CN109468287A

CN109468287A - A kind of hydroxylation enzyme mutant

Info

Publication number: CN109468287A
Application number: CN201811396041.1A
Authority: CN
Inventors: 范文超; 高书良; 王金刚; 梁岩; 袁圣伦; 任亮
Original assignee: Zhejiang Huari Biotechnology Co Ltd
Current assignee: Zhejiang Huari Biotechnology Co Ltd
Priority date: 2018-11-22
Filing date: 2018-11-22
Publication date: 2019-03-15
Anticipated expiration: 2038-11-22
Also published as: CN109468287B

Abstract

The present invention obtains the hydroxylation enzyme mutant SEQ ID NOs:3-4 of enzyme activity raising by the method for directed evolution, these mutant or its expression microorganism energy efficient catalytic dammarendiol prepare protopanoxadiol by hydroxylating, have industrialized developing and application prospect.

Description

A kind of hydroxylation enzyme mutant

Technical field

The invention belongs to biocatalysis fields, are related to a kind of hydroxylation enzyme mutant, specifically, being related to a kind of up to Ma alkene two Alcohol is hydroxylated enzyme mutant and its purposes in synthesis protopanoxadiol.

Background technique

Ginsenoside is the general name for the saponin(e being separated to from ginseng and its congener (such as Radix Notoginseng, American Ginseng), is belonged to It is the main effective ingredient in ginseng in Triterpene saponins.Currently, at least 60 kinds of saponin(es have been isolated from ginseng, In some ginsenosides be proven to have extensive physiological function and medical value: including antitumor, immunological regulation, it is antifatigue, The functions such as shield heart, protect liver.According to the form of aglycon aglucon, ginsenoside is typically divided into three classes, oleanane type, protoplast's ginseng two Alcohol type (PPD type) and protopanaxatriol type (PPT type).Protopanoxadiol (protopanaxadiol, PPD) is synthesis ginseng soap The important as precursors of glycoside compound generally passes through dammarendiol (dammarenediol-II, DMD) and closes through hydroxylase catalysis At.Reaction equation is as follows:

The hydroxylase for being catalyzed step reaction in ginseng Panax Ginseng at present has been cloned identification (Han, J.-Y., H.- S.Hwang,S.-W.Choi,H.-J.Kim and Y.E.Choi.Cytochrome P450 CYP716A53v2Catalyzes the Formation of Protopanaxatriol from Protopanaxadiol During Ginsenoside Biosynthesis in Panax Ginseng.Plant Cell Physiol.2012,53(9):1535–1545.).Ginseng The CYP716A47 albumen in the source Panax Ginseng, also known as protopanoxadiol synthase (protopanaxadiol synthase, PPDS) or dammarendiol hydroxylase (dammarenediol-II hydroxylase, DMD hydroxylase), the property of can choose Ground is catalyzed DMD substrate, in C-12 generation hydroxylatings, generates PPD.But the DMD hydroxylase catalysis efficiency is lower, if to its into Row transformation, increases substantially enzyme activity, it will provide feasibility for industrial applications.

Summary of the invention

In order to develop the dammarendiol hydroxylase (abbreviation hydroxylase herein) of high catalytic efficiency, the present invention is with ginseng (Genebank:AEY75213.1, i.e. SEQ ID NO:1, are named the dammarendiol hydroxylase in the source PanaxGinseng herein Based on DMDH), hydroxylase gene mutation library is established by the method for fallibility PCR, then obtain by screening-gene mutation library The hydroxylation enzyme mutant that enzyme activity significantly improves.

Therefore, the first purpose of this invention is to provide a kind of hydroxylase, amino acid sequence are as follows:

SEQ ID NO:3, for SEQ ID NO:1 the 186th M replaces with L, the 321st K replaces with the mutation of A Body, it may be assumed that

MVLFFSLSLLLLPLLLLFAYFSYTKRIPQKENDSKAPLPPGQTGWPLIGETLNYLSCVKSGVSENFVK YRKEKYSPKVFRTSLLGEPMAILCGPEGNKFLYSTEKKLVQVWFPSSVEKMFPRSHGESNADNFSKVRGKMMFLLK VDGMKKYVGLMDRVMKQFLETDWNRQQQINVHNTVKKYTVTLSCRVFMSIDDEEQVTRLGSSIQNIEAGLLAVPIN IPGTAMNRAIKTVKLLTREVEAVIKQRKVDLLENKQASQPQDLLSHLLLTANQDGQFLSESDIASHLIGLMQGGYT TLNGTITFVLNYLAEFPDVYNQVLAEQVEIANSKHPKELLNWEDLRKMKYSWNVAQEVLRIIPPGVGTFREAITDF TYAGYLIPKGWKMHLIPHDTHKNPTYFPSPEKFDPTRFEGNGPAPYTFTPFGGGPRMCPGIEYARLVILIFMHNVV TNFRWEKLIPNEKILTDPIPRFAHGLPIHLHPHN(SEQ ID NO:3)；Or

SEQ ID NO:4, for SEQ ID NO:1 the 120th F replaces with A, the 186th M replaces with L, the 279th The S of position replaces with the mutant of A, it may be assumed that

MVLFFSLSLLLLPLLLLFAYFSYTKRIPQKENDSKAPLPPGQTGWPLIGETLNYLSCVKSGVSENFVK YRKEKYSPKVFRTSLLGEPMAILCGPEGNKFLYSTEKKLVQVWFPSSVEKMAPRSHGESNADNFSKVRGKMMFLLK VDGMKKYVGLMDRVMKQFLETDWNRQQQINVHNTVKKYTVTLSCRVFMSIDDEEQVTRLGSSIQNIEAGLLAVPIN IPGTAMNRAIKTVKLLTREVEAVIKQRKVDLLENKQASQPQDLLSHLLLTANQDGQFLAESDIASHLIGLMQGGYT TLNGTITFVLNYLAEFPDVYNQVLKEQVEIANSKHPKELLNWEDLRKMKYSWNVAQEVLRIIPPGVGTFREAITDF TYAGYLIPKGWKMHLIPHDTHKNPTYFPSPEKFDPTRFEGNGPAPYTFTPFGGGPRMCPGIEYARLVILIFMHNVV TNFRWEKLIPNEKILTDPIPRFAHGLPIHLHPHN(SEQ ID NO:4)。

It is preferred that the amino acid sequence of above-mentioned hydroxylase is SEQ ID NO:3.

Second object of the present invention is to provide the gene for encoding above-mentioned hydroxylase.

Third object of the present invention is to provide the plasmid comprising said gene.

In view of belonging to cytochrome P 450 monooxygenases in DMD hydroxylase, when carrying out catalysis reaction, P450 is needed also There are competence exertion normal functions as auxilin for protoenzyme, so needing to co-express auxiliary while expressing DMD hydroxylase Enzyme P450 reductase.P450 reductase for example including but be not limited to the CPR2 in the source arabidopsis Arabidopsis thaliana Enzyme (Genebank:NP_849472.2), i.e. SEQ ID NO:5.

Therefore, in a preferred embodiment, the plasmid comprising said gene also includes P450 reductase SEQ The encoding gene SEQ ID NO:6 of IDNO:5, for co-expressing hydroxylase and P450 reductase.

Preferably, the skeleton of the co-expression plasmid is pYES2.1 carrier.

Fourth object of the present invention is to provide the plasmid for having converted above-mentioned coexpression hydroxylase and P450 reductase Microorganism.

