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

嗜热细菌Thermoanaerobacter sp.中参与酒精生产的酶。

• 批准号: 319182766
• 负责人: Professor Dr. Mirko Basen
• 金额: --
• 依托单位: Lehrstuhl Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/319182766?language=en
• 关键词: Enzymes; involved; alcohol; production; thermophilic

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.

热厌氧细菌是一种嗜热的解糖细菌，其最适温度在60°C或以上。乙醇是糖发酵过程中除醋酸、氢和乳酸外的主要最终产物。在一些物种中已经研究了参与乙醇形成的酶和途径，双功能醛/醇脱氢酶AdhE被认为是必不可少的，它最终以NAD(P)H作为电子供体将乙酰辅酶a还原为乙醇，而在它们的基因组中编码的其他醇脱氢酶可能参与也可能不参与。总之，目前还不清楚，哪些酶参与，途径可能因物种而异。此外，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。对热厌氧菌sp.菌株X514的初步实验表明，该菌株在纤维二糖的生长过程中也产生了醛铁氧还蛋白氧化还原酶（AOR）。最近，我发表了一个AOR是必要的乙醇生产的突变菌株的古细菌火球菌。该菌株还产热厌氧菌sp.菌株X514的乙醇脱氢酶AdhA。由此产生的AOR-AdhA途径导致糖产生乙醇，并将各种有机酸还原为相应的醇，并从糖中提取还原物。有趣的是，进一步的初步实验表明，热厌氧菌sp.菌株X514在纤维素二糖存在的情况下从异丁酸酯中产生异丁醇。基于这些初步实验，我推测AOR可能在热厌氧菌sp.菌株X514的酒精生成中起作用。我打算研究热厌氧菌sp.菌株X514中糖生成乙醇和有机酸还原为相应醇的途径和酶。我打算分离和表征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，我打算对铁氧还蛋白氧化酶进行纯化和表征。转录组分析将进行，以确定候选基因/酶必不可少的酒精形成。本研究将开发热厌氧菌菌株X514的遗传系统，并用于删除被认为必需酶的编码基因，如双功能醛/醇脱氢酶AdhE、一级醇脱氢酶AdhA、二级醇脱氢酶AdhB、AOR、Rnf或Nfn。缺失突变体的表型将在生长和细胞悬浮实验中进行研究。综上所述，转录组学、遗传学、生理和生化方法的结合将使我们全面了解热厌氧菌sp.菌株X514在有或没有外部添加有机酸的情况下在糖上生长过程中的碳和氧化还原代谢。