Beta-farnesene synthase mutant and application thereof

Abstract

The invention discloses a beta-farnesene synthase mutant, which is characterized in that the amino acid sequence of the beta-farnesene synthase mutant has the difference of one or more amino acid residues selected from A26T, A169E, Y251H, V323S, G325A and A438T relative to the amino acid sequence of wild type beta-farnesene synthase derived from walnuts. The beta-farnesene synthase mutant gene and the encoding protein thereof provided by the invention are effectively expressed in saccharomyces cerevisiae. Under the same condition, the walnut-derived beta-farnesene synthase mutant provided by the invention not only has higher activity compared with the beta-farnesene synthase derived from artemisia annua, German chamomile, peppermint and fragrant citrus, but also has significantly improved enzyme activity compared with the wild walnut beta-farnesene synthase; and the saccharomyces cerevisiae can have higher farnesene yield.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a β-farnesene synthase mutant and its applications. Background Technology

[0002] Farnesene (also known as farnesene) is a volatile sesquiterpene compound composed of three isoprene units. Farnesene is a precursor for the synthesis of vitamin E and vitamin K1; it can also be used as a biosynthetic fuel in diesel and aviation fuel applications; farnesene can be chemically catalyzed to become isosqualene, which can then be hydrogenated to become squalane, a substance widely used in the cosmetics industry. α-Farnesene and β-farnesene are two common isomers of farnesene. In 2011, Amyris in the United States achieved mass production of β-farnesene using yeast and used it as a raw material to prepare squalane. Hubei Nengte Technology Co., Ltd. achieved the synthesis of vitamin E using β-farnesene synthesized by yeast fermentation as a precursor in 2018, becoming the world's third-largest supplier of vitamin E raw materials.

[0003] Farnesene is present in very low amounts in plants, making microbial fermentation the most cost-effective method for its synthesis. In eukaryotes, farnesene is synthesized via the mevalonate (MVA) pathway, where farnesene synthase is the key rate-limiting enzyme, and its activity directly affects the yield of farnesene. Plant-derived farnesene synthases are currently the most extensively studied, with those from apples, pears, oranges, Asteraceae plants, mint, and legumes being the primary research subjects.

[0004] The activity of farnesene synthase and its expression level in Saccharomyces cerevisiae play a crucial role in the yield of farnesene biosynthesis, but currently only a few types of β-farnesene synthases have been studied and applied. Therefore, discovering new farnesene synthase resources and improving their activity through modification is of great significance for reducing costs and increasing efficiency in the farnesene synthesis process. Summary of the Invention

[0005] To address the issue that only a few types of β-farnesene synthases have been studied and applied in existing technologies, this invention provides a β-farnesene synthase mutant and its applications. Specifically, this invention modifies the wild-type β-farnesene synthase JrbFS from walnuts to obtain a mutant gene containing six amino acid residue substitutions, and expresses this mutant gene in *Saccharomyces cerevisiae*. Under the same conditions, the walnut-derived β-farnesene synthase mutant of this invention enables the host to possess a higher farnesene synthesis capacity.

[0006] The wild-type β-farnesene synthase from walnut described in this invention has the NCBI accession number XP_018849875.1 and its gene coding sequence is XM_018994330.2.

[0007] The first aspect of the present invention provides a β-farnesene synthase mutant, which has a difference of one or more amino acid residues selected from A26T, A169E, Y251H, V323S, G325A and A438T in the amino acid sequence relative to the wild-type β-farnesene synthase.

[0008] In some embodiments, the β-farnesene synthase mutant differs from the wild-type β-farnesene synthase in the amino acid sequences of A26T, A169E, Y251H, V323S, G325A, and A438T amino acid residues, as shown in SEQ ID NO:3.

[0009] A second aspect of the present invention provides a gene encoding β-farnesene synthase, comprising a β-farnesene synthase mutant as described in the first aspect of the present invention; preferably, the nucleotide sequence of the gene is shown in SEQ ID NO:2 or SEQ ID NO:4.

