CN116064639A - Monoterpene synthase, genetically engineered strain and application thereof - Google Patents

Monoterpene synthase, genetically engineered strain and application thereof Download PDF

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CN116064639A
CN116064639A CN202211340237.5A CN202211340237A CN116064639A CN 116064639 A CN116064639 A CN 116064639A CN 202211340237 A CN202211340237 A CN 202211340237A CN 116064639 A CN116064639 A CN 116064639A
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gene
saccharomyces cerevisiae
monoterpene
identity
seq
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戴宗杰
苏立秋
王钦宏
张媛媛
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Tianjin Institute of Industrial Biotechnology of CAS
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

The invention discloses a eucalyptol synthase gene, a recombinant saccharomyces cerevisiae, a construction method and application thereof. The construction method of the recombinant saccharomyces cerevisiae comprises the following steps: the coding gene of the monoterpene synthase with the amino acid sequence shown as SEQ ID No.10, 11, 12 or 13 is introduced into saccharomyces cerevisiae to obtain the recombinant saccharomyces cerevisiae for producing eucalyptol. The invention provides powerful strain and research foundation for the biosynthesis of monoterpenes, and has important significance for the excavation of high-activity monoterpene synthetase.

Description

Monoterpene synthase, genetically engineered strain and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to monoterpene synthase, a genetic engineering strain and application thereof.
Background
Monoterpenes are terpenes and derivatives thereof containing two isoprene units in the molecule. The structure classification comprises: acyclic monoterpenes, monocyclic monoterpenes, polycyclic monoterpenes. Eucalyptus (1, 8-cineol) is a monoterpene oxide, and is the main component of eucalyptus essential oil. Because of its medicinal value and its great potential for use in the food, cosmetic, and energy industries, it has attracted great attention.
At present, monoterpenes are mainly obtained by extraction from natural plants or chemical synthesis, but the yield of the methods is low and the method is highly dependent on the availability of raw materials. Meanwhile, the generated waste has complex components and pollutes the environment, thereby bringing difficulty to the subsequent treatment. Along with the development of synthetic biology technology and metabolic engineering, microorganisms are modified to become cell factories for synthesizing high-added-value chemicals, and a new green and clean production way is opened for synthesizing terpenoids.
Monoterpene synthase is one of the key factors affecting terpenoid biosynthesis. The currently excavated monoterpene synthases have low catalytic activity, which limits the application of monoterpene microbial synthesis (Leferrink NGH, jervis AJ, zebec Z, toogood HS, hay S, takano E, scrutton NS. A 'Plug and Play' Platform for the Production of Diverse Monoterpene Hydrocarbon Scaffolds in Escherichia coll. Chemistry select. 2016 Jun 21;1 (9): 1893-1896. Doi: 10.1002/slct. 20160563. PMID: 29756025; PMCID: PMC5954.; lei D, qiau Z, qiao J, zhao GR. Plasticity engineering of plant monoterpene synthases and application for microbial production of monotepenoids. Biotechnol. 2021 Jun 30;14 (1): 147. Doi: 10.1186/S13068-021-01998-8. PMID: 34193244; PMCID: PMC 8247113.).
Therefore, the excavation and acquisition of superior monoterpene synthases would be of great importance to advance monoterpene biosynthesis.
Disclosure of Invention
The invention provides a monoterpene synthase and an application of the same in producing eucalyptol.
It is an object of the present invention to provide monoterpene synthases.
The first aspect of the present invention relates to a monoterpene synthase comprising one of the amino acid sequences selected from the group consisting of:
a) SEQ ID NO:10 to SEQ ID NO:13;
b) At least 70% identical, preferably at least 80% identical, at least 90% identical, at least 95% identical, more preferably at least 99% identical to the sequence shown in a), still having the function.
The invention also provides nucleic acids encoding the above monoterpene synthases.
The invention also provides a genetically engineered bacterium expressing the monoterpene synthase, which comprises nucleic acid for encoding the monoterpene synthase.
According to an embodiment of the present invention, the genetically engineered bacterium is a recombinant strain obtained by constructing an expression cassette from the nucleic acid and introducing the expression cassette into a host bacterium.
It is an object of the present invention to provide a method for constructing a recombinant Saccharomyces cerevisiae producing monoterpene substances.
The invention provides a construction method of a recombinant saccharomyces cerevisiae for producing monoterpene substances, which comprises the following steps: and introducing the monoterpene synthase gene into the saccharomyces cerevisiae to obtain the recombinant saccharomyces cerevisiae for producing monoterpenes.
For example, the Saccharomyces cerevisiae is one that is capable of accumulating monoterpene synthesis precursor geranyl diphosphate (GPP).
Alternatively, according to the above construction method, the monoterpene synthetase gene encodes a monoterpene synthetase with the amino acid sequence shown in SEQ ID No.10, 11, 12, 13. Alternatively, according to the above construction method, the Saccharomyces cerevisiae is used for increasing the content and/or activity of farnesyl pyrophosphate synthase ERG20, 3-hydroxy-3-methylglutaryl-CoA tHMG1, acetyl-CoA acyltransferase mvaE, HMG-CoA synthase mvaS-m, isopentenyl pyrophosphate isomerase IDI1, mevalonate kinase ERG12, MVAP kinase ERG8 and/or MVAPP decarboxylase ERG19 in the starting Saccharomyces cerevisiae. For example, increasing the enzyme content and/or activity is achieved by increasing the copy number of the corresponding enzyme gene of Saccharomyces cerevisiae.
Optionally, the Saccharomyces cerevisiae is TIB H1 (China patent 202110934151.4), and the preservation number of the Saccharomyces cerevisiae in the China general microbiological culture Collection center is CGMCC No.21000.
Optionally, the construction method comprises the following steps: and (3) at least one of the following modifications is carried out on the saccharomyces cerevisiae to obtain the saccharomyces cerevisiae: a1, introducing a farnesyl pyrophosphoric acid synthetase gene 96-site and 127-site double-point mutant gene ERG20 F96W/N127W A gene; a2, introducing a tHMG1 gene; a3, introducing mvaE genes; a4, introducing mvaS-m genes; a5, introducing IDI1 gene; a6, introducing ERG12 genes; a7, introducing ERG8 genes; a8, ERG19 gene is introduced.
