Construction Of ADH Gene Knockout Vector And Inducible Expression Vectorin In Arthrobacter Sp.

Arthrobacter sp.is a kind of Actinomycete with high GC content widely present in the soil.It has strong environmental adaptability and can degrade the environmental pollutants by using various environmental pollutants as the sole carbon source or nitrogen source.With the rapid development of wholegenome sequencing and molecular biology technology,it has become a hot spot to explore the mechanism of thedegradation of environmental pollutants by Arthrobacter sp.at the molecular level.As aresearch tool of gene function,gene editing has played a huge role in microbial research.At present,the gene editing technology of Arthrobacter sp.is not perfect,and it has greatlimitations,making it impossible to use it widely in scientific research.In this study,Arthrobacter sp.HW08 is a high swainsonine(SW)degradation rate gram-positive bacterium isolated from the soil embedded with Oxytropis ochrocephala in this laboratory.First,the suicide plasmid pK18 mobsacB is used to construct the Arthrobacter sp.HW08 knockout vector,and the NADP-dependent alcohol dehydrogenase(ADH)gene associated with the degradation of SW in Arthrobacter sp.HW08 is knocked out.Secondly,in order to obtain a vector suitable for the construction of the Arthrobacter sp.HW08 CRISPR-Cas9 gene knockout system,the shuttle plasmid pART2 is transformed into an inducible expression vector pARTP using techniques such as overlapping PCR and site-directed mutagenesis.The results obtained are as follows:1.Bioinformatics analysis of ADH proteinBioinformatics analysis of ADH indicated that the enzyme is a stable non-secretory protein with a molecular weight of 36.638 kDa,which contains a enoyl reductase domain,an ADHs catalytic domain,a C-terminal,a conserved binding domain of a zinc atom,and a binding domain of a NAD(P)cofactor.NADP binds to the major groove in ADH and forms hydrogen bonds with the amino acid residues His42,Ser43,His63,Thr162,Gly184,Gly185,Ser246 and Arg338 of ADH;SW binds to the minor groove in ADH and forms hydrogen bonds with amino acid residues Arg24,His133 and Val135.2.Construction of pK18mobsacB-?ADH vectorThe Arthrobacter sp.HW08 gene knockout vector with the suicide plasmid pK18 mobsacB as the backbone is constructed by homologous recombination.The homologous arm sequence is designed based on the upstream and downstream DNA sequences of the Arthrobacter sp.HW08 ADH gene and PCR amplification is performed to construct the knockout vector pK18mobsacB-?ADH.3.Screening of ADH gene deletion strainsThe knockout vector is transferred into the competent cells of Arthrobacter sp.HW08 by electroporation,and the ADH gene deletion strain is screened by the characteristics of pK18 mobsacB vector.The transformants are identified by PCR and verified by sequencing,and finally the ADH gene deletion strain is obtained.4.Construction of an inducible expression vector pARTPIn order to obtain a vector for constructing the Arthrobacter sp.HW08 CRISPR-Cas9 gene knockout system.The shuttle vector pART2 is transformed into a constitutive expression vector pARTF by overlapping PCR and site-directed mutagenesis.Secondly,the expression vector pARTP is constructed by adding the lactose operon gene(laclq)to the pARTF vector.