Recombinant strain for producing beta-farnesene and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a recombinant strain for producing beta-farnesene as well as a construction method and application of the recombinant strain. According to the present invention, the beta-farnesene synthase MacbFS variant gene derived from chamomile is integrated to four sites such as YPRCDelta 15, YML059C, YDR448W and YIL009C, or is integrated to four sites such as YPRCDelta 15, YML059C, YBR128C and YHR178W, such that the recombinant strain with the high yield of beta-farnesene is obtained; experiments show that the combination of the sites has better heterologous gene expression potential, the yield of the beta-farnesene is obviously increased, the shake flask yield is up to 882.88-1054.10 mg / L, and a new way is provided for biosynthesis of the beta-farnesene.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a recombinant strain producing beta-farnesene and application thereof. Background Art

[0002] Farnesene includes two isomers, α-farnesene and β-farnesene, and was first discovered in apple peel in nature. As a chemical signal molecule, farnesene plays an important role in plant pollination, apple seed dispersal and plant defense. In the food industry, farnesene is widely used in the production of seasonings and spices, providing food with unique flavor and aroma. In the pharmaceutical field, farnesene is also used as an important intermediate in drug synthesis, with pharmacological activities such as antibacterial, anti-inflammatory and anti-tumor. Due to its suitable cetane number, density and low cloud point, farnesene is considered to be a potential biofuel and chemical raw material to promote the development of renewable energy. At the same time, after a simple hydrogenation reaction, it can generate high-energy fuel farnesane. The flash point of this biofuel is more than 100°C and it is less dangerous; the net heat of combustion is higher than that of existing military fuels, exceeding 44MJ / kg; compared with other fuels, it can reduce greenhouse gas emissions by 82%. Therefore, it has great potential as a jet fuel substitute and a high-performance polymer precursor, and is in high demand in industrial production (see Figure 1 ). In recent years, some researchers have envisioned a process of obtaining farnesyl acetone from farnesene and then converting it into isophylline alcohol through hydrogenation and addition reactions. In this process, the three-step reaction of farnesene to isophylline alcohol can simplify the synthesis process and reduce the use of expensive raw materials, which proves that the new method of synthesizing isophylline alcohol from farnesene obtained by microbial fermentation has broad application prospects in the production of vitamin E.

[0003] 1. Current status of β-farnesene biosynthesis

[0004] The research on biosynthesis of β-farnesene by metabolically engineered microbial hosts has received extensive attention. Compared with traditional chemical synthesis methods, biosynthesis of farnesene has some significant advantages. In addition to being able to effectively avoid complex chemical reaction steps and reduce the production of difficult-to-separate byproducts, biosynthesis can also utilize biological systems such as microorganisms or plants to directly synthesize farnesene through metabolic pathways, regulate the expression of related genes in the organism, achieve directional synthesis of β-farnesene, and improve the yield and purity of β-farnesene.

[0005] In the past few years, the biosynthesis of β-farnesene has been successfully achieved through host platforms such as Escherichia coli, Bacillus cerevisiae and Yarrowia lipolytica, and continuous exploration has been made to further increase its biosynthetic yield and realize the industrial scale of biosynthesis of high value-added product β-farnesene.

[0006] The biosynthesis of β-farnesene is mainly catalyzed by plant-derived β-farnesene synthase to convert the important precursor farnesyl pyrophosphate (FPP). In the Saccharomyces cerevisiae system, farnesyl diphosphate (FPP) is condensed from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through the mevalonate (MVA) pathway. Afterwards, FPP is further catalyzed to produce β-farnesene by heterologously introduced β-farnesene synthase. The synthetic metabolic pathways of β-farnesene are as follows: Figure 2 shown.

[0007] 2.β-Farnesene synthase copy number optimization strategy

[0008] In industrial production, integrating heterologous genes into the genome of the host organism can achieve more stable expression of the enzyme catalytic effect than the shuttle plasmid expression system. Therefore, for the construction of production strains, the modification of heterologous genes in the host strain is achieved through chromosomal integration. The copy number of the heterologous catalytic enzyme, β-farnesene synthase, in the host cell genome has different effects on the product yield.

