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

Philip D Weyman,Jianping Yu,Pin-Ching Maness,Hamilton O Smith,Walter A Vargas,Yingkai Tong,Qing Xu,Francisco Rodriguez-Valera

doi:10.1371/journal.pone.0020126

Philip D Weyman, Jianping Yu + Show 6 more

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https://doi.org/10.1371/journal.pone.0020126

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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 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.

Highlights

Hydrogen (H2) production from photosynthetic microorganisms is an attractive strategy to store solar energy as a fuel [1]
The uptake hydrogenases in cyanobacteria function largely in recycling H2 produced as a byproduct of nitrogen fixation while bidirectional hydrogenases have been implicated in disposing of excess reductant as H2 [5,6]
To eliminate background hydrogenase activity in S. elongatus to better detect the activity from heterologously-expressed enzymes, we knocked out the endogenous hydrogenase by transforming with a plasmid that would replace the hoxYH genes with an antibiotic cassette via DNA recombination

Summary

Introduction

Hydrogen (H2) production from photosynthetic microorganisms is an attractive strategy to store solar energy as a fuel [1]. Development of photobiological H2 production using photosynthetic microorganisms such as cyanobacteria and micro-algae can provide an alternative to fossil fuels by using the energy of the sun to convert H2O into H2. Two different groups of [NiFe] hydrogenases, the uptake hydrogenases and the bidirectional hydrogenases, have been found in many cyanobacterial genomes [2]. The uptake hydrogenases in cyanobacteria function largely in recycling H2 produced as a byproduct of nitrogen fixation while bidirectional hydrogenases have been implicated in disposing of excess reductant as H2 [5,6]

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