Metabolic fluxes and their control mechanisms are fundamental in cellular metabolism, offering in... more 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 cyanobacteriumSynechocystissp. 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 from 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 carbox...
bioRxiv (Cold Spring Harbor Laboratory), Sep 17, 2022
Cyanobacteria represent an attractive platform for the sustainable production of chemicals and fu... more Cyanobacteria represent an attractive platform for the sustainable production of chemicals and fuels. However, the obtained rates, yields, and titers are below those required for commercial application. Carbon metabolism alone cannot achieve maximal accumulation of end-products, since an efficient production of target molecules entails energy and redox balance, in addition to carbon flow. The interplay between cofactor regeneration and heterologous metabolite overproduction in cyanobacteria is not fully explored. Here, we applied stoichiometric metabolic modelling of the cyanobacterium Synechocystis sp. PCC 6803, in order to investigate the optimality of energy and redox metabolism, while overproducing bio-alkenes-isobutene, isoprene, ethylene and 1-undecene. Our network-wide analysis indicates that the rate of NADP+ reduction, rather than ATP synthesis, controls ATP/NADPH ratio, and thereby chemical production. The simulation implies that energy and redox balance necessitates gluconeogenesis, and that acetate metabolism via phosphoketolase serves as an efficient carbon-and energy-recycling pathway. Furthermore, we show that an auxiliary pathway, composed of serine, one-carbon and glycine metabolism, supports cellular redox homeostasis and ATP cycling, and that the Synechocystis metabolism is controlled by few key reactions carrying a high flux. The study also revealed non-intuitive metabolic pathways to enhance isoprene, ethylene and 1-undecene production. We conclude that metabolism of ATP and NAD(P)H is entwined with carbon and nitrogen metabolism, and cannot be assessed in isolation. We envision that the presented here in-depth metabolic analysis will guide the a priori design of Synechocystis as a host strain for an efficient manufacturing of target products.
Sustainable production of biofuels and other valuable compounds is one of our future challenges. ... more 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 yet 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.
Nostoc sp. PCC 7120 is an oxygen-evolving photoautotrophic N 2 fixing filamentous cyanobacterium.... more Nostoc sp. PCC 7120 is an oxygen-evolving photoautotrophic N 2 fixing filamentous cyanobacterium. Upon nitrogen starvation, a range of processes are initiated, such as differentiation of the heterocysts, specific cells where N 2 fixation takes place. We have characterized and quantified the proteome of the Nostoc sp. PCC 7120 wild-type strain grown under N 2 fixing and non-N 2 fixing conditions. To assess global proteome changes in response to environmental changes, measurements were made using the quantitative proteomics tool, iTRAQ, on a whole cell digest. From this approach, a total of 486 different proteins was accurately identified across 2 biological replicate experiments, where 226 identifications contained 2 or more distinct peptides. Results of metabolic regulation will be discussed to demonstrate that proteomics represents an important tool for the development of heterocystous cyanobacteria for future biological H 2 production.
Cyanobacteria represent an attractive platform for the sustainable production of chemicals and fu... more Cyanobacteria represent an attractive platform for the sustainable production of chemicals and fuels. However, the obtained rates, yields, and titers are below those required for commercial application. Carbon metabolism alone cannot achieve maximal accumulation of end-products, since an efficient production of target molecules entails energy and redox balance, in addition to carbon flow. The interplay between cofactor regeneration and heterologous metabolite overproduction in cyanobacteria is not fully explored. Here, we applied stoichiometric metabolic modelling of the cyanobacterium Synechocystis sp. PCC 6803, in order to investigate the optimality of energy and redox metabolism, while overproducing bio-alkenes - isobutene, isoprene, ethylene and 1-undecene. Our network-wide analysis indicates that the rate of NADP+ reduction, rather than ATP synthesis, controls ATP/NADPH ratio, and thereby chemical production. The simulation implies that energy and redox balance necessitates glu...
Photoautotrophic cyanobacteria often confront hydrogen peroxide (H2O2), a reactive oxygen species... more 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. Nostoc 29133 was better able to tolerate H2O2-induced inhibition of chlorophyll a and photosystem II performance, as compared to Anabaena 7120. The intracellular hydroperoxide level (indicator of oxidative status) also did not exhibit as much a rise in Nostoc 29133, as it did in Anabaena 7120 after H2O2 treatment. Accordingly, Nostoc 29133 showed higher intrinsic constitutive catalase activity than Anabaena 7120 indicating that the superior tolerance of Nostoc 29133 stems from its higher ability to decompose H2O2. It is suggested that difference in H2O2 tolerance between closely related filamentous cyanobacteria, as is borne out by this study...
