Thermodynamic limitations of PHB production from formate and fructose in Cupriavidus necator.

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.

Wide range of metabolic adaptations to the acquisition of the Calvin cycle revealed by comparison of microbial genomes.

Knowledge of the genetic basis for autotrophic metabolism is valuable since it relates to both the emergence of life and to the metabolic engineering challenge of incorporating CO2 as a potential substrate for biorefining. The most common CO2 fixation pathway is the Calvin cycle, which utilizes Rubisco and phosphoribulokinase enzymes. We searched thousands of microbial genomes and found that 6.0% contained the Calvin cycle. We then contrasted the genomes of Calvin cycle-positive, non-cyanobacterial microbes and their closest relatives by enrichment analysis, ancestral character estimation, and random forest machine learning, to explore genetic adaptations associated with acquisition of the Calvin cycle. The Calvin cycle overlaps with the pentose phosphate pathway and glycolysis, and we could confirm positive associations with fructose-1,6-bisphosphatase, aldolase, and transketolase, constituting a conserved operon, as well as ribulose-phosphate 3-epimerase, ribose-5-phosphate isomerase, and phosphoglycerate kinase. Additionally, carbohydrate storage enzymes, carboxysome proteins (that raise CO2 concentration around Rubisco), and Rubisco activases CbbQ and CbbX accompanied the Calvin cycle. Photorespiration did not appear to be adapted specifically for the Calvin cycle in the non-cyanobacterial microbes under study. Our results suggest that chemoautotrophy in Calvin cycle-positive organisms was commonly enabled by hydrogenase, and less commonly ammonia monooxygenase (nitrification). The enrichment of specific DNA-binding domains indicated Calvin-cycle associated genetic regulation. Metabolic regulatory adaptations were illustrated by negative correlation to AraC and the enzyme arabinose-5-phosphate isomerase, which suggests a downregulation of the metabolite arabinose-5-phosphate, which may interfere with the Calvin cycle through enzyme inhibition and substrate competition. Certain domains of unknown function that were found to be important in the analysis may indicate yet unknown regulatory mechanisms in Calvin cycle-utilizing microbes. Our gene ranking provides targets for experiments seeking to improve CO2 fixation, or engineer novel CO2-fixing organisms.

Kinetic modeling of the Calvin cycle identifies flux control and stable metabolomes in Synechocystis carbon fixation.

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.

Thermodynamic analysis of computed pathways integrated into the metabolic networks of E. coli and Synechocystis reveals contrasting expansion potential.

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.

The art of destruction: revealing the proteolytic capacity of bacterial caspase homologs.

Caspases are proteases that initiate and execute programmed cell death in animal tissues, thereby facilitating multicellular development and survival. While caspases are unique to metazoans and specifically cleave substrates at aspartic acid residues, homologs are found in protozoa, plants, algae, fungi, bacteria and archaea, and show specificity for basic residues. In this issue of Molecular Microbiology, Klemencic and colleagues present the first biochemical characterization of a bacterial caspase homolog, classified as an orthocaspase. By expressing the gene MaOC1 from the cyanobacterium Microcystis aeruginosa PCC 7806 in Escherichia coli, the authors discovered specificity for substrates with arginine in the P1 position. The protein requires autocatalytic processing to become active and is dependent on an intact histidine-cysteine dyad. These results significantly extend our knowledge of the specificities of bacterial caspase homologs, which are known to be highly diverse in protein domain architectures and active site mutations. Although bacterial programmed cell death is one possible area of action, the function of most bacterial caspase homologs remains unexplored. Cyanobacteria represent the best studied group in terms of prokaryotic caspase-like proteins both genomically and experimentally, and thereby provide a suitable platform for further investigations into activation, regulation and physiological roles of orthocaspases.

Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation.

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.

Slow Protein Turnover Explains Limited Protein-Level Response to Diurnal Transcriptional Oscillations in Cyanobacteria.

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.

Protein allocation and utilization in the versatile chemolithoautotroph Cupriavidus necator.

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.

Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes.

Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803, where we aim to target all genes for repression. We track the growth of all library members in multiple conditions and estimate gene fitness. The library reveals several clones with increased growth rates, and these have a common upregulation of genes related to cyclic electron flow. We challenge the library with 0.1 M L-lactate and find that repression of peroxiredoxin bcp2 increases growth rate by 49%. Transforming the library into an L-lactate-secreting Synechocystis strain and sorting top lactate producers enriches clones with sgRNAs targeting nutrient assimilation, central carbon metabolism, and cyclic electron flow. In many examples, productivity can be enhanced by repression of essential genes, which are difficult to access by transposon insertion.

Phylogenetic Distribution and Diversity of Bacterial Pseudo-Orthocaspases Underline Their Putative Role in Photosynthesis.

Orthocaspases are prokaryotic caspase homologs - proteases, which cleave their substrates after positively charged residues using a conserved histidine - cysteine (HC) dyad situated in a catalytic p20 domain. However, in orthocaspases pseudo-variants have been identified, which instead of the catalytic HC residues contain tyrosine and serine, respectively. The presence and distribution of these presumably proteolytically inactive p20-containing enzymes has until now escaped attention. We have performed a detailed analysis of orthocaspases in all available prokaryotic genomes, focusing on pseudo-orthocaspases. Surprisingly we identified type I metacaspase homologs in filamentous cyanobacteria. While genes encoding pseudo-orthocaspases seem to be absent in Archaea, our results show conservation of these genes in organisms performing either anoxygenic photosynthesis (orders Rhizobiales, Rhodobacterales, and Rhodospirillales in Alphaproteobacteria) or oxygenic photosynthesis (all sequenced cyanobacteria, except Gloeobacter, Prochlorococcus, and Cyanobium). Contrary to earlier reports, we were able to detect pseudo-orthocaspases in all sequenced strains of the unicellular cyanobacteria Synechococcus and Synechocystis. In silico comparisons of the primary as well as tertiary structures of pseudo-p20 domains with their presumably proteolytically active homologs suggest that differences in their amino acid sequences have no influence on the overall structures. Mutations therefore affect most likely only the proteolytic activity. Our data provide an insight into diversification of pseudo-orthocaspases in Prokaryotes, their taxa-specific distribution, and allow suggestions on their taxa-specific function.

