CN107365715A - A kind of preparation method and application of the Candida glycerolgenesis engineering bacteria of the benzyl carbinol of high yield 2 - Google Patents
A kind of preparation method and application of the Candida glycerolgenesis engineering bacteria of the benzyl carbinol of high yield 2 Download PDFInfo
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- CN107365715A CN107365715A CN201710702750.7A CN201710702750A CN107365715A CN 107365715 A CN107365715 A CN 107365715A CN 201710702750 A CN201710702750 A CN 201710702750A CN 107365715 A CN107365715 A CN 107365715A
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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
- 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
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
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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
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y102/0101—Acetaldehyde dehydrogenase (acetylating) (1.2.1.10)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/99—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with other acceptors (1.2.99)
- C12Y102/99003—Aldehyde dehydrogenase (pyrroloquinoline-quinone) (1.2.99.3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/99—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with other acceptors (1.2.99)
- C12Y102/99007—Aldehyde dehydrogenase (FAD-independent) (1.2.99.7)
Abstract
The invention discloses a kind of preparation method and application of the Candida glycerolgenesis engineering bacteria of the benzyl carbinol of high yield 2, belong to gene engineering technology field.Candida glycerolgenesis (Candida glycerinogenes CCTCC M 93018) is one plant of diploid strains, and fast with the speed of growth compared with haploid strains, viability is strong, inheritance stability, prevents lethal or detrimental mutation advantage.The Candida glycerolgenesis 2 benzyl carbinols production bacterial strain excellent as one plant simultaneously, and the characteristic for possessing the benzyl carbinol of enduring high-concentration 2 (4g/L) provides favourable basis for the Metabolically engineered of bacterial strain.The present invention utilizes gene Knockout, has knocked out the acetaldehyde dehydrogenase gene ALD3 of the benzyl carbinol of diploid Candida glycerolgenesis bacterial strain 2 synthesis competition approach.Compared with original Candida glycerolgenesis bacterial strain, ALD3 knockout mutationss bacterial strain 2 benzyl carbinol output increased 18.5%, reach 3.65g/L.
Description
Technical field
The present invention relates to a kind of preparation method and application of the Candida glycerolgenesis engineering bacteria of high yield 2 phenylethyl alcohol, belong to
Gene engineering technology field.
Background technology
2 phenylethyl alcohol, there is simple and elegant, fine and smooth and lasting rose fragrance, be the perfume base of most of essence, in essence and flavoring agent
Boundary's attention.In recent years, 2 phenylethyl alcohol was widely used in the industries such as medicine, food, cosmetics, tobacco and daily chemical product,
Market demand constantly increases.2 phenylethyl alcohol is primarily present in the plants essential oils such as rose, jasmine, narcissus in nature, naturally
Content is relatively low, now commonly uses steam distillation method extraction but loses larger, tends not to meet the needs of people.
The chemical synthesis of 2 phenylethyl alcohol, which exists, endangers many disadvantages such as human health and Environmental security, product purification difficulty
End.With the improvement of living standards and the concern to health, people increasingly pay attention to the security of food, more advocate " green " and
" natural ", natural additive is also increasingly inclined to use in food production.In US and European, the seasoning of " natural " can be marked as
Agent and aromatic must be extracted using physical method from natural material and enzymatic or Production by Microorganism Fermentation.Therefore
As people are increasingly heated up to green, safety, the demand of natural additive, exploitation micro-organisms natural 2-benzyl carbinol turns into one
Kind new trend.
The method of Microbe synthesis benzyl carbinol is concentrated mainly at present carries out biology turn using saccharomycete to L-phenylalanine
Change.L-phenylalanine synthesis 2 phenylethyl alcohol is catalyzed generation phenylpyruvic acid by amino acid transaminase first, then by phenylalanine decarboxylation
Enzymatic generates phenylacetaldehyde, and 2 phenylethyl alcohol, i.e. Ehrlich pathway are finally generated under alcohol dehydrogenase enzyme effect.But phenylacetaldehyde also may be used
Phenylacetate (metabolic pathway is shown in Fig. 1) is generated under the catalysis of acetaldehyde dehydrogenase, therefore 2- can be influenceed in the presence of competition approach
The generation of benzyl carbinol.But the 2 phenylethyl alcohol of high concentration has certain toxicity to microbial cell.Research shows 2.5g/L 2- benzene
Ethanol can cause saccharomyces cerevisiae biomass to reduce by 75%, 3g/L 2 phenylethyl alcohols and can almost completely inhibit Wine brewing yeast strain growth
(Seward RJ,Willets MG,Dinsdale,et al.The effects of ethanol,hexan-1-ol,and 2-
phenylethanol on cider yeast growth,viability,and energy status；Synergistic
inhibition[J].J I Brewing.1996,102(6):439-443.).Therefore 2 phenylethyl alcohol to the toxicity of thalline very
The strategy by Metabolically engineered raising 2 phenylethyl alcohol yield is hindered in big degree.
