title: Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Synechococcus elongatus.
abstract: Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H(2) evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.
authors.affiliation: Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute, Rockville, Maryland, United States of America.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Weyman
authors.forename: Walter A
authors.initials: WA
authors.lastname: Vargas
authors.forename: Yingkai
authors.initials: Y
authors.lastname: Tong
authors.forename: Jianping
authors.initials: J
authors.lastname: Yu
authors.forename: Pin-Ching
authors.initials: PC
authors.lastname: Maness
authors.forename: Hamilton O
authors.initials: HO
authors.lastname: Smith
authors.forename: Qing
authors.initials: Q
authors.lastname: Xu
mesh_terms: D020591:Alteromonas / Q000201:enzymology*; D015870:Gene Expression; D006864:Hydrogenase / Q000235:genetics* / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D021122:Protein Subunits / Q000378:metabolism; D046940:Synechococcus / Q000235:genetics*; D020616:Thiocapsa roseopersicina / Q000201:enzymology*
chemical_list: D021122:Protein Subunits; C043296:nickel-iron hydrogenase; D006864:Hydrogenase