Efficient Production Of Natural Vanillin By Amycolatopsis Sp. Using CRISPR-Cas12a Gene Editing System

Vanillin,known as the"Queen of Flavors",is one of the most widely produced synthetic flavors in the world and has a wide range of applications in the food,beverage,chemical,fragrance and pharmaceutical.Microbial production vanillin has the advantages of green,efficient and sustainable.The rapid development of CRISPR technology has provided many molecular biology tools for Amycolatopsis sp.,and the establishment and expansion of the Amycolatopsis sp.CRISPR gene editing system is of great value for the construction of highly productive strains.In this study,the vanillin-producing Amycolatopsis sp.CCTCC NO:M2011265 was used as the starting strain.In order to further improve the production of vanillin,the genetic transformation conditions of Amycolatopsis sp.was optimized,a CRISPR-Cas12a gene editing tool was established,and the high-yield vanillin mutants were obtained by metabolic engineering,as well as the fermentation performance of mutants was investigated.Details are as follows:（1）Development of gene editing tools for Amycolatopsis sp..Firstly,the genetic transformation conditions of Amycolatopsis sp.were optimized,and the results showed that the optimal conditions were shuttle plasmid p ULcr RNA demethylation,preparation of competent cell with OD₆₀₀between 0.6 and 0.8,and electroporation conditions of 1800 V for 5 ms.Secondly,the integration plasmid p DZLCas12a was used to assess the non-toxic effect of Cas12a nuclease on the growth of Amycolatopsis sp..Next,a p ULΔvdh plasmid was constructed using the vdh gene as the target gene repair-free template,and it was verified that the Cas12a nuclease was able to target cleavage of the target gene.（2）High yield vanillin mutant strains were obtained by metabolic modification of Amycolatopsis sp.using CRISPR-Cas12a gene editing technology.The genes encoding vanillin dehydrogenase（vdh）and HMPHP-Co A dehydrogenase（phd B）were identified as target genes for gene deletion through analysis of the metabolic pathway of ferulic acid.Firstly,the plasmid p ULΔvdh LR was constructed and successfully obtained the deletion vdh gene WGN01mutant with an editing efficiency of 21.4%after electroporation into the Amycolatopsis sp..Then,the plasmid p ULΔphd BLR gene was constructed by replacing the promoters expressing Cas12a and cr RNA with kas O*p and gapdh,using WGN01 as the starting strain,and successfully obtained vdh and phd B genes deletion WGN02 mutant with 58%efficiency of phd B gene deletion,and also showed that strong promoters to enhance Cas12a and cr RNA expression could improve the gene editing efficiency.The p ULwgn03 plasmid,in which overexpresses the key gene of vanillin synthesis（ech-fcs-ech2）,was further constructed and transferred into WGN02,and the WGN03 mutant was successfully obtained.（3）Comparison of vanillin production performance by fermentation of mutant strains.The results of shake flask fermentation showed that vanillin yield of WGN01mutant increased by25.9%compared to the wild type,and the concentration of vanillic acid as a by-product decreased by 80.3%.The vanillin yield of WGN02 mutant was 8.0%and 36.2%higher than that of WGN01 and the wild type,respectively;the by-product vanillic acid also decreased by2.25%and 84.8%,respectively;the vanillin yield of WGN03 mutant was 14.2%and 52.5%higher than that of WGN02 mutant and the wild type,respectively,and the fermentation time was reduced by half.Further,fed batch fermentation was carried out on a 3 L fermenter and the results showed that WGN03 mutant produced 21.87 g·L^-1vanillin at 44 h,and the molar conversion rate was 93.0%,while wild type,WGN01 mutant and WGN02 mutant produced10.6,14.6 and 20.4 g·L^-1 vanillin at 66 h,respectively.The fermentation time of WGN03 mutant was 22 h shorter than that of wild type,WGN01 mutant and WGN02 mutant,and the vanillin yield of WGN03 mutant was 107.3%,49.7%and 7.2%higher than that of wild type,WGN01mutant and WGN02 mutant,respectively,showing a good industrial production prospect.