title: (13)C metabolic flux analysis of the extremely thermophilic, fast growing, xylose-utilizing Geobacillus strain LC300.
abstract: 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 (13)C-labeling experiments and applied (13)C-metabolic flux analysis to quantify precise metabolic fluxes. Specifically, the complete set of singly labeled xylose tracers, [1-(13)C], [2-(13)C], [3-(13)C], [4-(13)C], and [5-(13)C]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 (13)C-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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, Newark, DE 19716, USA.
authors.forename: Lauren T
authors.initials: LT
authors.lastname: Cordova
authors.affiliation: Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, Newark, DE 19716, USA. Electronic address: mranton@udel.edu.
authors.forename: Maciek R
authors.initials: MR
authors.lastname: Antoniewicz
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D066241:Carbon-13 Magnetic Resonance Spectroscopy / Q000379:methods*; D049109:Cell Proliferation / Q000502:physiology; D002952:Citric Acid Cycle / Q000502:physiology; D056506:Geobacillus / Q000145:classification* / Q000302:isolation & purification / Q000378:metabolism*; D006019:Glycolysis / Q000502:physiology; D064688:Metabolic Flux Analysis / Q000379:methods*; D010427:Pentose Phosphate Pathway / Q000502:physiology; D013045:Species Specificity; D014994:Xylose / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D014994:Xylose
keywords: COMPLETE-MFA; Metabolic network model; Parallel labeling experiments; Thermophile; Xylose metabolism