D-glucose overflow metabolism in an evolutionary engineered high-performance D-xylose consuming Saccharomyces cerevisiae strain

Abstract

Co-consumption of D-xylose and D-glucose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. There is a need for improved sugar conversion rates to minimize fermentation times. Previously, we have employed evolutionary engineering to enhance D-xylose transport and metabolism in the presence of D-glucose in a xylose-fermenting S. cerevisiae strain devoid of hexokinases. Re-introduction of Hxk2 in the high performance xylose-consuming strains restored D-glucose utilization during D-xylose/D-glucose co-metabolism, but at rates lower than the non-evolved strain. In the absence of D-xylose, D-glucose consumption was similar to the parental strain. The evolved strains accumulated trehalose-6-phosphate during sugar co-metabolism, and showed an increased expression of trehalose pathway genes. Upon the deletion of TSL1, trehalose-6-phosphate levels were decreased and D-glucose consumption and growth on mixed sugars was improved. The data suggest that D-glucose/D-xylose co-consumption in high-performance D-xylose consuming strains causes the glycolytic flux to saturate. Excess D-glucose is phosphorylated enters the trehalose pathway resulting in glucose recycling and energy dissipation, accumulation of trehalose-6-phosphate which inhibits the hexokinase activity, and release of trehalose into the medium.

Keywords: D-xylose transporter; bioethanol; glycolysis; sugar transport; trehalose-6-phosphate; yeast.

Figure 1.

D-glucose (◆), D-xylose (▓) and total sugar (▲) consumption rates in mmol/gDW.hr (A), and in mmol/l.hr (B), of the DS71054 hexokinase deletion strain (DS) and the evolved derivatives DS71054-evoB, DS71054-evo3, DS71054-evo4 and DS71054-evo6, all complemented with the HXK2 gene. Cells were grown anaerobically on minimal medium with 7% D-glucose and 3% D-xylose, or with only 7% D-glucose (△). Error bars were obtained from biological triplicates.

Figure 2.

D-glucose (◆), D-xylose (▓) and total sugar consumption rates (in mmol/l.h) (▲) by the parental DS71054 hexokinase deletion strain (DS) and the evolved derivatives DS71054-evoB, DS71054-evo3, DS71054-evo4 and DS71054-evo6 grown anaerobically in minimal medium supplemented with 7% D-glucose and 3% D-xylose. The strains were complemented with Hxk1 (A), Glk1 (B), Hxk2-Y (C) (Bergdahl et al. 2013) and spHxk2 (D) (Bonini, Van Dijck and Thevelein 2003). Error bars were obtained from biological duplicates.

Figure 3.

D-glucose (grey bars) and D-xylose (white bars) consumption rates (in mmol/l.hr) by DS71054-evo6, DS71054-evo6-ΔTps3 and DS71054-evo6-ΔTsl1 complemented with the HXK2 gene. Cells were grown anaerobically on 7% D-glucose and 3% D-xylose. Error bars were obtained from biological duplicates.

Figure 4.

Intracellular trehalose-6-phosphate in the DS71054 hexokinase deletion strain (DS), the evolved derivative DS71054-evo6, and indicated deletion mutants of the enzymes of the trehalose pathway, all complemented with the HXK2 gene. The strains were grown anaerobically with 7% D-glucose and 3% D-xylose (grey bars), 7% D-glucose (white bar) or 3% D-xylose (white bar). The intracellular concentration was measured after 12 h and signal normalized for the total ion count. Error bars were obtained from biological duplicates.

Figure 5.

Schematic view of the DS71054-evo6 strain co-consuming D-glucose and D-xylose. D-glucose is transported into the cell via Hxt2 and Hxt4. Hxt37 N367I transports solely D-xylose. The metabolism of D-glucose is mediated via the enzymes hexokinase (Hxk2) and glucose-6-phosphate isomerase (Pgi1) to yield fructose-6-phosphate, which is further converted in the glycolytic pathway into ethanol. Accumulation of glucose-6-phosphate leads to accumulation of trehalose-6-phosphate which inhibits Hxk2 therefore decreasing the glycolysis rate. Furthermore, in in vitro studies, Hxk2 can also be inhibited by accumulation of D-xylose in the presence of MgATP, but in this study an expected increase in the phosphorylation of Hxk2 was not observed. The conversion of D-xylose into xylulose is in S. cerevisiae possible via the introduction of xylose isomerase (pirXI), followed by the phosphorylation of xylulose into xylulose-5-phosphate by xylulose kinase (Xks1). Xylulose-5-phosphate enters the pentose phosphate pathway to eventually yield fructose-6-phosphate and glyceraldehyde-3-phosphate (GAP) that can be converted into ethanol.