Genome-scale metabolic network reconstruction and in silico flux analysis of the thermophilic bacterium Thermus thermophilus HB27

Abstract

BackgroundThermus thermophilus, an extremely thermophilic bacterium, has been widely recognized as a model organism for studying how microbes can survive and adapt under high temperature environment. However, the thermotolerant mechanisms and cellular metabolism still remains mostly unravelled. Thus, it is highly required to consider systems biological approaches where T. thermophilus metabolic network model can be employed together with high throughput experimental data for elucidating its physiological characteristics under such harsh conditions.ResultsWe reconstructed a genome-scale metabolic model of T. thermophilus, iTT548, the first ever large-scale network of a thermophilic bacterium, accounting for 548 unique genes, 796 reactions and 635 unique metabolites. Our initial comparative analysis of the model with Escherichia coli has revealed several distinctive metabolic reactions, mainly in amino acid metabolism and carotenoid biosynthesis, producing relevant compounds to retain the cellular membrane for withstanding high temperature. Constraints-based flux analysis was, then, applied to simulate the metabolic state in glucose minimal and amino acid rich media. Remarkably, resulting growth predictions were highly consistent with the experimental observations. The subsequent comparative flux analysis under different environmental conditions highlighted that the cells consumed branched chain amino acids preferably and utilized them directly in the relevant anabolic pathways for the fatty acid synthesis. Finally, gene essentiality study was also conducted via single gene deletion analysis, to identify the conditional essential genes in glucose minimal and complex media.ConclusionsThe reconstructed genome-scale metabolic model elucidates the phenotypes of T. thermophilus, thus allowing us to gain valuable insights into its cellular metabolism through in silico simulations. The information obtained from such analysis would not only shed light on the understanding of physiology of thermophiles but also helps us to devise metabolic engineering strategies to develop T. thermophilus as a thermostable microbial cell factory.

Correlating enzyme annotations with a large set of microbial growth temperatures reveals metabolic adaptations to growth at diverse temperatures

“…However, a communality is that all three strains relied heavily on glycolysis and the TCA cycle. Studies on Thermus thermophilus, possibly the most well-studied thermophile, revealed alternative pathways of amino acids synthesis, with lysine being synthesized by alpha-aminoadipate pathway instead of diaminopimelate pathway (Kosuge and Hoshino 1998) and homocysteine produced from an alternative precursor, O-acetyl-L-homoserine (Lee et al 2014) . T. thermophilus is also known to produce a variety of polyamines, the most common ones, spermidine and spermine, are synthesised using a distinct pathway from L-arginine via aminopropyl agmatine (Oshima 2007) .…”

Co-utilization of glucose and xylose by evolved Thermus thermophilus LC113 strain elucidated by 13 C metabolic flux analysis and whole genome sequencing

“…In order to construct the lumped biomass equation the composition of biomass of T. thermophilus LC113 grown at 81 °C was measured using the methods described in (Long and Antoniewicz, 2014). Figure 3 compares the measured biomass composition for T. thermophilus LC113 and the biomass composition previously reported for T. thermophilus HB27 (Lee et al, 2014). Overall, we measured a higher protein content for T. thermophilus LC113 (68% of dry weight) compared to T. thermophilus HB27 (50 wt%), and a lower RNA content (10 wt%) compared to 21 wt% for T. thermophilus HB27 (note that the value of 21 wt% was not measured directly, but was assumed the same as for E. coli ).…”

“…These results suggest that different T. thermophilus strains grown at different temperatures may have a significantly different biomass composition, i.e. biomass composition of T. thermophilus HB27 was characterized at 70 °C (Lee et al, 2014). It is therefore important to measure biomass composition under relevant experimental conditions to ensure that the biomass reaction used for 13 C-MFA calculations is representative of the organism studied.…”

“…The model is based on the validated model described previously for T. thermophilus HB8 (Swarup et al, 2014). The model includes all major metabolic pathways of central carbon metabolism, including glycolysis, non-oxidative pentose phosphate pathway, TCA cycle, glyoxylate shunt, anaplorotic and cataplerotic reactions, reactions for xylose metabolism, as well as lumped amino acid biosynthesis pathways, and a lumped cell growth reaction that is based on the measured biomass composition and information provided in (Lee et al, 2014). The oxidative pentose phosphate pathway was not included in the model based on the results described in (Swarup et al, 2014).…”

“…Measured biomass composition for T. thermophilus LC113 (mean ± stdev, n =2) compared to T. thermophilus HB27 (Lee et al, 2014). …”