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

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. SIMILAR CONTENT BEING VIEWED BY OTHERS VERSATILE SELECTIVE EVOLUTIONARY PRESSURE USING SYNTHETIC DEFECT IN UNIVERSAL METABOLISM Article Open access 25 November 2021 LABORATORY

EVOLUTION OF SYNTHETIC ELECTRON TRANSPORT SYSTEM VARIANTS REVEALS A LARGER METABOLIC RESPIRATORY SYSTEM AND ITS PLASTICITY Article Open access 27 June 2022 AN AUTOMATED HIGH-THROUGHPUT

LIGHTING SYSTEM FOR SCREENING PHOTOSYNTHETIC MICROORGANISMS IN PLATE-BASED FORMATS Article Open access 14 March 2025 INTRODUCTION Biological H2 production is a promising source of renewable

energy. Microorganisms produce H2 by the activity of nitrogenases and hydrogenases1. Nitrogenases catalyze the reduction of N2 with the limiting stoichiometry N2+8 H+ + 

8e−+16MgATP+16H2O↔H2+2NH3+16MgADP+16Pi in a process known as biological nitrogen fixation, which produces H2 as a by-product2. On the other hand, hydrogenases catalyze the reversible 2 H+ +

  2e−↔H2 reaction. H2 metabolism has been well studied in purple non-sulfur bacteria (PNS), a group of microorganisms notable for their metabolic versatility. PNS can grow

photoautotrophically, photoheterotrophically, chemoorganotrophically, and chemolitotrophically with H2 as an electron donor and O2 as an electron acceptor3. H2 production by PNS generally

occurs during photoheterotrophic anaerobic growth and is mainly due to nitrogenase4. _Rhodobacter capsulatus_, the model PNS used in this study5, 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 medium7, and two hydrogenases, a membrane-bound

[Ni-Fe] hydrogenase for H2 uptake and a cytosolic [Ni-Fe] hydrogenase for H2 sensing8. Nitrogenases are two-component metalloproteins comprising an N2-reducing dinitrogenase (MoFe protein)

and a dinitrogenase reductase (Fe protein) acting as an obligate electron donor9. These components are encoded by _nifDK_ and _nifH_, respectively, in the case of the Mo-nitrogenases, and by

_anfDGK_ and _anfH_ in the case of the Fe-only nitrogenases10. The uptake hydrogenase is a heterodimer of the _hupA_ and _hupB_ (formerly _hupS_ and _hupL_) gene products11, in which

activity is linked to the respiratory chain by the cytochrome _b_-containing protein HupC. 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. The

H2-sensing system comprises three elements: a cytosolic [Ni-Fe] hydrogenase, HupUV; a histidine kinase, HupT; and a response regulator, HupR. In the absence of H2, HupUV and HupT interact,

causing HupT autophosphorylation and the transfer of a phosphate group to HupR, which in this state is unable to activate the transcription. 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. Similar regulatory systems are found in other bacterial species such as _Bradyrhizobium japonicum_14 and _Ralstonia eutropha_15. Due to its great commercial significance, nitrogenase

has been the subject of extensive biochemical, genetic and structural analyses. Nonetheless, it has proven difficult to find or engineer strains of microbes carrying nitrogenases with

significantly increased H2 production efficiency. In this work, we present a method for the high-throughput selection of nitrogenase variants with enhanced H2 production. An _R. capsulatus_

strain has been re-engineered to generate a fluorescent signal in response to nitrogenase-produced H2. A combination of _nifH_ random mutagenesis and fluorescence-activated cell sorting

(FACS) is then used to select the H2-overproducing nitrogenase variants in the _R. capsulatus_ sensor strain (Fig. 1A). RESULTS GENETIC MODULES FOR THE SELECTION OF H2-OVERPRODUCING

NITROGENASE VARIANTS 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. The reporter

module was constructed in four steps. In a first step, an 874-bp DNA fragment comprising a promoter sequence upstream of _hupA_16 was translationally fused to _lacZ_ in the replicative

vector pMP220 to generate pRHB502. As illustrated in Fig. 1A, the expression from _hupA_ promoter is activated by HupR in response to H2. The _in vivo_ β-galactosidase activity of _R.

capsulatus_ cells harboring pRHB502 (RC4) was 600-fold higher than that of the control strain RC3 (carrying pMP220) and responded positively to the presence of 10% H2 in the culture gas

phase, which confirmed the induction of the transcription from P_hupA_ by H2 (Fig. S2A). In a second step, the reporter dose was adjusted by integrating P_hupA::lacZ_ between _hypF_ and

