A santalene synthase mutant, an engineered bacterium and application thereof

Abstract

The application discloses a santalene synthase mutant, an engineering bacterium and application thereof, and belongs to the technical field of protein engineering and gene engineering. The total yield of Z-alpha-santalol, Z-beta-santalol, Z-alpha-myrcenol, Z-epi-beta-santalol, alpha-santalene, beta-santalene, alpha-myrcene and epi-beta-santalene can reach 1.9 g / L after 6-day fermentation culture of the Saccharomyces cerevisiae engineering strain SZ-1. The total yield of the eight products produced by the engineering strain SZ-2 can reach 0.7 g / L, wherein Z-alpha-santalol and Z-beta-santalol respectively account for 43.4% and 22% of the total products. The application can produce high-quality sandalwood essential oil meeting international standards by utilizing SanSyn F441V The Saccharomyces cerevisiae engineering strain constructed by the mutant can produce high-quality sandalwood essential oil meeting international standards, and can effectively alleviate the situation that the market demand cannot be met, and protect wild sandalwood resources.

Description

Technical Field

[0001] This invention belongs to the fields of protein engineering and genetic engineering technology, specifically relating to a santalene synthase mutant, engineered bacteria and their applications. Background Technology

[0002] Sandalwood volatile oil has a strong aroma and ranges in color from yellow to dark brown. Its main components are sesquiterpenoid compounds Z-α-santalol and Z-β-santalol. Sandalwood volatile oil has high medicinal and economic value, possessing pharmacological activities such as sedation, antibacterial and antiviral properties, anti-inflammatory and analgesic effects, antioxidant properties, and antitumor effects. In addition, it is also commonly used in the food, cosmetics, and perfume industries.

[0003] Currently, sandalwood volatile oil is mainly extracted from the heartwood and roots of 25-year-old sandalwood trees using steam distillation. Due to the long growth cycle of sandalwood and the complex extraction process, the supply of sandalwood essential oil cannot meet market demand. With the development of synthetic biology, breakthroughs have been made in the production of important active ingredients in traditional Chinese medicine, such as the production of artemisinin and protopanaxadiol using engineered strains of Saccharomyces cerevisiae. The biosynthetic pathways of the main components of sandalwood essential oil have been elucidated. In plants, farnesyl pyrophosphate (FPP) is catalyzed by santalene synthase to produce α-santalene, β-santalene, epi-β-santalene, exo-α-bergamotene, and other products. Subsequently, these compounds are hydroxylated by cytochrome P450 monooxidase to produce Z-α-santalol, Z-β-santalol, Z-epi-β-santalol, Z-epi-β-santalol, Z-exo-α-bergamotol, and other products. There are reports on the production of santalene and santalol using Saccharomyces cerevisiae (ACS Synth. Biol. 2020, 9, 2, 449–456).

[0004] According to international standards for sandalwood essential oil quality, the content of Z-α-santalol should reach 45% to 55%, and the content of Z-β-santalol should reach 15% to 25%. However, the Z-α-santalol content in the engineered strains of *Saccharomyces cerevisiae* currently reported to produce sandalwood essential oil has not yet met international standards. The main reason is that santalene synthase (STS), while catalyzing the production of santalene from fructospheric polyphenol oxidase (FPP), also produces a large amount of exo-α-bergamotene. Exo-α-bergamotene is further oxidized by cytochrome P450 to produce Z-exo-α-bergamotol, thus causing the produced sandalwood essential oil to fail to meet international standards. Summary of the Invention

[0005] The primary objective of this application is to provide a santalene synthase mutant.

[0006] Another objective of this application is to provide a brewing yeast strain that produces high levels of santalene and santalol.

[0007] Another objective of this application is to provide a method for constructing and applying the above-mentioned engineered brewer's yeast strain that produces high levels of santalene and santalol.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A santalene synthase mutant, comprising at least one of the santalene synthase SanSyn mutant and the SaSSy mutant; wherein the amino acid sequence of the santalene synthase SanSyn mutant is selected from any of the following mutant sequences:

[0010] SanSyn E297L The mutant, whose amino acid sequence is obtained by mutating glutamic acid at position 297 of the amino acid sequence shown in SEQ ID NO.1 to leucine, specifically by mutating the codon at position 297 of SEQ ID NO.1 from GAA in SEQ ID NO.3 to TTA;

[0011] SanSyn F441V The mutant, whose amino acid sequence is obtained by mutating phenylalanine at position 441 of the amino acid sequence shown in SEQ ID NO.1 to valine, specifically by mutating the codon at position 441 of SEQ ID NO.1 from TTT in SEQ ID NO.3 to GTT;

[0012] The amino acid sequence of the santalene synthase SaSSy mutant is selected from any of the following mutant sequences:

[0013] SaSSy L317CThe mutant, whose amino acid sequence is obtained by mutating leucine at position 317 of the amino acid sequence shown in SEQ ID NO.2 to cysteine, specifically, the codon at position 317 of SEQ ID NO.2 is mutated from CTC in SEQ ID NO.4 to TGC;

[0014] SaSSy L317I The mutant, whose amino acid sequence is obtained by mutating leucine at position 317 of the amino acid sequence shown in SEQ ID NO.2 to isoleucine, specifically, the codon at position 317 of SEQ ID NO.2 is mutated from CTC in SEQ ID NO.4 to ATC;

[0015] SaSSy L317T The mutant, whose amino acid sequence is obtained by mutating leucine at position 317 of the amino acid sequence shown in SEQ ID NO.2 to threonine, specifically, the codon at position 317 of SEQ ID NO.2 is mutated from CTC in SEQ ID NO.4 to ACT;

[0016] SaSSy L317V The mutant, whose amino acid sequence is obtained by mutating leucine at position 317 of the amino acid sequence shown in SEQ ID NO.2 to valine, specifically, the codon at position 317 of SEQ ID NO.2 is mutated from CTC in SEQ ID NO.4 to GTT;

[0017] SaSSy S459V The mutant, whose amino acid sequence is obtained by mutating serine at position 459 of the amino acid sequence shown in SEQ ID NO.2 to valine, specifically, the codon at position 459 of SEQ ID NO.2 is mutated from AGT in SEQ ID NO.4 to GTT;

[0018] SaSSy S459A The mutant, whose amino acid sequence is obtained by mutating serine at position 459 of the amino acid sequence shown in SEQ ID NO.2 to alanine, specifically by mutating the codon at position 459 of SEQ ID NO.2 from AGT in SEQ ID NO.4 to GCT;

[0019] SaSSy S459H The mutant, whose amino acid sequence is obtained by mutating serine at position 459 of the amino acid sequence shown in SEQ ID NO.2 to histidine, specifically, the codon at position 459 of SEQ ID NO.2 is mutated from AGT in SEQ ID NO.4 to CAC;

[0020] SaSSy S459IThe mutant, whose amino acid sequence is obtained by mutating serine at position 459 of the amino acid sequence shown in SEQ ID NO.2 to isoleucine, specifically, the codon at position 459 of SEQ ID NO.2 is mutated from AGT in SEQ ID NO.4 to ATC;

[0021] SaSSy S459T The mutant, whose amino acid sequence is obtained by mutating serine at position 459 of the amino acid sequence shown in SEQ ID NO.2 to threonine, specifically, the codon at position 459 of SEQ ID NO.2 is mutated from AGT in SEQ ID NO.4 to ACT;

[0022] SaSSy S535C The mutant, whose amino acid sequence is obtained by mutating serine at position 535 of the amino acid sequence shown in SEQ ID NO.2 to cysteine, specifically, the codon at position 535 of SEQ ID NO.2 is mutated from TCT in SEQ ID NO.4 to TGC;

[0023] SaSSy S535T The mutant, whose amino acid sequence is obtained by mutating serine at position 535 of the amino acid sequence shown in SEQ ID NO.2 to threonine, specifically, the codon at position 535 of SEQ ID NO.2 is mutated from TCT in SEQ ID NO.4 to ACT;

[0024] SaSSy F538W The mutant, whose amino acid sequence is obtained by mutating phenylalanine at position 538 of the amino acid sequence shown in SEQ ID NO.2 to tryptophan, specifically by mutating the codon at position 538 of SEQ ID NO.2 from TTC in SEQ ID NO.4 to TGG.

[0025] The gene encoding the above-mentioned santalene synthase mutant.

[0026] Recombinant expression vectors containing the above genes or engineered Saccharomyces cerevisiae strains that produce high levels of santalene.

[0027] A high-yield *Saccharomyces cerevisiae* engineered strain for santalene and santalol, characterized by the following features: Starting with a *Saccharomyces cerevisiae* strain, the OYE2, OYE3, ATF1, ATF2, LPP1, DPP1, and ROX1 genes are knocked out, and the endogenous promoter of the *Saccharomyces cerevisiae* ERG9 gene is replaced with P... HXT1 Promoters, overexpression of mSTS, ADH2, ALD6, and SeACS L641PIDI1, tHMG1, UPC2-1, CYP736A167, and SaCPR2 genes; among which:

[0028] The IDI1 and UPC2-1 genes were integrated into the DPP1 site on the chromosome of Saccharomyces cerevisiae.

[0029] ADH2, ALD6, SeACS L641P The gene was integrated into the LPP1 site on the chromosome of Saccharomyces cerevisiae.

[0030] mSTS and tHMG1 genes are integrated into the chromosome P of Saccharomyces cerevisiae. ERG9 site;

[0031] The CYP736A167, SaCPR2, and second copy of the mSTS gene were integrated into the MET17 site of Saccharomyces cerevisiae.

[0032] The third copy of the mSTS gene was integrated into the ROX1 site on the Saccharomyces cerevisiae chromosome;

[0033] The second copy of the CYP736A167 gene, the second copy of the tHMG1 gene, or the second and third copies of the CYP736A167 gene are integrated into the ROX1 site of the Saccharomyces cerevisiae chromosome.

[0034] The preferred brewing yeast is brewing yeast CEN.PK2-1D.

[0035] The nucleotide sequence of the mSTS gene is a nucleotide sequence of a santalene synthase mutant, specifically at least one of the nucleotide sequences of the santalene synthase SanSyn mutant and the SaSSy mutant; preferably SanSyn. F441V The nucleotide sequence of the mutant.

[0036] The method for knocking out the OYE2, OYE3, ATF1, and ATF2 genes is to use a CRISPR-Cas9 gene knockout system; preferably, the knockout is performed through the following steps:

[0037] S1. The recombinant plasmid pCRCT-OYE2-OYE3 was transformed into Saccharomyces cerevisiae, and after culture, expression and screening, strains with the OYE2 and OYE3 genes knocked out were obtained;

[0038] S2. The recombinant plasmid pCRCT-ATF1-ATF2 was transformed into a Saccharomyces cerevisiae strain with the OYE2 and OYE3 genes knocked out. After culture, expression and screening, strains with the OYE2, OYE3, ATF1 and ATF2 genes knocked out were obtained.

[0039] The brewing yeast mentioned in step S1 is preferably brewing yeast CEN.PK2-1D.

[0040] The culture medium used for screening in step S1 is preferably SD-URA-deficient plate medium; the formulation of the SD-URA-deficient plate medium is as follows: YNB medium 6.7 g / L, glucose 20 g / L, agar powder 20 g / L, uracil (URA)-deficient amino acids (100×) 10 mL / L. The uracil (URA)-deficient amino acids (100×) are prepared as follows: weigh out 0.9 g of valine, 0.25 g of adenine sulfate, 0.18 g of tyrosine, 0.12 g of arginine, 0.24 g of tryptophan, 0.6 g of aspartic acid, 1.2 g of threonine, 0.6 g of glutamic acid, 0.18 g of lysine, 0.3 g of phenylalanine, 0.12 g of methionine, 0.36 g of leucine, 2.25 g of serine, and 0.12 g of histidine, and bring the volume to 57 mL with ddH2O.

[0041] The culture conditions described in step S1 are preferably 28–32°C and 200–250 rpm; more preferably 30°C and 220 rpm.

[0042] The IDI1, ADH2, and ALD6 genes are preferably cloned from the genome of Saccharomyces cerevisiae CEN.PK2-1D. The nucleotide sequence of IDI1 can be found in GenBank: NM_001183931.1; the nucleotide sequence of ADH2 can be found in GenBank: NM_001182812.1; and the nucleotide sequence of ALD6 can be found in GenBank: NM_001183875.1.

[0043] The CYP736A167, SaCPR2, tHMG1, UPC2-1, and P mentioned above HXT1 These sequences are preserved for our laboratory. The nucleotide sequence of CYP736A167 is available in GenBank: KU169302.1; the nucleotide sequence of SaCPR2 is available in GenBank: KC842188.1; tHMG1, UPC2-1, and P... HXT1 The nucleotide sequence of the gene has been disclosed in patent application CN201911180415.0, "A high-yielding engineered Saccharomyces cerevisiae strain of santalene and santalol, its construction method and application." The SeACS mentioned above... L641P The gene was artificially synthesized using Saccharomyces cerevisiae as the host through codon optimization, and its nucleotide sequence is shown in SEQ ID NO.5.

[0044] The mSTS gene sequence is a nucleotide sequence of a santalene synthase mutant, specifically at least one of the nucleotide sequences of the santalene synthase SanSyn mutant and the SaSSy mutant; preferably SanSyn. F441V The nucleotide sequence of the mutant.

[0045] The method for constructing the above-mentioned engineered brewer's yeast strain that produces high levels of santalene and santalol includes the following steps:

[0046] (1) Gene knockout: The OYE2, OYE3, ATF1, and ATF2 genes were knocked out using the CRISPR-Cas9 gene knockout system. The nucleotide sequences of the OYE2 and OYE3 crRNA spacers used are shown in SEQ ID NO.6; the nucleotide sequences of the ATF1 and ATF2 crRNA spacers are shown in SEQ ID NO.7. After culture, expression, and screening, strains with the OYE2, OYE3, ATF1, and ATF2 genes knocked out were obtained.

