Biploid recombinant bacterium for producing bisabolol as well as construction method and application of biploid recombinant bacterium

Abstract

The invention relates to a diploid recombinant bacterium for producing bisabolol as well as a construction method and application of the diploid recombinant bacterium. The recombinant bacterium is used for over-expression of farnesyl pyrophosphate synthase, bisabolol synthase, an MVA pathway and an HO gene. According to the invention, a brand new gene modification strategy is designed, a bisabolol synthesis path is constructed in the saccharomycetes, and the HO gene is overexpressed, so that a diploid recombinant bacterium for producing the bisabolol is obtained, the production performance of the strain is further improved, the yield of the bisabolol can be improved by more than 30%, and a new method and a new thought are provided for producing the bisabolol.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and relates to a diploid recombinant bacterium for producing bisabolol, a construction method and an application thereof. Background Art

[0002] Terpenoids are a large class of isoprenoid derivatives found widely in nature, primarily isolated from plants, microorganisms, and marine organisms. Over 50,000 terpenoids have been discovered, including the monoterpene menthol, the sesquiterpene artemisinin, the diterpene paclitaxel, the triterpene ginsenosides, and the tetraterpene carotenoids. (-)-α-Bisabolol (abbreviated as bisabolol) is a monocyclic sesquiterpene alcohol isolated from chamomile and is a multifunctional raw material. Due to its environmentally friendly, versatile, and naturally safe properties, bisabolol has gained market favor in recent years and is widely used in pharmaceuticals, oral hygiene products, personal care products, and food.

[0003] Currently, bisabolol is mainly extracted by distillation from the leaves of eucalyptus trees. The extraction yield is low and the plant resources are limited. It is easily affected by season and place of origin, making it difficult to produce on a large scale. The chemical synthesis of bisabolol faces problems such as difficulty in chiral resolution, low activity and environmental unfriendliness. The rapid development of biotechnology has accelerated the construction of microbial cell factories and provided an effective alternative method for the production of natural products. At present, synthetic biology methods can use microbial hosts as chassis cells, design and modify appropriate metabolic pathways, and use cheap glucose, starch, corn steep liquor, etc. as raw materials to achieve the de novo synthesis of terpenoid products in microbial cells.

[0004] In recent years, the production of bisabolol using microbial cell factories has received increasing attention. For example, CN118638768A discloses an engineered yeast strain containing a (-)-α-bisabolol synthase mutant. The engineered yeast strain containing the (-)-α-bisabolol synthase mutant contains genes encoding the (-)-α-bisabolol synthase mutant Mut4(F324Y), farnesyl pyrophosphate synthase ERG20, isopentenyl pyrophosphate isomerase IDI1, 3-hydroxy-3-methylglutaryl-CoA reductase 1 tHMG1, mevalonate kinase ERG12, and mevalonate-5-diphosphate decarboxylase ERG19. The engineered yeast strain achieves a yield of nearly 70 mg / L in shake flask fermentation.

[0005] In summary, it is of great significance to develop and design engineered bacteria for producing bisabolol in order to increase the yield of bisabolol. Summary of the Invention

[0006] In response to the deficiencies of the existing technology and actual needs, the present invention provides a diploid recombinant bacterium for producing bisabolol, a construction method and application thereof, and constructs a new engineered bacterium for producing bisabolol, providing a new method and new ideas for the production of bisabolol.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a diploid recombinant bacterium for producing bisabolol, wherein the recombinant bacterium overexpresses farnesyl pyrophosphate synthase (ERG20), bisabolol synthase, MVA pathway, and HO genes.

[0009] In the present invention, a new genetic modification strategy was designed to construct a bisabolol synthesis pathway in the starting strain and overexpress the HO gene to obtain a diploid recombinant bacterium that produces bisabolol, further improving the production performance of the strain and providing a new method and new ideas for the production of bisabolol.

[0010] Preferably, the MVA pathway includes acetyl-CoA transferase (ERG10), 3-methyl-3-hydroxyglutaryl-CoA synthase (ERG13), 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1), mevalonate kinase (ERG12), mevalonate-5-phosphate kinase (ERG8), mevalonate-5-pyrophosphate decarboxylase (ERG19) and isopentenyl pyrophosphate isomerase (IDI1).

[0011] Preferably, the starting strain of the recombinant bacteria includes Saccharomyces cerevisiae.

[0012] Preferably, the Saccharomyces cerevisiae comprises Saccharomyces cerevisiae CEN.PK2-1C.

[0013] It can be understood that based on the genetic modification strategy designed in the present invention, a diploid recombinant bacterium that produces bisabolol can be obtained. The relevant amino acid sequences and gene sequences of each enzyme can be selected and adjusted according to actual needs. Sequences with the same function in the art are all suitable for the present invention.

[0014] Preferably, the amino acid sequence of the farnesyl pyrophosphate synthase includes the sequence shown in SEQ ID NO.1.

[0015] Preferably, the amino acid sequence of the bisabolol synthase includes the sequence shown in SEQ ID NO.9.

[0016] Preferably, the amino acid sequence encoded by the HO gene includes the sequence shown in SEQ ID NO.20;

[0017] Preferably, the nucleic acid sequence of the HO gene includes the sequence shown in SEQ ID NO.21.

[0018] Preferably, the nucleic acid sequence of the farnesyl pyrophosphate synthase gene includes the sequence shown in SEQ ID NO.10.

[0019] Preferably, the nucleic acid sequence of the bisabolol synthase gene includes the sequence shown in SEQ ID NO.18 and / or SEQ ID NO.19.

