High-yield GPP / FPP saccharomyces cerevisiae engineering strain as well as construction method and application thereof

Abstract

The invention provides a high-yield GPP / FPP saccharomyces cerevisiae engineering strain as well as a construction method and application thereof, and relates to the technical field of synthetic biology. Through a gene editing technology, heterologous mvaE and mvaS genes are introduced into saccharomyces cerevisiae, endogenous ERG12, ERG8, ERG19 and ERG20 genes are over-expressed, and the metabolic flux of an MVA pathway in saccharomyces cerevisiae cells is enhanced; meanwhile, a positioning signal tag of peroxisome is added to the gene, and an MVA pathway and a terpenoid precursor synthesis pathway are reconstructed in the peroxisome, so that the compartment synthesis of the terpenoid precursor is realized. Besides, a heterologous MthIPKs gene is further introduced, a branch pathway for IPP synthesis is constructed, higher energy efficiency and less enzymatic reactions are achieved, an MVA pathway and metabolic flux for terpenoid synthesis are concentrated, and the biosynthesis efficiency and conversion rate are further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of synthetic biology, and in particular to an engineering strain of high-yield GPP / FPP brewer's yeast, and a construction method and application thereof. Background Art

[0002] Terpenoids are extremely diverse in chemical structure, including more than 180,000 different substances. They are widely distributed in animals, plants, insects and microorganisms, and have diverse physical, chemical and physiological functions. They play an important role in the fields of spices, food, medicine, health products, cosmetics and bioenergy. The traditional method of extracting terpenoids from natural sources has the disadvantages of low yield, high cost and long cycle, making it difficult to achieve large-scale application. With the advancement of synthetic biology, the use of microorganisms to synthesize terpenoids has become a new trend.

[0003] Saccharomyces cerevisiae is the simplest model eukaryotic microorganism with advantages such as clear genetic background, high recombination efficiency and simple genetic manipulation. The high-yield artemisinic acid strain of Keasling's team confirmed the strong ability of Saccharomyces cerevisiae to synthesize terpenoids and is an ideal chassis cell for the synthesis of terpenoids. Saccharomyces cerevisiae synthesizes the common precursors IPP and DMAPP of terpenoids through the mevalonic acid (MVA) pathway. This pathway is a common terpenoid precursor synthesis pathway for eukaryotes, archaea and a small number of bacteria. The flux of synthetic precursors and intermediates generated by the naturally existing MVA pathway of Saccharomyces cerevisiae is far from enough to meet the needs of large-scale synthesis of terpenoids, and chassis cells with high precursor production are urgently needed. Since the endogenous MVA pathway of yeast exists in the cytoplasm, there are a large number of competing pathways for the substrates and products of the MVA pathway, which has a strong metabolic crosstalk effect; at the same time, some enzymes, cofactors and metabolites related to the synthesis of terpenoids are distributed in different subcellular compartments, and there are large spatial barriers, which seriously affect the synthesis efficiency of the target product; in addition, the intermediates or by-products produced in the metabolic pathway of terpenoids accumulate in the cytoplasm, directly causing serious interference to cell growth.

[0004] In recent years, researchers have begun to try to reconstruct the MVA pathway in mitochondrial organelles. Although mitochondrial engineering strategies can achieve compartmentalized synthesis and improve efficiency, they are prone to cause metabolic burden and cell apoptosis. Therefore, it is urgent to explore new organelle engineering strategies to overcome the above limitations. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a high-GPP / FPP-yielding Saccharomyces cerevisiae engineered strain and its construction method and application, and to reconstruct and optimize the MVA pathway in the peroxisome of Saccharomyces cerevisiae through gene editing technology to achieve efficient and compartmentalized synthesis of terpenoid compounds.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a method for constructing a high-yield GPP / FPP Saccharomyces cerevisiae engineered strain, the method comprising the following steps:

[0008] S1. Overexpression of MVA pathway, introduction of heterologous mvaE and mvaS genes into Saccharomyces cerevisiae, design and construct mvaE gene expression cassette EfmvaE and mvaS gene expression cassette EfmvaS, assemble EfmvaE and EfmvaS gene expression cassettes into vector pMYT039, and obtain recombinant vector M3_ConS-EfMvaS-EfMvaE-Con1_pMYT039;

