A sclareol synthase mutant, a recombinant genetically engineered strain and application thereof

Abstract

This invention discloses a perillaldehyde synthase mutant, a recombinant genetically engineered strain, and their applications, belonging to the field of biotechnology. This invention modifies perillaldehyde synthase through semi-rational design, resulting in a perillaldehyde synthase mutant with good catalytic activity, significantly increasing perillaldehyde yield; furthermore, by integrating the encoding of HMG-CoA reductase... HMG1 Gene, encoding isopentenyl pyrophosphate isomerase IDI1 Gene encoding geraniol geraniol pyrophosphate synthase SsGGPPS Gene, encoding lysine pyrophosphate diol ester synthase SsLPPS The recombinant genetically engineered strain obtained from the gene and nucleic acid molecule encoding the perillol synthase mutant can produce high yields of perillol using glucose as a substrate, and has significant industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a perilla frutescens alcohol synthase mutant, a recombinant genetically engineered strain, and their applications. Background Technology

[0002] Natural ambergris, as a top-grade fragrance, holds an extremely important position in the perfume and cosmetics industry. However, because it originates from the endangered sperm whale, ambergris is extremely scarce. Ambroxol, a synthetic fragrance with an ambergris-like aroma, can serve as a substitute for natural ambergris.

[0003] Currently, ambroxol relies on chemical synthesis, with perillyl alcohol as the raw material. It can be obtained through oxidation, saponification, dehydration, lactone formation, reduction, cyclization, and dehydration. Perillyl alcohol is primarily derived from the flowers and leaves of the perilla plant, obtained through plant extraction. However, due to limitations in climate and geographical environment, the plant extraction method makes perillyl alcohol prohibitively expensive. With advancements in metabolic engineering and synthetic biology, the microbial synthesis of perillyl alcohol could serve as an effective alternative.

[0004] Previous studies have shown that introducing heterologous pathways into Escherichia coli and Saccharomyces cerevisiae can be used to produce perillyl alcohol, but the biosynthesis of perillyl alcohol has problems such as insufficient perillyl alcohol synthase activity and low perillyl alcohol yield. Summary of the Invention

[0005] The purpose of this invention is to provide a perillaldehyde synthase mutant, a recombinant genetically engineered strain, and their applications. This addresses the problems of insufficient perillaldehyde synthase activity and low perillaldehyde yield in existing microbial production of perillaldehyde.

[0006] In a first aspect, the present invention provides a perillaldehyde synthase mutant, which is obtained by substituting and / or deleting and / or adding one or more amino acid residues at at least one of the following sites in the amino acid sequence shown in SEQ ID NO.4: position 87, position 113, position 167, position 212, position 221, position 292, position 326, position 431, position 484, position 489, position 558, and position 561.

[0007] The above-mentioned perilla alcohol synthase or its mutant provided by the present invention can be a natural, recombinant or synthetic active polypeptide. The active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants) using recombinant technology.

[0008] In some embodiments, the perilla ethanol synthase mutant has a mutation at at least one of the following sites in the amino acid sequence shown in SEQ ID NO.4: A1) Q87L, A2) V133S, A3) R167P, A4) Y212A, A5) I221K, A6) R292A, A7) T326I, A8) N431I, A9) E484G, A10) K489N, A11) D558V, A12) T561E.

[0009] In this invention, site-directed mutagenesis of amino acids in the substrate binding pocket can enhance the activity of the perillaldehyde synthase mutant, thereby increasing the yield of perillaldehyde.

[0010] In some embodiments, in addition to the aforementioned mutation occurring at at least one of the following sites: 87, 113, 167, 212, 221, 292, 326, 431, 484, 489, 558, and 561, the above-mentioned perillaldehyde synthase mutant may further have conserved amino acid substitutions at other sites, resulting in the mutated amino acids, such as those in SEQ ID NO. 4, exhibiting higher catalytic activity. Preferably, the conserved substitution of amino acids preserves the higher catalytic activity of the perillaldehyde synthase of the present invention. It will be apparent to those skilled in the art that such substitutions can occur in regions other than the aforementioned sites while still retaining the corresponding catalytic activity. Preferably, the conserved substitution variant has a conserved amino acid substitution at at least one position. Examples of conservative substitutions are those occurring within the following groups of amino acids: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine), and small molecule amino acids (such as glycine, alanine, serine, threonine, and methionine). The most common amino acid swaps are those between G and A; A and G, S; V and I, L, A, T, S; I and V, L, M; L and I, M, V; M and L, I, V; P and A, S, N; F and Y, W, H; Y and F, W, H; W and Y, F, H; R and K, E, D; K and R, E, D; H and Q, N, S; D and N, E, K, R, Q; E and Q, D, K, R, N; S and T, A; T and S, V, A; C and S, T, A; N and D, Q, H, S; Q and E, N, H, K, R, as well as their opposite swaps.