[0010] A third aspect of the present invention provides a recombinant expression vector comprising a gene encoding β-farnesene synthase as described in the second aspect of the present invention; preferably further comprising the ERG20 gene.

[0011] A fourth aspect of the present invention provides a genetically engineered bacterium comprising a gene encoding β-farnesene synthase as described in the second aspect of the present invention, or a recombinant expression vector as described in the third aspect of the present invention.

[0012] In some embodiments, the starting strain of the genetically engineered bacteria is yeast.

[0013] In some embodiments, the yeast is brewer's yeast.

[0014] In some embodiments, the brewer's yeast is a leucine auxotrophic yeast, and / or the recombinant expression vector further includes the LEU2 gene.

[0015] The fifth aspect of the present invention provides a reaction system or kit for producing farnesene, comprising a β-farnesene synthase mutant as described in the first aspect of the present invention, a gene encoding β-farnesene synthase as described in the second aspect of the present invention, a recombinant expression vector as described in the third aspect of the present invention, or a genetically engineered bacterium as described in the fourth aspect of the present invention.

[0016] In some embodiments, the reaction system or kit for producing farnesene also includes a culture medium.

[0017] In some embodiments, the culture medium is one or more of YPD medium, D-galactose, n-dodecane, polyolefin, and IPM isopropyl myristate.

[0018] The sixth aspect of the present invention provides a method for culturing genetically engineered bacteria as described in the fourth aspect of the present invention, wherein the genetically engineered bacteria are placed in a culture medium and cultured to obtain the bacteria.

[0019] The seventh aspect of the present invention provides a method for producing β-farnesene synthase, wherein the genetically engineered bacteria as described in the fourth aspect of the present invention are placed in a culture medium and cultured to obtain the synthase.

[0020] The eighth aspect of the present invention provides a method for producing farnesene by culturing genetically engineered bacteria as described in the fourth aspect of the present invention in a culture medium and fermenting them to produce farnesene.

[0021] In some implementations, the method includes one or more of the following conditions:

[0022] (i) The culture medium is YPD medium;

[0023] (ii) The incubation temperature is 25-35℃;

[0024] (iii) Shaking should be used during cultivation;

[0025] (iv) Culture the genetically engineered bacteria to the late logarithmic phase, and add D-galactose and / or n-dodecane.

[0026] In some embodiments, in step (iv) of the method, the final content of D-galactose is 0.5-2% by mass, and / or the final content of n-dodecane is 5-10% by mass; the mass concentration is the mass of a component in a unit volume of the mixture, and may also be written as w / v.

[0027] In some embodiments, the culture temperature is 30°C, and the oscillation is performed at 250 rpm.

[0028] In some specific embodiments, the final content of D-galactose is 1% by mass, and / or the final content of n-dodecane is 10% by mass.

[0029] The ninth aspect of the present invention provides a β-farnesene synthase mutant as described in the first aspect of the present invention, a gene encoding β-farnesene synthase as described in the second aspect of the present invention, a recombinant expression vector as described in the third aspect of the present invention, and the application of genetically engineered bacteria as described in the fourth aspect of the present invention in the production of farnesene.

[0030] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0031] The reagents and raw materials used in this invention are all commercially available.

[0032] The positive and progressive effects of this invention are as follows:

[0033] (1) The β-farnesene synthase gene and its encoded protein from walnuts provided by this invention are effectively expressed in Saccharomyces cerevisiae. Under the same conditions, compared with β-farnesene synthases from Artemisia annua, chamomile, peppermint and orange, the β-farnesene synthase from walnuts provided by this invention enables Saccharomyces cerevisiae to have a higher farnesene yield.

[0034] (2) The β-farnesene synthase mutant from walnut provided by the present invention has higher activity than wild-type β-farnesene synthase and has potential application value in farnesene biosynthesis. Attached Figure Description

[0035] Figure 1 Phylogenetic analysis (neighbor-joining method) of β-farnesene synthase derived from walnuts and previously reported β-farnesene synthases.

[0036] Figure 2 This is a plasmid map used to knock the walnut-derived β-farnesene synthase gene into Saccharomyces cerevisiae.