[0020] Preferably, the nucleic acid sequence of the HO gene includes the sequence shown in SEQ ID NO.20.

[0021] Preferably, the amino acid sequence of the acetyl-CoA transferase includes the sequence shown in SEQ ID NO.3.

[0022] Preferably, the amino acid sequence of the 3-methyl-3-hydroxyglutaryl-CoA synthase includes the sequence shown in SEQ ID NO.4.

[0023] Preferably, the amino acid sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase includes the sequence shown in SEQ ID NO.2.

[0024] Preferably, the amino acid sequence of the mevalonate kinase includes the sequence shown in SEQ ID NO.5;

[0025] Preferably, the amino acid sequence of the mevalonate 5-phosphate kinase includes the sequence shown in SEQ ID NO.6.

[0026] Preferably, the amino acid sequence of the mevalonate 5-pyrophosphate decarboxylase includes the sequence shown in SEQ ID NO.7.

[0027] Preferably, the amino acid sequence of the isopentenyl pyrophosphate isomerase includes the sequence shown in SEQ ID NO.8.

[0028] Preferably, the nucleic acid sequence of the acetyl-CoA transferase gene includes the sequence shown in SEQ ID NO.12.

[0029] Preferably, the nucleic acid sequence of the 3-methyl-3-hydroxyglutaryl-CoA synthase gene includes the sequence shown in SEQ ID NO.13.

[0030] Preferably, the nucleic acid sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase gene includes the sequence shown in SEQ ID NO.11.

[0031] Preferably, the nucleic acid sequence of the mevalonate kinase gene includes the sequence shown in SEQ ID NO.14.

[0032] Preferably, the nucleic acid sequence of the mevalonate 5-phosphate kinase gene includes the sequence shown in SEQ ID NO.15.

[0033] Preferably, the nucleic acid sequence of the mevalonate 5-pyrophosphate decarboxylase gene includes the sequence shown in SEQ ID NO.16.

[0034] Preferably, the nucleic acid sequence of the isopentenyl pyrophosphate isomerase gene includes the sequence shown in SEQ ID NO.17.

[0035] Preferably, the farnesyl pyrophosphate synthase gene is located at the lpp1 site on the genome of the recombinant bacterium.

[0036] Preferably, the bisabolol synthase gene is located at the abm1 site on the genome of the recombinant bacterium.

[0037] Preferably, the HO gene is located at the MAT site on the recombinant bacterial genome.

[0038] Preferably, the acetyl-CoA transferase gene is located at the dpp1 site on the genome of the recombinant bacterium.

[0039] Preferably, the 3-methyl-3-hydroxyglutaryl-CoA synthase gene is located at the dpp1 site on the genome of the recombinant bacterium.

[0040] Preferably, the 3-hydroxy-3-methylglutaryl-CoA reductase gene is located at the lpp1 site on the genome of the recombinant bacterium.

[0041] Preferably, the mevalonate kinase gene is located at the ho site on the genome of the recombinant bacterium.

[0042] Preferably, the mevalonate 5-phosphate kinase gene is located at the ho site on the genome of the recombinant bacterium.

[0043] Preferably, the mevalonate 5-pyrophosphate decarboxylase gene is located at the gal80 site on the recombinant bacterial genome.

[0044] Preferably, the isopentenyl pyrophosphate isomerase gene is located at the gal80 site on the recombinant bacterial genome.

[0045] Preferably, one copy of the farnesyl pyrophosphate synthase gene ERG20 (amino acid sequence of SEQ ID NO.1, nucleotide sequence of SEQ ID NO.10) and one copy of the truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 (amino acid sequence of SEQ ID NO.2, nucleotide sequence of SEQ ID NO.11) are inserted into the lpp1 site on the genome; one copy of the acetyl-CoA transferase ERG10 (amino acid sequence of SEQ ID NO.3, nucleotide sequence of SEQ ID NO.12) and one copy of the 3-methyl-3-hydroxyglutaryl-CoA synthase ERG13 (amino acid sequence of SEQ ID NO.4, nucleotide sequence of SEQ ID NO.13) are inserted into the dpp1 site; one copy of the mevalonate kinase ERG12 (amino acid sequence of SEQ ID NO.5, nucleotide sequence of SEQ ID NO.14) and one copy of the mevalonate-5-phosphate kinase ERG8 (amino acid sequence of SEQ ID NO.15) are inserted into the ho site on the genome. NO.6, nucleotide sequence SEQ ID NO.15); one copy of mevalonate-5-pyrophosphate decarboxylase ERG19 (amino acid sequence SEQ ID NO.7, nucleotide sequence SEQ ID NO.16) and one copy of isopentenyl pyrophosphate isomerase IDI1 (amino acid sequence SEQ ID NO.8, nucleotide sequence SEQ ID NO.17) were inserted into the gal80 locus on the genome; two copies of bisabolol synthase (amino acid sequence SEQ ID NO.9, nucleotide sequences SEQ ID NO.18 and SEQ ID NO.19) were inserted into the abm1 locus on the genome; and two copies of truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 (amino acid sequence SEQ ID NO.2, nucleotide sequence SEQ ID NO.11) were inserted into the ahp1 locus on the genome.

[0046] In the present invention, a specific genome integration expression strategy is designed to further improve production performance.

[0047] In a second aspect, the present invention provides a method for constructing the diploid recombinant bacterium for producing bisabolol according to the first aspect, the method comprising:

[0048] The farnesyl pyrophosphate synthase gene, the bisabolol synthase gene, the MVA pathway gene and the HO gene are overexpressed in the starting strain.

