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.

Differential catalase activity and tolerance to hydrogen peroxide in the filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Anabaena sp. PCC 7120.

Photoautotrophic cyanobacteria often confront hydrogen peroxide (H2O2), a reactive oxygen species potentially toxic to cells when present in sufficiently high concentrations. In this study, H2O2 tolerance ability of filamentous cyanobacteria Nostoc punctiforme ATCC 29133 (Nostoc 29133) and Anabaena sp. PCC 7120 (Anabaena 7120) was investigated at increasing concentrations of H2O2 (0-0.5 mM). In Nostoc 29133, 0.25 and 0.5 mM H2O2 caused a reduction in chlorophyll a content by 12 and 20%, respectively, whereas with similar treatments, a total loss of chlorophyll a was detected in Anabaena 7120. Further, Nostoc 29133 was able to maintain its photosystem II performance in the presence of H2O2 up to a concentration of 0.5 mM, whereas in Anabaena 7120, 0.25 mM H2O2 caused a complete reduction of photosystem II performance. The intracellular hydroperoxide level (indicator of oxidative status) did not increase to the same high level in Nostoc 29133, as compared to in Anabaena 7120 after H2O2 treatment. This might be explained by that Nostoc 29133 showed a 20-fold higher intrinsic constitutive catalase activity than Anabaena 7120, thus indicating that the superior tolerance of Nostoc 29133 to H2O2 stems from its higher ability to decompose H2O2. It is suggested that difference in H2O2 tolerance between closely related filamentous cyanobacteria, as revealed in this study, may be taken into account for judicious selection and effective use of strains in biotechnological applications.

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.

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

Photosynthetic production of molecular hydrogen (H2 ) 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 H2 downstream of Photosystem I. Although the process has a theoretical potential to displace fossil fuels, photosynthetic H2 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 H2 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 H2 production.

Doing synthetic biology with photosynthetic microorganisms.

The use of photosynthetic microbes as synthetic biology hosts for the sustainable production of commodity chemicals and even fuels has received increasing attention over the last decade. The number of studies published, tools implemented, and resources made available for microalgae have increased beyond expectations during the last few years. However, the tools available for genetic engineering in these organisms still lag those available for the more commonly used heterotrophic host organisms. In this mini-review, we provide an overview of the photosynthetic microbes most commonly used in synthetic biology studies, namely cyanobacteria, chlorophytes, eustigmatophytes and diatoms. We provide basic information on the techniques and tools available for each model group of organisms, we outline the state-of-the-art, and we list the synthetic biology tools that have been successfully used. We specifically focus on the latest CRISPR developments, as we believe that precision editing and advanced genetic engineering tools will be pivotal to the advancement of the field. Finally, we discuss the relative strengths and weaknesses of each group of organisms and examine the challenges that need to be overcome to achieve their synthetic biology potential.

Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions.

DNA-binding proteins from starved cells (Dps, EC: 1.16.3.1) have a variety of different biochemical activities such as DNA-binding, iron sequestration, and H2O2 detoxification. Most bacteria commonly feature one or two Dps enzymes, whereas the cyanobacterium Nostoc punctiforme displays an unusually high number of five Dps proteins (NpDps1-5). Our previous studies have indicated physiological differences, as well as cell-specific expression, among these five proteins. Three of the five NpDps proteins, NpDps1, -2, and -3, were classified as canonical Dps proteins. To further investigate their properties and possible importance for physiological function, here we characterized and compared them in vitro Nondenaturing PAGE, gel filtration, and dynamic light-scattering experiments disclosed that the three NpDps proteins exist as multimeric protein species in the bacterial cell. We also demonstrate Dps-mediated iron oxidation catalysis in the presence of H2O2 However, no iron oxidation with O2 as the electron acceptor was detected under our experimental conditions. In modeled structures of NpDps1, -2, and -3, protein channels were identified that could serve as the entrance for ferrous iron into the dodecameric structures. Furthermore, we could demonstrate pH-dependent DNA-binding properties for NpDps2 and -3. This study adds critical insights into the functions and stabilities of the three canonical Dps proteins from N. punctiforme and suggests that each of the Dps proteins within this bacterium has a specific biochemical property and function.

Harnessing transcription for bioproduction in cyanobacteria.

