DSF degrading enzyme encoding gene fadY and application thereof
A technology of encoding genes and degrading enzymes, which is applied to the DSF degrading enzyme encoding gene fadY and its application fields, can solve the problems of not being a degrading gene, not finding a degrading gene, affecting the degradation ability, etc., and achieving the effect of wide application prospects
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Embodiment 1
[0033] Example 1 Whole genome sequencing analysis of Acinetobacter lactucae strain QL-1
[0034] In recent years, high-throughput sequencing technology has developed rapidly, and bioinformatics has gradually become an important tool for auxiliary research in various fields. Using the genome information of the currently sequenced strains and some open database platforms, functional gene annotation and positioning, and looking for its possible participation Substrate degradation and metabolism genes, which is an effective strategy to obtain genes with related functions. In this embodiment, a whole-genome sequencing method is used to search for possible degradation genes in the strain QL-1 through the results of the analysis of the genome characteristics of the strain QL-1 and the functional gene annotation. The specific experimental methods and results are as follows:
[0035] (1) Genomic characteristics of strain QL-1
[0036] In order to understand the gene function and structure o...
Embodiment 2
[0040] Example 2 Obtaining the gene fadY encoding DSF degrading enzyme
[0041] In this example, based on the results of the genome feature analysis and functional gene annotation of the strain QL-1 of Example 1, possible degrading enzyme gene mutants were constructed for verification, in order to find highly efficient degrading enzymes. The specific experimental methods and results are as follows:
[0042] (1) Experimental method
[0043] According to reports, the rpfB gene located upstream of the DSF synthesis gene rpfF in X. campestris Xcc has been identified as an acyl-CoA ligase encoding gene, which participates in the degradation of DSF family signal molecules and regulates the expression of the QS system.
[0044] According to the results of the whole genome sequencing analysis of the strain QL-1 in Example 1, genes related to fatty acid degradation were found in the whole genome of the strain QL-1, and these genes were BLAST compared with the rpfB gene. The sequence homology ...
Embodiment 3
[0049] Example 3 Optimization, expression and purification of DSF degradation enzyme FadY
[0050] (1) Experimental method
[0051] According to the results of the whole genome sequencing analysis of the Acinetobacter lactucae strain QL-1 in Example 1, the gene fadY is presumed to encode a long-chain fatty acyl-CoA ligase, encoding a 559 amino acid (aa) protein, and the predicted molecular weight is 61.53kDa, isoelectric point is 8.03.
[0052] In order to obtain a higher yield and a more pure degrading enzyme, the gene fadY was codon optimized. The codon-optimized DNA fragment encoding the DSF degradation enzyme FadY was amplified by PCR, and subcloned into the expression vector pGEX-6p-1 to obtain the recombinant plasmid pGEX-fadY. The recombinant plasmid was transferred into E. coli BL21 to obtain the recombinant Bacteria E.coil BL21 / pGEX-fadY. Bacteria were inoculated in LB medium and cultured at 37℃, and 100mg / mL ampicillin was added. 600 When reaching about 0.6, IPTG inducer...
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