CN101633928B - Recombinant expression of aldehyde reductase and application thereof in bioconversion of glycerol into 1,3-propylene glycol - Google Patents
Recombinant expression of aldehyde reductase and application thereof in bioconversion of glycerol into 1,3-propylene glycol Download PDFInfo
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- CN101633928B CN101633928B CN2008102282685A CN200810228268A CN101633928B CN 101633928 B CN101633928 B CN 101633928B CN 2008102282685 A CN2008102282685 A CN 2008102282685A CN 200810228268 A CN200810228268 A CN 200810228268A CN 101633928 B CN101633928 B CN 101633928B
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- fermentation
- ammediol
- promotor
- aldehyde reductase
- recombinant
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 title claims abstract description 46
- 108010053754 Aldehyde reductase Proteins 0.000 title claims abstract description 25
- 102100027265 Aldo-keto reductase family 1 member B1 Human genes 0.000 title claims abstract description 16
- 238000003259 recombinant expression Methods 0.000 title claims abstract description 8
- YPFDHNVEDLHUCE-UHFFFAOYSA-N propane-1,3-diol Chemical compound OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 title abstract 6
- 238000000855 fermentation Methods 0.000 claims abstract description 33
- 230000004151 fermentation Effects 0.000 claims abstract description 32
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- 239000013604 expression vector Substances 0.000 claims abstract description 7
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- UXFQFBNBSPQBJW-UHFFFAOYSA-N 2-amino-2-methylpropane-1,3-diol Chemical compound OCC(N)(C)CO UXFQFBNBSPQBJW-UHFFFAOYSA-N 0.000 claims description 57
- 238000000034 method Methods 0.000 claims description 19
- 235000011187 glycerol Nutrition 0.000 claims description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
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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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种醛还原酶的重组表达及其在甘油生物转化为1，3-丙二醇中的应用，属于生物工程技术领域。其特征是通过一种含有上述醛还原酶基因序列的重组表达载体，将该酶表达到宿主细胞中，并应用于制备1，3-丙二醇。本发明的效果和益处是重组克雷伯氏菌(pDK-yqhD)以甘油为底物，发酵30h后，测得发酵液中的1，3-丙二醇浓度比对照菌株提高17.1％。本发明的效果和益处是：同原始菌株相比，利用本发明提供的重组菌株进行发酵，1，3-丙二醇的产量提高10～50％，为进一步构建以甘油为底物高产1，3-丙二醇的基因工程菌打下基础。The invention discloses a recombinant expression of aldehyde reductase and its application in the biotransformation of glycerol into 1,3-propanediol, belonging to the technical field of bioengineering. It is characterized in that the enzyme is expressed into host cells through a recombinant expression vector containing the above-mentioned aldehyde reductase gene sequence, and applied to prepare 1,3-propanediol. The effect and benefit of the present invention is that the recombinant Klebsiella (pDK-yqhD) uses glycerol as a substrate, and after 30 hours of fermentation, the concentration of 1,3-propanediol in the fermentation liquid is measured to be 17.1% higher than that of the control strain. The effects and benefits of the present invention are: compared with the original strain, the yield of 1,3-propanediol is increased by 10% to 50% by using the recombinant strain provided by the present invention for fermentation. Propylene glycol lays the groundwork for genetically engineered bacteria.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to the recombinant expression vector that contains the aldehyde reductase gene, and with its transformed host cells ferment glycerin high yield 1, the application of ammediol.
