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This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The Δldh Δpta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.

Pyrobaculum islandicum uses iron, thiosulfate, and elemental sulfur for anaerobic respiration, while Pyrobaculum aerophilum uses iron and nitrate; however, the constraints on these processes and their physiological mechanisms for iron and sulfur reduction are not well understood. Growth rates on sulfur compounds are highest at pH 5 to 6 and highly reduced (<-420-mV) conditions, while growth rates on nitrate and iron are highest at pH 7 to 9 and more-oxidized (>-210-mV) conditions. Growth on iron expands the known pH range of growth for both organisms. P. islandicum differs from P. aerophilum in that it requires direct contact with insoluble iron oxide for growth, it did not produce any extracellular compounds when grown on insoluble iron, and it lacked 2,6-anthrahydroquinone disulfonate oxidase activity. Furthermore, iron reduction in P. islandicum appears to be completely independent of c-type cytochromes. Like that in P. aerophilum, NADH-dependent ferric reductase activity in P. islandicum increased significantly in iron-grown cultures relative to that in non-iron-grown cultures. Proteomic analyses showed that there were significant increases in the amounts of a putative membrane-bound thiosulfate reductase in P. islandicum cultures grown on thiosulfate relative to those in cultures grown on iron and elemental sulfur. This is the first evidence of this enzyme being used in either a hyperthermophile or an archaeon. Pyrobaculum arsenaticum and Pyrobaculum calidifontis also grew on Fe(III) citrate and insoluble iron oxide, but only P. arsenaticum could grow on insoluble iron without direct contact.

The hyperthermophilic archaeon Pyrobaculum aerophilum used 20 mM Fe(III) citrate, 100 mM poorly crystalline Fe(III) oxide, and 10 mM KNO3 as terminal electron acceptors. The two forms of iron were reduced at different rates but with equal growth yields. The insoluble iron was reduced when segregated spatially by dialysis tubing, indicating that direct contact with the iron was not necessary for growth. When partitioned, there was no detectable Fe(III) or Fe(II) outside of the tubing after growth, suggesting that an electron shuttle, not a chelator, may be used as an extracellular mediator of iron reduction. The addition of 25 and 50% (vol vol(-1)) cell-free spent insoluble iron media to fresh media led to growth without a lag phase. Liquid chromatography analysis of spent media showed that cultures grown in iron, especially insoluble iron, produced soluble extracellular compounds that were absent or less abundant in spent nitrate medium. NADH-dependent ferric reductase activity increased approximately 100-fold, while nitrate reductase activity decreased 10-fold in whole-cell extracts from iron-grown cells relative to those from nitrate-grown cells, suggesting that dissimilatory iron reduction was regulated. A novel 2,6-anthrahydroquinone disulfonate oxidase activity was more than 580-fold higher in iron-grown cells than in nitrate-grown cells. The activity was primarily (>95%) associated with the membrane cellular fraction, but its physiological function is unknown. Nitrate-grown cultures produced two membrane-bound, c-type cytochromes that are predicted to be monoheme and part of nitrite reductase and a bc1 complex using genome analyses. Only one cytochrome was present in cells grown on Fe(III) citrate whose relative abundance was unchanged.