This study investigated the metabolism of Geobacillus sp. LC300, a promising biorefinery host organism with high substrate utilization rates. A new defined medium was designed and tested that allows for exponential growth to elevated cell densities suitable for quantitative physiological studies. Screening of the metabolic requirements of G. sp. LC300 revealed prototrophy for all essential amino acids and most vitamins and only showed auxotrophy for vitamin B12 and biotin. The effect of temperature and pH on growth rate was investigated, adjusting the optimal growth temperature to several degrees lower than previously reported. Lastly, studies on carbon source utilization revealed a capability for fast growth on several common carbon sources, including monosaccharides, oligosaccharides, and polysaccharides, and the highest ever reported growth rate in defined medium on glucose (2.20 h(-1)) or glycerol (1.95 h(-1)). These findings provide a foundation for further exploration of G. sp. LC300's physiology and metabolic regulation, and its potential use in bioproduction processes.

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.

 För att motverka klimatförändringar måste de globala utsläppen av växthusgaser halveras innan 2030. För att nå detta mål måste nya produktionsprocesser för bränslen och kemikalier utvecklas som är oberoende av fossila resurser. Industriell bioteknik utgör en nyckel-teknik i denna omställning på grund av dess förutsättningar för omvandling av biomassa till biobränslen och biokemikalier. Geobacillus sp. LC300 är en termofil bakterie som uppvisar påfallande höga tillväxthastigheter och metabol förmåga, vilket gör den lovande för att utvecklas till en mikrobiell cellfabrik för biokemikalieproduktion. Bakteriens metabolism är dock outforskad, och dess näringsbehov och optimala tillväxtförhållanden okända. Målet med denna avhandling var att utforska G. sp. LC300s snabba metabolism och därmed utvärdera dess potential och underlätta dess utveckling till en mikrobiell cellfabrik.En homologibaserad genomskalemodell konstruerades för att utforska dess metabolism. Genom att analysera modellens metabola vägar förutspåddes en prototrofi för alla 20 aminosyror, samt en auxotrofi för vitamin B12. Genom analys av transportprotein kunde tillväxtmöjligheter på flera olika kolkällor även förutspås, och modellen estimerade både intra- och extracellulära reaktions-hastigheter på både glukos och xylos med hög noggrannhet. Modellen är ett viktigt verktyg för att utöka förståelsen för G. sp. LC300s metabolism och som guide vid manipulering av metabolismen för att därmed utveckla organismen till en mikrobiell cellfabrik. De odlingsmedia som tidigare använts för odling av G. sp. LC300 innehåller komplexa komponenter, t. ex. jästextrakt, och saknar näringsinnehåll för odlingar med hög celldensitet. Detta komplicerar kvantitativa studier av metabolismen som kräver precis kontroll över odlingsbetingelser och höga celldensiteter för kvantifiering av hastigheter och utbyten. För att undgå detta problem utvecklades ett minimalt medium med definierad sammansättning, baserat på den cellmassakomposition som genomskalemodellen estimerat. Under utvecklingen av mediet bekräftades auxotrofin för vitamin B12, och en ytterligare auxotrofi för biotin upptäcktes. Det nya mediet tillät odlingar till hög celldensitet utan tillsats av komplexa komponenter. Vid undersökning av optimala odlingsbetingelser för G. sp. LC300 upptäcktes en flera grader lägre optimal tillväxttemperatur än den som tidigare rapporterats. Med ett definierat medium och optimala odlingsbetingelser kunde även ytterligare kolkällor utvärderas, till exempel glycerol och stärkelse, som är vanliga biprodukter från industrin. För att undersöka de underliggande faktorerna bakom den snabba substratkonsumptions-hastigheten, tillväxthastigheten och respirationen hos G. sp. LC300 användes glukosbegränsade kemostatodlingar. Odlingarna visade en kapacitet till fullt respirativ tillväxt vid hastigheter högre än den maximala specifika tillväxthastigheten hos de flesta andra mikroorganismer, och en lägre fraktion av substrat som konsumeras av metabola underhålls-funktioner än hos E. coli. Proteomikanalys visade att den höga respirativa kapaciteten kunde kopplas till en högre allokering av protein till respirationskedjan än i andra bakterier, vilket möjliggjordes av lägre proteinallokering till den centrala kolmetabolismen tack vare höga omsättningsnummer för dessa enzym. Slutligen indikerade enzymbegränsad modellering att överflödesmetabolismen som observerats vid höga tillväxthastigheter hos G. sp. LC300 beror på proteinbegränsing, vilket skapat en omställning från respirativ till respirofermentativ metabolism.Tillsammans ger dessa upptäckter fördjupad förståelse för den snabba metabolismen hos G. sp. LC300, och betonar dess potential som mikrobiell cellfabrik. Detta arbete kan vara en grund för utveckling av nya produktionsprocesser som kan komma att spela en viktig roll i framtidens cirkulära bioekonomi.

