CN101205541A - Recombinant expression carrier and method for high-yield of 1,3-propanediol by fermenting glycerin using host cell converted thereby - Google Patents

Abstract

The invention provides a recombinant expression vector which comprises a copied alcohol oxidoreductase gene or a plurality of copied alcohol oxidoreductase genes under the control of promoters, and proper host cells which are transformed by the vector. The invention also provides a method for using the host cells to ferment glycerol for high yield of 1,3-propanediols under aerobic or anaerobic condition, thereby output of the 1,3-propanediols is obviously improved.

Description

Recombinant expression vector and with its transformed host cells ferment glycerin high yield 1, the method for ammediol

Technical field

The invention belongs to technical field of bioengineering, relate in particular to the recombinant expression vector that contains pure oxidoreductase gene, and with its transformed host cells ferment glycerin high yield 1, the method for ammediol.

Background technology

1, (be called for short 1, be a kind of important chemical material 3-PD) to ammediol, can be used as organic solvent and be used for industries such as printing ink, printing and dyeing, coating, medicine, lubricant, antifreezing agent, goes back the useful as drug synthetic intermediate.1, the topmost purposes of ammediol is as polyester and urethane synthetic monomer, particularly the polytrimethylene terephthalate (PTT) with terephthalic acid polymerization generation is the novel thermoplastic polyester material of a kind of high-performance, easy processing, have good rebound resilience, good dyeing behavior and bulk performance and biodegradability, but the ptt fiber normal pressure is printed and dyed and is had good anti-soil dirt performance, wear resistance and antistatic property in addition, can be in carpet applications and nylon competition.The premium properties of PTT makes it be cited as one of 1998 six big petrochemical industry product innovations of the U.S..

The good market outlook of PTT widespread use have in recent years promoted 1 greatly, the research of ammediol production method.Only because of 1, ammediol production technology difficulty is big, cost is high and limited the large-scale industrial production of PTT.At present, have only Dupont and Shell company to adopt traditional chemical synthesis route, with propylene oxide or propylene be raw material production only for they synthetic PTT personal 1, ammediol.Chemical synthesis need be carried out under high temperature and valuable catalyst action, and by product is many, poor selectivity, and facility investment is huge, and production cost is corresponding higher.Biological process produces 1, and ammediol is to utilize microorganism disproportionation glycerine to produce.Compare with chemical synthesis, have mild condition, easy and simple to handle, by product is few, advantages such as non-environmental-pollution are subjected to special attention in recent years.

Occurring in nature can be 1 with transformation of glycerol, if the microbial host anaerobism or the facultative anaerobe of ammediol, wherein Klebsiella pneumonia (Klebsiella pneumoniae), clostridium butyricum (Clostridium butyricum) and C. freundii (Citrobacter freundii) have higher 1, the ammediol transformation efficiency, to substrate glycerine and product 1, ammediol has higher tolerance, therefore has higher development and is worth and application prospect.Microbial transformation glycerine produces 1, and ammediol is mainly by the regulation and control of Protosol (dha) operon, and glycerine is kept the redox equilibrium of cell along oxidation and reduction two paths metabolism.In oxidative pathway, glycerine is relying on NAD ⁺The katalysis of glycerol dehydrogenase (GDH) under dehydrogenation generate Protosol (DHA), Protosol phosphoric acid under the kinase whose effect of Protosol turns to phosphodihydroxyacetone, further metabolism generates pyruvic acid, metabolism is various small molecules products then.Oxidative pathway produces ATP and reducing equivalent NADH/NADPH, and follows the microorganism cells growth.In the reduction approach, glycerine is relying on coenzyme B ₁₂The effect of glycerol dehydratase (GDHt) under become the 3-hydroxy propanal, the NADH that utilizes oxidative pathway to produce is then depending on 1 of NADH, the effect of ammediol oxydo-reductase generates 1 down, ammediol.

In recent years, existing many companies and scientific research institution are at producing 1, and the biological process of ammediol is researched and developed.Such as, Dupont and Genercor company disclose a series of patents (US Patent6514733 in the recent period; US Patent 6013494), wherein having developed a kind of jointly is substrate with glucose, utilizes recombination bacillus coli fermentative production 1, the method of ammediol is gone into operation at present, produces 4.5 ten thousand tons per year, produce 1 for biological process, ammediol and chemical method contend with provides powerful competitive power.In order to improve 1, the throughput of ammediol, Dalian University of Technology (Wang Jianfeng etc., klebsiella micro-aerobe fermentation production 1, the research of ammediol.Modern chemical industry, 2001,21 (5): 28-31; Chinese patent ZL 01117282.7) adopted little oxygen condition bottom fermentation to produce 1, ammediol; (the Chinese patent ZL 03121946.2 of Tsing-Hua University; Chinese patent ZL 200510011917.2) adopts external source to add vitamins C, vitamin-E or FUMARIC ACID TECH GRADE and promote 1, ammediol production; People such as Biebl (Biebl, H., Marten, S.Fermentation of glycerol to 1,3-propanediol:use of cosubstrates.Appl.Microbiol.Biotechnol.1995,44:15-19.) then find to add glucose as auxiliary carbon source, can improve the transformation efficiency of glycerine greatly.Domestic also have the recombination bacillus coli of use ferment glycerin to produce 1, the report of ammediol, Southern Yangtze University (Chinese patent application 200610039670.X) has made up a strain recombination bacillus coli, in the single batch fermentation process of initial glycerol concentration 50g/l, final 1, ammediol output can reach 35～42g/l.In addition, in order to reduce cost, also have by biofuel and 1, the report of ammediol coproduction, Tsing-Hua University (Chinese patent ZL 200510011867.8) has developed and has a kind ofly utilized by-product glycerin in the production of biodiesel process as 1, and the carbon source of ammediol fermentation is to improve raw material availability and production efficiency.But biological process produces 1 at present, and there are problems such as production concentration is low, the production cycle is long and glycerol conversion yield is low, production intensity is low in ammediol.