Candida glycerolgenesis (Candida glycerinogenes CCTCC M 93018) is one plant of diploid strains,
Fast with the speed of growth compared with haploid strains, viability is strong, inheritance stability, prevents lethal or detrimental mutation advantage.
Candida glycerolgenesis has enduring high-concentration 2- benzene second as one plant of bacterial strain with excellent fermentation 2 phenylethyl alcohol performance simultaneously
The characteristics of alcohol (4g/L), the yield that 2 phenylethyl alcohol is improved for Metabolically engineered bacterial strain provide favourable basis.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of Candida glycerolgenesis engineered strain of high yield 2 phenylethyl alcohol,
Knock out the acetaldehyde dehydrogenase gene ALD3 of 2 phenylethyl alcohol synthesis competition approach.
Present invention also offers a kind of method for the Candida glycerolgenesis engineered strain for building high yield 2 phenylethyl alcohol, knock out
2 phenylethyl alcohol synthesizes the acetaldehyde dehydrogenase gene ALD3 of competition approach, obtains the Candida glycerolgenesis engineering bacteria of high yield benzyl carbinol
Strain.The invention solves another technical problem be to provide a kind of method of fermenting and producing benzyl carbinol.Content is as follows：
(1) access of the ring Candida glycerolgenesis of picking 1 seed culture medium (dusty yeast 10g/L, peptone 20g/L, glucose
20g/L, surplus are water) in, at 30 DEG C, under the conditions of 200r/min, shaken cultivation 18h, obtain liquid seed.
(2) the liquid seed that step (1) obtains is accessed into fermentation medium (L-phenylalanine by the inoculum concentration of 5% (v/v)
7g/L, glucose 90g/L, KH2PO45g/L, dusty yeast 1g/L, MgSO4·7H2O 0.5g/L, surplus are water) in, control hair
Ferment temperature is 30 DEG C, rotating speed 200r/min, time 72h, fermentation ends.
Beneficial effects of the present invention：
1. Candida glycerolgenesis (Candida glycerinogenes CCTCC M 93018) is one plant of diploid bacterium
Strain, fast with the speed of growth compared with haploid strains, viability is strong, inheritance stability, prevents lethal or detrimental mutation excellent
Gesture.
2. the Candida glycerolgenesis bacterial strain selected by the present invention is one plant of bacterial strain with excellent 2 phenylethyl alcohol fermenting property,
And the 2 phenylethyl alcohol (4g/L) of higher concentration can be resistant to compared with other 2 phenylethyl alcohols produce bacterial strain, effectively overcome 2- benzene second
The alcohol toxic action to bacterial strain during the fermentation, the yield that 2 phenylethyl alcohol is improved for Metabolically engineered bacterial strain provide favourable base
Plinth.
3. the acetaldehyde that the Candida glycerolgenesis bacterial strain selected by the present invention has knocked out 2 phenylethyl alcohol synthesis competition approach takes off
Hydrogenase gene ALD3, compared with original Candida glycerolgenesis bacterial strain, ALD3 knock-out bacterial strain 2 phenylethyl alcohol output increaseds
18.5%, reach 3.65g/L.
Brief description of the drawings
Fig. 1 L-phenylalanines bioconversion synthesizes the summary of 2 phenylethyl alcohol approach.
Fig. 2 plasmids pADHuha.
Fig. 3 plasmids pADHuhb.
Fig. 4 acetaldehyde dehydrogenase genes ALD3 knocks out principle schematic.
Fig. 5 engineering strains shake flask fermentation synthesizes 2 phenylethyl alcohol.
