Biology subjects

Yacoby, I.

Publications and source records attributed to Yacoby, I..

11 recordsLinked to original sources

bioRxiv2026

The anoxic switch: Strain variation in H2 exchange during repeated illumination after dark anoxia

Hydrogen (H2) exchange during transitions from dark incubation to sustained illumination provides a readout of physiological history in green microalgae. In Chlamydomonas reinhardtii, the first illumination after prolonged dark anoxia gives a higher H2 production rate than later illuminations, an anoxic switch linked to photosystem II (PSII) photosynthetic control. Membrane-inlet mass spectrometry was used to compare 54 independently grown cultures from 17 strains across sequential irradiances and 48 cultures from 15 strains during seven repeated cycles of 2 min of light and 3 min of darkness at strain-specific irradiances. For the primary early-response 30s endpoint, first sustained-light rates exceeded the mean of cycles 5-7 in 43 of 48 cultures. Ratios of strain mean first to later rates exceeded 1 in 14 of 15 strains and ranged from 0.63 to 17.48, and strain accounted for most of the variance in culture-level ratios. G1122 was the only strain with higher later rates. The predominance of higher first rates persisted across alternative rate windows and after excluding cultures with elevated pre-light O2, although exact magnitudes depended on the window. O2 and nominal CO2 signal changes varied among strains, without a detected cross-strain correlation with the H2 ratio. An enhanced first-light H2 response was therefore the predominant pattern in strains of the Chlorellaceae, Scenedesmaceae, Selenastraceae and Chlamydomonadales, but its magnitude differed by more than an order of magnitude, identifying candidate strains for testing whether PSII photosynthetic control explains this variation. Keywords: anoxia; green microalgae; hydrogen; illumination history; membrane-inlet mass spectrometry; photosynthetic induction

plant biology↗

bioRxiv2026

ATP synthase deficiency promotes prolonged hydrogenproduction in Chlamydomonas

Photosynthetic electron transport is tightly regulated to balance ATP and NADPH production and prevent over-reduction of the photosynthetic machinery. How this balance is maintained under severe ATP limitation, particularly under anoxia, is not fully resolved. Here, we used several deletion mutants of chloroplast ATP synthase subunits from Chlamydomonas reinhardtii to examine how electron flow is reorganized under anoxic conditions through the interplay between linear electron flow, cyclic electron flow, and alternative electron sinks. Using a combination of optical spectroscopy methods and membrane inlet mass spectrometry, we characterized changes in photosynthetic electron transport, proton motive force relaxation and gas exchange. Under oxic conditions, the {Delta}atpH mutant (lacking the c-subunit) showed strong acceptor-side limitation at photosystem I (PSI), whereas atpC1-2 (lacking the {gamma}-subunit) resembled the wild type. Under anoxia, this limitation was partially relieved, coinciding with sustained electron transport and prolonged hydrogen production. In particular, the {Delta}atpH mutant exhibited increased cyclic electron flow under oxic conditions and maintained PSI activity under anoxia despite impaired ATP synthesis. Prolonged hydrogen production was also observed in atpC1-2, which did not share the increased respiration of {Delta}atpH. Hydrogenase activity under anoxia is consistent with a role as an electron sink that may help alleviate PSI acceptor-side limitation and rebalance the redox state of the electron transport chain. These findings show that ATP synthase deficiency reorganizes photosynthetic electron flow and promotes prolonged hydrogen production, and point to hydrogenase as a candidate electron sink under energy-constrained conditions.

plant biology↗

bioRxiv2026

Expanding The Algal Hydrogen Toolbox: A Non-GMO Platform Reveals Multiple Physiological Routes To Sustained Hydrogen Production Across Microalgae

Sustainable hydrogen production from microalgae remains limited by intrinsic physiological constraints and the need to preserve biomass value for food and feed applications. Transgenic approaches to overcome these limitations were proven successful, yet result in genetically modified (GMO) strains that face major regulatory and deployment barriers. Here, we present a non-GMO experimental platform that enables systematic isolation of hydrogen-producing phenotypes through high-throughput UV mutagenesis pipline coupled with targeted physiological screening. Applying this approach across phylogenetically distinct algal species, including the industrial strain Chlorella vulgaris and the extremophile Chlorella ohadii, we achieve high discovery efficiency, recovering 0.4-0.6% validated hydrogen-producing mutants and achieving 6.7-25% validation rates among screen-positive candidates, indicating strong enrichment at the primary screening stage. We show that sustained hydrogen production represents a physiologically accessible state emerging across diverse genetic backgrounds. This state is consistently associated with reorganization of photosynthetic electron partitioning, yet arises through multiple distinct configurations that differentially balance hydrogen production, oxygen metabolism, and carbon fixation. This framework provides a scalable route to identify hydrogen-producing strains in industrially relevant algae without introducing foreign DNA and expands the accessible design space for photobiological hydrogen production.

