CN104830704A - Recombinant bacterium obtained by in-situ expression of (R)-carbonyl reductase in candida parapsilosis and method using same to produce (R)-phenyl glycol efficiently - Google Patents

Recombinant bacterium obtained by in-situ expression of (R)-carbonyl reductase in candida parapsilosis and method using same to produce (R)-phenyl glycol efficiently Download PDF

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CN104830704A
CN104830704A CN201510206914.8A CN201510206914A CN104830704A CN 104830704 A CN104830704 A CN 104830704A CN 201510206914 A CN201510206914 A CN 201510206914A CN 104830704 A CN104830704 A CN 104830704A
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plasmid
rcr
pcp
candida parapsilosis
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张荣珍
徐岩
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Jiangnan University
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract

The invention discloses a recombinant bacterium obtained by in-situ expression of (R)-carbonyl reductase in candida parapsilosis and a method using the same to produce (R)-phenyl glycol efficiently, and belongs to the technical field of bio-catalytic asymmetric conversion. The invention provides a strain of candida parapsilosis pCP-rcr, which has been preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC No. M2015106. The expression plasmid pCP of foreign protein in candida parapsilosis is established, then (R)-carbonyl reductase is cloned to the plasmid to obtain recombinant plasmid pCP-rcr, and finally the recombinant plasmid pCP-rcr is electrically converted into candida parapsilosis to obtain the recombinant strain candida parapsilosis pCP-rcr. The recombinant bacterium can be used to catalyze 2-hydroxy acetophenone, by optimizing the biological conversion reaction conditions, the optical purity of the product namely (R)-phenyl glycol can reach 99.9%, the yield can reach 99.6%, the biological conversion time is shortened from 48 hours to 13 hours, and an efficient and low-cost method is provided for preparing (R)-phenyl glycol.

Description

(R)-carbonyl reductase is expressed recombinant bacterium at Candida parapsilosis situ and is efficiently prepared the method for (R)-phenylglycol with it
Technical field
Utilize the expression plasmid of the Candida parapsilosis of design, realize ( rthe expression of)-carbonyl reductase, efficient preparation ( rthe method of)-phenylglycol, the present invention relates to genetic engineering means build ( rthe expressed in situ recombinant bacterium of)-carbonyl reductase in Candida parapsilosis, and with its efficiently preparation ( rthe method of)-phenylglycol, belongs to biocatalysis asymmetric transformation technical field.
Background technology
Chiral alcohol is the precursor substance synthesizing many high value chipal compounds, is all widely used in pharmaceutical industry, Fine Chemical Industry and information industry.Optical purity ( r)-phenylglycol is not only indispensable important chiral additives in liquid crystal material, and has become preparation and have the important intermediate of optically active medicine, agricultural chemicals and functional materials, carry out prepare high-optical-purity ( rthe research of)-phenylglycol is extremely meaningful.
( rthe chemical structure of)-phenylglycol is:
Stereoselectivity carbonyl reductase can the different prochiral ketone compounds of catalytic reduction, obtains corresponding chiral alcohol product.In order to improve the efficiency of carbonyl reductase catalyzed conversion chiral alcohol further, usually building recombination bacillus coli and improving object enzyme protein expression amount and function.But some carbonyl reductases, may incorrect folding due to protein molecular when intestinal bacteria intracellular expression, cause enzyme activity on the low side or almost there is no enzyme activity, thus do not possess bio-transformation function or the low phenomenon of transformation efficiency.In early-stage Study, derive from Candida parapsilosis c. parapsilosiscCTCC NO:M203011 ( r)-carbonyl reductase achieves expression in intestinal bacteria, and this recombinase can catalytic reduction 2-hydroxy acetophenone, produce ( r)-phenylglycol.But due to ( rexpression amount and the enzyme activity of)-carbonyl reductase are lower, cause the catalysis of recombinant bacterium unsatisfactory, bio-transformation ( rthe efficiency of)-phenylglycol and optical purity are only 61% and 81%.On this basis, devise the expression plasmid of enzyme in Candida parapsilosis cell, employing genetic engineering technique is incited somebody to action ( r)-carbonyl reductase is cloned in Candida parapsilosis cell, achieves the expressed in situ of this enzyme, recombinant bacterial strain catalytic substrate 2-hydroxy acetophenone, by optimizing bioconversion reaction condition, product ( r)-phenylglycol optical purity reaches 99.9%, and productive rate is 99.6%, the time shorten of bio-transformation 4 times.
Summary of the invention
(1) technical problem that will solve
The object of this invention is to provide a kind of genetic engineering technique design Candida parapsilosis expression plasmid, efficient expressed in situ ( r)-carbonyl reductase, utilize recombinant bacterial strain (culture presevation number: CCTCC NO:M 2015106) asymmetric transformation prepare ( rthe method of)-phenylglycol.The object of the invention is, according to the feature of Candida parapsilosis cell self, to utilize Protocols in Molecular Biology, and gene engineering research builds the expression plasmid in intestinal bacteria and Candida parapsilosis, realize ( rthe expressed in situ of)-carbonyl reductase.This recombinant bacterium asymmetric reduction 2-hydroxy acetophenone prepare optical activity ( r)-phenylglycol, the optical purity of product and productive rate are respectively up to 99.9% and 99.6%.( rafter)-carbonyl reductase expressed in situ, enhance the catalysis of this enzyme, for stereoselective oxidoreduction enzyme provides effective way in the expression of Candida parapsilosis situ and the preparation of efficient chirality.
