Pichia pastoris for efficient synthesis of cis-1,4-polyisoprene and a construction method thereof

Abstract

The application discloses a kind of high-efficiency synthesis cis-1,4-polyisoprene Pichia and its construction method, belong to synthetic biology field.Cis-1,4-polyisoprene Pichia strain heterologous expression Brazil rubber tree rubber synthesis pathway key gene CPT (HRT1 or HRT2), can further express REF and / or SRPP gene.Its construction method includes amplification target gene, constructs expression cassette, into Pichia and selects positive mutant strain.When cis-1,4-polyisoprene is synthesized using the strain, methanol can be used as carbon source to induce fermentation, and then extracted.The Pichia strain constructed by the method can de novo synthesize cis-1,4-polyisoprene, which meets the principles of green chemical production, and the yield can reach 10.81 mg / L.The exogenous gene has little effect on the growth of yeast, and has good industrial application prospect, which can be used for green production of cis-1,4-polyisoprene.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, and particularly relates to a Pichia pastoris for the efficient synthesis of cis-1,4-polyisoprene and its construction method. Background Technology

[0002] cis-1,4-polyisoprene is a crucial isoprene-like polymer in the rubber industry, used to produce over 40,000 products, such as tires and medical gloves. Industrially, polyisoprene is typically synthesized at high temperatures from isoprene (C5H8), a byproduct of petroleum cracking, or through various synthetic routes to obtain polymers similar to polyisoprene. However, these methods generally do not conform to the principles of green chemistry production because they are environmentally unfriendly, rely on non-renewable resources, and are subject to the uncertainty of oil supply and prices. Furthermore, the polymerization process can randomly generate trans-structures, leading to insufficient performance in rubber products.

[0003] Natural rubber, primarily composed of cis-1,4-polyisoprene, is a crucial industrial raw material relevant to both civilian and national defense needs. While over 2,500 plant species are known to synthesize natural rubber, the Brazilian rubber tree (H. brasiliensis) remains the primary commercial source of cis-1,4-polyisoprene due to its commercially viable yield and ability to produce high-polymerization-degree cis-1,4-polyisoprene. In recent years, demand for cis-1,4-polyisoprene has continued to grow; however, due to declining planted area, persistent pathogen infections, and a long growth cycle, the yield from the Brazilian rubber tree is increasingly unable to meet industrial requirements.

[0004] Pichia pastoris exhibits exceptional bioprocessing advantages, including: (1) the ability to perform ultra-high cell density fermentation under simple conditions; (2) an efficient and preferred chassis for producing high-value chemicals, including polyketides and terpenes; and (3) a unique ability to utilize methanol, establishing a sustainable C1 biomanufacturing platform. However, the microbial synthesis of cis-1,4-polyisoprene remains unrealized due to the high IPP requirements of polyterpene synthesis.

[0005] The cis-prenyltransferase (CPT) of the Brazilian rubber tree is a key gene in the rubber synthesis pathway, which condenses IPP into FPP or GGPP to obtain cis-1,4-polyisoprene during rubber synthesis. Therefore, this invention addresses the problems existing in the prior art by heterologously expressing the Brazilian rubber tree CPT and other potential functional genes in the rubber synthesis pathway in Pichia pastoris. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a Pichia pastoris strain for the efficient synthesis of cis-1,4-polyisoprene and its construction method, applicable to the green production of cis-1,4-polyisoprene. Furthermore, the present invention also provides a method for constructing this Pichia pastoris strain, a method for synthesizing cis-1,4-polyisoprene and its applications, as well as an expression plasmid for constructing this strain, thus solving the problems in the prior art.

[0007] This invention provides the following technical solution: A Pichia pastoris strain for efficient synthesis of cis-1,4-polyisoprene is disclosed. The Pichia pastoris strain heterologously expresses the key gene CPT in the rubber synthesis pathway of the Brazilian rubber tree, wherein the CPT gene is either the HRT1 gene or the HRT2 gene; wherein the amino acid sequence corresponding to the nucleotide sequence of the HRT1 gene is SEQ ID NO.1, and the amino acid sequence corresponding to the nucleotide sequence of the HRT2 gene is SEQ ID NO.2.

