CN104109651B - Utilize recombination bacillus coli and the construction method thereof of Pfansteihl synthesis S-1,2-propylene glycol - Google Patents
Utilize recombination bacillus coli and the construction method thereof of Pfansteihl synthesis S-1,2-propylene glycol Download PDFInfo
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Abstract
The invention discloses a kind of recombination bacillus coli utilizing L lactic acid synthesis S 1,2 propylene glycol and construction method thereof.The method, comprise the steps: 1) the lldD gene in escherichia coli BW25113 △ poxB, adheE gene and ackA pta gene are replaced with the encoding gene of 3 hydroxymalonate dehydrogenases, the encoding gene of coenzyme A dependent form succinic semialdehyde dehydrogenase and the encoding gene of propionyl-coa transferase respectively, the recombinant bacterium obtained is designated as BWPDO1；2) knock out ldhA gene and the dld gene of described BWPDO1, obtain recombinant bacterium BWPDO2；3) in described BWPDO2, import encoding gene and the encoding gene of NAD dependent form acetaldehyde coa dehydrogenase of pyruvic carboxylase, obtain recombinant bacterium BWPDO3, be described recombination bacillus coli.It is demonstrated experimentally that the recombination bacillus coli of gained of the present invention can utilize L lactic acid to be converted into S 1,2 propylene glycol, with 200mM sodium lactate as substrate, cultivate S 1,2 propylene glycol that can produce 3.4mM for 24 hours for 37 DEG C.The present invention is that the work of the yield and conversion ratio that improve biosynthesis S 1,2 propylene glycol lays the foundation.
Description
Technical field
The invention belongs to microorganism field, relate to one and utilize the recombination bacillus coli of Pfansteihl synthesis S-1,2-propylene glycol
And construction method.
Background technology
The structure of propylene glycol is divided into 1,2-propylene glycol and 1,3-propylene glycol.1,2-propylene glycol is mainly used to give birth to
Product unsaturated polyester resin, antifreezing agent, plasticizer substitute ethylene glycol and for antifreeze aircraft and make coolant and heat in food
Carriers etc., it can be additionally used in nonionic detergent or wetting agent.Propylene glycol or good solvent, can be used for food seasoning and
Spice, or the ointment ointment etc. of medical industry.
The active group of optical activity 1,2-PD, can be as chirality starting material for medicine, herbicide, pheromone
With the synthesis of liquid crystal, there is the highest researching value.Escherichia coli self there are the metabolic pathway generating 1,2-propylene glycol.
In metabolism L-rhamnose and L-fucose approach, the sugar after phosphorylation is generated L-lactic aldehyde and dihydroxyacetone phosphorus by aldolase catalysis
Acid (DHAP), after in the presence of the condition and NADH of anaerobism L-lactic aldehyde be catalyzed as L-1,2-propylene glycol, i.e. S-1.2-propylene glycol.
But owing to cost of material is too high, this path does not has fine economic benefit, so there is no good researching value.At thermophilic anaerobic
Bacterium (Thermoanaerobacterium thermosaccharolyticum) also has from methyl-glyoxal
(methylglyoxal) generate lactic aldehyde (Lactaldehyde) respectively or methylglyoxal (Acetol) ultimately generates 1,2-propylene glycol
Path, but the simply guess of some of which path, the most not evidence suggests that there is actual enzyme goes catalysis.
It is about the research of living things catalysis synthesis R-1,2-propylene glycol, at present, external existing about living things catalysis synthesis R-1,
The report of 2-propylene glycol, Tadashi etc. uses airlift fermentor that yeast reduction pyruvic alcohol is generated R-1,2-propylene glycol and ferment
Female oxidation resolution of racemic 1,2-PD is studied, but does not obtains industrial applications yet, and domestic rare report.
Summary of the invention
It is an object of the invention to provide a kind of recombination bacillus coli utilizing Pfansteihl synthesis S-1,2-propylene glycol and system thereof
Preparation Method and application.
Method provided by the present invention can be the preparation of the recombination bacillus coli A utilizing Pfansteihl synthesis S-1,2-propylene glycol
Method, specifically can include the step of following (1)-(3):
(1) lldD gene, adheE gene and the ackA-pta gene in escherichia coli BW25113-△ poxB is replaced respectively
It is changed to the encoding gene (mmsB gene) of 3-hydroxymalonate dehydrogenase, the coding base of coenzyme A dependent form succinic semialdehyde dehydrogenase
Because of (pdcD gene) and the encoding gene (pct gene) of propionyl-coa transferase, the recombinant bacterium obtained is designated as BWPDO1；
Described escherichia coli BW25113-△ poxB is by after the poxB gene knockout in escherichia coli BW25113 wild type
The bacterial strain of gained；
(2) knocking out ldhA gene and the dld gene of the BWPDO1 that step (1) obtains, the recombinant bacterium obtained is designated as BWPDO2；
(3) in the BWPDO2 that step (2) obtains, import encoding gene (ZpPDC gene) and the NAD of pyruvic carboxylase
The encoding gene (mhpf gene) of the acetaldehyde coa dehydrogenase of dependent form, the recombinant bacterium obtained is designated as BWPDO3；Described BWPDO3
It is described recombination bacillus coli A.
Wherein, described lldD gene is acetone acid and Pfansteihl conversion encoding gene, described ldhA gene and described dld base
Because the encoding gene of transforming protein between two different acetone acid and D-ALPHA-Hydroxypropionic acid.Described adheE gene and described ackA-
Pta gene is two different downstream genes affecting substrate acetyl CoA contents.
Method provided by the present invention alternatively utilizes the system of the recombination bacillus coli B of Pfansteihl synthesis S-1,2-propylene glycol
Preparation Method, including above step (1) and step (2)；It is big that the described BWPDO2 obtained in described step (2) is described restructuring
Enterobacteria B.
In the step (1) of said method, described 3-hydroxymalonate dehydrogenase is bacillus cereus (Bacillus
Cereus) 3-hydroxymalonate dehydrogenase, its aminoacid sequence is specifically as shown in sequence 12 in sequence table.Described coenzyme A relies on
Type succinic semialdehyde dehydrogenase is the coenzyme A dependent form of Yersinia enterocolitica (Yersinia enterocolitica)
Succinic semialdehyde dehydrogenase, its aminoacid sequence is specifically as shown in sequence 13 in sequence table.Described propionyl-coa transferase is third
The point mutation albumen of the propionyl-coa transferase of acid clostridium (Clostridium propionicum), its aminoacid sequence is concrete
As shown in sequence 14 in sequence table.
