Identification and characterization of putative xylose and cellobiose transporters in Aspergillus nidulans

One of the major drawbacks in biofuel production from lignocellulosic plant material is the inability of fermenting organisms to produce ethanol when growing on sugars other than glucose. Lignocellulose is composed of hexose (glucose) and pentose sugars (mainly xylose) and enzymatic deconstruction of it by, for example, filamentous fungi results in the release of these monosaccharides as well as in the release of oligosaccharides (e.g., the glucose dimer cellobiose) [6]. More specifically, most fermenting organisms are not very efficient at transporting pentose sugars and oligosaccharides into the cell. Complete conversion of all the sugars found in lignocellulose is desired to make 2G biofuel production an economically feasible process [14]. S. cerevisiae is one of the preferred organisms for fermentation as it is already applied in various industrial processes and is generally regarded as safe [10].

One successful strategy to improve non-glucose uptake in S. cerevisiae is to introduce transporters, from other organisms into its genome. The genomes of filamentous fungi, which are able to internalize a wide variety of mono- and oligosaccharides, are, therefore, screened to find transporters which are able to transport non-glucose sugars. Although this has greatly improved the ability of S. cerevisiae to take up pentose sugars such as xylose or cellodextrins such as cellobiose [13, 20, 21, 26], further genetic engineering is required to optimize non-glucose sugar transport. In addition, most transporters encoded by filamentous fungi have not been characterized yet, although these organisms also play a major role in 2G biofuel production. This work, therefore, aimed at identifying xylose- and cellobiose-specific transporters through screening the genome of the filamentous fungus A. nidulans and characterizing them when introduced into S. cerevisiae.

A BLAST analysis of XtrG, encoded by xtrG and identified as being upregulated in the presence of xylose [33], showed similarity to the N. crassa cellobiose transporter CDT-2. The name of XtrG was subsequently changed to CltA (cellobiose transporter A). Furthermore, another A. nidulans protein, encoded by AN2814, showed high identity to the N. crassa cellodextrin transporter CDT-1 and was, therefore, termed CltB. Cellooligosaccharides, such as cellobiose, released during enzymatic degradation of cellulose, have been shown to be important molecules for cellulase gene induction in filamentous fungi, such as T. reesei, N. crassa, and P. oxalicum [49–53]. The expression of cltA increased in the presence of cellobiose. Deletion of cltB and of cltA and cltB simultaneously, but not of cltA, resulted in reduced growth and cellulase secretion in the presence of cellobiose during the first 48 h, although this growth was restored after 72 h in the ?cltB strain. In contrast, when introduced into S. cerevisiae strain SC9721 together with a ?-glucosidase-encoding gene, CltB conferred only slow growth in the presence of different concentrations of cellobiose. These results suggest that the main function of CltB may not be cellobiose transport; it may also function as a transceptor involved in signaling the presence of lignocellulosic biomass. However, we have shown that the introduction of additional functional copies of CltB increases the growth in the presence of low concentrations of cellobiose, strongly indicating CltB is able to transport cellobiose. In N. crassa, CDT-1 and CDT-2, in addition to being cellobiose transporters, have been hypothetized to having a role in downstream signaling upon the detection of cellulose by the fungus [42]. Furthermore, the T. reesei transporters STP1 and CRT1 were also proposed to play an important role in the induction of cellulase-encoding genes [32]. This is the first time that a potential transceptor role has been identified for a protein in A. nidulans which is involved in the signaling process of cellulose.

In contrast, deletion of cltA did not result in reduced biomass accumulation and cellulase activity in the presence of cellobiose, but introduction of CltA into S. cerevisiae conferred growth in the presence of different concentrations of cellobiose. These results indicate that CltA (formerly XtrG) is a cellobiose transporter and this is the first time that a cellobiose-specific transporter has been identified in A. nidulans. The genome of A. nidulans encodes 357 MFS transporters and redundancy is very likely to exist between these transporters. This redundancy could compensate for the individual loss of CltA (growth not affected) and CltB (growth restored after 72 h), suggesting that other transporters exist with transceptor activities. Deletion of both CltA and CltB had a more severe impact on fungal growth in the presence of cellobiose, suggesting that these two proteins do play major roles in cellobiose signaling and uptake and may work together to ensure growth on cellobiose. An intriguing aspect of the biology of A. nidulans CltA is why it is also induced in the presence of xylose when it actually is a cellobiose transporter. It is possible that cellobiose transporters could also transport xylooligosaccharides or that the main transcriptional activator of genes encoding proteins required for xylose and xylan metabolism, XlnR (on which CltA was shown to be dependent) can also induce genes encoding cellobiose transporters. Actually, it has already been demonstrated that N. crassa CDT-2 is able to transport both cellodextrins and xylodextrins [54]. Indeed, in A. niger, cbhA, and cbhB, encoding cellobiohydrolases which catalyze the depolymerization of cellulose were shown to be expressed at much higher levels in the presence of xylose and xylan than when compared to sophorose and cellulose [55]. Furthermore, when grown in the presence of xylan or cellulose, A. nidulans always secretes both cellulases and xylanases (data not shown). This is probably due to the fact that cellulose and hemicelluloses are always found together as they make up the plant cell wall.

