A beta-farnesene synthase mutant and uses thereof

Abstract

The application provides a beta-farnesene synthetase mutant and application thereof, and belongs to the technical field of protein engineering modification and engineering bacteria. The application firstly performs semi-rational design on beta-farnesene synthetase AanFS by using protein engineering, adopts homologous modeling, uses molecular docking and molecular dynamics simulation, and calculates a mutant beneficial to improve the activity, obtains multiple active sites with improved activity after experimental verification, combines mutation, and obtains the optimal mutant AanFS K197T / F180H The yield of beta-farnesene is increased by 283.8% compared with the wild type. Meanwhile, a Yarrowia lipolytica strain Q26 strain expressing the beta-farnesene synthetase mutant is obtained by using genetic engineering technology, the strain can utilize oleic acid and kitchen waste oil as carbon sources to produce beta-farnesene, is an excellent strain for high-yield beta-farnesene, and therefore has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering and engineered bacteria technology, specifically relating to a β-farnesene synthase mutant and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Farnesene is a natural sesquiterpene produced by plants, comprising two isomers: α-farnesene (3,7,11-trimethyl-1,3,6,10-dodecanetraene) and β-farnesene (7,11-dimethyl-3-methylene-1,6,10-dodecanetriene). It was first isolated and identified from apple peels and is a major component of various plant essential oils in nature, such as *Mentha haplocalyx Briq*, *Chrysanthemum coronarium* L, and *Solanum tuberosum* L. Farnesene is a slightly viscous, oily liquid at room temperature and is highly volatile. It is insoluble in water but soluble in hydrocarbon organic solvents. With increasing understanding of its structure and function, its applications are becoming increasingly widespread, including agriculture, industry, and bioenergy, demonstrating its excellent properties and enormous application potential.

[0004] Farnesene can be obtained through plant extraction, chemical synthesis, and microbial fermentation. However, due to the low content of farnesene in plants and the significant impact of plant growth on factors such as season, region, and climate, the cost of extracting farnesene from plants is high, making it difficult to consistently meet market demand. Some studies have also explored chemical synthesis of farnesene using nerolidol, farnesol, myrcene, or geranyl bromide and butenol as raw materials; however, the availability of raw materials, cost, production efficiency, and environmental pollution are unavoidable problems associated with chemical synthesis. Microbial fermentation, overcoming the drawbacks of natural extraction and chemical synthesis, has become the optimal production method for obtaining farnesene.

[0005] The activity of terpene synthases is often the limiting factor in the heterologous synthesis of terpenes by microorganisms. The turnover rate of terpene synthases is usually less than 0.5 / s, which means that their catalytic efficiency is at a low level. Therefore, improving the catalytic activity of farnesene synthase is a key factor in ensuring high yields of farnesene. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a β-farnesene synthase mutant and its applications. First, this invention uses protein engineering to semi-rationally design the β-farnesene synthase AanFS. Homology modeling is employed, and molecular docking and molecular dynamics simulations are used to calculate mutants that enhance its activity. After experimental verification, multiple activity-enhancing sites are obtained and combined with mutations, resulting in the optimal mutant AanFS. K197T / F180H This study increased β-farnesene production by 283.8% compared to the wild type, significantly enhancing its activity. Simultaneously, using genetic engineering, the *Yarrowia lipolytica* strain Q26 expressing this β-farnesene synthase mutant was obtained. This strain can utilize oleic acid as a carbon source, achieving a β-farnesene production of 35.2 g / L after high-density cultivation; and using kitchen waste oil as a carbon source, the β-farnesene production reaches 31.9 g / L, making it an excellent strain for high-yielding β-farnesene. Based on these research findings, this invention was completed.

[0007] Specifically, the present invention relates to the following technical solutions:

[0008] In a first aspect, the present invention provides a β-farnesene synthase mutant, said β-farnesene synthase mutant having a mutation at one or more sites selected from the group consisting of: F180H, E184H, K197T, L326I, L326V, N332L and R482L, wherein the amino acid residue numbers are as shown in SEQ ID NO.2 (amino acid sequence of wild-type β-farnesene synthase).

[0009] The β-farnesene synthase mutant is a mutation based on the β-farnesene synthase shown in SEQ ID NO.2, specifically a combination of K197T and F180H. This invention has found that among all mutants, the K197T / F180H mutant (AanFS) exhibits the most significant mutation. K197T / F180H It exhibited the best activity, resulting in a 283.8% increase in β-farnesene yield compared to wild-type AanFS.

[0010] In a second aspect, the present invention provides a polynucleotide molecule that encodes the β-farnesene synthase mutant described in the first aspect above.

[0011] A third aspect of the present invention provides a recombinant expression vector containing the polynucleotide molecule described in the second aspect of the present invention.

[0012] In a fourth aspect, the present invention provides a host cell containing the vector described in the third aspect of the invention, a chromosome integrated with the polynucleotide molecule described in the second aspect of the invention, or expressing a β-farnesene synthase mutant described in the first aspect. The host cell possesses a high capacity for β-farnesene production.

[0013] The host cell can be a prokaryotic cell or a eukaryotic cell.

[0014] The host cell is any one or more of bacterial cells, fungal cells, or plant cells.

[0015] Using Yarrowia lipolytica strain Po1f as the starting strain, a strain of Yarrowia lipolytica Q26 was obtained through genetic engineering. This strain was deposited on August 12, 2022, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with the accession number CCTCC NO: M 20221274.

[0016] A fifth aspect of the present invention provides a method for culturing the host cells described above, the method comprising: culturing the host cells in a culture medium.

[0017] The culture medium can be any culture medium suitable for culturing bacterial cells, fungal cells, or plant cells.

[0018] In a sixth aspect, the present invention provides a microbial agent comprising the aforementioned bacterial cells, fungal cells, or their ferments or metabolites, particularly Yersinia lipolytica Q26 or its ferments or metabolites; specifically, the microbial agent contains a β-farnesene synthase mutant.

[0019] A seventh aspect of the present invention provides a method for producing β-farnesene, the method comprising a process of catalytic synthesis of β-farnesene using the above-described β-farnesene synthase mutant; or,

[0020] The process of culturing the above-mentioned host cells using fermentation substrates and then isolating and extracting β-farnesene.

[0021] The eighth aspect of the present invention provides applications of the β-farnesene synthase mutant described in the first aspect, the polynucleotide molecule described in the second aspect, the recombinant expression vector described in the third aspect, the host cell described in the fourth aspect, the culture method described in the fifth aspect, the bacterial agent described in the sixth aspect, and the method for producing β-farnesene described in the seventh aspect in the fields of cosmetics, agriculture, industry, and bioenergy.

