Valencene synthase mutants and valencene hyper-producing strains

Abstract

The present disclosure relates to a valencene synthase mutant and a valencene hyper-producing strain, which belongs to the field of synthetic biology. The synthase of valencene is derived from Eryngium glaciale, and the directed evolution of this enzyme has resulted in a valencene synthase mutant with improved enzyme function, and the yield of the strain containing this mutant is 3.15 times that of the strain containing the wild-type synthase. The valencene synthase mutant of the present disclosure enhances the valencene synthesis ability of the strain and lays a solid foundation for its industrial production. The present disclosure uses the valencene synthase mutant to construct a hyper-producing strain for synthesizing valencene, and the fermentation tank yield reaches 12.4 g / L, the highest level reported so far.

Description

[Technical Field]

[0001] The present disclosure is in the field of synthetic biology and relates to valencene synthase mutants and strains that produce high levels of valencene. [Background technology]

[0002] Valencene is a sesquiterpene compound with a citrus aroma and is one of the most valuable terpenes available commercially. It is widely used as a flavoring in the food and beverage industries and has high economic value. Currently, valencene is mainly extracted from plants, but this method is uneconomical due to its low content in plants and high extraction costs.

[0003] In recent years, microbial cell factories have been widely used for the production of chemical compounds. Currently, heterologous synthesis of valencene in microorganisms is possible by expressing valencene synthase in microorganisms. However, the achievable yield levels are still low. This is mainly due to the fact that currently available valencene synthases do not have high enzymatic activity. Therefore, complex metabolic engineering techniques are often required to achieve high valencene yields. Therefore, obtaining a valencene synthase with high enzymatic activity is a key factor in achieving high valencene yields. Summary of the Invention

[0004] An object of the present disclosure is to provide a high-performance valencene synthase mutant and its use in valencene production. It is also an object of the present disclosure to provide a high-production strain of valencene and a method for achieving increased valencene yield.

[0005] To achieve the above objectives, the present disclosure provides the following technical solutions:

[0006] The present disclosure provides a valencene synthase variant, the wild-type of which is derived from Eryngium glaciale and whose sequence is set forth in SEQ ID NO. 1, which has an amino acid substitution at at least one of positions 533, 336, 196, 176, 306, and 325 compared to the wild-type valencene synthase, wherein the positions are determined with reference to SEQ ID NO. 1, and the valencene synthase variant has improved enzymatic activity compared to the wild-type valencene synthase; In some embodiments, the valencene synthase mutant comprises at least one of the following mutations: I533V, R336K, H196R, D176E, R306K, K325E; In some embodiments, the valencene synthase mutant comprises a I533V mutation, and optionally at least one of R336K, H196R, D176E, R306K, and K325E mutations; In some embodiments, the valencene synthase mutant comprises an I533V, an R336K mutation, and optionally at least one of an H196R, a D176E, an R306K, and a K325E mutation.

[0007] The present disclosure also provides a valencene synthase mutant comprising one of the following mutation positions compared to wild-type valencene synthase (SEQ ID NO. 1): (1) I533V, R336K; (2) I533V, R336K, H196R, D176E; (3) I533V, R336K, R306K; (4) I533V, R336K, K325E; or (5) I533V, R336K, H196R, D176E, R306K, K325E; wherein the positions are determined with reference to SEQ ID NO. 1.

[0008] In some embodiments, the amino acid sequence of the valencene synthase variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID NO.1.

[0009] In some embodiments, the amino acid sequence of the valencene synthase variant is set forth in SEQ ID NOs. 92-96.

[0010] In some embodiments, compared to wild-type valencene synthase (SEQ ID NO. 1), the valencene synthase mutant shown in SEQ ID NO. 92 has I533V and R336K mutations, the valencene synthase mutant shown in SEQ ID NO. 93 has I533V, R336K, H196R and D176E mutations, the valencene synthase mutant shown in SEQ ID NO. 94 has I533V, R336K and R306K mutations, the valencene synthase mutant shown in SEQ ID NO. 95 has I533V, R336K and K325E mutations, and the valencene synthase mutant shown in SEQ ID NO. 96 has I533V, R336K, H196R, D176E, R306K and K325E mutations.

[0011] In some embodiments, the amino acid sequence of the valencene synthase variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity to the sequence set forth in SEQ ID NOs. 92-96.

