Analysis of metabolic pathways and fluxes in a newly discovered thermophilic and ethanol‐tolerant Geobacillus strain

Abstract

1A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius 2 M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and is tolerant to 3 high ethanol concentrations (10% v/v). We have investigated the central metabolism of this 4 bacterium using both in vitro enzyme assays and 13 C-based flux analysis to provide insights into 5 the physiological properties of this extremophile and explore its metabolism for bioethanol or 6 other bioprocess applications. Our findings show that glucose metabolism in G. 7 thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the 8 TCA cycle; the Entner-Doudoroff pathway and transhydrogenase activity were not detected. 9Anaplerotic reactions (including the glyoxylate shunt, pyruvate carboxylase and 10 phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not 11 be accurately determined using amino acid labelling. When growth conditions were switched 12 from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 13 100 units (gm DCW) -1 ·hr -1 ) through the TCA cycle and oxidative pentose phosphate pathway 14 were reduced from 64±3 to 25±2 and from 30±2 to 19±2, respectively. The carbon flux under 15 microaerobic growth was directed to ethanol, L-lactate (>99% optical purity), acetate, and 16formate. Under fully anaerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid 17 fermentation process and exhibited a maximum ethanol yield of 0.38±0.07 mol mol -1 glucose. In 18 silico flux balance modelling demonstrates that lactate and acetate production from G. 19 thermoglucosidasius M10EXG reduces the maximum ethanol yield by approximately three 20 folds, thus indicating that both pathways should be modified to maximize ethanol production. 21

Characterization of the Central Metabolic Pathways in Thermoanaerobacter sp. Strain X514 via Isotopomer-Assisted Metabolite Analysis

“…The preparation and isotopic analysis of proteogenic amino acids were performed as previously described (23,27). In brief, biomass was hydrolyzed in a solution containing 6 M HCl at 100°C for 24 h. The amino acid solution was dried under air flush overnight, and amino acid samples were derivatized in tetrahydrofuran and N-(tert-butyl dimethylsilyl)-N-methyl-trifluoroacetamide (Sigma-Aldrich, St. Louis, MO) at 70°C for 1 h. A gas chromatograph (Hewlett-Packard model 7890A; Agilent Technologies, CA) equipped with a DB5-MS column (J&W Scientific, Folsom, CA) and a mass spectrometer (model 5975C; Agilent Technologies, CA) was used for analyzing amino acid labeling profiles.…”

“…Based on 13 C-labeling patterns in key amino acids, the active pathways can be traced back, and new enzymes can be revealed. In this study, 13 C-based isotopic analysis was applied to accurately examine the annotated pathways in X514 and to investigate gaps in key biosynthetic pathways (25)(26)(27). Specifically, glucose (the first or sixth carbon labeled) and pyruvate (the first carbon labeled) were used, respectively, as the sole source of carbon to grow X514.…”

Correlation of Genomic and Physiological Traits of Thermoanaerobacter Species with Biofuel Yields

“…Isotopic analysis of proteogenic amino acids was performed (42,43). Biomass was hydrolyzed in 6 M HCl at 100°C.…”

Facultative Anaerobe Caldibacillus debilis GB1: Characterization and Use in a Designed Aerotolerant, Cellulose-Degrading Coculture with Clostridium thermocellum

“…Aerobic acetate production is commonly used to generate ATP and deal with the excess acetyl coenzyme A generated by the high carbon flux through glycolysis (29). A unique characteristic that makes C. debilis GB1 desirable for use for biofuel production via CBP is its low level of lactate production under all conditions tested, which is rare in wild-type thermophilic bacilli, such as Geobacillus species (27,30,31). While both C. debilis GB1 and strain DSM 16016 can grow aerobically using respirofermentative metabolism (32), only GB1 maintains a low-lactate phenotype.…”