As another optional embodiment, hydroxylase and P450 reductase can be located on different plasmids, The transformant obtained after the two plasmid cotransformation microorganisms can also express P450 reductase SEQ ID NO:5 and hydroxylase.

When hydroxylase and P450 reductase can be located on different plasmids, the skeleton of two kinds of plasmids can be all It is pUC57 carrier.

Preferably, mentioned microorganism is selected from saccharomyces cerevisiae or Yarrowia lipolytica.More preferable saccharomyces cerevisiae BY4742 (EUROSCARF)。

Of the invention the 5th is designed to provide the use of above-mentioned hydroxylase or microorganism in synthesis protopanoxadiol On the way.

In one embodiment, such use is to restore using dammarendiol as substrate raw material in hydroxylase and P450 Protopanoxadiol is prepared by hydroxylating under enzymatic.

Preferably, above-mentioned enzymic catalytic reaction is in pH7.4,30 DEG C of temperature progress.

The hydroxylation enzyme mutant SEQ ID NOs:3-4 that the present invention constructs significantly improves the catalysis activity of hydroxylating, Expeditiously protopanoxadiol can be generated by catalysis substrate dammarendiol, the enzyme-catalyzed reaction condition is mild, has industry and melts Hair and application prospect.

Specific embodiment

The present invention is described in further details below in conjunction with specific embodiment.It should be understood that following embodiment is only used for The bright present invention is not for limiting the scope of the invention.

Additive amount, content and the concentration of many kinds of substance is referred to herein, wherein the percentage composition, except special instruction Outside, all refer to mass percentage.

It in the embodiments herein, is illustrated if do not made for reaction temperature or operation temperature, the temperature is logical Often refer to room temperature (15-30 DEG C).

For simplicity, amino acid abbreviations herein both can be used English trigram, can also use English-word Mother, this be it is well known to those skilled in the art, these abbreviations are listed in the following table:

1 amino acid bilingual of table and abbreviation

Alanine	Alanine	A or Ala	Aliphatic category
				Arginine	Arginine	R or Arg	Basic amine group acids
Asparagine	Asparagine	N or Asn	Amides
				Aspartic acid	Aspartic acid	D or Asp	Acidic amino acid class
Cysteine	Cysteine	C or Cys	Sulfur-bearing class
				Glutamine	Glutamine	Q or Gln	Amides
Glutamic acid	Glutamic acid	E or Glu	Acidic amino acid class
				Glycine	Glycine	G or Gly	Aliphatic category
Histidine	Histidine	H or His	Basic amine group acids
				Isoleucine	Isoleucine	I or Ile	Aliphatic category
Leucine	Leucine	L or Leu	Aliphatic category
				Lysine	Lysine	K or Lys	Basic amine group acids
Methionine	Methionine	M or Met	Sulfur-bearing class
				Phenylalanine	Phenylalanine	F or Phe	Aromatic
Proline	Proline	P or Pro	Imino acid
				Serine	Serine	S or Ser	Hydroxy kind
Threonine	Threonine	T or Thr	Hydroxy kind
				Tryptophan	Tryptophan	W or Trp	Aromatic
Tyrosine	Tyrosine	Y or Tyr	Aromatic
				Valine	Valine	V or Val	Aliphatic category

As the foundation forms of building hydroxylation enzyme mutant, from the dammarendiol hydroxyl of ginseng Panax Ginseng The amino acid sequence for changing enzyme (Genebank:AEY75213.1 is named as DMDH herein) is SEQ ID NO:1:

MVLFFSLSLLLLPLLLLFAYFSYTKRIPQKENDSKAPLPPGQTGWPLIGETLNYLSCVKSGVSENFVK YRKEKYSPKVFRTSLLGEPMAILCGPEGNKFLYSTEKKLVQVWFPSSVEKMFPRSHGESNADNFSKVRGKMMFLLK VDGMKKYVGLMDRVMKQFLETDWNRQQQINVHNTVKKYTVTMSCRVFMSIDDEEQVTRLGSSIQNIEAGLLAVPIN IPGTAMNRAIKTVKLLTREVEAVIKQRKVDLLENKQASQPQDLLSHLLLTANQDGQFLSESDIASHLIGLMQGGYT TLNGTITFVLNYLAEFPDVYNQVLKEQVEIANSKHPKELLNWEDLRKMKYSWNVAQEVLRIIPPGVGTFREAITDF TYAGYLIPKGWKMHLIPHDTHKNPTYFPSPEKFDPTRFEGNGPAPYTFTPFGGGPRMCPGIEYARLVILIFMHNVV TNFRWEKLIPNEKILTDPIPRFAHGLPIHLHPHN(SEQ ID NO:1)。

The coding gene sequence of DMDH is SEQ ID NO:2:

atggttttgttcttttctttgtctttgttgttgttgccattgttattattgtttgcttatttttctta cactaaacgtattcctcaaaaggaaaacgactctaaggctccattgccacccggtcaaactggatggccattgatt ggtgaaactttaaattacttgtcttgtgttaagtctggtgtttctgaaaacttcgttaagtatagaaaggaaaaat attctccaaaggttttcagaacttctttattgggtgaaccaatggctattttgtgcggtccagaaggtaataagtt cttgtattcaactgaaaagaagttggtccaagtttggttcccttcttcagttgaaaaaatgttccctcgttctcac ggagagtcaaacgcagataacttttctaaagttagaggtaaaatgatgttcttattaaaagttgatggtatgaaaa aatatgttggtttgatggatagagttatgaaacaattcttggagactgattggaatagacaacaacaaattaatgt tcataacactgttaaaaagtacacagttactatgtcttgtagagttttcatgtcaattgacgacgaagagcaagtt acaagattgggttcttcaatacaaaatattgaagctggtttgttggctgttccaattaatattcccggtactgcaa tgaatagagctattaaaacagttaaattgttaactagagaagttgaagctgttataaaacaaagaaaagttgattt attggaaaacaagcaagcttcacaaccacaagatttgttatctcatttgttgttgactgctaatcaagatggtcaa tttttgtcagaatcagatattgcatctcatttgattggattgatgcaaggtggttacacaacattaaacggtacaa taacatttgttttaaattatttggctgaatttccagatgtttataatcaagttttgaaagaacaagttgaaattgc taattcaaaacatccaaaagaattgttaaattgggaagacttgagaaagatgaagtattcttggaacgtcgctcaa gaagtcttgaggattattccacccggtgttggtactttcagagaagctataactgattttacatacgctggttact tgattccaaaaggttggaaaatgcatttgattccacatgatactcataaaaatccaacttatttcccatctccaga aaaatttgacccaactagattcgaaggtaacggaccagctccatacactttcactccattcggtggtggacctagg atgtgccccggtattgagtatgctaggttagttatattgatatttatgcataatgttgttacaaattttagatggg aaaaattgataccaaatgaaaagattttgactgacccaattccaagattcgctcacggtttgccaatacatttaca tccacataattag

(SEQ ID NO:2)。

In order to obtain the higher hydroxylation enzyme mutant of enzymatic activity, the present invention to the gene order SEQ ID NO:2 of DMDH into Row point mutation.The variant amino acid sequence that one or more amino acid sites replace is obtained by fallibility round pcr, is filtered out Several sites that enzyme activity can be improved obtain the mutation that 2 plants of enzyme activities significantly improve then in a manner of pinpointing combinatorial mutagenesis Body.

