Effects Of Folate Pathway On Accumulation Of L-serine In Corynebacterium Glutamicum SYPS-062

As one of the most important coenzyme in the degradation pathway of L-serine, folate also plays an important role in one carbon unit cycle and cell proliferation. In this paper, a preliminary study has been carried on the effect of folate metabolism on the growth and L-serine accumulation of Corynebacterium glutamicum SYPS-062, and approached the impact of expression of folate metabolism-related gene on C. glutamicum SYPS-062.Folate metabolism pathway in C. glutamicum SYPS-062 was regulated through both endogenous and exogenous regulation. Sulfamethoxazole, methotrexate, folate and PABA were added to investigate the function of folate metabolism to C. glutamicum SYPS-062. As endogenous regulation, antisense RNA and overexpression techniques were applied to regulate the expression of the key enzyme in folate synthesis. Disturbance of sulfamethoxazole and methotrexate revealed that they could depress the growth of C. glutamicum SYPS-062 and increase the yield of L-serine. In contrast, PABA accelerated the growth of C. glutamicum SYPS-062 while greatly decreased the L-serine production capacity. However, the growth of C. glutamicum SYPS-062 wasn't accelerated by folate during the addition concentration range, instead of overdosage folate depressed the growth of C. glutamicum SYPS-062. It indicated that C. glutamicum SYPS-062 with the capacity of de novo folate synthesis can't uptake exogenous folate. Moreover, the analysis of flux distribution certificated that folate metabolism as controllable pathway was very important in C. glutamicum SYPS-062 growth and L-serine production.For further investigation, we compared the differences of pabAB between C. glutamicum ATCC 13032 and C. glutamicum SYPS-062. The sequence homology of pabAB in these two strains is 99.14%, 16 base pairs differences that resulted in the changes of 7 amino acids were found. The expression of pabAB was down-regulated by antisense RNA technique, the results show that slower growth rate and higher L-serine output have occurred in the genetic engineering bacteria.The pabAB genes from different sources were expressed in Escherichia coli, and the specific activity of aminodeoxychorismate synthase that pabAB encode were measured. Specific activity of aminodeoxychorismate synthase in C. glutamicum SYPS-062 was 46.6% lower than that in C. glutamicum ATCC 13032, which indicated that the concentration of coenzyme of SHMT in C. glutamicum SYPS-062 might be lower than model strain. The decrease of degradation might be the main reason of L-serine accumulation in C. glutamicum SYPS-062. Meanwhile, the low level of folate resulted in the slow growth rate of C. glutamicum SYPS-062.