In this work, we describe two new extremely thermophilic organisms that can efficiently ferment glucose and xylose at high temperatures. The first organism is new Geobacillus strain LC300 with extremely fast growth rates on glucose and xylose at 72 °C. Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced and detailed 13C-metabolic flux analysis (13C-MFA) was performed using parallel labeling experiments. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in fed-batch culture, where cells maintained high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications. The second strain we describe is an evolved Thermus thermophilus strain LC113 that efficiently co-utilizes glucose and xylose, the two most abundant sugars in lignocellulosic biomass, at high temperatures and without any carbon catabolite repression. To generate the strain, wild-type T. thermophilus HB8 was first evolved on glucose to improve its growth characteristics. The cells were then further evolved on xylose. The resulting strain, designated T. thermophilus LC113, was characterized in detail via growth studies and 13C-MFA. Compared to the starting strain, the evolved strain had an increased growth rate, increased biomass yield, increased tolerance to high temperatures up to 90 °C, and gained the ability to grow on xylose. At the optimal growth temperature of 81 °C the maximum growth rate of T. thermophilus LC113 on glucose and xylose was 0.44 and 0.46 h-1, respectively. In medium containing both glucose and xylose the strain efficiently co-utilized the two sugars. 13C-MFA results provided insights into the flexible metabolism of T. thermophilus LC113 that allows efficient glucose and xylose co-utilization. 13C-MFA revealed that metabolic fluxes in the upper part of metabolism adjusted flexibly to varying sugar availability. Fluxes in the lower part of metabolism including TCA cycle remained remarkably constant. This is the first time that 13C-MFA has been applied to elucidate co-utilization of glucose and xylose in any organism.

Extremely thermophilic organisms for efficient glucose and xylose fermentations

253rd National Meeting (2017)

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