Sustainable production of biofuels and other valuable compounds is one of our future challenges. One tempting possibility is to use photosynthetic cyanobacteria as production factories. Currently, tools for genetic engineering of cyanobacteria are not good enough to exploit the full potential of cyanobacteria. A wide variety of expression systems will be required to adjust both the expression of heterologous enzyme(s) and metabolic routes to the best possible balance, allowing the optimal production of a particular substance. In bacteria, transcription, especially the initiation of transcription, has a central role in adjusting gene expression and thus also metabolic fluxes of cells according to environmental cues. Here we summarize the recent progress in developing tools for efficient cyanofactories, focusing especially on transcriptional regulation.

The two Dps proteins, NpDps2 and NpDps5, are involved in light-induced oxidative stress tolerance in the N2-fixing cyanobacterium Nostoc punctiforme.

Cyanobacteria are photosynthetic prokaryotes that are considered biotechnologically prominent organisms for production of high-value compounds. Cyanobacteria are subject to high-light intensities, which is a challenge that needs to be addressed in design of efficient bio-engineered photosynthetic organisms. Dps proteins are members of the ferritin superfamily and are omnipresent in prokaryotes. They play a major role in oxidative stress protection and iron homeostasis. The filamentous, heterocyst-forming Nostoc punctiforme, has five Dps proteins. In this study we elucidated the role of these Dps proteins in acclimation to high light intensity, the gene loci organization and the transcriptional regulation of all five dps genes in N. punctiforme was revealed, and dps-deletion mutant strains were used in physiological characterization. Two mutants defective in Dps2 and Dps5 activity displayed a reduced fitness under increased illumination, as well as a differential Photosystem (PS) stoichiometry, with an elevated Photosystem II to Photosystem I ratio in the dps5 deletion strain. This work establishes a Dps-mediated link between light tolerance, H2O2 detoxification, and iron homeostasis, and provides further evidence on the non-redundant role of multiple Dps proteins in this multicellular cyanobacterium.

Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments.

BACKGROUND: In the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N2 fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N2-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts. RESULTS: Here we report the first cell-type specific proteome of newly formed heterocysts, compared to N2-fixing filaments, using the cysteine-specific selective ICAT methodology. The data set defined a good quantitative accuracy of the ICAT reagent in complex protein samples. The relative abundance levels of 511 proteins were determined and 74% showed a cell-type specific differential abundance. The majority of the identified proteins have not previously been quantified at the cell-type specific level. We have in addition analyzed the cell-type specific differential abundance of a large section of proteins quantified in both newly formed and steady-state diazotrophic cultures in N. punctiforme. The results describe a wide distribution of members of the putative redox regulated Cys-proteome in the central metabolism of both vegetative cells and heterocysts of N. punctiforme. CONCLUSIONS: The data set broadens our understanding of heterocysts and describes novel proteins involved in heterocyst physiology, including signaling and regulatory proteins as well as a large number of proteins with unknown function. Significant differences in cell-type specific abundance levels were present in the cell-type specific proteomes of newly formed diazotrophic filaments as compared to steady-state cultures. Therefore we conclude that by using our approach we are able to analyze a synchronized fraction of newly formed heterocysts, which enabled a better detection of proteins involved in the heterocyst specific physiology.

Cellular and functional specificity among ferritin-like proteins in the multicellular cyanobacterium Nostoc punctiforme.

Ferritin-like proteins constitute a remarkably heterogeneous protein family, including ferritins, bacterioferritins and Dps proteins. The genome of the filamentous heterocyst-forming cyanobacterium Nostoc punctiforme encodes five ferritin-like proteins. In the present paper, we report a multidimensional characterization of these proteins. Our phylogenetic and bioinformatics analyses suggest both structural and physiological differences among the ferritin-like proteins. The expression of these five genes responded differently to hydrogen peroxide treatment, with a significantly higher rise in transcript level for Npun_F3730 as compared with the other four genes. A specific role for Npun_F3730 in the cells tolerance against hydrogen peroxide was also supported by the inactivation of Npun_F3730, Npun_R5701 and Npun_R6212; among these, only the ΔNpun_F3730 strain showed an increased sensitivity to hydrogen peroxide compared with wild type. Analysis of promoter-GFP reporter fusions of the ferritin-like genes indicated that Npun_F3730 and Npun_R5701 were expressed in all cell types of a diazotrophic culture, while Npun_F6212 was expressed specifically in heterocysts. Our study provides the first comprehensive analysis combining functional differentiation and cellular specificity within this important group of proteins in a multicellular cyanobacterium.

