Discovery and characterization of novel d-xylose-specific transporters from Neurospora crassa and Pichia stipitis

Abstract

Saccharomyces cerevisiae is considered one of the most promising organisms for ethanol production from lignocellulosic feedstock. Unfortunately, pentose sugars, which comprise up to 30% of lignocellulose, cannot be utilized by wild type S. cerevisiae. Heterologous pathways were introduced into S. cerevisiae to enable utilization of d-xylose, the most abundant pentose sugar. However, the resulting recombinant S. cerevisiae strains exhibited a slow growth rate and poor sugar utilization efficiency when grown on d-xylose as the sole carbon source. d-xylose uptake is the first step of d-xylose utilization. d-xylose can only enter yeast cells through hexose transporters, which have two orders of magnitude lower affinity towards d-xylose compared to hexoses. It was also shown that inefficient pentose uptake is the limiting step in some d-xylose metabolizing yeast strains. Here we report the cloning and characterization of two novel d-xylose-specific transporters from Neurospora crassa and Pichia stipitis. These two transporters were identified from a total of 18 putative pentose transporters. They were functionally expressed and properly localized in S. cerevisiae as indicated by HPLC analysis and fluorescence confocal microscopy, respectively. Kinetic parameters of the d-xylose-specific transporters were determined using a (14)C-labeled sugar uptake assay. Use of pentose-specific transporters should improve d-xylose consumption and ethanol production in fast d-xylose assimilating strains, thereby lowering the cost of lignocellulosic ethanol production.

Engineering of yeast hexose transporters to transport d -xylose without inhibition by d -glucose

“…Transport is a bottleneck in xylose fermentation, at least at low concentrations, and certainly is exacerbated with improvements of intracellular xylose metabolism (15,16,21). Some heterologous xylose transporters have been identified (11,(31)(32)(33), and increased transporter expression has proven to be beneficial for xylose-fermentation performance (17,19,20). The drawback of all these transporters is that they are likewise inhibited by glucose.…”

“…Inhibition of transport has also been directly verified by uptake assays for single transporters (11,26), as well as for S. cerevisiae (9) and different xylose-using yeast species (e.g., Candida shehatae, which also shows a sequential consumption of glucose and xylose) (27)(28)(29)(30). All known xylose transporters that can functionally be expressed in S. cerevisiae are neither selective for xylose, nor do they have a higher affinity for xylose, leading to competitive inhibition by glucose (31)(32)(33). Transport affinity for glucose is often two orders of magnitude higher than for xylose.…”

Organic acid production in Aspergillus niger and other filamentous fungi

“…The affinity for xylose of the six transporters ranged from 90 µM (XltA) to 15 mM (XltB), being, in most cases, higher than those reported for most of the fungal xylose transporters characterized to date, with values between 80 µM and 150 mM (Du et al, 2010;Leandro et al, 2006;Vankuyk PA et al, 2004;Weierstall et al, 1999). A. niger XltA showed to have a very high affinity for xylose (0.09 ± 0.03 mM), which is even higher than reported for the E. coli xylose transporter XylE (0.47 mM) (Davis and Henderson, 1987;Farwick et al, 2014), and the A.…”

“…These yeast mutant strains, unable to grow on glucose, fructose, mannose and galactose as a single carbon source, have also subsequently been used as tools for the functional characterisation of sugar transporters from other fungal species (Colabardini et al, 2014;dos Reis et al, 2013;Du et al, 2010;Leandro et al, 2013;Polidori et al, 2007;Saloheimo et al, 2007;Vankuyk PA et al, 2004;Wahl et al, 2010) In contrast to S. cerevisiae, the only functionally validated sugar transporters in A. niger are the recently identified D-galacturonic acid transporter GatA (Martens-Uzunova and Schaap, 2008;Sloothaak et al, 2014), two fructose transporters (Coelho et al, 2013) and the high-affinity sugar/H + symporter MstA (Vankuyk PA et al, 2004). Furthermore, transcriptional data for the A. niger mstC gene suggests that it encodes a low-affinity glucose transporter (Jørgensen et al, 2007), but no experimental data supporting its role as a functional sugar transporter is publicly available.…”

“…Native industrially relevant xylose transporters that show high affinity and capacity for xylose uptake have been identified from Escherichia coli, Pichia stipitis and Candida intermedia (Leandro et al, 2006;Weierstall et al, 1999). Additionally several transporters from filamentous fungi have been reported to transport xylose, such as An25 and An29-2 from Neurospora crassa, XtrD from A. nidulans, MstA from A. niger and Xlt1 and Str1 from T. reesei (Colabardini et al, 2014;Du et al, 2010;Huang et al, 2015;Saloheimo et al, 2007). Of this list only An25, and MstA have been biochemically characterized.…”