Embodiment
The present invention is described in further detail below by embodiment.
Embodiment 1
The structure for the heterozygous mutation strain that ALD genes list copy knocks out
(1) synthesis of fragment is knocked out
PCR expands 2.5kb ALD3 genetic fragments, and the fragment includes ALD3 upstream region of gene 500bp-0bp nucleic acid sequence
Arrange (PloALDa, SEQ ID No.2), ALD3 genes and ALD3 downstream of gene 0p-500bp nucleotide sequence (TloALDa, SEQ
ID No.3).And be connected with pMD19-T simple, obtain recombinant plasmid 19T-ALDa.Then using 19T-Ald3a as template, instead
The PloALDa-19T-TloALDa fragments containing two sections of homology arms are obtained to PCR, pass through restriction enzyme site and selection markers HisG-
URA5-HisG (HUH) fragment connects, and obtains and knocks out plasmid 19T-PloALDa-HUH-TloALDa, is named as plasmid pADHuha,
The knockout fragment that restriction enzyme BamH I cuttings plasmid pADHuha is linearized
(2) knockout of first chain of ALD3 genes
In liquid YPD medium, 30 DEG C are incubated overnight the ring Candida glycerolgenesis inoculation of picking one.Take 100 μ L mistakes
Night nutrient solution is transferred in fresh YPD, 30 DEG C of culture 4-6 hours, makes nutrient solution OD600Reach 0.8-1.2, thalline is collected by centrifugation.
With lithium acetate transformation method, the knockout fragment of linearisation obtained above is converted into competence made of Candida glycerolgenesis bacterial strain
MM flat boards are coated in cell, after conversion, 30 DEG C of cultures obtain single bacterium colony in 2-3 days.Picking single bacterium colony extraction genome enters performing PCR
After checking is correct, the heterozygous mutation strain that ALD3 genes list copy knocks out is obtained.
(3) rejecting of selection markers
The heterozygous mutation strain that ALD3 genes list copy knocks out is cultivated in YPD culture mediums and arrives OD600During to 1.0, centrifugation
Thalline is collected, is cleaned with aseptic deionized water, is coated with 2 × FOA flat boards, 30 DEG C of cultures single bacterium colony occur in 2-3 days.Picking single bacterium colony
Genomic PCR checking is extracted, the mutant strain that the URA5 marker gene of acquisition is rejected carries out preservation, is knocked out as Article 2 chain
Convert Host Strains.
Embodiment 2
The structure for the homozygous mutation strain that ALD3 Gene Doubles copy knocks out
(1) synthesis of fragment is knocked out
PCR expands 1.5kb ALD3 genetic fragments, and is connected with pMD19-T simple, obtains recombinant plasmid 19T-
ALD3b.Then using 19T-Ald3b as template, inverse PCR obtains the PloALDb-19T-TloALDb pieces containing two sections of homology arms
Section, is connected by restriction enzyme site with selection markers HisG-URA5-HisG (HUH) fragment, is obtained and is knocked out plasmid 19T-PloALDb-
HUH-TloALDb, is named as plasmid pADHuhb, and what restriction enzyme BamH I cuttings plasmid pADHuhb was linearized strikes
Except fragment.
(2) knockout of ALD3 genes Article 2 chain
The heterozygous mutation strain that the ring ALD3 genes list of picking one copy knocks out is seeded in liquid YPD medium, and 30 DEG C overnight
Culture.Take 100ul to be incubated overnight liquid to be transferred in fresh YPD, 30 DEG C of culture 4-6 hours, make nutrient solution OD600Reach 0.8-
1.2, thalline is collected by centrifugation.With lithium acetate transformation method, the knockout fragment conversion ALD3 gene lists of linearisation obtained above are copied
In competent cell made of the heterozygous mutation strain that shellfish knocks out, MM flat boards are coated on after conversion, 30 DEG C of cultures obtain single for 2-3 days
Bacterium colony.After picking single bacterium colony extraction genome enters performing PCR checking correctly, the homozygous mutation that ALD3 Gene Doubles copy knocks out is obtained
Strain.
