CN100392071C - Engineered bacterium lacking lactic acid production path and its construction method and uses - Google Patents
Engineered bacterium lacking lactic acid production path and its construction method and uses Download PDFInfo
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- CN100392071C CN100392071C CNB2005101277440A CN200510127744A CN100392071C CN 100392071 C CN100392071 C CN 100392071C CN B2005101277440 A CNB2005101277440 A CN B2005101277440A CN 200510127744 A CN200510127744 A CN 200510127744A CN 100392071 C CN100392071 C CN 100392071C
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Abstract
The present invention discloses a strain of engineered bacteria lacking a lactic acid production path and a construction method and the application thereof. The present invention has the purpose of providing a strain of engineering bacteria lacking a lactic acid production path and the construction method thereof and the application of producing 1, 3-propylene glycol and/or 2, 3-butanediol. The engineering bacteria can be obtained by gene silencing lactate dehydrogenase in wild-type strains capable of producing the 1, 3-propylene glycol and/or the 2, 3-butanediol through a method of homologous recombination. The wild-type strains capable of producing the 1, 3-propylene glycol and/or the 2, 3-butanediol comprises the strains of Klebsiella, Citobacter, Enterobacter and Serratia. The present invention plays the important effect on producing the 1, 3-propylene glycol and the 2, 3-butanediol in a mode of industrialization, and has the wide application foreground.
Description
Technical field
The present invention relates to engineering bacteria and the construction process and the application of lacking lactic acid production path, particularly relate to the engineering bacteria of lacking lactic acid production path and construction process thereof and it is producing 1, ammediol and/or 2, the application in the 3-butyleneglycol.
Background technology
1, ammediol is a kind of important chemical material, can be used as organic solvent and prepare printing ink, printing and dyeing, coating, medicine, lubricant, antifreezing agent etc., its topmost purposes is the monomer that has potential using value in the production as polyester and polyurethanes and ring compound.Known have the number of chemical method can produce 1, ammediol, for example, with oxyethane be raw material through shortening again the method for acidylate can synthesize 1, ammediol; Obtain the 3-hydroxy propanal with acrolein hydration, the 3-hydroxy propanal can generate 1 through shortening, ammediol.Though these methods have been used for industrial production at present, these methods need high temperature, high pressure and complicated precious metal catalyst to realize, not only investment is big, separates purification difficult, also can cause serious environmental to pollute.
2, the 3-butyleneglycol can be used as fuel, also can be used as organic solvent or industrial chemicals and is used for synthetic other chemical, for example fragrance material diacetyl and use industrial chemicals methylethylketone quite widely.Because 2,3-butyleneglycol structure is special, the chemical method Synthetic 2,3-butyleneglycol cost is very high, never realizes suitability for industrialized production, thereby its purposes is not fully developed yet, but owing to have a good application prospect, at present in the world to 2, the demand of 3-butyleneglycol increases day by day.Although fermentative Production 2,3-butyleneglycol level is higher relatively, does not also realize industrialization.