[0012] The present disclosure also provides genes encoding the above-mentioned valencene synthase mutants, and in some embodiments, the nucleotide sequence of the genes can be optimized according to host codon bias.

[0013] The present disclosure also provides a recombinant plasmid comprising the gene, which is capable of expressing the valencene synthase mutant after being introduced into a host cell.

[0014] The present disclosure also provides recombinant cells containing the above genes, including bacteria (such as Escherichia coli), fungi (such as yeast (Saccharomyces cerevisiae) and actinomycetes) as hosts.

[0015] The present disclosure also provides the use of the above valencene synthase variant, gene, recombinant plasmid or recombinant cell in the production of valencene and nootkatone.

[0016] The present disclosure also provides a high-valencene producing strain comprising a gene encoding the above-mentioned valencene synthase mutant.

[0017] The present inventors have discovered that acetoacetyl-coenzyme A thiolase, hydroxymethylglutaryl-coenzyme A (HMG-CoA) synthase, hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, mevalonate kinase, mevalonate-5-phosphate kinase, mevalonate pyrophosphate decarboxylase, and isoprene pyrophosphate isomerase belong to the mevalonate pathway (MVA pathway), and that the mevalonate pathway produces isoprene pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMPO). They found that the recombinant bacterium can synthesize farnesyl pyrophosphate (APP), which can be used as a precursor to synthesize farnesyl pyrophosphate (FPP) through the catalytic action of farnesyl pyrophosphate synthase, and that FPP is a substrate for the biosynthesis of valencene (the synthetic pathway for valencene is shown in Figure 1). Therefore, they found that if the recombinant bacterium can express at least one of the enzymes in the mevalonate pathway and farnesyl pyrophosphate synthase, it will be advantageous for the synthesis of FPP and, ultimately, the biosynthesis of valencene.

[0018] In some embodiments, the valencene hyper-producing strain comprises the gene ERG20, which encodes farnesyl pyrophosphate synthase.

[0019] In some embodiments, the high-valencene producing strain includes at least one mevalonate pathway (MVA pathway) gene, and the MVA pathway genes include ERG10, a gene encoding acetoacetyl-coenzyme A thiolase, ERG13, a gene encoding HMG-CoA synthase, tHMG1, a gene encoding HMG-CoA reductase, ERG12, a gene encoding mevalonate kinase, ERG8, a gene encoding mevalonate-5-phosphate kinase, MVD1, a gene encoding mevalonate pyrophosphate decarboxylase, and IDI1, a gene encoding isoprene pyrophosphate isomerase.

[0020] In some embodiments, the valencene hyper-producing strain comprises the gene ERG20 encoding farnesyl pyrophosphate synthase and mevalonate pathway (MVA pathway) genes.

[0021] In some embodiments, the copy numbers of MVA pathway genes and farnesene pyrophosphate synthase genes, ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, and ERG20, in the high valencene producing strain are 2, 2, 3, 2, 2, 2, 2, and 2, respectively.

[0022] In some embodiments, the copy number of the gene encoding the valencene synthase variant is two or three, preferably two.

[0023] In some embodiments, the host for the valencene hyper-producing strain is Saccharomyces cerevisiae.

[0024] In some embodiments, when the host of the valencene hyper-producing strain is Saccharomyces cerevisiae, the valencene hyper-producing strain is one in which the GAL80 gene has been knocked out.

[0025] Advantages and Beneficial Effects of the Present Disclosure (1) The present disclosure provides a valencene synthase mutant with significantly improved performance, and the strain containing the mutant produces 3.15-fold higher valencene yield than the strain containing the wild-type synthase. The valencene synthase mutant of the present disclosure lays a strong foundation for the industrial production of valencene.

[0026] (2) This disclosure demonstrates the construction of a high-yielding valencene strain using a valencene synthase mutant, and the yield of the constructed strain in a fermentation tank reached 12.4 g / L, the highest level reported to date. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a diagram of the synthesis route of valencene. [Figure 2] 1 is a graph showing the amino acid alignment results of EGVS and the 5-epi-aristolochene synthase TEAS. [Figure 3] 1 shows the results of a docking simulation of EGVS and FPP. [Figure 4] 1 is a graph showing the results of amino acid residue bias from amino acid sequence alignment between valencene synthase EgVS and its homologous sequences. DETAILED DESCRIPTION OF THE INVENTION

[0028] The following embodiments are used to further illustrate the present disclosure, but should not be construed as limiting the present disclosure, and other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present disclosure should be considered as equivalent substitutions and fall within the scope of the claims of the present disclosure.