Background technology
1; ammediol (1; 3-propanediol; be called for short 1; 3-PD) be colourless, tasteless thick liquid; be a kind of important chemical material, can be used as solvent, antifreezing agent or protective material, fine chemical material and new polyester---the monomer of Poly(Trimethylene Terephthalate) (PTT) and urethane.PTT is a kind of new polyester material, can be used for the dress materials of carpet, engineering plastics, film and textile industry etc., and PTT have excellent rebound resilience, dyeability, pollution resistance, preferably the uvioresistant discoloration and be difficult for static, the characteristics such as less that absorb water, be cited as one of the U.S.'s six big petrochemical industry product innovations in 1998.1, the polymerized plasticizer that ammediol also can be used for producing thermoplastic polyurethane and is used as PVC.As dibasic alcohol, it can also replace 1, and 4-butyleneglycol and neopentyl glycol are as intermediate.U.S. Chem Systems company discovers, 1, and the functionalized with glycols group of ammediol makes it be used for production of polyurethane and has a lot of potentiality, as is used for the production of polyester polyol and as chain propagation agent.In the synthetic field of medicine, 1, oneself is applied ammediol, and some new purposes are also developed in this field.In recent years, with 1, oneself is more and more paid attention to ammediol as the organic synthesis raw material, and as 1, ammediol can synthesize 3-hydroxy-propionic acid and propanedioic acid through atmospheric oxidation, but with the urea reaction synthesizing annular carbonate.1, ammediol is considered to the industrial chemicals that have broad prospect of application this century most.
In view of 1, the extensive market outlook of ammediol, its production method is subjected to the extensive concern of domestic and international large-lot producer and scientific research institution for many years.World-renowned chemical industrial company of several families, for example Dutch Shell company, German Degussa company drops into large-scale production 1 in succession, and ammediol has also stepped up applied research and exploitation.It produces 1, and the method for ammediol mainly is a chemical synthesis.Synthesize 1 with chemical method, ammediol need and use valuable catalyzer could realize that cost is higher at high temperature, high pressure, facility investment is big, technical difficulty height, separation and purification of products difficulty, particularly Preparation of catalysts is difficult, and produces the waste gas of contaminate environment such as CO.Along with the development of modern biotechnology, people begin to attempt with Production by Microorganism Fermentation 1, the research of ammediol.Microbe fermentation method has mild condition, simple to operate, advantages such as by product is few, environmental protection, and the research of this respect becomes the focus of current domestic and international research.
The microbial method ferment glycerin produces 1, and the bacterial classification of ammediol comprises Klebsiella (Klebsiella), citric acid Pseudomonas (Citrobacter), fusobacterium (Clostridium) etc.Klebsiella (Klebsiellapneumoniae) has higher glycerine tolerance, higher transformation efficiency and 1, and therefore ammediol throughput be subjected to more concern.And the Klebsiella amphimicrobe, its biochemical characteristic and intestinal bacteria (E.coli) are very close, and this just is the improvement of genes of bacterial classification and utilizes genetically engineered to make up new bacterial classification and provide convenience.Klebsiella is that the pathways metabolism of substrate through anaerobic fermentation relates to two parallel routes of oxidative pathway and reduction approach with glycerine.By oxidative pathway, glycerine quilt and NAD
+Glycerol dehydrogenase (GDH) catalytic dehydrogenation that links to each other generates otan (DHA), and further metabolism is a pyruvic acid then, generates energy ATP and reducing equivalent NAD
+By products such as/NADH and acetate, ethanol, and be accompanied by the growth of microorganism cells; And by the reduction approach, glycerine then by with vitamins B
12The glycerol dehydratase that is associated (GDHt) catalytic dehydration generates 3-hydroxy propanal (3-HPA), again further by link to each other with NADH 1, ammediol oxydo-reductase (PDOR) is reduced to product 1, and ammediol has consumed the excessive DPNH (NADH) that generates in the oxidative pathway simultaneously.Oxidative pathway and reduction approach are by reducing equivalent NAD
+/ NADH is connected, and makes glycerine disproportionation and the coupling of cell growth phase.
