CN116064633A - Construction of efficient biosynthesis of vitamin K 2 Engineering bacteria method - Google Patents
Construction of efficient biosynthesis of vitamin K 2 Engineering bacteria method Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a method for constructing efficient biosynthesis of vitamin K 2 The engineering bacteria method comprises the following steps: step one, constructing and obtaining a delta pfue strain EC1; step two, using the whole genome of the strain EC1 as a template and using P degQ The promoter sequence replaces the original promoter of the bacillus subtilis farnesyl diphosphate synthase gene ispA to obtain a strain EC2; step three, taking the whole genome of the strain EC2 as a template, knocking out the 1-deoxyxylulose-5-phosphate synthase gene dxs, and obtaining the strain EC3; step four, using Klebsiella complete genesThe group is used as a template to amplify and obtain dxs gene by the method of pHY-P 43 The vector ligation was transformed into strain EC3 to give EC4. According to the invention, the bacillus subtilis with high MK-7 yield is preferably constructed through promoter replacement and key genes, and the MK-7 yield of the final strain EC4 is improved by 5.42 times compared with that of the original strain.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method for constructing efficient biosynthesis of vitamin K 2 The engineering bacteria method.
Background
Vitamin K 2 Is a fat-soluble vitamin, is a menaquinone series compound, and the structure of the fat-soluble vitamin is composed of a 2-methyl-1, 4-naphthoquinone mother nucleus and isoprene side chains with different lengths. The number of the side chains of the isoprene at the C-3 position on the molecular structure of the compound can be divided into different isomers. Wherein menaquinone-7 (MK-7) containing seven isoprene side chains is vitamin K 2 The structure form with the highest activity.
Vitamin K at present 2 Mainly uses chemical synthesis, but the traditional chemical synthesis method has the problems of limited sources of chemical raw materials, large amount of isomers produced by chemical reaction, more byproducts, low yield, environmental pollution and the like, and the synthesized vitamin K 2 The side chain of the isoprene is in a cis structure, and the activity is low. Thus, vitamin K is industrially produced by microbial fermentation 2 Is favored. The method has the greatest advantages that: can greatly simplify the production process, improve the labor condition, reduce the environmental pollution and simultaneously is beneficial to the development and the comprehensive utilization of resources. However, the existing strain is fermented to produce vitamin K 2 The yield of (2) is relatively low, the fermentation level is relatively low, and the cost is high, so that the fermentation is difficult to be accepted by common people. Therefore, excellent strains are bred efficiently, and the production of vitamin K by fermentation is improved 2 For preventing and treating osteoporosisThe diseases such as pine disease, cardiovascular diseases, arterial calcification and the like have important theoretical and application values.
Bacillus subtilis is one of the 40 edible probiotics approved by the FDA. Due to its high growth rate, menaquinone is high in content and has passed safety certification, it is considered to be the most potential strain for industrial production of menaquinone. The synthetic biology adopts the knowledge and materials obtained by the traditional biology as the basis, utilizes the systematic biological means to quantitatively analyze the metabolic products, designs a new biological system or deeply reforms the original biological system under the guidance of engineering and computer assistance, constructs a reconstructed biochemical synthesis network of a microbial cell factory or assembles an artificial metabolic pathway, and can realize the biosynthesis of important chemicals such as artemisinin, opium and the like. Vitamin K 2 The synthesis in bacillus subtilis is very challenging but at the same time quite pioneering, due to the about 38-step enzyme-catalyzed reaction involving 4 metabolic modules, and the presence of toxic intermediates and competing and inhibitory metabolic pathways, the adoption of synthetic biological methods to preferentially metabolize key enzymes and modulate the appropriate activities of key enzymes.
Disclosure of Invention
The present invention has for its object to propose a method for constructing a highly efficient biosynthesis of vitamin K 2 The engineering bacteria method aims at solving the problem that the existing biological method causes vitamin K due to the existence of toxic intermediates and competitive and inhibitory metabolic pathways 2 The yield is not high.