Optionally, the ERG20 F96W/N127W Gene encoded ERG20 F96W/N127W The sequence of the protein is shown as SEQ ID No. 14; and/or, the sequence of the tHMG1 protein coded by the tHMG1 gene is Genbank login number: AJS96703.1 sequence is shown at positions 530-1054; and/or the sequence of the mvaE protein coded by the mvaE gene is shown in Genbank accession number AAG 02439.1; and/or the sequence of the mvaS-m protein coded by the mvaS-m gene is shown by substituting glycine for alanine at 110 th position of Genbank accession number AAG 02438.1; and/or, the sequence of IDI1 protein coded by the IDI1 gene is Genbank login number: the sequence NP 015208.1 is shown; and/or, the sequence of the ERG12 protein coded by the ERG12 gene is Genbank login number: the sequence NP 013935.1 is shown; and/or, the sequence of the ERG8 protein coded by the ERG8 gene is Genbank login number: the sequence NP 013947.1 is shown; and/or, the sequence of the ERG19 protein coded by the ERG19 gene is Genbank login number: the NP-014441.1 sequence is shown.
Optionally, the ERG20 F96W/N127W The gene sequence is shown in 722 th to 1780 th positions of SEQ ID No. 1; and/or, the tHMG1 gene sequence is shown in 800 th-2383 th positions of SEQ ID No. 2; and/or the mvaE gene sequence is shown in the reverse complement of 270 th-2681 th positions of SEQ ID No. 3; and/or, the mVAS-m gene sequence is shown in 3358-4509 of SEQ ID No. 3; and/or, the IDI1 gene sequenceAs shown in the reverse complementation of 270 th to 1136 th positions of SEQ ID No. 4; and/or the ERG12 gene sequence is shown in 1813-3144 of SEQ ID No. 4; and/or the ERG8 gene sequence is shown in the reverse complement of 333-1688 th site of SEQ ID No. 5; and/or the ERG19 gene sequence is shown in positions 2365-3555 of SEQ ID No. 5.
Alternatively, according to the above construction method, the introducing a monoterpene synthase gene into the starting saccharomyces cerevisiae is achieved by introducing a monoterpene synthase gene expression cassette into the starting saccharomyces cerevisiae; and/or, the A1 is obtained by introducing ERG20 into the Saccharomyces cerevisiae F96W/N127W Realizing a gene expression cassette; and/or, said A2 is achieved by introducing a hmg1 gene expression cassette into said s.cerevisiae; and/or, the A3 is realized by introducing an mvaE gene expression cassette into the saccharomyces cerevisiae; and/or, the A4 is realized by introducing an mvaS-m gene expression cassette into the saccharomyces cerevisiae; and/or, the A5 is realized by introducing an IDI1 gene expression cassette into the saccharomyces cerevisiae; and/or, the A6 is realized by introducing an ERG12 gene expression cassette into the saccharomyces cerevisiae; and/or, the A7 is realized by introducing an ERG8 gene expression cassette into the saccharomyces cerevisiae; and/or, the A8 is realized by introducing an ERG19 gene expression cassette into the saccharomyces cerevisiae.
Alternatively, according to the above construction method, the monoterpene synthase gene expression cassette comprises a promoter, the monoterpene synthase gene and a terminator, the promoter being Gal2; the terminator is CYC1. For example, the sequence of the monoterpene synthase gene expression cassette is shown at positions 72-2159 of SEQ ID No.6, at positions 72-2117 of SEQ ID No.7, at positions 72-2117 of SEQ ID No.8, and at positions 72-2129 of SEQ ID No. 9.
Alternatively, said ERG20 according to the above construction method F96W/N127W The sequence of the gene expression cassette is shown in 54 th to 2141 th positions of SEQ ID No. 1; and/or, the sequence of the tHMG1 gene expression cassette is shown in 75-2769 positions of SEQ ID No. 2; and/or the mvaE gene expression cassette sequence is shown in the reverse complement of the 78 th-3357 th site of SEQ ID No. 3; and/or, the mva-m gene expression cassette sequence is shown in 2682-4728 of SEQ ID No. 3; and/or, the IDI1 gene expression cassette sequenceThe sequence is shown as the reverse complement of the 69 th bit to the 1812 th bit of SEQ ID No. 4; and/or the ERG12 gene expression cassette sequence is shown in 1137-3256 of SEQ ID No. 4; and/or the ERG8 gene expression cassette sequence is shown in the reverse complementation of the 74 th to 2364 th positions of SEQ ID No. 5; and/or the ERG19 gene expression cassette sequence is shown in SEQ ID No.5 at positions 1689-3774.
The invention also provides the recombinant saccharomyces cerevisiae constructed by adopting the construction method.
It is another object of the present invention to provide a method for producing monoterpenes.
The invention provides a method for producing monoterpene, which comprises the steps of culturing the recombinant saccharomyces cerevisiae to obtain a fermentation product, namely eucalyptol, a-pinene, sabinene, myrcene, carene, terpinolene, ocimene and a-terpineol.
The invention also provides the use of a monoterpene synthetase and its encoding gene in the preparation of a monoterpene, wherein the monoterpene synthetase has an amino acid sequence shown as SEQ ID No.10, 11, 12 or 13, or has at least 70% identity, preferably at least 80% identity, at least 90% identity, at least 95% identity, more preferably at least 99% identity with the nucleotide sequence shown as SEQ ID No.10, 11, 12 or 13, and still has the function; more preferably, the gene encoding the monoterpene synthase is derived from Cordyceps sinensis (Ophiocordyceps sinensis), and Tuber (Hypoxylon).
The invention also provides any one of the following application, wherein X1 and the application of the construction method in preparing and producing monoterpene products; x2, application of the construction method in producing monoterpenes; x3, application of the recombinant saccharomyces cerevisiae in preparing and producing monoterpene products; x4, application of the recombinant saccharomyces cerevisiae in production of monoterpenes; application of X5 and monoterpene synthase genes in preparing and producing eucalyptol products; application of X6 and monoterpene synthase genes in production of eucalyptol; application of X7 and monoterpene synthase in preparing eucalyptol products; application of X8 and monoterpene synthase in producing eucalyptol. Alternatively, in the above application, the monoterpene synthase gene encodes a monoterpene synthase having an amino acid sequence shown in (SEQ ID No. 10-13). For another example, the nucleotide sequence of the eucalypti synthase gene may be at positions 777-1940 of SEQ ID No. 6; 777-1898 of SEQ ID No. 7; 777-1898 of SEQ ID No. 8; SEQ ID No.9, positions 777-1910.