In recent years, existing many companies and scientific research institution produce 1 at biotransformation method, and ammediol is researched and developed.Biotransformation method produces 1, and the strategy a kind of commonly used of ammediol is to utilize original strain, improves 1 by the optimization for fermentation technology condition, the output of ammediol.Adopt little oxygen condition bottom fermentation to produce 1 as Dalian University of Technology, ammediol is also finished pilot scale and is amplified (Chinese patent ZL01117282.7); Tsing-Hua University adopts external source interpolation vitamins C, vitamin-E or FUMARIC ACID TECH GRADE to promote 1, ammediol production (Chinese patent ZL03121946.2; Chinese patent ZL200510011917.2); Southeast China University adopts the method for adding nonionogenic tenside in fermented liquid to change the permeability of cytolemma, thereby improves 1, the output of ammediol (Chinese patent CN200810020203.1).Though can improve 1 to a certain extent by the optimization for fermentation technology condition, the output of ammediol, owing to be subjected to many effects limit such as throughput of original strain self, and 1, the fermentation yield of ammediol can't obtain to increase considerably.In order to break the restriction of original strain self throughput, people turn to another kind of strategy gradually, promptly adopt recombinant bacterial strain fermentative production 1, ammediol.Adopting aspect the recombinant bacterial strain, U.S. Dupont company and second-biggest-in-the-world industrial enzyme manufacturer Genencor international corporation have applied for the fermentable carbon source being substrate usefulness genetic engineering bacterium direct production 1, the patent (USPatent7067300 of ammediol; US Patent6514733), 1, the ammediol production peak can reach 135g/L.Domestic each scientific research institutions also actively develop the research of recombinant bacterial strain structure aspect, made up a strain recombination bacillus coli as Southern Yangtze University, this bacterial strain is 50g/L at initial glycerol concentration, in the single batch fermentation maturing fermentation liquid 1, the ultimate density of ammediol can reach 35～42g/L (Chinese patent ZL200610039670.X).Beijing University of Chemical Technology's high expression level glycerol dehydratase and from colibacillary aldehyde reductase simultaneously in Cray Bai Shi bacillus makes 1, the output of ammediol be improved (Chinese patent CN200710176065.1).Aspect the key enzyme transformation, Shanghai University of Science and Technology adopts the fallibility round pcr to obtain 1, ammediol oxidoreductase isozyme variant enzyme (Chinese patent CN200710171758.1; Chinese patent CN200710171759.6).Remove Dupont company and adopt recombination bacillus coli to produce 1, outside ammediol output is greatly improved, adopt recombinant bacterial strain to produce 1 at present, still there are problems such as production concentration is low, glycerol conversion yield is low, production intensity is low in ammediol.
Discover that in the anaerobism batch fermentation when initial glycerol concentration reached 44.2g/L, the 3-hydroxy propanal accumulated in cell, cause cell to stop growing, (Barbirato, F.et al. are stagnated in glycerine consumption, Appl.Environ.Microbiol.1996,62 (4): 1448-1451).Also reflect simultaneously in thalline this moment 1, the relative shortage of ammediol oxydo-reductase.Even but in thalline high expression level 1, the ammediol oxydo-reductase still can't obtain expected effect (Zheng, P.etal., Process Biochem.2006,41 (10): 2160-2169).1, the ammediol oxydo-reductase is a two-way enzyme, can be converted into 1 by catalysis 3-hydroxy propanal, and ammediol again can be with 1 of generation, and ammediol is converted into the 3-hydroxy propanal, and like this to 1, the accumulation of ammediol is disadvantageous.And the aldehyde reductase among the klebsiella DSM2026 (German national microbial strains preservation center, numbering 2026) has converse zero the character that should be, the 3-hydroxy propanal can be converted into 1, ammediol, thereby the toxic action of releasing 3-hydroxy propanal, help 1, ammediol synthetic.