Based on the above object, the present invention provides a method for constructing a highly efficient biosynthesis of vitamin K 2 The engineering bacteria method comprises the following steps:
step one, taking a bacillus subtilis 168 genome as a template, knocking out a purine transporter gene pfuE, and constructing a delta pfuE strain EC1;
step two, using the whole genome of the strain EC1 as a template and using P degQ The promoter sequence replaces the original promoter of the bacillus subtilis farnesyl diphosphate synthase gene ispA to obtain a strain EC2;
step three, taking the whole genome of the strain EC2 as a template, knocking out the 1-deoxyxylulose-5-phosphate synthase gene dxs, and obtaining the strain EC3;
step four, using the whole genome of Klebsiella as a template, amplifying to obtain dxs gene, and performing pHY-P reaction 43 Vector ligation into strain EC3 to obtain vitamin K for biosynthesis 2 EC4 of the strain.
Preferably, the method for constructing the delta pbuE strain EC1 in the first step comprises the following steps:
a1, using a bacillus subtilis 168 genome as a template, and adopting a primer group shown as SEQ ID NO.1-6 to amplify to obtain a left homology arm, a right homology arm sequence and an intermediate fragment;
a2, performing overlap extension PCR on the obtained left homologous arm, right homologous arm sequences and intermediate fragments to obtain fusion gene fragments;
and A3, after purifying the amplified product, electrically transferring the fusion gene fragment into competent cells of the bacillus subtilis 168 to obtain the strain EC1.
Preferably, the method for obtaining the strain EC2 in the second step comprises the following steps:
b1, using bacillus subtilis 168 genome as a template, and amplifying by using a primer shown as SEQ ID NO.7-8 to obtain a left homology arm and P of the ispA1 original promoter degQ A sequence and a right homology arm sequence;
b2, using plasmid p7C6 as template, amplifying with primer shown as SEQ ID NO.25-26 to obtain chloramphenicol resistance gene Cm R A sequence;
b3, the sequence obtained in B1 and the chloramphenicol resistance gene Cm obtained in B2 R And (3) carrying out sequence row overlapping extension PCR, purifying the obtained fusion gene fragment by using an amplification product, and electrically transferring the fusion gene fragment into competent cells of the EC1 strain to obtain strains EC2, EC21 and EC22.
Preferably, the method for obtaining the strain EC3 in the third step comprises the following steps:
c1, using the whole genome of the strain EC2 as a template, and adopting primers shown as SEQ ID NO.27-32 to amplify to obtain left and right homologous arm sequences and intermediate fragments;
c2, performing overlap extension PCR on the obtained left homologous arm, right homologous arm sequences and intermediate fragments to obtain fusion gene fragments;
and C3, purifying the amplified product, and electrically transferring the fusion gene fragment into competent cells of the strain EC2 to obtain the strain EC3.
Preferably, the method for obtaining the strain EC4 in the fourth step comprises the following steps:
d1, culturing klebsiella to an exponential growth medium phase, and extracting whole genome DNA;
d2, using the whole genome of Klebsiella as a template, and adopting a primer shown as SEQ ID NO.33-34 to amplify to obtain dxs gene fragments;
d3, after purification of amplified product, constructing recombinant plasmid pHY-P 43 The dxs1 is transformed into strain EC3, giving strain EC4.
Preferably, the construction of the recombinant plasmid pHY-P 43 The method of-dxs 1 is to use XbaI and BglII to amplify the product and carrier pHY-P 43 Double enzyme digestion is carried out, and the recovered products of the double enzyme digestion and the recovery products are connected by T4 ligase to obtain the double enzyme digestion.
Preferably, the amplification conditions are: denaturation at 98℃for 3min; then denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃for 20s for a total of 34 cycles; finally, the extension is carried out for 5min at 72 ℃.
The invention has the beneficial effects that: according to the invention, a synthetic biology method is adopted, bacillus subtilis 168is taken as a mode microorganism, firstly, an enzyme gene which does not influence the growth of thalli and reduces energy and nutrient component diversion is knocked out, secondly, a promoter with appropriate strength for ispA transcription of a bacillus subtilis farnesyl diphosphate synthase coding gene is optimized, finally, according to computer simulation and structural analysis, a klebsiella 1-deoxyxylulose-5-phosphate synthase coding gene dxs is optimized, the dxs of an original strain is replaced, and the MK-7 yield is successfully increased to 5.42 times of the original strain.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only of the invention and that other drawings can be obtained from them without inventive effort for a person skilled in the art.
