Identification of membrane-bound [NiFe] and cytoplasmic [FeFe] hydrogenases
To identify potential hydrogenases, a BLAST search of the T. ethanolicus genome was conducted using the hydrogenases reported for T. saccharolyticum . The search identified four gene clusters with similarity to ech and hyd genes, as shown in Table 3. No matches were found in T. ethanolicus for the hfs genes of T. saccharolyticum. The genomic organization of the identified hydrogenase gene clusters in T. ethanolicus is shown in Figure 1. Based on the genetic grouping and similarity to known hydrogenases, the first two are likely to be membrane-bound [NiFe] hydrogenases while the last two are likely to be cytoplasmic [FeFe] hydrogenases. The first gene cluster similar to ech is composed of 12 genes with six genes for ech and six more matching the hyp [NiFe] maturation genes. The second gene cluster similar to T. saccharolyticum ech is composed of seven genes, similar to the mbh genes of P. furiosus, as described below. The five hyd genes of T. saccharolyticum match five similar genes in T. ethanolicus, and appear to encode hydABCD, the four-subunit cytoplasmic bifurcating hydrogenase. The gene named hydII in T. saccharolyticum has a match in T. ethanolicus but is the only hydrogenase in its genomic neighborhood. In T. saccharolyticum, hydII is 2 Kbp upstream of hyd.
In addition to hydrogenase genes from T. saccharolyticum, hydrogenase genes from two other species were used as BLAST queries as well. The C. thermocellum hydG maturase gene was used to identify a similar gene (TheetDRAFT_1696) in the T. ethanolicus genome. A BLAST search using the 14-gene mbh operon from P. furiosus identified a 13-gene cluster in T. ethanolicus, of which seven genes are the second cluster of ech-like genes previously identified using T. saccharolyticum sequences. Immediately upstream of those seven ech-like genes are a cluster of six genes annotated as cation/H+ antiporters. A similar genetic organization occurs for the mbh membrane-bound [NiFe] hydrogenase genes in P. furiosus . Besides mbh, an mbx hydrogenase and two four-subunit [NiFe] Soluble Hydrognases SHI and SHII have been reported in P. furiosus . A BLAST search using SHI and SHII as queries identified a single cluster of four genes which are annotated as sulfite reductase. The gene configuration of this cluster is shown in Figure 1. The coding sequences for the first two subunits (A and B) overlap, and in some other species occur as a single gene. Subunit C is an oxidoreductase FAD/NAD(P)-binding domain protein with electron transfer subunit and iron-sulfur cluster binding domain. Subunit D is a 4Fe-4S ferredoxin iron-sulfur binding domain-containing protein. The gene cluster was named shi for Soluble Hydrogenase I. It likely encodes an NADPH-linked cystoplasmic hydrogenase based on its similarity to the genes in P. furiosus,
The SHI and SHII hydrogenases have been well characterized in P. furiosus, and due to in vitro sulfur reductase activity were previously thought to play a role in sulfur metabolism . However, that is no longer the case due to observed down regulation of the corresponding genes when elemental sulfur is present . Our search for hydrogenases in T. ethanolicus led us to P. furiosus when we noticed the smell of hydrogen sulfide from spent cultures, but we believe that the shi genes are in fact unrelated to sulfur metabolism.
Deletion of all hydrogenase activities
Strains were constructed with deletions for the genes responsible for hydrogen production in T. ethanolicus (Table 1). All strains carry a deletion of the tdk gene encoding thymidine kinase, which allows counterselection against integrated gene cassettes and generation of markerless mutations . Rather than deleting the hyd and hydII clusters individually, a single deletion was made for hydG, which has been shown to effectively eliminate [FeFe] hydrogenase activity in C. thermocellum .