A high constitutive catalase activity confers resistance to methyl viologen-promoted oxidative stress in a mutant of the cyanobacterium Nostoc punctiforme ATCC 29133.

A spontaneous methyl viologen (MV)-resistant mutant of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133 was isolated and the major enzymatic antioxidants involved in combating MV-induced oxidative stress were evaluated. The mutant displayed a high constitutive catalase activity as a consequence of which, the intracellular level of reactive oxygen species in the mutant was lower than the wild type (N. punctiforme) in the presence of MV. The superoxide dismutase (SOD) activity that consisted of a SodA (manganese-SOD) and a SodB (iron-SOD) was not suppressed in the mutant following MV treatment. The mutant was, however, characterised by a lower peroxidase activity compared with its wild type, and its improved tolerance to externally added H2O2 could only be attributed to enhanced catalase activity. Furthermore, MV-induced toxic effects on the wild type such as (1) loss of photosynthetic performance assessed as maximal quantum yield of photosystem II, (2) nitrogenase inactivation, and (3) filament fragmentation and cell lysis were not observed in the mutant. These findings highlight the importance of catalase in preventing MV-promoted oxidative damage and cell death in the cyanobacterium N. punctiforme. Such oxidative stress resistant mutants of cyanobacteria are likely to be a better source of biofertilisers, as they can grow and fix nitrogen in an unhindered manner in agricultural fields that are often contaminated with the herbicide MV, also commonly known as paraquat.

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.

Modeling Photosystem I with the alternative reaction center protein PsaB2 in the nitrogen fixing cyanobacterium Nostoc punctiforme.

Five nitrogen fixing cyanobacterial strains have been found to contain PsaB2, an additional and divergent gene copy for the Photosystem I reaction center protein PsaB. In all five species the divergent gene, psaB2, is located separately from the normal psaAB operon in the genome. The protein, PsaB2, was recently identified in heterocysts of Nostoc punctiforme sp. strain PCC 73102. 12 conserved amino acid replacements and one insertion, were identified by a multiple sequence alignment of several PsaB2 and PsaB1 sequences. Several, including an inserted glutamine, are located close to the iron-sulfur cluster F(X) in the electron transfer chain. By homology modeling, using the Photosystem I crystal structure as template, we have found that the amino acid composition in PsaB2 will introduce changes in critical parts of the Photosystem I protein structure. The changes are close to F(X) and the phylloquinone (PhQ) in the B-branch, indicating that the electron transfer properties most likely will be affected. We suggest that the divergent PsaB2 protein produces an alternative Photosystem I reaction center with different structural and electron transfer properties. Some interesting physiologcial consequences that this can have for the function of Photosystem I in heterocysts, are discussed.

HupW protease specifically required for processing of the catalytic subunit of the uptake hydrogenase in the cyanobacterium Nostoc sp. strain PCC 7120.

The maturation process of [NiFe] hydrogenases includes a proteolytic cleavage of the large subunit. We constructed a mutant of Nostoc strain PCC 7120 in which hupW, encoding a putative hydrogenase-specific protease, is inactivated. Our results indicate that the protein product of hupW selectively cleaves the uptake hydrogenase in this cyanobacterium.

Design, engineering, and construction of photosynthetic microbial cell factories for renewable solar fuel production.

There is an urgent need to develop sustainable solutions to convert solar energy into energy carriers used in the society. In addition to solar cells generating electricity, there are several options to generate solar fuels. This paper outlines and discusses the design and engineering of photosynthetic microbial systems for the generation of renewable solar fuels, with a focus on cyanobacteria. Cyanobacteria are prokaryotic microorganisms with the same type of photosynthesis as higher plants. Native and engineered cyanobacteria have been used by us and others as model systems to examine, demonstrate, and develop photobiological H(2) production. More recently, the production of carbon-containing solar fuels like ethanol, butanol, and isoprene have been demonstrated. We are using a synthetic biology approach to develop efficient photosynthetic microbial cell factories for direct generation of biofuels from solar energy. Present progress and advances in the design, engineering, and construction of such cyanobacterial cells for the generation of a portfolio of solar fuels, e.g., hydrogen, alcohols, and isoprene, are presented and discussed. Possibilities and challenges when introducing and using synthetic biology are highlighted.