So produce 1 at biological process at present, need to improve cell 1 in the ammediol, ammediol synthetic to overcome problems such as production concentration is low, the production cycle is long and glycerol conversion yield is low, production intensity is low.

Summary of the invention

Originally discover, to producing 1, the glycerine pathways metabolism of the microorganism of ammediol, can utilize certain micro-organisms, such as the bacterial classification in Klebsiella (Klebsiella spp.) or the fusobacterium (Clostridium spp.), especially Klebsiella pneumonia (Klebsiellapneumoniae), aerogenesis klebsiella (Klebsiella aerogenes), clostridium pasteurianum (Clostridium pasteuianum), clostridium butylicum (Clostridium butyricum) waits itself not contain and can utilize NADPH synthetic 1, the characteristic of the gene of ammediol, at these microorganism expression in vivo alcohol oxydo-reductase (yqhD) genes, thereby can make this microorganism effectively utilize reducing power in the body, cause 1 thus, the concentration and the production intensity of ammediol significantly improve.Also find among the present invention,, significantly improve the enzymic activity of glycerol dehydratase, can further improve in the host living beings 1, the productive rate of ammediol by increasing the copy number of rate-limiting enzyme-glycerol dehydrase gene in the host organisms.

Therefore, one aspect of the present invention provides a kind of recombinant expression vector, wherein comprises one or the pure oxidoreductase gene under promotor control of multiple copied.Preferably, can also comprise one or the glycerol dehydrase gene under described same or different promoters control of multiple copied in the described carrier.

In some embodiments aspect this, described pure oxidoreductase gene can be external source or native gene, it can come from intestinal bacteria (Escherichia coli), subtilis (Bacillus subtilis), dysentery bacterium (Shigella flexneri) or salmonella (Salmonella spp.), preferred intestinal bacteria (Escherichia coli).Preferably, described pure oxidoreductase gene have sequence shown in SEQ ID NO:2, its degeneracy sequence or coding because of one or replacement, disappearance, insertion and/or the interpolation of several amino acid residue different but still have the nucleotide sequence of the active enzyme of same enzyme with the coded sequence of SEQ IDNO:2.

In some embodiments aspect this, described glycerol dehydrase gene can come from Klebsiella (Klebsiella spp.), fusobacterium (Clostridium spp.), preferred Klebsiella pneumonia (Klebsiella pneumoniae), aerogenesis klebsiella (Klebsiella aerogenes), clostridium pasteurianum (Clostridium pasteuianum), clostridium butylicum (Clostridium butyricum), most preferably Klebsiella pneumonia (Klebsiella pneumoniae).Preferably, described glycerol dehydrase gene have sequence shown in SEQ ID NO:1, its degeneracy sequence or coding because of one or replacement, disappearance, insertion and/or the interpolation of several amino acid residue different but still have the nucleotide sequence of the active enzyme of same enzyme with the coded sequence of SEQ ID NO:1.

In the present invention, activity for glycerol dehydratase adopts MBTH (3-methyl-2-[4-morpholinodithio ketone hydrazone) method to determine (Toraya, T., Kazutoshi, U., Fukui, S., Hogenkamp, H.P.C., Studies on the mechanism of the adenosyl-cobalamindependentdiol dehydratase reaction by the use of analogs of the coenzyme.J.Biol.Chem.1977,252,963-970); The activity of alcohol oxydo-reductase adopt spectrophotometry determine (Zhang Xiaomei etc., coding 1, the clone of ammediol oxidoreductase isozyme gene yqhD with efficiently express.Food and biotechnology journal, 2006,25 (4): 77-80).The glycerol dehydratase before and after transforming in the cell and the existence of pure oxydo-reductase are identified by polyacrylamide gel electrophoresis method well known to those of ordinary skill in the art.

In some embodiments aspect this, but described promotor can be constitutive promoter or inducible promoter, and the suitable promotor of prokaryotic gene is expressed in well known to those of ordinary skill in the art can being applied to.In some embodiments, described promotor is the constitutive promoter that is selected 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.

In some embodiments aspect this, described recombinant vectors can have any suitable carriers skeleton, for example can be selected from following vehicle group: pBR322, pUC18 or pET serial carrier.

In another aspect, the invention provides the host cell that contains aforementioned recombinant expression vector.

In the embodiment in this regard, described host cell can be selected from following group: Klebsiella (Klebsiella spp.), fusobacterium (Clostridium spp.), preferred Klebsiella pneumonia (Klebsiella pneumoniae), aerogenesis klebsiella (Klebsiella aerogenes), clostridium pasteurianum (Clostridium pasteuianum), clostridium butylicum (Clostridium butyricum), most preferably Klebsiella pneumonia (Klebsiella pneumoniae).

In the preferred embodiment in this regard, the host cell that contains recombinant vectors of the present invention as recombinant microorganism is Klebsiella pneumonia (Klebsiella pneumoniae) pKP-dhaBY, it has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 17th, 2007, it abbreviates CGMCC as, and deposit number is CGMCC 2111.

Still in another aspect, the invention provides recombinant microorganism of the present invention in preparation 1, the purposes in the ammediol.

The using microbe method that obtains product of fermenting is well-known for the person of ordinary skill of the art.In the present invention, fermentation can be carried out under aerobic or anaerobic condition, wherein preferred little oxygen condition.Can adopt batch fermentation, fed-batch fermentation or continuous fermentation method to realize the present invention in addition, and known any fermentation pattern all will be fit to.

In fermentation, contain carbon source, nitrogenous source, inorganic salt, VITAMIN and other required various compositions of strain growth under suitable temperature, pH condition in the substratum of cultivating.The preferred substratum of the present invention is the industrial fermentation substratum (as LB substratum, SD substratum, yeast powder phosphoric acid salt substratum) of common principal component, knows and suitable medium is microbiology or zymology those skilled in the art.