[0049] Preferably, the overexpression method comprises inserting the farnesyl pyrophosphate synthase gene, the bisabolol synthase gene, the MVA pathway gene and the HO gene into the genome of the starting strain.

[0050] Preferably, the method of inserting into the genome of the starting strain comprises the CRISPR / CAS method.

[0051] Preferably, the nucleic acid sequence of the gRNA of the CRISPR / CAS method includes the sequence shown in SEQ ID NO.22-SEQ ID NO.27.

[0052] In a third aspect, the present invention provides use of the diploid recombinant bacterium producing bisabolol described in the first aspect in the preparation of bisabolol.

[0053] In a fourth aspect, the present invention provides a method for producing bisabolol, comprising:

[0054] The bisabolol is obtained by fermenting and culturing the diploid recombinant bacteria for producing bisabolol described in the first aspect and purifying the product.

[0055] It is understood that the strain culture methods and bisabolol purification methods related to the art are applicable to the present invention without special limitation.

[0056] Compared with the prior art, the present invention has at least the following beneficial effects:

[0057] The present invention designs a new genetic modification strategy to construct a bisabolol synthesis pathway in yeast and overexpress the HO gene to obtain a diploid recombinant bacterium that produces bisabolol. This further improves the production performance of the strain, and the bisabolol yield can be increased by up to 30%. This provides a new method and new ideas for the production of bisabolol. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Schematic diagram of the design of the recombinant plasmid pYC7 containing the functional HO gene;

[0059] Figure 2 Figure 1 is the colony PCR verification result of diploid strains YC7 and YC8;

[0060] Figure 3 This is the gas chromatogram of bisabolol standard;

[0061] Figure 4 This is the gas chromatogram of the product after strain YC6 sample treatment. DETAILED DESCRIPTION

[0062] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0063] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0064] Example 1

[0065] In this example, a bisabolol-producing strain was constructed.

[0066] The wild-type yeast strain CEN.PK2-1C (purchased from Euroscarf) was used as the starting strain for bisabolol production. CRISPR-Cas9-mediated genome editing, a commonly used synthetic biology tool in Saccharomyces cerevisiae, was used to integrate key enzymes of the bisabolol biosynthesis pathway into the genome. One copy of the farnesyl pyrophosphate synthase gene ERG20 (amino acid sequence: SEQ ID NO. 1, nucleotide sequence: SEQ ID NO. 10) and one copy of the truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 (amino acid sequence: SEQ ID NO. 2, nucleotide sequence: SEQ ID NO. 11) were inserted into the lpp1 locus of the genome. The gRNA-N20 sequence used was ATGTAAAACTGACGTTCGAA (nucleotide sequence: SEQ ID NO. 22), and the corresponding gRNA-1 plasmid was constructed. One copy of acetyl-CoA transferase ERG10 (amino acid sequence of SEQ ID NO.3, nucleotide sequence of SEQ ID NO.12) and one copy of 3-methyl-3-hydroxyglutaryl-CoA synthase ERG13 (amino acid sequence of SEQ ID NO.4, nucleotide sequence of SEQ ID NO.13) were inserted into the dpp1 site, and the gRNA-N20 sequence used was CCAGGGATATCTCCGAATAG (nucleotide sequence of SEQ ID NO.23) to construct the corresponding gRNA-2 plasmid. One copy of mevalonate kinase ERG12 (amino acid sequence of SEQ ID NO.5, nucleotide sequence of SEQ ID NO.14) and one copy of mevalonate-5-phosphate kinase ERG8 (amino acid sequence of SEQ ID NO.6, nucleotide sequence of SEQ ID NO.15) were inserted into the ho site on the genome, and the gRNA-N20 sequence used was GCCGGCTTGATCGACTCAGA (nucleotide sequence of SEQ ID NO.24) to construct the corresponding gRNA-3 plasmid. One copy of mevalonate-5-pyrophosphate decarboxylase ERG19 (amino acid sequence of SEQ ID NO.7, nucleotide sequence of SEQ ID NO.16) and one copy of isopentenyl pyrophosphate isomerase IDI1 (amino acid sequence of SEQ ID NO.8, nucleotide sequence of SEQ ID NO.17) were inserted into the gal80 site on the genome, and the gRNA-N20 sequence used was ATAAGGCTGCTGCTGAACGT (nucleotide sequence of SEQ ID NO.25) to construct the corresponding gRNA-4 plasmid.Two copies of bisabolol synthase (amino acid sequence of SEQ ID NO. 9, nucleotide sequences of SEQ ID NO. 18 and SEQ ID NO. 19) were inserted into the abm1 locus on the genome, and the gRNA-N20 sequence used was ATAGTAAATACACTTGCCTG (nucleotide sequence of SEQ ID NO. 26) to construct the corresponding gRNA-5 plasmid. Two copies of truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 (amino acid sequence of SEQ ID NO. 2, nucleotide sequence of SEQ ID NO. 11) were inserted into the ahp1 locus on the genome, and the gRNA-N20 sequence used was AGTAAAATAGGAACTCCGCG (nucleotide sequence of SEQ ID NO. 27) to construct the corresponding gRNA-6 plasmid.

