Enhanced production of L-arginine by improving carbamoyl phosphate supply in metabolically engineered Corynebacterium crenatum.

Carbamoyl phosphate is an important precursor for L-arginine and pyrimidines biosynthesis. In view of this importance, the cell factory should enhance carbamoyl phosphate synthesis to improve related compound production. In this work, we verified that carbamoyl phosphate is essential for L-arginine production in Corynebacterium sp., followed by engineering of carbamoyl phosphate synthesis for further strain improvement. First, carAB encoding carbamoyl phosphate synthetase II was overexpressed to improve the synthesis of carbamoyl phosphate. Second, the regulation of glutamine synthetase increases the supply of L-glutamine, providing an effective substrate for carbamoyl phosphate synthetase II. Third, carbamate kinase, which catalyzes inorganic ammonia synthesis carbamoyl phosphate, was screened and selected to assist in carbamoyl phosphate supply. Finally, we disrupted ldh (encoding lactate dehydrogenase) to decrease by-production formation and save NADH to regenerate ATP through the electron transport chain. Subsequently, the resulting strain allowed a dramatically increased L-arginine production of 68.6 ± 1.2 g∙L-1, with an overall productivity of 0.71 ± 0.01 g∙L-1∙h-1 in 5-L bioreactor. Stepwise rational metabolic engineering based on an increase in the supply of carbamoyl phosphate resulted in a gradual increase in L-arginine production. The strategy described here can also be implemented to improve L-arginine and pyrimidine derivatives. KEY POINTS: • The L-arginine production strongly depended on the supply of carbamoyl phosphate. • The novel carbamoyl phosphate synthesis pathway for C. crenatum based on carbamate kinase was first applied to L-arginine synthesis. • ATP was regenerated followed with the disruption of lactate formation.

Early to Mid-Holocene Palaeoenvironment Change and Sedimentary Evolution in the Xianghu Area, Zhejiang.

A 2.5 m long sediment core (XH-2) obtained from Xianghu area, near the Kuahuqiao site, were analyzed for grain size, diatom index, and geochemistry of organic carbon. The results of the total organic carbon (TOC) and stable organic carbon isotope (δ13C) in sediment samples from core XH-2 in the Xianghu area in Zhejiang Province have revealed the evolution history of sedimentary environmental and climatic changes during the breeding-prosperity-decline period of the Kuahuqiao culture. During 9300-8200 cal a BP, TOC contents were relatively high and stable, whereas δ13C values tended to be negative. This condition indicated that the climate was humid, and the sedimentary environment in the Xianghu area was stable. During 8200-7500 cal a BP, TOC contents presented a fluctuating declining trend, and δ13C values were significantly high, implying that the climate was arid, and the Xianghu area was gradually reduced to land. Thus, conducive conditions were provided for the development of the Kuohuqiao culture (7700-7400 cal a BP). From 7500 cal a BP, TOC contents obviously declined, and δ13C values were partially low, suggesting strengthened hydrodynamic force and wet conditions in the Xianghu area. This condition was related to the rise in sea level at approximately 7400 cal a BP, and the Kuahuqiao site became obsolete due to the transgression event. The TOC contents in core XH-2 were remarkably influenced by grain size, whereas no significant correlation existed between the δ13C variability and grain size. Sedimentary environment changes in the Xianghu area from 9300 to 6600 cal a BP, which was reflected by the TOC and δ13C records in core XH-2, accorded with the diatom results in this core and those in the Baima Lake area.