Cineole is an important component of many different plant essential oils, which is a monoterpene substance. The invention provides a new way for producing cineole by digging and screening cineole synthetase with the capability of synthesizing monoterpene. Meanwhile, further strengthening MVA path in Saccharomyces cerevisiae and introducing monoterpene synthase gene to construct recombinant Saccharomyces cerevisiae for producing eucalyptol; provides powerful enzyme and strain research foundation for the biosynthesis of monoterpenes, and has important significance for the production of monoterpenes.
Drawings
FIG. 1A flask-shaking fermentation of eucalyptol levels by different sources of monoterpene synthase Saccharomyces cerevisiae strains.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Data were processed using SPSS11.5 statistical software and experimental results were expressed as averages.
Saccharomyces cerevisiaeSaccharomyces cerevisiae) TIB H1 was preserved in the China general microbiological culture collection center (CGMCC for short, address: the collection registration number is CGMCC No.21000 in the microbiological institute of the national academy of sciences of China, north Chen West Lu No.1, no.3 in the Chaoyang area of Beijing city.
YPD medium, comprising per L volume of YPD medium: 20g peptone, 10g yeast extract, 20g glucose, 20g agar powder (YPD solid medium addition).
SD plates without uracil addition: SD medium without uracil addition per L volume contains: 8g Ura minus meida (Beijing Pankeno technologies Co., ltd.), 20g of agar powder, 20g of glucose.
SD plates containing 5-fluoroorotic acid, SD containing 5-fluoroorotic acid per L volume: 8g Ura minus meida (Beijing pantuno technology Co., ltd.), 20g of agar powder, 20g of glucose, 60mg of uracil, 1g of 5-fluoroorotic acid.
Delft liquid Medium, per L volume Delft liquid Medium comprising: 20g glucose, 7.5g ammonium sulfate, 0.5 g g magnesium sulfate heptahydrate, 14.4 g potassium dihydrogen phosphate, 2ml trace metal salt mother liquor (1L volume contains 3.0g ferric sulfate heptahydrate, 4.5g zinc sulfate heptahydrate, 4.5g calcium chloride dihydrate, 0.84g manganese chloride dihydrate, 0.3g cobalt chloride hexahydrate, 0.3g copper sulfate pentahydrate, 0.4g sodium molybdate dihydrate, 1.0g boric acid, 0.1g potassium iodide, 19.0g disodium ethylenediamine tetraacetate), 1ml vitamin mother liquor (1L volume contains 0.05g D-biotin, 1.0g D-pantothenic acid, 1.0g vitamin B1,1.0g pyridoxine, 1.0g nicotinic acid, 0.2g 4-amino benzoic acid, 25.0g inositol), 60mg uracil.
The information on the gene fragments and protein sequences in the examples described below is given in the following table.
TABLE 1 Gene fragment and protein sequence related information
Figure 632290DEST_PATH_IMAGE001
TABLE 2 protein related information
Genbank accession number Update time
tHMG1 protein AJS96703.1, bits 530-1054 2016/5/23
mvaE protein AAG02439.1 2000/8/29
mVAS-m protein Substitution of alanine at position 110 of AAG02438.1 with glycine 2000/8/29
ERG12 proteins NP_013935.1 2020/10/2
IDI1 protein NP_015208.1 2020/10/2
ERG19 proteins NP_014441.1 2020/10/2
ERG8 proteins NP_013947.1 2020/10/2
Example 1 preparation of target Gene
1. Acquisition of MVA pathway modification related genes
ERG20 F96W-N127W -T ERG20 、tHMG-T hmg1 、IDI1-T IDI1 、ERG8-T ERG8 、ERG12-T ERG12 、ERG19-T ERG19 Genes and Gal1, gal2, gal7, gal10 promoters, ADH, CYC1 terminators.
Extracting yeast genome DNA as a template, and amplifying by using primers required for gene amplification in Table 3 to obtain fragments conforming to expected sizes
An amplification system (TAKARA corporation) was configured using PrimSTARHS DNA polymerase, and the amplification system was: 5 XPS Buffer 10. Mu.L, dntp Mix 4. Mu.L, 1. Mu.L of each primer, 1. Mu.L of genomic DNA template, 0.5. Mu.L of HS polymerase (2.5U/. Mu.L), and distilled water to a total volume of 50. Mu.L. The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, extension at 72℃for 2.5 minutes (30 cycles); extension at 72℃for 10 min (1 cycle).
Table 3 shows primer sequences
Figure 549430DEST_PATH_IMAGE002
2. Eucalyptus synthase and mva-m, mvaE gene acquisition
Previous literature studies reported that cordyceps sinensis Ophiocordyceps sinensis contains terpene synthase for synthesizing secondary metabolites through whole genome sequencing, eight terpene synthases annotated as being derived from Ophiocordyceps sinensis are found in NCBI database, and through screening and verification, cins_OCO (GenBank: EQL03520.1, from cordyceps sinensis Ophiocordyceps sinensis, SEQ ID No. 10) obtained has the capability of synthesizing monoterpene substances, and based on comparison with cins_OCO amino acid sequences in the database, proteins OTA93495.1 and 61.02 which have higher homology with the same are mined; OTA69335.1, 61.56%; OTB07499.1, 58.98%; OTB14322.1, 56.18%; xp_033432897.1, 59.72%; KAH8598469.1, 52.34%; OJJ40615.1, 48.81%; xp_025402928.1, 46.7%; xp_035358724.1, 43.7%; OJI81974.1, 43.42%. Through gene synthesis and enzyme function verification, the CinS_HCO (GenBank: OTA93495.1, derived from Hypoxylon of Ledocarpus, 61.02%, SEQ ID No. 11), the CinS_HEC (GenBank: OTA69335.1, derived from Hypoxylon of Ledocarpus, 61.56%, SEQ ID No. 12) and the CinS_HCI (GenBank: OTB07499.1, derived from Hypoxylon of Ledocarpus, 58.98%, SEQ ID No. 13) have the capability of synthesizing monoterpenes.
The gene sequences of the above genes were artificially synthesized by the Souzhou Jin Weizhi biotechnology Co., ltd according to the methods of CinS_OCO, cinS_HCO, cinS_HEC, cinS_HCI, mvaE (GenBank: AAG02439.1, HMG-CoA reductase, derived from Enterococcus), mvaS-m (Genbank: AAG02438.1, position 110 alanine was replaced with glycine, HMG-CoA synthase, derived from Enterococcus), and codon optimization, and then the double-stranded DNA was transformed and cloned into cloning vector pET28a (Souzhou Jin Weizhi biotechnology Co., ltd.) to construct plasmids containing the above genes, respectively.