[0067] Donor plasmid pYC1 containing the complete expression cassettes of ERG20 and tHMG1 and the upstream and downstream homology arms of the integration site lpp1 was constructed; donor plasmid pYC2 containing the complete expression cassettes of ERG10 and ERG13 and the upstream and downstream homology arms of the integration site dpp1; donor plasmid pYC3 containing the complete expression cassettes of ERG12 and ERG8 and the upstream and downstream homology arms of the integration site ho; donor plasmid pYC4 containing the complete expression cassettes of ERG19 and DID1 and the upstream and downstream homology arms of the integration site gal80; donor plasmid pYC5 containing two copies of bisabolol synthase and the upstream and downstream homology arms of the integration site abm1; and donor plasmid pYC6 containing two copies of 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 and the upstream and downstream homology arms of the integration site ahp1 were constructed.

[0068] The bisabolol synthase gene (MrBBS) from Matricaria recutita, available at NCBI, was codon-optimized to improve expression in Saccharomyces cerevisiae. Gene synthesis was performed by GeneWeiZhi, with the nucleotide sequences shown in SEQ ID NOs. 18 and 19, and inserted into the plasmid pUC-GW-kan (purchased from GeneWeiZhi), generating plasmids pUC-GW-kan-MrBBS-v1 and pUC-GW-kan-MrBBS-v2.

[0069] Using the CEN.PK2-1C genome as a template, primers were used to amplify the desired fragments. The required primers and fragments are shown in Table 1. For the construction of plasmid pYC5, the synthetic plasmids pUC-GW-kan-MrBBS-v1 and plasmid pUC-GW-kan-MrBBS-v2 were used as templates, and primers were used to amplify the required synthase fragments MrBBS-v1 and MrBBS-v2. The required primers and fragments are shown in Table 1. After gel recovery of the above fragments using the Tiangen Gum Recovery Kit, the fragments were ligated to the linearized backbone fragment of the expression vector pUC19 after HindIII digestion using the ClonExpress Multis One Step Cloning Kit, Vazyme, Catalog No.: C113-02 using homologous recombination. The reaction system and reaction conditions were all carried out according to the kit instructions. After seamless assembly, Trans1 T1 competent cells were transformed. After sequencing confirmation, donor plasmids pYC1, pYC2, pYC3, pYC4, pYC5 and pYC6 were obtained.

[0070] Table 1

[0071]

[0072]

[0073]

[0074]

[0075] Using primers PF1 / PR7 and plasmid pYC1 as a template, the Donor 1 fragment was amplified and transformed into the competent yeast strain CEN.PK2-1C with a plasmid containing Cas9 and a gRNA-1 plasmid using the PEG / LiAC method, respectively, to obtain strain YC1. Primers PF8 / PR14 were then used to amplify the Donor 2 fragment using plasmid pYC2 as a template. This fragment was then transformed into the competent yeast strain YC1 with a plasmid containing Cas9 and a gRNA-2 plasmid using the PEG / LiAC method, respectively, to obtain strain YC2. Primers PF15 / PR21 were then used to amplify the Donor 3 fragment using plasmid pYC3 as a template. This fragment was then transformed into the competent yeast strain YC2 with a plasmid containing Cas9 and a gRNA-3 plasmid using the PEG / LiAC method, respectively, to obtain strain YC3. Continuing with primers PF22 / PR28, plasmid pYC4 was used as a template to amplify the Donor4 fragment, which was then transformed into the competent yeast strain YC3 using the PEG / LiAC method with the plasmid containing Cas9 and the gRNA-4 plasmid, respectively, to obtain strain YC4. Continuing with primers PF29 / PR35, plasmid pYC5 was used as a template to amplify the Donor5 fragment, which was then transformed into the competent yeast strain YC4 using the PEG / LiAC method with the plasmid containing Cas9 and the gRNA-5 plasmid, respectively, to obtain strain YC5. Continuing with primers PF36 / PR42, plasmid pYC6 was used as a template to amplify the Donor6 fragment, which was then transformed into the competent yeast strain YC5 using the PEG / LiAC method with the plasmid containing Cas9 and the gRNA-6 plasmid, respectively, to obtain strain YC6.

[0076] SEQ ID NO. 1, ERG20 from Saccharomyces cerevisiae, amino acid sequence:

[0077] MASEKEIRRERFLNVFPKLVEELNASLLAYGMPKEACDWYAHSLNYNTPGGKLNRGLSVVDTYAILSNKTVEQLGQEEYEKVAILGWCIELLQAYFLVADDMMDKSITRRGQPCWYKVPEVGEIAINDAFMLEAAIYKLLKSHFRNEKYYIDITELFHEVTFQTELGQLMDLITAPEDKVDLSKFSLKKHSFIVTFKTAYYSFYLPVALAMYVAGITDEKDLKQARDVLIPLGEYFQIQDDYLDCFGTPEQIGKIGTDIQDNKCSWVINKALELASAEQRKTLDENYGKKDSVAEAKCKKIFNDLKIDQLYHEYEESVAKDLKAKISQVDESRGFKADVLTAFLNKVYKRSK。

[0078] SEQ ID NO.2, tHMG1 from Saccharomyces cerevisiae, amino acid sequence:

[0079] MAADQLVKTEVTKKSFTAPVQKASTPVLTNKTVISGSKVKSLSSAQSSSSGPSSSSEEDDSRDIESLDKKIRPLEELEALLSSGNTKQLKNKEVAALVIHGKLPLYALEKKLGDTTRAVAVRRKALSILAEAPVLASDRLPYKNYDYDRVFGACCENVIGYMPLPVGVIGPLVIDGTSYHIPMATTEGCLVASAMRGCKAINAGGGATTVLTKDGMTRGPVVRFPTLKRSGACKIWLDSEEGQNAIKKAFNSTSRFARLQHIQTCLAGDLLFMRFRTTTGDAMGMNMISKGVEYSLKQMVEEYGWEDMEVVSVSGNYCTDKKPAAINWIEGRGKSVVAEATIPGDVVRKVLKSDVSALVELNIAKNLVGSAMAGSVGGFNAHAANLVTAVFLALGQDPAQNVESSNCITLMKEVDGDLRISVSMPSIEVGTIGGGTVLEPQGAMLDLLGVRGPHATAPGTNARQLARIVACAVLAGELSLCAALAAGHLVQSHMTHNRKPAEPTKPNNLDATDINRLKDGSVTCIKS。