[0011] A perillaldehyde synthase mutant with a certain degree of amino acid homology to the above-mentioned perillaldehyde synthase mutant preferably has a homology between 70% and 99%, for example, it can be 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or any two or more of these values; more preferably, it has a homology between 80% and 99%, for example, it can be 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or any two or more of these values; even more preferably, it has a homology between 90% and 99%, for example, it can be 90%, 92%, 94%, 96%, 98%, 99%, or any two or more of these values; most preferably, it has a homology of 99%, which also falls within the protection scope of this invention.

[0012] In some implementations, the perillaldehyde synthase mutant includes the following combination of mutations: B1) R167P / K489N, B2) Y212A / K489N, B3) R292A / K489N, B4) R167P / R292A / K489N, B5) Y212A / R292A / K489N, and B6) R167P / Y212A / R292A / K489N.

[0013] In this invention, the inventors further discovered that, compared to single-site mutations, the catalytic activity of the perillaldehyde synthase mutant obtained by combined mutations is significantly improved, thereby further significantly increasing the yield of perillaldehyde.

[0014] In a second aspect, the present invention provides a nucleic acid molecule encoding any of the above-mentioned perillaldehyde synthase mutants.

[0015] In this invention, the nucleotide sequence of the nucleic acid molecule encoding the perillaldehyde synthase mutant is obtained by altering the corresponding sites in SEQ ID NO.3 based on the amino acid residue changes of the perillaldehyde synthase mutant.

[0016] The nucleic acid molecules provided by this invention can usually be obtained by PCR amplification or artificial synthesis.

[0017] In a third aspect, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecules.

[0018] In a fourth aspect, the present invention provides a recombinant genetically engineered strain that produces high levels of perillaldehyde, comprising the above-mentioned recombinant vector, or whose genome integrates the above-mentioned nucleic acid molecules.

[0019] In some implementations, the recombinant genetically engineered strain is obtained by introducing the above-mentioned recombinant vector into the host cell or by integrating the above-mentioned nucleic acid molecule into the host cell.

[0020] In some preferred embodiments, the host cell includes *Escherichia coli* (E. coli). Escherichia coli ), brewer's yeast ( Saccharomyces cerevisiae Pichia pastoris () Pichia pastoris ), Hansenula polymorpha ( Hansenula polymorpha ), Yarrowia lipolytica ( Yarrowia lipolytica At least one of the following. More preferably, the host cell includes *Yarrowia lipolytica* (Yarrowia lipolytica). Yarrowia lipolytica ).

[0021] In some implementations, the recombinant genetically engineered strain integrates the aforementioned nucleic acid molecules into its genome, and also integrates molecules encoding HMG-CoA reductase. HMG1 Gene, encoding isopentenyl pyrophosphate isomerase IDI1 Gene encoding geraniol geraniol pyrophosphate synthase SsGGPPS Gene, encoding lysine pyrophosphate diol ester synthase SsLPPS Gene.

[0022] In this invention, the inventors further integrated HMG-CoA reductase encoding a specific enzyme into a recombinant genetically engineered strain. HMG1 Gene, encoding isopentenyl pyrophosphate isomerase IDI1 Gene encoding geraniol geraniol pyrophosphate synthase SsGGPPS Gene, encoding lysine pyrophosphate diol ester synthase SsLPPS The recombinant genetically engineered strain obtained from the gene can produce high yields of perillaldehyde using glucose as a substrate.

