Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening.

Emma Barahona,Luis M Rubio,Emilio Jiménez-Vicente

doi:10.1038/srep38291

Abstract

When produced biologically, especially by photosynthetic organisms, hydrogen gas (H2) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H2-producing enzymes is the absence of methods for the high-throughput screening of H2 production in biological systems. Here, we re-engineered the natural H2 sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H2. A lacZ gene was placed under the control of the hupA H2-inducible promoter in a strain lacking the uptake hydrogenase and the nifH nitrogenase gene. This system was then used in combination with fluorescence-activated cell sorting flow cytometry to screen large libraries of nitrogenase Fe protein variants generated by random mutagenesis. Exact correlation between fluorescence emission and H2 production levels was found for all automatically selected strains. One of the selected H2-overproducing Fe protein variants lacked 40% of the wild-type amino acid sequence, a surprising finding for a protein that is highly conserved in nature. We propose that this method has great potential to improve microbial H2 production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.

Highlights

Rhodobacter capsulatus, the model purple non-sulfur bacteria (PNS) used in this study[5], carries two genetically distinct nitrogenases[6], which are differentially expressed depending on the Mo availability in the medium[7], and two hydrogenases, a membrane-bound [Ni-Fe] hydrogenase for H2 uptake and a cytosolic [Ni-Fe] hydrogenase for H2 sensing[8]
We present a method for the high-throughput selection of nitrogenase variants with enhanced H2 production
The construction of two genetic modules was required to perform high-throughput experiments to obtain nitrogenase variants with improved H2 production: a module expressing over a million random variants of dinitrogenase reductase (NifH) per experiment and a reporter module directing the emission of a visible signal in response to H2

Summary

Introduction

Rhodobacter capsulatus, the model PNS used in this study[5], carries two genetically distinct nitrogenases (a Mo-nitrogenase and an Fe-only nitrogenase)[6], which are differentially expressed depending on the Mo availability in the medium[7], and two hydrogenases, a membrane-bound [Ni-Fe] hydrogenase for H2 uptake and a cytosolic [Ni-Fe] hydrogenase for H2 sensing[8]. Nitrogenases are two-component metalloproteins comprising an N2-reducing dinitrogenase (MoFe protein) and a dinitrogenase reductase (Fe protein) acting as an obligate electron donor[9]. Structural, maturation-related and regulatory genes for the uptake hydrogenase are clustered in the genome of R. capsulatus[12] (Fig. S1). In R. capsulatus, a two-component signal transduction system activates the transcription of the hup gene cluster in the presence of H2 13. In the presence of H2, HupUV binds H2 and HupT is released In this state, phosphotransfer between HupT and HupR is not favored, and the unphosphorylated HupR binds to promoter DNA and activates the transcription of uptake hydrogenase genes. A combination of nifH random mutagenesis and fluorescence-activated cell sorting (FACS) is used to select the H2-overproducing nitrogenase variants in the R. capsulatus sensor strain (Fig. 1A)

Results

Discussion

Conclusion