Biochemical and physiological characterization of the pyruvate formate-lyase Pfl1 of Chlamydomonas reinhardtii, a typically bacterial enzyme in a eukaryotic alga

The unicellular green alga Chlamydomonas reinhardtii has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen-sensitive [Fe-Fe] hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and, in addition, a formate- and ethanol-producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl; EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes but only rarely in eukaryotes. Both the hydrogen- and the formate/ethanol-producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulfur depletion in illuminated cultures. Before now, the presence of a Pfl protein in C. reinhardtii was predicted from formate secretion and the homology of the deduced protein of the PFL1 gene model to known Pfl enzymes. In this study, we proved the formate-producing activity of the putative Pfl1 enzyme by heterologous expression of the C. reinhardtii PFL1 cDNA in Escherichia coli and subsequent in vitro activity tests of the purified protein. Furthermore, a Pfl-deficient E. coli strain secretes formate when expressing the PFL1 cDNA of C. reinhardtii. We also examined the Pfl1 fermentation pathway of C. reinhardtii under the physiological condition of sulfur depletion. Genetic and biochemical analyses show that sulfur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and also potentially ethanol-producing enzymes, such as pyruvate decarboxylase (EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.     

Enhanced hydrogen production from glucose by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

To utilize fermentative bacteria for producing the alternative fuel hydrogen, we performed successive rounds of P1 transduction from the Keio Escherichia coli K-12 library to introduce multiple, stable mutations into a single bacterium to direct the metabolic flux toward hydrogen production. E. coli cells convert glucose to various organic acids (such as succinate, pyruvate, lactate, formate, and acetate) to synthesize energy and hydrogen from formate by the formate hydrogen-lyase (FHL) system that consists of hydrogenase 3 and formate dehydrogenase-H. We altered the regulation of FHL by inactivating the repressor encoded by hycA and by overexpressing the activator encoded by fhlA, removed hydrogen uptake activity by deleting hyaB (hydrogenase 1) and hybC (hydrogenase 2), redirected glucose metabolism to formate by using the fdnG, fdoG, narG, focA, focB, poxB, and aceE mutations, and inactivated the succinate and lactate synthesis pathways by deleting frdC and ldhA, respectively. The best of the metabolically engineered strains, BW25113 hyaB hybC hycA fdoG frdC ldhA aceE, increased hydrogen production 4.6-fold from glucose and increased the hydrogen yield twofold from 0.65 to 1.3 mol H₂/mol glucose (maximum, 2 mol H₂/mol glucose).     

End-product induced metabolic shifts in <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> ATCC 27405

When attempting to increase yields of desirable end-products during fermentation, there is the possibility that increased concentrations of one product redirects metabolism towards the synthesis of less desired products. Changes in growth, final end-product concentrations, and activities of enzymes involved in pyruvate catabolism and fermentative end-product formation were studied in Clostridium thermocellum in response to the addition of individual end-products (H₂, acetate, ethanol, formate, and lactate) to the growth medium. These were added to the growth medium at concentrations ten times greater than those found at the end of growth in cultures grown under carbon-limited conditions using cellobiose (1.1 g l⁻¹) as model soluble substrate. Although growth rate and final cell biomass decreased significantly with the addition of all end-products, addition of individual end-products had less pronounced effects on growth. Metabolic shifts, represented by changes in final end-product concentrations, were observed; H₂ and acetate yields increased in the presence of exogenous ethanol and lactate, while ethanol yields increased in the presence of exogenous hydrogen (H₂), acetate, and lactate. Late exponential phase enzyme activity data of enzymes involved in pyruvate catabolism and end-product formation revealed no changes in enzyme levels greater than 2-fold in response to the presence of any given end-product, with the exception of pyruvate:formate lyase (PFL), ferredoxin-dependent hydrogenase (Fd-H₂ase), and pyruvate:ferredoxin oxidoreductase (PFO): PFL and Fd-H₂ase activities increased 2-fold in the presence of ethanol, while PFO activity decreased by 57% in the presence of sodium formate. Changes in enzyme levels did not necessarily correlate with changes in final end-product yields, suggesting that changes in final end-product yields may be governed by thermodynamic considerations rather than levels of enzyme expressed under the conditions tested. We demonstrate that bacterial metabolism may be manipulated in order to selectively improve desired product yields.     

