O2 Sensitivity Research Articles

Evidence from high-altitude acclimatization for an integrated cerebrovascular and ventilatory hypercapnic response but different responses to hypoxia.

Ventilation and cerebral blood flow (CBF) are both sensitive to hypoxia and hypercapnia. To compare chemosensitivity in these two systems, we made simultaneous measurements of ventilatory and cerebrovascular responses to hypoxia and hypercapnia in 35 normal human subjects before and after acclimatization to hypoxia. Ventilation and CBF were measured during stepwise changes in isocapnic hypoxia and iso-oxic hypercapnia. We used MRI to quantify actual cerebral perfusion. Measurements were repeated after 2 days of acclimatization to hypoxia at 3,800 m altitude (partial pressure of inspired O2 = 90 Torr) to compare plasticity in the chemosensitivity of these two systems. Potential effects of hypoxic and hypercapnic responses on acute mountain sickness (AMS) were assessed also. The pattern of CBF and ventilatory responses to hypercapnia were almost identical. CO2 responses were augmented to a similar degree in both systems by concomitant acute hypoxia or acclimatization to sustained hypoxia. Conversely, the pattern of CBF and ventilatory responses to hypoxia were markedly different. Ventilation showed the well-known increase with acute hypoxia and a progressive decline in absolute value over 25 min of sustained hypoxia. With acclimatization to hypoxia for 2 days, the absolute values of ventilation and O2 sensitivity increased. By contrast, O2 sensitivity of CBF or its absolute value did not change during sustained hypoxia for up to 2 days. The results suggest a common or integrated control mechanism for CBF and ventilation by CO2 but different mechanisms of O2 sensitivity and plasticity between the systems. Ventilatory and cerebrovascular responses were the same for all subjects irrespective of AMS symptoms. NEW & NOTEWORTHY Ventilatory and cerebrovascular hypercapnic response patterns show similar plasticity in CO2 sensitivity following hypoxic acclimatization, suggesting an integrated control mechanism. Conversely, ventilatory and cerebrovascular hypoxic responses differ. Ventilation initially increases but adapts with prolonged hypoxia (hypoxic ventilatory decline), and ventilatory sensitivity increases following acclimatization. In contrast, cerebral blood flow hypoxic sensitivity remains constant over a range of hypoxic stimuli, with no cerebrovascular acclimatization to sustained hypoxia, suggesting different mechanisms for O2 sensitivity in the two systems.

The Activity of JmjC Histone Lysine Demethylase KDM4A is Highly Sensitive to Oxygen Concentrations.

The JmjC histone lysine demethylases (KDMs) are epigenetic regulators involved in the removal of methyl groups from post-translationally modified lysyl residues within histone tails, modulating gene transcription. These enzymes require molecular oxygen for catalytic activity and, as 2-oxoglutarate (2OG)-dependent oxygenases, are related to the cellular oxygen sensing HIF hydroxylases PHD2 and FIH. Recent studies have indicated that the activity of some KDMs, including the pseudogene-encoded KDM4E, may be sensitive to changing oxygen concentrations. Here, we report detailed analysis of the effect of oxygen availability on the activity of the KDM4 subfamily member KDM4A, importantly demonstrating a high level of O2 sensitivity both with isolated protein and in cells. Kinetic analysis of the recombinant enzyme revealed a high KMapp(O2) of 173 ± 23 μM, indicating that the activity of the enzyme is able to respond sensitively to a reduction in oxygen concentration. Furthermore, immunofluorescence experiments in U2OS cells conditionally overexpressing KDM4A showed that the cellular activity of KDM4A against its primary substrate, H3K9me3, displayed a graded response to depleting oxygen concentrations in line with the data obtained using isolated protein. These results suggest that KDM4A possesses the potential to act as an oxygen sensor in the context of chromatin modifications, with possible implications for epigenetic regulation in hypoxic disease states. Importantly, this correlation between the oxygen sensitivity of the catalytic activity of KDM4A in biochemical and cellular assays demonstrates the utility of biochemical studies in understanding the factors contributing to the diverse biological functions and varied activity of the 2OG oxygenases.

