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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.

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HxtB confers growth of S. cerevisiae in the presence of xylose. a HxtB::GFP localizes to the plasma membrane in S. cerevisiae when grown in maltose-rich media. Microscopy pictures were taken in the absence (DIC) and presence of fluorescence (GFP). b Growth of S. cerevisiae strain EBY.VW4000 transformed with (hxtB) and without (control) the hexose transporter hxtB gene in the presence of low (0.1 %) and high (1 %) concentrations of xylose. Both the control and hxtB strains also contained the genes encoding enzymes of the xylose metabolic pathway. Yeast strains were first pre-grown in maltose-rich media before a serial dilution was made and cells were incubated on plates containing the different carbon sources at 30 °C for 144 h. c Xylose transport as determined by [14C] xylose uptake in the S. cerevisiae hxtB strain in the presence of different concentrations of xylose. A S. cerevisiae strain which did not contain the HxtB hexose transporter was used as the control strain. Standard deviations were obtained from three biological replicates. d Growth, as determined by optical density at 600 nm (OD600nm), ethanol production and xylose consumption of the S. cerevisiae control (does not contain hxtB) and HxtB strains when incubated in YNB supplemented with 1 % xylose
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Fig7: HxtB confers growth of S. cerevisiae in the presence of xylose. a HxtB::GFP localizes to the plasma membrane in S. cerevisiae when grown in maltose-rich media. Microscopy pictures were taken in the absence (DIC) and presence of fluorescence (GFP). b Growth of S. cerevisiae strain EBY.VW4000 transformed with (hxtB) and without (control) the hexose transporter hxtB gene in the presence of low (0.1 %) and high (1 %) concentrations of xylose. Both the control and hxtB strains also contained the genes encoding enzymes of the xylose metabolic pathway. Yeast strains were first pre-grown in maltose-rich media before a serial dilution was made and cells were incubated on plates containing the different carbon sources at 30 °C for 144 h. c Xylose transport as determined by [14C] xylose uptake in the S. cerevisiae hxtB strain in the presence of different concentrations of xylose. A S. cerevisiae strain which did not contain the HxtB hexose transporter was used as the control strain. Standard deviations were obtained from three biological replicates. d Growth, as determined by optical density at 600 nm (OD600nm), ethanol production and xylose consumption of the S. cerevisiae control (does not contain hxtB) and HxtB strains when incubated in YNB supplemented with 1 % xylose

Mentions: To confirm the presence and the cellular localization of HxtB, the HxtB::GFP and HxtE::GFP strains were constructed. Growth phenotypes of HxtB::GFP and HxtE::GFP were identical to the wild-type strain (data not shown). Both strains were grown for 10, 15, 20, and 24 h in minimal medium containing 0.1 or 1 % xylose. HxtB::GFP and HxtE::GFP were expressed in the presence of low and high concentrations of xylose upon which it localized to the fungal plasma membrane and small vacuoles (Fig. 6; Additional file 3). To confirm the xylose-transporting capacity of HxtB::GFP, it was introduced into S. cerevisiae EBY.VW4000 strain which was previously transformed with all the components necessary for the xylose metabolic pathway (see “Methods” section). The S. cerevisiae EBY.VW4000 strain lacks around 20 glucose transporters and is unable to grow on various hexose and pentose monosaccharides, including glucose, fructose, mannose, galactose, and xylose [48]. This strain is, therefore, a good tool for evaluating the ability of heterologous introduced transporter to take up various sugars thus conferring growth to S. cerevisiae in the presence of various pentose and hexose sugars. HxtB::GFP localized to the plasma membrane in S. cerevisiae when grown in maltose-rich conditions (Fig. 7a). Furthermore, when transferred from maltose-rich media to media containing low and high concentrations of xylose, S. cerevisiae strain HxtB::GFP was able to grow in both 0.1 and 1 % xylose, whereas the strain which lacked HxtB::GFP (control) was not able to do it (Fig. 7b). Furthermore, 14C-xylose uptake in S. cerevisiae HxtB::GFP followed single saturation kinetics with a Km value of 0.54 ± 0.08 mM and a Vmax of 1.14 ± 0.08 µm of xylose h−1 per mg cell dry weight (Fig. 7c). In contrast, the S. cerevisiae strain which does not contain hxtB was unable to transport xylose (Fig. 7c). In agreement, S. cerevisiae HxtB::GFP was able to grow and consume around 90 % of extracellular xylose after 192 h of growth in xylose-rich medium and at the same time produce ethanol (Fig. 7d). The control strain, which did not contain the HxtB transporter, did not grow in the presence of xylose and, hence, did not consume xylose and did not produce ethanol (Fig. 7d). In addition, as previously reported, S. cerevisiae strains containing the transporters HxtB, HxtC, and HxtE are able to grow in the presence of glucose, galactose, fructose, and mannose, while HxtD was not able to use any of these monosaccharides for growth [46]. In contrast, we were not able to see any xylose transport by S. cerevisiae strains harboring HxtC, -D, or -E (data not shown). Taken together, these results suggest that HxtB plays, in addition to being a glucose transporter, a major role in xylose uptake.Fig. 6