In the step (3) of above-mentioned method, described pyruvic carboxylase is Zymobacterpalmae (Zymobacter palmae)
Pyruvic carboxylase, its aminoacid sequence is specifically as shown in sequence 15 in sequence table.The acetaldehyde coenzyme A of described NAD dependent form
Dehydrogenase is from the colibacillary acetaldehyde coa dehydrogenase needing NAD, and its aminoacid sequence is concrete such as sequence 16 in sequence table
Shown in.
Further, in step (1), the nucleotide sequence of the encoding gene (mmsB gene) of described 3-hydroxymalonate dehydrogenase
It is specially the 116-994 position of sequence 1 in sequence table；The encoding gene of described coenzyme A dependent form succinic semialdehyde dehydrogenase
The nucleotide sequence of (pdcD gene) is specifically the 326-1714 position of sequence 2 in sequence table；Described propionyl-coa transferase
The nucleotide sequence of encoding gene (pct gene) is specifically the 326-1900 position of sequence 3 in sequence table.In step (3), described
The nucleotide sequence of the encoding gene (ZpPDC gene) of pyruvic carboxylase is specifically the 1001-2671 of sequence 4 in sequence table
Position；The nucleotide sequence of the encoding gene (mhpf gene) of the acetaldehyde coa dehydrogenase of described NAD dependent form is specifically sequence table
The 3-953 position of middle sequence 4.
It addition, in step (1), the nucleotide sequence of described poxB gene is specifically for shown in sequence in sequence table 18；Described
The nucleotide sequence of lldD gene is specifically sequence 5 in sequence table；The nucleotide sequence of described adheE gene is specifically sequence table
Middle sequence 6；The nucleotide sequence of described ackA-pta gene is specifically sequence 7 in sequence table.In step (2), described ldhA base
The nucleotide sequence of cause is specifically sequence 8 in sequence table；The nucleotide sequence of described dld gene is specifically sequence 9 in sequence table.
In the step (1) of described method, described escherichia coli BW25113-△ poxB, specifically walk as follows according to including
Rapid method obtains: proceed to sequence table in the competence containing the described escherichia coli BW25113 wild type of pKD46 plasmid
DNA fragmentation 17 shown in middle sequence 17, described DNA fragmentation 17 occurs with the genome of described escherichia coli BW25113 wild type
Homologous recombination, i.e. realizes knocking out the described poxB gene of described escherichia coli BW25113 wild type, it is thus achieved that described escherichia coli
BW25113-△poxB；It is true that after proceeding to described DNA fragmentation 17 in described escherichia coli BW25113 wild type, also wrap
Include and eliminate pKD46 plasmid and proceed to the step of pCP20 plasmid and eliminate the step of pCP20 plasmid and (be incorporated into for removal described
The Kan resistant gene on described DNA fragmentation 17 in escherichia coli BW25113 wild type gene group).
In the step (1) of described method, the described lldD gene in described escherichia coli BW25113-△ poxB is replaced
It is changed to the encoding gene of described 3-hydroxymalonate dehydrogenase, realizes particular by following method: to containing pKD46 plasmid
The competence of described escherichia coli BW25113-△ poxB proceeds to the DNA fragmentation 1 shown in sequence 1 in sequence table, described DNA sheet
Section 1 and the genome generation homologous recombination of described escherichia coli BW25113-△ poxB, i.e. realize replacing described lldD gene
Encoding gene for described 3-hydroxymalonate dehydrogenase.In the present invention, in described escherichia coli BW25113-△ poxB
After proceeding to described DNA fragmentation 1, also include eliminating the step of pKD46 plasmid and proceeding to the step of pCP20 plasmid and eliminate pCP20
The step of plasmid (is incorporated on the described DNA fragmentation 1 in described escherichia coli BW25113-△ poxB genome for removing
Kan resistant gene).
Described adheE gene in described escherichia coli BW25113-△ poxB is replaced with coenzyme A dependent form succinic acid
The encoding gene of semialdehyde dehydrogenase, realizes particular by following method: to the described escherichia coli containing pKD46 plasmid
The competence of BW25113-△ poxB proceeds to the DNA fragmentation 2 shown in sequence 2 in sequence table, described DNA fragmentation 2 with described greatly
The genome generation homologous recombination of enterobacteria BW25113-△ poxB, i.e. realizes described adheE gene is replaced with described coenzyme A
The encoding gene of dependent form succinic semialdehyde dehydrogenase.In the present invention, in described escherichia coli BW25113-△ poxB
After proceeding to described DNA fragmentation 2, also include eliminating the step of pKD46 plasmid and proceeding to the step of pCP20 plasmid and eliminate pCP20
The step of plasmid (is incorporated on the described DNA fragmentation 2 in described escherichia coli BW25113-△ poxB genome for removing
Kan resistant gene).
Described ackA-pta gene in described escherichia coli BW25113-△ poxB is replaced with propionyl-coa transferase
Encoding gene, realize particular by following method: to the described escherichia coli BW25113-△ containing pKD46 plasmid
The competence of poxB proceeds to the DNA fragmentation 3 shown in sequence 3 in sequence table, described DNA fragmentation 3 and described escherichia coli
The genome generation homologous recombination of BW25113-△ poxB, i.e. realizes described ackA-pta gene is replaced with described propionyl coenzyme
The encoding gene of A transferring enzyme.In the present invention, in described escherichia coli BW25113-△ poxB, described DNA fragmentation 3 is being proceeded to
After, also include the step eliminating pKD46 plasmid and proceed to the step of pCP20 plasmid and eliminate the step of pCP20 plasmid and (be used for
Except being incorporated into the Kan resistant gene on the described DNA fragmentation 3 in described escherichia coli BW25113-△ poxB genome).
In the step (2) of described method, knock out the described ldhA gene of described BWPDO1, particular by following method
Realize: in the competence containing the described BWPDO1 of pKD46 plasmid, proceed to the DNA fragmentation shown in sequence 10 in sequence table
10, described DNA fragmentation 10 and the genome generation homologous recombination of described BWPDO1, i.e. realize knocking out the described of described BWPDO1
LdhA gene.In the present invention, after proceeding to described DNA fragmentation 10 in described BWPDO1, also include eliminating pKD46 plasmid
Step and proceed to the step of pCP20 plasmid and eliminate the step of pCP20 plasmid and (be incorporated into described BWPDO1 genome for removal
In described DNA fragmentation 10 on Kan resistant gene).