[0047] (2) Module construction: ADH2, ALD6, and SeACS are respectively constructed. L641P IDI1, tHMG1, UPC2-1, CYP736A167, SaCPR2, mSTS and their corresponding promoters and terminators are sequentially connected to obtain the corresponding expression modules;

[0048] (3) Strain construction: The above-mentioned gene modules are integrated into the corresponding chromosomal loci of the brewing strain to obtain the brewing yeast engineered strain that produces high levels of santalene and santalol.

[0049] Among them, the IDI1 and UPC2-1 genes were integrated into the DPP1 site on the chromosome of Saccharomyces cerevisiae;

[0050] ADH2, ALD6, SeACS L641P The gene was integrated into the LPP1 site on the chromosome of Saccharomyces cerevisiae.

[0051] mSTS and tHMG1 genes are integrated into the chromosome P of Saccharomyces cerevisiae. ERG9 site;

[0052] The CYP736A167, SaCPR2, and second copy of the mSTS gene were integrated into the MET17 gene locus of Saccharomyces cerevisiae.

[0053] The third copy of the mSTS gene was integrated into the ROX1 site on the Saccharomyces cerevisiae chromosome.

[0054] The second copy of the CYP736A167 gene, the second copy of the tHMG1 gene, or the second and third copies of the CYP736A167 gene are integrated into the ROX1 site of the Saccharomyces cerevisiae chromosome.

[0055] Specifically, as a preferred embodiment, the method for constructing the engineered brewer's yeast strain that produces high levels of santalene and santalol includes the following steps:

[0056] (1) Gene knockout:

[0057] (a) The recombinant plasmid pCRCT-OYE2-OYE3 was transformed into Saccharomyces cerevisiae M, and after culture and screening, yeast strain M-1 with the OYE2 and OYE3 genes knocked out was obtained.

[0058] (b) The recombinant plasmid pCRCT-ATF1-ATF2 was transformed into the Saccharomyces cerevisiae M-1 strain, and after culture and screening, the yeast strain M-2 with the OYE2, OYE3, ATF1 and ATF2 genes knocked out was obtained.

[0059] (2) Module construction:

[0060] (a) P TEF2 UPC2-1 and T FBA1 By connecting sequentially, we obtain module P. TEF2 -UPC2-1-T FBA1 Name it Module 1;

[0061] (b) P TPI1 IDI1 and T PGK1 By connecting sequentially, we obtain module P. TPI1 -IDI1-T PGK1 Name it Module 2;

[0062] (c) P ENO2 ALD6, T TDH2 and P PDC1 By connecting sequentially, we obtain module P. ENO2 -ALD6-T TDH2 -P PDC1 Name it Module 3;

[0063] (d) ADH2, T ADH2 P TEF1 SeACS L641P and T PGI1 By connecting sequentially, we obtain module ADH2-T. ADH2 -P TEF1 -SeACS L641P -T PGI1 Name it Module 4;

[0064] (e) P TPI1 tHMG1, T PGK1 P TDH3 mSTS and T ENO2 By connecting sequentially, we obtain module P. TPI1 -tHMG1-T PGK1 -P TDH3 -mSTS-T ENO2 Name it Module 5;

[0065] (f) P PGK1CYP736A167, T ADH1 P TEF1 SaCPR2 and T CYC1 By connecting sequentially, we obtain module P. PGK1 -CYP736A167-T ADH1 -P TEF1 -SaCPR2-T CYC1 Name it Module 6;

[0066] (g) P FBA1 CYP736A167 and T PGI1 By connecting sequentially, we obtain module P. FBA1 -CYP736A167-T PGI1 Name it Module 7;

[0067] (h) LPP1 up HIS3 and LPP1 down By connecting them sequentially, we obtain module LPP1. up -HIS3-LPP1 down Name it Module 8;

[0068] (i) DPP1 up TRP1 and DPP1 down By connecting sequentially, we obtain module DPP1. up -TRP1-DPP1 down Name it Module 9;

[0069] (j) P ERG9up KILEU2, P HXT1 and P ERG9down By connecting sequentially, we obtain module P. ERG9up -KILEU2-P HXT1 -P ERG9down Name it Module 10;

[0070] (k) MET17 up URA3 and MET17 down By connecting them sequentially, we obtain module MET17. up -URA3-MET17 down Name it Module 11;

[0071] (l) ROX1 up MET17 and ROX1 down By connecting them sequentially, we obtain module ROX1. up -MET17-ROX1 down Name it Module 12;

[0072] (3) Construction of strain M-6

[0073] (I) Module 1, Module 2 and the selection marker TRP1 were co-transformed into strain M-2, with the integration site being the DPP1 site on the Saccharomyces cerevisiae chromosome. Then, strain M-3 was obtained by screening and culturing in a yeast auxotype medium.

[0074] (II) Module 3, Module 4 and screening marker HIS3 were co-transformed into strain M-3, with the integration site being the LPP1 site on the chromosome of Saccharomyces cerevisiae. Then, strain M-4 was obtained by screening and culturing in yeast auxotrophic medium.

[0075] (III) Module 5, P HXT1 Strain M-4 was co-transformed with the selection marker KILEU2, and the integration site was the P chromosome of Saccharomyces cerevisiae. ERG9 The site was identified, and then strain M-5 was obtained through screening and culture in a yeast auxotrophic medium.

[0076] (Ⅳ) P in module 5 TDH3 -mSTS-T ENO2 The screening marker URA3 of fragment, module 6 and module 11 was co-transformed into strain M-5, with the integration site being the MET17 site on the Saccharomyces cerevisiae chromosome. Then, strain M-6 was obtained by screening and culturing in yeast auxotrophic medium.

[0077] (4) Construction of strain SZ-1

[0078] P in Module 5 and Module 6 PGK1 -CYP736A167-T ADH1 The fragment and the selection marker MET17 were co-transformed into strain M-6, with the integration site being the ROX1 site on the Saccharomyces cerevisiae chromosome. Then, the strain was screened and cultured in a yeast auxotrophic medium to obtain strain SZ-1, which is the Saccharomyces cerevisiae engineered strain that produces high levels of santalene and santalol.

[0079] (5) Construction of strain SZ-2

[0080] P in module 5 TDH3 -mSTS-T ENO2 P in Fragment, Module 6 PGK1 -CYP736A167-T ADH1 Fragment, module 7, and screening marker MET17 were co-transformed into strain M-6, with the integration site being the ROX1 site on the Saccharomyces cerevisiae chromosome. After screening and culturing in a yeast auxotrophic medium, strain SZ-2 was obtained, which is the Saccharomyces cerevisiae engineered strain that produces high levels of santalene and santalol.

[0081] The yeast strain M is preferably Saccharomyces cerevisiae CEN.PK2-1D.

[0082] The mSTS gene sequence is a nucleotide sequence of a santalene synthase mutant, specifically at least one of the nucleotide sequences of the santalene synthase SanSyn mutant and the SaSSy mutant; preferably SanSyn. F441V The nucleotide sequence of the mutant.

[0083] In step (2), the P TEF2 P TPI1 P ENO2 P PDC1 P TEF1 P TDH3 P PGK1 and P FBA1 It can be cloned from the genome of Saccharomyces cerevisiae CEN.PK2-1D, in which P TEF2 The nucleotide sequence can be found in GenBank: CP020124.1, from position 477500 to 478059; P TPI1 The nucleotide sequence can be found in GenBank: CP020126.1, from position 564689 to 564189; P ENO2 The nucleotide sequence can be found in GenBank: CP020130.1, from position 469225 to 469824; P PDC1 The nucleotide sequence can be found in GenBank: CP020134.1, from position 234920 to 234120; P TEF1 The nucleotide sequence can be found in GenBank: CP020138.1, from position 700414 to 700832; P TDH3 The nucleotide sequence can be found in GenBank: CP020129.1, from position 884060 to 884735; P PGK1 The nucleotide sequence can be found in GenBank: CP020125.1, from position 142054 to 143036; P FBA1 The nucleotide sequence can be found in GenBank:CP020133.1, from position 327996 to position 327497.

[0084] In step (2), the T FBA1 T PGK1 T TDH2 T ADH2 T PGI1 T ENO2 T ADH1 T CYC1 The terminator can be cloned from the genome of *Saccharomyces cerevisiae* CEN.PK2-1D, in which T... FBA1The nucleotide sequence can be found in GenBank: CP020133.1, from position 327996 to 327497; T PGK1 The nucleotide sequence can be found in GenBank: CP020125.1, from position 144287 to 144714; T TDH2 The nucleotide sequence can be found in GenBank: CP020132.1, from position 459599 to 459200; T ADH2 The nucleotide sequence can be found in GenBank: CP020135.1, from position 879067 to 878668; T PGI1 The nucleotide sequence can be found in GenBank: CP020124.1, from position 612625 to 612226; T ENO2 The nucleotide sequence can be found in GenBank: CP020124.1, from position 612625 to 612226; T ADH1 The nucleotide sequence can be found in GenBank: CP020137.1, from position 159575 to 159411; T CYC1 The nucleotide sequence can be found in GenBank:LT727633.1, from position 8068 to position 8257.

[0085] In step (2), the LPP1 up The nucleotide sequence can be found in GenBank: CP020126.1, from position 1462217 to 1462518; the LPP1 described down The nucleotide sequence can be found in GenBank: CP020126.1, from position 1463344 to 1463625; the DPP1 described up The nucleotide sequence can be found in GenBank: CP020126.1, from position 1037979 to 1038277; the DPP1 described down The nucleotide sequence can be found in GenBank: CP020126.1, from position 1039148 to 1039481; the P... ERG9up The nucleotide sequence can be found in GenBank: CP020130.1, from position 502815 to position 503221; the P... ERG9down The nucleotide sequence can be found in GenBank: CP020126.1, from position 503341 to 503776; the MET17... up The nucleotide sequence can be found in GenBank: CP020134.1, from position 726291 to 726591; the MET17... downThe nucleotide sequence can be found in GenBank: CP020134.1, from position 727927 to position 728222; the ROX1 described up The nucleotide sequence can be found in GenBank: CP020138.1, from position 679325 to position 679688; the ROX1 described down The nucleotide sequence can be found in GenBank:CP020134.1, from position 680639 to position 681039.

[0086] In step (2), TRP1, HIS3, KILEU2, URA3, and MET17 are screening markers and are stored in our laboratory. The nucleotide sequence of TRP1 is shown in SEQ ID NO.8; the nucleotide sequence of HIS3 is shown in SEQ ID NO.9; the nucleotide sequence of KILEU2 is shown in SEQ ID NO.10; the nucleotide sequence of URA3 is shown in SEQ ID NO.11; and the nucleotide sequence of MET17 is shown in SEQ ID NO.12.

[0087] In steps (3), (4) and (5), the integration is performed using a yeast conversion kit; preferably, it is performed using a super yeast conversion kit.

[0088] In step (I), the yeast auxotrophic culture medium is formulated as follows: YNB medium 6.7 g / L, tryptophan (TRP) auxotrophic amino acid (100×) 10 mL / L, and glucose 20 g / L. The preparation of the solid culture medium requires the addition of 20 g / L agar powder. The TRP (tryptophan) auxotrophic amino acid (100×) formulation is as follows: uracil 0.12 g, adenine sulfate 0.25 g, valine 0.9 g, arginine 0.12 g, aspartic acid 0.6 g, threonine 1.2 g, tyrosine 0.18 g, methionine 0.12 g, glutamic acid 0.6 g, leucine 0.36 g, lysine 0.18 g, serine 2.25 g, phenylalanine 0.3 g, histidine 0.12 g, and diluted to 57 mL with ddH2O.

[0089] In step (II), the yeast auxotrophic culture medium is formulated as follows: YNB medium 6.7 g / L, tryptophan-histidine (TRP-HIS) auxotrophic amino acids (100×) 10 mL / L, and glucose 20 g / L. The preparation of the solid culture medium requires the addition of 20 g / L agar powder; wherein the tryptophan-histidine (TRP-HIS) auxotrophic amino acid (100×) formulation is as follows: uracil 0.12 g, adenine sulfate 0.25 g, valine 0.9 g, arginine 0.12 g, aspartic acid 0.6 g, threonine 1.2 g, tyrosine 0.18 g, methionine 0.12 g, glutamic acid 0.6 g, leucine 0.36 g, lysine 0.18 g, serine 2.25 g, phenylalanine 0.3 g, and diluted to 57 mL with ddH2O.

[0090] In step (III), the yeast auxotrophic culture medium is formulated as follows: 6.7 g / L YNB medium, 10 mL / L of tryptophan-histidine-leucine (TRP-HIS-LEU) auxotrophic amino acids (100×), and 20 g / L glucose. The preparation of the solid culture medium requires the addition of 20 g / L agar powder. The tryptophan-histidine-leucine (TRP-HIS-LEU) auxotrophic amino acid (100×) formulation is as follows: 0.12 g uracil, 0.25 g adenine sulfate, 0.9 g valine, 0.12 g arginine, 0.6 g aspartic acid, 1.2 g threonine, 0.18 g tyrosine, 0.12 g methionine, 0.6 g glutamic acid, 0.18 g lysine, 2.25 g serine, and 0.3 g phenylalanine, diluted to 57 mL with ddH2O.

[0091] In step (IV), the yeast auxotrophic culture medium is formulated as follows: 6.7 g / L YNB medium, 10 mL / L of tryptophan-histidine-leucine-uracil (TRP-HIS-LEU-URA) auxotrophic amino acids (100×), and 20 g / L glucose. The preparation of the solid culture medium requires the addition of 20 g / L agar powder. The TRP-HIS-LEU-URA (tryptophan-histidine-leucine-uracil) auxotrophic amino acids (100×) are formulated as follows: 0.9 g valine, 0.25 g adenine sulfate, 0.12 g arginine, 0.6 g aspartic acid, 0.18 g tyrosine, 0.6 g glutamic acid, 1.2 g threonine, 2.25 g serine, 0.3 g phenylalanine, 0.18 g lysine, and 0.12 g methionine, diluted to 57 mL with ddH2O.

