 protein in this application (green represents the transmembrane domain, and blue represents the HMG-CoA reductase functional domain). Figure 4 Analysis of the structure and functional domains of the EVM0017893 protein in this application (green represents the transmembrane domain, and blue represents the HMG-CoA reductase functional domain). Figure 5 The structure and functional domain analysis of the EVM0023638 protein in this application (green represents the transmembrane domain, blue represents the HMG-CoA reductase functional domain, and the N-terminus is marked with an additional 110 amino acids forming a cavity structure). Figure 6 The gene integration pattern of Pichia pastoris in this application is shown (the location of the two homologous arms is marked: ERG9 500F is the upstream distal homologous arm, and the one adjacent to ERG9 CDS is the downstream homologous arm, with the inserted expression element, HMGR gene, BleoR, and pSer1 in between). Figure 7 For the identification of the Pichia pastoris HMGR transformed strain of this application, A is the detection primer setup (marking the fragment ligation points corresponding to each primer), and B is the PCR detection result of the positive clone strain; Figure 8 The results of quantitative (blue) and biomass (gray) detection of Pichia pastoris in this application are shown. Figure 9 The predicted results for the protein HbHMGR2 (encoded by EVM0023638) in this application are shown in A, which shows the overall structure (with three N-terminal transmembrane domains marked), B, which shows the predicted structure of the pocket structure, and C, which shows the predicted domains, repeat sequences, motifs, and features. Detailed Implementation

[0012] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0013] Based on such Figure 1 The present invention demonstrates the synthetic pathways and precursor metabolism modification methods of the terpenoid compounds shown in the following experiments.

[0014] I. Experimental Materials 1. Strains and gene sources: wild-type strain of Pichia pastoris; latex tissue from the Brazilian rubber tree (used for RNA extraction and reverse transcription into cDNA); 2. Reagents: RNA extraction kit, reverse transcription kit, GeneJET genomic DNA purification kit (Thermo, catalog number K0721), 2×Hieff® PCR Master Mix (With Dye) (Yisheng, Shanghai), isopentenyl pyrophosphate (IPP) ELISA kit, bleomycin, YPD medium, yeast competent cell preparation kit; 3. Instruments: spectrophotometer, high-speed refrigerated centrifuge, PCR instrument, agarose gel electrophoresis system, gel imaging system, constant temperature shaker, incubator.

[0015] II. Experimental Methods 1. Selection, acquisition, and optimization of the HMGR gene: (1) RNA extraction and cDNA synthesis: Total RNA was extracted from the latex tissue of Brazilian rubber trees using an RNA extraction kit and then reverse transcribed into cDNA using a reverse transcription kit; (2) Gene amplification: Based on the three HMGR gene sequences obtained by transcription and sequencing, specific primers were designed and PCR amplification was performed using cDNA as a template. Only the original fragments of EVM0017893 and EVM0023638 were successfully obtained, while EVM0006956 was not amplified. (3) Codon optimization: The biotechnology company was commissioned to optimize the codons of EVM0006956 according to the codon usage preference of Pichia pastoris and to artificially synthesize the optimized sequence EVM0006956*p; the codons of EVM0017893 and EVM0023638 of the amplified butterfly were optimized to obtain EVM0017893*p and EVM0023638*p. The optimized gene sequences were submitted to the NCBI database for record.

[0016] 2. Bioinformatics analysis of the HMGR gene: (1) Sequence alignment: Homology analysis of the original and optimized sequences of the three HMGR genes was performed using the NCBI online BLAST tool (https: / / blast.ncbi.nlm.nil.gov / blast.cgi) from the National Center for Biotechnology Information (NCBI), matching known HMGR subtypes of the Brazilian rubber tree. The results are as follows: Figure 2 As shown; (2) Functional domain prediction: The signal peptide, transmembrane helix and low-complexity regions of the target protein were predicted using the SMART tool (https: / / smart.embl.de). Domains with an E value < 0.1 were considered significant. (3) 3D Structure Prediction: The 3D model structure of HMGR was predicted using the Robbetta prediction tool (https: / / robetta.bakerlab.org). The final rendered image was generated using PyMOL 2.5.5, and the structural differences of different HMGR proteins were analyzed. The results for EVM0006956, EVM0017893, and EVM00236638 are shown below. Figure 3-5 As shown.