Herein, term " hydroxylase ", " DMD hydroxylase ", " protopanoxadiol synthase ", " PPDS ", " dammarendiol Hydroxylase " indicates identical meaning.The hydroxylase that amino acid sequence is SEQ ID NO:1 is referred to as " DMDH ", is ginseng The CYP716A47 albumen in the source Panax Ginseng.DMD hydroxylase belongs to cytochrome P 450 monooxygenases, needs in catalysis Want the CPR2 enzyme (Genebank:NP_849472.2) in the P450 reductase such as source arabidopsis Arabidopsis thaliana As auxilin enzyme.

In one embodiment, the amino acid sequence of above-mentioned CPR2 enzyme (Genebank:NP_849472.2) is SEQ ID NO:5:

MSSSSSSSTSMIDLMAAIIKGEPVIVSDPANASAYESVAAELSSMLIENRQFAMIVTTSIAVLIGCIV MLVWRRSGSGNSKRVEPLKPLVIKPREEEIDDGRKKVTIFFGTQTGTAEGFAKALGEEAKARYEKTRFKIVDLDDY AADDDEYEEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEGNDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVD DILVEQGAQRLVQVGLGDDDQCIEDDFTAWREALWPELDTILREEGDTAVATPYTAAVLEYRVSIHDSEDAKFNDI NMANGNGYTVFDAQHPYKANVAVKRELHTPESDRSCIHLEFDIAGSGLTYETGDHVGVLCDNLSETVDEALRLLDM SPDTYFSLHAEKEDGTPISSSLPPPFPPCNLRTALTRYACLLSSPKKSALVALAAHASDPTEAERLKHLASPAGKV DEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAGVAPRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIHKG VCSTWMKNAVPYEKSENCSSAPIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVLFF GCRNRRMDFIYEEELQRFVESGALAELSVAFSREGPTKEYVQHKMMDKASDIWNMISQGAYLYVCGDAKGMARDVH RSLHTIAQEQGSMDSTKAEGFVKNLQTSGRYLRDVW(SEQ ID NO:5)。

Its coding gene sequence is SEQ ID NO:6:

atgtcttcttcttcatcatcttcaacttctatgattgacttgatggcagcaattattaagggagaacc agtcattgtctctgatccagctaacgcttctgcatatgagtctgtcgcagctgagttgtcttctatgttaattgaa aatagacagtttgctatgattgttactacatcaattgcagtcttgattggttgtattgttatgttggtttggagga ggtctggatctggtaattcaaagagagttgaacctttaaaacctttagttattaaaccaagagaagaagaaattga cgatggtcgtaaaaaagttactatcttttttggaactcagactggtactgctgagggtttcgctaaagctttgggt gaggaggctaaggctagatatgaaaaaactagattcaagattgttgatttggatgattacgctgcagacgacgacg aatacgaggaaaaattgaaaaaagaagatgtcgctttcttctttttagctacatatggtgacggtgaaccaacaga caatgctgcaagattttacaagtggttcacagaaggaaatgatcgtggtgagtggttgaaaaatttgaaatatggt gtttttggtttgggaaacagacaatacgagcatttcaataaagttgcaaaagtcgttgatgatattttagtcgaac aaggtgctcagagattggtccaagtcggtttgggtgacgatgatcaatgtatagaggatgacttcacagcttggag ggaggctttgtggccagaattggatactattttgagagaggaaggtgatacagctgtcgctacaccatacactgct gcagtcttggagtacagagtttcaattcatgattctgaagatgcaaaatttaacgatattaacatggctaatggta atggttatactgttttcgatgctcaacatccatacaaggcaaacgtcgcagtcaagagggagttgcacacaccaga atcagatagatcatgcattcatttagagtttgacatagctggttctggtttgacatacgaaactggtgaccacgtt ggtgtcttgtgcgacaacttgtcagagacagtcgatgaagctttgagattattagacatgtctccagacacttatt tctctttgcatgctgagaaagaagatggtactccaatttcatcttcattgcctcctccattcccaccttgcaactt aagaactgctttgactagatacgcttgtttgttgtcttctcctaagaaatctgctttggtcgctttggctgcacat gcttctgatccaacagaggcagagaggttaaagcacttggcatcaccagctggaaaggttgatgagtactctaagt gggtcgtcgagtcacagcgttctttgttagaagttatggcagagttcccttcagctaagccaccattgggtgtctt cttcgctggagttgcaccaaggttgcaaccaagattttattctatttcttcttctccaaagattgcagaaactagg attcatgttacttgtgctttggtttacgaaaaaatgccaactggtcgtattcataaaggtgtctgttctacatgga tgaagaacgctgttccttatgaaaaatctgaaaactgttcatcagctcctatattcgtcagacaatctaacttcaa gttaccttcagattcaaaggttccaattattatgattggtcccggtactggattggcaccttttcgtggtttcttg caagaaaggttggctttggttgagtctggtgtcgaattgggaccatcagtcttgtttttcggttgtaggaacagaa gaatggatttcatttatgaggaggaattgcaaaggtttgttgagtctggtgctttggcagagttgtctgtcgcttt ctctagggagggtcctacaaaggaatacgttcaacataaaatgatggataaagcatctgatatatggaatatgatt tcacaaggagcttatttgtatgtctgcggtgatgcaaagggtatggctagggacgtccatcgttctttacatacta ttgctcaagaacaaggttctatggattcaacaaaggctgaaggatttgttaagaacttgcaaacttctggaagata tttgagagatgtctggtag(SEQ ID NO:6)。

Herein, term " wild (type) ", " wild enzyme ", " wild-type enzyme " and " WT " indicates identical meaning, is all Refer to the hydroxylase DMDH (SEQ ID NO:1) not being genetically engineered.

2 hydroxylation enzyme mutant SEQ ID NOs:3-4 that the present invention filters out have the characteristics that it is common, i.e., relative to SEQID NO:1 has the forward mutation assay M186L in a site, implies that the rite-directed mutagenesis of at least the 186th amino acids may It can cause the variation of enzymatic activity.

Hydroxylation enzyme mutant (SEQ ID NOs:3-4) of the invention is since amino acid sequence is clear, art technology Personnel are readily available its encoding gene, the expression cassette comprising these genes and plasmid and the transformant comprising the plasmid.This A little genes, expression cassette, plasmid, transformant can be obtained by genetic engineering building mode well-known to those skilled in the art.

Above-mentioned transformant host can be any suitable expression hydroxylation enzyme mutant SEQ ID NOs:3-4 and P450 reduction The microorganism of enzyme, includes bacterium and fungi.Preferred microorganism is saccharomyces cerevisiae or Yarrowia lipolytica, more preferable saccharomyces cerevisiae BY4742。

When as being that biocatalyst is used to produce protopanoxadiol, hydroxylase of the invention and P450 reductase can be in The form of existing enzyme or the form of thallus.The form of the enzyme includes resolvase, immobilised enzymes, including purifying enzyme, thick enzyme, fermentation The fixed enzyme etc. of liquid, carrier；The form of the thallus includes survival thallus and dead thallus.

Hydroxylase and P450 reductase of the invention isolate and purify including immobilised enzymes technology of preparing is also this field skill Known to art personnel.

Embodiment

Material and method

Full genome synthesis herein is completed by Suzhou Jin Weizhi Biotechnology Co., Ltd；Expression vector is by Zhejiang Hua Rui Bioisystech Co., Ltd's subclone preparation.Primer synthesis and sequencing are all completed by Suzhou Jin Weizhi Biotechnology Co., Ltd.