[0023] In some implementations, the enzyme encoding HMG-CoA reductase is... HMG1 Gene, encoding isopentenyl pyrophosphate isomerase IDI1 The gene is an endogenous gene of *Yarrowia lipolytica*, encoding geranylgeranyl pyrophosphate synthase. SsGGPPS The nucleotide sequence of the gene is shown in SEQ ID NO.1, encoding lysine pyrophosphate diol ester synthase. SsLPPS The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0024] The genes provided by this invention can usually be obtained by PCR amplification or artificial synthesis.

[0025] In some implementations, the recombinant genetically engineered strain also includes an integrated leucine deficiency. LEU Gene.

[0026] In this invention, the inventors further discovered that by integrating leucine defects... LEU Genes can further increase the production of perillaldehyde.

[0027] In some embodiments, the method for constructing any of the above-mentioned recombinant genetically engineered strains producing high levels of perillaldehyde includes the following steps: encoding HMG-CoA reductase... HMG1 Gene, encoding isopentenyl pyrophosphate isomerase IDI1 The gene was integrated into the genome of *Yersinia lipolytica*, and simultaneously, the gene encoding geranylgeranyl pyrophosphate synthase was overexpressed in *Yersinia lipolytica*. SsGGPPS Gene, encoding lysine pyrophosphate diol ester synthase SsLPPS Genes and nucleic acid molecules encoding perilla ethanol synthase mutants were used to obtain recombinant genetically engineered strains.

[0028] The method for constructing recombinant genetically engineered strains provided by this invention is simple, and the yield of perillaldehyde can be significantly increased by optimizing the expression of key genes in the process of perillaldehyde synthesis.

[0029] In some implementations, the method further includes restoring the leucine deficiency to the resulting recombinant genetically engineered strain. LEU The steps of gene generation.

[0030] In this invention, by addressing the leucine deficiency LEU Gene replacement can further increase the production of perillaldehyde.

[0031] In some implementations, the construction method includes the following steps: 1) encoding HMG-CoA reductase... HMG1 Gene, encoding isopentenyl pyrophosphate isomerase IDI1 1) Integrate the gene into the A2 site of *Yarrowia lipolytica*; 2) Based on the strain obtained in step 1), overexpress the gene encoding gerany-gerany pyrophosphate synthase at the IntD10 site. SsGGPPS Gene, encoding lysine pyrophosphate diol ester synthase SsLPPS 3) Based on the strain obtained in step 2), overexpress the nucleic acid molecule encoding the perillaldehyde synthase mutant at the IntF site; 4) Based on the strain obtained in step 3), express leucine-deficient gene at the IntA1 site. LEU Gene.

[0032] In this invention, the integration of the above-mentioned genes can be performed using the Cre-loxP system.

[0033] In a fifth aspect, the present invention provides the use of any of the above-mentioned perillaldehyde synthase mutants, the above-mentioned nucleic acid molecules, the above-mentioned recombinant vectors, or the above-mentioned recombinant genetically engineered strains in the synthesis of perillaldehyde.

[0034] In a sixth aspect, the present invention provides a method for synthesizing perillyl alcohol, comprising the following steps: using glucose as a substrate, fermenting the above-mentioned recombinant genetically engineered strain to obtain perillyl alcohol.

[0035] In some implementations, the fermentation process also includes the addition of isopropyl myristate.

[0036] In some implementations, the amount of isopropyl myristate added is 5-15%.

[0037] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention modifies perillaldehyde synthase through semi-rational design, resulting in a perillaldehyde synthase mutant with better catalytic activity, significantly increasing perillaldehyde yield; furthermore, by integrating the encoding of HMG-CoA reductase... HMG1 Gene, encoding isopentenyl pyrophosphate isomerase IDI1 Gene encoding geraniol geraniol pyrophosphate synthase SsGGPPS Gene, encoding lysine pyrophosphate diol ester synthase SsLPPS The recombinant genetically engineered strain obtained from the gene and nucleic acid molecule encoding the perillol synthase mutant can produce high yields of perillol using glucose as a substrate, and has significant industrial application value. Attached Figure Description

[0038] Figure 1 This is the result of fermentation production of perillaldehyde by the single point mutant strain constructed in Example 2 of the present invention;

[0039] Figure 2 This is the result of fermentation production of perillaldehyde by the combined mutant strain constructed in Example 3 of the present invention. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0041] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0042] Example 1

