CN117987434A - Patchouli alcohol synthase coding gene and expression system thereof - Google Patents
Patchouli alcohol synthase coding gene and expression system thereof Download PDFInfo
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- CN117987434A CN117987434A CN202410404349.5A CN202410404349A CN117987434A CN 117987434 A CN117987434 A CN 117987434A CN 202410404349 A CN202410404349 A CN 202410404349A CN 117987434 A CN117987434 A CN 117987434A
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- GGHMUJBZYLPWFD-UHFFFAOYSA-N patchoulialcohol Chemical compound C1CC2(C)C3(O)CCC(C)C2CC1C3(C)C GGHMUJBZYLPWFD-UHFFFAOYSA-N 0.000 title claims abstract description 142
- GGHMUJBZYLPWFD-MYYUVRNCSA-N Patchouli alcohol Natural products O[C@@]12C(C)(C)[C@H]3C[C@H]([C@H](C)CC1)[C@]2(C)CC3 GGHMUJBZYLPWFD-MYYUVRNCSA-N 0.000 title claims abstract description 70
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 34
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 239000013598 vector Substances 0.000 claims description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 102100036826 Aldehyde oxidase Human genes 0.000 claims description 10
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 5
- 239000000834 fixative Substances 0.000 claims description 3
- 230000006698 induction Effects 0.000 claims description 3
- 239000002304 perfume Substances 0.000 claims description 3
- 230000001939 inductive effect Effects 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 239000013612 plasmid Substances 0.000 description 16
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 241000894006 Bacteria Species 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 241000235058 Komagataella pastoris Species 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 101150051118 PTM1 gene Proteins 0.000 description 5
- 235000011187 glycerol Nutrition 0.000 description 5
- 238000010276 construction Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000001738 pogostemon cablin oil Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 3
- 239000003205 fragrance Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 239000000600 sorbitol Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 244000146510 Pereskia bleo Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 241000235015 Yarrowia lipolytica Species 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 239000012159 carrier gas Substances 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 239000001307 helium Substances 0.000 description 2
- 229910052734 helium Inorganic materials 0.000 description 2
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- GGHMUJBZYLPWFD-CUZKYEQNSA-N patchouli alcohol Chemical compound C1C[C@]2(C)[C@@]3(O)CC[C@H](C)[C@@H]2C[C@@H]1C3(C)C GGHMUJBZYLPWFD-CUZKYEQNSA-N 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241001529821 Agastache Species 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241000490050 Eleutherococcus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 1
- 241000207923 Lamiaceae Species 0.000 description 1
- 229910004619 Na2MoO4 Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000002505 Pogostemon cablin Species 0.000 description 1
- 235000011751 Pogostemon cablin Nutrition 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001754 anti-pyretic effect Effects 0.000 description 1
- 239000002111 antiemetic agent Substances 0.000 description 1
- 229940125683 antiemetic agent Drugs 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- 229940125716 antipyretic agent Drugs 0.000 description 1
- 239000010692 aromatic oil Substances 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229910052927 chalcanthite Inorganic materials 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000013530 defoamer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000012526 feed medium Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000019837 monoammonium phosphate Nutrition 0.000 description 1
- 239000006012 monoammonium phosphate Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 229940094933 n-dodecane Drugs 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229930004725 sesquiterpene Natural products 0.000 description 1
- -1 sesquiterpene compound Chemical class 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 238000001256 steam distillation Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
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- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
- C12Y402/0307—Patchoulol synthase (4.2.3.70)
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
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Abstract
The invention provides a patchouli alcohol synthase coding gene and an expression system thereof, belonging to the technical field of patchouli alcohol production. The invention provides an optimized patchouli alcohol synthase coding gene and an evolved patchouli alcohol synthase coding gene, which can be used for heterologously synthesizing patchouli alcohol, and solve the problem that the patchouli alcohol yield needs to be further improved in the prior art. The invention effectively improves the yield of patchouli alcohol based on the designed coding gene and the expression system constructed by combining, and can be used for realizing the efficient patchouli alcohol production in the actual industry.
Description
Technical Field
The invention relates to the technical field of patchouli alcohol production, in particular to patchouli alcohol synthase coding genes and an expression system thereof.
Background
Herba Agastaches is plant of genus Eleutherococcus of family Labiatae, and is an aromatic stomach invigorating, antipyretic and antiemetic agent. The aromatic oil has strong fragrance, can be used as an excellent fragrance fixative, and is widely applied to the field of cosmetics. The main component of the patchouli with fragrance is its volatile oil, the content is about 1.5%.
Patchouli alcohol (Patchoulol), also known as patchouli alcohol, is the most important component of patchouli oil, accounting for over 50% of patchouli oil. About 1/3 of the higher perfumes are used worldwide with patchouli alcohol. Patchoulol is a tricyclic sesquiterpene compound with a spatial structure of tetrahedron with 5 chiral carbon molecules. The molecular formula is C 15H26 O, the molecular weight is 222.37, the boiling point is 280 ℃ (under normal pressure), and the relative density is 1.001g/cm 3; is insoluble in water, soluble in alcohols, ethers and common organic solvents.
