Expanding the substrates for a bacterial hydrogenlyase reaction.

Ciaran M Lamont,Tracy Palmer,Constanze Pinske,Ciarán L Kelly,Frank Sargent,Grant Buchanan

doi:10.1099/mic.0.000471

Abstract

Escherichia coli produces enzymes dedicated to hydrogen metabolism under anaerobic conditions. In particular, a formate hydrogenlyase (FHL) enzyme is responsible for the majority of hydrogen gas produced under fermentative conditions. FHL comprises a formate dehydrogenase (encoded by fdhF) linked directly to [NiFe]-hydrogenase-3 (Hyd-3), and formate is the only natural substrate known for proton reduction by this hydrogenase. In this work, the possibility of engineering an alternative electron donor for hydrogen production has been explored. Rational design and genetic engineering led to the construction of a fusion between Thermotoga maritima ferredoxin (Fd) and Hyd-3. The Fd-Hyd-3 fusion was found to evolve hydrogen when co-produced with T. maritima pyruvate :: ferredoxin oxidoreductase (PFOR), which links pyruvate oxidation to the reduction of ferredoxin. Analysis of the key organic acids produced during fermentation suggested that the PFOR/Fd-Hyd-3 fusion system successfully diverted pyruvate onto a new pathway towards hydrogen production.

Highlights

Escherichia coli produces enzymes dedicated to hydrogen metabolism under anaerobic conditions
Escherichia coli performs formate-dependent hydrogen production [1]. This is catalysed by the formate hydrogenlyase (FHL) complex [2,3,4], which is a membrane-bound enzyme comprising [NiFe]-hydrogenase-3 (Hyd-3) and a formate dehydrogenase component encoded by fdhF [5]
The E. coli Hyd-3 isoenzyme is unusual for a nickel-containing hydrogenase as it is apparently tuned towards proton reduction rather than H2 oxidation [2]

Summary

Introduction

Escherichia coli produces enzymes dedicated to hydrogen metabolism under anaerobic conditions. D N) MG16dZ and FTD300, and the strain MG059e1 (as MG1655, hycEHis), were grown anaerobically in M9 medium supplemented with 0.8 % (w/v) glucose for 24 h after which the OD600 was measured and the H2 content in the headspace quantified by gas chromatography. The FTF2013 and FTF2015[pREP4] strains were transformed with pUNI-PROM (empty control vector), pUNI-Tm-POR (encoding T. maritima PFOR) or pUNI-Tm-Fd-POR (encoding T. maritima PFOR and Fd) grown at 37 C for 24 h in anaerobic Hungate tubes containing 5 ml M9 medium supplemented with 0.8 % (w/v) glucose and 0.2 % (w/v) casamino acids.

Results

Conclusion