Flexible, Luminescent Metal-Organic Frameworks Showing Synergistic Solid-Solution Effects on Porosity and Sensitivity.

Mixing molecular building blocks in the solid solution manner is a valuable strategy to obtain structures and properties in between the isostructural parent metal-organic frameworks (MOFs). We report nonlinear/synergistic solid-solution effects using highly related yet non-isostructural, phosphorescent CuI triazolate frameworks as parent phases. Near the phase boundaries associated with conformational diversity and ligand heterogeneity, the porosity (+150 %) and optical O2 sensitivity (410 times, limit of detection 0.07 ppm) can be drastically improved from the best-performing parent MOFs and even exceeds the records hold by precious-metal complexes (3 ppm) and C70 (0.2 ppm).

Flexible, Luminescent Metal–Organic Frameworks Showing Synergistic Solid‐Solution Effects on Porosity and Sensitivity

AbstractMixing molecular building blocks in the solid solution manner is a valuable strategy to obtain structures and properties in between the isostructural parent metal–organic frameworks (MOFs). We report nonlinear/synergistic solid‐solution effects using highly related yet non‐isostructural, phosphorescent CuI triazolate frameworks as parent phases. Near the phase boundaries associated with conformational diversity and ligand heterogeneity, the porosity (+150 %) and optical O2 sensitivity (410 times, limit of detection 0.07 ppm) can be drastically improved from the best‐performing parent MOFs and even exceeds the records hold by precious‐metal complexes (3 ppm) and C70 (0.2 ppm).

Trityl radicals in perfluorocarbon emulsions as stable, sensitive, and biocompatible oximetry probes

EPR oximetry with the use of trityl radicals can enable sensitive O2 measurement in biological cells and tissues. However, in vitro cellular and in vivo biological applications are limited by rapid trityl probe degradation or biological clearance and the need to enhance probe O2 sensitivity. We synthesized novel perfluorocarbon (PFC) emulsions, ∼200nm droplet size, containing esterified perchlorinated triphenyl methyl (PTM) radicals dispersed in physiological aqueous buffers. These formulations exhibit excellent EPR signal stability, over 20-fold greater than free PTM probes, with high oxygen sensitivity ∼17mG/mmHg enabling pO2 measurement in aqueous solutions or cell suspensions with sensitivity >0.5mmHg. Thus, PFC-PTM probes hold great promise to enable combined O2 delivery and sensing as needed to restore or enhance tissue oxygenation in disease.

Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach

[FeFe] hydrogenases catalyze rapid H2 production but are highly O2-sensitive. Developing O2-tolerant enzymes is needed for sustainable H2 production technologies, but the lack of a quantitative and predictive assay for O2 tolerance has impeded progress. We describe a new approach to provide quantitative assessment of O2 sensitivity by using an assay employing ferredoxin NADP+ reductase (FNR) to transfer electrons from NADPH to hydrogenase via ferredoxins (Fd). Hydrogenase inactivation is measured during H2 production in an O2-containing environment. An alternative assay uses dithionite (DTH) to provide reduced Fd. This second assay measures the remaining hydrogenase activity in periodic samples taken from the NADPH-driven reaction solutions. The second assay validates the more convenient NADPH-driven assay, which better mimics physiological conditions. During development of the NADPH-driven assay and while characterizing the Clostridium pasteurianum (Cp) [FeFe] hydrogenase, CpI, we detected significant rates of direct electron loss from reduced Fd to O2 However, this loss does not interfere with measurement of first order hydrogenase inactivation, providing rate constants insensitive to initial hydrogenase concentration. We show increased activity and O2 tolerance for a protein fusion between Cp ferredoxin (CpFd) and CpI mediated by a 15-amino acid linker but not for a longer linker. We suggest that this precise, solution phase assay for [FeFe] hydrogenase O2 sensitivity and the insights we provide constitute an important advance toward the discovery of the O2-tolerant [FeFe] hydrogenases required for photosynthetic, biological H2 production.

Arteriolar oxygen reactivity: where is the sensor and what is the mechanism of action?

