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.

Metabolically engineered cyanobacteria have the potential to mitigate anthropogenic CO2 emissions by converting CO2 into renewable fuels and chemicals. Yet, better understanding of metabolic regulation in cyanobacteria is required to develop more productive strains that can make industrial scale-up economically feasible. The aim of this study was to find the cause for the previously reported inconsistency between oscillating transcription and constant protein levels under day-night growth conditions. To determine whether translational regulation counteracts transcriptional changes, Synechocystis sp. PCC 6803 was cultivated in an artificial day-night setting and the level of transcription, translation and protein was measured across the genome at different time points using mRNA sequencing, ribosome profiling and quantitative proteomics. Furthermore, the effect of protein turnover on the amplitude of protein oscillations was investigated through in silico simulations using a protein mass balance model. Our experimental analysis revealed that protein oscillations were not dampened by translational regulation, as evidenced by high correlation between translational and transcriptional oscillations (r = 0.88) and unchanged protein levels. Instead, model simulations showed that these observations can be attributed to a slow protein turnover, which reduces the effect of protein synthesis oscillations on the protein level. In conclusion, these results suggest that cyanobacteria have evolved to govern diurnal metabolic shifts through allosteric regulatory mechanisms in order to avoid the energy burden of replacing the proteome on a daily basis. Identification and manipulation of such mechanisms could be part of a metabolic engineering strategy for overproduction of chemicals.

Earth's climate has been upset by carbon dioxide (CO₂) emissions from human activities and the use of fossil resources. To prevent catastrophic events on the environment and on civilizations, we urgently need to develop alternative solutions that utilize renewable resources. One contributing solution is to exploit metabolically engineered cyanobacteria that convert CO₂ and sunlight into bioproducts, such as fuels or biodegradable plastics. However, these ancient bacteria have evolved a complex, robust, and self-regulated metabolic network that efficiently converts CO₂ to biomass rather than a foreign chemical, which limits the productivity of engineered strains and the potential to use them in an industrial setting. Extended knowledge is therefore needed regarding the regulatory processes that govern their metabolism, in order to develop more efficient producer strains. The present investigation took advantage of recently developed systems biology techniques to explore translational and post-translational regulation in the model cyanobacterium Synechocystis. The first study showed that CO₂-starved Synechocystis downregulated anabolic processes by post-translational ribosome inactivation. Ribosome profiling indicated that ribosomes that remained active were primarily synthesizing proteins involved in CO₂ uptake and stress mediation. The regulatory protein thioredoxin TrxC was upregulated by induced translation and may conduct post-translational redox regulation during CO₂ stress. In the second study, diurnal oscillations in the transcriptome, translatome and proteome were investigated using mRNA sequencing, ribosome profiling and quantitative proteomics. A stable proteome was observed, despite significant oscillations in protein synthesis. A model describing the dynamic relationship between protein synthesis and protein abundance suggested that a slow protein turnover is causing the observed effect. A stable proteome suggests that shifts between autotrophic and heterotrophic metabolism occurring during day-night cycles are controlled by post-translational regulation. The third study investigated proteome arrangement strategies during light and CO₂ limitation. Integration of proteomics data into a cellular protein economy model indicated that Synechocystis keeps underutilized protein reserves. These reserves may be activated by post-translational regulation to quickly adapt to changing growth conditions. In the fourth study, post-translational regulation by metabolite interactions was investigated using interaction proteomics and enzyme kinetic assays. Glyceraldehyde-3-phosphate activated the enzyme fructose/sedoheptulose-bisphosphatase, suggesting a feedforward activation mechanism in the Calvin cycle. AMP deactivates the same enzyme, which may facilitate glycogen catabolism during CO₂ starvation. The findings and data of this work could guide future studies and attempts to engineer cyanobacteria for a sustainable production of commodity chemicals.

