Improvement L-ornithine Production By Promoter Sequence Replacement

L-ornithine is a non-essential amino acid widely used in food,pharmaceutical and other fields.Corynebacterium glutamicum is the main producing bacterium of L-ornithine.N-acetyl-?-glutamyl-phosphate reductase?argC?is the first enzyme of the argCJBD gene cluster in the L-ornithine biosynthetic pathway in C.glutamicum.Overexpression of argC enhances the L-omithine terminal biosynthesis pathway,thereby increasing the flux of the L-omithine biosynthesis pathway,resulting in accumulation of L-ornithine and an increase in yield.The L-ornithine yield of the wild-type strain of C.glutamicum was 8.86±0.15 g·L-1.After replacing the high-intensity promoter sequence,the L-ornithine yield reached 10.50±0.03 g·L-1,which was 118%of the wild-type.In this study,sixteen different intensity promoter sequences were screened by proteomic abundance values of fermentation of C.glutamicum S9114 for 12 h.The eGFP were linked to the promoter sequences and expressed by plasmids in C.glutamicum.The fluorescence intensities were measured in the range of 52.0 RFU/OD to 5966.8 RFU/OD,and the fluorescence intensity varied more than 100 times,the strongest promoter sequence has a promoter strength 3.3 times that of the Ptac promoter.Eight promoter sequences were selected to replace the promoter sequence of the argC gene and the replacement strains were subjected to fermentation culture and yield determination.The results showed that the yield of L-ornithine increased from 0.87±0.04 g·L-1 to 10.50±0.03 g·L-1 as the intensity of the promoter sequence increased.The replacement strain of the strongest promoter sequence Pc624₀5210 was 118%of the wild type in the L-omithine production.In this experiment,the promoter sequences of different intensity in C.glutamicum S9114 were screened and validated,which provided regulatory elements for precise control of gene expression in metabolic engineering and laid a solid foundation for the increase of L-ornithine production.The rational design of the enzyme can use a few bases of mutation to bring about a huge change in the characteristics of the enzyme.This method of controlling the metabolism through minimal modification of the genome has broad application prospects.In order to lay the foundation for the minimally mutated metabolic engineering,this study carried out a rational design experiment of enzyme.In this study,computer-aided simulation mutagenesis was used to improve the esterase activity.Two positive mutants,P218R and A242R,were obtained with 2.5 and 2.1 folds higher than the WT Est3563 esterase.With the help of structural analysis,it was found that the mutations could deteriorate the seal of the binding pocket and expose the active site.Thus,the catalytic efficiency of the mutants became higher.This study provides a viable strategy to enhance enzyme activities.