CN104109651A - Recombinant Escherichia coli for synthesizing S-1,2-propanediol from L-lactic acid and construction method thereof - Google Patents
Recombinant Escherichia coli for synthesizing S-1,2-propanediol from L-lactic acid and construction method thereof Download PDFInfo
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Abstract
The invention discloses a recombinant Escherichia coli for synthesizing S-1,2-propanediol from L-lactic acid and a construction method thereof. The method comprises the following steps: 1) respectively substituting lldD gene, adheE gene and ackA-pta gene in Escherichia coli BW25113-DELTApoxB with coding gene of 3-hydroxypropionate dehydrogenase, coding gene of coenzyme A-dependent succinic semialdehyde dehydrogenase and coding gene of propionyl coenzyme A transferase to obtain recombinant bacterium BWPDO1; 2) knocking out ldhA gene and dld gene of the BWPDO1 to obtain recombinant bacterium BWPDO2; and 3) transferring coding gene of pyruvate decarboxylase and coding gene of NAD-dependent acetaldehyde coenzyme A dehydrogenase into the BWPDO2 to obtain recombinant bacterium BWPDO3 which is the recombinant Escherichia coli. The experiment proves that the recombinant Escherichia coli can generate S-1,2-propanediol by converting L-lactic acid; and by using 200 mM of sodium lactate as the substrate, the culture is performed at 37 DEG C for 24 hours to generate 3.4 mM of S-1,2-propanediol. The recombinant Escherichia coli lays foundation for enhancing the yield and conversion rate of the biosynthetic S-1,2-propanediol.
Description
Technical field
The invention belongs to microorganism field, relate to one and utilize Pfansteihl to synthesize S-1, the recombination bacillus coli of 2-propylene glycol and construction process thereof.
Background technology
The structure of propylene glycol is divided into 1,2-PD and 1,3-PD.1,2-PD is mainly used to produce unsaturated polyester resin, frostproofer, the alternative ethylene glycol of softening agent is for antifreeze aircraft and make refrigerant and thermal barrier etc. at food, and it also can be used for nonionic detergent or wetting Agent for Printing Inks.Propylene glycol or good solvent, can be used for food seasoning and spices, or the ointment ointment of medicine industry etc.
Optical activity 1,2-PD has active group, can be used as synthetic for medicine, weedicide, pheromone and liquid crystal of chirality initiator, has very high researching value.In intestinal bacteria, self have the metabolic pathway that generates 1,2-PD.In metabolism L-rhamnosyl and L-fucose approach, sugar after phosphorylation generates L-lactic aldehyde and dihydroxyacetone phosphate (DHAP) by zymohexase catalysis, after under the condition of anaerobism and NADH exist L-lactic aldehyde catalyzed be L-1,2-propylene glycol, i.e. S-1.2-propylene glycol.But because cost of material is too high, this path does not have fine economic benefit, so there is no good researching value.In thermophilc anaerobe (Thermoanaerobacterium thermosaccharolyticum), also have from methyl-glyoxal (methylglyoxal) and generate respectively lactic aldehyde (Lactaldehyde) or pyruvic aldehyde (Acetol) finally generates 1, the path of 2-propylene glycol, but wherein some path is just guessed, not evidence suggests at present and exist actual enzyme to go catalysis.
About the synthetic R-1 of biocatalysis, the research of 2-propylene glycol, at present, external existing about the synthetic R-1 of biocatalysis, the report of 2-propylene glycol, Tadashi etc. adopt airlift fermentor to generate R-1 to yeast reduction hydroxyacetone, 2-propylene glycol and yeast oxidation resolution of racemic 1,2-propylene glycol is studied, but obtains industrial applications not yet, and domestic rare report.
Summary of the invention
The object of this invention is to provide one and utilize Pfansteihl to synthesize S-1, recombination bacillus coli of 2-propylene glycol and preparation method thereof and application.
Method provided by the present invention can be utilizes Pfansteihl to synthesize S-1, and the preparation method of the recombination bacillus coli A of 2-propylene glycol specifically can comprise the step of (1)-(3) as follows:
(1) lldD gene, adheE gene and ackA-pta gene in intestinal bacteria BW25113-△ poxB are replaced with respectively to encoding gene (mmsB gene), the encoding gene (pdcD gene) of coenzyme A dependent form succinic semialdehyde dehydrogenase and the encoding gene (pct gene) of propionyl-coa transferase of 3-hydroxymalonate dehydrogenase, the recombinant bacterium obtaining is designated as BWPDO1;
Described intestinal bacteria BW25113-△ poxB is by the bacterial strain of gained after the poxB gene knockout in intestinal bacteria BW25113 wild-type;
(2) knock out ldhA gene and the dld gene of BWPDO1 that step (1) obtains, the recombinant bacterium obtaining is designated as BWPDO2;
(3) encoding gene (mhpf gene) that imports the encoding gene (ZpPDC gene) of pyruvic carboxylase and the acetaldehyde coa dehydrogenase of NAD dependent form in the BWPDO2 obtaining to step (2), the recombinant bacterium obtaining is designated as BWPDO3; Described BWPDO3 is described recombination bacillus coli A.
Wherein, described lldD gene is that pyruvic acid and Pfansteihl conversion encoding gene, described ldhA gene and described dld gene are the encoding gene of transforming protein between two different pyruvic acid and D-ALPHA-Hydroxypropionic acid.Described adheE gene and described ackA-pta gene are two different downstream genes that affect substrate acetyl-CoA content.
Method provided by the present invention also can be utilizes Pfansteihl to synthesize S-1, and the preparation method of the recombination bacillus coli B of 2-propylene glycol comprises above step (1) and step (2); The described BWPDO2 obtaining in described step (2) is described recombination bacillus coli B.
In the step (1) of aforesaid method, described 3-hydroxymalonate dehydrogenase is the 3-hydroxymalonate dehydrogenase of bacillus cereus (Bacillus cereus), and its aminoacid sequence is specifically as shown in sequence in sequence table 12.Described coenzyme A dependent form succinic semialdehyde dehydrogenase is the coenzyme A dependent form succinic semialdehyde dehydrogenase of Yersinia enterocolitica (Yersinia enterocolitica), and its aminoacid sequence is specifically as shown in sequence in sequence table 13.Described propionyl-coa transferase is the point mutation albumen of the propionyl-coa transferase of clostridium propionicum (Clostridium propionicum), and its aminoacid sequence is specifically as shown in sequence in sequence table 14.
In the step (3) of above-mentioned method, described pyruvic carboxylase is the pyruvic carboxylase of palm fermenting bacteria (Zymobacter palmae), and its aminoacid sequence is specifically as shown in sequence in sequence table 15.The acetaldehyde coa dehydrogenase of described NAD dependent form is the acetaldehyde coa dehydrogenase that needs NAD from colibacillary, and its aminoacid sequence is specifically as shown in sequence in sequence table 16.
