Construction And Fermentation Optimization Of The Recombinant Corynebacterium Crenatum Coproducing L-arginine And Poly-β-hydroxybutyrate

L-arginine is a kind of semi-essential amino acid. With its unique physiological and pharmacological effect, L-arginine is adopted in food, medicine and health products field widely. Production of arginine by fermentation has drawn much attention at home and abroad in recent years. Polyhydroxyalkanoates(PHAs) are a kind of thermoplastic polyesters stored in the cells as a source of carbon and energy storage by prokaryotic microorganisms under the imbalance of carbon and nitrogen. And poly-β-hydroxybutyrate(PHB), which is found earliest and researched the most, is one of the PHAs family. Thanks to its properties such as biodegradability and biocompatibility, PHB has wide application and has already preliminary entered the stage of commercial production at present. As reported, introducing the metabolic pathways of PHB into cells resulted in the coproduction of PHB and some chemical products with more production, such as succinate, L-glutamate and L-tryptophan. All of these improved the utilization rate of substrate. NAD kinase is an important enzyme which can adjust coenzyme I and II metabolism balance in bacterial. Overexpression of NAD kinase in strains can effectively increase the intracellular NADPH / NADP+, then promote the corresponding NADPH-dependent product yield. Corynebacterium crenatum SYPA 5-5 is an industrial strain for L-arginine production. Using this strain, we introduced the entire phb CAB operon of R. eutropha H16 into the cells to coproduce PHB and L-arginine simultaneously and overexpressed NAD kinase to regulate the intracellular coenzyme level, then investigated the effect of the yield of the two products. The main results of this research are as fallows:(1) Adopting genetic engineering technique, we introduced the entire phb CAB operon of R. eutropha H16 into the E. coli-Corynebacterium shuttle expression vector p DXW-10 to construct the recombinant plasmid p DXW-10-phb CAB. It was transferred into E. coli and expressed well. Then it was transferred into C. crenatum SYPA 5-5 to obtain the recombinant SYPA 5-5/p DXW-10-phb CAB. After assay of PHB synthesis enzyme activity, PHB content and transmission electron microscope, PHB accumulated obviously in the recombinant cells. Batch fermentation in 5-L fermentor showed that PHB content in the recombinant strains accounted for 12.8 % of DCW at 96 h, while only 2.1 % of the parrent strains. In the broth, L-arginine yield was 43.21 g/L, increased by 20.5 % comparing to the parrent strains. In C. crenatum SYPA 5-5 the coproduction of L-arginine and PHB was achieved and the accumulation of PHB in cells could promote the L-arginine production.(2) During the biosynthesis of L-arginine and PHB, the coenzyme NADPH both was necessary. So based on SYPA 5-5/p DXW-10-phb CAB, overexpressing of homologous NAD kinase to regulate intracellular coenzyme levels was tried out and then examined the effects on the two products yield. By genetic engineering technique, the recombinant SYPA 5-5/p DXW-10-phb CAB-ppn K was constructed. After assay of PHB synthesis enzyme activity, PHB content and transmission electron microscope, PHB content was further increased. 5-L fermentor batch fermentation experiments showed that comparing to the parrent strains(2.1 %), PHB content in SYPA 5-5/p DXW-10-phb CAB-ppn K reached 14.5 % of DCW at 96 h. Comparing to the parrent strains, L-arginine yield was 45.49 g/L(increased by 26.8 %). Analysing transcription level of key genes in L-arginine biosynthesis pathway, we found that PHB accumulation and NAD kinase overexpression could enhance the transcription and expression of several genes. Finally, carbon source concentration, C:N ratio, stirring speed and the fermentation strategy were optimized using SYPA 5-5/p DXW-10-phb CAB-ppn K in 5-L fermentor. The results: carbon source concentration 180 g/L, C:N ratio 9:1, stirring speed 600 rpm, the optimized L-arginine production reached 48.07 g/L, PHB content accounted for 15.7 % of DCW, which was significantly higher than that before optimization. Fermentation time was prolonged in fed-batch fermentation and the yields of two products were further enhanced.