Recombinant strains producing beta-farnesene and uses thereof

Abstract

The present application relates to the field of biotechnology, and more particularly to a kind of recombinant strain for producing beta-farnesene and its construction method and application.The present application obtains the recombinant strain for producing beta-farnesene by integrating the beta-farnesene synthase MacbFS variant gene from chamomile into four sites of YPRCdelta15, YML059C, YDR448W and YIL009C or four sites of YPRCdelta15, YML059C, YBR128C and YHR178W.The experiment shows that the above site combination has better heterologous gene expression potential, significantly improves the yield of beta-farnesene, and the yield in shake flask is as high as 882.88-1054.10 mg / L, which provides a new way for biosynthesis of beta-farnesene.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to recombinant strains that produce β-farnesene and their applications. Background Technology

[0002] Farnesene comprises two isomers, α-farnesene and β-farnesene, and was first discovered in nature in apple peel. As a chemical signaling molecule, farnesene plays a crucial role in plant pollination, apple seed dispersal, and plant defense. In the food industry, farnesene is widely used in the production of flavorings and spices, providing unique flavors and aromas to food. In the pharmaceutical field, farnesene is also used as an important intermediate in drug synthesis, exhibiting pharmacological activities such as antibacterial, anti-inflammatory, and antitumor activity. Due to its suitable cetane number, density, and low cloud point, farnesene is considered a potential biofuel and chemical feedstock, driving the development of renewable energy. Simultaneously, it can be converted into the high-energy fuel farnesane through a simple hydrogenation reaction. This biofuel has a high flash point exceeding 100°C, low hazard, and a net heat of combustion exceeding 44 MJ / kg, higher than existing military fuels. Compared to other fuels, it can reduce greenhouse gas emissions by 82%, making it a highly promising alternative to jet fuel and a high-performance polymer precursor, with significant demand in industrial production (see...). Figure 1 In recent years, researchers have envisioned a process to obtain farnesylacetone from farnesene, and then convert it into isophyllecithin via hydrogenation and addition reactions. In this process, the three-step reaction from farnesene to isophyllecithin simplifies the synthesis process and reduces the use of expensive raw materials, demonstrating that this new method of synthesizing isophyllecithin from farnesene obtained through microbial fermentation has broad application prospects in the production of vitamin E.

[0003] 1. Current Status of β-Farnese Biosynthesis

[0004] Research on the biosynthesis of β-farnesene through metabolic engineering microbial hosts has attracted widespread attention. Compared with traditional chemical synthesis methods, biosynthesis of farnesene has several significant advantages. Biosynthesis effectively avoids complex chemical reaction steps and reduces the generation of difficult-to-separate byproducts. Furthermore, it utilizes biological systems such as microorganisms or plants to directly synthesize farnesene through metabolic pathways, regulating the expression of related genes within the organism to achieve targeted synthesis of β-farnesene, thereby improving its yield and purity.

[0005] In the past few years, we have successfully achieved the biosynthesis of β-farnesene using host platforms such as Escherichia coli, Saccharomyces cerevisiae, and Yersinia lipolyticis. We are also constantly exploring ways to further increase the biosynthetic yield and achieve the industrial-scale biosynthesis of high-value-added β-farnesene.

[0006] The biosynthesis of β-farnesene primarily involves the conversion of the important precursor farnesyl pyrophosphate (FPP) catalyzed by plant-derived β-farnesene synthase. In the *Saccharomyces cerevisiae* system, farnesyl diphosphate (FPP) is formed by the condensation of isopentenyl diphosphate (IPP) and dimethylpropenyl diphosphate (DMAPP) via the mevalonate (MVA) pathway. Subsequently, through heterologously introduced β-farnesene synthase, FPP is further catalyzed to generate β-farnesene. The biosynthetic pathway of β-farnesene is as follows: Figure 2 As shown.

[0007] 2. Optimization strategy for β-farnesene synthase copy number

[0008] In industrial production, integrating heterologous genes into the host organism's genome is more effective than shuttle plasmid expression systems in achieving stable expression of enzyme catalysis. 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 varying effects on 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 host strain's metabolic pathway flux has not been achieved. To achieve stable expression of heterologous genes in host cells, auxotrophic tags in the host cell *Saccharomyces cerevisiae* are often used to integrate heterologous genes at corresponding auxotrophic sites, serving as a genomic integration screening condition. However, these conventional integration sites have certain limitations in the expression of heterologous genes, namely, the product yield is somewhat restricted.