[0029] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The plasmids involved in the following examples are plasmids known to those skilled in the art. If no specific techniques or conditions are indicated in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. If no manufacturer is indicated, the reagents or equipment used are conventional products that are commercially available. [Example]

[0030] Example 1: Construction of yeast expression vectors Characteristics of the plasmid pZY900 ΔLEU2:LEU2(URA3)_TCYC1_LacZ_pGAL10pGAL1_ERG20_tERG20, in which promoters GAL1 and GAL10 controlled the expression of genes ERG20 and LacZ, respectively, the screening marker was Leu2, and the inserted chromosomal position was Leu2.

[0031] Detailed construction process of plasmid pZY900: The genome of Saccharomyces cerevisiae S288c was used as a template, and the primers 900-1F / 1R, 900-2F / 2R, 900-6F / 6R, and 900-7F / 7R were used to amplify the fragments 9001 (left homologous arm of Leu2), 9002 (terminator tTDH2), 9006 (gene ERG20 and terminator tERG20), and 9007 (right homologous arm of Leu2). The genome of Saccharomyces cerevisiae CEN.PK2-1D was used as a template, and primers 900-3F / 3R and 900-5F / 5R were used to amplify fragments 9003 (terminator tCYC1) and 9005 (promoter pGAL1 and Pgal10), respectively. Primers 900-4F / 4R were used to amplify pCAS as a template to obtain fragment 9004 (a nonsense gene used for target gene replacement). The plasmid backbone was obtained by amplifying pRS426 with primers 900-8F / 8R (introduction of an MssI cleavage site and marker screening). The above fragments were recombined in Saccharomyces cerevisiae using the DNA Assembly method (yeast assembly method) to construct pZY900, which was then amplified in E. coli, digested, and sequenced. (For details on constructing pCAS, please refer to the paper "Zhang, Yueping et al., A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nature communications 10, 1 1053. 5 Mar. 2019, doi:10.1038 / s41467-019-09005-3.")

[0032] The sequences of the primers used in the construction of plasmid pZY900 are shown in Table 1 below.

[0033] [Table 1]

[0034] Characteristics of plasmid pYH300: ΔLEU2:LEU2(URA3)_TCYC1_LacZ_pGAL10pGAL1_ERG20_tERG20: Expression of the ERG20 and LacZ genes was controlled by the GAL1 and GAL10 promoters, respectively. The screening marker was Leu2, and the insertion site was Leu2. The difference from pZY900 is that the enzyme cleavage site between the homology arms and the plasmid backbone was NotI.

[0035] Construction of plasmid pYH300: Using pZY900 as a template, a fragment between the homology arms was obtained by amplification with primers P48-F / R, and a vector backbone containing a NotI cleavage site was obtained by amplification with primers P49-F / R, and the plasmid was obtained by homologous recombination.

[0036] [Table 2]

[0037] Example 2: Construction of a valencene synthetic vector The two valencene synthases that have been more extensively studied in existing research are the valencene synthase CnVS from Callitropsis nootkatensis, mentioned in the literature (Beekwilder, Jules et al., Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene., Plant biotechnology journal vol. 12, 2 (2014): 174-82. doi:10.1111 / pbi.12124), and the valencene synthase EgVS from Eryngium glaciale, described in Patent Publication US 2015 / 0007368 A1 (not reported in the literature). We first compared the wild-type valencene synthases derived from these. The coding sequences of CnVS and EgVS were synthesized after being optimized according to the codons of Saccharomyces cerevisiae, and their nucleotide sequences are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0038] A gene-specific primer pair CnVS-F / R was designed, and the synthesized gene (SEQ ID NO. 2) was used as a template. Using Takara's Prime STAR high-fidelity enzyme, a CnVS gene fragment was obtained by PCR amplification. The gel was recovered using the Tiangen gel recovery kit, and then ligated into the yeast expression vector pYH300, which had been digested with BsaI, using Yeasen's homologous recombination kit. The correct sequence was determined by sequencing, and a yeast expression vector containing the gene was obtained and named pYH329.