Fermention medium of the present invention must contain the glycerine carbon source.Auxiliary carbon source can include, but are not limited to such as glucose monose, such as the oligosaccharides of sucrose, such as the polysaccharide of starch, perhaps their mixture etc.The mixture of glycerine and glucose preferably.More preferably wherein the weight ratio of glycerine and glucose is 6: 1-10: 1, and especially preferably 8: 1.

Available nitrogenous source example comprises the ammonium salt of nitrogen, various organic or mineral acid, as the microorganism cells of ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, amine and other nitrogenous compound, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean protein hydrolyzate, soya-bean cake hydrolyzate and multiple fermentation and digestion product thereof etc.

Available inorganic salt example comprises potassium primary phosphate, dipotassium hydrogen phosphate, trimagnesium phosphate, sal epsom, sodium-chlor, ferric sulfate, sal epsom, copper sulfate and lime carbonate etc.

Can adopt lime carbonate, organic or mineral acid, basic solution (for example, KOH solution), ammonia or pH damping fluid to wait the pH value of regulating substratum.

In another aspect, the invention provides preparation 1, the method for ammediol, it is included in cultivates recombinant microorganism of the present invention in the substratum that contains glycerine, ferment, and reclaim 1, the step of ammediol from the back substratum that ferments.In the present invention, identify 1 from culture, ammediol can adopt known method, for example efficient liquid phase chromatographic analysis, ion exchange resin, concentrate, saltout and the precipitator method etc. (such as, Skraly, F.A., Lytle, B.L., Cameron, D.C.Construction and characterization of a 1,3-propanediol operon.Appl.Environ.Microbiol.1998,64:98-105).Reclaim 1, the method for ammediol is according to methods known in the art, for example by organic solvent extraction, concentrate and distillation etc. (such as, Zhou Peng etc., lower concentration 1 in the fermented liquid, and ammediol concentrates the purification Technology's Study progress.Chemistry and biotechnology, 2005,22 (2): 4-6).

According to aforesaid method, wherein said fermentation can be the aerobic fermentation that bubbling air carries out in microbial cultivation process, perhaps feeds the anaerobically fermenting that nitrogen carries out in microbial cultivation process.

In the embodiment aspect this, the fermentation inoculum size can be 1%-12%, preferred 5%-10%.Culture temperature can be 20 ℃-50 ℃, preferred 30-40 ℃.Air flow can be 0.1-0.8vvm during fermentation, preferred 0.2-0.6vvm.Can regulate pH in the fermenting process and maintain 5.0-9.0, preferred 6.0-8.0.The fermentor tank mixing speed can be 80rpm-350rpm, preferred 100rpm-250rpm.Can after beginning 3-5 hour, fermentation add 700-900g/l glycerine and 90-115g/l glucose mixing solutions by stream, glycerine wherein: glucose is 6: 1-10: 1 (w/w), preferred streams adds about 800g/l glycerine and about 100g/l glucose mixing solutions, wherein glycerine: glucose is 8: 1 (w/w).Can keep glycerol concentration in the fermenting process at 2-30g/l, preferred 5-20g/l.

Be with it should be appreciated by those skilled in the art, produce 1 to utilizing recombinant microorganism of the present invention in anaerobism and/or ferment under aerobic conditions glycerine, the method of ammediol, anaerobism that is applied and/or aerobic conditions and regulate the method for nutrient and somatomedin during the fermentation so that the product rate of formation reaches maximum technology is well-known in the industrial microorganism field, such as by bang army etc. and (bang armies etc. such as Zhu MM, the klebsiella micro-aerobe fermentation produces 1, the research of ammediol, modern chemical industry, 2001,21 (5): 28-31; Zhu MM, Lawman PD, Cameron DC.Improving 1,3-propanediol productions from glycerol in metabolicallyengineered E.coli by reducing accumulation of sn～glycerol～3～phosphate.Biotechnol Prog.2002,18 (4): the 694-699) method of being introduced.

Description of drawings

Fig. 1 shows the building process of recombinant vectors.

Embodiment

Below specify all respects of the present invention and feature by preferred implementation.It should be appreciated by those skilled in the art that these embodiment just are used for illustration purpose, and do not limit the scope of the invention.Protection scope of the present invention only is subjected to the restriction of claims.Under the condition that does not deviate from claims scope, those skilled in the art can carry out various modifications and improvement to various aspects of the present invention, and these modifications and improvement also belong to protection scope of the present invention.

In addition, unless it should be noted that and specialize, below among the embodiment used various materials and reagent all be material and reagent commonly used in this area, can obtain by conventional commercial sources; Method therefor is and well known to a person skilled in the art ordinary method.

The structure of reorganization Klebsiella pneumonia (Klebsiella pneumoniae)

Make up the process of recombinant expression vector

(1) clone of glycerol dehydrase gene dhaB:

Gene order according to disclosed Klebsiella pneumonia glycerol dehydratase (dhaB) among the GenBank (U30903) designs following required primer:

Bp1：5’-CGA GAATTCATGAAAAGATCAAAACGATTTGCAGT-3’(SEQ ID NO：3)

Bp2：5’-TTC AAGCTTTTAGCTTCCTTTACGCAGCTTATGCC-3’(SEQ ID NO：4)