[0092] The yeast-deficient culture medium formulation described in steps (4) and (5) is as follows: YNB 6.7 g / L, tryptophan-histidine-leucine-uracil-methionine (TRP-HIS-LEU-URA-MET) deficient amino acid (100×) 10 mL / L, glucose 20 g / L. The preparation of the solid culture medium requires the addition of 20 g / L of agar powder. The tryptophan-histidine-leucine-uracil-methionine (TRP-HIS-LEU-URA-MET) deficient amino acid (100×) formulation is as follows: valine 0.9 g, adenine sulfate 0.25 g, tyrosine 0.18 g, arginine 0.12 g, aspartic acid 0.6 g, threonine 1.2 g, glutamic acid 0.6 g, lysine 0.18 g, phenylalanine 0.3 g, serine 2.25 g, and adjusted to 57 mL with ddH2O.

[0093] The application of the above-mentioned santalene synthase mutants, recombinant expression vectors, or high-santalene-producing Saccharomyces cerevisiae engineered strains in the preparation of sandalwood volatile oil.

[0094] The application of the above-mentioned engineered Saccharomyces cerevisiae strain that produces high levels of santalene and santalol in the preparation of sandalwood volatile oil.

[0095] A method for preparing santalene and santalol involves inoculating the above-mentioned engineered brewer's yeast strain that produces high yields of santalene and santalol into a fermentation medium, adding feed medium when the dissolved oxygen value rises, fermenting in batches with the feed medium, and then extracting the fermentation broth to obtain santalene and santalol.

[0096] The fermentation time for the supplemental batch fermentation is preferably at least six days; more preferably six days.

[0097] The fermentation medium preferably comprises the following components: (NH4)2SO4 15g / L, KH2PO4 8g / L, MgSO4 3g / L, ZnSO4·7H2O 0.72g / L, vitamin solution 12ml / L, trace metal solution 10ml / L, and glucose 25g / L.

[0098] The feed culture medium preferably comprises the following components: 10 mL / L trace metal solution, 12 mL / L vitamin solution, 9 g / L KH2PO4, 2.5 g / L anhydrous MgSO4, 3.5 g / L K2SO4, 0.28 g / L Na2SO4, 150 g / L glucose, and 350 g / L ethanol.

[0099] The vitamin solution preferably comprises the following components: biotin 0.05 g / L, calcium pantothenate 1 g / L, niacin 1 g / L, inositol 25 g / L, thiamine hydrochloride 1 g / L, pyridoxine hydrochloride 1 g / L, and p-aminobenzoic acid 0.2 g / L.

[0100] The trace metal solution preferably comprises the following components: EDTA 15 g / L, ZnSO4·7H2O 10.2 g / L, MnCl2·4H2O 0.5 g / L, anhydrous CuSO4 0.5 g / L, CoCl2·6H2O 0.86 g / L, Na2MoO4·2H2O 0.56 g / L, CaCl2·2H2O 3.84 g / L, and FeSO4·7H2O 5.12 g / L.

[0101] The preferred culture conditions for the engineered brewer's yeast that produces high levels of santalene and santalol in the fermentation medium are: temperature 30℃, pH 5.5, dissolved oxygen 40%, stirring speed 250-800 rpm, aeration rate 2 L / min, and fermentation time 144 h.

[0102] The preferred inoculation amount of the engineered brewer's yeast strain that produces high levels of santalene and santalol is 10% (v / v).

[0103] The engineered Saccharomyces cerevisiae strain that produces high levels of santalene and santalol needs to be activated before being inoculated into the fermentation medium.

[0104] The activation is preferably multi-stage activation.

[0105] The activation method is more preferably as follows: a single colony of engineered Saccharomyces cerevisiae that produces high levels of santalene and santalol is inoculated into a test tube containing 5 mL of SD-TRP1-HIS-LEU-URA-MET deficient medium and cultured at 220-250 rpm and 30°C for 10-24 h; then the cultured bacterial solution is inoculated into a 250 mL shake flask containing 5 mL of SD-TRP-HIS-LEU-URA-MET deficient medium and cultured at 220-250 rpm and 30°C for 24 h.

[0106] The SD-TRP-HIS-LEU-URA-MET deficient culture medium preferably comprises the following components: YNB 6.7 g / L, tryptophan-histidine-leucine-uracil-methionine (TRP-HIS-LEU-URA-MET) deficient amino acids (100×) 10 mL / L, and glucose 20 g / L; wherein the components of the tryptophan-histidine-leucine-uracil-methionine (TRP-HIS-LEU-URA-MET) deficient amino acids (100×) are as follows: valine 0.9 g, adenine sulfate 0.25 g, tyrosine 0.18 g, arginine 0.12 g, aspartic acid 0.6 g, threonine 1.2 g, glutamic acid 0.6 g, lysine 0.18 g, phenylalanine 0.3 g, serine 2.25 g, and adjusted to 57 mL with ddH2O.

[0107] Compared with the prior art, this application has the following beneficial effects:

[0108] This invention uses the mutant SanSyn F441V For example, a high-yield *Saccharomyces cerevisiae* engineered strain of *Saccharomyces cerevisiae* was constructed. The obtained *Saccharomyces cerevisiae* engineered strain SZ-1 was fermented for 6 days, and the total yield of Z-α-santalol, Z-β-santalol, Z-α-bergamot, Z-epi-β-santalol, α-santalene, β-santalene, α-bergamot, and epi-β-santalene reached 1.9 g / L. The *Saccharomyces cerevisiae* engineered strain SZ-2 was fermented for 6 days, and the total yield of the above eight products reached 0.7 g / L, of which Z-α-santalol and Z-β-santalol accounted for 43.4% and 22% of the total products, respectively. This invention utilizes SanSyn... F441V The mutant strain of Saccharomyces cerevisiae can be used to produce high-quality sandalwood essential oil that meets international standards, which can effectively alleviate the supply shortage in the market and protect wild sandalwood resources. Attached Figure Description

[0109] Figure 1 Diagrams showing the biosynthetic pathways for the synthesis of santalene and santalol by strains SZ-1 and SZ-2.

[0110] Figure 2 The images show the gas chromatography-mass spectrometry (GC-MS) and total ion chromatograms of the fermentation products obtained from strains SZ-1 and SZ-2 after 6 days of fermentation. A represents the GC-MS and total ion chromatogram of the fermentation products obtained from strain SZ-1 after 6 days of fermentation; B represents the GC-MS and total ion chromatogram of the fermentation products obtained from strain SZ-2 after 6 days of fermentation; 1 represents α-santalene, 2 represents exo-α-bergamerene, 3 represents epi-β-santalene, 4 represents β-santalene, 5 represents Z-α-santalol, 6 represents Z-exo-α-bergamerol, 7 represents Z-epi-β-santalol, and 8 represents Z-β-santalol.

[0111] Figure 3 Figures show the biomass and yields of santalene and santalol during fed-batch fermentation of strains SZ-1 and SZ-2; Figure A shows the biomass and yields of santalene and santalol during fed-batch fermentation of strain SZ-1; Figure B shows the biomass and yields of santalene and santalol during fed-batch fermentation of strain SZ-2. Detailed Implementation

[0112] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0113] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following examples that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the reagents and raw materials used in this invention are commercially available.

[0114] OYE2-OYE3 and ATF1-ATF2 crRNA arrary, SeACS L641P DNA sequences were synthesized by Nanjing Genscript Biotech Co., Ltd.; genomic DNA extraction reagent (DNAiso Reagent) was purchased from Bio-Rad Biotechnology Co., Ltd.; Phanta Max Super-Fidelity DNA Polymerase cloning kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; agarose gel DNA recovery kit was purchased from Beijing Qingke Biotechnology Co., Ltd.; pEASY-Blunt vector was purchased from TransGen Biotech Co., Ltd.; super yeast transformation kit was purchased from Beijing Coolerbott Technology Co., Ltd.; ClonExpress II One Step Cloning Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; pCFB2988 vector was purchased from Addgene; pCRCT vector was purchased from Wuhan Miaoling Biotechnology Co., Ltd.; pESC-LEU vector was purchased from Addgene.

[0115] The IDI1, ADH2, and ALD6 genes were cloned from the genome of *Saccharomyces cerevisiae* CEN.PK2-1D. The nucleotide sequences of IDI1, ADH2, and ALD6 are available in GenBank: NM_001183931.1; ADH2: NM_001182812.1; and ALD6: NM_001183875.1. The nucleotide sequences of CYP736A167, SaCPR2, tHMG1, UPC2-1, and P are also available in GenBank. HXT1 These sequences are preserved for our laboratory. The nucleotide sequence of CYP736A167 is available in GenBank: KU169302.1; the nucleotide sequence of SaCPR2 is available in GenBank: KC842188.1; tHMG1, UPC2-1, and P... HXT1 The nucleotide sequence of the gene has been disclosed in patent application CN201911180415.0, "An engineered strain of Saccharomyces cerevisiae with high production of santalene and santalol, its construction method and application"; SeACS L641P The gene was artificially synthesized using Saccharomyces cerevisiae as the host through codon optimization, and its nucleotide sequence is shown in SEQ ID NO.5.

[0116] P TEF2 PTPI1 P ENO2 P PDC1 P TEF1 P TDH3 P PGK1 and P FBA1 It can be cloned from the genome of Saccharomyces cerevisiae CEN.PK2-1D, in which P TEF2 The nucleotide sequence can be found in GenBank: CP020124.1, from position 477500 to 478059; P TPI1 The nucleotide sequence can be found in GenBank: CP020126.1, from position 564689 to 564189; P ENO2 The nucleotide sequence can be found in GenBank: CP020130.1, from position 469225 to 469824; P PDC1 The nucleotide sequence can be found in GenBank: CP020134.1, from position 234920 to 234120; P TEF1 The nucleotide sequence can be found in GenBank: CP020138.1, from position 700414 to 700832; P TDH3 The nucleotide sequence can be found in GenBank: CP020129.1, from position 884060 to 884735; P PGK1 The nucleotide sequence can be found in GenBank: CP020125.1, from position 142054 to 143036; P FBA1 The nucleotide sequence can be found in GenBank:CP020133.1, from position 327996 to position 327497.

[0117] T FBA1 T PGK1 T TDH2 T ADH2 T PGI1 T ENO2 T ADH1 T CYC1 The terminator can be cloned from the genome of *Saccharomyces cerevisiae* CEN.PK2-1D, in which T... FBA1 The nucleotide sequence can be found in GenBank: CP020133.1, from position 327996 to 327497; T PGK1 The nucleotide sequence can be found in GenBank: CP020125.1, from position 144287 to 144714; T TDH2 The nucleotide sequence can be found in GenBank: CP020132.1, from position 459599 to 459200; T ADH2The nucleotide sequence can be found in GenBank: CP020135.1, from position 879067 to 878668; T PGI1 The nucleotide sequence can be found in GenBank: CP020124.1, from position 612625 to 612226; T ENO2 The nucleotide sequence can be found in GenBank: CP020124.1, from position 612625 to 612226; T ADH1 The nucleotide sequence can be found in GenBank: CP020137.1, from position 159575 to 159411; T CYC1 The nucleotide sequence can be found in GenBank: LT727633.1, from position 8068 to 8257. LPP1 up The nucleotide sequence can be found in GenBank: CP020126.1, from position 1462217 to 1462518; LPP1 down The nucleotide sequence can be found in GenBank: CP020126.1, from position 1463344 to 1463625; the DPP1 described up The nucleotide sequence can be found in GenBank: CP020126.1, from position 1037979 to 1038277; the DPP1 described down The nucleotide sequence can be found in GenBank: CP020126.1, from position 1039148 to 1039481; the P... ERG9up The nucleotide sequence can be found in GenBank: CP020130.1, from position 502815 to position 503221; the P... ERG9down The nucleotide sequence can be found in GenBank: CP020126.1, from position 503341 to 503776; the MET17... up The nucleotide sequence can be found in GenBank: CP020134.1, from position 726291 to 726591; the MET17... down The nucleotide sequence can be found in GenBank: CP020134.1, from position 727927 to position 728222; the ROX1 described up The nucleotide sequence can be found in GenBank: CP020138.1, from position 679325 to position 679688; the ROX1 described downThe nucleotide sequences are available in GenBank:CP020134.1, from position 680639 to 681039. TRP1, HIS3, KILEU2, URA3, and MET17 are screening markers and are stored in our laboratory. The nucleotide sequence of TRP1 is shown in SEQ ID NO.8; the nucleotide sequence of HIS3 is shown in SEQ ID NO.9; the nucleotide sequence of KILEU2 is shown in SEQ ID NO.10; the nucleotide sequence of URA3 is shown in SEQ ID NO.11; and the nucleotide sequence of MET17 is shown in SEQ ID NO.12.

[0118] Example 1: Heterologous expression of santalene synthase and its variants in Saccharomyces cerevisiae and detection of its products

[0119] 1. Construct expression vectors for wild-type santalene synthase SanSyn, SaSSy, SanSyn mutant, and SaSSy mutant Saccharomyces cerevisiae.

[0120] Based on the santalene synthase SanSyn gene (NCBI accession number HQ452480.1), it was synthesized by Genscript Biotech Co., Ltd. after codon optimization. The nucleotide sequence after codon optimization is shown in SEQ ID NO.3, and it is defined as wild-type santalene synthase SanSyn (SanSyn wild-type, SanSyn(wt)).

[0121] Based on the santalene synthase SaSSy gene (NCBI accession number HQ343276.1), it was synthesized by Genscript Biotech Co., Ltd. after codon optimization. The nucleotide sequence after codon optimization is shown in SEQ ID NO.4, and it is defined as wild-type santalene synthase SaSSy (SaSSy wild-type, SaSSy(wt)).

