Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

<p>Abstract</p> <p>Background</p> <p>The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either <it>hupW </it>or <it>hoxW</it>, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria <it>Nostoc punctiforme </it>ATCC 29133 and <it>Nostoc </it>sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively.</p> <p>Results</p> <p>In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of <it>hupW </it>in <it>Nostoc punctiforme </it>ATCC 29133 and <it>hupW </it>and <it>hoxW </it>in <it>Nostoc </it>sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (<it>hupW</it>) and LexA (<it>hoxW</it>). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase.</p> <p>Conclusion</p> <p>Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co-evolution could be the result of a close interaction between the protease and the large subunit of the [NiFe]-hydrogenases, a theory supported by protein-protein docking experiments performed with 3D-models. Finally we present data that may explain the specificity seen among hydrogenase specific proteases, the so called "HOXBOX"; an amino acid sequence specific for proteases of Hox-type. This opens the door for more detailed studies of the specificity found among hydrogenase specific proteases and the structural properties behind it.</p

Transcription of the extended hyp-operon in Nostoc sp. strain PCC 7120

<p>Abstract</p> <p>Background</p> <p>The maturation of hydrogenases into active enzymes is a complex process and e.g. a correctly assembled active site requires the involvement of at least seven proteins, encoded by <it>hypABCDEF </it>and a hydrogenase specific protease, encoded either by <it>hupW </it>or <it>hoxW</it>. The N₂-fixing cyanobacterium <it>Nostoc </it>sp. strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. The present study addresses the presence and expression of <it>hyp</it>-genes in <it>Nostoc </it>sp. strain PCC 7120.</p> <p>Results</p> <p>RT-PCRs demonstrated that the six <it>hyp</it>-genes together with one ORF may be transcribed as a single operon. Transcriptional start points (TSPs) were identified 280 bp upstream from <it>hypF </it>and 445 bp upstream of <it>hypC</it>, respectively, demonstrating the existence of several transcripts. In addition, five upstream ORFs located in between <it>hupSL</it>, encoding the small and large subunits of the uptake hydrogenase, and the <it>hyp</it>-operon, and two downstream ORFs from the <it>hyp</it>-genes were shown to be part of the same transcript unit. A third TSP was identified 45 bp upstream of asr0689, the first of five ORFs in this operon. The ORFs are annotated as encoding unknown proteins, with the exception of alr0692 which is identified as a NifU-like protein. Orthologues of the four ORFs asr0689-alr0692, with a highly conserved genomic arrangement positioned between <it>hupSL</it>, and the <it>hyp </it>genes are found in several other N₂-fixing cyanobacteria, but are absent in non N₂-fixing cyanobacteria with only the bidirectional hydrogenase. Short conserved sequences were found in six intergenic regions of the extended <it>hyp</it>-operon, appearing between 11 and 79 times in the genome.</p> <p>Conclusion</p> <p>This study demonstrated that five ORFs upstream of the <it>hyp</it>-gene cluster are co-transcribed with the <it>hyp</it>-genes, and identified three TSPs in the extended <it>hyp</it>-gene cluster in <it>Nostoc </it>sp. strain PCC 7120. This may indicate a function related to the assembly of a functional uptake hydrogenase, hypothetically in the assembly of the small subunit of the enzyme.</p

Characterization of the hupSL promoter activity in Nostoc punctiforme ATCC 29133

<p>Abstract</p> <p>Background</p> <p>In cyanobacteria three enzymes are directly involved in the hydrogen metabolism; a nitrogenase that produces molecular hydrogen, H₂, as a by-product of nitrogen fixation, an uptake hydrogenase that recaptures H₂and oxidize it, and a bidirectional hydrogenase that can both oxidize and produce H₂.<it>Nostoc punctiforme </it>ATCC 29133 is a filamentous dinitrogen fixing cyanobacterium containing a nitrogenase and an uptake hydrogenase but no bidirectional hydrogenase. Generally, little is known about the transcriptional regulation of the cyanobacterial uptake hydrogenases. In this study gel shift assays showed that NtcA has a specific affinity to a region of the <it>hupSL </it>promoter containing a predicted NtcA binding site. The predicted NtcA binding site is centred at 258.5 bp upstream the transcription start point (tsp). To further investigate the <it>hupSL </it>promoter, truncated versions of the <it>hupSL </it>promoter were fused to either <it>gfp </it>or <it>luxAB</it>, encoding the reporter proteins Green Fluorescent Protein and Luciferase, respectively.</p> <p>Results</p> <p>Interestingly, all <it>hupsSL </it>promoter deletion constructs showed heterocyst specific expression. Unexpectedly the shortest promoter fragment, a fragment covering 57 bp upstream and 258 bp downstream the tsp, exhibited the highest promoter activity. Deletion of the NtcA binding site neither affected the expression to any larger extent nor the heterocyst specificity.</p> <p>Conclusion</p> <p>Obtained data suggest that the <it>hupSL </it>promoter in <it>N. punctiforme </it>is not strictly dependent on the upstream NtcA cis element and that the shortest promoter fragment (-57 to tsp) is enough for a high and heterocyst specific expression of <it>hupSL</it>. This is highly interesting because it indicates that the information that determines heterocyst specific gene expression might be confined to this short sequence or in the downstream untranslated leader sequence.</p