[0009] Regarding the biosynthesis of β-farnesene, the fermentation results still have room for improvement in the biosynthesis of similar sesquiterpene natural products, and the maximum utilization of the metabolic pathway flux of the host strain has not been achieved. In order to achieve stable expression of heterologous genes in host cells, the nutritional deficiency tag of the host cell Saccharomyces cerevisiae is often used to integrate heterologous genes at the corresponding nutritional deficiency sites as a screening condition for genome integration, but such conventional integration sites have certain limitations in the expression effect of heterologous genes, that is, the product yield is subject to certain restrictions.

[0010] Different gene integration sites have different effects on the expression of heterologous genes due to differences in the 3D structure of chromosomes and the difference in the openness of local chromosome sites. In a recent study on the construction of β-farnesene biosynthetic strains in Saccharomyces cerevisiae, JCR27, which improves the flux of the MVA pathway, was used as the chassis, and the Saccharomyces cerevisiae LiAc / SS vector DNA / PEG method was used to perform gene deletion and genome integration on the engineered yeast. After obtaining a single clone, it was verified by PCR to achieve the integration of 5 copies of the β-farnesene synthase gene Mac-bFS (F11S, M35T, T319S, I434T, I460V), and the integration sites included Leu2, Ura3 and YPRCdelta15. The final fermentation yield of β-farnesene was 791 mg / L. Further improving the yield of β-farnesene has important application value and significance. Summary of the invention

[0011] In view of this, the present invention provides a recombinant strain producing β-farnesene and its application.

[0012] The present invention provides a recombinant strain, which comprises an MVA pathway, a β-farnesene synthase MacbFS variant gene is integrated into the YPRCdelta15 site, and the original gene at at least one site among YML059C, YDR448W, YIL009C, YBR128C, and YHR178W is replaced by the β-farnesene synthase MacbFS variant gene.

[0013] The β-farnesene synthase MacbFS variant gene is derived from chamomile (Matricaria chamomilla var. recutita).

[0014] The β-farnesene synthase MacbFS variant gene has a sequence as shown in SEQ ID NO: 1 or a sequence having at least 80% homology thereto.

[0015] The base bacteria of the recombinant strain of the present invention is yeast with enhanced MVA pathway.

[0016] Furthermore, the yeast includes Saccharomyces cerevisiae. In an embodiment of the present invention, the base bacteria of the recombinant strain is Saccharomyces cerevisiae enhanced by the MVA pathway, and more specifically, the Saccharomyces cerevisiae enhanced by the MVA pathway uses CEN.PK2-1D as the starting strain, and overexpresses ERG19, ERG8, IDI1, ERG12, ERG10, ERG13 and ERG20 genes, overexpresses two copies of tHMG, and its ERG5 and GAL1,7,10 are knocked out.

[0017] In some specific embodiments, the present invention provides a recombinant strain with high β-farnesene production, which uses the Saccharomyces cerevisiae enhanced by the MVA pathway as described above as the base strain, integrates the β-farnesene synthase MacbFS variant gene at its YPRCdelta15 site, and replaces the original genes at its YML059C, YDR448W, and YIL009C sites with the β-farnesene synthase MacbFS variant gene.

[0018] In other specific embodiments, the recombinant strain with high β-farnesene production provided by the present invention uses the Saccharomyces cerevisiae enhanced by the MVA pathway as described above as the base strain, integrates the β-farnesene synthase MacbFS variant gene at its YPRCdelta15 site, and replaces the original genes at its YML059C, YBR128C and YHR178W sites with the β-farnesene synthase MacbFS variant gene.

[0019] The β-farnesene synthase MacbFS variant gene is derived from Matricaria chamomilla var. recutita and then optimized according to the codon preference of Saccharomyces cerevisiae. The optimized β-farnesene synthase MacbFS variant gene has a sequence as shown in SEQ ID NO:1.

