A Preliminary Study In Optimization Oxygen-supplying Strategy Of L-arginine Production By Corynebacterium Crenatum

L-arginine is one kind of nonessential alkaline amino acids in human and animal bodies; it is also a critical material in the process of protein creatine synthesis, and plays a major role of intermediate metabolite in urea circulation, which has unique functions in physiology and pharmacology.Dissolved oxygen is usually nutrition and environmental factors for the aerobic fermentation of L-arginine, and has a significant impact on the bacterial growth and the formation of products. There are two ways to solve oxygen supplyment problem, one is to improve the dissolved oxygen level in fermentation process, the other one is to improve utilization rate of cell by genetic technology.The effect of oxygen supply level on fermentative charictorisics and metabolic flux distribution, by controlling different agitations on the performance of producing L-arginine using Corynebacterium crenatum SYPA5-5 was investigated in batch fermentation process. It indicated that: There is a higher requirement of oxygen supplyment in producing L-arginine by fermantation. In the fermantation, mass oxygen is consumed to maintain cell metabolism and L-arginine production. Otherwise; insufficient oxygen supplyment may lower the production of L-arginine.An overall enhanced L-arginine production could be obtained under the high oxygen supply (HOS) condition. However, during later production phases, a much more stable L-arginine production was achieved under the medium oxygen supply (MOS) condition rather than under the HOS condition. As a result, a two-stage oxygen supply strategy, which maintained HOS condition during early fermentation phase, and then reduced agitation step-wisely to keep a stable, smooth and moderate DO changing profile throughout the production phases, was proposed. With this proposed control strategy, the final L-arginine concentration was largely increased and reached to a high level of 36.6 g/L, which was 16% and 51% higher than those obtained under the HOS and MOS conditions. The two-stage oxygen supply strategy could also accelerate glucose consumption rate and thus shorten fermentation time under the same initial fermentation condition.C. crenatum SYPA5-5 is a gram-positive strain, which is used in industry area.There is no system used to express C. crenatum especially in the literature we had consulted. In this work, we use cat as a reporter gene to construct an expression system which has different promoters successfully. After the research in activity of E.coli and C. crenatum promoters, we have found that tac promoter had higher promoter strength both in E.coli and C. crenatum. Secondly, there is lacZ promoter and T7 promoter at the last.So we use tac promoter in further study as the results above.An expression vector pJC1-tac-vgb, which carries VHb gene from Vitreoscilla has been constructed in our research successfully and transducted to E.coli JM109 and C. crenatum SYPA5-5. We use the carbon monoxide differential spectrometry to determine the activity of VHb which is expressed by recombinant strain. The fermentive character of SYPA5-5 (pJC1-tac-vgb) is also primary analysed. The result shows that the acid producing ability of recombinant strain without IPTG added is improved by 13%, and also, the strain concentration is improved by 10.9%.