Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae

Abstract

In previous work, we developed a Saccharomyces cerevisiae strain (DLG-K1) lacking the main monosaccharide transporters (hxt-null) and displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities. This strain proved to be a useful chassis strain to study new glucose/xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Proteins with high amino acid sequence similarity (78–80%) to SsXUT1 were identified from Spathaspora passalidarum and Spathaspora arborariae genomes. The characterization of these putative transporter genes (SpXUT1 and SaXUT1, respectively) was performed in the same chassis strain. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested (including glucose and xylose), but after being grown in maltose, the uptake of 14C-glucose and 14C-xylose was detected. While SsXUT1 lacks lysine residues with high ubiquitinylation potential in its N-terminal domain and displays only one in its C-terminal domain, both SpXUT1 and SaXUT1 transporters have several such residues in their C-terminal domains. A truncated version of SpXUT1 gene, deprived of the respective 3′-end, was cloned in DLG-K1 and allowed growth and fermentation in glucose or xylose. In another approach, two arrestins known to be involved in the ubiquitinylation and endocytosis of sugar transporters (ROD1 and ROG3) were knocked out, but only the rog3 mutant allowed a significant improvement of growth and fermentation in glucose when either of the XUT permeases were expressed. Therefore, for the efficient heterologous expression of monosaccharide (e.g., glucose/xylose) transporters in S. cerevisiae, we propose either the removal of lysines involved in ubiquitinylation and endocytosis or the use of chassis strains hampered in the specific mechanism of membrane protein turnover.

Engineered yeasts and lignocellulosic biomaterials: shaping a new dimension for biorefinery and global bioeconomy

“…Based on numerous studies on the utilization of extracellular xylose via the xylulose isomer for efficient fermentation, six pathways have been identified and engineered in S. cerevisiae for xylose metabolism; introduction of xylose transporter into the cell, efficient xylose utilization through iterative cycles before selection [ 165 ], expression of xylose or xylitol dehydrogenase (XDH) or only the xylose isomerase gene to bring the intracellular xylose and xylulose [ 166 ], increasing the xylulose stage to xylulose-5-phosphate via the expression of endogenous xylulokinase gene [ 167 ], correction of redox imbalance by introduction of xylose reductase and xylitol dehydrogenase to change either their co-factor specificity or adjusting their NADH-/NADPH-producing steps [ 97 ], and lastly, expansion of PPP flux by over-expression of ribulose-5-phospho-3-epimerase enzyme (RPE), ribulose-5-phosphate isomerase enzyme (RPI), transketolase enzyme (TKL), and transaldolase enzyme (TAL) [ 168 , 169 ].…”