(2) technical scheme
One, genomic acquisition
Wild-type Candida parapsilosis ( c. parapsilosis) CCTCC NO:M203011 grow YPD substratum consist of: glucose 2%, yeast extract paste 1%, peptone 2%.Nearly level and smooth candidiasis CCTCC NO:M203011 is inoculated in substratum liquid amount is in 30 DEG C, 200 r/min shaking culture 48 h in the 250 mL shaking flasks of 30%.After cultivation terminates, by thalline in centrifugal 10 min of 8000 r/min, with brine twice rear collecting cell.Utilize genome DNA extracting reagent kit Genomic DNA Mini Preparation Kit(Takara company) extract genome.
Two, the structure of Candida parapsilosis expressed in situ plasmid pCP
This laboratory build Candida parapsilosis expression plasmid pCP be by pUC57 be starting template build come, comprise following element: promoter gene mAL2pwith aCT1p, terminator gene aCT1twith uRA3t(simultaneously as downstream homology arm) and expression cassette upstream homology arm gene uRA3p and resistant gene sAT1;
Plasmid pCP builds design of primers:
URA3p_1: 5’-ccg gaattcg tattgcaaac aaacg-3’ ( EcoR I)，
URA3p_2: 5’-cgcatccatt c agatcttgt atgaagacgg ca-3’ ( Bgl II)，
MAL2p_1: 5’-gtcttcatac a agatctgaa tggatgcggg-3’ ( Bgl II)，
MAL2p_2: 5’-attgacat ga gctcccgcgg aatagttgta gta -3’( Sac I)，
ACT1t_1: 5’-caccactag g gtaccgagtg aaattct -3’( Kpn I)，
ACT1t_2: 5’-gtcttc ctgc agattttatg atggaat -3’( Pst I)，
ACT1p_1: 5’-aaaat ctgca ggaagaccgt ccaac -3’( Pst I)，
ACT1p_2: 5’-tttaagctta tct gcggccg caccgttatc gataactaaa-3’ ( Not I)，
SAT1_1: 5’-cgataacggt gcggccgcat gaaaatttcg gtga -3’( Not I)，
SAT1_2: 5’-gattaaatat tc ggatcctt aggcgtcatc ct-3’ ( BamH I) ，
URA3t_1: 5’-gatgacgcct aa ggatccga atatttaatc at-3’ ( BamH I)，
URA3t_2: 5’-atccc aagct taacgatcaa gagaaa-3’ ( Hind III)，
Total length 4.5 Kb of expression plasmid pCP, the method adopting overlap-extension PCR and enzyme to cut connection builds, and concrete construction step is as follows:
Will act1psequence is cloned through TA, is connected on carrier pMD19-T, obtains T- act1pplasmid.Use primer SAT1_1 and URA3t_2, with sat1 He ura3t fragment is template, obtains through Overlap extension PCR amplification sat1-ura3t.PCR reaction system 5 × buffer 10.0 μ L, dNTP 5.0 μ L, sat1 fragment 5.0 μ L, ura3t fragment 5.0 μ L, DNA polysaccharase 0.5 μ L, ddH 2o 24.5 μ L; PCR condition is: 98 DEG C of thermally denature 10 s; 98 DEG C of 10 s, 54 DEG C of 15 s, 72 DEG C of 55 s.After 5-10 circulation, take out PCR reaction solution, add each 1 μ L of primer SAT1_1 and URA3t_2, then carry out 30 circulations, last 72 DEG C extend 10 min again.
By T- act1pplasmid and sat1-ura3tfragment uses restriction enzyme respectively noti and hind III carries out double digestion, after glue reclaims, and will sat1-ura3tfragment is connected to T- act1pon plasmid, obtain T-ASU plasmid.
T-ASU plasmid and pUC57 plasmid are used restriction enzyme respectively psti and hind III carries out double digestion, after glue reclaims, and will act1p- sat1- ura3tfragment is connected on pUC57 plasmid, obtains pUC57-ASU plasmid.
Use primer URA3p_1 and MAL2p_2, with ura3pwith mal2pfragment is template, obtains through Overlap extension PCR amplification ura3p-mal2p.
Will ura3p-mal2pfragment and pUC57-ASU plasmid use restriction enzyme respectively ecoRi and saci carries out double digestion, after glue reclaims, and will ura3p-mal2pfragment is connected on pUC57-ASU plasmid, obtains pUC57-UMASU plasmid.
Will act1tfragment and pUC57-UMASU plasmid use restriction enzyme respectively kpni and psti carries out double digestion, after glue reclaims, and will act1tfragment is connected on pUC57-UMASU plasmid, obtains for Candida parapsilosis cell situ expression plasmid pCP.
Three, contain rcrthe recombinant plasmid pCP-of gene rcrstructure
( r) -carbonyl reductase gene rcrclone:
With Candida parapsilosis genome rcrfor PCR reaction template, utilization contains sacIwith kpnIprimer RCR_1 and RCR_2 of restriction enzyme site:
Synthesis two ends primer:
RCR_1：5’-ctacaactat tccgcgggag ctcatgtcaa ttccatca-3’（ Sac I），
RCR_2：5’-cactcggtac cctagtggtg gtggtggtgg tgtggattaa aa-3’ ( Kpn I)，
PCR reaction system: ddH 2o 33.5 μ L, archaeal dna polymerase 0.5 μ L, RCR_1 1 μ L, RCR_2 1 μ L, 5 × buffer 10 μ L, dNTP 4 μ L.
PCR reacts: 98 DEG C of thermally denature 10 s, 98 DEG C of 10 s, 52 DEG C of 15 s, 72 DEG C of 70 s.Carry out 30 circulations, last 72 DEG C extend 10 min again.Finally obtain total length ( r)-carbonyl reductase rcrgene, rcrfull length gene is 1011 bp.
Gene rcrand the enzyme of pCP is cut:
Utilize the vast Tyke biological gene Technology Co., Ltd. in plasmid extraction kit Mini-Plasmid Rapid Isolation Kit(Beijing) extract plasmid pCP.