_hupA_ in the _R. capsulatus_ chromosome to generate the S1 strain (Fig. S1). The S1 strain exhibited a much lower β-galactosidase activity background level and a larger fold increase in the

activity in response to the external H2 than RC4 (Fig. S2B). In a third step, the reporter response to H2 was adjusted by mutating the genes involved in the H2 signal transduction pathway

and metabolism. S1 derivative strains lacking _hupAB_ structural genes for the uptake-hydrogenase (RC25-S1, also termed S2), the H2 response regulator encoding gene _hupR_ (RC54-S1), or the

histidine kinase gene _hupT_ (RC24-S1) were generated, and their responses to H2 were analyzed (Fig. 1B and Fig. S3), obtaining the following results. First, the response to 10% exogenous H2

improved 5-fold in the Δ_hupAB_ strain S2 compared to in S1; second, the constitutive activation of P_hupA_ was observed in the Δ_hupT_ strain RC24-S1; and third, no response to H2 was

observed in the Δ_hupR_ strain RC54-S1. These results were in agreement with previous reports13,16,17,18 indicating proper control by the _R. capsulatus_ H2-sensing system and therefore

permitting further H2 sensor development through the use of strain S2. The deletion of _hupAB_ genes in the RC25 strain completely eliminated its _in vivo_ uptake hydrogenase activity (nil

compared to 2504 ± 450 nmol H2 h−1 OD600−1 in the wild-type strain). Thus, in contrast to the wild-type strain, the S2 sensor strain (derived from RC25) evolved high levels of H2 under

diazotrophic growth conditions (Fig. S2C) in spite of having similar levels of _in vivo_ nitrogenase activity (Fig. S2D). In addition, the X-gal and H2-dependent signal-to-noise ratio was

much better in S2 than in S1 cultures (Fig. S2E). It was therefore concluded that the elimination of the uptake hydrogenase activity would facilitate the detection of intracellular H2

produced during the nitrogen fixation process. The S2 response to the H2 produced by the nitrogenase was evaluated in intact bacterial cells by using a fluorescent MUG-dependent

β-galactosidase activity assay19, which permitted high-throughput growth and screening in a 96-well format as well as the recovery of viable selected clones. _R. capsulatus_ strains were

grown in 96-well microplates both under non-diazotrophic and diazotrophic conditions inside a glovebox, and their β-galactosidase activities were determined. Figure 1C shows that the

β-galactosidase activity in S2 was 15-fold higher under diazotrophic conditions compared to non-diazotrophic conditions, validating its use as a biosensor. Importantly, all tested strains

with mutations in H2 the metabolism or signal transduction pathway responded identically to nitrogenase-produced H2 and exogenous added H2 (compare Fig. 1C to Fig. S3). Finally, the removal

of endogenous _nifH_ was necessary to use the sensor in combination with the genetic module consisting of an expression library of _nifH_ variants (Fig. S4). The S3 sensor strain (Δ_nifH_

Δ_hupAB_ P_hupA::lacZ_) β-galactosidase activity levels in response to H2 were identical to those of S2 (Fig. 1B), demonstrating that the absence of _nifH_ did not modify the capacity to

detect H2. Importantly, the introduction of the pRHB576 expression vector carrying a wild-type copy of _nifH_ under the control of its own promoter restored the _in vivo_ nitrogenase

activity of strain S3 (Fig. S5), validating its use to screen the activity of the _nifH_ variants in S3. Interestingly, a general decrease in the nitrogenase activities was observed in the

sensor S3-derived strains with respect to the wild-type _R. capsulatus_. This effect was associated with carrying the expression vector pBBR1MCS-3 (Fig. S6). The second genetic module

consisted of an expression library of randomly generated nitrogenase variants. The mutation analysis was initially constrained to _nifH_ because the limiting steps in the nitrogenase

catalysis are the dissociation of the NifH and NifDK components and the release of Pi from NifH2. Random _nifH_ variants (_nifHv_) having an average of 5 amino acid changes (Table S1) were

generated by error-prone PCR, ligated into pRHB602, and introduced into _E. coli_ DH5α to obtain P_nifH_::_nifH_ expression libraries (~ 4 × 106 clones per library). No obvious mutational

hotspots were observed after sequencing 20 _nifH_ variants per library. Finally, the two genetic modules were combined by introducing the _nifH_v-expression libraries into _R. capsulatus_

sensor strain S3. The mating process resulted in ~8 × 105 clones per library, a number that required high-throughput screening methods. HIGH-THROUGHPUT SELECTION OF H2-OVERPRODUCING _NIFH_