Knock out the described dld gene of described BWPDO1, realize particular by following method: to containing pKD46 plasmid
Described BWPDO1 competence in proceed to the DNA fragmentation 11 shown in sequence 11 in sequence table, described DNA fragmentation 11 is with described
The genome generation homologous recombination of BWPDO1, i.e. realizes knocking out the described dld gene of described BWPDO1.In the present invention, to
After described BWPDO1 proceeds to described DNA fragmentation 11, also include eliminating the step of pKD46 plasmid and proceeding to the step of pCP20 plasmid
Rapid and eliminate the step of pCP20 plasmid and (be incorporated on the described DNA fragmentation 11 in described BWPDO1 genome for removing
Kan resistant gene).
In the step (3) of described method, the encoding gene of described pyruvic carboxylase and the acetaldehyde of described NAD dependent form
The encoding gene of coa dehydrogenase is to be imported in described BWPDO2 by the form of recombinant expression carrier.
In one embodiment of the invention, described recombinant expression carrier is specially DNA sheet shown in sequence in sequence table 4
The recombiant plasmid that section obtains after being inserted into the multiple clone site of pBAD43 carrier.Described multiple clone site concretely NcoI (sequence
Being BspHI on row 4, both are isocaudarner) and SphI.
Described recombination bacillus coli A or the described recombination bacillus coli B utilizing described method to prepare falls within this
Bright protection domain.
Described recombination bacillus coli A or described recombination bacillus coli B is utilizing Pfansteihl or its salt synthesis S-1,2-the third two
Application in alcohol falls within protection scope of the present invention.
It is a further object to provide a kind of method utilizing Pfansteihl or its salt synthesis S-1,2-propylene glycol.
The method utilizing Pfansteihl or its salt synthesis S-1,2-propylene glycol provided by the present invention, can be as follows (A) or
(B):
(A) comprise the steps: to add the L-of final concentration of 200mM in the cultivating system of described recombination bacillus coli B
Lactic acid or its salt, as substrate, carry out fermentation culture in 37 DEG C, obtain S-1,2-propylene glycol from culture fluid；
(B) in the cultivating system of described recombination bacillus coli A, add Pfansteihl or its salt conduct of final concentration of 200mM
Substrate, cultivates between OD600 to 0.6-0.8 in 37 DEG C, adds the L-arabinose of final concentration of 2g/L as derivant, in
37 DEG C carry out fermentation culture, obtain S-1,2-propylene glycol from culture fluid.
In the process, in the cultivating system of described recombination bacillus coli A or described recombination bacillus coli B, culture medium
Carbon source be glucose, its content is 10g/L.
In the process, the training method of described fermentation culture can be shaken cultivation, and rotating speed can be 180rpm；Time is
24 hours.
In the process, recombination bacillus coli A's or described recombination bacillus coli B described in described cultivating system is initial
OD600 is 0.1.
It is demonstrated experimentally that the present invention reacts the one of path by Red recombinant means to affecting Pfansteihl to S-1,2-propylene glycol
A little genes have carried out the work of gene knockout, are inserted in colibacillary genome by the gene of three key enzymes simultaneously,
Utilize T5 promoter to express to it, and proceeded to the encoding gene of pyruvic carboxylase by recombinant expression carrier
The encoding gene (mhpf gene) of the acetaldehyde coa dehydrogenase of (ZpPDC gene) and NAD dependent form carries out reducing power to bacterial strain
Balance (the namely covering method of NADH), is finally obtained and can be converted into S-1,2-propylene glycol with biologically-derived Pfansteihl
Metabolic engineering bacterial strain.This recombination bacillus coli is with 200mM sodium lactate as substrate, and 37 DEG C of cultivations can produce 3.4mM in 24 hours
S-1,2-propylene glycol.The present invention is that base has been laid in the work of the yield and conversion ratio that improve biosynthesis S-1,2-propylene glycol
Plinth.
Accompanying drawing explanation
Fig. 1 is that the PCR of recombination bacillus coli BWPDO1 identifies.Wherein, A is for replacing upper mmsB while knocking out lldD gene
Gene；B is for replacing upper pdcD gene while knocking out adhE gene；C is for replacing upper pct base while knocking out ackA-pta gene
Cause.In A-C, 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 the result that recombination bacillus coli BWPDO1 utilizes Pfansteihl synthesis S-1,2-propylene glycol.
Fig. 3 is that the PCR of recombination bacillus coli BWPDO2 identifies.Wherein, A is for knocking out dld gene；B is for knocking out ldhA gene.
In A and B, 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 the result that recombination bacillus coli BWPDO2 utilizes Pfansteihl synthesis S-1,2-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 the result that recombination bacillus coli BWPDO3 utilizes Pfansteihl synthesis S-1,2-propylene glycol.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
PBAD43 carrier: Youbio Products, its catalog number is VT1293.
PKD46 plasmid (GenBank ID:AY048746.1), pCP20 plasmid (GenBank ID:HB393402.1):
ATCC (American Type Culture Collecti).List of references: 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.
Escherichia coli BW25113 wild type: Coli Genetics Stock Center, its catalog number is CGSC#
7636。
Escherichia coli BW25113-△ poxB: knocked out poxB gene (sequence 18) institute by escherichia coli BW25113 wild type
, concrete operations are as follows:
1, the DNA fragmentation shown in artificial synthesized sequence 17: 1-59 position is escherichia coli BW25113 wild type self
The upstream sequence (i.e. upstream homology arm) of poxB gene；80-113 position is FRT site sequence；488-1282 position is that Kan resists
Property gene；1473-1506 position is FRT site sequence；1527-1585 position is escherichia coli BW25113 wild type self
Downstream sequence (i.e. downstream homology arm) this DNA fragmentation of poxB gene is for knocking out escherichia coli BW25113 wild type self
PoxB gene.
2, carry out in accordance with the following steps
(1) with reference to the method described in (1) in embodiment 1 step one 1, to escherichia coli BW25113 wild type transfer
Enter pKD46 plasmid and prepare competence, thus obtaining the sense of the recombination bacillus coli BW25113 wild type containing pKD46 plasmid
By state cell.
(2) with reference to the method described in (2) in embodiment 1 step one 1, by the DNA fragmentation of above step 1 synthetic
Proceed to the competent cell of recombination bacillus coli BW25113 wild type containing pKD46 plasmid.
