利用CRISPR-Cas9技术构建敲除VDH的产香兰素拟无枝酸菌

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

【目的】建立适用于拟无枝酸菌(Amycolatopsis sp.)的CRISPR-Cas9基因编辑系统,敲除其编码香兰素脱氢酶基因(VDH),减少发酵副产物香草酸。【方法】以VDH为靶标基因,将pKCcas9dO质粒上的tipA、j23119启动子分别替换为pRLE6质粒Kmr启动子、链霉菌中常用的强启动子permE*,同时将sgRNA替换为能识别靶基因香兰素脱氢酶的特异性sgRNA,获得质粒pKCKmCas9VDH。然后将其与靶基因的上下游同源臂连接,获得敲除质粒pLYZYP01。将pLYZYP01质粒电转进Amycolatopsis sp.感受态细胞,筛选获得VDH的敲除突变体菌株。【结果】利用上述方法,成功获得VDH敲除菌株Amycolatopsis sp.ΔVDH。【结论】建立了适用于拟无枝酸菌CCTCC M 2011265的基因敲除系统,成功敲除VDH基因,在添加12 g/L底物阿魏酸的情况下,香兰素产量达到9.19 g/L,摩尔转化率由88.6%提高到97.7%。

[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 Kmr 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%.