Extremely Thermophilic Organisms for Efficient Glucose and Xylose Fermentations

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, 2.15 h^-1 on glucose and 1.52 h^-1 on xylose (doubling time less than 30 minutes). The corresponding specific glucose and xylose utilization rates are 5.55 g/g/h and 5.24 g/g/h, respectively. As such, 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 using PacBio’s RS II single-molecule real-time (SMRT) sequencing platform and detailed ¹³C-metabolic flux analysis (¹³C-MFA) was performed using parallel labeling experiments. Additionally, 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 with [1,6-¹³C]glucose, [5-¹³C]xylose, and [1,6-¹³C]glucose + [5-¹³C]xylose as isotopic tracers. 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, i.e. upper glycolysis and non-oxidative pentose phosphate pathway, adjusted flexibly to varying sugar availability. On the other hand, 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. Therefore, this study also serves as an important benchmark for future investigations of glucose and xylose co-utilization in other strains.