Further, in step (1), the nucleotide sequence of the encoding gene of described 3-hydroxymalonate dehydrogenase (mmsB gene) is specially the 116-994 position of sequence 1 in sequence table; The nucleotide sequence of the encoding gene (pdcD gene) of described coenzyme A dependent form succinic semialdehyde dehydrogenase is specially the 326-1714 position of sequence 2 in sequence table; The nucleotide sequence of the encoding gene (pct gene) of described propionyl-coa transferase is specially the 326-1900 position of sequence 3 in sequence table.In step (3), the nucleotide sequence of the encoding gene of described pyruvic carboxylase (ZpPDC gene) is specially the 1001-2671 position of sequence 4 in sequence table; The nucleotide sequence of the encoding gene (mhpf gene) of the acetaldehyde coa dehydrogenase of described NAD dependent form is specially the 3-953 position of sequence 4 in sequence table.
In addition, in step (1), the nucleotide sequence of described poxB gene is specially in sequence table shown in sequence 18; The nucleotide sequence of described lldD gene is specially sequence 5 in sequence table; The nucleotide sequence of described adheE gene is specially sequence 6 in sequence table; The nucleotide sequence of described ackA-pta gene is specially sequence 7 in sequence table.In step (2), the nucleotide sequence of described ldhA gene is specially sequence 8 in sequence table; The nucleotide sequence of described dld gene is specially sequence 9 in sequence table.
In the step (1) of described method, described intestinal bacteria BW25113-△ poxB, specifically obtain according to the method comprising the steps: in the competence of the described intestinal bacteria BW25113 wild-type that contains pKD46 plasmid, proceed to the DNA fragmentation 17 shown in sequence 17 in sequence table, the genome generation homologous recombination of described DNA fragmentation 17 and described intestinal bacteria BW25113 wild-type, realize the described poxB gene that knocks out described intestinal bacteria BW25113 wild-type, obtain described intestinal bacteria BW25113-△ poxB; In fact, proceeding to after described DNA fragmentation 17 in described intestinal bacteria BW25113 wild-type, also comprise and eliminate pKD46 plasmid and proceed to the step of pCP20 plasmid and eliminate the step (for removing the Kan resistant gene on the described DNA fragmentation 17 that is incorporated into described intestinal bacteria BW25113 wild type gene group) of pCP20 plasmid.
In the step (1) of described method, the encoding gene that is described 3-hydroxymalonate dehydrogenase by the described lldD Gene Replacement in described intestinal bacteria BW25113-△ poxB, specifically realize by the following method: in the competence of the described intestinal bacteria BW25113-△ poxB that contains pKD46 plasmid, proceed to the DNA fragmentation 1 shown in sequence 1 in sequence table, the genome generation homologous recombination of described DNA fragmentation 1 and described intestinal bacteria BW25113-△ poxB, the encoding gene that to realize described lldD Gene Replacement be described 3-hydroxymalonate dehydrogenase.In the present invention, proceeding to after described DNA fragmentation 1 in described intestinal bacteria BW25113-△ poxB, also comprise and eliminate the step of pKD46 plasmid and proceed to the step of pCP20 plasmid and eliminate the step (for removing the Kan resistant gene on the described DNA fragmentation 1 that is incorporated into described intestinal bacteria BW25113-△ poxB genome) of pCP20 plasmid.
The encoding gene that is coenzyme A dependent form succinic semialdehyde dehydrogenase by the described adheE Gene Replacement in described intestinal bacteria BW25113-△ poxB, specifically realize by the following method: in the competence of the described intestinal bacteria BW25113-△ poxB that contains pKD46 plasmid, proceed to the DNA fragmentation 2 shown in sequence 2 in sequence table, the genome generation homologous recombination of described DNA fragmentation 2 and described intestinal bacteria BW25113-△ poxB, realizing described adheE Gene Replacement is the encoding gene of described coenzyme A dependent form succinic semialdehyde dehydrogenase.In the present invention, proceeding to after described DNA fragmentation 2 in described intestinal bacteria BW25113-△ poxB, also comprise and eliminate the step of pKD46 plasmid and proceed to the step of pCP20 plasmid and eliminate the step (for removing the Kan resistant gene on the described DNA fragmentation 2 that is incorporated into described intestinal bacteria BW25113-△ poxB genome) of pCP20 plasmid.
The encoding gene that is propionyl-coa transferase by the described ackA-pta Gene Replacement in described intestinal bacteria BW25113-△ poxB, specifically realize by the following method: in the competence of the described intestinal bacteria BW25113-△ poxB that contains pKD46 plasmid, proceed to the DNA fragmentation 3 shown in sequence 3 in sequence table, the genome generation homologous recombination of described DNA fragmentation 3 and described intestinal bacteria BW25113-△ poxB, the encoding gene that to realize described ackA-pta Gene Replacement be described propionyl-coa transferase.In the present invention, proceeding to after described DNA fragmentation 3 in described intestinal bacteria BW25113-△ poxB, also comprise and eliminate the step of pKD46 plasmid and proceed to the step of pCP20 plasmid and eliminate the step (for removing the Kan resistant gene on the described DNA fragmentation 3 that is incorporated into described intestinal bacteria BW25113-△ poxB genome) of pCP20 plasmid.
In the step (2) of described method, knock out the described ldhA gene of described BWPDO1, specifically realize by the following method: in the competence of the described BWPDO1 that contains pKD46 plasmid, proceed to the DNA fragmentation 10 shown in sequence 10 in sequence table, the genome generation homologous recombination of described DNA fragmentation 10 and described BWPDO1, realizes the described ldhA gene that knocks out described BWPDO1.In the present invention, proceeding to after described DNA fragmentation 10 in described BWPDO1, also comprise and eliminate the step of pKD46 plasmid and proceed to the step of pCP20 plasmid and eliminate the step (for removing the Kan resistant gene on the described DNA fragmentation 10 that is incorporated into described BWPDO1 genome) of pCP20 plasmid.
Knock out the described dld gene of described BWPDO1, specifically realize by the following method: in the competence of the described BWPDO1 that contains pKD46 plasmid, proceed to the DNA fragmentation 11 shown in sequence 11 in sequence table, the genome generation homologous recombination of described DNA fragmentation 11 and described BWPDO1, realizes the described dld gene that knocks out described BWPDO1.In the present invention, proceeding to after described DNA fragmentation 11 in described BWPDO1, also comprise and eliminate the step of pKD46 plasmid and proceed to the step of pCP20 plasmid and eliminate the step (for removing the Kan resistant gene on the described DNA fragmentation 11 that is incorporated into described BWPDO1 genome) of pCP20 plasmid.