The hemiterpene isoprene is a volatile C5 hydrocarbon, with industrial applications. It is genera... more The hemiterpene isoprene is a volatile C5 hydrocarbon, with industrial applications. It is generated today from fossil resources, but can also be made in biological processes. We have utilized engineered photosynthetic cyanobacteria for direct, light-driven production of bio-isoprene from carbon dioxide, and show that isoprene in a subsequent photochemical step, using simulated or natural solar light, can be dimerized into limonene, paradiprene, and isomeric C10H16 hydrocarbons (monoterpenes) in very high yields (above 90% after 44 hours) under sensitized conditions. The optimal sensitizer in our experiments is di(naphth-1-yl)methanone which we can use with a loading of merely 0.1 mol%, and it is easily recycled for subsequent photodimerization cycles. The isoprene dimers generated are a mixture of [2+2], [4+2] and [4+4] cycloadducts, and after hydrogenation this mixture is nearly ideal as a jet fuel drop-in. Importantly, the photodimerization can be carried out at ambient condition...
Photosynthetic production of molecular hydrogen (H2) by cyanobacteria and green algae is a potent... more 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.
Cyanobacteria are photosynthetic prokaryotes that are considered biotechnologically prominent org... more 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) sto...
Iron limitation as a growth-restricted cultivation strategy in the diazotrophic cyanobacterium No... more Iron limitation as a growth-restricted cultivation strategy in the diazotrophic cyanobacterium Nostoc PCC 7120 ΔhupW for a new perspective on H2 production systems
An 8-plex iTRAQ Shotgun Proteomics Investigation of N2-fixing Heterocysts in the Cyanobacterium N... more An 8-plex iTRAQ Shotgun Proteomics Investigation of N2-fixing Heterocysts in the Cyanobacterium Nostoc sp. PCC 7120
Ethylene is a volatile hydrocarbon with a massive global market in the plastic industry. The ethy... more 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 th...
The current economic and environmental context requests an accelerating development of sustainabl... more 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 ...
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2019
Highlights The dodecamer stability of the two atypical NpDps are pH dependent. Both NpDps4 an... more Highlights The dodecamer stability of the two atypical NpDps are pH dependent. Both NpDps4 and NpDps5 show DNA binding properties. Both NpDps4 and NpDps5 show binding properties upon interaction with NpFdx proteins. NpDps5, with structural similarities to bacterioferritins, is also related to Dps proteins, due to its dodecameric structure and DNA binding ability.
Metabolic fluxes and their control mechanisms are fundamental in cellular metabolism, offering in... more 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 cyanobacteriumSynechocystissp. 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 from 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 carbox...
bioRxiv (Cold Spring Harbor Laboratory), Sep 17, 2022
Cyanobacteria represent an attractive platform for the sustainable production of chemicals and fu... more Cyanobacteria represent an attractive platform for the sustainable production of chemicals and fuels. However, the obtained rates, yields, and titers are below those required for commercial application. Carbon metabolism alone cannot achieve maximal accumulation of end-products, since an efficient production of target molecules entails energy and redox balance, in addition to carbon flow. The interplay between cofactor regeneration and heterologous metabolite overproduction in cyanobacteria is not fully explored. Here, we applied stoichiometric metabolic modelling of the cyanobacterium Synechocystis sp. PCC 6803, in order to investigate the optimality of energy and redox metabolism, while overproducing bio-alkenes-isobutene, isoprene, ethylene and 1-undecene. Our network-wide analysis indicates that the rate of NADP+ reduction, rather than ATP synthesis, controls ATP/NADPH ratio, and thereby chemical production. The simulation implies that energy and redox balance necessitates gluconeogenesis, and that acetate metabolism via phosphoketolase serves as an efficient carbon-and energy-recycling pathway. Furthermore, we show that an auxiliary pathway, composed of serine, one-carbon and glycine metabolism, supports cellular redox homeostasis and ATP cycling, and that the Synechocystis metabolism is controlled by few key reactions carrying a high flux. The study also revealed non-intuitive metabolic pathways to enhance isoprene, ethylene and 1-undecene production. We conclude that metabolism of ATP and NAD(P)H is entwined with carbon and nitrogen metabolism, and cannot be assessed in isolation. We envision that the presented here in-depth metabolic analysis will guide the a priori design of Synechocystis as a host strain for an efficient manufacturing of target products.
Sustainable production of biofuels and other valuable compounds is one of our future challenges. ... more 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 yet 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.
