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Powerful fermentative hydrogen evolution of photosynthate in the cyanobacterium Lyngbya aestuarii BL J mediated by a bidirectional hydrogenase.

ABSTRACT: Cyanobacteria are considered good models for biohydrogen production because they are relatively simple organisms with a demonstrable ability to generate H2 under certain physiological conditions. However, most produce only little H2, revert readily to H2 consumption, and suffer from hydrogenase sensitivity to O2. Strains of the cyanobacteria Lyngbya aestuarii and Microcoleus chthonoplastes obtained from marine intertidal cyanobacterial mats were recently found to display much better H2 production potential. Because of their ecological origin in environments that become quickly anoxic in the dark, we hypothesized that this differential ability may have evolved to serve a role in the fermentation of the photosynthate. Here we show that, when forced to ferment internal substrate, these cyanobacteria display desirable characteristics of physiological H2 production. Among them, the strain L. aestuarii BL J had the fastest specific rates and attained the highest H2 concentrations during fermentation of photosynthate, which proceeded via a mixed acid fermentation pathway to yield acetate, ethanol, lactate, H2, CO2, and pyruvate. Contrary to expectations, the H2 yield per mole of glucose was only average compared to that of other cyanobacteria. Thermodynamic analyses point to the use of electron donors more electronegative than NAD(P)H in Lyngbya hydrogenases as the basis for its strong H2 production ability. In any event, the high specific rates and H2 concentrations coupled with the lack of reversibility of the enzyme, at the expense of internal, photosynthetically generated reductants, makes L. aestuarii BL J and/or its enzymes, a potentially feasible platform for large-scale H2 production.

SUBMITTER: Kothari A

PROVIDER: S-EPMC4261827 | biostudies-other | 2014

REPOSITORIES: biostudies-other

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Powerful fermentative hydrogen evolution of photosynthate in the cyanobacterium Lyngbya aestuarii BL J mediated by a bidirectional hydrogenase.

Kothari Ankita A Parameswaran Prathap P Garcia-Pichel Ferran F

Frontiers in microbiology 20141210

Cyanobacteria are considered good models for biohydrogen production because they are relatively simple organisms with a demonstrable ability to generate H2 under certain physiological conditions. However, most produce only little H2, revert readily to H2 consumption, and suffer from hydrogenase sensitivity to O2. Strains of the cyanobacteria Lyngbya aestuarii and Microcoleus chthonoplastes obtained from marine intertidal cyanobacterial mats were recently found to display much better H2 productio ...[more]

PMID: 25540642

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Project description:The filamentous, non-heterocystous cyanobacterium Lyngbya aestuarii is an important contributor to marine intertidal microbial mats system worldwide. The recent isolate L. aestuarii BL J, is an unusually powerful hydrogen producer. Here we report a morphological, ultrastructural, and genomic characterization of this strain to set the basis for future systems studies and applications of this organism. The filaments contain circa 17 ?m wide trichomes, composed of stacked disk-like short cells (2 ?m long), encased in a prominent, laminated exopolysaccharide sheath. Cellular division occurs by transversal centripetal growth of cross-walls, where several rounds of division proceed simultaneously. Filament division occurs by cell self-immolation of one or groups of cells (necridial cells) at the breakage point. Short, sheath-less, motile filaments (hormogonia) are also formed. Morphologically and phylogenetically L. aestuarii belongs to a clade of important cyanobacteria that include members of the marine Trichodesmiun and Hydrocoleum genera, as well as terrestrial Microcoleus vaginatus strains, and alkalyphilic strains of Arthrospira. A draft genome of strain BL J was compared to those of other cyanobacteria in order to ascertain some of its ecological constraints and biotechnological potential. The genome had an average GC content of 41.1%. Of the 6.87 Mb sequenced, 6.44 Mb was present as large contigs (>10,000 bp). It contained 6515 putative protein-encoding genes, of which, 43% encode proteins of known functional role, 26% corresponded to proteins with domain or family assignments, 19.6% encode conserved hypothetical proteins, and 11.3% encode apparently unique hypothetical proteins. The strain's genome reveals its adaptations to a life of exposure to intense solar radiation and desiccation. It likely employs the storage compounds, glycogen, and cyanophycin but no polyhydroxyalkanoates, and can produce the osmolytes, trehalose, and glycine betaine. According to its genome, BL J strain also has the potential to produce a plethora of products of biotechnological interest such as Curacin A, Barbamide, Hemolysin-type calcium-binding toxin, the suncreens scytonemin, and mycosporines, as well as heptadecane and pentadecane alkanes. With respect to hydrogen production, initial comparisons of the genetic architecture and sequence of relevant genes and loci, and a comparative model of protein structure of the NiFe bidirectional hydrogenase, did not reveal conspicuous differences that could explain its unusual hydrogen producing capacity.

Project description:BACKGROUND: Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H+ + 2e- <--> H2 (g). RESULTS: Hydrogen yields were enhanced up to 41-fold by cloning the bidirectional hydrogenase (encoded by hoxEFUYH) from the cyanobacterium into E. coli. Using an optimized medium, E. coli cells expressing hoxEFUYH also produced twice as much hydrogen as the well-studied Enterobacter aerogenes HU-101, and hydrogen gas bubbles are clearly visible from the cultures. Overexpression of HoxU alone (small diaphorase subunit) accounts for 43% of the additional hydrogen produced by HoxEFUYH. In addition, hydrogen production in E. coli mutants with defects in the native formate hydrogenlyase system show that the cyanobacterial hydrogenase depends on both the native E. coli hydrogenase 3 as well as on its maturation proteins. Hydrogen absorption by cells expressing hoxEFUYH was up to 10 times lower than cells which lack the cloned cyanobacterial hydrogenase; hence, the enhanced hydrogen production in the presence of hoxEFUYH is due to inhibition of hydrogen uptake activity in E. coli. Hydrogen uptake by cells expressing hoxEFUYH was suppressed in three wild-type strains and in two hycE mutants but not in a double mutant defective in hydrogenase 1 and hydrogenase 2; hence, the active cyanobacterial locus suppresses hydrogen uptake by hydrogenase 1 and hydrogenase 2 but not by hydrogenase 3. Differential gene expression indicated that overexpression of HoxEFUYH does not alter expression of the native E. coli hydrogenase system; instead, biofilm-related genes are differentially regulated by expression of the cyanobacterial enzymes which resulted in 2-fold elevated biofilm formation. This appears to be the first enhanced hydrogen production by cloning a cyanobacterial enzyme into a heterologous host. CONCLUSION: Enhanced hydrogen production in E. coli cells expressing the cyanobacterial HoxEFUYH is by inhibiting hydrogen uptake of both hydrogenase 1 and hydrogenase 2.

Project description:BACKGROUND:Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "Deep ecotype" that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. RESULTS:We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. CONCLUSIONS:Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

Dataset Information

Powerful fermentative hydrogen evolution of photosynthate in the cyanobacterium Lyngbya aestuarii BL J mediated by a bidirectional hydrogenase.

Publications

Powerful fermentative hydrogen evolution of photosynthate in the cyanobacterium Lyngbya aestuarii BL J mediated by a bidirectional hydrogenase.

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