Arterioles in the peripheral microcirculation are exquisitely sensitive to changes in PO2 in their environment: increases in PO2 cause vasoconstriction while decreases in PO2 result in vasodilatation. However, the cell type that senses O2 (the O2 sensor) and the signalling pathway that couples changes in PO2 to changes in arteriolar tone (the mechanism of action) remain unclear. Many (but not all) ex vivo studies of isolated cannulated resistance arteries and large, first-order arterioles support the hypothesis that these vessels are intrinsically sensitive to PO2 with the smooth muscle, endothelial cells, or red blood cells serving as the O2 sensor. However, in situ studies testing these hypotheses in downstream arterioles have failed to find evidence of intrinsic O2 sensitivity, and instead have supported the idea that extravascular cells sense O2 . Similarly, ex vivo studies of isolated, cannulated resistance arteries and large first-order arterioles support the hypotheses that O2 -dependent inhibition of production of vasodilator cyclooxygenase products or O2 -dependent destruction of nitric oxide mediates O2 reactivity of these upstream vessels. In contrast, most in vivo studies of downstream arterioles have disproved these hypotheses and instead have provided evidence supporting the idea that O2 -dependent production of vasoconstrictors mediates arteriolar O2 reactivity, with significant regional heterogeneity in the specific vasoconstrictor involved. Oxygen-induced vasoconstriction may serve as a protective mechanism to reduce the oxidative burden to which a tissue is exposed, a process that is superimposed on top of the local mechanisms which regulate tissue blood flow to meet a tissue's metabolic demand.

Highly Efficient Triplet Photosensitizers: A Systematic Approach to the Application of Ir(III) Complexes containing Extended Phenanthrolines.

A series of Ir(III) complexes, based on 1,10-phenanthroline featuring aryl acetylene chromophores, were prepared and investigated as triplet photosensitizers. The complexes were synthesized by Sonogashira cross-coupling reactions using a "chemistry-on-the-complex" method. The absorption properties and luminescence lifetimes were successfully tuned by controlling the number and type of light-harvesting group. Intense UV/Vis absorption was observed for the Ir(III) complexes with two light-harvesting groups at the 3- and 8-positions of the phenanthroline. The asymmetric Ir(III) complex (with a triphenylamine (TPA) and a pyrene moiety attached) exhibited the longest lifetime. Red emission was observed for all the complexes in deaerated solutions at room temperature. Their emission at low temperature (77 K) and nanosecond time-resolved transient difference absorption spectra revealed the origin of their triplet excited states. The singlet-oxygen ((1) O2 ) sensitization and triplet-triplet annihilation (TTA)-based upconversion were explored. Highly efficient TTA upconversion (ΦUC =28.1 %) and (1) O2 sensitization (ΦΔ =97.0 %) were achieved for the asymmetric Ir(III) complex, which showed intense absorption in the visible region (λabs =482 nm, ϵ=50900 m(-1) cm(-1) ) and had a long-lived triplet excited state (53.3 μs at RT).

Gadolinium(III) Porpholactones as Efficient and Robust Singlet Oxygen Photosensitizers.

Construction of Gd(III) photosensitizers is important for designing theranostic agents owing to the unique properties arising from seven unpaired f electrons of the Gd(3+) ion. Combining these with the advantages of porpholactones with tunable NIR absorption, we herein report the synthesis of Gd(III) complexes Gd-1-4 (1, porphyrin; 2, porpholactone; 3 and 4, cis- and trans-porphodilactone, respectively) and investigated their function as singlet oxygen ((1) O2 ) photosensitizers. These Gd complexes displayed (1) O2 quantum yields (ΦΔ s) from 0.64-0.99 with the order Gd-1<Gd-2<Gd-3<Gd-4. The gradually enhanced (1) O2 sensitization after β-oxazolone moiety replacement was ascribed to the narrowing of the energy gap (ΔE) between the lowest triplet states (T1 ) of the ligand and the energy level of the (1) Δg →(3) Σg transition of (1) O2 . In particular, Gd-4 is capable of excitation in the visible to NIR region (400-700 nm) with a quantum yield near unity. These Gd complexes were first demonstrated as efficient photosensitizers in photocatalysis such as oxidative C-H bond functionalization of secondary or tertiary amines, and the oxygenation of the natural product cholesterol. Finally, after glycosylation, these water-soluble Gd complexes showed potential applications in photodynamic therapy (PDT) in HeLa cells. This work revealed that Gd(III) complexes of "bioinspired" β-modified porpholactones are efficient NIR photosensitizers and form a chemical basis to construct appealing photocatalysts and theranostic agents based on lanthanides.

