CN113046287A - Construction method and application of PHA synthase N-terminal deletion mutant - Google Patents
Construction method and application of PHA synthase N-terminal deletion mutant Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, in particular to a method for constructing an N-terminal deletion mutant of PHA synthase. Cloning Aeromonas hydrophila PHA synthase by designing a primer, and then constructing a recombinant plasmid of deletion mutation of amino acids 2 to 28 at the N end. After enzyme digestion sequencing confirms that the construction of the expression vector is successful, the expression vector is transformed into Alcaligenes eutrophus (Ralstonia eutropha) mutant strain PHB‑4Different carbon sources are selected for shake flask fermentation to verify the PHA synthase function activity, and then the PHA synthase function activity is compared with that of wild aeromonas hydrophila. Through the method, the synthase gene with the mutation at the N end has the function of generating PHA, the PHA yield is higher than that of wild bacteria, and the PHA yield is improved. This will be used in the future by gene engineering technologyThe construction of the chimeric enzyme for producing the novel PHA provides a theoretical basis and has certain value in promoting wider application of the PHA.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a method for constructing an N-terminal deletion mutant of PHA synthase.
Technical Field
Polyhydroxyalkanoate (polyhydroxyakanonate) is a substance synthesized by many bacteria under conditions of nutrient imbalance for intracellular energy and carbon source storage. PHA has good biocompatibility, biodegradability, hydrophobicity and unique properties brought by different functional groups, is used as a tissue engineering material drug sustained-release material, and is a biological material with good development potential. Water-insoluble high molecular weight PHAs exhibit thermoplastic and/or elastomeric properties, as well as other physical and material properties, including biodegradation, biocompatibility, piezoelectricity, etc., and have attracted increasing attention from the industry. PHAs have many potential uses, such as bulk commodity plastics, fishing lines, woven materials, and potential biomedical applications.
The existing PHA production has the problems of high production cost, low yield and single component. PHA synthase is a key enzyme in the synthesis of PHA, and its activity and substrate specificity determine the content and composition of PHA. In the study of deletion mutation of PHA synthase, it was found that the N-terminus of PHA synthase is related to the properties of the enzyme, such as substrate specificity, enzyme activity, heat resistance, and plays an important role in the composition of the final polymer for polymerization. The research aims to change the substrate specificity of PHA synthase, improve the catalytic activity of PHA synthase and improve the PHA yield by modifying the N end.
Alcaligenes eutropha (r. eutropha) PHA synthase has very high enzymatic activity but produces only PHB. The Aeromonas hydrophila WQ (A. hydrophila WQ) PHA synthase has a wide substrate range and produces P (3HB-co-3HHx), but its PHA-producing ability is low. Based on these two parents, engineering the N-terminus of PHA synthase to produce novel PHAs is of great theoretical and practical interest.
Disclosure of Invention
The invention aims to research the deletion mutation of the N end of the synthase gene, research the influence of the N end on the synthase property, change the substrate specificity of PHA synthase, improve the catalytic activity of PHA synthase, improve the PHA yield and facilitate the construction of PHA mosaic enzyme in the future.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for constructing a PHA synthase N-terminal deletion mutation by cloning and designing primers comprises the following steps: a method for constructing an N-terminal deletion mutant of PHA synthase comprises the following steps:
1) obtaining a sequence of aeromonas hydrophila WQ (A.hydrophila WQ) from NCBI, and designing primers P1 and P2 according to the sequence;
P1-5’-TTAAAGCTTCTAATGGAGCGCACCGCCCAG-3’P2-5’-TTTGAATTCTCATGCGGCGTCCTCCTCT-3’
2) in the form of pUC19-phaPCJWQUsing P1 and P2 as primers as a template, and obtaining phaPCJ by PCR amplificationWQ；
3) phaPCJ obtained in step 2)WQThe recombinant plasmid pBBR1MCS2-phaC 'J is obtained by adopting the steps of recovering the gel through cutting, performing double enzyme digestion through HindIII and EcoRI, inserting the gel-digested expression vector pBBR1MCS2, and connecting the gel-digested expression vector pBBR1MCS 2-phaC' J through T4 ligaseWQ；
4) The recombinant plasmid pBBR1MCS 2-phaC' JWQTransforming Escherichia coli S17-1 competent cells, spreading the obtained bacterial liquid on LB plate containing Kan resistance, culturing, selecting positive clone, culturing, and extracting recombinant plasmid pBBR1MCS 2-phaC' JWQPerforming double enzyme digestion by HindIII and EcoRI, screening positive transformants, and then performing deletion of the mutant PHB by the obtained positive transformants and PHA synthase-4Co-culturing, recombinant plasmid pBBR1MCS 2-phaC' JWQTransformation into PHB-4To obtain the recombinant strain.