In the step (3) of described method, the encoding gene of the acetaldehyde coa dehydrogenase of the encoding gene of described pyruvic carboxylase and described NAD dependent form is to import in described BWPDO2 by the form of recombinant expression vector.
In one embodiment of the invention, described recombinant expression vector is specially DNA fragmentation shown in sequence in sequence table 4 is inserted into the recombinant plasmid obtaining after the multiple clone site of pBAD43 carrier.Described multiple clone site specifically can be NcoI (be BspHI in sequence 4, both are isocaudarner) and SphI.
Utilize described recombination bacillus coli A or the described recombination bacillus coli B that described method prepares also to belong to protection scope of the present invention.
Described recombination bacillus coli A or described recombination bacillus coli B are utilizing Pfansteihl or its salt to synthesize S-1, and the application in 2-propylene glycol also belongs to protection scope of the present invention.
Another object of the present invention is to provide one and utilizes Pfansteihl or the synthetic S-1 of its salt, the method for 2-propylene glycol.
The synthetic S-1 of Pfansteihl or its salt that utilizes provided by the present invention, the method for 2-propylene glycol, can be following (A) or (B):
(A) comprise the steps: to add in the culture system of described recombination bacillus coli B final concentration be the Pfansteihl of 200mM or its salt as substrate, carry out fermentation culture in 37 DEG C, from nutrient solution, obtain S-1,2-propylene glycol;
(B) be that the Pfansteihl of 200mM or its salt are as substrate to adding final concentration in the culture system of described recombination bacillus coli A, be cultured between OD600 to 0.6-0.8 in 37 DEG C, adding final concentration is that the L-arabinose of 2g/L is as inductor, carry out fermentation culture in 37 DEG C, from nutrient solution, obtain S-1,2-propylene glycol.
In described method, in the culture system of described recombination bacillus coli A or described recombination bacillus coli B, the carbon source of substratum is glucose, and its content is 10g/L.
In described method, the training method of described fermentation culture can be shaking culture, and rotating speed can be 180rpm; Time is 24 hours.
In described method, the initial OD 600 of recombination bacillus coli A described in described culture system or described recombination bacillus coli B is 0.1.
Experimental results show that, the present invention passes through Red recombinant means to affecting Pfansteihl to S-1, some genes of 2-propylene glycol reaction path have carried out the work of gene knockout, the gene of three key enzymes is inserted in colibacillary genome and is gone simultaneously, utilize T5 promotor to regulate and control its expression, and the encoding gene (mhpf gene) that proceeds to the encoding gene (ZpPDC gene) of pyruvic carboxylase and the acetaldehyde coa dehydrogenase of NAD dependent form by recombinant expression vector reduces equilibrium of forces (the namely covering method of NADH) to bacterial strain, finally obtain and can transform generation S-1 by biologically-derived Pfansteihl, the metabolic engineering bacterial strain of 2-propylene glycol.This recombination bacillus coli, taking 200mM Sodium.alpha.-hydroxypropionate as substrate, is cultivated the S-1 that can produce 3.4mM for 24 hours, 2-propylene glycol for 37 DEG C.The present invention is for improving biosynthesizing S-1, and the output of 2-propylene glycol and the work of transformation efficiency lay the foundation.
Brief description of the drawings
Fig. 1 is the PCR qualification of recombination bacillus coli BWPDO1.Wherein, A replaces upper mmsB gene when knocking out lldD gene; B replaces upper pdcD gene when knocking out adhE gene; C replaces upper pct gene when knocking out ackA-pta gene.In A-C, the each band of swimming lane M1 is descending is followed successively by 10.0,8.0,6.0,5.0,4.0,3.0,2.0,1.5,1.0,0.5kb; The each band of swimming lane M2 is descending is followed successively by 4.5,3.0,2.0,1.2,0.8,0.5kb.
Fig. 2 is that recombination bacillus coli BWPDO1 utilizes Pfansteihl to synthesize S-1, the result of 2-propylene glycol.
Fig. 3 is the PCR qualification of recombination bacillus coli BWPDO2.Wherein, A is for knocking out dld gene; B is for knocking out ldhA gene.In A and B, the each band of swimming lane M is descending is followed successively by 4.5,3.0,2.0,1.2,0.8,0.5kb.
Fig. 4 is that recombination bacillus coli BWPDO2 utilizes Pfansteihl to synthesize S-1, the result of 2-propylene glycol.
Fig. 5 is the PCR the result of recombination bacillus coli BWPDO3.The each band of swimming lane M is descending is followed successively by 4.5,3.0,2.0,1.2,0.8,0.5kb.
Fig. 6 is that recombination bacillus coli BWPDO3 utilizes Pfansteihl to synthesize S-1, the result of 2-propylene glycol.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
PBAD43 carrier: Youbio company product, its catalog number is VT1293.
PKD46 plasmid (GenBank ID:AY048746.1), pCP20 plasmid (GenBank ID:HB393402.1): ATCC (American Type Culture Collecti).Reference: One-step inactivation ofchromosomal genes in Escherichia coli K-12using PCR products, DatsenkoKA, Wanner BL., Proc Natl Acad Sci USA.2000Jun6; 97 (12): 6640-5.
Intestinal bacteria BW25113 wild-type: Coli Genetics Stock Center, its catalog number is CGSC#7636.
Intestinal bacteria BW25113-△ poxB: knock out poxB gene (sequence 18) gained by intestinal bacteria BW25113 wild-type, concrete operations are as follows:
1, the DNA fragmentation shown in artificial synthesized sequence 17: 1-59 position is the upstream sequence (being upstream homology arm) of the poxB gene of intestinal bacteria BW25113 wild-type self; 80-113 position is FRT site sequence; 488-1282 position is Kan resistant gene; 1473-1506 position is FRT site sequence; 1527-1585 position is the poxB gene that this DNA fragmentation of downstream sequence (being downstream homology arm) of the poxB gene of intestinal bacteria BW25113 wild-type self is used for knocking out intestinal bacteria BW25113 wild-type self.
2, carry out in accordance with the following steps
(1) with reference to the method for recording in (1) in embodiment 1 step 11, in intestinal bacteria BW25113 wild-type, proceed to pKD46 plasmid and prepare competence, thus the competent cell of the recombination bacillus coli BW25113 wild-type that acquisition contains pKD46 plasmid.
(2), with reference to the method for recording in (2) in embodiment 1 step 11, the DNA fragmentation of above step 1 synthetic is proceeded to the competent cell of the recombination bacillus coli BW25113 wild-type that contains pKD46 plasmid.