FIG. 1 shows the verification of PCR products by agarose gel electrophoresis during the construction of EC1; lanes 1,2: a left homology arm segment; 3,4: chloramphenicol resistance fragments; 5,6: a right homology arm segment; m is marker;
FIG. 2 shows agarose gel electrophoresis of the recovered fragments of the validation gel during the construction of EC1; lanes 1,2,3: recovering the fragments by using glue; m is marker;
FIG. 3 shows the verification of PCR products by agarose gel electrophoresis during the construction of EC2; lanes 1,2: a left homology arm segment; 3,4: chloramphenicol resistance fragments; 5,6: a right homology arm segment; m is marker;
FIG. 4 shows agarose gel electrophoresis of the recovered fragments of the validation gel during the construction of EC2; lanes 1,2,3: recovering the fragments by using glue; m is marker;
FIG. 5 shows the verification of PCR products by agarose gel electrophoresis during the construction of EC3; lanes 1,2: a left homology arm segment; 3,4: chloramphenicol resistance fragments; 5,6: a right homology arm segment; 7,8: p (P) degQ Fragments;
FIG. 6 shows agarose gel electrophoresis of the recovered fragments of the validation gel during the construction of EC3; lanes 5,6: recovering the fragments by using glue; m is marker;
FIG. 7 is a liquid chromatography detection pattern of MK-7 produced by an original strain;
FIG. 8is a liquid chromatography detection pattern of MK-7 produced by EC4.
Detailed Description
The present invention will be further described in detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent.
It is to be noted that unless otherwise defined, technical or scientific terms used herein should be taken in a general sense as understood by one of ordinary skill in the art to which the present invention belongs.
Examples
Construction of recombinant bacterium EC1
(1) The bacillus subtilis 168 genome is used as a template, and primers pfuE-L-F (primer 1) and pfuE-L-R (primer 2), pfuE-p 7C6-F (primer 3) and pfuE-p 7C6-R (primer 4), pfuE-R-F (primer 5) and pfuE-R-R (primer 6) are respectively adopted for amplification to obtain a left homology arm, a chloramphenicol resistance fragment and a right homology arm sequence. Primers 1 to 6 are shown in Table 1.
TABLE 1 primer sequence listing
The PCR amplification systems of the left and right homology arms are as follows: ddH 2 O10 uL, whole genome template 0.5uL, upstream and downstream primers 1.0uL,Primer star 12.5uL each. PCR amplification conditions: denaturation at 98℃for 3min,34 cycles (98℃for 10s,55℃for 5s,72℃for 10 s) and finally extension at 72℃for 5min.
Intermediate fragment PCR amplification conditions: denaturation at 98℃for 3min,34 cycles (98℃for 10s,55℃for 5s,72℃for 12 s) and finally extension at 72℃for 5min.
(2) Performing overlap extension PCR on the three gene fragments obtained in the step (1), wherein the PCR conditions are as follows: pre-denaturing at 98 ℃ for 5min, then denaturing for 10s at 98 ℃, annealing for 5s at 55 ℃ and extending for 32s at 72 ℃, performing gel cutting for 34 cycles in total, and recovering fragments with correct sizes to obtain fusion gene fragments. The electrophoresis diagrams of the recovered fragments of the PCR products and the DNA fragment recovery kit are shown in figures 1 and 2.
After the target gene band amplified product is purified, the fusion gene fragment is electrically transferred into competent cells of the strain bacillus subtilis 168, a single colony grows on a flat plate, the single colony is selected for liquid culture, and the delta pbuE strain, namely the strain EC1, is obtained through PCR bacterial liquid verification.