[0020] Different from the prior art which uses nutritional deficiency gene integration sites (such as Leu2, Ura3, etc.) selected for β-farnesene biosynthesis, the present invention selects non-essential gene integration sites with excellent source gene expression potential, such as YML059C, YDR448W, YIL009C, YBR128C, and YHR178W, and performs site iterative integration after making specific combinations, ultimately obtaining a higher level of β-farnesene production.

[0021] The present invention constructs a synthetic pathway of β-farnesene in yeast by integrating a β-farnesene synthase mutant gene from chamomile (Matricaria chamomilla var. recutita) into a yeast genome. After research, the present invention has found two best combinations of synthesizing β-farnesene, a first combination of four different sites of YPRCdelta15, YML059C, YDR448W and YIL009C, and a second combination of four different sites of YPRCdelta15, YML059C, YBR128C and YHR178W. The β-farnesene synthase mutant gene is integrated into the site of the first combination, achieving a yield of β-farnesene up to 1054.10 mg / L in a shake flask. The β-farnesene synthase mutant gene is integrated into the site of the second combination, achieving a yield of β-farnesene up to 882.88 mg / L in a shake flask.

[0022] The above results show that the present invention achieves high yield of β-farnesene in a shake flask by selecting a specific site combination on the chromosome to integrate the β-farnesene synthase heterologous gene mutant expression frame from chamomile (Matricariachamomilla var.recutita).

[0023] The present invention also provides a method for constructing the recombinant strain, comprising: using a microorganism containing an MVA metabolic pathway as a base bacterium, integrating a β-farnesene synthase MacbFS variant gene at its YPRCdelta15 site, and replacing the original gene at at least one site of YML059C (abbreviated as M), YDR448W (abbreviated as D), YIL009C (abbreviated as I), YBR128C (abbreviated as B), and YHR178W with a β-farnesene synthase MacbFS variant gene.

[0024] In the present invention, the original genes at the YML059C, YDR448W, YIL009C, YBR128C, and YHR178W sites are NTE1, ADA2, EST3, ATG14, and STB5 genes, respectively.

[0025] In the present invention, the original gene at at least one site of YML059C (abbreviated as M), YDR448W (abbreviated as D), YIL009C (abbreviated as I), YBR128C (abbreviated as B), and YHR178W is replaced with the β-farnesene synthase MacbFS variant gene, thereby achieving the integration of the β-farnesene synthase MacbFS variant gene at the above-mentioned site.

[0026] In the present invention, the integration is iterative integration, which means that the monoclone that completes the integration of the first site is inoculated in a full nutrient medium and cultured, for discarding the Cas9 plasmid that realizes the cutting and screening functions, for the next iterative integration (i.e., the integration of the next site). The cultured bacterial liquid is streaked on a solid plate with double uracil and 5-fluorolactic acid (5-FOA) added for screening to ensure the loss state of the Cas9 plasmid. The strain obtained by reverse screening is used as a chassis for the next iteration (i.e., the iteration of the next site), and the aforementioned operation is repeated to finally obtain a saccharomyces cerevisiae strain that produces β-farnesene in which all four sites are successfully integrated into the genome.

[0027] In the present invention, the integration method includes the editing method mediated by the CRISPR-Cas9 system; the gRNA in the CRISPR-Cas9 system has a sequence as shown in SEQ ID NO: 2 to 7.

[0028] The present invention also provides a product for improving the yield of β-farnesene, including a product for promoting the expression of the β-farnesene synthase MacbFS variant or its encoding gene.

[0029] The products include any of the following:

[0030] 1) integrating the β-farnesene synthase MacbFS variant gene into a genetic element, expression cassette, expression vector or recombinant strain at the YPRCdelta15 site;

[0031] 2) knocking out a gene element, expression cassette, expression vector or recombinant strain of the original gene at at least one site of YML059C, YDR448W, YIL009C, YBR128C, or YHR178W.

[0032] The present invention also provides a method for preparing β-farnesene, which comprises: fermenting the recombinant strain of the present invention to obtain a product containing β-farnesene.