[0008] Preferably, the Pichia pastoris strain also expresses the rubber synthesis pathway genes REF and / or SRPP; the amino acid sequence corresponding to the nucleotide sequence of the REF gene is SEQ ID NO.3, and the amino acid sequence corresponding to the nucleotide sequence of the SRPP gene is SEQ ID NO.4.

[0009] Preferably, the Pichia pastoris strain simultaneously expresses the HRT1 gene, REF gene, and SRPP gene.

[0010] Preferably, a method for constructing a Pichia pastoris strain includes the following steps: S1, HRT1, HRT2, REF and SRPP genes were amplified from the latex cDNA of Brazilian rubber tree; S2, using plasmid pPIC3.5K as a vector, the genes amplified in step S1 were inserted into the multiple cloning site to construct a gene expression cassette regulated by a methanol-inducible promoter. S3. The expression cassette and HIS4 selection marker constructed in step S2 are transferred into the AOX1 site of Pichia pastoris to screen for positive mutant strains, namely the Pichia pastoris strain that efficiently synthesizes cis-1,4-polyisoprene.

[0011] Preferably, in step S2, when constructing the gene expression cassette, the REF gene and SRPP gene are inserted into the vector in different arrangements and combinations.

[0012] Preferably, in step S3, positive mutant strains are screened by PCR detection. The PCR reaction system is 10 μL, including 5 μL 2×Hieff® PCRMasterMix, 0.5 μL upstream / downstream primers, 3 μL sterile ddH2O, and 1 μL genomic DNA; after PCR amplification, electrophoresis is performed for verification.

[0013] Preferably, a method for efficiently synthesizing cis-1,4-polyisoprene using the aforementioned Pichia pastoris strain includes the following steps: inoculating the Pichia pastoris strain into a culture medium, inducing fermentation at 30°C and 230 rpm for 5 days, and then extracting cis-1,4-polyisoprene.

[0014] Preferably, the steps for extracting cis-1,4-polyisoprene are as follows: centrifuge the fermentation broth, suspend the precipitate in 10% NaOH, incubate in a boiling water bath for 1 hour, centrifuge, wash the precipitate with deionized water until neutral and dry, wash the precipitate with anhydrous ethanol and dry, finally extract with chloroform by ultrasonication, centrifuge, filter the supernatant, and dry to obtain cis-1,4-polyisoprene.

[0015] Preferably, the Pichia pastoris strain is used in the green production of cis-1,4-polyisoprene.

[0016] Preferably, an expression plasmid for constructing a Pichia pastoris strain is provided, wherein the expression plasmid uses pPIC3.5K as a vector and contains a methanol-inducible promoter, target genes: HRT1 gene, HRT2 gene, REF gene and / or SRPP gene, AOX1 terminator and HIS4 selection marker.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a Pichia pastoris strain capable of efficiently synthesizing cis-1,4-polyisoprene and its construction method. By heterologously expressing key genes from the Brazilian rubber tree in Pichia pastoris, a Pichia pastoris strain capable of de novo synthesis of cis-1,4-polyisoprene was successfully constructed. This strain utilizes methanol as a carbon source, conforming to the principles of green chemistry production, not relying on non-renewable resources, and avoiding the problem of insufficient rubber performance caused by the formation of trans structures in traditional petroleum-based synthesis methods. Simultaneously, by expressing the REF and SRPP genes, the yield of cis-1,4-polyisoprene was significantly increased. The strain simultaneously expressing HRT1, REF, and SRPP achieved a yield of 10.81 mg / L, 11.71 times that of the original strain. Furthermore, the biomass of the constructed mutant strain is not significantly different from that of the wild-type strain, and the introduction of exogenous genes has minimal impact on yeast growth, demonstrating good prospects for industrial application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the de novo synthesis pathway and precursor metabolism modification of cis-1,4-polyisoprene in Pichia pastoris according to the present invention.

[0020] Figure 2 The present invention relates to the Pichia pastoris CPT expression plasmid for the Brazilian rubber tree.

[0021] Figure 3 This invention relates to the identification of Pichia pastoris CPT-transformed strains. A represents the primer setup method, and B represents the PCR detection results of positive clone strains.

[0022] Figure 4 The results are obtained by gas chromatography-mass spectrometry analysis of the lysed product of the Pichia pastoris CPT transformed strain of the present invention.