Wherein, introduce EcoRI and HindIII site (shown in underscore) among primer Bp1 and the Bp2 respectively.(for example, the preparation method sees works such as F. Ao Sibai, fine works molecular biology experiment guide with the Klebsiella pneumonia genome.Science Press, 1998) finish the PCR reaction (for example, referring to Sambrook for template, J., Russel, D.W., Molecular Cloning:A Laboratory Manual, 3rd ed., Cold Spring Harbor, New York:Cold Spring Harbor LaboratoryPress, 2001.): in 200 μ l PCR reaction tubess, add following composition: Buffer 5 μ l (Tris-HCl 100mM, KCl 500mM, MgCl ₂15mM), 2.5mmol/l dNTPs 8 μ l, template DNA 2 μ l (50ng/ μ l), Taq archaeal dna polymerase 1 μ l (5U/ μ l), each 1 μ l of Bp1 and Bp2 (20 μ M, three rich polygala root Bioisystech Co., Ltd are synthetic by Beijing), moisturizing to 50 μ l; Reaction conditions: 94 ℃ of sex change 4min, through 94 ℃ of 1min, 55 ℃ of 1min, 30 circulations of 72 ℃ of 3min, the PCR product that obtains is confirmed through electrophoretic analysis, behind DNA purification kit purifying, EcoRI and HindIII double digestion, the fragment of recovery 2.7kb wherein, with be connected through the carrier pET28a of same double digestion (can available from Novagen company), transformed into escherichia coli Dh5 α screens positive recombinant chou, obtains recombinant plasmid pET28a-dhaB.

(2) 1, the clone of ammediol oxydo-reductase isozyme gene yqhD:

Gene order according to disclosed intestinal bacteria alcohol oxydo-reductase (yqhD) among the GenBank (U00096.2) designs following required primer:

Yp1：5’-GTC AAGCTTAAGGGAGCAAGTAATGAACAAC-3’(SEQ IDNO：5)

Yp2：5’-CTC AAGCTTTTAGCGGGCGGCTTCGTATA-3’(SEQ IDNO：6)

The HindIII site is all introduced at the primer two ends, and (for example, the preparation method sees works such as F. Ao Sibai, fine works molecular biology experiment guide with the bacillus coli gene group.Science Press, 1998) finish the PCR reaction (for example, referring to Sambrook for template, J., Russel, D.W., Molecular Cloning:ALaboratory Manual, 3rd ed., Cold Spring Harbor, New York:Cold SpringHarbor Laboratory Press, 2001.): in 200 μ l PCR reaction tubess, add following composition: Buffer 5 μ l (Tris-HCl 100mM, KCl 500mM, MgCl ₂15mM), 2.5mmol/ldNTPs 8 μ l, template DNA 2 μ l (50ng/ μ l), Taq archaeal dna polymerase 1 μ l (5U/ μ l), each 1 μ l of Yp1 and Yp2 (20 μ M, three rich polygala root Bioisystech Co., Ltd are synthetic by Beijing), moisturizing to 50 μ l; Reaction conditions: 94 ℃ of sex change 4min, through 94 ℃ of 1min, 58 ℃ of 1min, 30 circulations of 72 ℃ of 90s, the PCR product that obtains is confirmed through electrophoretic analysis, behind DNA purification kit purifying, link cloning vector pMD18T (can available from Takara company) by T-A clone, obtain plasmid pMD18T-yqhD, HindIII digested plasmid pMD18T-yqhD, reclaim the fragment of 1.16kb wherein, be connected with the carrier pET28a-dhaB that cuts through same enzyme, identify the correct carrier of yqhD direction of insertion by order-checking, transformed into escherichia coli Dh5 α screens positive recombinant chou, obtains recombinant plasmid pET28a-dhaB-yqhD.

(3) structure of the clone of promoter gene sequence pk and recombinant plasmid pKP-dhaBY:

Design following required primer according to Klebsiella pneumonia strain constitutive promoter pk dna sequence dna:

Kp1：3’-GTAC AGATCTCGTTATTTTGTCGCCCGCCAT-5’(SEQ IDNO：7)

Kp2：3’-CAT GAATTCTCGGCTCAAAAGGTGAAATCCG-5’(SEQ IDNO：8)

Introduce BglII and EcoRI site (shown in underscore) among primer Kp1 and the Kp2 respectively.(for example, the preparation method sees works such as F. Ao Sibai, fine works molecular biology experiment guide with the Klebsiella pneumonia genome.Science Press, 1998) finish the PCR reaction for template: in 200 μ l PCR reaction tubess, add following composition: Buffer 5 μ l (Tris-HCl 100mM, KCl 500mM, MgCl ₂15mM), 2.5mmol/l dNTPs 2 μ l, template DNA 2 μ l (50ng/ μ l), TaqDNA polysaccharase 1 μ l (5U/ μ l), each 1 μ l of Kp1 and Kp2 (20 μ M, three rich polygala root Bioisystech Co., Ltd are synthetic by Beijing), moisturizing to 50 μ l; Reaction conditions: 94 ℃ of sex change 4min, through 94 ℃ of 1min, 60 ℃ of 30s, 30 circulations of 72 ℃ of 30s, the PCR product that obtains is confirmed through electrophoretic analysis, behind DNA purification kit purifying, BglII and EcoRI double digestion, the fragment of recovery 0.3kb wherein, be connected with carrier pET28a-dhaB-yqhD through same double digestion, transformed into escherichia coli Dh5 α screens positive recombinant chou, obtains recombinant plasmid pKP-dhaBY.