Embodiment 3
The homozygous mutation strain access that the original Candida glycerolgenesis of the ring of picking 1, ALD3 Gene Doubles copy knock out respectively
In seed culture medium (dusty yeast 10g/L, peptone 20g/L, glucose 20g/L, surplus are water), at 30 DEG C, 200r/min bars
Under part, shaken cultivation 18h, liquid seed is obtained.Obtained liquid seed is contained by 5% (v/v) inoculum concentration access access
30mL fermentation mediums (L-phenylalanine 7g/L, glucose 90g/L, KH2PO45g/L, dusty yeast 1g/L, MgSO4·7H2O
0.5g/L, surplus are water) 250mL conical flasks in, control fermentation temperature be 30 DEG C, rotating speed 200r/min, time 72h,
Fermentation ends.
The assay method of 2 phenylethyl alcohol in zymotic fluid, analyzed using high performance liquid chromatography (HPLC), it is specific as follows：Will hair
Zymotic fluid is centrifuged, and 10,000r/min, handled again with 0.45 μm of filtering with microporous membrane after centrifugation, filtrate goes up efficient liquid phase immediately
Chromatograph (Agilent, USA), chromatographic column be C18 posts (250mm × 4.6mm, 10 μm；Ailite, China), mobile phase is first
Alcohol:Water=50:50 (v/v), flow rate of mobile phase 0.7mL/min, 30 DEG C, Detection wavelength 260nm of column temperature, the μ L of sample introduction 10.Fermentation knot
The zymotic fluid of gained carries out the detection of 2 phenylethyl alcohol content after beam.Compared with original Candida glycerolgenesis bacterial strain, ALD3 strikes
18.5% except mutant strain bacterial strain 2 phenylethyl alcohol output increased, reach 3.65g/L.
Sequence table
Southern Yangtze University
A kind of preparation method and application of the Candida glycerolgenesis engineering bacteria of high yield 2 phenylethyl alcohol
7
1
1509
DNA
1
ATGATGTCAGCACTGTTCAGAACCATTGAGACTCCAAACGGTAAAACCCTGGAACAACCACTGGGTCTCTTCA
TCGACAATGAGTGGGTGAAAACAAACCGTACTTTTGAGACCATTAATCCGTCCACAGGTGAGGCGATCTGTCATGTT
TACCGTGCTGGGGTCCAGGAGGTGAACGACGCTGTCGAAGCTGCAAATAGAGCATTTAGAAACGAATCTTGGTCAGG
TCTAACTGGTTCTCAACGTGGCGATTTACTGTATCGCATGTACCAAGTTATCAAAAGAGACGCCGAGAGCATTGCAT
CGATTGAGTCCATGGATAATGGTAAACCGTATGCTGCAGAATGCCTAGATGGAGATTTAGGTGAAGCTGCTGACGTT
TTCAAATATTATGCCGGTTGGGCCGACAAGATCACCGGTGAACTCATTGGCTCGAGTGTATTAGGTAAGAATAAGAT
