Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305, USA.
Plant Physiol. 2012 Mar;158(3):1293-305. doi: 10.1104/pp.111.191569. Epub 2012 Jan 23.
The green alga Chlamydomonas reinhardtii has numerous genes encoding enzymes that function in fermentative pathways. Among these, the bifunctional alcohol/acetaldehyde dehydrogenase (ADH1), highly homologous to the Escherichia coli AdhE enzyme, is proposed to be a key component of fermentative metabolism. To investigate the physiological role of ADH1 in dark anoxic metabolism, a Chlamydomonas adh1 mutant was generated. We detected no ethanol synthesis in this mutant when it was placed under anoxia; the two other ADH homologs encoded on the Chlamydomonas genome do not appear to participate in ethanol production under our experimental conditions. Pyruvate formate lyase, acetate kinase, and hydrogenase protein levels were similar in wild-type cells and the adh1 mutant, while the mutant had significantly more pyruvate:ferredoxin oxidoreductase. Furthermore, a marked change in metabolite levels (in addition to ethanol) synthesized by the mutant under anoxic conditions was observed; formate levels were reduced, acetate levels were elevated, and the production of CO(2) was significantly reduced, but fermentative H(2) production was unchanged relative to wild-type cells. Of particular interest is the finding that the mutant accumulates high levels of extracellular glycerol, which requires NADH as a substrate for its synthesis. Lactate production is also increased slightly in the mutant relative to the control strain. These findings demonstrate a restructuring of fermentative metabolism in the adh1 mutant in a way that sustains the recycling (oxidation) of NADH and the survival of the mutant (similar to wild-type cell survival) during dark anoxic growth.
莱茵衣藻（Chlamydomonas reinhardtii）拥有许多编码在发酵途径中起作用的酶的基因。其中，二功能醇/乙醛脱氢酶（ADH1）与大肠杆菌的 AdhE 酶高度同源，被认为是发酵代谢的关键组成部分。为了研究 ADH1 在黑暗缺氧代谢中的生理作用，我们生成了衣藻的 adh1 突变体。当这个突变体被置于缺氧条件下时，我们没有检测到它合成乙醇；衣藻基因组上的另外两个 ADH 同源物似乎没有在我们的实验条件下参与乙醇的产生。在野生型细胞和 adh1 突变体中，丙酮酸甲酸裂解酶、乙酰激酶和氢化酶的蛋白水平相似，而突变体中丙酮酸：铁氧还蛋白氧化还原酶的水平显著更高。此外，在缺氧条件下，突变体合成的代谢物水平（除了乙醇）发生了明显的变化；甲酸水平降低，乙酸水平升高，CO2 的产生显著减少，但相对于野生型细胞，发酵性 H2 的产生没有变化。特别值得注意的是，突变体积累了高水平的细胞外甘油，这需要 NADH 作为其合成的底物。与对照株相比，突变体中乳酸的产生也略有增加。这些发现表明，adh1 突变体中发酵代谢发生了重构，以维持 NADH 的循环（氧化）和突变体的存活（类似于野生型细胞的存活），使其在黑暗缺氧生长过程中得以生存。
