Due to its inevitable formation during biodiesel production and its relatively high degree of reduction, glycerol is an attractive carbon source for microbial fermentation processes. However, glycerol is catabolized in a fully respiratory manner by the eukaryotic platform organism Saccharomyces cerevisiae. We previously engineered S. cerevisiae strains to favor fermentative metabolism of glycerol by replacing the native FAD-dependent glycerol catabolic pathway with the NAD-dependent ‘DHA pathway’. In addition, a heterologous aquaglyceroporin (Fps1 homolog) was expressed to facilitate glycerol uptake. The current study was launched to scrutinize the formation of S. cerevisiae’s natural fermentation product ethanol from glycerol caused by the conducted genetic modifications. This understanding is supposed to facilitate future engineering of this yeast for fermenting glycerol into valuable products more reduced than ethanol. A strain solely exhibiting the glycerol catabolic pathway replacement produced ethanol at concentrations close to the detection limit. The expression of the heterologous aquaglyceroporin caused significant ethanol production (8.5 g L−1 from 51.5 g L−1 glycerol consumed) in a strain catabolizing glycerol via the DHA pathway but not in the wild-type background. A reduction of oxygen availability in the shake flask cultures further increased the ethanol titer up to 15.7 g L−1 (from 45 g L−1 glycerol consumed). The increased yield of cytosolic NADH caused by the glycerol catabolic pathway replacement seems to be a minimal requirement for the occurrence of alcoholic fermentation in S. cerevisiae growing in synthetic glycerol medium. The remarkable metabolic switch to ethanol formation in the DHA pathway strain with the heterologous aquaglyceroporin supports the assumption of a much stronger influx of glycerol accompanied by an increased rate of cytosolic NADH production via the DHA pathway. The fact that a reduction of oxygen supply increases ethanol production in DHA pathway strains is in line with the hypothesis that a major part of glycerol in normal shake flask cultures still enters the catabolism in a respiratory manner.

中文翻译：

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酿酒酵母表现出改良的甘油摄取和分解代谢途径，从这种被认为“不可发酵”的碳源中形成大量乙醇

由于其在生物柴油生产过程中不可避免的形成及其相对高度的还原，甘油是微生物发酵过程中有吸引力的碳源。然而，甘油被真核平台生物酿酒酵母以完全呼吸的方式分解代谢。我们之前设计了酿酒酵母菌株，通过用 NAD 依赖性“DHA 途径”代替天然 FAD 依赖性甘油分解代谢途径来促进甘油的发酵代谢。此外，表达了一种异源水甘油通道蛋白（Fps1 同源物）以促进甘油的摄取。目前的研究旨在审查由进行的基因改造引起的从甘油中形成酿酒酵母的天然发酵产物乙醇。这种理解应该有助于这种酵母的未来工程设计，用于将甘油发酵成比乙醇更少的有价值的产品。仅表现出甘油分解代谢途径替代的菌株在接近检测限的浓度下产生乙醇。异源水甘油通道蛋白的表达在通过 DHA 途径分解甘油的菌株中导致显着的乙醇产生（消耗 51.5 g L-1 甘油中的 8.5 g L-1），但在野生型背景中没有。摇瓶培养物中氧气可用性的降低进一步将乙醇滴度提高到 15.7 g L-1（消耗 45 g L-1 甘油）。由甘油分解代谢途径替代引起的细胞溶质 NADH 产量增加似乎是 S. 中发生酒精发酵的最低要求。在合成甘油培养基中生长的酿酒酵母。异源水甘油通道蛋白在 DHA 途径菌株中向乙醇形成的显着代谢转变支持了以下假设：甘油流入量更大，同时通过 DHA 途径产生细胞溶质 NADH 的速率增加。氧气供应减少会增加 DHA 途径菌株中乙醇产量的事实与正常摇瓶培养物中甘油的主要部分仍以呼吸方式进入分解代谢的假设一致。