Corynebacterium Glutamicum ATCC 13032 Gene Knockout And The Exploration Of Point Mutation Methods Mediated By Recombination Engineering

Gene recombination is a molecular biology technique that can be used for gene cloning as well as gene modification.It is simple,efficient,and is of great value and significance for gene characterization.This thesis focuses on the Corynebacterium glutamicum ATCC 13032 gene deletion and recombineering-mediated point mutation.C.glutamicum ATCC 13032 and its derivatives are industrially important amino acid producer.Chromosomal gene deletion study will be helpful for gene characterization and protein function study.To overcome the low efficiency of current methods,we explore various systems and compare the knock-out efficiency,to establish an efficient and convenient gene editing method for Corynebacterium glutamicum ATCC 13032.Firstly the ribosome binding site before the I-Scel gene was adjusted for higher level gene expression.Though the targeting DNA fragment was successfully replaced the antibiotic resistance gene via recombineeering;however,the second antibiotic resistance gene removal step was not achieved via short homology arm.Secondly,by extending the homology arm to 1 kb,the scarless deletion was successfully achieved via suicide vector mediated gene deletion.The high deletion efficiency shows its potential as an effective deletion strategy.Another gene deletion system,the Cre/loxP-mediated gene deletion system,was preliminarily tested by constructing the Cre-inducible expression vector and crtI2 gene deletion vector.Lastly,IPTG-inducible recT-cas9 gene expression vector was constructed which will be used for single-stranded DNA assisted CRISPR/Cas9-mediated chromosomal gene editing.The second part of the thesis is the study of recombineering-mediated gene point mutation.The N-acetylneuraminic acid aldolase gene nanA was selected for the S208G and E192 saturation mutagenesis.Co-transformation of the nanA gene harboring plasmid pLS122 and the mutagenic DNA fragment in to recombineering-proficient Escherichia coli cells rendered the nucleotide mutations.By adjusting the molar ratio of 1:1 of the pLS122 and the mutagenic DNA fragment,the S208G mutation efficiency at the 5 sites increased from 44.2%to 79.73%.For E192 saturation mutagenesis,19 amino acids were successfully obtained;however the amino acid asparagines was not obtained through many trials,showing the limitation of the method.We found that the length of the homology arm had no obvious effect on the mutation efficiency,whereas the chromosome-based recombineering system was more suitable for point mutation.Further improvement is needed to make it a robust and feasible gene modification strategy.