VARIANTS _R. capsulatus_ S3 libraries expressing _nifH_ variants (_nifH_v-S3) were grown under nitrogenase-derepressing conditions inside a glovebox. After treatment with fluorescein

di-β-D-galactopyranoside (FDG), culture samples were screened by FACS flow cytometry. Approximately 2 × 105 events were processed per sample. _R. capsulatus_ cells emitting fluorescence at

levels significantly higher than the main population (0.024% of total population, see P2 area in Fig. 2A) were sorted by the cytometer into separate wells of 96-well microplates containing

growth medium. S3 derivatives transformed with empty expression vector (pRHB602-S3) or wild-type _nifH_-containing expression vector (pRHB576-S3) were used as negative and positive controls,

respectively. pRHB602-S3 populations emitted the lowest average level of fluorescence, and only 0.008% of the population was found above the set threshold in the P2 area. On the other hand,

the average fluorescence emission in the pRHB576-S3 populations was the highest, although only 0.0039% of the population was above the selective threshold. The _nifH_v-S3 cells sorted by

FACS were then cultured under nitrogenase-derepressing conditions using serine as a poor nitrogen source inside a glovebox for a secondary MUG-based fluorescence screen. Growth was observed

in approximately 25% of the inoculated wells. Figure 2B shows the β-galactosidase activity levels of 67 _nifH_v-S3 cultures along with those of the pRHB602-S3 (blue) and pRHB576-S3 (red)

cultures used as controls. The β-galactosidase activity was 2 to 12-fold higher in the _nifH_v-S3 cultures than in the pRHB576-S3 strain (carrying wild-type _nifH_). Importantly, 85% of the

_nifH_v-S3 clones harbored an expression vector with a _nifHv_ gene, as determined by PCR analysis. The strong correlation of the results from the FACS primary screening and the secondary

MUG screening in the 96-well format validates the use of flow cytometry as a high-throughput method to screen _nifH_v-S3 libraries. NITROGENASE-DEPENDENT H2 OVERPRODUCTION IN SELECTED

STRAINS WITH _NIFH_ VARIANTS Based on the β-galactosidase activity levels, _R. capsulatus_ strains V1, V7, V8, V10, V17, V18, V20 and V21 (Fig. 2B, black bars), carrying the corresponding

_nifH_ variants, were selected for _in vivo_ H2 production assays. All these strains produced more H2 than the pRHB576-S3 strain carrying wild-type _nifH_ (Fig. 3A), with the V1 and V7

strains consistently producing up to 10-fold more H2. Importantly, the acetylene reduction activity was almost abolished in the strains carrying H2-overproducing _nifH_ variants (Fig. 3B), a

result consistent with the random mutagenesis process designed to select only for improved H2 production. The ratio of H2 to ethylene production in V7 strain was 6000-fold higher than in

pRHB576-S3. Neither H2 nor ethylene was produced in the absence of _nifH_ (see strain pRHB602-S3 in Fig. 3), indicating that the H2 production was dependent on the _nifH_ expression. The

nature of the mutations in the _nifH_-V1 and _nifH_-V7 variants was determined by DNA sequencing (Table S2 and Fig. S7). A frame shift mutation was found in codon 172 of _nifH_-V7 that would

result in a NifH protein lacking 124 amino acids at its C-terminus. In addition, NifH-V7 would carry G32C and A107T amino acid substitutions. This was surprising because NifH is a highly

conserved protein, and such a drastic truncation would be expected to yield an inactive protein. An overlap of the three-dimensional models of _R. capsulatus_ NifH and NifH-V7 is shown in

Fig. 3C. The overall protein overlap (4–171 amino acid residues) and an RMSD value of 0.900 Angstroms for the protein backbone atoms indicate very similar model architectures. However,

subtle differences were observed in the loops near the [4Fe-4S] cluster, where residues K43, A44, A100, G101, R102 and G115 did not overlap. To confirm that the H2 overproduction in V7 was

dependent on the activity of both Mo-nitrogenase component proteins, the following two experiments were performed. First, the _nifH_-V7 expression plasmid was cured from the V7 strain by

passing five times under a nonselective media. Plasmid elimination was confirmed by PCR analysis and by growth inhibition on media supplemented with tetracycline (Tc). The resulting strain,

