A Vanillin Producing Amycolatopsis Sp.with The Vdh Gene Knockout By CRISPR-Cas9 Technology

Vanillin is one of the most widely used food deodorants in the world.It has been widely used in the fields of food,beverage,perfume and medicine.Because the chemical synthesis of vanillin is not green enough,biotechnology has become an alternative method to synthesize vanillin.Amycolatopsis CCTCC NO:M2011265,a producing vanillin strain,was screened in our preliminary study.Here,to further improve vanillin production,the gene and function of vanillin dehydrogenase（VDH）were determined by genome sequencing analysis and gene mining of key enzymes in vanillin metabolism pathway.As well as,a mutant strain that knocked out vdh by CRISPR/Cas9 technology was obtained and its fermentation performance was investigated.Details are listed as follows:（1）Analysis of Amycolatopsis CCTCC NO:M2011265 genome sequencing.De novo sequence was performed on the whole genome of Amycolatopsis,results showed that 8.43 Mbp genomic sequence,64 scaffolds and N50 of 0.34 Mbp were obtained,8185 genes were predicted.Four vanillin-related candidate genes were mined from annotated data:FCS（z4344）,ECH（z4343）,ECH₂（z7967）and VDH（z8076）by comparing blast2GO algorithm,rpsblast tool and KOBAS tool with GO,COG and KEGG database.It was speculated that the metabolic pathway of vanillin in Amycolatopsis is that vanillin was synthesized from ferulic acid by FCS-ECH/ECH₂and further degraded by VDH.（2）Verification of the vdh function.By designing primers according to the genome sequencing,the vdh gene was expressed in p ET-28a to construct a recombinant strain E.coli BL21-vdh.Through the catalytic reaction of vanillin and vanillic acid respectively,it is concluded that the ability of vanillin dehydrogenase to degrade vanillin to produce vanillic acid is much stronger than its reverse reaction.（3）Determination of the conditions for genetic transformation of Amycolatopsis CCTCC NO:M2011265.The three periods（the mycelium,spores and their mixing）of cells cultured in the seed medium,TSB medium and YEME medium were selected to prepare the competent state,respectively.And three voltages of 1500 V,2000 V and2500 V were selected for the electric conversion.The results showed that the transformers strains could be obtained only from the mycelium of cells in YEME medium,and the conversion rate was 1.7×10³ CFU·μg^-1 DNA when the electric intensity was 1500 V.（4）Establishment of CRISPR/Cas9 editing system and construction of mutant strains.The specific sgRNA was designed with vdh gene as the target gene.The original promoter and sgRNA on the p KCcas9d O plasmid were replaced with Km^r promoter,erm E*promoter and specific sgRNA to obtain the plasmid p KCKmcas9VDH.Then,the knockout plasmid p LYZYP01 of vdh gene was obtained by linking the upstream and downstream homologous arm fragment of vdh gene to the plasmid p KCKmcas9VDH.The mutant Amycolatopsis sp.Δvdh was obtained by electroporating the knockout plasmid p LYZYP01 into the Amycolatopsis.（5）Analysis of fermentation performance of mutant strains.The comparison of the fermentation performance between the original strain and the mutant strain showed that the vanillin concentration of the mutant strain was 9.2 g·L^-1,which was increased by10.3%,the byproduct concentration was 154.0 mg·L^-1,which was 10.3%higher than that of the original strain,and the molar conversion rate was increased from 88.6%to97.7%.The byproduct vanillic acid concentration was 154.0 mg·L^-1,decreased by 9.4%.This study provides an effective tool for further metabolic engineering of vanillin-producing Amycolatopsis.