Rewiring yeast sugar transporter preference through modifying a conserved protein motif

Abstract

Utilization of exogenous sugars found in lignocellulosic biomass hydrolysates, such as xylose, must be improved before yeast can serve as an efficient biofuel and biochemical production platform. In particular, the first step in this process, the molecular transport of xylose into the cell, can serve as a significant flux bottleneck and is highly inhibited by other sugars. Here we demonstrate that sugar transport preference and kinetics can be rewired through the programming of a sequence motif of the general form G-G/F-XXX-G found in the first transmembrane span. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose. Through saturation mutagenesis and subsequent rational mutagenesis, four transporter mutants unable to confer growth on glucose but able to sustain growth on xylose were engineered. Specifically, Candida intermedia gxs1 Phe(38)Ile(39)Met(40), Scheffersomyces stipitis rgt2 Phe(38) and Met(40), and Saccharomyces cerevisiae hxt7 Ile(39)Met(40)Met(340) all exhibit this phenotype. In these cases, primary hexose transporters were rewired into xylose transporters. These xylose transporters nevertheless remained inhibited by glucose. Furthermore, in the course of identifying this motif, novel wild-type transporters with superior monosaccharide growth profiles were discovered, namely S. stipitis RGT2 and Debaryomyces hansenii 2D01474. These findings build toward the engineering of efficient pentose utilization in yeast and provide a blueprint for reprogramming transporter properties.

Keywords: metabolic engineering; protein engineering; transporter engineering; xylose metabolism.

Fig. 1.

Sequence categorization and phenotypic classification of native and heterologous transporters. (A) The distribution of phenotypic classes for all 46 transporters. (B) The distribution of each sequence category present in each phenotypic class. Transporters containing the conserved motif are enriched in the phenotypic classes that confer growth on xylose. (C) Weblogos of the phenotypic classes illustrate enrichment of the G-G/F-XXXG motif in TMS1. µ_all = 0 represents no growth in the five carbon sources tested; µ_X = 0 represents growth on hexoses but not xylose, µ_X < µ_G represents growth on xylose is less than that on glucose, and µ_X > µ_G represents growth on xylose is greater than that on glucose.

Fig. 2.

Classification tree of fractional change in carbon source growth profile. This figure depicts hypothetical fractional change data to demonstrate how these phenotypes were classified. Little fractional change across all sugars indicates that the substitution does not control efficiency or selectivity in this background. Amplification or attenuation of growth rates across all carbon sources indicates an efficiency substitution. Amplification of growth on one sugar, ideally xylose, and attenuation of all others indicates a selectivity substitution.

Fig. 3.

Fractional change of saturation mutagenesis libraries of C. intermedia GXS1. Fractional change in growth by substitutions at positions 38 (A), 39 (B), and 40 (C). The solid line is the confidence line for no growth based on the negative control sample. (A–C) Error bars were calculated using the sum of least squares method.

Fig. 4.

Growth characterization of C. intermedia gxs1 triple mutants. (A) Fractional change from wild type for the two triple mutants and an empty vector control. A bar chart of the exponential growth rates from which fractional change was calculated can be found in SI Appendix. Error bars were calculated using the sum of least squares method. (B) Average growth curves on xylose based on OD₆₀₀. (C) Average growth curves on glucose based on OD₆₀₀. Calculated exponential growth rates are shown in SI Appendix, Fig. S7.

Fig. 5.

Further characterization of C. intermedia gxs1 Phe³⁸Ile³⁹Met⁴⁰ triple mutant. (A) Glucose uptake at high cell density for S. cerevisiae EX.12 expressing wild type, Phe³⁸Ile³⁹Met⁴⁰, and empty vector. (B) Xylose uptake at high cell density for S. cerevisiae EX.12 expressing wild type, Phe³⁸Ile³⁹Met⁴⁰, and empty vector. (C) Inhibition of the growth rate on xylose with increasing glucose concentration. (D) V_max of both the wild type and the mutant. (E) K_M of both the wild type and triple mutant. (A–E) Error is based on SD of biological replicates.

Fig. 6.

Growth characterization of S. stipitis RGT2 and mutants. (A) Fractional change from wild type for the two single mutants and an empty vector control. Error bars were calculated using the sum of least squares method. A bar chart of the exponential growth rates from which fractional change was calculated can be found in SI Appendix. (B) Average growth curves on xylose based on OD₆₀₀. (C) Average growth curves on glucose based on OD₆₀₀. Calculated maximum exponential growth rates are shown in SI Appendix, Fig. S7.

Fig. 7.

Growth characterization of S. cerevisiae HXT7 and mutants. (A) Fractional change from wild type for the mutants and an empty vector control. Error bars were calculated using the sum of least squares method. A bar chart of the exponential growth rates from which fractional change was calculated can be found in SI Appendix. (B) Average growth curves on xylose based on OD₆₀₀. (C) Average growth curves on glucose based on OD₆₀₀. Calculated maximum exponential growth rates are shown in SI Appendix, Fig. S7.