V7C, was unable to produce H2 above the levels of the pRHB602-S3 control strain (0.42 nmol H2 h−1 OD600−1 ml−1) (Fig. 3D). The reintroduction of the _nifH_-V7 expression plasmid by mating

completely restored the H2-producing activity (compare H2 productions of V7 and V7′ in Fig. 3D). The presence of a truncated form of NifH in V7 and its elimination in V7C were confirmed by

an immunoblot analysis (Fig. 3E). This analysis also showed that all strains accumulated similar levels of nitrogenase component proteins, ruling out the possibility of increased

accumulation underlying the H2 overproduction by V7. Second, _R. capsulatus_ sensor strains lacking either _nifH_ (strain S3) or _nifHDK_ (strain S5) were complemented with an expression

plasmid harboring _nifH_-V7. While the S3-V7 cultures produced large amounts of H2, background levels were detected in the S5-V7 cultures (Fig. S8). This result demonstrates that the NifH-V7

dependent H2 production also requires the presence of NifDK and is not due to the activity of other cellular proteins, such as the Fe-only nitrogenase. DISCUSSION There are many reports of

metabolic engineering aiming to increase microbial H2 production. Genetic approaches include deleting hydrogenase and/or nitrogenase structural and regulatory genes20,21,22,23,24,25,

eliminating Rubisco26, lowering intracellular O2 levels27, re-engineering hydrogenase to increase its tolerance to O228, and modifying nitrogenase substrate selectivity by site-directed

mutagenesis29,30. Nonetheless, it has been difficult to find or engineer vastly improved H2-overproducing enzymes or microbial strains. Directed evolution mimics biological evolution in the

laboratory and is an effective approach to change enzyme properties such as catalytic turnover31. Directed evolution requires a strategy to generate libraries with a large number of variants

and a method capable of screening or selecting for the best variants in a large pool. In this work, we have combined the random mutagenesis of _nifH_ and high-throughput screening to

improve nitrogenase H2-producing activity. We hypothesized that the nitrogenase H2 production could be greatly improved if no selection was applied to maintain the N2-reducing activity. The

_nifH_ gene was selected to obtain a proof of concept because the limiting steps in nitrogenase catalysis are the NifH/NifDK complex dissociation and the release of Pi from NifH2. FACS flow

cytometry was used as a high-throughput method to select H2-overproducing NifH variants from the pool. A genetic module endowing a NifH-expressing sensor strain with the capacity to emit

light in proportion to the amount of H2 detected was required for this selection. The screening of bacterial libraries by FACS flow cytometry had been performed before in directed evolution

procedures32. A number of H2-overproducing NifH variants were obtained. Perhaps the most interesting was the V7 variant, which lacks 124 amino acid residues at the C-terminus of the protein

(Fig. 3C). In wild-type NifH, ATP binding and hydrolysis is required for electron transfer to NifDK during catalysis2. Interestingly, NifH-V7 lacks all residues shown to enable hydrogen

bonding to the adenine base (Asp185, Gln218, and Gln236 in the _Azotobacter vinelandii_ NifH), suggesting that NifH-V7 might have lost its specificity for Mg·ATP over other nucleotides33.

The mechanism by which NifH-V7 is capable of sustaining H+ reduction merits further investigation. Other biological and chemical methods have also been developed to detect H2-producing

microorganisms. Chemochromic transparent sensor films that turn blue in the presence of H2 and indicators consisting of a coloring agent and a water-soluble derivative of Wilkinson’s

catalyst have been used for phenotypic screenings of _Chlamydomonas reinhardtii_34,35 and _R. capsulatus_36, respectively. Moreover, a biosensor to screen algal H2 production was developed

starting from the _R. capsulatus_ H2-sensing system37,38. However, all these methods suffer from limitations in the number of clones that could be screened per experiment, making directed

evolution techniques impossible to apply. CONCLUSIONS In this work, we have re-engineered the natural H2 sensing system of _R. capsulatus_ and combined it with FACS for the high-throughput

selection of nitrogenase variants with enhanced H2 production that independently retain their N2 fixation activity. This method allows screening 105–106 variants per experiment, thus

permitting enzyme improvement by directed evolution. This technology possesses great potential to identify nitrogenase amino acid substitutions leading to H2-overproducing variants that

could be mimicked in nitrogenases from other microorganisms, expanding the impact of the findings. In addition, it might be used for the genome-wide screening of mutations leading to

enhanced H2 production in _R. capsulatu_s. MATERIALS AND METHODS Β-GALACTOSIDASE ACTIVITY ASSAYS Transcription from P_hupA_ was estimated by measuring β-galactosidase activity of _R.

capsulatus_ strains carrying P_hupA_::_lacZ_ transcriptional fusions. Cells were cultured at 30 °C for 7 h, transferred to assay tubes (0.7 ml of culture), permeabilized by addition of 20 μl

of chloroform and 10 μl of 0.1% SDS, and β-galactosidase activity estimated at 28 °C as described39. When the 96-well microplate format was used, β-galactosidase activity assays were

carried out as described in19 with modifications. _R. capsulatus_ cultures were incubated overnight under diazotrophic conditions inside a glove box in a 96-well plate (black/clear Optilux™

flat bottom; BD Biosciences) covered with a transparent adhesive sealer. One hundred and twenty μl of each culture were transferred to a 96-well microplate containing 100 μl of Z-Buffer in

each well39, then supplemented with 25 μl of 4-methylumbelliferone β-D-galacto-pyranoside (MUG; 1 mg/ml stock solution in dimethyl sulfoxide), and incubated at room temperature for 2 h in

darkness. MUG hydrolysis by β-galactosidase was quantified by fluorescence emission at 445 nm (372 nm excitation wavelength) in a Genios Pro (Tecan) microplate fluorometer. CONSTRUCTION OF