Goal gene rcrand expression plasmid pCP uses respectively ecor I enzyme is cut, and endonuclease reaction system forms: 10 × Buffer H 4 μ L, DNA 10 μ L, saci 2 μ L, kpni 2 μ L, ddH 2system is supplied 40 μ L by O.Be added in Eppendorf pipe according to the order of water, damping fluid, plasmid DNA, enzyme, build pipe lid, vibration makes liquid fully mix, being placed in centrifugal 2 s in whizzer makes liquid concentrate at the bottom of pipe, 37 DEG C of 3 h, pipe is maybe placed in 65 DEG C of insulation 10 min by the Loading Buffer adding 1/10 volume in pipe, stops endonuclease reaction.Digestion products carries out agarose gel electrophoresis analysis and crosses column purification, cuts glue and reclaims and concentrate.
Gene rcrwith the connection of plasmid pCP:
Reaction system is composed as follows: plasmid pCP 0.8 μ L, gene rcr4.2 μ L, Ligation Solution 5 μ L, is placed in 16 DEG C of incubators and connects 12-16 h by Hybrid connections liquid.
Connection product conversion intestinal bacteria ( escherich coli) JM109
At 100 μ L of every pipe e. coliadd 10 μ L in JM109 competent cell suspension and connect product, mix gently, in ice bath, leave standstill 30 min.Proceed in 42 DEG C of water-baths, thermal shock 90 s.2 min are cooled in fast transfer to ice bath.Often add 700 μ L LB liquid nutrient mediums (peptone 10 g/L, yeast extract paste 5 g/L, NaCl 10 g/L) in pipe, 37 DEG C of 100 r/min shaking table incubation cultivates 1 h.Centrifugal 2 min of bacterium liquid 3,000 r/min after cultivating, abandon supernatant 600 μ L, LB flat board (peptone 10 g/L, yeast extract paste 5 g/L, NaCl 10 g/L containing 100 μ g/mL penbritins is applied to after the mixing of residue bacterium liquid, agar 20 g/L) on, be inverted overnight incubation for 37 DEG C.
The selection of positive colony:
Picking 4 clone, transfer be equipped with 5 mL containing 100 μ g/mL penbritins LB substratum in, cultivate 12 h, utilize the vast Tyke biological gene Technology Co., Ltd. in plasmid extraction kit Mini-Plasmid Rapid Isolation Kit(Beijing for 37 DEG C) extract plasmid.Digestion verification is carried out: 10 × Buffer H 2 μ L, plasmid DNA 5 μ L by following reaction system, saci 0.5 μ L, kpni0.5 μ L, ddH 2system is supplied 20 μ L by O.Obtain positive plasmid pCP- rcr.Recombinant plasmid pCP-simultaneously rcrdeliver to Shanghai Sheng Gong biotechnology company limited to carry out checking order and determine further.
Four, contain rcrthe structure of the restructuring Candida parapsilosis bacterium of gene
Plasmid pCP- rcrsingle endonuclease digestion:
Utilize the vast Tyke biological gene Technology Co., Ltd. in plasmid extraction kit Mini-Plasmid Rapid Isolation Kit(Beijing) from e. coliplasmid pCP-is extracted in JM109 rcr.
Be added in Eppendorf pipe according to the order of water, damping fluid, gene or plasmid DNA, enzyme, build pipe lid, vibration makes liquid fully mix, being placed in centrifugal 10 s in whizzer makes liquid concentrate at the bottom of pipe, 37 DEG C of water-bath 1 h, pipe is maybe placed in 65 DEG C of insulation 10 min by the QuickCut Buffer adding 1/10 volume in pipe, stops endonuclease reaction.Digestion products carries out agarose gel electrophoresis analysis and crosses column purification.Sample Vacuumdrier after purifying is concentrated into 5 μ L.
Reaction system forms: 10 × QuickCut Buffer 5 μ L, DNA 20 μ L, QuickCut ecor I1.5 μ L, ddH 2system is supplied 50 μ L by O.
Single endonuclease digestion linearization plasmid transforms Candida parapsilosis:
In 80 μ L Candida parapsilosis competent cell suspensions of every pipe, add the dense linearizing product of 5-10 ng single endonuclease digestion, proceed in the electric revolving cup of 1 mm after mixing gently, leave standstill 3 min on ice.Dry steam, be placed on electroporation, select yeast parameter (shock parameters: 1500 V, 200 Ω, 25 μ F), electricity transforms.Add the sorbyl alcohol of 1 M of 1 mL ice bath after electric shock immediately, proceed in 1.5 mL centrifuge tubes, 30 DEG C, quiescent culture 2 h.Then by nutrient solution in centrifugal 5 min of 5000 r/min, abandon supernatant 900 μ L.After re-suspended cell, the YPD screening coated containing 40 μ g/mL Knowles rhzomorphs is dull and stereotyped, is inverted 2 d in 30 DEG C of incubators.
The selection of positive colony:
After will abandoning supernatant, resuspended bacterium liquid is drawn in the Knowles rhzomorph resistant panel that 100 μ L are applied to containing 40 μ g/mL.Cultivate after 2 days, the clone that choosing colony is larger transfers in 4 mL YPD liquid nutrient mediums, and 30 DEG C, 220 r/min cultivate 24 h, gets 1 mL bacterium liquid and extracts genome.With restructuring c.parapsilosisgenome is template, uses primer SAT1_1 and URA3t_2 to carry out PCR checking, obtains positive colony candida parapsilosispCP- rcr.
