Construction and application of (-)-carvone strain for positioned biosynthesis by utilizing saccharomyces cerevisiae and peroxisome

Abstract

The invention relates to a strain for positioning biosynthesis of (-)-carvone by using saccharomyces cerevisiae and peroxisome and application of the strain, and relates to the technical field of biology. The strain is prepared by the following steps: (1) carrying out fusion expression on GPP synthetase (ERG20WW) and (-)-limonene synthetase (MsLimS), and co-expressing NPP enzyme (SlNDPS1); (2) performing fusion expression on CYP71D18 and ATR2, and performing overexpression on an ISPD gene; and (3) the enzyme is localized and expressed on peroxisome, and a peroxisome localized synthesized (-)-carvone strain is constructed. The strain provided by the invention not only realizes biosynthesis of carvone in saccharomyces cerevisiae, but also greatly improves the fermentation yield of (-)-carvone and intermediate products (-)-limonene and (-)-carvol thereof through a peroxisome isolation effect, so that a foundation is laid for industrial production of synthesis of carvone by a fermentation method.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and application of a strain for biosynthesizing (-)-carvone by using saccharomyces cerevisiae and peroxisome. Background Art

[0002] Carvone is a monoterpene ketone compound, which is mainly found in aromatic and medicinal plants such as Lamiaceae and Asteraceae. In nature, carvone exists in two enantiomers, (-)-carvone and (-)-carvone, which have different aromas. (-)-carvone has a minty aroma, while (-)-carvone has a parsley / dill flavor. Carvone has multiple physiological activities such as antiviral activity, antibacterial activity, antifungal activity, antiparasitic activity, antineuraminidase activity, antioxidant activity, anti-inflammatory activity and anticancer activity. At the same time, it is also the starting material for the synthesis of many natural products and has a variety of pharmacological and industrial application prospects. Carvone has a wide range of uses in food, cosmetics, medicine, agriculture and other aspects. At present, carvone in the market mainly comes from plant extraction and chemical synthesis. Extraction from plants is affected by climate, origin and other environmental factors, and the cost is relatively high. The chemical synthesis method may cause serious environmental pollution during the production process. Therefore, using the strategies of synthetic biology and metabolic engineering, carvone was synthesized in Saccharomyces cerevisiae, providing a new production method for the source of carvone.

[0003] At present, the biological synthesis of carvone mainly adopts the whole-cell catalysis method, and there is no example of de novo synthesis of carvone in microbial cells. Summary of the invention

[0004] The technical problem to be solved by the present invention is to use Saccharomyces cerevisiae as a chassis cell combination to synthesize (-)-carvone by peroxisome biosynthesis. The technical problem to be solved is not limited to the technical subject described, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] To achieve the above purpose, the technical solution adopted by the present invention mainly includes:

[0007] The invention provides a recombinant bacterium, wherein the recombinant bacterium (an engineered bacterium) can synthesize (-)-carvone using (-)-limonene as a raw material.

[0008] Furthermore, the following metabolic reactions exist in the recombinant bacteria:

[0009] (A1) converting (-)-limonene into (-)-trans-carveol;

[0010] (A2) Conversion of (-)-trans-carveol to (-)-carvone.

[0011] Furthermore, the recombinant bacteria also have metabolic reactions (A3) and / or (A4):

[0012] (A3) converting isopentenyl pyrophosphate (IPP) into geranyl pyrophosphate (GPP), and then converting geranyl pyrophosphate (GPP) into (-)-limonene;

[0013] (A4) Converting dimethylallyl pyrophosphate (DMAPP) into neryl pyrophosphate (NPP), and then converting neryl pyrophosphate (NPP) into (-)-limonene.

[0014] Furthermore, the recombinant bacteria also have metabolic reactions (A5) and / or (A6):

[0015] (A5) converting 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) into mevalonate;

[0016] (A6) converting isopentenyl pyrophosphate (IPP) into dimethylallyl pyrophosphate (DMAPP) or converting dimethylallyl pyrophosphate (DMAPP) into isopentenyl pyrophosphate (IPP).

[0017] The mevalonate can generate isopentenyl pyrophosphate (IPP) under the action of ERG12, ERG8 and ERG19.

[0018] Furthermore, the specific pathways in the recombinant bacteria are as follows:

[0019] Said (A1) is catalyzed by cytochrome P450 limonene-6-hydroxylase (CYP71D18) and cytochrome P450 reductase (ATR2);

[0020] Said (A2) is catalyzed by carveol dehydrogenase (ISPD);

[0021] In (A3), isopentenyl pyrophosphate (IPP) is converted into geranyl pyrophosphate (GPP) by GPP synthase (ERG20 WW ) catalyzes the conversion of geranyl pyrophosphate (GPP) into (-)-limonene catalyzed by (-)-limonene synthase (MsLimS);

[0022] In said (A4), the conversion of dimethylallyl pyrophosphate (DMAPP) into NPP is catalyzed by orange acyl diphosphate synthase (SlNDPS1), and the conversion of NPP into (-)-limonene is catalyzed by (-)-limonene synthase (MsLimS);

[0023] The (A5) is catalyzed by 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1);

[0024] The (A6) is catalyzed by isoprene pyrophosphate isomerase (IDI1).

[0025] Furthermore, the recombinant bacteria is recombinant bacteria R1, recombinant bacteria R2 or recombinant bacteria R3;

[0026] The recombinant bacterium R1 has the following characteristics B1)-B3):

[0027] B1) containing cytochrome P450 limonene-6-hydroxylase (CYP71D18) gene or / and containing substances that regulate the expression of cytochrome P450 limonene-6-hydroxylase (CYP71D18) gene or / and containing substances that regulate the activity or content of cytochrome P450 limonene-6-hydroxylase (CYP71D18);

[0028] B2) containing cytochrome P450 reductase (ATR2) gene or / and containing substances that regulate the expression of cytochrome P450 reductase (ATR2) gene or / and containing substances that regulate the activity or content of cytochrome P450 reductase (ATR2);

[0029] B3) containing carveol dehydrogenase (ISPD) gene or / and containing substances that regulate the expression of carveol dehydrogenase (ISPD) gene or / and containing substances that regulate the activity or content of carveol dehydrogenase (ISPD).

[0030] In addition to the characteristics of B1) to B3), the recombinant bacterium R2 also has the following characteristics B4) and / or B5):

[0031] B4) contains GPP synthase (ERG20 WW ) gene or / and the gene that regulates GPP synthase (ERG20 WW ) gene expression or / and substances that regulate GPP synthase (ERG20 WW ) activity or content; containing (-)-limonene synthase (MsLimS) gene or / and containing substances that regulate the expression of (-)-limonene synthase (MsLimS) gene or / and containing substances that regulate the activity or content of (-)-limonene synthase (MsLimS);

[0032] B5) containing the orange acyl diphosphate synthase (SlNDPS1) gene or / and containing substances that regulate the expression of the orange acyl diphosphate synthase (SlNDPS1) gene or / and containing substances that regulate the activity or content of orange acyl diphosphate synthase (SlNDPS1); containing the (-)-limonene synthase (MsLimS) gene or / and containing substances that regulate the expression of the (-)-limonene synthase (MsLimS) gene or / and containing substances that regulate the activity or content of (-)-limonene synthase (MsLimS);

[0033] In addition to the characteristics of the recombinant bacterium R2, the recombinant bacterium R3 also has the following characteristics B6) and / or B7):

[0034] B6) containing 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1) gene or / and containing substances that regulate the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1) gene or / and containing substances that regulate the activity or content of 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1);

[0035] B7) contains an isoprene pyrophosphate isomerase (IDI1) gene or / and contains a substance that regulates the expression of the isoprene pyrophosphate isomerase (IDI1) gene or / and contains a substance that regulates the activity or content of isoprene pyrophosphate isomerase (IDI1).

[0036] The substance contains P450 limonene-6-hydroxylase (CYP71D18), cytochrome P450 reductase (ATR2), carveol dehydrogenase (ISPD), GPP synthase (ERG20 WW ), acyl diphosphate synthase (SlNDPS1), orange acyl diphosphate synthase (SlNDPS1), 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1) and isoprene pyrophosphate isomerase (IDI1) encoding one or more enzymes.

[0037] The yeast may be Saccharomyces cerevisiae. The Pichia pastoris may be Saccharomyces cerevisiae BY4742.

[0038] The cytochrome P450 limonene-6-hydroxylase (CYP71D18) and cytochrome P450 reductase (ATR2) were from spearmint, and the carveol dehydrogenase (ISPD) was from peppermint.

[0039] The (-)-limonene synthase MsLimS is derived from lemon (Citrus lemon), the NPP synthase SlNDPS1 is derived from tomato (Solanum lycopersicum), the cytochrome P450 limonene-6-hydroxylase gene CYP71D18 is derived from spearmint (Mentha spicata), the cytochrome P450 reductase ATR2 is derived from Arabidopsis thaliana, and the carveol dehydrogenase gene ISPD is derived from mint.

[0040] The chassis cells for constructing the recombinant bacteria include but are not limited to Saccharomyces cerevisiae strains, and all other proteins that can be expressed by expression systems such as bacteria, yeast, and cells are included in the application scope of the present invention.