As far back as 1881, Freund just found that clostridium pasteurianum can produce 1, ammediol by ferment glycerin.So far found 1, the ammediol producer is bacterium, mainly contain Klebsiella pneumonia, citrobacter freundii and enterobacter agglomerans in the intestinal bacteria, the short lactobacillus of clostridium butylicum of fusobacterium and clostridium pasteurianum and lactobacillus and Lactobacillus buchneri, they can only utilize glycerine cheap carbon sources such as (and can not utilize) carbohydrates to produce 1, ammediol.Producing 1, in the process of ammediol, glycerine generation disproportionation, the product in the oxidative pathway is consistent with the carbohydrate fermentation product, and produces for the cell necessary ATP that grows, release reducing power NADH when some product forms
2The reduction approach then consumes reducing power unnecessary in the oxidative pathway, generates 1, ammediol.It is identical generating being reflected in each bacterium of pyruvic acid in the oxidative pathway, and the whereabouts of pyruvic acid is then different because of microbe species.In intestinal bacteria, pyruvic acid is acetyl-CoA and formic acid by the pyruvate formate-lyase catalytic decomposition, and formic acid often can be decomposed into CO again
2And H
2Acetyl-CoA generates excessive ATP in the process that forms acetate through acetylphosphate, and will consume 2 moles of reducing powers in the reaction of acetaldehyde formation alcoholic acid; Pyruvic acid also may be converted into 2, the 3-butyleneglycol; In addition, also have lactic acid and succsinic acid in the product of intestinal bacteria ferment glycerin, the process that generates lactic acid will consume 1 reducing power, and the process of generation succsinic acid will consume 2 reducing powers.In clostridium butylicum, two typical oxidation productss are arranged: acetate and butyric acid.Butyric acid is by 2 NADH of two molecule acetyl-CoA oxidations
2After a succession of reaction in generate, and be accompanied by the generation of ATP.Butanols is main oxidation products in clostridium pasteurianum, and the process that generates butanols will consume 4 reducing powers, in addition, also has a spot of ethanol to produce.The reduction approach comprises two-step reaction: the first step, and by depending on coenzyme B
12The glycerol dehydratase dehydrating glycerin with catalyst generate the 3-hydroxy propanal; In second step, by 1, the reduction of ammediol redox enzyme catalysis 3-hydroxy propanal generates 1, ammediol, and this process consumes a part reducing power.
Can produce 2, the bacterial classification of 3-butyleneglycol mainly contains Klebsiella, bacillus, Aeromonas and serratia.The Klebsiella pneumonia bacillus polymyxa is tool industrialization potentiality.They can utilize carbohydrate also finally to be converted into 2 through pyruvic acid, α-acetylactis, 3-oxobutanol, the 3-butyleneglycol, and this process consumes a part reducing power.
Produce 1 at fermentation using bacteria glycerine, in the process of ammediol, the growth of cell is subjected to the restraining effect of substrate glycerine and multiple product.In klebsiella pneumoniae, by product lactic acid is maximum.Wild-type klebsiella pneumoniae ferment glycerin produces 1, and lactic acid production can reach more than the 40g/L in the process of ammediol, and not only cell growth is unfavorable in the generation of so a large amount of lactic acid, also can cause the waste of substrate glycerine.Can fight for reducing power owing to produce the approach of lactic acid, the generation of lactic acid must cause 1, and ammediol output reduces.Produce 1 as the klebsiella pneumoniae ferment glycerin, the by product of ammediol, 2,3-butyleneglycol cell growth and 1, the side effect that ammediol produces is far smaller than other by product, and its value is far longer than other by products, therefore reduces other by product output, make 2, the primary product that the 3-butyleneglycol becomes oxidative pathway is unusual rational and effective approach.
For the transformation efficiency and 1 that improves glycerine, ammediol and 2, the output of 3-butyleneglycol, someone takes bacterial screening, bacterial classification chemomorphosis, optimizes methods such as substratum and fermentation condition, though these methods to glycerol conversion yield and 1, ammediol and 2,3-butyleneglycol output increases, but the toxic action that can't fundamentally solve multiple byproducts build-up pair cell is to 1, ammediol and 2, the huge negative impact that the production of 3-butyleneglycol is brought.
Summary of the invention
The engineering bacteria that the purpose of this invention is to provide lacking lactic acid production path.
The engineering bacteria of lacking lactic acid production path provided by the present invention is will produce 1 by the method for homologous recombination, ammediol and/or 2, the engineering bacteria that the reticent back of the lactate dehydrogenase gene in the wild type strain of 3-butyleneglycol (ldhA) obtains.
Describedly can be used for producing 1, ammediol and/or 2, the wild type strain of 3-butyleneglycol comprises the bacterial strain of Pseudomonas such as Klebsiella, Citrobacter, enterobacter and husky Lei Shi.