[0022] A ninth aspect of the present invention provides a method for obtaining the above-described β-farnesene synthase mutant, the method comprising:

[0023] Semi-rational design of the β-farnesene synthase AanFS (its amino acid sequence is shown in SEQ ID NO.2) was performed using protein engineering techniques. Since information on the three-dimensional structure of farnesene synthase is currently unavailable, homology modeling was used to simulate the spatial structure of AanFS. A complex involving the binding of AanFS to the substrate FPP was constructed using molecular docking and molecular dynamics (MD) simulations, and key amino acid residues involved in FPP binding were calculated. Calculations of these amino acid residues simulated mutants that enhance AanFS activity, and these mutants were experimentally verified to have increased activity. Furthermore, based on molecular docking and molecular dynamics simulations, the possible β-farnesene release region of AanFS was calculated, and key amino acid residues affecting β-farnesene release were predicted. Subsequently, acidic, basic, and amino acids with different side chain types were selected as substitute amino acids to screen for mutants that improve β-farnesene release efficiency, and these mutants were experimentally verified to have increased activity. To further enhance the catalytic activity of AanFS, this invention employs combined mutations of beneficial amino acid residues near the substrate binding region and the product release center, resulting in the optimal mutant AanFS. K197T / F180H This greatly enhances its activity.

[0024] The beneficial technical effects of one or more of the above technical solutions:

[0025] 1. The above technical solution provides a novel β-farnesene synthase mutant, AanFS. K197T / F180H The enzyme was then applied.

[0026] 2. The above technical solution provides multiple beneficial mutation sites for β-farnesene synthase AanFS: F180, E184, K197, L326, L326, N332 and R482.

[0027] 3. The above technical solution provides a novel *Yarrowia lipolytica* yeast that can be used for high-yield β-farnesene production and has broad industrial application prospects; simultaneously, the construction of the engineered strain CCTCC NO: M 20221274 involves a novel protein engineering mutant, AanFS. K197T / F180H , as well as the peroxisome and cytoplasm co-localization of the synthetic pathway.

[0028] 4. The Yarrowia lipolytica β-farnesene production engineered strain constructed above can efficiently synthesize high-value-added β-farnesene using inexpensive oils. Further research shows that adding dodecane during β-farnesene fermentation for two-phase fermentation can effectively extract β-farnesene, saving the cost of product separation and purification in industrial production, and further saving production capacity. Therefore, it has good practical application value. Attached Figure Description

[0029] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0030] Figure 1 This is a structural diagram of the plasmid used in Example 1 of the present invention.

[0031] Figure 2 This is an effect diagram of the AanFS beneficial mutant in Example 2 of the present invention.

[0032] Figure 3 This is a graph showing the mevalonic acid yield of the strain in Example 3 of the present invention.

[0033] Figure 4 This is a graph showing the β-farnesene yield of the strain in Example 3 of the present invention.

[0034] Figure 5 This is a graph showing the oleic acid fermentation yield of the engineered strain Q26 in Example 4 of the present invention.

[0035] Figure 6 This is a graph showing the fermentation yield of kitchen waste oil by engineered strain Q26 in Example 4 of the present invention. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0038] As mentioned earlier, the activity of terpene synthases is often the limiting factor in heterologous terpene synthesis in microorganisms. The turnover rate of terpene synthases is usually less than 0.5 / s, which means that their catalytic efficiency is at a low level. Therefore, improving the catalytic activity of farnesene synthase is a key factor in ensuring high yields of farnesene.

[0039] In view of this, in a typical embodiment of the present invention, a β-farnesene synthase mutant is provided, wherein the β-farnesene synthase mutant is selected from one or more sites of the following group: F180H, E184H, K197T, L326I, L326V, N332L and R482L, wherein the amino acid residue numbers are as shown in SEQ ID NO.2 (amino acid sequence of wild-type β-farnesene synthase).

[0040] The amino acid sequence of the β-farnesene synthase mutant has at least 80% homology with SEQ ID NO.2; more preferably, at least 90% homology; most preferably, at least 95% homology; such as at least 96%, 97%, 98%, or 99% homology.

[0041] The number of mutation sites in the β-farnesene synthase mutant is 1-7, more preferably 1-3, such as 1, 2 or 3.

[0042] Furthermore, the β-farnesene synthase mutant is a mutation based on the β-farnesene synthase shown in SEQ ID NO.2, specifically a combination of K197T and F180H. This invention has found that among all mutants, the K197T / F180H mutant (AanFS) exhibits the most significant mutation. K197T / F180H It exhibited the best activity, resulting in a 283.8% increase in β-farnesene yield compared to wild-type AanFS.

[0043] In another specific embodiment of the present invention, a polynucleotide molecule is provided, the polynucleotide molecule encoding the above-mentioned β-farnesene synthase mutant.

[0044] In another specific embodiment of the present invention, a recombinant expression vector is provided, the recombinant expression vector containing the above-mentioned polynucleotide molecules.

[0045] Specifically, the recombinant expression vector is obtained by effectively linking the above-mentioned polynucleotide molecules to the expression vector. The expression vector is any one or more of viral vectors, plasmids, bacteriophages, kinases, or artificial chromosomes. The viral vector may include adenovirus vectors, retrovirus vectors, or adeno-associated virus vectors. The artificial chromosome includes bacterial artificial chromosomes (BAC), bacteriophage P1-derived vectors (PAC), yeast artificial chromosomes (YAC), or mammalian artificial chromosomes (MAC).

[0046] In another specific embodiment of the present invention, a host cell is provided, wherein the host cell contains the above-mentioned vector, has the above-mentioned polynucleotide molecule integrated into its chromosome, or expresses the above-mentioned β-farnesene synthase mutant. The above-mentioned host cell has a high production capacity of β-farnesene.

[0047] The host cell can be a prokaryotic cell or a eukaryotic cell.

[0048] More specifically, the host cell is any one or more of bacterial cells, fungal cells, or plant cells;

[0049] The bacterial cells mentioned include, but are not limited to, any species of Escherichia coli, Agrobacterium, Bacillus, Streptomyces, Pseudomonas, or Staphylococcus.

[0050] More specifically, the bacterial cells are Escherichia coli, Agrobacterium tumefaciens, Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, and Pseudomonas fluorescens.

[0051] The plants mentioned include, but are not limited to, Artemisia annua plants, Arabidopsis thaliana plants, corn plants, sorghum plants, potato plants, tomato plants, wheat plants, rapeseed plants, rapeseed plants, soybean plants, rice plants, barley plants, and tobacco plants.

[0052] The fungal cells include, but are not limited to, yeast.

[0053] The yeast strain was modified to have peroxisome and cytoplasmic co-localization of the mevalonate synthesis pathway compared to its endogenous activity, and to possess enhanced β-farnesene synthase mutants (including AanFS). K197T / F180HIt possesses the activity of all genes involved in metabolic pathways and the enzymes encoded by the β-oxidation gene POT1, while exhibiting reduced fatty acid metabolism due to the absence of DGA1 and DGA2.

[0054] More specifically, the yeast is Yersinia lipolytica.

[0055] Using Yarrowia lipolytica strain Po1f as the starting strain, a strain of Yarrowia lipolytica Q26 was obtained through genetic engineering. This strain was deposited on August 12, 2022, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with the accession number CCTCC NO: M 20221274.