The primers required for gene amplification in Table 4 were amplified using the above plasmids as templates to obtain fragments of the desired size, and the products were recovered and stored by tapping. Thus obtained a CinS-OCO gene (represented by 777-1940 of SEQ ID No. 6), a CinS-HCO gene (represented by 777-1898 of SEQ ID No. 7), a CinS-HEC gene (represented by 777-1898 of SEQ ID No. 8), a CinS-HCI gene (represented by 777-1910 of SEQ ID No. 9) andmvaEgene (reverse complement of 270-2681 bits of SEQ ID No. 3) andmvaS-mgene (shown in positions 3358-4509 of SEQ ID No. 3).
Table 4 shows primer sequences
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3、P Gal1 -ERG20 F96W-N127W -T Eer20 Construction of expression elements
Fragment P obtained above was subjected to the above-described PCR technique using the Overlap PCR technique Gal1 ，ERG20 F96W-N127W -T Eer20 Ligation amplification was performed using PrimSTARHS DNA polymerase to prepare an amplification system (TAKARA Co.), which was: 5 XPS Buffer 10. Mu.L, dntp Mix 4. Mu.L, primer P Gal1 1. Mu.L each of-f and TErg20-r, 1. Mu.L of genomic DNA template (P Gal1 ，ERG20 F96W-N127W -T Eer20 The addition mole ratio of each segment is 1: 1) HS polymerase (2.5U/. Mu.L) 0.5. Mu.L, distilled water was added theretoThe total volume was 50. Mu.L. The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, extension at 72℃for 3 minutes (30 cycles); the mixture was extended at 72℃for 10 minutes (1 cycle), and the product was recovered and stored by tapping. Obtaining P Gal1 -ERG20 F96W-N127W -T Eer20 An expression element. P (P) Gal1 -ERG20 F96W-N127W -T Eer20 Expression element contains ERG20 F96W-N127W Expression cassette for expressing ERG20 F96W-N127W Gene, expression product is ERG20 F96W-N127W And (3) protein.
4、P Gal7 -tHMG1-T hmg1 Construction of expression elements
Fragment P obtained above was subjected to the above-described PCR technique using the Overlap PCR technique Gal7 ，tHMG1-T hmg1 Ligation amplification was performed using PrimSTARHS DNA polymerase to prepare an amplification system (TAKARA Co.), which was: 5 XPS Buffer 10. Mu.L, dntp Mix 4. Mu.L, primer P Gal7 1. Mu.L each of-f and Thmg1-r, 1. Mu.L of genomic DNA template (P) Gal7 ，tHMG-T hmg1 The addition mole ratio of each segment is 1: 1) HS polymerase (2.5U/. Mu.L) 0.5. Mu.L, distilled water was added to a total volume of 50. Mu.L. The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, extension at 72℃for 3 minutes (30 cycles); extending at 72 deg.c for 10 min (1 cycle), and recovering and storing the product to obtain P Gal7 -tHMG1-T hmg1 An expression element. P (P) Gal7 -tHMG1-T hmg1 The expression element contains a tHMG1 expression cassette which expresses the tHMG1 gene and the expression product is the tHMG1 protein.
4、T ADH -mvaE-P GAL1 -P GAL10 -mvaS-m-T CYC1 Construction of expression elements
Fragment T obtained above was subjected to the above-described PCR technique using the Overlap PCR technique ADH ，mvaE，P GAL1 ，P GAL10 ，mvaS-m，T CYC1 Ligation amplification was performed using PrimSTAR HS DNA polymerase to prepare an amplification system (TAKARA Co.), which was: 5 XPS Buffer 10. Mu.L, dntp Mix 4. Mu.L, primer T ADH1 -r and T CYC1 R 1. Mu.L each, 1. Mu.L of genomic DNA template (T ADH ，mvaE，P GAL1 ，P GAL10 ，mvaS-m，T CYC1 Molar ratio of each fragment 1:3:5:5:3: 1) HS polymerase (2.5U/. Mu.L) 0.5. Mu.L, distilled water was added to a total volume of 50. Mu.L. The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, extension at 72℃for 6 minutes (30 cycles); extending at 72 deg.C for 10 min (1 cycle), and recovering and storing the product by tapping to obtain T ADH -mvaE-P GAL1 -P GAL10 -mvaS-m-T CYC1 An expression element. T (T) ADH -mvaE-P GAL1 -P GAL10 -mvaS-m-T CYC1 The expression element comprises a mvaE expression cassette and a mvaS-m expression cassette. The mvaE expression cassette expresses mvaE gene and the expression product is mvaE protein. The mvaS-m expression cassette expresses mvaS-m gene and the expression product is mvaS-m protein.
5、T IDI1 -IDI1-P GAL1 -P GAL10 -ERG12-T ERG12 Construction of expression elements
The fragment IDI1-T obtained above was subjected to the above-described PCR technique using the overlay PCR IDI1 ，P GAL1 ，P GAL10 ，ERG12-T ERG12 Ligation amplification was performed using PrimSTAR HS DNA polymerase to prepare an amplification system (TAKARA Co.), which was: 5 XPS Buffer 10. Mu.L, dntp Mix 4. Mu.L, primer T IDI1 -r and T ERG20 1. Mu.L each of r, 1. Mu.L of genomic DNA template (IDI 1-T) IDI1 ，P GAL1 ，P GAL10 ，ERG12-T ERG12 Molar ratio of each fragment 1:3:3: 1) HS polymerase (2.5U/. Mu.L) 0.5. Mu.L, distilled water was added to a total volume of 50. Mu.L. The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, extension at 72℃for 6 minutes (30 cycles); extending at 72 deg.C for 10 min (1 cycle), and recovering and storing the product by tapping to obtain T IDI1 -IDI1-P GAL1 -P GAL10 -ERG12 –T ERG12 An expression element. T (T) IDI1 -IDI1-P GAL1 -P GAL10 -ERG12-T ERG12 The expression element contains an IDI1 expression cassette and an ERG12 expression cassette. The IDI1 expression cassette expresses IDI1 gene and the expression product is IDI1 protein. The ERG12 expression cassette expresses ERG12 gene and the expression product is ERG12 protein.
