Identification of improved Gal2 mutants
To identify Gal2 variants with improved xylose transport properties in the presence of glucose, the GAL2 wildtype open reading frame was subjected to error-prone PCR (epPCR) as described in “Materials and methods”. The obtained PCR products were co-transformed with the linearized p426H7 plasmids into the strain AFY10X to allow for in vivo plasmid assembly and the transformants were initially plated on the permissive, ethanol-containing (SCE-Ura) agar plates. The resulting colonies were subsequently replica-plated on media containing glucose/xylose mixtures and screened for fastest growth based on the colony size. One of fastest growing clones (designated ep3.1) was selected for further characterization. The GAL2-plasmid was isolated and sequenced, revealing five distinct mutations causing amino acid exchanges, namely M107K, V239L, N376Y, M435I and L558S. The substitution N376Y was already described in our previous publication to enable glucose-insensitive xylose transport, but its transport activity for the pentose was rather low17. We therefore concluded that at least one of the additional mutations is responsible for the improved phenotype of the ep3.1 mutant. To test this assumption, we combined each of the four mutations individually with N376Y and tested the growth conferred by the resulting plasmids on different carbon sources. For growth tests on hexoses (glucose and galactose) the strain EBY.VW4000, which is deficient for all transporters capable of hexose transport in S. cerevisiae16 was used, whereas the growth on pentoses was assayed in the screening strain AFY10X. In accordance with our previous observations17, the constructs containing the N376Y mutations were not able to confer growth on glucose (Supplementary Fig. S1). On xylose or xylose/glucose mixtures, the superior properties of the ep3.1 variant compared to the N376Y single mutant could be clearly attributed to the M435I mutation (Supplementary Fig. S2). The N376Y/M435I variant was the only double mutant that conferred strong growth on pure xylose and glucose/xylose mixture.
To see more subtle differences between the different variants than is possible on agar plates, we grew liquid cultures and measured the consumption of xylose in AFY10X cells transformed with the wildtype, ep3.1 and the N376Y/M435I variants (Fig. 1). The media contained 0.5% (w/v) xylose with or without glucose addition (2% w/v). Comparing the growth and xylose consumption rates, the N376Y/M435I variant performed even better than the ep3.1 construct, suggesting that at least one of the remaining ep3.1 mutations (M107K, V239L and L558S) has a negative impact on the xylose transport activity.
Fermentation of the screening strain AFY10X transformed with different Gal2 variants. The Gal2 constructs (wildtype, the selected mutant ep3.1 and the N376Y/M435I double mutant) were expressed from multicopy plasmids in the hxt0/hxk0 strain AFY10X. The consumption of xylose (0.5% w/v; open symbols, dashed lines) in the absence (A) or presence (B) of glucose (2% w/v) and growth curves (OD600; closed symbols, full lines) are shown. The mean values of three independent cultures are shown. The error bars are omitted for clarity.
Interestingly, the M435I mutation alone appears to have a negative influence on the transport of both glucose and xylose (Supplementary Fig. S3), which implies that there is a synergistic effect of the N376Y and M435I mutations in improving the uptake of xylose. We therefore also combined M435I with N376F, which has a lower vmax for xylose compared to wildtype Gal217. In growth tests on agar plates containing pure xylose or a mixture of xylose and glucose, the N376F/M435I variant performed comparably to the N376F single mutant, but slightly worse than the N376Y/M435I double mutant (Supplementary Fig. S4).
Xylose uptake capacity of mutant Gal2 variants
To better understand the results described above and to analyze the transport activity of the Gal2 variants independently of cell growth, which is influenced by multiple factors, we performed sugar uptake assays with radiolabeled (14C) xylose (Fig. 2). To estimate the transport capacity (defined by the maximum velocity, vmax), the assays were performed at nearly saturating xylose concentrations (500 mM). Strikingly, the measured transport velocities mirror the growth behavior of the transformants described above. Whereas the N376Y mutation alone negatively affects xylose transport17 (see also Supplementary Fig. S2), this defect is more than compensated by the additional M435I mutation. The lower transport rate by the N376F and N376F/M435I variants is consistent with the results shown in Supplementary Fig. S4.
Uptake of 14C-labelled xylose by selected Gal2 variants. Gal2 wildtype (WT) and indicated mutants were expressed in the hxt0 strain EBY.VW4000 and the uptake velocity was measured at 500 mM total xylose. The uptake velocity is shown as nmol xylose taken up per minute per mg cell dry weight (nmol min−1 mgCDW−1). Mean values and standard deviation of triplicate measurements are shown.
Taken together, the results show that the M435I substitution increases the transport capacity in combination with tyrosine (but not phenylalanine) at position 376 in a synergistic manner.