Prepared by Candida parapsilosis bacterium asymmetric transformation of five, recombinating ( r)-phenylglycol
Utilize restructuring Candida parapsilosis candida parapsilosispCP- rcrcatalytic asymmetric reduction reacts:
Candida parapsilosis ( c. parapsilosis) CCTCC NO:M203011 substratum consists of: glucose 4%, yeast extract paste 0.5%, (NH 4) 2hPO 41.3%, KH 2pO 40.7%, ZnSO 47H 2o 0.03%, NaCl 0.01%, pH7.0.
Picking positive colony list colony inoculation is in the yeast growth medium of the Knowles rhzomorph 5 mL YPD containing 40 μ g/mL, and in 28 DEG C, 200 r/min shaking culture are spent the night.Getting 1 mL nutrient solution transfers in 50 mL Candida parapsilosis fermention mediums, in 28 DEG C, and 200 r/min shaking culture 36 h.
Centrifugal 10 min of restructuring Candida parapsilosis cell 8,000 r/min after cultivation also collect with after brine three times.Asymmetric transformation reaction is carried out: 1 mL 0.1 mol/L acetate buffer solution (pH4.0 ~ 6.0) or 1 mL 0.1 mol/L phosphoric acid buffer (pH 6.0 ~ 7.0) in following system, or 1 mL 0.1 mol/L Tris-HCl damping fluid (pH 7.5 ~ 9.0), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g/mL to recombinate Candida parapsilosis wet thallus, after mixing on 35 DEG C of constant-temperature tables oscillatory reaction 25 h.
After reaction terminates, by centrifugal for reaction mixture removing thalline, supernatant liquor adds 2 times of volume of ethylacetate extractions, and organic phase is used for analyzing.Product is analyzed by Chiral stationary phase liquid chromatography (Agillent HP1100), and condition is Chiralcel OB-H post (4.6 mm × 25 cm; Daicel Chemical Ind., Ltd., Japan), moving phase is normal hexane/Virahol (9/1, v/v), flow velocity 0.4 mL/min, and determined wavelength is 215 nm.The optical purity of product is weighed by mapping excessive value.
Product ( rthe calculating of)-phenylglycol mapping excessive value: mapping excessive value (e.e.%)=[( c r- c s)/( c r+ c s)] ' 100%,
Product ( rthe calculating of)-phenylglycol productive rate: productive rate (%)=( c r/ c 0) ' 100%,
In formula c sfor after reacting ( sthe concentration of)-enantiomorph, c rfor after reacting ( rthe concentration of)-enantiomorph, c 0for reacting the concentration of front substrate 2-hydroxy acetophenone.
With full cell for catalyzer, after asymmetric bioconversion reaction, product is ( r)-phenylglycol.
(3) beneficial effect
Success build Candida parapsilosis expressed in situ plasmid pCP and ( rthe recombinant plasmid pCP-of)-carbonyl reductase rcr, recombinant plasmid transformed Candida parapsilosis competent cell, successfully obtains recombinant type Candida parapsilosis candida parapsilosispCP- rcr.
Reconstitution cell candida parapsilosispCP- rcrasymmetric transformation 2-hydroxy acetophenone, obtain high-optical-purity and high yield product ( r)-benzoglycols.By Optimal reaction conditions, at 30 DEG C, in the acetate buffer solution of pH6.5, utilize 0.1 g/mL reconstitution cell catalyzed conversion 6 g/L substrate 2-hydroxy acetophenone, product ( rthe optical purity of)-phenylglycol and productive rate are respectively 99.9% and 99.6%.Compare wild-type Candida parapsilosis cell asymmetric transformation, the reaction times shortens 4 times.These work are not only ( rthe efficient preparation of)-phenylglycol provides effective way, and contributes to the stereoselectivity being familiar with enzyme from protein folding mode different hosts, and the high-quality biological catalyst exploitation for chiral inversion has more important meaning.
Biological material specimens preservation: Candida parapsilosis expressed in situ recombinant bacterium candida parapsilosispCP- rcr, be preserved in China typical culture collection center, be called for short CCTCC, address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M 2015106, preservation date: on March 12nd, 2015.
Accompanying drawing explanation
Fig. 1 represents each element of Candida parapsilosis expressed in situ plasmid pCP in the present invention.
Embodiment
Embodiment 1
Candida parapsilosis ( c. parapsilosis) CCTCC NO:M203011(is in the patent No.: open in CN 03132140.2) yeast culture, its growth medium YPD consists of: glucose 2%, yeast extract paste 1%, peptone 2%.Candida parapsilosis is inoculated in 30 DEG C in 5 mL test tubes, 200 r/min shaking culture 24 h.
Embodiment 2
Candida parapsilosis ( c. parapsilosis) the genomic extraction of CCTCC NO:M203011: thalline embodiment 1 cultivated is in 8, centrifugal 10 min of 000 r/min, with brine twice rear collecting cell, utilize genome DNA extracting reagent kit Genomic DNA Mini Preparation Kit(Takara company) extract genome.
Embodiment 3
Goal gene is cloned from Candida parapsilosis genome rcr.
Synthesis two ends primer:
RCR_1：5’-ctacaactat tccgcgggag ctcatgtcaa ttccatca-3’（ Sac I），
RCR_2：5’-cactcggtac cctagtggtg gtggtggtgg tgtggattaa aa-3’ ( Kpn I)，
PCR reaction system: ddH 2o 33.5 μ L, DNA polysaccharase 0.5 μ L, RCR_1 1 μ L, RCR_2 1 μ L, 5 × buffer 10 μ L, dNTP 4 μ L;
PCR condition: 98 DEG C of thermally denature 10 s; 98 DEG C of 10 s, 52 DEG C of 15 s, 72 DEG C of 1 min.After 30 circulations, last 72 DEG C extend 10 min again.Utilize 3S Spin Agarose Gel DNA Purification Kit(Shanghai Shenergy Biocolor BioScience & Technology Company) purify DNA segment.Obtain rcrgene, rcrfull length gene is 1011 bp.