[0023] Figure 5 This is the relative quantitative result of gas chromatography-mass spectrometry of the product of the Pichia pastoris CPT transformed strain of the present invention.

[0024] Figure 6 The results are obtained by gel permeation chromatography of the product of the Pichia pastoris CPT transformed strain of the present invention.

[0025] Figure 7 The present invention provides the 1H NMR spectra of wild-type Pichia pastoris, Pichia pastoris mutant strains, and standards in the range of 1.4-5.2 ppm.

[0026] Figure 8 This is the quantitative result of cis-1,4-polyisoprene from the Pichia pastoris CPT transformed strain product of the present invention.

[0027] Figure 9 This invention relates to the arrangement and combination of the REF and SRPP genes of the Brazilian rubber tree in Pichia pastoris.

[0028] Figure 10 This is the quantitative result of cis-1,4-polyisoprene in the products of the Pichia pastoris CPT, REF and SRPP transformed strains of the present invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0030] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0031] A Pichia pastoris strain for efficient synthesis of cis-1,4-polyisoprene is disclosed. The Pichia pastoris strain heterologously expresses the key gene CPT in the rubber synthesis pathway of the Brazilian rubber tree, wherein the CPT gene is either the HRT1 gene or the HRT2 gene; wherein the amino acid sequence corresponding to the nucleotide sequence of the HRT1 gene is SEQ ID NO.1, and the amino acid sequence corresponding to the nucleotide sequence of the HRT2 gene is SEQ ID NO.2.

[0032] The Pichia pastoris strain also expresses the rubber synthesis pathway genes REF and / or SRPP; the nucleotide sequence of the REF gene corresponds to the amino acid sequence of SEQ ID NO.3, and the nucleotide sequence of the SRPP gene corresponds to the amino acid sequence of SEQ ID NO.4.

[0033] The corresponding amino acid sequence encoded by the HRT1 gene in SEQ ID NO.1 is as follows: MELYNGERPSVFRLLGKYMRKGLYSILTQGPIPTHIAFILDGNRRFAKKHKLPEGGGHKAGFLALLNVLTYCYELGVKYATIYAFSIDNFRRKPHEVQYVMDLMLEKIEGMIMEESIINAYDICVRFVGNLKLLSEPVKTAADKI MRATANNSKCVLLIAVCYTSTDEIVHAVEESSELNSNEVCNNQELEEANATGSGTVIQIENMESYSGIKLVDLEKNTYINPYPDVLIRTSGETRLSNYLLWQTTNCILYSPHALWPEIGLRHVVWAVINCQRHYSYLEKHKEYLK.

[0034] SEQ ID NO.2: The amino acid sequence encoded by the HRT2 gene is as follows: MELYNGERPSVFRLLGKYMRKGLYSILTQGPIPTHIAFILDGNGRFAKKHKLPEGGGHKAGFLALLNVLTYCYELGVKYATIYAFSIDNFRRKPHEVQYVMNLMLEKIEGMIMEESIINAYDICVRFVGNLKLLDEPLKTAA DKIMRATAKNSKFVLLLAVCYTSTDEIVHAVEESSKDKLKSDEICNDGNGDCVIKIEEMEPYSEIKLVELERNTYINPYPDVLIRTSGETRLSNYLLWQTTNCILYSPHALWPEIGLRHVVWAVINCQRHYSYLEKHKEYLK.

[0035] SEQ ID NO.3: The amino acid sequence encoded by the REF gene is as follows: MAEDEDNQQGQGEGLKYLGFVQDAATYAVTTFSNVYLFAKDKSGPLQPGVDIIEGPVKNVAVPLYNRFSYIPNGALKFVDSTVVASVTIIDDRSLPPIVKDASIQVVSAIRAAPEAARSLASSLPGQTKILAKVFYGEN.

[0036] SEQ ID NO.4: The amino acid sequence encoded by the SRPP gene is as follows: MAEEVEEERLKYLDFVRAAGVYAVDSFSTLYLYAKDISGPLKPGVDTIENVVKTVVTPVYYIPLEAVKFVDKTVDVSVTSLDGVVPPVIKQVSAQTYSVAQD APRIVLDVASSVFNTGVQEGAKALYANLEPKAEQYAVITWRALNKLPLVPQVANVVVPTAVYFSEKYNDVVRGTTEQGYRVSSYLPLLPTEKITKVFGDEAS.