[0041] Furthermore, the recombinant bacteria is a recombinant yeast, and the recombinant yeast is prepared by performing the following operations on the yeast as the recipient bacteria:

[0042] C1) introduction of cytochrome P450 limonene-6-hydroxylase (CYP71D18) gene or / and introduction of substances that regulate the expression of cytochrome P450 limonene-6-hydroxylase (CYP71D18) gene or / and introduction of substances that regulate the activity or content of cytochrome P450 limonene-6-hydroxylase (CYP71D18);

[0043] C2) introducing a cytochrome P450 reductase (ATR2) gene or / and introducing a substance that regulates the expression of the cytochrome P450 reductase (ATR2) gene or / and introducing a substance that regulates the activity or content of the cytochrome P450 reductase (ATR2);

[0044] C3) introducing a carveol dehydrogenase (ISPD) gene or / and introducing a substance that regulates the expression of the carveol dehydrogenase (ISPD) gene or / and introducing a substance that regulates the activity or content of carveol dehydrogenase (ISPD);

[0045] C4) to introduce GPP synthase (ERG20 WW ) gene or / and the introduction of regulatory GPP synthase (ERG20 WW ) gene expression or / and the introduction of substances regulating GPP synthase (ERG20 WW ) activity or content of a substance;

[0046] C5) introducing a (-)-limonene synthase (MsLimS) gene or / and introducing a substance that regulates the expression of the (-)-limonene synthase (MsLimS) gene or / and introducing a substance that regulates the activity or content of the (-)-limonene synthase (MsLimS);

[0047] C6) introducing a gene for orange acyl diphosphate synthase (SlNDPS1) or / and introducing a substance that regulates the expression of the gene for orange acyl diphosphate synthase (SlNDPS1) or / and introducing a substance that regulates the activity or content of orange acyl diphosphate synthase (SlNDPS1);

[0048] C7) introducing a 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1) gene or / and introducing a substance that regulates the expression of the 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1) gene or / and introducing a substance that regulates the activity or content of 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1);

[0049] C8) Introduction of an isoprene pyrophosphate isomerase (IDI1) gene or / and introduction of a substance that regulates the expression of the isoprene pyrophosphate isomerase (IDI1) gene or / and introduction of a substance that regulates the activity or content of isoprene pyrophosphate isomerase (IDI1).

[0050] Furthermore, the protein information in the above recombinant bacteria is as follows

[0051] M1-1) amino acid sequence is a protein consisting of 527 amino acids encoded by SEQ ID No. 1 (3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1));

[0052] M1-2) a protein derived from M1-1) or having 80% or more identity with the protein shown in M1-1) obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 527 amino acids encoded by SEQ ID No. 1;

[0053] M1-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M1-1) or M1-2);

[0054] M2) The isoprene pyrophosphate isomerase (IDI1) is any one of the following:

[0055] M2-1) amino acid sequence is a protein consisting of 288 amino acids encoded by SEQ ID No. 2;

[0056] M2-2) a protein derived from M2-1) or having 80% or more identity with the protein shown in M2-1) obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence of the protein consisting of 288 amino acids encoded by SEQ ID No. 2;

[0057] M2-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M2-1) or M2-2);

[0058] M3) The (-)-limonene synthase (MsLimS) is any one of the following:

[0059] M3-1) The amino acid sequence is a protein consisting of 544 amino acids encoded by SEQ ID No. 3;

[0060] M3-2) a protein having the same function as (-)-limonene synthase (MsLimS) obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 544 amino acids encoded by SEQ ID No. 3, and derived from M3-1) or having 80% or more identity with the protein shown in M3-1);

[0061] M3-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M3-1) or M3-2);

[0062] M4) GPP synthase (ERG20 WW ) is any of the following:

[0063] M4-1) amino acid sequence is a protein consisting of 352 amino acids encoded by SEQ ID No. 4;

[0064] M4-2) The amino acid sequence of the protein consisting of three amino acids encoded by SEQ ID No. 4 is substituted and / or deleted and / or added to obtain a protein having the ability to bind to GPP synthase (ERG20 WW ) a protein having the same function as that derived from M4-1) or having more than 80% identity with the protein shown in M4-1);

[0065] M4-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M4-1) or M4-2);

[0066] M5) The orange acyl diphosphate synthase (S1NDPS1) is any one of the following:

[0067] M5-1) The amino acid sequence is a protein consisting of 260 amino acids encoded by SEQ ID No.5;

[0068] M5-2) a protein having the same function as orange acyl diphosphate synthase (S1NDPS1) obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 260 amino acids encoded by SEQ ID No.5, and derived from M5-1) or having 80% or more identity with the protein shown in M5-1);

[0069] M5-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M5-1) or M5-2);

[0070] M6) The cytochrome P450 limonene-6-hydroxylase (CYP71D18) is any one of the following:

[0071] M6-1) The amino acid sequence is a protein consisting of 496 amino acids encoded by SEQ ID No.6;

[0072] M6-2) a protein derived from M6-1) or having 80% or more identity with the protein shown in M6-1) obtained by replacing and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 496 amino acids encoded by SEQ ID No. 6, and having the same function as cytochrome P450 limonene-6-hydroxylase (CYP71D18);

[0073] M6-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M6-1) or M6-2);

[0074] M7) The cytochrome P450 reductase (ATR2) is any one of the following:

[0075] M7-1) The amino acid sequence is a protein consisting of 646 amino acids encoded by SEQ ID No.7;

[0076] M7-2) a protein having the same function as cytochrome P450 reductase (ATR2) obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 646 amino acids encoded by SEQ ID No. 7, and derived from M7-1) or having 80% or more identity with the protein shown in M7-1);

[0077] M7-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M7-1) or M7-2);

[0078] M8) The carveol dehydrogenase (ISPD) is any one of the following:

[0079] M8-1) The amino acid sequence is a protein consisting of 265 amino acids encoded by SEQ ID No. 8;

[0080] M8-2) a protein having the same function as carveol dehydrogenase (ISPD) obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 265 amino acids encoded by SEQ ID No. 8, and derived from M8-1) or having 80% or more identity with the protein shown in M8-1);

[0081] M8-3) A fusion protein having the same function is obtained by connecting a tag to the N-terminus and / or C-terminus of M8-1) or M8-2).

[0082] The gene encoding the 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1) may be SEQ ID No. 1, the gene encoding the isoprene pyrophosphate isomerase (IDI1) may be SEQ ID No. 2, the gene encoding the (-)-limonene synthase (MsLimS) may be SEQ ID No. 3, the gene encoding the GPP synthase (ERG20 WW ) may be SEQ ID No.4, the coding gene of the orange acyl diphosphate synthase (SlNDPS1) may be SEQ ID No.5, the coding gene of the cytochrome P450 limonene-6-hydroxylase (CYP71D18) may be SEQ ID No.6, the coding gene of the cytochrome P450 reductase (ATR2) may be SEQ ID No.7, and the coding gene of the carveol dehydrogenase (ISPD) may be SEQ ID No.8.

[0083] Furthermore, the P450 limonene-6-hydroxylase (CYP71D18), cytochrome P450 reductase (ATR2), carveol dehydrogenase (ISPD), GPP synthase (ERG20 WW ), (-)-limonene synthase (MsLimS), orange diphosphate synthase (SlNDPS1), 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1) and isoprene pyrophosphate isomerase (IDI1) can add peroxisomal protein localization expression signal guide peptides, including but not limited to these enzymes, which can be fusion products of these enzymes, or other enzymes involved in the synthesis of (-)-carvone.

[0084] The use of the aforementioned recombinant bacteria in the preparation of (-)-carvone also falls within the protection scope of the present invention.

[0085] The present invention also provides a method for preparing (-)-carvone, which comprises using the aforementioned recombinant bacteria as a fermentation strain and (-)-limonene as a raw material to prepare (-)-carvone.

[0086] The invention optimizes the MVA pathway by overexpressing tHMG1 and IDI1, thereby increasing the flux of (-)-limonene synthesis precursors. The limonene synthase MsLimS and the GPP synthase (Erg20p double mutant N127W-F96W) are fused, and a pathway for synthesizing limonene from NPP using NPP synthase (SlNDPS1) is introduced to increase the fermentation yield of (-)-limonene. At the same time, a carvone biosynthesis pathway is constructed in the cytoplasm, and a cytochrome P450 limonene-6-hydroxylase gene CYP71D18 from spearmint, a cytochrome P450 reductase gene ATR2 from mint, and a carveol dehydrogenase gene ISPD from mint are introduced into saccharomyces cerevisiae, thereby realizing the biosynthesis of (-)-carvone in vivo by the MVA pathway of saccharomyces cerevisiae via (-)-limonene and (-)-trans-carvol. The production method for synthesizing (-)-carvone by using saccharomyces cerevisiae and its peroxisomal subcellular organelles disclosed in the present invention will lay a foundation for the industrial production of carvone. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 It is a synthetic pathway from the precursors IPP and DMAPP to (-)-limonene.

[0088] Figure 2 This is the synthetic pathway from (-)-limonene to (-)-carvone.

[0089] Figure 3 Schematic diagram of the (-)-carvone biosynthetic pathway constructed in Saccharomyces cerevisiae.

[0090] Figure 4 The gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) detection and analysis diagrams of (-)-carveol in the fermentation products of strains CElp1 and CElp2, wherein A is the gas chromatography detection result of (-)-carveol; B is the mass spectrometry identification result of (-)-carveol in the fermentation product of strain CElp2; C is the standard mass spectrum of (-)-carveol.