[0017] 3. Construction of homologous arms and expression boxes: (1) Homologous arm amplification: Genomic DNA was extracted from wild-type Pichia pastoris strains. Based on the known sequence of the Pichia pastoris ERG9 gene (GenBank accession number: XM_002481443.1), two pairs of primers were designed to amplify the 500bp fragment upstream of the ERG9 CDS (ERG9 500F) and the 500bp fragment adjacent to the start of the ERG9 CDS, respectively. The amplification products were recovered. (2) Acquisition of expression elements: Amplify PGAP, pHis4 promoter and tAOX1, tCyc1 terminator fragments from Pichia pastoris genomic DNA; synthesize bleomycin resistance gene (BleoR) and pSer1 promoter fragment; (3) Expression cassette assembly: Using the Gibson assembly method, homologous arms, expression elements, HMGR gene (optimized or original fragment), BleoR, and pSer1 were assembled into complete expression cassettes in the order of "upstream homologous arm (ERG9 500F) - promoter - HMGR gene - terminator - BleoR - pSer1 - downstream homologous arm (adjacent to ERG9 CDS)". Five expression cassettes were constructed, corresponding to P1-P5 mutant strains, as follows: Figure 6 As shown.

[0018] 4. Yeast transformation and positive clone screening: (1) Preparation of competent yeast cells: Pichia pastoris competent cells were prepared according to the instructions using a yeast competent cell preparation kit; (2) Transformation: Five expression cassettes were introduced into competent cells by electrotransformation. After being incubated on ice for 30 min, they were electrotransformed at 1.5 kV. (3) Resistance screening: The transformed cells were plated on a substrate containing... Bleomycin YPD selection plates were incubated at 30℃ for 48-72 h, and single colonies with good growth were selected.

[0019] 5. PCR identification of positive mutant strains: (1) Genomic DNA extraction: The resistant single colony was inoculated into YPD liquid medium containing bleomycin and cultured overnight at 30°C and 230 rpm. The bacterial cells were collected and genomic DNA was extracted using the GeneJET genomic DNA purification kit. (2) PCR amplification: Using genomic DNA as a template, PCR amplification was performed using 8 pairs of specific primers. The reaction system includes: , upstream primer, Downstream primers, Sterilized double-distilled water Genomic DNA; Reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ de-denaturation for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min; (3) Electrophoresis verification: Take The product was subjected to 1% agarose gel electrophoresis, and the results were observed using a gel imaging system. If the size of the target fragment was consistent with the expectation, it was determined to be a positive clone. Figure 7 ).

[0020] 6. Strain growth assay: (1) Seed culture: Select positive clones and inoculate them into YPD-free liquid medium and culture overnight at 30°C and 230 rpm; (2) Expanded culture: The seed culture was inoculated into fresh YPD liquid medium at a ratio of 1:100, the initial OD600 was adjusted to 0.1, and cultured at 30℃ and 230 rpm for 48 h. (3) Biomass detection: Sterile YPD medium was used as a blank control. The absorbance (OD600) of the yeast culture was measured at a wavelength of 600 nm using a spectrophotometer. Each strain was replicated three times and the average value was taken.