Construction of recombinant bacterium EC2
TABLE 2 primer sequence listing
Based on the obtained strain EC1, P was used degQ 、P 43 、P hag The specific construction process of the promoter for replacing the original promoter of the ispA gene of bacillus subtilis is as follows:
the position of the bacillus subtilis 168IspA coding gene on the whole genome and the 1000bp upstream gene sequence are found out through NCBI online websites, and the promoter position of the IspA gene is predicted by using a Softberry website. The Bacillus subtilis 168 genome is used as a template, and the primers 7-12 in the table 2 are used for amplification to obtain the left homology arm and P of the ispA1 original promoter degQ A sequence and a right homology arm sequence; amplification by primer 13-18 to obtain left homology arm and P of ispA2 original promoter 43 A sequence and a right homology arm sequence; amplification with primers 19-24 to give the left homology arm, P, of ispA3 original promoter hag Sequences and right homology arm sequences.
The chloramphenicol resistance gene Cm is obtained by using the plasmid p7C6 as a template and using the primers 25-26 for amplification R Sequence. And (3) after the obtained PCR product is identified to be correct through agarose gel electrophoresis, the target band is recovered through gel.
Left and right homology arms and P degQ The PCR amplification system is as follows: ddH 2 O10 uL, bacillus subtilis 168 whole genome template 0.5uL, upstream and downstream primers 1.0uL,Primer star 12.5uL each. PCR amplification conditions: denaturation at 98℃for 3min,34 cycles (98℃for 10s,58℃for 5s,72℃for 10 s) and finally extension at 72℃for 5min. P (P) 43 And P hag The amplification method is the same.
Middle segment Cm R The amplification system is as follows: ddH 2 O10 uL, p7C6 plasmid template 0.5uL, upstream and downstream primers 1.0uL,Primer star 12.5uL each. PCR amplification conditions: denaturation at 98℃for 3min,34 cycles (98℃for 10s,53℃for 5s,72℃for 15 s) and finally extension at 72℃for 5min.
Overlapping extension PCR is carried out on the four gene fragments obtained in the steps, and PCR conditions are as follows: pre-denaturation at 98℃for 5min, denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃for 35s, totalAnd (3) carrying out 34 cycles in total, and cutting gel to recover fragments with correct sizes to obtain fusion gene fragments. Left homology arm of ispA1 original promoter, P degQ Sequence, right homology arm sequence PCR product, middle fragment Cm R And DNA fragment recovery kit recovery fragment electrophoresis diagrams are shown in FIG. 3 and FIG. 4.
After the target gene band amplified product is purified, the fusion gene fragment is electrically transferred into competent cells of the EC1 strain, a single colony grows on a flat plate, the single colony is selected for liquid culture, and PCR bacterial liquid is verified to obtain the target strain, and finally the strain Bacillus subtilis, delta pfue P is obtained degQ ispA. Strain Bacillus subtilis Δpfue P 43 ispA and Δpfue P hag The ispA construction method is as above, and is designated EC2, EC21 and EC22, respectively.
Construction of recombinant bacterium EC3
TABLE 3 primer sequence listing
| Primer(s) | Sequence (5 '-3') | Numbering device |
| dxs-L-F | GTCTCCTCCCGTGATTGG | SEQ ID NO.27 |
| dxs-L-R | CCCGGGTCGTCAAAGAAAGAACGATTAGATGT | SEQ ID NO.28 |
| dxs-p7C6-F | GTTCTTTCTTTGACGACCCGGGGATCCTCT | SEQ ID NO.29 |
| dxs-p7C6-R | AGTTGATCCGCTGTTCAAGCGAAAACATACCAC | SEQ ID NO.30 |
| dxs-R-F | GTTTTCGCTTGAACAGCGGATCAACTCACTTTCA | SEQ ID NO.31 |
| dxs-R-R | TGCCGGACTCATTAAAGAAATCTAT | SEQ ID NO.32 |
The primers dxs-L-F (SEQ ID NO. 27) and dxs-L-R (SEQ ID NO. 28), dxs-p7C6-F (SEQ ID NO. 29) and dxs-p7C6-R (SEQ ID NO. 30), dxs-R-F (SEQ ID NO. 31) and dxs-R-R (SEQ ID NO. 32) in the above Table 3 were used as templates for amplification to obtain the right and left homology arm sequences and chloramphenicol resistance fragments, respectively.
The PCR amplification systems of the left and right homology arms are as follows: ddH 2 O10 uL, bacillus subtilis 168 whole genome template 0.5uL, upstream and downstream primers 1.0uL,Primer star 12.5uL each. PCR amplification conditions: denaturation at 98℃for 3min,34 cycles (98℃for 10s,58℃for 5s,72℃for 10 s) and finally extension at 72℃for 5min.