[0091] Figure 5 The gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) detection and analysis diagrams of (-)-carvone in the fermentation products of strains CVlp1 and CVlp2, wherein A is the gas chromatography detection result of (-)-carveol; B is the mass spectrometry identification result of (-)-carvone in the fermentation product of strain CVlp2; C is the standard mass spectrum of (-)-carvone. DETAILED DESCRIPTION

[0092] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0093] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0094] The strains used in the present invention are shown in Table 1, the primers used are shown in Table 2, and the plasmids involved are shown in Table 3.

[0095] Table 1 Strains used in the present invention

[0096]

[0097] Table 2 Primers used in the present invention

[0098]

[0099]

[0100] Table 3 Plasmids used and constructed in the present invention

[0101]

[0102] Example 1: Biosynthesis pathway of (-)-limonene in Saccharomyces cerevisiae and plasmid construction

[0103] The chassis cells used in this embodiment include but are not limited to Saccharomyces cerevisiae strains, and all other proteins that can be expressed by expression systems such as bacteria, yeast, and cells are included in the application scope of the present invention.

[0104] (1) Biosynthesis of (-)-limonene in Saccharomyces cerevisiae

[0105] In this example, Saccharomyces cerevisiae BY4742 was used as a chassis cell to construct a heterologous biosynthetic pathway for (-)-limonene in Saccharomyces cerevisiae, which mainly included the following steps: (1) overexpressing two rate-limiting enzyme genes tHMG1 and IDI1 in the MVA pathway to increase the supply of precursors IPP and DMAPP; (2) introducing a double mutant of GPP synthase (ERG20 WW), NPP synthase from tomato (SlNDPS1) and (-)-limonene synthase from lemon (MsLimS) were used for the synthesis of (-)-limonene. The limonene synthase gene (MsLimS) and the GPP synthase gene (ERG20 WW ) co-expression of the NPP synthase gene (SlNDPS1) to achieve a synthetic pathway for (-)-limonene using both GPP and NPP as substrates (e.g. Figure 1 ) to maximize the amount of (-)-limonene.

[0106] (2) Construction of tHMG1 and IDI1 co-expression plasmid pY26TEF-GPD-tHMG1-IDI1

[0107] Both GPP and NPP can serve as direct precursors for (-)-limonene synthesis. In order to increase the supply of DMAPP and IPP for GPP and NPP biosynthesis, IDI1 (isopentenyl diphosphate isomerase) and tHMG1 (truncated HMG1, 3-hydroxy-3-methylglutaryl coenzyme reductase) were overexpressed in Saccharomyces cerevisiae, and the IDI1 and tHMG1 co-expression plasmid pY26TEF-GPD-tHMG1-IDI1 was constructed.

[0108] The genome of Saccharomyces cerevisiae BY4742 was used as a template, and the target gene tHMG1 was amplified using the primer pair tHMG1-F-SpeI / tHMG1-R-XhoI), and the target gene IDI1 was amplified using the primer pair IDI1-F-NotI / IDI11-R-BglII. Afterwards, the plasmid pY26TEF-GPD (purchased from Miaoling Biotechnology, catalog number P1616) was double-digested with XhoI and BglII to recover a fragment with a sequence length of about 6217 bp, and the plasmid pY26TEF-GPD was double-digested with NotI and SpeI to recover a fragment with a sequence length of about 1138 bp. The tHMG1 gene fragment, the IDI1 gene fragment, the fragment with a sequence length of about 6217 bp and the fragment with a sequence length of about 1138 bp obtained by double digestion of pY26TEF-GPD are connected by seamless cloning, and the connected plasmid is pY26TEF-GPD-tHMG1-IDI1. pY26TEF-GPD-tHMG1-IDI1 is a recombinant expression vector obtained by replacing the nucleotide sequence between the XhoI and SpeI restriction enzyme recognition sites of pY26TEF-GPD with the nucleotide sequence (CDS) of tHMG1, and replacing the nucleotide sequence between the NotI and BglII restriction enzyme recognition sites of pY26TEF-GPD with the nucleotide sequence (CDS) of IDI1, while keeping other nucleotide sequences unchanged, and the vector expresses the tHMG1 gene and the IDI1 gene.

[0109] (3) Plasmid pESC-LEU-MsLimS-GGGS-ERG20 related to (-)-limonene synthesis WW -SlNDPS1 build

[0110] An orthogonal limonene biosynthetic pathway was constructed by overexpressing the limonene synthase MsLimS and the orange acyl diphosphate synthase SlNDPS1. Since ERG20 and ERG9 (squalene synthases) have high catalytic activity for the conversion of DMAPP to squalene, this may greatly reduce the metabolic flux of GPP to limonene. The yeast Erg20p mutant N127W-F96W (ERG20 WW ) has good activity in catalyzing GPP to form monoterpenoid chemicals. Since a large amount of GPP will still be rapidly converted by the FPP synthesis subunit of Erg20p before diffusion, the Erg20 mutant ERG20 WW Fusion with limonene synthase MsLimS to overcome the space restriction between them and allow the GPP form to be sequestered into FPP as much as possible. ERG20 WW MsLimS can be expressed by fusion with the linker GGGS or other linkers with similar mechanisms.

[0111] Gene ERG20 WW The plasmid pY26TEF-GPD-ERG20 was synthesized by Gene Company and constructed on the pY26TEF-GPD vector. WW The genes MsLimS and SlNDPS1 were synthesized by Gene Company and constructed on the pESC-LEU vector (purchased from Miaoling Biotechnology, product number P0238) to obtain plasmids pESC-Leu-MsLimS and pESC-Leu-SlNDPS1.

[0112] The plasmid pESC-LEU-MsLimS was double-digested with NotI and SacI to recover the large fragment containing LEU-MsLimS by gel excision, and the plasmid pESC-LEU-SlNDPS was double-digested with NotI and SacI to recover the small fragment containing SlNDPS1 by gel excision, which was transformed into Escherichia coli DH5α after T4 DNA Ligase ligation. The clone was picked and sequenced to verify the correctness of the sequence to obtain the plasmid pESC-LEU-MsLimS-SlNDPS1. The plasmid pESC-LEU-MsLimS-SlNDPS1 is a recombinant expression vector obtained by replacing the nucleotide sequence between the restriction recognition sites NotI and SacI of the plasmid pESC-Leu with SlNDPS1, replacing the nucleotide sequence between the restriction recognition sites NcoI and SacII of the plasmid pESC-Leu with MsLimS, and keeping other nucleotide sequences unchanged.

[0113] Plasmid pY26TEF-GPD-ERG20 WW As template, use primers MsLimS-GGGS-ERG20 WW -F and MsLimS-GGGS-ERG20 WW -R amplified the target gene GGGS-ERG20 WW The vector pESC-LEU-MsLimS-SlNDPS1 was digested with restriction endonuclease SacII and the target fragment GGGS-ERG20 was obtained. WW Through seamless cloning and ligation, E. coli DH5α was transformed, and the clone was selected for sequencing to verify the sequence was correct to obtain the plasmid pESC-LEU-MsLimS-GGGS-ERG20 WW -SlNDPS1. pESC-LEU-MsLimS-GGGS-ERG20 WW -SlNDPS1 is based on MsLimS and ERG20 WW The fusion sequence MsLimS-GGGS-ERG20 WW The nucleotide sequence between the NcoI and SacII restriction enzyme recognition sites of pESC-LEU is replaced, and the nucleotide sequence between the NotI and SacI restriction enzyme recognition sites of pESC-LEU is replaced with the nucleotide sequence (CDS) of SlNDPS1, while keeping other nucleotide sequences unchanged to obtain a recombinant expression vector, which expresses MsLimS and ERG20 WW The fusion gene MsLimS-GGGS-ERG20 WW and SlNDPS1 gene.

[0114] MsLimS-GGGS-ERG20 WW The nucleotide sequence is based on the coding region sequence of MsLimS gene (CDS, 1635bp), the nucleotide sequence of GGGS (12bp, the specific base is GGTGGTGGTTCT), ERG20 WW The nucleotide sequences of the coding region sequence (CDS, 1059 bp) of the gene were connected in the 5'-3' direction.

[0115] Example 2: Biosynthesis pathway of (-)-carveol and (-)-carvone in Saccharomyces cerevisiae and plasmid construction

[0116] (1) Biosynthesis of (-)-carveol and (-)-carvone in Saccharomyces cerevisiae

[0117] Saccharomyces cerevisiae was used as a chassis cell to construct a cytoplasmic (-)-carveol and (-)-carvone synthesis strain, and de novo biosynthesis of (-)-carveol and (-)-carvone was achieved in Saccharomyces cerevisiae cells. Carveol and carvone can be converted from limonene. (-)-limonene is hydroxylated to generate (-)-trans-carveol, and then (-)-carvone is generated by carveol dehydrogenase. In order to achieve the synthesis of (-)-trans-carveol and (-)-carvone from (-)-limonene in Saccharomyces cerevisiae, the cytochrome P450 limonene-6-hydroxylase gene CYP71D18 and the cytochrome P450 reductase gene ATR2 from spearmint were selected to hydroxylate (-)-limonene to (-)-trans-carveol, and then the carveol dehydrogenase gene ISPD from mint oxidized (-)-trans-carveol to (-)-carvone, such as Figure 2 shown.