[0039] A gene-specific primer pair, EgVS-F / R, was designed, and the synthesized gene (SEQ ID NO. 3) was used as a template for PCR amplification using Takara's Prime STAR high-fidelity enzyme. The EgVS gene fragment was recovered from the gel using Tiangen's gel recovery kit, and then ligated into BsaI-cleaved yeast expression vector pYH300 using Yeasen's homologous recombination kit. After sequencing to determine the correct sequence, a yeast expression vector containing the gene was obtained and named pYH327.

[0040] [Table 3]

[0041] Example 3: Construction of a valencene-synthesizing strain Plasmids pYH327 and pYH329 were linearized with NotI to obtain pYH327-NotI fragments and pYH329-NotI fragments, which were then transformed into competent cells of the yeast JCR27 strain using the PEG / LiAC method (for the construction of the yeast JCR27 strain, see Siemon, Thomas et al., Semisynthesis of Plant-Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block., Journal of the American Chemical Society 142,6 (2020):2760-2765. doi:10.1021 / jacs.9b12940). The resulting positive bacteria were designated JGH29 and JGH31. Strain JGH29 was derived from Saccharomyces cerevisiae CEN.PK2-1D by first enhancing the MVA pathway and then introducing a fragment containing the farnesyl pyrophosphate synthase gene and EgVS. Strain JGH31 was derived from Saccharomyces cerevisiae CEN.PK2-1D by first enhancing the MVA pathway and then introducing a fragment containing the farnesyl pyrophosphate synthase gene and CnVS.

[0042] Example 4: Shake flask fermentation of JGH29 and JGH31 strains Strains JGH29 and JGH31 were inoculated into seed medium (peptone (20 g / L), yeast powder (10 g / L), glucose (20 g / L)) and cultured at 30°C and 200 rpm for 20–24 h. The cultures were then transferred to fermentation medium (peptone (20 g / L), yeast powder (10 g / L), glucose (10 g / L), galactose (10 g / L)). An organic phase (n-dodecane or isopropyl myristate) was added at 20% by volume to the fermentation broth, and the fermentation was continued at 30°C and 200 rpm for 72 h. GCMS analysis revealed that the valencene yield of strain JGH29 was 78 mg / L, while that of strain JGH31 was 22 mg / L. This experiment demonstrated that the valencene synthase from Eryngium glaciale exhibited superior performance.

[0043] Example 5: Obtaining a valencene synthase mutant vector These experiments demonstrated that the valencene synthase from Eryngium glaciale has good performance, but the yield was not enough to reach 100 mg / L. To achieve an industrial production level of valencene, the performance of the valencene synthase must be further improved.

[0044] First, we simulated the enzyme's three-dimensional structure using the Swiss model and used 4RNQ as the optimal template. Sequence alignment of the two enzymes revealed that the I533 position in the enzyme corresponds to the V residue in the template 4RNQ (Fig. 2). Structural simulation revealed that I533 is located at the bottom of the substrate-binding cavity (Fig. 3). Therefore, we speculated that the I533V mutation might increase the volume of the binding cavity, making it more suitable for substrate binding. Finally, we selected this mutation for experimental verification and constructed the valencene mutant I533V.

[0045] Using the synthesized gene (SEQ ID NO. 3) as a template, a gene-specific primer pair P4-F / P50-R was designed. A portion of the EgVS gene fragment was amplified by PCR using Takara's Prime STAR high-fidelity enzyme, and the remaining portion of EgVS was amplified by PCR using P51-F / P4-R. The gel was recovered using Tiangen's gel recovery kit, and then ligated into the yeast expression vector pYH300, which had been cut with BsaI, using Yeasen's homologous recombination kit. The correct sequence was determined by sequencing, and a yeast expression vector containing the gene was obtained and named pYH332.

[0046] [Table 4]

[0047] During the construction of pYH332, we accidentally isolated a plasmid containing both the I533V and R336K mutations, which we named pYH340. These two plasmids were linearized with NotI and then transformed into the yeast strain JCR27 to obtain strains JGH37 and JGH44. In shake flask fermentation (under the same conditions as in Example 4), the yields of valencene reached 69 mg / L and 100 mg / L, respectively. The enzyme performance of the I533V and R336K mutations was improved compared to that of the wild-type EgVS.