[0056] The morphological characteristics of the strain are as follows: the strain exhibits yeast and pseudohyphae dimorphism, the cell has a pale white matte surface, and wrinkles appear after prolonged storage.

[0057] The Yarrowia lipolytica Q26 strain was modified to have enhanced β-farnesene synthase mutants (including AanFS) with peroxisome and cytoplasmic co-localization of the mevalonate synthesis pathway compared to its endogenous activity. K197T / F180H It possesses the activity of all genes involved in metabolic pathways and the enzymes encoded by the β-oxidation gene POT1, while exhibiting reduced fatty acid metabolism due to the absence of DGA1 and DGA2.

[0058] Specifically, the construction method of Yarrowia lipolytica Q26 includes:

[0059] Using the uracil- and leucine-auxotrophic Yarrowia lipolytica Po1f as the starting strain, the key enzymes in the mevalonate synthesis pathway, AtoB, HMGR, and HMGS, were co-localized in peroxisomes (each gene's encoded protein has an enhanced peroxisome localization signal ePTS1 fused to its C-terminus) and cytoplasm, and multiple copies of ERG12, IDI, and ERG20AanFS were integrated. K197T / F180H Overexpress all genes in the metabolic pathway and POT1, and knock out DGA1 and DGA2.

[0060] The Yarrowia lipolytica Po1f, with ATCC number MYA-2613 and genotype MATAura3-3021leu2-270xpr2-322axp2-deltaNU49XPR2::SUC2, is described.

[0061] The AanFS is a β-farnesene synthase, its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2; the AanFS K197T / F180H The AanFS mutant has the nucleotide sequence shown in SEQ ID NO.3 and the amino acid sequence shown in SEQ ID NO.4; AtoB is acetyl-CoA acetyltransferase, with the nucleotide sequence shown in SEQ ID NO.5; HMGR is NADH-dependent HMG-CoA reductase, with the nucleotide sequence shown in SEQ ID NO.6; ePTS1 is an enhanced peroxisome localization signal, with the nucleotide sequence shown in SEQ ID NO.7; the enzymes encoded by all genes in the metabolic pathway include, but are not limited to, HMGS, ERG12, IDI, ERG20, GPPS, ERG8, and ERG19; POT1 is 3-ketoacyl-CoA thioase; DGA1 and DGA2 are diacylglycerol acyltransferase 1 / 2; the AanFS... K197T / F180H The number of copies in the genome is 9 or more.

[0062] Specifically, (1) Yarrowia lipolytica Po1f strain was cultured overnight, transformed with linearized plasmid pki-AtoB-HMGR-HMGS based on non-homologous recombination (NHEJ), and AHH12 strain was obtained after the screening marker was recovered by the Cre-LoxP system; (2) CP7 strain was obtained by integrating linearized plasmid pki-AtoB-HMGR-HMGS-ePTS1 into AHH12 strain; (3) Q4 strain was obtained by integrating linearized plasmid YLEP-Leu-ERG20mAan into CP7 strain; (4) linearized plasmid p was integrated into Q4 strain. (5) After integrating the linearized plasmids pki-ERG12-IDI-ERG20mAan and 114-GPPS-ERG8-ERG19 into the Q5 strain, the Q6 strain was obtained; (6) After integrating the linearized plasmid pki-ERG12-IDI-ERG20mAan into the Q6 strain, the Q7 strain was obtained; (7) After overexpressing the POT1 gene in the Q7 strain, the Q12 strain was obtained; (8) After knocking out the DGA1 and DGA2 genes in the Q12 strain, the Q26 strain was obtained.

[0063] In another specific embodiment of the present invention, a method for culturing the above-mentioned host cells is provided, the method comprising: placing the host cells in a culture medium for culturing.

[0064] The culture medium can be any culture medium suitable for culturing bacterial cells, fungal cells, or plant cells.

[0065] When the fungus is *Yarrowia lipolytica*, especially *Yarrowia lipolytica* Q26, it can provide ideal production capacity as an engineered strain for β-farnesene. To better realize the industrial application of the above strain, this invention studies the fermentation culture method of the engineered strain, providing a more suitable industrial continuous fermentation method for the above strain. Furthermore, research on this fermentation method shows that adding dodecane for two-phase fermentation during the culture process can effectively extract β-farnesene, saving the cost of product separation and purification in industrial production and further saving production capacity.

[0066] Therefore, in another specific embodiment of the present invention, a fermentation culture method for Yarrowia lipolytica Q26 is provided. Specifically, the fermentation culture method includes, but is not limited to, a method using oleic acid fermentation culture and a method using waste cooking oil (WCO) fermentation culture.

[0067] In another specific embodiment of the present invention, the oleic acid fermentation culture method includes fermentation culture using 2×YPO and YPO medium;

[0068] The 2×YPO medium comprises 4% peptone, 2% yeast extract, and 5% oleic acid;

[0069] In another specific embodiment of the present invention, the oleic acid fermentation culture method is as follows: the bacterial culture is inoculated into YPO medium and cultured for a period of time to obtain single clones, which are then cultured in shake flasks at 30°C and 220 rpm to obtain seed culture; during the fermentation culture stage, the temperature is 30°C, the pH is 6.5, the aeration rate / stirring rate parameter is 1.5 / 700 vvm / rpm, and sterilized oleic acid is added when the oleic acid content in the fermentation broth is insufficient to maintain 12h.

[0070] In another specific embodiment of the present invention, the fermentation culture method for kitchen waste oil (WCO) includes fermentation culture using 2×YP+WCO and YPO medium; preferably, the 2×YP+WCO medium includes 4% peptone, 2% yeast extract, and 5% kitchen waste oil (WCO).

[0071] In another specific embodiment of the present invention, the cultivation method is as follows: the bacterial culture is inoculated into YPO medium and cultured for a period of time to obtain single clones, which are then cultured in shake flasks at 30°C and 220 rpm to obtain seed culture; during the fermentation culture stage, the temperature is 30°C, the pH is 6.5, the aeration rate / stirring rate parameter is 1.5 / 700 vvm / rpm, and sterilized waste cooking oil (WCO) is added when the content of waste cooking oil (WCO) in the fermentation broth is insufficient to maintain 12h.

[0072] In a sixth aspect, the present invention provides a microbial agent comprising the aforementioned bacterial cells, fungal cells, or their ferments or metabolites, particularly Yersinia lipolytica Q26 or its ferments or metabolites; specifically, the microbial agent contains a β-farnesene synthase mutant.

[0073] In this invention, the term "fermentation product" is used to refer to fermentation products. The corresponding fermentation product can be a liquid obtained from the fermentation culture of the aforementioned bacterial cells or fungal cells (including *Yarrowia lipolytica* Q26), and therefore can also be called a fermentation broth; the liquid may contain bacteria or fungi (cells), but is not necessarily required to contain bacteria or fungi; the liquid preferably contains metabolites produced by *Yarrowia lipolytica* Q26 of this invention, particularly a β-farnesene synthase mutant.

