Bi-directional electron transfer between H2 and NADPH mitigates light fluctuation responses 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 inter-organellar communication. Under anaerobic conditions, green algae also accumulate molecular hydrogen (H2), a promising alternative for fossil fuels. However, to scale-up its accumulation, a firm understanding of its integration in the photosynthetic apparatus is still required. While it is generally accepted that NADPH metabolism correlates to H2 accumulation, the mechanism of this collaboration is still vague and relies on indirect measurements. Here, we investigated this connection in Chlamydomonas reinhardtii 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 initially eventuates in H2 evolution, which initiates the photosynthetic electron flow. Later on, as illumination continues the majority of NADPH is diverted to the Calvin-Benson-Bassham cycle. Dark onset triggers re-assimilation of H2, which produces NADPH and so, enables initiation of dark fermentative metabolism.

ChimeraUGEM: unsupervised gene expression modeling in any given organism.

MOTIVATION: Regulation of the amount of protein that is synthesized from genes has proved to be a serious challenge in terms of analysis and prediction, and in terms of engineering and optimization, due to the large diversity in expression machinery across species. RESULTS: To address this challenge, we developed a methodology and a software tool (ChimeraUGEM) for predicting gene expression as well as adapting the coding sequence of a target gene to any host organism. We demonstrate these methods by predicting protein levels in seven organisms, in seven human tissues, and by increasing in vivo the expression of a synthetic gene up to 26-fold in the single-cell green alga Chlamydomonas reinhardtii. The underlying model is designed to capture sequence patterns and regulatory signals with minimal prior knowledge on the host organism and can be applied to a multitude of species and applications. AVAILABILITY AND IMPLEMENTATION: Source code (MATLAB, C) and binaries are freely available for download for non-commercial use at http://www.cs.tau.ac.il/~tamirtul/ChimeraUGEM/, and supported on macOS, Linux and Windows. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Enhancing heterologous expression in Chlamydomonas reinhardtii by transcript sequence optimization.

Various species of microalgae have recently emerged as promising host-organisms for use in biotechnology industries due to their unique properties. These include efficient conversion of sunlight into organic compounds, the ability to grow in extreme conditions and the occurrence of numerous post-translational modification pathways. However, the inability to obtain high levels of nuclear heterologous gene expression in microalgae hinders the development of the entire field. To overcome this limitation, we analyzed different sequence optimization algorithms while studying the effect of transcript sequence features on heterologous expression in the model microalga Chlamydomonas reinhardtii, whose genome consists of rare features such as a high GC content. Based on the analysis of genomic data, we created eight unique sequences coding for a synthetic ferredoxin-hydrogenase enzyme, used here as a reporter gene. Following in silico design, these synthetic genes were transformed into the C. reinhardtii nucleus, after which gene expression levels were measured. The empirical data, measured in vivo show a discrepancy of up to 65-fold between the different constructs. In this work we demonstrate how the combination of computational methods and our empirical results enable us to learn about the way gene expression is encoded in the C. reinhardtii transcripts. We describe the deleterious effect on overall expression of codons encoding for splicing signals. Subsequently, our analysis shows that utilization of a frequent subset of preferred codons results in elevated transcript levels, and that mRNA folding energy in the vicinity of translation initiation significantly affects gene expression.

Green Algal Hydrogenase Activity Is Outcompeted by Carbon Fixation before Inactivation by Oxygen Takes Place.

Photoproduction of hydrogen by green algae is considered a transitory release valve of excess reducing power and a potential carbon-free source of sustainable energy. It is generally accepted that the transitory production of hydrogen is governed by fast inactivation of hydrogenase by oxygen. However, our data suggest that photosynthetic electron loss to competing processes, mainly carbon fixation, stops hydrogen production, supports hydrogen uptake, and precedes the inevitable inactivation by oxygen. Here, we show that when transitioning from dark anaerobiosis to light, hydrogen production ceases within 2 min, regardless of the presence of oxygen. Simultaneous monitoring of the active hydrogenase pool size shows that it remains entirely intact up to 4 min after illumination and is inactivated only later. Thus, our data reveal a window of 4 min in which the hydrogenase pool is not being degraded by oxygen. Furthermore, we show that electron loss, prominently to carbon fixation, outcompetes hydrogen production and leads to hydrogen uptake. Indeed, supplying additional reducing power to hydrogenase at the cessation point regenerates the accumulation of hydrogen. Our results imply the fast cessation of hydrogen production is governed by electron loss rather than oxygen inactivation, which takes place minutes later.

Machine learning for comprehensive prediction of high risk for Alzheimer's disease based on chromatic pupilloperimetry.