6、T ERG8 -ERG8-P GAL1 -P GAL10 -ERG19-T ERG19 Construction of expression elements
The fragment ERG8-T obtained above was amplified by using the overlay PCR technique ERG8 ，P Gal1 ，P Gal10 ，ERG19-T ERG19 Ligation amplification was performed using PrimSTAR HS DNA polymerase to prepare an amplification system (TAKARA Co.), which was: 5 XPS Buffer 10. Mu.L, dntp Mix 4. Mu.L, primer T ERG8 -r and T CYC1 1. Mu.L each of r, 1. Mu.L of genomic DNA template (ERG 8-T) ERG8 ，P GAL1 ，P GAL10 ，ERG19-T ERG19 Molar ratio of each fragment 1:3:3: 1) HS polymerase (2.5U/. Mu.L) 0.5. Mu.L, distilled water was added to a total volume of 50. Mu.L. The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, extension at 72℃for 6 minutes (30 cycles); extending at 72 deg.C for 10 min (1 cycle), and recovering and storing the product by tapping to obtain T ERG8 -ERG8-P GAL1 -P GAL10 -ERG19- T erg19 An expression element. T (T) ERG8 -ERG8-P GAL1 -P GAL10 -ERG19- T erg19 The expression element comprises an ERG8 expression cassette and an ERG19 expression cassette. The ERG8 expression cassette expresses ERG8 gene and the expression product is ERG8 protein. The ERG19 expression cassette expresses ERG19 gene and the expression product is ERG19 protein.
11. Construction of PGal 2-CinS-OCO-Tcyc 1 expression element
The fragments PGal2, cinS_OCO, tcyc1 obtained above were subjected to ligation amplification using the overlay PCR technique, and an amplification system (TAKARA Co.) was configured using PrimSTAR HS DNA polymerase, the amplification system being: 5 XPS Buffer 10. Mu.L, dntp Mix 4. Mu.L, primers PGal2-f and TCYC1-r 1. Mu.L each, genomic DNA template 1. Mu.L (PGal 2, cinS-OCO, tcyc1 each fragment molar ratio 1:3:1), HS polymerase (2.5U/. Mu.L) 0.5. Mu.L, distilled water was added to a total volume of 50. Mu.L. The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, extension at 72℃for 3 minutes (30 cycles); the product was recovered and stored by tapping at 72℃for 10 minutes (1 cycle), and the PGal 2-CinS-OCO-Tcyc 1 expression element was obtained. The PGal 2-CinS_OCO-Tcyc1 expression element contains a CinS_OCO expression cassette, expresses the CinS_OCO gene and the expression product is the CinS_OCO protein.
8、P Gal2 -CinS_HCO-T cyc1 Construction of expression elements
Fragment P obtained above was subjected to the above-described PCR technique using the Overlap PCR technique Gal2 ，CinS_HCO，T cyc1 Ligation amplification was performed using PrimSTAR HS DNA polymerase to prepare an amplification system (TAKARA Co.), which was: 5 XPS Buffer 10. Mu.L, dntp Mix 4. Mu.L, primers PGal2-f and TCYC1-r 1. Mu.L each, genomic DNA template 1. Mu.L (PGal 2, cinS-HCO, tcyc1 each fragment molar ratio 1:3:1), HS polymerase (2.5U/. Mu.L) 0.5. Mu.L, distilled water was added to a total volume of 50. Mu.L. The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, extension at 72℃for 3 minutes (30 cycles); extending at 72 deg.c for 10 min (1 cycle), and recovering and storing the product to obtain P Gal2 -CinS_HCO-T cyc1 An expression element. P (P) Gal2 -CinS_HCO-T cyc1 The expression element contains a CinS_HCO expression cassette, expresses the CinS_HCO gene and the expression product is the CinS_HCO protein.
9、P Gal2 -CinS_HEC-T cyc1 Construction of expression elements
Fragment P obtained above was subjected to the above-described PCR technique using the Overlap PCR technique Gal2 ，CinS_HEC，T cyc1 Ligation amplification was performed using PrimSTAR HS DNA polymerase to prepare an amplification system (TAKARA Co.), which was: 5 XPS Buffer 10. Mu.L, dntp Mix 4. Mu.L, primers PGal2-f and TCYC1-r 1. Mu.L each, genomic DNA template 1. Mu.L (PGal 2, cinS_HEC, tcyc1 each fragment molar ratio 1:3:1), HS polymerase (2.5U/. Mu.L) 0.5. Mu.L, distilled water was added to a total volume of 50. Mu.L. The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, extension at 72℃for 3 minutes (30 cycles); extending at 72 deg.c for 10 min (1 cycle), and recovering and storing the product to obtain P Gal2 -CinS_HEC-T cyc1 An expression element. P (P) Gal2 -CinS_HEC-T cyc1 The expression element contains a cins_hec expression cassette, expresses a cins_hec gene and the expression product is cins_hec protein.
10、P Gal2 - CinS_HCI -T cyc1 Construction of expression elements
By using OverlaThe fragment P obtained above was subjected to the P PCR technique Gal2 ，CinS_HCI，T cyc1 Ligation amplification was performed using PrimSTAR HS DNA polymerase to prepare an amplification system (TAKARA Co.), which was: 5 XPS Buffer 10. Mu.L, dntp Mix 4. Mu.L, primers PGal2-f and TCYC1-r 1. Mu.L each, genomic DNA template 1. Mu.L (PGal 2, cinS_HCI, tcyc1 each fragment molar ratio 1:3:1), HS polymerase (2.5U/. Mu.L) 0.5. Mu.L, distilled water was added to a total volume of 50. Mu.L. The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 55℃for 5 seconds, extension at 72℃for 3 minutes (30 cycles); extending at 72 deg.c for 10 min (1 cycle), and recovering and storing the product to obtain P Gal2 -CinS_HCI -T cyc1 An expression element. P (P) Gal2 -CinS_HCI -T cyc1 The expression element contains a CinS_HCI expression cassette, expresses the CinS_HCI gene and the expression product is the CinS_HCI protein.
Example 2 construction of recombinant bacteria
1. Preparation of Yeast competence
Single colonies of the starting bacteria were grown overnight at 30℃and 250rpm in YPD medium and the cell densities of the overnight cultures were counted to give final OD 600nm Cell turbidity of 0.1 was inoculated with 20ml of YPD medium. Culturing at 30℃and 250rpm to OD 600nm 0.8. Cells were collected by centrifugation at 2500rpm for 5 minutes using a sterile centrifuge tube. The culture medium was discarded, and the cells were suspended in sterile water and centrifuged as above. Discarding water, suspending cells in 1mL of 100mM lithium acetate, transferring the suspension to a sterile centrifuge tube; precipitating cells at a high speed for a short time, and removing lithium acetate; cells were suspended in 4-fold system of 100mM lithium acetate and sub-packaged to obtain competent cells.
