Engineering Escherichia coll for FermentativeDihydrogen Production: Potential Role of NADH-Ferredoxin Oxidoreductase from the Hydrogenosome of Anaerobic Protozoa

摘要

Trichomonas vaginalis generates reduced ferredoxin within a unique subcellularorganelle, hydrogenosome that is used as a reductant for H2 production. Pyruvateferredoxin oxidoreductase and NADH dehydrogenase (NADH-DH) are the two enzymescatalyzing the production of reduced ferredoxin. The genes encoding the two subunits ofNADH-DH were cloned and expressed in Escherichia coli. Kinetic properties of therecombinant heterodimer were similar to that of the native enzyme from the hydro-genosome. The recombinant holoenzyme contained 2.15 non-heme iron and 1.95 acid-labile sulfur atoms per heterodimer. The EPR spectrum of the dithionite-reduced proteinrevealed a [2Fe-2S] cluster with a rhombic symmetry of g_(xyz)=1.917,1.951, and 2.009corresponding to cluster N l a of the respiratory complex I. Based on the Fe content,absorption spectrum, and the EPR spectrum of the purified small subunit, the [2Fe-2S]cluster was located in the small subunit of the holoenzyme. This recombinant NADH-DHoxidized NADH and reduced low redox potential electron carriers, such as viologen dyes aswell as Clostridium ferredoxin that can couple to hydrogenase for H2 production fromNADH. These results show that this unique hydrogenosome NADH dehydrogenase with acritical role in H2 evolution in the hydrogenosome can be produced with near-nativeproperties in E. coli for metabolic engineering of the bacterium towards developing a darkfermentation process for conversion of biomass-derived sugars to H2 as an energy source.