[0010] Different gene integration sites have varying effects on the expression of heterologous genes due to differences in chromosomal 3D structure and the openness of local chromosomal sites. In a recent study on the construction of a *Saccharomyces cerevisiae* β-farnesene biosynthetic strain, JCR27, which enhances MVA pathway throughput, was used as the chassis. Gene deletion and genome integration were performed on engineered yeast using the *Saccharomyces cerevisiae* LiAc / SS vector DNA / PEG method. After obtaining single clones, PCR verification confirmed the integration of five copies of the β-farnesene synthase gene Mac-bFS (F11S, M35T, T319S, I434T, I460V), with integration sites including Leu2, Ura3, and YPRCdelta15. The final fermentation yield of β-farnesene was 791 mg / L. Further increasing β-farnesene yield has significant application value and implications. Summary of the Invention

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

[0012] The present invention provides a recombinant strain containing the MVA pathway, wherein the β-farnesene synthase MacbFS variant gene is integrated at 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.

[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 with it.

[0015] The chassis bacteria of the recombinant strain described in this invention are MVA pathway-enhanced yeasts.

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

[0017] In some specific embodiments, the present invention provides a recombinant strain that produces high levels of β-farnesene, using the MVA pathway-enhanced Saccharomyces cerevisiae as the chassis strain, integrating the β-farnesene synthase MacbFS variant gene at its YPRCdelta15 site, and replacing 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 MVA pathway-enhanced Saccharomyces cerevisiae as the chassis 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 was derived from chamomile (Matricaria chamomilla var. recutita) and then optimized according to the codon preference of Saccharomyces cerevisiae. The optimized β-farnesene synthase MacbFS variant gene has the sequence shown in SEQ ID NO: 1.

[0020] Unlike existing technologies that use nutrient deficiency gene integration sites (such as Leu2, Ura3, etc.) for β-farnesene biosynthesis, this invention selects non-essential gene integration sites with superior source gene expression potential, such as YML059C, YDR448W, YIL009C, YBR128C, and YHR178W, and performs iterative integration of these sites after specific combinations, ultimately obtaining a higher level of β-farnesene yield.

[0021] This invention constructs a β-farnesene synthesis pathway in yeast by integrating a β-farnesene synthase mutant gene derived from chamomile (Matricaria chamomilla var. recutita) into the yeast genome. Through research, this invention discovered two optimal combinations for β-farnesene synthesis: a first combination of four different sites (YPRCdelta15, YML059C, YDR448W, and YIL009C) and a second combination of four different sites (YPRCdelta15, YML059C, YBR128C, and YHR178W). Integrating the β-farnesene synthase mutant gene into the first combination site achieved a β-farnesene yield as high as 1054.10 mg / L in shake flasks. Integrating the β-farnesene synthase mutant gene into the second combination site achieved a β-farnesene yield as high as 882.88 mg / L in shake flasks.

[0022] The above results demonstrate that the present invention achieves high yield of β-farnesene in shake flasks by integrating the expression cassette of a heterologous β-farnesene synthase mutant from chamomile (Matricariachamomilla var. recutita) through the selection of specific sites on chromosomes.

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

[0024] In this invention, the original genes at the YML059C, YDR448W, YIL009C, YBR128C, and YHR178W loci are, in order, NTE1, ADA2, EST3, ATG14, and STB5 genes.

[0025] In this invention, the β-farnesene synthase MacbFS variant gene is integrated into the above-mentioned sites by replacing the original gene at at least one of the sites YML059C (M), YDR448W (D), YIL009C (I), YBR128C (B), and YHR178W with the β-farnesene synthase MacbFS variant gene.

[0026] In this invention, the integration is iterative integration, which means that a single clone that has completed the integration of the first site is inoculated into a complete nutrient medium for culture, which is used to discard the Cas9 plasmid that performs the cleavage and screening functions, so as to facilitate the next iteration of integration (i.e., integration of the next site). The cultured bacterial solution is streaked onto a solid plate supplemented with double uracil and 5-fluoroalbumin (5-FOA) for screening, ensuring that the Cas9 plasmid is lost. The strain obtained by reverse screening is used as the chassis for the next iteration (i.e., iteration of the next site), and the above operation is repeated, finally obtaining a Saccharomyces cerevisiae strain that produces β-farnesene with all four sites successfully integrated into the genome.

