Metabolic Engineering Of Corynebacterium Glutamicum For Improved Cofactor Supply And For L-arginine Synthesis

L-arginine,a semi-essential amino acid with a variety of special physiological and biochemical functions,is widely used in medicine,food,health products etc.Corynebacterium glutamicum SNK118 is an L-arginine industrial production strain obtained through multiple mutagenesis screening,and could produce 40.47 g·L^?1 arginine in 5-L fed-batch fermentation.To better undertstand its genetic background,the whole genome of C.glutamicum SNK118 was sequenced.Furthermore,based on the L-arginine synthesis pathway and regulatory mechanism,cofactor engineering of C.glutamicum SNK118 were implemented to improve its L-arginine production.?1?Nicotinamide Adenine Dinucleotide Phosphate?NADPH?is a key coenzyme in the L-arginine production pathway.Two NADP⁺/NADPH producing genes,pntAB?encoding pyrimidine nucleotide transhydrogenase?and ppnK?encoding NAD⁺kinase?,were co-expressed to increase the intracellular NADPH pool,resulting in strain JML03.After 80 h of fed-batch fermentation in 5-L bioreactor,JML03 strain allowed production of 61.13 g·L^?1 of L-arginine with yield up to 0.34 g·g^-1 glucose.The titer and the yield of L-arginine was increased by 47%and 78.9%than those of control strain JML00?harboring pXMJ-19?,respectively;?2?To remove genes encoding for repressors in L-arginine synthesis pathway,argR and farR,encoding strict regulatory repressors,were consecutively knocked out by Cre/loxP-assisted-homologous system and the resulted strains?JML04 and JML05?displayed higher L-arginine production.The titer of L-arginine was increased by 19.2%and 28.4%respectively compared with the original strains.Furthermore,the competitive branch pathway was blocked by knocking out ldh gene?encoding lactate dehydrogenase?and its L-arginine titer was further increased.In fed-batch fermentation,strain JML06 allowed production of 52.70 g·L^–1 of L-arginine with yield of 0.31 g·g^-1 glucose;?3?Finally,strain JML07 was constructed by transforming plasmid harboring pntAB and ppnK into strain JML06.After 75 h of fermentation,67.01 g·L^–1 of L-arginine with a volumetric productivity of 0.89 g·L^–1·h^-1 and yield of 0.35 g·g^-1 glucose was achieved by JML07.In comparison,C.glutamicum SNK 118 could only produce 40.47 g·L^–11 arginine with a yield of0.24 g·g^–11 and productivity of 0.56 g·L^–1·h^-1 after 72 h.This sutdy provides a high L-arginine production strain,as well as an effective cofactor manipulating strategy for promoting the synthesis of NADPH-dependent metabolites;?4?To provide guidance for reasonable selection of gene knockout systems,the advantages and disadvantages of CRISPR-Cpf1 and Cre/loxP-recombination knockout are evaluated.Gene argR of C.glutamicum ATCC 13032 was knocked out by CRISPR-Cpf1 and Cre/loxP-recombination knockout systems respectively.Compared with Cre/loxP system,CRISPR-Cpf1is time-and labor-saving,and theoretically,it can be employed in multiple gene knockout at a time.Consequently,CRISPR-Cpf1-assisted system is overall more efficiencient.However,its homologous recombination efficiency could be further improved.