[0033] The present invention integrates the β-farnesene synthase MacbFS variant gene from chamomile into a specific combination of sites, and obtains a recombinant strain with high β-farnesene production, with a shake flask yield of up to 882.88-1054.10 mg / L, which is significantly higher than the yield of other site combinations. This shows that the above site combination has a better potential for heterologous expression of the β-farnesene synthase MacbFS variant gene, and ultimately significantly increases the yield of β-farnesene. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Illustration of the application of farnesene;

[0035] Figure 2 The metabolic pathway of β-farnesene biosynthesis is shown;

[0036] Figure 3 Schematic diagram of the chromosome genome integration site of the present invention is shown;

[0037] Figure 4 The YPRCdelta15 locus heterologous gene integration map is shown;

[0038] Figure 5 The YML059C locus heterologous gene integration map is shown;

[0039] Figure 6 The YDR448W locus heterologous gene integration map is shown;

[0040] Figure 7 Shown is the heterologous gene integration map at the YIL009C site. DETAILED DESCRIPTION

[0041] The present invention provides a recombinant strain producing β-farnesene and a construction method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0043] In the present invention, the genotype of the base bacteria SyBE_Sc0125XJ01 of the recombinant strain is shown in Table 1.

[0044] Table 1 Genotype information of chassis strains

[0045]

[0046] The present invention integrates the β-farnesene synthase MacbFS variant gene (VMacbFS for short) from chamomile into a specific combination of sites in the genome of the chamomile fungus after codon optimization, thereby obtaining a recombinant strain with high β-farnesene production. The nucleic acid sequence of the optimized VMacbFS is SEQ ID NO: 1:

[0047]

[0048] In the present invention, the integration gene site includes YPRCdelta15 and at least one of the following sites: YML059C, YDR448W, YIL009C, YBR128C, and YHR178W. The present invention uses CRISPR / Cas9 technology for iterative integration, and the sequences of the gRNAs corresponding to YPRCdelta15, YML059C, YDR448W, and YIL009C are shown in SEQ ID NOs: 2 to 5 (see Table 4). The sequences of YBR128C and YHR178W are shown in SEQ ID NOs: 6 to 7, respectively (see Table 6). In the present invention, the integration site YHR142W is also involved in the construction of the comparison strain, and the sequence of the gRNA corresponding to it is shown in SEQ ID NO: 8 (see Table 6).

[0049] The primer sequences involved in the present invention are shown in Table 2.

[0050] Table 2 Primer sequences

[0051]

[0052]

[0053]

[0054] The present invention will be further described below in conjunction with embodiments:

[0055] Example 1 Construction of β-farnesene biosynthetic pathway

[0056] The gene of the chamomile-derived β-farnesene synthase MacbFS variant (hereinafter abbreviated as VMacbFS) used in the present invention was codon-optimized for the Saccharomyces cerevisiae chassis and synthesized at Nine-Sky Gene Technology (Tianjin) Co., Ltd.

[0057] In this example, the cerevisiae SyBE_Sc0125XJ01 with enhanced carbon flux in the MVA pathway was used as the base strain, and four gene loci were selected for iterative integration using CRISPR / Cas9 technology: YPRCdelta15, YML059C, YDR448W, and YIL009C. The information of heterologous genes integrated at different gene loci is shown in Table 3, and the schematic diagram of the integration site is shown in Figure 3 .

[0058] Table 3 Strain gene integration information

[0059]

[0060]