(3) resistance screening and PCR identify
Recombinant bacterium after step (2) electricity is turned DNA fragmentation coats the monoclonal that Kan resistant panel grows, and carries out PCR inspection
Survey.With the monoclonal chosen as template, carry out PCR amplification with primer poxB-F and poxB-R, select that empirical tests is correct (to be obtained big
The little band being about 1720bp mesh) clone carry out continuing streak culture.
PoxB-F:5 '-ATTAACGGTAGGGTCGTCTCCG-3 '；
PoxB-R:5 '-TCATCGGGCTATTTAACCGTTAGTG-3 '.
(4) pCP20 Plasmid elimination resistance is utilized
Carry out with reference to the method described in (4) in embodiment 1 step one 1.For the monoclonal of picking, with primer
PoxB-F and poxB-R carries out PCR amplification, selects the clone of empirical tests correct (obtaining the band of size about 327bp mesh) to continue
Continuous streak culture.
(5) step (4) is identified that positive clone is inoculated in LB fluid medium, 42 DEG C of cultivations, pass on twice, draw
Monoclonal, to remove pCP20 plasmid, last simultaneously at ammonia benzyl resistance and the flat lining out of nonreactive, ammonia benzyl flat board can not grow
Bacterial strain, for knocking out successful nonreactive deletant, is escherichia coli BW25113-△ poxB.
Escherichia coli BW25113-△ poxB is entered by the present inventor further with primer poxB-F and poxB-R
Row examining order, determines the correctness of inserted gene order.
Result shows, uses primer poxB-F and poxB-R to check order escherichia coli BW25113-△ poxB, order-checking
Result display escherichia coli BW25113-△ poxB genome portion of sequence 17 in corresponding sequence table has lacked sequence 17
80-1472 position, it is achieved that knocking out of poxB gene.
Embodiment 1, the structure utilizing the recombination bacillus coli of Pfansteihl synthesis S-1,2-propylene glycol and Function Identification
One, the structure of recombination bacillus coli BWPDO1 and Function Identification
1, the structure of recombination bacillus coli BWPDO1
Three key enzymes participating in catalysis are determined: be third respectively according to the approach utilizing Pfansteihl synthesis S-1,2-propylene glycol
Acyl coenzyme A transferring enzyme (Pct, aminoacid sequence is sequence 14) catalysis is relied on by the reaction of Pfansteihl to L-lactyl-CoA A, coenzyme A
Type succinic semialdehyde dehydrogenase (PdcD, aminoacid sequence is sequence 13) is catalyzed by L-lactyl-CoA A to L-lactic aldehyde approach and 3-
Hydroxymalonate dehydrogenase (MmsB, aminoacid sequence is sequence 12) catalysis L-lactic aldehyde reaction ultimately generates S-1,2-propylene glycol.
Mentality of designing: replace while knocking out the lldD gene (sequence 5) of escherichia coli BW25113-△ poxB self
MmsB gene, replaces upper pdcD gene while knocking out the adhE gene (sequence 6) of escherichia coli BW25113-△ poxB self,
Upper pct gene is replaced while knocking out the ackA-pta gene (sequence 7) of escherichia coli BW25113-△ poxB self.And will
Three key gene pct, pdcD and mmsB genes use T5 promoter to control to express, and are followed by SD sequence in T5 promoter
Row.
According to as above mentality of designing, according to Red recombination system operation principle, synthetic contains homology arm as follows and is used for striking
Three DNA fragmentations except replacing:
DNA fragmentation A: its nucleotide sequence is as shown in sequence 1 in sequence table.Wherein, 1-40 position is escherichia coli
The upstream sequence (i.e. upstream homology arm) of the lldD gene of BW25113-△ poxB self；41-100 position is T5 promoter sequence
Row；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 escherichia coli
The downstream sequence (i.e. downstream homology arm) of the lldD gene of BW25113-△ poxB self.This DNA fragmentation A is used for knocking out large intestine bar
The lldD gene of bacterium BW25113-△ poxB self replaces mmsB gene simultaneously.
DNA fragmentation B: its nucleotide sequence is as shown in sequence 2 in sequence table.Wherein, 1-250 position is escherichia coli
The upstream sequence (i.e. upstream homology arm) of the adhE gene of BW25113-△ poxB self；251-310 position is T5 promoter sequence
Row；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 escherichia coli
The downstream sequence (i.e. downstream homology arm) of the adhE gene of BW25113-△ poxB self.This DNA fragmentation B is used for knocking out large intestine bar
The adhE gene of bacterium BW25113-△ poxB self replaces pdcD gene simultaneously.
DNA fragmentation C: its nucleotide sequence is as shown in sequence 3 in sequence table.Wherein, 1-250 position is escherichia coli
The upstream sequence (i.e. upstream homology arm) of the ackA-pta gene of BW25113-△ poxB self；251-310 position is that T5 starts
Subsequence；311-325 position is SD sequence；326-1900 position is pct gene order；1922-1955 position is FRT site sequence
Row；2330-3124 position is Kan resistant gene；3315-3348 position is FRT site sequence；3349-3598 position is large intestine
The downstream sequence (i.e. downstream homology arm) of the ackA-pta gene of bacillus BW25113-△ poxB self.This DNA fragmentation C is used for striking
Except the ackA-pta gene of escherichia coli BW25113-△ poxB self replaces upper pct gene simultaneously.
Three above DNA fragmentation (DNA fragmentation A, DNA fragmentation B and DNA fragmentation C) is proceeded to large intestine by the way of electricity turns
The competent cell of bacillus BW25113-△ poxB, experiment proceeds in three times, carries out successively, thus realizes knocking out of genes of interest
With replacement.By DNA fragmentation A proceed to experiment as a example by illustrate to specifically comprise the following steps that
(1) in pKD46 Plastid transformation to the competent cell of escherichia coli BW25113-△ poxB, 30 DEG C of shaking tables
180rpm, incubated overnight.Culture fluid is inoculated into LB new for 100mL in the ratio of inoculum concentration 1% (volume fraction) and cultivates by next day
In the conical flask of base (100 μ g/mL ampicillin), 30 DEG C of shaking table 180rpm, cultivate；Final concentration 0.2% is added during OD=0.1
(0.2g/100mL) L-arabinose；When OD=0.6-0.7, bacterium solution is distributed into 50mL/ pipe, is placed in cooled on ice to 4 DEG C
Below；6 DEG C, 4000rpm, centrifugal 10min, abandon supernatant, collect thalline；Often to add 25mL10% (volume fraction) aseptic pre-cooling sweet for pipe
Oil, suspends gently, 4 DEG C, 4000rpm, centrifugal 10min, abandons supernatant；Often to add 15mL10% (volume fraction) aseptic pre-cooling sweet for pipe
Oil, suspends gently, 4 DEG C, 4000rpm, centrifugal 10min, abandons supernatant；Often pipe adds 2mL10% (volume fraction) aseptic pre-cooling glycerol,
Suspend gently, 4 DEG C, 4000rpm, centrifugal 10min, abandon supernatant；Often pipe adds 200 μ L aseptic pre-cooling glycerol, suspends gently, is distributed into
50 μ L/ pipes.Put into-80 DEG C frozen standby.The competence obtaining escherichia coli BW25113-△ poxB containing pKD46 plasmid is thin
Born of the same parents.