Identification and characterization of putative xylose and cellobiose transporters in Aspergillus nidulans
HxtB confers growth of S. cerevisiae in the presence of xylose. a HxtB::GFP localizes to the plasma membrane in S. cerevisiae when grown in maltose-rich media. Microscopy pictures were taken in the absence (DIC) and presence of fluorescence (GFP). b Growth of S. cerevisiae strain EBY.VW4000 transformed with (hxtB) and without (control) the hexose transporter hxtB gene in the presence of low (0.1 %) and high (1 %) concentrations of xylose. Both the control and hxtB strains also contained the genes encoding enzymes of the xylose metabolic pathway. Yeast strains were first pre-grown in maltose-rich media before a serial dilution was made and cells were incubated on plates containing the different carbon sources at 30 °C for 144 h. c Xylose transport as determined by [14C] xylose uptake in the S. cerevisiae hxtB strain in the presence of different concentrations of xylose. A S. cerevisiae strain which did not contain the HxtB hexose transporter was used as the control strain. Standard deviations were obtained from three biological replicates. d Growth, as determined by optical density at 600 nm (OD600nm), ethanol production and xylose consumption of the S. cerevisiae control (does not contain hxtB) and HxtB strains when incubated in YNB supplemented with 1 % xylose
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Fig7: HxtB confers growth of S. cerevisiae in the presence of xylose. a HxtB::GFP localizes to the plasma membrane in S. cerevisiae when grown in maltose-rich media. Microscopy pictures were taken in the absence (DIC) and presence of fluorescence (GFP). b Growth of S. cerevisiae strain EBY.VW4000 transformed with (hxtB) and without (control) the hexose transporter hxtB gene in the presence of low (0.1 %) and high (1 %) concentrations of xylose. Both the control and hxtB strains also contained the genes encoding enzymes of the xylose metabolic pathway. Yeast strains were first pre-grown in maltose-rich media before a serial dilution was made and cells were incubated on plates containing the different carbon sources at 30 °C for 144 h. c Xylose transport as determined by [14C] xylose uptake in the S. cerevisiae hxtB strain in the presence of different concentrations of xylose. A S. cerevisiae strain which did not contain the HxtB hexose transporter was used as the control strain. Standard deviations were obtained from three biological replicates. d Growth, as determined by optical density at 600 nm (OD600nm), ethanol production and xylose consumption of the S. cerevisiae control (does not contain hxtB) and HxtB strains when incubated in YNB supplemented with 1 % xylose
Mentions: To confirm the presence and the cellular localization of HxtB, the HxtB::GFP and HxtE::GFP strains were constructed. Growth phenotypes of HxtB::GFP and HxtE::GFP were identical to the wild-type strain (data not shown). Both strains were grown for 10, 15, 20, and 24 h in minimal medium containing 0.1 or 1 % xylose. HxtB::GFP and HxtE::GFP were expressed in the presence of low and high concentrations of xylose upon which it localized to the fungal plasma membrane and small vacuoles (Fig. 6; Additional file 3). To confirm the xylose-transporting capacity of HxtB::GFP, it was introduced into S. cerevisiae EBY.VW4000 strain which was previously transformed with all the components necessary for the xylose metabolic pathway (see “Methods” section). The S. cerevisiae EBY.VW4000 strain lacks around 20 glucose transporters and is unable to grow on various hexose and pentose monosaccharides, including glucose, fructose, mannose, galactose, and xylose [48]. This strain is, therefore, a good tool for evaluating the ability of heterologous introduced transporter to take up various sugars thus conferring growth to S. cerevisiae in the presence of various pentose and hexose sugars. HxtB::GFP localized to the plasma membrane in S. cerevisiae when grown in maltose-rich conditions (Fig. 7a). Furthermore, when transferred from maltose-rich media to media containing low and high concentrations of xylose, S. cerevisiae strain HxtB::GFP was able to grow in both 0.1 and 1 % xylose, whereas the strain which lacked HxtB::GFP (control) was not able to do it (Fig. 7b). Furthermore, 14C-xylose uptake in S. cerevisiae HxtB::GFP followed single saturation kinetics with a Km value of 0.54 ± 0.08 mM and a Vmax of 1.14 ± 0.08 µm of xylose h−1 per mg cell dry weight (Fig. 7c). In contrast, the S. cerevisiae strain which does not contain hxtB was unable to transport xylose (Fig. 7c). In agreement, S. cerevisiae HxtB::GFP was able to grow and consume around 90 % of extracellular xylose after 192 h of growth in xylose-rich medium and at the same time produce ethanol (Fig. 7d). The control strain, which did not contain the HxtB transporter, did not grow in the presence of xylose and, hence, did not consume xylose and did not produce ethanol (Fig. 7d). In addition, as previously reported, S. cerevisiae strains containing the transporters HxtB, HxtC, and HxtE are able to grow in the presence of glucose, galactose, fructose, and mannose, while HxtD was not able to use any of these monosaccharides for growth [46]. In contrast, we were not able to see any xylose transport by S. cerevisiae strains harboring HxtC, -D, or -E (data not shown). Taken together, these results suggest that HxtB plays, in addition to being a glucose transporter, a major role in xylose uptake.Fig. 6

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.


Related in: MedlinePlus