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Cellobionic acid utilization: from Neurospora crassa to Saccharomyces cerevisiae.

Li X, Chomvong K, Yu VY, Liang JM, Lin Y, Cate JH - Biotechnol Biofuels (2015)

Bottom Line: Intracellular cellobionic acid was further cleaved to glucose 1-phosphate and gluconic acid by CAP.However, it remains unclear how N. crassa utilizes extracellular gluconic acid.The aldonic acid pathway was successfully implemented in Saccharomyces cerevisiae when N. crassa gluconokinase was co-expressed, resulting in cellobionic acid consumption in both aerobic and anaerobic conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA USA.

ABSTRACT

Background: Economical production of fuels and chemicals from plant biomass requires the efficient use of sugars derived from the plant cell wall. Neurospora crassa, a model lignocellulosic degrading fungus, is capable of breaking down the complex structure of the plant cell wall. In addition to cellulases and hemicellulases, N. crassa secretes lytic polysaccharide monooxygenases (LPMOs), which cleave cellulose by generating oxidized sugars-particularly aldonic acids. However, the strategies N. crassa employs to utilize these sugars are unknown.

Results: We identified an aldonic acid utilization pathway in N. crassa, comprised of an extracellular hydrolase (NCU08755), cellobionic acid transporter (CBT-1, NCU05853) and cellobionic acid phosphorylase (CAP, NCU09425). Extracellular cellobionic acid could be imported directly by CBT-1 or cleaved to gluconic acid and glucose by a β-glucosidase (NCU08755) outside the cells. Intracellular cellobionic acid was further cleaved to glucose 1-phosphate and gluconic acid by CAP. However, it remains unclear how N. crassa utilizes extracellular gluconic acid. The aldonic acid pathway was successfully implemented in Saccharomyces cerevisiae when N. crassa gluconokinase was co-expressed, resulting in cellobionic acid consumption in both aerobic and anaerobic conditions.

Conclusions: We successfully identified a branched aldonic acid utilization pathway in N. crassa and transferred its essential components into S. cerevisiae, a robust industrial microorganism.

No MeSH data available.


Related in: MedlinePlus

Cellobionic acid cleavage by secreted β-glucosidase NCU08755. a SDS-PAGE gel of proteins secreted during N. crassa growth on different carbon sources. The arrow indicates the expected size of NCU08755. Lanes are: molecular weight markers (M), tenfold concentrated secretomes of cultures grown on glucose (G1), cellobiose (G2), gluconic acid (A1) and cellobionic acid (A2), and a threefold concentrated secretome of a culture grown on Avicel cellulose (Av). b Cellobionic acid consumption profile of the four N. crassa strains with secreted β-glucosidases individually deleted.
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Fig2: Cellobionic acid cleavage by secreted β-glucosidase NCU08755. a SDS-PAGE gel of proteins secreted during N. crassa growth on different carbon sources. The arrow indicates the expected size of NCU08755. Lanes are: molecular weight markers (M), tenfold concentrated secretomes of cultures grown on glucose (G1), cellobiose (G2), gluconic acid (A1) and cellobionic acid (A2), and a threefold concentrated secretome of a culture grown on Avicel cellulose (Av). b Cellobionic acid consumption profile of the four N. crassa strains with secreted β-glucosidases individually deleted.

Mentions: We next tested whether the β-1,4 glycosidic bond in cellobionic acid is targeted by β-glucosidase family enzymes. The N. crassa genome encodes at least seven β-glucosidases, four of which are highly upregulated when N. crassa is grown on cellulose [9]. To identify β-glucosidases responsible for degrading cellobionic acid, the secretome of N. crassa grown on cellobionic acid was analyzed by LC–MS/MS. Only one of the four major β-glucosidases, NCU08755, was identified in the secretome of cells grown in cellobionic acid (Fig. 2a, see Additional file 1: Figure S2). The protein band for NCU08755 was absent in the secretome of cells grown on gluconic acid (Fig. 2a). We then tested cellobionic acid consumption by strains of N. crassa with the four β-glucosidases knocked out individually. Only the ΔNCU08755 strain showed a decrease in cellobionic acid consumption in comparison to the wild-type strain (Fig. 2b). These results suggested that NCU08755 is the major β-glucosidase involved in cellobionic acid depolymerization in N. crassa. In addition, a phylogenetic analysis of NCU08755 fungal homologs suggested that extracellular cellobionic acid cleavage might be a common strategy among fungi (see Additional file 1: Figure S3).Fig. 2


Cellobionic acid utilization: from Neurospora crassa to Saccharomyces cerevisiae.

