Establishment Of Genetic Manipulation Method And Construction Of Genetic Manipulation Tool For Corynebacterium Stationis

Corynebacterium stationis is a non-spore-forming Gram-positive bacterium isolated from infant feces and has long been used in the fermentation of vitamin B12,5′-inosinic acid(IMP)and other products,and is an important industrial microbial chassis.As a non-model and difficult genetic manipulation strain,there are few reports on its development,therefore,the aim of this study is to optimize the transformation of C.sta into an efficient microbial cell factory through metabolic engineering,based on which there is an urgent need to establish relevant genetic manipulation methods and manipulation tools in C.stagnans.We used C.sta ATCC 6872 as a starting strain to establish an exogenous plasmid transformation method and knock out the restriction-modificaton system(RM system)related genes in the cells to improve the transformation rate of heterologous plasmids.Tools for strain transformation were provided through the construction of heterologous gene expression vectors,promoter libraries and CRISPR-Cas(Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein)based gene editing toolboxes.The main studies and results are as follows:(1)Firstly,to establish a heterologous plasmid transformation method for C.sta,we chose to perform heterologous gene transformation by electroporation transformation,and prepared receptor cells by optimizing the formulation of electrotransformation receptor medium,adding glycine and isonicotinic acid hydrazide to the receptor medium to weaken the cell wall and increase the cell wall fluidity with the maximum tolerance concentration of these two substances in C.sta,and we also optimized other factors that affect the efficiency of electrotransformation Other factors affecting the electrotransformation efficiency such as electric field strength and replication medium were optimized.Finally,we performed electrotransformation with 1.8 Kv voltage,220Ωresistance and 25μF capacitance,and selected LBHis medium with high osmotic pressure as the replication medium,and successfully increased the heterologous plasmid transformation efficiency to 2.3±0.3×10~3cfu/μg DNA electrotransformation efficiency.In addition,we subjected the plasmid to methylation modification by multiple transformation extraction,and the transformation efficiency could be enhanced approximately 3.3-fold by this method.For the knockdown of RM system-related genes in C.sta and measuring the transformation efficiency,the knockdown of AW169＿06220 gene increased 4.9-fold compared with the wild type,and the knockdown of AW169＿03655 gene increased8.2-fold compared with the wild type,and the combined knockdown increased the transformation efficiency 14.8-fold,and the transformation efficiency of C.sta was increased to 3.1±0.1×10~4cfu/μg DNA by the above RM system-related gene knockdown.(2)Secondly,shuttle Shuttle plasmids are important tools for genetic manipulation,and we constructed expression vectors in C.sta that can be genetically replicated stably in the cell and can express heterologous genes.We constructed the IPTG-inducible shuttle plasmid p ZLGZ with the p CG1 replicon of the p CG1 family and used it to express the green fluorescent protein gene for testing.The small number of promoters reported in C.sta and the large differences could not meet the demand for metabolic modification.We used the promoter sequence of Corynebacterium.glutamicum ATCC 13032 pyruvate carboxylase Ppyc as a template to fix its-35 and-10 regions to construct an artificial promoter plasmid library with green fluorescent protein as a reporter gene,and transformed this plasmid library into C.sta,and obtained through multiple rounds of screening A series of promoter libraries with 30-fold intensity difference were obtained through multiple rounds of screening,and the promoter libraries were sequenced.This study provides genetic manipulation tools and expression elements for metabolic engineering of C.sta.(3)Finally,for targeted modification of C.sta,CRISPR-based genetic manipulation tools were constructed in this bacterium,including CRISPRi(CRISPR interference),Target-AID(Target activation-induced cytidine deaminase),and CRISPR/The CRISPRi tool is a gene repression tool,and we eventually validated the functionality of this tool in C.sta by interfering with exogenous green protein genes and endogenous genes related to carotenoid synthesis.We used this system to validate the intracellular AW169＿03655 gene,which is more than 70%efficient in editing bases on the intracellular genome and easy for plasmid loss.We constructed this tool and modified it to reduce the expression of Cas protein through the optimization of induction system and ribosome binding site,and to reduce cytotoxicity and improve cell survival rate.The recombinant enzyme expression was regulated by rec ET,and the recombinant enzyme functionality was verified by using streptomycin resistance as a screening marker.