(3) resistance screening and PCR qualification
The recombinant bacterium that step (2) electricity is turned after DNA fragmentation is coated the mono-clonal that Kan resistant panel grows, and carries out PCR detection.Taking the mono-clonal chosen as template, carry out pcr amplification with primer poxB-F and poxB-R, select the clone of empirical tests correct (obtain size and be about 1720bp object band) to continue streak culture.
poxB-F：5’-ATTAACGGTAGGGTCGTCTCCG-3’；
poxB-R：5’-TCATCGGGCTATTTAACCGTTAGTG-3’。
(4) utilize pCP20 plasmid to eliminate resistance
Carry out with reference to the method for recording in (4) in embodiment 1 step 11.For the mono-clonal of picking, carry out pcr amplification with primer poxB-F and poxB-R, select the clone of empirical tests correct (obtaining big or small about 327bp object band) to continue streak culture.
(5) step (4) is identified to positive clone is inoculated in LB liquid nutrient medium, 42 DEG C of cultivations, go down to posterity twice, draw mono-clonal, to remove pCP20 plasmid, last simultaneously at ammonia benzyl resistance and the flat lining out of nonreactive, the bacterial strain that ammonia benzyl flat board can not be grown, for knocking out successful nonreactive deletant, is intestinal bacteria BW25113-△ poxB.
The present inventor further utilizes primer poxB-F and poxB-R to carry out examining order to intestinal bacteria BW25113-△ poxB, determines the exactness of the gene order of inserting.
Result shows, adopt primer poxB-F and poxB-R to check order to intestinal bacteria BW25113-△ poxB, sequencing result show intestinal bacteria BW25113-△ poxB sequence 17 in corresponding sequence table genome excalation the 80-1472 position of sequence 17, realized knocking out of poxB gene.
Embodiment 1, utilize Pfansteihl to synthesize S-1, the structure of the recombination bacillus coli of 2-propylene glycol and Function Identification
One, the structure of recombination bacillus coli BWPDO1 and Function Identification
1, the structure of recombination bacillus coli BWPDO1
According to utilizing Pfansteihl to synthesize S-1, the approach of 2-propylene glycol is determined three key enzymes that participate in catalysis: be respectively propionyl-coa transferase (Pct, aminoacid sequence is sequence 14) catalysis by Pfansteihl to L-lactoyl coenzyme A reaction, coenzyme A dependent form succinic semialdehyde dehydrogenase (PdcD, aminoacid sequence is sequence 13) catalysis by L-lactoyl coenzyme A to L-lactic aldehyde approach and 3-hydroxymalonate dehydrogenase (MmsB, aminoacid sequence is sequence 12) final S-1, the 2-propylene glycol of generating of catalysis L-lactic aldehyde reaction.
Mentality of designing: replace upper mmsB gene when knocking out the lldD gene (sequence 5) of intestinal bacteria BW25113-△ poxB self, when knocking out the adhE gene (sequence 6) of intestinal bacteria BW25113-△ poxB self, replace upper pdcD gene, when knocking out the ackA-pta gene (sequence 7) of intestinal bacteria BW25113-△ poxB self, replace upper pct gene.And adopt the control of T5 promotor to express three key gene-pct, pdcD and mmsB gene, and after T5 promotor, connect SD sequence.
According to as above mentality of designing, according to Red recombination system principle of work, synthetic contains homology arm as follows for knocking out three DNA fragmentations of replacement:
DNA fragmentation A: its nucleotide sequence is as shown in sequence in sequence table 1.Wherein, 1-40 position is the upstream sequence (being upstream homology arm) of the lldD gene of intestinal bacteria BW25113-△ poxB self; 41-100 position is T5 promoter sequence; 101-115 position is SD sequence; 116-994 position is mmsB gene order; 1016-1049 position is FRT site sequence; 1424-2218 position is Kan resistant gene; 2409-2442 position is FRT site sequence; 2443-2482 position is the downstream sequence (being downstream homology arm) of the lldD gene of intestinal bacteria BW25113-△ poxB self.This DNA fragmentation A replaces mmsB gene for the lldD gene that knocks out intestinal bacteria BW25113-△ poxB self simultaneously.
DNA fragmentation B: its nucleotide sequence is as shown in sequence in sequence table 2.Wherein, 1-250 position is the upstream sequence (being upstream homology arm) of the adhE gene of intestinal bacteria BW25113-△ poxB self; 251-310 position is T5 promoter sequence; 311-325 position is SD sequence; 326-1714 position is pdcD gene order; 1736-1769 position is FRT site sequence; 2144-2938 position is Kan resistant gene; 3129-3162 position is FRT site sequence; 3163-3412 position is the downstream sequence (being downstream homology arm) of the adhE gene of intestinal bacteria BW25113-△ poxB self.This DNA fragmentation B replaces pdcD gene for the adhE gene that knocks out intestinal bacteria BW25113-△ poxB self simultaneously.
DNA fragmentation C: its nucleotide sequence is as shown in sequence in sequence table 3.Wherein, 1-250 position is the upstream sequence (being upstream homology arm) of the ackA-pta gene of intestinal bacteria BW25113-△ poxB self; 251-310 position is T5 promoter sequence; 311-325 position is SD sequence; 326-1900 position is pct gene order; 1922-1955 position is FRT site sequence; 2330-3124 position is Kan resistant gene; 3315-3348 position is FRT site sequence; 3349-3598 position is the downstream sequence (being downstream homology arm) of the ackA-pta gene of intestinal bacteria BW25113-△ poxB self.This DNA fragmentation C replaces pct gene for the ackA-pta gene that knocks out intestinal bacteria BW25113-△ poxB self simultaneously.
Above three DNA fragmentations (DNA fragmentation A, DNA fragmentation B and DNA fragmentation C) mode turning by electricity is proceeded to the competent cell of intestinal bacteria BW25113-△ poxB, divide three experiments to proceed to, carry out successively, thereby realize knocking out and replacing of goal gene.Taking DNA fragmentation A proceed to experiment as example explanation concrete steps as follows:
(1) pKD46 plasmid is transformed in the competent cell of intestinal bacteria BW25113-△ poxB to 30 DEG C of shaking table 180rpm, incubated overnight.In the ratio of inoculum size 1% (volume fraction), nutrient solution is inoculated into next day in the Erlenmeyer flask of the LB substratum that 100mL is new (100 μ g/mL penbritin), 30 DEG C of shaking table 180rpm, cultivate; When OD=0.1, add final concentration 0.2% (0.2g/100mL) L-arabinose; In the time of OD=0.6-0.7, bacterium liquid is distributed into 50mL/ pipe, be placed in below cooled on ice to 4 DEG C; 6 DEG C, 4000rpm, centrifugal 10min, abandons supernatant, collects thalline; Every pipe adds the aseptic precooling glycerine of 25mL10% (volume fraction), suspends gently, and 4 DEG C, 4000rpm, centrifugal 10min, abandons supernatant; Every pipe adds the aseptic precooling glycerine of 15mL10% (volume fraction), suspends gently, and 4 DEG C, 4000rpm, centrifugal 10min, abandons supernatant; Every pipe adds the aseptic precooling glycerine of 2mL10% (volume fraction), suspends gently, and 4 DEG C, 4000rpm, centrifugal 10min, abandons supernatant; Every pipe adds the aseptic precooling glycerine of 200 μ L, suspends gently, is distributed into 50 μ L/ pipes.Put into-80 DEG C frozen for subsequent use.Obtain the competent cell of the intestinal bacteria BW25113-△ poxB that contains pKD46 plasmid.