[0037] Figure 3 Gas chromatographic identification of β-farnesene in fermentation products of Saccharomyces cerevisiae containing the jujube-derived β-farnesene synthase gene.

[0038] Figure 4 Comparison of farnesene production in Saccharomyces cerevisiae containing β-farnesene synthase genes from different sources. Detailed Implementation

[0039] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0040] Example 1: Expression and synthesis of farnesene from jujube β-farnesene synthase and its mutants in Saccharomyces cerevisiae.

[0041] (1) Synthesis and codon optimization of jujube β-farnesene synthase gene

[0042] We selected a possible β-farnesene synthase sequence from walnut (NCBI accession number XP_018849875.1, gene coding sequence XM_018994330.2) as the initial sequence. Phylogenetic analysis was performed with previously reported β-farnesene synthase genes, such as... Figure 1 As shown, the amino acid sequence of β-farnesene synthase from walnut (SEQ ID NO: 1) is most similar to that of β-farnesene synthase from orange, but the sequence similarity between the two is only 48.8%.

[0043] To improve the expression of the β-farnesene synthase gene in Saccharomyces cerevisiae, codon optimization was performed on XM_018994330.2 to obtain SEQ ID NO:2, and then the gene was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0044] SEQ ID NO:1:

[0045] MSFPISAVPSSTQIEASGKLVGRNLAHFSPSVWGSHFLSYASDSNVLDADDDHKIMQQVQELKDEVKRMLIISPGSTDQTLSEKLDLIDAIQHLGVSYHFESEIDEILQKAHKINPPCFNNINIMDHAADDQLKLYTISLWFRLL RQQGYDVSCDIFNEFKDDKGSFKASLISDVKGMLSLYEAAHLGINGEDILDEALAFTTTHLELAVNHIRPQLAKKVKHALNRPIRKALPRLEGLYYISIYKEEDSYSETLLKFAKLDFNVLQSQHQKEIGGITRCWKNLDFTANLP YARDRIVEGYFWTMGVFFEPQYSLARRIMTKVIGMTSILDDTYDAYGSYAELKLFTEAIERWDVSAIDILPEYMKLIYKALLDVYDEIEAETAKDGRPFCVHYAKESMKKLIQAYFIEAKWCNEGYAPTMEEYMSNAMTTSAYQML APTSFLGMGNIADEEVFKWVFNDPKILRASTIICRLMDDIKSHKFEQSRAHAVSAVECYMKQYGVSAEEEVYKLLGKEIVNAWKDINEELLMNRTADHHVPMPILERVLNLARVIDLVYEDGDAYTDSNMMKDYIASLLVNPLVLQ

[0046] SEQ ID NO:2:

[0047]

[0048] (2) Construction of integrative plasmids for JrbFS gene knock-in

[0049] like Figure 2 As shown in Table 1 below, each gene element is described as follows:

[0050] Table 1 Integral plasmid gene elements

[0051]

[0052] Each gene element was amplified by high-fidelity PCR and the fragments were ligated into a plasmid pUC-JrbFS using the Gibson ligation method. The core of this plasmid consists of three parts: First, the left and right arms of homologous recombination, which are responsible for inserting the target gene into the genome via homologous recombination. The left arm of homologous recombination contains the LEU2 gene, which encodes 3-isopropylmalate dehydrogenase, a key enzyme in the leucine synthesis pathway. The Saccharomyces cerevisiae CEN.PK2-1D used in this invention is a leucine auxotrophic yeast and cannot grow in leucine-deficient media. Therefore, if the gene knock-in is successful, the host can grow in leucine-free media. Second, the ERG20 gene, which encodes (2E,6E)-farnesyl diphosphate synthase, which is responsible for the synthesis of farnesyl pyrophosphate, a precursor for farnesene synthesis. The gene already has one copy in the host, and here ERG20 is induced to be expressed via the galactose promoter pGal10 to synthesize more farnesyl pyrophosphate; third, JrbFS, which encodes walnut-derived β-farnesene synthase for the synthesis of β-farnesene.