Molecular biology experiment herein includes plasmid construction, digestion, connection, competent cell preparation, conversion, culture Basigamy etc., referring especially to " Molecular Cloning:A Laboratory guide " (third edition), J. Pehanorm Brooker, D.W. Russell (beauty) is compiled Write, Huang Peitang etc. is translated, Science Press, Beijing, 2002) it carries out.Specific experiment item can be determined by simple experiment when necessary Part.

The reaction condition or kit specification that PCR amplification experiment is provided according to plasmid or DNA profiling supplier carry out.It must It can be adjusted by simple experiment when wanting.

The high performance liquid chromatography (HPLC) of 1.DMD and PPD content measures

Shimadzu LC-20A liquid chromatograph, Shodex C18-120-5 4E column (5 μm, 4.6mm × 250mm) chromatographic column, 35 DEG C of column temperature, mobile phase A water, Mobile phase B acetonitrile, gradient elution: 0min (35%B), 55min (90% ), B 50-55min (90%B), 55-65min (35%B).Flow velocity: 0.8ml/min, wavelength 203nm.

DMD and PPD is purchased from Wuhan ChemFaces Bioisystech Co., Ltd.

2. hydroxylase enzyme activity determination

The preparation of albumen microbody is carried out to fermentation thalli, is reacted for enzyme activity determination.500 μ l reaction systems include: 100mM Potassium phosphate, pH7.4, the NADPH containing 1mM, 50 micromole substrate DMD and 1mg albumen microbodies.Reaction system is incubated at 30 DEG C 2h is stripped twice with n-hexane.After sample vacuum drying, with molten, the liquor charging facies analysis of 500 μ l methanol weight.

Enzyme activity definition: under the conditions of pH7.4,30 DEG C of temperature, it is catalyzed DMD per minute and generates needed for 1 micromole (mol) PPD The enzyme amount wanted is defined as 1 unit (U).

3. culture medium

YPD culture medium: 10g/L yeast extract, 20g/L tryptone, 20g/L glucose.(solid medium separately adds 20g/L agar powder.)

The building of 1 wild type DMDH and CPR2 gene co-expressing plasmid of embodiment

DMDH genes of SEQ ID NO:2 (or PgPPDS) used in 1. and CPR2 genes of SEQ ID NO:6 (or AtCPR2), synthesized by Suzhou Jin Weizhi biotechnology company, target gene is loaded on pUC57 carrier, is obtained respectively PUC57-DMDH and pUC57-CPR2 plasmid is used for subsequent PCR amplification template.

2. the building of wild type DMDH and CPR2 gene co-expressing plasmid includes the following steps:

2.1 using the carrier pYES2.1/V5-his-TOPO of Invitrogen as template, using following primer pair (5 ' -3 '), Amplification vector segment.

PYES2.1-vector-F:AAGCTGCGGCCCTGCATTAA,

pYES2.1-vector-R:ACGCGCCCTGTAGCGCCCCA。

2.2 use following primer pair (5 ' -3 '), using saccharomyces cerevisiae BY4742 genomic DNA as template amplification promoter TDH3p。

TDH3p-F:ctgttccagagaacccccatg gtttaaacaccctggtcgacCAGTTCGAGTTTATCATT ATC,

TDH3p-cpr2-R:GATGATGAAGAAGAAGACATTTTGTTTGTTTATGTGTGTT.

2.3 use following primer pair, using the AtCPR2 gene of synthesis as template, amplification gene AtCPR2.

CPR2-tdh3-F:acacacataaacaaacaaaATGTCTTCTTCTTCATCATC,

CPR2-tpi1t-R:AATTATATTAATCCTACCAGACATCTCTCAAAT。

2.4 use following primer pair, using saccharomyces cerevisiae BY4742 genomic DNA as template amplification terminator TPI1t.

TPI1t-cpr2-F:gatatttgagagatgtctggtagGATTAATATAATTATATAAA AA,

TPI1t-tef1p-R:GTAAGGATTCGCGGTCCTCG GCTCTTCTATATAACAGTTG。

2.5 pairs of above three segments carry out overlapPCR, obtain expression cassette TDH3p-AtCPR2-TPI1t.

2.6 use following primer pair, using saccharomyces cerevisiae BY4742 genomic DNA as template amplification promoter TEF1p.

TEF1p-tpi1t-F:ctgttatatagaagagcCGAGGACCGCGAATCCTTAC,

TEF1p-DMDH-R:GAAAAGAACAAAACCATTTTGTAATTAAAACTTAGATTAGA。

2.7 use following primer pair, using the PgPPDS gene of synthesis as template amplification gene PgPPDs.

DMDH-tef1p-F:ctaagttttaattacaaaATGGTTTTGTTCTTTTCTTT

DMDH-cyc1t-R:GGAAAAGGGGCCTGTCTAATTATGTGGATGTAAATGT

2.8 use following primer pair, using saccharomyces cerevisiae BY4742 genomic DNA as template amplification terminator CYC1t.

CYC1t-DMDH-F:catttacatccacataattagACAGGCCCCTTTTCCTTTGT,

CYC1t-R:GAGGTTTTCACCGTCATCACCGGTTACATGCGTACACGCGTT。

2.9 pairs of above three segments carry out overlap PCR, obtain expression cassette TEF1p-DMDH-CYC1t.

2.10 use Gibson method, to carrier segments obtained above, TDH3p-CPR2-TPI1t and TEF1p-DMDH- CYC1t carries out assembly and connection, completes plasmid construction, obtains DMDH and CPR2 co-expression plasmid, is named as pYES2.1-DMDH- CPR2。

2 fallibility PCR method of embodiment constructs hydroxylase random mutant library

Using the plasmid pYES2.1-DMDH-CPR2 obtained in embodiment 1 as template, constructed using fallibility round pcr random The primer pair of mutant library, use is as follows:

Forward primer Errorpcr-F:ATGGTTTTGTTCTTTTCTTT,

Reverse primer Errorpcr-R:AATTATGTGGATGTAAATGT.

50 μ L fallibility PCR reaction systems include: 50ng plasmid template pYES2.1-DMDH-CPR2,30pmol pair of primers Errorpcr-F and Errorpcr-R, 1X Taq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mMdTTP, 7mM MgCl₂, (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM) MnCl₂, the Taq enzyme (Fermentas) of 2.5 units.PCR Reaction condition are as follows: 95 DEG C of 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min/kbp；30 circulations；72℃ 10min.Glue returns 1.5kb random mutation segment is received as big primer, is MegaPrimer PCR:94 DEG C with KOD-plus archaeal dna polymerase 5min,；98 DEG C of 10s, 60 DEG C of 30s, 68 DEG C of 2min/kbp, 25 circulations；68℃ 10min.DpnI digested plasmid template turns Change saccharomyces cerevisiae BY4742 (EUROSCARF), obtains more than 10⁴The random mutation library of a clone.