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.

Metabolic adaptations in a H2 producing heterocyst-forming cyanobacterium: potentials and implications for biological engineering.

Nostoc punctiforme ATCC 29133 is a photoautotrophic cyanobacterium with the ability to fix atmospheric nitrogen and photoproduce hydrogen through the enzyme nitrogenase. The H(2) produced is reoxidized by an uptake hydrogenase. Inactivation of the uptake hydrogenase in N. punctiforme leads to increased H(2) release but unchanged rates of N(2) fixation, indicating redirected metabolism. System-wide understanding of the mechanisms of this metabolic redirection was obtained using complementary quantitative proteomic approaches, at both the filament and the heterocyst level. Of the total 1070 identified and quantified proteins, 239 were differentially expressed in the uptake hydrogenase mutant (NHM5) as compared to wild type. Our results indicate that the inactivation of uptake hydrogenase in N. punctiforme changes the overall metabolic equilibrium, affecting both oxygen reduction mechanisms in heterocysts as well as processes providing reducing equivalents for metabolic functions such as N(2) fixation. We identify specific metabolic processes used by NHM5 to maintain a high rate of N(2) fixation, and thereby potential targets for further improvement of nitrogenase based H(2) photogeneration. These targets include, but are not limited to, components of the oxygen scavenging capacity and cell envelope of heterocysts and proteins directly or indirectly involved in reduced carbon transport from vegetative cells to heterocysts.

Isobutene production in Synechocystis sp. PCC 6803 by introducing α-ketoisocaproate dioxygenase from Rattus norvegicus.

Cyanobacteria can be utilized as a platform for direct phototrophic conversion of CO2 to produce several types of carbon-neutral biofuels. One promising compound to be produced photobiologically in cyanobacteria is isobutene. As a volatile compound, isobutene will quickly escape the cells without building up to toxic levels in growth medium or get caught in the membranes. Unlike liquid biofuels, gaseous isobutene may be collected from the headspace and thus avoid the costly extraction of a chemical from culture medium or from cells. Here we investigate a putative synthetic pathway for isobutene production suitable for a photoautotrophic host. First, we expressed α-ketoisocaproate dioxygenase from Rattus norvegicus (RnKICD) in Escherichia coli. We discovered isobutene formation with the purified RnKICD with the rate of 104.6 â€‹± â€‹9 â€‹ng (mg protein)-1 min-1 using α-ketoisocaproate as a substrate. We further demonstrate isobutene production in the cyanobacterium Synechocystis sp. PCC 6803 by introducing the RnKICD enzyme. Synechocystis strain heterologously expressing the RnKICD produced 91 â€‹ng â€‹l-1 OD750 -1 â€‹h-1. Thus, we demonstrate a novel sustainable platform for cyanobacterial production of an important building block chemical, isobutene. These results indicate that RnKICD can be used to further optimize the synthetic isobutene pathway by protein and metabolic engineering efforts.

Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme.

Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes. We could identify a significant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled Photosystem II complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.

The CyAbrB transcription factor CalA regulates the iron superoxide dismutase in Nostoc sp. strain PCC 7120.

In the present investigation the results of induced over-production of the CyAbrB transcription factor CalA (Cyanobacterial AbrB-like, annotated as Alr0946) in the cyanobacterium Nostoc sp. PCC 7120 were analysed. The CalA overexpression strain showed a bleaching phenotype with lower growth rate and truncated filaments 2 days after induction of overexpression. The phenotype was even more pronounced when illumination was increased from 35 to 125 µmol m(-2) s(-1). Using gel-based quantitative proteomics, the induced overexpression of CalA was shown to downregulate the abundance of FeSOD, one of two types of superoxide dismutases in Nostoc sp. PCC 7120. The change in protein abundance was also accompanied by lower transcript as well as activity levels. Purified recombinant CalA from Nostoc sp. PCC 7120 was shown to interact with the promoter region of alr2938, encoding FeSOD, indicating a transcriptional regulation of FeSOD by CalA. The bleaching phenotype is in line with a decreased tolerance against oxidative stress and indicates that CalA is involved in regulation of cellular responses in which FeSOD has an important and specific function in the filamentous cyanobacterium Nostoc sp. PCC 7120.