[0074] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is separated from the liquid by centrifugation, filtration, sedimentation, or other means known in the art. The liquid remaining after removing the bacterial cells is called the "supernatant." In the present invention, the supernatant contains extracellular metabolites of bacteria or fungi (including Yersinia lipophila Q26). In embodiments of the present invention, the bacterial agent may also contain the supernatant; the supernatant preferably contains a β-farnesene synthase mutant.

[0075] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is centrifuged, filtered, settled, or otherwise known in the art to separate the bacterial cells grown in the fermentation broth or culture medium from the liquid to obtain bacterial cells. The bacterial cells can be broken up to obtain bacterial fragments. The breaking method can be ultrasound (e.g., ice bath ultrasound to break up cells) or other methods known in the art. Alternatively, the bacterial fragments can be centrifuged to collect the supernatant, which is designated as the cell-free extract. In the present invention, the bacterial fragments or cell-free extract contain intracellular metabolites of bacteria or fungi (including Yersinia lipophila Q26). In embodiments of the present invention, the bacterial agent may also contain the bacterial fragments or cell-free extract.

[0076] Furthermore, in embodiments of the present invention, for ease of storage and transportation, and to improve the survival rate of the bacterial strain, the bacterial agent may also be a solid, and more preferably a lyophilized powder. That is, it is obtained by further freeze-drying the aforementioned bacteria or fungi (including Yersinia lipolyticis Q26) or their ferments or metabolites. The freeze-drying technology (including vacuum freeze-drying technology) can be carried out using conventional methods, and will not be elaborated further here.

[0077] In another specific embodiment of the present invention, the microbial agent may further include excipients acceptable to the microbial agent.

[0078] In another specific embodiment of the present invention, the excipients are selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers, and solvents. The present invention does not impose any special restrictions on the sources of acceptable excipients for the bacterial agent; generally, commercially available products are sufficient. No specific limitations are made here.

[0079] In another specific embodiment of the present invention, a method for producing β-farnesene is provided, the method comprising a process of catalytic synthesis of β-farnesene using the above-mentioned β-farnesene synthase mutant; or,

[0080] The process of culturing the above-mentioned host cells using fermentation substrates and then isolating and extracting β-farnesene.

[0081] When the host cell is a cell or fungus (especially Yersinia lipolytica Q26), the addition of dodecane for two-phase fermentation can effectively extract β-farnesene, thereby saving the cost of product separation and purification in industrial production and further saving production capacity.

[0082] Furthermore, the concentration of dodecane added is 1-20%, such as 1%, 5%, 10%, 15% or 20%, preferably 10%, where the percentage is the volume percentage of the dodecane to the fermentation broth before the addition of the dodecane.

[0083] In another specific embodiment of the present invention, the above-mentioned β-farnesene synthase mutant, polynucleotide molecule, recombinant expression vector, host cell, culture method, bacterial agent or method for producing β-farnesene is provided for application in the fields of cosmetics, agriculture, industry and bioenergy.

[0084] In another specific embodiment of the present invention, a method for obtaining the above-mentioned β-farnesene synthase mutant is provided, the method comprising:

[0085] Semi-rational design of the β-farnesene synthase AanFS (its amino acid sequence is shown in SEQ ID NO.2) was performed using protein engineering techniques. Since information on the three-dimensional structure of farnesene synthase is currently unavailable, homology modeling was used to simulate the spatial structure of AanFS. A complex involving the binding of AanFS to the substrate FPP was constructed using molecular docking and molecular dynamics (MD) simulations, and key amino acid residues involved in FPP binding were calculated. Using the online tool HotSpot Wizard 3.0, calculations of these amino acid residues were performed to simulate mutants that enhance AanFS activity, and these mutants were experimentally verified to have improved activity. Furthermore, this invention calculated the possible β-farnesene release region of AanFS based on molecular docking and molecular dynamics simulations, predicting key amino acid residues affecting β-farnesene release. Subsequently, acidic, basic, and amino acids with different side chain types were selected as substitute amino acids to screen for mutants that improve β-farnesene release efficiency, and these mutants were experimentally verified to have improved activity. To further enhance the catalytic activity of AanFS, this invention employs combined mutations of beneficial amino acid residues near the substrate binding region and the product release center, resulting in the optimal mutant AanFS. K197T / F180H This greatly enhances its activity.

[0086] In another specific embodiment of the present invention, the beneficial mutation sites obtained by the protein engineering technology include, but are not limited to, F180, E184, K197, L326, L326, N332 and R482, but are not limited to the mutants F180H, E184H, K197T, L326I, L326V, N332L and R482L. Any form of alteration to this site should be included within the scope of protection of the present invention.

[0087] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0088] Example 1

[0089] I. Materials and Methods

[0090] 1. Gene synthesis in this invention was completed by Universal Biosystems (Anhui) Co., Ltd.; primer synthesis and sequencing in this invention were completed by Beijing Qingke Biotechnology Co., Ltd.

[0091] 2. Unless otherwise specified, the experimental methods used in the following examples, including plasmid construction, enzyme digestion, preparation of competent cells, and transformation, are all conventional methods. Specific experimental conditions can be determined through simple experiments if necessary.

[0092] 3. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0093] 4. The original strain of Yersinia lipolytica involved in this embodiment, Po1f (ATCC number MYA-2613; genotype MATAura3-3021leu2-270xpr2-322axp2-deltaNU49XPR2::SUC2), was purchased from ATCC.

[0094] 5. The genes involved in this embodiment are derived from AanFS of Artemisia annua, which were synthesized after codon optimization (General Biotechnology, Anhui, China); AtoB, HMGR, HMGS, ERG12, IDI, ERG20, GPPS, ERG8, ERG19, POT1, DGA1 and DGA2 genes were cloned from the genome of Yersinia lipolytica.

[0095] 6. LB solid medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride, 2% agar powder.

[0096] LB liquid medium: 1% peptone, 0.5% yeast extract, 1% sodium chloride.

[0097] YPD medium: 2% peptone, 1% yeast extract, 2% glucose.

[0098] YPD solid medium: 2% peptone, 1% yeast extract, 2% glucose, 2% agar powder.

