Biological synthesis method of xylosic acid
A technology of xylonic acid and xylose, applied in the field of recombinant engineering acceptor bacteria, can solve problems such as restricting application, restricting commercial application, environmental pollution, etc., and achieves the effects of reducing production cost, reducing oxidation, and avoiding reaction conditions
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[0057] Example 1:
[0058] The construction of the co-expression vector of xylose dehydrogenase gene (xylB) and xylon lactonase gene (xylC), the specific process is as follows:
[0059] Use oligonucleotide 5'-CAT GCC ATG GGC ATG TCC TCA GCC ATC TATCCC AG-3' and 5'-CGC GGA TCC TCA ACG CCA GCC GGC GTC GAT C-3' as primers, use C. crescentus genomic DNA As a template, the polymerase chain reaction (PCR) method was used to amplify the xylose dehydrogenase gene (xylB gene) (SEQ ID No: 1), and NcoI and BamHI were introduced at the 5'end and 3'end, respectively. Site, and then use the above restriction site to clone this gene into pACYCduet-1 (purchased from Novagen) vector to obtain the recombinant plasmid pA-xylB;
[0060] Use oligonucleotide 5'-GGG AAT TCC ATA TGA CCG CTC AGG TTA CATGCG-3' and 5'-CCG CTC GAG TTAAAC CAG ACG AAC TTC GTG C-3' as primers and use C. crescentus genomic DNA as template , Using polymerase chain reaction (PCR) method to amplify the xylonolactonase gene (xylC gen...
Example Embodiment
[0061] Example 2:
[0062] The preparation of a recombinant engineered E. coli strain for synthesizing xylonic acid, and using the strain to fermentatively transform D-xylose to produce xylonic acid, the specific process is as follows:
[0063] The recombinant plasmid pA-xylBC constructed in Example 1 was extracted by alkaline lysis method, and 10μl of recombinant plasmid pA-xylBC was transformed into E. coli BL21(DE3) competent cells by heat shock transformation method, and then 50μl of the transformed bacterial solution Cloth contains 34μg·mL -1 The positive clones were screened on the LB plate of chloramphenicol, and the colonies grown were the recombinant E. coli strains co-expressing xylose dehydrogenase and xylonic lactonase. The recombinant E. coli strain containing the recombinant plasmid pA-xylBC can be further verified by sequencing.
[0064] Pick the constructed single colony of recombinant E. coli and inoculate it to contain 34μg·mL -1 Chloramphenicol in LB liquid medium...
Example Embodiment
[0066] Example 3:
[0067] The preparation of a recombinant engineered E. coli strain for synthesizing xylonic acid, and using the strain to fermentatively transform D-xylose to produce xylonic acid, the specific process is as follows:
[0068] The recombinant plasmid pA-xylBC constructed in Example 1 was extracted by alkaline lysis method, and 1 μl of the recombinant plasmid pA-xylBC was transformed into E. coli BL21 (DE3) competent cells by the heat shock transformation method, and then 100 μl of the transformed bacterial solution was coated Cloth contains 34μg·mL -1 The positive clones were screened on the LB plate of chloramphenicol, and the colonies grown were the recombinant E. coli strains co-expressing xylose dehydrogenase and xylonic lactonase. The recombinant E. coli strain containing the recombinant plasmid pA-xylBC can be further verified by sequencing.
[0069] Pick the constructed single colony of recombinant E. coli and inoculate it to contain 34μg·mL -1 Chloramphenic...
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