Embodiment 4
The structure of pCP expression plasmid:
Plasmid pCP builds design of primers:
URA3p_1: 5’-ccg gaattcg tattgcaaac aaacg-3’ ( EcoR I)，
URA3p_2: 5’-cgcatccatt c agatcttgt atgaagacgg ca -3’( Bgl II)，
MAL2p_1: 5’-gtcttcatac a agatctgaa tggatgcggg -3’( Bgl II)，
MAL2p_2: 5’-attgacat ga gctcccgcgg aatagttgta gta -3’( Sac I)，
ACT1t_1: 5’-caccactag g gtaccgagtg aaattct -3’( Kpn I)，
ACT1t_2: 5’-gtcttc ctgc agattttatg atggaat-3’ ( Pst I)，
ACT1p_1: 5’-aaaat ctgca ggaagaccgt ccaac -3’( Pst I)，
ACT1p_2: 5’-tttaagctta tct gcggccg caccgttatc gataactaaa-3’ ( Not I)，
SAT1_1: 5’-cgataacggt gcggccgcat gaaaatttcg gtga -3’( NotI)，
SAT1_2: 5’-gattaaatat tc ggatcctt aggcgtcatc ct -3’( BamH I)，
URA3t_1: 5’-gatgacgcct aa ggatccga atatttaatc at-3’ ( BamH I)，
URA3t_2: 5’-atccc aagct taacgatcaa gagaaa-3’ ( Hind III)，
Expression plasmid pCP total length 4.5 Kb, the method adopting overlap-extension PCR and enzyme to cut connection builds:
Will act1psequence is cloned through TA, is connected on carrier pMD19-T, obtains T- act1pplasmid.Use primer SAT1_1 and URA3t_2, with sat1with ura3tfragment is template, obtains through Overlap extension PCR amplification sat1- ura3t.PCR reaction system 5 × buffer 10.0 μ L, dNTP 5.0 μ L, sat1 fragment 5.0 μ L, ura3t fragment 5.0 μ L, DNA polysaccharase 0.5 μ L, ddH 2o 24.5 μ L, PCR condition is: 98 DEG C of thermally denature 10 s; 98 DEG C of 10 s, 54 DEG C of 15 s, 72 DEG C of 55 s.After 5-10 circulation, take out PCR reaction solution, add each 1 μ L of primer SAT1_1 and URA3t_2, then carry out 30 circulations, last 72 DEG C extend 10 min again.
By T- act1pplasmid and sat1-ura3tfragment uses restriction enzyme respectively noti and hind III carries out double digestion, after glue reclaims, and will sat1-ura3tfragment is connected to T- act1pon plasmid, obtain T-ASU plasmid.
T-ASU plasmid and pUC57 plasmid are used restriction enzyme respectively psti and hind III carries out double digestion, after glue reclaims, and will act1p- sat1- ura3tfragment is connected on pUC57 plasmid, obtains pUC57-ASU plasmid.
Use primer URA3p_1 and MAL2p_2, with ura3pwith mal2pfragment is template, obtains through Overlap extension PCR amplification ura3p-mal2p.
Will ura3p-mal2pfragment and pUC57-ASU plasmid use restriction enzyme respectively ecor I and saci carries out double digestion, after glue reclaims, and will ura3p-mal2pfragment is connected on pUC57-ASU plasmid, obtains pUC57-UMASU plasmid.
Will act1tfragment and pUC57-UMASU plasmid use restriction enzyme respectively kpni and psti carries out double digestion, after glue reclaims, and will act1tfragment is connected on pUC57-UMASU plasmid, obtains expression plasmid pCP.
Embodiment 5
PCP- rcrthe structure of recombinant plasmid: goal gene rcrand the endonuclease reaction system of plasmid pCP: 10 × Buffer H 2 μ L, DNA 5 μ L, saci 0.5 μ L, kpni0.5 μ L, ddH 2system is supplied 20 μ L by O, 37 DEG C of water-bath 1 h, and pipe is maybe placed in 65 DEG C of insulation 10 min by the 10 × QuickCut Buffer adding 1/10 volume in pipe, stops endonuclease reaction.Digestion products through agarose gel electrophoresis analysis and cross column purification.Adopt following ligation system: plasmid pCP 1.5 μ L, gene rcr7.0 μ L, 10 × Ligation buffer 1 μ L, T4 ligase 0.5 μ L, be placed in 16 DEG C of incubators and connect 16 h by Hybrid connections liquid.
At every pipe 100 μ L e. coliadd 10 μ L in JM109 competent cell suspension and connect product, mix gently, in ice bath, leave standstill 30 min.Proceed in 42 DEG C of water-baths, thermal shock 90 s.Fast transfer to ice bath cools 2 min.Often add 700 μ L LB liquid nutrient mediums in pipe, 37 DEG C of 100 r/min shaking table incubation cultivates 1 h.Centrifugal 2 min of bacterium liquid 5,000 r/min after cultivating, abandon supernatant 600 μ L, be applied on the LB flat board containing 100 μ g/mL penbritins, be inverted overnight incubation for 37 DEG C after the mixing of residue bacterium liquid.
Picking 4 clone, transfer into LB substratum (peptone 10 g/L containing 100 μ g/mL penbritins that 4 mL are housed, yeast extract paste 5 g/L, NaCl 10 g/L) in, cultivate 12 h, utilize the vast Tyke biological gene Technology Co., Ltd. in plasmid extraction kit Mini-Plasmid Rapid Isolation Kit(Beijing for 37 DEG C) extract plasmid.With saci and kpni carries out double digestion checking, obtains positive plasmid pCP- rcr.