By producing 1, the microorganism glycerine pathways metabolism of ammediol as seen, the amount of NADH is restriction 1 in the thalline, the important factor that ammediol generates.Consider and have a large amount of NADPH in the thalline, and certain micro-organisms, such as the bacterial classification in Klebsiella (Klebsiella), citric acid Pseudomonas (Citrobacter), the fusobacterium (Clostridium) etc., wherein 1, the ammediol oxydo-reductase can not effectively utilize NADPH to synthesize 1, ammediol.And aldehyde reductase be the natural NADPH that can utilize as coenzyme, the aldehyde reductase that obtains of we clone both can utilize NADPH simultaneously, can utilize NADH again, thereby strengthen the catalytic capability of aldehyde reductase.If use aldehyde reductase in these microbies, to realize efficiently expressing, the balance of oxidative pathway and reduction approach not only can be maintained like this, and reducing power in the thalline can be effectively utilized, help 1, the concentration of ammediol and the raising of production intensity.
Summary of the invention
The technical problem to be solved in the present invention provide a kind of can high yield 1, the recombinant bacterial strain of ammediol improves 1, the output of ammediol.
Technical scheme of the present invention is as follows:
The first step: the gene of aldehyde reductase of the present invention comes from Klebsiella (Klebsiella), intestinal bacteria (Escherichia coli), subtilis (Bacillus subtilis), dysentery bacterium (Shigellaflexneri) or salmonella (Salmonella).Described aldehyde reductase have shown in SEQ ID NO:1 aminoacid sequence or because of replace, lack, insert and/or add one or the several amino acid residue different with the sequence of SEQ ID NO:1, but still have the aminoacid sequence of the active enzyme of same enzyme.Described aldehyde reductase gene has the nucleotide sequence shown in SEQ ID NO:2, its degeneracy sequence or coding because of replace, lack, insert and/or add one or the several amino acid residue different with the coded sequence of SEQ ID NO:2, but still have the nucleotide sequence of the active enzyme of same enzyme.
Second step: the invention provides a kind of recombinant expression vector, wherein comprise the aldehyde reductase gene under promotor control of one or more copies.
What described promotor was well known to those skilled in the art can be applied to the suitable promotor that prokaryotic gene is expressed.Described promotor is selected from the constitutive promoter among pk (protein kinase) promotor, nif (fixed nitrogen) promotor or dha (Protosol) promotor, or is selected from lac (lactose) promotor, T7 (phage) promotor, tac promotor (hybrid promoter of lactose and tryptophane) or trp (tryptophane) but inducible promoter among the promotor.
Described recombinant vectors has any suitable carriers skeleton, for example can be selected from pBR322, pUC series, pET series or pDK serial carrier.
The 3rd step: the invention provides a kind of host cell that contains aforementioned recombinant expression vector.
Described host cell is selected from Klebsiella (Klebsiella), citric acid Pseudomonas (Citrobacter) or fusobacterium (Clostridium) etc.
The 4th step: the invention provides above-mentioned recombinant bacterial strain in preparation 1, the application in the ammediol.
Fermentation mode adopts batch fermentation, batch formula stream to add fermentation or continuously ferments etc.
Wherein said fermentation is aerobic fermentation, the micro-aerobe fermentation that bubbling air carries out in microbial cultivation process or feeds the anaerobically fermenting that nitrogen carries out in microbial cultivation process.
Contain other required various compositions of carbon source, nitrogenous source, inorganic salt, VITAMIN and strain growth in seed and the fermention medium.
Fermentation inoculum size 1～12%, 20～50 ℃ of culture temperature, air flow is 0.1～1vvm during fermentation, regulates pH and maintains 5.0～9.0, mixing speed is 80～350rpm, incubation time 10～50h.
Effect of the present invention with benefit is: compares with original strain, utilizes recombinant bacterial strain provided by the invention to ferment, and 1, the output of ammediol improves 10～50%.
Embodiment
Be described in detail specific embodiments of the invention below in conjunction with technical scheme.
