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

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.


Supplemental Materials and Methods
Bacterial strains and plasmids. Bacterial strains and plasmids used in this study are listed in Table S3.
R. capsulatus cultures growing with exogenous H 2 were set up either in shake flasks (200 rpm) inside a glove box (Coy Labs, Michigan, USA) with 90% N 2 / 10% H 2 , or by injecting 10% H 2 in the N 2 headspace of capped culture vials. Addition of exogenous H 2 was used to measure β-galactosidase activity and H 2 consumption.
Plasmid constructions and DNA manipulations. DNA purification, restriction enzyme digestion, plasmid constructions, PCR, E. coli transformation and other DNA techniques were carried out by standard methods 2 . Plasmids used in this study are listed in Table S3, and primers used for PCR amplifications are listed in Table S4.
The plasmid to introduce an in-frame nifH deletion into the R. capsulatus chromosome was generated as follows. Flanking regions of nifH gene were amplified by PCR using the oligonucleotides P1 and P2 for the region upstream of nifH, and P3 and P4 for the region downstream of nifH. The resulting PCR products were digested with BamHI and EcoRI and cloned into the BamHI site of pK18mob suicide vector 3 by quadruple-ligations together with an EcoRI-digested Gm-resistance cassette to generate pRHB541.
The plasmid to introduce an in-frame nifHDK deletion into the R. capsulatus chromosome was generated as follows. Upstream region of nifH gene was amplified by PCR using the oligonucleotides P1 and P2 and downstream region of nifK gene was also amplified using P21 and P22. PCR products were digested with BamHI and EcoRI and cloned into the BamHI site of pK18mob suicide vector by quadruple-ligations together with an EcoRI-digested Gm-resistance cassette to generate pRHB704.
The plasmid to introduce an in-frame hupAB deletion into the R. capsulatus chromosome was generated as follows. Flanking regions of hupAB genes were amplified by PCR using the oligonucleotides P5 and P6 for the region upstream and P7 and P8 for the region downstream. The resulting PCR products were digested with BamHI and HindIII and cloned into the BamHI site of a modified version of pK18mobsacB (pSm18mobsacB) suicide vector previously digested with BamHI 3 by triple-ligation to generate pRHB577.
To introduce an in-frame hupT deletion from R. capsulatus chromosome, hupT flanking regions were amplified by PCR using oligonucleotides P9 and P10 for the region upstream and P11 and P12 for the region downstream. The resulting PCR products were digested with XbaI and BamHI and cloned into the XbaI site of pK18mobsacB plasmid by quadruple-ligations together with a BamHI-digested Gm-resistance cassette to generate plasmid pRHB627.
To introduce an in-frame hupR deletion from R. capsulatus chromosome, regions flanking hupR were PCR-amplified using oligonucleotides P13 and P14 for the region upstream and P15 and P16 for the region downstream. The resulting PCR products were digested with SalI and HindIII and cloned into SalI and HindIII sites of pK18mobsacB to generate plasmid pRHB552.
To construct the PhupA::lacZ transcriptional fusion, an 874-bp DNA fragment containing the hupA promoter (PhupA) 4 was amplified using the oligonucleotides P17 and P18 and cloned into the KpnI and XbaI sites of replicative plasmid pMP220, yielding pRHB502. In addition, the digested PCR product was cloned into the KpnI and XbaI sites of pVIK112 suicide vector 5 to generate pRHB501, which carries a chromosomal translational fusion.
A 681-bp DNA fragment containing R. capsulatus nifH promoter (PnifH) followed by NdeI and XbaI restriction sites 6 and flanked by transcriptional terminators was synthesized (GenScript, USA) and cloned into the KpnI and SacI sites of the broadhost-range cloning vector pBBR1MCS-3 7 to generate pRHB602. R. capsulatus nifH gene was amplified using primers P19 and P20, digested using NdeI and XbaI restriction enzymes and cloned into pRHB602 to generate pRHB576. The same PCR product comprising the amplification of a full nifH gene was cloned into the NdeI and XbaI sites of pUC18 to generate pRHB529, used as a template in Error-Prone assays. The nifH, nifD and nifK were amplified together from R. capsulatus genomic DNA using the oligonucleotides P19 and P23 digested using NdeI and XbaI restriction enzymes and cloned into pRHB602 to generate pRHB618. All DNA constructions were confirmed by restriction analysis and DNA sequencing. When pK18mobsacB or pSm18mobsacB were used to generate mutants, putative single recombinants were selected by their resistance to Sm or Km, and then isolated from the agar surface and inoculated in YP liquid medium. Cells were passaged five times under no selective pressure to undergo a second recombination event. After passaging, different dilutions (10 -1 , 10 -2 , 10 -3 , and 10 -4 ) of the final culture were spread on YPS plates containing 10% (w/v) of sucrose. Double recombinant colonies were selected due to their capacity to grow in presence of 10% of sucrose.   S2. Characterization of R. capsulatus H 2 reporter strains. β-galactosidase activity determinations in the absence or presence of 10% H 2 in the culture gas phase (A and B). Strain RC3 carries reporter plasmid pMP220; strain RC4 carries plasmid pRHB502 (PhupA::lacZ in pMP220). Strain S1 carries the PhupA::lacZ fusion integrated into the chromosome. (C) In vivo nitrogenase activity of WT and S2 measured as levels of ethylene production (nmol min ) by WT and S2 in non-diazotrophic and diazotrophic conditions. (E) Colorimetric assays using X-gal to measure the response to H 2 in WT, S1, RC25 and S2 cultures growing in the presence of 10% of H 2 . WT and RC25 parental strains are used as controls.  Fig. S3. β-Galactosidase activity determination in R. capsulatus reporter strains with mutations in H 2 signal transduction and metabolism pathways. βgalactosidase activity was determined either in the absence or presence of 10% of H 2 in the gas phase. Strain genotypes are described in Supplementary Table 2.  S4. NifH expression and diazotrophic growth analysis of RC20 strain. (A) NifH Immunodetection in WT and RC20 (ΔnifH) strains grown under non-diazotrophic or diazotrophic conditions. Lower panel shows Comassie-blue general staining of proteins as protein loading control. RC20 was used to generate S3 reporter strain (S3 genealogy is WT!RC20!RC31!S3). (B) RC20 genetic complementation assay. Plasmids pRHB602 (PnifH) and pRHB576 (PnifH::nifH) were introduced into WT and RC20 strains. Transformed cultures were then plated on RCV media supplemented with ammonium (non-diazotrophic) or lacking ammonium (diazotrophic).