At present, the preparation method of patchouli alcohol mainly comprises a plant extraction method, a chemical synthesis method and an organic solvent separation method. At present, the main production mode of patchouli oil in China is still the traditional plant extraction method which uses steam distillation for extraction. The production method is time-consuming, consumes a large amount of energy, has high cost and pollution, and is unstable due to the influence of environmental factors on the quality of the produced patchouli oil. The chemical method has complex synthesis steps, more byproducts, lower yield and higher cost; the chemical separation method can obtain high-purity patchouli alcohol, but the yield is extremely low, and the industrial production is difficult.
Chinese patent CN113549562A discloses an engineering bacterium for efficiently producing patchouli alcohol, a construction method and application thereof, wherein the engineering bacterium is obtained by taking yarrowia lipolytica as an original strain and inserting optimized patchouli alcohol synthase coding genes PS1 and tHMGR coding genes into the genome of the yarrowia lipolytica; the engineering bacteria can synthesize patchouli alcohol, and the construction method is efficient and simple to operate.
However, the yield of patchouli alcohol in the prior art needs to be further improved.
Disclosure of Invention
The invention aims to provide a patchouli alcohol synthase coding gene and an expression system thereof, which can effectively improve the yield of patchouli alcohol.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an optimized patchouli alcohol synthase coding gene, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
The invention also provides an evolutionary patchouli alcohol synthase coding gene, and the nucleotide sequence of the gene is shown as SEQ ID NO. 4.
The invention also provides a recombinant vector, which comprises an initial vector and the optimized patchouli alcohol synthase coding gene.
The invention also provides another recombinant vector, which comprises an initial vector and the gene for encoding the patchouli alcohol synthase.
Preferably, the initial vector is a pPICZA vector, a pHIL-S1 vector, a pYAM P vector, a pPIC9 vector or a pPIC9K vector.
Preferably, the initial vector contains an AOX1 promoter and an AOX1 terminator, and the optimized patchouli alcohol synthase encoding gene or the evolved patchouli alcohol synthase encoding gene is inserted between the AOX1 promoter and the AOX1 terminator.
The invention also provides a recombinant strain for expressing patchouli alcohol synthase, wherein any one of the recombinant vectors is transformed into the recombinant strain.
Preferably, the recombinant strain expresses patchouli alcohol synthase by methanol induction.
The invention also provides application of the optimized patchouli alcohol synthase encoding gene, the evolved patchouli alcohol synthase encoding gene, the recombinant vector or the recombinant strain in patchouli alcohol production and/or preparation of a perfume fixative.
The invention also provides a preparation method of patchouli alcohol, which comprises the following steps:
(1) Transforming a host cell with any one of the recombinant vectors described above to obtain a recombinant strain;
(2) Culturing recombinant strain, and inducing to synthesize patchouli alcohol;
(3) Recovering and purifying the expressed patchouli alcohol.
The invention has the beneficial effects that:
The invention provides an optimized patchouli alcohol synthase coding gene and an evolved patchouli alcohol synthase coding gene, which can be used for heterologously synthesizing patchouli alcohol, effectively improves the yield of patchouli alcohol, and can be used for realizing high-efficiency patchouli alcohol production in actual industry.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The culture medium and the preparation thereof in the embodiment of the invention:
YPD medium: 20g/L peptone, 10g/L yeast extract, 20g/L glucose, and 20g/L agar powder.
Fermentation medium: 5g/L of monopotassium phosphate, 0.27g/L of calcium sulfate, 5g/L of magnesium sulfate, 10g/L of potassium sulfate, 40g/L of monoammonium phosphate, 1.65g/L of potassium hydroxide, 20g/L of glycerin, 0.33g/L of defoamer, 2g/L of histidine and 12ml/L of PTM1.
Feed medium: 50% (v/v) glycerol, 12ml/L PTM1.
The formulation of the trace metal solution PTM1 solution is ：CuSO4·5H2O 6g/L,KI 0.09g/L,MnSO4·H2O 3g/L,Na2MoO4·2H2O 0.2g/L, boric acid 0.02g/L, coCl 2·6H2O 0.5g/L,FeSO4·7H2O 65g/L,ZnCl2 g/L, sulfuric acid 5ml and biotin 0.2g/L.
The patchouli alcohol detection method in the embodiment of the invention comprises the following steps:
1. Standard samples of patchouli alcohol were purchased from Shanghai leaf Biotechnology Inc.
2, Preparing patchouli alcohol standard substance solution: weighing 20mg patchouli alcohol standard substance, dissolving in n-dodecane, and fixing volume in 1L volumetric flask, wherein the concentration of standard substance mother liquor is 20mg/L.