[0009] S2, overexpressing the IPP synthesis pathway, introducing the heterologous MthIPK encoding gene MTH47 into Saccharomyces cerevisiae, designing and constructing the ERG12, ERG8, ERG19 and MTH47 gene expression cassettes, assembling the ERG12 and ERG8 gene expression cassettes into the vector pMYT041, assembling the ERG19 and MTH47 gene expression cassettes into the vector pMYT043, and obtaining the recombinant vectors M3_Con1-ERG12-ERG8-Con2_pMYT041 and M3_Con2-ERG19-IPK-Con3_pMYT043;

[0010] S3, overexpression of GPP / FPP synthesis pathway, design and construction of IDI1 and ERG20 gene expression cassettes, assembly into vector pMYT044, and obtain recombinant vector M3_Con3-IDI1-ERG20-ConE_pMYT044;

[0011] S4, adding the peroxisome localization signal PTS PX1 to the gene expression frame constructed in steps S1, S2, and S3 to obtain a recombinant vector with a localization signal tag;

[0012] S5, constructing an integration plasmid, using EcoRV single restriction enzyme to digest the vector pZX8-Int.7, using NotI endonuclease to digest the recombinant vectors obtained in steps S1, S2, S3, and S4, dividing them into 3 groups and co-transforming them with the linearized vector pZX8-Int.7 into Saccharomyces cerevisiae cells, screening positive clones by PCR and sequencing to obtain an integration plasmid for the full GPP / FPP production pathway;

[0013] S6. Linearize the integration plasmid and transfer it into Saccharomyces cerevisiae cells, screen the positive integration strain clones by PCR and sequence them to obtain the Saccharomyces cerevisiae engineering strain with high GPP / FPP production.

[0014] Preferably, in step S4, the peroxisome localization signal PTS PX1 is added to the C-terminus of the gene expression frame.

[0015] Preferably, the MVA pathway in step S1 comprises a fusion protein expression cassette.

[0016] Preferably, the fusion protein expression frame utilizes a 2×GGGGS flexible protein linker to connect the EfmvaE and EfmvaS genes and assemble them into the vector pMYT039.

[0017] Preferably, the GPP / FPP synthesis pathway in step S3 comprises a fusion protein expression cassette.

[0018] Preferably, the fusion protein expression frame uses a 2×GGGGS flexible protein linker to connect IDI1 and ERG20 genes and assemble them into the vector pMYT044.

[0019] In a second aspect, the present invention provides a high-yield GPP / FPP Saccharomyces cerevisiae engineered strain, wherein the Saccharomyces cerevisiae engineered strain is constructed by the construction method described in the first aspect.

[0020] In a third aspect, the present invention provides the use of the engineered strain of Saccharomyces cerevisiae described in the second aspect in the large-scale production of terpenoid compound precursor GPP / FPP.

[0021] Beneficial effects of the present invention:

[0022] 1. The present invention can achieve compartmentalized synthesis of terpenoid compound precursors by reconstructing the MVA pathway, IPP synthesis pathway and GPP / FPP synthesis pathway in the peroxisome organelles of Saccharomyces cerevisiae strains, thereby greatly increasing the yield of the precursor GPP / FPP.

[0023] 2. The present invention shortens the spatial distance between different enzymes through fusion protein technology, and realizes sequential catalytic activity in a smaller space; it not only shortens the metabolite transfer process and increases the reaction rate, but also effectively reduces the loss of intermediates in the competitive pathway and improves the conversion efficiency of the target pathway, thereby further increasing the yield of terpenoid precursor GPP / FPP in peroxisomes.

[0024] 3. The present invention constructs a branch pathway for IPP synthesis by introducing heterologous MthIPKs, which has higher energy efficiency and fewer enzymatic reactions, concentrates the metabolic flow of the MVA pathway and terpenoid synthesis, and improves the efficiency and conversion rate of biosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Schematic diagram of the modification of the GPP / FPP pathway for enhanced synthesis of terpenoid precursors in Saccharomyces cerevisiae cells.