Second purpose of the present invention provides a kind of construction process of engineering bacteria of above-mentioned lacking lactic acid production path.
Construction process provided by the present invention may further comprise the steps:
1) the portion homologous sequence of pcr amplification lactate dehydrogenase gene;
2) the portion homologous sequence of the lactate dehydrogenase gene that step 1 is increased imports in this hybridization of the parents F+strain;
3) with step 2) the reorganization bacterium of the portion homologous sequence that carries lactate dehydrogenase gene that obtains with produce 1, ammediol and/or 2, the wild type strain of 3-butyleneglycol carries out this hybridization of parents, obtains the engineering bacteria of lacking lactic acid production path behind resistance screening.
According to principle of homologous recombination, the portion homologous sequence of the lactate dehydrogenase gene of described step 1) amplification is which partial sequence of this gene is unimportant, so long as its homologous sequence gets final product, as to produce 1, ammediol and/or 2, the genomic dna of the wild type strain of 3-butyleneglycol is a template, and via SEQ ID № in the sequence table: 1 and SEQ ID №: 2 or by SEQ ID № in the sequence table: 3 and SEQ ID №: 4 primers of forming are to the sequence of pcr amplification.
The suicide vector of the portion homologous sequence that the portion homologous sequence of lactate dehydrogenase gene can be by containing described lactate dehydrogenase gene described step 2) imports to the F+strain that is used for this hybridization of parents, described suicide vector can be pGPCm (Zhao Dehua, Li Jilun. the structure of klebsiella pneumoniae nitrogenase double-mutant strain and to substrate reductive characteristic. Science Bulletin, 2004,49 (15): 1512-1518), pGP704 (Miller VL and Mekalanos JJ.Anovel suicide vector and its use in construction of insertion mutations:Osmoregulation of outer membrane proteins and virulence determinants in VibrioCholerae requires toxR.J Bacteriol 1988,170 (6): 2575-2583.), pUX19 (ZhangYP, et al.Functional Characterization of Three GlnB Homologs in thePhotosynthetic Bacterium Rhodospirillum rubrum:Roles in Sensing Ammonium andEnergy Status.J Bacteriol 2001,183 (21): 6159-6168.), pSUP202 (Simon R, PrieferU, P ü hler A (1983) A broad-host range mobilization system in vivo geneticengineering:transposon mutagenesis in Gram-negative bacteria.Biotechnology1:784-791.), pJQ200SK (Quadt, J., and M.F.Hynes.1993.Versatile suicidevectors which allow direct select ion for gene replacement in Gram-negativebacteria.Gene 127:15-21.) or pPHU281 (H ü bner, P., B.Masepohl, W.Klipp, andT.A.Bickle.1993.nif gene expression studies in Rhodobacter capsulatus:ntrC-independent repression by high ammonium concentrations.Mol.Microbiol.10:123-132.) etc.
Be the carrier that sets out with pGPCm, the suicide vector that carries lactate dehydrogenase gene portion homologous sequence of structure is pGP-ldhA or pGP704-ldhAk12.
The suicide vector that carries lactate dehydrogenase gene portion homologous sequence can transform the host bacterium by using ordinary methods such as heat shock method, electrotransformation, joint conversion method.
Described step 2) bacterial strain that is used for this hybridization of parents in is the coli strain that contains λ pir, as intestinal bacteria SM10 (λ pir) (Miller VL and Mekalanos JJ.A novel suic ide vector and its usein construction of insertion mutations:Osmoregulation of outer membraneproteins and virulence determinants in Vibrio Cholerae requires toxR.JBacteriol 1988,170 (6): 2575-2583.), S17-1 (λ pir) (Simon R, Priefer U, P ü hlerA (1983) A broad-host range mobilization system in vivo genetic engineering:transposon mutagenesis in Gram-negat ive bacteria.Biotechnology 1:784-791.) etc.