Middle segment Cm R The amplification system is as follows: ddH 2 O10 uL, p7C6 plasmid template 0.5uL, upstream and downstream primers 1.0uL,Primer star 12.5uL each. PCR amplification conditions: denaturation at 98℃for 3min,34 cycles (98℃for 10s,53℃for 5s,72℃for 15 s) and finally extension at 72℃for 5min.
Overlapping extension PCR is carried out on the three gene fragments obtained in the steps, and the PCR conditions are as follows: pre-denaturing at 98 ℃ for 5min, then denaturing for 10s at 98 ℃, annealing for 5s at 55 ℃ and extending for 32s at 72 ℃, performing gel cutting for 34 cycles in total, and recovering fragments with correct sizes to obtain fusion gene fragments. The electrophoresis patterns of the recovered fragments of the PCR products and the DNA fragment recovery kit are shown in FIG. 5 and FIG. 6.
And (3) purifying the target gene band amplified product, electrically transferring the fusion gene fragment into competent cells of the strain EC2, and after single colony grows on a plate, picking the single colony for liquid culture, and verifying the PCR bacterial liquid to obtain the strain EC 2-delta dxs, namely the strain EC3.
Construction of recombinant bacterium EC4
TABLE 4 primer sequence listing
| Primer(s) | Sequence (5 '-3') | Numbering device |
| Dxs1-F | GCTCTAGAGATTGTACCGTTCGTATAGCATAC | SEQ ID NO.33 |
| Dxs1-R | GAAGATCTAAATCAAGGCCTGGCTGGCATAA | SEQ ID NO.34 |
| Dxs2-F | GCTCTAGAAACTGACAAACATCACCCTC | SEQ ID NO.35 |
| Dxs2-R | GAAGATCTGGCTTCCATACCAGCGGCAT | SEQ ID NO.36 |
| Dxs3-F | GCTCTAGAGGCTGTTTGCGTTCTTG | SEQ ID NO.37 |
| Dxs3-R | GAAGATCTGCCCTAGACGCCATCAA | SEQ ID NO.38 |
Based on the obtained strain EC3, P was used 43 The promoter is used for over-expressing 1-deoxyxylulose-5-phosphate synthase gene dxs on chromosome of Klebsiella Klebsiella variicola, escherichia coli and bifidobacterium Bifidobacterium longum, and the specific construction process is as follows:
klebsiella variicola was cultured to the medium-term of exponential growth, 3mL of the bacterial liquid was centrifuged at 10,000Xg for 5min, and the supernatant was discarded, and genomic DNA was extracted according to the kit instructions. The promoter positions of dxs genes were predicted using the Softberry website, respectively, using P as templates for the complete genomes of Klebsiella variicola, escherichia coli and Bifidobacterium longum 43 Promoter replaces the original promoter of dxs gene, pHY plasmid carries P 43 A promoter portion. By using primers 33 and 34, 35 and 36, 37 and 38, wherein the upstream primer cleavage site is XbaI (underlined in dxs-F) and the downstream primer cleavage site is BglII (underlined in dxs-R). PCR amplification conditions: denaturation at 98℃for 3min,34 cycles (98℃for 10s,55℃for 5s,72℃for 20 s) and finally extension at 72℃for 5min, and amplification to obtain dxs gene fragment. The PCR products obtained were identified by agarose gel electrophoresis.
After purification of the amplified product, the PCR product and the vector pHY-P were subjected to XbaI and BglII 43 Double enzyme digestion is carried out, and the recovered products of the double enzyme digestion and the recovery products are connected for 16 hours at the temperature of 4 ℃ by using T4 ligase. Recombinant plasmid pHY-P 43 Transformation of dxs into EC3, obtaining positive clones by screening and identification, and finally obtaining strain EC3-P 43 -dxs1，EC3-P 43 -dxs2 and EC3-P 43 -dxs3 to hit itNamed EC4, EC41 and EC42.