(2) impression of step (1) preparation escherichia coli BW25113-△ poxB containing pKD46 plasmid is taken out from-80 DEG C
State cell, puts and thaws on ice, electric shock cup (1mm slit) dry rearmounted on ice；Take 200ng DNA fragmentation (DNA sheet achieved above
Section A), join in competence, gentle mixing, it is added in the pole cup of pre-cooling；1800V shocks by electricity, 6ms, adds 1mL LB immediately
Culture medium；Being taken out by liquid puts in EP pipe, 150rpm constant temperature culture 1h；Take 200 μ L to be applied to containing card that antibiotic (35 μ g/
ML) LB flat board；It is inverted flat board, incubated overnight in constant incubator.
(3) resistance screening and PCR identify
Coat, after step (2) electricity is turned DNA fragmentation A, the monoclonal that Kan resistant panel grows, carry out PCR detection.To choose
The monoclonal gone out is template, with table 1 is given numbered 1 primer to carrying out PCR amplification respectively, select empirical tests correct
Clone carries out continuing streak culture.PCR amplification arranges the escherichia coli BW25113-△ poxB work not proceeding to DNA fragmentation A simultaneously
For wild type control (WT).The theoretical length of amplified production is shown in Table 1 (wild type and saltant type KmR)。
Table 1 gene knockout checking primer and theoretical amplification length
(4) eliminate pKD46 plasmid and utilize pCP20 Plasmid elimination resistance
Picking monoclonal, 42 DEG C of cultivations in test tube, continue 1% (volume fraction) and are forwarded to newly after 180rpm, 4-6 hour
LB culture medium continues cultivate 6-8 hour.On the flat board of final concentration of 50 μ g/mL kanamycin, line, it is placed in 42 DEG C, mistake
Night cultivates.Rear picking monoclonal, respectively at ammonia benzyl resistance and the flat lining out of Ka Na resistance, selects ammonia benzyl flat board length but card
The bacterium colony of that flat board length, is used for preparing electricity and turns competence.PCP20 plasmid is proceeded to after preparation competence.Prepare electricity turn competence and
The method converted is ibid.It is coated with the flat board of ammonia benzyl resistance after converting plasmid pCP20, is placed in overnight incubation in 30 DEG C of incubators.Picking
Monoclonal, utilizes the checking primer of in table 1 numbered 1 to carry out PCR amplification, and the clone selecting empirical tests correct carries out continuing to draw
Line is cultivated.PCR amplification arranges escherichia coli BW25113-△ poxB as wild type control (WT) simultaneously.The theory of amplified production
Length is shown in Table 1 (wild type and saltant type KmS)。
(5) step (4) is identified that correct positive colony is inoculated in LB fluid medium, 42 DEG C of cultivations, pass on two
Secondary, draw monoclonal, to remove pCP20 plasmid, last simultaneously at ammonia benzyl resistance and the flat lining out of nonreactive, ammonia benzyl flat board can not be given birth to
Long bacterial strain is for knocking out successful nonreactive deletant.
Complete lldD gene through above step (1)-(5) knocks out and inserts mmsB gene；Then start to continue to complete
Become and the recombinant bacterium knocking out and inserting mmsB gene of lldD gene proceeds to pKD46 plasmid, continue to make competence, i.e. with reference to upper
State step (1)-(5) and proceed to DNA fragmentation B, checking, complete adhE gene and replace with pdcD gene；Then proceed to reference to above-mentioned step
Suddenly (1)-(5) proceed to DNA fragmentation C in the recombinant bacterium completing adhE gene and replacing with pdcD gene, complete ackA-pta gene
Replace pct gene.The recombinant bacterium named recombinant bacterium BWPDO1 that will finally obtain.
Testing result corresponding to above step (3) is as shown in swimming lane W and swimming lane 1 in Fig. 1, and in figure, swimming lane W is wild matrix
Duan great little, but swimming lane 1 is to knock out corresponding gene after exchange carries the PCR fragment size of screening resistance Kan.Can from figure
Go out, acquired results all with table 1 predicts the outcome consistent.The correct clone of empirical tests is selected to carry out continuing streak culture.
Testing result corresponding to above step (4) is as shown in swimming lane W and swimming lane 2 in Fig. 1, and in figure, swimming lane W is wild matrix
Duan great little, swimming lane 2 is the clip size after the Kan resistance utilizing pCP20 to eliminate between two FRT sites.Can from figure
Go out, acquired results all with table 1 predicts the outcome consistent.
Further with three couple of 1-3 numbered in table 1, the present inventor verifies that primer carries out examining order, determine
The correctness of inserted gene order.
Result shows, the primer of numbered 1 in employing table 1 checks order to recombinant bacterium BWPDO1, and sequencing result shows
Show that recombinant bacterium BWPDO1 genome portion of sequence 1 in corresponding sequence table has lacked the 1016-2408 position of sequence 1, it is achieved
The replacement of mmsB gene pairs lldD gene.The primer of numbered 2 in employing table 1 checks order to recombinant bacterium BWPDO1,
Sequencing result display recombinant bacterium BWPDO1 genome portion of sequence 2 in corresponding sequence table has lacked the 1736-of sequence 2
3128, it is achieved that the replacement of pdcD gene pairs adheE gene.The primer of numbered 3 in employing table 1 is to recombinant bacterium
BWPDO1 checks order, the genome portion disappearance of sequencing result display recombinant bacterium BWPDO1 sequence 3 in corresponding to sequence table
The 1922-3314 position of sequence 3, it is achieved that 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 culture medium, after be seeded in new LB culture medium with 1% (volume ratio),
180rpm, 37 DEG C, cultivates 12-16h, measures OD600, receive bacterium, 4000rpm, 4 DEG C of centrifugal 10min.With brine 2 times,
Thalline is collected by similarity condition is centrifugal.By resuspended for thalline to the PBS of pH7.2-7.4, final about OD600 to 25 to 30, add
Entering Pfansteihl sodium as substrate, final concentration is respectively 200mM and 50mM, and adds the glucose of 1% (1g/100ml), carries out not
Only cell effect, reaction temperature arranges 30 DEG C and 37 DEG C, reacts 24h.It is sampled with reaction, centrifugal, take supernatant, and survey
Determine L-lactic acid and S-1,2-propanediol.In triplicate, result takes three meansigma methodss repeated in experiment.