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

[0028] The present invention also provides products that increase β-farnesene production, including products that promote the expression of β-farnesene synthase MacbFS variants or their encoding genes.

[0029] The product includes any one of the following:

[0030] 1) Integrating the β-farnesene synthase MacbFS variant gene into gene elements, expression cassettes, expression vectors, or recombinant strains at the YPRCdelta15 site;

[0031] 2) Knock out the gene element, expression cassette, expression vector or recombinant strain of the original gene at at least one of the following sites: YML059C, YDR448W, YIL009C, YBR128C, and YHR178W.

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

[0033] This invention integrates the β-farnesene synthase MacbFS variant gene from chamomile into specific site combinations, obtaining a recombinant strain with high β-farnesene production. The shake-flask yield reached 882.88–1054.10 mg / L, significantly higher than that of other site combinations. This indicates that the above site combinations have superior potential for heterologous expression of the β-farnesene synthase MacbFS variant gene, ultimately significantly increasing β-farnesene yield. Attached Figure Description

[0034] Figure 1 Application diagram of sifanilide;

[0035] Figure 2 This illustrates the β-farnesene biosynthetic metabolic pathway.

[0036] Figure 3 This diagram illustrates the chromosome genome integration site of the present invention.

[0037] Figure 4 This shows the integration map of heterologous genes at the YPRCdelta15 locus;

[0038] Figure 5 This shows the integration map of heterologous genes at the YML059C locus;

[0039] Figure 6 This shows the integration map of heterologous genes at the YDR448W locus;

[0040] Figure 7 This shows the heterologous gene integration map at the YIL009C locus. Detailed Implementation

[0041] This invention provides a recombinant strain producing β-farnesene, its construction method, and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments; those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

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

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

[0044] Table 1 Genotypic information of chassis strains

[0045]

[0046] This invention integrates a variant gene of β-farnesene synthase MacbFS (VMacbFS) from chamomile into a specific combination of sites in the genome of substrate bacteria after codon optimization, thereby obtaining a recombinant strain that produces high levels of β-farnesene. The optimized VMacbFS nucleic acid sequence SEQ ID NO: 1 is as follows:

[0047]

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

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

[0050] Table 2 Primer sequences

[0051]

[0052]

[0053]

[0054] The present invention will be further illustrated below with reference to the embodiments:

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

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

[0057] In this embodiment, *Saccharomyces cerevisiae* SyBE_Sc0125XJ01, which enhances carbon flux in the MVA pathway, was used as the chassis strain. Four gene loci were selected for iterative integration using CRISPR / Cas9 technology: YPRCdelta15, YML059C, YDR448W, and YIL009C. Information on the heterologous genes integrated at different gene loci is shown in Table 3, and a schematic diagram of the integration sites is shown below. 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 in our laboratory and contains a Cas9 protein expression cassette, the E. coli selection tag kanamycin (kanR), the Saccharomyces cerevisiae selection tag URA3, and a designed single guide RNA (gRNA). The gRNA is expressed via... https: / / www.atum.bio / eCommerce / cas9 / input The website design yielded artificially designed gRNA fragments via oligo annealing PCR. The oligo annealing PCR system was prepared by adding 100 μM of YPRCdelta15-gRNA-F / YPRCdelta15-gRNA-R, YML059C-gRNA-F / YML059C-gRNA-R, YDR448W-gRNA-F / YDR448W-gRNA-R, and YIL009C-gRNA-F / YIL009C-gRNA-R, along with annealing buffer. The PCR reaction program was as follows: 95℃, 5 min; 90℃, 5 min; 85℃, 5 min; 80℃, 5 min; 75℃, 5 min; 70℃, 5 min; 65℃, 5 min; 60℃, 5 min; then decreasing to 25℃ for 5 minutes. The final product was diluted 100-fold and subjected to T4 ligation. The CRISPR plasmid vector was digested with Esp3I (BsmBI) restriction endonuclease, ligated with diluted oligo annealed PCR product using a T4 ligation reaction, transformed into E. coli DH5α, and single clones were selected from the corresponding antibiotic plates to extract plasmids and sequenced to obtain CRISPR plasmids for the corresponding gene loci.