[0099] II. Amplification of Gene Elements and Preparation of Target Plasmids

[0100] (I) Preparation of target gene

[0101]

[0102] 2. Based on the nucleotide sequence of the β-farnesene synthase gene AanFS (GenBank: AY835398.1) from Artemisia annua provided on NCBI, after codon optimization, it was synthesized and optimized by General Biosystems (Anhui) Co., Ltd. The optimized amino acid sequence of AanFS is shown in SEQ ID NO.2 (MSTLPISSVSFSSSTSPLVVDDKVSTKPDVIRHTMNFNASIWGDQFLTYDEPEDLVMKKQLVEELKEEVK). KELITIKGSNEPMQHVKLIELIDAVQRLGIAYHFEEEIEEALQHIHVTYGEQWVDKENLQSISLWFRLLRQQGFNVSSGVFKDFMDEKGKFKESLCNDAQGILALYEAAFMRVEDETILDNALEFTK VHLDIIAKDPSCDSSLRTQIHQALKQPLRRRLARIEALHYMPIYQQETSHDEVLLKLAKLDFSVLQSMHKKELSHICKWWKDLDLQNKLPYVRDRVVEGYFWILSIYYEPQHARTRMFLMKTCMWLV VLDDTFDNYGTYEELEIFTQAVERWSISCLDMLPEYMKLIYQELVNLHVEMEESLEKEGKTYQIHYVKEMAKELVRNYLVEARWLKEGYMPTLEEYMSVSMVTGTYGLMIARSYVGRGDIVTEDTFKWVSSYPPIIKASCVIVRLMDDIVSHKEEQERGHVASSIECYSKESGASEEEACEYISRKVEDAWKVINRESLRPTAVPFPLLMPAINLARMCEVLYSVNDGFTHAEGDMKSYMKSFFVHPMVV*) is shown in the figure.

[0103] 3. Based on the highly active AanFS mutant AanFS screened through protein engineering. K197T / F180H

[0104] 4. The highly active AanFS mutant AanFS screened by protein engineering K197T / F180H , whose amino acid sequence is shown in SEQ ID NO.4 (MSTLPISSVSFSSSTSPLVVDDKVSTKPDVIRHTMNFNASIWGDQFLTYDEPEDLVMKKQLVEELKEEVK KELITIKGSNEPMQHVKLIELIDAVQRLGIAYHFEEEIEEALQHIHVTYGEQWVDKENLQSISLWFRLLRQQGFNVSSGVFKDFMDEKGKFKESLCNDAQGILALYEAAHMRVEDETILDNALEFTTVHLDIIAKDPSCDSSLRTQIHQALKQPLRRRLARIEALHYMPIYQQETSHDEVLLKLAKLDFSVLQSMHKKELSHICKWWKDLDLQNKLPYVRDRVVEGYFWILSIYYEPQHARTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSISCLDMLPEYMKLIYQELVNLHVEMEESLEKEGKTYQIHYVKEMAKELVRNYLVEARWLKEGYMPTLEEYMSVSMVTGTYGLMIARSYVGRGDIVTEDTFKWVSSYPPIIKASCVIVRLMDDIVSHKEEQERGHVASSIECYSKESGASEEEACEYISRKVEDAWKVINRESLRPTAVPFPLLMPAINLARMCEVLYSVNDGFTHAEGDMKSYMKSFFVHPMVV*)

[0105]

[0106]

[0107] 7. According to the literature DeLoache, WC, Russ, ZN, and Dueber, JE (2016) Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways, Nature communications 7, 11152, the enhanced peroxisome localization signal ePTS1 has the nucleotide sequence shown in SEQ ID NO.7 (CTGGGCCGAGGACGACGATCCAAGCTG).

[0108] 8. Based on the different AanFS mutants designed by protein engineering, the primers in Table 1 were used to reverse PCR from the plasmid YLEP-Leu-AanFS (Table 1 only lists the primers used for the beneficial mutants).

[0109] 9. Based on the HMG-CoA synthase gene HMGS (GenBank: YALI0_F30481g), mevalonate kinase gene ERG12 (GenBank: YALI0_B16038g), IPP isomerase gene IDI (GenBank: YALI0_F04015g), FPP synthase gene ERG20 (GenBank: YALI0_E05753g), GPP synthase gene GPPS (GenBank: YALI0_D17050g), and mevalonate kinase gene from Yarrowia lipolytica provided on NCBI, The nucleotide sequences of the enzyme gene ERG8 (GenBank: YALI0_E06193g), the mevalonate pyrophosphate decarboxylase gene ERG19 (GenBank: YALI0_F05632g), the 3-ketoacyl-CoA thioase gene POT1 (GenBank: YALI0_E18568g), and the diacylglycerol acyltransferase 1 / 2 genes DGA1 (GenBank: YALI0_E32769g) and DGA2 (GenBank: YALI0_D07986g) were obtained by PCR amplification from the Yarrowia lipolytica genome using the primers in Table 1.

[0110] Table 1 Primer sequences

[0111]

[0112]

[0113] (II) Construction of plasmids

[0114] The structures of the plasmids used in this embodiment are shown in [reference needed]. Figure 1 .

[0115] 1. The gene knockout or knock-in system used in this embodiment is based on the existing CRISPR / Cas9 operating system. Plasmid construction for different sgRNAs was based on the purchased plasmid pCRISPRyl, which has an AvrII restriction site. Upstream of this site is the promoter SCR1'-tRNAGly, used to initiate sgRNA expression. Inserting a 20bp sequence of the sgRNA into this site yields the sgRNA plasmid. The sgRNA sequence used for knocking out gene DGA1 is 5'-ccccgctatcgacaccattactc-3', and the sgRNA sequence used for knocking out gene DGA2 is 5'-tcgtccaccctgtcgctcagcgg-3'.

[0116] 2. The backbone plasmid used in this embodiment

[0117] The backbone plasmids pki-2, pki-1, YLEP-Leu, JMP-114, and 113-GPD-TEF were prepared according to the method described in Cui, Z., Jiang, X., Zheng, H., Qi, Q., and Hou, J. (2019) Homology-independent genome integration enables rapid library construction for enzyme expression and pathway optimization in Yarrowia lipolytica, Biotechnology and bioengineering 116, 354-363.

[0118] 3. Construction of plasmid YLEP-Leu-AanFS

[0119] The plasmid YLEP-Leu-AanFS was prepared using YLEP-Leu as its backbone. The AanFS gene was inserted into the ut8 expression frame of the YLEP-Leu plasmid using the conventional Gibson assembly method to obtain the YLEP-Leu-AanFS plasmid.

[0120] 4. Construction of plasmid YLEP-Leu-ERG20mAan

[0121] The plasmid YLEP-Leu-ERG20mAan was prepared using YLEP-Leu-AanFS as its backbone. The ERG20 gene was fused to the N-terminus of the protein encoded by AanFS using standard Gibson assembly. AanFS was then constructed via inverse PCR and standard Gibson assembly. K197T / F180H The mutant plasmid is YLEP-Leu-ERG20mAan.

[0122] 5. Construction of plasmid pki-ERG12-IDI-ERG20mAan

[0123] The plasmid pki-ERG12-IDI-ERG20mAan was prepared using pki-2 and pki-1 as a backbone. The IDI gene was inserted downstream of the TEFin promoter in the pki-2 plasmid using standard Gibson assembly. The ERG20AanFS gene in the YLEP-Leu-ERG20mAan plasmid was then extracted. K197T / F180H The gene was inserted downstream of the hp4d promoter in the pki-2 plasmid; ERG12 was constructed into the ut8 expression frame of the pki-1 plasmid and inserted into the NheI restriction site of the pki-2 plasmid to obtain the pki-ERG12-IDI-ERG20mAan plasmid.