In the method for constructing the deletion mutant at the N-terminal of the PHA synthase, the reaction system in the step 2) is 50 μ l: ddH2O32.5μl；10×Pfu Buffer5μl；2.5mM dNTP5μl；Mg 2+4 mu l of the solution; 1 μ l of template DNA; 25 μ M primer 11 μ l; 25 μ M primer 21 μ l; 5U/. mu.l Pfu enzyme 0.5. mu.l;
in the method for constructing the deletion mutant at the N-terminal of the PHA synthase, the amplification conditions in the step 2) are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30 sec; annealing at 62 ℃ for 30 sec; extension at 72 ℃ for 2.5min for 25 cycles; extension at 72 ℃ for 10 min.
In the method for constructing the N-terminal deletion mutant of PHA synthase, in step 4), the culture is carried out at 37 ℃ overnight.
The recombinant strain produced by the method for constructing the PHA synthase N-terminal deletion mutant is applied to PHA production.
The invention has the beneficial effects that: by the method, the deletion of the N end of the PHA synthase is successfully constructed, the substrate specificity of the PHA synthase is changed by modifying the N end, the catalytic activity of the PHA synthase is improved, and the PHA yield is improved. Solves the problem of single PHA monomer composition. The effect on PHA synthase substrate specificity in different host bacteria after N-terminal deletion was also investigated. Has certain value in promoting the wider application of PHA.
Drawings
FIG. 1 is a schematic diagram of the construction process of pBBR1MCS 2-phaC' JWQ expression plasmid.
FIG. 2 is an agarose gel electrophoresis of plasmid pUC19-phaPCJWQ, wherein M is (1kb DNA Ladder (10000bp,8000bp,6000bp,5000bp,4000bp,3500bp,3000bp,2000bp,1000bp,750bp,500bp,250bp), 1 is (plasmid pUC19-phaPCJWQ), and 2 is (plasmid pUC 19-phaPCJWQ).
FIG. 3 is a photograph of phaC ' JWQ agarose gel electrophoresis, in which M is (1kb DNA Ladder (10000bp,8000bp,6000bp,5000bp,4000bp,3500bp,3000bp,2000bp,1000bp,750bp,500bp,250 bp);), 1 is (phaC ' JWQ PCR electrophoresis result.), and 2 is (phaC ' JWQ PCR electrophoresis result.).
FIG. 4 is a diagram of agarose gel electrophoresis of the recombinant plasmid digestion, wherein M is (1kb DNA Ladder (10000bp,8000bp,6000bp,5000bp,4000bp,3500bp,3000bp,2000bp,1000bp,750bp,500bp,250 bp);), 1 is (empty plasmid electrophoresis result), 2 is (recombinant plasmid electrophoresis result), 3 is (recombinant plasmid Hind III single digestion electrophoresis result), and 4 is (recombinant plasmid Hind III, EcoRI single digestion electrophoresis result).
FIG. 5 shows the results of Nile red fluorescent staining.
Detailed Description
A method for preparing an N-terminal deletion mutant of PHA synthase comprises the following steps:
example 1 uses alcaligenes eutrophus (r. eutropha) PHA synthase and aeromonas hydrophila WQ (a. hydrophila WQ) PHA synthase.
1. Cloning of PHA synthase Gene:
obtaining the sequence of Aeromonas hydrophila WQ (A. hydrophila WQ) from NCBI, and designing primers P1-5'-TTAAAGCTTCTAATGGAGCGCACCGCCCAG-3', P2-5 '-TTTGAATTCTCATGCGGCGTCCTCCTCT-3'. Plasmid pUC19-phaPCJ registered at NCBIWQUsing P1 and P2 as primers as a template, and carrying out PCR amplification to obtain phaPCJWQ. The amplification system is: the reaction system was 50. mu.l: ddH2O32.5μl；10×Pfu Buffer5μl；2.5mM dNTP5μl；Mg 2+4 mu l of the solution; 1 μ l of template DNA; 25 μ M primer 11 μ l; 25 μ M primer 21 μ l; 5U/. mu.l Pfu enzyme 0.5. mu.l; the amplification conditions were: pre-denaturation at 5 deg.C for 5 min; denaturation at 94 ℃ for 30 sec; annealing at 62 ℃ for 30 sec; extension at 72 ℃ for 2.5min for 25 cycles; extension at 72 ℃ for 10 min.
The size of the PCR product is basically consistent with the expected result, phaC' JWQThe size is 2186bp, as shown in FIG. 2.
2. Constructing a plasmid:
obtaining phaPCJ by PCR amplificationWQThe recombinant plasmid pBBR1MCS 2-phaC' J is obtained by adopting the steps of recovering the cutting glue, performing double enzyme digestion by HindIII and EcoRI, inserting the cut enzyme pBBR1MCS2, and performing overnight connection at 16 ℃ by using T4 ligaseWQ. The recombinant plasmid pBBR1MCS 2-phaC' JWQTransforming Escherichia coli S17-1 competent cells, spreading the bacterial liquid on LB plate containing Kan resistance, culturing at 37 deg.C overnight, selecting positive clone, culturing, and extracting recombinant plasmid pBBR1MCS 2-phaC' JWQPositive transformants were screened by double digestion with HindIII and EcoRI. Then the obtained positive transformant and PHA synthase deletion mutant PHB are subjected to-4Co-culture, transformation of recombinant plasmids into PHB-4Obtaining recombinant bacteria, and carrying out quality improvement and enzyme cutting verification.