The strain with all four identified hydrogenase systems inactivated was named H0. Fermentation products were measured in batch bottle fermentations for H0 and other intermediate strains and compared to the wildtype (WT) strain and the tdk deletion strain (WT_tdk). As shown in Table 4, hydrogen production is eliminated in strain H0, and acetate levels are 10-fold lower than the controls. Lactate was the major product of strain H0 with a mass yield about 84%, while ethanol was much lower than the control, at 7.6 mM vs. 18-34 mM. The shift from ethanol and acetate production to lactate production implies pyruvate is not being efficiently converted to acetyl-coA via pyruvate:ferredoxin oxidoreductase in strain H0. Hydrogen production by ferredoxin-linked hydrogenases results in re-oxidation of reduced ferredoxin , which is required by pyruvate:ferredoxin oxidoreductase. There is no pyruvate formate lyase or pyruvate dehydrogenase in T. ethanolicus. Thus, removing hydrogen production may shift the flux of pyruvate from acetyl-coA to lactate.
Analysis of strains with one intact hydrogenase
Fermentation profiles of intermediate strains with three out of four hydrogenase systems deleted are also listed in Table 4. These strains retain only one hydrogenase while the other three are deleted. As shown in Table 4, strains H1, H2, H3, and H4 retain only ech, mbh, hyd, and shi, respectively. All these strains produce significant amount of hydrogen, suggesting that all four hydrogenases are potentially active in WT and that any of them can compensate for deletion of the others. Production of ethanol, lactate, acetate, and hydrogen is similar for H1, H2, and H4, while H3 produces about 20% less ethanol and more organic acid and hydrogen. The hydrogenases in H1, H2, and H4 are likely to be [NiFe] hydrogenases while those in H3 are [FeFe] hydrogenases. [FeFe] hydrogenases typically possess higher hydrogen evolution rates than [NiFe] hydrogenases and are often the targets of studies for biohydrogen production .
Relative to WT, the intermediate strains and WT_tdk showed lower lactate and higher ethanol levels. It is unknown why the fermentation products of WT_tdk differ somewhat from WT, but wide variation in the fermentation product profile was observed in the original species description of T. ethanolicus .
Methyl viologen hydrogenase activity
During efforts to create an ethanologenic strain of T. ethanolicus, the authors noticed high in vitro hydrogenase activity, up to 50 times greater than that of Thermoanaerobacterium saccharolyticum reported previously . Methyl viologen (MV) hydrogenase activity was assayed with cleared lysate cell extracts for strains H0, H1, H2, H3, H4, WT, and WT_tdk. MV acts as a universal electron acceptor-donor and can interact with hydrogenases that have either NAD(P)H or ferredoxin as a natural substrate . Strain H0, which does not produce hydrogen, exhibited a small background hydrogenase activity. Strain H3, producing the highest amount of hydrogen, shows the highest hydrogenase activity among the mutants (H1, H2, H3, H4) with only one hydrogenase. However, with around 10% higher hydrogen production for H3 compared to the other three strains, its hydrogenase activity is 10 times higher. Although it is expected that the majority of enzymatic activity present in whole cells is also present in the cleared lysates, it is possible that enzymatic activity present in the membrane fraction (H1 and H2) could be underrepresented . For strains WT and WT_tdk, they both have all the hydrogenases and produce comparable amount of hydrogen. However, the WT strain shows a hydrogenase activity about five times that of the WT_tdk strain. The WT strain gives twice the hydrogenase activity of H3, while the WT_tdk strain has 40% hydrogenase activity of H3. Nevertheless, WT, WT_tdk, and H3 produce almost the same amount of hydrogen. High hydrogenase activity does not correlate to high hydrogen production in T. ethanolicus, which has also been reported for T. saccharolyticum . The hydrogenases in T. ethanolicus, all capable of producing hydrogen, seem to be at standby mode to balance redox reactions for pyruvate metabolism to acetyl-CoA. When fermentation goes in the direction of ethanol production, a small amount of hydrogen is produced. When fermentation goes in the direction of organic acids, more hydrogen is produced. This might explain why variations in product ratios occur in the fermentation of wild-type T. ethanolicus [2, 20].