By biparent cross, make the portion homologous sequence and product 1, ammediol and/or 2 of lactate dehydrogenase gene, the purpose bacterial strain generation homologous recombination of 3-butyleneglycol, produce 1 thereby make, ammediol and/or 2, the lactate dehydrogenase gene silence in the wild type strain of 3-butyleneglycol.
Describedly can be used for producing 1, ammediol and/or 2, the wild type strain of 3-butyleneglycol comprises the bacterial strain of Pseudomonas such as Klebsiella, Citrobacter, enterobacter, Escherichia, Shigella, salmonella and husky Lei Shi.Wherein, the klebsiella pneumoniae lactic acid producing is maximum, and therefore method of the present invention also is applicable to other product 1, ammediol and/or 2, the wild type strain of 3-butyleneglycol.
The present invention utilizes the method for homologous recombination to make and produces 1, ammediol and/or 2, and the lactate dehydrogenase gene silence in the wild type strain of 3-butyleneglycol, thus obtain the genetic engineering bacterium that the lactic acid producing approach is blocked.Carry out 1 with engineering bacteria of the present invention, ammediol and/or 2, the fermentative production of 3-butyleneglycol does not produce lactic acid, makes the toxic action of by product pair cell significantly reduce; In addition, because the by product kind reduces, also simplified back extraction process.Experiment showed, engineering bacteria of the present invention was fermented 40 hours according to a conventional method, 1, ammediol concentration can reach more than the 50g/L, and 2,3-butyleneglycol concentration can reach more than the 40g/L; Fermented 60 hours, 1, ammediol concentration can reach more than the 80g/L, and 2,3-butyleneglycol concentration can reach more than the 70g/L.The present invention will be 1, and ammediol and 2 plays a significant role in the suitability for industrialized production of 3-butyleneglycol, has broad application prospects.
Below in conjunction with specific embodiment the present invention is described in further detail.
Figure of description
Fig. 1 is the physical map of carrier pGPCm
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and the primer synthetic work is given birth to the worker by Shanghai and finished, and the glycerine percentage concentration is a mass percent concentration, and the ammonium sulfate percentage concentration is a mass percent concentration.
The structure of the engineering bacteria of embodiment 1, lacking lactic acid production path
One, the clone of lactate dehydrogenase gene ldhA partial sequence
According to the complete dna sequence dna of lactate dehydrogenase gene ldhA (GenBank number: WUGSC 573) design primer PCR its portion homologous sequence that increases, primer sequence is as follows:
Primer1 (upstream primer): 5 '-ACGGTTGCGAACGGTATGTA-3 ' (SEQ ID № in the sequence table: 1)
Primer2 (downstream primer): 5 '-AGTGGTCTCCGAAATGCTGA-3 ' (SEQ ID № in the sequence table: 2)
With wild-type klebsiella pneumoniae genomic dna is template, under the guiding of primer primer1 and primer2, and the partial sequence of pcr amplification lactate dehydrogenase gene ldhA, the pcr amplification condition is: 94 ℃ of 5min of elder generation; 94 ℃ of 1min then, 56 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations; Last 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out 0.8% agarose gel electrophoresis, reclaim the also purpose fragment of the about 800bp of purifying, it is cloned among the carrier pGEM-T easy (TaKaRa company), recombinant products transformed into escherichia coli JM109 competent cell, the screening positive transformant, the upgrading grain carries out enzyme with restriction enzyme EcoRI and cuts evaluation, cuts the endonuclease bamhi that has obtained 805bp through enzyme, show and obtained the correct recombinant vectors of insertion sequence, called after pT-ldhA.
Two, the structure of lactate dehydrogenase gene ldhA suicide vector pGP-ldhA
Cut the pT-ldhA plasmid with restriction enzyme EcoRI enzyme, the length that reclaims also purification step one amplification is the part fragment of the ldhA of 805bp, again it is connected with the T4 dna ligase with the carrier pGPCm that cuts through the EcoRI enzyme (its physical map as shown in Figure 1), to connect product transformed into escherichia coli SM10 (λ pir) competent cell, the screening positive transformant, the upgrading grain obtains the suicide vector of lactate dehydrogenase gene ldhA, called after pGP-ldhA.