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

[0063]

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

[0065] Construction of two expression cassettes: Using existing fragments or plasmids carrying the pGPD promoter as templates, and pGPD-F / pGPD-R as primers, the pGPD promoter was amplified using KOD enzyme; using existing fragments or plasmids carrying the pGal1 promoter as templates, and pGal-F / pGal-R as primers, the pGal1 promoter was amplified using KOD enzyme; using gene plasmids synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. as templates, 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 homologous arm regions; using existing fragments or plasmids carrying the tGPD terminator as templates, and tGPD-F / tGPD-R as primers, the tGPD terminator was amplified using KOD enzyme. The primer sequences are shown in Table 2. The PCR reaction procedure was set according to the KOD enzyme instruction manual. After the PCR procedure, DNA agarose gel electrophoresis was performed. The DNA marker was from Beijing TransGen Biotech Co., Ltd. Plus II DNA Marker. After electrophoresis, bright bands of the target length were selected for DNA agarose gel recovery. After recovery, overlap extension PCR (OVERLAP OCR) was used to ligate the two different expression cassettes. The pGPD1 promoter, the β-farnesene synthase variant VMacbFS gene, and the tGPD terminator were used as one group for OVERLAP PCR to obtain the pGPD_VMacbFS_tGPD expression cassette; the pGal1 promoter, the β-farnesene synthase variant VMacbFS gene, and the tGPD terminator were used as another group for OVERLAP PCR to obtain the pGal_VMacbFS_tGPD expression cassette.

[0066] Constructing heterologous gene fragments inserted at different gene integration sites: Taking the YPRCdelta15 site as an example, the gRNA sequence corresponding to YPRCdelta15 in the Saccharomyces cerevisiae genome was located, and the 500 bp before and after it was used as homologous arms for homologous recombination of the inserted fragment. Host cells from the substrate were picked up with a toothpick and boiled in 50 μL of 20 mM NaOH at 99°C for 5 min and then at 4°C for 1 min for three cycles. Using the supernatant after boiling as a template, and using YPRCdelta15-upstream500-F / YPRCdelta15-upstream500-R and YPRCdelta15-downstream500-F / YPRCdelta15-downstream500-R as primers, KOD enzyme was used to amplify the 500 bp homologous arms before and after the YPRCdelta15 integration site (YPRCdelta15-upstream500 and YPRCdelta15-downstream500, respectively).

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

[0068] Construction of a 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 constructed CRISPR plasmid corresponding to the YPRCdelta15 site and the insert gene with two homologous arms were co-transformed into the chassis strain using ssDNA / LiOAc transformation. Colony PCR interface verification was performed using YPRCdelta-vF-1 / YPRCdelta-vR-1 and YPRCdelta-vF-2 / YPRCdelta-vR-2. Single colonies showing positive bands at both interfaces in nucleic acid electrophoresis were selected for sequencing, yielding a *Saccharomyces cerevisiae* strain that successfully integrated the β-farnesene expression cassette at the YPRCdelta15 site. The heterologous gene integration map at the YPRCdelta15 site is shown below. Figure 4 The heterologous gene integration maps of YML059C, YDR448W, and YIL009C sites are shown in the following figures. Figures 5-7 .

[0070] Using CRISPR-Cas9 technology, iterative integration of gene loci was performed sequentially to obtain target strains YPRCdelta15, YML059C, YDR448W, and YIL009C, all of which integrated the target gene (VMacbFS gene). Single clones that successfully integrated the heterologous gene at the YPRCdelta15 locus were inoculated into fully nutritious YPD medium and cultured for at least 24 hours. This step was to discard the Cas9 plasmid, which performs the cutting and screening functions, to facilitate the next iteration (i.e., integration at the next locus). The cultured bacterial solution was streaked onto a solid plate supplemented with double uracil and 5-fluoroalbumin (5-FOA) for screening, ensuring the loss of the Cas9 plasmid. Using the strains obtained from reverse screening as the chassis, the next iteration was performed, repeating the aforementioned ssDNA / LiAcO transformation method to co-transform the chassis strain and subsequent experimental procedures. Finally, a *Saccharomyces cerevisiae* strain producing β-farnesene with all four loci successfully integrated into the genome was obtained, resulting in strain 1 of this invention.

