Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering for biotechnology. Here we apply limited proteolysis-small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions shows that some metabolites interact in a species-specific manner. We estimate that approximately 35% of interacting metabolites affect enzyme activity in vitro, and the effect is often minor. Using LiP-SMap data as a guide, we find that the Calvin cycle intermediate glyceraldehyde-3-phosphate enhances activity of fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) from Synechocystis sp. PCC 6803 and Cupriavidus necator in reducing conditions, suggesting a convergent feed-forward activation of the cycle. In oxidizing conditions, glyceraldehyde-3-phosphate inhibits Synechocystis F/SBPase by promoting enzyme aggregation. In contrast, the glycolytic intermediate glucose-6-phosphate activates F/SBPase from Cupriavidus necator but not F/SBPase from Synechocystis. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated.

Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering to improve industrial phenotypes. Recently developed chemoproteomics workflows allow for genome-wide detection of metabolite-protein interactions that may regulate pathway activity. We applied limited proteolysis small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions showed that some metabolites interacted in a species-specific manner, such as interactions of glucose-6-phosphate in Cupriavidus necator and of glyoxylate in Synechocystis sp PCC 6803. These are interpreted in light of the different central carbon conversion pathways present. Metabolites interacting with the Calvin cycle enzymes fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) and transketolase were tested for effects on catalytic activity in vitro. The Calvin cycle intermediate glyceraldehyde-3-phosphate activated both Synechocystis and Cupriavidus F/SBPase, which suggests a feed-forward activation of the cycle in both photoautotrophs and chemolithoautotrophs. In contrast to the stimulating effect in reduced conditions, glyceraldehyde-3-phosphate inactivated the Synechocystis F/SBPase in oxidized conditions by accelerating protein aggregation. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated and may act in addition to redox regulation.

The chemolithotroph Cupriavidus necator H16 is known as a natural producer of the bioplastic-polymer PHB, as well as for its metabolic versatility to utilize different substrates, including formate as the sole carbon and energy source. Depending on the entry point of the substrate, this versatility requires adjustment of the thermodynamic landscape to maintain sufficiently high driving forces for biological processes. Here we employed a model of the core metabolism of C. necator H16 to analyze the thermodynamic driving forces and PHB yields from formate for different metabolic engineering strategies. For this, we enumerated elementary flux modes (EFMs) of the network and evaluated their PHB yields as well as thermodynamics via Max-min driving force (MDF) analysis and random sampling of driving forces. A heterologous ATP:citrate lyase reaction was predicted to increase driving force for producing acetyl-CoA. A heterologous phosphoketolase reaction was predicted to increase maximal PHB yields as well as driving forces. These enzymes were then verified experimentally to enhance PHB titers between 60 and 300% in select conditions. The EFM analysis also revealed that PHB production from formate may be limited by low driving forces through citrate lyase and aconitase, as well as cofactor balancing, and identified additional reactions associated with low and high PHB yield. Proteomics analysis of the engineered strains confirmed an increased abundance of aconitase and cofactor balancing. The findings of this study aid in understanding metabolic adaptation. Furthermore, the outlined approach will be useful in designing metabolic engineering strategies in other non-model bacteria.

