How E. coli produces hydrogen

大肠杆菌如何产生氢气

• 批准号: BB/I02008X/1
• 负责人: Frank Sargent
• 金额: $ 40.4万
• 依托单位: University of Dundee
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI02008X%2F1
• 关键词: How; E.; coli; produces; hydrogen

Prokaryotes are the simplest living organisms on planet Earth. They include the single-celled bacteria and their cousins the archaea, which are the closest surviving examples of the earliest life-forms that ever existed. Many of these organisms can grow without oxygen, and instead utilise other chemicals from the environment to generate energy for life. Sometimes the chemicals used are unusual and bacteria can use one of the simplest molecules in the Universe to gain energy for growth; hydrogen. Moreover, a range of microorganisms, from photosynthetic algae to strictly anaerobic bacteria, can actually produce hydrogen as a by-product. For example, in the absence of oxygen some bacteria, such as the gut-dwelling Escherichia coli, grow by a process known as fermentation. This initially results in formic acid being produced, which is ultimately used by the cell to generate hydrogen gas. This process requires the action of a complicated enzyme called formate hydrogenlyase (FHL), which comprises at least seven different proteins together with iron, sulphur, nickel and molybdenum atoms. The activity of E. coli FHL was first described as long ago as 1932 by Marjory Stephenson, the first female Fellow of the Royal Society. In the years that have followed, no scientist has been able to isolate FHL in order to study it more closely. In this research, we now describe an innovative new approach that has allowed the purification of FHL for the first time. The overall aim of this project is to understand how FHL works at the molecular level, and modify this activity so it will be suitable for industrial applications. Biological approaches to hydrogen production (so-called 'biohydrogen') are growing in importance as fossil fuel resources verge on the limits of economical extraction, and the environmental impact of carbon emissions gains long-overdue recognition. Hydrogen has the highest energy per weight of any fuel, and its use (particularly in a fuel cell) is clean and efficient. At present 99% of hydrogen is produced by reforming fossil fuels and 1% comes from electrolysis, with most being used as a feedstock by the chemical industry. Most importantly, biohydrogen offers the prospect of FULLY RENEWABLE hydrogen, freed from any dependence on fossil fuel, and the scope for taping into this resource is enormous. The biochemistry of hydrogen production depends upon normally oxygen-sensitive enzymes known as hydrogenases. FHL contains a hydrogenase (the so-called 'Hyd-3' enzyme) that is responsible for all of the hydrogen produced by E. coli. The active site of Hyd-3 contains nickel, iron, carbon monoxide and cyanide molecules (which can be studied using the advanced spectroscopy available in Oxford), and is thus termed a [NiFe]hydrogenase. Indeed, we and others have proposed that the active sites of such hydrogenases are as active in hydrogen chemistry as platinum catalysts - an expensive and limited resource. Hyd-3 is rapidly inactivated by oxygen, and this may be a reason why its isolation has proven problematic for so long. Our recent studies of [NiFe]hydrogenases, together with that of others, has identified an important subset of enzymes that can function in air (so-called 'oxygen-tolerant hydrogenases'). These enzymes hold the key to technological developments of biohydrogen and we now have fresh insight into the molecular mechanism of their oxygen tolerance. Another important aim of this project, therefore, is to use this new knowledge to engineer oxygen tolerance into FHL. The Oxford and Dundee groups are superbly complementary. Dundee has expertise in studying the molecular cell biology of hydrogenases in E. coli, and Oxford has pioneered biophysical methods for studying hydrogenases, most notably protein film electrochemistry (PFE) and spectroscopy. PFE is the most powerful of all techniques for studying the properties of hydrogenases and has been instrumental in understanding the mechanistic details of their chemistry.

