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

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ABSTRACT

Background: The conversion of lignocellulosic biomass to biofuels (second-generation biofuel production) is an environmentally friendlier alternative to petroleum-based energy sources. Enzymatic deconstruction of lignocellulose, catalyzed by filamentous fungi such as Aspergillus nidulans, releases a mixture of mono- and polysaccharides, including hexose (glucose) and pentose (xylose) sugars, cellodextrins (cellobiose), and xylooligosaccharides (xylobiose). These sugars can subsequently be fermented by yeast cells to ethanol. One of the major drawbacks in this process lies in the inability of yeast, such as Saccharomyces cerevisiae, to successfully internalize sugars other than glucose. The aim of this study was, therefore, to screen the genome of A. nidulans, which encodes a multitude of sugar transporters, for transporters able to internalize non-glucose sugars and characterize them when introduced into S. cerevisiae.

Results: This work identified two proteins in A. nidulans, CltA and CltB, with roles in cellobiose transport and cellulose signaling, respectively. CltA, when introduced into S. cerevisiae, conferred growth on low and high concentrations of cellobiose. Deletion of cltB resulted in reduced growth and extracellular cellulase activity in A. nidulans in the presence of cellobiose. CltB, when introduced into S. cerevisiae, was not able to confer growth on cellobiose, suggesting that this protein is a sensor rather than a transporter. 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. Furthermore, a previously identified glucose transporter, HxtB, was also found to be a major xylose transporter in A. nidulans. In S. cerevisiae, HxtB conferred growth on xylose which was accompanied by ethanol production.

Conclusions: This work identified a cellobiose transporter, a xylose transporter, and a putative cellulose transceptor in A. nidulans. This is the first time that a sensor role for a protein in A. nidulans has been proposed. Both transporters are also able to transport glucose, highlighting the preference of A. nidulans for this carbon source. This work provides a basis for future studies which aim at characterizing and/or genetically engineering Aspergillus spp. transporters, which, in addition to glucose, can also internalize other carbon sources, to improve transport and fermentation of non-glucose sugars in S. cerevisiae.

Electronic supplementary material: The online version of this article (doi:10.1186/s13068-016-0611-1) contains supplementary material, which is available to authorized users.

No MeSH data available.


HxtB is a xylose transporter. Fungal dry weight of the wild-type, ΔhxtB, ΔhxtC, ΔhxtD, and ΔhxtE strains was measured after strains were grown for 24 or 48 h in the presence of 1 % glucose (a) or in the presence of 0.2 and 2 % xylose (b). Xylose transport, as assessed by 14C-xylose uptake, was measured in the wild-type and ΔhxtB strains in the presence of different concentrations of xylose (c)
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Fig5: HxtB is a xylose transporter. Fungal dry weight of the wild-type, ΔhxtB, ΔhxtC, ΔhxtD, and ΔhxtE strains was measured after strains were grown for 24 or 48 h in the presence of 1 % glucose (a) or in the presence of 0.2 and 2 % xylose (b). Xylose transport, as assessed by 14C-xylose uptake, was measured in the wild-type and ΔhxtB strains in the presence of different concentrations of xylose (c)

Mentions: The A. nidulans wild-type strain was first grown from spores in fructose-rich media before being transferred to media containing either 0.2 or 2 % xylose for 6, 12, 18 and 24 h (Fig. 4). Gene expression of hxtB-E was assessed by RT-qPCR in these conditions. All four genes were induced to a different extent in the presence of low concentrations of xylose (0.2 %) but not in the presence of 2 % xylose (Fig. 4a–d). Next, the four transporter-encoding genes were knocked out in A. nidulans and growth of these strains in the presence of glucose and xylose was assessed. The wild-type and the four deletion strains were grown in liquid minimal medium supplemented with 1 % glucose, 0.2 % xylose or 2 % xylose for 24 and 48 h before fungal dry weight was measured (Fig. 5). All strains showed a similar biomass when grown in 1 % glucose for 24 and 48 h (Fig. 5a). The ΔhxtB and ΔhxtE strains showed significantly reduced biomass when grown in 2 % xylose for 48 h (Fig. 5b). However, after 72 h of growth, all the mutant strains had a similar dry weight to the wild-type strain (data not shown). To further characterize xylose uptake, the concentration of xylose was measured in the supernatants of the wild-type, ΔhxtB and ΔhxtE strains when grown for 72 h in medium supplemented with either 1 or 2 % xylose. After 72 h, the wild-type strain and ΔhxtE strains had consumed all the xylose in the extracellular medium, whereas xylose consumption was much slower in the ΔhxtB strain and residual xylose could still be detected after 72 h in the supernatant of this strain (Table 1).Fig. 4