Earth has entered a new geological epoch, the Anthropocene, defined by humanity’s impact on the environment with increased emissions of CO2 due to burning of fossil resource as a major contributor. To ensure a sustainable future, humanity has to move towards a circular economy, where released CO2 is re-captured and turned into resources. Biological CO2 fixation performed by autotrophic microorganisms using renewable energy can thereby play an important role, but requires improvement in capacity and efficiency. To enable targeted improvements, computational methods in systems biology and metabolic engineering were used in this thesis to identify thermodynamic and kinetic constraints of autotrophic microorganisms using the Calvin cycle as their primary CO2 fixation pathway. In Paper I, the different metabolic networks of the photoautotrophic cyanobacterium Synechocystis and the heterotrophic E. coli were compared, revealing network- specific intracellular metabolite concentration ranges and thermodynamic driving forces, causing different capabilities for production of industrially relevant chemicals. For Paper II, a kinetic metabolic model of the Calvin cycle in Synechocystis was developed and analyzed, exposing factors favoring a stable operation, such as a low concentration of Ribulose 1,5-phosphate or low saturation states of many enzymes towards their substrates. It furthermore revealed that control over the reaction rates in the Calvin cycle was distributed, but the CO2 fixation rate could be increased by higher rates through enzymes such as fructose 1,6-bisphosphatase or phosphoglycerate kinase. In Paper III, experimentally determined interactions between metabolites and proteins in several autotrophic microorganisms were tested for their regulatory functions. For Synechocystis, these interactions were interpreted in the metabolic context by integrating them in an expanded kinetic model, revealing significant shifts in metabolome stability when biochemical regulation was added to transketolase, an enzyme central to the Calvin cycle, but only minor effects on flux control. Lastly, for Paper IV the thermodynamic landscape of Cupriavidus necator and its natural capacity of producing the bioplastic PHB were evaluated. Different substrate utilization scenarios and metabolic engineering strategies were simulated using a metabolic model, revealing substrate-independent thermodynamic constraints and contrasting effects of the engineering efforts. This work provides the knowledge for further studies and targeted engineering efforts aiming to alleviate constraints on autotrophic metabolism to improve its performance in transforming CO2 into usable resources.

Jorden har gått in i en ny geologisk epok, Antropocenen, definierad av mänsklighetens påverkan på miljön. Förbränningen av fossila resurser och det resulterande utsläppet av koldioxid driver klimatförändringar, en av mänsklighetens största utmaningar någonsin. För att säkerställa en hållbar framtid måste mänskligheten sträva till en cirkulär ekonomi, där utsläppt koldioxid återfångas och återförvandlas till resurser. Biologisk koldioxid-fixering, utförd av autotrofa mikroorganismer med förnybar energi, kan spela en viktig roll i denna processen, men kräver förbättring av kapacitet och effektivitet av den autotrofa metabolismen. Beräkningsmetoder inom systembiologi har använts i denna avhandling för att identifiera termodynamiska och kinetiska begränsningar för autotrofa mikroorganismer med calvincykeln som deras primära koldioxid-fixeringsväg, som kan bidra att möjliggöra konkreta förbättringar av metabolismen. Först jämfördes de olika metaboliska nätverken för den fotoautotrofa cyanobakterien Synechocystis och den heterotrofa bakterien E. coli, som resulterade i nätverksspecifika metabolitkoncentrationer och termodynamiska drivkrafter, vilket orsakade olika kapacitet för produktionen av industriellt relevanta kemikalier. För det andra utvecklades och analyserades en kinetisk metabolisk modell av calvincykeln i Synechocystis, som avslöjade faktorer som möjliggör en stabil funktion av metabolismen, såsom en låg koncentration av Ribulose 1,5-fosfat eller låga enzymsatureringsnivåer för många enzymer för deras substrat. Det visades dessutom att kontrollen över reaktionshastigheterna i calvincykeln var distribuerad, men CO2- fixeringshastigheten kan ökas med högre hastigheter genom enzymer som fruktos 1,6-bisfosfatas eller fosfoglyceratkinas. För det tredje testades experimentellt bestämda interaktioner mellan metaboliter och proteiner i flera autotrofa mikroorganismer för deras reglerande funktioner. För Synechocystis tolkades dessa interaktioner i det metaboliska sammanhanget genom att integrera dem i en utökad kinetisk modell, vilket avslöjade betydande förändringar i metabolomstabilitet när biokemisk reglering tillsattes till transketolas, ett enzym som är centralt för clavincykeln, men endast mindre effekter på kontroll över reaktionshastigheterna. Slutligen utvärderades för det fjärde det termodynamiska landskapet i Cupriavidus necator och dess naturliga kapacitet att producera bioplasten PHB. Olika substratanvändningsscenarier och strategier för att modifiera metabolismen simulerades med hjälp av en metabolisk modell, vilket avslöjade substratoberoende termodynamiska begränsningar och kontrasterande effekter av de modifierarna. Denna avhandling ger kunskap för ytterligare studier och riktade modifierar av metabolismen som syftar till att lindra begränsningar för den autotrofa metabolismen för att förbättra dess prestanda vid omvandling av CO2 till användbara resurser.