原核生物是地球上最简单的生物。它们包括单细胞细菌和它们的表亲古细菌，它们是有史以来最早的生命形式的最接近的幸存例子。这些生物中的许多可以在没有氧气的情况下生长，而是利用环境中的其他化学物质来产生生命所需的能量。有时使用的化学物质是不寻常的，细菌可以使用宇宙中最简单的分子之一来获得生长所需的能量：氢。此外，从光合藻类到严格意义上的厌氧细菌等一系列微生物实际上都可以产生氢气作为副产品。例如，在没有氧气的情况下，一些细菌，如肠道大肠杆菌，通过一种称为发酵的过程生长。这最初导致产生甲酸，甲酸最终被电池用于产生氢气。这个过程需要一种复杂的酶的作用，称为甲酸氢解酶（FHL），它包括至少七种不同的蛋白质以及铁，硫，镍和钼原子。E.早在1932年，皇家学会的第一位女性研究员Marjory斯蒂芬森就首次描述了大肠杆菌FHL。在随后的几年里，没有科学家能够分离出FHL，以便更仔细地研究它。在这项研究中，我们现在描述了一种创新的新方法，首次允许FHL的纯化。该项目的总体目标是了解FHL如何在分子水平上工作，并修改这种活性，使其适合工业应用。随着化石燃料资源濒临经济开采的极限，生物制氢方法（所谓的“生物氢”）的重要性越来越大，碳排放对环境的影响也得到了早就应该得到的认识。氢是所有燃料中单位重量能量最高的，它的使用（特别是在燃料电池中）是清洁和高效的。目前，99%的氢是通过重整化石燃料生产的，1%来自电解，其中大部分被用作化学工业的原料。最重要的是，生物氢提供了完全可再生氢的前景，摆脱了对化石燃料的任何依赖，而且利用这种资源的范围是巨大的。生物化学的氢生产取决于通常氧敏感的酶称为氢化酶。FHL含有一种氢化酶（所谓的“Hyd-3”酶），负责E.杆菌Hyd-3的活性位点含有镍、铁、一氧化碳和氰化物分子（可以使用牛津大学的先进光谱学进行研究），因此被称为[NiFe]氢化酶。事实上，我们和其他人已经提出，这种氢化酶的活性位点在氢化学中的活性与铂催化剂一样-一种昂贵且有限的资源。Hyd-3被氧气迅速灭活，这可能是为什么它的分离长期以来一直存在问题的原因。我们最近对[NiFe]氢化酶的研究，以及其他人的研究，已经确定了一个重要的可以在空气中发挥作用的酶子集（所谓的“耐氧氢化酶”）。这些酶是生物制氢技术发展的关键，我们现在对它们耐氧的分子机制有了新的认识。因此，该项目的另一个重要目的是利用这些新知识将耐氧性工程化到FHL中。牛津集团和邓迪集团是极好的互补。邓迪在研究大肠杆菌氢化酶的分子细胞生物学方面有专长。牛津大学开创了研究氢化酶的生物物理方法，最著名的是蛋白质膜电化学（PFE）和光谱学。PFE是研究氢化酶性质的所有技术中最强大的技术，并且有助于理解其化学的机械细节。

项目成果

Exploring the directionality of Escherichia coli formate hydrogenlyase: a membrane-bound enzyme capable of fixing carbon dioxide to organic acid.

• DOI: 10.1002/mbo3.365
• 发表时间: 2016-10
• 期刊: MICROBIOLOGYOPEN
• 影响因子: 3.4
• 作者: Pinske, Constanze;Sargent, Frank
• 通讯作者: Sargent, Frank

The importance of iron in the biosynthesis and assembly of [NiFe]-hydrogenases.

• DOI: 10.1515/bmc-2014-0001
• 发表时间: 2014-03-01
• 期刊: Biomolecular concepts
• 作者: Pinske, Constanze;Sawers, R Gary
• 通讯作者: Sawers, R Gary

Integration of an [FeFe]-hydrogenase into the anaerobic metabolism of Escherichia coli.

• DOI: 10.1016/j.btre.2015.10.002
• 发表时间: 2015-12
• 期刊: Biotechnology reports (Amsterdam, Netherlands)
• 作者: Kelly CL;Pinske C;Murphy BJ;Parkin A;Armstrong F;Palmer T;Sargent F
• 通讯作者: Sargent F

Zymographic differentiation of [NiFe]-hydrogenases 1, 2 and 3 of Escherichia coli K-12.

• DOI: 10.1186/1471-2180-12-134
• 发表时间: 2012-07-06
• 期刊: BMC microbiology
• 影响因子: 4.2
• 作者: Pinske C;Jaroschinsky M;Sargent F;Sawers G
• 通讯作者: Sawers G

Expanding the substrates for a bacterial hydrogenlyase reaction.

• DOI: 10.1099/mic.0.000471
• 发表时间: 2017-05
• 期刊: Microbiology (Reading, England)
• 作者: Lamont CM;Kelly CL;Pinske C;Buchanan G;Palmer T;Sargent F
• 通讯作者: Sargent F