CN116064633B - Construction of efficient biosynthesis of vitamin K2Engineering bacteria method - Google Patents
Construction of efficient biosynthesis of vitamin K2Engineering bacteria method Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a method for constructing high-efficiency biological vitamin K 2 engineering bacteria, which comprises the following steps: step one, constructing and obtaining a delta pbuE strain EC1; step two, replacing an original promoter of a bacillus subtilis farnesyl diphosphate synthase gene ispA by a P degQ promoter sequence by taking the whole genome of the strain EC1 as a template 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; and step four, amplifying to obtain dxs genes by taking the whole genome of the klebsiella as a template, and carrying out connection with a pHY-P 43 carrier to transform the dxs genes into a strain EC3 to obtain 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 biological vitamin K 2 engineering bacteria.
Background
Vitamin K 2 is a fat-soluble vitamin, is a menaquinone series compound, and has a structure 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. Among them, menaquinone-7 (MK-7) containing seven isoprene side chains is the most active structural form of vitamin K 2.
At present, vitamin K 2 is mainly chemically synthesized, but the traditional chemical synthesis method has the problems of limited sources of chemical raw materials, a large amount of isomers produced by chemical reaction, a large amount of byproducts, low yield, environmental pollution and the like, and the side chain of isoprene in the synthesized vitamin K 2 is in a cis structure and has low activity. Therefore, the industrial use of microbial fermentation to produce vitamin K 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 has relatively low yield and low fermentation level in the fermentation production of vitamin K 2, so that the cost is too high and the price is high, and the method is difficult to be accepted by common people. Therefore, the method has important theoretical and application values for effectively breeding excellent strains, improving the production performance of vitamin K 2 produced by fermentation and preventing and treating diseases such as osteoporosis, cardiovascular diseases, arterial calcification and the like.
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 is very challenging but at the same time quite pioneering as it is synthesized in Bacillus subtilis by the fact that it involves about 38 enzymatic reactions of 4 metabolic modules and there are toxic intermediates and competing and inhibitory metabolic pathways, with the adoption of synthetic biological methods to preferentially metabolize key enzymes and modulate the appropriate activities of key enzymes.
Disclosure of Invention
Therefore, the invention aims to provide a method for constructing high-efficiency biological vitamin K 2 engineering bacteria, which aims to solve the problem that the yield of vitamin K 2 is low due to toxic intermediates and competitive and inhibitory metabolic pathways in the existing biological method.
Based on the above purpose, the invention provides a method for constructing high-efficiency biological vitamin K 2 engineering bacteria, which comprises the following steps:
Step one, taking a bacillus subtilis 168 genome as a template, knocking out a purine transporter gene pbuE, and constructing to obtain a delta pbuE strain EC1;
Step two, replacing an original promoter of a bacillus subtilis farnesyl diphosphate synthase gene ispA by a P degQ promoter sequence by taking the whole genome of the strain EC1 as a template 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;
And step four, using the whole genome of the klebsiella as a template, amplifying to obtain dxs genes, and connecting the dxs genes with a pHY-P 43 carrier to transform the dxs genes into a strain EC3 to obtain EC4 for biosynthesis of vitamin K 2 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 a 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 sequence, a P degQ sequence and a right homology arm sequence of an ispA1 original promoter;
B2, using plasmid p7C6 as a template, and amplifying by using a primer shown as SEQ ID NO.25-26 to obtain a chloramphenicol resistance gene Cm R sequence;
B3, overlapping and extending the sequence obtained in the B1 with the chloramphenicol resistance gene Cm R sequence obtained in the B2, purifying the obtained fusion gene fragment through amplification products, 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;
and D3, after purifying the amplified product, constructing a recombinant plasmid pHY-P 43 -dxs1, and converting the recombinant plasmid pHY-P 43 -dxs1 into the strain EC3 to obtain the strain EC4.
Preferably, the method for constructing the recombinant plasmid pHY-P 43 -dxs1 is to use XbaI and BglII to carry out double digestion on an amplification product and a vector pHY-P 43, and the recovery products of the two are obtained after being connected by T4 ligase.
