Tian Liang, Lo Jonathan, Shao Xiongjun, Zheng Tianyong, Olson Daniel G, Lynd Lee R
Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, USA.
Bioenergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
Appl Environ Microbiol. 2016 Nov 21;82(24):7134-7141. doi: 10.1128/AEM.02130-16. Print 2016 Dec 15.
Ferredoxin:NAD oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway in Thermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activity in vitro with a ferredoxin-based FNOR assay. To determine its role in metabolism, the tsac_1705 gene was deleted in different strains of T. saccharolyticum In wild-type T. saccharolyticum, deletion of tsac_1705 resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR in T. saccharolyticum When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. Finally, we tested the effect of heterologous expression of Tsac_1705 in Clostridium thermocellum and found improvements in both the titer and the yield of ethanol.
Redox balance plays a crucial role in many metabolic engineering strategies. Ferredoxins are widely used as electron carriers for anaerobic microorganism and plants. This study identified the gene responsible for electron transfer from ferredoxin to NAD, a key reaction in the ethanol production pathway of this organism and many other metabolic pathways. Identification of this gene is an important step in transferring the ethanol production ability of this organism to other organisms.
铁氧化还原蛋白：NAD氧化还原酶（NADH - FNOR）催化电子从还原型铁氧化还原蛋白转移至NAD。据推测，该酶是嗜热解糖栖热厌氧菌基于NADH的乙醇代谢途径中铁氧化还原蛋白氧化的主要负责酶；然而，相应基因尚未被鉴定。在此，我们基于其功能域的同源性将Tsac_1705蛋白鉴定为候选FNOR。然后，我们通过基于铁氧化还原蛋白的FNOR测定在体外证实了其活性。为确定其在代谢中的作用，在嗜热解糖栖热厌氧菌的不同菌株中删除了tsac_1705基因。在野生型嗜热解糖栖热厌氧菌中，tsac_1705的缺失导致NADH - FNOR活性丧失75%，这表明Tsac_1705是嗜热解糖栖热厌氧菌中的主要NADH - FNOR。当NADH和NADPH连接的FNOR基因均被删除时，乙醇滴度降低，乙醇与乙酸的比例接近1，表明FNOR活性缺失。最后，我们测试了Tsac_1705在热纤梭菌中的异源表达效果，发现乙醇的滴度和产量均有所提高。
氧化还原平衡在许多代谢工程策略中起着关键作用。铁氧化还原蛋白广泛用作厌氧微生物和植物的电子载体。本研究鉴定了负责铁氧化还原蛋白至NAD电子转移的基因，这是该生物体乙醇生产途径及许多其他代谢途径中的关键反应。该基因的鉴定是将该生物体的乙醇生产能力转移至其他生物体的重要一步。