[0021] 7. Extraction and quantification of IPP: (1) IPP extraction: Take 1 mL of the bacterial culture medium that has been expanded for 48 h and extract it through the IPP extract. Filter the solution through a nylon filter, and immediately wash the cells twice with 2 mL of ultrapure water preheated to 30°C. Immerse the filter in 5 mL of ice-cold extraction buffer (40% methanol + 40% acetonitrile + 20% water, v / v) and incubate at -20°C for 20 min. Remove the filter, transfer the extract to a centrifuge tube, centrifuge at 16000 rpm for 10 min, aspirate the supernatant to a new tube, and dry under nitrogen at 40°C for 30 min. Resuspend the dried material in ultrapure water, centrifuge at 16,000 rpm for 10 min, and collect the supernatant for later use; (2) Quantitative analysis of IPP: The isopentenyl pyrophosphate (IPP) ELISA kit was used. The manufacturer’s instructions were followed to set up a standard curve and a blank control. Each sample was tested in triplicate. The absorbance value was read by an ELISA reader. The IPP content in the sample was calculated based on the standard curve. The average value of the results was taken.

[0022] 8. Further analysis of the EVM0023638 protein: The physicochemical properties of the EVM0023638 protein were analyzed using ExPASy ProtParam. Its transmembrane domains and bag-like structure were further verified using SWISS-MODEL and PyMOL software to explore the relationship between structure and catalytic activity.

[0023] III. Experimental Results 1. Bioinformatics analysis results: such as Figure 9 As shown, the C-terminus of all three HMGR genes has a conserved HMG-CoA reductase domain; the protein encoded by EVM0023638 contains three N-terminal transmembrane domains and a unique bag-like structure, with a molecular weight of approximately 76 kDa and an isoelectric point of 6.2. 2. Expression cassettes and transformation results: All five expression cassettes were successfully constructed, and electrophoresis verification showed that the fragment size was consistent with the expectation. After transformation, a large number of resistant single colonies were obtained on the selection plates, and PCR identification results showed that all the selected single colonies were positive clones and the gene insertion site was correct. 3. Results of strain growth assay: The OD600 values ​​of the P1-P5 mutant strains ranged from 1.8 to 2.2, while the OD600 value of the wild-type strain was 2.0. There was no significant difference between the two, which proves that the integration of exogenous genes and metabolic modification did not affect yeast growth. 4. Quantitative results of IPP: such as Figure 8 As shown, the IPP content of all mutant strains was significantly higher than that of the wild type, with strain P5 (containing the original EVM0023638) having the highest IPP content, reaching [value missing]. More wild type The IPP content increased by 430%, followed by strain P3 (containing EVM0023638*p), which had an IPP content 380% higher than the wild type.

Claims

1. A Pichia pastoris chassis for efficiently supplying the terpene precursor IPP, characterized in that, The strain of the chassis was constructed by heterologously expressing the HMGR gene, which is highly expressed in the latex tissue of the Brazilian rubber tree, in Pichia pastoris. The HMGR gene is selected from at least one of EVM0006956, EVM0017893, and EVM0023638. The original promoter of the ERG9 gene of Pichia pastoris was replaced by the weak promoter pSer1. After the HMGR gene, selection marker gene, and expression element were assembled into an expression cassette, it was integrated into the Pichia pastoris genome through homologous recombination.

2. The Pichia pastoris chassis according to claim 1, characterized in that, The protein encoded by the EVM0023638 gene includes three N-terminal transmembrane domains and a unique pouch-like structure near the first transmembrane domain, and the C-terminus of the protein has an HMG-CoA reductase functional domain.

3. The Pichia pastoris chassis according to claim 1, characterized in that, The HMGR gene was integrated into the Pichia pastoris genome after codon optimization. Among them, the EVM0006956 gene was not obtained from the latex cDNA of Brazilian rubber tree, and its optimized sequence was obtained by artificial synthesis and is denoted as EVM0006956*p. The optimized sequences of the amplified EVM0017893 and EVM0023638 are denoted as EVM0017893*p and EVM0023638*p, respectively.

4. The Pichia pastoris chassis according to claim 1, characterized in that, The homologous arms of the homologous recombination are two 500 bp fragments upstream of the CDS of the endogenous ERG9 gene in Pichia pastoris: the first homologous arm is a 500 bp fragment distal to the upstream of the ERG9 CDS, located upstream of the original ERG9 promoter, denoted as ERG9 500F; the second homologous arm is a 500 bp fragment adjacent to the start of the ERG9 CDS, located downstream of the original ERG9 promoter. The two homologous arms together define the region of the original ERG9 promoter.