(2) take out steps (1) from-80 DEG C and prepare the competent cell of the intestinal bacteria BW25113-△ poxB that contains pKD46 plasmid, put on ice and thaw, electric shock glass (a 1mm slit) is dried postposition on ice; Get 200ng DNA fragmentation (the above DNA fragmentation A obtaining), join in competence, tenderness mixes, and is added in the pole cup of precooling; 1800V electric shock, 6ms, adds 1mL LB substratum immediately; Liquid is taken out and puts into EP pipe, 150rpm constant temperature culture 1h; Get 200 μ L and be applied to the LB flat board containing that microbiotic of card (35 μ g/mL); Be inverted flat board, incubated overnight in constant incubator.
(3) resistance screening and PCR qualification
After step (2) electricity is turned to DNA fragmentation A, coat the mono-clonal that Kan resistant panel grows, carry out PCR detection.Taking the mono-clonal chosen as template, carry out respectively pcr amplification with 1 the primer pair of being numbered providing in table 1, select the correct clone of empirical tests to continue streak culture.Pcr amplification arranges the intestinal bacteria BW25113-△ poxB that does not proceed to DNA fragmentation A as wild-type contrast (WT) simultaneously.The theoretical length of amplified production is in table 1 (wild-type and saltant type Km
r).
Table 1 gene knockout checking primer and theoretical amplification length
(4) eliminate pKD46 plasmid and utilize pCP20 plasmid to eliminate resistance
Picking mono-clonal, 42 DEG C of cultivations in test tube, 180rpm, continues 1% (volume fraction) and is forwarded to continuation cultivation 6-8 hour in new LB substratum after 4-6 hour.Be on the flat board of 50 μ g/mL kantlex at final concentration, line, is placed in 42 DEG C, incubated overnight.Rear picking mono-clonal, respectively at the flat lining out of ammonia benzyl resistance and Ka Na resistance, selects ammonia benzyl flat board and does not grow but block that dull and stereotyped long bacterium colony, turns competence for the preparation of electricity.After preparation competence, proceed to pCP20 plasmid.Preparing electricity, to turn the method for competence and conversion the same.The flat board that is coated with ammonia benzyl resistance after conversion plasmid pCP20, is placed in 30 DEG C of incubator overnight incubation.Picking mono-clonal, utilizes 1 the checking primer of being numbered in table 1 to carry out pcr amplification, selects the correct clone of empirical tests to continue streak culture.Pcr amplification arranges intestinal bacteria BW25113-△ poxB as wild-type contrast (WT) simultaneously.The theoretical length of amplified production is in table 1 (wild-type and saltant type Km
s).
(5) step (4) is identified to correct positive colony is inoculated in LB liquid nutrient medium, 42 DEG C of cultivations, go down to posterity twice, draw mono-clonal, to remove pCP20 plasmid, last simultaneously at ammonia benzyl resistance and the flat lining out of nonreactive, the bacterial strain that ammonia benzyl flat board can not be grown is for knocking out successful nonreactive deletant.
Complete knocking out of lldD gene and insert mmsB gene through above step (1)-(5); Then start to continue to proceed to pKD46 plasmid to completing in the recombinant bacterium that knocks out and insert mmsB gene of lldD gene, continue to make competence, proceed to DNA fragmentation B with reference to above-mentioned steps (1)-(5), checking, completing adhE Gene Replacement is pdcD gene; Then continue to proceed to DNA fragmentation C with reference to above-mentioned steps (1)-(5) to completing in the recombinant bacterium that adhE Gene Replacement is pdcD gene, complete ackA-pta Gene Replacement pct gene.By the recombinant bacterium called after recombinant bacterium BWPDO1 finally obtaining.
But the detected result of above step (3) correspondence is as shown in swimming lane W in Fig. 1 and swimming lane 1, and in figure, swimming lane W is wild-type fragment size, the PCR clip size of swimming lane 1 for knocking out corresponding gene after exchanging carry screening resistance Kan.As can be seen from the figure, acquired results all with in table 1, predict the outcome consistent.Select the correct clone of empirical tests to continue streak culture.
The detected result of above step (4) correspondence is as shown in swimming lane W in Fig. 1 and swimming lane 2, and in figure, swimming lane W is wild-type fragment size, and swimming lane 2 is for to utilize pCP20 to eliminate the clip size after the Kan resistance between two FRT sites.As can be seen from the figure, acquired results all with in table 1, predict the outcome consistent.
The present inventor further utilizes the three couples checking primer that is numbered 1-3 in table 1 to carry out examining order, determines the exactness of the gene order of inserting.
Result shows, 1 the primer pair of being numbered in employing table 1 checks order to recombinant bacterium BWPDO1, sequencing result show recombinant bacterium BWPDO1 sequence 1 in corresponding sequence table genome excalation the 1016-2408 position of sequence 1, realized the replacement of mmsB gene pairs lldD gene.2 the primer pair of being numbered in employing table 1 checks order to recombinant bacterium BWPDO1, sequencing result show recombinant bacterium BWPDO1 sequence 2 in corresponding sequence table genome excalation the 1736-3128 position of sequence 2, realized the replacement of pdcD gene pairs adheE gene.3 the primer pair of being numbered in employing table 1 checks order to recombinant bacterium BWPDO1, sequencing result show recombinant bacterium BWPDO1 correspond to sequence 3 in sequence table genome excalation the 1922-3314 position of sequence 3, realized the replacement of pct gene pairs ackA-pta gene.