[0037] The Pichia pastoris strain simultaneously expresses the HRT1 gene, REF gene, and SRPP gene.

[0038] A method for constructing a Pichia pastoris strain, comprising the following steps: S1, HRT1, HRT2, REF and SRPP genes were amplified from the latex cDNA of Brazilian rubber tree; S2, using plasmid pPIC3.5K as a vector, the genes amplified in step S1 were inserted into the multiple cloning site to construct a gene expression cassette regulated by a methanol-inducible promoter. S3. The expression cassette and HIS4 selection marker constructed in step S2 are transferred into the AOX1 site of Pichia pastoris to screen for positive mutant strains, namely the Pichia pastoris strain that efficiently synthesizes cis-1,4-polyisoprene.

[0039] In step S2, when constructing the gene expression cassette, the REF gene and SRPP gene are inserted into the vector in different arrangements.

[0040] In step S3, positive mutant strains were screened by PCR detection. The PCR reaction system was 10 μL, including 5 μL 2×Hieff® PCRMasterMix, 0.5 μL upstream / downstream primers, 3 μL sterile ddH2O, and 1 μL genomic DNA. Electrophoresis was performed after PCR amplification to verify the results.

[0041] A method for efficiently synthesizing cis-1,4-polyisoprene using the aforementioned Pichia pastoris strain includes the following steps: inoculating the Pichia pastoris strain into a culture medium, inducing fermentation at 30°C and 230 rpm for 5 days, and then extracting cis-1,4-polyisoprene.

[0042] The steps for extracting cis-1,4-polyisoprene are as follows: centrifuge the fermentation broth, suspend the precipitate in 10% NaOH, incubate in a boiling water bath for 1 hour, centrifuge, wash the precipitate with deionized water until neutral and dry, wash the precipitate with anhydrous ethanol and dry, and finally extract with chloroform by ultrasonication, centrifuge, filter the supernatant, and dry to obtain cis-1,4-polyisoprene.

[0043] Application of the Pichia pastoris strain in the green production of cis-1,4-polyisoprene.

[0044] An expression plasmid for constructing Pichia pastoris strains, the expression plasmid using pPIC3.5K as a vector, comprising a methanol-inducible promoter, target genes: HRT1 gene, HRT2 gene, REF gene and / or SRPP gene, AOX1 terminator and HIS4 selection marker.

[0045] Unless otherwise specified, the test reagents and instruments used in the following examples are all available from conventional commercial sources.

[0046] One possible implementation method: I. De novo synthesis of polyisoprene from Pichia pastoris Key genes CPT, specifically HRT1 and HRT2, from the rubber synthesis pathway of *Rhizopus brevicornu* were heterologously expressed in *Pichia pastoris*. Using IPP synthesized via the endogenous MVA pathway in *Pichia pastoris*, a de novo polyisoprene synthesis pathway was constructed in *Pichia pastoris*. Furthermore, polyisoprene yield was further increased by expressing the rubber synthesis pathway genes REF and SRPP. The endogenous metabolic reactions involved are as follows: Figure 1 As shown, the rubber synthesis pathway genes HRT1 and HRT2 from the Brazilian rubber tree were transformed into Pichia pastoris, and CPT with higher efficiency was obtained by screening. Then, REF and SRPP were expressed on the selected CPT mutant to increase the yield of cis-1,4-polyisoprene in Pichia pastoris.

[0047] II. Detection of gene integration and positive mutants Gene integration method: HRT1 and HRT2 were amplified from the latex cDNA of Brazilian rubber tree. Using the commercially available plasmid pPIC3.5K as a vector, HRT1 and HRT2 were inserted into the multiple cloning site, respectively. A methanol-inducible promoter-regulated gene expression cassette was constructed. The expression cassette and HIS4 selection marker were transformed into the AOX1 site of Pichia pastoris to obtain mutant strains P7 and P8. The constructed expression plasmid is shown below. Figure 2 As shown.

