Biological production of 5‐aminolevulinic acid (5‐ALA) has received growing attention over the years. However, there is the tradeoff between 5‐ALA biosynthesis and cell growth because the fermentation broth will become acidic due to the production of 5‐ALA. To address this limitation, we engineered an acid‐tolerant yeast, Issatchenkia orientalis SD108, for 5‐ALA production. We first discovered that the cell growth rate of I. orientalis SD108 was boosted by 5‐ALA and its endogenous ALA synthetase (ALAS) showed higher activity than those homologs from other yeasts. The titer of 5‐ALA was improved from 28 mg/L to 120‐, 150‐, and 300 mg/L, by optimizing plasmid design, overexpressing a transporter, and increasing gene copy number, respectively. After redirecting the metabolic flux using the pyruvate decarboxylase (PDC) knockout strain (SD108ΔPDC) and culturing with urea, we increased the titer of 5‐ALA to 510 mg/L, a 13‐fold enhancement, proving the importance of the newly identified IoALAS with higher activity and the strategic selection of nitrogen sources for knockout strains. This study demonstrates the acid‐tolerant I. orientalis SD108ΔPDC has a high potential for 5‐ALA production at a large scale in the future.

中文翻译：

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非模型酵母 Issatchenkia orientalis SD108 用于 5-氨基乙酰丙酸生产的代谢工程


多年来，5-氨基乙酰丙酸 （5-ALA） 的生物生产越来越受到关注。然而，5-ALA 生物合成和细胞生长之间存在权衡，因为发酵液会因产生 5-ALA 而变成酸性。为了解决这一限制，我们设计了一种耐酸酵母 Issatchenkia orientalis SD108，用于生产 5-ALA。我们首先发现 5-ALA 促进了侧方鸢尾菌 SD108 的细胞生长速率，其内源性 ALA 合成酶 （ALAS） 显示出比其他酵母的同源物更高的活性。通过优化质粒设计、过表达转运蛋白和增加基因拷贝数，5-ALA 的滴度分别从 28 mg/L 提高到 120-、150-和 300-。在使用丙酮酸脱羧酶 （PDC） 敲除菌株 （SD108ΔPDC） 重定向代谢通量并用尿素培养后，我们将 5-ALA 的滴度提高到 510 mg/L，提高了 13 倍，证明了新发现的具有更高活性的 IoALAS 的重要性以及基因敲除菌株的氮源的战略选择。本研究表明，耐酸的 I. orientalis SD108ΔPDC 在未来大规模生产 5-ALA 的潜力很大。