Embodiment 6
( rthe acquisition of the restructuring Candida parapsilosis of)-carbonyl reductase expressed in situ: utilize the vast Tyke biological gene Technology Co., Ltd. in plasmid extraction kit Mini-Plasmid Rapid Isolation Kit(Beijing) from e.coliplasmid pCP-is extracted in JM109 rcr.The plasmid extracted carries out single endonuclease digestion linearization process, reacts to be: 10 × QuickCut Buffer 5 μ L, DNA 15 μ L, QuickCut ecor I1.5 μ L, ddH 2o complements to 50 μ L.37 DEG C of water-bath 1 h, pipe is maybe placed in 65 DEG C of insulation 10 min by the 10 × QuickCut Buffer adding 1/10 volume in pipe, stops endonuclease reaction.Digestion products carries out agarose gel electrophoresis analysis and crosses column purification.Sample Vacuumdrier after purifying is concentrated into 0-5 μ L.
Add in 80 μ L Candida parapsilosis competent cell suspensions of every pipe 5-10 ng concentrate after single endonuclease digestion linearizing product pCP- rcr, proceed in the electric revolving cup of 1 μm after mixing gently, leave standstill 3 min on ice.Dry steam, be placed on electroporation, select yeast parameter (shock parameters: 1500 V, 200 Ω, 25 μ F), electricity transforms.Add the sorbyl alcohol of 1 M of 1 mL ice bath after electric shock immediately, proceed in 1.5 mL centrifuge tubes, 30 DEG C, quiescent culture 2 h.Then by nutrient solution in centrifugal 5 min of 5000 r/min, abandon supernatant 900 μ L.After resuspended for bacterium liquid, the Knowles rhzomorph resistance YPD be applied to containing 40 μ g/mL is dull and stereotyped, and cultivate after 2 days, the clone that choosing colony is larger transfers in 4 mL YPD liquid nutrient mediums, and 30 DEG C, 220 r/min cultivate 24 h, gets 1 mL bacterium liquid and extracts genome.With Candida parapsilosis genome of recombinating for template, primer SAT1_1 and URA3t_2 is used to carry out PCR checking.Preservation positive colony c. parapsilosispCP- rcr.
Embodiment 7
The cultivation of recombinant bacterial strain Candida parapsilosis and thalline obtain: Candida parapsilosis ( c. parapsilosis) CCTCC NO:M203011 substratum consists of: glucose 4%, yeast extract paste 0.5%, (NH 4) 2hPO 41.3%, KH 2pO 40.7%, ZnSO 47H 2o 0.03%, NaCl 0.01%, pH7.0.Recombinant type Candida parapsilosis cultivates the Knowles rhzomorph also needing interpolation 40 μ g/mL.
Picking positive colony list colony inoculation in the YPD yeast growth medium of 5 mL containing the Knowles rhzomorph of 40 μ g/mL, in 30 DEG C, 200 r/min shaking culture spend the night.Getting 1 mL nutrient solution transfers in 50 mL Candida parapsilosis fermention mediums, in 30 DEG C, 200 r/min shaking culture 48 h.Recombinant type Candida parapsilosis cell after cultivation is in 8, and centrifugal 10 min of 000 r/min also collect with after brine three times.
Embodiment 8
In 2 mL 0.1 mol/L phosphoric acid buffers (pH 5.0), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g respectively and to recombinate Candida parapsilosis cell, after mixing on 30 DEG C of constant-temperature tables oscillatory reaction 24 h.After reaction, mixture is centrifugal, gets supernatant liquor extraction, product ( rthe optical purity of)-phenylglycol and yield are respectively 69.3% e.e. and 68.1%.
Embodiment 9
In 2 mL 0.1 mol/L phosphoric acid buffers (pH 5.5), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g respectively and to recombinate Candida parapsilosis cell, after mixing on 30 DEG C of constant-temperature tables oscillatory reaction 24 h.After reaction, mixture is centrifugal, gets supernatant liquor extraction, product ( rthe optical purity of)-phenylglycol and yield are respectively 85.4% e.e. and 82.4%.
Embodiment 10
In 2 mL 0.1 mol/L phosphoric acid buffers (pH 6.0), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g respectively and to recombinate Candida parapsilosis cell, after mixing on 30 DEG C of constant-temperature tables oscillatory reaction 24 h.After reaction, mixture is centrifugal, gets supernatant liquor extraction, product ( rthe optical purity of)-phenylglycol and yield are respectively 93.6% e.e. and 94.0%.
Embodiment 11
In 2 mL 0.1 mol/L phosphoric acid buffers (pH 6.5), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g respectively and to recombinate Candida parapsilosis cell, after mixing on 20 DEG C of constant-temperature tables oscillatory reaction 19 h.After reaction, mixture is centrifugal, gets supernatant liquor extraction, product ( rthe optical purity of)-phenylglycol and yield are respectively 86.5% e.e. and 75.2%.
Embodiment 12
In 2 mL 0.1 mol/L phosphoric acid buffers (pH 6.5), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g respectively and to recombinate Candida parapsilosis cell, after mixing on 25 DEG C of constant-temperature tables oscillatory reaction 15 h.After reaction, mixture is centrifugal, gets supernatant liquor extraction, product ( rthe optical purity of)-phenylglycol and yield are respectively 89.9% e.e. and 90.3%.
Embodiment 13
In 2 mL 0.1 mol/L phosphoric acid buffers (pH 6.5), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g respectively and to recombinate Candida parapsilosis cell, after mixing on 30 DEG C of constant-temperature tables oscillatory reaction 13 h.After reaction, mixture is centrifugal, gets supernatant liquor extraction, product ( rthe optical purity of)-phenylglycol and yield are respectively 99.9% e.e. and 99.6%.
