Proteomic Research Of L-arginine Hyper Producing Corynebacterium Crenatum In Different Oxygen-supplying Strategy

L-Arginine, a substantial intermediate metabolite in ornithine cycle, is one kind of semi-dispensable amino acids. It is also a critical material in the process of protein and creatine phosphate synthesis, and has significant effect in maintaining normal physiologic function in organism. Therefore, L-arginine has numerous applications in pharmaceuticals, food flavor and cosmetics.The stain Corynebacterium crenatum SYPA5-5, researched in this thesis is an L-arginine hyper-producing mutant strain stored in our lab. Preliminary study showed that C. crenatum SYPA5-5 was an aerobic bacterium, that inadequately supplying of oxygen not only inhibited cell growth, but also severely inhibited the synthesis of L-arginine. There are two ways to solve oxygen supplying problem. The one is to improve the dissolved oxygen level during fermentation process, and the other is to improve utilization rate of cells by genetic technology.In this article, we established two oxygen supplying models and detected their fermentative characteristics. Proteomic approaches including two-dimensional electrophoresis (2-DE) and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to analyze differential expression of proteins in C. crenatum in different oxygen supplying conditions during fermentation process. The results showed that 109 different protein points were detected in the pH4-7 gels. Database research showed that the proteins performed different functions in carbohydrate metabolism, amino acid metabolism and energy metabolism, etc. Most of the differential expressed proteins concentrated in the pentose phosphate pathway, TCA cycle and the urea cycle, especially in the pentose phosphate pathway. The result shows consistent with preliminary study.The HMP not only regulates glucose metabolism, but also provides NADPH for cells. Therefore, the enhancement of HMP pathway provides more NADPH to alleviate oxygen toxicity and to facilitate synthesis of L-arginine. So, in this work, we constructed an expression vector pJC1-tac-ppnK, which carried the gene of NAD kinase, and successfully transducted it into E.coli JM109 and C. crenatum SYPA5-5. The fermentive characteristics of C.creatum (pJC1-tac-ppnK) were also primary analyzed. The result showed that the L-arginine producing ability of recombinant strain has improved from 45.17 g·L^-1 to 52.25 g·L^-1 and the YP/S has also improved from 33.48% to 36.62%.