Farnesyl transferase mutants and uses thereof

Abstract

The application discloses a farnesol synthase mutant and application thereof. Through directional evolution on farnesol synthase PAH1 from Saccharomyces cerevisiae, a mutant containing at least one of G154R, E171D, E222V, F246Y, L273S, S589C, E614D, T632F or V642I is obtained. The farnesol yield of a strain expressing the mutant is up to 5.31 times (531%) of the wild type. The application also relates to a nucleic acid molecule encoding the mutant, a recombinant vector, a recombinant cell and application thereof in biosynthesis of farnesol. The mutant significantly improves the production efficiency of farnesol, and provides a new strategy for industrial applications such as fungicides and spices.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically relating to a farnesol synthase mutant and its applications. Background Technology

[0002] Farnesol (molecular formula: C15H26O), also known as farnesol or farnesol, is an aromatic, noncyclic sesquiterpenol. It is widely distributed in many plant essential oils and also exists in animals and microorganisms. It plays an important role in signal transduction, quorum sensing, and apoptosis induction, and can be used as a bactericidal cleaning agent, insecticide, and insect attractant.

[0003] Currently, the yield of farnesol synthesized by microbial engineered strains is relatively low, making it difficult to meet market demand. The main limiting factor is the low activity of farnesol synthase. Therefore, obtaining highly active farnesol synthase is a major bottleneck and research hotspot in the efficient biosynthesis of farnesene. Developing highly active farnesol synthase mutants is of great significance for the production and application of farnesol. The pathway for yeast to synthesize farnesol is as follows: Figure 1 As shown.

[0004] Guo Junqi et al. enhanced the expression of truncated forms of HMG-CoA reductase (tHMGR1) and FPP synthase (ERG20) in the mevalonate pathway in CEN.PK2-1D (farnesol yield <0.1 mg / L), increasing farnesol yield by approximately 50.8 times to 5.08 mg / L. Replacing the squalene synthase encoding gene ERG9 promoter with the HXT1 promoter to downregulate its expression level further increased farnesol yield by 47.1 times to 239.17 mg / L. Based on enhancing the precursor enzymes and inhibiting the competitive pathway, the expression of endogenous phosphatase and heterologous farnesol synthase using galactose-inducible promoters both promoted farnesol synthesis. The enhanced expression of endogenous phosphatase PAH1 had the most significant effect on increasing farnesol yield (see Guo Junqi et al., Metabolic Engineering to Improve Farnesol Yield in Saccharomyces cerevisiae, Acta Microbiologica Sinica, 2021, 61(05)).

[0005] Existing technology CN113969288A discloses a method for constructing a farnesol-producing genetically engineered strain. This method utilizes homologous recombination to integrate and express the truncated form of the HMG-CoA reductase gene (tHMG1), the rate-limiting enzyme of the MVA pathway, and the fused expression of the FPP synthase gene (ERG20) and the endogenous phosphatase gene (PAH1) of *Saccharomyces cerevisiae*, thereby enhancing the metabolic intensity of the MVA pathway and increasing farnesol expression. Simultaneously, the copper ion-inducible promoter pCUP1 is used to replace the squalene synthase promoter ERG9, downregulating the ergosterol competitive pathway and increasing farnesol production; ultimately, a farnesol-producing genetically engineered strain is obtained. The farnesol yield of this genetically engineered strain can reach approximately 573 mg / L in shake-flask fermentation and approximately 21 g / L in fermenter, fully meeting commercial production standards and showing good industrial application prospects.

[0006] However, the existing technologies mentioned above all utilize PAH1 for the synthesis of farnesol, and none of them have studied its mutants. Mutants are an important technical means of modifying genes and enzymes. Therefore, it is urgent to develop farnesol synthase mutants to improve the production performance of farnesol synthesized by strains and further expand the application prospects of biosynthetic farnesol. Summary of the Invention

[0007] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a farnesol synthase mutant with an amino acid sequence of any of the following: (1) The sequence shown in SEQ ID NO: 1 obtained by mutation of at least one of G154R, E171D, E222V, F246Y, L273S, S589C, E614D, T632F or V642I; (2) A sequence which is functionally equivalent to the sequence described in (1) by substitution, deletion or addition of 1-2 amino acid residues; (3) A sequence that has ≥90% sequence homology and is functionally equivalent to (1) or (2).

[0008] In some embodiments, the mutation is a combination of G154R / L273S, G154R / E614D, L273S / E614D, or L273S.