[0026] Figure 2 Schematic diagram of the expression cassettes of acetyl-CoA acyltransferase / HMG-CoA synthetase (mvaE) and mevalonate synthase (mvaS).

[0027] Figure 3 Schematic diagram of the expression frames of ERG12, ERG8, ERG19 and MTH47 genes in the IPP overexpression pathway.

[0028] Figure 4 Schematic diagram of the expression frames of IDI1 and ERG20 genes in the GPP / FPP overexpression pathway.

[0029] Figure 5 Schematic diagram of the recombinant protein expression frame of the peroxisome localization signal (PTS), taking the EfmvaE protein as an example.

[0030] Figure 6 Schematic diagram of the expression frame for functional protein fusion transformation.

[0031] Figure 7 This is the PCR identification result of the recombinant strain of Saccharomyces cerevisiae that highly expresses GPP / FPP.

[0032] Figure 8 This is a sequence alignment result of the recombinant strain of Saccharomyces cerevisiae that highly expresses GPP / FPP.

[0033] Fig. 9 This is a comparison of lycopene production results of recombinant strains of Saccharomyces cerevisiae that highly express GPP / FPP. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0035] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0036] The primer sequences used in the examples are shown in Table 1:

[0037] Table 1

[0038]

[0039]

[0040] Example 1: Overexpression of the MVA pathway

[0041] HMGR is the main rate-limiting enzyme in Saccharomyces cerevisiae. Researchers have increased the number of copies of the HMGR gene or mutated the HMGR gene to remove feedback inhibition to enhance the synthesis of MVA. In addition, heterologous MVA pathways or isozymes of the MEP pathway from bacteria or archaea also provide more options and possibilities for overexpression of the MVA pathway. The two genes mvaE and mvaS from Enterococcus faecalis can not only complete the conversion of acetyl-CoA to MVA that can only be completed by the three genes of Saccharomyces cerevisiae ERG10, ERG13 and HMGR, but also do not have the feedback inhibition of HMGR.

[0042] The DNA sequences of mvaE and mvaS were downloaded from NCBI, codons were optimized for the host Saccharomyces cerevisiae, and the expression cassettes of the two genes were redesigned and constructed, such as Figure 1 As shown. The segmentation scheme of two complete expression frames was designed using internal software, overlapping single-stranded oligonucleotides were synthesized, and the full length was spliced ​​by overlap extension PCR. Using the PCR product as a template, the two gene expression frames were amplified using primers EfmvaE-F / EfmvaE-R and EfmvaS-F / EfmvaS-R, respectively, and the PCR products were recovered as DNA fragments for subsequent cloning and assembly. The EfmvaE and EfmvaS fragments and plasmid pMYT039 were mixed in a ratio of 2:2:1, BsaI, T4 ligase and buffer were added, and 20ul of Golden Gate reaction system was prepared. The reaction was carried out at 37℃ for 1min, 16℃ for 1min, and 60℃ for 5min after 30 cycles according to the following procedure. After the reaction, add 100ul of E. coli competent cells to the mixture, place on ice for 30min, heat shock at 42℃ for 30s, place on ice again for 5min, add 1ml of preheated LB culture medium, incubate at 37℃ for 60min, apply 100ul of bacterial solution to Amp-resistant LB plate, and culture at 37℃ overnight. The next day, PCR screened positive clones and prepared plasmids for sequencing verification. The final correct clone was named M3_ConS-EfMvaS-EfMvaE-Con1_pMYT039.

[0043] Example 2: Overexpression of IPP synthesis pathway

[0044] The endogenous MVA to IPP conversion process in yeast includes three functional enzymes: MVA kinase (MK), MVA-5-P kinase (PMK) and MVA-PP decarboxylase (DPMD), which are encoded by ERG12, ERG8 and ERG19 genes, respectively. In addition, in addition to the specific catalytic activity of MVA-PP to IPP, DPMD also has a non-specific activity of partially catalyzing the conversion of MVA-P to isopentenyl phosphate (IP), resulting in partial leakage of metabolic flux. In recent years, through genomic research, researchers have discovered that some archaea have alternative pathways for terpenoid compounds other than conventional MVA and MEP, among which IPKs have the functional activity of using IP and DMAP to generate IPP and DMAPP. The present invention introduces the heterologous MthIPK encoding gene MTH47, thereby reducing the loss of this part of the metabolic flux.