[0124] 6. Construction of plasmid pki-AtoB-HMGR-HMGS

[0125] The plasmid pki-AtoB-HMGR-HMGS was prepared using pki-2 and pki-1 as a backbone. The AtoB gene was inserted downstream of the TEFin promoter in the pki-2 plasmid using the standard Gibson assembly method, and HMGS was inserted downstream of the hp4d promoter in the pki-2 plasmid. HMGR was constructed into the ut8 expression frame of the pki-1 plasmid and inserted into the NheI restriction site of the pki-2 plasmid to obtain the pki-AtoB-HMGR-HMGS plasmid.

[0126] 7. Construction of plasmid pki-AtoB-HMGR-HMGS-ePTS1

[0127] The plasmid pki-AtoB-HMGR-HMGS-ePTS1 was obtained by fusing the enhanced peroxisome localization signal ePTS1 to the C-terminus of the protein encoded by each gene through reverse PCR and conventional Gibson assembly.

[0128] 8. Construction of plasmid 114-GPPS-ERG8-ERG19

[0129] Plasmid 114-GPPS-ERG8-ERG19 was prepared using JMP-114 and 113-GPD-TEF as its backbone. The ERG8 gene was inserted downstream of the GPD promoter in the 113-GPD-TEF plasmid using standard Gibson assembly, and the ERG19 gene was inserted downstream of the TEFin promoter in the 113-GPD-TEF plasmid. The GPPS gene was constructed downstream of the EXP promoter in the JMP-114 plasmid. Finally, the ERG8 and ERG19 expression cassettes were inserted into the JMP-114 plasmid to obtain the 114-GPPS-ERG8-ERG19 plasmid.

[0130] 9. Construction of plasmid YLEP-nat-POT1

[0131] The plasmid YLEP-nat-POT1 was prepared using YLEP-Leu as its backbone. The norovirus resistance gene nat was replaced with the Leu gene in the YLEP-Leu plasmid using the conventional Gibson assembly method to obtain the YLEP-nat plasmid. Then, POT1 was inserted downstream of the ut8 promoter in the YLEP-nat plasmid to obtain the YLEP-nat-POT1 plasmid.

[0132] Example 2: Obtaining the β-farnesene synthase mutant AanFS K197T / F180H

[0133] The wild-type β-farnesene synthase AanFS has low catalytic activity. In this example, protein engineering was used to semi-rationally design AanFS.

[0134] Since information on the three-dimensional structure of farnesene synthase is currently unavailable, this embodiment employs homology modeling, using four known protein structures with PDB IDs of 5EAU, 1N1B, 4GAX, and 4FJQ as templates to simulate the spatial structure of AanFS. Molecular docking and molecular dynamics (MD) simulations were used to construct the complex of AanFS binding to the substrate FPP, and F180, E184, K197, Y288, V289, G296, T319, C320, V325, L326, D327, F330, N332, Y336, C464, I466, R482, and T554 were identified as key amino acid residues involved in FPP binding. Using the online tool HotSpotWizard 3.0, mutants that enhance AanFS activity were simulated, as follows: F180H, E184H, K197T, Y288F, V289A, G296C, T319V, C320I, V325I, L326I, L326V, D327S, F330Y, N332S, Y336L, C464A, C464S, I466V, R482K, and T554S.

[0135] These amino acid mutations were introduced into the YLEP-Leu-AanFS free expression plasmid and transformed into Yersinia lipolytica. By comparing the β-farnesene yield of transformants containing different plasmids through fermentation, it was found that the F180H, E184H, K197T, L326I, and L326V mutants increased the β-farnesene yield by 89.1%, 15%, 193.9%, 27.5%, and 66% respectively compared with the wild type. Other mutations led to varying degrees of reduction in β-farnesene yield or complete loss of β-farnesene synthesis ability.

[0136] On the other hand, the release of farnesene may be a key factor limiting the activity of farnesene synthase. Therefore, in this embodiment, the possible β-farnesene release region of AanFS was calculated based on molecular docking and molecular dynamics simulations. D327, N332, R482, and T554 were found to be key amino acid residues affecting β-farnesene release. Subsequently, acidic, basic, and amino acid residues with different side chain types were selected as substitutes to screen for mutants that could improve β-farnesene release efficiency. These amino acids included phenylalanine, histidine, lysine, aspartic acid, leucine, serine, and alanine.

[0137] After introducing these amino acids into D327, N332, R482, and T554, respectively, the yield of β-farnesene was compared by transforming *Yarrowia lipolytica*, selecting transformants, and fermentation. It was found that only the N332L and R482L mutants increased the yield of β-farnesene by 34% and 102.2%, respectively, compared to the wild type. It is speculated that these two mutations may have hindered the formation of ionic bonds in the farnesene release region, thus allowing for more efficient release of β-farnesene from *AanFS*. Other mutations resulted in the near-complete loss of β-farnesene synthesis ability in *AanFS*.

[0138] To further enhance the catalytic activity of AanFS, this embodiment employed combined mutations of beneficial amino acid residues near the substrate binding region and the product release center. Among all combinations, the K197T / F180H mutant (AanFS) showed the best performance. K197T / F180H The assay exhibited the best activity, increasing the yield of β-farnesene by 283.8% compared to AanFS. The results showed that all mutants combined with the product release regions N332L and R482L lost the ability to synthesize β-farnesene, possibly because the introduction of N332L and R482L made the structure and function of AanFS more fragile.

[0139] Subsequently, this study purified AanFS. K197T / F180H The mutant and AanFS proteins were further confirmed by in vitro catalysis. K197T / F180H The effects of the mutants are consistent with the in vivo catalytic activity trend. This result indicates that the introduction of the K197T / F180H mutant residues does indeed improve the catalytic activity of AanFS. This experiment also demonstrates that semi-rational protein engineering is an effective method for improving the catalytic activity of terpene synthases, especially for terpene synthases lacking three-dimensional structural information and high-throughput screening methods.

[0140] Example 3: Construction of β-farnesene-producing Yersinia lipolytica engineered strain Q26

[0141] 1. Yarrowia lipolytica Po1f strain was cultured overnight, transformed into linearized plasmid pki-AtoB-HMGR-HMGS based on non-homologous recombination (NHEJ), and AHH12 strain was obtained after the marker was recovered and screened using the Cre-LoxP system.