Thermophilic microorganisms show high potential for use as biorefinery cell factories. Their high growth temperatures provide fast conversion rates, lower risk of contaminations, and facilitated purification of volatile products. To date, only a few thermophilic species have been utilized for microbial production purposes, and the development of production strains is impeded by the lack of metabolic engineering tools. In this study, we constructed a genome-scale metabolic model, an important part of the metabolic engineering pipeline, of the fast-growing thermophile Geobacillus sp. LC300. The model (iGEL604) contains 604 genes, 1249 reactions and 1311 metabolites, and the reaction reversibility is based on thermodynamics at the optimum growth temperature. The growth phenotype is analyzed by batch cultivations on two carbon sources, further closing balances in carbon and degree-of-reduction. The predictive ability of the model is benchmarked against experimentally determined growth characteristics and internal flux distributions, showing high similarity to experimental phenotypes.

Decoupling growth from product synthesis is a promising strategy to increase carbon partitioning and maximize productivity in cell factories. However, reduction in both substrate uptake rate and metabolic activity in the production phase are an underlying problem for upscaling. Here, we used CRISPR interference to repress growth in lactate-producing Synechocystis sp. PCC 6803. Carbon partitioning to lactate in the production phase exceeded 90%, but CO2 uptake was severely reduced compared to uptake during the growth phase. We characterized strains during the onset of growth arrest using transcriptomics and proteomics. Multiple genes involved in ATP homeostasis were regulated once growth was inhibited, which suggests an alteration of energy charge that may lead to reduced substrate uptake. In order to overcome the reduced metabolic activity and take advantage of increased carbon partitioning, we tested a novel production strategy that involved alternating growth arrest and recovery by periodic addition of an inducer molecule to activate CRISPRi. Using this strategy, we maintained lactate biosynthesis in Synechocystis for 30 days in a constant light turbidostat cultivation. Cumulative lactate titers were also increased by 100% compared to a constant growth-arrest regime, and reached 1 g/L. Further, the cultivation produced lactate for 30 days, compared to 20 days for the non-growth arrest cultivation. Periodic growth arrest could be applicable for other products, and in cyanobacteria, could be linked to internal circadian rhythms that persist in constant light.

Photoautotrophic production of fuels and chemicals by cyanobacteria typically gives lower volumetric productivities and titers than heterotrophic production. Cyanobacteria cultures become light limited above an optimal cell density, so that this substrate is not supplied to all cells sufficiently. Here, we investigate genetic strategies for a two-phase cultivation, where biofuel-producing Synechocystis cultures are limited to an optimal cell density through inducible CRISPR interference (CRISPRi) repression of cell growth. Fixed CO2 is diverted to ethanol or n-butanol. Among the most successful strategies was partial repression of citrate synthase gltA. Strong repression (>90%) of gitA at low culture densities increased carbon partitioning to n-butanol 5-fold relative to a nonrepression strain, but sacrificed volumetric productivity due to severe growth restriction. CO2 fixation continued for at least 3 days after growth was arrested. By targeting sgRNAs to different regions of the gitA gene, we could modulate GItA expression and carbon partitioning between growth and product to increase both specific and volumetric productivity. These growth arrest strategies can be useful for improving performance of other photoautotrophic processes.

 Geobacillus LC300 is a thermophilic bacterium displaying exceptionally fast growth and substrate utilization rates.  Despite its potential, fundamental understanding of its metabolism and fast growth is lacking. Here, the metabolism of G. sp. LC300 was studied through a combination of chemostat cultivations, proteomics, and enzyme-constrained modeling. Glucose-limited chemostat cultivations revealed an unprecedented respiratory capacity of 48 mmolO2 gDW-1 h-1 and concomitant complete respiratory metabolism until very high growth rates. Respiro-fermentative metabolism, i.e. formation of acetate in addition to respiration, only occurred at growth rates above 1.7 h-1 and above glucose uptake rates of 23 mmolglc gDW-1 h-. Proteome analysis of batch cultures showed an optimization of central carbon metabolism, with high apparent catalytic rates allowing a redistribution of protein resources to respiration and biosynthetic pathways. An enzyme-constrained genome-scale model was constructed, able to accurately simulate chemostat and batch growth. Proteome allocation analysis at varying growth rates was studied in the model, and the overflow metabolism observed at growth rates above 1.7 h-1 was explained by a limited protein supply causing a downregulation of large respiratory enzymes in favor of ATP generation through acetate formation. These insights into G. sp. LC300’s metabolic capabilities enhance our understanding of fast-growing thermophilic microorganisms, which also paves the way for more efficient biomanufacturing applications.