[0080] SEQ ID NO. 3, ERG10 from Saccharomyces cerevisiae, amino acid sequence:

[0081] MSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEIIFGNVLSANLGQAPARQVALAAGLSNHIVASTVNKVCASAMKAIILGAQSIKCGNADVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAEKCARDWDITREQQDNFAIESYQKSQKSQKEGKFD NEIVPVTIKGFRGKPDTQVTKDEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLKPLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLSILQQEGGKIGVAAICNGGGGASSIVIEKI.

[0082] SEQ ID NO. 4, ERG13 from Saccharomyces cerevisiae, amino acid sequence:

[0083] MKLSTKLCWCGIKGRLRPQKQQQLHNTNLQMTELKKQKTAEQKTRPQNVGIKGIQIYIPTQCVNQSELEKFDGVSQGKYTIGLGQTNMSFVNDREDIYSMSLTVLSKLIKSYNIDTNKIGRLEVGTETLIDKSKSVKSVLMQLFGENTDVEGIDTLNACYGGTNALFNSLNWIESNAWDGRDAIVVCGDIAIYDKGAARPTGGAGTVAMWIGPDAPIVFDSVRASYMEHAYDFYKPDFTSEYPYVDGHFSLTCYVKALDQVYKSYSKKAISKGLVSDPAGSDALNVLKYFDYNVFHVPTCKLVTKSYGRLLYNDFRANPQLFPEVDAELATRDYDESLTDKNIEKTFVNVAKPFHKERVAQSLIVPTNTGNMYTASVYAAFASLLNYVGSDDLQGKRVGLFSYGSGLAASLYSCKIVGDVQHIIKELDITNKLAKRITETPKDYEAAIELRENAHLKKNFKPQGSIEHLQSGVYYLTNIDDKFRRSYDVKK。

[0084] SEQ ID NO.5, ERG12 from Saccharomyces cerevisiae, amino acid sequence:

[0085] MSLPFLTSAPGKVIIFGEHSAVYNKPAVAASVSALRTYLLISESSAPDTIELDFPDISFNHKWSINDFNAITEDQVNSQKLAKAQQATDGLSQELVSLLDPLLAQLSESFHYHAAFCFLYMFVCLCPHAKNIKFSLKSTLPIGAGLGSSASISVSLALAMAYLGGLIGSNDLEKLSENDKHIVNQWAFIGEKCIHGTPSGIDNAVATYGNALLFEKDSHNGTINTNNFKFLDDFPAIPMILTYTRIPRSTKDLVARVRVLVTEKFPEVMKPILDAMGECALQGLEIMTKLSKCKGTDDEAVETNNELYEQLLELIRINHGLLVSIGVSHPGLELIKNLSDDLLRIGIGSTKLTGAGGGGCSLTLLRRDITQEQIDSFKKKLQDDFSYETFETDLGGTGCCLLSAKNLNKDLKIKSLVFQLFENKTTTKQQIDDLLLPGNTNLPWTS。

[0086] SEQ ID NO.6, ERG8 from Saccharomyces cerevisiae, amino acid sequence:

[0087] MSELRAFSAPGKALLAGGYLVLDPKYEAFVVGLSARMHAVAHPYGSLQESDKFEVRVKSKQFKDGEWLYHISPKTGFIPVSIGGSKNPFIEKVIANVFSYFKPNMDDYCNRNLFVIDIFSDDAYHSQEDSVTEHRGNRRLSFHSHRIEEVPKTGLGSSAGLVTVLTTALASFFVSDLENNVDKYREVIHNLSQVAHCQAQGKIGSGFDVAAAAYGSIRYRRFPPALISNLPDIGSATYGSKLAHLVNEEDWNITIKSNHLPSGLTLWMGDIKNGSETVKLVQKVKNWYDSHMPESLKIYTELDHANSRFMDGLSKLDRLHETHDDYSDQIFESLERNDCTCQKYPEITEVRDAVATIRRSFRKITKESGADIEPPVQTSLLDDCQTLKGVLTCLIPGAGGYDAIAVIAKQDVDLRAQTADDKRFSKVQWLDVTQADWGVRKEKDPETYLDK。

[0088] SEQ ID NO.7, ERG19 from Saccharomyces cerevisiae, amino acid sequence:

[0089] MTVYTASVTAPVNIATLKYWGKRDTKLNLPTNSSISVTLSQDDLRTLTSAATAPEFERDTLWLNGEPHSIDNERTQNCLRDLRQLRKEMESKDASLPTLSQWKLHIVSENNFPTAAGLASSAAGFAALVSAIAKLYQLPQSTSEISRIARKGSGSACRSLFGGYVAWEMGKAEDGHDSMAVQIADSSDWPQMKACVLVVSDIKKDVSSTQGMQLTVATSELFKERIEHVVPKRFEVMRKAIVEKDFATFAKETMMDSNSFHATCLDSFPPIFYMNDTSKRIISWCHTINQFYGETIVAYTFDAGPNAVLYYLAENESKLFAFIYKLFGSVPGWDKKFTTEQLEAFNHQFESSNFTARELDLELQKDVARVILTQVGSGPQETNESLIDAKTGLPKE。