[0043] In this embodiment, a chassis strain producing perillaldehyde was constructed using *Yarrowia lipolytica* as the starting strain. Specifically, the process includes the following steps:

[0044] 1) Mitochondrial localization of HMG1 and IDI1 in the MVA pathway

[0045] The MVA pathway HMG-CoA reductase HMG1 and isopentenyl pyrophosphate isomerase IDI1 were located in mitochondria, and the specific procedures are as follows:

[0046] With Po1f- ΔKu70 Using the genome as a template, amplification was performed using primers HMG1-F / R (sequences shown in SEQ ID NO. 5-6) and IDI1-F / R (sequences shown in SEQ ID NO. 7-8), respectively. HMG1 Gene (GenBank sequence number: XM503558.3), IDI1 Gene (GenBank sequence number: XM504974.3); with Po1f- ΔKu70 Using the genome as a template, promoter P was amplified separately. TEFin P FABin and Termination T PEX20 T CYC Using pUC19 as a template, the pUC19 plasmid backbone was amplified. The promoter, target gene, terminator, and pUC19 plasmid backbone fragments were ligated using the Uniclone One Step Seamless Cloning Kit to construct the expression cassette plasmid pUC19-P. TEFin -MLS- HMG1 -T PEX20 and pUC19-P FABin -MLS- IDI1 -T CYC .

[0047] In order to HMG1 Genes and IDI1The gene was integrated into the A2 locus on the yeast chromosome. 1000 bp upstream and downstream of the A2 locus were selected as upstream and downstream homologous arms to construct A2 homologous arm plasmids; using Po1f- ΔKu70 Using the genome as a template, homologous arms upstream and downstream of the A2 site were amplified using primers A2-F1 / R1 (sequences shown in SEQ ID NO. 9-10) and A2-F2 / R2 (sequences shown in SEQ ID NO. 11-12), respectively. The URA3 fragment containing the LoxP / loxR sequence was amplified using primer URA3-F / R (sequences shown in SEQ ID NO. 13-14). The pUC19-A2-URA3 plasmid was constructed using the Uniclone One Step Seamless Cloning Kit. Amplification was then performed using primers P1-F / R (sequences shown in SEQ ID NO. 15-16), P2-F / R (sequences shown in SEQ ID NO. 17-18), and P3-F / R (sequences shown in SEQ ID NO. 19-20), respectively. HMG1 Genes and IDI1 Gene expression cassette P TEFin -MLS- HMG1 -T PEX20 P FABin -MLS- IDI1 -T CYC And the pUC19-A2-URA3 fragment with a homologous arm, which will P TEFin -MLS- HMG1 -T PEX20 / P FABin -MLS- IDI1 -T CYC The gene expression cassette and the pUC19-A2-URA3 fragment were cloned in one step to construct the plasmid pUC19-A2-MLS-HMG1-MLS-IDI1.

[0048] Using plasmid pUC19-A2-MLS-HMG1-MLS-IDI1 as a template, homologous arms and gene expression cassettes were amplified and recovered using primers P4-F / R (sequences shown in SEQ ID NO. 21-22); the recovered fragments were transformed into Po1f- according to the Frozen-EZ Yeast Transformation II kit method. ΔKu70 The strain was plated on YNB-Ura plates; after culturing in a 30℃ incubator for 4 days, positive transformants were selected after PCR verification to obtain engineered strain SC-1.

[0049] 2) Overexpression of precursor synthesis genes SsGGPPS , SsLPPS

[0050] Overexpression of the geranylgeranyl pyrophosphate synthase gene derived from geranium SsGGPPS Lysandrin diol pyrophosphate synthase gene SsLPPS The specific steps are as follows:

[0051] Artificial synthesis based on codon preference of Yersinia lipophila. SsGGPPS , SsLPPS The genes, whose nucleotide sequences are shown in SEQ ID NO.1-2, were used as templates to amplify the following genes: SsGGPPS , SsLPPS Gene fragment; using the plasmid pUC19-P obtained in step 1). TEFin -MLS- HMG1 -T PEX20 and pUC19-P FABin -MLS- IDI1 -T CYC Using pUC19 as a template, the pUC19 backbone with promoter and terminator were amplified separately and ligated using the Uniclone One Step Seamless Cloning Kit to construct the expression cassette plasmid pUC19-P. TEFin - SsGGPPS -T PEX20 and pUC19-P FABin - SsLPPS- T CYC Amplification was performed using primers P1-F / R (same as above), P2-F / R (same as above), and P3-F / R (same as above), respectively. SsGGPPS , SsLPPS The gene expression cassette and the pUC19-A2-URA3 plasmid with homologous arms (constructed in the same way as in step 1) are identical to the pUC19-A2-URA3 plasmid, except that the A2 site is replaced with the IntD10 site. 1000 bp upstream and downstream of the IntD10 site are selected as homologous arms, and the homologous arms upstream and downstream of the IntD10 site are amplified using primers IntD10-F1 / R1 (sequences shown in SEQ ID NO. 23-24) and IntD10-F2 / R2 (sequences shown in SEQ ID NO. 25-26), respectively. TEFin - SsGGPPS -T PEX20 / P FABin - SsLPPS- T CYC The gene expression cassette and the pUC19-IntD10-URA3 fragment were cloned in one step to construct the plasmid pUC19-IntD10- SsGGPPS - SsLPPS .

[0052] With plasmid pUC19-IntD10- SsGGPPS- SsLPPS Using the template, homologous arms and gene expression cassettes were amplified and recovered using primers P4-F / R (as above); the recovered fragments were transformed into the recovered URA3-tagged SC-1 strain according to the Frozen-EZ Yeast Transformation II kit method and plated on YNB-Ura plates; after culturing in a 30℃ incubator for 4 days, positive transformants were selected after PCR verification to obtain engineered strain SC-2.

[0053] 3) Overexpression of key genes SsSCS

[0054] Overexpression of the perilla alcohol synthase gene derived from perilla SsSCS The specific steps are as follows:

[0055] Artificial synthesis based on codon preference of Yersinia lipophila. SsSCS The gene, whose nucleotide sequence is shown in SEQ ID NO.3 and whose amino acid sequence is shown in SEQ ID NO.4; SsSCS Gene as template, amplification SsSCS Gene fragment; using plasmid pUC19-P from step 2). FABin - SsLPPS- T CYC Using pUC19 as a template, a pUC19 backbone with promoter and terminator was amplified and ligated using the Uniclone One Step Seamless Cloning Kit to construct the expression cassette plasmid pUC19-P. FABin - SsSCS- T CYC Amplification was performed using primers P2-F2 / R (sequences shown in SEQ ID NO. 31 and 18) and P3-F / R (same as above), respectively. SsSCS The gene expression cassette and the plasmid pUC19-A2-URA3 with homologous arms (constructed in the same way as in step 1) are identical, except that the A2 site is replaced with the IntF site. 1000 bp upstream and downstream of the IntF site are selected as homologous arms, and the homologous arms upstream and downstream of the IntF site are amplified using primers IntF-F1 / R1 (sequences shown in SEQ ID NO. 27-28) and IntF-F2 / R2 (sequences shown in SEQ ID NO. 29-30), respectively. FABin - SsSCS- T CYC The gene expression cassette and the pUC19-IntF-URA3 fragment were cloned in one step to construct the plasmid pUC19-IntF- SsSCS .

[0056] With plasmid pUC19-IntF-SsSCS Using the template, homologous arms and gene expression cassettes were amplified and recovered using primers P4-F / R (as above); the recovered fragments were transformed into the recovered URA3-tagged SC-2 strain according to the Frozen-EZ Yeast Transformation II kit method and plated on YNB-Ura plates; after 4 days of incubation in a 30℃ incubator, positive transformants were selected after PCR verification to obtain engineered strain SC-3.

[0057] A single colony of engineered strain SC-3 was inoculated into a 10 mL centrifuge tube containing 1 mL of YPD medium and cultured in a shaker at 250 rpm and 30 °C for 24 h. The primary seed culture was inoculated into a 100 mL Erlenmeyer flask containing 15 mL of YPD medium and cultured for 24 h at an inoculation rate of 10% (v / v). The secondary seed culture was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of YPD-60 medium at an inoculation rate of 5% (v / v). 10% (v / v) isopropyl myristate was added, and fermentation was carried out under the same conditions for 120 h. The yield of perillaldehyde was 130.8 mg / L.