Li X, Chomvong K, Yu VY, Liang JM, Lin Y, Cate JH - Biotechnol Biofuels (2015)

Cellobionic acid cleavage by secreted β-glucosidase NCU08755. a SDS-PAGE gel of proteins secreted during N. crassa growth on different carbon sources. The arrow indicates the expected size of NCU08755. Lanes are: molecular weight markers (M), tenfold concentrated secretomes of cultures grown on glucose (G1), cellobiose (G2), gluconic acid (A1) and cellobionic acid (A2), and a threefold concentrated secretome of a culture grown on Avicel cellulose (Av). b Cellobionic acid consumption profile of the four N. crassa strains with secreted β-glucosidases individually deleted.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4537572&req=5

Fig2: Cellobionic acid cleavage by secreted β-glucosidase NCU08755. a SDS-PAGE gel of proteins secreted during N. crassa growth on different carbon sources. The arrow indicates the expected size of NCU08755. Lanes are: molecular weight markers (M), tenfold concentrated secretomes of cultures grown on glucose (G1), cellobiose (G2), gluconic acid (A1) and cellobionic acid (A2), and a threefold concentrated secretome of a culture grown on Avicel cellulose (Av). b Cellobionic acid consumption profile of the four N. crassa strains with secreted β-glucosidases individually deleted.
Mentions: We next tested whether the β-1,4 glycosidic bond in cellobionic acid is targeted by β-glucosidase family enzymes. The N. crassa genome encodes at least seven β-glucosidases, four of which are highly upregulated when N. crassa is grown on cellulose [9]. To identify β-glucosidases responsible for degrading cellobionic acid, the secretome of N. crassa grown on cellobionic acid was analyzed by LC–MS/MS. Only one of the four major β-glucosidases, NCU08755, was identified in the secretome of cells grown in cellobionic acid (Fig. 2a, see Additional file 1: Figure S2). The protein band for NCU08755 was absent in the secretome of cells grown on gluconic acid (Fig. 2a). We then tested cellobionic acid consumption by strains of N. crassa with the four β-glucosidases knocked out individually. Only the ΔNCU08755 strain showed a decrease in cellobionic acid consumption in comparison to the wild-type strain (Fig. 2b). These results suggested that NCU08755 is the major β-glucosidase involved in cellobionic acid depolymerization in N. crassa. In addition, a phylogenetic analysis of NCU08755 fungal homologs suggested that extracellular cellobionic acid cleavage might be a common strategy among fungi (see Additional file 1: Figure S3).Fig. 2

Bottom Line: Intracellular cellobionic acid was further cleaved to glucose 1-phosphate and gluconic acid by CAP.However, it remains unclear how N. crassa utilizes extracellular gluconic acid.The aldonic acid pathway was successfully implemented in Saccharomyces cerevisiae when N. crassa gluconokinase was co-expressed, resulting in cellobionic acid consumption in both aerobic and anaerobic conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA USA.

ABSTRACT

Background: Economical production of fuels and chemicals from plant biomass requires the efficient use of sugars derived from the plant cell wall. Neurospora crassa, a model lignocellulosic degrading fungus, is capable of breaking down the complex structure of the plant cell wall. In addition to cellulases and hemicellulases, N. crassa secretes lytic polysaccharide monooxygenases (LPMOs), which cleave cellulose by generating oxidized sugars-particularly aldonic acids. However, the strategies N. crassa employs to utilize these sugars are unknown.

Results: We identified an aldonic acid utilization pathway in N. crassa, comprised of an extracellular hydrolase (NCU08755), cellobionic acid transporter (CBT-1, NCU05853) and cellobionic acid phosphorylase (CAP, NCU09425). Extracellular cellobionic acid could be imported directly by CBT-1 or cleaved to gluconic acid and glucose by a β-glucosidase (NCU08755) outside the cells. Intracellular cellobionic acid was further cleaved to glucose 1-phosphate and gluconic acid by CAP. However, it remains unclear how N. crassa utilizes extracellular gluconic acid. The aldonic acid pathway was successfully implemented in Saccharomyces cerevisiae when N. crassa gluconokinase was co-expressed, resulting in cellobionic acid consumption in both aerobic and anaerobic conditions.

Conclusions: We successfully identified a branched aldonic acid utilization pathway in N. crassa and transferred its essential components into S. cerevisiae, a robust industrial microorganism.

No MeSH data available.


Related in: MedlinePlus