Result is as in figure 2 it is shown, this it appears that along with the carrying out of full cell effect from figure, under lactic acid content does not has
Fall and risen, analyzing reason is probably and does not knock out acetone acid to two gene dld and ldhA of D-ALPHA-Hydroxypropionic acid metabolism,
Owing in metabolic pathway, this approach flux is higher, so the acetone acid that 1% glucose added produces through glycolysis passes through this
Approach produces D-ALPHA-Hydroxypropionic acid, causes required generation S-1,2-propanediol pathway metabolic flux to reduce, and lactic acid pathways reaction needs
NADH, as coenzyme, causes required approach NADH offer amount to reduce, the most indirectly causes yield to decline.Later stage propanediol declines
Reason be probably cyton intracellular metabolite and in order to maintain intracellular NADH level to be caused.For solving this problem, the present invention's
What inventor had proceeded transforming protein gene ldhA and dld between acetone acid and D-ALPHA-Hydroxypropionic acid knocks out work.
Two, the structure of recombination bacillus coli BWPDO2 and Function Identification
1, the structure of recombination bacillus coli BWPDO2
According to Red recombination system operation principle, synthetic contains homology arm as follows and is used for knocking out ldhA gene (sequence 8)
Two DNA fragmentations with dld gene (sequence 9):
DNA fragmentation D: its nucleotide sequence is as shown in sequence 10 in sequence table.Wherein, 1-59 position is escherichia coli
The upstream sequence (i.e. upstream homology arm) of the ldhA gene of 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 escherichia coli
The downstream sequence (i.e. downstream homology arm) of the ldhA gene of BW25113-△ poxB self.This DNA fragmentation D is used for knocking out large intestine bar
The ldhA gene of bacterium BWPDO1 self.
DNA fragmentation E: its nucleotide sequence is as shown in sequence 11 in sequence table.Wherein, 1-59 position is escherichia coli
The upstream sequence (i.e. upstream homology arm) of the dld gene of 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 escherichia coli
The downstream sequence (i.e. downstream homology arm) of the dld gene of BW25113-△ poxB self.This DNA fragmentation E is used for knocking out large intestine bar
The dld gene of bacterium BWPDO1 self.
Two above DNA fragmentation (DNA fragmentation D and DNA fragmentation E) is proceeded to by the way of electricity turns the weight that step one builds
The competent cell of group escherichia coli BWPDO1, experiment proceeds at twice, carries out successively, thus realizes knocking out of genes of interest.
By DNA fragmentation D proceed to experiment as a example by illustrate to specifically comprise the following steps that
(1) with reference to the method described in (1) in step one 1, the recombination bacillus coli containing pKD46 plasmid is prepared
The competent cell of BWPDO1.
(2) with reference to the method described in (2) in step one 1, DNA fragmentation D achieved above is proceeded to containing pKD46 matter
The competent cell of the recombination bacillus coli BWPDO1 of grain.
(3) resistance screening and PCR identify
Coat, after step (2) electricity is turned DNA fragmentation E, the monoclonal that Kan resistant panel grows, carry out PCR detection.To choose
The monoclonal gone out is template, with table 1 is given numbered 4 primer to carrying out PCR amplification, select the correct clone of empirical tests
Carry out continuing streak culture.PCR amplification arranges the escherichia coli BW25113-△ poxB conduct not proceeding to two DNA fragmentations simultaneously
Wild type control (WT).The theoretical length of amplified production is shown in Table 1 (wild type and saltant type KmS)。
(4) pCP20 Plasmid elimination resistance is utilized
Carry out with reference to the method described in (4) in step one 1.For the monoclonal of picking, utilize in table 1 is numbered
The checking primer of 4 carries out PCR amplification, and the clone selecting empirical tests correct carries out continuing streak culture.PCR amplification is arranged not simultaneously
Proceed to the escherichia coli BW25113-△ poxB of two DNA fragmentations as wild type control (WT).The theoretical length of amplified production
It is shown in Table 1 (wild type and saltant type KmS)。
(5) step (4) is identified that correct positive colony is inoculated in LB fluid medium, 42 DEG C of cultivations, pass on two
Secondary, draw monoclonal, to remove pCP20 plasmid, last simultaneously at ammonia benzyl resistance and the flat lining out of nonreactive, ammonia benzyl flat board can not be given birth to
Long bacterial strain is for knocking out successful nonreactive deletant.
Knocking out of ldhA gene is completed through above step (1)-(5)；Then start to continue to having knocked out ldhA gene
Recombinant bacterium proceeds to pKD46 plasmid, continues to make competence, i.e. proceed to DNA fragmentation E with reference to above-mentioned steps (1)-(5), checking,
Complete knocking out of dld gene.The recombinant bacterium named recombinant bacterium BWPDO2 that will finally obtain.
Testing result corresponding to above step (3) is as shown in swimming lane W and swimming lane 1 in Fig. 3, and in figure, swimming lane W is wild matrix
Duan great little, but swimming lane 1 is to knock out corresponding gene after exchange carries the PCR fragment size of screening resistance Kan.Can from figure
Go out, acquired results all with table 1 predicts the outcome consistent.
Testing result corresponding to above step (4) is as shown in swimming lane W and swimming lane 2 in Fig. 3, and in figure, swimming lane W is wild matrix
Duan great little, swimming lane 2 is the clip size after the Kan resistance utilizing pCP20 to eliminate between two FRT sites.Can from figure
Go out, acquired results all with table 1 predicts the outcome consistent.
The present inventor carries out examining order, really further with two couples checking primer of numbered 4 and 5 in table 1
The correctness of fixed inserted gene order.
