Study Of Cultrue Conditions And NADPH Supply For Lipid Accumulation In Trichosporon Cutaneum ACCC20271

Development of efficient and environment-friendly bioenergy is important in order to solve the problems of energy shortage. Recently, biodiesel produced from microbial lipid has been received wide attention around the world for its sustainability and similar contents with plant oil. In our study, we focused on an oleaginous yeast Trichosporon cutaneum ACCC20271for its conditions of lipid production. Several culture conditions including carbon/nitrogen ratio, dissolved oxygen level and initial pH value were optimized in the Erlenmeyer flask. The source of reducing power (NADPH) was intensively investigated at enzyme, transcription and biochemistry levels. Contributions of the malic enzyme (ME) and pentose phosphate (PP) pathways to NADPH generation were compared to explore possible mechanisms underlying microbial lipid production.(1) The fermentation conditions for T. cutaneum were studied. The optimal ratio of carbon and nitrogen source (C-mol/N-mol) was found to be82.5. The final biomass concentration was11.7g/L, approximately a40%increase compared to that obtained before the optimization. The lipid yield was correspondingly increased by more than30%(approximately3.76g/L). We found these culture conditions are important cell growth as well as lipid produciton. Besides, the compositions of fatty acids produced by T. cutaneum ACCC20271was found to be similar with that of sunflower and corn lipids.(2) The correlation between lipid accumulation and activities of ME and G6PDH was investigated to further discover the mechanism for lipid accumulation. The results showed that lipids started to accumulate after nitrogen depletion at about18h. Under nitrogen limitation condition, an approximately2.5-fold increase in ME activity was observed during rapid lipid accumulation phase in comparison to that obtained in exponential growth phase. When nitrogen source in the medium was sufficient, only a slight increase in ME activity was obtained, indicating that ME was closely related to the oil accumulation. The changes of G6DPH activity between16h and28h in nitrogen sufficient cultures were, however, very similar with those under nitrogen-limited conditions, implying that the increase of G6PDH might be mainly responsible for the cell growth other than lipid accumulation in T. cutaneum.(3) The genes of me and zwf coding ME and G6DPH respectively were cloned through CODEHOP primer design and degenerate PCR. Partial sequence information of these two genes was obtained, which therefore provided some basic information for further study of these genes and therefore possible mechanisms involved in lipid accumulation.(4) The qRT-PCR results showed that the transcriptional level of the me gene was not regulated by the nitrogen limitation, implying the ME activity might be regulated by a series of complex regulations instead of the transcription of a single gene. The transcriptional level of the zwf gene was not regulated by the nitrogen condition either. In addition, the transcription of the zwf gene decreased significantly during lipid accumulation, suggesting an important role of G6PDH during rapid growth phase of this strain.