[0061] Construction of CRISPR plasmid for genome integration at selected gene loci: The CRISPR plasmid was constructed by our laboratory and contains the Cas9 protein expression frame, the E. coli screening tag kanamycin kanR, the S. cerevisiae screening tag URA3, and an artificially designed single guide RNA (gRNA). https: / / www.atum.bio / eCommerce / cas9 / input The website was designed, and the artificially designed gRNA fragments were obtained by oligo annealing PCR. 100uM YPRCdelta15-gRNA-F / YPRCdelta15-gRNA-R, YML059C-gRNA-F / YML059C-gRNA-R, YDR448W-gRNA-F / YDR448W-gRNA-R, YIL009C-gRNA-F / YIL009C-gRNA-R and annealing buffer were added to the oligo annealing PCR system for Oligo annealing PCR. The PCR reaction program was as follows: 95℃, 5 minutes; 90℃, 5 minutes; 85℃, 5 minutes; 80℃, 5 minutes; 75℃, 5 minutes; 70℃, 5 minutes; 65℃ 5 minutes; 60℃ 5 minutes; successively reduced to 25℃; 5 minutes. The final product was diluted 100 times and subjected to T4 ligation reaction. The CRISPR plasmid vector was digested with Esp3I (BsmBI) restriction endonuclease, and subjected to T4 ligation reaction with the diluted oligo annealed PCR product, and then transformed into Escherichia coli DH5α. The single clone on the corresponding resistance plate was selected to extract the plasmid and sequenced to obtain the CRISPR plasmid of the corresponding gene site.

[0062] Table 4 gRNA corresponding to different gene integration sites

[0063]

[0064] Two VMacbFS gene expression cassettes with different promoters were constructed: (1) β-farnesene synthase variant gene VMacbFS matched with constitutive promoter pGPD and constitutive terminator tGPD; (2) β-farnesene synthase variant gene VMacbFS matched with inducible promoter pGal1 and constitutive terminator tGPD.

[0065] Construction of two expression frames: using the existing fragment or plasmid carrying the pGPD promoter as a template, pGPD-F / pGPD-R as primers, and KOD enzyme to amplify the pGPD promoter; using the existing fragment or plasmid carrying the pGal1 promoter as a template, pGal-F / pGal-R as primers, and KOD enzyme to amplify the pGal1 promoter; using the gene plasmid synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. as a template, two sets of primers pGPD-VMacbFS-F / pGal-VMacbFS-F and VMacbFS-R were used to amplify the β-farnesene synthase variant VMacbFS gene carrying different homology arm regions respectively; using the existing fragment or plasmid carrying the tGPD terminator as a template, tGPD-F / tGPD-R as primers, and KOD enzyme to amplify the tGPD terminator. The sequences of each primer are shown in Table 2. The PCR reaction program was set according to the KOD enzyme instruction manual. After the PCR program, DNA agarose gel electrophoresis was performed. The DNA marker was produced by Beijing Quanshijin Biotechnology Co., Ltd. Plus IIDNA Marker. After electrophoresis, the bright band of the target length was selected for DNA agarose gel recovery. After recovery, the two different expression frames were connected using overlap extension PCR technology (OVERLAP OCR). The pGPD1 promoter, β-farnesene synthase variant VMacbFS gene, and tGPD terminator were used as a group for OVERLAP PCR to obtain the pGPD_VMacbFS_tGPD expression frame; the pGal1 promoter, β-farnesene synthase variant VMacbFS gene, and tGPD terminator were used as a group for OVERLAP PCR to obtain the pGal_VMacbFS_tGPD expression frame.

[0066] Construct heterologous gene fragments inserted into different gene integration sites: Taking the YPRCdelta15 site as an example, locate the gRNA sequence corresponding to YPRCdelta15 in the Saccharomyces cerevisiae genome, and use the 500bp before and after it as the homologous arms for homologous recombination of the inserted fragment. Pick the chassis host cells with a toothpick and boil them in 50ul 20mM NaOH at 99℃ for 5min and 4℃ for 1min for three cycles. Use the supernatant after boiling as a template, use YPRCdelta15-upstream500-F / YPRCdelta15-upstream500-R and YPRCdelta15-downstream500-F / YPRCdelta15-downstream500-R as primers, and use KOD enzyme to amplify the upper and lower 500 homologous arms of the YPRCdelta15 integration site (YPRCdelta15-upstream500 and YPRCdelta15-downstream500, respectively).

[0067] The three fragments YPRCdelta15-upstream500, pGPD_VMacbFS_tGPD, and YPRCdelta15-downstream500 were used as a group for OVERLAP PCR to obtain the insertion fragment of the YPRCdelta15 site of the Saccharomyces cerevisiae chromosome. The primer sequences are shown in Table 2.