[0118] (2) Construction of plasmid pESC-HIS-CYP71D18-GGGS-ATR2 related to (-)-carveol synthesis

[0119] Genes CYP71D18, ATR2 and ISPD were synthesized by GeneWeizhi Biotechnology Co., Ltd. and constructed on the pESC-LEU vector to obtain plasmids pESC-LEU-CYP71D18, pESC-LEU-ATR2 and pESC-LEU-ISPD, respectively. Using pESC-LEU-ATR2 as a template, PCR amplification was performed using primer pair CYP-GGGS-ATR2-F / CYP-GGGS-ATR2-R to obtain the target fragment GGGS-ATR2, and then the plasmid pESC-Leu-CYP71D18 was digested with restriction endonuclease SacI, and the large fragment was recovered by gel excision. The large fragment was connected with the target fragment GGGS-ATR2 by seamless cloning, and Escherichia coli DH5α was transformed. After the clone was picked and sequenced to verify the correct sequence, the plasmid pESC-LEU-CYP71D18-GGGS-ATR2 was obtained. The plasmid was a recombinant expression vector obtained by replacing the nucleotide sequence between the NcoI and SacI restriction recognition sites of pESC-LEU with the fusion sequence CYP71D18 and ATR2 CYP71D18-GGGS-ATR2, and keeping other nucleotide sequences unchanged.

[0120] The vector screening marker was changed to HIS3, and the plasmid pESC-LEU-CYP71D18-GGGS-ATR2 was double-digested with restriction endonucleases NotI and SacI to obtain the fusion sequence CYP71D18-GGGS-ATR2, and the fusion sequence CYP71D18-GGGS-ATR2 was ligated between the NotI and SacI restriction sites of pESC-HIS (purchased from Miaoling Biotechnology, catalog number P1269) to obtain the plasmid pESC-HIS-CYP71D18-GGGS-ATR2. pESC-HIS-CYP71D18-GGGS-ATR2 is a recombinant expression vector obtained by replacing the nucleotide sequence between the NotI and SacI restriction recognition sites of pESC-HIS with the fusion sequence CYP71D18-GGGS-ATR2 of CYP71D18 and ATR2, while keeping other nucleotide sequences unchanged. The vector expresses the fusion gene CYP71D18-GGGS-ATR2 of CYP71D18 and ATR2.

[0121] The nucleotide sequence of CYP71D18-GGGS-ATR2 is formed by connecting the coding region sequence of CYP71D18 gene (CDS, 1488bp), the nucleotide sequence of GGGS (12bp, specific bases are GGTGGTGGTTCT), and the coding region sequence of ATR2 gene (CDS, 1941bp) in the 5'-3' direction.

[0122] (3) Construction of plasmid pESC-HIS-CYP-GGGS-ATR2-ISPD related to (-)-carvone synthesis

[0123] The genes ISPD were synthesized by Gene Company and constructed on vectors to obtain plasmid pESC-LEU-ISPD. The synthesis of carvone from limonene requires the participation of three enzymes, namely cytochrome P450 limonene-6-hydroxylase (CYP71D18), cytochrome P450 reductase (ATR2) and carveol dehydrogenase (ISPD). These three enzymes were recombined into plasmid pESC-HIS to form plasmid pESC-HIS-CYP71D18-GGGS-ATR2-ISPD. The specific method is as follows:

[0124] Plasmid pESC-HIS-CYP71D18-GGGS-ATR2 and plasmid pESC-LEU-ISPD were double-digested with NotI and SacI to obtain large and small fragments, respectively. The fragments were connected with T4 ligase and transformed into Escherichia coli DH5α. After the clones were picked and sequenced to verify the correctness of the sequence, plasmid pESC-HIS-CYP71D18-GGGS-ATR2-ISPD was obtained. pESC-HIS-CYP71D18-GGGS-ATR2-ISPD is a recombinant expression vector obtained by replacing the nucleotide sequence between the NotI and SacI restriction recognition sites of pESC-HIS with the fusion sequence CYP71D18 and ATR2 CYP71D18-GGGS-ATR2, and replacing the nucleotide sequence between the NcoI and SacII restriction recognition sites of pESC-HIS with the nucleotide sequence (CDS) of ISPD, while keeping other nucleotide sequences unchanged. The vector expresses the fusion gene CYP71D18 and ATR2 CYP71D18-GGGS-ATR2 and ISPD gene.

[0125] Example 3: Construction of peroxisome-localized expression vector for Saccharomyces cerevisiae

[0126] The present invention not only increases the yield of monoterpene compounds limonene, carveol and carvone by increasing the precursor supply and fusion expression of key enzymes, but also utilizes organelles to isolate biochemical pathways and control metabolic complexity through localized expression of enzymes, thereby improving the synthesis level of target products.

[0127] The present invention selects peroxisome as the site for enzyme localization, adds peroxisome signal peptide SKL to the C-terminus of the key enzyme, and guides the enzyme into the peroxisome through the signal peptide to exert its effect.

[0128] The peroxisome signal peptide (SKL) nucleotide sequence selected in this example is: TCTAAACTA.

[0129] The amino acid sequence of the peroxisome signal peptide (SKL) selected in this example is: SKL.

[0130] (1) Construction of peroxisomal expression plasmid pY26TEF-GPD-tHMG1-SKL-IDI1-SKL

[0131] Using the MVA pathway overexpression plasmid pY26TEF-GPD-tHMG1-IDI1 as a template, the primer pair tHMG1-SKL-R / IDI1-SKL-R was used to amplify the target fragment tHMG1-SKL-IDI1-SKL, which was connected with pY26TEF-GPD by seamless cloning and transformed into Escherichia coli DH5α. After selecting clones for sequencing to verify the correctness of the sequence, the peroxisome recombinant plasmid pY26TEF-GPD-tHMG1-SKL-IDI1-SKL was obtained. pY26TEF-GPD-tHMG1-SKL-IDI1-SKL is a recombinant expression vector obtained by replacing the nucleotide sequence between the SpeI and XhoI restriction enzyme recognition sites of pY26TEF-GPD with tHMG1-SKL, replacing the nucleotide sequence between the NotI and BglII restriction enzyme recognition sites of pY26TEF-GPD with IDI1-SKL, and keeping other nucleotide sequences unchanged. The vector expresses tHMG1-SKL gene and IDI1-SKL gene.

[0132] tHMG1-SKL is a nucleotide sequence obtained by adding TCTAAACTA to the 3' end of the coding region sequence of the tHMG1 gene.

[0133] IDI1-SKL is a nucleotide sequence obtained by adding TCTAAACTA to the 3' end of the coding region sequence of the IDI1 gene.

[0134] (2) Peroxisome expression plasmid pESC-LEU-MsLimS-GGGS-ERG20 WW - Construction of SKL-SlNDPS1-SKL

[0135] The plasmid pESC-LEU-MsLimS-GGGS-ERG20 overexpressing the limonene biosynthesis pathway WW -SlNDPS1 was used as a template, and the target fragment MsLimS-GGGS-ERG20 was amplified using primer pairs NPPS-SKL-F / Erg20-SKL-R. WW -SKL-SlNDPS1-SKL, the large fragment (6924 bp) was recovered from the plasmid pESC-LEU using the SacI and SacII double restriction tool, and then connected to MsLimS-GGGS-ERG20 using the seamless cloning kit WW -SKL-SlNDPS1-SKL and the aforementioned large fragment were transformed into Escherichia coli DH5α, and clones were selected for sequencing to verify the correct sequence to obtain the peroxisome recombinant plasmid pESC-LEU-MsLimS-GGGS-ERG20 WW -SKL-SlNDPS1-SKL.

[0136] pESC-LEU-MsLimS-GGGS-ERG20 WW -SKL-SlNDPS1-SKL is a peroxisomal signal peptide containing MsLimS and ERG20 WW The fusion sequence MsLimS-GGGS-ERG20 WW -SKL replaces the nucleotide sequence between the NcoI and SacII restriction enzyme recognition sites of pESC-LEU, and replaces the nucleotide sequence between the NotI and SacI restriction enzyme recognition sites of pESC-LEU with the nucleotide sequence of SlNDPS1-SKL, while keeping other nucleotide sequences unchanged to obtain a recombinant expression vector, which can express MsLimS and ERG20 with peroxisomal signal peptide sequences. WW Fusion gene MsLimS-GGGS-ERG20 WW -SKL and SlNDPS1-SKL genes.

[0137] MsLimS-GGGS-ERG20 WW -SKL is the coding sequence of MsLimS gene, the nucleotide sequence of GGGS (GGTGGTGGTTCT), ERG20 WW The coding region sequence of the gene and the SKL nucleotide sequence (TCTAAACTA) were connected in a 5'-3' direction.

[0138] SlNDPS1-SKL is a nucleotide sequence obtained by adding TCTAAACTA to the 3' end of the coding region sequence of the SlNDPS1 gene.