5. The Pichia pastoris chassis according to claim 1, characterized in that: The selection marker gene is the bleomycin resistance gene BleoR, and the expression elements include the promoters PGAP and pHis4 and the terminators tAOX1 and tCyc1.

6. A method for constructing a Pichia pastoris chassis as described in any one of claims 1-5, characterized in that: Includes the following steps: (1) Screening and acquisition of HMGR genes: Three highly expressed HMGR genes, EVM0006956, EVM0017893, and EVM0023638, were screened from the latex tissue of Brazilian rubber tree by transcriptome sequencing; the original fragments of EVM0017893 and EVM0023638 were amplified from the cDNA of Brazilian rubber tree latex, and the optimized sequence of EVM0006956, EVM0006956*p, was artificially synthesized. At the same time, the codons of EVM0017893 and EVM0023638 were optimized by Pichia pastoris to obtain EVM0017893*p and EVM0023638*p. (2) Construction of expression cassette: The 500 bp fragment ERG9 500F from the distal end of the CDS of the endogenous ERG9 gene in Pichia pastoris and the 500 bp fragment adjacent to the start end of the ERG9 CDS are used as homologous arms. Expression elements, HMGR gene EVM0006956*p / EVM0017893*p / EVM0023638*p / EVM0017893 original fragment / EVM0023638 original fragment, selection marker BleoR and pseudopromoter pSer1 are inserted sequentially between the two homologous arms to construct a complete expression cassette. The expression elements are assembled in the order of "promoter-target gene-terminator". The promoter is selected from PGAP or pHis4 and the terminator is selected from tAOX1 or tCyc1. (3) Homologous recombination integration: The expression cassette constructed in step (2) is introduced into Pichia pastoris competent cells through homologous recombination, so that the expression cassette is integrated into the ERG9 locus of the Pichia pastoris genome and replaces the original ERG9 promoter; (4) Screening and identification of positive clones: Transformed yeast cells were plated on screening plates containing bleomycin to screen for resistant single colonies; genomic DNA of resistant single colonies was extracted and PCR was performed using specific primers designed for the junction of homologous arms and exogenous fragments to verify whether the fragment was completely inserted and to obtain positive mutant strains. (5) Strain screening: Biomass and IPP were measured for positive mutant strains, and Pichia pastoris chassis strains with no significant difference in growth rate from wild type and significantly increased IPP content were screened.

7. The construction method according to claim 6, characterized in that, The basis for codon optimization in step (1) is the codon usage preference of Pichia pastoris. The optimization goal is to remove rare codons, improve the stability of mRNA secondary structure, and enhance the translation efficiency of HGMR gene in Pichia pastoris.

8. The construction method according to claim 6, characterized in that, The specific primers in step (4) include Primer8F / R, Primer9F / R, Primer10F / R, Primer11F / R, Primer12F / R, Primer13F / R, Primer14F / R, and Primer15F / R, which correspond to the connection between the first homologous arm and the promoter, the promoter and the HMGR gene, the HMGR1 gene and the terminator, the terminator and the promoter, the promoter and BleoR, BleoR and the terminator, the terminator and pSer1, and the connection between pSer1 and the second homologous arm, respectively.

9. The construction method according to claim 6, characterized in that: In step (5), biomass detection is performed by measuring the absorbance of yeast culture medium at a wavelength of 600 nm using a spectrophotometer; the quantitative detection of IPP is performed by using an isopentenyl pyrophosphate ELISA kit, setting a standard curve and a blank control, reading the absorbance value using an enzyme-linked immunosorbent assay (ELISA) reader, and calculating the IPP content in the sample based on the standard curve.

10. The use of the strain of Pichia pastoris chassis as described in any one of claims 1-5 or obtained by the construction method of any one of claims 6-9 in the heterologous synthesis of terpenoid compounds.

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