RANDOM MUTAGENESIS LIBRARIES Random mutagenesis of _nifH_ was carried out by Error-Prone PCR using PCR GeneMorph® II Random Mutagenesis Kit (Stratagene) according to the manufacturer’s

instructions. Reaction mixtures contained 1 μl DNA template (0.4 ng of pRHB529 including 0.1 ng of _nifH_), 5 μl of 10X Mutazyme II reaction buffer, 1 μl of 40 mM dNTP mix (200 μM each

final), 0.5 μl of primer mix (250 ng/μl of each primer), 1 μl of Mutazyme II DNA polymerase (2.5 U/μl), and 41.5 μl of H2O. Primers P19 and P20 were used to amplify _nifH_ gene by Mutazyme

II DNA polymerase. PCR conditions used were: 95 °C for 2 min, followed by 30 cycles of 95 °C for 30 sec, 55 °C for 30 sec, and 72 °C for 1 min, and finished with an incubation at 72 °C for

10 min. Amplified DNA was digested with _Nde_I and _Xba_I, ligated into pRHB602, and introduced into _E. coli_ DH5α competent cells (NEB, C2987I) by heat shock to generate a expression

library of _nifH_ variants. On average, ~ 4 × 106 transformants were recovered per 100 ng DNA. _IN VIVO_ HYDROGENASE ACTIVITY ASSAYS Hydrogenase activity was measured by using a Clark-type

hydrogen microelectrode (Unisense) with O2 as electron acceptor40. When necessary, hydrogenase expression was induced by injecting 9 ml of H2 into 100 ml-capped vials containing 10 ml of _R.

capsulatus_ cultures and incubating under culture conditions for 6 h. _IN VIVO_ NITROGENASE ACTIVITY ASSAYS To determine acetylene reduction activity in _R. capsulatus_ cultures grown under

diazotrophic conditions, 1-ml samples were transferred to 9-ml sealed vials with a 94% N2/6% acetylene gas phase and incubated at 30 °C in the light for 1 h. Ethylene formation was detected

in 50 μl samples withdrawn from the gas phase by using a Shimadzu GC-2014 gas chromatographer equipped with a 9-ft long, 1/8-in diameter Porapak R column. _In vivo_ nitrogenase activity

units are defined as nmol ethylene formed per min per ml of culture at an OD600 equal to 1. To determine H2 production in _R. capsulatus_ cultures grown under diazotrophic conditions, 16-ml

samples were transferred to 23-ml sealed vials with a 100% N2 atmosphere and incubated at 30 °C in the light for 48 h. H2 formation was detected in 250 μl samples withdrawn from the gas

phase by using a Shimadzu GC-8A gas chromatographer equipped with a 6-ft long, 1/8-in diameter Molecular Sieve column. Activity units are defined as nmol H2 formed per h in a culture at an

OD600 equal to 1. PROTEIN METHODS Protein concentration was determined by the bicinchoninic acid method (Pierce) with bovine serum albumin as the standard41. For SDS-PAGE, cells from 1-ml

culture samples were collected by centrifugation, resuspended in 2 × Laemmli sample buffer supplemented with 0.1 M dithiothreitol (to a concentration equivalent to an OD600 of 4), and

electrophoresed in 12% acrylamide/bisacrylamide (29:1) gels. For immunoblot analysis proteins were transferred to nitrocellulose membranes for 40 min at 20 V using a Transfer-Blot® Semi Dry

system (Bio-Rad). Immunoblot analyses were carried out with antibodies raised against a 1:1 mixture of _A. vinelandii_ and _Rhodospirillum rubrum_ NifH proteins (1:2,500 dilution) or with

antibodies raised against _R. capsulatus_ NifDK (1:2,000 dilution; antibody kindly donated by Yves Jouanneau, CNRS, Grenoble). Secondary alkaline phosphatase-conjugated anti-rabbit

antibodies (Sigma, A3687) were used at 1:5,000 dilution. FLOW CYTOMETRY Cells from 50-ml _R. capsulatus_ cultures under diazotrophic conditions inside a glove box were collected by

centrifugation in Falcon tubes for 15 min at 4 °C, 4500 × _g_, resuspended in 5 ml PBS supplemented with 10% glycerol, and incubated for 30 min at 4 °C. Cells were then collected,

resuspended in 1 ml of an 8:1:1 mixture of PBS, fluorescein di-β-D-galactopyranoside (FDG) and propidium iodide (PI), and incubated at 37 °C for 30 min to facilitate FDG entrance into the

cells. FDG releases fluorescein when cleavage by β-galactosidase42. Cells were collected by centrifugation, resuspended in RCV medium supplemented with Tc, and analyzed in a FACSVantage

