Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae

Abstract

f Saccharomyces cerevisiae cannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1) and intracellular ␤-glucosidase (gh1-1) genes from Neurospora crassa. Here, we report that an engineered S. cerevisiae strain expressing the putative hexose transporter gene HXT2.4 from Scheffersomyces stipitis and gh1-1 can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter when HXT2.4 is overexpressed in S. cerevisiae. However, cellobiose fermentation by the engineered strain expressing HXT2.4 and gh1-1 was much slower and less efficient than that by an engineered strain that initially expressed cdt-1 and gh1-1. The rate of cellobiose fermentation by the HXT2.4-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolved S. cerevisiae strain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higher K m and 4-fold higher V max values than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed in S. cerevisiae are suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineered S. cerevisiae strains. Ethanol production from cellulosic biomass not only is sustainable but also does not cause ethical issues like corn-or sugarbased ethanols do (1). The economic production of ethanol from cellulosic biomass requires substantial improvements in all unit operations, such as physical pretreatment, chemical or enzymatic depolymerization, and fermentation. The efficient hydrolysis of cellulose and the utilization of mixed sugars (glucose and xylose) present in cellulosic hydrolysates are especially necessary for producing cellulosic ethanol on a commercial scale (2-4).Fungal cellulases, which are widely used for degrading cellulose, exhibit weak ␤-glucosidase activity. The low ␤-glucosidase activity can lead to an accumulation of cellobiose because of its slow degradation of cellobiose into glucose during simultaneous saccharification and fermentation (SSF). The accumulation of cellobiose during SSF reduces the overall rate and efficiency of cellulose hydrolysis because cellobiose is an inhibitor of cellulases. Therefore, supplementation with bacterial or fungal ␤-glucosidase is necessary to enhance cellulose hydrolysis in spite of the disadvantages in the cost of doing so. In order to reduce the enzyme costs from supplementation with ␤-glucosidase, engineered Saccharomyce...

Rewiring yeast sugar transporter preference through modifying a conserved protein motif

“…These results demonstrated that mutations at specific residues (e.g., Phe 40 in C. intermedia GXS1) can have a significant impact on the carbohydrate selectivity of these MFS transporters. The fact that single amino acid substitutions can have such a significant impact on transport phenotype (38)(39)(40) indicates how simple homology based searches can be ineffective at identifying efficient xylose transporters (35,36). However, evidence of natural xylose exclusivity is seen in the Escherichia coli xylE transporter that has recently been crystallized (41).…”

“…As an alternative to bioprospecting, we have previously reported that xylose affinity and exponential growth rates on xylose can be improved via directed evolution of Candida intermedia glucose-xylose symporter 1 (GXS1) and Scheffersomyces stipitis xylose uptake 3 (XUT3) (38). These results demonstrated that mutations at specific residues (e.g., Phe 40 in C. intermedia GXS1) can have a significant impact on the carbohydrate selectivity of these MFS transporters. The fact that single amino acid substitutions can have such a significant impact on transport phenotype (38)(39)(40) indicates how simple homology based searches can be ineffective at identifying efficient xylose transporters (35,36).…”

“…Previously, we reported on single amino acid substitutions at Phe 40 in transmembrane span 1 (TMS1) that can alter monosaccharide transport profiles of C. intermedia GXS1 (38). The potency of this residue as well as its proximity to the outer pore of the transporter suggested it could be part of an important contact and recognition site for monosaccharides.…”

“…In this work, we report on the discovery of a conserved Gly 36 40 residue of C. intermedia GXS1 that controls transporter efficiency and selectivity. 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, taking the general form G-G/F-XXX-G. We conduct saturation mutagenesis on Val 38 , Leu 39 , and Phe 40 within the variable region of this motif in C. intermedia GXS1 to demonstrate control of sugar selectivity.…”

Leveraging transcription factors to speed cellobiose fermentation by

“…By contrast, cellobiose is an unusual substrate for S. cerevisiae, and is therefore not recognized as a readily fermentable sugar like glucose. Efforts to optimize cellobiose fermentation in engineered S. cerevisiae through combinatorial transcriptional engineering [11], experimental evolution [29], or by exploring and evolving new cellodextrin transporters [12,14,30,31] or an alternative cellobiose phosphorylase Figure 1 Suboptimal cellobiose metabolism in engineered Saccharomyces cerevisiae. (A) Fermentation profiles of recombinant cellobiose-utilizing S. cerevisiae with plasmid pRS426-BT on cellobiose or glucose in anaerobic conditions with an initial OD 600 of 1.…”