[0090] SEQ ID NO. 8, IDI1 from Saccharomyces cerevisiae, amino acid sequence:

[0091] MTADNNSMPHGAVSSYAKLVQNQTPEDILEEFPEIIPLQQRPNTRSSETSNDESGETCFSGHDEEQIKLMNENCIVLDWDDNAIGAGTKKVCHLMENIEKGLLHRAFSVFIFNEQGELLLQQRATEKITFPDLWTNTCCSHPLC IDDELGLKGKLDDKIKGAITAAVRKLDHELGIPEDETKTRGKFHFLNRIHYMAPSNEPWGEHEIDYILFYKINAKENLTVNPNVNEVRDFKWVSPNDLKTMFADPSYKFTPWFKIICENYLFNWWEQLDDLSEVENDRQIHRML.

[0092] SEQ ID NO.9, MrBBS from Matricaria recutita, amino acid sequence:

[0093] MSTLSVSTPSFSSSPLSSVNKNSTKQHVTRNSVIFHDSIWGDQFLEYKEKFNVATEKQLIEELKEEVRNELMIRACNEASRYIKLIQLIDVVERLGLAYHFEKEIEESLQHIYVTYGHKWTNYNNIESLSLWFRLLRQNGFNVSSDIFENHIDEKGNFQESLCNDPQGMLALYEAAYMRVEGEIILDKALEFTKLHLGIISNDPSCDSSLRTEIKQALKQPLRRRLPRLEAVRYIAIYQQKASHSEVLLKLAKLDFNVLQEMHKDELSQICKWWKDLDIRNKLPYVRDRLIEGYFWILGIYFEPQHSRTRMFLMKTCMWLIVLDDTFDNYGTYEELEIFTQAVERWSITCLDELPEYMKLIYHEQFRVHQEMEESLEKEGKAYQIHYIKEMAKEGTRSLLLEAKWLKEGYMPTLDEYLSNSLVTCGYALMTARSYVARDDGIVTEDAFKWVATHPPIVKAACKILRLMDDIATHKEEQERGHIASSIECYRKETGASEEEACMDFLKQVEDGWKVINQESLMPTDVPFPLLIPAINLARADLYKDNDGYNHADKEVIGYIKSLFVHPMIV。

[0094] SEQ ID NO.10, ERG20 from Saccharomyces cerevisiae, nucleotide sequence:

[0095]

[0096] Example 2

[0097] In this example, a diploid strain producing bisabolol was constructed.

[0098] The production performance of the strain was further improved by mating the haploid MATa yeast strain in the hope of obtaining the diploid MATα / MATa strain.

[0099] (1) Construction of diploid transformation plasmid

[0100] Gene synthesis of a functional HO gene (amino acid sequence of SEQ ID NO. 20, nucleotide sequence of SEQ ID NO. 21) was performed by GENEWIZ, and the gene was inserted into the plasmid pUC-GW-kan (purchased from GENEWIZ) to obtain the plasmid pUC-GW-kan-HO.

[0101] First, using the Saccharomyces cerevisiae CEN.PK2-1C genome as a template, PCR amplification was performed using gene-specific primer pairs PF43 / PR43 and PF44 / PR44 (using 2× Phanta Max Master Mix (Dye Plus), Vazyme) to obtain the promoter pGAL10 fragment and the terminator tHO fragment, respectively. Using the plasmid pUC-GW-kan-HO as a template, PCR amplification was performed using gene-specific primer pair PF45 / PR45 to obtain the HO gene fragment. These fragments were gel-recovered using a Tiangen Gum Recovery Kit and ligated into the linearized backbone fragment of the pYES2 vector digested with NheI. Homologous recombination was performed using the ClonExpress Multis One Step Cloning Kit (Vazyme, Catalog No. C113-02), using the kit's instructions for reaction system and conditions. After seamless assembly, Trans1T1 competent cells were transformed. After sequencing confirmation, the recombinant plasmid pYC7 containing the functional HO gene was obtained. The structural diagram is shown in Figure 1 shown.

[0102] (2) Construction of diploid production strains

[0103] The recombinant plasmid pYC7 containing the functional HO gene was transformed into the competent yeast strains YC5 and YC6 by the PEG / LiAC method. After overnight recovery in YPD (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L) medium, it was spread on a YPD solid medium plate containing the selection antibiotic hygromycin (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, agar powder 20 g / L, hygromycin 300 mg / L) and cultured at 30 ° C for 48 hours. The DNA of the MAT site was quickly detected by polymerase chain reaction (PCR) to clearly identify the mating type of the haploid strain and confirm the diploid strain. The specific steps of the PCR method are as follows: colony PCR verification was performed using three primers DP1 / DP2 / DP3 at the same time ( Figure 2 ), DNA at the MATα site will produce a 404bp product, while DNA at the MATa site will produce a 544bp product. Haploid colonies of commonly used laboratory yeast strains will produce corresponding MATα- or MATA-specific products, while diploid colonies will produce both products. Colony PCR can be performed directly on yeast colonies without the need for DNA purification steps, and ultimately the diploid strains YC7 and YC8 were successfully screened.

[0104] Table 2

[0105]

[0106] Example 3

[0107] This example provides a fermentation process for producing bisabolol by Saccharomyces cerevisiae and a method for detecting the product.

