Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae.
Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae.
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DOI：
10.1186/s13068-017-0791-3
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发表时间：
2017
影响因子：
6.3
通讯作者：
Pronk JT
中科院分区：
文献类型：
--
作者：
Papapetridis I;van Dijk M;van Maris AJA;Pronk JT
甘油的形成有助于酿酒酵母的细胞氧化还原平衡和渗透调节，是酵母生物乙醇生产的重要副产品。通过工程化途径替代甘油途径来减少 NAD+ 依赖性乙酸盐已被证明可以提高乙醇产量并有助于含乙酸盐介质的解毒。然而，不产生甘油的菌株的渗透敏感性限制了它们在高渗透压工业过程中的应用。本研究探索了在保持渗透压耐受性的同时最大限度地减少乙酸盐还原菌株的甘油产量的工程策略。 GPD2 编码 NAD+ 依赖性甘油 3-磷酸脱氢酶 (G3PDH) 的两种酿酒酵母同工酶之一。在醋酸盐还原菌株中删除该基因会导致厌氧、低渗透压培养物中的甘油产量降低四倍，但几乎不影响高渗透压下的甘油产量。用古菌 NADP+ 偏好酶替换两种天然 G3PDH，并结合 ALD6 的删除，产生了一种乙酸盐还原菌株，其表型类似于低渗透压培养物中的甘油阴性 gpd1Δ gpd2Δ 菌株。该菌株在高渗透压（1 mol L−1 葡萄糖）下厌氧生长，同时消耗乙酸盐并且几乎不产生细胞外甘油。其在高渗透压培养物中的乙醇产量比表达天然甘油途径的乙酸盐还原菌株高 13%。 GPD2 的缺失提供了一种有吸引力的策略，可以提高醋酸盐还原性酿酒酵母菌株在低渗透压培养基中的产量，但在高渗透压培养基中则不然。用异源 NADP+ 偏好酶替换天然酵母 G3PDH，结合删除 ALD6，实际上消除了高渗透培养物中甘油的产生，同时能够有效地将乙酸盐还原为乙醇。经过进一步优化生长动力学后，这种解偶联甘油形成在氧化还原稳态和渗透压耐受性中的作用的策略可适用于提高工业菌株在高比重含乙酸盐工艺中的性能。本文的在线版本 (doi:10.1186/s13068-017-0791-3) 包含补充材料，可供授权用户使用。
Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD+-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining osmotolerance. GPD2 encodes one of two S. cerevisiae isoenzymes of NAD+-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP+-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1Δ gpd2Δ strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L−1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway. Deletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP+-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes. The online version of this article (doi:10.1186/s13068-017-0791-3) contains supplementary material, which is available to authorized users.