Photosynthetic hydrogen production: Novel protocols, promising engineering approaches and application of semi-synthetic hydrogenases

Photosynthetic production of molecular hydrogen (H-2) by cyanobacteria and green algae is a potential source of renewable energy. These organisms are capable of water biophotolysis by taking advantage of photosynthetic apparatus that links water oxidation at Photosystem II and reduction of protons to H-2 downstream of Photosystem I. Although the process has a theoretical potential to displace fossil fuels, photosynthetic H-2 production in its current state is not yet efficient enough for industrial applications due to a number of physiological, biochemical, and engineering barriers. This article presents a short overview of the metabolic pathways and enzymes involved in H-2 photoproduction in cyanobacteria and green algae and our present understanding of the mechanisms of this process. We also summarize recent advances in engineering photosynthetic cell factories capable of overcoming the major barriers to efficient and sustainable H-2 production

Photoautotrophic production of renewable ethylene by engineered cyanobacteria: Steering the cell metabolism towards biotechnological use

Ethylene is a volatile hydrocarbon with a massive global market in the plastic industry. The ethylene now used for commercial applications is produced exclusively from nonrenewable petroleum sources, while competitive biotechnological production systems do not yet exist. This review focuses on the currently developed photoautotrophic bioproduction strategies that enable direct solar-driven conversion of CO2 into ethylene, based on the use of genetically engineered photosynthetic cyanobacteria expressing heterologous ethylene forming enzyme (EFE) from Pseudomonas syringae. The emphasis is on the different engineering strategies to express EFE and to direct the cellular carbon flux towards the primary metabolite 2-oxoglutarate, highlighting associated metabolic constraints, and technical considerations on cultivation strategies and conditional parameters. While the research field has progressed towards more robust strains with better production profiles, and deeper understanding of the associated metabolic limitations, it is clear that there is room for significant improvement to reach industrial relevance. At the same time, existing information and the development of synthetic biology tools for engineering cyanobacteria open new possibilities for improving the prospects for the sustainable production of renewable ethylene

Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133

<p>Abstract</p> <p>Background</p> <p>Cyanobacteria harbor two [NiFe]-type hydrogenases consisting of a large and a small subunit, the Hup- and Hox-hydrogenase, respectively. Insertion of ligands and correct folding of nickel-iron hydrogenases require assistance of accessory maturation proteins (encoded by the <it>hyp</it>-genes). The intergenic region between the structural genes encoding the uptake hydrogenase (<it>hupSL</it>) and the accessory maturation proteins (<it>hyp </it>genes) in the cyanobacteria <it>Nostoc </it>PCC 7120 and <it>N. punctiforme </it>were analysed using molecular methods.</p> <p>Findings</p> <p>The five ORFs, located in between the uptake hydrogenase structural genes and the <it>hyp</it>-genes, can form a transcript with the <it>hyp</it>-genes. An identical genomic localization of these ORFs are found in other filamentous, N₂-fixing cyanobacterial strains. In <it>N. punctiforme </it>and <it>Nostoc </it>PCC 7120 the ORFs upstream of the <it>hyp</it>-genes showed similar transcript level profiles as <it>hupS </it>(hydrogenase structural gene), <it>nifD </it>(nitrogenase structural gene), <it>hypC </it>and <it>hypF </it>(accessory hydrogenase maturation genes) after nitrogen depletion. <it>In silico </it>analyzes showed that these ORFs in <it>N. punctiform</it>e harbor the same conserved regions as their homologues in <it>Nostoc </it>PCC 7120 and that they, like their homologues in <it>Nostoc </it>PCC 7120, can be transcribed together with the <it>hyp</it>-genes forming a larger extended <it>hyp-</it>operon. DNA binding studies showed interactions of the transcriptional regulators CalA and CalB to the promoter regions of the extended <it>hyp</it>-operon in <it>N. punctiforme </it>and <it>Nostoc </it>PCC 7120.</p> <p>Conclusions</p> <p>The five ORFs upstream of the <it>hyp</it>-genes in several filamentous N₂-fixing cyanobacteria have an identical genomic localization, in between the genes encoding the uptake hydrogenase and the maturation protein genes. In <it>N. punctiforme </it>and <it>Nostoc </it>PCC 7120 they are transcribed as one operon and may form transcripts together with the <it>hyp</it>-genes. The expression pattern of the five ORFs within the extended <it>hyp</it>-operon in both <it>Nostoc punctiforme </it>and <it>Nostoc </it>PCC 7120 is similar to the expression patterns of <it>hupS</it>, <it>nifD</it>, <it>hypF </it>and <it>hypC</it>. CalA, a known transcription factor, interacts with the promoter region between <it>hupSL </it>and the five ORFs in the extended <it>hyp</it>-operon in both <it>Nostoc </it>strains.</p

