Modeling and analysis of the rapid aerobic metabolism of Geobacillus sp. LC300

QC 2024-08-20

Abstract

To mitigate climate change, global greenhouse gas emissions must be halved before 2030. To achieve this goal, alternative routes for fuel and chemical production that do not rely on fossil resources must be explored. Industrial biotechnology has been identified as a key technology in this transition, allowing the sustainable valorization of biomass to biofuels and biochemicals. Geobacillus sp. LC300 is a thermophilic microorganism displaying remarkable growth rates and metabolic capabilities, thus showing promise for development into a microbial cell factory for sustainable production of biochemicals. However, the metabolism of the organism is unexplored, and its metabolic requirements and optimal growth conditions unknown. The aim of this thesis was to investigate the fast metabolism of Geobacillus sp. LC300 and thereby evaluate the potential and facilitate the development of the organism as a microbial cell factory. To explore the metabolic landscape of G. sp. LC300, a homology-based genome-scale metabolic model was constructed. By analyzing the model-predicted metabolic pathways, a prototrophy for all amino acids was predicted, along with an auxotrophy for vitamin B12. Analysis of transporters further predicted growth on several carbon sources, and the model showed accurate predictions of intracellular flux distributions and growth yields on both glucose and xylose. This model serves as a crucial tool for understanding the G. sp. LC300’s metabolism and guiding metabolic engineering efforts to optimize it for industrial use. Growth media previously used for the cultivation of G. sp. LC300 contained complex components, such as yeast extract, and was unable to support growth to high cell densities. This complicated quantitative studies of metabolism where controlled conditions and high cell densities are important for quantification of rates and yields. A minimal medium was developed based on the biomass composition predicted by the genome-scale model. In this development, the predicted auxotrophy for vitamin B12 was confirmed, and an additional auxotrophy for biotin revealed. The modified medium supported growth to high cell densities without the addition of complex components. An investigation of the optimal growth conditions of G. sp. LC300 revealed an optimal growth temperature several degrees lower than earlier reported values, providing a more accurate basis for the development of future production process settings. The range of carbon source utilization was further investigated, revealing fast growth on substrates like glycerol and starch that are common byproducts and in waste-streams from industry.To investigate the keys to the rapid substrate consumption rate, growth, and respiration of G. sp. LC300, glucose-limited chemostat cultivations were performed. The cultivations revealed a capacity of fully respiratory growth at a rate higher than the maximum specific growth rate of most other microorganisms, and a lower fraction of substrate consumed by maintenance than E. coli. Proteomics analysis further revealed an unusually low allocation of protein to the central carbon metabolism and translation, made possible by high turnover numbers of these enzymes allowing a larger allocation to respiratory enzymes. Finally, enzyme-constrained modeling indicated limited protein availability as the cause of overflow metabolism at growth rates above critical, with a switch from respiratory to respiro-fermentative pathways. Together, these findings provide insights into the rapid metabolism of G. sp. LC300 and highlights its potential as a microbial cell factory. This work can provide the basis for the development of new production processes that play an important role in the bioeconomy of the future and help circularize greenhouse gas emissions to net-zero.

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