Artificial pheomelanin nanoparticles and their photo-sensitization properties

Pheomelanin-type nanoparticles (PMNPs) were synthesized through a simple oxidative polymerization of 3,4-dihydroxyphenylalanine (DOPA) in the presence of cysteine by KMnO4. The synthesized PMNPs had a diameter of approximately 100nm, exhibited high dispersion stability in neutral water and various culture media and possessed similar morphology to naturally occurring pheomelanins. The efficiency of photoinduced generation of hydroxyl radicals from PMNPs was determined and related in vitro cell experiments that were carried out, with data being compared to those from eumelanin-type nanoparticles (EMNPs) and natural sepia melanin nanoparticles. Endocytosed PMNPs showed the highest phototoxicity (~50% viability) to UV-irradiated HeLa cells, confirming the direct relationship between phototoxic efficiency and the generation of hydroxyl radicals through the complex processes of the O2 sensitization.

Redox-Active Metal-Organic Composites for Highly Selective Oxygen Separation Applications.

A redox-active metal-organic composite material shows improved and selective O2 adsorption over N2 with respect to individual components (MIL-101 and ferrocene). The O2 sensitivity of the composite material arises due to the formation of maghemite nanoparticles with the pore of the metal-organic framework material.

Reactive Oxygen Species Mediated Activation of a Dormant Singlet Oxygen Photosensitizer: From Autocatalytic Singlet Oxygen Amplification to Chemicontrolled Photodynamic Therapy.

Here we show the design, preparation, and characterization of a dormant singlet oxygen ((1)O2) photosensitizer that is activated upon its reaction with reactive oxygen species (ROS), including (1)O2 itself, in what constitutes an autocatalytic process. The compound is based on a two segment photosensitizer-trap molecule where the photosensitizer segment consists of a Br-substituted boron-dipyrromethene (BODIPY) dye. The trap segment consists of the chromanol ring of α-tocopherol, the most potent naturally occurring lipid soluble antioxidant. Time-resolved absorption, fluorescence, and (1)O2 phosphorescence studies together with fluorescence and (1)O2 phosphorescence emission quantum yields collected on Br2B-PMHC and related bromo and iodo-substituted BODIPY dyes show that the trap segment provides a total of three layers of intramolecular suppression of (1)O2 production. Oxidation of the trap segment with ROS restores the sensitizing properties of the photosensitizer segment resulting in ∼40-fold enhancement in (1)O2 production. The juxtaposed antioxidant (chromanol) and prooxidant (Br-BODIPY) antagonistic chemical activities of the two-segment compound enable the autocatalytic, and in general ROS-mediated, activation of (1)O2 sensitization providing a chemical cue for the spatiotemporal control of (1)O2.The usefulness of this approach to selectively photoactivate the production of singlet oxygen in ROS stressed vs regular cells was successfully tested via the photodynamic inactivation of a ROS stressed Gram negative Escherichia coli strain.

Water-soluble octahedral molybdenum cluster compounds Na2[Mo6I8(N3)6] and Na2[Mo6I8(NCS)6]: Syntheses, luminescence, and in vitro studies