[0071] Example 2: Two-phase fermentation for the production of β-farnesene

[0072] A single colony of *Saccharomyces cerevisiae* (i.e., strain 1 of this invention) constructed in Example 1 was picked from the YPD solid plate and inoculated into three separate shake flasks during the transfer process, serving as a parallel group experiment for this strain to ensure the parallelism of the biological experimental results. The colonies were cultured in 5 ml of YPD liquid medium for 24 hours (primary seed culture) to determine the initial OD value. 600 =0.2% was added to 5ml of YPD liquid medium as a secondary seed culture; after incubation for about 15 hours, the initial OD was used as the seed culture. 600 =0.1 was inoculated into a 250ml shake flask containing 50ml YPD liquid medium and placed in a shaker at 30℃. After culturing for about 12 hours, 10g / L galactose and 20% (10ml) organic phase isopropyl myristate (IPM) were added for product extraction, and the mixture was placed in a shaker at 30℃ for further culturing. The fermentation process was terminated after 72 hours of culturing.

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

[0074] Detection results: Fermentation results showed that the recombinant strain (labeled as strain 1) obtained by integrating the β-farnesene synthase variant expression cassette into four genomic loci in SyBE_Sc0125XJ01, obtained by shake-flask fermentation, had an average β-farnesene yield of 1054.10±32.35 mg / L, which is the highest yield reported in the 50 ml shake-flask system. Among them, YPRCdelta15-MD-YIL009C-1, YPRCdelta15-MD-YIL009C-2 and YPRCdelta15-MD-YIL009C-3 represent three parallel groups.

[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 and corresponding gRNA sequences of the gene integration sites YBR128C, YHR142W, and YHR178W involved in this embodiment are shown in Table 6.

[0080] Table 6. Gene Integration Information of Strains

[0081]

[0082] Following the method of Example 1, the β-farnesene synthase MacbFS variant gene was integrated into SyBE_Sc0125XJ01 according to the integration sites shown in Table 7. The integrated gene information is shown in Tables 3 and 6. Comparative strains 1-4 and strain 2 of the present invention were obtained respectively. Single colonies of each strain were picked and inoculated. Then, the same fermentation experiment was performed according to the method of Example 2. The three parallel biological 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 yield of β-farnesene in recombinant strains obtained from other site combinations was significantly lower than that of the strain of this invention. This indicates that the gene integration site combination selected in this invention has good potential for β-farnesene production and may have similar effects in the production of other similar natural products.

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

[0085]

[0086]

[0087] Where M is the abbreviation for YML059C, B is the abbreviation for YBR128C, and D is the abbreviation for YDR448W.

[0088] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant strain, characterized in that, It contains the MVA pathway and integrates the β-farnesene synthase MacbFS gene at the YPRCdelta15 site; and contains any of the mutations shown in (1) to (2): (1) The original genes at the YML059C, YDR448W and YIL009C sites were replaced by the β-farnesene synthase MacbFS gene; (2) The original genes at the YML059C, YBR128C and YHR178W sites were replaced by the β-farnesene synthase MacbFS gene; The β-farnesene synthase MacbFS gene is derived from chamomile ( Matricaria chamomilla var. recutita ); The recombinant strain originated from *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae ).

2. The recombinant strain according to claim 1, characterized in that, The nucleotide sequence of the β-farnesene synthase MacbFS gene is shown in SEQ ID NO:

1.

3. The recombinant strain according to claim 1 or 2, characterized in that, Its substrate bacteria are MVA-enhanced Saccharomyces cerevisiae.

4. The recombinant strain according to claim 3, characterized in that, The MVA pathway-enhanced Saccharomyces cerevisiae used CEN.PK2-1D as the starting strain and overexpressed ERG19, ERG8, IDI1, ERG12, ERG10, ERG13 and ERG20 genes, overexpressed two copies of tHMG, and its ERG5 and GAL1,7,10 were knocked out.

5. The method for constructing the recombinant strain according to any one of claims 1 to 4, characterized in that, Using *Saccharomyces cerevisiae* containing the MVA metabolic pathway as the substrate strain, the β-farnesene synthase MacbFS gene was integrated at its YPRCdelta15 site, and any one of the following combinations of mutations was performed: (1) The original genes at the YML059C, YDR448W and YIL009C sites were replaced by the β-farnesene synthase MacbFS gene; (2) The original genes at the YML059C, YBR128C and YHR178W sites were replaced by the β-farnesene synthase MacbFS gene.

6. The construction method according to claim 5, characterized in that, The integration is iterative integration, and the integration method includes CRISPR-Cas9 system-mediated editing; the sequence of gRNA in the CRISPR-Cas9 system is shown in SEQ ID NO: 2~7.

7. A method for preparing β-farnesene, characterized in that, Fermentation of the recombinant strain according to any one of claims 1 to 4 yields a product containing β-farnesene.

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