Machine learning predicts system-wide metabolic flux control in cyanobacteria

Metabolic fluxes and their control mechanisms are fundamental in cellular metabolism, offering insights for the study of biological systems and biotechnological applications. However, quantitative and predictive understanding of controlling biochemical reactions in microbial cell factories, especially at the system level, is limited. In this work, we present ARCTICA, a computational framework that integrates constraint-based modelling with machine learning tools to address this challenge. Using the model cyanobacterium Synechocystis sp. PCC 6803 as chassis, we demonstrate that ARCTICA effectively simulates global-scale metabolic flux control. Key findings are that (i) the photosynthetic bioproduction is mainly governed by enzymes within the Calvin-Benson-Bassham (CBB) cycle, rather than by those involve in the biosynthesis of the end-product, (ii) the catalytic capacity of the CBB cycle limits the photosynthetic activity and downstream pathways and (iii) ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a major, but not the most, limiting step within the CBB cycle. Predicted metabolic reactions qualitatively align with prior experimental observations, validating our modelling approach. ARCTICA serves as a valuable pipeline for understanding cellular physiology and predicting rate-limiting steps in genome-scale metabolic networks, and thus provides guidance for bioengineering of cyanobacteria

Optimal energy and redox metabolism in the cyanobacterium Synechocystis sp. PCC 6803

Understanding energy and redox homeostasis and carbon partitioning is crucial for systems metabolic engineering of cell factories. Carbon metabolism alone cannot achieve maximal accumulation of metabolites in production hosts, since an efficient production of target molecules requires energy and redox balance, in addition to carbon flow. The interplay between cofactor regeneration and heterologous production in photosynthetic microorganisms is not fully explored. To investigate the optimality of energy and redox metabolism, while overproducing alkenes-isobutene, isoprene, ethylene and 1-undecene, in the cyanobacterium Synechocystis sp. PCC 6803, we applied stoichiometric metabolic modelling. Our network-wide analysis indicates that the rate of NAD(P)H regeneration, rather than of ATP, controls ATP/NADPH ratio, and thereby bioproduction. The simulation also implies that energy and redox balance is interconnected with carbon and nitrogen metabolism. Furthermore, we show that an auxiliary pathway, composed of serine, one-carbon and glycine metabolism, supports cellular redox homeostasis and ATP cycling. The study revealed non-intuitive metabolic pathways required to enhance alkene production, which are mainly driven by a few key reactions carrying a high flux. We envision that the presented comparative in-silico metabolic analysis will guide the rational design of Synechocystis as a photobiological production platform of target chemicals

Synthetic biology in marine cyanobacteria : Advances and challenges

The current economic and environmental context requests an accelerating development of sustainable alternatives for the production of various target compounds. Biological processes offer viable solutions and have gained renewed interest in the recent years. For example, photosynthetic chassis organisms are particularly promising for bioprocesses, as they do not require biomass-derived carbon sources and contribute to atmospheric CO2 fixation, therefore supporting climate change mitigation. Marine cyanobacteria are of particular interest for biotechnology applications, thanks to their rich diversity, their robustness to environmental changes, and their metabolic capabilities with potential for therapeutics and chemicals production without requiring freshwater. The additional cyanobacterial properties, such as efficient photosynthesis, are also highly beneficial for biotechnological processes. Due to their capabilities, research efforts have developed several genetic tools for direct metabolic engineering applications. While progress toward a robust genetic toolkit is continuously achieved, further work is still needed to routinely modify these species and unlock their full potential for industrial applications. In contrast to the understudied marine cyanobacteria, genetic engineering and synthetic biology in freshwater cyanobacteria are currently more advanced with a variety of tools already optimized. This mini-review will explore the opportunities provided by marine cyanobacteria for a greener future. A short discussion will cover the advances and challenges regarding genetic engineering and synthetic biology in marine cyanobacteria, followed by a parallel with freshwater cyanobacteria and their current genetic availability to guide the prospect for marine species