The expression and preparation of 3 hydroxylase of embodiment

Expressive host selects 0 bacterial strain of saccharomyces cerevisiae BY4742MATa his3 Δ 1leu2 Δ 0met15 Δ 0ura3 Δ.Turn Change, expression and enzyme solution preparation method bibliography (Han, J.-Y., H.-S.Hwang, S.-W.Choi, H.-J.Kim and Y.E.Choi.Cytochrome P450 CYP716A53v2Catalyzes the Formation of Protopanaxatriol from Protopanaxadiol During Ginsenoside Biosynthesis in Panax Ginseng.Plant Cell Physiol.2012,53 (9): 1535-1545.), the specific method is as follows:

Host strain BY4742 strain is crossed on YPD solid medium, 30 DEG C are cultivated 2 days, are chosen single colonie switching and are equipped with The test tube of 4ml YPD fluid nutrient medium.30 DEG C, 220rpm is incubated overnight, and switching is equipped with the 250ml of 25ml YPD fluid nutrient medium Shaking flask, 30 DEG C, 220rpm cultivates 4~6h, culture to OD₆₀₀It is 0.8~1.0, bacterium solution is used to prepare the conversion impression of saccharomyces cerevisiae State.The production and conversion of competence are carried out using Frozen-EZ Yeast Transformation II Kit, stringent to refer to Its operation instructions.Converted product be coated with SC-leu (the basic nitrogen source 1.7g/l of glucose 20g/l, YNB, histidine, leucine and Each 50mg/l of methionine), 30 DEG C are cultivated 4 days.Picking mutation library transformant is inoculated with YPD culture medium, cultivates thallus.Culture knot Thalline were collected by centrifugation by Shu Hou, 2000g 10min, uses 50ml TEK buffer (100mM KCl, 50mM Tris-HCl, 1mM EDTA thallus) is resuspended, 6100g 3min centrifugation uses 2ml Extraction buffer (20mM beta -mercaptoethanol, 1%BSA, 0.6M sorb Sugar alcohol, 50mM TrisHCl, 1mM EDTA) thallus is resuspended, bead is added, it is broken to carry out concussion.Extraction buffer 6100g 15min centrifugation, supernatant filtering, is added the MgCl of final concentration of 50mM₂, continue ice bath 1h, 12500g is centrifuged 5min, and particulate matter makes It is dissolved with the TEG (30% glycerol, 50mM Tris-HCl, 1mM EDTA) of 1ml, and carries out homogeneity with polytetrafluoroethylene (PTFE), completed The small liquid solution of albumen.

The screening of the hydroxylation enzyme mutant of embodiment 4

Picking mutation library transformant one by one carries out the preparation of albumen microbody, reacts for enzyme activity determination.It is hydroxylated enzyme mutant Enzyme activity determination method referring to " hydroxylase enzyme activity determination " part above-mentioned.

In random mutation library, by being screened to about 2000 mutant clones, the outstanding mutant such as institute in table 1 of screening Show.To this two plant mutants body, clone is cooked further verifying, after carrying out plasmid extraction, Suzhou Jin Weizhi biotech company is sent to carry out Sequencing confirmation, identifies amino acid mutation site.

Table 1, mutant enzyme activity

Compared to wild type hydroxylase DMDH it can be seen from the experimental result of table 1, mutant SEQ ID NOs:3-4 is bright Aobvious to improve enzyme activity, wherein the enzyme activity of SEQ ID NO:3 improves more than 4 times, expeditiously catalysis substrate can reach Ma alkene two Alcohol DMD generates protopanoxadiol PPD, has industrialized developing potentiality.