And 3, carrying out qualitative and quantitative analysis on patchouli alcohol by adopting gas phase mass spectrometry: the GC system (Agilent 7820A, USA) was equipped with HP-5 chromatography column HP-5 (30 m*0.25 mm*0.25 μm film thickness) and Flame Ionization Detector (FID). The initial temperature was 100deg.C, maintained for 1min, at 14deg.C/min to 240deg.C, maintained for 1min, at 35deg.C/min to 280 deg.C, and maintained for 1 min. The carrier gas is high-purity helium, the flow rate is 1.0 mL/min, the sample injection is 1.0 mu L, and no flow division is performed. MS conditions: using the EI ion source, electron Multiplication Voltage (EMV) mode, the gain factor was 1.01, and the actual EMV was 1.576×10 3 V.
Example 1
Construction of patchouli alcohol producing Strain
Obtaining target genes: the patchouli alcohol synthase coding gene PTS is synthesized by Nanjing Jinsri biotechnology Co., ltd, and inserted into plasmid pPICZA. The nucleotide sequence of the optimized PTS is shown in SEQ ID NO. 1:
ATGGAATTGTACGCTCAATCCGTTGGAGTTGGTGCTGCCAGTAGACCATTGGCTAACTTTCATCCATGTGTTTGGGATGATAAGTTTATTGTTTACAACCCTCAATCCTGTCAAGCTGGTGAAAGAGAAGAAGCTGAAGAATTGAAGGTTGAATTGAAGAGAGAATTGAAGGAGGCGTCCGATAACTACATGAGACAATTGAAGATGGTTGATGCTATTCAAAGATTGGGTATTGATTACTTGTTTGTTGAAGATGTTGATGAGGCATTGAAGAACTTGTTTGAAATGTTTGATGCTTTTTGTAAGAACAACCATGATATGCACGCTACTGCTTTGTCCTTTAGATTGTTGAGACAACATGGATACAGAGTTTCCTGTGAAGTTTTTGAAAAGTTTAAGGATGGTAAGGATGGATTTAAGGTTCCAAACGAAGATGGTGCTGTTGCTGTTTTGGAATTTTTTGAAGCTACTCATTTGAGAGTTCATGGTGAAGATGTTTTGGATAACGCTTTTGATTTTACTAGAAACTACTTGGAATCCGTTTACGCTACTTTGAACGATCCAACTGCTAAGCAAGTTCATAACGCTTTGAACGAATTTTCATTTAGAAGAGGATTGCCTAGAGTTGAAGCTAGAAAGTACATTTCTATTTACGAACAATACGCTTCACATCATAAGGGATTGTTGAAGTTGGCTAAGTTGGATTTTAACTTGGTTCAAGCGTTGCATAGAAGAGAATTGTCCGAAGATTCCAGATGGTGGAAAACTTTGCAAGTTCCAACTAAGTTGTCCTTTGTTAGAGATAGATTGGTTGAATCATACTTTTGGGCTTCAGGATCATACTTTGAACCTAACTACTCCGTTGCTAGAATGATTTTGGCTAAGGGATTGGCTGTTTTGTCCTTGATGGATGATGTTTACGATGCTTACGGTACTTTTGAAGAATTGCAAATGTTTACTGATGCTATTGAAAGATGGGATGCTTCCTGTTTGGATAAGTTGCCAGATTACATGAAGATTGTTTACAAGGCTTTGTTGGATGTTTTTGAAGAAGTTGATGAAGAATTGATTAAGTTGGGAGCACCATACAGAGCTTACTACGGTAAGGAAGCTATGAAGTACGCTGCTAGAGCTTACATGGAAGAAGCTCAATGGAGAGAACAAAAGCATAAGCCAACTACTAAGGAATACATGAAGTTGGCTACTAAGACTTGTGGTTACATTACTTTGATTATTTTGTCCTGTTTGGGAGTTGAAGAAGGTATTGTTACTAAGGAGGCATTTGATTGGGTTTTTAGCAGACCACCATTTATTGAAGCTACTTTGATTATTGCTAGATTGGTTAACGATATTACTGGTCATGAATTTGAAAAGAAGAGAGAACATGTTAGAACTGCTGTTGAATGTTACATGGAAGAACATAAGGTTGGTAAGCAAGAAGTTGTTTCCGAATTTTACAACCAAATGGAATCCGCTTGGAAGGATATTAACGAAGGATTTTTGAGACCAGTTGAATTTCCTATTCCATTGTTGTACTTGATTTTGAACTCCGTTAGAACTTTGGAAGTTATTTACAAGGAAGGTGATTCCTACACTCATGTTGGTCCAGCTATGCAAAACATTATTAAGCAATTGTACTTGCATCCAGTTCCATACTGA, As shown in SEQ ID NO. 1.
In the invention, PTS is inserted between an AOX1 promoter and an AOX1 terminator to obtain plasmid pPICZA-P AOX1-PTS-TAOX1.
Preparation of pichia pastoris electrotransformation competent cells
(1) Activating. Taking out frozen P.pastoris GS115 glycerol bacteria, sucking 100 mu L of stock solution by a pipetting gun, transferring to a shake flask, placing in a constant-temperature shaking incubator at 30 ℃, and shaking and culturing at 200 r/min to obtain about 12: 12h, wherein the thallus is turbid.
