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首页> 外文期刊>Applied Microbiology >Understanding and Eliminating the Detrimental Effect of Thiamine Deficiency on the Oleaginous Yeast Yarrowia lipolytica
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Understanding and Eliminating the Detrimental Effect of Thiamine Deficiency on the Oleaginous Yeast Yarrowia lipolytica

机译：理解和消除硫胺素缺乏对含油酵母Yarrawia Lipolytica的不利影响

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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 de novo , the oleaginous yeast Yarrowia lipolytica 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 Y. lipolytica . 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 de novo thiamine biosynthesis in Y. lipolytica 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 Saccharomyces cerevisiae (scTHI13), we engineered a thiamine-prototrophic Y. lipolytica 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 Y. lipolytica strain.IMPORTANCE 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. Yarrowia lipolytica , a nonconventional oleaginous yeast with broad biotechnological applications, is a native thiamine auxotroph whose affected cellular metabolism is not well understood. Therefore, Y. lipolytica 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 Y. lipolytica and identifies genes and novel thiamine-regulated elements that eliminate thiamine auxotrophy in Y. lipolytica . Furthermore, the discovery of thiamine-regulated elements enables the development of thiamine biosensors with useful applications in synthetic biology and metabolic engineering.
机译：硫胺素是一种用作所有活细胞中碳和能量代谢中的关键酶的辅助因子的维生素。虽然大多数植物，真菌和细菌可以合成硫胺素de novo，但含油酵母Yarrowia lipolytica不能。在这项研究中，我们使用蛋白质组学与生理学特征一起，以阐明受硫胺素可用性影响和调节的关键代谢过程，并鉴定Y. Lipolytica中硫胺素滋巢的遗传基础。具体而言，我们发现硫胺素耗竭导致脂质生物合成途径和能量代谢（即，ATP合酶）降低的蛋白质丰富，从而导致我们研究中观察到的可忽略不计的糖和糖同化。使用比较基因组学，我们鉴定了缺少的4-氨基-5-羟甲基-2-甲基吡啶胺磷酸盐合成酶（Thi13）基因在Y.Lipolytica中的De Novo Thiamine Biosynatheris，并发现了一种特殊的启动子，P3，其具有强烈的活化和紧张镇压分别由低硫胺素浓度。利用我们的硫胺素调节启动子（P3）的优势来表达Saccharomyces Cerevisiae（SCTHI13）的缺失基因，我们设计了一种硫胺素 - proTotrophic Y. Lipolytica菌株。通过将该工程化菌株与野生型菌株进行比较，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，并表现出具有硫胺素补充剂的增强的脂质生成，在工程化硫胺素 - prodotoOphic y.pipolytica菌株中.Importance thiamine发挥着至关重要的作用所有活细胞中涉及碳和能量代谢的酶的必需辅助因子。硫胺素缺乏对细胞健康产生不利影响。 Yarrowia Lipolytica，一种具有宽阔的生物技术应用的非共同抗性酵母，是一种天然硫胺素滋巢，其受影响的细胞代谢尚未得到很好的理解。因此，Y. Lipolytica是硫胺素代谢研究的理想真核宿主，特别是因为哺乳动物细胞也是硫胺素营养营养和硫胺素缺乏症涉及几种人类疾病。本研究阐明了硫胺素缺乏对Y. Lipolytica细胞代谢的根本作用，并鉴定了消除Y. Lipolytica中硫胺素毒细胞的基因和新的硫胺素调节元素。此外，硫胺素调节元件的发现能够在合成生物学和代谢工程中发育具有有用应用的硫胺素生物传感器。

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