TABLE 5 genotype of strains
| Strain | Features (e.g. a character) |
| Bacillus subtilis | Bacillus subtilis 168 |
| EC1 | Bacillus subtilis,ΔpbuE |
| EC2 | Bacillus subtilis,ΔpbuE P degQ -ispA |
| EC21 | Bacillus subtilis,ΔpbuE P 43 -ispA |
| EC22 | Bacillus subtilis,ΔpbuE P hag -ispA |
| EC3 | Bacillus subtilis,ΔpbuE P degQ -ispAΔdxs |
| EC4 | Bacillus subtilis,ΔpbuE P degQ -ispAΔdxs P 43 -dxs1 |
| EC41 | Bacillus subtilis,ΔpbuE P degQ -ispAΔdxs P 43 -dxs2 |
| EC42 | Bacillus subtilis,ΔpbuE P degQ -ispAΔdxs P 43 -dxs3 |
Related P 43 Promoter sequence, P degQ Promoter sequence, P hag The promoter sequence, the pbuE gene fragment and the dxs gene fragment are shown in table 6 below.
Table 6 promoter sequence and gene fragment sequence table
| Sequence(s) | Numbering device |
| P 43 Promoter sequence | SEQ ID NO.39 |
| P degQ Promoter sequence | SEQ ID NO.40 |
| P hag Promoter sequence | SEQ ID NO.41 |
| PbuE gene fragment | SEQ ID NO.42 |
| dxs gene fragment | SEQ ID NO.43 |
Determination of MK-7 yield by Strain fermentation
Bacillus subtilis Bacillus subtilis 168 and recombinant bacteria EC1-EC4 obtained by construction are respectively inoculated into a seed culture medium. Seed culture medium formula (mass percent): yeast extract 0.5%, peptone 1%, sodium chloride 0.5%. Culturing at 37 ℃ and 220rpm for 12 hours to obtain bacillus subtilis seed liquid.
The obtained seed solution was transferred to a fermentation medium for cultivation at an inoculum size of 5%. Fermentation medium formula (mass percent): 1% of yeast extract, 1% of peptone, 0.4% of disodium hydrogen phosphate, 1% of potassium dihydrogen phosphate, 0.25% of ammonium sulfate, 0.05% of ammonium chloride, 0.02% of sodium citrate, 1% of glucose and 0.1% of magnesium sulfate heptahydrate. After 3 days of culture at 37℃and resting conditions, the fermentation broth was taken to determine MK-7 content. The results are shown in Table 7 below.
TABLE 7 fermentation production of MK-7 by strains
After the purine transporter gene pfuE is knocked out, the MK-7 content is increased by 20.2% compared with the original strain. The ispA gene promoter was used as P degQ After promoter replacement, MK-7 yield increased significantly, with a yield of 160.7.+ -. 2.81mg/L, 2.73 times that of the original strain. In use P 43 Or P hag When the promoter replaces the promoter of ispA gene, MK-7 yields were only 1.55 and 1.40 times that of the original strain. Then we knock out dxs gene in EC2 strain to obtain EC3, transform dxs gene in Klebsiella into bacillus subtilis strain to obtain strain EC4, and make MK-7 yield (liquid chromatogram as shown in FIG. 8, peak area as 34566) reach maximum 319.2 + -2.05 mg/L, which is 5.42 times of original strain (liquid chromatogram as shown in FIG. 7, peak area as 6377). As a control, dxs genes of E.coli and bifidobacteria were transformed into Bacillus subtilis strains, respectivelyAfter that, MK-7 production was increased compared to the original strain, 112.6.+ -. 1.21 and 108.9.+ -. 1.45mg/L, respectively, but much less than MK-7 production by the EC4 strain.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to suggest that the scope of the invention (including the claims) is limited to these examples; the technical features of the above embodiments or in the different embodiments may also be combined within the idea of the invention, the steps may be implemented in any order and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.
The present invention is intended to embrace all such alternatives, modifications and variances which fall within the broad scope of the appended claims. Therefore, any omission, modification, equivalent replacement, improvement, etc. of the present invention should be included in the scope of the present invention.