plant biology↗

bioRxiv2026

A Conserved Mechanism for Positioning Ferredoxin NADP+ Reductase at Photosystem I in Green Algae

The association of ferredoxin-NADP+ reductase (FNR) with thylakoid membranes constitutes a central regulatory node in photosynthetic electron transport, governing NADPH production essential for carbon fixation. In cyanobacteria and higher plants, this interaction is mediated by an intrinsic FNR domain or specialized proteins, yet the mode of recruitment in green algae has remained enigmatic. Here, we show that in the green microalga Chlamydomonas reinhardtii, FNR is directly tethered to photosystem I-LHCI (PSI-LHCI) through a conserved N-terminal -helix of the antenna protein Lhca4. Cryogenic electron microscopy localizes FNR to the stromal side of PSI proximal to Lhca4, while AlphaFold modeling identifies a specific interaction interface, which we validate using isothermal titration calorimetry. Structural modeling further reveals that the spatial separation between PSI-bound FNR and ferredoxin (Fd) is incompatible with direct electron transfer, indicating that it occurs sequentially rather than through a stable PSI-Fd-FNR complex. Comparative analysis demonstrates that the FNR-binding N-terminal motif of Lhca proteins is conserved across diverse green microalgae, suggesting an evolutionarily conserved strategy for positioning FNR at PSI. Collectively, our results uncover a novel mechanism for FNR recruitment and establish a new principle by which photosynthetic electron partitioning is regulated through spatial organization of electron transfer components.

plant biology↗

bioRxiv2025

Photobiological Hydrogen Production at Scale: Integrating Bioprocess Optimization and Techno-Economic Modeling

Microalgal hydrogen production driven by solar energy offers significant promise as a sustainable energy alternative, yet remains economically challenging due to issues of scalability from laboratory to industrial applications. Here, we demonstrate scalable hydrogen production at semi-industrial volumes using the Chlamydomonas reinhardtii pgr5 mutant, employing an optimized cultivation protocol and photobioreactor design. This approach achieves a fivefold increase in hydrogen yield. Notably, the post-production biomass maintains a high-quality protein and nutrients profile, emphasizing microalgae as a "green coin" with energy security on one side and food security on the other. Techno-economic analysis suggests, achievable hydrogen production costs could reach $2.70/kg H2 under projected improvements, and potentially decrease furthur to $1.48/kg with full optimization. By effectively bridging laboratory research and practical industrial implementation, our study establishes a dual-purpose algal hydrogen production platform aligned with circular economy principles, positioning microalgae prominently within sustainable energy and food frameworks.

bioengineering↗

bioRxiv2024

Alanine-scanning of the yeast killer toxin K2 reveals key residues for activity, gain-of-function variants, and supports prediction of precursor processing and 3D structure.

Yeast killer toxins (YKTs) are antimicrobial proteins secreted by yeast with potential applications ranging from food preservation to therapeutic agents in human health. However, the practical use of many YTKs is limited by specific pH requirements, low temperature stability, low production yields, and narrow target specificity. While protein engineering could potentially overcome these challenges, progress is hindered by a lack of detailed knowledge about sequence-function relationships and structural data for these often multi-step processed proteins. In this study, we focused on the YKT K2, encoded by the M2 dsRNA satellite virus in Saccharomyces cerevisiae. Using alanine scanning mutagenesis of the full open reading frame and structure predictions combined with molecular dynamics simulations, we generated a comprehensive sequence-function map, refined the model for the proteolytic processing of the K2 precursor, and predicted the mature toxin structure. Our findings also demonstrate that K2 can be engineered towards enhanced toxicity and altered target specificity through single-site mutations. Furthermore, we identified structural homology between K2 and the SMK toxin from the yeast Millerozyma farinosa. Our cost-effective workflow provides a platform to broadly map YKT sequence-structure-function relationships, facilitating the engineering towards toxin-based technologies. The workflow could also serve as a template to resolve the processing and conformations of other proteins within the secretory pathway - a dynamic multi-step process that is challenging to structurally capture by purification and solving structures of intermediates.

