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When carbohydrates are fermented by the hyperthermophilic anaerobe Thermotoga maritima , molecular hydrogen (H 2 ) 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 2 through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded by ldh ) with a truncated ldh fused to a kanamycin resistance cassette expressed from a native P  groESL  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 2 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 malK3 , had undergone repeated mutation, and high-temperature anaerobic [ 14 C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of the malK3 mutation into a clean genetic background also conferred increased H 2 production, confirming that the mutant allele was sufficient for increased H 2 synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H 2 production, changing fermentation efficiency and shifting energy management. IMPORTANCE 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 2 ) because of the competing needs for cell mass synthesis and metabolite formation. Here we describe cell lines of the extremophile Thermotoga maritima that exceed the physiologic limits for H 2 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 2 .

Uncoupling Fermentative Synthesis of Molecular Hydrogen from Biomass Formation in Thermotoga maritima