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

Cellobionic acid phosphorylase NCU09425 (CAP). a In vitro activity assay of purified intracellular β-glucosidase gh1-1 (NCU00130) on cellobionic acid. The purified NCU00130 was omitted in the ‘control’ condition. b In vitro activity assay of purified NCU09425 (CAP) and Saccharophagus degradans cellobiose phosphorylase (SdCBP) on cellobionic acid and cellobiose. A2 cellobionic acid, G2 cellobiose.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Cellobionic acid phosphorylase NCU09425 (CAP). a In vitro activity assay of purified intracellular β-glucosidase gh1-1 (NCU00130) on cellobionic acid. The purified NCU00130 was omitted in the ‘control’ condition. b In vitro activity assay of purified NCU09425 (CAP) and Saccharophagus degradans cellobiose phosphorylase (SdCBP) on cellobionic acid and cellobiose. A2 cellobionic acid, G2 cellobiose.

Mentions: After cellobionic acid is transported into N. crassa, it must be processed to monomeric units for further utilization. An intracellular β-glucosidase NCU00130 was previously reported to cleave cellobiose to two molecules of glucose [2]. Purified NCU00130 was tested for its ability to hydrolyze cellobionic acid, and was able to release gluconic acid from cellobionic acid (Fig. 4a). However, the reaction rate was poor (with apparent turnover number of 0.11 s−1) in comparison to reactions with cellobiose as the substrate [13]. We hypothesized that N. crassa likely uses another pathway to consume cellobionic acid rather than relying on NCU00130 activity.Fig. 4


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 phosphorylase NCU09425 (CAP). a In vitro activity assay of purified intracellular β-glucosidase gh1-1 (NCU00130) on cellobionic acid. The purified NCU00130 was omitted in the ‘control’ condition. b In vitro activity assay of purified NCU09425 (CAP) and Saccharophagus degradans cellobiose phosphorylase (SdCBP) on cellobionic acid and cellobiose. A2 cellobionic acid, G2 cellobiose.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Cellobionic acid phosphorylase NCU09425 (CAP). a In vitro activity assay of purified intracellular β-glucosidase gh1-1 (NCU00130) on cellobionic acid. The purified NCU00130 was omitted in the ‘control’ condition. b In vitro activity assay of purified NCU09425 (CAP) and Saccharophagus degradans cellobiose phosphorylase (SdCBP) on cellobionic acid and cellobiose. A2 cellobionic acid, G2 cellobiose.
Mentions: After cellobionic acid is transported into N. crassa, it must be processed to monomeric units for further utilization. An intracellular β-glucosidase NCU00130 was previously reported to cleave cellobiose to two molecules of glucose [2]. Purified NCU00130 was tested for its ability to hydrolyze cellobionic acid, and was able to release gluconic acid from cellobionic acid (Fig. 4a). However, the reaction rate was poor (with apparent turnover number of 0.11 s−1) in comparison to reactions with cellobiose as the substrate [13]. We hypothesized that N. crassa likely uses another pathway to consume cellobionic acid rather than relying on NCU00130 activity.Fig. 4

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