Metabolic engineering to enhance bacterial hydrogen production

Hydrogen fuel is renewable, efficient and clean, and fermentative bacteria hold great promise for its generation. Here we use the isogenic Escherichia coli K‐12 KEIO library to rapidly construct multiple, precise deletions in the E. coli genome to direct the metabolic flux towards hydrogen production. Escherichia coli has three active hydrogenases, and the genes involved in the regulation of the formate hydrogen lyase (FHL) system for synthesizing hydrogen from formate via hydrogenase 3 were also manipulated to enhance hydrogen production. Specifically, we altered regulation of FHL by controlling the regulators HycA and FhlA, removed hydrogen consumption by hydrogenases 1 and 2 via the hyaB and hybC mutations, and re‐directed formate metabolism using the fdnG, fdoG, narG, focA, fnr and focB mutations. The result was a 141‐fold increase in hydrogen production from formate to create a bacterium (BW25113 hyaB hybC hycA fdoG/pCA24N‐FhlA) that produces the largest amount of hydrogen to date and one that achieves the theoretical yield for hydrogen from formate. In addition, the hydrogen yield from glucose was increased by 50%, and there was threefold higher hydrogen production from glucose with this strain.     

Compartmentalisation of [FeFe]‐hydrogenase maturation in Chlamydomonas reinhardtii

Molecular hydrogen (H₂) can be produced in green microalgae by [FeFe]‐hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA1 contains a catalytic site comprising a classic [4Fe4S] cluster linked to a unique 2Fe sub‐cluster. From in vitro studies it appears that the [4Fe4S] cluster is incorporated first by the housekeeping FeS cluster assembly machinery, followed by the 2Fe sub‐cluster, whose biosynthesis requires the specific maturases HYDEF and HYDG. To investigate the maturation process in vivo, we expressed HYDA1 from the C. reinhardtii chloroplast and nuclear genomes (with and without a chloroplast transit peptide) in a hydrogenase‐deficient mutant strain, and examined the cellular enzymatic hydrogenase activity, as well as in vivo H₂ production. The transformants expressing HYDA1 from the chloroplast genome displayed levels of H₂ production comparable to the wild type, as did the transformants expressing full‐length HYDA1 from the nuclear genome. In contrast, cells equipped with cytoplasm‐targeted HYDA1 produced inactive enzyme, which could only be activated in vitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA1 FeS cluster can only be built by the chloroplastic FeS cluster assembly machinery. Further, the expression of a bacterial hydrogenase gene, CPI, from the C. reinhardtii chloroplast genome resulted in H₂‐producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA1 in vivo and that overexpression of foreign hydrogenases in C. reinhardtii is possible. All chloroplast transformants were stable and no toxic effects were seen from HYDA1 or CPI expression.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities

In the past, it has been difficult to discriminate between hydrogen synthesis and uptake for the three active hydrogenases in Escherichia coli (hydrogenase 1, 2, and 3); however, by combining isogenic deletion mutations from the Keio collection, we were able to see the role of hydrogenase 3. In a cell that lacks hydrogen uptake via hydrogenase 1 (hyaB) and via hydrogenase 2 (hybC), inactivation of hydrogenase 3 (hycE) decreased hydrogen uptake. Similarly, inactivation of the formate hydrogen lyase complex, which produces hydrogen from formate (fhlA) in the hyaB hybC background, also decreased hydrogen uptake; hence, hydrogenase 3 has significant hydrogen uptake activity. Moreover, hydrogen uptake could be restored in the hyaB hybC hycE and hyaB hybC fhlA mutants by expressing hycE and fhlA, respectively, from a plasmid. The hydrogen uptake results were corroborated using two independent methods (both filter plate assays and a gas-chromatography-based hydrogen uptake assay). A 30-fold increase in the forward reaction, hydrogen formation by hydrogenase 3, was also detected for the strain containing active hydrogenase 3 activity but no hydrogenase 1 or 2 activity relative to the strain lacking all three hydrogenases. These results indicate clearly that hydrogenase 3 is a reversible hydrogenase.     