[0045] The sequences of the above four genes ERG12, ERG8, ERG19 and MTH47 were also downloaded from NCBI, codons were optimized for the host Saccharomyces cerevisiae, and the expression cassettes of the four genes were redesigned and constructed, such as Figure 3 As shown. ERG12 and ERG8 were assembled into a vector pMYT041, and ERG19 and MTH47 were assembled into another vector pMYT043. The specific operation method and process were the same as in Example 1, and the primer pairs were ERG12-F / ERG12-R, ERG8-F / ERG8-R, ERG19-F / ERG19-R and MTH47-F / MTH47-R to amplify each fragment. The final correct clones were named M3_Con1-ERG12-ERG8-Con2_pMYT041 and M3_Con2-ERG19-IPK-Con3_pMYT043, respectively.

[0046] Example 3: Overexpression of GPP / FPP synthesis pathway

[0047] The conversion of endogenous IPP to GPP / FPP in yeast involves two functional enzymes: IPP generates isomers of DMAPP under the catalysis of isomerase IDI. One molecule of DMAPP polymerizes different numbers of IPP molecules under the catalysis of the corresponding isoprene pyrophosphate synthase to form geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP), which are encoded by IDI1 and ERG20 genes, respectively.

[0048] The sequences of the above two genes IDI1 and ERG20 were downloaded from NCBI, codons were optimized for the host Saccharomyces cerevisiae, and the expression cassettes of the two genes were redesigned and constructed, such as Figure 4As shown. The IDI1 and ERG20 gene expression cassettes were assembled into the vector pMYT044. The specific operation method and process were the same as in Example 1. The primer pairs IDI1-F / IDI1-R and ERG20-F / ERG20-R were used to amplify each fragment. The final correct clone was named M3_Con3-IDI1-ERG20-ConE_pMYT044.

[0049] Example 4: Addition of peroxisome targeting signal (PTS)

[0050] All the above proteins are not added with localization signals and are distributed in the cytoplasm after expression. In order to concentrate the entire pathway in the peroxisome, the present invention adds PTS PX1 derived from the PEX8 gene to each protein.

[0051] The gene sequence of the PX1 signal peptide is TCCAAGCTG, which is added to the C-terminus of the above protein by PCR. Each protein (taking EfmvaE as an example) is amplified with two pairs of primers EfmvaE-F / EfmvaE-SKL-R and EfmvaE-SKL-F / EfmvaE-R, and then the full-length fragment is amplified using the head and tail primers EfmvaE-F / EfmvaE-R. The primers for the remaining proteins are similar to those above, and the specific operation method and assembly process are the same as in Example 1. The final correct clones were named M3_ConS-EfMvaS-PX1-EfMvaE-PX1-Con1_pMYT039, M3_Con1-ERG12-PX1-ERG8-PX1-Con2_pMYT041, M3_Con2-ERG19-PX1-IPK-PX1-Con3_pMYT043, and M3_Con3-IDI1-PX1-ERG20-PX1-ConE_pMYT044.

[0052] Example 5: Construction of fusion protein expression cassette

[0053] The present invention selects the key functional enzymes of the MVA pathway and the GPP / FPP synthesis pathway for protein fusion modification, shortens the spatial distance between the functional enzymes of the metabolic pathway, shortens the reaction distance, and improves the reaction efficiency. In this example, the flexible protein linker of 2×GGGGS is used to connect EfmvaE and EfmvaS, and assembles them into the vector pMYT039; similarly, the 2×GGGGS protein linker is used to connect IDI1 and ERG20 proteins, and assembles them into the vector pMYT044. The schematic diagram of the two fusion proteins is shown in Figure 2. Figure 6 shown.