[0142] The specific methods are as follows: (1) Yarrowia lipolytica Po1f was cultured overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose), and competent cells were prepared using the conventional method for preparing competent cells of yeast lithium acetate. (2) 1-5 μg of pki-AtoB-HMGR-HMGS plasmid linearized by SspI restriction endonuclease was added to 40 μL of competent cells, followed by 2-5 μL of salmon sperm DNA, and incubated at 30℃ for 15 min. (3) 280 μL of 50% PEG4000, 70 μL of 0.5M lithium acetate (pH 6.0) and 16 μL of 1MDTT (final concentration 40 mM) were added to the above system, and incubated at 30℃ for 1 h. (4) 40 μL of LDMSO (final concentration about 10%) was added to the above system, and heat-shocked at 39℃ for 10 min. (5) Add 600 μL of 0.1 M lithium acetate (pH 6.0) and incubate at 30 °C for 1 h. (6) Centrifuge to collect the bacterial cells, spread them on screening plates containing the corresponding antibiotics or auxotrophic medium, and incubate at 30 °C for 2-3 days. (7) Randomly select 12-24 transformants, inoculate them on YPD medium and incubate for 96 h, and detect the mevalonic acid production in the transformants. In summary, the method for detecting mevalonic acid is as follows: Take 1 mL of fermentation broth, centrifuge at 13000 rpm for 5 min. When glucose is used as the carbon source, aspirate the supernatant and filter it through a 0.22 μm aqueous filter membrane. Transfer the supernatant to a liquid chromatography vial for quantitative detection by liquid chromatography. When oleic acid or other oils are used as the carbon source, transfer the supernatant to a new 1.5 mL centrifuge tube, add 200 μL of n-hexane, and shake at 220 rpm for 20 min to extract residual oils in the culture medium. Centrifuge at 13000 rpm for 5 min to remove the upper organic phase. Add another 200 μL of n-hexane and repeat once. Centrifuge at 13000 rpm for 5 min to remove the upper organic phase. Filter the supernatant through a 0.22 μm aqueous filter membrane and transfer the supernatant to a liquid chromatography vial for quantitative detection by liquid chromatography. The liquid chromatography detection procedure is as follows: Aminex HPX-87H column (300×7.8mm) was used, the mobile phase was 5mM H2SO4, the flow rate was 0.6mL / min, the detector was a differential detector, and the total program duration was 25min.

[0143] Compared to the original strain Po1f, strain AHH12 exhibited a strong mevalonic acid synthesis capacity, achieving a yield of 1.96 g / L. This strain can serve as a starting strain for β-farnesene synthesis and can also be used as a production platform for other terpenes.

[0144] 2. Furthermore, when oleic acid was used as a carbon source, strain AHH12 accumulated only 0.34 g / L of mevalonic acid under the same fermentation conditions. To construct a platform strain for efficient mevalonic acid synthesis using lipids, this embodiment localized the mevalonic acid synthesis pathway to the peroxisome. Similarly, strain CP7 was obtained by integrating the linearized plasmid pki-AtoB-HMGR-HMGS-ePTS1 into strain AHH12 based on non-homologous recombination (NHEJ). The transformation and detection methods were the same as described above.

[0145] The CP7 strain accumulated 1.90 g / L of mevalonic acid after 96 h of fermentation in a medium using oleic acid as a carbon source, which was 5.3 times higher than that accumulated by the AHH12 strain under the same fermentation conditions. Furthermore, the CP7 strain accumulated 1.84 g / L of mevalonic acid using glucose as a carbon source, which was essentially consistent with the accumulation of the AHH12 strain using glucose as a carbon source. This indicates that using lipids as a carbon source and localizing the mevalonic acid synthesis pathway within the peroxidase significantly improves the conversion efficiency of acetyl-CoA to mevalonic acid within the peroxidase. Thus, a platform strain, CP7, was obtained that efficiently synthesizes mevalonic acid using lipids, providing abundant mevalonic acid precursors for the synthesis of β-farnesene.

[0146] 3. To enhance the metabolic intensity of downstream β-farnesene synthesis, this embodiment uses AanFS. K197T / F180H The plasmid YLEP-Leu-ERG20 was fused with ERG20 in the CP7 strain to obtain the Q4 strain. The transformation method was the same as described above.

[0147] 12–24 transformants were randomly selected and inoculated into YPO medium supplemented with 10% dodecane for two-phase fermentation for 96 h. The yield of β-farnesene in the transformants was then detected. The β-farnesene detection method is as follows: 1–2 mL of fermentation broth was centrifuged at 13000 rpm for 5 min, and the upper dodecane layer was collected and transferred to a gas chromatography vial for quantitative detection by gas chromatography. The gas chromatography detection procedure is as follows: The gas chromatograph is equipped with a flame ionization detector (FID) and an Rtx-5 column (30.0 m, 0.25 mm ID, 0.25 μm df; RESTEK, USA); the injection volume is 1 μL, the injection port temperature is set to 280 °C, the detector temperature is set to 290 °C, the column oven initial temperature is 80 °C and held for 1 min, the temperature is increased to 250 °C at a rate of 10 °C / min and held for 1 min, the temperature is increased to 280 °C at a rate of 10 °C / min and held for 10 min, the carrier gas used is nitrogen, the flow rate is 30 mL / min, and the split ratio is set to 50.

[0148] ERG20AanFS K197T / F180HThe fusion form is more conducive to the synthesis of β-farnesene, and the resulting Q4 strain accumulated 0.52 g / L of β-farnesene after 96 h of fermentation.

[0149] 4. Further, the key rate-limiting enzymes IDI1 and ERG12 of the mevalonate pathway were overexpressed in strain Q4. Specifically, strain Q5 was obtained by integrating the linearized plasmid pki-ERG12-IDI-ERG20mAan into strain Q4. The β-farnesene yield of strain Q5 was increased by 51.9% compared to Q4, reaching 0.79 g / L. The transformation and detection methods were the same as those described above.

[0150] 5. Furthermore, the linearized plasmids pki-ERG12-IDI-ERG20mAan and 114-GPPS-ERG8-ERG19 were integrated into strain Q5, enabling overexpression of all genes in the mevalonate pathway. The resulting strain Q6 exhibited a significantly increased β-farnesene yield, reaching 1.21 g / L, a 53.1% increase compared to strain Q5. The transformation and detection methods were the same as described above.

[0151] 6. The ERG12, IDI, and ERG20mAan gene expression cassettes were randomly integrated again into strain Q6, i.e., the linearized plasmid pki-ERG12-IDI-ERG20mAan was integrated, resulting in strain Q7, which further increased the β-farnesene yield by 28.9%, reaching 1.56 g / L. The transformation and detection methods were the same as those described above.

[0152] 7. Fatty acids taken from the culture medium by *Yarrowia lipolytica* are converted to acetyl-CoA via β-oxidation. This process requires the participation of a series of enzymes. In this example, multiple related enzymes were overexpressed to enhance the strain's ability to utilize fatty acids, thereby providing a richer source of acetyl-CoA precursors for the synthesis of β-farnesene. When 3-ketoacyl-CoA thioase POT1 was overexpressed in strain Q7, the yield of β-farnesene increased by 69%, with the most significant effect, reaching 2.52 g / L, thus obtaining strain Q12. The transformation and detection methods were the same as those described above.

