When carbohydrates are fermented by the hyperthermophilic anaerobe Thermotoga maritima, molecular hydrogen (H2) 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 H2 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 PgroESL 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 H2 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 [14C]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 H2 production, confirming that the mutant allele was sufficient for increased H2 synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H2 production, changing fermentation efficiency and shifting energy management.
- Adaptive laboratory evolution
- Bacterial genetics
- Thermotoga maritima
- Transient gene inactivation
ASJC Scopus subject areas
- Food Science
- Applied Microbiology and Biotechnology