GTGTTATGTCGAGCCTACACCACTGGGTGCCGTTGGCGGTATAGTCCCTTGGAATTTCCCGTTTACCATGATGGCAT
GGAAAATTGCCCCGGCACTGGCGACGGGTTGTACAGTGGTTATGAAGTCAAGTGAAGTCACACCGTTGACGGCATTA
TGGTATGGCAAGATTGCACTTGAAGTGGGTCTACCTAAAGGTGTACTTAACATCCTCTCCGGTTTTGGATCGGATGT
TGGATCGGCCATGGCTTCACATCCAAAGTTGGCTAAGATAGCGTTCACTGGCTCAACTGCAACTGGTAAAAAAATCA
TGGAAGCAGCAGGTGGTTCCAACTTGAAAAAGGTTACACTAGAGTGTGGTGGTAAATCTCCTTACATTGTTTTTGAT
GATGCTGACTTAGAATTGGCAGTAGAATGGGCATATTGGGGTATTTGGTATAACAAAGGTGAGGTTTGTACTTCAAC
TTCGAGATTTTTGATTCAGGAAGACATTTACGATAAGTTTGTTGAGAGTTTTGTTGAGTTGACCAAGACGAGAGCAA
TCACTGCTGATCCGTTTGATGATAGATGCACTATCGGGCCTTTGGTTTCTAGCTCACAGTACGAAAAAGTCAAAAAG
TACGTTGAAATAGGTAAAAATGAAGGAGCAAAGCTACTAACTGGCAAATTCATCGACGGGCCAGGCTATTTCTATGA
GCCATTTATCTTCAGTGAATGCACTGACGATATGACAATCATGAAAGAGGAAATCTTTGGCCCTGTTGTGGGGATTA
CTAAATTCTCAACGGTTAAAGAGGCGATCGAGAGAGCCAATGCTACGACTTACGGTTTAGGAGCTGCGTTGTTTTCC
TCTAACATAACAAAGGCACATTCTGTGGCTGCCAAGTTGGAGGCTGGAATGGTGTGGATCAATTCTAATGGTGATTC
TGATATCCACATTCCATTTGGTGGTTCCAAAATGAGTGGTATAGGTAGGGAGTTGGGGCCATACGCACTAGACTTGT
TTACTGAGAAAAAGGCAGTTCATGTCAACTTATCGCTTCCGGTCAAGTGA
2
500
DNA
2
GTGATTCGGCATCCAATACGTTCTCTGAAATAAGCAGTCTGCCTCCATTGGTAGATTTTATGGGTGGTTTCTT
AGTATTGACGGTTTTTTCATATCCGAGATCTTCCTTTTGCATCTTATAGAATCTCTCTATCTACCAGTTGACCTAAA
CGTGTAATGAGAGGAGAAGGTTCTGCTCGAGGTTGATGCACAGCTCTCCATTCCACGGTCTTTCCACTAACCCATTG
TAATTACCAGAGAAAAATTCCATAGAAAAGAAAAAAAAAAAGAAAAAGAAAAAAAAATGCCGCCGCACGATATCCGC
TTTCACTCGTATTCACTTGCCCCATTGGGCAGGTAAAGCTATACTTGACAATGATCAAGAGGGGTCCCGCCAGAATG
CTTATCTAGCCGGTAGGGGAGAAGCCACTGATAACCAGCNCTGCTGATGTTGGATGAAATATCCAGTATTGAGCAAG
CTTGTAGAATATAGGATATCATTGGATATTATTTCTAATAGA
3
500
DNA
3
TCAAGTACATGAATACGATTGCATATAGGTACGTAATTACTATAGTCTTCTCTATATGTAAACTCAAAATGGA
AATATCTGGATTTCTGGAAAGTTCTCTACACACGCGTACATGGTATTTTGAAATCTCAACTGCACTTAGATACGAAT
TAAATAGAATTTCCTTTAATTTAGAATGTACTTGCTTTCCTTCTTCTCCTTTAACATACACAATTGGCTTCCAACCG
CTATCAAGGAAACAAAATGGTTGTTCAAAACCCAAATAACTGGCACTGGGTCGACAAGAATTGTATTGACTGGGCTC
GTGCCTACTTTGATGAGAAATTGAAGAATGTTGAAGCTCAGGATGATTCAACTACAGTCAAATTGACTTCTGTGAAG
AAGGTAGATGGCGATTGTGAGGTTTGCCAACGCAAGGGCAAGGTCATTTCGTTATTTGACATGTCGATTGAAATTAA
TTATGAATCTAGCACAGGTCACAAAGGGGTCATTAGTATTCC
4
500
DNA
4
ATGATGTCAGCACTGTTCAGAACCATTGAGACTCCAAACGGTAAAACCCTGGAACAACCACTGGGTCTCTTCA
TCGACAATGAGTGGGTGAAAACAAACCGTACTTTTGAGACCATTAATCCGTCCACAGGTGAGGCGATCTGTCATGTT