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 168 is 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 EC 1; 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 EC 1; 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 EC 2; 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 EC 2; 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 EC 3; lanes 1,2: a left homology arm segment; 3,4: chloramphenicol resistance fragments; 5,6: a right homology arm segment; 7,8: a P degQ fragment;
FIG. 6 shows agarose gel electrophoresis of the recovered fragments of the validation gel during the construction of EC 3; 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. 8 is a liquid chromatography detection pattern of MK-7 produced by EC 4.
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 pbuE-L-F (primer 1) and pbuE-L-R (primer 2), pbuE-p7C6-F (primer 3) and pbuE-p7C6-R (primer 4), pbuE-R-F (primer 5) and pbuE-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, and 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 strain delta pbuE is obtained through PCR bacterial liquid verification, namely the strain EC1.
Construction of recombinant bacterium EC2
TABLE 2 primer sequence listing
Based on the obtained strain EC1, the specific construction process for replacing the original promoter of the bacillus subtilis ispA gene by using the P degQ、P43、Phag promoter 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 Softberry websites are utilized to predict the promoter position of the IspA gene. Using bacillus subtilis 168 genome as a template, and amplifying by using primers 7-12 in table 2 to obtain a left homology arm, a P degQ sequence and a right homology arm sequence of the ispA1 original promoter; amplifying by using the primers 13-18 to obtain a left homology arm sequence, a P 43 sequence and a right homology arm sequence of the ispA2 original promoter; the left homology arm, P hag sequence and right homology arm sequence of ispA3 original promoter are obtained by using the primer 19-24 amplification.
The chloramphenicol resistance gene Cm R sequence is obtained by using the plasmid p7C6 as a template and using the primers 25-26 for amplification. And (3) after the obtained PCR product is identified to be correct through agarose gel electrophoresis, the target band is recovered through gel.
The left and right homology arms and the P degQ PCR amplification system are as follows: ddH 2 O10 uL, bacillus subtilis 168 whole genome template 0.5uL, and upstream and downstream primers 1.0uL,Primer star 12.5uL. 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. The amplification methods of P 43 and P hag are the same.
The intermediate fragment Cm R amplification system was: ddH 2 O10 uL, p7C6 plasmid template 0.5uL, and 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-denaturing at 98 ℃ for 5min, then denaturing for 10s at 98 ℃, annealing for 5s at 55 ℃ and extending for 35s at 72 ℃, performing gel cutting and recovering fragments with correct sizes for 34 cycles in total, and obtaining fusion gene fragments. The left homology arm, P degQ sequence and right homology arm sequence PCR products of ispA1 original promoter, the middle fragment Cm R and the recovery fragment electrophoresis diagram of the DNA fragment recovery kit are shown in fig. 3 and 4.
And (3) purifying the target gene band amplified product, electrically transferring the fusion gene fragment into competent cells of the EC1 strain, and after single colony grows on a flat plate, selecting the single colony for liquid culture, verifying by PCR bacterial liquid to obtain the target strain, and finally obtaining the strain Bacillus subtilis, delta pbuE P degQ -ispA. Strains Bacillus subtilis. DELTA. pbuE P 43 -ispA and. DELTA. pbuE P hag -ispA were constructed as described above and 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, and upstream and downstream primers 1.0uL,Primer star 12.5uL. 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.