[0108] (1) Fermentation process

[0109] The fermentation process is carried out in a 2L fermenter, and the strain needs to be cultured in a seed shake flask before entering the fermenter.

[0110] The seed shake flask medium is LGM medium, and the components of LGM medium are as follows: ammonium sulfate 15g / L, potassium dihydrogen phosphate 8g / L; magnesium sulfate heptahydrate 6.15g / L; biotin 0.05g / L; calcium pantothenate 1g / L; niacin 1g / L; inositol 25g / L; thiamine hydrochloride 1g / L; pyridoxal hydrochloride 1g / L; p-aminobenzoic acid 0.2g / L; zinc sulfate heptahydrate 0.0575g / L; manganese chloride tetrahydrate 0.0032g / L; copper sulfate pentahydrate 0.0032g / L; cobalt chloride hexahydrate 0.0047g / L; sodium molybdate dihydrate 0.0048g / L; ferrous sulfate heptahydrate 0.028g / L; calcium chloride dihydrate 0.029g / L; 0.5M EDTA (80mL / L); succinic acid 5.9g / L, and the pH was adjusted to pH 6.0.

[0111] The basic culture medium of the fermentation tank is LGM culture medium, and the composition and content of the culture medium are the same as those of the seed shake flask.

[0112] The seed culture medium and fermentation medium were sterilized by autoclaving (121°C, 20 min). When preparing solid plates, 2% agar was added to the culture medium.

[0113] Inoculate a single colony of the biological strain to be verified constructed in Examples 1 and 2 into a sterile seed shake flask and culture it at 30°C and 150rpm for 24h. Transfer the grown seed shake flask to a fermenter for culture. The initial liquid volume of the fermenter is 500mL, and the inoculation volume is 10%. After inoculation, the temperature of the fermenter is controlled at 30°C; use ammonia water to adjust the pH to 6.0±0.1; the dissolved oxygen is controlled at 40%, which is adjusted by adjusting the rotation speed and ventilation volume. After the initial sugar in the fermenter is exhausted, the dissolved oxygen rebounds and enters the feeding stage. The feeding medium is 600g / L glucose solution, and the feeding rate is controlled at 6g / L / h. As the feeding proceeds, the volume of the fermentation broth gradually increases. When the volume of the fermentation broth reaches about 1.5L, the fermentation ends.

[0114] Take an appropriate amount of bacterial liquid sample from the tank, add an appropriate amount of methanol, shake vigorously for 30 minutes, and then centrifuge. Then add an appropriate amount of n-heptane, shake vigorously for 30 minutes, and then centrifuge. Take an appropriate amount of supernatant to determine the titer of the product after culture.

[0115] (2) Detection of product titer

[0116] Analysis was performed on an Agilent GC8890 gas chromatograph equipped with an Agilent HP-5 capillary column (30.0 m × 0.32 mm × 0.25 μm). The sample injection volume was 1 μL, the split ratio was 50:1, the air flow rate was 300 mL / min, the hydrogen flow rate was 30 mL / min, and the carrier gas flow rate was 25 mL / min. The injector and FID detector temperatures were set to 280°C and 300°C, respectively. The gas flow rate through the column was set at 2 mL / min for the first 3 minutes and 3 mL / min for the next 5.375 minutes. The column temperature was initially maintained at 150°C for 1.5 minutes, then increased at a rate of 16°C / min to 180°C, held for 0 minutes, and then increased at a rate of 120°C / min to 300°C and held for 4 minutes. Quantification was performed using an internal standard method. A standard curve was established based on the gas phase to determine the concentration of bisabolol in the intact culture medium.

[0117] (3) Fermentation testing of haploid and diploid production strains

[0118] The diploid strain YC7 constructed in Examples 1 and 2, as well as its mother haploid strain YC5, diploid strain YC8, and its mother haploid strain YC6 were fermented in a fermenter. When the fermentation liquid volume reached about 1.5 L, the fermentation was terminated. After the fermentation tank sample was processed, gas chromatography was performed. The gas chromatogram of the bisabolol standard is shown in FIG. Figure 3 As shown in Figure 2, the peak time of bisabolol is 4.217 min. Figure 4 As shown (using fermentation products from strain YC6 as an example), fermentation samples tested revealed that the bisabolol yield of the diploid strain YC7 increased by 31.8% compared to the haploid strain (YC5). Furthermore, the bisabolol yield of the diploid strain YC8 increased by 15.8% compared to the haploid strain (YC6). This indicates that switching yeast zygosity and automatically forming diploid yeasts can improve strain production performance. The magnitude of the overall yield increase is shown in Table 3.

[0119] Table 3

[0120] Strain name Bisabolol yield (g / L) Output increase (%) YC5 15.8 / YC6 22.6 / YC7 20.8 31.6 YC8 26.1 15.5

[0121] The present invention designs a new genetic modification strategy to construct a bisabolol synthesis pathway in yeast and overexpress the HO gene to obtain a diploid recombinant bacterium that produces bisabolol. This further improves the production performance of the strain, and the bisabolol yield can be increased by up to 30%. This provides a new method and new ideas for the production of bisabolol.

[0122] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A diploid recombinant bacterium for producing bisabolol, characterized in that: The recombinant bacteria overexpress farnesyl pyrophosphate synthase, bisabolol synthase, MVA pathway and HO genes.

2. The diploid recombinant bacterium for producing bisabolol according to claim 1, characterized in that: The MVA pathway includes one or more of acetyl-CoA transferase, 3-methyl-3-hydroxyglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, mevalonate-5-phosphate kinase, mevalonate-5-pyrophosphate decarboxylase, or isopentenyl pyrophosphate isomerase.