TACCGTGCTGGGGTCCAGGAGGTGAACGACGCTGTCGAAGCTGCAAATAGAGCATTTAGAAACGAATCTTGGTCAGG
TCTAACTGGTTCTCAACGTGGCGATTTACTGTATCGCATGTACCAAGTTATCAAAAGAGACGCCGAGAGCATTGCAT
CGATTGAGTCCATGGATAATGGTAAACCGTATGCTGCAGAATGCCTAGATGGAGATTTAGGTGAAGCTGCTGACGTT
TTCAAATATTATGCCGGTTGGGCCGACAAGATCACCGGTGAACTCATTGGCTCGAGTGTATTAGGTAAGAATAAGAT
GTGTTATGTCGAGCCTACACCACTGGGTGCCGTTGGCGGTAT
5
509
DNA
5
CTGCTGATCCGTTTGATGATAGATGCACTATCGGGCCTTTGGTTTCTAGCTCACAGTACGAAAAAGTCAAAAA
GTACGTTGAAATAGGTAAAAATGAAGGAGCAAAGCTACTAACTGGCAAATTCATCGACGGGCCAGGCTATTTCTATG
AGCCATTTATCTTCAGTGAATGCACTGACGATATGACAATCATGAAAGAGGAAATCTTTGGCCCTGTTGTGGGGATT
ACTAAATTCTCAACGGTTAAAGAGGCGATCGAGAGAGCCAATGCTACGACTTACGGTTTAGGAGCTGCGTTGTTTTC
CTCTAACATAACAAAGGCACATTCTGTGGCTGCCAAGTTGGAGGCTGGAATGGTGTGGATCAATTCTAATGGTGATT
CTGATATCCACATTCCATTTGGTGGTTCCAAAATGAGTGGTATAGGTAGGGAGTTGGGGCCATACGCACTAGACTTG
TTTACTGAGAAAAAGGCAGTTCATGTCAACTTATCGCTTCCGGTCAAGTGA
6
1611
DNA
6
CTCGAAAACGGCGACGGTATTAGACGTCCCGATTGTAATTGACTTAGTCCTCTTAGGTTCACTATTTGCCTCT
TGTGGTTCTTATCAAAATTGTTGCCGGTTGTGGCAGCTGGAGTAGTGCTTATAGTACTGAATGATGATGACGATGAC
AATCTCCTCTTTGGCCTGATTGACTTTGGCAGTGAATGAAAATGCTGTAGTGATGATTTATTGGACCTTTGAGAAGT
AGATAGCCCTGTTATTATTGGCGTAACTCCATTTACTTCATAAGGTGAGCCTGGTGGTGATATCGAAATCTGCTGTA
ATATATTCATAATATTATTAGTGGTCAATGATGTCTCATTATACACGTTCTCACTTGACATAATGTAATTGTGCTTC
CTGCCTTGTTCCTTAGAGTATATTCTAAATTACTATAGTAAACACCTTTAAATGTATTCCAAAATTTGTCAAAAGTG
ATCAAACCAATCAGTTGGGCGGCCAAGTTCCCCTCTGATTTCTGTCTTTGTCGATAAGTAGGGAATAACCGATAGAG
TGGATATTTTTATTCGTGATGATTTTTTTTTTCTCGCCATTTCTCATTTTTGCTATGATTCATGAGAGAAAAAAAGT
GTTTTTGTCTAATCCAGAAACTATCTTTAAAAGTTAATTTTCATATAATTGAGTGTCTTGAATCACCTGGTCAAGTC
AGATCATTCAGATCGCATATATTTAATTAGCATGCCTTCATACAAGGAAACATTTCTTCAAGCTGCTTTAGATGCTG
AAGCTCTTAAATTTGGTACTTTCACTTTAAAGAGTGGAAGAATATCTCCATATTTCTTCAATATGGGATTATTCAGC
ACTGCAAAGACCTTGAGTACATTAGGTGAATCTTATGCACGTGCCATCGTTGAGTCGGGAATTGAATTTGATATATT
ATTTGGTCCAGCTTATAAGGGTATCCCACTAGCGGCAATCACTGTTACAAAATTGTACGAAATCGGCGGTGCAAAAT
ATGCCAACATTGGCTATTCTTTCAACAGGAAGGAAAAGAAAGACCATGGTGAAGGAGGTTCTATTGTCGGATGCAAT
ATGAAGGGTAAAAAGATTCTAATCATTGATGACGTTATGACCGCAGGTACTGCCATCAATGAAGCATTTGGTATTAT
TTCTGCAGAAGGTGGTAATGCTGTTGGTTGCATTATTGCTTTGGATAGAATGGAAACTACCAAGGACTCCAATGACT
CTGCAACTAACATTGTTGCAAAAAGATACGGCGTCCCTGTTTTCTCTATCGTTTGCTTTGATGACATTATTGAGGTC
TTGAAAGATCAGCTTTCTGAAGAACAAATGGAGAAAATCAACGAATACAGGAAACAGTATGTTCCAGCTAAATAGAG