The intermediate fragment Cm R amplification system was: ddH 2 O10 uL, p7C6 plasmid template 0.5uL, and 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, the P 43 promoter is utilized to over express 1-deoxyxylulose-5-phosphate synthase gene dxs on the chromosome of Klebsiella Klebsiella variicola, escherichia coli ESCHERICHIA COLI and bifidobacterium Bifidobacterium longum, and the specific construction process is as follows:
Klebsiella variicola was cultured to the medium-term exponential growth phase, 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 Softberry site was used to predict the promoter position of dxs gene, the whole genome of Klebsiella variicola, ESCHERICHIA COLI and Bifidobacterium longum was used as template, the P 43 promoter was used to replace the original promoter of dxs gene, and the pHY plasmid had the P 43 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 43 were digested with XbaI and BglII, and the recovered products were ligated with T4 ligase at 4℃for 16h. The recombinant plasmid pHY-P 43 -dxs is transformed into EC3, positive clones are obtained through screening and identification, and finally strains EC3-P 43-dxs1,EC3-P43 -dxs2 and EC3-P 43 -dxs3 are obtained and named as 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 PdegQ-ispA |
| EC21 | Bacillus subtilis,ΔpbuE P43-ispA |
| EC22 | Bacillus subtilis,ΔpbuE Phag-ispA |
| EC3 | Bacillus subtilis,ΔpbuE PdegQ-ispAΔdxs |
| EC4 | Bacillus subtilis,ΔpbuE PdegQ-ispAΔdxs P43-dxs1 |
| EC41 | Bacillus subtilis,ΔpbuE PdegQ-ispAΔdxs P43-dxs2 |
| EC42 | Bacillus subtilis,ΔpbuE PdegQ-ispAΔdxs P43-dxs3 |
The P 43 promoter sequence, the P degQ promoter sequence, the P hag 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 knocking out the purine transporter gene pbuE, the MK-7 content is increased by 20.2% compared with the original strain. After replacement of the ispA gene promoter with the P degQ promoter, a significant increase in MK-7 production occurred, with a yield of 160.7.+ -. 2.81mg/L, which was 2.73 times that of the original strain. When the promoter of ispA gene was replaced with the P 43 or P hag promoter, 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 is 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 is 6377). As a control, after transformation of the dxs genes of E.coli and bifidobacteria, respectively, into the B.subtilis strain, MK-7 production was increased compared to the original strain, respectively, at 112.6.+ -. 1.21 and 108.9.+ -. 1.45mg/L, but far 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 (6)
1. The method for constructing the high-efficiency biological vitamin K 2 engineering bacteria is characterized by comprising the following steps of:
Step one, taking a bacillus subtilis 168 genome as a template, knocking out a purine transporter gene pbuE, and constructing to obtain a delta pbuE strain EC1;
Step two, replacing an original promoter of a bacillus subtilis farnesyl diphosphate synthase gene ispA by a P degQ promoter sequence by taking the whole genome of the strain EC1 as a template 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 the klebsiella as a template, amplifying to obtain dxs genes, and connecting the dxs genes with a pHY-P 43 carrier to transform the dxs genes into a strain EC3 to obtain EC4 for biosynthesis of vitamin K 2 strain;
the method for obtaining the strain EC2 in the second step comprises the following steps:
B1, using a bacillus subtilis 168 genome as a template, and amplifying by using a primer shown as SEQ ID NO.7-12 to obtain a left homology arm sequence, a P degQ sequence and a right homology arm sequence of an ispA1 original promoter;
B2, using plasmid p7C6 as a template, and amplifying by using a primer shown as SEQ ID NO. 25-26 to obtain a chloramphenicol resistance gene Cm R sequence;
B3, overlapping and extending the sequence obtained in the B1 with the chloramphenicol resistance gene Cm R sequence obtained in the B2, purifying the obtained fusion gene fragment through amplification products, and electrically transferring the fusion gene fragment into competent cells of the EC1 strain to obtain strains EC2, EC21 and EC22.
2. The method for constructing an engineering bacterium for efficiently biosynthesizing vitamin K 2 according to claim 1, wherein the method for constructing the strain EC1 of delta pbuE in the first step includes 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.
3. The method for constructing an engineering bacterium for efficiently biosynthesizing vitamin K 2 according to claim 1, wherein 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.
4. The method for constructing an engineering bacterium for efficiently biosynthesizing vitamin K 2 according to claim 1, wherein 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;
and D3, after purifying the amplified product, constructing a recombinant plasmid pHY-P 43 -dxs1, and converting the recombinant plasmid pHY-P 43 -dxs1 into the strain EC3 to obtain the strain EC4.
5. The method for constructing efficient biosynthetic vitamin K 2 engineering bacteria according to claim 4, wherein the method for constructing recombinant plasmid pHY-P 43 -dxs1 is to use XbaI and BglII to carry out double digestion on amplified products and vector pHY-P 43, and the recovered products of the two are connected by T4 ligase.
6. The method for constructing an engineering bacterium for efficiently biosynthesizing vitamin K 2 according to claim 1, wherein the amplification conditions are as follows: denaturation at 98℃3 min; then denaturation at 98℃for 10s, annealing at 55℃for 5s, elongation at 72℃for 20 s, for a total of 34 cycles; finally, the extension is carried out at 72 ℃ for 5 min.
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