Currently there are no reliable biomarkers for early detection of Alzheimer's disease (AD) at the preclinical stage. This study assessed the pupil light reflex (PLR) for focal red and blue light stimuli in central and peripheral retina in 125 cognitively normal middle age subjects (45-71 years old) at high risk for AD due to a family history of the disease (FH+), and 61 age-similar subjects with no family history of AD (FH-) using Chromatic Pupilloperimetry coupled with Machine Learning (ML). All subjects had normal ophthalmic assessment, and normal retinal and optic nerve thickness by optical coherence tomography. No significant differences were observed between groups in cognitive function and volumetric brain MRI. Chromatic pupilloperimetry-based ML models were highly discriminative in differentiating subjects with and without AD family history, using transient PLR for focal red (primarily cone-mediated), and dim blue (primarily rod-mediated) light stimuli. Features associated with transient pupil response latency (PRL) achieved Area Under the Curve Receiver Operating Characteristic (AUC-ROC) of 0.90 ± 0.051 (left-eye) and 0.87 ± 0.048 (right-eye). Parameters associated with the contraction arm of the rod and cone-mediated PLR were more discriminative compared to parameters associated with the relaxation arm and melanopsin-mediated PLR. Significantly shorter PRL for dim blue light was measured in the FH+ group in two test targets in the temporal visual field in right eye that had highest relative weight in the ML algorithm (mean ± standard error, SE 0.449 s ± 0.007 s vs. 0.478 s ± 0.010 s, p = 0.038). Taken together our study suggests that subtle focal changes in pupil contraction latency may be detected in subjects at high risk to develop AD, decades before the onset of AD clinical symptoms. The dendrites of melanopsin containing retinal ganglion cells may be affected very early at the preclinical stages of AD.

Re-routing photosynthetic energy for continuous hydrogen production in vivo.

BACKGROUND: Hydrogen is considered a promising energy vector that can be produced from sustainable resources such as sunlight and water. In green algae, such as Chlamydomonas reinhardtii, photoproduction of hydrogen is catalyzed by the enzyme [FeFe]-hydrogenase (HydA). Although highly efficient, this process is transitory and thought to serve as a release valve for excess reducing power. Up to date, prolonged production of hydrogen was achieved by the deprivation of either nutrients or light, thus, hindering the full potential of photosynthetic hydrogen production. Previously we showed that the enzyme superoxide dismutase (SOD) can enhance HydA activity in vitro, specifically when tied together to a fusion protein. RESULTS: In this work, we explored the in vivo hydrogen production phenotype of HydA-SOD fusion. We found a sustained hydrogen production, which is dependent on linear electron flow, although other pathways feed it as well. In addition, other characteristics such as slower growth and oxygen production were also observed in Hyd-SOD-expressing algae. CONCLUSIONS: The Hyd-SOD fusion manages to outcompete the Calvin-Benson cycle, allowing sustained hydrogen production for up to 14 days in non-limiting conditions.

The Integration of Multiple Nuclear-Encoded Transgenes in the Green Alga Chlamydomonas reinhardtii Results in Higher Transcription Levels.

The integration of genes into the nuclear genome of Chlamydomonas reinhardtii is mediated by Non-Homologous-End-Joining, thus resulting in unpredicted insertion locations. This phenomenon defines 'the position-effect', which is used to explain the variation of expression levels between different clones transformed with the same DNA fragment. Likewise, nuclear transgenes often undergo epigenetic silencing that reduces their expression; hence, nuclear transformations require high-throughput screening methods to isolate clones that express the foreign gene at a desirable level. Here, we show that the number of integration sites of heterologous genes results in higher mRNA levels. By transforming both a synthetic ferredoxin-hydrogenase fusion enzyme and a Gaussia-Luciferase reporter protein, we were able to obtain 33 positive clones that exhibit a wide range of synthetic expression. We then performed a droplet-digital polymerase-chain-reaction for these lines to measure their transgene DNA copy-number and mRNA levels. Surprisingly, most clones contain two integration sites of the synthetic gene (45.5%), whilst 33.3% contain one, 18.1% include three and 3.1% encompass four. Remarkably, we observed a positive correlation between the raw DNA copy-number values to the mRNA levels, suggesting a general effect of which transcription of transgenes is partially modulated by their number of copies in the genome. However, our data indicate that only clones harboring at least three copies of the target amplicon show a significant increment in mRNA levels of the reporter transgene. Lastly, we measured protein activity for each of the reporter genes to elucidate the effect of copy-number variation on heterologous expression.