(sorter) flow cytometer using an argon ion laser to excite the fluorochrome (488 nm). Cells exhibiting high fluorescence levels were sorted and recovered in 96-well microplates containing

YPS medium supplemented with Tc. NIFH 3-D MODELS 3-D model of _R. capsulatus_ NifH and the V7 variant were generated by homology modeling (http://swissmodel.expasy.org/) using the _A.

vinelandii_ NifH structures as template (1NIP for wild-type NifH and 1G5P for NifH-V7, respectively). Both models yielded NifH homodimer with capability to ligate [4Fe-4S] clusters.

ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Barahona, E. _et al_. Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening. _Sci. Rep._ 6, 38291;

doi: 10.1038/srep38291 (2016). PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. REFERENCES *

Hallenbeck, P. C. & Ghosh, D. Advances in fermentative biohydrogen production: the way forward? Trends Biotechnol. 27, 287–297 (2009). Article  CAS  Google Scholar  * Hoffman, B. M.,

Lukoyanov, D., Yang, Z. Y., Dean, D. R. & Seefeldt, L. C. Mechanism of nitrogen fixation by nitrogenase: the next stage. Chem. Rev. 114, 4041–4062 (2014). Article  CAS  Google Scholar  *

Hunter, C. N., Daldal, F. C. T. M. & Beatty, J. T. In Advances in Photosynthesis and Respiration Vol. 28 (Springer, Dordrecht, The Netherlands, 2008). * Kranz, R. G. & Haselkorn, R.

Anaerobic regulation of nitrogen-fixation genes in _Rhodopseudomonas capsulata_. Proc. Natl. Acad. Sci. USA. 83, 6805–6809 (1986). Article  CAS  ADS  Google Scholar  * Strnad, H. et al.

Complete genome sequence of the photosynthetic purple nonsulfur bacterium _Rhodobacter capsulatus_ SB 1003. J. Bacteriol. 192, 3545–3546 (2010). Article  CAS  Google Scholar  * Scolnik, P.

A. & Haselkorn, R. Activation of extra copies of genes coding for nitrogenase in _Rhodopseudomonas capsulata_. Nature 307, 289–292 (1984). Article  CAS  ADS  Google Scholar  * Masepohl,

B. & Hallenbeck, P. C. Nitrogen and molybdenum control of nitrogen fixation in the phototrophic bacterium _Rhodobacter capsulatus_. Adv. Exp. Med. Biol. 675, 49–70 (2010). Article  CAS 

Google Scholar  * Vignais, P. M. & Billoud, B. Occurrence, classification, and biological function of hydrogenases: an overview. Chem. Rev. 107, 4206–4272 (2007). Article  CAS  Google

Scholar  * Bulen, W. A. & LeComte, J. R. The nitrogenase system from Azotobacter: two-enzyme requirement for N2 reduction, ATP-dependent H2 evolution, and ATP hydrolysis. Proc. Natl.

Acad. Sci. USA. 56, 979–986 (1966). Article  CAS  ADS  Google Scholar  * Loveless, T. M. & Bishop, P. E. Identification of genes unique to Mo-independent nitrogenase systems in diverse

diazotrophs. Can. J. Microbiol. 45, 312–317 (1999). Article  CAS  Google Scholar  * Vignais, P. M. & Colbeau, A. Molecular biology of microbial hydrogenases. Curr. Issues Mol. Biol. 6,

159–188 (2004). CAS  PubMed  Google Scholar  * Colbeau, A. et al. Organization of the genes necessary for hydrogenase expression in _Rhodobacter capsulatus_. Sequence analysis and

identification of two _hyp_ regulatory mutants. Mol. Microbiol. 8, 15–29 (1993). Article  CAS  Google Scholar  * Vignais, P. M., Elsen, S. & Colbeau, A. Transcriptional regulation of the

uptake [NiFe]hydrogenase genes in _Rhodobacter capsulatus_. Biochem. Soc. Trans. 33, 28–32 (2005). Article  CAS  Google Scholar  * Durmowicz, M. C. & Maier, R. J. Roles of HoxX and HoxA

in biosynthesis of hydrogenase in _Bradyrhizobium japonicum_. J. Bacteriol. 179, 3676–3682 (1997). Article  CAS  Google Scholar  * Buhrke, T., Lenz, O., Krauss, N. & Friedrich, B.

