Orotic acid (OA) is an intermediate of the pyrimidine biosynthesis with high industrial relevance due to its use as precursor for production of biochemical pyrimidines or its use as carrier molecule in drug formulations. It can be produced by fermentation of microorganisms with engineered pyrimidine metabolism. In this study, we surprisingly discovered the yeast Yarrowia lipolytica as a powerful producer of OA. The overproduction of OA in the Y. lipolytica strain PO1f was found to be caused by the deletion of the URA3 gene which prevents the irreversible decarboxylation of OA to uridine monophosphate. It was shown that the lack of orotidine-5′-phosphate decarboxylase was the reason for the accumulation of OA inside the cell since a rescue mutant of the URA3 deletion in Y. lipolytica PO1f completely prevented the OA secretion into the medium. In addition, pyrimidine limitation in the cell massively enhanced the OA accumulation followed by secretion due to intense overflow metabolism during bioreactor cultivations. Accordingly, supplementation of the medium with 200 mg/L uracil drastically decreased the OA overproduction by 91%. OA productivity was further enhanced in fed-batch cultivation with glucose and ammonium sulfate feed to a maximal yield of 9.62 ± 0.21 g/L. Y. lipolytica is one of three OA overproducing yeasts described in the literature so far, and in this study, the highest productivity was shown. This work demonstrates the potential of Y. lipolytica as a possible production organism for OA and provides a basis for further metabolic pathway engineering to optimize OA productivity.

中文翻译：

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限嘧啶条件下解脂耶氏酵母生产乳清酸

乳清酸 (OA) 是嘧啶生物合成的中间体，由于其用作生产生化嘧啶的前体或用作药物制剂中的载体分子，因此具有高度的工业相关性。它可以通过具有工程化嘧啶代谢的微生物发酵产生。在这项研究中，我们惊奇地发现酵母解脂耶氏酵母是一种强大的 OA 生产者。发现解脂耶氏酵母菌株 PO1f中 OA 的过量产生是由URA3基因的缺失引起的，该基因阻止了 OA 不可逆地脱羧为尿苷一磷酸。结果表明，缺乏乳清蛋白-5'-磷酸脱羧酶是 OA 在细胞内积累的原因，因为解脂耶氏酵母 PO1f 中的URA3缺失完全阻止了 OA 分泌到培养基中。此外，由于生物反应器培养过程中强烈的溢出代谢，细胞中的嘧啶限制大量增强了 OA 的积累和分泌。因此，在培养基中添加 200 mg/L 尿嘧啶可显着降低 91% 的 OA 过量生产。使用葡萄糖和硫酸铵进料的分批补料培养进一步提高了 OA 生产力，最大产量为 9.62 ± 0.21 g/L。解脂耶氏酵母是迄今为止文献中描述的三种过度生产 OA 的酵母之一，在本研究中，显示出最高的生产力。这项工作证明了解脂耶氏酵母的潜力作为一种可能的 OA 生产有机体，并为进一步的代谢途径工程优化 OA 生产力提供了基础。