(2) And (5) switching. And (3) absorbing 100 mu L of seed culture solution, transferring the seed culture solution to a fresh YPD liquid shake flask, placing the fresh YPD liquid shake flask in a constant-temperature shaking incubator at 30 ℃ for culturing for 3-6 hours, and waiting for the growth concentration of thalli to reach OD 600 = 1.3-1.5.
(3) And (5) centrifuging. The bacterial solution is placed in a sterilized 50 mL precooling centrifuge tube, and centrifuged at 4 ℃ and 5000: 5000 r/min for 5: 5min, and the supernatant is decanted.
(4) And (5) re-suspending and centrifuging. The strain from the previous step was resuspended in LDST solution pre-chilled by 30 mL, left at room temperature for 30min, and centrifuged at 4℃in a low temperature and high speed centrifuge at 5000 r/min for 5min, after which the supernatant was decanted.
(5) The centrifugation was re-suspended again. The bacterial pellet is resuspended by gentle blowing with 5 mL sterilized 1M sorbitol solution, and centrifuged at low temperature and high speed at 5000 r/min at 4℃for 5 min. (repeatable 2-3 times)
(6) And (5) sub-packaging. And finally, taking 1 mL sorbitol solution by a pipette gun, re-suspending the bacterial precipitate, and sub-packaging according to the amount of 100 mu L of each tube for later use.
Pichia pastoris electric transformation and transformant identification
(1) In an ultra clean bench, 3 mu L of pPICZA-P AOX1-PTS-TAOX1 plasmid is sucked into pichia pastoris competent cells by a liquid-transferring gun, gently blown and evenly mixed, and then transferred into a precooled 2 mm electric rotating cup for ice bath.
(2) After a few minutes, the electrotransport device is opened, and parameters of the electrotransport device are set as follows: the electric shock is carried out by wiping off water stains on the surface of the electric rotating cup with paper at the voltage of 1500V, the resistance of 200 omega and the capacitance of 50F (BTX) and electric shock of 7-9 ms, and then placing the electric rotating cup into an electric rotating instrument.
(3) And immediately adding 1mL precooled 1M sorbitol solution after electric shock is finished, fully mixing, sucking out the mixed solution by using a pipetting gun, transferring the mixed solution into a new sterilization EP tube, and incubating for 1.5-2 h in a constant temperature incubator at 30 ℃.
(4) After incubation, the bacterial liquid is centrifuged at 5000 rpm ℃ and 5: 5 min, part of the supernatant is removed, 100 mu L of liquid is reserved for re-suspending the bacterial liquid, the bacterial liquid is coated on a screening culture medium containing Bleo, and the bacterial liquid is cultured for 3-4 days at 30 ℃ until transformants grow out.
(5) Picking the transformant obtained in the step (4) as a template, and performing colony PCR amplification by using a verification primer (plasmid verification primer F: ACGCTGTCTTGGAACCTAATATG, shown as SEQ ID NO.6, plasmid verification primer R: TGAGGAACAGTCATGTCTAAGGC, shown as SEQ ID NO. 7) to identify positive transformants in which plasmid transformation was successful. The PCR reaction system is shown in Table 1, and the PCR reaction conditions are as follows: 1) Pre-denaturation at 98 ℃ for 5min; 2) Denaturation at 98℃for 30s, annealing at 58℃for 30s, extension at 72℃for 1.5min, and three steps were performed for 30 cycles, followed by extension at 72℃for a further 10min.
TABLE 1 PCR reaction system
Example 2
Non-directed evolution of PTS sequences
The pPICZA-P AOX1-PTS-TAOX1 plasmid is used as a template for PCR amplification by using error-prone PCR primers (linear plasmid primer F CGACCATCATCATCATCATCATTGAG is shown as SEQ ID NO.8, linear plasmid primer R GAATTCCTCGTTTCGAATAATTAGTTGT is shown as SEQ ID NO.9, error-prone PTS amplification primer F AGGAATTCATGGAATTGTACGCTCAATCCGTTGG is shown as SEQ ID NO.10, error-prone PTS amplification primer R ATGATGGTCGACGGCGCTATTCAGATCC is shown as SEQ ID NO. 11) respectively to obtain the linear plasmid pPICZA-P AOX1-TAOX1 and the mutated PTS fragment. The error-prone PCR reaction system is shown in Table 2, and the PCR reaction conditions are as follows: 1) Pre-denaturation at 94℃for 3min; 2) Denaturation at 94℃for 1min, annealing at 55℃for 30s, extension at 72℃for 2min, and three steps were performed for 35 cycles and extension at 72℃for a further 5min.