[0053] (3) Rational modification of the JrbFS gene

[0054] The primers used for point mutation of the JrbFS gene are shown in Table 2 below:

[0055] Table 2. Primers used for point mutation of the JrbFS gene.

[0056]

[0057]

[0058] The specific process of point mutation is as follows:

[0059] Taking the A26T point mutation as an example, firstly, using the pUC-JrbFS plasmid as a template, a 6118 bp plasmid backbone fragment was amplified by high-fidelity PCR using LEU2RF and ERG20R primers. Then, a 1.88 kbp nucleic acid fragment containing the downstream JrbFS sequence was amplified using A26TF and LEU2RR primers, and a 1.45 kbp nucleic acid fragment containing the upstream JrbFS sequence was amplified using A26TR and ERG20F primers. The success of PCR amplification was determined by agarose gel electrophoresis. The three fragments were then mixed and recovered using a PCR product recovery kit. The resulting mixed fragment was re-circularized into a plasmid using a Gibson ligation reaction. The Gibson ligation product was then transformed into *E. coli* DH5α cells and plated on plates containing ampicillin. Positive transformants can be obtained after 20 hours of culture. Several colonies are identified by PCR using ERG20F and LEU2RR primers, and the PCR products are sequenced. Clones with successful mutations are selected and streaked on culture medium plates to obtain single colonies.

[0060] Subsequent point mutations all used the preceding mutant plasmid as a template. For example, after obtaining the pUC-JrbFS-A26T plasmid, this plasmid was used as a PCR template to construct the pUC-JrbFS-A26T-A169E mutant plasmid according to the above method. This process of accumulating mutations was repeated until the construction of the pUC-JrbFS-A26T-A169E-Y251H-V323S-G325A-A438T mutant plasmid was completed. The protein encoded by the JrbFS gene on this plasmid contains the above-mentioned 6 mutated residues (named JrbFS_M6, the complete amino acid sequence of JrbFS_M6 is shown in SEQ ID NO:3, and its gene coding sequence is shown in SEQ ID NO:4).

[0061] SEQ ID NO:3:

[0062] MSFPISAVPSSTQIEASGKLVGRNLTHFSPSVWGSHFLSYASDSNVLDADDDHKIMQQVQELKDEVKRMLIISPGSTDQTLSEKLDLIDAIQHLGVSYHFESEIDEILQKAHKINPPCFNNINIMDHAADDQLKLYTISLWFRLLRQQG YDVSCDIFNEFKDDKGSFKESLISDVKGMLSLYEAAHLGINGEDILDEALAFTTTHLELAVNHIRPQLAKKVKHALNRPIRKALPRLEGLYYISIYKEEDSHSETLLKFAKLDFNVLQSQHQKEIGGITRCWKNLDFTANLPYARDRIVEGYFWTMGVFFEPQYSLARRIMTKSIAMTSILDDTYDAYGSYAELKLFTEAIERWDVSAIDILPEYMKLIYKALLDVYDEIEAETAKDGRPFCVHYAKESMKKLIQAYFIEAKWCNEGYAPTMEEYMSNAMTTSAYQMLTPTSFLGMGNIADEEVFKWVFNDPKILRASTIICRLMDDIKSHKFEQSRAHAVSAVECYMKQYGVSAEEEVYKLLGKEIVNAWKDINEELLMNRTADHHVPMPILERVLNLARVIDLVYEDGDAYTDSNMMKDYIASLLVNPLVLQ

[0063] SEQ ID NO:4:

[0064] ATGTCATTTCCAATCTCTGCTGTTCCATCTTCTACTCAAATCGAAGCTAGTGGTAAGTTGGTTGGTAGAAATCTGACTCATTTCTCTCCATCTGTTTGGGGTTCTCATTTCTTGAGCTATGCTTCTGATTCCAATGTCTTGGATGCTGATGACGATCATAAGATCATGCAACAAGTTCAAGAATTGAAAGATGAGGTTAAGAGAATGTTGATCATCTCTCCAGGTTCTACTGATCAAACTCTGTCTGAGAAGTTGGACTTGATTGATGCTATCCAACATCTAGGTGTTAGCTACCATTTCGAATCCGAAATCGATGAAATTCTACAGAAGGCTCATAAGATTAATCCACCATGTTTCAACAACATCAACATCATGGATCATGCAGCAGATGACCAATTGAAGTTGTACACCATCTC TTTGTGGTTTAGATTGTTGAGACAACAAGGTTATGACGTTTCTTGTGACATCTTTAATGAGTTCAAAGACGATAAAGGCTCTTTCAAGGAATCTTTGATCTCTGATGTTAAAGGTATGTTGTCTTTGTACGAAGCTGCTCATTTGGGTATCAATGGTGAAGATATATTGGATGAAGCTCTAGCTTTCACTACCACTCATTTGGAATTGGCTGTCAATCATATCAGACCACAATTGGCTAAGAAAGTCAAACACGCTTTGAATAGACCAATCAGAAAGGCTTTGCCAAGATTGGAAGGTTTGTACTACATCTCCATCTACAAAGAAGAAGATTCTCATTCTGAAACTTTGTTGAAGTTCGCTAAATTGGACTTTAACGTTCTACAATCTCAACATCAGAAAGAGATTGGTGGTATCACTAGATGCTGGAAGAATCTGGACTTTACTGCAAACTTGCCATACGCTAGAGATAGAATCGTTGAAGGTTACTTCTGGACTATGGGTGTCTTTTTCGAACCACAATACTCTTTGGCTAGAAGAATCATGACCAAGTCCATTGCTATGACTTCTATCTTGGATGATACCTACGATGCATACGGTAGTTATGCTGAATTAAAGTTGTTCACTGAAGCTATCGAAAGATGGGATGTTTCTGCTATCGACATCTTGCCTGAATACATGAAGTTGATCTACAAGGCTTTGTTGGATGTCTACGATGAAATAGAAGCTGAAACTGCTAAAGATGGTAGACCATTCTGTGTTCATTACGCTAAAGAGTCCATGAAGAAATTGATTCAAGCCTATTTCATTGAAGCTAAATGGTGTAATGAGGGTTATGCTCCAACTATGGAAGAGTACATGTCTAATGCTATGACTACTTCTGCTTACCAAATGTTGACTCCAACCTCTTTCTTGGGTATGGGTAATATCGCTGATGAAGAAGTCTTTAAGTGGGTGTTCAACGATCCAAAGATTCTAAGAGCTTCTACCATCATCTGTAGATTGATGGATGACATCAAGTCTCACAAGTTCGAACAATCTCGTGCTCATGCAGTTTCTGCTGTTGAATGCTACATGAAGCAATATGGTGTCTCTGCTGAAGAAGAGGTTTACAAGTTGTTGGGTAAAGAAATCGTCAATGCTTGGAAAGACATCAACGAAGAACTGTTGATGAACAGAACTGCTGATCATCATGTTCCAATGCCAATCTTAGAAAGAGTTCTGAACTTGGCTAGAGTTATCGACTTGGTTTACGAAGATGGTGATGCTTATACTGACTCCAATATGATGAAAGATTACATTGCTTCTTTGCTGGTTAATCCATTGGTCTTGCAATGA

[0065] (4) Transfer the walnut β-farnesene synthase gene into Saccharomyces cerevisiae cells

[0066] The nucleotide fragment containing the JrbFS gene or its mutant form, constructed above, was transformed into *Saccharomyces cerevisiae* CEN.PK2-1D. The specific process was as follows: 1. Obtain the pLEU2-LEU2-tCYC1-ERG20-JrbFS-tGPM-LEU2R nucleic acid fragment using high-fidelity PCR. 2. Prepare competent *Saccharomyces cerevisiae* cells, transform the amplified nucleic acid fragment into the yeast cells using the LiAc / PEG method, and then perform resuscitation culture. 3. Screen the resuscitated cells on a yeast culture medium without leucine; positive clones were obtained after approximately three days. 4. Isolate the positive clones into single colonies by streak plating, then extract the genome of each single colony, and verify whether the target gene has been inserted into the genome using PCR and sequencing analysis.