3. The diploid recombinant bacterium for producing bisabolol according to claim 1 or 2, characterized in that: The starting strain of the recombinant bacteria includes Saccharomyces cerevisiae; Preferably, the Saccharomyces cerevisiae comprises Saccharomyces cerevisiae CEN.PK2-1C.

4. The diploid recombinant bacterium for producing bisabolol according to claim 1, characterized in that: The amino acid sequence of the farnesyl pyrophosphate synthase includes the sequence shown in SEQ ID NO.1; Preferably, the amino acid sequence of the bisabolol synthase includes the sequence shown in SEQ ID NO.9; Preferably, the amino acid sequence encoded by the HO gene includes the sequence shown in SEQ ID NO.20; Preferably, the nucleic acid sequence of the HO gene includes the sequence shown in SEQ ID NO.21; Preferably, the nucleic acid sequence of the farnesyl pyrophosphate synthase gene includes the sequence shown in SEQ ID NO.10; Preferably, the nucleic acid sequence of the bisabolol synthase gene includes the sequence shown in SEQ ID NO.18 and / or SEQ ID NO.19; Preferably, the nucleic acid sequence of the HO gene includes the sequence shown in SEQ ID NO.

20.

5. The diploid recombinant bacterium for producing bisabolol according to any one of claims 2 to 4, characterized in that: The amino acid sequence of the acetyl-CoA transferase includes the sequence shown in SEQ ID NO.3; Preferably, the amino acid sequence of the 3-methyl-3-hydroxyglutaryl-CoA synthase includes the sequence shown in SEQ ID NO.4; Preferably, the amino acid sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase includes the sequence shown in SEQ ID NO.2; Preferably, the amino acid sequence of the mevalonate kinase includes the sequence shown in SEQ ID NO.5; Preferably, the amino acid sequence of the mevalonate-5-phosphate kinase includes the sequence shown in SEQ ID NO.6; Preferably, the amino acid sequence of the mevalonate-5-pyrophosphate decarboxylase comprises the sequence shown in SEQ ID NO.7; Preferably, the amino acid sequence of the isopentenyl pyrophosphate isomerase includes the sequence shown in SEQ ID NO.8; Preferably, the nucleic acid sequence of the acetyl-CoA transferase gene includes the sequence shown in SEQ ID NO.12; Preferably, the nucleic acid sequence of the 3-methyl-3-hydroxyglutaryl-CoA synthase gene includes the sequence shown in SEQ ID NO.13; Preferably, the nucleic acid sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase gene includes the sequence shown in SEQ ID NO.11; Preferably, the nucleic acid sequence of the mevalonate kinase gene includes the sequence shown in SEQ ID NO.14; Preferably, the nucleic acid sequence of the mevalonate-5-phosphate kinase gene includes the sequence shown in SEQ ID NO.15; Preferably, the nucleic acid sequence of the mevalonate-5-pyrophosphate decarboxylase gene includes the sequence shown in SEQ ID NO.16; Preferably, the nucleic acid sequence of the isopentenyl pyrophosphate isomerase gene includes the sequence shown in SEQ ID NO.

17.

6. The diploid recombinant bacterium for producing bisabolol according to any one of claims 2 to 5, characterized in that: The farnesyl pyrophosphate synthase gene is located at the lpp1 site on the recombinant bacterial genome; Preferably, the bisabolol synthase gene is located at the abm1 site on the recombinant bacterial genome; Preferably, the HO gene is located at the MAT site on the recombinant bacterial genome; Preferably, the acetyl-CoA transferase gene is located at the dpp1 site on the recombinant bacterial genome; Preferably, the 3-methyl-3-hydroxyglutaryl-CoA synthase gene is located at the dpp1 site on the recombinant bacterial genome; Preferably, the 3-hydroxy-3-methylglutaryl-CoA reductase gene is located at the lpp1 site on the recombinant bacterial genome; Preferably, the mevalonate kinase gene is located at the ho site on the recombinant bacterial genome; Preferably, the mevalonate-5-phosphate kinase gene is located at the ho site on the recombinant bacterial genome; Preferably, the mevalonate-5-pyrophosphate decarboxylase gene is located at the gal80 site on the recombinant bacterial genome; Preferably, the isopentenyl pyrophosphate isomerase gene is located at the gal80 site on the recombinant bacterial genome.

7. A method for constructing a diploid recombinant bacterium for producing bisabolol according to any one of claims 1 to 6, characterized in that: The method comprises: The farnesyl pyrophosphate synthase gene, the bisabolol synthase gene, the MVA pathway gene and the HO gene are overexpressed in the starting strain.

8. The method for constructing a diploid recombinant bacterium for producing bisabolol according to claim 7, wherein: The overexpression method comprises inserting the farnesyl pyrophosphate synthase gene, the bisabolol synthase gene, the MVA pathway gene and the HO gene into the genome of the starting strain; Preferably, the method of inserting into the genome of the starting strain comprises CRISPR / CAS method; Preferably, the nucleic acid sequence of the gRNA of the CRISPR / CAS method includes the sequence shown in SEQ ID NO.22-SEQ ID NO.

27.

9. Use of the diploid recombinant bacterium producing bisabolol according to any one of claims 1 to 6 in the preparation of bisabolol.

10. A method for producing bisabolol, characterized in that: The method for producing bisabolol comprises: Fermentation and culturing the diploid recombinant bacterium for producing bisabolol according to any one of claims 1 to 6, and purifying the product to obtain the bisabolol.

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