Fermentation performance of engineered strains expressing mutant Gal2 variants
After having demonstrated the superior properties of the N376Y/M435I mutant in the screening strain AFY10X, which consumes xylose very slowly (see Fig. 1), we reasoned that the “true” utility of the new transporter variant for mixed-sugar fermentations could be best challenged in a system highly optimized for pentose fermentation. We selected the strain SRY027 (see “Materials and methods” and Table 1 for details), derived from the robust, diploid industrial strain HDY.GUF12 (Ethanol Red background), which was genetically modified by integrating overexpression cassettes necessary for xylose and arabinose utilization, and further optimized to consume xylose through adaptive laboratory evolution23,24. In SRY027, the genomic GAL2 alleles were deleted to avoid any interferences with the engineered GAL2 constructs. In this background, we overexpressed different Gal2 variants from a multicopy (2µ) plasmid and performed fermentations in glucose/xylose mixtures (30 g L−1 and 10 g L−1, respectively). All transformants consumed the sugars sequentially, with a fast glucose consumption phase (up to 12 h), followed by a slower, albeit considerably efficient xylose utilization, where the pentose sugar was consumed within approximately 36 h. Despite the presence of glucose-insensitive transporter variants (all mutants in which N376 was substituted by either Y or F), none of the transformants was able to significantly co-consume both sugars. Nevertheless, they did differ in the rate of xylose consumption (Fig. 3) in a manner that mirrors the xylose uptake capacities shown in Fig. 2, i.e. the N376Y/M435I exhibiting the highest and N376F/M435I the lowest velocity.
Fermentation of the industrial strain SRY027 expressing different Gal2 variants. The Gal2 constructs (wildtype—WT or the indicated mutants) were expressed from multicopy plasmids in the strain SRY027. (A) The consumption of xylose (starting concentration 10 g L−1, solid lines; plotted on the left Y-axis) and glucose (starting concentration 30 g L−1; symbols only, plotted on the right Y-axis) was measured via HPLC analysis. The mean values and standard deviation of three independent cultures are shown. The error bars may be smaller than the symbols. (B) Residual xylose concentration measured after 24 h of fermentation is shown. All values differ significantly from the wildtype (Tukey’s multiple comparisons test, P < 0.01).
One possible explanation for the inability of the cells to co-consume glucose and xylose could be the glucose-induced internalization and degradation of Gal2 in the vacuole33. This process is induced by phosphorylation and ubiquitination of the N-terminal cytoplasmic tail and can be abolished by mutating six serine residues at positions 32, 35, 39, 48, 53 and 55 to alanine. The resulting Gal26SA is stable at the plasma membrane even in the presence of glucose28. Therefore, we combined the 6SA N-terminal tail with the N376Y/M435I double mutation. To investigate if the 6SA tail is stabilizing the N376Y/M435I mutant, we also generated green florescent protein (GFP) fusion constructs as previously described28. As shown by fluorescence microscopy, the N376Y/M435I variant is internalized in glucose/xylose mixtures, whereas the 6SA/N376Y/M435I is nearly exclusively localized at the plasma membrane (Fig. 4A), as expected. Therefore, we transformed the stabilized constructs (without fused GFP) into the SRY027 strain, and performed a fermentation experiment in mixed-sugar medium. Again, the N376Y/M435I variant enabled faster xylose consumption compared to the control (Fig. 4B,C), reducing the overall duration of total xylose consumption by approximately 20 h (corresponding to 40% of the total fermentation time). Still, the consumption of glucose and xylose was largely sequential. This suggests that the reason for the inability of the strain for the simultaneous fermentation of both sugars does not primarily lie at the level of transport but might be rather due to metabolic constraints (see “Discussion”).
Stabilization of Gal2 variants by the 6SA N-terminal tail. The sextuple mutation within the N-terminal tail (6SA) conferring resistance to glucose-induced internalization was combined with the indicated Gal2 variants. (A) N376Y/M435I (left) and 6SA/N376Y/M435I (right) Gal2 variants were expressed as GFP fusions from single copy plasmids in CEN.PK2-1C and the localization of the transporters was analyzed by fluorescence microscopy. (B) The consumption of xylose (starting concentration 10 g L−1, solid lines; plotted on the left Y axis) and glucose (starting concentration 30 g L−1; symbols only, plotted on the right Y axis) was measured via HPLC analysis. The mean values and standard deviation of three independent cultures are shown. The error bars may be smaller than the symbols. (C) Residual xylose concentration measured after 24 h of fermentation is shown. The values differ significantly (unpaired two-tailed t test, P < 0.01). The fermentation experiments in (B) and (C) were performed with the strain SRY027, in which the Gal2 constructs were expressed from multicopy plasmids.
The N376Y/M435I variant confers improved growth on arabinose as well
Besides xylose, arabinose is a relevant constituent of plant biomass. Since Gal2 is well-known to transport arabinose, we wanted to test if the N376Y/M435I double mutation is beneficial for the utilization of this pentose as well. AFY10 was enabled to metabolize arabinose by transforming it with plasmids encoding L-arabinose isomerase (araA), L-ribulokinase (araB), L-ribulose-5-P 4-epimerase (araD) and different Gal2 variants (wildtype, N376F, N376F/M435I and N376Y/M435I). Growth tests were performed on solid media containing arabinose or arabinose/glucose mixtures with these transformants. Expectedly, all N376 mutants conferred glucose-resistant growth on arabinose. Moreover, on all media, the N376Y/M435I variant conferred the strongest growth (Fig. 5), similar to what was observed with xylose (see Supplementary Fig. S4). This demonstrates that the Gal2N376Y/M435I could also be used to improve the fermentation of hydrolysates that are rich in arabinose and glucose, such as pectin-rich biomass hydrolysates.
Growth conferred by different Gal2 variants on arabinose-containing media. The strain AFY10 was transformed with 2μ plasmids expressing the indicated Gal2 variants in combination with three additional 2μ plasmid expressing araA, araB and araD. The transformants were pre-grown in liquid selective SC medium with ethanol as a permissive carbon source and spotted onto selective SC solid medium containing the indicated carbon sources. Cells were grown at 30 °C for 6 days.