Diversity and Expression of Bacterial Metacaspases in an Aquatic Ecosystem.

Metacaspases are distant homologs of metazoan caspase proteases, implicated in stress response, and programmed cell death (PCD) in bacteria and phytoplankton. While the few previous studies on metacaspases have relied on cultured organisms and sequenced genomes, no studies have focused on metacaspases in a natural setting. We here present data from the first microbial community-wide metacaspase survey; performed by querying metagenomic and metatranscriptomic datasets from the brackish Baltic Sea, a water body characterized by pronounced environmental gradients and periods of massive cyanobacterial blooms. Metacaspase genes were restricted to ~4% of the bacteria, taxonomically affiliated mainly to Bacteroidetes, Alpha- and Betaproteobacteria and Cyanobacteria. The gene abundance was significantly higher in larger or particle-associated bacteria (>0.8 µm), and filamentous Cyanobacteria dominated metacaspase gene expression throughout the bloom season. Distinct seasonal expression patterns were detected for the three metacaspase genes in Nodularia spumigena, one of the main bloom-formers. Clustering of normalized gene expression in combination with analyses of genomic and assembly data suggest functional diversification of these genes, and possible roles of the metacaspase genes related to stress responses, i.e., sulfur metabolism in connection to oxidative stress, and nutrient stress induced cellular differentiation. Co-expression of genes encoding metacaspases and nodularin toxin synthesis enzymes was also observed in Nodularia spumigena. The study shows that metacaspases represent an adaptation of potentially high importance for several key organisms in the Baltic Sea, most prominently Cyanobacteria, and open up for further exploration of their physiological roles in microbes and assessment of their ecological impact in aquatic habitats.

Functional tradeoffs underpin salinity-driven divergence in microbial community composition.

Bacterial community composition and functional potential change subtly across gradients in the surface ocean. In contrast, while there are significant phylogenetic divergences between communities from freshwater and marine habitats, the underlying mechanisms to this phylogenetic structuring yet remain unknown. We hypothesized that the functional potential of natural bacterial communities is linked to this striking divide between microbiomes. To test this hypothesis, metagenomic sequencing of microbial communities along a 1,800 km transect in the Baltic Sea area, encompassing a continuous natural salinity gradient from limnic to fully marine conditions, was explored. Multivariate statistical analyses showed that salinity is the main determinant of dramatic changes in microbial community composition, but also of large scale changes in core metabolic functions of bacteria. Strikingly, genetically and metabolically different pathways for key metabolic processes, such as respiration, biosynthesis of quinones and isoprenoids, glycolysis and osmolyte transport, were differentially abundant at high and low salinities. These shifts in functional capacities were observed at multiple taxonomic levels and within dominant bacterial phyla, while bacteria, such as SAR11, were able to adapt to the entire salinity gradient. We propose that the large differences in central metabolism required at high and low salinities dictate the striking divide between freshwater and marine microbiomes, and that the ability to inhabit different salinity regimes evolved early during bacterial phylogenetic differentiation. These findings significantly advance our understanding of microbial distributions and stress the need to incorporate salinity in future climate change models that predict increased levels of precipitation and a reduction in salinity.

Prokaryotic caspase homologs: phylogenetic patterns and functional characteristics reveal considerable diversity.

Caspases accomplish initiation and execution of apoptosis, a programmed cell death process specific to metazoans. The existence of prokaryotic caspase homologs, termed metacaspases, has been known for slightly more than a decade. Despite their potential connection to the evolution of programmed cell death in eukaryotes, the phylogenetic distribution and functions of these prokaryotic metacaspase sequences are largely uncharted, while a few experiments imply involvement in programmed cell death. Aiming at providing a more detailed picture of prokaryotic caspase homologs, we applied a computational approach based on Hidden Markov Model search profiles to identify and functionally characterize putative metacaspases in bacterial and archaeal genomes. Out of the total of 1463 analyzed genomes, merely 267 (18%) were identified to contain putative metacaspases, but their taxonomic distribution included most prokaryotic phyla and a few archaea (Euryarchaeota). Metacaspases were particularly abundant in Alphaproteobacteria, Deltaproteobacteria and Cyanobacteria, which harbor many morphologically and developmentally complex organisms, and a distinct correlation was found between abundance and phenotypic complexity in Cyanobacteria. Notably, Bacillus subtilis and Escherichia coli, known to undergo genetically regulated autolysis, lacked metacaspases. Pfam domain architecture analysis combined with operon identification revealed rich and varied configurations among the metacaspase sequences. These imply roles in programmed cell death, but also e.g. in signaling, various enzymatic activities and protein modification. Together our data show a wide and scattered distribution of caspase homologs in prokaryotes with structurally and functionally diverse sub-groups, and with a potentially intriguing evolutionary role. These features will help delineate future characterizations of death pathways in prokaryotes.