Embodiment:
(1) clone of aldehyde reductase gene yqhD:
According to the aldehyde reductase gene order of disclosed klebsiella among the GenBank (Klebsiella pneumoniae) AS1.1736 (yqhD, ID:EU012494) the design primer is as follows:
P1:5’-ATGAATAATTTCGACCTGCATACCC-3’(SEQ?ID?NO：3)
P2：5’-TTAGCGTGCAGCCTCGTAAATAC-3’(SEQ?ID?NO：4)
(preparation method sees works such as F. Ao Sibai with klebsiella (Klebsiella pneumoniae) DSM2026 genome, " fine works molecular biology experiment guide ", Science Press, 1998) for template finish the PCR reaction (referring to Sambrook, J., Russel, D.W., Molecular Cloning:A Laboratory Maunal3rd ed., Cold Spring Harbor, New York:Cold Spring Harbor Laboratory Press, 2001).In the PCR reaction tubes, add following composition: genomic dna 1 μ l, dNTPs4 μ l, each 1 μ l of primer P1 and P2 (synthetic), 5 μ l10 * Ex Taq buffer, 1U Ex Taq archaeal dna polymerase, moisturizing to 50 μ l by precious biotechnology (Dalian) company limited; Reaction conditions: 95 ℃ of sex change 60s, through 95 ℃ of 30s, 58 ℃ of 1min, 72 ℃ 2min30 circulation.The PCR product that obtains confirms that through electrophoretic analysis after gel reclaimed the test kit purifying, the product after the recovery was connected into carrier pMD18-T, obtains recombinant plasmid pMD18-T-yqhD.Thermal shock method Transformed E .coli DH5 α screens positive recombinant chou containing on the resistant panel of penbritin, and the picking mono-clonal extracts plasmid then, and double digestion is identified correct recombinant plasmid, the sample presentation order-checking.
(2) subclone of aldehyde reductase gene yqhD:
Utilize the correct recombinant vectors pMD18-T-yqhD of order-checking to be template, introduce restriction enzyme site by PCR, the design primer is as follows:
P3:5’-CCTGCAGGTCGACG
CATATGAATAATT-3’(SEQ?ID?NO：5)
P4:5’-ACCCGG
GGATCCTCTAGAGATTTTAGCGTG-3’(SEQ?ID?NO：6)
The primer two ends are introduced Nde I and BamH I restriction enzyme site respectively.With recombinant plasmid pMD18-T-yqhD is template, is primer with P3 and P4, and amplification obtains having the yqhD gene of restriction enzyme site.The PCR product that obtains confirms that through electrophoretic analysis double digestion after gel reclaims the test kit purifying, the enzyme after the recovery are cut product and be connected into the carrier pET23a (+) that cuts through same enzyme, obtains recombinant plasmid pET23a (+)-yqhD.Thermal shock method Transformed E .coli DH5 α screens positive recombinant chou containing on the resistant panel of penbritin, and the picking mono-clonal extracts plasmid then, and double digestion is identified correct recombinant plasmid, the sample presentation order-checking.
(3) expression of aldehyde reductase in intestinal bacteria:
Recombinant plasmid pET23a (+)-yqhD thermal shock Transformed E .coli BL21 (DE3) that order-checking is correct, the picking mono-clonal is connected to the LB substratum, and 37 ℃ of following 170r/min cultivated 12 hours, and switching is cultured to OD
600Be 0.6, add IPTG to final concentration 1mM, 20 ℃ of following 150r/min induced 12 hours.Get 4ml bacterium liquid, 4 ℃, the centrifugal 15min of 12000r/min collects thalline, and is resuspended, ultrasonication, and 4 ℃, the centrifugal 20min of 8000r/min gets supernatant and carries out enzyme activity determination and SDS-PAGE.Concentrated gum concentration is 4%, and resolving gel concentration is 12%, and electrophoretic buffer adopts Tris-glycine system.