Said process specifically, as shown in fig. 1, at first utilizing round pcr is the gene (dhaB) that template amplification obtains the encoding glycerol dehydratase with Klebsiella pneumonia (Klebsiella pneumoniae) genome, arrow refers to the gene transcription direction, dna fragmentation top italic is restriction endonuclease sites (EcoR I and Hind III), carrier pET28a and gene dhaB are digested back (italic E and H represent EcoR I and HindIII respectively) with restriction enzyme EcoR I and Hind III respectively, reclaim fragment and connect, obtain carrier pET28a-dhaB.In addition, with the bacillus coli gene group is the template pcr amplification pure oxidoreductase gene (yqhD) that obtains encoding, link cloning vector by the T-A clone, obtain plasmid pMD18T-yqhD, arrow refers to the gene transcription direction, dna fragmentation top italic is restriction endonuclease sites (Hind III), carrier pET28a-dhaB and pMD18T-yqhD are digested back (italic H represents Hind III) with restriction enzyme Hind III respectively, reclaiming the purpose fragment connects, by the direction of insertion of order-checking evaluation yqhD, called after carrier pET28a-dhaB-yqhD in the right direction.In addition, with Klebsiella pneumonia (Klebsiella pneumoniae) genome is the gene order that template amplification obtains one section constitutive promoter (pk), two ends are introduced Bgl II and EcoR I site respectively, carrier pET28a-dhaB-yqhD and promoter gene Pk are digested back (italic B and H represent Bgl II and Hind III respectively) with restriction enzyme Bgl II and EcoR I respectively, reclaim fragment and connect, obtain recombinant vectors pKP-dhaBY.

The conversion of recombinant plasmid pKP-dhaBY and expression

Use method well-known to those having ordinary skill in the art to transform Klebsiella pneumonia the recombinant plasmid pKP-dhaBY of gained, the competent preparation method of Klebsiella pneumonia with the competent preparation method of intestinal bacteria referring to Sambrook J, Russel DW (2001) Molecular Cloning:ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY..Coat the LB culture medium flat plate (sulphuric acid kanamycin concentration 25 μ g/ml) of sulfur acid kantlex, the picking positive transformant, extract plasmid, cut with BglII, EcoRI and HindIII enzyme, the electrophoresis qualification result shows that used plasmid can be cut into four bands, is respectively 5.1kb, 2.7kb, 1.16kb and 0.3kb, this recombinant plasmid is expression vector pKP-dhaBY, and the reorganization bacterium of gained is reorganization Klebsiella pneumonia (Klebsiella pneumoniae) (pKP-dhaBY).

Recombinant microorganism aerobic and anaerobically fermenting glycerine produce 1, ammediol

Anaerobically fermenting is cultivated

(1) bacterial classification: Klebsiella pneumonia (Klebsiella pneumoniae) DSM2026, reorganization klebsiella pneumoniae (Klebsiella pneumoniae) (pKP-dhaBY).

(2) substratum:

The LB slant medium: every liter of substratum contains yeast powder 5.0g, peptone 10.0g, and NaCl 10.0g, agar powder 15.0g, pH transfers to 7.0.Seed and fermention medium see the following form 1.

Table 1. seed and fermention medium

Nutrient media components	Seed culture medium (/l)	Fermention medium (/l)
			Glycerine K 2HPO 4·3H 2O KH 2PO 4 (NH 4) 2SO 4 MgSO 4Yeast powder trace element solution CaCO 3Glucose	20g 3.4g 1.3g 2.0g 0.24g 2.0g 2.0ml 0.5g 5g	20g 1.5g 0.5g 2.0g 0.24g 2.0g 5.0ml 8g

Wherein concrete component and the content of going up the trace element solution in the table sees the following form 2.

Table 2. trace element solution component and content thereof

Solution component	Content (/l)
		ZnCl 2·6H 2O MnCl 2·4H 2O CoCl 2·6H 2O NiCl·6H 2O FeCl 2·4H 2O H 3BO 3 Na 2MoO 4·2H 2O CuCl 2·H 2O concentrated hydrochloric acid (37%)	0.68g 0.17g 0.47g 0.025g 5.4g 0.06g 0.005g 0.47g 10ml

Regulating pH before the medium sterilization is 7.0, adds sulphuric acid kanamycin 20 μ g/ml before the inoculation.

(3) training method

A. seed culture

Picking glycerine guarantees that the bacterial classification of Tibetan is forwarded to the LB inclined-plane, and 30 ℃ activate 12 hours down.Seed culture adopts aerobic to cultivate, and uses the 250ml triangular flask, liquid amount 50ml.37 ℃ of culture temperature, shaking speed 150rpm, incubation time 14-16 hour.

B. fermentation culture

The 5L fermentor tank is used in fermentation, initial loading liquid measure 2L, and 37 ℃ of culture temperature, inoculum size 10% feeds nitrogen during fermentation, and air flow is 0.2-0.6vvm, regulates pH with 5M KOH and maintains 7.0.The fermentor tank mixing speed is 100rpm-250rpm.Fermentation beginning after 3-5 hour stream add 800g/l glycerine and 100g/l glucose mixing solutions, wherein glycerine: glucose=8: 1 (w/w), keep glycerol concentration in the fermenting process at 5～20g/l.Control canisters uses original Klebsiella pneumonia as bacterial classification, and experimental tank uses described reorganization Klebsiella pneumonia as bacterial classification.

C. the result of fermenting

Fermentation was carried out 36 hours altogether, and in the control canisters fermented liquid 1, ammediol concentration is 58.4g/l during fermentation ends, consume glycerine 385.5g altogether, 1, the ammediol yield is 0.55mol/mol, the glycerine wear rate is 3.57g/ (lh), 1, and the ammediol production intensity is 1.62g/ (lh).In the experimental tank fermented liquid 1, ammediol concentration is 65.8g/l, consumes glycerine 404.9g altogether during fermentation ends, 1, and the ammediol yield is 0.59mol/mol, the glycerine wear rate is 3.75g/ (lh), 1, the ammediol production intensity is 1.83g/ (lh).This shows, use the reorganization Klebsiella pneumonia to carry out anaerobically fermenting as fermented bacterium, 1, the contrast of ammediol concentration ratio improves 13%, and the comparison of glycerine spending rate is according to improving 5.0%, and the transformation efficiency comparison is according to improving 7.3%, and the production intensity comparison is according to improving 13%.

Aerobic fermentation is cultivated

(1) bacterial classification: Klebsiella pneumonia (Klebsiella pneumoniae) DSM2026, reorganization Klebsiella pneumonia (Klebsiella pneumoniae) (pKP-dhaBY).

