title: Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Escherichia coli.
abstract: HynSL from Alteromonas 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, hupH and hypCABDFE) 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, cyt and orf1) was cloned into an IPTG-inducible expression vector and transferred into an Escherichia coli mutant strain lacking its native hydrogenases. Upon induction, HynSL from AltDE was expressed in E. coli and was active, as determined by an in vitro hydrogen evolution assay. Subsequent genetic analysis revealed that orf2, cyt, orf1 and hupH are not essential for assembling an active hydrogenase. However, hupH and orf2 can enhance the activity of the heterologously expressed hydrogenase. We used this genetic system to compare maturation mechanisms between AltDE HynSL and its Thiocapsa roseopersicina homologue. When the structural genes for the T. roseopersicina hydrogenase, hynSL, were expressed along with known T. roseopersicina accessory genes (hynD, hupK, hypC1C2 and hypDEF), no active hydrogenase was produced. Further, co-expression of AltDE accessory genes hypA and hypB with the entire set of the T. roseopersicina genes did not produce an active hydrogenase. However, co-expression of all AltDE accessory genes with the T. roseopersicina structural genes generated an active T. roseopersicina hydrogenase. This result demonstrates that the accessory genes from AltDE can complement their counterparts from T. roseopersicina and that the two hydrogenases share similar maturation mechanisms.
authors.affiliation: Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute, Rockville, MD 20850, USA.
authors.forename: P D
authors.initials: PD
authors.lastname: Weyman
authors.affiliation: Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute, Rockville, MD 20850, USA.
authors.forename: W A
authors.initials: WA
authors.lastname: Vargas
authors.affiliation: Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute, Rockville, MD 20850, USA.
authors.forename: R-Y
authors.initials: RY
authors.lastname: Chuang
authors.affiliation: Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute, Rockville, MD 20850, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Chang
authors.affiliation: Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute, Rockville, MD 20850, USA.
authors.forename: H O
authors.initials: HO
authors.lastname: Smith
authors.affiliation: Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute, Rockville, MD 20850, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Xu
mesh_terms: D020591:Alteromonas / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006864:Hydrogenase / Q000235:genetics* / Q000378:metabolism; D020616:Thiocapsa roseopersicina / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C043296:nickel-iron hydrogenase; D006864:Hydrogenase