The construction principle of the recombinant strain is that an expression element capable of expressing Cas9 protein is transferred into a strain TIB H1 in advance, then recombinant plasmids (plasmids 1-6) expressing gRNA are transformed into the strain together with the expression element or homologous recombinant fragments, the recombinant plasmids expressing the gRNA recognize and bind specific PAM regions of corresponding sites, simultaneously activate and guide the Cas9 protein to perform a shearing function, double-strand DNA of the corresponding sites is broken, and at the moment, the expression element or the homologous recombinant fragments containing the homologous regions are integrated into the strain DNA through homologous recombination repair.
2. Construction of recombinant plasmids containing gRNA
Construction of plasmid pBGR: amplifying a fragment 1 of the AmpR expression frame and ori fragment of the lozenge segment by using a primer 1 and a primer 3 by using a pET32a vector as a template, and amplifying a fragment Ura3 expression box by using a primer 8 and a primer 9 by using a saccharomyces cerevisiae TIB H1 genome as a template; the gRNA expression cassette 1 was amplified with primers 4 and 5, the gRNA expression cassette 2 with primers 10 and 11, the 2. Mu. Ori with primers 6 and 7, and the fragment 2 with primers 4 and 10 using overlap PCR with the gRNA expression cassette 1, the fragment of gRNA expression cassette 2, 2. Mu. Ori with use of overlap PCR with use of DiCarlo JE, norville JE, mali P, et al Genome engineering in Saccharomyces cerevisiae using CRISPR-Cassystems Nucleic Acids Res 2013b; 41:4336-43) as template; fragment 1 and fragment 2 were recombined in vitro using the Cloneexpress II kit (Norflu), transformed into DH 5. Alpha. Competent cells for expansion, and plasmids were extracted to obtain plasmid pBGR.
Plasmid 1: the plasmid pBGR is used as a template, the primers gRNA1-f and gRNA1-r are used for amplifying the gene fragment gRNA1, the primer pBGR-2-f is used for amplifying the gene fragment pBGR-2, and the gene fragments gRNA1 and pBGR-2 obtained above are recombined by using a Cloneexpress II kit (Norpran) to obtain the plasmid 1. Plasmid 1 expresses gRNA1 with target sequence tgaaactctaatcctactat, tagaaacgcggacacaggag.
Plasmid 2: the gene fragment gRNA2 is amplified by using pBGR as a template and the primer gRNA2-f, the gene fragment pBGR-2 is amplified by using the primer pBGR-2-f, and the gene fragments gRNA2 and pBGR-2 obtained above are recombined by using Cloneexpress II kit (Norpran) to obtain the plasmid 2. Plasmid 2 expresses gRNA2 with target sequence gcaatgcgatgttagtttag.
Plasmid 3: the gene fragment gRNA3 and pBGR-2 obtained above are recombined by using a Cloneexpress II kit (Norpran) to obtain a plasmid 3 by using pBGR as a template, using a primer gRNA3-f to amplify the gene fragment gRNA3 and using a primer pBGR-2-f to amplify the gene fragment pBGR-2. Plasmid 3 expresses gRNA3 with target sequence cgccattcaagagcagcaac.
Plasmid 4: the gene fragment gRNA4 and pBGR-2 obtained above are recombined by using a Cloneexpress II kit (Noruzan) to obtain a plasmid 4 by using pBGR as a template, using a primer gRNA4-f to amplify the gene fragment gRNA4 and using a primer pBGR-2-f to amplify the gene fragment pBGR-2. Plasmid 4 expresses gRNA4 with target sequence ttgtcacagtgtcacatcag.
Plasmid 5: the gene fragment gRNA6 and pBGR-2 obtained above are recombined by using pBGR as a template, the primer gRNA6-f and the primer pBGR-2-f to amplify the gene fragment pBGR-2, and a Cloneexpress II kit (Norpran) to obtain the plasmid 6. Plasmid 6 expresses gRNA6 with target sequence atatgtctctaattttggaa.
The primers used above are shown in Table 5.
TABLE 5 primer sequences
Figure 193218DEST_PATH_IMAGE005
3. Construction of GPP-1 Strain
The preparation method comprises the steps of preparing competent cells after the saccharomyces cerevisiae TIB H1 serving as a starting strain is cultured overnight in YPD liquid medium, and adding the competent cells according to the following sequence: 240. Mu.L PEG (50% w/v), 36. Mu.L 1.0 mol/L lithium acetate, 25. Mu.L salmon sperm DNA (sigma) DNA (2 mg/mL), 50. Mu.L water and gene (P) Gal1 -ERG20 F96W-N127W -T Erg20 Expression element, P Gal7 -tHMG1-T hmg1 Expression element, plasmid 1); shaking vigorously until the cells are completely mixed, standing at 30deg.C for 30 min, and standing in 4deg.C water bath for 20 min; centrifuging at 6000-8000rpm for 15s, and removing the conversion mixed solution; 100. Mu.L of sterile water was pipetted into the reaction tube, precipitated by gentle suspension and plated on SD plates without uracil addition, colonies were picked up and colonies were picked up on SD plates containing 5-fluoroorotic acid, and the colonies grown on the plates were designated GPP-1 and stored.
4. Construction of GPP-2 strains
The preparation method comprises the steps of preparing competent cells after the saccharomyces cerevisiae GPP-1 serving as a starting strain is cultured overnight in YPD liquid medium, and adding the competent cells according to the following sequence: 240. Mu.L PEG (50% w/v), 36. Mu.L 1.0 mol/L lithium acetate, 25. Mu.L salmon sperm DNA (sigma) (2 mg/mL), 50. Mu.L water and gene (T) ADH -mvaE-P GAL1 -P GAL10 -mvaS-m-T CYC1 Expression element, plasmid 2); shaking vigorously until the cells are completely mixed, standing at 30deg.C for 30 min, and standing in 4deg.C water bath for 20 min; centrifuging at 6000-8000rpm for 15s, and removing the conversion mixed solution; 100. Mu.L of sterile water was pipetted into the reaction tube, precipitated by gentle suspension and plated on SD plates without uracil addition, colonies were picked up and colonies were picked up on SD plates containing 5-fluoroorotic acid, and the colonies grown on the plates were designated GPP-2 and stored.