(4) purifying of aldehyde reductase and enzyme activity determination:
The purifying of aldehyde reductase at first adopts ion exchange chromatography (Q-Sepharose FF).Sample is the cell crude extract that cytoclasis is obtained, and needs before the last sample to handle with the filtering with microporous membrane of 0.45 μ m.50mM Tris-HCl damping fluid with pH7.4 (contains 0.1mM Mn
2+, 2mM DTT) go up sample, the protein that flow velocity 1ml/min, flush away do not combine closely carries out linear gradient elution with the above-mentioned Tris-HCl damping fluid that contains 1M KCl, flow velocity is 5ml/min.Adopt Fraction Collector to collect elutriant continuously, every pipe is collected 3ml.Judge protein wash-out situation by absorbancy under the observation 280nm in the chromatography process, selecting has the part of absorption peak to carry out enzyme activity determination.Protein concn need adopt the Xylene Brilliant Cyanine G method quantitative measurement.
Measure the collected elutriant of ion exchange chromatography, get its enzyme comparatively significant part alive and merge, with the further separation and purification of gel permeation chromatography (Sephacryl S-300).(contain 0.1mM Mn with the 50mMTris-HCl damping fluid that contains pH7.4
2+, 2mM DTT) carry out wash-out, flow velocity is 0.5ml/min, substep is collected every pipe 1ml.
Through behind the purifying, obtain the single pure enzyme of electrophoretic band.With the 3-hydroxy propanal is substrate, and the ratio vigor of this enzyme that records is 3.8U/mg; And with 1, when ammediol is substrate, detect less than enzyme activity.Simultaneously, find that this enzyme has handiness in the utilization of coenzyme, can utilize NADPH, can utilize NADH again.
Initial velocity method is measured enzyme activity: in the quartz colorimetric utensil of optical path 0.5cm, and 1.5mL reaction solution (27mM3-hydroxy propanal, 0.37mM NADH or NADPH, 1 μ M ZnCl
2, 100mM Tris-HCl buffer, pH7.4), the enzyme liquid that adds 0.1ml starts reaction, the absorbancy changing value of timing assaying reaction liquid immediately.Enzyme work is defined as: under 37 ℃, catalysis 3-hydroxy propanal is converted into 1, during ammediol, and micromole's number that per minute NADH of 340nm place or NADPH consume.
(5) structure of recombinant klebsiella (Klebsiellapneumoniae):
1) structure of recombinant expression vector:
Recombinant plasmid pET23a (+)-yqhD is cloned among the expression vector pDK after same enzyme is cut after Xba I enzyme is cut, thermal shock method Transformed E .coli DH5 α.The picking mono-clonal extracts plasmid pDK-yqhD, carries out double digestion and identifies.
2) electricity transforms klebsiella:
Double digestion is identified that correct recombinant plasmid pDK-yqhD electric shock transforms klebsiella (Klebsiellapneumoniae) DSM2026.
3) expression of recombinant klebsiella:
The picking mono-clonal is connected to the LB substratum, and 37 ℃ of following 170r/min are cultured to 12 hours, and switching once is cultured to OD
600Be 0.6, add IPTG to final concentration 1mM, 37 ℃ of following 120r/min induced 5 hours.Respectively the supernatant liquor after whole-cell protein after inducing and the fragmentation is carried out SDS-PAGE.After electrophoresis finishes, there is the band of 42.3kD protein protomer to occur, illustrates that aldehyde reductase realized expression in klebsiella.
(6) reorganization bacterium ferment glycerin produces 1, ammediol:
1) bacterial classification: klebsiella (Klesiella pneumoniae) DSM2026, recombinant klebsiella (Klebsiella pneumoniae) (pDK-yqhD).
2) substratum:
Seed culture medium (/l): glycerine 20g, K
2HPO
43H
2O4.454g, KH
2PO
41.3g, (NH
4)
2SO
42.0g, MgSO
47H
2O0.2g, yeast powder 1.0g, CaCO
32g, Fe
2+Solution 1ml, Ca
2+Solution 1ml, micro-A2ml.