The verification is shown in FIG. 3, and the sequencing result shows that the expression vector pBBR1MCS 2-phaC' JWQThe construction was successful.
3. And (3) verifying the function of the recombinant bacteria:
plasmid-containing R.eutropha PHB-4The recombinant bacteria are cultured in two steps, a single colony is picked by a toothpick and cultured in 10ml LB overnight, when seed liquid grows well, the bacterial colony is transferred into 100ml mineral salt culture medium, corresponding antibiotics are added, the bacterial colony is cultured for 72h at 200rpm under the condition of 30 ℃, the bacterial colony is collected by centrifugation for 10min at 8000rpm, the bacterial colony is washed once by water and once by alcohol, the bacterial colony is freeze-dried to constant weight, and 50mg of the bacterial colony is weighed and esterified for gas chromatography analysis.
In the research, the activity of PHA synthase is preliminarily and qualitatively detected by Nile red which is a fluorescent dye and can be used for dyeing lipids, proteins and the like. Preparing a solid culture medium for fermenting all mineral salt solid culture mediums into a solid plate, adding 0.5mg/ml Nile red, culturing for 3 days in a dark place, and photographing with an ultraviolet lamp to observe whether fluorescence exists or not so as to preliminarily detect the PHA synthase activity. As a result, as shown in FIG. 4, it was preliminarily confirmed that the constructed PHA synthase had PHA synthase activity by the deletion of the N-terminus.
The results of the molecular composition and content of PHA produced by the recombinant bacteria are shown in Table 1.
TABLE 1
The results are shown in Table 1 with pBBR1MCS 2-phaC' JWQThe recombinant strain of (a) is capable of producing PHA in a medium with three different carbon sources, and the R.eutropha synthase deletion mutant and the recombinant strain do not contain pBBR1MCS 2-phaC' JWQThe recombinant strain can not produce PHA, thereby proving that the expression plasmid is successfully constructed and the synthase gene has function. PHA monomers generated by three different carbon sources have different compositions, the thallus grows best when lauric acid is used as a carbon source, the PHA yield is highest and accounts for 65.05 percent of the dry weight of cells, and the generated 3HB content is 60.29 percent at most. With fructose as the carbon source, only 3HB monomer was produced, and 3HHX was not produced. And the two groups of control groups do not generate PHA, which proves that the constructed plasmids are successful and the PHA synthase gene has the function of synthesizing PHA.
Claims (5)
1. A method for constructing an N-terminal deletion mutant of PHA synthase is characterized by comprising the following steps:
1) obtaining a sequence of aeromonas hydrophila WQ (A.hydrophila WQ) from NCBI, and designing primers P1 and P2 according to the sequence;
P1-5’-TTAAAGCTTCTAATGGAGCGCACCGCCCAG-3’P2-5’-TTTGAATTCTCATGCGGCGTCCTCCTCT-3’
2) in the form of pUC19-phaPCJWQUsing P1 and P2 as primers as a template, and obtaining phaPCJ by PCR amplificationWQ；
3) phaPCJ obtained in step 2)WQThe recombinant plasmid pBBR1MCS2-phaC 'J is obtained by adopting the steps of recovering the gel through cutting, performing double enzyme digestion through HindIII and EcoRI, inserting the gel-digested expression vector pBBR1MCS2, and connecting the gel-digested expression vector pBBR1MCS 2-phaC' J through T4 ligaseWQ；
4) The recombinant plasmid pBBR1MCS 2-phaC' JWQTransforming Escherichia coli S17-1 competent cells, spreading the obtained bacterial liquid on LB plate containing Kan resistance, culturing, selecting positive clone, culturing, and extracting recombinant plasmid pBBR1MCS 2-phaC' JWQPerforming double enzyme digestion by HindIII and EcoRI, screening positive transformants, and then performing deletion of the mutant PHB by the obtained positive transformants and PHA synthase-4Co-culturing, recombinant plasmid pBBR1MCS 2-phaC' JWQTransformation into PHB-4To obtain the recombinant strain.
2. The method as claimed in claim 1, wherein the reaction system in step 2) is 50 μ l: ddH2O32.5μl；10×Pfu Buffer5μl；2.5mM dNTP5μl；Mg2+4 mu l of the solution; 1 μ l of template DNA; 25 μ M primer 11 μ l; 25 μ M primer 21 μ l; 5U/. mu.l Pfu enzyme 0.5. mu.l;
3. the method as claimed in claim 1, wherein the amplification conditions in step 2) are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30 sec; annealing at 62 ℃ for 30 sec; extension at 72 ℃ for 2.5min for 25 cycles; extension at 72 ℃ for 10 min.
4. The method as set forth in claim 1, wherein the cultivation in step 4) is carried out at 37 ℃ overnight.
5. The use of the recombinant bacterium produced by the method for constructing the deletion mutant of the N-terminal of PHA synthase described in claim 1 in the production of PHA.
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