microbiology↗

bioRxiv2022

Calredoxin regulates the chloroplast NADPH-dependent thioredoxin reductase in Chlamydomonas reinhardtii

Calredoxin (CRX) is a calcium (Ca2+)-dependent thioredoxin (TRX) in the chloroplast of Chlamydomonas reinhardtii with largely unclear physiological role. We elucidated the CRX functionality by performing in-depth quantitative proteomics of wild type cells in comparison with crx insertional mutant (IMcrx), two CRISPR/Cas9 KO mutants and CRX rescues. These analyses revealed that the chloroplast NADPH-dependent TRX reductase (NTRC) is co-regulated with CRX. Electron transfer measurements revealed that CRX inhibits NADPH-dependent reduction of oxidized chloroplast 2-Cys peroxiredoxin (PRX1) via NTRC and that the function of the NADPH-NTRC complex is under strict control of CRX. Via non-reducing SDS-PAGE assays and mass spectrometry, our data also demonstrated that PRX1 is more oxidized under high light (HL) conditions in the absence of CRX. The redox tuning of PRX1 and control of the NADPH-NTRC complex via CRX interconnects redox control with active photosynthetic electron transport and metabolism as well as Ca2+ signaling. In this way, an economic use of NADPH for PRX1 reduction is ensured. The finding, that the absence of CRX under HL conditions severely inhibited light-driven CO2 fixation underpins the importance of CRX for redox tuning as well as for efficient photosynthesis. One-sentence summaryCalredoxin dependent redox regulation ensures efficient photosynthesis.

plant biology↗

bioRxiv2021

A novel PSII photosynthetic control is activated in anoxic cultures of green algae

Photosynthetic green algae face an ever-changing environment of fluctuating light as well as unstable oxygen levels, which via the production of free radicals constantly challenges the integrity of the photosynthetic complexes. To face such challenges, a complex photosynthetic control network monitors and tightly control the membrane redox potential. Here, we show that not only that the photosynthetic control set the rate limiting step of photosynthetic linear electron flow, but also, upon its ultimate dissipation, it triggers intrinsic alternations in the activity of the photosynthetic complexes. These changes have a grave and prolonged effect on the activity of photosystem II, leading to a massive 3-fold decrease in its electron output. We came into this conclusion via studying a variety of green algae species and applying advance mass-spectrometry and diverse spectroscopic techniques. Our results shed new light on the mechanism of photosynthetic regulation, and provide new target for improving photosynthesis.

plant biology↗

bioRxiv2020

Bi-directional electron transfer between H2 and NADPH mitigates the response to light fluctuations in green algae

The metabolism of green algae has been the focus of much research over the last century. These photosynthetic organisms can thrive under various conditions and adapt quickly to changing environments by concomitant usage of several metabolic apparatuses. The main electron coordinator in their chloroplasts, nicotinamide adenine dinucleotide phosphate (NADPH), participates in many enzymatic activities and is also responsible for interorganelle communication. Under anaerobic conditions, green algae also accumulate molecular hydrogen (H2), a promising alternative for fossil fuels. However, in order to scale-up its accumulation, a firm understanding of its integration in the photosynthetic apparatus is still lacking. While it is generally accepted that NADPH metabolism correlates to H2 accumulation, the mechanism of this collaboration is still vague and rely on indirect measurements. Here, we investigated this connection using simultaneous measurements of both dissolved gases concentration, NADPH fluorescence and electrochromic shifts at 520-546 nm. Our results indicate that energy transfer between H2 and NADPH is bi-directional and crucial for the maintenance of redox balance under light fluctuations. At light onset, NADPH consumption is initially eventuated in H2 evolution, which initiate the photosynthetic electron flow. Later on, as illumination continues the majority of NADPH is recycled by Nda2 rather than consumed by terminal sinks such as CBB cycle and H2 production. Dark onset triggers re-assimilation of H2, which produces NADPH and so, enables initiation of dark fermentative metabolism. One sentence summaryEnergy transfer between H2 and NADPH is bi-directional and crucial for the maintenance of redox balance under light fluctuations.