CACCTCCTTCTTAGTATACGTCTCTTATTATACAGAAATATTTGCTTAGATTTTTTACTTATCATATATATAATTCC
AATTGTTGACTAACCTCTAATTCTTTGGATTTTATGTTTTATCTTTTTGGCTTCAACGGGCTTCTCTGTCTCAGCGG
CAGATTTCATATAAGCACCTGCAGTTTCTGGGTGTTTCATAATGAGGAATGAGTGCATTGGTACAATGGTGTTCA
7
1145
DNA
7
TTCCAGTGGTGCATGAACGCATGAGAAAGCCCCCGGAAGATCATCTTCCGGGGGCTTTTTTTTTGGCGCGCGA
TACAGACCGGTTCAGACAGGATAAAGAGGAACGCAGAATGTTAGACAACACCCGCTTACGCATAGCTATTCAGAAAT
CAGGCCGTTTAAGCGATGATTCACGAGAATTGCTGGCCCGCTGCGGCATAAAAATTAATTTACACACTCAGCGCCTG
ATTGCGATGGCGGAAAACATGCCGATTGATATCCTGCGCGTGCGTGATGATGACATTCCGGGTCTGGTAATGGATGG
CGTGGTCGATCTCGGTATTATCGGCGAAAACGTGCTGGAAGAAGAGCTACTCAACCGCCGCGCACAGGGCGAAGATC
CACGCTATTTAACCCTGCGCCGTCTTGACTTCGGCGGCTGCCGTTTATCGCTGGCAACACCGGTTGACGAAGCCTGG
GACGGCCCGGCCGCGCTGGACGGTAAACGTATCGCTACCTCATATCCGCACCTCCTCAAACGCTACCTCGACCAGAA
AGGCGTCTCTTTTAAATCGTGTCTGTTAAATGGTTCTGTCGAAGTCGCGCCGCGCGCGGGGCTGGCCGACGCTATCT
GCGATTTGGTCTCTACCGGCGCGACGCTTGAAGCTAACGGCCTGCGTGAAGTCGAAGTTATCTACCGCTCTAAAGCC
TGTCTGATTCAGCGCGACGGTGAGATGGCACAGAGCAAGCAAGAGCTGATCGATAAATTGCTGACCCGTATTCAGGG
CGTGATTCAGGCGCGCGAATCGAAATACATCATGATGCACGCGCCAAGTGAACGCCTGGAAGAGGTTATCGCCCTGC
TGCCAGGCGCCGAAAGGCCGACAATTCTGCCGCTGGCAGGCGAGCAACAGCGCGTGGCGATGCACATGGTCAGCAGC
GAAACGTTGTTCTGGGAAACCATGGAGAAACTGAAAGCGCTTGGCGCCAGCTCGATTCTGGTACTGCCGATCGAGAA
GATGATGGAGTGATCTGACGCCTGATGGCGCTGCGCTTATCAGGCCTACGTAATGCGTTGATATTTTGGGTTCTGTA
GGCCGGATAAGGCGGAACCCTGTGATGGAGTAAAGACCATGAGCTTCAATACCCTGATTGACTGGAACAGC
Claims (6)
1. a kind of preparation method of the Candida glycerolgenesis engineering bacteria of high yield 2 phenylethyl alcohol, it is characterised in that knock out production glycerine
The ALD3 genes (SEQ ID No.1) of Candida genome.
2. a kind of preparation method of the Candida glycerolgenesis engineering bacteria of high yield 2 phenylethyl alcohol as described in right 1, its feature exist
In Candida glycerolgenesis used is diploid strains, realizes that the knockout of diploid strains comprises the following steps.
Step 1:ALD3 upstream region of gene 500bp-0bp nucleotide sequence (PloALDa, SEQ ID No.2) and coding will be encoded
ALD3 downstream of gene 0p-500bp nucleotide sequence (TloALDa, SEQ ID No.3) is connected with selection markers, obtains ALD3 bases
Because of the knockout frame of first chain.