[0139] (3) Construction of peroxisomal expression plasmid pESC-HIS-CYP71D18-GGGS-ATR2-SKL

[0140] A peroxisomal signal peptide SKL was added to the C-terminus of the key enzyme gene by PCR. The recombinant plasmid pESC-HIS-CYP71D18-GGGS-ATR-ISPD was used as a template and the primer pair CYP-SKL-F / ATR2-SKL-R was used to obtain the target fragment CYP71D18-GGGS-ATR2-SKL by PCR. The large fragment was recovered by using the SacI and SacII double restriction tool plasmid pESC-LEU, and then connected using a seamless cloning kit and transformed into Escherichia coli DH5α. After the clone was picked and sequenced to verify the correct sequence, the peroxisomal recombinant plasmid pESC-HIS-CYP71D18-GGGS-ATR2-SKL was obtained. pESC-HIS-CYP71D18-GGGS-ATR2-SKL is a recombinant expression vector obtained by replacing the nucleotide sequence between the NotI and SacI restriction recognition sites of pESC-HIS with CYP71D18-GGGS-ATR2-SKL while keeping other nucleotide sequences unchanged. The vector expresses the fusion gene CYP71D18 and ATR2 CYP71D18-GGGS-ATR2-SKL with a peroxisomal signal peptide sequence.

[0141] The nucleotide sequence of CYP71D18-GGGS-ATR2-SKL is formed by connecting the coding region sequence of CYP71D18 gene (CDS, 1488 bp), the nucleotide sequence of GGGS (12 bp), the coding region sequence of ATR2 gene (CDS, 1941 bp) and the nucleotide sequence of SKL in the 5'-3' direction.

[0142] (4) Construction of peroxisomal expression plasmid pESC-HIS-CYP71D18-GGGS-ATR2-SKL-ISPD-SKL

[0143] A peroxisomal signal peptide SKL was added to the C-terminus of the key enzyme gene by PCR. The recombinant plasmid pESC-HIS-CYP71D18-GGGS-ATR-ISPD was used as a template and the primer pair CYP-SKL-F / ISPD-SKL-R was used to obtain the target fragment CYP71D18-GGGS-ATR2-SKL-ISPD-SKL by PCR. The large fragment was recovered by using the SacI and SacII double restriction tool plasmid pESC-LEU, and then connected using a seamless cloning kit and transformed into Escherichia coli DH5α. After the clone was picked and sequenced to verify the correctness of the sequence, the peroxisomal recombinant plasmid pESC-HIS-CYP71D18-GGGS-ATR2-SKL-ISPD-SKL was obtained. pESC-HIS-CYP71D18-GGGS-ATR2-SKL-ISPD-SKL is a recombinant expression vector obtained by replacing the nucleotide sequence between the NotI and SacI restriction recognition sites of pESC-HIS with CYP71D18-GGGS-ATR2-SKL, replacing the nucleotide sequence between the NcoI and SacII restriction recognition sites of pESC-HIS with ISPD-SKL, and keeping other nucleotide sequences unchanged. The vector expresses the CYP71D18 and ATR2 fusion gene CYP71D18-GGGS-ATR2-SKL and ISPD-SKL gene with a peroxisomal signal peptide sequence.

[0144] ISPD-SKL is a nucleotide sequence obtained by adding TCTAAACTA to the 3' end of the coding region sequence of the ISPD gene.

[0145] Example 4: Construction of Saccharomyces cerevisiae and peroxisome-localized expression strain for biosynthesis of (-)-carvone

[0146] (1) Saccharomyces cerevisiae transformation method

[0147] In this example, the lithium acetate method was used to prepare Saccharomyces cerevisiae competent cells, and the plasmid was transferred into the yeast cells by electroporation.

[0148] Take 50 μL of the glycerol-preserved bacteria to be transformed and inoculate into 4 mL YPD liquid medium for overnight culture. Transfer the bacterial solution to 100 mL YPD liquid medium with a final concentration of about 0.2 and culture in a constant temperature shaker at 30°C and 250 rpm for 5-8 hours. 600=0.6-1.0) was transferred to a 50 mL sterile centrifuge tube, centrifuged at 3500 rpm for 5 min, the supernatant was removed, the precipitate was washed twice with sterile deionized water, centrifuged at 3500 rpm for 5 min, 30 mL of yeast treatment solution (0.6 M sorbitol, 10 mM Tris-HCl, 10 mM LiAc, 10 mM DTT, sterilized by 0.22 μm filter membrane filtration, prepared and used immediately) was added after discarding the supernatant, and the mixture was placed in a metal bath at 25° C. for 20 min, the supernatant was removed, the precipitate was washed twice with 30 mL of 1 M sorbitol solution, centrifuged at 3500 rpm for 5 min, the bacteria were collected, about 1000 μL of 1 M sorbitol solution was added to resuspend the bacteria, and the prepared yeast competent cells were placed on ice for use.

[0149] Take 60μL yeast competent cell solution and add 100ng of the plasmid to be transformed. After mixing, transfer to a 1mm electroporation cup, place in an ice bath for 5min, and electroporate once at 1.5kV. The electroporation time should not be less than 5.0ms. Immediately after electroporation, add 1mL of pre-cooled 1M sorbitol and transfer to a new 1.5mL EP tube. According to the nutritional deficiency screening marker on the transformed plasmid or fragment, the electroporation solution is directly spread on the corresponding SD selection medium containing 1M sorbitol after centrifugation, and placed in a 30℃ constant temperature incubator for 2-3 days. Extract the yeast plasmid or genome from the grown clones, and then perform colony PCR verification.

[0150] (2) Screening and verification of Saccharomyces cerevisiae transformants

[0151] The Saccharomyces cerevisiae transformants obtained after electroporation were screened again using a new solid selection medium. After cell lysis of the clones with good growth, the transformants were further verified by PCR. Pick a single colony on the plate coated with electroporation and streak it on the solid plate of the selection medium again, and place it in a 30℃ incubator for 2-3 days. Use TaKaralysis buffer for microorganism to direct PCR to verify whether the plasmid is introduced into the Saccharomyces cerevisiae cells. Take 30μL buffer in an EP tube, pick more colonies with the tip of the gun, and mix them; heat denature at 80℃ for 20min, centrifuge at low speed for 30s, and take 1-5μL of the supernatant after lysis as a PCR reaction template. Then perform PCR reaction. Like the ordinary PCR reaction system, after the PCR reaction is completed, take 5μL of the reaction system and add 1μL of 6× loading Buffer. After mixing, perform agarose gel electrophoresis. The length of the amplified target gene is obtained by comparison with the Marker, and compared with the designed amplification length to determine whether the PCR reaction is successful.

[0152] (3) Construction of Saccharomyces cerevisiae and its peroxisome-localized (-)-limonene synthesis strain

[0153] The successfully constructed peroxisomal subcellular localization overexpression vectors pY26TEF-GPD-tHMG1-SKL-IDI1-SKL and pESC-LEU-MsLimS-GGGS-ERG20 WW -SKL-SlNDPS1-SKL was transformed into the Saccharomyces cerevisiae chassis strain BY4742 by electroporation to obtain LMlp1. The electroporated bacterial solution was spread on 1M sorbitol-containing uracil and leucine-deficient yeast SD selection medium, cultured at 30°C for 3 days, and single clone cells were picked and lysed. Using the single clone cell lysate and the control strain BY4742 lysate as templates and the primer pair tHMG1-SKL-R / IDI1-SKL-R as primers, PCR amplified a 3667bp gene fragment, and using the primer pair GAL10-R / GAL1-RI as primers, PCR amplified a 4389bp gene fragment strain, which is the (-)-limonene synthesis strain LMlp1.

[0154] The successfully constructed cytoplasmic expression vector pY26TEF-GPD-tHMG1-IDI1 and peroxisomal subcellular localization overexpression vector pESC-LEU-MsLimS-GGGS-ERG20 WW -SKL-SlNDPS1-SKL was transformed into the Saccharomyces cerevisiae chassis strain BY4742 by electroporation to obtain LMlp2. The electroporated bacterial solution was spread on 1M sorbitol-containing uracil and leucine-deficient yeast SD selection medium, cultured at 30°C for 3 days, and single clone cells were picked and lysed. Using the single clone cell lysate and the control strain BY4742 lysate as templates and the primer pair tHMG1-R-XhoI / IDI1-R-BglII as primers, PCR amplified a 3641bp gene fragment, and using the primer pair GAL10-R / GAL1-RI as primers, PCR amplified a 4389bp gene fragment strain, which is the (-)-limonene synthesis strain LMlp2.

[0155] The main difference between the (-)-limonene strains LMlp1 and LMlp2 is that LMlp1 contains the peroxisomal expression plasmid pY26TEF-GPD-tHMG1-SKL-IDI1-SKL of tHMG1 and IDI1, while LMlp2 contains the cytoplasmic expression plasmid pY26TEF-GPD-tHMG1-IDI1 of tHMG1 and IDI1.

[0156] The limonene-synthesizing strain LMlp1 can express the enzyme tHMG1 encoded by the nucleotide sequence SEQ ID No.1, the enzyme IDI1 encoded by the nucleotide sequence SEQ ID No.2, the enzyme MsLimS encoded by the nucleotide sequence SEQ ID No.3, and the enzyme ERG20 encoded by the nucleotide sequence SEQ ID No.4. WW and the enzyme SlNDPS1 encoded by the nucleotide sequence SEQ ID No.5, and tHMG1, IDI1, MsLimS, ERG20 WW and SlNDPS1 can be localized on peroxisomes.