5. Construction of GPP-3 Strain
The preparation method comprises the steps of preparing competent cells after the saccharomyces cerevisiae GPP-2 serving as a starting strain is cultured overnight in YPD liquid medium, and adding the competent cells according to the following sequence: 240. Mu.L PEG (50% w/v), 36. Mu.L 1.0 mol/L lithium acetate, 25. Mu.L salmon sperm DNA (sigma) (2 mg/mL), 50. Mu.L water and gene (T) IDI1 -IDI1-P GAL1 -P GAL10 -ERG12-T ERG12 Expression element, plasmid 3); shaking vigorously until the cells are completely mixed, standing at 30deg.C for 30 min, and standing in 4deg.C water bath for 20 min; centrifuging at 6000-8000rpm for 15s, and removing the conversion mixed solution; 100. Mu.L of sterile water was pipetted into the reaction tube, precipitated by gentle suspension and plated on SD plates without uracil addition, colonies were picked up and colonies were picked up on SD plates containing 5-fluoroorotic acid, and the colonies grown on the plates were designated GPP-3 and stored.
6. Construction of GPP-4 Strain
The preparation method comprises the steps of preparing competent cells after the saccharomyces cerevisiae GPP-3 serving as a starting strain is cultured overnight in YPD liquid medium, and adding the competent cells according to the following sequence: 240. Mu.L PEG (50% w/v), 36. Mu.L 1.0 mol/L lithium acetate, 25. Mu.L salmon sperm DNA (sigma) (2 mg/mL), 50. Mu.L water and gene (T) ERG8 -ERG8-P GAL1 -P GAL10 -ERG19-T ERG19 Expression element, plasmid 4); shaking vigorously until the cells are completely mixed, standing at 30deg.C for 30 min, and standing in 4deg.C water bath for 20 min; centrifuging at 6000-8000rpm for 15s, and removing the conversion mixed solution; 100. Mu.L of sterile water was pipetted into the reaction tube, precipitated by gentle suspension and plated on SD plates without uracil addition, colonies were picked up and colonies were picked up on SD plates containing 5-fluoroorotic acid, and the colonies grown on plates were designated GPP-4 and stored.
7. Construction of SQ-1 Strain
The preparation method comprises the steps of preparing competent cells after the saccharomyces cerevisiae GPP-4 serving as a starting strain is cultured overnight in YPD liquid medium, and adding the competent cells according to the following sequence: 240. Mu.L PEG (50% w/v), 36. Mu.L 1.0 mol/L lithium acetate, 25. Mu.L salmon sperm DNA (sigma) (2 mg/mL), 50. Mu.L water and gene (P) Gal2 - CinS_OCO -T cyc1 Expression element, plasmid 5); shaking vigorously until the cells are completely mixed, standing at 30deg.C for 30 min, and standing in 4deg.C water bath for 20 min; centrifuging at 6000-8000rpm for 15s, and removing the conversion mixed solution; 100. Mu.L of sterile water was pipetted into the reaction tube, the pellet was gently suspended and plated on SD plates without uracil addition, colonies were picked up and colonies were picked up on SD plates containing 5-fluoroorotic acid, and the colonies grown on the plates were designated SQ-1 and stored.
8. Construction of SQ-2 Strain
The preparation method comprises the steps of preparing competent cells after the saccharomyces cerevisiae GPP-4 serving as a starting strain is cultured overnight in YPD liquid medium, and adding the competent cells according to the following sequence: 240. Mu.L PEG (50% w/v), 36. Mu.L 1.0 mol/L lithium acetate, 25. Mu.L salmon sperm DNA (sigma) (2 mg/mL), 50. Mu.L water and gene (P) Gal2 - CinS_HCO -T cyc1 Expression element, plasmid 5); shaking vigorously until the cells are completely mixed, standing at 30deg.C for 30 min, and standing in 4deg.C water bath for 20 min; centrifuging at 6000-8000rpm for 15s, and removing the conversion mixed solution; 100. Mu.L of sterile water was pipetted into the reaction tube, the pellet was gently suspended and plated on SD plates without uracil addition, colonies were picked up and colonies were picked up on SD plates containing 5-fluoroorotic acid, and the colonies grown on plates were designated SQ-2 and stored.
9. Construction of SQ-3 Strain
The preparation method comprises the steps of preparing competent cells after the saccharomyces cerevisiae GPP-4 serving as a starting strain is cultured overnight in YPD liquid medium, and adding the competent cells according to the following sequence: 240. Mu.L PEG (50% w/v), 36. Mu.L 1.0 mol/L lithium acetate, 25. Mu.L salmon sperm DNA (sigma) (2 mg/mL), 50. Mu.L water and gene (P) Gal2 -CinS_HEC -T cyc1 Expression element, plasmid 5); shaking vigorously until the cells are completely mixed, standing at 30deg.C for 30 min, and standing in 4deg.C water bath for 20 min; centrifuging at 6000-8000rpm for 15s, and removing the conversion mixed solution; 100. Mu.L of sterile water was pipetted into the reaction tube, precipitated by gentle suspension and plated on SD plates without uracil addition, colonies were picked up until they had grown and colonies were 5-foldSD plates of fluoroorotic acid, in which the grown colonies were designated SQ-3 and stored.
10. Construction of SQ-4 Strain
The preparation method comprises the steps of preparing competent cells after the saccharomyces cerevisiae GPP-4 serving as a starting strain is cultured overnight in YPD liquid medium, and adding the competent cells according to the following sequence: 240. Mu.L PEG (50% w/v), 36. Mu.L 1.0 mol/L lithium acetate, 25. Mu.L salmon sperm DNA (sigma) (2 mg/mL), 50. Mu.L water and gene (P) Gal2 -CinS_HCI -T cyc1 Expression element, plasmid 5); shaking vigorously until the cells are completely mixed, standing at 30deg.C for 30 min, and standing in 4deg.C water bath for 20 min; centrifuging at 6000-8000rpm for 15s, and removing the conversion mixed solution; 100. Mu.L of sterile water was pipetted into the reaction tube, the pellet was gently suspended and plated on SD plates without uracil addition, colonies were picked up and colonies were picked up on SD plates containing 5-fluoroorotic acid, and the colonies grown on plates were designated SQ-4 and stored.