TABLE 2 PCR reaction system (30. Mu.l)
After sequencing the obtained sequence, four mutant PTS sequences were obtained as follows:
Nucleotide sequence of PTS 1:
ATGGAATTGTACGCTCAATCCGTTGGAGTTGGTGCTGCCAGTAGACCATTGGCTAACTTTCATCCATGTGTTTGGGATGATAAGTTTATTGTTTACAACCCTCAATCCTGACAAGCTGGTGAAAGAGAAGAAGCTGAAGAATTGAAGGTTGAATTGAAGAGAGAATTGAAGGAGGCGTCCGATAACTACATGAGACAATTGAAGATGGTTGATGCTATTCAAAGATTGGGTATTGATTACTTGTTTGTTGAAGATGTTGATGAGGCATTGAAGAACTTGTTTGAAATGTTTGATGCTTTTTGTAAGAACAATCATGATATGCACGCTACTGCTTTGTCCTTTAGATTGTTGAGACAACATGGATACAGAGTTTCCTGTGAAGTTTTTGAAAAGTTTAAGGATGGTAAGGATGGATTTAAGGTTCCAAACGAAGATGGTGCTGTTGCTGTTTTGGAATTTTTTGAAGCTACTCATTTGAGAGTTCATGGTGAGGATGTTTTGGATAACGCTTTTGATTTTACCAGAAACTACTTGGAATCCGTTTACGCTACTTTGAACGATCCAACTGCTAAGCAAGTTCATAACGCTTTGAACGAATTTTCATTTAGAAGAGGATTGCCTAGAGTTGAAGCTAGAAAGTACATCTCTATTTACGAACAATACGCTTCACATCATAAGGGATTGTTGAAGTTGGCTAAGTTGGATTTTAACTTGGTTCAAGCGTTGCATAGAAGAGAATTGTCCGAAGATTCCAGATGGTGGAAAACTTTGCAAGTTCCAACTAAGTTGTCCTTTGTTAGAGATAGATTGGTTGAATCATACTTTTGGGCTTCAGGATCATACTTTGAACTAACTACTCCGTTGCTAGAATGATTTTGGCTAAGGGATTGGCTGTTTTGTCCTTTGATGGATGATGTTTACGATGCTTACGGTACTTTTGAAGAATTGCAAATGTTTGCTGATGCTATTGAAAGATGGGATGCTTCCTGTTTGGATAAGTTGCCAGATTACATGAAGATTGTTTACAAGGCTTTGTTGGATGTTGTTGAAGAAGTTGATGAAGAGTTGATTAAGTTGGGAGCACCATACAGAGCTTACTACCGTAAGGAAGCTATGAAGTACGCTGCTAGAGCTTACATGGAAGAAGCTCAATGGAGAGAACAAAAGCATAAGCCAACTACTAAGGAATACATGAAGTTGGCTACTAAGACTTGTGGTTACATTACTTTGATTATTTTGTCCTGTTTGGGAGTTGAAGAAGGTATTGTTACTAAGGAGGCATTTGATTGGGTTTTTAGCAGACCACCATTTATTGAAGCTACTTTGATTATTGCTAGATTGGTTAACGATATTACTGGTCATGAATTTGAAAAGAGGAGAGAACATGTTAGAACTGCTGTTGAATGTTACATGGAAGAACATAAGGTTGGTAAGCAAGAAGTTGTTTCCGAATTTTACAACCAAATGGAATCCGCTTGGAAGGATACTAACGAAGGATTTTTGAGACCAGTTGAATTTCCTATTCCATTGTTGTACTTGATTTTGAACTCCGTTAGAACTTTGGAAGTTATTTACAAGGAAGGTGATTCCTACACTCATGTTGGTCCAGCTATGCAAAACATTATTAAGCAATTGTACTTGCATCCAGTTCCATACTGA, As shown in SEQ ID NO. 2.
Nucleotide sequence of PTS 2:
ATGGAATTGTACGCTCAATCCGTTGGAGTTGGTGCTGCCAGTAGACCATTGGCTAACTTTCATCCATGTGTTTGGGATGATAAGTTTATTGTTTACAACCCTCAATCCTGTCAAGCTGGTGAAAGAGAAGAAGCTGAAGAATTGAAGGTTGAATTGAAGAGAGAATTGAAGGAGGCGTCCGATAACTACATGAGACAATTGAAGATGGTTGATGCTATTCAAAGATTGGGTATTGATTACTTGTTTGTTGAAGATGTTGATGAGGCATTGAAGAACTTGTTTGAAATGTTTGATGCTTTTTGTAAGAACAACCATGATATGCACGCTACTGCTTTGTCCTTTAGATTGTTGAGACAACATGGATACAGAGTTTCCTGTGAAGTTTTTGAAAAGTTTAAGGATGGTAAGGATGGATTTAAGGTTCCAAACGAAGATGGTGCTGTTGCTGTTTTGGAATTTTTTGAAGCTACTCATTTGAGAGTTCATGGTGAAGATGTTTTGGATAACGCTTTTGATTTTACTAGAAACTACTTGGAATCCGTTTACGCTACTTTGAACGATCCAACTGCTAAGCAAGTTCATAACGCTTTGAACGAATTTTCATTTAGAAGAGGATTGCCTAGAGTTGAAGCTAGAAAGTACATTTCTATTTACGAACAATACGCTTCACATCATAAGGGATTGTTGAAGTTGGCTAAGTTGGATTTTAACTTGGTTCAAGCGTTGCATAGAAGAGAATTGTCCGAAGATTCCAGATGGTGGAAAACTTTGCAAGTTCCAACTAAGTTGTCCTTTGTTAGAGATAGATTGGTTGAATCATACTTTTGGGCTTCAGGATCATACTTTGAACCTAACTACTCCGTTGCTAGAATGATTTTGGCTAAGGGATTGGCTGTTTTGTCCTTGATGGATGATGTTTACGATGCTTACGGTACTTTTGAAGAATTGCAAATGTTTACTGATGCTATTGAAAGATGGGATGCTTCCTGTTTGGATAAGTTGCCAGATTACATGAAGATTGTTTACAAGGCTTTGTTGGATGTTTTTGAAGAAGTTGATGAAGAATTGATTAAGTTGGGAGCACCATACAGAGCTTACTACGGTAAGGAAGCTATGAAGTACGCTGCTAGAGCTTACATGGAAGAAGCTCAATGGAGAGAACAAAAGCATAAGCCAACTACTAAGGAATACATGAAGTTGGCTACTAAGACTTGTGGTTACATTACTTTGATTATTTTGTCCTGTTTGGGAGTTGAAGAAGGTATTGTTACTAAGGAGGCATTTGATTGGGTTTTTAGCAGACCACCATTTATTGAAGCTACTTTGATTATTGCTAGATTGGTTAACGATATTACTGGTCATGAATTTGAAAAGAAGAGAGAACATGTTAGAACTGCTGTTGAATGTTACATGGAAGAACATAAGGTTGGTAAGCAAGAAGTTGTTTCCGAATTTTACAACCAAATGGAATCCGCTTGGAAGGATATTAACGAAGGATTTTTGAGACCAGTTGAATTTCCTATTCCATTGTTGTACTTGATTTTGAACTCCGTTAGAACTTTGGAAGTTATTTACAAGGAAGGTGATTCCTACACTCATGTTGGTCCAGCTATGCAAAACAATATAAAGCAATTGTACTTGCATCCAGTTCCATACTGA, As shown in SEQ ID NO. 3.
Nucleotide sequence of PTS 3:
ATGGAATTGTACGCTCAATCCGTTGGAGTTGGTGCTGCCAGTAGACCATTGGCTAACTTTCATCCATGTGTTTGGGATGATAAGTTTATTGTTTACAACCCTCAATCCTGTCAAGCTGGTGAAAGAGAAGAAGCTGAAGAATTGAAGGTTGAATTGAAGAGAGAATTGAAGGAGGCGTCCGATAACTACATGAGACAATTGAAGATGGTTGATGCTATTCAAAGATTGGGTATTGATTACTTGTTTGTTGAAGATGTTGATGAGGCATTGAAGAACTTGTTTGAAATGTTTGATGCTTTTTGTAAGAACAACCATGATATGCACGCTACTGCTTTGTCCTTTAGATTGTTGAGACAACATGGATACAGAGTTTCCTGTGAAGTTTTTGAAAAGTTTAAGGATGGTAAGGATGGATTTAAGGTTCCAAACGAAGATGGTGCTGTTGCTGTTTTGGAATTTTTTGAAGCTACTCATTTGAGAGTTCATGGTGAAGATGTTTTGGATAACGCTTTTGATTTTACTAGAAACTACTTGGAATCCGTTTACGCTACTTTGAACGATCCAACTGCTAAGCAAGTTCATAACGCTTTGAACGAATTTTCATTTAGAAGAGGATTGCCTAGAGTTGAAGCTAGAAAGTACATTTCTATTTACGAACAATACGCTTCACATCATAAGGGATTGTTGAAGTTGGCTAAGTTGGATTTTAACTTGGTTCAAGCGTTGCATAGAAGAGAATTGTCCGAAGATTCCAGATGGTGGAAAACTTTGCAAGTTCCAACTAAGTTGTCCTTTGTTAGAGATAGATTGGTTGAATCATACTTTTGGGCTTCAGGATCATACTTTGAACTAACTACTCCGTTGCTAGAATGA（ Here truncated ）TTTTGGCTAAGGGATTGGCTGTTTTGTCCTTTGATGGATGATGTTTACGATGCTTACGGTACTTTTGAAGAATTGCAAATGTTTACTGATGCTATTGAAAGATGGGATGCTTCCTGTTTGGATAAGTTGCCAGATTACATGAAGATTGTTTACAAGGCTTTGTTGGATGTTTTTGAAGAAGTTGATGAAGAATTGATTAAGTTGGGAGCACCATACAGAGCTTACTACGGTAAGGAAGCTATGAAGTACGCTGCTAGAGCTTACATGGAAGAAGCTCAATGGAGAGAACAAAAGCATAAGCCAACTACTAAGGAATACATGAAGTTGGCTACTAAGACTTGTGGTTACATTACTTTGATTATTTTGTCCTGTTTGGGAGTTGAAGAAGGTATTGTTACTAAGGAGGCATTTGATTGGGTTTTTAGCAGACCACCATTTATTGAAGCTACTTTGATTATTGCTAGATTGGTTAACGATATTACTGGTCATGAATTTGAAAAGAAGAGAGAACATGTTAGAACTGCTGTTGAATGTTACATGGAAGAACATAAGGTTGGTAAGCAAGAAGTTGTTTCCGAATTTTACAACCAAATGGAATCCGCTTGGAAGGATATTAACGAAGGATTTTTGAGACCAGTTGAATTTCCTATTCCATTGTTGTACTTGATTTTGAACTCCGTTAGAACTTTGGAAGTTATTTACAAGGAAGGTGATTCCTACACTCATGTTGGTCCAGCTATGCAAAACATTATTAAGCAATTGTACTTGCATCCAGTTCCATACTGA, is shown as SEQ ID NO. 4.