[0157] The limonene-synthesizing strain LMlp2 can express the enzyme tHMG1 encoded by the nucleotide sequence SEQ ID No.1, the enzyme IDI1 encoded by the nucleotide sequence SEQ ID No.2, the enzyme MsLimS encoded by the nucleotide sequence SEQ ID No.3, and the enzyme ERG20 encoded by the nucleotide sequence SEQ ID No.4. WW and the enzyme SlNDPS1 encoded by the nucleotide sequence SEQ ID No.5, and MsLimS, ERG20 WW and SlNDPS1 can be localized on peroxisomes.

[0158] In this embodiment, plasmid pY26TEF-GPD-tHMG1-IDI1 and plasmid pESC-LEU-MsLimS-GGGS-ERG20 can also be sequentially added. WW -SlNDPS1 was transformed into Saccharomyces cerevisiae, or the vector containing tHMG1, IDI1, MsLimS-GGGS-ERG20 WW (or MsLimS, ERG20 WW The recombinant expression plasmids of the five DNA fragments of SlNDPS1 were recombined into Saccharomyces cerevisiae to obtain an engineered strain for synthesizing (-)-limonene.

[0159] (4) Construction of Saccharomyces cerevisiae and a strain that synthesizes (-)-carveol in a peroxisome-localized manner

[0160] The successfully constructed peroxisomal subcellular localization overexpression vector pESC-HIS-CYP71D18-GGGS-ATR2-SKL was transformed into Saccharomyces cerevisiae LMlp1 to obtain the (-)-carveol peroxisomal localization biosynthesis strain CElp1. The electroporated bacterial solution was spread on 1M sorbitol-deficient yeast SD selection medium, cultured at 30°C for 3 days, and single clone cells were picked and lysed. Using the single clone cell lysate and the control strain LMlp1 lysate as templates, PCR amplified a 3667bp gene fragment using the primer pair tHMG1-SKL-R / IDI1-SKL-R, and PCR amplified a 3632bp gene fragment using the primer pair GAL10-R / GAL10pro-F, which is the (-)-carveol synthesis strain CElp1.

[0161] The successfully constructed peroxisomal subcellular localization overexpression vector pESC-HIS-CYP71D18-GGGS-ATR2-SKL was transformed into Saccharomyces cerevisiae LMlp2 to obtain the (-)-carveol peroxisome localization biosynthesis strain CElp2. The electroporated bacterial solution was spread on 1M sorbitol-deficient yeast SD selection medium, cultured at 30°C for 3 days, and single clone cells were picked and lysed. Using the single clone cell lysate and the control strain LMlp1 lysate as templates, the primer pair tHMG1-R-XhoI / IDI1-R-BglII was used as primers, and PCR amplified a 3641bp gene fragment. Using the primer pair GAL10-R / GAL10pro-F as primers, PCR amplified a 4398bp gene fragment, which was the cytoplasmic (-)-carveol synthesis strain CElp2.

[0162] The main difference between the (-)-carveol strains CElp1 and CElp2 is that CElp1 contains the peroxisomal expression plasmid pY26TEF-GPD-tHMG1-SKL-IDI1-SKL of tHMG1 and IDI1, while CElp2 contains the cytoplasmic expression plasmid pY26TEF-GPD-tHMG1-IDI1 of tHMG1 and IDI1.

[0163] The carveol-synthesizing strain CElp1 can express the enzyme tHMG1 encoded by the nucleotide sequence SEQ ID No.1, the enzyme IDI1 encoded by the nucleotide sequence SEQ ID No.2, the enzyme MsLimS encoded by the nucleotide sequence SEQ ID No.3, and the enzyme ERG20 encoded by the nucleotide sequence SEQ ID No.4. WW, enzyme SlNDPS1 encoded by nucleotide sequence SEQ ID No.5, enzyme CYP71D18 encoded by nucleotide sequence SEQ ID No.6, enzyme ATR2 encoded by nucleotide sequence SEQ ID No.7, and tHMG1, IDI1, MsLimS, ERG20 WW , SlNDPS1, CYP71D18, and ATR2 can be expressed on peroxisomes.

[0164] The carveol-synthesizing strain CElp2 can express the enzyme tHMG1 encoded by the nucleotide sequence SEQ ID No.1, the enzyme IDI1 encoded by the nucleotide sequence SEQ ID No.2, the enzyme MsLimS encoded by the nucleotide sequence SEQ ID No.3, and the enzyme ERG20 encoded by the nucleotide sequence SEQ ID No.4. WW , enzyme SlNDPS1 encoded by nucleotide sequence SEQ ID No.5, enzyme CYP71D18 encoded by nucleotide sequence SEQ ID No.6, enzyme ATR2 encoded by nucleotide sequence SEQ ID No.7, and MsLimS, ERG20 WW , SlNDPS1, CYP71D18, and ATR2 can be expressed on peroxisomes.

[0165] In this embodiment, either plasmid transfer or recombinant related genes can be used to obtain strains with corresponding functions.

[0166] (5) Construction of Saccharomyces cerevisiae and a strain that synthesizes (-)-carvone in peroxisomes

[0167] The successfully constructed peroxisomal subcellular localization overexpression vector pESC-HIS-CYP71D18-GGGS-ATR2-SKL-ISPD-SKL was transformed into Saccharomyces cerevisiae LMlp1 to obtain the (-)-carveol peroxisomal localization biosynthesis strain CVlp1. The electroporated bacterial solution was spread on 1M sorbitol-deficient yeast SD selection medium, cultured at 30°C for 3 days, and single clone cells were picked and lysed. Using the single clone cell lysate and the control strain LMlp1 lysate as templates, the primer pair tHMG1-SKL-R / IDI1-SKL-R was used as primers, and the PCR amplified a 3667bp gene fragment. Using the primer pair GAL10-R / GAL1-RI as primers, the strain that could amplify a 5142bp gene fragment by PCR was the (-)-carveol synthesis strain CVlp1.

[0168] The successfully constructed peroxisomal subcellular localization overexpression vector pESC-HIS-CYP71D18-GGGS-ATR2-SKL-ISPD-SKL was transformed into Saccharomyces cerevisiae LMlp2 to obtain the (-)-carveol peroxisomal localization biosynthesis strain CVlp2. The electroporated bacterial solution was spread on 1M sorbitol-deficient yeast SD selection medium, cultured at 30°C for 3 days, and single clone cells were picked and lysed. Using the single clone cell lysate and the control strain LMlp1 lysate as templates, the primer pair tHMG1-R-XhoI / IDI1-R-BglII was used as primers, and PCR amplified a 3641bp gene fragment. Using the primer pair GAL10-R / GAL1-RI as primers, PCR amplified a 5142bp gene fragment, which was the (-)-carveol synthesis strain CVlp2.

[0169] The main difference between the (-)-carvone strains CVlp1 and CVlp2 is that CVlp1 contains the peroxisomal expression plasmid pY26TEF-GPD-tHMG1-SKL-IDI1-SKL of tHMG1 and IDI1, while CVlp2 contains the cytoplasmic expression plasmid pY26TEF-GPD-tHMG1-IDI1 of tHMG1 and IDI1.

[0170] The carvone synthesis strain CVlp1 can express the enzyme tHMG1 encoded by the nucleotide sequence SEQ ID No.1, the enzyme IDI1 encoded by the nucleotide sequence SEQ ID No.2, the enzyme MsLimS encoded by the nucleotide sequence SEQ ID No.3, and the enzyme ERG20 encoded by the nucleotide sequence SEQ ID No.4. WW , enzyme SlNDPS1 encoded by nucleotide sequence SEQ ID No.5, enzyme CYP71D18 encoded by nucleotide sequence SEQ ID No.6, enzyme ATR2 encoded by nucleotide sequence SEQ ID No.7, enzyme ISPD encoded by nucleotide sequence SEQ ID No.8, and tHMG1, IDI1, MsLimS, ERG20 WW , SlNDPS1, CYP71D18, ATR2, and ISPD can be expressed on peroxisomes.

[0171] The carvone synthesis strain CVlp2 can express the enzyme tHMG1 encoded by the nucleotide sequence SEQ ID No.1, the enzyme IDI1 encoded by the nucleotide sequence SEQ ID No.2, the enzyme MsLimS encoded by the nucleotide sequence SEQ ID No.3, and the enzyme ERG20 encoded by the nucleotide sequence SEQ ID No.4. WW, enzyme SlNDPS1 encoded by nucleotide sequence SEQ ID No.5, enzyme CYP71D18 encoded by nucleotide sequence SEQ ID No.6, enzyme ATR2 encoded by nucleotide sequence SEQ ID No.7, enzyme ISPD encoded by nucleotide sequence SEQ ID No.8, and MsLimS, ERG20 WW , SlNDPS1, CYP71D18, ATR2, and ISPD can be expressed on peroxisomes.

[0172] In this embodiment, either plasmid transfer or recombinant related genes can be used to obtain strains with corresponding functions.