[0122] The wild-type santalene synthase SanSyn (primers: pESC-LEU-SanSyn-F / pESC-LEU-SanSyn-R), wild-type santalene synthase SaSSy (primers: pESC-LEU-SaSSy-F / pESC-LEU-SaSSy-R), and the SanSyn mutant (i.e., SanSyn) with homologous arms were cloned. F441V mutant or SanSyn E297L The mutant (primers: pESC-LEU-SanSyn-F / pESC-LEU-SanSyn-R) and the SaSSy mutant (i.e., SaSSy) L317V Or SaSSy L317T Or SaSSy L317I Or SaSSy L317C Or SaSSyS459V Or SaSSy S459T Or SaSSy S459I Or SaSSy L459H Or SaSSy S459A Or SaSSy S535T Or SaSSy S535C Or SaSSy F538W (Primers: pESC-LEU-SaSSy-F / pESC-LEU-SaSSy-R) were used to construct the BamHI / XhoI site of the pESC-LEU vector using the ClonExpressII One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.), resulting in wild-type santalene synthase SanSyn expression vector, wild-type santalene synthase SaSSy expression vector, SanSyn mutant expression vector, and SaSSy mutant expression vector. The ligation system and method are described in the ClonExpress II One Step Cloning Kit product manual.

[0123] 2. Heterologous expression of brewer's yeast

[0124] (1) The wild-type santalene synthase SanSyn expression vector, the wild-type santalene synthase SaSSy expression vector, the SanSyn mutant expression vector, and the SaSSy mutant expression vector were respectively transformed into strain WL07 using a super yeast transformation kit (this strain WL07 has been disclosed in Zha W, An T, Li T, Zhu J, Gao K, Sun Z, Xu W, Lin P, Zi J. Reconstruction of the Biosynthetic Pathway of Santalols under Control of the GAL Regulatory System in Yeast. ACS Synth. Biol. 2020, 9, 449-456.). The specific transformation method was as described in the instructions for the super yeast transformation kit. The screening plate was SD-LEU plate medium, and the culture was carried out at 30℃ for 3 days.

[0125] The SD-LEU plate culture medium formula is as follows: YNB 6.7 g / L, LEU (leucine) deficient amino acid (100×) 10 mL / L, and glucose 20 g / L. The preparation of the solid culture medium requires the addition of 20 g / L of agar powder.

[0126] The LEU (leucine) deficient amino acid (100×) formulation is as follows: uracil 0.12g, adenine sulfate 0.25g, valine 0.9g, arginine 0.12g, aspartic acid 0.6g, threonine 1.2g, tyrosine 0.18g, methionine 0.12g, glutamic acid 0.6g, tryptophan 0.24g, lysine 0.18g, serine 2.25g, phenylalanine 0.3g, histidine 0.12g, and diluted to 57mL with ddH2O.

[0127] (2) Pick a single clone of the bacterial strain from the plate and culture it in 5 mL of SD-LEU medium for 24 h to obtain a bacterial solution; inoculate 1 mL of the bacterial solution into 50 mL of SG-LEU medium for expression for 4 days;

[0128] The SG-LEU culture medium formula is as follows: YNB 6.7g / L, LEU (leucine) deficient amino acid (100×) 10mL / L, glucose 2g / L, galactose 18g / L.

[0129] (3) Extraction and detection of the product

[0130] After 4 days of fermentation, the fermentation broth was added to 50 mL of ethyl acetate and sonicated for 1 h. After standing for 3 days, the organic phase was obtained. 500 μL of the organic phase was analyzed by GC-MS. The instrument used was an Agilent GC-MS system 7890B-5977B. Detection method: injection volume 1 μL, solvent delay 11 min, helium carrier gas, flow rate 1 mL / min. Column: HP-5MS. Chromatographic conditions: 50℃ for 3 min, temperature ramped to 70℃ at 20℃ / min and held for 1 min, temperature ramped to 300℃ at 15℃ / min and held for 3 min.

[0131] Compared with SEQ ID NO.1 or SEQ ID NO.2, the amino acid mutations of the santalene synthase mutants and the proportions of each component of the expression product of each santalene synthase mutant are shown in Table 1.

[0132] Table 1:

[0133]

[0134]

[0135] a : SanSyn wild type (nucleotide sequence as shown in SEQ ID NO.3).

[0136] b SaSSy wild type (nucleotide sequence as shown in SEQ ID NO.4).

[0137] cThe parentheses :() indicate the codon for the amino acid at that site in wild-type santalene synthase SanSyn.

[0138] d The parentheses :() indicate the codon used when the site is mutated to the specified amino acid.

[0139] e The part in parentheses () indicates the percentage content of each component in the product.

[0140] The results in Table 1 indicate that SanSyn F441V The mutant is most similar to the proportions of components found in sandalwood plants.

[0141] Example 2: CRISPR-Cas9 system knockout of OYE2, OYE3, ATF1, and ATF2 genes in Saccharomyces cerevisiae CEN.PK2-1D

[0142] 1. pCRCT vector transformation of Saccharomyces cerevisiae to knock out the target gene

[0143] (1) The pCRCT-OYE2-OYE3 vector (constructed by Nanjing Genscript Biotech Co., Ltd., with the crRNA spacer nucleic acid sequences of the OYE2 and OYE3 genes as shown in SEQ ID NO.6) was transformed into Saccharomyces cerevisiae CEN.PK2-1D (purchased from Euroscarf, Germany) using a super yeast transformation kit. The specific transformation method was referred to the instructions of the super yeast transformation kit to obtain single-clone transformants.

[0144] (2) Screening was performed using SD-URA-deficient plate medium, which was incubated at 30℃ for five days. The formulation of the SD-URA-deficient plate medium was as follows: YNB medium 6.7 g / L, glucose 20 g / L, agar powder 20 g / L, and uracil (URA)-deficient amino acids (100×) 10 mL / L. The uracil (URA)-deficient amino acids (100×) were prepared as follows: 0.9 g of valine, 0.25 g of adenine sulfate, 0.18 g of tyrosine, 0.12 g of arginine, 0.24 g of tryptophan, 0.6 g of aspartic acid, 1.2 g of threonine, 0.6 g of glutamic acid, 0.18 g of lysine, 0.3 g of phenylalanine, 0.12 g of methionine, 0.36 g of leucine, 2.25 g of serine, and 0.12 g of histidine were weighed and diluted to 57 mL with ddH2O.

[0145] (3) Take the single-clone transformant from step (1) into 4 mL of SD-URA defect plate medium and culture it in a shaker at 30℃ and 220 rpm.

[0146] (4) After two days of culture, take 100 μL of bacterial culture and inoculate it into fresh SD-URA defective plate medium to continue expression for two more days.

[0147] (5) Extract the genome of the single colony strain in step (4) by diluting by plating, amplify the target gene and sequence it to verify that the gene has been knocked out.

[0148] (6) Using YPD medium, pCRCT-OYE2-OYE3 was lost during subculturing to construct strain M-1. The YPD medium formula was: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, and 20 g / L agar powder.

[0149] (7) The pCRCT-ATF1-ATF2 vector (constructed by Nanjing Genscript Biotech Co., Ltd., with the crRNA spacer nucleic acid sequence of ATF1 and ATF2 genes as shown in SEQ ID NO.7) was used to transform strain M-1 using a super yeast transformation kit. Based on strain M-1, the above steps (1)-(6) were repeated to knock out ATF1 and ATF2 genes, and Saccharomyces cerevisiae M-2 was constructed.

[0150] Example 3: Construction of carriers and modules (using SanSyn) F441V (Taking the mutant as an example, other mutant construction methods are as described in this embodiment.)

[0151] 1. Overlap PCR construction module

[0152] (1) The DNA fragments required for building the module were obtained by the first round of PCR amplification. The reaction conditions and conditions are described in the instructions of the PhantaMax Super-Fidelity DNA Polymerase Cloning Kit. The DNA fragments were then added with 40-50 bp overlapping sequences for the second round of overlap PCR.

[0153] (2) Take 50-100 ng of the DNA fragment required for the construction module and perform a second round of PCR reaction. The reaction system and conditions are as described in (1), and the number of cycles is changed to 10 cycles.

[0154] (3) Take the second round PCR reaction solution as the template for the third round PCR reaction. The reaction system and conditions are as described in (1).

[0155] Construct the following modules using the overlap PCR steps described above:

[0156] (a) P TEF2 UPC2-1 and T FBA1 By connecting sequentially, we obtain module P. TEF2 -UPC2-1-T FBA1 Named module 1, where clone PTEF2 The primer is OE-P TEF2 -UPC2-1-T FBA1 -1F / OE-P TEF2 -UPC2-1-T FBA1 -1R, the primer for cloning UPC2-1 is OE-P. TEF2 -UPC2-1-T FBA1 -2F / OE-P TEF2 -UPC2-1-T FBA1 -2R, Cloned T FBA1 The primer is OE-P TEF2 -UPC2-1-T FBA1 -3F / OE-P TEF2 -UPC2-1-T FBA1 -3R;

[0157] (b) P TPI1 IDI1 and T PGK1 By connecting sequentially, we obtain module P. TPI1 -IDI1-T PGK1 Named module 2, where clone P TPI1 The primer is OE-P TPI1 -IDI1-T PGK1 -1F / OE-P TPI1 -IDI1-T PGK1 -1R, the primer for cloning IDI1 is OE-P. TPI1 -IDI1-T PGK1 -2F / OE-P TPI1 -IDI1-T PGK1 -2R, Cloned T PGK1 The primer is OE-P TPI1 -IDI1-T PGK1 -3F / OE-P TPI1 -IDI1-T PGK1 -3R;

[0158] (c) P ENO2 ALD6, T TDH2 and P PDC1 By connecting sequentially, we obtain module P. ENO2 -ALD6-T TDH2 -P PDC1 Named module 3, which contains clone P ENO2 The primer is OE-P ENO2 -ALD6-T TDH2 -P PDC1 -1F / OE-P ENO2 -ALD6-T TDH2 -P PDC1-1R, the primers for cloning ALD6 are OE-P. ENO2 -ALD6-T TDH2 -P PDC1 -2F / OE-P ENO2 -ALD6-T TDH2 -P PDC1 -2R, Cloned T TDH2 The primer is OE-P ENO2 -ALD6-T TDH2 -P PDC1 -3F / OE-P ENO2 -ALD6-T TDH2 -P PDC1 -3R, clone P PDC1 The primer is OE-P ENO2 -ALD6-T TDH2 -P PDC1 -4F / OE-P ENO2 -ALD6-T TDH2 -P PDC1 -4R;

[0159] (d) ADH2, T ADH2 P TEF1 SeACS L641P and T PGI1 By connecting sequentially, we obtain module ADH2-T. ADH2 -P TEF1 -SeACS L641P -T PGI1 It is named Module 4, in which the primer for cloning ADH2 is OE-ADH2-T. ADH2 -P TEF1 -SeACS L641P -T PGI1 -1F / OE-ADH2-T ADH2 -P TEF1 -SeACS L641P -T PGI1 -1R, Cloned T ADH2 The primers are OE-ADH2-T ADH2 -P TEF1 -SeACS L641P -T PGI1 -2F / OE-ADH2-T AD H2 -P TEF1 -SeACS L641P -T PGI1 -2R, clone P TEF1 The primers are OE-ADH2-T ADH2 -P TEF1 -SeACS L641P -TPGI1 -3F / OE-ADH2-T A DH2 -P TEF1 -SeACS L641P -T PGI1 -3R, clone SeACS L641P Primer OE-ADH2-T ADH2 -P TEF1 -SeACS L641P -T PGI1 -4F / OE-ADH2-T ADH2 -P TEF1 -SeACS L641P -T PGI1 -4R, Cloned T PGI1 Primer OE-ADH2-T ADH2 -P TEF1 -SeACS L641P -T PGI1 -5F / OE-ADH2-T ADH2 -P TEF1 -SeACS L641P -T PGI1 -5R;

[0160] (e) P TPI1 tHMG1, T PGK1 P TDH3 SanSyn F441V and T ENO2 By connecting sequentially, we obtain module P. TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 Named module 5, which contains clone P TPI1 The primer is OE-P TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -1F / OE-P TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -1R, where the primers for cloning tHMG1 are OE-P. TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -2F / OE-P TPI1 -tHMG1-TPGK1 -P TDH3 -SanSyn F441V -T ENO2 -2R, where clone T PGK1 The primer is OE-P TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -3F / OE-P TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -3R, clone P TDH3 Primer OE-P TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -4F / OE-P TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -4R, a clone of SanSyn F441V Primer OE-P TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -5F / OE-P TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -5R, Cloned T ENO2 Primer OE-P TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -6F / OE-P TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -6R;

[0161] (f) P FBA1 CYP736A167 and T PGI1 By connecting sequentially, we obtain module P. FBA1 -CYP736A167-TPGI1 Named module 7, which contains clone P FBA1 The primer is OE-P FBA1 -CYP736A167-T PGI1 -1F / OE-P FBA1 -CYP736A167-T PGI1 -1R, the primers for cloning CYP736A167 are OE-P. FBA1 -CYP736A167-T PGI1 -2F / OE-P FBA1 -CYP736A167-T PGI1 -2R, Cloned T PGI1 The primer is OE-P FBA1 -CYP736A167-T PGI1 -3F / OE-P FBA1 -CYP736A167-T PGI1 -3R;

[0162] (g) LPP1 up HIS3 and LPP1 down By connecting them sequentially, we obtain module LPP1. up -HIS3-LPP1 down Named module 8, which clones LPP1 up The primer is OE-LPP1 up -HIS3-LPP1 down -1F / OE-LPP1 up -HIS3-LPP1 down -1R, the primer for cloning HIS3 is OE-LPP1. up -HIS3-LPP1 down -2F / OE-LPP1 up -HIS3-LPP1 down -2R, clone LPP1 down The primer is OE-LPP1 up -HIS3-LPP1 down -3F / OE-LPP1 up -HIS3-LPP1 down -3R;

[0163] (h) DPP1 up TRP1 and DPP1 down By connecting sequentially, we obtain module DPP1. up -TRP1-DPP1 down Name it module 9, clone DPP1 up The primer is OE-DPP1 up-TRP1-DPP1 down -1F / OE-DPP1 up -TRP1-DPP1 down -1R, the primer for cloning TRP1 is OE-DPP1. up -TRP1-DPP1 down -2F / OE-DPP1 up -TRP1-DPP1 down -2R, clone DPP1 down The primer is OE-DPP1 up -TRP1-DPP1 down -3F / OE-DPP1 up -TRP1-DPP1 down -3R;