Der Planet Erde ist in eine neue geologische Epoche eingetreten, das Anthropozän, definiert durch die Auswirkungen der Menschheit auf die Umwelt. Der Anstieg der CO2-Konzentration in der Atmosphäre, versursacht durch die Verbrennung fossiler Ressourcen, ist hauptverantwortlich für die Klimakrise. Um eine nachhaltige Zukunft zu gewährleisten ist eine zirkuläre Wirtschaft essenziell, in der freigesetztes CO2 wieder aufgefangen und in Ressourcen umgewandelt wird. Die biologische Aufnahme von CO2 durch autotrophe Mikroorganismen kann dabei eine wichtige Rolle spielen, erfordert aber eine Verbesserung der Kapazität und Effizienz des Stoffwechsels. In der vorliegenden Arbeit wurden computergestützte Methoden der Systembiologie verwendet um thermodynamische und kinetische Limitationen autotropher Mikroorganismen zu identifizieren, die den Calvin-Zyklus als primären CO2-Fixierungsweg verwenden. Mit diesen Informationen können Strategien entwickelt werden um diese Limitationen gezielt aufzuheben oder abzuschwächen. Dazu wurden zuerst die unterschiedlichen metabolischen Netzwerke des photoautotrophen Cyanobakteriums Synechocystis und des heterotrophen Bakteriums E. coli verglichen, wobei netzwerkspezifische Metabolitkonzentrationen und thermodynamische Triebkräfte aufgezeigt wurden. Daraus folgte die unterschiedliche Fähigkeiten zur Produktion industriell relevanter Chemikalien. Als zweites wurde ein kinetisches Modell des Calvin-Zyklus für Synechocystis entwickelt und analysiert. Faktoren für eine robuste Funktion des Stoffwechsels wurden aufgezeigt, wie zum Beispiel eine niedrige Konzentration von Ribulose 1,5-Phosphatoder niedrige Sättigungszustände vieler Enzyme. Es zeigte sich außerdem, dass die Kontrolle über die Reaktionsgeschwindigkeiten im Calvin-Zyklus auf mehrere Reaktionen verteilt war, die CO2-Fixierungsgeschwindigkeit jedoch durch höhere Reaktionsgeschwindigkeiten durch Enzyme wie Fructose-1,6-Bisphosphatase erhöht werden konnte. Als drittes wurden experimentell ermittelte Interaktionen zwischen Metaboliten und Proteinen in mehreren autotrophen Mikroorganismen auf ihre regulatorischen Funktionen getestet. Für Synechocystis wurden diese Interaktionen in ihrem metabolischen Kontext interpretiert, indem sie in ein erweitertes kinetisches Modell integriert wurden. Es wurden signifikante Verschiebungen in der Metabolomstabilität aufgezeigt, wenn die biochemische Regulation zu Transketolase, einem zentralen Enzym des Calvin-Zyklus, hinzugefügt wurde. Auswirkungen auf die Kontrolle der Reaktionsgeschwindigkeiten waren nur gering. Viertens wurde die Verteilung von thermodynamische Triebkräften in Cupriavidus necator und dessen natürliche Fähigkeit zur Produktion des Biokunststoffs PHB evaluiert. Unter Verwendung eines Computermodells wurden verschiedene Substratnutzungsszenarien und Interventionsstrategien simuliert, wodurch substratunabhängige thermodynamische Limitationen und gegensätzliche Auswirkungen der Interventionsstrategien identifiziert wurden. Diese Arbeit liefert die Grundlage für weitere Studien und gezielte technische Bemühungen mit dem Ziel die Grenzen des autotrophen Stoffwechsels zu erweitern und um die Umwandlung von CO2 in nutzbare Ressourcen zu verbessern.

Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile 'knallgas' bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO2-reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy.

