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On thermodynamic and kinetic constraints in autotrophic metabolism

Time: Fri 2021-11-26 09.30

Location: Air & Fire, Science for Life Laboratory, Tomtebodavägen 23, Solna

Language: English

Subject area: Biotechnology

Doctoral student: Markus Janasch , Systembiologi, Science for Life Laboratory, SciLifeLab, Hudson Group (Microbial Metabolic Engineering)

Opponent: Professor Bas Teusink, Vrije Universiteit Amsterdam

Supervisor: Assoc. Professor Elton P. Hudson, Systembiologi, Science for Life Laboratory, SciLifeLab

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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.