Another characteristic of sugar transporters of the MFS is that they very often accept multiple monosaccharides and are, therefore, capable of transporting different sugars into the fungal cells; for example, XtrD transports various monosaccharides [33]. Previously, four glucose transporters were identified in A. nidulans (HxtB-HxtE) and we decided to verify if they could be involved in xylose transport. Deletion of hxtB, and to some extent hxtE, resulted in significantly reduced growth in the presence of high (2 % w/v) concentrations of xylose after 48 h. HxtB (also named MstC) has previously been shown to be a high-affinity glucose transporter which also appears to be able to translocate mannose, galactose, fructose, and xylose [56]. The expression of hxtB is not induced in the presence of glucose but rather in the absence of it, and this gene is also subject to CreA-mediated carbon catabolite repression [56]. This difference in growth and gene expression between high and low concentrations of xylose may therefore be explained by the high-affinity uptake system in which HxtB plays a role or due to the different time points at which both assays were carried out. Furthermore, gene transcription does not necessarily reflect protein secretion and growth. Growth on glucose was not affected, probably, because HxtB is a high affinity glucose transporter which is expressed when glucose is present in low concentrations. Glucose uptake in the presence of high concentrations of this sugar occurs via low affinity transporters such as MstE, which is induced by glucose in A. nidulans [57]. Furthermore, deletion of hxtB resulted in decreased affinity for and decreased transport capacity of xylose, indicating that HxtB is also a xylose transporter in addition to being a glucose transporter. In agreement, when HxtB::GFP was introduced into S. cerevisiae, where it located to the plasma membrane, it conferred growth of S. cerevisiae in the presence of different concentrations of xylose and was capable of successfully transporting xylose into the yeast cell. Furthermore, the S. cerevisiae HxtB::GFP strain produced ethanol when growing in xylose-rich media. This work, therefore, identified an A. nidulans transporter which in addition to taking up glucose was also efficient at transporting xylose.

Glucose is the preferred carbon source for most microorganisms as it provides rapid energy for survival and niche colonization. Hence, most fungi, including A. nidulans are specialized in taking up glucose as soon as it is detected in the environment through high and low affinity uptake systems. As shown in this work, A. nidulans transporters have preferentiality for glucose but when this sugar is not available, switch to transporting other sugars, such as xylose, arabinose, or galactose. In addition to the search for transporters which can also translocate pentose or cellodextrins, the focus of research should be directed to the molecular engineering of individual transporters to render them “blind” to glucose and increase affinity and specificity for alternative, non-glucose sugars as was done by [23, 58]. In addition, yeast strains, which already harbor heterologous introduced transporters, can be genetically modified through directed evolution to improve growth in the presence of pentose sugars or cellodextrins [33]. At the same time, introducing components required for the efficient transport and metabolism of various different sugars into S. cerevisiae, thus allowing the co-fermentation of multiple carbohydrates, has also proved to be a successful strategy [59, 60]. Furthermore, although the genome of A. nidulans (and other filamentous fungi) encodes a multitude of MFS sugar transporters, they have scarcely been characterized and further studies, including those on carbon source sensing and signaling, are required to confirm or reject the above proposed hypothesis. This work identified a cellobiose transporter and a potential cellobiose transceptor in A. nidulans, a role which has also been associated with cellobiose transporters in N. crassa and T. reesei. Furthermore, this study provided further characterization of a glucose/xylose transporter. Taken together, this work provides a preliminary screening and characterization of MFS transporters in A. nidulans and lays a basis for further exploration of sugar sensing and transport in industrially relevant fungi.