Three, the structure of klebsiella pneumoniae lacking lactic acid production path mutant strain
The intestinal bacteria SM10 (λ pir) (donor bacterium) and the wild-type klebsiella pneumoniae (recipient bacterium) that carry carrier pGP-ldhA are carried out this hybridization of parents, and concrete grammar is: incubated overnight donor and recipient bacterium in containing the LB liquid nutrient medium of paraxin; Donor bacterium and recipient bacterium are pressed 3: 1 mixed in the MgSO of 10mM
4In the solution, filter, filter membrane is placed on the LB flat board, cultivated 8-12 hour for 37 ℃; MgSO with 10mM
4Solution washes long lawn on filter membrane, coats behind the gradient dilution on the chlorampenicol resistant flat board, and 37 ℃ of cultivations are screened.Finally obtain having the recombinant bacterial strain of paraxin (Cm) resistance, be the klebsiella pneumoniae mutant strain that the lactic acid producing approach is blocked.
The structure of the engineering bacteria of embodiment 2, lacking lactic acid production path
According to e. coli k12 strain ldhA gene (GenBank number: U36928) design following primer:
Primer3 (upstream primer): 5 '-CGAGTCCTTTGGCTTTGAGC-3 ' (SEQ ID № in the sequence table: 3)
Primer4 (downstream primer): 5 '-TCAGTGCTTCTGCTGTCAGG-3 ' (SEQ ID № in the sequence table: 4)
With the e. coli k12 strain genomic dna is template, under the guiding of primer primer3 and primer4, and the partial sequence of pcr amplification e. coli k12 strain lactate dehydrogenase gene ldhA, the pcr amplification condition is: 94 ℃ of 5min of elder generation; 94 ℃ of 1min then, 58 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations; Last 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out 0.8% agarose gel electrophoresis, recovery obtains the fragment of 854bp size, this fragment is connected into the suicide vector that obtains lactate dehydrogenase gene ldhA among the suicide vector pGP704, called after pGP704-ldhAk12, with this recombinant plasmid transformed intestinal bacteria SM10 (λ pir) competent cell, the screening positive transformant, carry out this hybridization of parents with the intestinal bacteria SM10 that carries suicide plasmid pGP704-ldhAk12 (λ pir) with e. coli k12 strain, selection has the bacterial strain of penbritin (Ap) resistance, is the e. coli k12 strain mutant strain that the lactic acid producing approach is blocked.
The activity of the serum lactic dehydrogenase of the engineering bacteria of embodiment 3, lacking lactic acid production path detects
The activity that the engineering bacteria of the lacking lactic acid production path that embodiment 1 and embodiment 2 made up carries out serum lactic dehydrogenase detects, and is contrast with the wild type strain, and concrete grammar may further comprise the steps:
(1) engineering bacteria is inoculated in the 200mL rich broth substratum (containing Tryptones 10g in every L water, yeast powder 1g, NaCl 5g, pH7.0,121 ℃ of sterilization 30min), 37 ℃ of following shaking culture 12 hours, centrifugal collection thalline;
(2) with 100mL phosphoric acid buffer (0.1M, pH7.5) suspend washing thalline 2 times;
(3) with 2.5mL phosphoric acid buffer (0.1M, pH7.5) suspension thalline;
(4) ultrasonic disruption thalline;
(5) 1270g is centrifugal 1 hour, gets supernatant mensuration enzyme and lives, and measuring method is for measuring OD with BECKMAN DU640 ultraviolet-visible pectrophotometer
340nmVariation in 1min, wherein the serum lactic dehydrogenase reaction system comprises: NADH0.33mM, Sodium.alpha.-ketopropionate 30mM, enzyme liquid 20 μ l mend to 1mL with phosphoric acid buffer.Add Sodium.alpha.-ketopropionate and begin reaction.
