Understanding and Eliminating the Detrimental Effect of Thiamine Deficiency on the Oleaginous Yeast Yarrowia lipolytica.

Thiamine is a vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism in all living cells. While most plants, fungi, and bacteria can synthesize thiamine , the oleaginous yeast cannot. In this study, we used proteomics together with physiological characterization to elucidate key metabolic processes influenced and regulated by thiamine availability and to identify the genetic basis of thiamine auxotrophy in Specifically, we found that thiamine depletion results in decreased protein abundance for the lipid biosynthesis pathway and energy metabolism (i.e., ATP synthase), leading to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THI13) gene for the thiamine biosynthesis in and discovered an exceptional promoter, P3, that exhibits strong activation and tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene from (scTHI13), we engineered a thiamine-prototrophic strain. By comparing this engineered strain to the wild-type strain, we revealed the tight relationship between thiamine availability and lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic strain. Thiamine plays a crucial role as an essential cofactor for enzymes involved in carbon and energy metabolism in all living cells. Thiamine deficiency has detrimental consequences for cellular health. , a nonconventional oleaginous yeast with broad biotechnological applications, is a native thiamine auxotroph whose affected cellular metabolism is not well understood. Therefore, is an ideal eukaryotic host for the study of thiamine metabolism, especially because mammalian cells are also thiamine auxotrophic and thiamine deficiency is implicated in several human diseases. This study elucidates the fundamental effects of thiamine deficiency on cellular metabolism in and identifies genes and novel thiamine-regulated elements that eliminate thiamine auxotrophy in Furthermore, the discovery of thiamine-regulated elements enables the development of thiamine biosensors with useful applications in synthetic biology and metabolic engineering.