[0173] Example 5: Preparation of (-)-limonene, (-)-carvone and (-)-carveol by fermentation of Saccharomyces cerevisiae strains

[0174] (1) Fermentation and culture method of Saccharomyces cerevisiae strain

[0175] Yeast SC medium formula: yeast basal nitrogen base (YNB) 1.7 g / L, ammonium sulfate 5 g / L, D-glucose 20 g / L, L-arginine 0.1 g / L, L-cysteine ​​0.1 g / L, L-lysine 0.1 g / L, L-threonine 0.1 g / L, L-aspartic acid 0.05 g / L, L-isoleucine 0.05 g / L, L-phenylalanine 0.05 g / L, L-proline 0.05 g / L, L-serine 0.05 g / L, L-tyrosine 0.05 g / L, L-valine 0.05 g / L, L-methionine 0.05 g / L, L-tryptophan 0.1 g / L, L-histidine 0.05 g / L, L-uracil 0.1 g / L, L-leucine 0.1 g / L, L-adenine 0.1 g / L, pH 6.5. If solid culture medium is prepared, add 2% agar powder. Yeast SC medium does not contain any carbon source such as glucose. When used, glucose, galactose, raffinose, etc. are added as yeast carbon sources as needed. Nutritionally deficient yeast SC medium is a yeast SC medium that lacks essential nutrients such as corresponding essential amino acids or uracil.

[0176] YPD liquid fermentation medium formula: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, pH 6.5. If making solid culture medium, add 2% agar powder.

[0177] The fermentation culture method of the Saccharomyces cerevisiae strain is as follows: the strain to be tested is inoculated into the corresponding nutritionally deficient yeast SC solid medium, cultured for 2 to 3 days, and then the colony is picked from the solid plate and inoculated into 5 mL of the corresponding nutritionally deficient liquid yeast SD medium, cultured at 30°C for 24-48 hours, and when the OD 600When the OD value reached about 2.0, the culture solution was centrifuged and inoculated into a 250 mL shake flask containing 20 mL YP liquid fermentation medium for further cultivation. The initial OD value of the culture solution was 600 The concentration of galactose is about 0.4, and galactose with a final concentration of 2% and raffinose with a final concentration of 1% are added to induce protein expression. In addition, isopropyl myristate with a final concentration of 10% (v / v) is added to reduce the volatilization of target products such as limonene, carveol and carvone, and reduce the toxicity of organic target products to yeast cells. At the same time, during the fermentation process, target products such as limonene, carveol and carvone are extracted into the isopropyl myristate organic phase. The shake flask containing the above culture solution is placed in a shaker at 30°C for culture, and the culture is continued at 220rpm for 160h. After the fermentation is completed, the fermentation solution is centrifuged to collect the upper organic phase for analysis and detection of the fermentation product.

[0178] (2) Extraction and detection of fermentation products of (-)-limonene, (-)-carveol and (-)-carvone

[0179] The fermentation broth was placed in a 50 mL centrifuge tube and centrifuged. The upper organic layer was taken out and placed in a 2 mL EP tube for further centrifugation. The upper organic layer was then taken out and diluted with n-hexane in a ratio of 1:1. The prepared samples were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS).

[0180] Detection instrument: Agilent GC-MS / MS Agilent 7890A / 5975C.

[0181] Chromatographic column: HP-5ms (30m*0.25mM)

[0182] The GC detection conditions were as follows: injection volume 1 μL, 45 °C, 5 °C / min to 130 °C, 10 °C / min to 250 °C, and maintained for 3 min, injection port temperature 250 °C.

[0183] (3) Preparation of (-)-limonene, (-)-carvone and (-)-carveol by fermentation of Saccharomyces cerevisiae

[0184] By using selective medium screening and colony PCR verification, the (-)-limonene-producing strains LMlp1 and LMlp2, (-)-carveol-producing strains CElp1 and CElp2, and (-)-carvone-producing strains CVlp1 and CVlp2 of Saccharomyces cerevisiae were obtained. tHMG1 and IDI1 that enhance the flux of the MVA pathway in the (-)-limonene strain LMlp1 (containing the peroxisome-localized expression plasmid pY26TEF-GPD-tHMG1-SKL-IDI1-SKL) contain peroxisome-localized expression signal peptides, while tHMG1 and IDI1 in the (-)-limonene strain LMlp2 (containing the cytoplasmic expression plasmid pY26TEF-GPD-tHMG1-IDI1) do not have peroxisome-localized expression signal peptides. Carveol and carvone synthesis strains were constructed on limonene synthesis strains, among which carveol synthesis strain CElp1 and carvone synthesis strain CVlp1 were constructed with limonene synthesis strain LMlp1 as chassis cells, and carveol synthesis strain CElp2 and carvone synthesis strain CVlp2 were constructed with limonene synthesis strain LMlp2 as chassis cells. tHMG1 and IDI1 in LMlp1, CElp1 and CVlp1 can be expressed in peroxisomes, while tHMG1 and IDI1 in LMlp2, CElp2 and CVlp2 are not expressed in peroxisomes.

[0185] The GC and GC-MS test results of the fermentation products of carveol-synthesizing strains CElp1 and CElp2 are as follows: Figure 4 As shown, Figure 4 A in the figure is the gas chromatography test result of the fermentation product of the strain. Figure 4 Figure B is the mass spectrometry identification result of (-)-carveol in the fermentation product of strain CElp2. Figure 4 C is the standard mass spectrum of (-)-carveol. Figure 4 It can be seen that the precursor substances (-)-limonene (retention time 7.8min) and (-)-carveol (retention time 12.9min) were detected in the fermentation products of carveol-synthesizing strains CElp1 and CElp2. Figure 4 As shown in A, the standard sample carveol contains trans-carvone and cis-carveol. The peak at 12.9 min is trans-carveol, and the peak at 13.2 min is cis-carveol. The fermentation products of strains CElp1 and CElp2 that convert (-)-limonene to (-)-carveol both detected (-)-carveol at 12.9 min. Mass spectrometry analysis spectrum Figure 4 The product B was determined to be carveol by comparison with the database. The standard spectrum of carveol is as follows Figure 4As shown in C. The amount of limonene in the fermentation products of carveol synthesis strains CElp1 and CElp2 was 2.59 mg / L and 4.78 mg / L, respectively. The fermentation yield of (-)-carveol in the fermentation products of CElp1 and CElp2 was 0.89 mg / L and 1.16 mg / L, respectively.

[0186] The GC and GC-MS test results of the fermentation products of the carvone-producing strains CVlp1 and CVlp2 of Saccharomyces cerevisiae are as follows Figure 5 As shown, Figure 5 A in the figure is the gas chromatography test result of the fermentation product of the strain. Figure 5 Figure B is the mass spectrometry identification result of (-)-carvone in the fermentation product of strain CVlp2. Figure 5 C in the figure is the standard mass spectrum of (-)-carvone. Figure 5 It can be seen that the precursor (-)-limonene (retention time 7.8min), the precursor (-)-carveol (retention time 12.9min) and the target product (-)-carvone (retention time 13.6min) were detected in the fermentation products of carvone-synthesizing strains CVlp1 and CVlp2. Figure 5 As shown in A, the standard sample 10 mg / L carvone peaks at 13.6 min, and the fermentation products of carvone-synthesizing strains CVlp1 and CVlp2 also have peaks detected at 13.6 min. Figure 5 The product B was determined to be carvone after comparison with the database. The standard spectrum of carvone is as follows Figure 5 As shown in C. The amount of limonene in the fermentation products of carveol synthesis strains CElp1 and CElp2 was 3.32 mg / L and 3.52 mg / L, respectively. The fermentation yield of (-)-carvone in the fermentation products of CVlp1 and CVlp2 was 0.16 mg / L and 0.29 mg / L, respectively.

[0187] The present invention provides an example of using Saccharomyces cerevisiae as a chassis cell to biosynthesize (-)-carvone. The present invention uses Saccharomyces cerevisiae as a chassis cell, and successfully constructs a pathway for de novo biosynthesis of (-)-carvone via intermediate products (-)-limonene and (-)-carveol in Saccharomyces cerevisiae through overexpression, fusion expression and peroxisome localization expression of key enzymes. When only the key enzymes in the (-)-limonene to (-)-carvone synthesis pathway are guided to the peroxisome using a signal peptide, Saccharomyces cerevisiae still maintains a good (-)-carvone synthesis ability. The peroxisome may be a preferred organelle for synthesizing monoterpenes and their derivatives. The production method for synthesizing (-)-carvone using Saccharomyces cerevisiae and its peroxisomal subcellular organelles disclosed in the present invention will lay a foundation for the industrial production of carvone.

[0188] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

[0189] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A recombinant bacterium, characterized in that: The recombinant bacteria can synthesize (-)-carvone using (-)-limonene as a raw material.

2. The recombinant bacterium according to claim 1, characterized in that The following metabolic reactions exist in the recombinant bacteria: (A1) converting (-)-limonene into (-)-trans-carveol; (A2) Conversion of (-)-trans-carveol to (-)-carvone.

3. The recombinant bacterium according to claim 2, characterized in that The recombinant bacteria also have metabolic reactions (A3) and / or (A4): (A3) converting isopentenyl pyrophosphate into geranyl pyrophosphate, and then converting geranyl pyrophosphate into (-)-limonene; (A4) Converting dimethylallyl pyrophosphate into neryl pyrophosphate, and then converting neryl pyrophosphate into (-)-limonene.