2, the Function Identification of recombination bacillus coli BWPDO1
BWPDO1 is linked into incubated overnight in LB substratum, after be seeded in new LB substratum with 1% (volume ratio), 180rpm, 37 DEG C, cultivate 12-16h, measure OD600, receive bacterium, 4000rpm, 4 DEG C of centrifugal 10min.With physiological saline washing 2 times, by the centrifugal collection thalline of similarity condition.By resuspended thalline to the PBS of pH7.2-7.4, final OD600 to 25 is to 30 left and right, add Pfansteihl sodium as substrate, final concentration is respectively 200mM and 50mM, and add the glucose of 1% (1g/100ml), carry out rest cells reaction, temperature of reaction arranges 30 DEG C and 37 DEG C, reaction 24h.Sample with reaction, centrifugal, get supernatant, and measure Pfansteihl content and S-1,2-propanediol.In triplicate, result is got the mean value repeating three times in experiment.
Result as shown in Figure 2, from figure, can obviously find out the carrying out along with full cell response, lactic acid content does not decline and all rises to some extent, analyzing reason may be not knock out two gene dld and the ldhA of pyruvic acid to D-ALPHA-Hydroxypropionic acid metabolism, because this approach flux in metabolic pathway is higher, so the pyruvic acid that 1% glucose adding produces through glycolysis-produces D-ALPHA-Hydroxypropionic acid through this approach, cause required generation S-1, 2-propylene glycol approach metabolic flux reduces, and the reaction of lactic acid approach needs NADH as coenzyme, cause the required approach NADH amount of providing to reduce, also indirectly cause production declining.The reason that later stage propanediol declines may be cell paste intracellular metabolite and cause in order to maintain NADH level in cell.For head it off, the present inventor has proceeded the work that knocks out of transforming protein gene ldhA and dld between pyruvic acid and D-ALPHA-Hydroxypropionic acid.
Two, the structure of recombination bacillus coli BWPDO2 and Function Identification
1, the structure of recombination bacillus coli BWPDO2
According to Red recombination system principle of work, synthetic contains homology arm as follows for knocking out two DNA fragmentations of ldhA gene (sequence 8) and dld gene (sequence 9):
DNA fragmentation D: its nucleotide sequence is as shown in sequence in sequence table 10.Wherein, 1-59 position is the upstream sequence (being upstream homology arm) of the ldhA gene of intestinal bacteria BW25113-△ poxB self; 80-113 position is FRT site sequence; 488-1282 position is Kan resistant gene; 1473-1506 position is FRT site sequence; 1527-1585 position is the downstream sequence (being downstream homology arm) of the ldhA gene of intestinal bacteria BW25113-△ poxB self.This DNA fragmentation D is for knocking out the ldhA gene of intestinal bacteria BWPDO1 self.
DNA fragmentation E: its nucleotide sequence is as shown in sequence in sequence table 11.Wherein, 1-59 position is the upstream sequence (being upstream homology arm) of the dld gene of intestinal bacteria BW25113-△ poxB self; 80-113 position is FRT site sequence; 488-1282 position is Kan resistant gene; 1473-1506 position is FRT site sequence; 1527-1585 position is the downstream sequence (being downstream homology arm) of the dld gene of intestinal bacteria BW25113-△ poxB self.This DNA fragmentation E is for knocking out the dld gene of intestinal bacteria BWPDO1 self.
The mode that above two DNA fragmentations (DNA fragmentation D and DNA fragmentation E) are turned by electricity proceeds to the competent cell of the recombination bacillus coli BWPDO1 of step 1 structure, and experiment proceeds at twice, carries out successively, thereby realizes knocking out of goal gene.Taking DNA fragmentation D proceed to experiment as example explanation concrete steps as follows:
(1), with reference to the method for recording in (1) in step 11, prepare the competent cell of the recombination bacillus coli BWPDO1 that contains pKD46 plasmid.
(2), with reference to the method for recording in (2) in step 11, the DNA fragmentation D obtaining is above proceeded to the competent cell of the recombination bacillus coli BWPDO1 that contains pKD46 plasmid.
(3) resistance screening and PCR qualification
After step (2) electricity is turned to DNA fragmentation E, coat the mono-clonal that Kan resistant panel grows, carry out PCR detection.Taking the mono-clonal chosen as template, carry out pcr amplification with 4 the primer pair of being numbered providing in table 1, select the correct clone of empirical tests to continue streak culture.Pcr amplification arranges the intestinal bacteria BW25113-△ poxB that does not proceed to two DNA fragmentations as wild-type contrast (WT) simultaneously.The theoretical length of amplified production is in table 1 (wild-type and saltant type Km
s).
(4) utilize pCP20 plasmid to eliminate resistance
Carry out with reference to the method for recording in (4) in step 11.For the mono-clonal of picking, utilize 4 the checking primer of being numbered in table 1 to carry out pcr amplification, select the correct clone of empirical tests to continue streak culture.Pcr amplification arranges the intestinal bacteria BW25113-△ poxB that does not proceed to two DNA fragmentations as wild-type contrast (WT) simultaneously.The theoretical length of amplified production is in table 1 (wild-type and saltant type Km
s).
(5) step (4) is identified to correct positive colony is inoculated in LB liquid nutrient medium, 42 DEG C of cultivations, go down to posterity twice, draw mono-clonal, to remove pCP20 plasmid, last simultaneously at ammonia benzyl resistance and the flat lining out of nonreactive, the bacterial strain that ammonia benzyl flat board can not be grown is for knocking out successful nonreactive deletant.
Complete knocking out of ldhA gene through above step (1)-(5); Then start to continue to proceed to pKD46 plasmid to having knocked out in the recombinant bacterium of ldhA gene, continue to make competence, proceed to DNA fragmentation E with reference to above-mentioned steps (1)-(5), checking, completes knocking out of dld gene.By the recombinant bacterium called after recombinant bacterium BWPDO2 finally obtaining.
But the detected result of above step (3) correspondence is as shown in swimming lane W in Fig. 3 and swimming lane 1, and in figure, swimming lane W is wild-type fragment size, the PCR clip size of swimming lane 1 for knocking out corresponding gene after exchanging carry screening resistance Kan.As can be seen from the figure, acquired results all with in table 1, predict the outcome consistent.
The detected result of above step (4) correspondence is as shown in swimming lane W in Fig. 3 and swimming lane 2, and in figure, swimming lane W is wild-type fragment size, and swimming lane 2 is for to utilize pCP20 to eliminate the clip size after the Kan resistance between two FRT sites.As can be seen from the figure, acquired results all with in table 1, predict the outcome consistent.
The present inventor further utilizes and in table 1, is numbered two couples checking primer of 4 and 5 and carries out examining order, determines the exactness of the gene order of inserting.