[0058] Example 2

[0059] In this embodiment, the engineered strain SC-3(WT) constructed in Example 1 was used as the starting strain, and a single point mutation was performed on the perillol synthase in the engineered strain SC-3 to construct a single point mutant strain that produces high perillol.

[0060] Specifically, site-directed mutagenesis was performed on amino acids selected from the substrate binding pocket, using the plasmid pUC19-IntF- constructed in Example 1. SsSCS Using a template, primers were designed for plasmid amplification and mutagenesis. After obtaining the linearized plasmid vector with the mutated bases, it was transformed into *E. coli* DH5α, and after in vivo repair and circularization, the plasmid with the mutated bases was obtained. The expression cassette carrying the mutated gene was then transformed into a yeast substrate strain to obtain a highly efficient mutant strain of *Perilla frutescens* alcohol synthase. The specific procedures are as follows:

[0061] With plasmid pUC19-IntF- SsSCSUsing these as templates, 12 pairs of primers (Q87L-F / R, V133S-F / R, R167P-F / R, Y212A-F / R, I221K-F / R, R292A-F / R, T326I-F / R, N431I-F / R, E484G-F / R, K489N-F / R, D558V-F / R, T561E-FR) were used, with sequences as shown in SEQ ID. PCR amplification was performed on (as shown in NO.32-55), yielding 12 mutant sequences. The mutation methods were as follows: glutamine at position 87 of SsSCS was mutated to leucine, valine at position 133 to serine, arginine at position 167 to proline, tyrosine at position 212 to alanine, isoleucine at position 221 to lysine, arginine at position 292 to alanine, threonine at position 326 to isoleucine, asparagine at position 431 to isoleucine, glutamic acid at position 484 to glycine, lysine at position 489 to asparagine, aspartic acid at position 558 to valine, and threonine at position 561 to glutamic acid.

[0062] The above PCR amplification reaction system is as follows: 1 μL template, 2 μL each of upstream and downstream primers, 25 μL PrimeSTAR Max DNA polymerase, and 20 μL sterile double-distilled water;

[0063] The above PCR amplification program is as follows: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles, and 72℃ extension for 7 min.

[0064] After the reaction, the PCR product was purified using a PCR product purification kit. 8.5 μL of the purified PCR product was added to 0.5 μL of Dpn I restriction endonuclease and 1 μL of 10×CutSmart, and incubated at 37°C for 4 hours.

[0065] The 12 linearized plasmids treated with Dpn I were transformed into *E. coli* DH5α and plated onto LB agar plates containing ampicillin. The plates were then incubated overnight at 37°C with the plates inverted. Transformants grown on the plates were picked and sequenced for verification. After successful sequencing verification, plasmids were extracted using a plasmid extraction kit to obtain the mutant plasmid: pUC19-IntF- SsSCS (Q87L), pUC19-IntF- SsSCS (V133S), pUC19-IntF- SsSCS (R167P), pUC19-IntF- SsSCS (Y212A), pUC19-IntF- SsSCS (I221K), pUC19-IntF- SsSCS(R292A), pUC19-IntF- SsSCS (T326I), pUC19-IntF- SsSCS (N431I), pUC19-IntF- SsSCS (E484G), pUC19-IntF- SsSCS (K489N), pUC19-IntF- SsSCS (D558V), pUC19-IntF- SsSCS (T561E).

[0066] Using the aforementioned point mutant plasmid as a template, and following the method described in Example 1, the homologous arms and gene expression cassette were amplified and recovered using primers P4-F / R. The recovered fragments were then transformed into the recovered URA3-tagged SC-2 strain according to the Frozen-EZ Yeast Transformation II kit method and plated on YNB-Ura plates. After 4 days of incubation at 30°C, positive transformants were selected after PCR verification, yielding mutant strains SC-3(Q87L), SC-3(V133S), SC-3(R167P), SC-3(Y212A), SC-3(I221K), SC-3(R292A), SC-3(T326I), SC-3(N431I), SC-3(E484G), SC-3(K489N), SC-3(D558V), and SC-3(T561E).

