Universal motifs and the diversity of autocatalytic systems

Autocatalysis is essential for the origin of life and chemical evolution. However, the lack of a unified framework so far prevents a systematic study of autocatalysis. Here, we derive, from basic principles, general stoichiometric conditions for catalysis and autocatalysis in chemical reaction networks. This allows for a classification of minimal autocatalytic motifs called cores. While all known autocatalytic systems indeed contain minimal motifs, the classification also reveals hitherto unidentified motifs.We further examine conditions for kinetic viability of such networks, which depends on the autocatalytic motifs they contain and is notably increased by internal catalytic cycles. Finally, we show how this framework extends the range of conceivable autocatalytic systems, by applying our stoichiometric and kinetic analysis to autocatalysis emerging from coupled compartments. The unified approach to autocatalysis presented in this work lays a foundation toward the building of a systems-level theory of chemical evolution

Promiscuous phosphoketolase and metabolic rewiring enables novel non-oxidative glycolysis in yeast for high-yield production of acetyl-CoA derived products

Carbon-conserving pathways have the potential of increasing product yields in biotechnological processes. The aim of this project was to investigate the functionality of a novel carbon-conserving pathway that produces 3 mol of acetyl-CoA from fructose-6-phosphate without carbon loss in the yeast Saccharomyces cerevisiae. This cyclic pathway relies on a generalist phosphoketolase (Xfspk), which can convert xylulose-5-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate (S7P) to acetyl phosphate. This cycle is proposed to overcome bottlenecks from the previously reported non-oxidative glycolysis (NOG) cycle. Here, in silico simulations showed accumulation of S7P in the NOG cycle, which was resolved by blocking the non-oxidative pentose phosphate pathway and introducing Xfspk and part of the riboneogenesis pathway. To implement this, a transketolase and transaldolase deficient S. cerevisiae was generated and a cyclic pathway, the Glycolysis AlTernative High Carbon Yield Cycle (GATHCYC), was enabled through xfspk expression and sedoheptulose bisphosphatase (SHB17) overexpression. Flux through the GATHCYC was demonstrated in vitro with a phosphoketolase assay on crude cell free extracts, and in vivo by constructing a strain that was dependent on a functional pathway to survive. Finally, we showed that introducing the GATHCYC as a carbon-conserving route for 3-hydroxypropionic acid (3-HP) production resulted in a 109% increase in 3-HP titers when the glucose was exhausted compared to the phosphoketolase route only

Fast pyrolysis of agricultural residues: Reaction mechanisms and effects of feedstock properties and Microwave operating conditions on the yield and product composition

Fundamental understanding of the pyrolysis process plays an indispensable role in valorization of wastes and the development of novel sustainable technologies. This study introduces a novel approach by investigating the reaction mechanisms involve in Microwave-Assisted Fast Pyrolysis (MAFP) to unveil the thermal decomposition of agricultural residues: pecan nutshell (NS), sugarcane bagasse (SB), and orange seed (OS) biomasses. The holistic understanding of the pyrolysis process for these biomasses was analyzed based on the final chemical compositions and yields of bio-oil, biochar and biogas and correlated to the microwave processing conditions and feedstock’s chemical composition. The findings revealed that the bio-oil is enhanced at moderated microwave energy (<5 GJ/t) as result of endothermic reactions such as heterolytic fragmentation, Maccoll elimination, Friedel-Craft acylation, Piancatelli rearrangement and methoxylation. The maximum yield of bio-oil for protein-rich biomass was due to selective heating (Paal-Knorr pyrrole synthesis, Baeyer-Villiger oxidation, Maillard reaction, and ring conversion of γ-butyrolactone). The formation of biochar and biogas is attributed to the repolymerization of aromatic aldehydes, hydrocarbons, amines, and ethers, as well as dehydroxymethylation and dealkylation processes. This study provides a comprehensive understanding of the reaction mechanisms for several wastes using microwave pyrolysis, to establish the bases for effective valorization and agricultural waste management

Chemical Transformation Motifs—Modelling Pathways as Integer Hyperflows

Andersen JL, Flamm C, Merkle D, Stadler PF. Chemical Transformation Motifs—Modelling Pathways as Integer Hyperflows. IEEE/ACM Transactions on Computational Biology and Bioinformatics. 2019;16(2):510-523.We present an elaborate framework for formally modelling pathways in chemical reaction networks on a mechanistic level. Networks are modelled mathematically as directed multi-hypergraphs, with vertices corresponding to molecules and hyperedges to reactions. Pathways are modelled as integer hyperflows and we expand the network model by detailed routing constraints. In contrast to the more traditional approaches like Flux Balance Analysis or Elementary Mode analysis we insist on integer-valued flows. While this choice makes it necessary to solve possibly hard integer linear programs, it has the advantage that more detailed mechanistic questions can be formulated. It is thus possible to query networks for general transformation motifs, and to automatically enumerate optimal and near-optimal pathways. Similarities and differences between our work and traditional approaches in metabolic network analysis are discussed in detail. To demonstrate the applicability of the mathematical framework to real-life problems we first explore the design space of possible non-oxidative glycolysis pathways and show that recent manually designed pathways can be further optimized. We then use a model of sugar chemistry to investigate pathways in the autocatalytic formose process. A graph transformation-based approach is used to automatically generate the reaction networks of interest

Engineering central carbon metabolism with phosphoketolase pathways in Saccharomyces cerevisiae

We need more efficient biocatalysts to make sustainable microbial production of chemicals and fuels more profitable before they can replace petroleum-based sources. Rewiring the metabolic pathways in the biocatalysts to avoid the loss of carbon as CO2 can aid in improving product yields and thereby the profitability of the process. In this thesis, I investigated the use of phosphoketolase (PK) pathways in the yeast Saccharomyces cerevisiae to produce the precursor metabolite acetyl-CoA without loss of carbon as CO2. Firstly, we investigated the effect of acetyl-phosphate (AcP) accumulation from the linear PK pathway when downstream product formation is limited. Accumulated AcP was degraded to acetate, which limited the benefit of the PK pathway. Furthermore, we investigated a combinatorial strategy to supply acetyl-CoA and NADPH for fatty acid (FA) production. We combined the PK strategy with overexpression of the transcription factor Stb5 to activate NADPH generating pathways. This strategy increased the FA titer in the glucose phase, but with a counteractive response that possibly arose from the lack of an effective NADPH sink. Secondly, we expanded the linear PK pathway to a novel configuration of the cyclic non-oxidative glycolysis (NOG) that can recycle all the carbon from glucose into acetyl-CoA, thus potentially increasing product yields even further. We showed through kinetic modeling that the new configuration resolves potential bottlenecks in the previous configuration. We verified both in vitro and in vivo functionality of the cycle in S.\ua0cerevisiae. Furthermore, we demonstrated increased titers of an acetyl-CoA-derived product in the glucose phase compared to the linear PK pathway, indicating increased precursor supply from the cycle. Finally, we further characterized the S.\ua0cerevisiae strain with the cycle, using omics. Most notably, the cycle strain yielded respiro-fermentative growth in chemostat cultures with acetate as the main overflow metabolite. This points to a metabolic imbalance and extensive AcP degradation to acetate, which needs to be resolved before the cycle can be efficiently utilized. This thesis highlights the status of this novel NOG configuration and will aid in the further development of cell factories with high-yield production of acetyl-CoA-derived products