Fermention medium (/l): glycerine 50g, K
2HPO
43H
2O4.454g, KH
2PO
41.3g, (NH
4)
2SO
42.0g, MgSO
47H
2O0.2g, yeast powder 1.0g, CaCO
32g, Fe
2+Solution 1ml, Ca
2+Solution 1ml, micro-A2ml.
Fe
2+Solution (/l): FeSO
47H
2O5g, saturated hydrochloric acid 4ml.
Ca
2+Solution (/l): CaCl
220g.
Trace element A solution (/l): ZnCl
20.07g, MnCl
24H
2O0.1g, CoCl
26H
2O0.2g, NiCl
26H
2O0.025g, CuCl
22H
2O0.02g, NaMoO
42H
2O0.035g, H
3BO
30.06g, saturated hydrochloric acid 0.9ml.
3) training method:
A. seed culture
Picking mono-clonal from the solid medium, 37 ℃ are used the seed culture mediums activation down.Seed culture is used 50ml triangular flask, liquid amount 10ml.37 ℃ of culture temperature, shaking speed 170rpm, incubation time 12 hours.
B. fermentation culture
Control group uses klebsiella as bacterial classification, and experimental group uses recombinant klebsiella (pDK-yqhD) as bacterial classification.250ml triangular flask, liquid amount 100ml, inoculum size 2%, 37 ℃ of culture temperature, shaking speed 120rpm are used in fermentation.The fermentation beginning is experimental group adding IPTG abduction delivering after 2 hours.
4) fermentation result:
Fermentation was carried out 30 hours altogether, and in the control group fermented liquid 1, ammediol concentration is 16.89g/l during fermentation ends, and in the experimental group fermented liquid 1, ammediol concentration is 19.77g/l.This shows, use recombinant klebsiella to ferment as fermented bacterium, 1, the contrast of ammediol concentration ratio improves 17.1%.
Sequence table
SEQ?ID?NO：1
SEQ?ID?NO：2
SEQ?ID?NO：3
SEQ?ID?NO：4
SEQ?ID?NO：5
SEQ?ID?NO：6
Claims (1)
1. the recombinant expressed of an aldehyde reductase is 1 in the glycerine bio-transformation, and the application in the ammediol is characterized in that:
The nucleotide sequence of the aldehyde reductase gene that (1) uses is shown in SEQ ID NO:2 or be the degeneracy sequence of SEQ ID NO:2;
(2) recombinant expression vector of Shi Yonging contains the above-mentioned aldehyde reductase gene under promotor control of a copy; Wherein but promotor is selected from constitutive promoter or inducible promoter; Wherein constitutive promoter is selected from protein kinase promotor, fixed nitrogen promotor or Protosol promotor; But inducible promoter is selected from the hybrid promoter or the trp promoter of lactose promotor, phage promoter, lactose and tryptophane; The skeleton of carrier is selected from pBR322, pUC series, pET series or pDK serial carrier;
(3) host cell that uses is Klebsiella;
(4) fermentation mode adopts batch fermentation, batch formula stream to add fermentation or continuously ferments; Fermentation is the micro-aerobe fermentation that bubbling air carries out in microbial cultivation process or feeds the anaerobically fermenting that nitrogen carries out in microbial cultivation process; Contain carbon source, nitrogenous source, inorganic salt and VITAMIN in seed and the fermention medium; Fermentation inoculum size 1～12%, 20～50 ℃ of culture temperature, air flow is 0.1～1.0vvm during fermentation, regulates pH and maintains 5.0～9.0, mixing speed is 80～350rpm, incubation time 10～50h.
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| Yang,G.et.al.ACCESSION：EU012494.《GenBank》.2007, * |
| Yang,G.et.al.ACCESSION:EU276022.《GenBank》.2007, * |
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