[0164] (i) P ERG9up KILEU2, P HXT1 and P ERG9down By connecting sequentially, we obtain module P. ERG9up -KILEU2-P HXT1 -P ERG9down Named module 10, which contains clone P ERG9up The primer is OE-P ERG9up -KILEU2-P HXT1 -P ERG9down -1F / OE-P ERG9up -KILEU2-P HXT1 -P ERG9down -1R, the primers for cloning KILEU2 are OE-P. ERG9up -KILEU2-P HXT1 -P ERG9down -2F / OE-P ERG9up -KILEU2-P HXT1 -P ERG9down -2R, clone P HXT1 The primer is OE-P ERG9up -KILEU2-P HXT1 -P ERG9down -3F / OE-P ERG9up -KILEU2-P HXT1 -P ERG9down -3R, clone P ERG9down The primer is OE-P ERG9up -KILEU2-P HXT1 -P ERG9down -4F / OE-P ERG9up -KILEU2-P HXT1 -P ERG9down -4R;

[0165] (j) MET17 up URA3 and MET17 down By connecting them sequentially, we obtain module MET17. up -URA3-MET17 down Named module 11, which clones MET17 up The primer is OE-MET17 up -URA3-MET17 down -1F / OE-MET17 up -URA3-MET17 down -1R, the primer for cloning URA3 is OE-MET17. up -URA3-MET17 down -2F / OE-MET17 up -URA3-MET17 down -2R, clone MET17 down The primer is OE-MET17 up -URA3-MET17 down -3F / OE-MET17 up -URA3-MET17 down -3R;

[0166] (k) ROX1 up MET17 and ROX1 down By connecting them sequentially, we obtain module ROX1. up -MET17-ROX1 down Named module 12, which clones ROX1 up The primer is OE-ROX1 up -MET17-ROX1 down -1F / OE-ROX1 up -MET17-ROX1 down -1R, the primer for cloning MET17 is OE-ROX1. up -MET17-ROX1 down -2F / OE-ROX1 up -MET17-ROX1 down -2R, clone ROX1 down The primer is OE-ROX1 up -MET17-ROX1 down -3F / OE-ROX1 up -MET17-ROX1 down -3R;

[0167] The nucleotide sequences of the primers described in this embodiment are shown in Table 2.

[0168] Table 2. Primer Sequences

[0169]

[0170]

[0171]

[0172] 2. Constructing a carrier through homologous recombination

[0173] The CYP736A167 gene with a homologous arm was cloned (primers: CYP736A167-F / CYP736A167-R), and constructed into the NotⅢ / SacI site of the vector pSP-GM2 (purchased from BioVector Plasmid Vector Bacterial Cell Gene Preservation Center) using the ClonExpress II One Step Cloning Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The ligation system and method are as described in the product instructions. The pSP-GM2-CYP736A167 recombinant vector was constructed.

[0174] As described above, SaCPR2 (primers: SaCPR2-F / SaCPR2-R) was constructed into the BamHI / NheI site of the pSP-GM2-CYP736A167 recombinant vector to construct the vector pSP-GM2-CYP736A167-SaCPR2, thus obtaining P PGK1 -CYP736A167-T ADH1 -P TEF1 -SaCPR2-T CYC1 Module 6.

[0175] 3. Construction of integrated carriers

[0176] (1) Module 9 was constructed into the HindⅢ / NheI site of the pCFB2988 vector (purchased from BioVector plasmid vector strain cell gene preservation center) to construct the pCFB2988-DPP1 vector. Module 1 was then constructed into the BamHI site of the pCFB2988-DPP1 vector to construct the vector pCFB2988-DPP1-UPC2-1. Finally, Module 2 was constructed into the BamHI site of pCFB2988-DPP1-UPC2-1 to construct the vector pSZ-1.

[0177] (2) As described above, modules 8, 3 and 4 are connected to the pCFB2988 carrier in sequence to construct carrier pSZ-2;

[0178] (3) Modules 10 and 5 are sequentially constructed onto the pCFB2988 vector to construct vector pSZ-3;

[0179] (4) Using the pSP-GM2-CYP736A167-SaCPR2 vector as a template, module 6 was cloned (primers: P PGK1 -CYP736A167-T ADH1 -P TEF1 -SaCPR2-T CYC1 -F / P PGK1 -CYP736A167-T ADH1 -P TEF1 -SaCPR2-T CYC1 -R), clone P using module 5 as a template. TDH3 -SanSyn F441V -T ENO2 Fragment (primer: pSZ13-P) TDH3 -SanSyn F441V -T ENO2 -F / pSZ13-P TDH3 -SanSyn F441V -T ENO2 -R), sequentially connect module 11, module 6 and P TDH3 -SanSyn F441V -T ENO2 The vector pSZ-4 was constructed on pCFB2988.

[0180] (5) Using the pSP-GM2-CYP736A167-SaCPR2 vector as a template, P was cloned. PGK1 -CYP736A167-T ADH1 Fragment (primer: P) PGK1 -CYP736A167-T ADH1 -F / P PGK1 -CYP736A167-T ADH1 -R), sequentially connect module 12, module 5 and P PGK1 -CYP736A167-T ADH1 The vector pSZ-5 was constructed by inserting it into the pCFB2988 vector.

[0181] (6) Cloning P using pSZ-5 vector as a template TDH3 -SanSyn F441V -T ENO2 -P PGK1 -CYP736A167-T ADH1 Fragment (primer: P) TDH3 -SanSyn F441V -T ENO2 -PPGK1 -CYP736A167-T ADH1 -F / P TDH3 -SanSyn F441V -T ENO2 -P PGK1 -CYP736A167-T ADH1 -R), sequentially add module 12, module 7 and P TDH3 -SanSyn F441V -T ENO2 -P PGK1 -CYP736A167-T ADH1 Connect to the pCFB2988 vector to construct the vector pSZ-6.

[0182] Example 4: Modular construction of engineered yeast (using SanSyn) F441V (Taking the mutant as an example, other mutant construction methods are as described in this embodiment.)

[0183] (1) Amplification of DNA integration fragment DPP1 using pSZ-1 vector as template up -TRP1-P TEF2 -UPC2-1-T FBA1 -P TPI1 -IDI1-T PGK1 -DPP1 down The amplification primers were DPP1-F / DPP1-R. The integrated fragment was transformed into Saccharomyces cerevisiae M-2 using a super yeast transformation kit. The integration site was DPP1. The strain was screened using SD-HIS plate medium to obtain strain M-3. The preparation method of SD-TRP plate medium is as follows: YNB medium 6.7g / L, tryptophan (TRP) deficient amino acid (100×) 10mL / L, glucose 20g / L, agar powder 20g / L; wherein the formula of tryptophan (TRP) deficient amino acid (100×) is as follows: uracil 0.12g, adenine sulfate 0.25g, valine 0.9g, arginine 0.12g, aspartic acid 0.6g, threonine 1.2g, tyrosine 0.18g, methionine 0.12g, glutamic acid 0.6g, leucine 0.36g, lysine 0.18g, serine 2.25g, phenylalanine 0.3g, histidine 0.12g, and ddH2O is used to make up to 57mL.

[0184] (2) Amplification of LPP1 using pSZ-2 as a template up -HIS3-P ENO2 -ALD6-T TDH2 -P PDC1 -ADH2-T ADH2 -P TEF1 -SeACS L641P-T PGI1 -LPP1 down The amplification primers were LPP1-F / LPP1-R. The above-mentioned integrated fragment was transformed into strain M-3, with the integration site being LPP1. The strain was screened using tryptophan-histidine (SD-TRP-HIS) plate medium to obtain strain M-4. The preparation method of tryptophan-histidine (SD-TRP-HIS) plate medium is as follows: YNB medium 6.7 g / L, tryptophan-histidine (TRP-HIS) deficient amino acid (100×) 10 mL / L, glucose 20 g / L, agar powder 20 g / L; wherein, the formula of the tryptophan-histidine (TRP-HIS) deficient amino acid (100×) is as follows: uracil 0.12 g, adenine sulfate 0.25 g, valine 0.9 g, arginine 0.12 g, aspartic acid 0.6 g, threonine 1.2 g, tyrosine 0.18 g, methionine 0.12 g, glutamic acid 0.6 g, leucine 0.36 g, lysine 0.18 g, serine 2.25 g, phenylalanine 0.3 g, and ddH2O to a final volume of 57 mL.

[0185] (3) Amplify P using pSZ-3 as a template ERG9up -KILEU2-P TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -P HXT1 -P ERG9down The integrated fragment was amplified using primer P. ERG9 -F / P ERG9 -R. The above-mentioned integrated fragment was transformed into strain M-4, with the integration site being P. ERG9 Strain M-5 was obtained by screening using SD-TRP-HIS-LEU agar plates. The SD-TRP-HIS-LEU agar plate was prepared as follows: 6.7 g / L YNB medium, 10 mL / L tryptophan-histidine-leucine (TRP-HIS-LEU) deficient amino acids (100×), 20 g / L glucose, and 20 g / L agar powder. The formula for the tryptophan-histidine-leucine (TRP-HIS-LEU) deficient amino acids (100×) was as follows: 0.12 g uracil, 0.25 g adenine sulfate, 0.9 g valine, 0.12 g arginine, 0.6 g aspartic acid, 1.2 g threonine, 0.18 g tyrosine, 0.12 g methionine, 0.6 g glutamic acid, 0.18 g lysine, 2.25 g serine, and 0.3 g phenylalanine, and the volume was adjusted to 57 mL with ddH2O.

[0186] (4) Amplification of MET17 using pSZ-4 as a template up-URA3-P TDH3 -SanSyn F441V -T ENO2 -P PGK1 -CYP736A167-T ADH1 -P TEF1 -SaCPR2-T CYC1 -MET17 down The integrated fragment was amplified using primers MET17-F / MET17-R. This integrated fragment was then transformed into strain M-5, with the integration site at MET17. Strain M-6 was obtained through screening on SD-TRP-HIS-LEU-URA plates. The preparation method of SD-TRP-HIS-LEU-URA plate medium is as follows: YNB medium 6.7g / L, tryptophan-histidine-leucine-uracil (TRP-HIS-LEU-URA) deficient amino acid (100×) 10mL / L, glucose 20g / L, agar powder 20g / L; the formula of SD-TRP-HIS-LEU-URA (tryptophan-histidine-leucine-uracil) deficient amino acid (100×) is as follows: valine 0.9g, adenine sulfate 0.25g, arginine 0.12g, aspartic acid 0.6g, tyrosine 0.18g, glutamic acid 0.6g, threonine 1.2g, serine 2.25g, phenylalanine 0.3g, lysine 0.18g, methionine 0.12g, and ddH2O to a final volume of 57mL.

[0187] (5) Amplification using vector pSZ-5 as a template

[0188] ROX1 up -MET17-P TPI1 -tHMG1-T PGK1 -P TDH3 -SanSyn F441V -T ENO2 -P PGK1 -CYP736A167-T ADH1 -ROX1 downThe integrated fragment was amplified using primers ROX1-F / ROX1-R. The M-6 strain was transformed, with the integration site being ROX1. The SZ-1 strain was obtained by screening on SD-TRP-HIS-LEU-URA-MET plates. The preparation method of SD-TRP-HIS-LEU-URA-MET plate medium is as follows: YNB medium 6.7g / L, TRP-HIS-LEU-URA-MET (tryptophan-histidine-leucine-uracil-methionine) deficient amino acids (100×) 10mL / L, glucose 20g / L, agar powder 20g / L; the formula of TRP-HIS-LEU-URA-MET (tryptophan-histidine-leucine-uracil-methionine) deficient amino acids (100×) is as follows: valine 0.9g, adenine sulfate 0.25g, tyrosine 0.18g, arginine 0.12g, aspartic acid 0.6g, threonine 1.2g, glutamic acid 0.6g, lysine 0.18g, phenylalanine 0.3g, serine 2.25g, and adjusted to 57mL with ddH2O.

[0189] (6) ROX1 was amplified using the pSZ-6 vector template. up -MET17-P TDH3 -SanSyn F441V -T ENO2 -P PGK1 -CYP736A167-T ADH1 -P FBA1 -CYP736A167-T PGI1 -ROX1 down The integrated fragment was amplified using primers ROX1-F / ROX1-R. The M-6 strain was transformed, with the integration site at ROX1. The SZ-2 strain was obtained by screening on SD-TRP-HIS-LEU-URA-MET plates. The preparation method of SD-TRP-HIS-LEU-URA-MET plate medium is as follows: YNB medium 6.7g / L, TRP-HIS-LEU-URA-MET (tryptophan-histidine-leucine-uracil-methionine) deficient amino acids (100×) 10mL / L, glucose 20g / L, agar powder 20g / L; the formula of TRP-HIS-LEU-URA-MET (tryptophan-histidine-leucine-uracil-methionine) deficient amino acids (100×) is as follows: valine 0.9g, adenine sulfate 0.25g, tyrosine 0.18g, arginine 0.12g, aspartic acid 0.6g, threonine 1.2g, glutamic acid 0.6g, lysine 0.18g, phenylalanine 0.3g, serine 2.25g, and adjusted to 57mL with ddH2O.

[0190] Example 5: Fermentation of santalene and santalol by yeast strains SZ-1 and SZ-2

[0191] 1. Fermentation culture of SZ-1 and SZ-2 strains

[0192] (1) Select single colonies of strains SZ-1 and SZ-2 obtained in Example 5 into test tubes containing 5 mL of SD-URA-LEU-TRP-MET-HIS medium and culture them in a shaker for 24 h at a temperature of 30℃ and a rotation speed of 220-250 rpm to obtain SZ-1 and SZ-2 bacterial solutions respectively.

[0193] (2) The SZ-1 and SZ-2 bacterial cultures obtained in step (1) were respectively inoculated into four 250 mL shake flasks containing 50 mL SD-TRP-HIS-LEU-URA-MET medium and cultured at 220-250 rpm and 30 °C for 24 h.

