Heterologous Expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] Hydrogenases in Synechococcus elongatus

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 H2 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.

耐氧型[NiFe]氢化酶（[NiFe] hydrogenase）若能在目标宿主生物中实现异源表达（heterologous expression），即可应用于未来的光生物制氢系统。为实现[NiFe]氢化酶在蓝细菌（cyanobacteria）中的异源表达，研究人员将深海交替单胞菌（Alteromonas macleodii）深海生态型（AltDE）来源的两个氢化酶结构基因hynS与hynL，以及HynSL操纵子（gene operon）的侧翼基因，克隆至带有异丙基-β-D-硫代半乳糖苷（IPTG）诱导型启动子的载体，并转入集胞藻Synechococcus elongatus PCC7942。经IPTG诱导后，氢化酶蛋白以预期分子量正确表达。异源表达的HynSL氢化酶经体外产氢试验（in vitro H₂ evolution assay）检测后呈现活性，表明该催化中心在蓝细菌宿主中实现了正确组装。采用相似的表达系统，研究人员将玫瑰色硫囊菌（Thiocapsa roseopersicina）来源的氢化酶结构基因hynSL，以及全部已知的辅助基因，转入集胞藻S. elongatus。成功表达出分子量符合预期的蛋白，但未检测到酶活性。然而，当将AltDE来源的11个辅助基因与hynSL共表达时，经体外试验检测发现玫瑰色硫囊菌的氢化酶恢复了活性。本研究首次报道了在蓝细菌中实现异源活性表达的[NiFe]氢化酶。