Embodiment 14
In 2 mL 0.1 mol/L phosphoric acid buffers (pH 6.5), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g respectively and to recombinate Candida parapsilosis cell, after mixing on 35 DEG C of constant-temperature tables oscillatory reaction 15 h.After reaction, mixture is centrifugal, gets supernatant liquor extraction, product ( rthe optical purity of)-phenylglycol and yield are respectively 98.7% e.e. and 97.0%.
Embodiment 11
In 2 mL 0.1 mol/L phosphoric acid buffers (pH 6.5), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g respectively and to recombinate Candida parapsilosis cell, after mixing on 40 DEG C of constant-temperature tables oscillatory reaction 20 h.After reaction, mixture is centrifugal, gets supernatant liquor extraction, product ( rthe optical purity of)-phenylglycol and yield are respectively 78.7% e.e. and 67.5%.
Embodiment 12
In 2 mL 0.1 mol/L phosphoric acid buffers (pH 7.0), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g respectively and to recombinate Candida parapsilosis cell, after mixing on 30 DEG C of constant-temperature tables oscillatory reaction 24 h.After reaction, mixture is centrifugal, gets supernatant liquor extraction, product ( rthe optical purity of)-phenylglycol and yield are respectively 94.4% e.e. and 95.7%.
Embodiment 13
In 2 mL 0.1 mol/L phosphoric acid buffers (pH 8.0), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g respectively and to recombinate Candida parapsilosis cell, after mixing on 30 DEG C of constant-temperature tables oscillatory reaction 24 h.After reaction, mixture is centrifugal, gets supernatant liquor extraction, product ( rthe optical purity of)-phenylglycol is 87.9% and 82.9%.
Embodiment 14
In 2 mL 0.1 mol/L phosphoric acid buffers (pH 9.0), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g respectively and to recombinate Candida parapsilosis cell, after mixing on 30 DEG C of constant-temperature tables oscillatory reaction 24 h.After reaction, mixture is centrifugal, gets supernatant liquor extraction, product ( rthe optical purity of)-phenylglycol is 63.7%. with 60.1%.
Embodiment 15
In 2 mL 0.1 mol/L phosphoric acid buffers (pH 10.0), add 6 g/L substrate 2-hydroxy acetophenones and 0.1 g respectively and to recombinate Candida parapsilosis cell, after mixing on 30 DEG C of constant-temperature tables oscillatory reaction 24 h.After reaction, mixture is centrifugal, gets supernatant liquor extraction, product ( rthe optical purity of)-phenylglycol is 57.3% e.e. and 42.6%.

Claims (8)

  1. A 1. strain asymmetric transformation preparation ( rthe expressed in situ recombinant bacterium of)-phenylglycol, its Classification And Nomenclature be Candida parapsilosis bacterium/( r)-carbonyl reductase candida parapsilosispCP- rcr, be preserved in China typical culture collection center, deposit number: CCTCC NO:M2015106.
  2. 2. utilize the expressed in situ recombinant bacterium asymmetric transformation described in claim 1 prepare ( rthe method of)-phenylglycol, it is characterized in that by ( r)-carbonyl reductase, after Candida parapsilosis situ is expressed, utilizes the recombinant bacterial strain after cultivating candida parapsilosispCP- rcr, with 2-hydroxy acetophenone for substrate, catalysis asymmetric transformation reacts: in the phosphoric acid buffer of 2 mL 0.1 mol/L pH 6.5, add reconstitution cell concentration 0.1 g/mL, 2-hydroxy acetophenone concentration of substrate is 6 g/L, temperature of reaction 30 DEG C, reaction times 13 h.
  3. 3. method according to claim 2, is characterized in that recombinant bacterial strain candida parapsilosispCP- rcrcultivation
    Growth medium YPD is in g/L: Tryptones 20, yeast extract 10, and glucose 20 is prepared with deionized water; Solid medium adds 15g/L agar powder;
    Fermention medium is in g/L: yeast extract 5, glucose 40, (NH 4) 2hPO 413, KH 2pO 47, ZnSO 47H 2o 0.3, NaCl 0.1, pH7.0, prepares with deionized water;
    Culture condition: picking recombinant bacterial strain candida parapsilosispCP- rcrsingle colony inoculation in 5 mL YPD liquid nutrient mediums, in 30 DEG C, 200 r/min shaking culture 12 h; Getting 1 mL nutrient solution transfers in 50 mL fermention mediums, in 30 DEG C, 200 r/min shaking culture cultivate 48 h.
  4. 4. method according to claim 2, is characterized in that recombinant bacterial strain candida parapsilosispCP- rcrstructure:
    By goal gene rcrinsert in Candida parapsilosis expressed in situ plasmid pCP, construction recombination plasmid pCP- rcr, recombinant plasmid pCP- rcrtransform Candida parapsilosis competent cell, by the YPD plate screening object recombinant bacterial strain containing 40 μ g/mL Knowles rhzomorphs c. parapsilosispCP- rcr.