Result shows, the primer of numbered 4 in employing table 1 checks order to recombinant bacterium BWPDO2, and sequencing result shows
Show that recombinant bacterium BWPDO2 genome portion of sequence 10 in corresponding sequence table has lacked the 80-1472 position of sequence 10, it is achieved
The knocking out of ldhA gene.The primer of numbered 5 in employing table 1 checks order to recombinant bacterium BWPDO2, and sequencing result shows
Show that recombinant bacterium BWPDO2 genome portion of sequence 11 in corresponding sequence table has lacked the 80-1472 position of sequence 11, it is achieved
The knocking out of dld gene.
2, the Function Identification of recombination bacillus coli BWPDO2
BWPDO2 is accessed in LB culture medium, 180rpm, 37 DEG C of incubated overnight.After with 1% (volume ratio) inoculum concentration access
Containing different glucose initial concentrations (0.5%, 1%, 2%, unit g/100ml) and different Pfansteihl na concn (50,200mM)
M9 culture medium in (reaction initial OD 600 be about 0.1), 180rpm, 37 DEG C of cultivations, 24h.Substrate L-under the conditions of sampling and measuring is each
Sodium lactate and the content of product S-1,2-propylene glycol.In triplicate, result takes three meansigma methodss repeated in experiment.
As shown in Figure 4, different gradient glucose condition and the produced production concentration of concentration of substrate all differ result, its
In, through fermentation in 24 hours, the highest condition of yield was that under 1% glucose condition, Pfansteihl concentration of substrate is 200mM, S-1,2-the third two
Alcohol yield is about 2mM, 2.8 times of relatively resting cell system yield.
Three, the structure of recombination bacillus coli BWPDO3 and Function Identification
1, the structure of recombination bacillus coli BWPDO3
In order to improve Pfansteihl further to the conversion ratio of S-1,2-propylene glycol and yield, the present inventor is to restructuring
Escherichia coli BWPDO2 bacterial strain proceeds transformation, and finding two enzymes is from Zymobacterpalmae (Zymobacter respectively
Palmae) pyruvic carboxylase (pyruvate decarboxylase) (ZpPDC, sequence in aminoacid sequence such as sequence table
Shown in 15) and from colibacillary NAD dependent form acetaldehyde coa dehydrogenase (acetaldehyde-CoA
Dehydrogenase II, NAD-binding) (mhpf, aminoacid sequence is as shown in sequence 16 in sequence table), utilize pBAD43
Carrier carries out expressing in series to the gene of its two enzyme.
(1) synthesis ZpPDC-mhpf genetic fragment
DNA fragmentation shown in sequence 4 in artificial synthesized sequence table.Wherein, 1-6 position is the identification of restriction enzyme site BspHI
Sequence, 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) two ends synthesized with restricted enzyme BspHI and SphI double digestion step (1) are with restriction enzyme site
ZpPDC-mhpf genetic fragment, glue carries with the pBAD43 through NcoI (being isocaudarner with BspHI) and SphI double digestion after reclaiming
The skeleton large fragment of body is connected, and obtains recombiant plasmid.To show restriction enzyme site NcoI and SphI of pBAD43 carrier through order-checking
Between small fragment to replace with the recombiant plasmid obtained after DNA fragmentation shown in the 2-2671 position of sequence 4 in sequence table named
pBAD-MP。
(3) the recombinant vector pBAD-MP of step (2) gained is proceeded to by the way of electricity turns step 2 and builds the weight obtained
Group escherichia coli BWPDO2 competent cell in (concrete operations see above), picking monoclonal, use primer pBAD-F and
PBAD-R (two primer sequences are pBAD43 carrier and carry sequence) carries out PCR amplification for ZpPDC-mhpf genetic fragment.
PBAD-F:5 '-CTGTTTCTCCATACCCGTT-3 '；
PBAD-R:5 '-CTCATCCGCCAAAACAG-3 '.
By through PCR amplification obtain the monoclonal of the band (Fig. 5) that size is about 2746bp mesh use as above primer to carrying out
Order-checking.Sequencing result is shown containing the named BWPDO3 of recombinant bacterium of sequence 4 in ordered list.
2, the Function Identification of recombination bacillus coli BWPDO3
Being accessed by BWPDO3 in LB culture medium, 180rpm, 37 DEG C of incubated overnight, for kind of a daughter bacteria.After connect with 1% (volume ratio)
The amount of kind accesses in the M9 culture medium containing final concentration 1% (1g/100ml) glucose and 200mM Pfansteihl sodium (reacts initially
OD600 is about 0.1), 180rpm, 37 DEG C of cultivations, between OD600 to 0.6-0.8, add variable concentrations L-arabinose and lure
Leading, Concentraton gradient is respectively 0.05%, and 0.1%, 0.2%, 0.4% (0.05% is i.e. equivalent to 0.05g/100ml, successively class
Push away), cultivate 24h afterwards, carry out BWPDO2 simultaneously and compare with CMC model.Under the conditions of sampling and measuring is each substrate Pfansteihl sodium and
The content of product S-1,2-propylene glycol.In triplicate, result takes three meansigma methodss repeated in experiment.
Result is as shown in Figure 6, it can be seen that Pfansteihl is synthesized S-1,2-propanediol by recombination bacillus coli BWPDO3
Having risen, the suitableeest derivant addition is 0.2%, and yield is about 3.4mM, and (i.e. every liter fermentation liquid obtains 3.4mmol's
S-1,2-propylene glycol), improve about 1.5 times than recombination bacillus coli BWPDO2 yield.