[0048] To further improve the performance of EgVS, alignment of EgVS with homologous sequences showed biases in amino acid residues ( Figure 4 ), and eight positions, N81D, D176E, E216G, R306K, K325E, G347E, H491E, and R350K, were selected, followed by site-directed mutagenesis of EgVS (I533V, R336K).

[0049] Using the pYH340 plasmid (containing the EgVS (I533V, R336K) mutation) as a template, gene-specific primer pair P4-F / P52-R was designed. A portion of the EgVS gene fragment was amplified by PCR using Takara's Prime STAR high-fidelity enzyme. The remaining EgVS gene fragment was amplified by PCR using P53-F / P4-R. The resulting fragment was gel-collected using a Tiangen gel recovery kit. This fragment was then ligated into BsaI-cleaved yeast expression vector pZY900 by homologous recombination using a Yeasen homologous recombination kit. After sequencing, the correct sequence was determined. This yeast expression vector containing the EgVS (I533V, R336K, N81D) coding sequence was obtained and designated pYH355.

[0050] Using the pYH340 plasmid as a template, we designed the gene-specific primer pair P4-F / P54-R. We obtained a partial EgVS gene fragment by PCR amplification using Takara Prime STAR high-fidelity enzyme. We then amplified the remaining EgVS gene fragment by PCR using P55-F / P4-R. The resulting fragment was then recovered using a Tiangen gel recovery kit. This fragment was then ligated into the BsaI-cleaved yeast expression vector pZY900 by homologous recombination using a Yeasen homologous recombination kit. After sequencing, we obtained a yeast expression vector containing the EgVS (I533V, R336K, H196R, D176E) coding sequence, designated pYH356. The H196R mutation was accidentally introduced during construction.

[0051] Using the pYH340 plasmid as a template, a gene-specific primer pair, P4-F / P56-R, was designed. A portion of the EgVS gene fragment was amplified by PCR using Takara's Prime STAR high-fidelity enzyme. The remaining portion of the EgVS gene fragment was amplified by PCR using P57-F / P4-R. The resulting gel fragment was recovered using a Tiangen gel recovery kit. The resulting fragment was ligated into the yeast expression vector pZY900, which had been cut with BsaI, by homologous recombination using a Yeasen homologous recombination kit. The correct sequence was determined by sequencing, and the resulting yeast expression vector containing the EgVS (I533V, R336K, E216G) coding sequence was designated pYH358.

[0052] Using the pYH340 plasmid as a template, a gene-specific primer pair, P4-F / P58-R, was designed. A portion of the EgVS gene fragment was amplified by PCR using Takara's Prime STAR high-fidelity enzyme. The remaining portion of the EgVS gene fragment was amplified by PCR using P59-F / P4-R. The resulting fragment was then recovered using a Tiangen gel recovery kit. The resulting fragment was then ligated into BsaI-cleaved yeast expression vector pZY900 by homologous recombination using a Yeasen homologous recombination kit. The correct sequence was determined by sequencing, and the resulting yeast expression vector containing the EgVS (I533V, R336K, R306K) coding sequence was designated pYH361.

[0053] Using the pYH340 plasmid as a template, a gene-specific primer pair, P4-F / P60-R, was designed. A portion of the EgVS gene fragment was amplified by PCR using Takara's Prime STAR high-fidelity enzyme. The remaining portion of the EgVS gene fragment was amplified by PCR using P61-F / P4-R. The resulting gel fragment was then recovered using a Tiangen gel recovery kit. The resulting fragment was then ligated into the BsaI-cleaved yeast expression vector pZY900 using a Yeasen homologous recombination kit. After sequencing, the correct sequence was confirmed. This yeast expression vector, designated pYH362, contained the EgVS (I533V, R336K, K325E) coding sequence.

[0054] Using the pYH340 plasmid as a template, a gene-specific primer pair, P4-F / P62-R, was designed. A portion of the EgVS gene fragment was amplified by PCR using Takara's Prime STAR high-fidelity enzyme. The remaining portion of the EgVS gene fragment was amplified by PCR using P63-F / P4-R. The resulting gel was recovered using a Tiangen gel recovery kit, and then ligated into BsaI-cleaved yeast expression vector pZY900 using a Yeasen homologous recombination kit. After sequencing, the correct sequence was confirmed. This yeast expression vector, designated pYH363, contained the EgVS (I533V, R336K, G347E) coding sequence.

