Metabolic Engineeirng Of L-arginine Biosynthesis By Corynebacterium Crenatum SYPH5-8

L-Arginine (L-Arg) is a conditionally essential amino acid with various physiologicalfunctions, which is widely used in the fields of pharmaceutical, food and chemical industry.Corynebacterium crenatum SYPH5-8was an L-arginine producer obtained from multiplemutation-screening steps and could produce high concentration of L-arginine using glucose.This study aimed to engineer the limiting steps of L-arginine biosynthetic pathway inSYPH5-8to strengthen metabolic flux toward L-arginine and explore the related regulatorymechanism. The main results were described as follow.(1) The activity of N-acetylglutamate kinase of SYPH5-8(CcNAGK, argB) withaddition of L-arginine was detected in vitro. The concentration of L-arginine needed for50%inhibition (I0.5) was1.36mM and the residual activity was only3.0%after adding5mML-arginine. Although SYPH5-8was obtained from multiple mutation-screening of variousL-arginine analogs, CcNAGK was also inhibited by L-arginine. The14target mutation siteswere designed based on alignment of the amino acid sequence of CcNAGK with thehomologous protein sequences from L-arginine sensitive NAGK which L-arginine bindingsites were analyzed and verified. The mutants were constructed and screened respectively invitro. Neither H268N nor R209A significantly affected the specific activities of CcNAGK.Meanwhile, the I0.5of the two mutants increased about40-fold when compared with wild typeand residual activities after adding15mM L-arginine was more than99.0%respectively.These results indicated that the point mutations of H268N or R209A in CcNAGK deregulatedits feedback-inhibition to L-arginine.(2) We introduced the mutations of H268N and R209A into the chromosome ofSYPH5-8respectively based on homologous recombination. The recombinantsSYPH5-8-NAGKR209A(designated as R-8) and SYPH5-8-NAGKH268N(designated as H-7)were obtained from screening. The biomass and L-arginine production were lower than theirparental strain over the fermentation course. Surprisingly, a large amount of L-citrulline andL-ornithine, as the intermediates of L-arginine biosynthetic pathway, was accumulated. Toanalysis the machnism of L-citrulline and L-ornithine accumulation, transcription levels ofarg gene cluster were determined in two recombinants, using SYPH5-8as control.Unexpectedly, the transcription levels of argG and argH decreased remarkably with thespecific activities of argininosuccinate synthase (ASS) and argininosuccinase (ASL) alsodecreased. The lower transcription levels of argGH might be the reason for intermediatesaccumulation.(3) To overexpress argGH genes, the recombinant plasmid pDXW-10-argGH wasconstructed and transformed to H-7and R-8to obtain the recombinants R-8-argGH(designated as R-8-GH) and H-7-argGH (designated as H-7-GH). H-7-GH showed about1.4-fold increase in L-arginine production, about82.8%reduction in L-citrulline formationand about79.3%reduction in L-ornithine formation, when compared with H-7. R-8-GHshowed similar effect to H-7-GH. Compared with SYPH5-8, the fermentation time ofH-7-GH reduced16h, L-arginine yield on glucose improved84.6%and the overall productivity improved1-fold. Furthermore, the two by-products L-lysine and L-isoleucinedecreased51.7%and53.4%respectively. By increasing concentration of yeast extract to14g/L and adding8g/L beef extract significantly improved L-arginine production. The biomassand L-arginine concentration was19.9g/L and45.1g/L, which improved41.1%and49.8%respectively after culturing64h when compared with minimum medium. The fed-batchstetagy of carbon and nitrogenous source was also optimized. The biomass and L-arginineproduction was22.7g/L and51.7g/L respectively after culturing80h with optimized stetagy.