[0153] 8. *Yarrowia lipolytica* exhibits strong fatty acid synthesis metabolism, which consumes a large amount of carbon source and reduces the carbon metabolic flux directed towards the synthesis of target products. When oleic acid was used as the carbon source, after 96 hours of fermentation in shake flasks, the intracellular lipid accumulation of strain Q12 was 96.3 mg / g DCW, lower than the 142.9 mg / g DCW accumulation of strain CP7, and even lower than the 179.8 mg / g DCW accumulation of strain Po1f. This result indicates that enhancing the farnesene synthesis pathway in strain Q12 is beneficial for reducing the allocation of carbon source to fatty acid synthesis. Diacylglycerol acyltransferases DGA1 and DGA2 catalyze the synthesis of TAGs from diacylglycerols (DAGs). Based on the existing CRISPR / Cas9 system, simultaneously knocking out DGA1 and DGA2 in strain Q12 yielded strain Q26, which reduced lipid accumulation to 24.1 mg / g DCW and significantly increased β-farnesene yield by 33.1%, reaching 3.34 g / L. When using waste cooking oil (WCO) as a carbon source, strain Q26 accumulated 3.64 g / L β-farnesene under the same fermentation conditions, indicating that this engineered strain has excellent prospects for industrial application.

[0154] Example 4: Fed-feed fermentation culture of strain Q26

[0155] The high-yield β-farnesene-producing Yarrowia lipolytica Q26, constructed through genetic engineering as described in Example 3, was subjected to a fed-batch fermentation experiment in a 5-L fermenter.

[0156] In this embodiment, the oleic acid fermentation method used 2×YPO medium (4% peptone, 2% yeast extract, 5% oleic acid) with a working volume of 4L. The bacterial culture was inoculated into YPO medium and cultured for a period of time. Single clones were obtained and cultured in shake flasks at 30℃ and 220rpm to obtain seed culture, which was then transferred to a fermenter. The fermentation temperature was 30℃, pH was 6.5, and the aeration / stirring rate parameters were 1.5 / 700 vvm / rpm. When the oleic acid content in the fermentation broth was insufficient to sustain fermentation for 12 hours, an appropriate amount of sterilized oleic acid was added. Samples were taken every 24 hours to detect the β-farnesene content and the biomass of the bacteria in the fermentation broth. Figure 4 The fermentation results showed that the β-farnesene yield reached 35.2 g / L after 216 h of fermentation, with a yield of 0.17 g / g oleic acid, and continued to accumulate. This is the highest β-farnesene yield reported to date in Yersinia lipolytica.

[0157] In this embodiment, the culture medium used for the fermentation of waste cooking oil (WCO) was 2×YP+WCO medium (4% peptone, 2% yeast extract, 5% waste cooking oil (WCO)), with a working volume of 4L. The bacterial culture was inoculated into YPO medium and cultured for a period of time. Single clones were obtained and cultured in shake flasks at 30℃ and 220rpm to obtain seed culture, which was then transferred to a fermenter. The fermentation temperature was 30℃, the pH was 6.5, and the aeration / stirring rate parameters were 1.5 / 700 vvm / rpm. When the waste cooking oil (WCO) content in the fermentation broth was insufficient to maintain the fermentation for 12 hours, an appropriate amount of sterilized waste cooking oil (WCO) was added. Samples were taken every 24 hours to detect the β-farnesene content and the biomass of the bacteria in the fermentation broth. Figure 5 The fermentation results showed that the concentration reached 31.9 g / L after 216 h of fermentation, with a yield of 0.16 g / g WCO. The Q26 strain has great potential for the sustainable synthesis of high-value-added β-farnesene from waste oils, which can help improve the economic value of waste oils.

[0158] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A β-farnesene synthase mutant, characterized in that, The β-farnesene synthase mutants are selected from the following groups of sites with mutations: F180H, K197T / F180H, wherein the amino acid sequence of the wild-type β-farnesene synthase is shown in SEQ ID NO.

2.

2. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the β-farnesene synthase mutant of claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the polynucleotide molecule of claim 2; The recombinant expression vector is obtained by effectively linking the polynucleotide molecule of claim 2 to the expression vector.

4. A host cell, characterized in that, The host cell contains the recombinant expression vector of claim 3, the chromosome is integrated with the polynucleotide molecule of claim 2, or expresses the β-farnesene synthase mutant of claim 1; The host cell can be any one or more of bacterial cells and fungal cells; The bacterial cells are Escherichia coli, Agrobacterium tumefaciens, Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, and Pseudomonas fluorescens. The fungal cells are yeasts; The yeast strain is modified to have, compared to its endogenous activity, co-localization of the peroxisome and cytoplasmic regions of the mevalonate synthesis pathway, and possesses enhanced β-farnesene synthase mutants as described in claim 1, as well as all genes of the metabolic pathway and β-oxidation genes. POT1 The activity of the encoded enzyme, while having the deficiency DGA1 and DGA2 And weakened fatty acid metabolism; The yeast strain is *Yarrowia lipolytica*. Yersinia lipophila strain ( Yarrowia lipolytica Using Po1f as the starting strain, a strain of Yersinia lipophila was obtained through genetic engineering. Yarrowia lipolytica strain Q26 was deposited at the China Center for Type Culture Collection on August 12, 2022, with the following accession number: CCTCC NO: M 20221274.

5. The method for culturing the host cells according to claim 4, characterized in that, The method includes: culturing the host cells in a culture medium; The culture medium is any culture medium suitable for culturing bacterial cells, fungal cells or plant cells; When the fungus Yersinia lipophila ( Yarrowia lipolytica In Q26, the fermentation culture method includes a method using oleic acid fermentation culture and a method using kitchen waste oil fermentation culture; The oleic acid fermentation culture method includes fermentation culture using 2×YPO and YPO medium; The 2×YPO medium comprises 4% peptone, 2% yeast extract, and 5% oleic acid; The method for fermenting and culturing kitchen waste oil includes fermentation culture using 2×YP+WCO and YPO medium. The 2×YP+WCO medium comprises 4% peptone, 2% yeast extract, and 5% kitchen waste oil.

6. A microbial agent, characterized in that, The bacterial agent comprises the host cell described in claim 4; The microbial agent also includes excipients acceptable to the microbial agent.

7. A method for producing β-farnesene, characterized in that, The method includes a process of catalytically synthesizing β-farnesene using the β-farnesene synthase mutant of claim 1; or... The process of culturing the host cells of claim 4 using a fermentation substrate and then isolating and extracting β-farnesene.

8. The method as described in claim 7, characterized in that, When the host cell is a cell or fungus, dodecane is added during culture; The concentration of dodecane added is 1-20%.

9. The use of the β-farnesene synthase mutant of claim 1, the polynucleotide molecule of claim 2, the recombinant expression vector of claim 3, the host cell of claim 4, the culture method of claim 5, the bacterial agent of claim 6, or the method for producing β-farnesene according to any one of claims 7-8 in the fields of cosmetics, agriculture, industry, and bioenergy.

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