The octahedral molybdenum cluster compounds Na2[Mo6I8(N3)6] and Na2[Mo6I8(NCS)6] were synthesized and their luminescent properties and biological activity were investigated. Most molybdenum cluster compounds reported so far have had low solubility in water and/or underwent hydrolysis connected with aggregation, sedimentation, and diminishing luminescence and singlet oxygen-sensitizing properties. This behavior prevented the investigation of their photophysical properties and in vitro biological activities in aqueous media. Title compounds behave differently. They are well soluble in water and have red luminescence, which is quenched by oxygen to produce singlet oxygen, O2(1Δg), in high quantum yields. Even though [Mo6I8(N3)6]2− and [Mo6I8(NCS)6]2− gradually hydrolyze to mono-hydroxy derivatives, they do not form sedimenting aggregates, luminescence properties are well-defined, and the quantum yields of O2(1Δg) remain unchanged. These attractive features, suitable for luminescent probes or O2(1Δg) sensitizers in living cells, are overshadowed by the toxicity, especially of [Mo6I8(NCS)6]2−, and by the limited uptake by the HeLa and HEK 293T cell lines.

Multifunctional Logic in a Photosensitizer with Triple-Mode Fluorescent and Photodynamic Activity.

Herein we describe a photosensitizer (PS) with the capacity to perform multiple logic operations based on a pyrene-containing phthalocyanine (Pc) derivative. The system presents three output signals (fluorescence at 377 and 683 nm, and singlet oxygen ((1)O2) production), which are dependent on three inputs: two chemical (concentration of dithiothreitol (DTT) and acidic pH) and one physical (visible light above 530 nm for (1)O2 sensitization). The multi-input/multioutput nature of this PS leads to single-, double-, and triple-mode activation pathways of its fluorescent and photodynamic functions, through the interplay of various interrelated AND, ID, and INHIBIT gates. Dual fluorescence emissions are potentially useful for orthogonal optical imaging protocols while (1)O2 is the main reactive species in photodynamic therapy (PDT). We thus expect that this kind of PS logic system will be of great interest for multimodal cellular imaging and therapeutic applications.

Moderate inhibition of mitochondrial function augments carotid body hypoxic sensitivity.

A functional role for the mitochondria in acute O2 sensing in the carotid body (CB) remains undetermined. Whilst total inhibition of mitochondrial activity causes intense CB stimulation, it is unclear whether this response can be moderated such that graded impairment of oxidative phosphorylation might be a mechanism that sets and modifies the O2 sensitivity of the whole organ. We assessed NADH autofluorescence and [Ca2+]i in freshly dissociated CB type I cells and sensory chemoafferent discharge frequency in an intact CB preparation, in the presence of varying concentrations of nitrite (NO2 −), a mitochondrial nitric oxide (NO) donor and a competitive inhibitor of mitochondrial complex IV. NO2 − increased CB type I cell NADH in a manner that was dose-dependent and rapidly reversible. Similar concentrations of NO2 − raised type I cell [Ca2+]i via L-type channels in a PO2-dependent manner and increased chemoafferent discharge frequency. Moderate inhibition of the CB mitochondria by NO2 − augmented chemoafferent discharge frequency during graded hypoxia, consistent with a heightened CB O2 sensitivity. Furthermore, NO2 − also exaggerated chemoafferent excitation during hypercapnia signifying an increase in CB CO2 sensitivity. These data show that NO2 − can moderate the hypoxia sensitivity of the CB and thus suggest that O2 sensitivity could be set and modified in this organ by interactions between NO and mitochondrial complex IV.

Role of TrkB during the postnatal development of the rat carotid body

Brain-derived neurotrophic factor (BDNF) supports innervation of the carotid body by neurons projecting from the petrosal ganglion. Although carotid body glomus cells also express TrkB, BDNF's high affinity receptor, the role of BDNF in carotid body growth and O2 sensitivity has not been studied. Neonatal rats were treated with the TrkB antagonist K252a (100μgkg−1, i.p., b.i.d.) or vehicle on postnatal days P0–P6 and studied on P7. Carotid body volume was decreased by 35% after chronic K252a (P<0.001); a reduction in carotid body size was also elicited using the more selective TrkB antagonist ANA-12 (500μgkg−1, i.p., b.i.d.). In contrast, single-unit chemoafferent responses to 5% O2, measured in vitro, were unaffected by chronic K252a administration. Normoxic and hypoxic ventilation, measured by head–body plethysmography, were also normal after chronic K252a administration, but acute K252a administration produced a slower, deeper breathing pattern during the transition into hypoxia. These data suggest that BDNF regulates postnatal carotid body growth but does not influence the development of glomus cell O2 sensitivity.