[0055] Using the pYH340 plasmid as a template, a gene-specific primer pair, P4-F / P64-R, was designed. A portion of the EgVS gene fragment was amplified by PCR using Takara's Prime STAR high-fidelity enzyme. The remaining portion of the EgVS gene fragment was amplified by PCR using P65-F / P4-R. The resulting fragment was then recovered using a Tiangen gel recovery kit. The resulting fragment was then ligated into BsaI-cleaved yeast expression vector pZY900 by homologous recombination using a Yeasen homologous recombination kit. The correct sequence was determined by sequencing, and the resulting yeast expression vector containing the EgVS (I533V, R336K, H491E) coding sequence was designated pYH372.

[0056] Using the pYH340 plasmid as a template, a gene-specific primer pair, P4-F / P66-R, was designed. A portion of the EgVS gene fragment was amplified by PCR using Takara's Prime STAR high-fidelity enzyme. The remaining portion of the EgVS gene fragment was amplified by PCR using P67-F / P4-R. The resulting fragment was then recovered using a Tiangen gel recovery kit. The resulting fragment was then ligated into BsaI-cleaved yeast expression vector pZY900 by homologous recombination using a Yeasen homologous recombination kit. The correct sequence was determined by sequencing, and the resulting yeast expression vector containing the EgVS (I533V, R336K, R350K) coding sequence was designated pYH375.

[0057] [Table 5]

[0058] Example 6: Identification of a strain containing a valencene synthase mutant and shake flask fermentation The thus obtained plasmids pYH355, 356, 358, 361, 362, 363, 372, and 375 were linearized with MssI and then inserted into the yeast JCR27 strain to obtain JGH55, 56, 57, 58, 59, 60, 63, and 64 strains.

[0059] The shake flask fermentation method was consistent with that described in Example 4. Compared with strain JGH44, the yields of strains JGH57 and JGH60, which contained the E216G and G347E mutations, were significantly reduced to 13 mg / L and 38 mg / L, respectively. The yields of strains JGH55, JGH63, and JGH64, which contained the N81D, H491E, and R350K mutations, were slightly reduced to 94 mg / L, 86 mg / L, and 64 mg / L, respectively. However, the yields of strains JGH56, JGH58, and JGH59, which contained the D176E mutation (an unexpected mutation H196R introduced during construction), R306K, and K325E mutations, were increased to 117 mg / L, 143 mg / L, and 114 mg / L, respectively. The beneficial mutations were combined to construct the plasmid pYH383. After linearization with MssI, PYH383 was transformed into strain JCR27, resulting in strain JGH71, which further increased yield. After 72 hours of shake flask fermentation, mutants containing I533V, R336K, H196R, D176E, R306K, and K325E produced valencene at 248 mg / L, 3.15-fold higher than the wild-type, significantly improving enzyme performance. Furthermore, the ratio of valencene to aristrene, a by-product, decreased from 2.97:1 to 4.18:1. The final yield of the mutants was increased compared to the wild-type, demonstrating that, except for I533, which is located within the active pocket, all other positions are located outside the active pocket, far from the pocket, indicating that distal residues are also crucial for protein performance.

[0060] Construction of pYH383: Using pYH362 as a template, gene-specific primer pair P4-F / P68-R was designed and a portion of the EgVS gene fragment was obtained by PCR amplification using Takara Prime STAR high-fidelity enzyme. Using pYH356 as a template, PCR amplification using P69-F / P4-R was performed to obtain the remaining EgVS gene fragment. The resulting gel was recovered using a Tiangen gel recovery kit, and then ligated into BsaI-cleaved yeast expression vector pZY900 using a Yeasen homologous recombination kit. After sequencing, the correct sequence was determined. This yeast expression vector, designated pYH383, contained the coding sequence EgVS (I533V, R336K, K325E, R306K, D176E, H196R).

[0061] [Table 6]

[0062] Example 7: Optimization of metabolic pathways to increase the yield of valencene To further increase the yield of valencene, plasmids pYH384 and pYH385 were constructed to increase the copy number of valencene synthase.

