A vanillin producing Amycolatopsis sp. with the VDH gene knockout by CRISPR-Cas9 technology

Abstract:

[Objective] In order to reduce the fermentation by-product vanillic acid, we established a CRISPR-Cas9 gene editing system for Amycolatopsis sp. to knock out the vanillin dehydrogenase gene (VDH). [Methods] Using VDH as the target gene, we replaced the promoters of tipA and j23119 on the pKCcas9dO plasmid with the Km^r promoter of the pRLE6 plasmid and the strong promoter permE* commonly used in Streptomyces, respectively. At the same time, we constructed the plasmid pKCKmCas9VDH by replacing the sgRNA with the specific sgRNA sequence that could recognize the target gene vanillin dehydrogenase, and linked the plasmid with the upstream and downstream homologous arms of the target gene to construct the knockout plasmid pLYZYP01. Then we transformed pLYZYP01 into Amycolatopsis sp. CCTCC M 2011265 and selected the VDH knockout mutant strains. [Results] We successfully obtained a knockout strain Amycolatopsis sp. ΔVDH with higher yield of vanillin. [Conclusion] We established a knockout system for Amycolatopsis sp. CCTCC M 2011265, and successfully knocked out the VDH gene. Under the condition of adding 12 g/L substrate ferulic acid, the yield of vanillin reached 9.19 g/L, and the conversion rate increased from 88.6% to 97.7%.