(2) substratum:

The LB slant medium: every liter of substratum contains yeast powder 5.0g, peptone 10.0g, and NaCl 10.0g, agar powder 15.0g, pH transfers to 7.0.Seed and fermention medium see the following form 3.

Table 3. seed and fermention medium

Nutrient media components	Seed culture medium (/l)	Fermention medium (/l)
			Glycerine K 2HPO 4·3H 2O KH 2PO 4 (NH 4) 2SO 4 MgSO 4The yeast powder trace element solution	20g 3.4g 1.3g 2.0g 0.24g 2.0g 2.0ml	20g 1.5g 0.5g 2.0g 0.24g 2.0g 5.0ml

CaCO 3Glucose

0.5g 5g

8g

Wherein concrete component and the content of going up the trace element solution in the table sees the following form 4.

Table 4. trace element solution component and content thereof

Solution component	Content (/l)
		ZnCl 2·6H 2O MnCl 2·4H 2O CoCl 2·6H 2O NiCl·6H 2O FeCl 2·4H 2O H 3BO 3 Na 2MoO 4·2H 2O CuCl 2·H 2O concentrated hydrochloric acid (37%)	0.68g 0.17g 0.47g 0.025g 5.4g 0.06g 0.005g 0.47g 10ml

Regulating pH before the medium sterilization is 7.0.

(3) training method

A. seed culture

Picking glycerine guarantees that the bacterial classification of Tibetan is forwarded to the LB inclined-plane, and 30 ℃ activate 12 hours down.Seed culture adopts aerobic to cultivate, and uses the 250ml triangular flask, liquid amount 50ml.37 ℃ of culture temperature, shaking speed 150rpm, incubation time 14-16 hour.

B. fermentation culture

The 5L fermentor tank is used in fermentation, initial loading liquid measure 2L, and 37 ℃ of culture temperature, inoculum size 10%, bubbling air during fermentation, air flow are 0.2-0.6vvm, regulate pH with 5M KOH and maintain 7.0.The fermentor tank mixing speed is 100rpm-250rpm.Fermentation beginning after 3-5 hour stream add 800g/l glycerine and 100g/l glucose mixing solutions, wherein glycerine: glucose=8: 1 (w/w), keep glycerol concentration in the fermenting process at 5～20g/l.Control canisters uses original Klebsiella pneumonia as bacterial classification, and experimental tank uses the reorganization Klebsiella pneumonia as bacterial classification.

C. the result of fermenting

Fermentation was carried out 30 hours altogether, and in the control canisters fermented liquid 1, ammediol concentration is 67.4g/l during fermentation ends, consume glycerine 407.9g altogether, 1, the ammediol yield is 0.60mol/mol, the glycerine wear rate is 4.53g/ (lh), 1, and the ammediol production intensity is 2.25g/ (lh).In the experimental tank fermented liquid 1, ammediol concentration is 84.8g/l, consumes glycerine 473.7g altogether during fermentation ends, 1, and the ammediol yield is 0.65mol/mol, the glycerine wear rate is 5.26g/ (lh), 1, the ammediol production intensity is 2.83g/ (lh).This shows, use the reorganization Klebsiella pneumonia to carry out aerobic fermentation as fermented bacterium, 1, the contrast of ammediol concentration ratio improves 26%, and the comparison of glycerine spending rate is according to improving 16%, and the transformation efficiency comparison is according to improving 8.3%, and the production intensity comparison is according to improving 26%.

3.30L fermentor tank amplifies fermentation

(1) bacterial classification:

Reorganization Klebsiella pneumonia (Klebsiella pneumoniae) (pKP-dhaBY).

(2) substratum:

Medium component is with the aerobic fermentation substratum.

(3) training method

A. seed culture

With aerobic fermentation seed culture mode.

B. fermentation culture

30L Germany Braun fermentor tank is used in fermentation, initial loading liquid measure 15L, and 37 ℃ of culture temperature, inoculum size 10%, bubbling air during fermentation, air flow are 0.2-0.6vvm, with the KOH/NH of 5M/0.5M ₃H ₂O regulates pH and maintains 7.0.The fermentor tank mixing speed is 120rpm.Fermentation beginning after 4-6 hour stream add 800g/l glycerine and 100g/l glucose mixing solutions, wherein glycerine: glucose=8: 1 (w/w), keep glycerol concentration in the fermenting process at 5～20g/l.

C. the result of fermenting

Fermentation was carried out 32 hours altogether, and in the ferment tank liquid 1, ammediol concentration is 89.8g/l during fermentation ends, consume glycerine 3344g altogether, 1, the ammediol yield is 0.65mol/mol, the glycerine wear rate is 5.23g/ (lh), 1, and the ammediol production intensity is 2.81g/ (lh).This shows, in the fermentor tank of 10 times of amplifications, obtained higher 1, ammediol concentration, 1, the production intensity of ammediol improves, and has reduced fermentation costs, for later industrialization is laid a good foundation.

In sum, compared with the prior art, in fermentation result of the present invention, product 1, ammediol concentration has improved more than 20%.

The front has been described the present invention by way of example.Those of ordinary skill is fully aware of, can make various changes and modification by the present invention, and without departing from the spirit and scope of the present invention.Scope of the present invention is not limited only to these specific embodiments, but is as the criterion with appended claims.