Sequence table

<110>Zhejiang Hua Rui Bioisystech Co., Ltd

<120>a kind of hydroxylation enzyme mutant

<130> SHPI1812133

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 482

<212> PRT

<213> Panax Ginseng

<400> 1

Met Val Leu Phe Phe Ser Leu Ser Leu Leu Leu Leu Pro Leu Leu Leu

1 5 10 15

Leu Phe Ala Tyr Phe Ser Tyr Thr Lys Arg Ile Pro Gln Lys Glu Asn

20 25 30

Asp Ser Lys Ala Pro Leu Pro Pro Gly Gln Thr Gly Trp Pro Leu Ile

35 40 45

Gly Glu Thr Leu Asn Tyr Leu Ser Cys Val Lys Ser Gly Val Ser Glu

50 55 60

Asn Phe Val Lys Tyr Arg Lys Glu Lys Tyr Ser Pro Lys Val Phe Arg

65 70 75 80

Thr Ser Leu Leu Gly Glu Pro Met Ala Ile Leu Cys Gly Pro Glu Gly

85 90 95

Asn Lys Phe Leu Tyr Ser Thr Glu Lys Lys Leu Val Gln Val Trp Phe

100 105 110

Pro Ser Ser Val Glu Lys Met Phe Pro Arg Ser His Gly Glu Ser Asn

115 120 125

Ala Asp Asn Phe Ser Lys Val Arg Gly Lys Met Met Phe Leu Leu Lys

130 135 140

Val Asp Gly Met Lys Lys Tyr Val Gly Leu Met Asp Arg Val Met Lys

145 150 155 160

Gln Phe Leu Glu Thr Asp Trp Asn Arg Gln Gln Gln Ile Asn Val His

165 170 175

Asn Thr Val Lys Lys Tyr Thr Val Thr Met Ser Cys Arg Val Phe Met

180 185 190

Ser Ile Asp Asp Glu Glu Gln Val Thr Arg Leu Gly Ser Ser Ile Gln

195 200 205

Asn Ile Glu Ala Gly Leu Leu Ala Val Pro Ile Asn Ile Pro Gly Thr

210 215 220

Ala Met Asn Arg Ala Ile Lys Thr Val Lys Leu Leu Thr Arg Glu Val

225 230 235 240

Glu Ala Val Ile Lys Gln Arg Lys Val Asp Leu Leu Glu Asn Lys Gln

245 250 255

Ala Ser Gln Pro Gln Asp Leu Leu Ser His Leu Leu Leu Thr Ala Asn

260 265 270

Gln Asp Gly Gln Phe Leu Ser Glu Ser Asp Ile Ala Ser His Leu Ile

275 280 285

Gly Leu Met Gln Gly Gly Tyr Thr Thr Leu Asn Gly Thr Ile Thr Phe

290 295 300

Val Leu Asn Tyr Leu Ala Glu Phe Pro Asp Val Tyr Asn Gln Val Leu

305 310 315 320

Lys Glu Gln Val Glu Ile Ala Asn Ser Lys His Pro Lys Glu Leu Leu

325 330 335

Asn Trp Glu Asp Leu Arg Lys Met Lys Tyr Ser Trp Asn Val Ala Gln

340 345 350

Glu Val Leu Arg Ile Ile Pro Pro Gly Val Gly Thr Phe Arg Glu Ala

355 360 365

Ile Thr Asp Phe Thr Tyr Ala Gly Tyr Leu Ile Pro Lys Gly Trp Lys

370 375 380

Met His Leu Ile Pro His Asp Thr His Lys Asn Pro Thr Tyr Phe Pro

385 390 395 400

Ser Pro Glu Lys Phe Asp Pro Thr Arg Phe Glu Gly Asn Gly Pro Ala

405 410 415

Pro Tyr Thr Phe Thr Pro Phe Gly Gly Gly Pro Arg Met Cys Pro Gly

420 425 430

Ile Glu Tyr Ala Arg Leu Val Ile Leu Ile Phe Met His Asn Val Val

435 440 445

Thr Asn Phe Arg Trp Glu Lys Leu Ile Pro Asn Glu Lys Ile Leu Thr

450 455 460

Asp Pro Ile Pro Arg Phe Ala His Gly Leu Pro Ile His Leu His Pro

465 470 475 480

His Asn

<210> 2

<211> 1449

<212> DNA

<213> Panax Ginseng

<400> 2

atggttttgt tcttttcttt gtctttgttg ttgttgccat tgttattatt gtttgcttat 60

ttttcttaca ctaaacgtat tcctcaaaag gaaaacgact ctaaggctcc attgccaccc 120

ggtcaaactg gatggccatt gattggtgaa actttaaatt acttgtcttg tgttaagtct 180

ggtgtttctg aaaacttcgt taagtataga aaggaaaaat attctccaaa ggttttcaga 240

acttctttat tgggtgaacc aatggctatt ttgtgcggtc cagaaggtaa taagttcttg 300

tattcaactg aaaagaagtt ggtccaagtt tggttccctt cttcagttga aaaaatgttc 360

cctcgttctc acggagagtc aaacgcagat aacttttcta aagttagagg taaaatgatg 420

ttcttattaa aagttgatgg tatgaaaaaa tatgttggtt tgatggatag agttatgaaa 480

caattcttgg agactgattg gaatagacaa caacaaatta atgttcataa cactgttaaa 540

aagtacacag ttactatgtc ttgtagagtt ttcatgtcaa ttgacgacga agagcaagtt 600

acaagattgg gttcttcaat acaaaatatt gaagctggtt tgttggctgt tccaattaat 660

attcccggta ctgcaatgaa tagagctatt aaaacagtta aattgttaac tagagaagtt 720

gaagctgtta taaaacaaag aaaagttgat ttattggaaa acaagcaagc ttcacaacca 780

caagatttgt tatctcattt gttgttgact gctaatcaag atggtcaatt tttgtcagaa 840

tcagatattg catctcattt gattggattg atgcaaggtg gttacacaac attaaacggt 900

acaataacat ttgttttaaa ttatttggct gaatttccag atgtttataa tcaagttttg 960

aaagaacaag ttgaaattgc taattcaaaa catccaaaag aattgttaaa ttgggaagac 1020

ttgagaaaga tgaagtattc ttggaacgtc gctcaagaag tcttgaggat tattccaccc 1080

ggtgttggta ctttcagaga agctataact gattttacat acgctggtta cttgattcca 1140

aaaggttgga aaatgcattt gattccacat gatactcata aaaatccaac ttatttccca 1200

tctccagaaa aatttgaccc aactagattc gaaggtaacg gaccagctcc atacactttc 1260

actccattcg gtggtggacc taggatgtgc cccggtattg agtatgctag gttagttata 1320

ttgatattta tgcataatgt tgttacaaat tttagatggg aaaaattgat accaaatgaa 1380

aagattttga ctgacccaat tccaagattc gctcacggtt tgccaataca tttacatcca 1440

cataattag 1449

<210> 3

<211> 482

<212> PRT

<213>artificial sequence ()

<400> 3

Met Val Leu Phe Phe Ser Leu Ser Leu Leu Leu Leu Pro Leu Leu Leu

1 5 10 15

Leu Phe Ala Tyr Phe Ser Tyr Thr Lys Arg Ile Pro Gln Lys Glu Asn

20 25 30

Asp Ser Lys Ala Pro Leu Pro Pro Gly Gln Thr Gly Trp Pro Leu Ile

35 40 45

Gly Glu Thr Leu Asn Tyr Leu Ser Cys Val Lys Ser Gly Val Ser Glu

50 55 60

Asn Phe Val Lys Tyr Arg Lys Glu Lys Tyr Ser Pro Lys Val Phe Arg

65 70 75 80

Thr Ser Leu Leu Gly Glu Pro Met Ala Ile Leu Cys Gly Pro Glu Gly

85 90 95

Asn Lys Phe Leu Tyr Ser Thr Glu Lys Lys Leu Val Gln Val Trp Phe

100 105 110

Pro Ser Ser Val Glu Lys Met Phe Pro Arg Ser His Gly Glu Ser Asn

115 120 125

Ala Asp Asn Phe Ser Lys Val Arg Gly Lys Met Met Phe Leu Leu Lys

130 135 140

Val Asp Gly Met Lys Lys Tyr Val Gly Leu Met Asp Arg Val Met Lys

145 150 155 160

Gln Phe Leu Glu Thr Asp Trp Asn Arg Gln Gln Gln Ile Asn Val His

165 170 175

Asn Thr Val Lys Lys Tyr Thr Val Thr Leu Ser Cys Arg Val Phe Met

180 185 190

Ser Ile Asp Asp Glu Glu Gln Val Thr Arg Leu Gly Ser Ser Ile Gln

195 200 205

Asn Ile Glu Ala Gly Leu Leu Ala Val Pro Ile Asn Ile Pro Gly Thr

210 215 220

Ala Met Asn Arg Ala Ile Lys Thr Val Lys Leu Leu Thr Arg Glu Val

225 230 235 240

Glu Ala Val Ile Lys Gln Arg Lys Val Asp Leu Leu Glu Asn Lys Gln

245 250 255

Ala Ser Gln Pro Gln Asp Leu Leu Ser His Leu Leu Leu Thr Ala Asn

260 265 270

Gln Asp Gly Gln Phe Leu Ser Glu Ser Asp Ile Ala Ser His Leu Ile

275 280 285

Gly Leu Met Gln Gly Gly Tyr Thr Thr Leu Asn Gly Thr Ile Thr Phe

290 295 300

Val Leu Asn Tyr Leu Ala Glu Phe Pro Asp Val Tyr Asn Gln Val Leu

305 310 315 320

Ala Glu Gln Val Glu Ile Ala Asn Ser Lys His Pro Lys Glu Leu Leu

325 330 335

Asn Trp Glu Asp Leu Arg Lys Met Lys Tyr Ser Trp Asn Val Ala Gln

340 345 350

Glu Val Leu Arg Ile Ile Pro Pro Gly Val Gly Thr Phe Arg Glu Ala

355 360 365

Ile Thr Asp Phe Thr Tyr Ala Gly Tyr Leu Ile Pro Lys Gly Trp Lys

370 375 380

Met His Leu Ile Pro His Asp Thr His Lys Asn Pro Thr Tyr Phe Pro

385 390 395 400

Ser Pro Glu Lys Phe Asp Pro Thr Arg Phe Glu Gly Asn Gly Pro Ala

405 410 415

Pro Tyr Thr Phe Thr Pro Phe Gly Gly Gly Pro Arg Met Cys Pro Gly

420 425 430

Ile Glu Tyr Ala Arg Leu Val Ile Leu Ile Phe Met His Asn Val Val

435 440 445

Thr Asn Phe Arg Trp Glu Lys Leu Ile Pro Asn Glu Lys Ile Leu Thr

450 455 460

Asp Pro Ile Pro Arg Phe Ala His Gly Leu Pro Ile His Leu His Pro

465 470 475 480

His Asn

<210> 4

<211> 482

<212> PRT

<213>artificial sequence ()