[0194] The SD-TRP-HIS-LEU-URA-MET culture medium is prepared as follows: YNB 6.7 g / L, TRP-HIS-LEU-URA-MET (tryptophan-histidine-leucine-uracil-methionine) deficient amino acids (100×) 10 mL / L, and glucose 20 g / L. The TRP-HIS-LEU-URA-MET (tryptophan-histidine-leucine-uracil-methionine) deficient amino acid (100×) formulation is as follows: valine 0.9 g, adenine sulfate 0.25 g, tyrosine 0.18 g, arginine 0.12 g, aspartic acid 0.6 g, threonine 1.2 g, glutamic acid 0.6 g, lysine 0.18 g, phenylalanine 0.3 g, serine 2.25 g, and diluted to 57 mL with ddH2O.

[0195] (3) 200 mL of each of the four bottles of bacterial solution of SZ-1 and SZ-2 were connected to a 5L fermenter containing 1.8L of fermentation medium. The temperature was controlled at 30℃, the pH was controlled at 5.5 with ammonia water, the dissolved oxygen value was controlled at 40%, the rotation speed was 250-800 rpm, and the aeration rate was 2L / min.

[0196] (4) When the dissolved oxygen value begins to rise, start the feeding system to feed (i.e. add feeding medium) at a feeding rate of 2-14 mL / h.

[0197] The fermentation medium consists of the following components: (NH4)2SO4 15g / L, KH2PO4 8g / L, MgSO4 3g / L, ZnSO4·7H2O 0.72g / L, vitamin solution 12ml / L, trace metal solution 10ml / L, and glucose 25g / L.

[0198] The supplemental culture medium comprises the following components: 10 mL / L trace metal solution, 12 mL / L vitamin solution, 9 g / L KH2PO4, 2.5 g / L anhydrous MgSO4, 3.5 g / L K2SO4, 0.28 g / L Na2SO4, 150 g / L glucose, and 350 g / L ethanol.

[0199] The vitamin solution comprises the following components: biotin 0.05 g / L, calcium pantothenate 1 g / L, niacin 1 g / L, inositol 25 g / L, thiamine hydrochloride 1 g / L, pyridoxine hydrochloride 1 g / L, and para-aminobenzoic acid 0.2 g / L.

[0200] The trace metal solution comprises the following components: EDTA 15 g / L, ZnSO4·7H2O 10.2 g / L, MnCl2·4H2O 0.5 g / L, anhydrous CuSO4 0.5 g / L, CoCl2·6H2O 0.86 g / L, Na2MoO4·2H2O 0.56 g / L, CaCl2·2H2O 3.84 g / L, and FeSO4·7H2O 5.12 g / L.

[0201] 2. Extraction and detection of the product

[0202] Fermentation broths of strains SZ-1 and SZ-2 at 24h, 48h, 72h, 96h, 120h, and 144h were taken respectively, and 40mL of ethyl acetate was added. The mixture was sonicated for 1h and allowed to stand for 5d to separate the ethyl acetate organic phase. 500μL of the organic phase was analyzed by GC-MS. The instrument used was an Agilent GC-MS system 7890B-5977B. Detection method: injection volume 1μL, solvent delay 10min, helium carrier gas, flow rate 1mL / min. Column: HP-5MS. Chromatographic conditions: 50℃ for 3min, ramped to 70℃ at a rate of 20℃ / min and held for 1min, ramped to 160℃ at 3℃ / min, and ramped to 300℃ at 20℃ / min.

[0203] Chromatograms of the fermentation products from strains SZ-1 and SZ-2 after six days of fermentation are shown below. Figure 2 As shown; a schematic diagram of the yields of santalene and santalol on day 6 of fed-batch fermentation of strains SZ-1 and SZ-2 is shown below. Figure 3 As shown.

[0204] from Figure 3As can be seen, after 6 days of fermentation culture, the engineered Saccharomyces cerevisiae strain SZ-1 produced a total yield of 1.9 g / L of Z-α-santalol, Z-β-santalol, Z-α-bergamotol, Z-epi-β-santalol, α-santalene, β-santalene, α-bergamotol, and epi-β-santalene. After 6 days of fermentation culture, the engineered Saccharomyces cerevisiae strain SZ-2 produced a total yield of 0.7 g / L of the above eight products, with Z-α-santalol and Z-β-santalol accounting for 43.4% and 22% of the total products, respectively.