Pyruvate fermentation in Rhodospirillum rubrum and after transfer from aerobic to anaerobic conditions in the dark

The fermentative metabolism of Rhodospirillum rubrum (strain Ha, F₁, S₁) was studied after transfering the cells from aerobic to anaerobic dark culture conditions. Pyruvate was metabolized mainly to acetate and formate, and to a lesser extent to CO₂ and propionate, by all strains. Therefore, pyruvate formate lyase would appear to be the characteristic key enzyme of the dark anaerobic fermentation metabolism in R. rubrum. Strain F₁ and S₁ metabolized the formate further to H₂ and CO₂. It is concluded that this cleavage was catalysed by a formate hydrogen lyase system. Strain Ha was unable to metabolize formate. The cleavage of formate and the synthesis of poly--hydroxy-butyric acid were increased by a low pH value (6.5). Fermentation equations and schemes of the pyruvate metabolism are discussed.     

Dependence on the F<Subscript>0</Subscript>F<Subscript>1</Subscript>-ATP synthase for the activities of the hydrogen-oxidizing hydrogenases 1 and 2 during glucose and glycerol fermentation at high and low pH in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli has four [NiFe]-hydrogenases (Hyd); three of these, Hyd-1, Hyd-2 and Hyd-3 have been characterized well. In this study the requirement for the F₀F₁-ATP synthase for the activities of the hydrogen-oxidizing hydrogenases Hyd-1 and Hyd-2 was examined. During fermentative growth on glucose at pH 7.5 an E. coli F₀F₁-ATP synthase mutant (DK8) lacked hydrogenase activity. At pH 5.5 hydrogenase activity was only 20% that of the wild type. Using in-gel activity staining, it could be demonstrated that both Hyd-1 and Hyd-2 were essentially inactive at these pHs, indicating that the residual activity at pH 5.5 was due to the hydrogen-evolving Hyd-3 enzyme. During fermentative growth in the presence of glycerol, hydrogenase activity in the mutant was highest at pH 7.5 attaining a value of 0.76 U/mg, or ~50% of wild type activity, and Hyd-2 was only partially active at this pH, while Hyd-1 was inactive. Essentially no hydrogenase activity was measured at pH 5.5 during growth with glycerol. At this pH the mutant had a hydrogenase activity that was maximally only ~10% of wild type activity with either carbon substrate but a weak activity of both Hyd-1 and Hyd-2 could be detected. Taken together, these results demonstrate for the first time that the activity of the hydrogen-oxidizing hydrogenases in E. coli depends on an active F₀F₁-ATP synthase during growth at high and low pH.     

Induction and regulation of ribulose bisphosphate carboxylase activity in Rhizobium japonicum during formate-dependent growth

Rhizobium japonicum CJ1 was capable of growing using formate as the sole source of carbon and energy. During aerobic growth on formate a cytoplasmic NAD⁺-dependent formate dehydrogenase and ribulose bisphosphate carboxylase activity was demonstrated in cell-free extracts, but hydrogenase enzyme activity could not be detected. Under microaerobic growth conditions either formate or hydrogen metabolism could separately or together support ribulose bisphosphate carboxylase-dependent CO₂ fixation. A number of R. japonicum strains defective in hydrogen uptake activity were shown to metabolise formate and induce ribulose bisphosphate carboxylase activity. The induction and regulation of ribulose bisphosphate carboxylase is discussed.Abbreviations hup hydrogen uptake - MOPS 3-(N-morpholino)-propanesulphonate - TSA tryptone soya agar - RuBP ribulose 1,5-bisphosphate - FDH formate dehydrogenase     

Mutational analysis and characterization of the Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1.