Koldioxidutsläpp från mänskliga aktiviteter och utnyttjandet av fossila råvaror har satt jordens klimat ur balans. För förhindra katastrofala konsekvenser på civilisationer och naturliga miljöer behöver vi snarast utveckla alternativa lösningar som baserar sig på förnybara råvaror. En potentiell lösning är att använda genetiskt modifierade cyanobakterier som omvandlar koldioxid och solljus till värdefulla produkter, såsom bränslen och biologiskt nedbrytbar plast. Men dessa uråldriga bakterier har utvecklat ett komplext, robust och självreglerat metaboliskt nätverk för att effektivt omvandla koldioxid till sin egen biomassa, snarare än en främmande kemikalie. Detta begränsar de modifierade cyanobakteriernas produktivitet och möjligheten att använda dem för industriellt bruk. Därför krävs det utökad kunskap om hur deras metabolism är reglerad för att kunna utveckla högproduktiva stammar. Denna undersökning har utnyttjat nyligen utvecklade systembiologi-tekniker för att utforska hur metabolismen regleras i cyanobakterien Synechocystis. Resultaten från den första studien visade att Synechocystis nedreglerar anabola processer genom posttranslationell inaktivering av ribosomer när de svältes på koldioxid. Ribosomprofilering visade att ribosomer som fortfarande var aktiva tillverkade proteiner involverade i koldioxidupptag och stresshantering. Det regulatoriska proteinet thioredoxin TrxC ökade i mängd genom uppreglerad translation och utför möjligen posttranslationell redox-reglering under koldioxidstress. I den andra studien undersöktes dagliga svängningar i transkriptom, translatom och proteom med hjälp av mRNA-sekvensering, ribosomprofilering och kvantitativ proteomik. Ett stabilt proteom observerades, trots signifikanta svängningar i translation. En matematisk modell som beskrev det dynamiska förhållandet mellan proteinsyntes och proteinnivåer i cellen antydde att en långsam proteinomsättning orsakar den observerade effekten. Observationen av ett stabilt proteom antydde att omställningen mellan autotrof och heterotrof metabolism som sker mellan dag och natt regleras på posttranslationell nivå. Den tredje studien undersökte proteomallokeringsstrategier under ljus- och koldioxidbegränsad tillväxt. Integrering av proteomikdata i en cellulär proteinekonomimodell antydde att Synechocystis håller på underutnyttjade proteinreserver. Dessa reserver aktiveras förmodligen av posttranslationell reglering för att snabbt anpassa sig till förändringar i tillväxtmiljön. I den fjärde studien undersöktes posttranslationell reglering av metabolitinteraktioner med hjälp av interaktionsproteomik och enzymkinetiska analyser. Glyceraldehyd-3-fosfat aktiverade enzymet fruktos/sedoheptulosbisfosfatas, vilket föreslår en feedforward-aktiveringsmekanism i Calvin cykeln. AMP hämmar samma enzym, vilket skulle kunna styra glykogenkatabolism under koldioxidsvält. Resultaten och datan från detta arbete kan vägleda framtida forskning och försök att modifiera cyanobakterier för en hållbar produktion av råvarukemikalier.

Cyanobacteria must balance separate demands for energy generation, carbon assimilation, and biomass synthesis. We used shotgun proteomics to investigate proteome allocation strategies in the model cyanobacterium Synechocystis sp. PCC 6803 as it adapted to light and inorganic carbon (C-i) limitation. When partitioning the proteome into seven functional sectors, we find that sector sizes change linearly with growth rate. The sector encompassing ribosomes is significantly smaller than in E. coli, which may explain the lower maximum growth rate in Synechocystis. Limitation of light dramatically affects multiple proteome sectors, whereas the effect of C-i limitation is weak. Carbon assimilation proteins respond more strongly to changes in light intensity than to C-i. A coarse-grained cell economy model generally explains proteome trends. However, deviations from model predictions suggest that the large proteome sectors for carbon and light assimilation are not optimally utilized under some growth conditions and may constrain the proteome space available to ribosomes.

Cyanobacteria experience both rapid and periodic fluctuations in light and inorganic carbon (C-i) and have evolved regulatory mechanisms to respond to these, including extensive posttranscriptional gene regulation. We report the first genome-wide ribosome profiling data set for cyanobacteria, where ribosome occupancy on mRNA is quantified with codon-level precision. We measured the transcriptome and translatome of Synechocystis during autotrophic growth before (high carbon [HC] condition) and 24 h after removing CO2 from the feedgas (low carbon [LC] condition). Ribosome occupancy patterns in the 5' untranslated region suggest that ribosomes can assemble there and slide to the Shine-Dalgarno site, where they pause. At LC, total translation was reduced by 80% and ribosome pausing was increased at stop and start codons and in untranslated regions, which may be a sequestration mechanism to inactivate ribosomes in response to rapid C-i depletion. Several stress response genes, such as thioredoxin M (sll1057), a putative endonuclease (slr0915), protease HtrA (slr1204), and heat shock protein HspA (sll1514) showed marked increases in translational efficiency at LC, indicating translational control in response to Ci depletion. Ribosome pause scores within open reading frames were mostly constant, though several ribosomal proteins had significantly altered pause score distributions at LC, which might indicate translational regulation of ribosome biosynthesis in response to Ci depletion. We show that ribosome profiling is a powerful tool to decipher dynamic gene regulation strategies in cyanobacteria. IMPORTANCE Ribosome profiling accesses the translational step of gene expression via deep sequencing of ribosome-protected mRNA footprints. Pairing of ribosome profiling and transcriptomics data provides a translational efficiency for each gene. Here, the translatome and transcriptome of the model cyanobacterium Synechocystis were compared under carbon-replete and carbon starvation conditions. The latter may be experienced when cyanobacteria are cultivated in poorly mixed bioreactors or engineered to be product-secreting cell factories. A small fraction of genes (<200), including stress response genes, showed changes in translational efficiency during carbon starvation, indicating condition-dependent translation-level regulation. We observed ribosome occupancy in untranslated regions, possibly due to an alternative translation initiation mechanism in Synechocystis. The higher proportion of ribosomes residing in untranslated regions during carbon starvation may be a mechanism to quickly inactivate superfluous ribosomes. This work provides the first ribosome profiling data for cyanobacteria and reveals new regulation strategies for coping with nutrient limitation.

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.