The Feedback Inhibition Mechanism In L-arginine Synthesis Pathway Of Corynebacterium Glutamicum ATCC 14067 And Related Biosynthesis Pathway Metabolic Engineering

Corynebacterium glutamicum?C.glutamicum?is not only the workhorse of biotechnological amino acid production but also a model organism to study the metabolic regulation mechanism within the Corynebacteriales.Genome editing technology plays an essential role in current biotechnology,whereas the current general gene editing tools are still limited in C.glutamicum ATCC 14067.In C.glutamicum the L-arginine synthesis pathway is feedback inhibition by the final product of the route,L-arginine,to avoid the over-production of L-arginine in vivo.Since the allosteric regulation of NAGK activity is directly controlled by its product L-arginine,efforts have been made to prevent or alleviate the feedback inhibition.However,the mechanism of NAGK deregulation remains unresolved.This study focused on the L-arginine synthesis pathway by C.glutamicum ATCC 14067.We employed the RecET recombineering system for dsDNA recombination,and besides we developed an effective and simple gene editing system in C.glutamicum,firstly.Secondly,the allosteric regulation mechanism of CgNAGK was explored.Finally,the L-arginine biosynthesis pathway was engineered for production of L-arginjne based on the above study.The specific research content and results can be summarized as follows:?i?RecET assisted gene markerless deletion system.Several orthologous exonuclease-recombinase pairs were selected to test the recombineering activity in C.glutamicum,firstly.The RecET pair showed the highest recombination activity,and the recombination efficiency was enhanced 17 times by condition optimization,such as the homology length,dsDNA quantity,ds DNA phosphothiolated,recovery time and induction time of RecET.Besides,the gene replacement frequency can reach up to?94%via RecET.Finally,we developed a system containing a linear self-excisable dsDNA cassette and RecET system?RecET-Cre/lox system?for gene markerless deletion in ATCC 14067.This strategy provides a simple and effective strategy for C.glutamicum genomic markerless deletion.?ii?Feedback regulation mechanism of CgNAGK in L-arginine biosynthesis pathway.L-arginine synthesis is controlled by feed-back inhibition,exerted by the final product of the route,L-arginine.L-arginine sensitive NAGK owns an allosteric regulation region for the binding of L-arginine which is far away from the catalytic region.The monomeric CgNAGK mutations,V16G,V16E,L17G,L17R and A18K were obtained by homology modeling,analysis of the hydrophobic force between subunits and rational site-directed mutagenesis.The L-arginie inhibition kinetic curve fitting analysis and the specific activities analysis found that the monomeric mutants retained a similar sensitivity to L-arginine as the hexameric form,but lost the strongly cooperative nature of inhibition by L-arginine.Meanwhile,the monomeric one onle remain¹0%catalytic activity of the hexameric one.These results verified that hexameric architecture is required for the positive cooperativity of inhibition by L-arginine and for efficient catalysis,but that it is not the determinant of inhibition by L-arginine.To identify the amino acid residues of the N-helix required for inhibition by L-arginine,the N-helix was truncated to different lengths.Activity assays showed that the?2-13 and?14-17 mutants are as sensitive to L-arginine inhibition as wild-type hexameric CgNAGK,and the deletion mutants in 18-23 residues were more resistant to feedback inhibition.These results indicated that residues 18-23 in the N-helix of CgNAGK were required for the allosteic regulation.Except for that,the amino acid E19 was mutated and the results showed that the hydrophilic of E19 played an essential role in the allosteric regulation.The potential binding sites of arginine and the residues essential for its inhibition were identified by homology modeling,inhibitor docking,and sitedirected mutagenesis.Analysis of the mutant enzymes and docking analysis demonstrates that residue W23 positions the inhibitor of L-arginine,and the interaction between arginine and residues L282,T284 may play an important role in the forming of L-arginine binding cavity,and the H-bond between E281,L282,T284 and L-arginine is also essential for the allosteric regulation.Based on the results we propose a linkage mechanism for the remote allosteric regulation of NAGK activity,in which residue R209 may play an essential role.In addition to that,the mutation of the conserved residue,H268E,not only is arginine-insensitive but also increased the specific activity of CgNAGK by 7.8%.?iii?L-arginine biosynthesis pathway metabolic engineering.L-arginine biosynthetic pathway flux was increased through overexpression the arginine-insensitive mutation,H268E(NAGK_H268E),and replacement the native promoter of the L-arginine operon,argC and argGH,with eftu via the RecET-Cre/lox system,and amplification expression of argininosuccinate synthase?encoded by argG?and argininosuccinate lyase?encoded by argH?,which can enhance and optimize L-arginine biosynthesis pathway.Channeling carbon flux into TCA by overexpression anaplerotic enzyme,pyruvate carboxylase?encoded by pyc?,replacement of the native promoter of icd with eftu to enhance precursor?-ketoglutarate synthesis and increase L-arginine production.The final strain,GHCI-BGHP,which the L-arginine pathway was preliminarily engineering in this study,can produce 11.96 g/L L-arginine in fed-batch fermentation.