Claims (6)
1. utilize the preparation method of the recombination bacillus coli A of a Pfansteihl synthesis S-1,2-propylene glycol, including following (1)-(3)
Step:
(1) by escherichia coli BW25113-△poxBInlldDGene,adhEGene andackA-ptaGene
Replace with respectively 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 obtained is designated as BWPDO1；
Described escherichia coli BW25113-△poxBFor by escherichia coli BW25113 wild typepoxBGene knockout
The bacterial strain of rear gained；
The aminoacid sequence of described 3-hydroxymalonate dehydrogenase is sequence 12 in sequence table；Described coenzyme A dependent form succinic acid half
The aminoacid sequence of aldehyde dehydrogenase is sequence 13 in sequence table；The aminoacid sequence of described propionyl-coa transferase is sequence table
Middle sequence 14；
The nucleotides sequence of the encoding gene of described 3-hydroxymalonate dehydrogenase is classified as the 116-994 position of sequence 1 in sequence table；Institute
The nucleotides sequence of the encoding gene stating coenzyme A dependent form succinic semialdehyde dehydrogenase is classified as the 326-of sequence 2 in sequence table
1714；The nucleotides sequence of the encoding gene of described propionyl-coa transferase is classified as the 326-1900 of sequence 3 in sequence table
Position；
DescribedpoxBThe nucleotides sequence of gene is classified as in sequence table shown in sequence 18；DescribedlldDThe nucleotides sequence of gene
It is classified as sequence 5 in sequence table；DescribedadhEThe nucleotides sequence of gene is classified as sequence 6 in sequence table；DescribedackA-pta
The nucleotides sequence of gene is classified as sequence 7 in sequence table；
Described escherichia coli BW25113-ΔpoxBIt is to obtain according to the method comprised the steps: to containing pKD46 matter
The competence of the described escherichia coli BW25113 wild type of grain proceeds to the DNA fragmentation 17 shown in sequence 17 in sequence table, described
DNA fragmentation 17 and the genome generation homologous recombination of described escherichia coli BW25113 wild type, i.e. realize knocking out described large intestine bar
Bacterium BW25113 wild type describedpoxBGene, it is thus achieved that described escherichia coli BW25113-ΔpoxB；
By described escherichia coli BW25113-△poxBIn describedlldDGene replaces with described 3-hydracrylic acid dehydrogenation
The encoding gene of enzyme, realizes by the following method: to the described escherichia coli BW25113-△ containing pKD46 plasmidpoxBCompetence in proceed to the DNA fragmentation 1 shown in sequence 1 in sequence table, described DNA fragmentation 1 and described escherichia coli
BW25113-△poxBGenome generation homologous recombination, i.e. realize by describedlldDGene replaces with described 3-hydroxyl
The encoding gene of propanoic acid dehydrogenase；
By described escherichia coli BW25113-△poxBIn describedadhEGene replaces with coenzyme A dependent form succinic acid
The encoding gene of semialdehyde dehydrogenase, realizes by the following method: to the described escherichia coli containing pKD46 plasmid
BW25113-△poxBCompetence in proceed to the DNA fragmentation 2 shown in sequence 2 in sequence table, described DNA fragmentation 2 is with described
Escherichia coli BW25113-△poxBGenome generation homologous recombination, i.e. realize by describedadhEGene replaces with institute
State the encoding gene of coenzyme A dependent form succinic semialdehyde dehydrogenase；
By described escherichia coli BW25113-△poxBIn describedackA-ptaGene replaces with propionyl coenzyme A
The encoding gene of transferring enzyme, realizes by the following method: to the described escherichia coli BW25113-△ containing pKD46 plasmidpoxBCompetence in proceed to the DNA fragmentation 3 shown in sequence 3 in sequence table, described DNA fragmentation 3 and described escherichia coli
BW25113-△poxBGenome generation homologous recombination, i.e. realize by describedackA-ptaGene replaces with described
The encoding gene of propionyl-coa transferase；
(2) knock out the BWPDO1's that step (1) obtainsldhAGene anddldGene, the recombinant bacterium obtained is designated as BWPDO2；
DescribedldhAThe nucleotides sequence of gene is classified as sequence 8 in sequence table；DescribeddldThe nucleotides sequence of gene is classified as sequence
Sequence 9 in table；
Knock out the described of described BWPDO1ldhAGene, realizes by the following method: to the institute containing pKD46 plasmid
State and the competence of BWPDO1 proceeds to the DNA fragmentation 10 shown in sequence 10 in sequence table, described DNA fragmentation 10 and described BWPDO1
Genome generation homologous recombination, i.e. realize knock out the described of described BWPDO1ldhAGene；
Knock out the described of described BWPDO1dldGene, realizes by the following method: to containing described in pKD46 plasmid
Proceeding to the DNA fragmentation 11 shown in sequence 11 in sequence table in the competence of BWPDO1, described DNA fragmentation 11 is with described BWPDO1's
Genome generation homologous recombination, i.e. realizes knocking out the described of described BWPDO1dldGene；
(3) in the BWPDO2 that step (2) obtains, import encoding gene and the NAD dependent form acetaldehyde coenzyme A of pyruvic carboxylase
The encoding gene of dehydrogenase, the recombinant bacterium obtained is designated as BWPDO3；Described BWPDO3 is described recombination bacillus coli A；
The aminoacid sequence of described pyruvic carboxylase is sequence 15 in sequence table；Described NAD dependent form acetaldehyde coa dehydrogenase
Aminoacid sequence be sequence 16 in sequence table；
The nucleotides sequence of the encoding gene of described pyruvic carboxylase is classified as the 1001-2671 position of sequence 4 in sequence table；Described
The nucleotides sequence of the encoding gene of NAD dependent form acetaldehyde coa dehydrogenase is classified as the 3-953 position of sequence 4 in sequence table；
The encoding gene of described pyruvic carboxylase and the encoding gene of described NAD dependent form acetaldehyde coa dehydrogenase are to pass through
The form of recombinant expression carrier imports in described BWPDO2；
Described recombinant expression carrier is specially the polyclone that DNA fragmentation shown in sequence in sequence table 4 is inserted into pBAD43 carrier
The recombiant plasmid obtained behind site.
2. utilize the preparation method of the recombination bacillus coli B of a Pfansteihl synthesis S-1,2-propylene glycol, including in claim 1
Step (1) and step (2)；The described BWPDO2 obtained in described step (2) is described recombination bacillus coli B.
3. utilize the described recombination bacillus coli A that method described in claim 1 prepares.
4. utilize the described recombination bacillus coli B that method described in claim 2 prepares.
5. the recombination bacillus coli A described in the claim 3 or recombination bacillus coli B described in claim 4 is utilizing Pfansteihl
Or the application in its salt synthesis S-1,2-propylene glycol.
6. utilize Pfansteihl or the method for its salt synthesis S-1,2-propylene glycol, for as follows (A) or (B):
(A) comprise the steps: to add in the cultivating system of the recombination bacillus coli B described in claim 4 final concentration of
The Pfansteihl of 200mM or its salt, as substrate, carry out fermentation culture in 37 DEG C, obtain S-1,2-propylene glycol from culture fluid；
(B) add in the cultivating system of the recombination bacillus coli A described in claim 3 final concentration of 200mM Pfansteihl or
Its salt, as substrate, is cultivated to OD in 37 DEG C600Between 0.6-0.8, add the L-arabinose of final concentration of 2g/L as luring
Lead agent, carry out fermentation culture in 37 DEG C, from culture fluid, obtain S-1,2-propylene glycol.
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