[0054] Each gene fragment was amplified by PCR and the DNA sequence of 2×GGGGS protein linker was added, and the full-length sequence of the expression frame was obtained in two rounds of amplification. Taking EfmvaE as an example, the fragments were amplified with 2 pairs of primers EfmvaE-F / EfmvaE-linker-R and EfmvaS-linker-F / EfmvaS-R, and then the full-length fragment was amplified using the head and tail primers EfmvaE-F / EfmvaS-R. The primers for the remaining proteins are similar to those above, and the specific operation methods and assembly processes are the same as in Example 1. The final correct clones were named M3_ConS-EfMvaS-PX1 fusing EfMvaE-PX1-Con1_pMYT039 and M3_Con3-IDI1-PX1 fusingERG20-PX1-ConE_pMYT044.

[0055] Example 6: Complete pathway for assembling terpenoid precursor GPP / FPP

[0056] In order to achieve the conversion of yeast from acetyl-CoA to terpenoid precursors GPP / FPP, this example assembled all the above pathways into one vector to facilitate subsequent gene editing operations. This example assembled the above complete pathways through the transformation associated recombination (TAR) technology of Saccharomyces cerevisiae.

[0057] The assembly vector pZX8-Int.7 was constructed with reference to the design in the MYT yeast toolkit of William et al. Using pMYT081 as a template, primers Int.7-5HA-F / Int.7-5HA-R and Int.7-3HA-F / Int.7-3HA-R were used to amplify the two fragments of 5HA and 3HA respectively, and then Int.7-5HA-F / Int.7-3HA-R were used to splice into one fragment Int.7-HA. The PCR product was purified and recovered by agarose gel as a DNA fragment for subsequent cloning and assembly. Int.7-HA was mixed with the linearized pZX8 plasmid in a ratio of 5:1, the volume was supplemented to 10ul with sterile water, and then 10ul of Gibson reaction solution was added to prepare 20ul of Gibson reaction solution, and the reaction was carried out at a constant temperature of 50°C for 45min. After the reaction was completed, the mixture was added to 100ul of Escherichia coli competent cells for transformation, and the specific method process was the same as in Example 1. The above correct clone plasmid and plasmid pMYT035 were subjected to Golden Gate reaction, and the specific method and process were the same as in Example 1. The final correct clone was named pZX8-Int.7.

[0058] The plasmids M3_ConS-EfMvaS-EfMvaE-Con1_pMYT039, M3_Con1-ERG12-ERG8-Con2_pMYT041, M3_Con2-ERG19-IPK-Con3_pMYT043, M3_Con3-IDI1-ERG20-ConE_pMYT044, M3_Con S-EfMvaS-PX1-EfMvaE-PX1-Con1_pMYT039, M3_Con1-ERG12-PX1-ERG8-PX1-Con2_pMYT041, M 3_Con2-ERG19-PX1-IPK-PX1-Con3_pMYT043, M3_Con3-IDI1-PX1-ERG20-PX1-ConE_pMYT044,

[0059] M3_ConS-EfMvaS-PX1 fusing EfMvaE-PX1-Con1_pMYT039 and M3_Con3-IDI1-PX1 fusing ERG20-PX1-ConE_pMYT044, the target band was recovered by agarose gel electrophoresis, and the DNA fragment of the target gene was purified by nucleic acid column. EcoRV single restriction enzyme digestion vector pZX8-Int.7, the DNA fragment of the vector was purified by nucleic acid column. The above target genes were divided into three groups and co-transfected into Saccharomyces cerevisiae cells with the linearized vector pZX8-Int.7, specifically grouped as (1) M3_ConS-EfMvaS-EfMvaE-Con1_pMYT039, M3_Con1-ERG12-ERG8-Con2_pMYT041, M3_Con2-ERG19-IPK-Con3_pMYT043 and M3_Con3-IDI1-ERG20-ConE_pMYT044, (2) M 3_ConS-EfMvaS-PX1-EfMvaE-PX1-Con1_pMYT039, M3_Con1-ERG12-PX1-ERG8-PX1-Con2_pMYT041, M3_Con2-E RG19-PX1-IPK-PX1-Con3_pMYT043 and M3_Con3-IDI1-PX1-ERG20-PX1-ConE_pMYT044, (3)M3_ConS-EfMvaS-PX1 fusing EfMvaE-PX1-Con1_pMYT039, M3_Con1-ERG12-PX1-ERG8-PX1-Con2_pMYT041, M3_Con2-ERG19-PX1-IPK-PX1-Con3_pMYT043 and M3_Con3-IDI1-PX1 fusing ERG20-PX1-ConE_pMYT044. Yeast competent cell preparation and transformation methods are as follows:

[0060] Preparation of competent yeast: Take yeast cells VL6-48 frozen at -80℃ and streak them on a sterile YPD plate for activation, and culture at 30℃ for 36-48h; when a single colony visible to the naked eye appears on the plate, extract the single colony and inoculate it into a sterile liquid YPD medium, and culture it at 30℃ overnight; inoculate the bacterial solution into 50ml fresh YPD medium, and the initial OD600 of the fresh bacterial solution is about 0.4-0.5; place it at 30℃, 220rpm and culture it at a constant temperature until the OD600 of the culture solution reaches 0.9-1.0. Transfer the bacterial solution to a centrifuge tube, and collect the yeast cells by centrifugation at 4000rpm for 10min; resuspend and wash the cells with 0.5 times the volume (25mL) of sterile water, and centrifuge at 4000rpm for 10min to collect the yeast cells; repeat the above steps once; resuspend the yeast cells with 25mL of 0.1M LiAc and 0.25ml of 1M DTT solution, and culture at 30℃ and 100rpm for 1h; collect the yeast cells by centrifugation at 4000rpm for 10min; resuspend and wash the cells with 25mL of 1M sorbitol solution, and collect the yeast cells by centrifugation at 4000rpm for 10min; discard the supernatant, resuspend the cells with the remaining sorbitol, and dispense the cells into 1.5ml centrifuge tubes at a specification of 100ul / tube, which are yeast competent cells.

[0061] The yeast transformation method is as follows: prepare the transformation system according to Table 2:

[0062] Table 2

[0063] Element Volume(ul) PEG3350 (50%) 520 1MLiAc 72 ssDNA (2mg / ml) 80 DNA 48 Total volume 720

[0064] The competent cells were centrifuged at 4000rpm for 2min, and the supernatant was removed; the mixture prepared in the previous step was added, the yeast cells were resuspended by pipetting, and the mixture was mixed by inversion; incubated at 42℃ for 40min, and the mixture was mixed by inversion every 10min; placed on ice for 5min; centrifuged at 4000rpm for 2min, and the supernatant was discarded to collect the yeast cells; 6ml YPD culture medium was added and cultured at 30℃ overnight; the yeast cells were centrifuged at 4000rpm for 2min to collect the yeast cells; the cells were resuspended in sterile water and then spread on SC-His selective plates; cultured at 30℃ for 48h, a single colony was picked and inoculated into fresh SD-His liquid culture medium for culture, centrifuged after 2 days, and the bacteria were resuspended with appropriate amount of sterile water, and PCR was used to screen positive clones. The yeast plasmid was extracted and transformed into E. coli DH5α, and the positive clones were screened by PCR and sequenced in the same way. The final correct clones were named pZX8-Int.7-M3, pZX8-Int.7-M3-PX1 and pZX8-Int.7-M3-PX1-Up_DownFusion.

[0065] Example 7: Construction of Saccharomyces cerevisiae strains that highly express GPP / FPP

[0066] Plasmids pZX8-Int.7-M3, pZX8-Int.7-M3-PX1 and pZX8-Int.7-M3-PX1-Up_DownFusion were digested with NotI endonuclease, the target bands were recovered by agarose gel electrophoresis, and the DNA fragments of the target pathway were purified by nucleic acid column. The present invention is based on the principle of homologous recombination to perform genome editing and transformation of yeast strains. Int.7-5HA and Int.7-3HA described above are used as recombination arms for homologous recombination, and G418 is used as a screening marker for the recombinant strains after genetic modification. Saccharomyces cerevisiae BY4741 was used as the starting strain, and the preparation and transformation of Saccharomyces cerevisiae competence were performed according to the method flow of Example 6. 500fmol of the DNA fragments of the target pathway were added to the transformation system and transformed according to the method of Example 6. After overnight incubation, 100uL of the resuspended cells were drawn and coated on a G418-resistant YPD plate. The culture medium for subsequent screening and other steps is also G418-resistant YPD. PCR screening was performed using two pairs of primers: Int.75'HA-JJFP / Int.75'HA-JJRP and Int.73'HA-JJFP / Int.73'HA-JJRP. Figure 7 Three clones were randomly selected for each treatment and the sequences were verified by Sanger sequencing. The sequence alignment results (partial) are shown in Figure 8 Finally, the correct recombinant strains were obtained in each treatment and named as HXSBD-TP-1, HXSBD-TP-2 and HXSBD-TP-3.

