Department of Chemical and Biomolecular Engineering, The University of Tennessee, Knoxville, Tennessee, USA.
Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
Appl Environ Microbiol. 2020 Jan 21;86(3). doi: 10.1128/AEM.02299-19.
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.
硫胺素是一种维生素，作为所有活细胞中碳和能量代谢关键酶的辅因子发挥作用。虽然大多数植物、真菌和细菌都可以合成硫胺素，但油脂酵母却不能。在这项研究中，我们使用蛋白质组学和生理特征描述相结合的方法，阐明了受硫胺素可用性影响和调节的关键代谢过程，并确定了硫胺素营养缺陷在 中的遗传基础。具体来说，我们发现硫胺素耗尽会导致脂质生物合成途径和能量代谢（即 ATP 合酶）的蛋白质丰度降低，导致我们的研究中观察到的生长不良和糖同化不良。通过比较基因组学，我们确定了缺失的 4-氨基-5-羟甲基-2-甲基嘧啶磷酸合酶（THI13）基因用于硫胺素生物合成，并发现了一个特殊的启动子 P3，它分别由低和高硫胺素浓度强烈激活和紧密抑制。利用我们的硫胺素调控启动子（P3）的优势，从 （scTHI13）表达缺失的基因，我们构建了一个硫胺素原养型 菌株。通过比较这个工程菌株和野生型菌株，我们揭示了硫胺素可用性和脂质生物合成之间的紧密关系，并证明了在工程硫胺素原养型 菌株中补充硫胺素可增强脂质生产。硫胺素作为所有活细胞中参与碳和能量代谢的酶的必需辅因子发挥着至关重要的作用。硫胺素缺乏对细胞健康有不利影响。 ，一种具有广泛生物技术应用的非传统油脂酵母，是一种天然的硫胺素营养缺陷型，其受影响的细胞代谢尚不清楚。因此， 是研究硫胺素代谢的理想真核宿主，特别是因为哺乳动物细胞也是硫胺素营养缺陷型，硫胺素缺乏与几种人类疾病有关。这项研究阐明了硫胺素缺乏对 细胞代谢的基本影响，并确定了消除 硫胺素营养缺陷的基因和新的硫胺素调控元件。此外，硫胺素调控元件的发现使开发具有在合成生物学和代谢工程中有用应用的硫胺素生物传感器成为可能。