Sequence table

＜110〉Beijing University of Chemical Technology

＜120〉recombinant expression vector and with its transformed host cells ferment glycerin high yield 1, the method for ammediol

<130>

<160>8

<170>PatentIn version3.2

<210>1

<211>2693

<212>DNA

＜213〉Klebsiella pneumonia

<400>1

atgaaaagat caaaacgatt tgcagtactg gcccagcgcc ccgtcaatca ggacgggctg 60

attggcgagt ggcctgaaga ggggctgatc gccatggaca gcccctttga cccggtctct 120

tcagtaaaag tggacaacgg tctgatcgtc gaactggacg gcaaacgccg ggaccagttt 180

gacatgatcg accgatttat cgccgattac gcgatcaacg ttgagcgcac agagcaggca 240

atgcgcctgg aggcggtgga aatagcccgt atgctggtgg atattcacgt cagccgggag 300

gagatcattg ccatcactac cgccatcacg ccggccaaag cggtcgaggt gatggcgcag 360

atgaacgtgg tggagatgat gatggcgctg cagaagatgc gtgcccgccg gaccccctcc 420

aaccagtgcc acgtcaccaa tctcaaagat aatccggtgc agattgccgc tgacgccgcc 480

gaggccggga tccgcggctt ctcagaacag gagaccacgg tcggtatcgc gcgctacgcg 540

ccgtttaacg ccctggcgct gttggtcggt tcgcagtgcg gccgccccgg cgtgttgacg 600

cagtgctcgg tggaagaggc caccgagctg gagctgggca tgcgtggctt aaccagctac 660

gccgagacgg tgtcggtcta cggcaccgaa gcggtattta ccgacggcga tgatacgccg 720

tggtcaaagg cgttcctcgc ctcggcctac gcctcccgcg ggttgaaaat gcgctacacc 780

tccggcaccg gatccgaagc gctgatgggc tattcggaga gcaagtcgat gctctacctc 840

gaatcgcgct gcatcttcat tactaaaggc gccggggttc agggactgca aaacggcgcg 900

gtgagctgta tcggcatgac cggcgctgtg ccgtcgggca ttcgggcggt gctggcggaa 960

aacctgatcg cctctatgct cgacctcgaa gtggcgtccg ccaacgacca gactttctcc 1020

cactcggata ttcgccgcac cgcgcgcacc ctgatgcaga tgctgccggg caccgacttt 1080

attttctccg gctacagcgc ggtgccgaac tacgacaaca tgttcgccgg ctcgaacttc 1140

gatgcggaag attttgatga ttacaacatc ctgcagcgtg acctgatggt tgacggcggc 1200

ctgcgtccgg tgaccgaggc ggaaaccatt gccattcgcc agaaagcggc gcgggcgatc 1260

caggcggttt tccgcgagct ggggctgccg ccaatcgccg acgaggaggt ggaggccgcc 1320

acctacgcgc acggcagcaa cgagatgccg ccgcgtaacg tggtggagga tctgagtgcg 1380

gtggaagaga tgatgaagcg caacatcacc ggcctcgata ttgtcggcgc gctgagccgc 1440

agcggctttg aggatatcgc cagcaatatt ctcaatatgc tgcgccagcg ggtcaccggc 1500

gattacctgc agacctcggc cattctcgat cggcagttcg aggtggtgag tgcggtcaac 1560

gacatcaatg actatcaggg gccgggcacc ggctatcgca tctctgccga acgctgggcg 1620

gagatcaaaa atattccggg cgtggttcag cccgacacca ttgaataagg cggtattcct 1680

gtgcaacaga caacccaaat tcagccctct tttaccctga aaacccgcga gggcggggta 1740

gcttctgccg atgaacgcgc cgatgaagtg gtgatcggcg tcggccctgc cttcgataaa 1800

caccagcatc acactctgat cgatatgccc catggcgcga tcctcaaaga gctgattgcc 1860

ggggtggaag aagaggggct tcacgcccgg gtggtgcgca ttctgcgcac gtccgacgtc 1920

tcctttatgg cctgggatgc ggccaacctg agcggctcgg ggatcggcat cggtatccag 1980

tcgaagggga ccacggtcat ccatcagcgc gatctgctgc cgctcagcaa cctggagctg 2040

ttctcccagg cgccgctgct gacgctggag acctaccggc agattggcaa aaacgctgcg 2100

cgctatgcgc gcaaagagtc accttcgccg gtgccggtgg tgaacgatca gatggtgcgg 2160

ccgaaattta tggccaaagc cgcgctattt catatcaaag agaccaaaca tgtggtgcag 2220

gacgccgagc ccgtcaccct gcacatcgac ttagtaaggg agtgaccatg agcgagaaaa 2280

ccatgcgcgt gcaggattat ccgttagcca cccgctgccc ggagcatatc ctgacgccta 2340

ccggcaaacc attgaccgat attaccctcg agaaggtgct ctctggcgag gtgggcccgc 2400

aggatgtgcg gatctcccgc cagacccttg agtaccaggc gcagattgcc gagcagatgc 2460

agcgccatgc ggtggcgcgc aatttccgcc gcgcggcgga gcttatcgcc attcctgacg 2520

agcgcattct ggctatctat aacgcgctgc gcccgttccg ctcctcgcag gcggagctgc 2580

tggcgatcgc cgacgagctg gagcacacct ggcatgcgac agtgaatgcc gcctttgtcc 2640

gggagtcggc ggaagtgtat cagcagcggc ataagctgcg taaaggaagc taa 2693

<210>2

<211>1164

<212>DNA

＜213〉intestinal bacteria

<400>2

atgaacaact ttaatctgca caccccaacc cgcattctgt ttggtaaagg cgcaatcgct 60

ggtttacgcg aacaaattcc tcacgatgct cgcgtattga ttacctacgg cggcggcagc 120

gtgaaaaaaa ccggcgttct cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180

gaatttggcg gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg 240

gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt actggacggc 300

accaaattta tcgccgcagc ggctaactat ccggaaaata tcgatccgtg gcacattctg 360

caaacgggcg gtaaagagat taaaagcgcc atcccgatgg gctgtgtgct gacgctgcca 420

gcaaccggtt cagaatccaa cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480

caggcgttcc attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc 540

tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt acacaccgtg 600

gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg accgtttcgc agaaggcatt 660