[0067] (5) Fermentation preparation of farnesene

[0068] The aforementioned transgenic yeast was inoculated into shake flasks containing YPD medium (1% w / v yeast extract, 2% w / v peptone, 2% w / v glucose) and cultured at 30°C and 250 rpm until the late logarithmic phase. Then, D-galactose was added to a final concentration of 1% (w / v), and n-dodecane was added to a final concentration of 10% (v / v), and the culture was continued for 48 hours. After the culture was completed, the culture was centrifuged; n-dodecane remained on top, indicating that farnesene synthesized by the yeast during fermentation was extracted by n-dodecane. Saccharomyces cerevisiae CEN.PK2-1D was also fermented using the same method, and samples were prepared as a control group.

[0069] (6) Qualitative and quantitative determination of β-farnesene synthesized by fermentation

[0070] The gas chromatography-mass spectrometry (GC-2014C) and DB-5I.D. 30m×0.25mm×0.25μm columns were used as analytical instruments to analyze the n-dodecane extract of the transgenic yeast obtained above, as well as n-dodecane and β-farnesene standards. Specific analytical parameters are shown in Table 3 below.

[0071] Table 3 Gas Chromatography Analysis Parameters

[0072]

[0073] like Figure 3 As shown, the retention time of the β-farnesene standard in our method was 12.8 minutes, and β-farnesene was successfully synthesized by transgenic yeast.

[0074] Example 2: Comparison of the ability of β-farnesene synthases from different sources to synthesize farnesene

[0075] (1) Knocking β-farnesene synthase from different sources into Saccharomyces cerevisiae strains

[0076] The β-farnesene synthases from four plants—Artemisia annua, German chamomile, peppermint, and sweet orange—are widely studied and applied in this field. Therefore, we selected the β-farnesene synthase genes from these four plants for comparison with the β-farnesene synthase gene from walnut. The relevant gene information is shown in Table 4 below:

[0077] Table 4. β-Farnese synthase genes from different sources

[0078] Gene code Biological source NCBI Protein Registry AabFS Artemisia annua, yellow wormwood AAX39387.1 McbFS Matricaria chamomilla, German chamomile AIW60869.1 MpbFS Mentha piperita, peppermint O48935.1 CjbFS Citrus junos, orange Q94JS8.1

[0079] The first step was to construct β-farnesene synthase gene integration plasmids. We replaced the JrbFS element of the pUC-JrbFS plasmid described in Example 1 with the four β-farnesene synthase genes mentioned above, respectively, to obtain four β-farnesene synthase gene integration plasmids, namely pUC-AabFS, pUC-McbFS, pUC-MpbFS, and pUC-CjbFS.

[0080] The second step is to transform the nucleotide fragments containing β-farnesene synthase genes from four different biological sources constructed above into Saccharomyces cerevisiae CEN.PK2-1D. The specific process is as follows: First, four nucleic acid fragments, pLEU2-LEU2-tCYC1-ERG20-JrbFS-tGPM-LEU2R, pLEU2-LEU2-tCYC1-ERG20-AabFS-tGPM-LEU2R, pLEU2-LEU2-tCYC1-ERG20-McbFS-tGPM-LEU2R, pLEU2-LEU2-tCYC1-ERG20-MpbFS-tGPM-LEU2R, and pLEU2-LEU2-tCYC1-ERG20-CjbFS-tGPM-LEU2R, are obtained by high-fidelity PCR. Second, prepare competent cells of *Saccharomyces cerevisiae* CEN.PK2-1D. Transform the amplified nucleic acid fragments obtained above into the yeast cells using the LiAc / PEG method, followed by resuscitation culture. Third, screen the resuscitated cells on yeast culture medium without leucine. Successfully transformed positive clones can be obtained in approximately three days. Fourth, isolate single-clone colonies from the positive clones by streak plating. Extract the genome of each single-clone colony and verify whether the target gene has been inserted into the genome using PCR and sequencing analysis.