Oxygen tolerance of the H2-sensing [NiFe] hydrogenase from _Ralstonia eutropha_ H16 is based on limited access of oxygen to the active site. J. Biol. Chem. 280, 23791–23796 (2005). Article 

CAS  Google Scholar  * Colbeau, A. & Vignais, P. M. Use of hupS::lacZ gene fusion to study regulation of hydrogenase expression in _Rhodobacter capsulatus_: stimulation by H2 . J.

Bacteriol. 174, 4258–4264 (1992). Article  CAS  Google Scholar  * Colbeau, A. et al. _Rhodobacter capsulatus_ HypF is involved in regulation of hydrogenase synthesis through the HupUV

proteins. Eur. J. Biochem. 251, 65–71 (1998). Article  CAS  Google Scholar  * Dischert, W., Vignais, P. M. & Colbeau, A. The synthesis of _Rhodobacter capsulatus_ HupSL hydrogenase is

regulated by the two-component HupT/HupR system. Mol. Microbiol. 34, 995–1006 (1999). Article  CAS  Google Scholar  * Vidal-Aroca, F. et al. One-step high-throughput assay for quantitative

detection of β-galactosidase activity in intact Gram-negative bacteria, yeast, and mammalian cells. BioTechniques 40, 433–440 (2006). Article  CAS  Google Scholar  * Jahn, A., Keuntje, B.,

Dörffler, M., Klipp, W. & Oelze, J. Optimizing photoheterotrophic H2 production by _Rhodobacter capsulatus_ upon interposon mutagenesis in the _hupL_ gene. Appl. Microbiol. Biotechnol.

40, 687–690 (1994). Article  CAS  Google Scholar  * Liu, T., Li, X. & Zhou, Z. Improvement of hydrogen yield by hupR gene knock-out and nifA gene overexpression In Rhodobacter

sphaeroides 6016. Int. J. Hydrogen Energy 35, 9603–9610 (2010). CAS  Google Scholar  * Masukawa, H., Mochimaru, M. & Sakurai, H. Disruption of the uptake hydrogenase gene, but not of the

bidirectional hydrogenase gene, leads to enhanced photobiological hydrogen production by the nitrogen-fixing cyanobacterium _Anabaena_ sp. PCC 7120. Appl. Microbiol. Biotechnol. 58, 618–624

(2002). Article  CAS  Google Scholar  * Zhu, R., Wang, D., Zhang, Y. & Li, J. Hydrogen production by _draTGB hupL_ double mutant of _Rhodospirillum rubrum_ under different light

conditions. Chinese Science Bulletin 51, 2611–2618 (2006). Article  CAS  ADS  Google Scholar  * Kim, E., Lee, M., Kim, M. & Lee, J. Molecular hydrogen production by nitrogenase of

_Rhodobacter sphaeroides_ and by Fe-only hydrogenase of _Rhodospirillum rubrum_. Int. J. Hydrogen Energy 33, 1516–1521 (2008). Article  CAS  Google Scholar  * Rey, F. E., Heiniger, E. K.

& Harwood, C. S. Redirection of metabolism for biological hydrogen production. Applied and environmental microbiology 73, 1665–1671 (2007). Article  CAS  Google Scholar  * Wang, D.,

Zhang, Y., Welch, E., Li, J. & Roberts, G. P. Elimination of Rubisco alters the regulation of nitrogenase activity and increases hydrogen production in _Rhodospirillum rubrum_. Int. J.

Hydrogen Energy 35, 7377–7385 (2010). Article  CAS  Google Scholar  * Melis, A., Zhang, L., Forestier, M., Ghirardi, M. L. & Seibert, M. Sustained photobiological hydrogen gas production

upon reversible inactivation of oxygen evolution in the green alga _Chlamydomonas reinhardtii_. Plant Physiol. 122, 127–136 (2000). Article  CAS  Google Scholar  * Flanagan, L. A., Wright,

J. J., Roessler, M. M., Moir, J. W. & Parkin, A. Re-engineering a NiFe hydrogenase to increase the H2 production bias while maintaining native levels of O2 tolerance. Chem. Commun. 52,

9133–9136 (2016). Article  CAS  Google Scholar  * Christiansen, J., Cash, V. L., Seefeldt, L. C. & Dean, D. R. Isolation and characterization of an acetylene-resistant nitrogenase. J.