Nucleotide sequence of PTS 4:
ATGGAATTGTACGCTCAATCCGTTGGAGTTGGTGCTGCCAGTAGACCATTGGCTAACTTTCATCCATGTGTTTGGGATGATAAGTTTATTGTTTACAACCCTCAATCCTGTCAAGCTGGTGAAAGAGAAGAAGCTGAAGAATTGAAGGTTGAATTGAAGAGAGAATTGAAGGAGGCGTCCGATAACTACATGAGACAATTGAAGATGGTTGATGCTATTCAAAGATTGGGTATTGATTACTTGTTTGTTGAAGATGTTGATGAGGCATTGAAGAACTTGTTTGAAATGTTTGATGCTTTTTGTAAGAACAACCATGATATGCACGCTACTGCTTTGTCCTTTAGATTGTTGAGACAACATGGATACAGAGTTTCCTGTGAAGTTTTTGAAAAGTTTAAGGATGGTAAGGATGGATTTAAGGTTCCAAACGAAGATGGTGCTGTTGCTGTTTTGGAATTTTTTGAAGCTACTCATTTGAGAGTTCATGGTGAAGATGTTTTGGATAACGCTTTTGATTTTACTAGAAACTACTTGGAATCCGTTTACGCTACTTTGAACGATCCAACTGCTAAGCAAGTTCATAACGCTTTGAACGAATTTTCATTTAGAAGAGGATTGCCTAGAGTTGAAGCTAGAAAGTACATTTCTATTTACGAACAATACGCTTCACATCATAAGGGATTGTTGAAGTTGGCTAAGTTGGATTTTAACTTGGTTCAAGCGTTGCATAGAAGAGAATTGTCCGAAGATTCCAGATGGTGGAAAACTTTGCAAGTTCCAACTAAGTTGTCCTTTGTTAGAGATAGATTGGTTGAATCATACTTTTGGGCTTCAGGATCATACTTTGAACCTAACTACTCCGTTGCTAGAATGATTTTGGCTAAGGGATTGGCTGTTTTGTCCTTGATGGATGATGTTTACGATGCTTACGGTACTTTTGAAGAATTGCAAATGTTTACTGATGCTATTGAAAGATGGGATGCTTCCTGTTTGGATAAGTTGCCAGATTACATGAAGATTGTTTACAAGGCTTTGTTGGATGTTTTTGAAGAAGTTGATGAAGAATTGATTAAGTTGGGAGCACCATACAGAGCTTACTACGGTAAGGAAGCTATGAAGTACGCTGCTAGAGCTTACATGGAAGAAGCTCAATGGAGAGAACAAAAGCATAAGCCAACTACTAAGGAATACATGAAGTTGGCTACTAAGACTTGTGGTTACATTACTTTGATTATTTTGTCCTGTTTGGGAGTTGAAGAAGGTATTGTTACTAAGGAGGCATTTGATTGGGTTTTTAGCAGACCACCATTTATTGAAGCTACTTTGATTATTGCTAGATTGGTTAACGATATTACTGGTCATGAATTTGAAAAGAAGAGAGGACATGTTAGAACTGCTGTTGAATGTTACATGGAAGAACATAAGGTTGGTAAGCAAGAAGTTGTTTCCGAATTTTACAACCAAATGGAATCCGCTTGGAAGGATATTAACGAAGGATTTTTGAGACCAGTTGAATTTCCTATTCCATTGTTGTACTTGATTTTGAACTCCGTTAGAACTTTGGAAGTTATTTACAAGGAAGGTGATTCCTACACTCATGTTGGTCCAGCTATGCAAAACAATATTAAGCAATTGTACTTGCATCCAGTTCCATACTGA, As shown in SEQ ID NO. 5.
Example 3
Construction of mutant PTS sequence plasmid
The PTS mutant sequences obtained from the pPICZA-P AOX1-TAOX1 linear plasmid and the error-prone pcr primer were ligated, the ligation system is shown in Table 3, and the ligation conditions were 37℃for 30min.
TABLE 3 plasmid ligation system (20 μl)
The ligation products were transformed into E.coli by chemical means and screened using bleomycin (Bleo) -resistant LB plates. The positive clones were then subjected to plasmid extraction.
