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DNA encompassing the structural genes of an Escherichia coli [NiFe] hydrogenase has been cloned and sequenced. The genes were identified as those encoding the large and small subunits of hydrogenase isozyme 1 based on NH2-terminal sequences of purified subunits (kindly provided by K. Francis and K. T. Shanmugam). The structural genes formed part of a putative operon that contained four additional open reading frames. We have designated the operon hya and the six open reading frames hyaA through F. hyaA and hyaB encode the small and large structural subunits, respectively. The nucleotide-derived amino acid sequence of hyaC has a calculated molecular mass of 27.6 kilodaltons, contains 20% aromatic residues, and has four potential membrane-spanning regions. Open reading frames hyaD through F could encode polypeptides of 21.5, 14.9, and 31.5 kilodaltons, respectively. These putative peptides have no homology to other reported protein sequences, and their functions are unknown.The anaerobic hydrogen metabolism of Escherichia coli and other enterobacteria is intricately regulated with regard to both hydrogen production during fermentation and hydrogen oxidation during anaerobic respiration (13,14). This complex regulatory system responds to specific substrates as well as to global regulatory signals and results in the biosynthesis of discrete hydrogenases specific to a given metabolic pathway. Three E. coli hydrogenases have been described which are synthesized in response to different physiological conditions. Hydrogenases 1 and 2 have been biochemically characterized and are immunologically distinct membranebound nickel-containing proteins (2, 3, 31). The existence of hydrogenase 3 was originally inferred from the fact that hydrogenases 1 and 2 immunoprecipitated from cell lysates did not account for total hydrogenase activity (30). Hydrogenase 3 activity is very labile and has been only partially characterized (34). Hydrogenases 1 and 3 are induced to higher levels by growth on glucose and formate. Hydrogenase 3 has also been shown to have a role in the formate hydrogenlyase pathway and accounts for about 60 to 70% of the total hydrogenase activity in the cell. The physiological role of hydrogenase 1 has not been defined. Hydrogenase 2 has been implicated as a respiratory uptake hydrogenase coupled to fumarate reduction and is induced to a higher level by growth in the presence of glycerol and fumarate (30).The presence of three different enzymes catalyzing the same reactions makes the biochemical, physiological, and genetic analyses of their metabolic roles technically difficult. Toward understanding the physiological roles of the different hydrogenases, a large number of E. coli mutants defective in hydrogenase activities have been analyzed. Most of these mutants lack all three hydrogenase activities; based on genetic analysis, the mutations appear to be exclusively at loci affecting regulation and do not encompass the hydrogenase structural genes. These genetic loci have been designated as hydA through F (8, 16...

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Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two-phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5 mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway-only control.

, DNA 6:539-551, 1987) were analyzed by the codon usage method of Staden and McLachlan. The reported reading frames were found to contain regions of low codon probability which are matched by more probable sequences in other frames. Renewed nucleotide sequencing showed the probable frames to be correct. The corrected sequences of the two small and large subunits share a significant degree of sequence homology. The small subunit, which contains 10 conserved cysteine residues, is likely to coordinate at least 2 iron-sulfur clusters, while the finding of a selenocysteine codon (TGA) near the 3' end of the [NiFeSe] large-subunit gene matched by a regular cysteine codon (TGC) in the [NiFe] large-subunit gene indicates the presence of some of the ligands to the active-site nickel in the large subunit.Our knowledge of the structure and function of hydrogenases is increasing rapidly because of work on the molecular biology of the genes encoding these proteins. These investigations have provided the primary structure of the [Fe] hydrogenase from Desulfovibrio vulgaris subsp. vulgaris Hildenborough (12,23,24) [Fe] hydrogenase from D. vulgaris (63% G+C) was used as the standard in initial calculations, which indicated that the gene for the large subunit of D. baculatus (57% G+C) and both genes for the small and large subunits of D. gigas (63% G+C) contained regions of low coding probability. These were matched by regions of high coding probability in other frames, indicating the possibility of frameshifts due to errors in the original nucleotide sequence data. Comparable results were obtained when the codon usage table for the smallsubunit gene of the [NiFeSe] hydrogenase from D. baculatus, which displayed a high coding probability throughout its reading frame in these initial calculations, was used as the standard. Use of this table is preferred for calculations on the D. baculatus genes in view of the different G + C contents indicated above, and the results of the calculations on the previously published sequences (7,9) are shown in Fig. 1A through F.The large-subunit gene of D. baculatus hydrogenase, which contained three regions of codon improbability (Fig. 1C, I through III), was next resequenced, and this confirmed the correctness of the codon probability analysis. An erratum with the complete, corrected sequence has been pub-