[0067] Example 8: Construction of lycopene synthesis pathway

[0068] Lycopene is the main pigment of ripe tomatoes and a kind of carotenoid. It is a simple terpenoid compound, with FPP as substrate, and lycopene can be synthesized only through the catalytic action of three functional proteins: GGPP synthase, phytoene synthase and phytoene dehydrogenase. At the same time, lycopene, as a natural pigment, presents a red color visible to the naked eye and has a characteristic absorption peak at 474nm. Therefore, this embodiment uses lycopene as an important characterization indicator of the concentration of terpenoid precursor GPP / FPP. Using pESC-His as an expression vector, crtE (encoding GGPP synthase), crtB (encoding phytoene synthase) and crtI (encoding phytoene dehydrogenase) are assembled into a complete plasmid step by step. The DNA sequences of the three functional genes crtE, crtB and crtI are synthesized by our company Hongxun Biology. In the first round, the pESC-His plasmid was digested with SacI / SpeI, and the DNA fragment of the vector was purified by a nucleic acid column; the crtB gene and the linearized pESC-His were subjected to Gibson homologous recombination at a ratio of 5:1, and the specific experimental methods and procedures were the same as in Example 6. In the second round, the pESC-crtB plasmid from the previous round was digested with BamHI / SalI, and the DNA fragment of the vector was purified by a nucleic acid column; the crtI gene was assembled onto the vector by Gibson homologous recombination, and the specific experimental methods and procedures were the same as above. In the third round, the pESC-crtB-crtI plasmid from the previous round was digested with XhoI / SacII, and the DNA fragment of the vector was purified by a nucleic acid column; the final crtE gene was assembled onto the vector, and the specific experimental methods and procedures were the same as above. The final correct clones were named pESC-crtEBI.

[0069] Example 9: Lycopene production test in different recombinant strains of Saccharomyces cerevisiae

[0070] According to the method and process of Example 6, the competent cells of Saccharomyces cerevisiae were prepared and transformed, and the lycopene synthesis plasmid pESC-crtEBI was transformed into the high-yield GPP / FPP Saccharomyces cerevisiae recombinant strains HXSBD-TP-1, HXSBD-TP-2 and HXSBD-TP-3. The positive single clone was picked and inoculated into 4 ml of His medium, and cultured at 30°C for 48 h as seed liquid; then, 250 μL of seed liquid was inoculated into 25 ml of His medium, and the culture container was 250 ml, and the culture was cultured at 30°C and 220 rpm for 3 days.

[0071] The lycopene synthesized by fermentation was extracted by acetone extraction, and then the lycopene was detected by ultraviolet spectrophotometry. The bacterial solution was transferred to a centrifuge cup, centrifuged at 11340*g for 10 minutes, and the supernatant was discarded, being careful not to disturb the cell pellet. The bacteria were then washed with pre-cooled sterile deionized water, centrifuged at 11340*g for 10 minutes, the supernatant was discarded, being careful not to disturb the cell pellet, and the washing was repeated twice. Lycopene was extracted with acetone under dark conditions, and the extraction reaction was carried out at 55°C for 15 minutes to ensure that the bacteria turned from red to white. Subsequently, the sample was centrifuged at 11340*g for 10 minutes to remove the bacterial cells. The lycopene content in the extract was determined using an HPLC system (Shimadzu LC-20AT), which was equipped with a C18 column and a UV / VIS detector with a detection wavelength of 474nm. The mobile phase was acetonitrile-methanol-isopropanol (volume ratio of 50:30:20), the flow rate was 1 mL / min, and the column temperature was 40 °C.