[0205] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention. sequence list <110> Zhoushan Lanyin Biotechnology Co., Ltd. <120> A mutant of santalene synthase, an engineered bacterium and its application <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 551 <212> PRT <213> Artificial Sequence <223> The amino acid sequence of wild-type santalene synthase SanSyn <400> 1 Met Ser Thr Gln Gln Val Ser Ser Glu Asn Ile Val Arg Asn Ala Ala 1 5 10 15 Asn Phe His Pro Asn Ile Trp Gly Asn His Phe Leu Thr Cys Pro Ser 20 25 30 Gln Thr Ile Asp Ser Trp Thr Gln Gln His His Lys Glu Leu Lys Glu 35 40 45 Glu Val Arg Lys Met Met Val Ser Asp Ala Asn Lys Pro Ala Gln Arg 50 55 60 Leu Arg Leu Ile Asp Thr Val Gln Arg Leu Gly Val Ala Tyr His Phe 65 70 75 80 Glu Lys Glu Ile Asp Asp Ala Leu Glu Lys Ile Gly His Asp Pro Phe 85 90 95 Asp Asp Lys Asp Asp Leu Tyr Ile Val Ser Leu Cys Phe Arg Leu Leu 100 105 110 Arg Gln His Gly Ile Lys Ile Ser Cys Asp Val Phe Glu Lys Phe Lys 115 120 125 Asp Asp Asp Gly Lys Phe Lys Ala Ser Leu Met Asn Asp Val Gln Gly 130 135 140 Met Leu Ser Leu Tyr Glu Ala Ala His Leu Ala Ile His Gly Glu Asp 145 150 155 160 Ile Leu Asp Glu Ala Ile Val Phe Thr Thr Thr His Leu Lys Ser Thr 165 170 175 Val Ser Asn Ser Pro Val Asn Ser Thr Phe Ala Glu Gln Ile Arg His 180 185 190 Ser Leu Arg Val Pro Leu Arg Lys Ala Val Pro Arg Leu Glu Ser Arg 195 200 205 Tyr Phe Leu Asp Ile Tyr Ser Arg Asp Asp Leu His Asp Lys Thr Leu 210 215 220 Leu Asn Phe Ala Lys Leu Asp Phe Asn Ile Leu Gln Ala Met His Gln 225 230 235 240 Lys Glu Ala Ser Glu Met Thr Arg Trp Trp Arg Asp Phe Asp Phe Leu 245 250 255 Lys Lys Leu Pro Tyr Ile Arg Asp Arg Val Val Glu Leu Tyr Phe Trp 260 265 270 Ile Leu Val Gly Val Ser Tyr Gln Pro Lys Phe Ser Thr Gly Arg Ile 275 280 285 Phe Leu Ser Lys Ile Ile Cys Leu Glu Thr Leu Val Asp Asp Thr Phe 290 295 300 Asp Ala Tyr Gly Thr Phe Asp Glu Leu Ala Ile Phe Thr Glu Ala Val 305 310 315 320 Thr Arg Trp Asp Leu Gly His Arg Asp Ala Leu Pro Glu Tyr Met Lys 325 330 335 Phe Ile Phe Lys Thr Leu Ile Asp Val Tyr Ser Glu Ala Glu Gln Glu 340 345 350 Leu Ala Lys Glu Gly Arg Ser Tyr Ser Ile His Tyr Ala Ile Arg Ser 355 360 365 Phe Gln Glu Leu Val Met Lys Tyr Phe Cys Glu Ala Lys Trp Leu Asn 370 375 380 Lys Gly Tyr Val Pro Ser Leu Asp Asp Tyr Lys Ser Val Ser Leu Arg 385 390 395 400 Ser Ile Gly Phe Leu Pro Ile Ala Val Ala Ser Phe Val Phe Met Gly 405 410 415 Asp Ile Ala Thr Lys Glu Val Phe Glu Trp Glu Met Asn Asn Pro Lys 420 425 430 Ile Ile Ile Ala Ala Glu Thr Ile Phe Arg Phe Leu Asp Asp Ile Ala 435 440 445 Gly His Arg Phe Glu Gln Lys Arg Glu His Ser Pro Ser Ala Ile Glu 450 455 460 Cys Tyr Lys Asn Gln His Gly Val Ser Glu Glu Glu Ala Val Lys Ala 465 470 475 480 Leu Ser Leu Glu Val Ala Asn Ser Trp Lys Asp Ile Asn Glu Glu Leu 485 490 495 Leu Leu Asn Pro Met Ala Ile Pro Leu Pro Leu Leu Gln Val Ile Leu 500 505 510 Asp Leu Ser Arg Ser Ala Asp Phe Met Tyr Gly Asn Ala Gln Asp Arg 515 520 525 Phe Thr His Ser Thr Met Met Lys Asp Gln Val Asp Leu Val Leu Lys 530 535 540 Asp Pro Val Lys Leu Asp Asp 545 550 <210> 2 Met Asp Ser Ser Thr Ala Thr Ala Met Thr Ala Pro Phe Ile Asp Pro 1 5 10 15 Thr Asp His Val Asn Leu Lys Thr Asp Thr Asp Ala Ser Glu Asn Arg 20 25 30 Arg Met Gly Asn Tyr Lys Pro Ser Ile Trp Asn Tyr Asp Phe Leu Gln 35 40 45 Ser Leu Ala Thr His His Asn Ile Val Glu Glu Arg His Leu Lys Leu 50 55 60 Ala Glu Lys Leu Lys Gly Gln Val Lys Phe Met Phe Gly Ala Pro Met 65 70 75 80 Glu Pro Leu Ala Lys Leu Glu Leu Val Asp Val Val Gln Arg Leu Gly 85 90 95 Leu Asn His Leu Phe Glu Thr Glu Ile Lys Glu Ala Leu Phe Ser Ile 100 105 110 Tyr Lys Asp Gly Ser Asn Gly Trp Trp Phe Gly His Leu His Ala Thr 115 120 125 Ser Leu Arg Phe Arg Leu Leu Arg Gln Cys Gly Leu Phe Ile Pro Gln 130 135 140 Asp Val Phe Lys Thr Phe Gln Asn Lys Thr Gly Glu Phe Asp Met Lys 145 150 155 160 Leu Cys Asp Asn Val Lys Gly Leu Leu Ser Leu Tyr Glu Ala Ser Tyr 165 170 175 Leu Gly Trp Lys Gly Glu Asn Ile Leu Asp Glu Ala Lys Ala Phe Thr 180 185 190 Thr Lys Cys Leu Lys Ser Ala Trp Glu Asn Ile Ser Glu Lys Trp Leu 195 200 205 Ala Lys Arg Val Lys His Ala Leu Ala Leu Pro Leu His Trp Arg Val 210 215 220 Pro Arg Ile Glu Ala Arg Trp Phe Ile Glu Ala Tyr Glu Gln Glu Ala 225 230 235 240 Asn Met Asn Pro Thr Leu Leu Lys Leu Ala Lys Leu Asp Phe Asn Met 245 250 255 Val Gln Ser Ile His Gln Lys Glu Ile Gly Glu Leu Ala Arg Trp Trp 260 265 270 Val Thr Thr Gly Leu Asp Lys Leu Ala Phe Ala Arg Asn Asn Leu Leu 275 280 285 Gln Ser Tyr Met Trp Ser Cys Ala Ile Ala Ser Asp Pro Lys Phe Lys 290 295 300 Leu Ala Arg Glu Thr Ile Val Glu Ile Gly Ser Val Leu Thr Val Val 305 310 315 320 Asp Asp Gly Tyr Asp Val Tyr Gly Ser Ile Asp Glu Leu Asp Leu Tyr 325 330 335 Thr Ser Ser Val Glu Arg Trp Ser Cys Val Glu Ile Asp Lys Leu Pro 340 345 350 Asn Thr Leu Lys Leu Ile Phe Met Ser Met Phe Asn Lys Thr Asn Glu 355 360 365 Val Gly Leu Arg Val Gln His Glu Arg Gly Tyr Asn Ser Ile Pro Thr 370 375 380 Phe Ile Lys Ala Trp Val Glu Gln Cys Lys Ser Tyr Gln Lys Glu Ala 385 390 395 400 Arg Trp Phe His Gly Gly His Thr Pro Pro Leu Glu Glu Tyr Ser Leu 405 410 415 Asn Gly Leu Val Ser Ile Gly Phe Pro Leu Leu Leu Ile Thr Gly Tyr 420 425 430 Val Ala Ile Ala Glu Asn Glu Ala Ala Leu Asp Lys Val His Pro Leu 435 440 445 Pro Asp Leu Leu His Tyr Ser Ser Leu Leu Ser Arg Leu Ile Asn Asp 450 455 460 Ile Gly Thr Ser Pro Asp Glu Met Ala Arg Gly Asp Asn Leu Lys Ser 465 470 475 480 Ile His Cys Tyr Met Asn Glu Thr Gly Ala Ser Glu Glu Val Ala Arg 485 490 495 Glu His Ile Lys Gly Val Ile Glu Glu Asn Trp Lys Ile Leu Asn Gln 500 505 510 Cys Cys Phe Asp Gln Ser Gln Phe Gln Glu Pro Phe Ile Thr Phe Asn 515 520 525 Leu Asn Ser Val Arg Gly Ser His Phe Phe Tyr Glu Phe Gly Asp Gly 530 535 540 Phe Gly Val Thr Asp Ser Trp Thr Lys Val Asp Met Lys Ser Val Leu 545 550 555 560 Ile Asp Pro Ile Pro Leu Gly Glu Glu 565 <210> 3 atgtcaacac aacaagtttc atctgaaaat attgttagaa atgctgctaa ttttcatcca 60 aatatttggg gtaatcattt tttaacttgt ccatctcaaa ctattgattc ttggactcaa 120 caacatcata aagaattgaa agaagaagtt agaaaaaatga tggtttctga tgctaaataa 180 ccagcacaaa gattaagatt gattgatact gttcaaagat tgggtgttgc ttatcatttt 240 gaaaagaaa ttgatgatgc attagaaaaa attggtcatg atccatttga tgataaagat 300 gatttatata ttgtttcttt atgttttaga ttattaagac aacatggtat taaaatttct 360 tgtgatgttt ttgaaaaatt taaagatgat gatggtaaat ttaaagctag tttaatgaat 420 gatgttcaag gtatgttatc tttgtatgaa gcagctcatt tggctattca tggtgaagat 480 attttggatg aagctattgt ttttactaca actcatttaa aatctactgt ttctaattct 540 ccagttaatt ctacttttgc agaacaaatt agacattctt taagagttcc attgagaaaa 600 gctgttccaa gattagaatc tcgttatttt ttggatattt attctcgtga tgatttacat 660 gataaaactt tattaaattt tgctaaatta gattttaata ttttacaagc tatgcatcaa 720 aaagaagcta gtgaaatgac tagatggtgg agagattttg attttttgaa aaaattgcca 780 tatattagag atagagttgt tgaattatat ttttggattt tagttggtgt ttcttatcaa 840 ccaaaatttt ctactggtag aattttttta tctaaaatta tttgtttaga aactttagtt 900 gatgatacat ttgatgctta tggtacattt gatgaattgg ctatttttac agaagctgtt 960 acaagatggg attaggtca tagagatgca ttgccagaat atatgaaatt tatttttaaa 1020 acattaattg atgtttattc tgaagctgaa caagaattag ctaaagaagg tagatcatat 1080 tctattcatt atgctattag atcatttcaa gaattagtta tgaaatattt ttgtgaagct 1140 aaatggttaa ataaagtta tgttccatca ttggatgatt ataaatctgt ttcattaaga 1200 tcaatcggtt ttttaccaat cgctgttgct tcttttgttt ttatgggtga tattgcaaca 1260 aaagaagttt ttgaatggga aatgaataat ccaaaaatta ttattgcagc agaaacaatt 1320 tttagatttt tggatgatat tgcaggtcat agatttgaac aaaaaagaga acattcacca 1380 tcagcaatcg aatgttataa aaatcaacat ggtgtttcag aagaagaagc agttaaagca 1440 ttgtcattgg aagttgcaaa ttcatggaaa gatattaatg aagaattgtt gttgaatcca 1500 atggcaattc cattgccatt gttgcaagtt attttggatt tgtcaagatc agcagatttt 1560 atgtatggta atgcacaaga tagattttaca cattcaacaa tgatgaaaga tcaagttgat 1620 ttggttttga aagatccagt taaattggat gattaa 1656 <210> 4 atggattctt ctactgctac tgctatgact gctcctttta ttgatccaac cgatcacgtt 60 aacttgaaaa ctgatactga tgcctccgaa aacagaagaa tgggtaatta caaaccctcc 120 atctggaact acgatttctt gcaatctttg gctacccatc acaacatcgt tgaagaaaga 180 catttgaagt tggccgaaaa gttgaagggt caagttaagt ttatgttcgg tgctccaatg 240 gaaccattgg ctaaattgga attggttgat gttgtgcaga gattgggttt gaaccatttg 300 ttcgaaaccg aaatcaaaga ggccttgttc tctatctaca aggatggttc taatggttgg 360 tggtttggtc acttgcatgc tacatctttg agattcagac tgttgagaca atgcggtttg 420 ttcattccac aagatgttt caagaccttc caaaacaaga ccggtgaatt cgatatgaag 480 ttgtgcgata atgtcaaggg cttgttgtcc ttgtatgaag cttcttactt aggttggaag 540 ggtgagaaca ttttggatga agctaaagct ttcaccacca agtgtttgaa atctgcctgg 600 gaaaacattt ccgaaaaatg gttggctaag agagttaagc acgctttggc tttgccattg cattggagag ttccaagaat tgaagctaga tggttcattg aagcttacga acaagaggct aatatgaacc caactttgtt gaattggcc aagttggatt tcaacatggt ccaatccatc 840. caccaaaaag aaattggtga attggcaaga tggtgggtta ctactggttt ggataagttg gcttttgcca gaaacaactt gttgcaatcc tatatgtggt cttgcgctat tgcttctgat ccaaagttta agttggccag agaaaccatt gtggaaatcg gttctgtttt gaccgttgtt 1020. gatgatggtt atgatgtcta cggttccata gatgattgg acttgtacac ctcttctgtc gaagatggt cttgtgtcga aattgataag ttgccaaaca ccttgaagct gatcttcatg tctatgttca acaagaccaa cgaagttggt ttgagagttc aacacgaaag aggttacaac 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200. 1200 cgttggtttc acggtggtca tactccacca ttggaat attcattga cggcttggtt tccattggtt tccctttgtt gttgattaca ggttacgttg ctattgctga aaacgaagct gctttggaca aagttcatcc attgccagat ttgttgcact actcatcttt gttgtccaga 1380 ttgatcaacg acattggtac ttctccagac gaaatggcta gaggtgataa cttgaagtct 1440 atccattgct acatgaacga aactggtgct tctgaagaag ttgctagaga acatattaag 1500 ggtgtcatcg aagaaaactg gaagattttg aatcaatgct gcttcgacca atcgcaattt 1560 caagaaccat tcatcacctt caacctgaac tctgttagag gttctcattt cttctacgaa 1620 ttcggtgatg gtttcggtgt tactgattct tggacaaaag ttgacatgaa gtccgttttg 1680 atcgacccaa ttccattggg tgaagaatga 1710 <210> 5 atgtcacaaa ctcataaaca tgctattcca gcaaacatcg ctgatagatg tttgattaat 60 ccagaacaat acgaaactaa gtacaagcaa tctattaatg atccagatac attttggggt 120 gaacaaggta aaatcttgga ttggatcact ccataccaaa aggttaaaaa tacatcattt 180 gctcctggta atgtttctat taaatggtac gaagatggta ctttgaattt ggctgcaaac 240 tgtttggata gacatttgca agaaaatggt gacagaactg caattatttg ggaaggtgac 300 gatacatcac aatctaagca tatctcttac agagaattgc atagagatgt ttgtagattc 360 gcaaacacat tgttggattt gggtattaag aaaggtgacg ttgttgctat ctatatgcca 420 atggttccag aagctgcagt tgcaatgtta gcttgtgcaa gaattggtgc tgttcattca 480 gttattttg gtggttttc tccagaagct gttgcaggta gaatcatcga ttcttcatct 540 agattggtta ttacagcaga tgaaggtgtt agagctggta gatcaatccc attgaagaaa 600 aatgttgatg atgctttgaa aaatccaaac gttacttcag ttgaacatgt tatcgttttg 660 aaaagaacag gttctgatat tgattggcaa gaaggtagag atttgtggtg gagagatttg 720 attgaaaaag cttctccaga acatcaacca gaagcaatga acgctgaaga tccattgttt 780 attttgtaca cttcaggttc tacaggtaaa ccaaaaggtg ttttacatac tacaggtggt 840 tatttggttt acgctgcaac tacttttaaa tacgttttcg attaccatcc aggtgacatc 900 tattggtgta ctgctgatat gggttgggtt acaggtcatt catatttgtt atacggtcca 960 ttagcatgtg gtgctactac attgatgttt gaaggtgttc caaattggcc aactccagct 1020 agaatgtgtc aagttgttga taagcatcaa gttaacatct tgtacactgc accaacagct 1080 attagagcat tgatggctga aggtgacaaa gcaattgaag gtacagatag atcatctttg 1140 agaattttag gttctgttgg tgaaccaatt aatccagaag cttgggaatg gtactggaag 1200 aaaattggta aagaaaagtg tccagttgtt gatacttggt ggcaaactga aacaggtggt 1260 tttatgatta caccattgcc aggtgctatt gaattaaaag caggttcagc tactagacca 1320 tttttcggtg ttcaaccagc attagttgat aatgaaggtc atccacaaga aggtgctact 1380 gagggtaatt tggttattac agattcttgg ccaggtcaag caagaacatt gtttggtgac 1440 catgaaagat ttgaacaaac ttacttctca acttttaaaa acatgtactt ttctggtgac 1500 ggtgctagaa gagatgaaga tggttattac tggatcactg gtagagttga tgatgttttg 1560 aacgtttcag gtcatagatt gggtacagca gaaattgaat ctgcattggt tgctcatcca 1620 aaaattgcag aagctgcagt tgttggtatt ccacatgcta ttaaaggtca agcaatctat 1680 gcttacgtta ctttaaatca tggtgaagaa ccatcaccag aattgtatgc agaagttaga 1740 aactgggtta gaaaggaaat tggtccattg gctacaccag atgttttaca ttggactgat 1800 tcattgccaa agacaagatc tggtaaaatc atgagaagaa tcttgagaaa gattgctgca 1860 ggtgacactt caaatttggg tgacacttct acattggctg atccaggtgt tgttgaaaaa 1920 ccattggaag aaaaacaagc tattgcaatg ccatcttaa 1959 <210> 6 ccaaaacgtt gaatactacg ctcaacgtgc tcaaagacca ggaaccttga tttcaccct 60 ttccctctcc acaatctggg ggttacgaca atgctccagg tatctgggga accttgatta 120 tcactgagtt ttagagctat gctgttttga atggtcccaa aacgctgtgt attatggtca 180 gcgtgctcaa agacctggta ccatgatcat caccgttat ttcccctcaa gccggcggct 240 atgacaacgc ccctgggatt tggggtacca tgatcatcac ggagttttag ag 292 <210> 7 ccaaaaccaa caagatggcc aaatcatgaa aattattatc gcagttccga atactataca 60 tccagtgcat gattatattt cagtattaca agaattgaaa ctgagtggca gttccgaata 120 ctattcagtt ttagagctat gctgttttga atggtcccaa aactcgaatt gattagccct 180 gtaatcatac ctctgggtaa tccgaagagg cctattgatt tgtttaccag gtaaggatac 240 tgatgggttt gaaacgtgga aaagtaatcc gaagaggcct aatgttttag ag 292 <210> 8 aacgacatta ctatatatat atataggaa gcatttaata gacagcatcg tatatatgt 60 gtactttgca gttatgacgc cagatggcag tagtggaaga tattctttat tgaaaatag 120 cttgtcacct tacgtacaat cttgatccgg agctttcttt ttttgccga ttaagaatta 180 attcggtcga aaaaagaaaa ggagagggcc aagagggagg gcattggtga ctattgagca 240 cgtgagtata cgtgattaag cacacaaagg cagcttggag tatgtctgtt attaatttca 300 caggtagttc tggtccattg gtgaaagtttt gcggcttgca gagcacagag gccgcagaat 360 gtgctctaga ttccgatgct gacttgctgg gtattatatg tgtgcccaat agaaagagaa 420 caattgacccc ggttattgca aggaaaattt caagtcttgt aaaagcatat aaaatatagtt 480 caggcactcc gaaatacttg gttggcgtgt ttcgtaatca acctaaggag gatgttttgg 540 ctctggtcaa tgattacggc attgatatcg tccaactgca tggagatgag tcgtggcaag 600 aataccaaga gttcctcggt ttgccagtta ttaaaagact cgtatttcca aaagactgca 660 acatactact cagtgcagct tcacagaaac ctcattcgtt tattcccttg tttgattcag 720 aagcaggtgg gacaggtgaa cttttggatt ggaactcgat ttctgactgg gttggaaggc 780 aagagagcc cgaaagctta cattttatgt tagctggtgg actgacgcca gaaaatgttg 840 gtgatgcgct tagattaaat ggcgttattg gtgttgatgt aagcggaggt gtggagacaa 900 atggtgtaaa agactctaac aaaatagcaa atttcgtcaa aaatgctaag aaataggtta 960 ttactgagta gtatttattt aagtattgtt tgtgcacttg cctatgcggt gtgaaatacc 1020 gcacagatgc gtaaggagaa aataccgcat caggaaattg taaacgttaa tattttgtta 1080 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc 1140 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtg 1186 <210> 9 cttaactatg cggcatcaga gcagattgta ctgagagtgc accataaatt cccgttttaa 60 gagcttggtg agcgctagga gtcactgcca ggtatcgttt gaacacggca ttagtcaggg 120 aagtcataac acagtccttt cccgcaattt tctttttcta ttactcttgg cctcctctag 180 tacactctat attttttat gcctcggtaa tgatttcat tttttttttt cccctagcgg 240 atgactcttt ttttttctta gcgattggca tttcacata atgaattata cattatataa 300 agtaatgtga tttcttcgaa gaatatacta aaaaatgagc aggcaagata aacgaaggca 360 aagatgacag agcagaaagc cctagtaaag cgtattacaa atgaaaccaa gattcagatt 420 gcgatctct taaagggtgg tccctagcg atagagcact cgatcttccc agaaaaagag 480 gcagaagcag tagcagaaca ggccacacaa tcgcaagtga ttaacgtcca cacaggtata 540 gggttctgg accatatgat acatgctctg gccaagcatt ccggctggtc gctaatcgtt 600 gagtgcattg gtgacttaca catagacgac catcacacca ctgaagactg cgggattgct 660 ctcggtcaag cttttaaaga ggccctactg gcgcgtggag taaaaaggtt tggatcagga 720 tttgcgcctt tggatgaggc actttccaga gcggtggtag atctttcgaa caggccgtac 780 gcagttgtcg aacttggttt gcaaagggag aaagtaggag atctctcttg cgagatgatc 840 ccgcatttc ttgaaagctt tgcagaggct agcagaatta ccctccacgt tgattgtctg 900 cgaggcaaga atgatcatca ccgtagtgag agtgcgttca aggctcttgc ggttgccata 960 agagaagcca cctcgcccaa tggtaccaac gatgttccct ccaccaaagg tgttcttatg 1020 tagtgacacc gattatttaa agctgcagca tacgatatat atacatgtgt atatatgtat 1080 acctatgaat gtcagtaagt atgtatacga acagtatgat actgaagatg acaaggtaat 1140 gcatcattct atacgtgtca ttctgaacga ggcgcgcttt ccttttttct tttgcttttt 1200 tcttttttt tctcttgaac tcgacggatc tatgcggtgt gaaataccgc acagatgcgt 1260 aaggagaaaa taccgcatca 1280 <210> 10 agctcgctgt gaagatccca gcaaaggctt acaaagtgtt atctcttttg agacttgttg 60 agttgaacac tggtgttttc atcaaactta ccaaggacgt gtacccattg ttgaaacttg 120 tatcaccata tattgttatc ggacaacctt cacttgcatc tatccgttct ttaatccaaa 180 agagatctag aataatgtgg caaaggccag aagataaaga accaaaagag ataatcttga 240 atgaacaa tatcgttgaa gagaatag gtgatgaagg tgtcatttgt atcgaggata 300 tcatccatga gatttcgacg ttgggcgaaa atttctcgaa atgtactttc ttcctattac 360 420 aaatgcgcga acaaaacaag gagactcgtc aaatttcaaa cgctgccacg gctccagtta 480 540 atctgtacaa tagacatcgg gctcccattg gccctaccca catatgtaga aatacattac 600 tctattcact actgcattta gttatgttta acatttgata tagcagacta ccgccaggca 660 caatatattc cccttccctc ttgccattcg ctgtacttgt ggtggattcc aattcagcgc 720 agtcacgtgc tagtaatcac cgcattttt tcttttcctt tcaggctaaa accggttccg 780 ggcctgatcc ctgcactcat tttctaacgg aaaaccttca gaagcatac tacccattcc 840 agtttagagt catgacaggt tcaacatcag atgcttcata tacttttata tattgaatta 900 tataaatata tctatgtact ctaagtaagt acatctgctt taacgcattc ctacatttgc 960 ttcgattat ttttattgtt gatacctatt tgaagaagta aaaagtatcc cacactacac 1020 agattatacc atgtctaaga atatcgttgt cctaccgggt gatcacgtcg gtaaagaagt 1080 tactgacgaa gctattaagg tcttgaatgc cattgctgaa gtccgtccag aaattaagtt 1140 caatttccaa catcacttga tcgggggtgc tgccatcgat gccactggca ctcctttacc 1200 agatgaagct ctagaagcct ctaagaaagc cgatgctgtc ttactaggtg ctgttggtgg 1260 tccaaaatgg ggtacgggcg cagttagacc agaacaaggt ctattgaaga tcagaaagga 1320 attgggcta tacgccaact taagaccatg taactttgct tctgattctt tactagatct 1380 ttctcctttg aagcctgaat atgcaaaggg taccgatttc gtcgtcgtta gagaattggt 1440 tggtggtatc tactttggtg aaagaaaaga agatgaaggt gacggagttg cttgggactc 1500 tgagaaatac agtgttcctg aagttcaaag attacaaga atggctgctt tcttggcatt 1560 gcaacaaaac ccaccattac caatctggtc acttgacaag gctaacgtgc ttgcctcttc 1620 cagattgtgg agaaagactg ttgaagaaac catcaagact gagttcccac aattaactgt 1680 tcagcaccaa ttgatcgact ctgctgctat gattttggtt aaatcaccaa ctaagctaaa 1740 cggtgttgtt attaccaaca acatgtttgg tgatattatc tccgatgaag cctctgttat 1800 tccaggttct ttgggttat taccttctgc atctctagct tccctacctg acactaacaa 1860 ggcattcggt ttgtacgaac catgtcatgg ttctgcccca gatttaccag caaacaaggt 1920 taacccaatt gctaccatct tatctgcagc tatgatgttg aagttatcct tggatttggt 1980 tgaagaaggt agggctcttg aagaagctgt tagaaatgtc ttggatgcag gtgtcagaac 2040 cggtgacctt ggtggttcta actctaccac tgaggttggc gatgctatcg ccaaggctgt 2100 caaggaaatc ttggcttaat tatacaggaa acttaataga acaaatcaca tatttaatct 2160 aatagccacc tgcattggca cggtgcaaca ctcacttcaa cttcatctta caaaagatca 2220 cgtgatctgt tgtattggga tc 2242 <210> 11 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg 60 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta 120 ctgagagtgc accataccac agctttcaa ttcattcat catttttt ttattcttt 180 ttttgatttc ggttctttg aaattttt gattcggtaa tctccgaaca gaaggaagaa 240 cgaaggaagg agcacagact tagattggta tatatacgca tatgtagt tgaagaaaca 300 tgaaattgcc cagtattctt aacccactg cacagaaaaacctgcag gaaacgaga 360 taaatcatgt cgaaagctac ataggaa cgtgctgcta ctcatcctag tcctgttgct 420 gccaagctat ttaatcat gcacgaaaag caacaact tgtgtgcttc attggatgtt 480 cgtaccacca aggaattact ggagttagtt gaagcattag gtcccaaat ttgtttacta 540 aaaacacatg tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag 600 gcattatccg ccaagtacaa tttttactc ttcgagaca gaaaatttgc tgacattggt 660 atacagtca aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt 720 acgaatgcac acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa 780 gaagtaaca aggaactag aggccttttg atgttagcag attgtcatg caagggctcc 840 900 gttatcggct ttattgctca aagagacatg ggtggagaag atgaaggtta cgattggttt 960 attatgacac ccggtgtggg tttagatgac aagggagacg cattgggtca agagtaga 1020 accgtggatg atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt 1080 ccaaagggaa gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca 1140 tatttgagaa gatgcggcca gcaaaactaa aaaactgtat tataagaaa tgcatgtata 1200 ctaaactcac aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga 1260 aataccgcac agatgcgtaa ggaagaaata ccgcatcagg 1300 <210> 12 ttatttttg ctttttctct tgaggtcaca tgatcgcaaa atggcaaatg gcacgtgaag 60 ctgtcgatat tggggaactg tggtggttgg caaatgacta attaagttag tcaaggcgcc 120 atcctcatga aaactgtgta acataataac cgaagtgtcg aaaaggtggc accttgtcca 180 240 aaaggaagtt tttcctttt cttgctctct tgtcttttca tctactattt cttcgtgta 300 atacagggtc gtcagataca tagatacaat tctattaccc ccatccatac aatgccatct 360 cattcgata ctgttcaact acacgccggc caagagaacc ctggtgacaa tgctcacaga 420 tccagagctg taccaattta cgccaccact tcttatgttt tcgaaaactc taagcatggt 480 tcgcaattgt ttggtctaga agttccaggt tacgtctatt cccgtttcca aaacccaacc 540 agtaatgttt tggaagaaag aattgctgct ttagaaggtg gtgctgctgc tttggctgtt 600 tcctccggtc aagccgctca aacccttgcc atccaaggtt tggcacacac tggtgacaac 660 atcgtttcca cttcttactt atacggtggt acttataacc agttcaaaat ctcgttcaaa 720 agatttggta tcgaggctag atttgttgaa ggtgacaatc cagaagaatt cgaaaaggtc 780 tttgatgaaa gaaccaaggc tgttatttg gaaaccattg gtaatccaaa gtacaatgtt 840 ccggattttg aaaaaattgt tgcaattgct cacaaacacg gtattccagt tgtcgttgac 900 aacacatttg gtgccggtgg ttacttctgt cagccaatta aatacggtgc tgatattgta 960 acacattctg ctaccaaatg gattggtggt catggtacta ctatcggtgg tattattgtt 1020 gactctggta agttcccatg gaaggactac ccagaaaagt tccctcaatt ctctcaacct 1080 gccgaaggat atcacggtac tatctacaat gaagcctacg gtaacttggc atacatcgtt 1140 catgttagaa ctgaactatt aagagatttg ggtccattga tgaacccatt tgcctctttc 1200 ttgctactac aaggtgttga aacattatct ttgagagctg aaagacacgg tgaaaatgca 1260 ttgaagttag ccaaatggtt agaacaatcc ccatacgtat cttgggtttc ataccctggt 1320 ttagcatctc attctcatca tgaaaatgct aagaagtatc tatctaacgg ttcggtggt 1380 gtcttatctt tcggtgtaaa agacttacca aatgccgaca aggaaactga cccattcaaa 1440 cttctggtg ctcaagttgt tgacaattta aagcttgcct ctaacttggc caatgttggt 1500 gatgccaaga ccttagtcat tgctccatac ttcactaccc acaaacaatt aaatgacaaa 1560 gaaaagttgg catctggtgt taccaaggac ttaattcgtg tctctgttgg tatcgaattt 1620 attgatgaca ttattgcaga cttccagcaa tcttttgaaa ctgttttcgc tggccaaaaa 1680 ccatgagtgt gcgtaatgag ttgtaaaatt atgtataaac ctactttctc tcacaagtac 1740 tatactttta taaaacgaac tttattgaaa tgaatatcct ttttttccct tgttacatgt 1800 cgtgactcgt actttgaacc taaattgttc taacatcaaa gaacagtgtt aattcgcagt 1860 cgagaagaaa aatatggtga acaagactca tctacttcat gagactactt tacgcctcct 1920 ataaagctgt cacactggat aaatttattg taggaccaag ttacaaaaga ggatgatgga 1980 ggtttc 1986