Example 4
Gas-phase mass spectrum detection of pichia pastoris engineering bacteria products
GC-MS detection conditions: capillary column: HP-5 (30 m*0.25 mm*0.25 μm film thickness); the initial temperature was 100deg.C, maintained for 1min, at 14deg.C/min to 240deg.C, maintained for 1min, at 35deg.C/min to 280 deg.C, and maintained for 1 min. The carrier gas is high-purity helium, the flow rate is 1.0 mL/min, the sample injection is 1.0 mu L, and no flow division is performed.
MS conditions: using the EI ion source, electron Multiplication Voltage (EMV) mode, the gain factor was 1.01, and the actual EMV was 1.576×10 3 V. Patchouli alcohol was quantified by external standard method.
The results show that: at the shake flask fermentation level, the average yield of patchouli alcohol produced by the expression of the optimized PTS plasmid in pichia pastoris is 0.87mg/L; in the mutated PTS sequence, the average yield of patchouli alcohol of the PTS3 sequence plasmid (containing the coding gene of the evolutionary patchouli alcohol synthase shown in SEQ ID NO. 4) is increased to 1.17mg/L, and the other three are lethal mutations.
Based on analysis of the PTS3 sequence, it was found that the sequence was terminated in translation at 873bp (the stop codon encoded the 291 th amino acid), which was in fact the centrally truncated mutant sequence.
Example 5
Amplified culture of pichia pastoris engineering bacteria
In order to evaluate whether patchouli alcohol producing engineering bacteria can be used for production, the invention performs fed-batch fermentation in a 5L tank fermenter. First, single colonies were inoculated into 250mL of shake flask medium containing 50mL of YPD, and cultured at 30℃and 250rpm for 20 hours. Then 8% of the seed culture was inoculated into a 2L-charged 5L fermenter for fed-batch fermentation. When the culture was carried out until OD 600 =200, fermentation was carried out using a methanol solution containing 12ml/L PTM1 as an inducer.
The fermentation process of the invention is divided into three stages:
The first stage mainly maintains strain growth: the temperature was controlled at 30℃and pH 5.5, the stirring speed was 700rpm, the air flow rate was 2L/(L.min), and the Dissolved Oxygen (DO) concentration was maintained at about 30%, and this stage was mainly for the purpose of accumulating rapid growth of the strain, and culturing was carried out for about 24 hours;
The second stage is a glycerol feeding process: controlling the flow rate of the feed to be 13-23 g/(L.min), and properly adjusting the flow rate according to the real-time change of the DO value; stopping feeding when OD 600 reaches 200 until the glycerol in the culture medium is completely consumed;
The third stage is induction of methanol: adding a small amount of methanol solution containing PTM1 for multiple times to carry out adaptive domestication; 10% dodecane was then added thereto; and then the flow rate of the methanol is timely regulated according to the DO change, so that the condition that excessive accumulation of the methanol in the culture medium is avoided is ensured.
According to the invention, the 5L tank is fed-batch fermented, and the optimized PTS sequence patchouli alcohol yield is 28.44mg/L; the yield of the PTS3 sequence patchouli alcohol after mutation is 35.82mg/L.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. An optimized patchouli alcohol synthase coding gene is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
2. The gene for encoding the patchouli alcohol synthase is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 4.
3. A recombinant vector comprising a primary vector and the optimized patchouli alcohol synthase encoding gene of claim 1.
4. A recombinant vector comprising a starting vector and the gene encoding the evolutionary patchouli alcohol synthase of claim 2.
5. The recombinant vector according to claim 3 or 4, wherein the initial vector is a pPICZA vector, a pHIL-S1 vector, a pYAM P vector, a pPIC9 vector or a pPIC9K vector.
6. The recombinant vector according to claim 5, wherein the initial vector comprises an AOX1 promoter and an AOX1 terminator, and the optimized patchouli alcohol synthase encoding gene or the evolved patchouli alcohol synthase encoding gene is inserted between the AOX1 promoter and the AOX1 terminator.
7. A recombinant strain expressing patchouli alcohol synthase, characterized in that the recombinant strain is transformed with the recombinant vector according to any one of claims 3 to 6.
8. The recombinant strain of claim 7, wherein the recombinant strain expresses patchouli alcohol synthase by methanol induction.
9. Use of the optimized patchouli alcohol synthase encoding gene of claim 1, the evolved patchouli alcohol synthase encoding gene of claim 2, the recombinant vector of any one of claims 3 to 6 or the recombinant strain of any one of claims 7 to 8 in patchouli alcohol production and/or in the preparation of a perfume fixative.
10. The preparation method of patchouli alcohol is characterized by comprising the following steps:
(1) Transforming a host cell with the recombinant vector of any one of claims 3-6 to obtain a recombinant strain;
(2) Culturing recombinant strain, and inducing to synthesize patchouli alcohol;
(3) Recovering and purifying the expressed patchouli alcohol.
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