Beadle Center for Genetics, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
Appl Environ Microbiol. 2018 Aug 17;84(17). doi: 10.1128/AEM.00998-18. Print 2018 Sep 1.
When carbohydrates are fermented by the hyperthermophilic anaerobe , molecular hydrogen (H) is formed in strict proportion to substrate availability. Excretion of the organic acids acetate and lactate provide an additional sink for removal of excess reductant. However, mechanisms controlling energy management of these metabolic pathways are largely unexplored. To investigate this topic, transient gene inactivation was used to block lactate production as a strategy to produce spontaneous mutant cell lines that overproduced H through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded by ) with a truncated fused to a kanamycin resistance cassette expressed from a native P promoter. Passage of the unstable recombinant resulted in loss of the genetic marker and recovery of evolved cell lines, including strain Tma200. Relative to the wild type, and considering the mass balance of fermentation substrate and products, Tma200 grew more slowly, produced H at levels above the physiologic limit, and simultaneously consumed less maltose while oxidizing it more efficiently. Whole-genome resequencing indicated that the ABC maltose transporter subunit, encoded by , had undergone repeated mutation, and high-temperature anaerobic [C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of the mutation into a clean genetic background also conferred increased H production, confirming that the mutant allele was sufficient for increased H synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H production, changing fermentation efficiency and shifting energy management. Biorenewable energy sources are of growing interest to mitigate climate change, but like other commodities with nominal value, require innovation to maximize yields. Energetic considerations constrain production of many biofuels, such as molecular hydrogen (H) because of the competing needs for cell mass synthesis and metabolite formation. Here we describe cell lines of the extremophile that exceed the physiologic limits for H formation arising from genetic changes in fermentative metabolism. These cell lines were produced using a novel method called transient gene inactivation combined with adaptive laboratory evolution. Genome resequencing revealed unexpected changes in a maltose transport protein. Reduced rates of sugar uptake were accompanied by lower rates of growth and enhanced productivity of H.
当碳水化合物被嗜热厌氧菌发酵时，分子氢（H）会严格按照底物的可用性形成。乙酸盐和乳酸盐等有机酸的排泄为去除多余还原剂提供了另一个汇。然而，控制这些代谢途径能量管理的机制在很大程度上仍未得到探索。为了研究这个课题，我们使用瞬时基因失活来阻断乳酸的产生，作为一种策略，通过突变未预测的遗传靶点，产生自发突变的细胞系，从而过度产生 H。利用单交叉同源染色体重组，用融合到卡那霉素抗性盒的截断产物破坏乳酸脱氢酶（由编码），该基因与来自天然 P 启动子的表达载体相连。不稳定重组体的传递导致遗传标记的丢失和进化细胞系的恢复，包括 Tma200 菌株。与野生型相比，考虑到发酵底物和产物的质量平衡，Tma200 生长缓慢，产生的 H 水平高于生理极限，同时消耗的麦芽糖更少，而氧化效率更高。全基因组重测序表明，ABC 麦芽糖转运体亚基，由编码，经历了多次突变，高温厌氧 [C]麦芽糖转运测定表明麦芽糖转运速率降低。将突变转移到一个干净的遗传背景中也赋予了更高的 H 产量，证实突变等位基因足以增加 H 合成。这些数据表明，麦芽糖摄取率的降低伴随着 H 产量的增加，改变了发酵效率并改变了能量管理。生物可再生能源越来越受到气候变化的关注，但与其他具有名义价值的商品一样，需要创新来最大限度地提高产量。由于细胞质量合成和代谢物形成的竞争需求，能量因素限制了许多生物燃料的生产，例如分子氢（H）。在这里，我们描述了极端微生物的细胞系，这些细胞系通过发酵代谢的遗传变化，超过了 H 形成的生理限制。这些细胞系是使用一种称为瞬时基因失活与适应性实验室进化相结合的新方法产生的。基因组重测序揭示了麦芽糖转运蛋白的意外变化。糖摄取率降低伴随着生长率降低和 H 生产力提高。