[0048] Detection of mutants: Single colonies of *Pichia pastoris* were picked from screening plates, inoculated into culture medium, and cultured overnight. The cells were then collected. Genomic DNA was extracted according to the instructions of the GeneJET Genomic DNA Purification Kit K0721 (Thermo). PCR detection was performed using a Hieff® PCR Master Mix (WithDye) from Yisheng (Shanghai). The 10 μL reaction mixture included: 5 μL 2×Hieff® PCR Master Mix, 0.5 μL upstream / downstream primers, 3 μL sterile ddH2O, and 1 μL genomic DNA. PCR amplification was performed according to the reaction procedure recommended in the product instructions, and the products were verified by electrophoresis. The specific primer settings for each detection site of *Pichia pastoris* strains P7 and P8 transformed into HRT1 and HRT2, respectively, are as follows: Figure 3 As shown in Figure A, PCR was used to detect the presence of fragment junctions to determine whether the fragments were completely inserted. The gel electrophoresis results of colony PCR are shown. Figure 3 B) shows that all constructed mutant strains are positive clones, which were confirmed to be correct after sequencing and can be used for subsequent experiments.

[0049] III. Extraction of polyisoprene from Pichia pastoris After induced fermentation for 5 days, the fermentation broth was centrifuged. Each gram of precipitate was resuspended in 3 mL of 10% NaOH, incubated in boiling water for 1 hour, and then centrifuged at 16,000 rpm. The precipitate was washed with deionized water until neutral and dried at 40 °C. The precipitate was then washed with anhydrous ethanol to remove small molecules and dried again at 40 °C. 5 mL of chloroform was added to the dried sample, and ultrasonic extraction was performed for 1 hour at 40 Hz, 80% power, and 25 °C. The mixture was then centrifuged at 4,000 rpm, and the supernatant was collected in a new microcentrifuge tube. The supernatant was filtered through a 13 mm 0.22 μm nylon filter, and the dried sample was used for subsequent analysis.

[0050] IV. Pyrolysis Gas Chromatography-Mass Spectrometry Detection Cis-polyisoprene standards were purchased from Sigma-Aldridge (St. Louis, Missouri, USA). Semi-quantitative analysis of polyisoprene in the samples was performed using a Py-GC-MS system consisting of an Agilent 7890B5975CGC-MS and a Frontier 3030D pyrolysis instrument. 0.2 mg of sample was pyrolyzed at an initial furnace temperature of 25 °C, with a final temperature of 800 °C, a pyrolysis time of 0.1 seconds, and a transfer line temperature of 350 °C. Separation was performed on a DB-1 column (30 m × 0.25 mm × 0.25 m) under the following conditions: inlet temperature 300 °C, split ratio 120:1, and helium (99.999%) as carrier gas. The temperature program started at 40 °C, held for 2 minutes, then increased to 140 °C at a rate of 10 °C / min, and finally held at 20 °C. Mass spectrometry analysis was performed in scanning mode with the following parameters: ion source, EI; transmission line temperature, 300℃; ionization energy, 70 eV; ion source temperature, 230℃; quadrupole temperature, 150℃; scan range, 10–550 m / Hz. The results showed that isoprene monomers were present in both wild-type and *Pichia pastoris* strains P7 and P8 transformed with HRT1 and HRT2. Figure 4 The peak elution time was 3.37 min. However, the isoprene monomer content in the mutant strain was 4.36 and 3.49 times that of the wild type, respectively. Figure 5 This means that the expression of HRT1 and HRT2 enabled the successful synthesis of polyisoprene in Pichia pastoris.

[0051] V. GPC Analysis The molecular size distribution of yeast extracts was analyzed by GPC. Chromatography was performed at 35 °C with tetrahydrofuran as the eluent at a flow rate of 1.0 mL / min. The molecular weight of the yeast extracts was determined by comparison with major standards and by calculation using Agilent OpenLabCDSv2.7 Workstation / WSPlus and Agilent GPC / SEC. The results showed that strains expressing HRT1 and HRT2 produced significantly more molecules with molecular weights of approximately 1,000–1,200 Da compared to wild-type strains. Figure 6 This demonstrates that HRT1 and HRT2 can correctly perform CPT function and synthesize low molecular weight polyisoprene in Pichia pastoris.

