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- Asplund Samuelsson, Johannes, 1987-
(författare)
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Adaptations and constraints associated with autotrophy in microbial metabolism
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Carbon dioxide (CO2) emissions from human activities are driving climate change, but the pending crisis could be mitigated by a circular carbon economy where released CO2 is recycled into commodity chemicals. Autotrophic microbes can make a contribution by producing chemicals, such as biofuels, from CO2 and renewable energy. The primary natural CO2 fixation pathway is the Calvin cycle, in which the enzyme Rubisco carboxylates ribulose-1,5-bisphosphate. The present investigation used computational systems biology methods to map adaptations and constraints in autotrophic microbial metabolism based on the Calvin cycle. First, the metabolic network of the Calvin cycle-capable photoautotrophic cyanobacterium Synechocystis was contrasted with that of heterotrophic E. coli. Intracellular metabolite concentration ranges differed, leading to different capacity to provide thermodynamic driving forces to chemical production pathways. Second, the Calvin cycle in Synechocystis was modeled kinetically, showing that certain enzyme saturation and metabolite levels, for example high ribulose-1,5-bisphosphate concentration, were detrimental to stability. Control over reaction rates was distributed, but making certain enzymes faster, for example fructose-1,6-bisphosphatase, could increase overall carbon fixation rate. Third, Synechocystis was starved of CO2 and ribosome profiling was used to track the effect on translation. Stress response and CO2 uptake were upregulated, but constant Rubisco expression and ribosome pausing in 5' untranslated regions indicated readiness for reappearance of CO2. Finally, microbial genomes with and without the Calvin cycle were contrasted, revealing metabolic, energetic, and regulatory adaptations that describe the properties of a functional autotroph. These findings provide a background for future study and engineering of autotrophs for direct conversion of CO2 into commodity chemicals.
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| 2. |
- Kuil, Teun, et al.
(författare)
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Functional Analysis of H+-Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum
- 2022
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Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 0099-2240 .- 1098-5336. ; 88:4
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Tidskriftsartikel (refereegranskat)abstract
- The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PPi) as a phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PPi was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H+-pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk-malate shunt cycle, and acetate cycling in generating PPi. Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum, and heterologous expression in Escherichia coli. Unexpectedly, individually targeted gene deletions of the four putative PPi sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PPi sources reduced the growth rate by 22% (0.30 +/- 0.01 h(-1)) and the biomass yield by 38% (0.18 +/- 0.00 g(biomass) g(substrate)-1), this change was much smaller than what would be expected for stoichiometrically essential PPi-supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose, indicating that the unknown PPi-supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PPi altogether was falsified by enzyme assays, heterologous expression of candidate genes, and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PPi sources individually and combined play no significant PPi-supplying role, and the true source(s) of PPi, or alternative phosphorylating mechanisms, that drive(s) glycolysis in C. thermocellum remain(s) elusive. IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H+-pumping membrane-bound PPase, glycogen cycling, a Ppdk-malate shunt cycle, and acetate cycling are not significant sources of PPi supply, this study adds functional annotation of four genes and availability of an updated PP, stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PPi or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PPi and ATP-dependent glycolysis.
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| 3. |
- Schroeder, Wheaton L., et al.
(författare)
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A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis
- 2023
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 77, s. 306-322
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Tidskriftsartikel (refereegranskat)abstract
- Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is cumbersome in conventional processes. A promising, and increasingly studied, candidate for lignocellulose bioprocessing is the thermophilic anaerobe Clostridium thermocellum given its potential to produce ethanol, organic acids, and hydrogen gas from lignocellulosic biomass under high substrate loading. Possessing an atypical glycolytic pathway which substitutes GTP or pyrophosphate (PPi) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PPi sources are key to engineering its metabolism. Previous efforts to identify the primary pyrophosphate have been unsuccessful. Here, we explore pyrophosphate metabolism through reconstructing, updating, and analyzing a new genome-scale stoichiometric model for C. thermocellum, iCTH669. Hundreds of changes to the former GEM, iCBI655, including correcting cofactor usages, addressing charge and elemental balance, standardizing biomass composition, and incorporating the latest experimental evidence led to a MEMOTE score improvement to 94%. We found agreement of iCTH669 model predictions across all available fermentation and biomass yield datasets. The feasibility of hundreds of PPi synthesis routes, newly identified and previously proposed, were assessed through the lens of the iCTH669 model including biomass synthesis, tRNA synthesis, newly identified sources, and previously proposed PPi-generating cycles. In all cases, the metabolic cost of PPi synthesis is at best equivalent to investment of one ATP suggesting no direct energetic advantage for the cofactor substitution in C. thermocellum. Even though no unique source of PPi could be gleaned by the model, by combining with gene expression data two most likely scenarios emerge. First, previously investigated PPi sources likely account for most PPi production in wild-type strains. Second, alternate metabolic routes as encoded by iCTH669 can collectively maintain PPi levels even when previously investigated synthesis cycles are disrupted. Model iCTH669 is available at github.com/maranasgroup/iCTH669.
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