[0063] Using the Saccharomyces cerevisiae CEN.PK2-1D genome as a template, gene-specific primer pair P11-F / P11-R was designed and PCR amplified to obtain the left arm of the URA3 homology arm. The commercially available plasmid pRS423 was amplified with primers P12-F / R to obtain a histidine screening marker. Tcyc1 was obtained by amplification using primers P13-F / R from the CEN.PK2-1D genome as a template. Thmg1 was obtained by amplification using primers P14-F / R from the Saccharomyces cerevisiae S288C genome as a template. pGAL1-pGAL10 was obtained by amplification using primers P15-F / R from the CEN.PK2-1D genome as a template. The EgVS (I533V, R336K, K325E, R306K, D176E, H196R) coding sequence was obtained by amplification using primers P16-F / R from pYH383 as a template. tPGK1 was obtained by amplification using primers P17-F / R from the CEN.PK2-1D genome as a template. The right arm of the URA3 homology arm was obtained by amplification using primers P18-F / R from the CEN.PK2-1D genome as a template. The vector backbone was obtained by amplification using primers P19-F / R from the commercially available plasmid pRS426 as a template. The above fragments were recombined in Saccharomyces cerevisiae using the DNA assembly method (enzyme assembly method) to construct pYH384, which was then amplified in E. coli, digested, and sequenced to obtain pYH384. This plasmid contained the genes tHMG1 and EgVS (I533V, R336K, K325E, R306K, D176E, H196R).

[0064] Using the CEN.PK2-1D genome as a template, the gene-specific primer pair P20-F / P20-R was designed and PCR-amplified to obtain the left arm of the HIS3 homology arm. The commercially available plasmid pRS424 was used as a template and amplified with primers P21-F / R to obtain a tryptophan screening marker. TADH1 was obtained by amplification with primers P22-F / R using the CEN.PK2-1D genome as a template, and the EgVS (I533V, R336K, K325E, R306K, D176E, H196R) coding sequence was obtained by amplification with primers P23-F / R. pGAL1-pGAL10 was obtained by amplification with primers P24-F / R using the CEN.PK2-1D genome as a template. The right arm of the tCPS1 homology arm was obtained by amplification with primers P25-F / R using the CEN.PK2-1D genome as a template. The right arm of the HIS3 homology arm was obtained by amplification using primers P26-F / R from the CEN.PK2-1D genome as a template. The vector backbone was obtained by amplification using primers P27-F / R from the commercially available plasmid pRS426 as a template. The above fragments were recombined in Saccharomyces cerevisiae using the DNA assembly method (enzymatic assembly method), and amplified in E. coli to construct pYH385. After enzymatic digestion and sequencing, pYH385 was obtained. This plasmid contained the EgVS gene (I533V, R336K, K325E, R306K, D176E, H196R).

[0065] [Table 7-1]

[0066] [Table 7-2]

[0067] Plasmid pYH384 was linearized with MssI and inserted into strain JGH71 to obtain strain JGH72, which was based on CEN.PK2-1D and contained the MVA pathway genes and farnesene pyrophosphate synthase genes ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, and ERG20 with copy numbers of 2, 2, 3, 2, 2, 2, 2, and 2, respectively, and the gene encoding the valencene synthase mutant was in copy number 2.

[0068] Plasmid pYH385 was linearized with MssI and then inserted into strain JGH72 to obtain strain JGH73, which was based on CEN.PK2-1D and contained the MVA pathway genes and farnesene pyrophosphate synthase genes ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, and ERG20 with copy numbers of 2, 2, 3, 2, 2, 2, 2, and 2, respectively, and the gene encoding the valencene synthase mutant was in copy number 3.

[0069] Shake flask fermentation using this strain showed that when one copy of tHMG1 and valencene synthase were increased, the yield of strain JGH72 reached 393 mg / L. However, further increase in the amount of valencene synthase reduced the yield of the strain, and the shake flask yield of JGH73 was 377 mg / L. This indicated that the strain containing two copies of the valencene synthase mutant had a higher yield. Using JGH72 as a base, we further integrated pZY528 to knock out GAL80, thereby complementing the nutritional deficiency and eliminating the need for galactose induction, resulting in strain JGH78, which achieved a yield of 515 mg / L.

[0070] Construction of the pZY528 knockout cassette: Fragment 5281 (left homology arm containing the Gal80 locus) was obtained by PCR amplification using primers 5281-1F and 5281-1R from the CEN.PK2-1D genome as a template, and fragment 5284 (right homology arm containing the Gal80 locus) was obtained by PCR amplification using primers 5284-4F and 5284-4R. Fragment 5282 (containing a uracil screening marker) was obtained by PCR amplification using plasmid pRS426-ura (ATCC 87333) as a template and primers 5282-2F and 5282-2R. Fragment 5283 (containing a uracil screening marker) was obtained by PCR amplification using plasmid pRS424 as a template and primers 5283-3F and 5283-3R. Furthermore, fragments 5281, 5282, 5283 and 5284 were ligated by OE-PCR using primers 5281-1F and 5284-4R to obtain pZY528.