Claims

1. A Sansyn mutant of santalene synthase F441V Its characteristics are, Its amino acid sequence was obtained by mutating phenylalanine at position 441 of the amino acid sequence shown in SEQ ID NO.1 to valine.

2. Encoding the SanSyn mutant of santalene synthase as described in claim 1 F441V The genes.

3. A recombinant expression vector comprising the gene of claim 2.

4. A high-production santalene-producing engineered Saccharomyces cerevisiae containing the gene of claim 2.

5. A brewing yeast strain that produces high yields of santalene and santalol, characterized in that, The engineered Saccharomyces cerevisiae strain that produces high levels of santalene and santalol has the following characteristics: It uses Saccharomyces cerevisiae as the starting strain, knocks out the OYE2, OYE3, ATF1, ATF2, LPP1, DPP1, and ROX1 genes, and replaces the endogenous promoter of the ERG9 gene in Saccharomyces cerevisiae with P... HXT1 Promoters, overexpression of mSTS, ADH2, ALD6, and SeACS L641P IDI1, tHMG1, UPC2-1, CYP736A167, and SaCPR2 genes; among which: The IDI1 and UPC2-1 genes were integrated into the DPP1 site on the chromosome of Saccharomyces cerevisiae. ADH2, ALD6, SeACS L641P The gene was integrated into the LPP1 site on the chromosome of Saccharomyces cerevisiae. mSTS and tHMG1 genes are integrated into the chromosome P of Saccharomyces cerevisiae. ERG9 site; The CYP736A167, SaCPR2, and second copy of the mSTS gene were integrated into the MET17 site of Saccharomyces cerevisiae. The third copy of the mSTS gene was integrated into the ROX1 site on the Saccharomyces cerevisiae chromosome; The second copy of the CYP736A167 gene, the second copy of the tHMG1 gene, or the second and third copies of the CYP736A167 gene are integrated into the ROX1 site of the Saccharomyces cerevisiae chromosome. The sequence of the mSTS gene is the SanSyn mutant of santalene synthase as described in claim 1. F441V The nucleotide sequence.

6. The engineered brewer's yeast strain for high production of santalene and santalol according to claim 5, characterized in that, The crRNA spacer nucleic acid sequences of the OYE2 and OYE3 genes are shown in SEQ ID NO. 6; The crRNA spacer nucleic acid sequences of the ATF1 and ATF2 genes are shown in SEQ ID NO.

7.

7. The method for constructing the engineered Saccharomyces cerevisiae strain with high yield of santalene and santalol as described in claim 5 or 6, characterized in that, Includes the following steps: (1) Gene knockout: The OYE2, OYE3, ATF1 and ATF2 genes were knocked out using the CRISPR-Cas9 gene knockout system. The nucleotide sequences of the OYE2 and OYE3 crRNA spacers used are shown in SEQ ID NO.6; the nucleotide sequences of the ATF1 and ATF2 crRNA spacers are shown in SEQ ID NO.

7. After culture expression screening, strains with the OYE2, OYE3, ATF1 and ATF2 genes knocked out were obtained. (2) Module construction: ADH2, ALD6, and SeACS are respectively constructed. L641P IDI1, tHMG1, UPC2-1, CYP736A167, SaCPR2, mSTS and their corresponding promoters and terminators are sequentially connected to obtain the corresponding expression modules; (3) Strain construction: The above-mentioned gene modules are integrated into the corresponding chromosomal loci of the brewing strain to obtain the brewing yeast engineered strain that produces high levels of santalene and santalol; Among them, the IDI1 and UPC2-1 genes were integrated into the DPP1 site on the chromosome of Saccharomyces cerevisiae; ADH2, ALD6, SeACS L641P The gene was integrated into the LPP1 site on the chromosome of Saccharomyces cerevisiae. mSTS and tHMG1 genes are integrated into the chromosome P of Saccharomyces cerevisiae. ERG9 site; The CYP736A167, SaCPR2, and second copy of the mSTS gene were integrated into the MET17 gene locus of Saccharomyces cerevisiae. The third copy of the mSTS gene was integrated into the ROX1 site on the Saccharomyces cerevisiae chromosome; The second copy of the CYP736A167 gene, the second copy of the tHMG1 gene, or the second and third copies of the CYP736A167 gene are integrated into the ROX1 site of the Saccharomyces cerevisiae chromosome.

8. The application of the santalene synthase mutant of claim 1, the recombinant expression vector of claim 3, or the Saccharomyces cerevisiae engineered strain that produces high levels of santalene and santalol of claim 5 or 6 in the preparation of sandalwood volatile oil.

9. A method for preparing santalene and santalol, characterized in that, By inoculating the engineered brewer's yeast strain that produces high levels of santalene and santalol as described in claim 5 or 6 into a fermentation medium, and then adding feed medium as the dissolved oxygen level rises, and fermenting the feed in batches, the fermentation broth is extracted to obtain santalene and santalol.

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