Insights into the rapid metabolism of Geobacillus sp. LC300: unraveling metabolic requirements and optimal growth conditions.

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.

Genome-scale reconstruction and metabolic modelling of the fast-growing thermophile Geobacillus sp. LC300.

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.

Cycling between growth and production phases increases cyanobacteria bioproduction of lactate.

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.

Targeted Repression of Essential Genes To Arrest Growth and Increase Carbon Partitioning and Biofuel Titers in Cyanobacteria.

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 gltA 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 gltA gene, we could modulate GltA 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.