[0071] [Table 8]

[0072] Example 8: Fermentation tank fermentation of valencene hyper-producing strains The constructed JGH78 strain was subjected to fed-batch fermentation using a fermentation medium described in the literature (van Hoek, P.; de Hulster, E.; van Dijken, JP; Pronk, JT). Functional capacity in high-cell-density fed-batch cultures of baker's yeast. Biotechnol. Bioeng. 2000, 68, 517-523). A capping agent was added during the fermentation process for in situ extraction. The capping agent used was isopropyl myristate. During the fermentation process, the dissolved oxygen concentration was controlled at 20% or higher, the pH at 5, the glucose concentration at 1-2 g / L, and the ethanol concentration at 5 g / L or lower. Finally, in a 15 L steel fermentation tank, the yield of valencene product by JGH78 was 12.4 g / L, the highest level reported to date.

Claims

1. (1) I533V, R336K; (2) I533V, R336K, H196R, D176E; (3) I533V, R336K, R306K; (4) I533V, R336K, K325E; (5) I533V, R336K, H196R, D176E, R306K, K325E A valencene synthase mutant comprising an amino acid sequence having a set of mutations selected from the group consisting of: The position is determined by reference to the amino acid sequence of the wild-type valencene synthase shown in SEQ ID NO. 1, and the valencene synthase mutant has improved enzymatic activity compared to the wild-type valencene synthase; The amino acid sequence of the valencene synthase variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence set forth in SEQ ID NO. 1; Valencene synthase mutants.

2. A valencene synthase mutant, whose amino acid sequence is the amino acid sequence shown in SEQ ID NO. 92-96.

3. A gene encoding the valencene synthase mutant of claim 1.

4. A recombinant plasmid comprising the gene according to claim 3.

5. A recombinant cell comprising the gene according to claim 3.

6. Use of the valencene synthase mutant of claim 1 or 2, the gene of claim 3, the recombinant plasmid of claim 4, or the recombinant cell of claim 5 in the production of valencene and nootkatone.

7. A valencene hyper-producing strain comprising the gene according to claim 3.

8. the gene ERG20 encoding farnesyl pyrophosphate synthase and / or at least one of mevalonate pathway (MVA pathway) genes, wherein the mevalonate pathway genes include the gene ERG10 encoding acetoacetyl coenzyme A thiolase, the gene ERG13 encoding HMG-CoA synthase, the gene tHMG1 encoding HMG-CoA reductase, the gene ERG12 encoding mevalonate kinase, the gene ERG8 encoding mevalonate 5-phosphate kinase, the gene MVD1 encoding mevalonate pyrophosphate decarboxylase, and the gene IDI1 encoding isoprene pyrophosphate isomerase; The high-producing valencene strain according to claim 7.

9. the gene ERG20 encoding farnesyl pyrophosphate synthase and mevalonate pathway genes, the mevalonate pathway genes including the gene ERG10 encoding acetoacetyl coenzyme A thiolase, the gene ERG13 encoding HMG-CoA synthase, the gene tHMG1 encoding HMG-CoA reductase, the gene ERG12 encoding mevalonate kinase, the gene ERG8 encoding mevalonate-5-phosphate kinase, the gene MVD1 encoding mevalonate pyrophosphate decarboxylase, and the gene IDI1 encoding isoprene pyrophosphate isomerase; The high-producing valencene strain according to claim 7.

10. The high-production strain of valencene described in claim 9, characterized in that the copy numbers of MVA pathway genes and farnesene pyrophosphate synthase genes ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, and ERG20 are 2, 2, 3, 2, 2, 2, 2, and 2, respectively.

11. The high-producing valencene strain according to claim 7, characterized in that the copy number of the gene according to claim 3 is 2 or 3.

12. The valencene-rich strain according to claim 7, characterized in that the host is Saccharomyces cerevisiae.

13. The valencene high-producing strain according to claim 12, characterized in that the GAL80 gene is knocked out.

Images