Limits...
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

Specific cellobionic acid transporter NCU05853 (CBT-1). a Accumulation of cellobionic acid (A2) in the supernatant of β-glucosidase and NCU05853 mutant strains, providing Avicel as carbon source. Δ3BG, triple β-glucosidase deletion strain; Δ3BGΔ5853, triple β-glucosidase deletion plus ΔNCU05853 deletion strain. b Percentage of the remaining sugars after incubation with S. cerevisiae strains expressing different transporters. c Homology model of CBT-1, showing the conserved arginine residue R438. G2 cellobiose, X2 xylobiose, A2 cellobionic acid.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4537572&req=5

Fig3: Specific cellobionic acid transporter NCU05853 (CBT-1). a Accumulation of cellobionic acid (A2) in the supernatant of β-glucosidase and NCU05853 mutant strains, providing Avicel as carbon source. Δ3BG, triple β-glucosidase deletion strain; Δ3BGΔ5853, triple β-glucosidase deletion plus ΔNCU05853 deletion strain. b Percentage of the remaining sugars after incubation with S. cerevisiae strains expressing different transporters. c Homology model of CBT-1, showing the conserved arginine residue R438. G2 cellobiose, X2 xylobiose, A2 cellobionic acid.

Mentions: Deletion of NCU08755 slowed N. crassa growth on cellobionic acid but did not eliminate it, which implies that N. crassa expresses a parallel intracellular depolymerization pathway. Indeed, in a recent analysis of the N. crassa phosphoproteome, we identified a cellobionic acid transporter (NCU05853, or CBT-1) [10], a major facilitator superfamily (MFS) transporter previously noted to be highly upregulated in cellulolytic conditions [11], and recently identified as important in cellulase induction [12]. We investigated whether NCU05853 is the predominant cellobionic acid transporter in N. crassa using the triple extracellular β-glucosidase knockout background Δ3BG (ΔNCU08755, ΔNCU00130 and ΔNCU04952). In the Δ3BG strain, providing Avicel as carbon source, cellobionic acid accumulated as expected because this strain lacks the extracellular β-glucosidase NCU08755 responsible for cellobionic acid cleavage to gluconic acid and glucose (Fig. 3a). Notably, when NCU05853 was also deleted in addition to the three extracellular β-glucosidases, extracellular cellobionic acid accumulated in the growth media to a much higher level (Fig. 3a). To investigate the substrate specificities of NCU05853, the transporter was cloned and expressed in S. cerevisiae. Transport assays showed that only NCU05853, but not the previously studied NCU00801 (CDT-1) nor NCU08114 (CDT-2), was capable of transporting cellobionic acid into the cell (Fig. 3b). However, NCU05853 was not capable of transporting cellobiose or xylobiose (Fig. 3b). These results suggest that NCU05853 (CBT-1, hereafter) is a specific cellobionic acid transporter.Fig. 3


Cellobionic acid utilization: from Neurospora crassa to Saccharomyces cerevisiae.

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

Specific cellobionic acid transporter NCU05853 (CBT-1). a Accumulation of cellobionic acid (A2) in the supernatant of β-glucosidase and NCU05853 mutant strains, providing Avicel as carbon source. Δ3BG, triple β-glucosidase deletion strain; Δ3BGΔ5853, triple β-glucosidase deletion plus ΔNCU05853 deletion strain. b Percentage of the remaining sugars after incubation with S. cerevisiae strains expressing different transporters. c Homology model of CBT-1, showing the conserved arginine residue R438. G2 cellobiose, X2 xylobiose, A2 cellobionic acid.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4537572&req=5

Fig3: Specific cellobionic acid transporter NCU05853 (CBT-1). a Accumulation of cellobionic acid (A2) in the supernatant of β-glucosidase and NCU05853 mutant strains, providing Avicel as carbon source. Δ3BG, triple β-glucosidase deletion strain; Δ3BGΔ5853, triple β-glucosidase deletion plus ΔNCU05853 deletion strain. b Percentage of the remaining sugars after incubation with S. cerevisiae strains expressing different transporters. c Homology model of CBT-1, showing the conserved arginine residue R438. G2 cellobiose, X2 xylobiose, A2 cellobionic acid.
Mentions: Deletion of NCU08755 slowed N. crassa growth on cellobionic acid but did not eliminate it, which implies that N. crassa expresses a parallel intracellular depolymerization pathway. Indeed, in a recent analysis of the N. crassa phosphoproteome, we identified a cellobionic acid transporter (NCU05853, or CBT-1) [10], a major facilitator superfamily (MFS) transporter previously noted to be highly upregulated in cellulolytic conditions [11], and recently identified as important in cellulase induction [12]. We investigated whether NCU05853 is the predominant cellobionic acid transporter in N. crassa using the triple extracellular β-glucosidase knockout background Δ3BG (ΔNCU08755, ΔNCU00130 and ΔNCU04952). In the Δ3BG strain, providing Avicel as carbon source, cellobionic acid accumulated as expected because this strain lacks the extracellular β-glucosidase NCU08755 responsible for cellobionic acid cleavage to gluconic acid and glucose (Fig. 3a). Notably, when NCU05853 was also deleted in addition to the three extracellular β-glucosidases, extracellular cellobionic acid accumulated in the growth media to a much higher level (Fig. 3a). To investigate the substrate specificities of NCU05853, the transporter was cloned and expressed in S. cerevisiae. Transport assays showed that only NCU05853, but not the previously studied NCU00801 (CDT-1) nor NCU08114 (CDT-2), was capable of transporting cellobionic acid into the cell (Fig. 3b). However, NCU05853 was not capable of transporting cellobiose or xylobiose (Fig. 3b). These results suggest that NCU05853 (CBT-1, hereafter) is a specific cellobionic acid transporter.Fig. 3

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