[0067] The above-mentioned single-point mutant strain was inoculated into a 10mL centrifuge tube containing 1mL YPD medium and cultured in a shaker at 250rpm and 30℃ for 24h. The primary seed culture was inoculated into a 100mL Erlenmeyer flask containing 15mL YPD medium and cultured for 24h at an inoculation volume of 10% (v / v). The secondary seed culture was inoculated into a 250mL Erlenmeyer flask containing 50mL YPD-60 medium at an inoculation volume of 5% (v / v). 10% (v / v) isopropyl myristate was added, and fermentation was carried out under the same conditions for 120h. The results of perillaldehyde production are as follows: Figure 1 As shown.

[0068] from Figure 1 As can be seen, the perillol yield of the single point mutant strain was higher than that of the engineered strain SC-3(WT), and the highest perillol yield of the single point mutant strain SC-3(K489N) was 383.2 mg / L.

[0069] Example 3

[0070] In this embodiment, based on the single mutant engineered strain SC-3(K489N) with the best perillol yield, a combined mutant engineered strain was further constructed; a high-yielding perillol combined mutant strain was constructed using the four single-point mutations with the highest activity (R167P, Y212A, R292A, K489N).

[0071] Specifically, the construction method is the same as in Example 2. The single mutant plasmid pUC19-IntF- SsSCS Based on (K489N), a double mutant plasmid pUC19-IntF- was constructed using primers R167P-F / R. SsSCS (R167P / K489N); Double mutant plasmid pUC19-IntF- was constructed using primers Y212A-F / R. SsSCS (Y212A / K489N); Double mutant plasmid pUC19-IntF- was constructed using primers R292A-F / R. SsSCS (R292A / K489N). In the double mutant plasmid pUC19-IntF- SsSCS Based on (R292A / K489N), a triple mutant plasmid pUC19-IntF- was constructed using primers R167P-F / R. SsSCS (R167P / R292A / K489N); Triple mutant plasmid pUC19-IntF- was constructed using primers Y212A-F / R. SsSCS (Y212A / R292A / K489N). In the triple mutant plasmid pUC19-IntF- SsSCS Based on (Y212A / R292A / K489N), a quadruple mutant plasmid pUC19-IntF- was constructed using primers R167P-F / R. SsSCS (R167P / Y212A / R292A / K489N).

[0072] Using the aforementioned combined mutant plasmid as a template, and following the method described in Example 1, the homologous arms and gene expression cassette were amplified and recovered using primers P4-F / R. The recovered fragments were then transformed into the recovered URA3-tagged SC-2 strain according to the Frozen-EZ Yeast Transformation II kit method and plated on YNB-Ura plates. After incubation at 30°C for 4 days, positive transformants were selected after PCR verification, resulting in the combined mutant strains SC-3(R167P / K489N), SC-3(Y212A / K489N), SC-3(R292A / K489N), SC-3(R167P / R292A / K489N), SC-3(Y212A / R292A / K489N), and SC-3(R167P / Y212A / R292A / K489N).

[0073] The above-mentioned combined mutant strains were inoculated into 10 mL centrifuge tubes containing 1 mL of YPD medium and cultured in a shaker at 250 rpm and 30 °C for 24 h. The primary seed culture was inoculated into a 100 mL Erlenmeyer flask containing 15 mL of YPD medium and cultured for 24 h at an inoculation volume of 10% (v / v). The secondary seed culture was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of YPD-60 medium at an inoculation volume of 5% (v / v). 10% (v / v) isopropyl myristate was added, and fermentation was carried out under the same conditions for 120 h. The results of perillaldehyde production are as follows: Figure 2 As shown.

[0074] from Figure 2 As can be seen, the yield of perillaldehyde from the combined mutant strain was higher than that of the engineered strain SC-3 (WT) and the single point mutant strain SC-3 (K489N), and the highest yield of perillaldehyde from the combined mutant strain SC-3 (R167P / Y212A / R292A / K489N) was 730.1 mg / L.

[0075] Example 4

[0076] In this embodiment, the leucine deficiency was corrected in the combined mutant strain SC-3 (R167P / Y212A / R292A / K489N) constructed in Example 3. LEU Genes were used to construct a leucine-deficient strain that produces high levels of perillaldehyde.