Nostoc sp. PCC 7120 is an oxygen-evolving photoautotrophic N 2 fixing filamentous cyanobacterium.... more Nostoc sp. PCC 7120 is an oxygen-evolving photoautotrophic N 2 fixing filamentous cyanobacterium. Upon nitrogen starvation, a range of processes are initiated, such as differentiation of the heterocysts, specific cells where N 2 fixation takes place. We have characterized and quantified the proteome of the Nostoc sp. PCC 7120 wild-type strain grown under N 2 fixing and non-N 2 fixing conditions. To assess global proteome changes in response to environmental changes, measurements were made using the quantitative proteomics tool, iTRAQ, on a whole cell digest. From this approach, a total of 486 different proteins was accurately identified across 2 biological replicate experiments, where 226 identifications contained 2 or more distinct peptides. Results of metabolic regulation will be discussed to demonstrate that proteomics represents an important tool for the development of heterocystous cyanobacteria for future biological H 2 production.
Cyanobacteria represent an attractive platform for the sustainable production of chemicals and fu... more Cyanobacteria represent an attractive platform for the sustainable production of chemicals and fuels. However, the obtained rates, yields, and titers are below those required for commercial application. Carbon metabolism alone cannot achieve maximal accumulation of end-products, since an efficient production of target molecules entails energy and redox balance, in addition to carbon flow. The interplay between cofactor regeneration and heterologous metabolite overproduction in cyanobacteria is not fully explored. Here, we applied stoichiometric metabolic modelling of the cyanobacterium Synechocystis sp. PCC 6803, in order to investigate the optimality of energy and redox metabolism, while overproducing bio-alkenes - isobutene, isoprene, ethylene and 1-undecene. Our network-wide analysis indicates that the rate of NADP+ reduction, rather than ATP synthesis, controls ATP/NADPH ratio, and thereby chemical production. The simulation implies that energy and redox balance necessitates glu...
Photoautotrophic cyanobacteria often confront hydrogen peroxide (H2O2), a reactive oxygen species... more 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. Nostoc 29133 was better able to tolerate H2O2-induced inhibition of chlorophyll a and photosystem II performance, as compared to Anabaena 7120. The intracellular hydroperoxide level (indicator of oxidative status) also did not exhibit as much a rise in Nostoc 29133, as it did in Anabaena 7120 after H2O2 treatment. Accordingly, Nostoc 29133 showed higher intrinsic constitutive catalase activity than Anabaena 7120 indicating that the superior tolerance of Nostoc 29133 stems from its higher ability to decompose H2O2. It is suggested that difference in H2O2 tolerance between closely related filamentous cyanobacteria, as is borne out by this study...
The hemiterpene isoprene is a volatile C5 hydrocarbon, with industrial applications. It is genera... more The hemiterpene isoprene is a volatile C5 hydrocarbon, with industrial applications. It is generated today from fossil resources, but can also be made in biological processes. We have utilized engineered photosynthetic cyanobacteria for direct, light-driven production of bio-isoprene from carbon dioxide, and show that isoprene in a subsequent photochemical step, using simulated or natural solar light, can be dimerized into limonene, paradiprene, and isomeric C10H16 hydrocarbons (monoterpenes) in very high yields (above 90% after 44 hours) under sensitized conditions. The optimal sensitizer in our experiments is di(naphth-1-yl)methanone which we can use with a loading of merely 0.1 mol%, and it is easily recycled for subsequent photodimerization cycles. The isoprene dimers generated are a mixture of [2+2], [4+2] and [4+4] cycloadducts, and after hydrogenation this mixture is nearly ideal as a jet fuel drop-in. Importantly, the photodimerization can be carried out at ambient condition...
Photosynthetic production of molecular hydrogen (H2) by cyanobacteria and green algae is a potent... more 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.
Cyanobacteria are photosynthetic prokaryotes that are considered biotechnologically prominent org... more 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) sto...
Iron limitation as a growth-restricted cultivation strategy in the diazotrophic cyanobacterium No... more Iron limitation as a growth-restricted cultivation strategy in the diazotrophic cyanobacterium Nostoc PCC 7120 ΔhupW for a new perspective on H2 production systems
An 8-plex iTRAQ Shotgun Proteomics Investigation of N2-fixing Heterocysts in the Cyanobacterium N... more An 8-plex iTRAQ Shotgun Proteomics Investigation of N2-fixing Heterocysts in the Cyanobacterium Nostoc sp. PCC 7120
Ethylene is a volatile hydrocarbon with a massive global market in the plastic industry. The ethy... more 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 th...
The current economic and environmental context requests an accelerating development of sustainabl... more 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 ...
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2019
Highlights The dodecamer stability of the two atypical NpDps are pH dependent. Both NpDps4 an... more Highlights The dodecamer stability of the two atypical NpDps are pH dependent. Both NpDps4 and NpDps5 show DNA binding properties. Both NpDps4 and NpDps5 show binding properties upon interaction with NpFdx proteins. NpDps5, with structural similarities to bacterioferritins, is also related to Dps proteins, due to its dodecameric structure and DNA binding ability.
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Papers by Karin Stensjo