Step 2:Frame will be knocked out obtained by step 1 as lithium acetate (LiAC) conversion method and import diploid Candida glycerolgenesis,
By MM plate screenings, picking single bacterium colony enters performing PCR checking, obtains the heterozygous mutation strain that ALD3 genes list copy knocks out.
Step 3:25 μ L are taken to apply after Shaking culture in YPD the heterozygous mutation strain that the ALD3 genes list copy of step 2 knocks out
Cloth 2 × FOA flat boards, 30 DEG C culture 2-3 days after picking single bacterium colony enter performing PCR checking selection markers rejecting.
Step 4:The 0bp-500bp of the ORFs of ALD3 genes nucleotide sequence (PloALDb, SEQ ID will be encoded
No.4) and 1000bp-1509bp nucleotide sequence (TloALDb, SEQ ID No.5) is connected with selection markers, obtains ALD3 bases
Because of the knockout frame of Article 2 chain.
Step 5:Frame steps for importing 3 will be knocked out obtained by step 4 as lithium acetate (LiAC) conversion method, and rejecting screening marks
In the heterozygous mutation strain that ALD3 genes list copy knocks out, by MM plate screenings, picking single bacterium colony enters performing PCR checking, obtains
The homozygous mutation strain that ALD3 Gene Doubles copy knocks out.
3. the preparation method as described in right 2, it is characterised in that Candida glycerolgenesis is uracil auxotrophy bacterial strain,
Selection markers are nutrient defect type mark URA5 (SEQ ID No.6).
4. the preparation method as described in right 2, it is characterised in that the rejecting of selection markers is derived from two sections of difference of URA5 genes
HisG (SEQ ID No.7) repetitive sequence of one section of salmonella is inserted, and passes through the HisG repetitive sequences at URA5 genes both ends
Homologous recombination realize.
5. the Candida glycerolgenesis engineering bacteria of the high yield 2 phenylethyl alcohol of the method structure as described in right 2.
6. application of the Candida glycerolgenesis engineering bacteria in 2 phenylethyl alcohol production as described in right 5, step are as follows：
Step 1：The ring Candida glycerolgenesis of picking 1 accesses seed culture medium (dusty yeast 10g/L, peptone 20g/L, glucose
20g/L, surplus are water) in, at 30 DEG C, under the conditions of 200r/min, shaken cultivation 18h, obtain liquid seed.
Step 2：The liquid seed that step (1) obtains is accessed into fermentation medium (L-phenylalanine by the inoculum concentration of 5% (v/v)
7g/L, glucose 90g/L, KH2PO45g/L, dusty yeast 1g/L, MgSO4·7H2O 0.5g/L, surplus are water) in, control hair
Ferment temperature is 30 DEG C, rotating speed 200r/min, time 72h, fermentation ends.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107723300A (en) * | 2017-11-29 | 2018-02-23 | 江南大学 | It is overexpressed CgGsh1 genes and improves the benzyl carbinol tolerance of Candida glycerolgenesis 2 and yield |
| CN107858361A (en) * | 2017-12-12 | 2018-03-30 | 江南大学 | Candida glycerolgenesis heat shock protein gene CgHsp10 and its application |
| CN110079468A (en) * | 2019-05-09 | 2019-08-02 | 江南大学 | A method of enhancing Candida glycerolgenesis 2 phenylethyl alcohol tolerance |
| CN112322514A (en) * | 2020-11-09 | 2021-02-05 | 江南大学 | Method for enhancing tolerance and yield of glycerol-producing candida 2-phenethyl alcohol |
-
2017
- 2017-08-16 CN CN201710702750.7A patent/CN107365715A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107723300A (en) * | 2017-11-29 | 2018-02-23 | 江南大学 | It is overexpressed CgGsh1 genes and improves the benzyl carbinol tolerance of Candida glycerolgenesis 2 and yield |
| CN107858361A (en) * | 2017-12-12 | 2018-03-30 | 江南大学 | Candida glycerolgenesis heat shock protein gene CgHsp10 and its application |
| CN110079468A (en) * | 2019-05-09 | 2019-08-02 | 江南大学 | A method of enhancing Candida glycerolgenesis 2 phenylethyl alcohol tolerance |
| CN112322514A (en) * | 2020-11-09 | 2021-02-05 | 江南大学 | Method for enhancing tolerance and yield of glycerol-producing candida 2-phenethyl alcohol |
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