  5. 5. method according to claim 4, is characterized in that recombinant plasmid pCP- rcrstructure:
    Goal gene rcrand expression plasmid pCP uses respectively ecor I enzyme is cut, digestion products through agarose gel electrophoresis analysis and cross column purification, the digestion products after purifying rcrand expression plasmid pCP adopts ligase enzyme to connect, and connects 12-16 h in 16 DEG C of incubators; Connect product conversion e. colijM109 competent cell, is applied on the LB flat board containing 100 μ g/mL penbritins, and LB is dull and stereotyped: peptone 10 g/L, yeast extract paste 5 g/L, NaCl 10 g/L and agar 20 g/L; Be inverted overnight incubation for 37 DEG C;
    Picking 4 clone, transfer be equipped with 5 mL containing 100 μ g/mL penbritins LB liquid nutrient medium in, LB liquid nutrient medium: peptone 10 g/L, yeast extract paste 5 g/L and NaCl 10 g/L; Cultivate 12 h for 37 DEG C, utilize the plasmid extraction kit Mini-Plasmid Rapid Isolation Kit of the vast Tyke biological gene Technology Co., Ltd. in Beijing to extract plasmid; Recombinant plasmid adopts enzymatic cleavage methods to verify: 10 × QuickCut Buffer 2 μ L, plasmid DNA 5 μ L, saci 0.5 μ L, kpni 0.5 μ L, ddH 2system is supplied 20 μ L by O, obtains positive plasmid pCP- rcr.
  6. 6. method according to claim 4, is characterized in that rcrthe acquisition of gene
    With Candida parapsilosis genome rcrfor PCR reaction template, utilization contains sacIwith kpnIprimer RCR_1 and RCR_2 of restriction enzyme site:
    RCR_1:5 '-ctacaactat tccgcgg gag ctcatgtcaa ttccatca-3 ', is scribed ss saci,
    RCR_2:5 '-cactc ggtac cctagtggtg gtggtggtgg tgtggattaa aa-3 ', is scribed ss kpni,
    PCR reaction system: ddH 2o 33.5 μ L, DNA polysaccharase 0.5 μ L, RCR_1 1 μ L, RCR_2 1 μ L, 5 × buffer 10 μ L, dNTP 4 μ L;
    PCR condition is: 98 DEG C of thermally denature 10 s; 98 DEG C of 10 s, 52 DEG C of 15 s, 72 DEG C of 70s;
    After 30 circulations, last 72 DEG C extend 10 min again, obtain rcrgene, rcrfull length gene is 1011 bp.
  7. 7. method according to claim 4, is characterized in that the structure of expressed in situ plasmid pCP:
    Plasmid pCP be by pUC57 be starting template build come, comprise following element: promoter gene mAL2pwith aCT1p, terminator gene aCT1twith uRA3t,simultaneously as downstream homology arm, and expression cassette upstream homology arm gene uRA3p and resistant gene sAT1;
    Plasmid pCP builds design of primers:
    URA3p_1:5 '-ccg gaattcg tattgcaaac aaacg-3 ', is scribed ss ecor I,
    URA3p_2:5 '-cgcatccatt c agatcttgt atgaagacgg ca-3 ', is scribed ss bgliI,
    MAL2p_1:5 '-gtcttcatac a agatctgaa tggatgcggg-3 ', is scribed ss bgliI,
    MAL2p_2:5 '-attgacat ga gctcccgcgg aatagttgta gta-3 ', is scribed ss saci,
    ACT1t_1:5 '-caccactag g gtaccgagtg aaattct-3 ', is scribed ss kpni,
    ACT1t_2:5 '-gtcttc ctgc agattttatg atggaat-3 ', is scribed ss psti,
    ACT1p_1:5 '-aaaat ctgca ggaagaccgt ccaac-3 ', is scribed ss psti,
    ACT1p_2:5 '-tttaagctta tct gcggccg caccgttatc gataactaaa-3 ', is scribed ss noti,
    SAT1_1:5 '-cgataacggt gcggccgcat gaaaatttcg gtga-3 ', is scribed ss noti,
    SAT1_2:5 '-gattaaatat tc ggatcctt aggcgtcatc ct-3 ', is scribed ss bamh I,
    URA3t_1:5 '-gatgacgcct aa ggatccga atatttaatc at-3 ', is scribed ss bamh I,
    URA3t_2:5 '-atccc aagct taacgatcaa gagaaa-3 ', is scribed ss hind III,
    Expression plasmid pCP total length 4.5 Kb, the method adopting overlap-extension PCR and enzyme to cut connection builds:
    Will act1psequence is cloned through TA, is connected on carrier pMD19-T, obtains T- act1pplasmid; Use primer SAT1_1 and URA3t_2, with sat1with ura3tfragment is template, obtains through Overlap extension PCR amplification sat1- ura3t;
    By T- act1pplasmid and sat1-ura3tfragment uses restriction enzyme respectively noti and hind III carries out double digestion, after glue reclaims, and will sat1-ura3tfragment is connected to T- act1pon plasmid, obtain T-ASU plasmid;
    T-ASU plasmid and pUC57 plasmid are used restriction enzyme respectively psti and hind III carries out double digestion, after glue reclaims, and will act1p- sat1- ura3tfragment is connected on pUC57 plasmid, obtains pUC57-ASU plasmid;
    Use primer URA3p_1 and MAL2p_2, with ura3pwith mal2pfragment is template, obtains through Overlap extension PCR amplification ura3p-mal2p;
    Will ura3p-mal2pfragment and pUC57-ASU plasmid use restriction enzyme respectively ecor I and saci carries out double digestion, after glue reclaims, and will ura3p-mal2pfragment is connected on pUC57-ASU plasmid, obtains pUC57-UMASU plasmid;
    Will act1tfragment and pUC57-UMASU plasmid use restriction enzyme respectively kpni and psti carries out double digestion, after glue reclaims, and will act1tfragment is connected on pUC57-UMASU plasmid, obtains expression plasmid pCP.
  8. 8. method according to claim 6, is characterized in that for obtaining gene rcrrequired extract genomic bacterial classification be Candida parapsilosis ( c. parapsilosis) CCTCC NO:M203011.
CN201510206914.8A 2015-04-28 2015-04-28 Recombinant bacterium obtained by in-situ expression of (R)-carbonyl reductase in candida parapsilosis and method using same to produce (R)-phenyl glycol efficiently Pending CN104830704A (en)

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