13C metabolic flux analysis of the extremely thermophilic, fast growing, xylose-utilizing Geobacillus strain LC300

• 13C-Flux analysis of Geobacillus LC300 was performed using COMPLETE-MFA.
• Parallel experiments performed with [1-13C], [2-13C], [3-13C], [4-13C], and [5-13C]xylose.
• Two optimal isotopic tracers were identified, [2-13C]xylose and [5-13C]xylose.
• Pentose phosphate pathway, glycolysis and TCA cycle are highly active in Geobacillus LC300.
• Oxidative pentose phosphate pathway is also active and contributes to NADPH production.

Thermophiles are increasingly used as versatile hosts in the biotechnology industry. One of the key advantages of thermophiles is the potential to achieve high rates of feedstock conversion at elevated temperatures. The recently isolated Geobacillus strain LC300 grows extremely fast on xylose, with a doubling time of less than 30 min. In the accompanying paper, the genome of Geobacillus LC300 was sequenced and annotated. In this work, we have experimentally validated the metabolic network model using parallel 13C-labeling experiments and applied 13C-metabolic flux analysis to quantify precise metabolic fluxes. Specifically, the complete set of singly labeled xylose tracers, [1-13C], [2-13C], [3-13C], [4-13C], and [5-13C]xylose, was used for the first time. Isotopic labeling of biomass amino acids was measured by gas chromatography mass spectrometry (GC–MS). Isotopic labeling of carbon dioxide in the off-gas was also measured by an on-line mass spectrometer. The 13C-labeling data was then rigorously integrated for flux elucidation using the COMPLETE-MFA approach. The results provided important new insights into the metabolism of Geobacillus LC300, its efficient xylose utilization pathways, and the balance between carbon, redox and energy fluxes. The pentose phosphate pathway, glycolysis and TCA cycle were found to be highly active in Geobacillus LC300. The oxidative pentose phosphate pathway was also active and contributed significantly to NADPH production. No transhydrogenase activity was detected. Results from this work provide a solid foundation for future studies of this strain and its metabolic engineering and biotechnological applications.