Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Escherichia coli

HynSL fromAlteromonas macleodii‘deep ecotype’ (AltDE) is an oxygen-tolerant and thermostable [NiFe] hydrogenase. Its two structural genes (hynSL), encoding small and large hydrogenase subunits, are surrounded by eight genes (hynD,hupHandhypCABDFE) predicted to encode accessory proteins involved in maturation of the hydrogenase. A 13 kb fragment containing the ten structural and accessory genes along with three additional adjacent genes (orf2,cytandorf1) was cloned into an IPTG-inducible expression vector and transferred into anEscherichia colimutant strain lacking its native hydrogenases. Upon induction, HynSL from AltDE was expressed inE. coliand was active, as determined by anin vitrohydrogen evolution assay. Subsequent genetic analysis revealed thatorf2,cyt,orf1andhupHare not essential for assembling an active hydrogenase. However,hupHandorf2can enhance the activity of the heterologously expressed hydrogenase. We used this genetic system to compare maturation mechanisms between AltDE HynSL and itsThiocapsa roseopersicinahomologue. When the structural genes for theT. roseopersicinahydrogenase,hynSL, were expressed along with knownT. roseopersicinaaccessory genes (hynD, hupK, hypC1C2andhypDEF), no active hydrogenase was produced. Further, co-expression of AltDE accessory geneshypAandhypBwith the entire set of theT. roseopersicinagenes did not produce an active hydrogenase. However, co-expression of all AltDE accessory genes with theT. roseopersicinastructural genes generated an activeT. roseopersicinahydrogenase. This result demonstrates that the accessory genes from AltDE can complement their counterparts fromT. roseopersicinaand that the two hydrogenases share similar maturation mechanisms.