4. The recombinant bacterium according to claim 3, characterized in that The recombinant bacteria also have metabolic reactions (A5) and / or (A6): (A5) converting 3-hydroxy-3-methylglutaryl-CoA into mevalonate; (A6) Converting isopentenyl pyrophosphate into dimethylallyl pyrophosphate or converting dimethylallyl pyrophosphate into isopentenyl pyrophosphate.

5. The recombinant bacterium according to claim 4, characterized in that Said (A1) is catalyzed by cytochrome P450 limonene-6-hydroxylase and cytochrome P450 reductase; The (A2) is catalyzed by carveol dehydrogenase; In said (A3), the conversion of isopentenyl pyrophosphate into geranyl pyrophosphate is catalyzed by GPP synthase, and the conversion of geranyl pyrophosphate into (-)-limonene is catalyzed by (-)-limonene synthase; In said (A4), the conversion of dimethylallyl pyrophosphate into NPP is catalyzed by orange acyl diphosphate synthase, and the conversion of NPP into (-)-limonene is catalyzed by (-)-limonene synthase; The (A5) is catalyzed by 3-hydroxy-3-methylglutaryl-CoA reductase; The (A6) is catalyzed by isoprene pyrophosphate isomerase.

6. The recombinant bacterium according to any one of claims 1 to 5, characterized in that The recombinant bacteria is recombinant bacteria R1, recombinant bacteria R2 or recombinant bacteria R3; The recombinant bacterium R1 has the following characteristics B1)-B3): B1) containing a cytochrome P450 limonene-6-hydroxylase gene or / and a substance that regulates the expression of the cytochrome P450 limonene-6-hydroxylase gene or / and a substance that regulates the activity or content of the cytochrome P450 limonene-6-hydroxylase; B2) containing cytochrome P450 reductase gene or / and containing substances that regulate the expression of cytochrome P450 reductase gene or / and containing substances that regulate the activity or content of cytochrome P450 reductase; B3) containing carveol dehydrogenase gene or / and containing substances that regulate the expression of carveol dehydrogenase gene or / and containing substances that regulate the activity or content of carveol dehydrogenase; In addition to the characteristics of B1) to B3), the recombinant bacterium R2 also has the following characteristics B4) and / or B5): B4) containing a GPP synthase gene or / and a substance that regulates the expression of the GPP synthase gene or / and a substance that regulates the activity or content of the GPP synthase; Containing (-)-limonene synthase gene or / and containing substances that regulate the expression of (-)-limonene synthase gene or / and containing substances that regulate the activity or content of (-)-limonene synthase; B5) containing a gene for orange acyl diphosphate synthase or / and a substance that regulates the expression of the gene for orange acyl diphosphate synthase or / and a substance that regulates the activity or content of orange acyl diphosphate synthase; containing a gene for (-)-limonene synthase or / and a substance that regulates the expression of the gene for (-)-limonene synthase or / and a substance that regulates the activity or content of (-)-limonene synthase; In addition to the characteristics of the recombinant bacterium R2, the recombinant bacterium R3 also has the following characteristics B6) and / or B7): B6) containing a 3-hydroxy-3-methylglutaryl-CoA reductase gene or / and a substance that regulates the expression of the 3-hydroxy-3-methylglutaryl-CoA reductase gene or / and a substance that regulates the activity or content of 3-hydroxy-3-methylglutaryl-CoA reductase; B7) contains an isoprene pyrophosphate isomerase gene or / and contains a substance that regulates the expression of the isoprene pyrophosphate isomerase gene or / and contains a substance that regulates the activity or content of isoprene pyrophosphate isomerase.

7. The recombinant bacterium according to any one of claims 1 to 6, characterized in that The recombinant bacteria is a recombinant yeast, and the recombinant yeast is prepared by performing the following operations on the yeast as the recipient bacteria: C1) introducing a cytochrome P450 limonene-6-hydroxylase gene or / and introducing a substance that regulates the expression of the cytochrome P450 limonene-6-hydroxylase gene or / and introducing a substance that regulates the activity or content of the cytochrome P450 limonene-6-hydroxylase; C2) introducing a cytochrome P450 reductase gene or / and introducing a substance that regulates the expression of the cytochrome P450 reductase gene or / and introducing a substance that regulates the activity or content of the cytochrome P450 reductase; C3) introducing a carveol dehydrogenase gene or / and introducing a substance that regulates the expression of the carveol dehydrogenase gene or / and introducing a substance that regulates the activity or content of carveol dehydrogenase; C4) introducing a GPP synthase gene or / and introducing a substance that regulates the expression of the GPP synthase gene or / and introducing a substance that regulates the activity or content of the GPP synthase; C5) introduction of a (-)-limonene synthase gene or / and introduction of a substance that regulates the expression of the (-)-limonene synthase gene or / and introduction of a substance that regulates the activity or content of the (-)-limonene synthase; C6) introducing a gene for orange acyl diphosphate synthase or / and introducing a substance that regulates the expression of the gene for orange acyl diphosphate synthase or / and introducing a substance that regulates the activity or content of orange acyl diphosphate synthase; C7) introducing a 3-hydroxy-3-methylglutaryl-CoA reductase gene or / and introducing a substance that regulates the expression of the 3-hydroxy-3-methylglutaryl-CoA reductase gene or / and introducing a substance that regulates the activity or content of 3-hydroxy-3-methylglutaryl-CoA reductase; C8) Introduction of an isoprene pyrophosphate isomerase gene or / and introduction of a substance that regulates the expression of the isoprene pyrophosphate isomerase gene or / and introduction of a substance that regulates the activity or content of isoprene pyrophosphate isomerase.

8. The recombinant bacterium according to claim 7, characterized in that M1-1) amino acid sequence is a protein consisting of 527 amino acids encoded by SEQ ID No. 1 (3-hydroxy-3-methylglutaryl-CoA reductase); M1-2) a protein derived from M1-1) or having 80% or more identity with the protein shown in M1-1) having the same function as 3-hydroxy-3-methylglutaryl-CoA reductase, obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 527 amino acids encoded by SEQ ID No. 1; M1-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M1-1) or M1-2); M2) The isoprene pyrophosphate isomerase is any one of the following: M2-1) amino acid sequence is a protein consisting of 288 amino acids encoded by SEQ ID No. 2; M2-2) a protein having the same function as isoprene pyrophosphate isomerase, obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 288 amino acids encoded by SEQ ID No. 2, and derived from M2-1) or having 80% or more identity with the protein shown in M2-1); M2-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M2-1) or M2-2); M3) The (-)-limonene synthase is any one of the following: M3-1) The amino acid sequence is a protein consisting of 544 amino acids encoded by SEQ ID No. 3; M3-2) a protein having the same function as (-)-limonene synthase, obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 544 amino acids encoded by SEQ ID No. 3, and derived from M3-1) or having 80% or more identity with the protein shown in M3-1); M3-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M3-1) or M3-2); M4) The GPP synthetase is any one of the following: M4-1) amino acid sequence is a protein consisting of 352 amino acids encoded by SEQ ID No. 4; M4-2) a protein having the same function as GPP synthase obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 352 amino acids encoded by SEQ ID No. 4, and derived from M4-1) or having 80% or more identity with the protein shown in M4-1); M4-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M4-1) or M4-2); M5) The orange acyl diphosphate synthase is any one of the following: M5-1) The amino acid sequence is a protein consisting of 260 amino acids encoded by SEQ ID No.5; M5-2) a protein derived from M5-1) or having 80% or more identity with the protein shown in M5-1) obtained by replacing and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 260 amino acids encoded by SEQ ID No.5; M5-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M5-1) or M5-2); M6) The cytochrome P450 limonene-6-hydroxylase is any one of the following: M6-1) The amino acid sequence is a protein consisting of 496 amino acids encoded by SEQ ID No.6; M6-2) a protein derived from M6-1) or having 80% or more identity with the protein shown in M6-1) obtained by replacing and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 496 amino acids encoded by SEQ ID No. 6; M6-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M6-1) or M6-2); M7) The cytochrome P450 reductase is any one of the following: M7-1) The amino acid sequence is a protein consisting of 646 amino acids encoded by SEQ ID No.7; M7-2) a protein having the same function as cytochrome P450 reductase obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 646 amino acids encoded by SEQ ID No. 7, and derived from M7-1) or having 80% or more identity with the protein shown in M7-1); M7-3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of M7-1) or M7-2); M8) The carveol dehydrogenase is any one of the following: M8-1) The amino acid sequence is a protein consisting of 265 amino acids encoded by SEQ ID No. 8; M8-2) a protein derived from M8-1) or having 80% or more identity with the protein shown in M8-1) obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence of the protein consisting of 265 amino acids encoded by SEQ ID No. 8; M8-3) A fusion protein having the same function is obtained by connecting a tag to the N-terminus and / or C-terminus of M8-1) or M8-2).

9. The recombinant bacterium according to any one of claims 1 to 8, characterized in that Any one or more of the P450 limonene-6-hydroxylase, cytochrome P450 reductase, carveol dehydrogenase, GPP synthase, (-)-limonene synthase, orange acyl diphosphate synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase and isoprene pyrophosphate isomerase can be added with a peroxisomal protein localization expression signal guide peptide.

10. A method for preparing (-)-carvone, characterized in that The method comprises using the recombinant bacteria according to any one of claims 1 to 9 as a fermentation strain and using (-)-limonene as a raw material to prepare (-)-carvone.

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