What the lactate dehydrogenase activity of the wherein 4 strain lacking lactic acid production path mutant strains that acetonideexample 1 and embodiment 2 make up was minimum is 3.85% of wild type strain, the highest is 6.92% of wild type strain, show and obtained the engineering bacteria that lactate dehydrogenase activity is almost lost, can be used for fermentative production 1, ammediol and/or 2, the 3-butyleneglycol.
The fermentation of the engineering bacteria of embodiment 4, lacking lactic acid production path
(1) seed culture based formulas (1L): K
2HPO
4(dipotassium hydrogen phosphate) 3.4g, KH
2PO
4(potassium primary phosphate) 1.3g, (NH
4)
2SO
4(ammonium sulfate) 2.0g, MgSO
47H
2O (sal epsom) 0.2g, CaCl
22H
2O (calcium chloride) 0.02g, yeast extract (yeast powder) 1.0g, sucrose (sucrose) 20g, Fe solution (ferrous solution) 2.0mL, trace element solution (trace element solution) 1.0mL.
(2) fermentative medium formula (1L): K
2HPO
4(dipotassium hydrogen phosphate) 1.0g, KH
2PO
4(potassium primary phosphate) 0.5g, (NH
4)
2SO
4(ammonium sulfate) 2.0g, MgSO
47H
2O (sal epsom) 0.2g, CaCl
22H
2O (calcium chloride) 0.02g, yeast extract (yeast powder) 1.0g, glycerol (glycerine) 20g, Fe solution (ferrous solution) 1.0mL, trace element solution (trace element solution) 1.0mL.
Ferrous solution (1L): FeSO
47H
2O 5g, HCl (37%) 4mL.
Trace element solution (1L): ZnCl
270mg, CuCl
22H
2O 20mg, MnCl
24H
2O 0.1g, NiCl
26H
2O25mg, H
3BO
360mg, Na
2MO
42H
2O 35mg, CoCl
22H
2O 0.2g, HCl (37%) 4mL.
One, seed culture
Single bacterium colony of the klebsiella pneumoniae mutant strain of the lacking lactic acid production path that picking detects through embodiment 3 from the flat board is linked in the 500mL triangular flask that the 100mL seed culture medium is housed, and 37 ℃, 180r/min are cultivated 14h, make OD
60nmReach more than 3.0.
Two, fermentation
The seed liquor of step 1 being cultivated by 10% inoculum size inserts and is equipped with in the automatic fermentor tank of the 7.5L of NBS company of 4L fermention medium, adds 80% glycerine and 40% ammonium sulfate in the fermenting process, and is 7.0 with potassium hydroxide control pH value.Respectively at fermentation sampling after 40 and 60 hours, measure in the tunning 1, ammediol concentration and 2, the concentration of 3-butyleneglycol.The result was fermented 40 hours, and 1, ammediol concentration can reach more than the 50g/L, 2,3-butyleneglycol concentration can reach more than the 40g/L, ferments 60 hours, 1, ammediol concentration can reach more than the 80g/L, and 2,3-butyleneglycol concentration can reach more than the 70g/L, in this process, there is not lactic acid to produce, show that the engineering bacteria that the present invention makes up can be used for 1, ammediol and 2, the fermentative production of 3-butyleneglycol.
Sequence table
<160>4
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
acggttgcga?acggtatgta 20
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
agtggtctcc?gaaatgctga 20
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
cgagtccttt?ggctttgagc 20
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
tcagtgcttc?tgctgtcagg 20
Claims (10)
1. the engineering bacteria of lacking lactic acid production path is will produce 1 by the method for homologous recombination, ammediol and/or 2, the engineering bacteria that obtains after the lactate dehydrogenase gene silence in the wild type strain of 3-butyleneglycol.