[0072] The results are as follows Fig. 9 As shown in the figure, the lycopene production of the HXSBD-TP-1 recombinant strain overexpressing the complete GPP / FPP pathway in the cytoplasm was only 1.46 mg / g DCW, while the lycopene production of the HXSBD-TP-2 recombinant strain overexpressing the complete GPP / FPP pathway in the peroxisome was 5.61 times that of the HXSBD-TP-1 strain, and the lycopene production of the HXSBD-TP-3 recombinant strain modified with fusion protein was 1.45 times that of the HXSBD-TP-2 strain. After two-factor statistical analysis, the lycopene production of different recombinant strains was significantly different.

[0073] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for constructing a high-yield GPP / FPP Saccharomyces cerevisiae engineered strain, characterized in that: The method comprises the following steps: S1. Overexpression of MVA pathway, introduction of heterologous mvaE and mvaS genes into Saccharomyces cerevisiae, design and construct mvaE gene expression cassette EfmvaE and mvaS gene expression cassette EfmvaS, assemble EfmvaE and EfmvaS gene expression cassettes into vector pMYT039, and obtain recombinant vector M3_ConS-EfMvaS-EfMvaE-Con1_pMYT039; S2, overexpressing the IPP synthesis pathway, introducing the heterologous MthIPK encoding gene MTH47 into Saccharomyces cerevisiae, designing and constructing the ERG12, ERG8, ERG19 and MTH47 gene expression cassettes, assembling the ERG12 and ERG8 gene expression cassettes into the vector pMYT041, assembling the ERG19 and MTH47 gene expression cassettes into the vector pMYT043, and obtaining the recombinant vectors M3_Con1-ERG12-ERG8-Con2_pMYT041 and M3_Con2-ERG19-IPK-Con3_pMYT043; S3, overexpression of GPP / FPP synthesis pathway, design and construction of IDI1 and ERG20 gene expression cassettes, assembly into vector pMYT044, and obtain recombinant vector M3_Con3-IDI1-ERG20-ConE_pMYT044; S4, adding the peroxisome localization signal PTS PX1 to the gene expression frame constructed in steps S1, S2, and S3 to obtain a recombinant vector with a localization signal tag; S5, constructing an integration plasmid, using EcoRV single restriction enzyme to digest the vector pZX8-Int.7, using NotI endonuclease to digest the recombinant vectors obtained in steps S1, S2, S3, and S4, dividing them into 3 groups and co-transforming them with the linearized vector pZX8-Int.7 into Saccharomyces cerevisiae cells, screening positive clones by PCR and sequencing to obtain an integration plasmid for the full GPP / FPP production pathway; S6. Linearize the integration plasmid and transfer it into Saccharomyces cerevisiae cells, screen the positive integration strain clones by PCR and sequence them to obtain the Saccharomyces cerevisiae engineering strain with high GPP / FPP production.

2. The construction method according to claim 1, characterized in that: In step S4, the peroxisome localization signal PTSPX1 is added to the C-terminus of the gene expression frame.

3. The construction method according to claim 1, characterized in that: The MVA pathway in step S1 comprises a fusion protein expression cassette.

4. The construction method according to claim 3, characterized in that: The fusion protein expression frame uses a 2×GGGGS flexible protein linker to connect the EfmvaE and EfmvaS genes and assemble them into the vector pMYT039.

5. The construction method according to claim 1, characterized in that: The GPP / FPP synthesis pathway in step S3 comprises a fusion protein expression cassette.

6. The construction method according to claim 5, characterized in that: The fusion protein expression frame uses a 2×GGGGS flexible protein linker to connect IDI1 and ERG20 genes and assemble them into the vector pMYT044.

7. A high-yield GPP / FPP brewer's yeast engineered strain, characterized in that: The engineered strain of Saccharomyces cerevisiae is constructed by the construction method according to any one of claims 1 to 6.

8. Use of the engineered strain of Saccharomyces cerevisiae as claimed in claim 7 in the large-scale production of terpenoid precursor GPP / FPP.

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