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

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.


CltA and CltB are cellobiose transporters which are involved in the signaling response to cellulose. The expression of cltA (a) and cltB (b) was assessed by RT-qPCR in the presence of 1 % cellobiose. The effect of the single deletions of cltA and cltB and the double deletion cltA cltB was assessed on fungal biomass accumulation (DW dry weight) when grown for 48 or 72 h in the presence of 1 % glucose (c) or 1 % cellobiose (d). Cellulase activity (e) was measure in the supernatants of the same strains when grown in minimal medium supplemented with 1 % fructose for 16 h and then transferred to minimal medium containing 1 % avicel for 5 days. Error bars indicate standard deviation for three biological replicates
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Fig1: CltA and CltB are cellobiose transporters which are involved in the signaling response to cellulose. The expression of cltA (a) and cltB (b) was assessed by RT-qPCR in the presence of 1 % cellobiose. The effect of the single deletions of cltA and cltB and the double deletion cltA cltB was assessed on fungal biomass accumulation (DW dry weight) when grown for 48 or 72 h in the presence of 1 % glucose (c) or 1 % cellobiose (d). Cellulase activity (e) was measure in the supernatants of the same strains when grown in minimal medium supplemented with 1 % fructose for 16 h and then transferred to minimal medium containing 1 % avicel for 5 days. Error bars indicate standard deviation for three biological replicates

Mentions: To further characterize these potential cellobiose transporter-encoding genes (here now named cltA and cltB, respectively), we evaluated their expression patterns in the presence of 1 % cellobiose (Fig. 1a, b). The expression of cltA increased gradually (about 4.8-fold) over a time period of 4 h, whereas expression of cltB varied during the same time period (Fig. 1a, b). Next, both genes were deleted in A. nidulans and a ΔcltA ΔcltB double deletion strain was constructed. The wild-type, ΔcltA, ΔcltB, and the double ΔcltA ΔcltB strains were grown in 1 % glucose and 1 % cellobiose for 48 and 72 h, and biomass was determined (Fig. 1c, d). All the mutant strains had a similar biomass than the wild-type strain when grown in 1 % glucose (Fig. 1c). However, in the presence of cellobiose, the ΔcltB strain showed a ~50 % reduction in biomass after 48 h growth when compared to the wild-type strain, whereas there was no significant difference between the ΔcltA and wild-type strains (Fig. 1d). The double mutant showed a ~75 % reduction in biomass when compared to the wild-type strain after 48-h growth in 1 % cellobiose (Fig. 1d). These results suggest that CltA and CltB could collaborate towards cellobiose transport. Interestingly, there is also a reduction in cellulase activity in the ΔcltB and ΔcltA ΔcltB mutants of 50 and 70 %, respectively, than when compared to the wild-type strain (Fig. 1e), suggesting that these transporters play a role in the regulation/signaling of cellulase production.Fig. 1


Identification and characterization of putative xylose and cellobiose transporters in Aspergillus nidulans
CltA and CltB are cellobiose transporters which are involved in the signaling response to cellulose. The expression of cltA (a) and cltB (b) was assessed by RT-qPCR in the presence of 1 % cellobiose. The effect of the single deletions of cltA and cltB and the double deletion cltA cltB was assessed on fungal biomass accumulation (DW dry weight) when grown for 48 or 72 h in the presence of 1 % glucose (c) or 1 % cellobiose (d). Cellulase activity (e) was measure in the supernatants of the same strains when grown in minimal medium supplemented with 1 % fructose for 16 h and then transferred to minimal medium containing 1 % avicel for 5 days. Error bars indicate standard deviation for three biological replicates
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037631&req=5

Fig1: CltA and CltB are cellobiose transporters which are involved in the signaling response to cellulose. The expression of cltA (a) and cltB (b) was assessed by RT-qPCR in the presence of 1 % cellobiose. The effect of the single deletions of cltA and cltB and the double deletion cltA cltB was assessed on fungal biomass accumulation (DW dry weight) when grown for 48 or 72 h in the presence of 1 % glucose (c) or 1 % cellobiose (d). Cellulase activity (e) was measure in the supernatants of the same strains when grown in minimal medium supplemented with 1 % fructose for 16 h and then transferred to minimal medium containing 1 % avicel for 5 days. Error bars indicate standard deviation for three biological replicates
Mentions: To further characterize these potential cellobiose transporter-encoding genes (here now named cltA and cltB, respectively), we evaluated their expression patterns in the presence of 1 % cellobiose (Fig. 1a, b). The expression of cltA increased gradually (about 4.8-fold) over a time period of 4 h, whereas expression of cltB varied during the same time period (Fig. 1a, b). Next, both genes were deleted in A. nidulans and a ΔcltA ΔcltB double deletion strain was constructed. The wild-type, ΔcltA, ΔcltB, and the double ΔcltA ΔcltB strains were grown in 1 % glucose and 1 % cellobiose for 48 and 72 h, and biomass was determined (Fig. 1c, d). All the mutant strains had a similar biomass than the wild-type strain when grown in 1 % glucose (Fig. 1c). However, in the presence of cellobiose, the ΔcltB strain showed a ~50 % reduction in biomass after 48 h growth when compared to the wild-type strain, whereas there was no significant difference between the ΔcltA and wild-type strains (Fig. 1d). The double mutant showed a ~75 % reduction in biomass when compared to the wild-type strain after 48-h growth in 1 % cellobiose (Fig. 1d). These results suggest that CltA and CltB could collaborate towards cellobiose transport. Interestingly, there is also a reduction in cellulase activity in the ΔcltB and ΔcltA ΔcltB mutants of 50 and 70 %, respectively, than when compared to the wild-type strain (Fig. 1e), suggesting that these transporters play a role in the regulation/signaling of cellulase production.Fig. 1

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.