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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.


CltB overexpression increases the growth on cellobiose. a The expression of cltB was assessed by qRT-PCR in the presence of 0.1 % cellobiose. b Germlings of oCltB3::GFP were grown for 16 h in fructose 1 % and transferred to 0.1 or 1 % cellobiose. c The fungal biomass accumulation (dry weight) in the wild-type (TN02A3 and GR5), ΔcltB, ΔcltB::cltB+, and oCltB3::GFP strains was assessed for 24 h in the presence of 0.5 or 1 % cellobiose. *p < 0.005
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Fig2: CltB overexpression increases the growth on cellobiose. a The expression of cltB was assessed by qRT-PCR in the presence of 0.1 % cellobiose. b Germlings of oCltB3::GFP were grown for 16 h in fructose 1 % and transferred to 0.1 or 1 % cellobiose. c The fungal biomass accumulation (dry weight) in the wild-type (TN02A3 and GR5), ΔcltB, ΔcltB::cltB+, and oCltB3::GFP strains was assessed for 24 h in the presence of 0.5 or 1 % cellobiose. *p < 0.005

Mentions: We decided to investigate in more detail the phenotype provided by ΔcltB by complementing and overexpressing the cltB. First, we complemented the ΔcltB with a wild-type copy of cltB integrated ectopically, creating a strain ΔcltB::cltB+. Subsequently, we transformed the wild-type GR5 strain with CltB::GFP and selected for transformants with a single homologous integration and multiple ectopic integrations (Additional file 1). We selected single candidates for homologous (named CltB::GFP) and multiple ectopic integrations (named oCltB3::GFP). Growth phenotypes of ΔcltB::cltB+, CltB::GFP, and oCltB3::GFP were identical to the wild-type strain on MM with glucose as single carbon source (data not shown). Expression measured by qRT-PCR experiments showed that oCltB3::GFP has about eightfold more cltB expression than the wild-type strain in the presence of cellobiose (Fig. 2a). To verify the cellular localization and expression of CltB:GFP, the GFP strain was grown for 16 h in fructose and transferred to either 0.1 or 1 % cellobiose for 4 or 8 h (Fig. 2b). We have not observed any fluorescence in fructose (data not shown), but in contrast in 1 % cellobiose, we were able to see a weak fluorescence in oCltB3::GFP, mostly localized in the cytoplasm and in the cell membrane (Fig. 2b). To evaluate the impact of overexpressing cltB+ on growth in the presence of 0.5 and 1 % cellobiose as a single carbon source, the wild-type, ΔcltB, ΔcltB::cltB+, and oCltB3::GFP were grown for 24 h in MM + 0.5 or 1 % cellobiose (Fig. 2c). There is no significant difference in the growth (as evalutated by dry weight) of the wild type and ΔcltB::cltB+ in both 0.5 and 1 % cellobiose (Fig. 2c); in contrast, as it is also shown in Fig. 1d, we have observed a significant differential reduced growth in ΔcltB in both cellobiose concentrations (Fig. 2c). The overexpression strain oCltB3::GFP has shown more growth than the wild type only in 0.5 % but not in 1 % cellobiose (Fig. 2c). Taken together, these results suggest that CltB is able to transport cellobiose.Fig. 2


Identification and characterization of putative xylose and cellobiose transporters in Aspergillus nidulans
CltB overexpression increases the growth on cellobiose. a The expression of cltB was assessed by qRT-PCR in the presence of 0.1 % cellobiose. b Germlings of oCltB3::GFP were grown for 16 h in fructose 1 % and transferred to 0.1 or 1 % cellobiose. c The fungal biomass accumulation (dry weight) in the wild-type (TN02A3 and GR5), ΔcltB, ΔcltB::cltB+, and oCltB3::GFP strains was assessed for 24 h in the presence of 0.5 or 1 % cellobiose. *p < 0.005
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Fig2: CltB overexpression increases the growth on cellobiose. a The expression of cltB was assessed by qRT-PCR in the presence of 0.1 % cellobiose. b Germlings of oCltB3::GFP were grown for 16 h in fructose 1 % and transferred to 0.1 or 1 % cellobiose. c The fungal biomass accumulation (dry weight) in the wild-type (TN02A3 and GR5), ΔcltB, ΔcltB::cltB+, and oCltB3::GFP strains was assessed for 24 h in the presence of 0.5 or 1 % cellobiose. *p < 0.005
Mentions: We decided to investigate in more detail the phenotype provided by ΔcltB by complementing and overexpressing the cltB. First, we complemented the ΔcltB with a wild-type copy of cltB integrated ectopically, creating a strain ΔcltB::cltB+. Subsequently, we transformed the wild-type GR5 strain with CltB::GFP and selected for transformants with a single homologous integration and multiple ectopic integrations (Additional file 1). We selected single candidates for homologous (named CltB::GFP) and multiple ectopic integrations (named oCltB3::GFP). Growth phenotypes of ΔcltB::cltB+, CltB::GFP, and oCltB3::GFP were identical to the wild-type strain on MM with glucose as single carbon source (data not shown). Expression measured by qRT-PCR experiments showed that oCltB3::GFP has about eightfold more cltB expression than the wild-type strain in the presence of cellobiose (Fig. 2a). To verify the cellular localization and expression of CltB:GFP, the GFP strain was grown for 16 h in fructose and transferred to either 0.1 or 1 % cellobiose for 4 or 8 h (Fig. 2b). We have not observed any fluorescence in fructose (data not shown), but in contrast in 1 % cellobiose, we were able to see a weak fluorescence in oCltB3::GFP, mostly localized in the cytoplasm and in the cell membrane (Fig. 2b). To evaluate the impact of overexpressing cltB+ on growth in the presence of 0.5 and 1 % cellobiose as a single carbon source, the wild-type, ΔcltB, ΔcltB::cltB+, and oCltB3::GFP were grown for 24 h in MM + 0.5 or 1 % cellobiose (Fig. 2c). There is no significant difference in the growth (as evalutated by dry weight) of the wild type and ΔcltB::cltB+ in both 0.5 and 1 % cellobiose (Fig. 2c); in contrast, as it is also shown in Fig. 1d, we have observed a significant differential reduced growth in ΔcltB in both cellobiose concentrations (Fig. 2c). The overexpression strain oCltB3::GFP has shown more growth than the wild type only in 0.5 % but not in 1 % cellobiose (Fig. 2c). Taken together, these results suggest that CltB is able to transport cellobiose.Fig. 2

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.