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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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CltA and CltB confer growth of S. cerevisiae in the presence of low and high concentrations of cellobiose. a CltA and CltB localize to the plasma membrane in S. cerevisiae. Strains were grown for 16 h at 30 °C in YNB supplemented with 2 % glucose before pictures were taken without (DIC) and with (GFP) fluorescence. bS. cerevisiae strains containing A. nidulans cltA, cltB, and N. crassa cdt-1 with and without the β-glucosidase-encoding (gh1-1) gene were pre-grown for 24 h in YNB media containing 1 % glucose before a serial dilution was made (1:10 dilution starting at optical density OD600nm 1.0) and diluted cells were grown on YNB plates containing different concentrations of cellobiose (0.1–2 %). c Growth curves of S. cerevisiae strains containing the N. crassa cdt-1 and the A. nidulans cltA transporter-encoding genes. Both strains also contained the β-glucosidase-encoding gene gh1-1. Strains were grown for 144 h in the presence of different concentrations of cellobiose (0.1, 1, and 2 %) at 30 °C. Growth was assessed by measuring the OD at 600 nm. The yeast strain expressing CltB has not grown in liquid medium, and it was not used in this experiment
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Fig3: CltA and CltB confer growth of S. cerevisiae in the presence of low and high concentrations of cellobiose. a CltA and CltB localize to the plasma membrane in S. cerevisiae. Strains were grown for 16 h at 30 °C in YNB supplemented with 2 % glucose before pictures were taken without (DIC) and with (GFP) fluorescence. bS. cerevisiae strains containing A. nidulans cltA, cltB, and N. crassa cdt-1 with and without the β-glucosidase-encoding (gh1-1) gene were pre-grown for 24 h in YNB media containing 1 % glucose before a serial dilution was made (1:10 dilution starting at optical density OD600nm 1.0) and diluted cells were grown on YNB plates containing different concentrations of cellobiose (0.1–2 %). c Growth curves of S. cerevisiae strains containing the N. crassa cdt-1 and the A. nidulans cltA transporter-encoding genes. Both strains also contained the β-glucosidase-encoding gene gh1-1. Strains were grown for 144 h in the presence of different concentrations of cellobiose (0.1, 1, and 2 %) at 30 °C. Growth was assessed by measuring the OD at 600 nm. The yeast strain expressing CltB has not grown in liquid medium, and it was not used in this experiment

Mentions: To evaluate the ability of CltA and CltB to transport cellobiose, both genes were cloned into S. cerevisiae SC9721_pGH1-1, a SC9721 strain previously transformed with the N. crassa β-glucosidase-encoding gene gh1-1 (NCU00130) [26]. Both cltA and cltB were fused to gfp, and plasma membrane localization of CltA and CltB was confirmed by fluorescence microscopy when grown for 24 h in YNB supplemented with 1 % glucose medium (Fig. 3a). The S. cerevisiae CltA::GFP and CltB::GFP strains were then grown in liquid YNB medium supplemented with 1 % glucose for 24 h at 30 °C, before cells were washed and spotted in a serial dilution onto YNB solid medium containing either 1 % glucose or varying concentrations of cellobiose. Yeast strains containing only CltA or CltB (no β-glucosidase) were used as negative controls as they were unable to grow on cellobiose as sole carbon source. S. cerevisiae transformed with N. crassa cdt-1 and the β-glucosidase-encoding gene (gh1-1) was used as a positive control (Fig. 3b). The drop-out assay clearly shows that CltA, and to a lesser extent CltB, is able to transport cellobiose and, thus, enable S. cerevisiae to grow on cellobiose as sole carbon source.Fig. 3


Identification and characterization of putative xylose and cellobiose transporters in Aspergillus nidulans
CltA and CltB confer growth of S. cerevisiae in the presence of low and high concentrations of cellobiose. a CltA and CltB localize to the plasma membrane in S. cerevisiae. Strains were grown for 16 h at 30 °C in YNB supplemented with 2 % glucose before pictures were taken without (DIC) and with (GFP) fluorescence. bS. cerevisiae strains containing A. nidulans cltA, cltB, and N. crassa cdt-1 with and without the β-glucosidase-encoding (gh1-1) gene were pre-grown for 24 h in YNB media containing 1 % glucose before a serial dilution was made (1:10 dilution starting at optical density OD600nm 1.0) and diluted cells were grown on YNB plates containing different concentrations of cellobiose (0.1–2 %). c Growth curves of S. cerevisiae strains containing the N. crassa cdt-1 and the A. nidulans cltA transporter-encoding genes. Both strains also contained the β-glucosidase-encoding gene gh1-1. Strains were grown for 144 h in the presence of different concentrations of cellobiose (0.1, 1, and 2 %) at 30 °C. Growth was assessed by measuring the OD at 600 nm. The yeast strain expressing CltB has not grown in liquid medium, and it was not used in this experiment
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Fig3: CltA and CltB confer growth of S. cerevisiae in the presence of low and high concentrations of cellobiose. a CltA and CltB localize to the plasma membrane in S. cerevisiae. Strains were grown for 16 h at 30 °C in YNB supplemented with 2 % glucose before pictures were taken without (DIC) and with (GFP) fluorescence. bS. cerevisiae strains containing A. nidulans cltA, cltB, and N. crassa cdt-1 with and without the β-glucosidase-encoding (gh1-1) gene were pre-grown for 24 h in YNB media containing 1 % glucose before a serial dilution was made (1:10 dilution starting at optical density OD600nm 1.0) and diluted cells were grown on YNB plates containing different concentrations of cellobiose (0.1–2 %). c Growth curves of S. cerevisiae strains containing the N. crassa cdt-1 and the A. nidulans cltA transporter-encoding genes. Both strains also contained the β-glucosidase-encoding gene gh1-1. Strains were grown for 144 h in the presence of different concentrations of cellobiose (0.1, 1, and 2 %) at 30 °C. Growth was assessed by measuring the OD at 600 nm. The yeast strain expressing CltB has not grown in liquid medium, and it was not used in this experiment
Mentions: To evaluate the ability of CltA and CltB to transport cellobiose, both genes were cloned into S. cerevisiae SC9721_pGH1-1, a SC9721 strain previously transformed with the N. crassa β-glucosidase-encoding gene gh1-1 (NCU00130) [26]. Both cltA and cltB were fused to gfp, and plasma membrane localization of CltA and CltB was confirmed by fluorescence microscopy when grown for 24 h in YNB supplemented with 1 % glucose medium (Fig. 3a). The S. cerevisiae CltA::GFP and CltB::GFP strains were then grown in liquid YNB medium supplemented with 1 % glucose for 24 h at 30 °C, before cells were washed and spotted in a serial dilution onto YNB solid medium containing either 1 % glucose or varying concentrations of cellobiose. Yeast strains containing only CltA or CltB (no β-glucosidase) were used as negative controls as they were unable to grow on cellobiose as sole carbon source. S. cerevisiae transformed with N. crassa cdt-1 and the β-glucosidase-encoding gene (gh1-1) was used as a positive control (Fig. 3b). The drop-out assay clearly shows that CltA, and to a lesser extent CltB, is able to transport cellobiose and, thus, enable S. cerevisiae to grow on cellobiose as sole carbon source.Fig. 3

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