Nitrogen fixation and hydrogen production by photosynthetic bacteria

非硫紫色光合细菌是研究光合作用、固氮和生物技术转化等一系列基础和应用生物学领域的优良模式生物。因此，光合细菌是研究固氮及其调节的一般和独特方面的优秀生物。光合细菌除了在转录水平上对大多数固氮生物所共有的氧和固定氮进行调控外，还能够通过固氮酶的共价和代谢调控来调控其预制酶的活性。我们已经表明，固氮酶的调节相对于突然的外部添加铵需要膜结合的氨传感器/转运蛋白AmtB。Rh/Rh家族成员几乎存在于所有测序的生物体中，并且存在于古细菌、细菌、原生生物、植物、真菌和无脊椎动物中。我们将继续研究AmtB的结构/功能及其在控制固氮酶调节系统中的作用。特别是，我们将应用定点诱变来了解特定氨基酸在氨传感和/或运输中的作用。 固氮酶的酶机制也负责光合细菌的氢生产。这些生物体具有使用各种原料的潜在能力，以其将有机酸转化为氢气和二氧化碳的光驱动转化而闻名。因此，它们是从黑暗发酵的流出物中获得额外氢气的两阶段或共培养系统的理想候选者。此外，各种富含有机酸的工业和农业废物流可以潜在地用作光发酵的底物。我们最近已经证明了在氢气生产中的许多改进，包括作为生物柴油制造的废物产物产生的甘油的化学计量转化为氢气的证明，以及从各种糖蜜馏分中产生可观量的氢气。在一系列研究中，我们最近还表明，在葡萄糖的一步、单阶段发酵中可以获得显著量的氢气，每摩尔葡萄糖高达9摩尔H2。我们还首次展示了微氧发酵生产氢气的方法。这一过程，这承诺，使更多的氢从底物的释放耦合进一步降解与有限的呼吸产生的能量，将进一步研究根据本授权。这项技术在更广泛的工业生物技术背景下引起了人们的兴趣，因为它可以实现“不平衡”发酵的发展。将采用两种办法;生物反应器技术的发展，使用氧化还原状态来控制氧水平以实现最大底物转化，以及代谢工程来增加所需方向上的代谢通量。代谢工程也将与代谢建模结合使用，以进一步增加光发酵产氢。因此，目前的研究是基础和应用的兴趣。对AmtB参与固氮酶调节的分子细节的进一步研究应该对这种独特的代谢调节形式有更多的了解。此外，通过生理操纵和代谢工程增加氢气产量将为这些生物体生产其他化学品铺平道路，并可能提供从各种废物中可持续产生氢气的实用方法。

The non-sulfur purple photosynthetic bacteria serve as excellent model organism for research on a series of fundamental and applied basic biological areas of interest; photosynthesis, nitrogen fixation, and biotechnological conversions. The photosynthetic bacteria are thus excellent organisms for studying both general and unique aspects of nitrogen fixation and its regulation. Besides the regulation at the transcriptional level with respect to oxygen and fixed nitrogen shared by most nitrogen fixing organisms, the photosynthetic bacteria are capable of regulating the activity of preformed enzyme through the covalent and metabolic regulation of nitrogenase. We have shown that the regulation of nitrogenase with respect to the sudden external addition of ammonium requires the membrane bound ammonia sensor/transporter AmtB. Amt/Rh family members are found in almost every sequenced organism and are present in archaea, bacteria, protists, plants, fungi and invertebrates. We will continue our investigations into the structure/function of AmtB and its role in controlling the the nitrogenase regulatory system. In particular, we will be applying site directed mutagenesis to understanding the role that specific amino acids play in ammonia sensing and/or transport. The nitrogenase enzymatic machinery is also responsible for hydrogen production by photosynthetic bacteria. These organisms, which have the potential capacity to use a variety of feedstocks, are well known for their light driven conversion of organic acids to hydrogen and carbon dioxide. Thus, they are ideal candidates for two stage or co-culture systems which derive additional hydrogen from the effluents of dark fermentations. In addition, various industrial and agricultural waste streams rich in organic acids can potentially serve as substrates for photofermentation. We have recently demonstrated a number of improvements in hydrogen production including the demonstration of the stoichiometric conversion to hydrogen of glycerol produced as a waste product of biodiesel manufacture and the generation of appreciable amounts of hydrogen from various molasses fractions. In a series of investigations we have also recently shown that significant amounts of hydrogen, up to 9 moles of H2 per mole of glucose, can be obtained in a one step, single stage fermentation of glucose. Of some interest, we have also demonstrated for the first time the production of hydrogen thought microaerobic fermentation. This process, which promises to enable the release of additional hydrogen from substrates by coupling further degradation with the generation of energy from limited respiration, will be further investigated under the present grant. This technology is of interest in the broader context of industrial biotechnology as it would allow the development of “unbalanced’ fermentations. Two approaches will be used; development of bioreactor technology using a redox stat to control the oxygen levels for achieving maximum substrate conversion, and metabolic engineering to increase metabolic flux in the desired direction. Metabolic engineering will also be used, in conjunction with metabolic modeling to further increase photofermentative hydrogen production. Thus, the current research is of both fundamental and applied interest. Further research on the molecular details of the involvement of AmtB in nitrogenase regulation should shed more light on this unique form of metabolic regulation. In addition, increasing hydrogen production through both physiological manipulation and metabolic engineering will pave the way for the production of other chemicals by these organisms as well as possibly providing a practical means of sustainable hydrogen generation from various waste materials.