plant biology↗

bioRxiv2020

The Mehler reaction site is the Phylloquinone within Photosystem I

Photosynthesis is a vital process, responsible for fixing carbon dioxide, and producing most of the organic matter on the planet. However, photosynthesis has some inherent limitations in utilizing solar energy. Up to a third of the energy absorbed is lost in the reduction of O2 to produce the superoxide radical (O2*-), which occurs principally within photosystem I (PSI) via the Mehler reaction. Strikingly, the precise location as well as the evolutionary role of the reaction have long been a matter of debate. For decades, O2 reduction was assumed to take place solely in the distal iron-sulfur clusters of PSI rather than within the two asymmetrical cofactor branches. Here we demonstrate that under high irradiance, O2 photoreduction by PSI takes place at the phylloquinone of one of the branches (the A-branch). This conclusion derives from the light dependency of the O2 photoreduction rate constant, and from the high rates of O2 photoreduction in PSI complexes lacking iron-sulfur clusters and in a mutant PSI, in which the lifetime of this phyllosemiquinone state is extended 100-fold. On these grounds, we suggest that the Mehler reaction serves as a release valve, functioning only when needed, under conditions where both the distal iron-sulfur clusters of PSI and the mobile ferredoxin pool are over reduced. SIGNIFICANCE STATEMENTPhotosynthesis is the process responsible for the oxygenation of the ancient anoxic atmosphere, and the transformation of inorganic carbon to most of the organic matter on Earth. However, it is less commonly appreciated that the appearance of oxygen in the atmosphere led to the unavoidable opposite process in which oxygen is consumed, thereby producing deleterious oxygen radicals such as the superoxide radical. For almost half a decade, the location of the main site of superoxide radical production in chloroplasts has been a matter of debate. We now provide conclusive evidence that it is located in the phylloquinones(s) within photosystem I.

plant biology↗

bioRxiv2018

Solving the riddle of the evolution of Shine-Dalgarno based translation in chloroplasts

The chloroplast, a photosynthetic organelle found in all plant and algae species, originates from an ancient event in which a cyanobacterium was engulfed by a larger eukaryote. Thus, modern chloroplasts still harbor a bacterial-like genome and carry out all stages of gene expression, including mRNA translation by a 70S ribosome. However, the Shine-Dalgarno model, which predominantly regulates translation initiation by base-pairing between the ribosomal RNA and the mRNA in model bacteria genera, was reported to have ambiguous effects on chloroplast gene expression. Here we show that while the Shine-Dalgarno motif is clearly conserved in proteobacterial mRNAs, its general absence from chloroplast mRNAs is observed in cyanobacteria as well, promoting the idea that the evolutionary process of reducing the centrality of the Shine-Dalgarno mechanism began well before plastid endosymbiosis. As plastid ribosomal RNA anti-Shine-Dalgarno elements are highly similar to their bacterial counterparts, these sites alone cannot explain the decline in plastid Shine-Dalgarno generality. However, by computational simulation we show that upstream point mutations modulate the local structure of ribosomal RNA in chloroplasts, creating significantly tighter structures around the anti-Shine-Dalgarno locus, which in-turn reduce the probability of ribosome binding via the Shine-Dalgarno mechanism. To validate our model, we expressed a mCherry gene harboring a Shine-Dalgarno motif in the Chlamydomonas reinhardtii chloroplast. We show that co-expressing it with a 16S ribosomal RNA, modified according to our model, significantly enhances its expression compared to co-expression with an endogenous 16S gene.\n\nSignificance statementChloroplasts are fascinating intracellular organelles which have evolved from an ancient cyanobacterium engulfed by a larger eukaryote. Surprisingly, the canonical mechanism regulating bacterial translation initiation - Shine-Dalgarno - has been shown to play a reduced role in chloroplasts. Here, we show that mutations upstream from the anti-Shine-Dalgarno element decrease the probability of spontaneous ribosome binding by modulating the secondary structure of the ribosomal RNA. These mutations constitute a regulatory step which acclimates the Shine-Dalgarno mechanism to the translational regulation regime of chloroplasts. Interestingly, we show that these chloroplast features occur in modern cyanobacteria as well, promoting the idea that they have evolved prior to endosymbiosis.

plant biology↗