Claims (7)
1. Construction of efficient biosynthesis of vitamin K 2 The method for engineering bacteria is characterized by comprising the following steps:
step one, taking a bacillus subtilis 168 genome as a template, knocking out a purine transporter gene pfuE, and constructing a delta pfuE strain EC1;
step two, using the whole genome of the strain EC1 as a template and using P degQ The promoter sequence replaces the original promoter of the bacillus subtilis farnesyl diphosphate synthase gene ispA to obtain a strain EC2;
step three, taking the whole genome of the strain EC2 as a template, knocking out the 1-deoxyxylulose-5-phosphate synthase gene dxs, and obtaining the strain EC3;
step four, using the whole genome of Klebsiella as a template, amplifying to obtain dxs gene, and performing pHY-P reaction 43 Vector ligation into strain EC3 to obtain vitamin K for biosynthesis 2 EC4 of the strain.
2. Construction of highly efficient biosynthesis of vitamin K according to claim 1 2 Engineering bacteriaThe method for constructing the delta pbuE strain EC1 in the first step comprises the following steps:
a1, using a bacillus subtilis 168 genome as a template, and adopting a primer group shown as SEQ ID NO.1-3 to amplify to obtain a left homology arm, a right homology arm sequence and an intermediate fragment;
a2, performing overlap extension PCR on the obtained left homologous arm, right homologous arm sequences and intermediate fragments to obtain fusion gene fragments;
and A3, after purifying the amplified product, electrically transferring the fusion gene fragment into competent cells of the bacillus subtilis 168 to obtain the strain EC1.
3. Construction of highly efficient biosynthesis of vitamin K according to claim 1 2 The method for obtaining the strain EC2 in the second step comprises the following steps:
b1, using bacillus subtilis 168 genome as a template, and amplifying by using a primer shown as SEQ ID NO.7-8 to obtain a left homology arm and P of the ispA1 original promoter degQ A sequence and a right homology arm sequence;
b2, using plasmid p7C6 as template, amplifying with primer shown as SEQ ID NO.25-26 to obtain chloramphenicol resistance gene Cm R A sequence;
b3, the sequence obtained in B1 and the chloramphenicol resistance gene Cm obtained in B2 R And (3) carrying out sequence row overlapping extension PCR, purifying the obtained fusion gene fragment by using an amplification product, and electrically transferring the fusion gene fragment into competent cells of the EC1 strain to obtain strains EC2, EC21 and EC22.
4. Construction of highly efficient biosynthesis of vitamin K according to claim 1 2 The method for obtaining the strain EC3 in the third step comprises the following steps:
c1, using the whole genome of the strain EC2 as a template, and adopting primers shown as SEQ ID NO.27-28 to amplify to obtain left and right homologous arm sequences and intermediate fragments;
c2, performing overlap extension PCR on the obtained left homologous arm, right homologous arm sequences and intermediate fragments to obtain fusion gene fragments;
and C3, purifying the amplified product, and electrically transferring the fusion gene fragment into competent cells of the strain EC2 to obtain the strain EC3.
5. Construction of highly efficient biosynthesis of vitamin K according to claim 1 2 The method for obtaining the strain EC4 in the fourth step comprises the following steps:
d1, culturing klebsiella to an exponential growth medium phase, and extracting whole genome DNA;
d2, using the whole genome of Klebsiella as a template, and adopting a primer shown as SEQ ID NO.33-34 to amplify to obtain dxs gene fragments;
d3, after purification of amplified product, constructing recombinant plasmid pHY-P 43 The dxs1 is transformed into strain EC3, giving strain EC4.
6. Construction of highly efficient biosynthesis of vitamin K according to claim 5 2 The engineering bacteria method is characterized in that the construction of the recombinant plasmid pHY-P 43 The method of-dxs 1 is to use XbaI and BglII to amplify the product and carrier pHY-P 43 Double enzyme digestion is carried out, and the recovered products of the double enzyme digestion and the recovery products are connected by T4 ligase to obtain the double enzyme digestion.
7. Construction of highly efficient biosynthesis of vitamin K according to claim 1 2 The method for engineering bacteria is characterized in that the amplification conditions are as follows: denaturation at 98℃for 3min; then denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃for 20s for a total of 34 cycles; finally, the extension is carried out for 5min at 72 ℃.
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