2. engineering bacteria according to claim 1, it is characterized in that: described product 1, ammediol and/or 2, the wild type strain of 3-butyleneglycol comprises the bacterial strain of Klebsiella, Citrobacter, enterobacter, Escherichia, Shigella, salmonella and serratia.
3. the construction process of the engineering bacteria of a lacking lactic acid production path may further comprise the steps:
1) the portion homologous sequence of pcr amplification lactate dehydrogenase gene;
2) the portion homologous sequence of the lactate dehydrogenase gene that step 1 is increased imports in this hybridization of the parents F+strain;
3) with step 2) the reorganization bacterium of the portion homologous sequence that carries lactate dehydrogenase gene that obtains with produce 1, ammediol and/or 2, the wild type strain of 3-butyleneglycol carries out this hybridization of parents, obtains the engineering bacteria of lacking lactic acid production path behind resistance screening.
4. construction process according to claim 3, it is characterized in that: the portion homologous sequence of lactate dehydrogenase gene is to produce 1 in the described step 1), ammediol and/or 2, the genomic dna of the wild type strain of 3-butyleneglycol is a template, and via SEQ ID № in the sequence table: 1 and SEQ ID №: 2 or by SEQ ID № in the sequence table: 3 and SEQ ID №: 4 primers of forming are to the sequence of pcr amplification.
5. according to claim 3 or 4 described construction processs, it is characterized in that: the suicide vector of the portion homologous sequence of the portion homologous sequence of lactate dehydrogenase gene by containing described lactate dehydrogenase gene imports to the F+strain that is used for this hybridization of parents described step 2); Described suicide vector is pGPCm, pGP704, pUX19, pSUP202, pJQ200SK or pPHU281.
6. construction process according to claim 5 is characterized in that: the described suicide vector that carries lactate dehydrogenase gene portion homologous sequence is pGP-ldhA or pGP704-ldhAk12.
7. according to claim 3 or 4 described construction processs, it is characterized in that: the F+strain that described step 2) is used for this hybridization of parents is the coli strain that contains λ pir.
8. construction process according to claim 7 is characterized in that: the intestinal bacteria of the described λ of containing pir are intestinal bacteria SM10 (λ pir) or S17-1 (λ pir).
9. according to claim 3 or 4 described construction processs, it is characterized in that: be used to produce 1 in the described step 3), ammediol and/or 2, the wild type strain of 3-butyleneglycol are the bacterial strain of Klebsiella, Citrobacter, enterobacter and serratia.
10. the engineering bacteria of the described lacking lactic acid production path of claim 1 is producing 1, ammediol and/or 2, the application in the 3-butyleneglycol.
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| CA3042565A1 (en) * | 2009-04-30 | 2010-11-04 | Genomatica, Inc. | Microorganism expressing exogenous crotonase for producing 1,3-butanediol |
| CN102952826B (en) * | 2012-04-01 | 2015-02-04 | 中国科学院上海高等研究院 | Method for eliminating capability of klebsiella pneumoniae in synthesizing 2,3-butanediol and acetoin |
| CN103305543A (en) * | 2013-06-09 | 2013-09-18 | 中国科学院南海海洋研究所 | Engineering bacteria of inactivated acetolactate synthetase, and applications thereof in producing 1,3-propanediol |
| CN104278003B (en) * | 2013-07-12 | 2018-12-18 | 中国科学院天津工业生物技术研究所 | Produce recombination bacillus coli and its application of D-ALPHA-Hydroxypropionic acid |
| CN106148255B (en) * | 2015-04-10 | 2019-05-24 | 张家港美景荣化学工业有限公司 | Engineering bacteria lacking organic acid production way and application thereof in co-production of 1, 3-propylene glycol, 2, 3-butanediol and ethanol |
| CN105936915A (en) * | 2016-03-24 | 2016-09-14 | 中国科学院南海海洋研究所 | Double-gene knockout engineering bacteria and construction method and application thereof in fermentation production of 1,3-propylene glycol |
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