[0077] Specifically, it includes the following steps:

[0078] Amplification of the pUC19 backbone with promoter and terminator, and amplification of... LEU The gene (GenBank sequence number: AF260230.1) was cloned in one step to construct the expression cassette plasmid pUC19-P. TEFin - LEU -T PEX20 Amplification was performed using primers P1-F / R2 (sequences shown in SEQ ID NO.15, 56) and P3-F / R (same as above). LEUThe gene expression cassette and the plasmid pUC19-IntA1-URA3 with homologous arms (constructed in the same way as in step 1) are identical to the pUC19-A2-URA3 plasmid, except that the A2 site is replaced with the IntA1 site. 1000bp upstream and downstream of the IntA1 site are selected as homologous arms, and the homologous arms upstream and downstream of the IntA1 site are amplified using primers IntA1-F1 / R1 (sequences shown in SEQ ID NO. 57-58) and IntA1-F2 / R2 (sequences shown in SEQ ID NO. 59-60), respectively. The expression cassette plasmid pUC19-IntA1-URA3 is constructed through one-step cloning. LEU .

[0079] With plasmid pUC19-IntA1- LEU Using a template, the fragment with homologous arms and gene expression cassette was amplified and recovered using primers P4-F / R (as above); the recovered fragment was transformed into SC-3 (R167P / Y212A / R292A / K489N) strain according to the Frozen-EZ Yeast Transformation II kit method, and plated on YNB-Leu leucine-deficient screening plate; after culturing in a 30℃ incubator for 4 days, positive transformants were selected after PCR verification to obtain engineered strain SC-4.

[0080] Following the method described in Example 1, after two rounds of activation, the engineered strain SC-4 was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of YPD-60 medium at an inoculation rate of 5% (v / v). 10% (v / v) of isopropyl myristate was added, and fermentation was carried out at 30°C and 250 rpm for 120 h. The yield of perillyl alcohol was 1107.6 mg / L.

[0081] In summary, this invention modifies perillyl alcohol synthase through semi-rational design. Furthermore, the recombinant genetically engineered strain obtained by integrating key genes in the perillyl alcohol synthesis process can produce high yields of perillyl alcohol using glucose as a substrate.

[0082] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A perilla frutescens alcohol synthase mutant, characterized in that, The perilla ethanol synthase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.4 at any of the following sites: A1) Q87L, A2) V133S, A3) R167P, A4) Y212A, A5) I221K, A6) R292A, A7) T326I, A8) N431I, A9) E484G, A10) K489N, A11) D558V, A12) T561E.

2. The perilla frutescens alcohol synthase mutant according to claim 1, characterized in that, The perilla ethanol synthase mutants include the following combination mutations: B1) R167P / K489N, B2) Y212A / K489N, B3) R292A / K489N, B4) R167P / R292A / K489N, B5) Y212A / R292A / K489N, and B6) R167P / Y212A / R292A / K489N.

3. A nucleic acid molecule encoding the perillaldehyde synthase mutant of any one of claims 1-2.

4. A recombinant vector, characterized in that, Includes the nucleic acid molecule as described in claim 3.

5. A recombinant genetically engineered strain that produces high levels of perillaldehyde, characterized in that, This includes the recombinant vector of claim 4, or the nucleic acid molecule of claim 3 integrated into its genome.

6. The use of the perillaldehyde synthase mutant as described in any one of claims 1-2, the nucleic acid molecule as described in claim 3, the recombinant vector as described in claim 4, or the recombinant genetically engineered strain as described in claim 5 in the synthesis of perillaldehyde.

7. A method for synthesizing perillyl alcohol, characterized in that, The process includes the following steps: using glucose as a substrate, fermenting the recombinant genetically engineered strain described in claim 5 to obtain perillyl alcohol; The genome of the recombinant genetically engineered strain also integrates an enzyme encoding HMG-CoA reductase. HMG1 Gene, encoding isopentenyl pyrophosphate isomerase IDI1 Gene encoding geraniol geraniol pyrophosphate synthase SsGGPPS Gene, encoding lysine pyrophosphate diol ester synthase SsLPPS Gene.

8. The method according to claim 7, characterized in that, The fermentation process also includes the step of adding isopropyl myristate.

9. The method according to claim 8, characterized in that, The amount of isopropyl myristate added is 5-15%.

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