Identification and characterization of putative xylose and cellobiose transporters in Aspergillus nidulans
HxtB is a xylose transporter. Fungal dry weight of the wild-type, ΔhxtB, ΔhxtC, ΔhxtD, and ΔhxtE strains was measured after strains were grown for 24 or 48 h in the presence of 1 % glucose (a) or in the presence of 0.2 and 2 % xylose (b). Xylose transport, as assessed by 14C-xylose uptake, was measured in the wild-type and ΔhxtB strains in the presence of different concentrations of xylose (c)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037631&req=5

Fig5: HxtB is a xylose transporter. Fungal dry weight of the wild-type, ΔhxtB, ΔhxtC, ΔhxtD, and ΔhxtE strains was measured after strains were grown for 24 or 48 h in the presence of 1 % glucose (a) or in the presence of 0.2 and 2 % xylose (b). Xylose transport, as assessed by 14C-xylose uptake, was measured in the wild-type and ΔhxtB strains in the presence of different concentrations of xylose (c)
Mentions: The A. nidulans wild-type strain was first grown from spores in fructose-rich media before being transferred to media containing either 0.2 or 2 % xylose for 6, 12, 18 and 24 h (Fig. 4). Gene expression of hxtB-E was assessed by RT-qPCR in these conditions. All four genes were induced to a different extent in the presence of low concentrations of xylose (0.2 %) but not in the presence of 2 % xylose (Fig. 4a–d). Next, the four transporter-encoding genes were knocked out in A. nidulans and growth of these strains in the presence of glucose and xylose was assessed. The wild-type and the four deletion strains were grown in liquid minimal medium supplemented with 1 % glucose, 0.2 % xylose or 2 % xylose for 24 and 48 h before fungal dry weight was measured (Fig. 5). All strains showed a similar biomass when grown in 1 % glucose for 24 and 48 h (Fig. 5a). The ΔhxtB and ΔhxtE strains showed significantly reduced biomass when grown in 2 % xylose for 48 h (Fig. 5b). However, after 72 h of growth, all the mutant strains had a similar dry weight to the wild-type strain (data not shown). To further characterize xylose uptake, the concentration of xylose was measured in the supernatants of the wild-type, ΔhxtB and ΔhxtE strains when grown for 72 h in medium supplemented with either 1 or 2 % xylose. After 72 h, the wild-type strain and ΔhxtE strains had consumed all the xylose in the extracellular medium, whereas xylose consumption was much slower in the ΔhxtB strain and residual xylose could still be detected after 72 h in the supernatant of this strain (Table 1).Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: The conversion of lignocellulosic biomass to biofuels (second-generation biofuel production) is an environmentally friendlier alternative to petroleum-based energy sources. Enzymatic deconstruction of lignocellulose, catalyzed by filamentous fungi such as Aspergillus nidulans, releases a mixture of mono- and polysaccharides, including hexose (glucose) and pentose (xylose) sugars, cellodextrins (cellobiose), and xylooligosaccharides (xylobiose). These sugars can subsequently be fermented by yeast cells to ethanol. One of the major drawbacks in this process lies in the inability of yeast, such as Saccharomyces cerevisiae, to successfully internalize sugars other than glucose. The aim of this study was, therefore, to screen the genome of A. nidulans, which encodes a multitude of sugar transporters, for transporters able to internalize non-glucose sugars and characterize them when introduced into S. cerevisiae.

Results: This work identified two proteins in A. nidulans, CltA and CltB, with roles in cellobiose transport and cellulose signaling, respectively. CltA, when introduced into S. cerevisiae, conferred growth on low and high concentrations of cellobiose. Deletion of cltB resulted in reduced growth and extracellular cellulase activity in A. nidulans in the presence of cellobiose. CltB, when introduced into S. cerevisiae, was not able to confer growth on cellobiose, suggesting that this protein is a sensor rather than a transporter. 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. Furthermore, a previously identified glucose transporter, HxtB, was also found to be a major xylose transporter in A. nidulans. In S. cerevisiae, HxtB conferred growth on xylose which was accompanied by ethanol production.

Conclusions: This work identified a cellobiose transporter, a xylose transporter, and a putative cellulose transceptor in A. nidulans. This is the first time that a sensor role for a protein in A. nidulans has been proposed. Both transporters are also able to transport glucose, highlighting the preference of A. nidulans for this carbon source. This work provides a basis for future studies which aim at characterizing and/or genetically engineering Aspergillus spp. transporters, which, in addition to glucose, can also internalize other carbon sources, to improve transport and fermentation of non-glucose sugars in S. cerevisiae.

Electronic supplementary material: The online version of this article (doi:10.1186/s13068-016-0611-1) contains supplementary material, which is available to authorized users.

No MeSH data available.