Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA.
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, 27695, USA.
Microb Biotechnol. 2017 Nov;10(6):1535-1545. doi: 10.1111/1751-7915.12486. Epub 2017 Feb 14.
Ethanol is an important target for the renewable production of liquid transportation fuels. It can be produced biologically from pyruvate, via pyruvate decarboxylase, or from acetyl-CoA, by alcohol dehydrogenase E (AdhE). Thermophilic bacteria utilize AdhE, which is a bifunctional enzyme that contains both acetaldehyde dehydrogenase and alcohol dehydrogenase activities. Many of these organisms also contain a separate alcohol dehydrogenase (AdhA) that generates ethanol from acetaldehyde, although the role of AdhA in ethanol production is typically not clear. As acetyl-CoA is a key central metabolite that can be generated from a wide range of substrates, AdhE can serve as a single gene fuel module to produce ethanol through primary metabolic pathways. The focus here is on the hyperthermophilic archaeon Pyrococcus furiosus, which grows by fermenting sugar to acetate, CO and H . Previously, by the heterologous expression of adhA from a thermophilic bacterium, P. furiosus was shown to produce ethanol by a novel mechanism from acetate, mediated by AdhA and the native enzyme aldehyde oxidoreductase (AOR). In this study, the AOR gene was deleted from P. furiosus to evaluate ethanol production directly from acetyl-CoA by heterologous expression of the adhE gene from eight thermophilic bacteria. Only AdhEs from two Thermoanaerobacter strains showed significant activity in cell-free extracts of recombinant P. furiosus and supported ethanol production in vivo. In the AOR deletion background, the highest amount of ethanol (estimated 61% theoretical yield) was produced when adhE and adhA from Thermoanaerobacter were co-expressed.
乙醇是可再生液体运输燃料生产的重要目标。它可以通过丙酮酸经丙酮酸脱羧酶生物合成，或通过乙酰辅酶 A 经醇脱氢酶 E（AdhE）合成。嗜热细菌利用 AdhE，它是一种双功能酶，同时具有乙醛脱氢酶和醇脱氢酶活性。许多此类生物还含有一种单独的醇脱氢酶（AdhA），可将乙醛转化为乙醇，尽管 AdhA 在乙醇生产中的作用通常并不明确。由于乙酰辅酶 A 是一种关键的中心代谢物，可由多种底物生成，因此 AdhE 可以作为单一基因燃料模块，通过初级代谢途径生产乙醇。本文重点关注的是嗜热古菌 Pyrococcus furiosus，它通过发酵糖产生乙酸、CO 和 H 来生长。以前，通过异源表达来自嗜热细菌的 adhA，证明 P. furiosus 可以通过一种新机制从乙酸中产生乙醇，该机制由 AdhA 和天然酶醛氧化还原酶（AOR）介导。在这项研究中，从 P. furiosus 中删除了 AOR 基因，以评估通过异源表达来自 8 种嗜热细菌的 adhE 基因直接从乙酰辅酶 A 生产乙醇的情况。只有来自两种 Thermoanaerobacter 菌株的 AdhE 在重组 P. furiosus 的无细胞提取物中表现出显著活性，并支持体内乙醇生产。在 AOR 缺失背景下，当共表达来自 Thermoanaerobacter 的 adhE 和 adhA 时，可生产出最高量的乙醇（估计理论产率为 61%）。