Biol. Chem. 275, 11459–11464 (2000). Article  CAS  Google Scholar  * Masukawa, H., Inoue, K., Sakurai, H., Wolk, C. P. & Hausinger, R. P. Site-directed mutagenesis of the _Anabaena_ sp.

strain PCC 7120 nitrogenase active site to increase photobiological hydrogen production. Appl. Environ. Microbiol. 76, 6741–6750 (2010). Article  CAS  Google Scholar  * Packer, M. S. &

Liu, D. R. Methods for the directed evolution of proteins. Nat. Rev. Genet. 16, 379–394 (2015). Article  CAS  Google Scholar  * Griswold, K. E. et al. Evolution of highly active enzymes by

homology-independent recombination. Proc. Natl. Acad. Sci. USA. 102, 10082–10087 (2005). Article  CAS  ADS  Google Scholar  * Peters, J. W., Boyd, E. S., Hamilton, T. & Rubio, L. M. In

Nitrogen cycling In bacteria: molecular analysis (ed Moir, J. W. B. ) 59–99 (Caister Academic Press, 2011). * Flynn, T. M. Accumulation of O2-tolerant phenotypes in H2-producing strains of

_Chlamydomonas reinhardtii_ by sequential applications of chemical mutagenesis and selection. Int. J. Hydrogen Energy 27, 1421–1430 (2002). Article  CAS  Google Scholar  * Seibert, M.,

Benson, D. K. & Flynn, T. M. Method and apparatus for rapid biohydrogen phenotypic screening of microorganisms using a chemochromic sensor. US patent 6, 277,589 B1 (2001). * Katsuda, T.,

Ooshima, H., Azuma, M. & Kato, J. New detection method for hydrogen gas for screening hydrogen-producing microorganisms using water-soluble wilkinson’s catalyst derivative. J. Biosci.

Bioeng. 102, 220–226 (2006). Article  CAS  Google Scholar  * Wecker, M. S. A. & Ghirardi, M. L. High-throughput biosensor discriminates between different algal H2-photoproducing strains.

Biotechnol. Bioeng. 111, 1332–1340 (2014). Article  CAS  Google Scholar  * Wecker, M. S. A., Meuser, J. E., Posewitz, M. C. & Ghirardi, M. L. Design of a new biosensor for algal H2

production based on the H2-sensing system of _Rhodobacter capsulatus_. Int. J. Hydrogen Energy 36, 11229–11237 (2011). Article  CAS  Google Scholar  * Miller, J. H. Experiments in molecular

genetics. Cold Spring Harbor Laboratory Press. 466 (1972). * Albareda, M. et al. Dual role of HupF in the biosynthesis of [NiFe] hydrogenase in _Rhizobium leguminosarum_. BMC Microbiol. 12,

256 (2012). Article  CAS  Google Scholar  * Smith, P. K. et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85 (1985). Article  CAS  Google Scholar  * Plovins,

A., Alvarez, A. M., Ibañez, M., Molina, M. & Nombela, C. Use of fluorescein-di-beta-D-galactopyranoside (FDG) and C12-FDG as substrates for beta-galactosidase detection by flow cytometry

in animal, bacterial, and yeast cells. Appl. Microbiol. Biotechnol. 60, 4638–4641 (1994). CAS  Google Scholar  Download references ACKNOWLEDGEMENTS Flow cytometry was performed at the Flow

Cytometry Unit of the Interdepartmental Investigation Service (SIdI)- Faculty of Medicine (Universidad Autónoma de Madrid). We thank Laura Molero Martín for help in the design of the flow

cytometry protocol. We thank Luis Fernández Pacios for developing the model of the V7 NifH variant. This work was supported by the European Research Council Starting Grant 205442 and by

MINECO Grant BIO2014-59131-R. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de

Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, 28223-Pozuelo de Alarcón, Madrid, Spain Emma Barahona, Emilio Jiménez-Vicente & Luis M. Rubio Authors *

Emma Barahona View author publications You can also search for this author inPubMed Google Scholar * Emilio Jiménez-Vicente View author publications You can also search for this author

inPubMed Google Scholar * Luis M. Rubio View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS E.B. performed molecular biology, cellular biology,

flow cytometry and biochemical assays. E.J.V. contributed biochemical assays. E.B. and L.M.R performed the experimental design and data analysis and wrote the paper. L.M.R initiated and

directed this research. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. ELECTRONIC SUPPLEMENTARY MATERIAL SUPPLEMENTARY INFORMATION RIGHTS AND

PERMISSIONS This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s

Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the

license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Barahona, E., Jiménez-Vicente, E. & Rubio, L. Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening. _Sci Rep_ 6, 38291 (2016).

https://doi.org/10.1038/srep38291 Download citation * Received: 26 August 2016 * Accepted: 07 November 2016 * Published: 02 December 2016 * DOI: https://doi.org/10.1038/srep38291 SHARE THIS

ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard

Provided by the Springer Nature SharedIt content-sharing initiative