Deletion mutants of Escherichia coli specific for hydrogenase isoenzyme 1 (HYD1) have been constructed and characterized. The hya operon, which contains genes for the two HYD1 structural subunits and four additional genes, was mapped at 22 min on the E. coli chromosome. The total hydrogenase activities of the HYD1-negative mutant and wild-type strains were similar. However, the formate dehydrogenase activity associated with the formate hydrogen lyase pathway was lower in the mutant. The hya mutant (strain AP1), complemented with only the hydrogenase structural genes (hyaAB), produced antigenically identifiable but inactive HYD1 protein. The first five genes of hya (hyaA to hyaE) were required for the synthesis of active HYD1, but wild-type levels of HYD1 activity were restored only when mutant cells were transformed with all six genes of the operon. When AP1 was complemented with hya carried on a high-copy-number plasmid, the HYD1 structural subunits were overexpressed, but the excess protein was unprocessed and localized in the soluble fraction of the cell. The products of hyaDEF are postulated to be involved in the processing of nascent structural subunits (HYAA and HYAB). This processing takes place only after the subunits are inserted into the cell membrane. It is concluded that the biosynthesis of active HYD1 is a complex biochemical process involving the cellular localization and processing of nascent structural subunits, which are in turn dependent on the insertion of nickel into the nascent HYD1 large subunit.     

H2 production of Rhodospirillum rubrum during adaptation to anaerobic dark conditions

Rhodospirillum rubrum is able to produce H₂ during fermentation anaerobically in the dark in two ways, namely through formate hydrogen lyase and through the nitrogenase. After chemotrophic preculture aerobically in the dark formate hydrogen lyase was synthesized after a lag phase, whilst after phototrophic preculture a slight activity was present from the beginning of the anaerobic dark culture. During fermentation metabolism its activity increased noticeably. Hydrogen production through the nitrogenase occurred if the nitrogenase had been activated during phototrophic preculture. It ceased during fermentation metabolism after about 3 1/2 h anaerobic dark culture. The CO insensitive H₂ production by the nitrogenase could be partially inhibited by N₂. Potential activity of this system, however, remained and could be increased under conditions of nitrogenase induction. It seems therefore possible that synthesis of nitrogenase under N-deficiency can occur during fermentation metabolism in the same way as the formation of the photosynthetic apparatus in order to prepare for subsequent phototrophic metabolism.Abbreviations CAP chloramphenicol - DSM Deutsche Sammlung von Mikroorganismen, Göttingen - FHL formate hydrogen lyase - O.D optical density - PFL pyruvate formate lyase     

Dissecting the roles of Escherichia coli hydrogenases in biohydrogen production.

Escherichia coli can perform at least two modes of anaerobic hydrogen metabolism and expresses at least two types of hydrogenase activity. Respiratory hydrogen oxidation is catalysed by two 'uptake' hydrogenase isoenzymes, hydrogenase -1 and -2 (Hyd-1 and -2), and fermentative hydrogen production is catalysed by Hyd-3. Harnessing and enhancing the metabolic capability of E. coli to perform anaerobic mixed-acid fermentation is therefore an attractive approach for bio-hydrogen production from sugars. In this work, the effects of genetic modification of the genes encoding the uptake hydrogenases, as well as the importance of preculture conditions, on hydrogen production and fermentation balance were examined. In suspensions of resting cells pregrown aerobically with formate, deletions in Hyd-3 abolished hydrogen production, whereas the deletion of both uptake hydrogenases improved hydrogen production by 37% over the parent strain. Under fermentative conditions, respiratory H2 uptake activity was absent in strains lacking Hyd-2. The effect of a deletion in hycA on H2 production was found to be dependent upon environmental conditions, but H2 uptake was not significantly affected by this mutation.     