Biological fixation of atmospheric CO2 via the Calvin-Benson-Bassham cycle has massive ecological impact and offers potential for industrial exploitation, either by improving carbon fixation in plants and autotrophic bacteria, or by installation into new hosts. A kinetic model of the Calvin-Benson-Bassham cycle embedded in the central carbon metabolism of the cyanobacterium Synechocystis sp. PCC 6803 was developed to investigate its stability and underlying control mechanisms. To reduce the uncertainty associated with a single parameter set, random sampling of the steady-state metabolite concentrations and the enzyme kinetic parameters was employed, resulting in millions of parameterized models which were analyzed for flux control and stability against perturbation. Our results show that the Calvin cycle had an overall high intrinsic stability, but a high concentration of ribulose 1,5-bisphosphate was associated with unstable states. Low substrate saturation and high product saturation of enzymes involved in highly interconnected reactions correlated with increased network stability. Flux control, that is the effect that a change in one reaction rate has on the other reactions in the network, was distributed and mostly exerted by energy supply (ATP), but also by cofactor supply (NADPH). Sedoheptulose 1,7-bisphosphatase/fructose 1,6-bisphosphatase, fructose-bisphosphate aldolase, and transketolase had a weak but positive effect on overall network flux, in agreement with published observations. The identified flux control and relationships between metabolite concentrations and system stability can guide metabolic engineering. The kinetic model structure and parameterizing framework can be expanded for analysis of metabolic systems beyond the Calvin cycle.

Introducing biosynthetic pathways into an organism is both reliant on and challenged by endogenous biochemistry. Here we compared the expansion potential of the metabolic network in the photoautotroph Synechocystis with that of the heterotroph E. coli using the novel workflow POPPY (Prospecting Optimal Pathways with PYthon). First, E. coli and Synechocystis metabolomic and fluxomic data were combined with metabolic models to identify thermodynamic constraints on metabolite concentrations (NET analysis). Then, thousands of automatically constructed pathways were placed within each network and subjected to a network-embedded variant of the max-min driving force analysis (NEM). We found that the networks had different capabilities for imparting thermodynamic driving forces toward certain compounds. Key metabolites were constrained differently in Synechocystis due to opposing flux directions in glycolysis and carbon fixation, the forked tri-carboxylic acid cycle, and photorespiration. Furthermore, the lysine biosynthesis pathway in Synechocystis was identified as thermodynamically constrained, impacting both endogenous and heterologous reactions through low 2-oxoglutarate levels. Our study also identified important yet poorly covered areas in existing metabolomics data and provides a reference for future thermodynamics-based engineering in Synechocystis and beyond. The POPPY methodology represents a step in making optimal pathway-host matches, which is likely to become important as the practical range of host organisms is diversified. 

Metabolite-level regulation of enzyme activity is important for coping with environmental shifts. Recently developed proteomics methodologies allow for mapping of post-translational interactions, including metabolite-protein interactions, that may be relevant for quickly regulating pathway activity. While feedback and feedforward regulation in glycolysis has been investigated, there is relatively little study of metabolite-level regulation in the Calvin cycle, particularly in bacteria. Here, we applied limited proteolysis small molecule mapping (LiP-SMap) to identify metabolite-protein interactions in four Calvin-cycle harboring bacteria, including two cyanobacteria and two chemolithoautotrophs. We identified widespread protein interactions with the metabolites GAP, ATP, and AcCoA in all strains. Some species-specific interactions were also observed, such as sugar phosphates in Cupravidus necator and glyoxylate in Synechocystis sp. PCC 6803. We screened some metabolites with LiP interactions for their effects on kinetics of the enzymes F/SBPase and transketolase, two enzymatic steps of the Calvin cycle. For both Synechocystis and Cupriavidus F/SBPase, GAP showed an activating effect that may be part of feed-forward regulation in the Calvin cycle. While we verified multiple enzyme inhibitors on transketolase, the effect on kinetics was often small. Incorporation of F/SBPase and transketolase regulations into a kinetic metabolic model of Synechocystis central metabolism resulted in a general decreased stability of the network, and altered flux control coefficients of transketolase as well as other reactions. The LiP-SMap methodology is promising for uncovering new modes of metabolic regulation, but will benefit from improved peptide quantification and higher peptide coverage of enzymes, as known interactions are often not detected for low-coverage proteins. . Furthermore, not all LiP interactions appear to be relevant for catalysis, as 4/8 (transketolase) and 5/6 (F/SBPase) of the tested LiP effectors had an effect in in vitroassays.