Investigation of non-mevalonate-like pathway for terpene biosynthesis in Yarrowia lipolytica　

(Yarrowia lipolytica のテルペン生合成のための非メバロン酸様経路に関わる研究)

Yarrowia is one of the fungal genera in the family Dipodascaceae. For some time, the genus was monotypic, containing the single species Yarrowia lipolytica. This yeast is an attractive host for converting the various carbon source to high-value chemicals in an economical and environmentally friendly way. During the survey on the Y. lipolytica metabolome response to different carbon to nitrogen ratio, a chromatographic peak annotated as MEP was observed in the intracellular extract of Y. lipolytica cultured in a nitrogen limiting condition. This phenomenon is inconsistent with a conventional understanding regarding isoprenoid biosynthesis in yeast. This result raises an intriguing hypothesis of whether Y. lipolytica could utilize the MEP pathway for isoprenoid biosynthesis. Therefore, the objective of this study is to investigate the existence of the MEP pathway in Y. lipolytica. Four independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, the ¹³C labeling incorporation analysis, and the Y. lipolytica reference genome analysis.

In this study the time-course sampling of one Y. lipolytica strain was conducted to probe the suitable time points for multiple strains study. Since the variable changes in this study highly affect the growth of Y. lipolytica, it might not be suitable to choose the sampling point based on the growth phase alone. Therefore, the metabolome profile was considered to help understand the dynamics of metabolic changes occurring during the cultivation with several different C:N ratios and determined the crucial sampling time point and C:N ratio for multiple strains study. Further studied on the metabolic responses of several Yarrowia strains to nitrogen-limiting conditions was conducted, including Y. lipolytica PO1d, Y. lipolytica JCM 2304, Y. lipolytica JCM 21924, Y. lipolytica JCM 8061, and two recently identified species, Y. deformans JCM 1694 and Y. keelungensis JCM 14894. This study found that cultivation time of 36 h and carbon to nitrogen ratio of 4:1 and 5:0 was suitable for studying the effects of nitrogen-limiting conditions on Yarrowia and these conditions were applied to six strains of Yarrowia. These six strains of Yarrowia showed similar responses to nitrogen-limiting conditions; however, each strain had a unique metabolomic profile. Purine and pyrimidine metabolism were the most highly affected biological pathways in nitrogen-limiting conditions, indicating that these conditions affect energy availability within cells. This stress leads to a shift in cells to the utilization of a less ATP-dependent biological pathway.

In the next chapter, a more in-depth investigation of the existence of the MEP pathway in Y. lipolytica was presented. Four independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, the ¹³C labeling incorporation analysis, and the Y. lipolytica reference genome analysis. The study suggested that the mevalonate and MEP pathways co‑exist in Y. lipolytica and the nitrogen limiting condition triggers the utilization of the MEP pathway in Y. lipolytica.

In conclusion, the elucidation of cellular functions is an enormous effort and requires various strategies to capture the entire system. The metabolomics-based analysis offers a complete view of the metabolic shifts under environmental perturbations. This study demonstrated that the metabolomics analysis of oleaginous yeast Yarrowia spp. can yield known and unknown metabolic behavior, which raised an interesting follow-up experiment. The metabolome dataset presented in this study does not only provide information about key metabolites but also represents a useful resource for future research regarding non-mevalonate-like pathway in yeast. With the current data, this study proposed that the mevalonate pathway and the non-mevalonate-like pathway co-exist in Y. lipolytica. Notably, the nitrogen limiting condition triggered the utilization of the non-mevalonate-like pathway.