[0068] Construction of β-farnesene-producing Saccharomyces cerevisiae strain: The yeast strain chassis used in this example is Saccharomyces cerevisiae SyBE_Sc0125XJ01 with enhanced carbon flux in the MVA pathway.

[0069] The CRISPR plasmid corresponding to the constructed YPRCdelta15 locus and the inserted gene with upper and lower homology arms were co-transformed into the chassis strain by ssDNA / LiOAc transformation method, and YPRCdelta-vF-1 / YPRCdelta-vR-1 and YPRCdelta-vF-2 / YPRCdelta-vR-2 were used for colony PCR interface verification. Single colonies with positive bands on both interfaces in nucleic acid electrophoresis were selected for sequencing to obtain the YPRCdelta15 locus integrated with the β-farnesene expression cassette. Among them, the YPRCdelta15 locus heterologous gene integration map is shown in Figure 4 , the heterologous gene integration maps of YML059C, YDR448W and YIL009C loci are shown in Figures 5 to 7 .

[0070] CRISPR-Cas9 technology was used to perform iterative integration of gene sites in turn to obtain target strains in which YPRCdelta15, YML059C, YDR448W and YIL009C were all integrated with the target gene (VMacbFS gene): the monoclone that completed the heterologous gene integration of the YPRCdelta15 site was inoculated in a full nutrition YPD medium and cultured for more than 24 hours. The purpose of this experimental operation was to discard the Cas9 plasmid that realized the cutting and screening functions, so as to facilitate the next iterative integration (i.e., the integration of the next site). The cultured bacterial liquid was streaked on a solid plate with double uracil and 5-fluorolactic acid (5-FOA) added for screening to ensure the loss state of the Cas9 plasmid. The strain obtained by reverse screening was used as the chassis for the next iteration, and the aforementioned ssDNA / LiAcO transformation method was repeated to co-transfer to the chassis strain and the subsequent series of experimental operations, and finally the four sites were successfully integrated into the genome to produce β-farnesene in the Saccharomyces cerevisiae strain, and strain 1 of the present invention was obtained.

[0071] Example 2 Production of β-farnesene by two-phase fermentation

[0072] A single colony of the Saccharomyces cerevisiae constructed in Example 1 (i.e., strain 1 of the present invention) was picked up on the YPD solid plate and inoculated into three shake flasks during the transfer process as a parallel group experiment of the strain to ensure the parallelism of the biological experiment results. The colony was cultured in 5 ml YPD liquid medium (primary seed liquid) for 24 hours and the initial OD was 0. 600 =0.2 in 5 ml YPD liquid medium as secondary seed solution; after culturing for about 15 hours, the initial OD 600 =0.1 was inoculated into a 250 ml shake flask containing 50 ml YPD liquid culture medium and cultured in a 30°C shaker; after culture for about 12 h, 10 g / L galactose and 20% (10 ml) organic phase isopropyl myristate (IPM) were added for product extraction, and culture was continued in a 30°C shaker; the fermentation process was terminated after culture for 72 h.

[0073] Product extraction and detection: Take about 50ml of fermentation broth and centrifuge at 12,000rpm for 10min, take 1ml of the upper organic phase and put it in a 1.5ml centrifuge tube; take a new 1.5ml centrifuge tube, add 950ul of n-heptane, take 50ml of the organic phase and mix it several times, that is, dilute the sample 20 times, filter the diluted organic phase through a filter membrane and load it, and use gas chromatography tandem mass spectrometry (GC-MS) to determine the yield.

[0074] Test results: The fermentation results showed that the recombinant strain (marked as strain 1) obtained by integrating the β-farnesene synthase variant expression frame at four genomic sites in SyBE_Sc0125XJ01 of the present invention had an average β-farnesene yield of 1054.10±32.35 mg / L by shake flask fermentation, which is the highest yield in the 50 ml shake flask system reported in the prior art. Among them, YPRCdelta15-MD-YIL009C-1, YPRCdelta15-MD-YIL009C-2 and YPRCdelta15-MD-YIL009C-3 represent three parallel groups, respectively.

