Metabolic Engineering Of Corynebacterium Crenatum SYPA 5-5 For The L-arginine Production

Corynebacterium crenatum SYA 5 is an aerobic, Gram-positive, non-sporulating coryneform bacterium and the mutant C. crenatum strain SYPA 5-5 could produce more L-arginine than its WT strain SYA 5. Strategies for developing amino acid producers are now in transition towards systems metabolic engineering from random mutagenesis. Recently, several amino acid producers have been successfully developed by systems metabolic engineering. There are some efficient strategies and elevations to improve the L-arginine producing ability. The main results were described as follows:(1) The genes within the cluster of genes involved in arginine biosynthesis are organized as two separate parts, which are argCJBDFR and argGH operons that encode all of the enzymes required to convert glutamate to arginine via ornithine in Corynebacterium sp. The corresponding argCH genes and the argCH 9039 cluster for arginine biosynthesis were cloned and expressed in E.coli BL21 (DE3) and the origin strain C. crenatum SYPA 5-5. Then the corresponding enzymes were purified by affinity chromatography and the enzymatic characterizations were determined. The genes argC, J, B, D, F, G, H were over-expressed in C. crenatum SYPA 5-5. These results exhibited that the L-arginine producing abilities of the recombinant strains were improved by 9.3%-23.0%. Nevertheless, when the argCH 9039 cluster was over-expressed in C. crenatum SYPA 5-5 under its native promoter Parg, the L-arginine production was increased by 40.9% in the fask fermentation. In the 5 L jar, the L-arginine yield was 45.9 g/L, which is increased 26.5% than the strain SYPA 5-5. Surprisingly, the results demonstrated an increasing utilization of oxygen and the distinct enhancement of unit cell L-arginine yields with the cluster argCH-bearing in C. crenatum SYPA 5-5-9039.(2) Working with the genomic data of C. glutamicum ATCC 13032, we investigated the evolution of the arginine biosynthesis gene cluster of C. crenatum SYA 5. However, the argCH cluster of the C. crenatum SYPA 5-5 which is an industrialized L-arginine producer had a lethal mutation occurred in the ArgR repressor encoding gene. Additionally, the arginine repressor ArgR, the product of the argR gene, acts in conjunction with L-arginine to control the synthesis of arginine biosynthesis enzymes. The arginine hyper-production strain was examined for elevated arginine biosynthesis through the inactivation of the ArgR repressor by constructing an argR-auxotrophic mutant of C. glutamicum. The inactivation of ArgR resulted in increased enzyme activities involved in arginine biosynthesis and increased L-arginine production in C. crenatum SYPA 5-5. In contrast, constructing a strain C. crenatum SYPA 5-5-TR with overexpressing argR, a complete and functional ArgR decreased the expression of enzymes, depressed transcriptional level of the argCH cluster, and reduced the production of L-arginine in C. crenatum. In this study, we analyzed the cellular response of C. crenatum to the repressor ArgR using 2-DE and MS-MS. ArgR expression of 29 proteins from C. crenatum, whereas 22 proteins were repressed and 7 proteins were induced, respectively, with the presence of L-arginine. The relational level of proteins, enzymes involved in the arginine biosynthesis metabolism, as well as the other amino acids biosynthesis (such as L-valine, L-isoleucine, L-lysine, etal.), the tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP).(3) Arginine biosynthesis in C. crenatum SYPA 5-5 proceeds via a pathway that is controlled by arginine through feedback inhibition of NAGK, the enzyme that converts N-acetyl-L-glutamate (NAG) to N-acety-L-glutamy-L-phosphate. The gene argB encoded Ccre_NAGK was site-directed and the L-arginine binding sites of feedback inhibition were described. The N-helix and C-terminal residues were firstly deleted and the results indicated that they are both necessary for Ccre_NAGK. And the impact on these functions of 11 site-directed mutations (E19A,E19R,W23A,H26E,H268A,H268N,R209A,R209K,E281A,G287A,G287D) affecting 7 residues of Ccre_NAGK were studied, chosen on the basis of homology structural alignment. Consequently, we have performed site-directed mutagenesis of the key enzyme (NAGK) and the three mutations (E19R, H26E and H268N) exhibited the increase of I_0.5^R efficiently. To get a feedback-resistant and robust L-arginine producer, the multi-mutated NAGK36 (including E19R/H26E/H268N) was generated the engineered strains SYPA 5-5-CCB36 were constructed. The L-arginine synthesis was largely enhanced due to the overexpression of the argB36 which is resistant to feedback resistant by L-arginine. Thus L-arginine production could reach 45.6 g/l, about 25.7% higher compared with the initial strain. This is an example of up-modulation of the flux through the L-arginine metabolic pathway by deregulating the key enzyme of the pathway.(4) Then the proB, glnA and lysC genes was deleted, respectively, through the method of two homologous recombinations in C. crenatum SYPA 5-5. Distinguishedly, when the glnA or lysC genes was deleted, the biosynthesis of L-glutamine or L-lysine were destructed. Instead of the increased L-arginine production, the L-arginine biosynthesis was decreased. However, when the proB was deleted, the resulting strain, C. crenatum SYPA 5-5â–³proB, the L-arginine was accumulated up 36.0% with a low rate of L-proline formation compared with them in the C. crenatum wild type SYPA 5-5. Therefore, L-arginine metabolic flux from glutamate could be improved only by deleting the proB of the proline in the central metabolism.(5) To get a hyper production L-arginine strain, the forward effective metabolic engineering of C. crematum SYPA 5-5 for L-arginine production were recombined together. Consequently, the argB gene involved in the cluster argCH 9039 was site-directed and the recombinant plasmid pJC-9039SDB was constructed into the recombinant C. crenatum SYPA 5-5â–³proB. The characteristics of the recombinant C. crenatum SYPA 5-5â–³proB-9039SDB are resistant feedback inhibition, overexpressed of L-arginine biosynthesis and distructed of the L-proline biosynthesis. After the development of the recombinant hyper strain, the distribution of the metabolic was analysed and compared with the WT strain by flux analysis. The superiority of the C. crenatum SYPA 5-5â–³proB-9039SDB was that the flux of the L-arginine biosynthesis was increased directly in response to over expression of genetic metabolic modifications. Through strain engineering formation of proline was reduced due to deletion of its key enzyme coding gene proB. Overall, the arginine yield increased up to 38.2% through the combination of the different genetic modifications. The yield of L-arginine was 50.12 g/L and the rate of the convertion from L-glucose to arginine was 33.4%. Additionally, the results revealed an increase arginine unit yield per cell overall a reductive biomass resulted. Our strategy not only successfully improved arginine production by genetically modified C. crenatum strains but also revealed new constraints in attaining high productivity.