Overcoming the expression barrier of the ferredoxin‑hydrogenase chimera in Chlamydomonas reinhardtii supports a linear increment in photosynthetic hydrogen output

Weiner, Iddo; Shahar, Noam; Feldman, Yael; Landman, Shira; Milrad, Yuval; Ben-Zvi, Oren; Avitan, Meital; Dafni, Eyal; Schweitzer, Shira; Eilenberg, Haviva; Atar, Shimshi; Diament, Alon; Tuller, Tamir; Yacoby, Iftach

doi:10.1016/j.algal.2018.06.011

Cited by 21 publications

(32 citation statements)

References 32 publications

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“…Our results show that the operon formation is widely preferred across all protein-coding groups, while RNA genes tend to be found in the monocistronic form (Figure 4B). This negligible role that operons play in regulation of plastid RNA gene expression could be explained by the overall high levels of transcription in chloroplasts (35,38,52) and the rate limiting nature of chloroplast translation. According to this hypothesis, co-transcription of functionally related CDSs is expected to be more beneficial as they are subject to similar translation regulation (Figure 2D and E).…”

Section: Discussionmentioning

confidence: 99%

Prediction and large-scale analysis of primary operons in plastids reveals unique genetic features in the evolution of chloroplasts

Shahar

Weiner

Stotsky

et al. 2019

Nucleic Acids Research

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While bacterial operons have been thoroughly studied, few analyses of chloroplast operons exist, limiting the ability to study fundamental elements of these structures and utilize them for synthetic biology. Here, we describe the creation of a plastome-specific operon database (link provided below) achieved by combining experimental tools and predictive modeling. Using a Reverse-Transcription-PCR based method and published data, we determined the transcription-state of 213 gene pairs from four plastomes of evolutionary distinct organisms. By analyzing sequence-based features computed for our dataset, we were able to highlight fundamental characteristics differentiating between operon pairs and non-operon pairs. These include an interesting tendency toward maintaining similar messenger RNA-folding profiles in operon gene pairs, a feature that failed to yield any informative separation in cyanobacteria, suggesting that it catches unique traits of operon gene expression, which have evolved post-endosymbiosis. Subsequently, we used this feature set to train a random-forest classifier for operon prediction. As our results demonstrate the ability of our predictor to obtain accurate (84%) and robust predictions on unlabeled datasets, we proceeded to building operon maps for 2018 sequenced plastids. Our database may now present new opportunities for promoting metabolic engineering and synthetic biology in chloroplasts.

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Section: Discussionmentioning

confidence: 99%

Prediction and large-scale analysis of primary operons in plastids reveals unique genetic features in the evolution of chloroplasts

Shahar

Weiner

Stotsky

et al. 2019

Nucleic Acids Research

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“…Since the Rhodo assay only provides a rough estimation for the expression level of the enzyme, selected clones were further analyzed by the methyl viologen (MV) assay. The MV assay measures the kinetics of H 2 production and, therefore, can accurately quantity the active pool of the enzyme, (i.e., the mature enzyme pool) [37, 43]. To induce maturation, cultures were incubated in the dark under mild room temperature conditions.…”

Section: Resultsmentioning

confidence: 99%

“…Glucose oxidase (40 units mL −1 ), catalase (40 units mL −1 ), and glucose (20 mM) were added for complete anoxia. Chemical reaction buffer [43] 3× stock (for final concentration of 100 mM Tris–HCl, pH 7.2, 1 M NaCl, 10 mM methyl viologen, 20 mM sodium dithionite, and 0.2% [V/V] Triton X-100) was prepared in an anaerobic glove box (H 2 /N 2 ). The buffer was sealed in a vial and flushed with Ar for 10 min to remove residual H 2 traces.…”

Section: Methodsmentioning

confidence: 99%

Re-routing photosynthetic energy for continuous hydrogen production in vivo

Ben-Zvi

Dafni

Feldman

et al. 2019

Biotechnol Biofuels

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BackgroundHydrogen 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.ResultsIn 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.ConclusionsThe Hyd–SOD fusion manages to outcompete the Calvin–Benson cycle, allowing sustained hydrogen production for up to 14 days in non-limiting conditions.

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“…To test our software, we performed the R. capsulatus assay on 20 engineered strains of Chlamydomonas reinhardtii with known expression levels of a synthetic H 2 -producing enzyme (29). To produce images for the software, these strains were plated on four replica plates of 20 clones each.…”

Section: Resultsmentioning

confidence: 99%

“…In that assay, plates containing algal colonies are overlaid with engineered bacteria which produce GFP in the presence of gaseous hydrogen (H 2 ). This system, which generates a luminescence image (GFP) alongside a colony image (chlorophyll), is typically used as a qualitative phenotypic screen that reports on desirable genetic traits in heterogeneous populations (25–29). This assay represents a classical large-scale experiment in which the output is a colony luminescence image with an array of biological noise data which have so far prevented a quantitative analysis.…”

Section: Introductionmentioning

confidence: 99%

Image-Processing Software for High-Throughput Quantification of Colony Luminescence

Dafni

Weiner

Shahar

et al. 2019

mSphere

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Luminescent markers are widely used as reporters for various biologically interesting traits. In colony luminescence assays, the levels of luminescence around each colony can be used to compare the levels of traits of interest for different strains, treatments, etc., using quantitative measurements of the luminescence. However, automatic methods of obtaining this data are underdeveloped, making this a laborious manual process, especially in analyzing large numbers of colonies. The significance of this work is in developing an automatic, high-throughput tool for quantitative analysis of colony luminescence assays, which will allow fast collection of qualitative data from these assays and thus increase their overall usability.

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Overcoming the expression barrier of the ferredoxin‑hydrogenase chimera in Chlamydomonas reinhardtii supports a linear increment in photosynthetic hydrogen output

Cited by 21 publications

References 32 publications

Prediction and large-scale analysis of primary operons in plastids reveals unique genetic features in the evolution of chloroplasts

Prediction and large-scale analysis of primary operons in plastids reveals unique genetic features in the evolution of chloroplasts

Re-routing photosynthetic energy for continuous hydrogen production in vivo

Image-Processing Software for High-Throughput Quantification of Colony Luminescence

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