Impact of Substrate Glycoside Linkage and Elemental Sulfur on Bioenergetics of and Hydrogen Production by the Hyperthermophilic Archaeon Pyrococcus furiosus

Abstract

Glycoside linkage (cellobiose versus maltose) dramatically influenced bioenergetics to different extents and by different mechanisms in the hyperthermophilic archaeon Pyrococcus furiosus when it was grown in continuous culture at a dilution rate of 0.45 h ؊1 at 90°C. In the absence of S 0 , cellobiose-grown cells generated twice as much protein and had 50%-higher specific H 2 generation rates than maltose-grown cultures. Addition of S 0 to maltose-grown cultures boosted cell protein production fourfold and shifted gas production completely from H 2 to H 2 S. In contrast, the presence of S 0 in cellobiose-grown cells caused only a 1.3-fold increase in protein production and an incomplete shift from H 2 to H 2 S production, with 2.5 times more H 2 than H 2 S formed. Transcriptional response analysis revealed that many genes and operons known to be involved in ␣-or ␤-glucan uptake and processing were up-regulated in an S 0 -independent manner. Most differentially transcribed open reading frames (ORFs) responding to S 0 in cellobiose-grown cells also responded to S 0 in maltose-grown cells; these ORFs included ORFs encoding a membrane-bound oxidoreductase complex (MBX) and two hypothetical proteins (PF2025 and PF2026). However, additional genes (242 genes; 108 genes were up-regulated and 134 genes were down-regulated) were differentially transcribed when S 0 was present in the medium of maltose-grown cells, indicating that there were different cellular responses to the two sugars. These results indicate that carbohydrate characteristics (e.g., glycoside linkage) have a major impact on S 0 metabolism and hydrogen production in P. furiosus. Furthermore, such issues need to be considered in designing and implementing metabolic strategies for production of biofuel by fermentative anaerobes.Because of problems with sufficient access to petroleum and natural gas resources and the emerging threat of global warming, there is increasing interest in alternative energy options to supplement or replace fossil fuels (43). One prospect that has received considerable attention is the conversion of renewable resources (i.e., biomass) to ethanol using biological routes (20). While availability of bioprocess-based ethanol could offset current demands to some extent, problems with significant CO 2 emissions upon energy conversion would remain (37). A longer-term option being considered is the production of molecular hydrogen from biomass using fermentative, anaerobic microorganisms (38). For example, many mesophilic Clostridium and Enterobacter species can grow on fermentable sugars and produce hydrogen as a by-product of energy metabolism (8,11,16,28,38).Studies suggest that biohydrogen production rates may be enhanced at higher temperatures (9, 23). In fact, the production and consumption of molecular hydrogen drive the microbial physiology and bioenergetics of many hyperthermophilic bacteria and archaea inhabiting hydrothermal environments (1). The potential of these microorganisms for biofuel processes has not gone unnoticed (25)....

Novel Multiprotein Complexes Identified in the Hyperthermophilic Archaeon Pyrococcus furiosus by Non-denaturing Fractionation of the Native Proteome

“…3). In support of this, the expression of the genes encoding both components of PC-35 (PF0972/PF0973) are up-regulated when P. furiosus is grown on peptides (33,38,89). Another potential PC (PC-7) contains a component that is a paralog of the KOR ␥-subunit, although its function is unknown.…”

Distinct Physiological Roles of the Three [NiFe]-Hydrogenase Orthologs in the Hyperthermophilic Archaeon Thermococcus kodakarensis

“…Our genetic results revealing decreased alanine production and increased NH 3 formation in Hyh-deficient T. kodakarensis cells indicate that significant amounts of NADPH for alanine synthesis are provided through the oxidation of H 2 . The metabolic link between H 2 uptake and alanine production seems to exist in other Thermococcales members, as cultivation of cells under an increased H 2 partial pressure resulted in increased alanine production in P. furiosus (3,17) and T. litoralis (35).…”

Thermococcus kodakarensis as a Host for Gene Expression and Protein Secretion

“…Besides the thermostable DNA polymerase used in PCR, a vast range of enzymes from hyperthermophiles have been examined, including various lipases/esterases, proteases, sugar-modifying enzymes, and dehydrogenases. In contrast to the large number of studies on the enzymes from hyperthermophiles, attempts to utilize the hyperthermophile cells themselves are limited (9,23). This may be in part due to the fact that genetic manipulation systems have not been developed in most of the hyperthermophiles, which makes it difficult to utilize metabolic and cell-engineering strategies that are commonplace in mesophilic microorganisms.…”