[0052] VI. Quantitative analysis of cis-1,4-polyisoprene ¹H NMR spectra were recorded on a Bruker UltraShield spectrometer (500 MHz), with PEG6000 added as an internal control. Measurements were performed at 25 °C, with a total of 32 scans, obtaining ¹H NMR spectra of wild-type, mutant, and standard. Figure 7 The data were processed and analyzed using the Mestrenova, and the characteristic peaks of cis-1,4-polyisoprene were integrated and quantified. Figure 8 Expression of HRT1 and HRT2 resulted in the production of 0.99 mg / L and 0.69 mg / L of cis-1,4-polyisoprene in yeast, respectively. This demonstrates that HRT1 and HRT2 can act as CPTs in yeast and synthesize cis-1,4-polyisoprene.

[0053] REF (14.7 kDa) and SRPP (22.4 kDa) were transformed into strain P7 according to different assembly methods, thereby obtaining P701, P702 and P703 (… Figure 9 Quantitative results confirmed that SRPP expression alone had little effect on the yield of cis-1,4-polyisoprene, while the content of cis-1,4-polyisoprene was significantly increased in mutant strains expressing REF. Figure 10 This means that REF can promote the synthesis of cis-1,4-polyisoprene in Pichia pastoris. In particular, the strain expressing both REF and SRPP showed an increased cis-1,4-polyisoprene yield of 10.81 mg / L, which was 11.71 times that of the original strain.

[0054] VII. Biomass determination of mutant strains Monoclonal yeast cells were inoculated from the plate onto antibiotic-free medium and cultured overnight. They were then re-inoculated onto the medium to achieve an initial OD600 of 0.1 and continuously cultured at 30°C and 230 rpm. After 120 hours, a suitable amount of yeast culture was taken, with sterile medium used as a blank control. The absorbance of the yeast culture was measured at 600 nm using a spectrophotometer to determine the biomass. Results are as follows: Figure 5 , Figure 7 and Figure 9 The results, shown in the gray bar chart, indicate that the 120-hour biomass of all constructed Pichia pastoris mutant strains was not significantly different from that of the wild-type strain, demonstrating that the introduction of exogenous genes had little impact on yeast growth. These mutant strains can be used as production strains for polyisoprene.

[0055] This technical solution fills a research gap in the field of synthetic biology regarding the heterologous synthesis of cis-1,4-polyisoprene using microorganisms. For the first time, by heterologously expressing key genes (HRT1 and HRT2) from the Brazilian rubber tree in Pichia pastoris, a microbial strain capable of de novo synthesis of cis-1,4-polyisoprene was successfully constructed. This breaks through the limitations of traditional reliance on plant cultivation or petroleum-based chemical synthesis, providing a novel biosynthetic pathway for this key rubber raw material. Using Pichia pastoris as the chassis strain and leveraging its unique methanol (C1 compound) utilization capabilities, a sustainable C1 biomanufacturing platform is established. This platform is independent of non-renewable resources such as petroleum, mitigating the risks associated with petroleum supply fluctuations and price uncertainties. The production process eliminates the need for polluting processes such as high-temperature polymerization, avoiding the negative environmental impacts of traditional petroleum-based synthesis methods. Furthermore, it avoids the random generation of trans structures, ensuring the performance stability of rubber products from the source.

[0056] The core genes HRT1 and HRT2 can effectively exert CPT function in Pichia pastoris, achieving a yield of 0.99 mg / L and 0.69 mg / L of cis-1,4-polyisoprene, respectively. Furthermore, by introducing the combined expression of REF and SRPP genes, the yield can be increased to 10.81 mg / L, which is 11.71 times that of the original strain, significantly improving the synthesis efficiency. Pichia pastoris itself has an ultra-high cell density fermentation capacity, making it a preferred chassis for the production of high-value chemicals. Its fermentation conditions are simple and easy to scale up, which facilitates subsequent industrial mass production.

[0057] All constructed Pichia pastoris mutant strains (P7, P8, P701, P702, P703, etc.) showed no significant difference in 120h biomass compared to the wild-type strain, demonstrating that the introduction of exogenous genes did not significantly inhibit normal yeast growth. The strains possess good growth adaptability and genetic stability, and can be stably used for the large-scale production of cis-1,4-polyisoprene over a long period of time.