<400> 4

Met Val Leu Phe Phe Ser Leu Ser Leu Leu Leu Leu Pro Leu Leu Leu

1 5 10 15

Leu Phe Ala Tyr Phe Ser Tyr Thr Lys Arg Ile Pro Gln Lys Glu Asn

20 25 30

Asp Ser Lys Ala Pro Leu Pro Pro Gly Gln Thr Gly Trp Pro Leu Ile

35 40 45

Gly Glu Thr Leu Asn Tyr Leu Ser Cys Val Lys Ser Gly Val Ser Glu

50 55 60

Asn Phe Val Lys Tyr Arg Lys Glu Lys Tyr Ser Pro Lys Val Phe Arg

65 70 75 80

Thr Ser Leu Leu Gly Glu Pro Met Ala Ile Leu Cys Gly Pro Glu Gly

85 90 95

Asn Lys Phe Leu Tyr Ser Thr Glu Lys Lys Leu Val Gln Val Trp Phe

100 105 110

Pro Ser Ser Val Glu Lys Met Ala Pro Arg Ser His Gly Glu Ser Asn

115 120 125

Ala Asp Asn Phe Ser Lys Val Arg Gly Lys Met Met Phe Leu Leu Lys

130 135 140

Val Asp Gly Met Lys Lys Tyr Val Gly Leu Met Asp Arg Val Met Lys

145 150 155 160

Gln Phe Leu Glu Thr Asp Trp Asn Arg Gln Gln Gln Ile Asn Val His

165 170 175

Asn Thr Val Lys Lys Tyr Thr Val Thr Leu Ser Cys Arg Val Phe Met

180 185 190

Ser Ile Asp Asp Glu Glu Gln Val Thr Arg Leu Gly Ser Ser Ile Gln

195 200 205

Asn Ile Glu Ala Gly Leu Leu Ala Val Pro Ile Asn Ile Pro Gly Thr

210 215 220

Ala Met Asn Arg Ala Ile Lys Thr Val Lys Leu Leu Thr Arg Glu Val

225 230 235 240

Glu Ala Val Ile Lys Gln Arg Lys Val Asp Leu Leu Glu Asn Lys Gln

245 250 255

Ala Ser Gln Pro Gln Asp Leu Leu Ser His Leu Leu Leu Thr Ala Asn

260 265 270

Gln Asp Gly Gln Phe Leu Ala Glu Ser Asp Ile Ala Ser His Leu Ile

275 280 285

Gly Leu Met Gln Gly Gly Tyr Thr Thr Leu Asn Gly Thr Ile Thr Phe

290 295 300

Val Leu Asn Tyr Leu Ala Glu Phe Pro Asp Val Tyr Asn Gln Val Leu

305 310 315 320

Lys Glu Gln Val Glu Ile Ala Asn Ser Lys His Pro Lys Glu Leu Leu

325 330 335

Asn Trp Glu Asp Leu Arg Lys Met Lys Tyr Ser Trp Asn Val Ala Gln

340 345 350

Glu Val Leu Arg Ile Ile Pro Pro Gly Val Gly Thr Phe Arg Glu Ala

355 360 365

Ile Thr Asp Phe Thr Tyr Ala Gly Tyr Leu Ile Pro Lys Gly Trp Lys

370 375 380

Met His Leu Ile Pro His Asp Thr His Lys Asn Pro Thr Tyr Phe Pro

385 390 395 400

Ser Pro Glu Lys Phe Asp Pro Thr Arg Phe Glu Gly Asn Gly Pro Ala

405 410 415

Pro Tyr Thr Phe Thr Pro Phe Gly Gly Gly Pro Arg Met Cys Pro Gly

420 425 430

Ile Glu Tyr Ala Arg Leu Val Ile Leu Ile Phe Met His Asn Val Val

435 440 445

Thr Asn Phe Arg Trp Glu Lys Leu Ile Pro Asn Glu Lys Ile Leu Thr

450 455 460

Asp Pro Ile Pro Arg Phe Ala His Gly Leu Pro Ile His Leu His Pro

465 470 475 480

His Asn

<210> 5

<211> 712

<212> PRT

<213> Arabidopsis thaliana

<400> 5

Met Ser Ser Ser Ser Ser Ser Ser Thr Ser Met Ile Asp Leu Met Ala

1 5 10 15

Ala Ile Ile Lys Gly Glu Pro Val Ile Val Ser Asp Pro Ala Asn Ala

20 25 30

Ser Ala Tyr Glu Ser Val Ala Ala Glu Leu Ser Ser Met Leu Ile Glu

35 40 45

Asn Arg Gln Phe Ala Met Ile Val Thr Thr Ser Ile Ala Val Leu Ile

50 55 60

Gly Cys Ile Val Met Leu Val Trp Arg Arg Ser Gly Ser Gly Asn Ser

65 70 75 80

Lys Arg Val Glu Pro Leu Lys Pro Leu Val Ile Lys Pro Arg Glu Glu

85 90 95

Glu Ile Asp Asp Gly Arg Lys Lys Val Thr Ile Phe Phe Gly Thr Gln

100 105 110

Thr Gly Thr Ala Glu Gly Phe Ala Lys Ala Leu Gly Glu Glu Ala Lys

115 120 125

Ala Arg Tyr Glu Lys Thr Arg Phe Lys Ile Val Asp Leu Asp Asp Tyr

130 135 140

Ala Ala Asp Asp Asp Glu Tyr Glu Glu Lys Leu Lys Lys Glu Asp Val

145 150 155 160

Ala Phe Phe Phe Leu Ala Thr Tyr Gly Asp Gly Glu Pro Thr Asp Asn

165 170 175

Ala Ala Arg Phe Tyr Lys Trp Phe Thr Glu Gly Asn Asp Arg Gly Glu

180 185 190

Trp Leu Lys Asn Leu Lys Tyr Gly Val Phe Gly Leu Gly Asn Arg Gln

195 200 205

Tyr Glu His Phe Asn Lys Val Ala Lys Val Val Asp Asp Ile Leu Val

210 215 220

Glu Gln Gly Ala Gln Arg Leu Val Gln Val Gly Leu Gly Asp Asp Asp

225 230 235 240

Gln Cys Ile Glu Asp Asp Phe Thr Ala Trp Arg Glu Ala Leu Trp Pro

245 250 255

Glu Leu Asp Thr Ile Leu Arg Glu Glu Gly Asp Thr Ala Val Ala Thr

260 265 270

Pro Tyr Thr Ala Ala Val Leu Glu Tyr Arg Val Ser Ile His Asp Ser

275 280 285

Glu Asp Ala Lys Phe Asn Asp Ile Asn Met Ala Asn Gly Asn Gly Tyr

290 295 300

Thr Val Phe Asp Ala Gln His Pro Tyr Lys Ala Asn Val Ala Val Lys

305 310 315 320

Arg Glu Leu His Thr Pro Glu Ser Asp Arg Ser Cys Ile His Leu Glu

325 330 335

Phe Asp Ile Ala Gly Ser Gly Leu Thr Tyr Glu Thr Gly Asp His Val

340 345 350

Gly Val Leu Cys Asp Asn Leu Ser Glu Thr Val Asp Glu Ala Leu Arg

355 360 365

Leu Leu Asp Met Ser Pro Asp Thr Tyr Phe Ser Leu His Ala Glu Lys

370 375 380

Glu Asp Gly Thr Pro Ile Ser Ser Ser Leu Pro Pro Pro Phe Pro Pro

385 390 395 400

Cys Asn Leu Arg Thr Ala Leu Thr Arg Tyr Ala Cys Leu Leu Ser Ser

405 410 415

Pro Lys Lys Ser Ala Leu Val Ala Leu Ala Ala His Ala Ser Asp Pro

420 425 430

Thr Glu Ala Glu Arg Leu Lys His Leu Ala Ser Pro Ala Gly Lys Val

435 440 445

Asp Glu Tyr Ser Lys Trp Val Val Glu Ser Gln Arg Ser Leu Leu Glu

450 455 460

Val Met Ala Glu Phe Pro Ser Ala Lys Pro Pro Leu Gly Val Phe Phe

465 470 475 480

Ala Gly Val Ala Pro Arg Leu Gln Pro Arg Phe Tyr Ser Ile Ser Ser

485 490 495

Ser Pro Lys Ile Ala Glu Thr Arg Ile His Val Thr Cys Ala Leu Val

500 505 510

Tyr Glu Lys Met Pro Thr Gly Arg Ile His Lys Gly Val Cys Ser Thr