ttgctgacgc taatcgaaga tggtccgaaa gccctgaaag agccagaaaa ctacgatgtg 720

cgcgtcaacg tcatgtgggc ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780

ccgcaggact gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat 840

cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg cgataccaag 900

cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca tcactgaagg ttccgatgat 960

gagcgtattg acgccgcgat tgccgcaacc cgcaaattct ttgagcaatt aggcgtgccg 1020

acccacctct ccgactacgg tctggacggc agctccatcc cggctttgct gaaaaaactg 1080

gaagagcacg gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc 1140

cgtatatacg aagccgcccg ctaa 1164

<210>3

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer Bp1

<400>3

cgagaattca tgaaaagatc aaaacgattt gcagt 35

<210>4

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer Bp2

<400>4

ttcaagcttt tagcttcctt tacgcagctt atgcc 35

<210>5

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer Yp1

<400>5

gtcaagctta agggagcaag taatgaacaa c 31

<210>6

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer Yp2

<400>6

ctcaagcttt tagcgggcgg cttcgtata 29

<210>7

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer Kp1

<400>7

gtacagatct cgttattttg tcgcccgcca t 31

<210>8

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer Kp2

<400>8

catgaattct cggctcaaaa ggtgaaatcc g 31

Claims (17)

1. recombinant expression vector, it comprises one or the pure oxidoreductase gene under promotor control of multiple copied.

2. according to the recombinant expression vector of claim 1, it also comprises one or the glycerol dehydrase gene under described same or different promoters control of multiple copied.

3. according to the recombinant expression vector of claim 2, wherein said glycerol dehydrase gene comes from Klebsiella (Klebsiella spp.), fusobacterium (Clostridium spp.), preferred Klebsiella pneumonia (Klebsiella pneumoniae), aerogenesis klebsiella (Klebsiellaaerogenes), clostridium pasteurianum (Clostridium pasteuianum), clostridium butylicum (Clostridiumbutyricum), most preferably Klebsiella pneumonia (Klebsiella pneumoniae).

4. according to each recombinant expression vector of aforementioned claim, wherein said glycerol dehydrase gene have shown in SEQ ID NO:1 sequence, its degeneracy sequence or coding because of one or replacement, disappearance, insertion and/or the interpolation of several amino acid residue different but still have the nucleotide sequence of the active enzyme of same enzyme with the coded sequence of SEQ ID NO:1.

5. according to each recombinant expression vector of aforementioned claim, wherein said pure oxidoreductase gene comes from intestinal bacteria (Escherichia coli), subtilis (Bacillussubtilis), dysentery bacterium (Shigella flexneri) or salmonella (Salmonella spp.), most preferably intestinal bacteria (Escherichia coli).

6. according to each recombinant expression vector of aforementioned claim, wherein said pure oxidoreductase gene have shown in SEQ ID NO:2 sequence, its degeneracy sequence or coding because of one or replacement, disappearance, insertion and/or the interpolation of several amino acid residue different but still have the nucleotide sequence of the active enzyme of same enzyme with the coded sequence of SEQ ID NO:2.

7. according to each recombinant expression vector of aforementioned claim, but wherein said promotor is constitutive promoter or inducible promoter.

8. according to each recombinant expression vector of aforementioned claim, wherein said promotor is the constitutive promoter that is selected 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.

9. according to each recombinant expression vector in the aforementioned claim, the skeleton of wherein said carrier is selected from the group that comprises following carrier: pBR322, pUC18, pET serial carrier.

10. the host cell that contains each described recombinant expression vector among the claim 1-9.

11. according to the described host cell of claim 10, it is selected from following group: Klebsiella (Klebsiella spp.), fusobacterium (Clostridium spp.), preferred Klebsiella pneumonia (Klebsiella pneumoniae), aerogenesis klebsiella (Klebsiella aerogenes), clostridium pasteurianum (Clostridium pasteuianum), clostridium butylicum (Clostridium butyricum), most preferably Klebsiella pneumonia (Klebsiella pneumoniae).

12. according to the recombinant microorganism of claim 10 or 11, it is Klebsiella pneumonia (Klebsiella pneumoniae) pKP-dhaBY, its biological deposit number is CGMCC2111.

13. each recombinant microorganism is in preparation 1, the purposes in the ammediol among the claim 10-12.

14. prepare 1, the method for ammediol, it is included in the substratum that contains glycerine cultivates among the claim 10-12 each described recombinant microorganism and reclaim 1, the step of ammediol from the back substratum that ferments.

15. according to the method for claim 14, wherein said fermentation is carried out under the following conditions:

Fermentation inoculum size 1%-12%, and/or 20 ℃-50 ℃ of culture temperature, and/or when fermentation air flow be 0.1-0.8vvm, and/or adjusting pH maintains 5.0-9.0, and/or the fermentor tank mixing speed is 80rpm-350rpm, and/or fermentation begins, and stream adds 700-900g/l glycerine and 90-115g/l glucose mixing solutions, wherein glycerine after 3-5 hour: glucose is 6: 1-10: 1 (w/w), and/or keep glycerol concentration in the fermenting process at 2-30g/l.

16. according to the method for claim 15, the preferred following condition of wherein said fermentation is carried out:

Fermentation inoculum size 5%-10%, and/or 30 ℃-40 ℃ of culture temperature, and/or the fermentation air flow is 0.2-0.6vvm, and/or pH maintains 6.0-8.0, and/or the fermentor tank mixing speed is 100rpm-250rpm, and/or stream adds 800g/l glycerine and 100g/l glucose, wherein glycerine: glucose is 8: 1 (w/w), and/or fermenting process is kept glycerol concentration at 5-20g/l.

17. according to each method among the claim 14-16, wherein said fermentation is the aerobic fermentation that bubbling air carries out in microbial cultivation process, perhaps feeds the anaerobically fermenting that nitrogen carries out in microbial cultivation process.

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