[0009] The present invention also provides a nucleic acid molecule encoding a farnesol synthase mutant as described in any of the preceding claims.

[0010] The present invention also provides a recombinant vector comprising the above-described nucleic acid molecules.

[0011] The present invention also provides a recombinant cell comprising the above-described recombinant vector or whose genome integrates the above-described nucleic acid molecules.

[0012] In some embodiments, the cells are Saccharomyces cerevisiae.

[0013] In some embodiments, the brewer's yeast is a CEN.PK2-1D strain or a derivative thereof.

[0014] The present invention also provides a method for producing farnesol, which utilizes the recombinant cell fermentation described in any of the above-mentioned methods to produce farnesol.

[0015] The present invention also provides the use of any of the mutants described above in the preparation of bactericides, fragrances or insect attractants.

[0016] Finally, this invention provides a method for increasing farnesol production by introducing the above-mentioned mutant into host cells, resulting in a maximum increase of 531% in farnesol production after fermentation.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) Significantly enhanced activity: The mutant increases enzyme activity by up to 531%, breaking through the existing technical bottleneck; (2) Expanding application prospects: Providing core enzyme elements for the large-scale production of farnesol in detergents, fragrances and other fields; (3) Outstanding industrial value: The yield of recombinant bacteria in shake flasks is 5.31 times that of wild type, which greatly reduces production costs and lays an important foundation for the industrial application of farnesol. Attached Figure Description

[0018] Figure 1 This is a diagram showing the pathway for yeast to synthesize farnesol. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0020] The whole genome synthesis, primer synthesis and sequencing in this article were all completed by Wuhan Tianyi Huayu Gene Technology Co., Ltd. The high-fidelity enzyme (PrimeSTAR GXL DNA Polymerase), ordinary Taq enzyme (Premix Taq), pMD19-T Vector and other products used were purchased from Wuhan Youming Biotechnology Co., Ltd., and the restriction endonuclease was purchased from Hubei Jingmao Biotechnology Co., Ltd.

[0021] The construction method of the Saccharomyces cerevisiae strain YZL141 is described in Shi Bin et al., “Systematic Metabolic Engineering of Saccharomyces cerevisiae for Lycopene Overproduction.” Journal of Agricultural and Food Chemistry vol. 67,40(2019): 11148-11157. doi:10.1021 / acs.jafc.9b04519. It involves overexpression of the HMG-COA reductase gene, a key rate-limiting enzyme in the MVA pathway, in Saccharomyces cerevisiae CEN.PK2-1D. tHMG1 .

[0022] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder; YPD medium: 10 g / L yeast extract, 20 g / L tryptone, 20 g / L glucose; 10% IPM (isopropyl myristate) is added during fermentation to prevent product volatilization; YPD solid medium: 10 g / L yeast extract, 20 g / L tryptone, 20 g / L glucose, 20 g / L agar powder.

[0023] Method for determining the concentration of farnesol in fermentation broth: Testing process; Place 45 mL of fermentation broth into a 50 mL centrifuge tube and centrifuge at 10000 r / min for 10 min. Carefully aspirate the upper IPM (isopropyl myristate) liquid and dilute it with chromatographic grade n-hexane to an appropriate concentration for detection.

[0024] Instrumentation: An Agilent 7890A gas chromatograph was used. The column was an Agilent HP-5 (30 m x 0.32 mm x 0.25 μm). The column oven temperature was: initial temperature 100℃, hold for 2 min, then increased to 280℃ at 10℃ / min, hold for 3 min. The injection port temperature was 280℃. The injection volume was 1 μL. The column flow rate was 1 mL / min. The injection split ratio was 1:50. The detector (FID) temperature was 280℃. Nitrogen was used as the carrier gas. The inlet pressure was 12-18 psi. The mode was constant flow mode. The standard was farnesol (SIGMA).

[0025] SEQ ID NO: 1

[0026] Example 1 (1) Gene mutation synthesis The gene synthesis and mutation in this experiment were designed and synthesized by Wuhan Tianyi Huayu Gene Technology Co., Ltd., and the genes were used after sequencing was confirmed to be correct.

[0027] (2) Construction of recombinant expression vector After re-PCR cloning and gel recovery of the target gene vector with no sequencing errors, the target gene vector was ligated with the yeast linearized expression vector using the homologous recombination kit from Nanjing Novizan Biotechnology Co., Ltd. (37℃, 30 min). The vector was then transformed into DH5α Escherichia coli competent cells for screening. After verification by bacterial PCR, the yeast expression vector containing the correct target gene was obtained and named pPAH1-15, according to Table 1.