[0075] Table 5 Fermentation results of strain 1 of the present invention

[0076]

[0077]

[0078] Example 3 Iterative integration of fermentation information at other sites

[0079] The heterologous gene information of the gene integration sites YBR128C, YHR142W, and YHR178W involved in this example and the corresponding gRNA sequences are shown in Table 6.

[0080] Table 6 Strain gene integration information

[0081]

[0082] Referring to the method of Example 1, the β-farnesene synthase MacbFS variant gene was integrated into SyBE_Sc0125XJ01 according to the integration site combination shown in Table 7. The integrated gene information is shown in Tables 3 and 6. Comparative strains 1 to 4 and strain 2 of the present invention were obtained, respectively. A single colony was picked for inoculation, and then the same fermentation experiment was performed according to the method of Example 2. The three biological parallel yields of each strain were counted, and the average value was calculated. The results are shown in Table 7.

[0083] The results showed that the β-farnesene production of the recombinant strains obtained by other site combinations was significantly lower than that of the strain of the present invention, indicating that the gene integration site combination selected by the present invention has a good β-farnesene production potential and may have similar effects in the production of other similar natural products.

[0084] Table 7 Fermentation yields of strains with different integration site combinations

[0085]

[0086]

[0087] Among them, M is the abbreviation of YML059C, B is the abbreviation of YBR128C, and D is the abbreviation of YDR448W.

[0088] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant strain, characterized in that: It comprises the MVA pathway, and the β-farnesene synthase MacbFS variant gene is integrated into the YPRCdelta15 site, and the original gene at at least one of the sites YML059C, YDR448W, YIL009C, YBR128C, and YHR178W is replaced by the β-farnesene synthase MacbFS variant gene.

2. The recombinant strain according to claim 1, characterized in that The β-farnesene synthase MacbFS variant gene is derived from chamomile (Matricaria chamomilla var. recutita).

3. The recombinant strain according to claim 2, characterized in that The β-farnesene synthase MacbFS variant gene has a sequence as shown in SEQ ID NO: 1 or a sequence having at least 80% homology thereto.

4. The recombinant strain according to any one of claims 1 to 3, characterized in that The base bacteria is yeast with enhanced MVA pathway. The recombinant strain according to claim 4 , wherein the yeast comprises Saccharomyces cerevisiae.

6. The recombinant strain according to claim 5, characterized in that The MVA pathway-enhanced Saccharomyces cerevisiae uses CEN.PK2-1D as the starting strain, overexpresses ERG19, ERG8, IDI1, ERG12, ERG10, ERG13 and ERG20 genes, overexpresses two copies of tHMG, and its ERG5 and GAL1,7,10 are knocked out.

7. The method for constructing the recombinant strain according to any one of claims 1 to 6, characterized in that: Using a microorganism containing the MVA metabolic pathway as the base bacteria, the β-farnesene synthase MacbFS variant gene was integrated at its YPRCdelta15 site, and the original gene at at least one of its YML059C, YDR448W, YIL009C, YBR128C, and YHR178W sites was replaced with the β-farnesene synthase MacbFS variant gene.

8. The construction method according to claim 7, characterized in that: The integration is iterative integration, and the integration method includes CRISPR-Cas9 system-mediated editing; the gRNA in the CRISPR-Cas9 system has a sequence as shown in SEQ ID NO: 2 to 7.

9. A product for increasing the yield of β-farnesene, characterized in that The invention relates to a product that promotes the expression of a β-farnesene synthase MacbFS variant or a gene encoding the same; the product comprises any one of the following: 1) integrating the β-farnesene synthase MacbFS variant gene into a genetic element, expression cassette, expression vector or recombinant strain at the YPRCdelta15 site; 2) knocking out a gene element, expression cassette, expression vector or recombinant strain of the original gene at at least one site of YML059C, YDR448W, YIL009C, YBR128C, or YHR178W.

10. A method for preparing β-farnesene, characterized in that: Fermenting the recombinant strain according to any one of claims 1 to 6 to obtain a product containing β-farnesene.

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