Investigations by Protein Film Electrochemistry of Alternative Reactions of Nickel-Containing Carbon Monoxide Dehydrogenase.

Protein film electrochemistry has been used to investigate reactions of highly active nickel-containing carbon monoxide dehydrogenases (CODHs). When attached to a pyrolytic graphite electrode, these enzymes behave as reversible electrocatalysts, displaying CO2 reduction or CO oxidation at minimal overpotential. The O2 sensitivity of CODH is suppressed by adding cyanide, a reversible inhibitor of CO oxidation, or by raising the electrode potential. Reduction of N2O, isoelectronic with CO2, is catalyzed by CODH, but the reaction is sluggish, despite a large overpotential, and results in inactivation. Production of H2 and formate under highly reducing conditions is consistent with calculations predicting that a nickel-hydrido species might be formed, but the very low rates suggest that such a species is not on the main catalytic pathway.

Exploring mechanisms of resistance to dimethachlone in Sclerotinia sclerotiorum.

The dicarboximide fungicide dimethachlone has been widely used in China for more than 12 years to control the Sclerotinia stem rot caused by Sclerotinia sclerotiorum disease. First signs of resistance in the field are reported at low frequency. In this study, four resistant isolate/mutants were used to explore still unknown mechanisms leading to dimethachlone resistance. The resistant isolate/mutants had significantly higher EC50 values compared with the sensitive control isolates. Cross-resistance was confirmed between dimethachlone and procymidone, iprodione and fludioxonil. The resistant isolate/mutants revealed a decreased mycelial growth rate, were less pathogenic on leaves of oilseed rape, were more sensitive to osmotic pressure and oxidative stress and released more electrolytes compared with the sensitive isolates. Only in one lab mutant did we find a point mutation (V238A) in the SsOs1 gene of the high-osmolarity glycerol (HOG) signalling pathway. The expression of this gene was lost in the field resistant isolate HN456-1-JBJ and decreased in mycelium that was subjected to either high osmotic pressure or dimethachlone; however, another key gene in the HOG pathway, SsHog1, could be induced in the resistant isolate and mutants with NaCl treatment. This study demonstrates that resistance to dicarboximide fungicide dimethachlone in S. sclerotiorum is emerging in China. Several fitness parameters, including mycelial growth rate, sclerotia formed in vitro, aggressiveness on leaves and osmotic and H2 O2 sensitivity, indicate that the resistant strains may not effectively compete with sensitive isolates in the field in the absence of selection pressure. Lost expression or the V238A point mutation in the SsOs1 gene may confer resistance to dicarboximide fungicide dimethachlone in S. sclerotiorum, but this study illustrates that other, yet unknown mechanisms also exist.

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2.

Implementation of photobiological H2 production: the O 2 sensitivity of hydrogenases.

The search for the ultimate carbon-free fuel has intensified in recent years, with a major focus on photoproduction of H2. Biological sources of H2 include oxygenic photosynthetic green algae and cyanobacteria, both of which contain hydrogenase enzymes. Although algal and cyanobacterial hydrogenases perform the same enzymatic reaction through metallo-clusters, their hydrogenases have evolved separately, are expressed differently (transcription of algal hydrogenases is anaerobically induced, while bacterial hydrogenases are constitutively expressed), and display different sensitivity to O2 inactivation. Among various physiological factors, the sensitivity of hydrogenases to O2 has been one of the major factors preventing implementation of biological systems for commercial production of renewable H2. This review addresses recent strategies aimed at engineering increased O2 tolerance into hydrogenases (as of now mainly unsuccessful), as well as towards the development of methods to bypass the O2 sensitivity of hydrogenases (successful but still yielding low solar conversion efficiencies). The author concludes with a description of current approaches from various laboratories to incorporate multiple genetic traits into either algae or cyanobacteria to jointly address limiting factors other than the hydrogenase O2 sensitivity and achieve more sustained H2 photoproduction activity.