Result shows, 4 the primer pair of being numbered in employing table 1 checks order to recombinant bacterium BWPDO2, sequencing result show recombinant bacterium BWPDO2 sequence 10 in corresponding sequence table genome excalation the 80-1472 position of sequence 10, realized knocking out of ldhA gene.5 the primer pair of being numbered in employing table 1 checks order to recombinant bacterium BWPDO2, sequencing result show recombinant bacterium BWPDO2 sequence 11 in corresponding sequence table genome excalation the 80-1472 position of sequence 11, realized knocking out of dld gene.
2, the Function Identification of recombination bacillus coli BWPDO2
BWPDO2 is accessed in LB substratum to 180rpm, 37 DEG C of incubated overnight.After contain different glucose starting point concentrations (0.5%, 1%, 2% with 1% (volume ratio) inoculum size access, the g/100ml of unit) and the M9 substratum of Different L-Sodium.alpha.-hydroxypropionate concentration (50,200mM) in (reaction initial OD 600 is about 0.1), 180rpm, 37 DEG C of cultivations, 24h.The content of substrate Pfansteihl sodium and product S-1,2-PD under the each condition of sampling and measuring.In triplicate, result is got the mean value repeating three times in experiment.
Result as shown in Figure 4, different gradient glucose conditions are all not identical with production concentration that concentration of substrate produces, wherein, through fermentation in 24 hours, the highest condition of output is that under 1% glucose condition, Pfansteihl concentration of substrate is 200mM, S-1,2-propanediol is about 2mM, compared with 2.8 times of resting cell system output.
Three, the structure of recombination bacillus coli BWPDO3 and Function Identification
1, the structure of recombination bacillus coli BWPDO3
In order further to improve Pfansteihl to S-1, the transformation efficiency of 2-propylene glycol and output, the present inventor proceeds transformation to recombination bacillus coli BWPDO2 bacterial strain, finding two enzymes is respectively pyruvic carboxylase (pyruvate the decarboxylase) (ZpPDC from palm fermenting bacteria (Zymobacter palmae), aminoacid sequence is as shown in sequence in sequence table 15) and from colibacillary NAD dependent form acetaldehyde coa dehydrogenase (acetaldehyde-CoA dehydrogenase II, NAD-binding) (mhpf, aminoacid sequence is as shown in sequence in sequence table 16), utilize pBAD43 carrier to carry out tandem expression to the gene of its this two enzymes.
(1) synthetic ZpPDC-mhpf gene fragment
DNA fragmentation shown in sequence 4 in artificial synthesized sequence table.Wherein, 1-6 position is the recognition sequence of restriction enzyme site BspHI, and 3-953 position is the sequence of mhpf gene, and 1001-2671 position is the sequence of ZpPDC gene, and 2672-2677 position is the recognition sequence of restriction enzyme site SphI.
(2) use the ZpPDC-mhpf gene fragment of the synthetic two ends of restriction enzyme BspHI and SphI double digestion step (1) with restriction enzyme site, after glue reclaims, be connected with the skeleton large fragment of the pBAD43 carrier through NcoI (being isocaudarner with BspHI) and SphI double digestion, obtain recombinant plasmid.The recombinant plasmid called after pBAD-MP obtaining after DNA fragmentation shown in the 2-2671 position of sequence 4 in sequence table will be shown the small segment between the restriction enzyme site NcoI of pBAD43 carrier and SphI to replace with through order-checking.
(3) mode recombinant vectors pBAD-MP of step (2) gained being turned by electricity proceeds in the competent cell that step 2 builds the recombination bacillus coli BWPDO2 obtaining (concrete operations are referring to above), picking mono-clonal, adopts primer pBAD-F and pBAD-R (two primer sequences are pBAD43 carrier and carry sequence) to carry out pcr amplification for ZpPDC-mhpf gene fragment.
pBAD-F：5’-CTGTTTCTCCATACCCGTT-3’；
pBAD-R：5’-CTCATCCGCCAAAACAG-3’。
Adopt as above primer pair to check order the mono-clonal that obtains size through pcr amplification and be about 2746bp object band (Fig. 5).Sequencing result is shown to the recombinant bacterium called after BWPDO3 containing sequence 4 in ordered list.
2, the Function Identification of recombination bacillus coli BWPDO3
BWPDO3 is accessed in LB substratum, 180rpm, 37 DEG C of incubated overnight, are kind of a daughter bacteria.After access in the M9 substratum that contains final concentration 1% (1g/100ml) glucose and 200mM Pfansteihl sodium (reaction initial OD 600 is about 0.1) with 1% (volume ratio) inoculum size, 180rpm, 37 DEG C of cultivations, between OD600 to 0.6-0.8, add different concns L-arabinose to induce, concentration gradient is respectively 0.05%, 0.1%, 0.2%, 0.4% (0.05% be equivalent to 0.05g/100ml, the like), rear cultivation 24h, carries out BWPDO2 simultaneously and cultivates and contrast with condition.The content of substrate Pfansteihl sodium and product S-1,2-PD under the each condition of sampling and measuring.In triplicate, result is got the mean value repeating three times in experiment.
Result as shown in Figure 6, can find out, recombination bacillus coli BWPDO3 is by synthetic Pfansteihl S-1,2-propanediol rises to some extent, wherein the suitableeest inductor add-on is 0.2%, output is about 3.4mM (being the S-1 that every liter of fermented liquid obtains 3.4mmol, 2-propylene glycol), has promoted approximately 1.5 times than recombination bacillus coli BWPDO2 output.
Claims (10)
1. utilize Pfansteihl to synthesize a S-1, the preparation method of the recombination bacillus coli A of 2-propylene glycol, comprises the step of (1)-(3) as follows:
(1) lldD gene, adheE gene and ackA-pta gene in intestinal bacteria BW25113-△ poxB are replaced with respectively to the encoding gene of 3-hydroxymalonate dehydrogenase, the encoding gene of coenzyme A dependent form succinic semialdehyde dehydrogenase and the encoding gene of propionyl-coa transferase, the recombinant bacterium obtaining is designated as BWPDO1;
Described intestinal bacteria BW25113-△ poxB is by the bacterial strain of gained after the poxB gene knockout in intestinal bacteria BW25113 wild-type;
(2) knock out ldhA gene and the dld gene of BWPDO1 that step (1) obtains, the recombinant bacterium obtaining is designated as BWPDO2;
(3) in the BWPDO2 obtaining to step (2), import the encoding gene of pyruvic carboxylase and the encoding gene of NAD dependent form acetaldehyde coa dehydrogenase, the recombinant bacterium obtaining is designated as BWPDO3; Described BWPDO3 is described recombination bacillus coli A.
2. utilize Pfansteihl to synthesize a S-1, the preparation method of the recombination bacillus coli B of 2-propylene glycol, comprises step (1) and step (2) in claim 1; The described BWPDO2 obtaining in described step (2) is described recombination bacillus coli B.