[0058] Verification through multi-dimensional detection using pyrolysis gas chromatography-mass spectrometry, gel permeation chromatography, and nuclear magnetic resonance hydrogen spectroscopy confirmed that the synthesized product is cis-1,4-polyisoprene with a molecular weight concentrated in the range of 1000-1200 Da and good structural uniformity. This avoids the problem of mixed product structures in traditional synthesis methods and can meet the stringent requirements of the rubber industry for the purity and performance of raw materials. It can be widely used in the production of various products such as tires and medical gloves.

[0059] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can be modified and varied in various ways. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Pichia pastoris strain for efficient synthesis of cis-1,4-polyisoprene, characterized in that, The Pichia pastoris strain heterologously expresses the key gene CPT in the rubber synthesis pathway of the Brazilian rubber tree, wherein the CPT gene is either the HRT1 gene or the HRT2 gene; wherein the amino acid sequence corresponding to the nucleotide sequence of the HRT1 gene is SEQ ID NO.1, and the amino acid sequence corresponding to the nucleotide sequence of the HRT2 gene is SEQ ID NO.

2.

2. A Pichia pastoris strain for efficient synthesis of cis-1,4-polyisoprene according to claim 1, characterized in that, The Pichia pastoris strain also expresses the rubber synthesis pathway genes REF and / or SRPP; the nucleotide sequence of the REF gene corresponds to the amino acid sequence of SEQ ID NO.3, and the nucleotide sequence of the SRPP gene corresponds to the amino acid sequence of SEQ ID NO.

4.

3. A Pichia pastoris strain for efficient synthesis of cis-1,4-polyisoprene according to claim 2, characterized in that, The Pichia pastoris strain simultaneously expresses the HRT1 gene, REF gene, and SRPP gene.

4. A method for constructing a Pichia pastoris strain, used to construct the Pichia pastoris strain as described in any one of claims 1-3, characterized in that, Includes the following steps: S1, HRT1, HRT2, REF and SRPP genes were amplified from the latex cDNA of Brazilian rubber tree; S2, using plasmid pPIC3.5K as a vector, the genes amplified in step S1 were inserted into the multiple cloning site to construct a gene expression cassette regulated by a methanol-inducible promoter. S3. The expression cassette and HIS4 selection marker constructed in step S2 are transferred into the AOX1 site of Pichia pastoris to screen for positive mutant strains, namely the Pichia pastoris strain that efficiently synthesizes cis-1,4-polyisoprene.

5. A method for constructing a Pichia pastoris strain according to claim 4, characterized in that, In step S2, when constructing the gene expression cassette, the REF gene and SRPP gene are inserted into the vector in different arrangements.

6. A method for constructing a Pichia pastoris strain according to claim 4, characterized in that, In step S3, positive mutant strains are screened by PCR detection. The PCR reaction system is 10 μL, including 5 μL 2×Hieff® PCRMasterMix, 0.5 μL upstream / downstream primers, 3 μL sterile ddH2O, and 1 μL genomic DNA. PCR amplification was followed by electrophoresis verification.

7. A method for efficiently synthesizing cis-1,4-polyisoprene, using the Pichia pastoris strain as described in any one of claims 1-3, characterized in that, The process includes the following steps: inoculating the Pichia pastoris strain into a culture medium, inducing fermentation at 30°C and 230 rpm for 5 days, and then extracting cis-1,4-polyisoprene.

8. A method for efficiently synthesizing cis-1,4-polyisoprene according to claim 7, characterized in that, The steps for extracting cis-1,4-polyisoprene are as follows: centrifuge the fermentation broth, suspend the precipitate in 10% NaOH, incubate in a boiling water bath for 1 hour, centrifuge, wash the precipitate with deionized water until neutral and dry, wash the precipitate with anhydrous ethanol and dry, and finally extract with chloroform by ultrasonication, centrifuge, filter the supernatant, and dry to obtain cis-1,4-polyisoprene.

9. The application of any one of the Pichia pastoris strains described in claims 1-3 in the green production of cis-1,4-polyisoprene.

10. An expression plasmid for constructing any one of the Pichia pastoris strains according to claims 1-3, characterized in that, The expression plasmid uses pPIC3.5K as a vector and contains a methanol-inducible promoter, target genes: HRT1 gene, HRT2 gene, REF gene and / or SRPP gene, AOX1 terminator and HIS4 selection marker.

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