Enzymes involved in alcohol production in the thermophilic bacterium Thermoanaerobacter sp. strain X514

嗜热厌氧细菌是嗜热糖解细菌，最适温度为60 °C或以上。乙醇是糖发酵的主要终产物，除乙酸盐、氢气和乳酸盐外。参与乙醇形成的酶和途径已在少数物种中进行了研究，双功能醛/醇脱氢酶AdhE（以NAD（P）H作为电子供体将乙酰辅酶A最终还原为乙醇）被认为是必不可少的，而其基因组中编码的其他醇脱氢酶可能参与或可能不参与。总之，目前还不清楚，哪些酶参与和途径可能因物种而异。此外，目前还不清楚丙酮酸氧化过程中产生的还原铁氧还蛋白是如何被氧化的。用嗜热厌氧微生物菌株X514进行的初步实验表明，该物种在纤维二糖上生长期间也产生醛铁氧还蛋白氧化还原酶（AOR）。最近，我发表了AOR是一个突变菌株的古菌Pyrococcus furiosus的乙醇生产所必需的。该菌株还产生来自嗜热厌氧杆菌属菌株X514的伯醇脱氢酶AdhA。所得到的AOR-AdhA途径导致从糖生产乙醇，并导致各种有机酸还原成其相应的醇，还原当量衍生自糖。有趣的是，进一步的初步实验表明，热厌氧微生物菌株X514在纤维二糖的存在下从异丁酸酯产生异丁醇。基于这些初步的实验，我推测，AOR可能发挥作用，在酒精的形成热厌氧细菌菌株X514。我打算研究的途径和酶参与乙醇形成从糖和还原有机酸到其相应的醇在热厌氧微生物菌株X514。我打算分离和鉴定所涉及的醛和醇脱氢酶。AOR和/或铁氧还蛋白：NAD（P）H氧化还原酶如Rnf或Nfn可能是铁氧还蛋白再氧化所必需的。因此，我打算纯化和表征铁氧还蛋白氧化酶。将进行转录组分析，以确定酒精形成所必需的候选基因/酶。将开发热厌氧杆菌属菌株X514的遗传系统并用于删除编码嘌呤必需酶的基因，所述嘌呤必需酶例如双功能醛/醇脱氢酶AdhE、伯醇脱氢酶AdhA、仲醇脱氢酶AdhB、AOR、Rnf或Nfn。将在生长和细胞悬浮实验中研究缺失突变体的表型。 总之，转录组学，遗传，生理和生化方法的组合将导致全面了解碳和氧化还原代谢的热厌氧微生物菌株X514在糖的存在或不存在外部添加的有机酸的生长过程中。

Thermoanaerobacter species are thermophilic saccharolytic bacteria with a temperature optimum at or above 60 °C. Ethanol is the major end product of sugar fermentation, besides acetate, hydrogen and lactate. The enzymes and pathways involved in ethanol formation have been studied in a few species, and the bifunctional aldehyde/alcohol dehydrogenase AdhE, reducing acetyl-CoA ultimately to ethanol with NAD(P)H as electron donor, has been suggested to be essential, while other alcohol dehydrogenases encoded in their genomes may or may not be involved. In conclusion, it is unclear, which enzymes are involved and the pathway may vary from species to species. Moreover, it is unclear how reduced ferredoxin, produced during pyruvate oxidation, is oxidized. Preliminary experiments with Thermoanaerobacter sp. strain X514 revealed that this species also produced an aldehyde ferredoxin oxidoreductase (AOR) during growth on cellobiose. Recently, I have published that an AOR is essential for ethanol production of a mutant strain of the archaeon Pyrococcus furiosus. The strain additionally produced the primary alcohol dehydrogenase AdhA from Thermoanaerobacter sp. strain X514. The resulting AOR-AdhA pathway lead to the production of ethanol from sugars and to the reduction of a variety of organic acids to their corresponding alcohols, with reducing equivalents derived from sugars. Interestingly, further preliminary experiments showed that Thermoanaerobacter sp. strain X514 produced isobutanol from isobutyrate in the presence of cellobiose. Based on these preliminary experiments, I hypothesize that AOR may play a role in alcohol formation in Thermoanaerobacter sp. strain X514. I intend to study the pathways and enzymes involved in ethanol formation from sugar and in reduction of organic acids to their corresponding alcohols in Thermoanaerobacter sp. strain X514. I intend to isolate and characterize the aldehyde and alcohol dehydrogenases involved. AOR and/or a ferredoxin:NAD(P)H oxidoreductase such as Rnf or Nfn may be essential for ferredoxin re-oxidation. Therefore, I intend to purify and characterize the ferredoxin oxidizing enzymes. A transcriptome analysis will be carried out in order to identify candidate genes / enzymes essential for alcohol formation. A genetic system for Thermoanaerobacter sp. strain X514 will be developed and used to delete genes coding for putatively essential enzymes such as the bifunctional aldehyde/alcohol dehydrogenase AdhE, the primary alcohol dehydrogenase AdhA, the secondary alcohol dehydrogenase AdhB, AOR, Rnf or Nfn. The phenotype of the deletion mutants will be studied in growth and cell suspension experiments. In conclusion, the combination of transcriptomic, genetic, physiological and biochemical approaches will lead to a comprehensive understanding of carbon and redox metabolism in Thermoanaerobacter sp. strain X514 during growth on sugars in the presence or absence of externally added organic acids.