3. method according to claim 1 and 2, is characterized in that: in step (1), the aminoacid sequence of described 3-hydroxymalonate dehydrogenase is sequence 12 in sequence table; The aminoacid sequence of described coenzyme A dependent form succinic semialdehyde dehydrogenase is sequence 13 in sequence table; The aminoacid sequence of described propionyl-coa transferase is sequence 14 in sequence table; Or
In step (3), the aminoacid sequence of described pyruvic carboxylase is sequence 15 in sequence table; The aminoacid sequence of described NAD dependent form acetaldehyde coa dehydrogenase is sequence 16 in sequence table.
4. according to arbitrary described method in claim 1-3, it is characterized in that: in step (1), the nucleotides sequence of the encoding gene of described 3-hydroxymalonate dehydrogenase is classified the 116-994 position of sequence 1 in sequence table as; The nucleotides sequence of the encoding gene of described coenzyme A dependent form succinic semialdehyde dehydrogenase is classified the 326-1714 position of sequence 2 in sequence table as; The nucleotides sequence of the encoding gene of described propionyl-coa transferase is classified the 326-1900 position of sequence 3 in order-list as; Or
In step (3), the nucleotides sequence of the encoding gene of described pyruvic carboxylase is classified the 1001-2671 position of sequence 4 in sequence table as; The nucleotides sequence of the encoding gene of described NAD dependent form acetaldehyde coa dehydrogenase is classified the 3-953 position of sequence 4 in sequence table as; Or
In step (1), the nucleotides sequence of described poxB gene is classified as in sequence table shown in sequence 18; The nucleotides sequence of described lldD gene is classified sequence 5 in sequence table as; The nucleotides sequence of described adheE gene is classified sequence 6 in sequence table as; The nucleotides sequence of described ackA-pta gene is classified sequence 7 in sequence table as; Or
In step (2), the nucleotides sequence of described ldhA gene is classified sequence 8 in sequence table as; The nucleotides sequence of described dld gene is classified sequence 9 in sequence table as.
5. according to arbitrary described method in claim 1-4, it is characterized in that: in step (1),
Described intestinal bacteria BW25113-Δ poxB obtains according to the method comprising the steps: in the competence of the described intestinal bacteria BW25113 wild-type that contains pKD46 plasmid, proceed to the DNA fragmentation 17 shown in sequence 17 in sequence table, the genome generation homologous recombination of described DNA fragmentation 17 and described intestinal bacteria BW25113 wild-type, realize the described poxB gene that knocks out described intestinal bacteria BW25113 wild-type, obtain described intestinal bacteria BW25113-Δ poxB;
The encoding gene that is described 3-hydroxymalonate dehydrogenase by the described lldD Gene Replacement in described intestinal bacteria BW25113-△ poxB, realize by the following method: in the competence of the described intestinal bacteria BW25113-△ poxB that contains pKD46 plasmid, proceed to the DNA fragmentation 1 shown in sequence 1 in sequence table, the genome generation homologous recombination of described DNA fragmentation 1 and described intestinal bacteria BW25113-△ poxB, the encoding gene that to realize described lldD Gene Replacement be described 3-hydroxymalonate dehydrogenase;
The encoding gene that is coenzyme A dependent form succinic semialdehyde dehydrogenase by the described adheE Gene Replacement in described intestinal bacteria BW25113-△ poxB, realize by the following method: in the competence of the described intestinal bacteria BW25113-△ poxB that contains pKD46 plasmid, proceed to the DNA fragmentation 2 shown in sequence 2 in sequence table, the genome generation homologous recombination of described DNA fragmentation 2 and described intestinal bacteria BW25113-△ poxB, realizing described adheE Gene Replacement is the encoding gene of described coenzyme A dependent form succinic semialdehyde dehydrogenase;
The encoding gene that is propionyl-coa transferase by the described ackA-pta Gene Replacement in described intestinal bacteria BW25113-△ poxB, realize by the following method: in the competence of the described intestinal bacteria BW25113-△ poxB that contains pKD46 plasmid, proceed to the DNA fragmentation 3 shown in sequence 3 in sequence table, the genome generation homologous recombination of described DNA fragmentation 3 and described intestinal bacteria BW25113-△ poxB, the encoding gene that to realize described ackA-pta Gene Replacement be described propionyl-coa transferase.
6. according to arbitrary described method in claim 1-5, it is characterized in that: in step (2), knock out the described ldhA gene of described BWPDO1, realize by the following method: in the competence of the described BWPDO1 that contains pKD46 plasmid, proceed to the DNA fragmentation 10 shown in sequence 10 in sequence table, the genome generation homologous recombination of described DNA fragmentation 10 and described BWPDO1, realizes the described ldhA gene that knocks out described BWPDO1;
Knock out the described dld gene of described BWPDO1, realize by the following method: in the competence of the described BWPDO1 that contains pKD46 plasmid, proceed to the DNA fragmentation 11 shown in sequence 11 in sequence table, the genome generation homologous recombination of described DNA fragmentation 11 and described BWPDO1, realizes the described dld gene that knocks out described BWPDO1.
7. according to arbitrary described method in claim 1-6, it is characterized in that: in step (3), the encoding gene of the encoding gene of described pyruvic carboxylase and described NAD dependent form acetaldehyde coa dehydrogenase is to import in described BWPDO2 by the form of recombinant expression vector;
Described recombinant expression vector is specially DNA fragmentation shown in sequence in sequence table 4 is inserted into the recombinant plasmid obtaining after the multiple clone site of pBAD43 carrier.
8. utilize described recombination bacillus coli A or described recombination bacillus coli B that in claim 1-7, arbitrary described method prepares.
9. described in claim 8, recombination bacillus coli A or described recombination bacillus coli B are utilizing Pfansteihl or its salt to synthesize S-1, the application in 2-propylene glycol.
10. utilize Pfansteihl or its salt to synthesize S-1, the method for 2-propylene glycol, for following (A) or (B):
(A) comprise the steps: to add in the culture system of described recombination bacillus coli B final concentration be the Pfansteihl of 200mM or its salt as substrate, carry out fermentation culture in 37 DEG C, from nutrient solution, obtain S-1,2-propylene glycol;
(B) be that the Pfansteihl of 200mM or its salt are as substrate to adding final concentration in the culture system of described recombination bacillus coli A, be cultured between OD600 to 0.6-0.8 in 37 DEG C, adding final concentration is that the L-arabinose of 2g/L is as inductor, carry out fermentation culture in 37 DEG C, from nutrient solution, obtain S-1,2-propylene glycol.
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| CN116478893B (en) * | 2023-03-02 | 2024-05-10 | 天津大学 | Construction and application of engineering probiotics for producing propionic acid |
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