Development Of CRISPR-Cpf1 Genome Editing System In Corynebacterium Glutamicum

Corynebacterium glutamicum has wide applications in the industry for producing a variety of valuable products such as amino acids,organic acids and vitamins,etc.Although CRISPR-based genome editing technologies have been immensely developed in recent years,the predominant approaches for C.glutamicum genomic manipulation are still pk18mobsac B reverse selection gene editing system,which are complicated and time-consuming.In previous studies,we developed a one-step Rec ET-assisted CRISPR-Cpf1 genome editing technology for C.glutamicum.In the process,Cpf1 cleavage was found to disrupt Rec ET-mediated homologous recombination.In this study,we developed a two-step genome editing technology for C.glutamicum,Recombin Ation Prior to Induced Double-strand-break（RAPID）.Two compatible plasmids of p XMJ19 and p EC-XK99E were used in the RAPID genome editing system,in the p EC-XK99E plasmid expression Rec ET and Cpf1,and the p XMJ19 plasmid expression cr RNA and provide donor DNA molecules.Firstly,we optimized an inducible system for Cpf1 expression,determined IPTG induction system as the best strategy.Then we used sac B-mediated sucrose selection for plasmid curing and high efficiency.For the curing of p EC-XK99E derivatives,the gene encoding distribution protein Per1 was deleted to achieve its high-efficiency plasmid curing without antibiotics.Through the comparison of various operating procedure,we found that recombination between the donor DNA and chromosome completed during the recovery process.and optimized the recovery time and found that a 5 h recovery resulted in the highest gene deletion efficiency.Then,we optimized promoters for Rec ET expression,resulted in the highest gene deletion efficiency was almost 100%.We then investigated the integration of DNA fragments with different lengths,and found that RAPID technology achieved efficient integration of large fragments.The integration efficiency of the 1 kb DNA fragment reached 96%,which gradually decreased with the increasing length of DNA fragments.Surprisingly,a 4 kb DNA was integrated into the genome with an efficiency higher than72%,and a 5 kb DNA fragment could be successfully integrated with 24%efficiency.In addition,gene deletion and insertion were investigated using linear DNA fragments as the donor templates.The deletion efficiency was almost 100%,but integration was inefficienct.Therefore,we had to use a plasmid-borne DNA template for gene insertion,and a linear template for gene deletion.Owing to the transcribed RNA-processing capability of Cpf1 enabled simplified plasmid construction.In addition,recombination during the recovery process,this simplifies the operating procedure of the RAPID system.We used RAPID to modulate the C.glutamicum genome to construction of a series of D-Pantothenic acid producers.In this study,wild-type C.glutamicum ATCC 13032 was used as the starting strain.First,to enhance the synthetic pathway toα-ketoisovalerate,we integrated ilv B*N*C operon from C.glutamicum XV into the C.glutamicum genome.The ilv E and avt A were deleted,reducing the competition ofα-ketoisovalerate consumption.Subsequently,asp B and asp A_Eco were successively integrated into the C.glutamicum genome to increase the supply of the precursorβ-alanine.We deleted ilv A to blockα-ketobutyrate synthesis.The p Xtuf-pan BCD_Bsu plasmid was introduced into these strains to generate the D-Pantothenic acid producers.The final C.glutamicum strain produced18.62 g/L D-Pantothenic acid,which is currently the highest value obtained by microbial de novo synthesis from glucose withoutβ-alanine supplementation.