Example 3 use of recombinant bacteria in the production of monoterpenes
1. Engineering bacteria culture and product extraction
The yeast engineering strains SLQ-1, SLQ-2, SLQ-3 and SLQ-4 prepared in example 2 were activated in Delft liquid medium, seed solution (30 ℃,250rpm,16 h) was prepared in Delft liquid medium, inoculated in an inoculum size of 1% into a 100mL triangular flask containing 20mL of liquid medium and 2mL of sec-butylbenzene n-dodecane, cultured at 30 ℃,250rpm for 2-3 days, finally, the liquid in the triangular flask was transferred to a 50mL centrifuge tube, centrifuged at 5000rpm for 5min, and the organic phase was collected for use.
2. Qualitative and quantitative analysis of monoterpene substances produced by thalli
The organic phase material collected in step 1 was diluted 50-fold with n-hexane and detected by GC-MS. GC-MS measurement conditions: the temperature of the sample inlet is 260 ℃, the sample inlet volume is 1 mu L, no flow division is performed, and the solvent is delayed for 3min; chromatographic column: HP-5ms (30 m.0.25 mM); chromatographic conditions: preserving heat at 60 ℃,3min,40 ℃/min to 150 ℃,20 ℃/min to 220 ℃,40 ℃/min to 260 ℃ for 2min; MS conditions: full Scan 50-750 amu. Qualitative and quantitative analysis was performed with mixed standards of different monoterpene substances.
As a result, as shown in FIG. 1, the yield of each engineering bacterium was as follows when fermented for 3 days:
the eucalyptol yields of SQ-1, SQ-2, SQ-3, SQ-4 were 134.3+ -3.14 mg/L, 154.3+ -11.81 mg/L, 153.3+ -2.32 mg/L, 148.1+ -6.39 mg/L, respectively. Besides the main production of eucalyptol, the production of alpha-pinene, sabinene, myrcene, carene, terpinolene, ocimene and alpha-terpineol monoterpene substances is also carried out.
This example demonstrates that expression of a monoterpene synthase gene in Saccharomyces cerevisiae can produce eucalyptol a-pinene, sabinene, myrcene, carene, terpinolene, ocimene, a-terpineol.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (10)

1. A construction method of recombinant saccharomyces cerevisiae producing monoterpene substances is characterized by comprising the following steps: comprising the following steps: the coding gene of the monoterpene synthase is introduced into saccharomyces cerevisiae to obtain recombinant saccharomyces cerevisiae producing eucalyptol, and the amino acid sequence of the monoterpene synthase coded by the coding gene of the monoterpene synthase is shown as SEQ ID No.10, 11, 12 or 13, or has at least 70 percent of identity, preferably at least 80 percent of identity, at least 90 percent of identity, at least 95 percent of identity, more preferably at least 99 percent of identity with the nucleotide sequence shown as SEQ ID No.10, 11, 12 or 13, and still has the functions.
2. The method of claim, wherein: the monoterpene synthetase gene is derived from Cordyceps sinensis (Ophiocordyceps sinensis) and Tuber (Hypoxylon).
3. The construction method according to claim 1 or 2, characterized in that: the starting strain of the saccharomyces cerevisiae is the saccharomyces cerevisiae capable of accumulating monoterpene synthesis precursor geranyl diphosphate GPP.
4. A method of construction according to claim 3, wherein: the starting strain of the saccharomyces cerevisiae also comprises modification for improving the content and/or activity of farnesyl pyrophosphate synthetase ERG20, 3-hydroxy-3-methylglutaryl coenzyme A tHMG1, acetyl coenzyme A acyltransferase mvaE, HMG-CoA synthetase mvaS-m, isopentenyl pyrophosphate isomerase IDI1, mevalonate kinase ERG12, MVAP kinase ERG8 and/or MVAPP decarboxylase ERG19 in the starting saccharomyces cerevisiae.
5. The construction method according to claim 4, wherein: the saccharomyces cerevisiae starting strain is subjected to at least one of the following transformation to obtain the saccharomyces cerevisiae:
a1, introducing a farnesyl pyrophosphoric acid synthetase gene 96-site and 127-site double-point mutant gene ERG20 F96W/N127W A gene;
a2, introducing a tHMG1 gene;
a3, introducing mvaE genes;
a4, introducing mvaS-m genes;
a5, introducing IDI1 gene;
a6, introducing ERG12 genes;
a7, introducing ERG8 genes;
a8, ERG19 gene is introduced.
6. Recombinant s.cerevisiae constructed by the construction method according to any one of claims 1 to 5.
7. A method for producing a monoterpene, characterized by: comprising culturing the recombinant Saccharomyces cerevisiae of claim 6 to obtain a fermentation product, collecting eucalyptol, or a byproduct thereof, said byproduct being a-pinene, sabinene, myrcene, carene, terpinolene, ocimene, and/or a-terpineol;
preferably, the temperature of the culture is 25-35 ℃; the culture time is 24-72 hours;
more preferably, the culturing is performed under stirring or shaking conditions, for example shaking at 100-800rpm;
further preferably, an extractant is added to the medium; the extractant is selected from: n-dodecane, butyl oleate, dioctyl phthalate, dibutyl phthalate, isopropyl myristate; particularly preferably, the content of the additive extractant is 10 to 30%.
8. Use of a monoterpene synthase and its encoding gene for the preparation of a monoterpene, characterized in that the amino acid sequence of the monoterpene synthase is shown as SEQ ID No.10, 11, 12 or 13 or has at least 70% identity, preferably at least 80% identity, at least 90% identity, at least 95% identity, more preferably at least 99% identity with the nucleotide sequence shown as SEQ ID No.10, 11, 12 or 13, still having said function; more preferably, the gene encoding the monoterpene synthase is derived from Cordyceps sinensis (Ophiocordyceps sinensis), and Tuber (Hypoxylon).
9. The use according to claim 8, wherein the monoterpene is eucalyptol.
10. An application, characterized by any one of the following applications:
use of X1, the method of any one of claims 1-5 for the preparation of a product for the production of eucalyptol;
use of X2, the method of any one of claims 1-5 for the production of eucalyptol;
use of the recombinant s.cerevisiae according to claim 6 for the production of eucalyptol products;
use of X4, the recombinant saccharomyces cerevisiae of claim 6 for the production of eucalyptol.
CN202211340237.5A 2022-10-27 2022-10-27 Monoterpene synthase, genetically engineered strain and application thereof Pending CN116064639A (en)

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