[0081] (2) Fermentation preparation of farnesene

[0082] Saccharomyces cerevisiae containing β-farnesene synthase genes from four different sources (Artemisia annua, German chamomile, peppermint, and orange) and Saccharomyces cerevisiae containing JrbFS and JrbFS_M6 genes were inoculated into shake flasks containing YPD medium and cultured at 30°C and 250 rpm until the late logarithmic phase. D-galactose was then added to a final concentration of 1% (w / v), followed by n-dodecane to a final concentration of 10% (v / v), and the culture was continued for 48 hours. After culture, the cultures were centrifuged; n-dodecane remained on top, indicating that farnesene synthesized by the yeast during fermentation was extracted by n-dodecane.

[0083] (3) Quantitative determination of β-farnesene synthesized by fermentation

[0084] Using the gas chromatography method described in Example 1 as the analytical tool, a standard curve was first obtained by analyzing β-farnesene standards at different concentrations. Then, the n-dodecane extracts of the six transgenic yeasts obtained above were analyzed, and the farnesene content was calculated. Figure 4 As shown, under the same conditions, the farnesene yield of *Saccharomyces cerevisiae* containing walnut-derived β-farnesene synthase was 225 mg / L. After rational modification, the farnesene yield of the β-farnesene synthase mutant JrbFS_M6 increased to 286.4 mg / L (p = 0.001387 < 0.05, indicating a significant difference), demonstrating that the enzyme activity was enhanced after rational modification. Therefore, this β-farnesene synthase mutant has a comparative advantage among similar β-farnesene synthases and has potential application value in farnesene biosynthesis.

Claims

1. A β-farnesene synthase mutant, characterized in that, Its amino acid sequence differs from that of wild-type β-farnesene synthase from walnuts by one or more amino acid residues selected from A26T, A169E, Y251H, V323S, G325A, and A438T.

2. The β-farnesene synthase mutant as described in claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

3.

3. A gene encoding β-farnesene synthase, characterized in that, It comprises the β-farnesene synthase mutant as described in claim 1 or 2; preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO:2 or SEQ ID NO:

4.

4. A recombinant expression vector, characterized in that, It contains the gene encoding β-farnesene synthase as described in claim 3; preferably, it also contains the ERG20 gene.

5. A genetically engineered bacterium, characterized in that, It contains the gene encoding β-farnesene synthase as described in claim 3, or the recombinant expression vector as described in claim 4.

6. The genetically engineered bacteria as described in claim 5, characterized in that, Its origin is yeast; Preferably, the yeast is brewer's yeast; More preferably, the brewer's yeast is a leucine auxotrophic yeast, and / or the recombinant expression vector further includes the LEU2 gene.

7. A reaction system or kit for producing farnesene, characterized in that, It includes the genetically engineered bacteria as described in claim 5 or 6; preferably, it also includes one or more selected from the group consisting of: (1) Culture medium, such as YPD medium; (2) D-galactose; (3) One or more of n-dodecane, polyolefin and IPM isopropyl myristate.

8. A method for culturing genetically engineered bacteria as described in claim 5 or 6, characterized in that, The genetically engineered bacteria are placed in a culture medium and cultured to obtain the final product.

9. A method for producing β-farnesene synthase, characterized in that, The genetically engineered bacteria as described in claim 5 or 6 are placed in a culture medium and cultured to obtain the product.

10. A method for producing farnesene, characterized in that, The genetically engineered bacteria as described in claim 5 or 6 are cultured in a culture medium and fermented to produce farnesene. Preferably, the method includes one or more of the following conditions: (i) The culture medium is YPD medium; (ii) The incubation temperature is 25-35℃; (iii) Shaking should be used during cultivation; (iv) Culture the genetically engineered bacteria to the late logarithmic phase, and add D-galactose and / or n-dodecane; More preferably, in (iv), the final content of D-galactose is 0.5-2% by mass, and / or the final content of n-dodecane is 5-15% by mass.

11. The use of the β-farnesene synthase mutant as described in claim 1 or 2, the gene encoding β-farnesene synthase as described in claim 3, the recombinant expression vector as described in claim 4, and the genetically engineered bacteria as described in claim 5 or 6 in the production of farnesene.

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