515 520 525

Trp Met Lys Asn Ala Val Pro Tyr Glu Lys Ser Glu Asn Cys Ser Ser

530 535 540

Ala Pro Ile Phe Val Arg Gln Ser Asn Phe Lys Leu Pro Ser Asp Ser

545 550 555 560

Lys Val Pro Ile Ile Met Ile Gly Pro Gly Thr Gly Leu Ala Pro Phe

565 570 575

Arg Gly Phe Leu Gln Glu Arg Leu Ala Leu Val Glu Ser Gly Val Glu

580 585 590

Leu Gly Pro Ser Val Leu Phe Phe Gly Cys Arg Asn Arg Arg Met Asp

595 600 605

Phe Ile Tyr Glu Glu Glu Leu Gln Arg Phe Val Glu Ser Gly Ala Leu

610 615 620

Ala Glu Leu Ser Val Ala Phe Ser Arg Glu Gly Pro Thr Lys Glu Tyr

625 630 635 640

Val Gln His Lys Met Met Asp Lys Ala Ser Asp Ile Trp Asn Met Ile

645 650 655

Ser Gln Gly Ala Tyr Leu Tyr Val Cys Gly Asp Ala Lys Gly Met Ala

660 665 670

Arg Asp Val His Arg Ser Leu His Thr Ile Ala Gln Glu Gln Gly Ser

675 680 685

Met Asp Ser Thr Lys Ala Glu Gly Phe Val Lys Asn Leu Gln Thr Ser

690 695 700

Gly Arg Tyr Leu Arg Asp Val Trp

705 710

<210> 6

<211> 2139

<212> DNA

<213> Arabidopsis thaliana

<400> 6

atgtcttctt cttcatcatc ttcaacttct atgattgact tgatggcagc aattattaag 60

ggagaaccag tcattgtctc tgatccagct aacgcttctg catatgagtc tgtcgcagct 120

gagttgtctt ctatgttaat tgaaaataga cagtttgcta tgattgttac tacatcaatt 180

gcagtcttga ttggttgtat tgttatgttg gtttggagga ggtctggatc tggtaattca 240

aagagagttg aacctttaaa acctttagtt attaaaccaa gagaagaaga aattgacgat 300

ggtcgtaaaa aagttactat cttttttgga actcagactg gtactgctga gggtttcgct 360

aaagctttgg gtgaggaggc taaggctaga tatgaaaaaa ctagattcaa gattgttgat 420

ttggatgatt acgctgcaga cgacgacgaa tacgaggaaa aattgaaaaa agaagatgtc 480

gctttcttct ttttagctac atatggtgac ggtgaaccaa cagacaatgc tgcaagattt 540

tacaagtggt tcacagaagg aaatgatcgt ggtgagtggt tgaaaaattt gaaatatggt 600

gtttttggtt tgggaaacag acaatacgag catttcaata aagttgcaaa agtcgttgat 660

gatattttag tcgaacaagg tgctcagaga ttggtccaag tcggtttggg tgacgatgat 720

caatgtatag aggatgactt cacagcttgg agggaggctt tgtggccaga attggatact 780

attttgagag aggaaggtga tacagctgtc gctacaccat acactgctgc agtcttggag 840

tacagagttt caattcatga ttctgaagat gcaaaattta acgatattaa catggctaat 900

ggtaatggtt atactgtttt cgatgctcaa catccataca aggcaaacgt cgcagtcaag 960

agggagttgc acacaccaga atcagataga tcatgcattc atttagagtt tgacatagct 1020

ggttctggtt tgacatacga aactggtgac cacgttggtg tcttgtgcga caacttgtca 1080

gagacagtcg atgaagcttt gagattatta gacatgtctc cagacactta tttctctttg 1140

catgctgaga aagaagatgg tactccaatt tcatcttcat tgcctcctcc attcccacct 1200

tgcaacttaa gaactgcttt gactagatac gcttgtttgt tgtcttctcc taagaaatct 1260

gctttggtcg ctttggctgc acatgcttct gatccaacag aggcagagag gttaaagcac 1320

ttggcatcac cagctggaaa ggttgatgag tactctaagt gggtcgtcga gtcacagcgt 1380

tctttgttag aagttatggc agagttccct tcagctaagc caccattggg tgtcttcttc 1440

gctggagttg caccaaggtt gcaaccaaga ttttattcta tttcttcttc tccaaagatt 1500

gcagaaacta ggattcatgt tacttgtgct ttggtttacg aaaaaatgcc aactggtcgt 1560

attcataaag gtgtctgttc tacatggatg aagaacgctg ttccttatga aaaatctgaa 1620

aactgttcat cagctcctat attcgtcaga caatctaact tcaagttacc ttcagattca 1680

aaggttccaa ttattatgat tggtcccggt actggattgg caccttttcg tggtttcttg 1740

caagaaaggt tggctttggt tgagtctggt gtcgaattgg gaccatcagt cttgtttttc 1800

ggttgtagga acagaagaat ggatttcatt tatgaggagg aattgcaaag gtttgttgag 1860

tctggtgctt tggcagagtt gtctgtcgct ttctctaggg agggtcctac aaaggaatac 1920

gttcaacata aaatgatgga taaagcatct gatatatgga atatgatttc acaaggagct 1980

tatttgtatg tctgcggtga tgcaaagggt atggctaggg acgtccatcg ttctttacat 2040

actattgctc aagaacaagg ttctatggat tcaacaaagg ctgaaggatt tgttaagaac 2100

ttgcaaactt ctggaagata tttgagagat gtctggtag 2139

Claims

1. a kind of hydroxylase, amino acid sequence are as follows:

SEQ ID NO:3, for SEQ ID NO:1 the 186th M replaces with L, the 321st K replaces with the mutant of A；Or Person

SEQ ID NO:4, for SEQ ID NO:1 the 120th F replaces with A, the 186th M replaces with the S of L, the 279th Replace with the mutant of A.

2. encoding the gene of hydroxylase as described in claim 1.

3. including the plasmid of gene as claimed in claim 2.

4. plasmid as claimed in claim 3, which is characterized in that it also includes P450 reduction enzyme coding gene SEQ ID NO:6, For co-expressing hydroxylase and P450 reductase SEQ ID NO:5.

5. having converted plasmid as claimed in claim 3 and cotransformation for expressing the matter of P450 reductase SEQ ID NO:5 The microorganism of grain.

6. having converted the microorganism of plasmid as claimed in claim 5.

7. the microorganism is selected from saccharomyces cerevisiae or Yarrowia lipolytica such as microorganism described in claim 5 or 6.

8. microorganism as claimed in claim 7, which is characterized in that be saccharomyces cerevisiae BY4742.

9. hydroxylase as described in claim 1 or as described in claim 5 or 6 microorganism synthesis protopanoxadiol in Purposes.

10. purposes as claimed in claim 9, which is characterized in that using dammarendiol as substrate raw material, in hydroxylase and P450 Protopanoxadiol is prepared by hydroxylating under reduction enzymatic.