Nitrogen deprivation results in photosynthetic hydrogen production in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The unicellular green alga Chlamydomonas reinhardtii is able to use photosynthetically provided electrons for the production of molecular hydrogen by an [FeFe]-hydrogenase HYD1 accepting electrons from ferredoxin PetF. Despite the severe sensitivity of HYD1 towards oxygen, a sustained and relatively high photosynthetic hydrogen evolution capacity is established in C. reinhardtii cultures when deprived of sulfur. One of the major electron sources for proton reduction under this condition is the oxidation of starch and subsequent non-photochemical transfer of electrons to the plastoquinone pool. Here we report on the induction of photosynthetic hydrogen production by Chlamydomonas upon nitrogen starvation, a nutritional condition known to trigger the accumulation of large deposits of starch and lipids in the green alga. Photochemistry of photosystem II initially remained on a higher level in nitrogen-starved cells, resulting in a 2-day delay of the onset of hydrogen production compared with sulfur-deprived cells. Furthermore, though nitrogen-depleted cells accumulated large amounts of starch, both hydrogen yields and the extent of starch degradation were significantly lower than upon sulfur deficiency. Starch breakdown rates in nitrogen or sulfur-starved cultures transferred to darkness were comparable in both nutritional conditions. Methyl viologen treatment of illuminated cells significantly enhanced the efficiency of photosystem II photochemistry in sulfur-depleted cells, but had a minor effect on nitrogen-starved algae. Both the degradation of the cytochrome b ₆ f complex which occurs in C. reinhardtii upon nitrogen starvation and lower ferredoxin amounts might create a bottleneck impeding the conversion of carbohydrate reserves into hydrogen evolution.     

Hydrogen as an energy source for the human pathogen <Emphasis Type="Italic">Bilophila wadsworthia</Emphasis>

The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H₂, formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia.     

Genetic analysis of mutants of Salmonella typhimurium deficient in formate dehydrogenase activity

Summary A genetical study of mutants of Salmonella typhimurium deficient in formate dehydrogenase activity was performed. The affected gene was designated fdh A and mapped at 116 min, the order of genes in that region being xyl-fdh A-mtl-cys E.Abbreviations FHL formate hydrogenylase - FDH (PMS) formate dehydrogenase (phenazine methosulfate) - FDH (BV) formate dehydrogenase (benzyl viologen) - HYD hydrogenase - NR nitrate-reductase - TTR tetrathionate-reductase     

Overexpression of <Emphasis Type="Italic">ADH1</Emphasis> and <Emphasis Type="Italic">HXT1</Emphasis> genes in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> improves the fermentative efficiency during tequila elaboration

This work assessed the effect of the overexpression of ADH1 and HXT1 genes in the Saccharomyces cerevisiae AR5 strain during fermentation of Agave tequilana Weber blue variety must. Both genes were cloned individually and simultaneously into a yeast centromere plasmid. Two transformant strains overexpressing ADH1 and HXT1 individually and one strain overexpressing both genes were randomly selected and named A1, A3 and A5 respectively. Overexpression effect on growth and ethanol production of the A1, A3 and A5 strains was evaluated in fermentative conditions in A. tequilana Weber blue variety must and YPD medium. During growth in YPD and Agave media, all the recombinant strains showed lower cell mass formation than the wild type AR5 strain. Adh enzymatic activity in the recombinant strains A1 and A5 cultivated in A. tequilana and YPD medium was higher than in the wild type. The overexpression of both genes individually and simultaneously had no significant effect on ethanol formation; however, the fermentative efficiency of the A5 strain increased from 80.33% to 84.57% and 89.40% to 94.29% in YPD and Agave medium respectively.     

Characterization and physiological roles of membrane-bound hydrogenase isoenzymes from Salmonella typhimurium.