[0028] (3) Construction and functional characterization of engineered strains (3.1) Preparation of yeast competent cells After overnight culture, yeast strain YZL141 was transferred to 50 mL of fresh YPD medium and cultured at 30°C until OD500. 600 At approximately 0.6, after centrifugation at 500g for 5 min, the cells are suspended in TE / LiAC solution, centrifuged at high speed for 5 min, and the collected cells are resuspended in 1 mL TE / LiAC solution to obtain competent yeast transformation.

[0029] The expression vector pPAH1-15 was transformed into competent YZL141 yeast cells using the PEG-LiAc transformation method. The cells were then incubated at 30℃ for 30 min, heat-shocked at 42℃ for 10 min, centrifuged for 5 min, and then plated on SD-URA selection plates. After incubation at 30℃ for 3 days, colony PCR was performed using primer pairs to verify the colony. The positive bacteria transformed into YZL141 were named P1-15.

[0030] (4) Evaluation of shake-flask fermentation The strain was inoculated into 50 mL of YPD medium, with the addition of 1% galactose and 10% IPM (isopropyl myristate). After incubation at 30 °C for 3 days, the organic phase was collected by centrifugation at 4000 rpm for 8 min. Samples were then prepared for GC-MS detection and quantification. The yields in the shake flasks are shown in Table 1 below.

[0031] Table 1. Genotype and Yield Comparison of Various Mutants (Wild Type Ratio) P1 wild type 100% P2 G154R 115% P3 E171D 56% P4 E222V 12% P5 F246Y 22% P6 L273S 278% P7 S589C 39% P8 E614D 124% P9 T632F 7% P10 V642I 65% P11 G154R and L273S 378% P12 G154R and E614D 531% P13 L273S and E614D 291% P14 E614D and V642I 196% P15 L273S and E171D 172% Note: The yield of wild type is 20 mg / L.

[0032] According to the results in Table 1: (1) The best mutant was strain P12 (G154R+E614D): the yield was 531% higher than that of the wild type (5.31 times that of the wild type), which was the highest activity among all mutants; followed by strain P6 (L273S single-point mutation): the single-point mutation was the best, and the yield was 278% higher than that of the wild type, indicating that L273S is the core synergistic site.

[0033] (2) Synergistic effect of combined mutation Significant synergistic effect: Double mutants are generally superior to single-point mutants (e.g., P11 strain / P12 strain / P13 strain all >250%). Site specificity: The combination of G154R, L273S, and E614D (P11 strain / P12 strain) showed the strongest synergistic effect, while the combination of E171D with other sites (P15 strain) showed a weaker synergistic effect.

[0034] (3) Industrial value classification High-efficiency level (>300%): P12 strain (531%), P11 strain (378%). Medium-efficiency grade (100%-300%): P6 (278%), P13 strain (291%), P8 strain (124%). Low efficiency level (<100%): Other single-point mutations (such as P3 strain / P4 strain / P5 ​​strain / P7 strain / P9 strain / P10 strain).

[0035] (4) Biological significance: Key mutation sites: G154R, L273S, and E614D are the core amino acid sites that drive the enhancement of enzyme activity; Value of directed evolution: The breakthrough effect (531%) of the double mutant P12 confirms the additive effect of combined mutations, providing optimization direction for subsequent enzyme engineering.

[0036] Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 farnesol synthase mutant, characterized in that, Its amino acid sequence is the sequence obtained by L273S mutation of SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, Encodes the farnesol synthase mutant of claim 1.

3. A recombinant vector, characterized in that, It includes the nucleic acid molecule as described in claim 2.

4. A recombinant cell, characterized in that, The cell comprises the recombinant vector of claim 3 or the genome integrated with the nucleic acid molecule of claim 2, wherein the cell is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae ).

5. The recombinant cell as described in claim 4, characterized in that, The brewer's yeast is strain CEN.PK2-1D.

6. A method for producing farnesol, characterized in that, Farnesol is produced by fermentation of recombinant cells according to any one of claims 4-5.

7. The use of the mutant of claim 1 in the preparation of farnesol.

8. A method for increasing farnesol yield, characterized in that, The step includes introducing the mutant of claim 1 into Saccharomyces cerevisiae for fermentation.

Images