We found that Salmonella typhimurium strain LT2 (Z) possessed two immunologically distinct, membrane-bound hydrogenase isoenzymes, which were similar in electrophoretic mobilities and apoprotein contents to hydrogenase isoenzymes 1 and 2 of Escherichia coli. The S. typhimurium enzymes cross-reacted with antibodies raised to the respective hydrogenase isoenzymes of E. coli. As for E. coli, an additional membrane-bound hydrogenase activity (termed hydrogenase 3), which did not cross-react with antibodies raised against either hydrogenase 1 or 2, was also present in detergent-dispersed membrane preparations. The physiological role of each of the three isoenzymes in E. coli has remained unclear owing to the lack of mutants specifically defective for individual isoenzymes. However, analysis of two additional wild-type isolates of S. typhimurium revealed specific defects in their hydrogenase isoenzyme contents. S. typhimurium LT2 (A) lacked isoenzyme 2 but possessed normal levels of hydrogenases 1 and 3. S. typhimurium LT7 lacked both isoenzymes 1 and 2 but retained normal hydrogenase 3 activity. Characterization of hydrogen metabolism by these hydrogenase-defective isolates allowed us to identify the physiological role of each of the three isoenzymes. Hydrogenase 3 activity correlated closely with formate hydrogenlyase-dependent hydrogen evolution, whereas isoenzyme 2 catalyzed hydrogen uptake (oxidation) during anaerobic, respiration-dependent growth. Isoenzyme 1 also functioned as an uptake hydrogenase but only during fermentative growth. We postulate that this enzyme functions in a hydrogen-recycling reaction which operates during fermentative growth.     

Altered fermentative metabolism in Chlamydomonas reinhardtii mutants lacking pyruvate formate lyase and both pyruvate formate lyase and alcohol dehydrogenase

Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H(2) production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H(2) production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.     

Laboratoire de Chimie Bactérienne C.N.R.S., Marsielle, France.

Summary Mutants of E. coli, completely devoid of nitrite reductase activity with glucose or formate as donor were studied. Biochemical analysis indicates that they are simultaneously affected in nitrate reductase, nitrite reductase, fumarate reductase and hydrogenase activities as well as in cytochrome c₅₅₂ biosynthesis. The use of an antiserum specific for nitrate reductase shows that the nitrate reductase protein is probably missing. A single mutation is responsible for this phenotype: the gene affected, nir R, is located close to tyr R i.e. at 29 min on the chromosomal map.Abbreviations BV Benzyl-Viologen - NTG N-methyl-N-nitro-N-nitrosoguanidine - NR nitrate reductase - NIR nitrite reductase - FR fumarate reductase - HYD hydrogenase - CYT c₅₅₂ cytochrome c₅₅₂     

Enhanced hydrogen production from glucose using ldh- and frd-inactivated Escherichia coli strains

We improved the hydrogen yield from glucose using a genetically modified Escherichia coli. E. coli strain SR15 (ΔldhA, ΔfrdBC), in which glucose metabolism was directed to pyruvate formate lyase (PFL), was constructed. The hydrogen yield of wild-type strain of 1.08 mol/mol glucose, was enhanced to 1.82 mol/mol glucose in strain SR15. This figure is greater than 90 % of the theoretical hydrogen yield of facultative anaerobes (2.0 mol/mol glucose). Moreover, the specific hydrogen production rate of strain SR15 (13.4 mmol h⁻¹ g⁻¹ dry cell) was 1.4-fold higher than that of wild-type strain. In addition, the volumetric hydrogen production rate increased using the process where cells behaved as an effective catalyst. At 94.3 g dry cell/l, a productivity of 793 mmol h⁻¹ l⁻¹ (20.2 l h⁻¹ l⁻¹ at 37 °C) was achieved using SR15. The reported productivity substantially surpasses that of conventional biological hydrogen production processes and can be a trigger for practical applications.