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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

Reconstituted cellobionic acid utilization pathway in S. cerevisiae.a Aerobic cellobionic acid consumption of S. cerevisiae expressing CBT-1 and CAP. b In vitro assays of the purified gluconokinases from N. crassa (NCU07626, GnK) and S. cerevisiae (YDR248C). c Aerobic cellobionic acid consumption of S. cerevisiae expressing CBT-1, CAP and GnK. d Anaerobic cellobionic acid consumption of S. cerevisiae expressing CBT-1, CAP and GnK, providing cellobionic acid and glucose or xylose as an additional carbon source.
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Fig5: Reconstituted cellobionic acid utilization pathway in S. cerevisiae.a Aerobic cellobionic acid consumption of S. cerevisiae expressing CBT-1 and CAP. b In vitro assays of the purified gluconokinases from N. crassa (NCU07626, GnK) and S. cerevisiae (YDR248C). c Aerobic cellobionic acid consumption of S. cerevisiae expressing CBT-1, CAP and GnK. d Anaerobic cellobionic acid consumption of S. cerevisiae expressing CBT-1, CAP and GnK, providing cellobionic acid and glucose or xylose as an additional carbon source.

Mentions: With the identification and characterization of the cellobionic acid transporter (CBT-1) and the intracellular cellobionic acid phosphorylase (CAP), we aimed to transfer the cellobionic acid pathway from N. crassa to the industrial yeast S. cerevisiae. However, S. cerevisiae expressing CBT-1 and CAP did not consume cellobionic acid (Fig. 5a). Since the activity assays of the CBT-1 and CAP revealed that they were both functionally expressed in S. cerevisiae, the failure to consume cellobionic acid indicated that there are likely other components in the cellobionic acid utilization pathway missing in S. cerevisiae. Whereas the glucose 1-phosphate released by CAP is likely consumed directly by conversion to glucose 6-phosphate by phosphoglucomutase (Pgm2 or Pgm1 in yeast), we hypothesized that the activity of the putative endogenous S. cerevisiae gluconokinase (YDR248C) responsible for converting gluconic acid to 6-phosphogluconate was limited, resulting in the failure of the cellobionic acid consumption pathway to function. To test this hypothesis, gluconokinases from S. cerevisiae (YDR248C) and N. crassa (NCU07626) were purified and tested for activity in vitro. In comparison to YDR248C, N. crassa gluconokinase (GnK, hereafter) was capable of converting more gluconic acid to 6-phosphogluconate at all enzyme concentrations tested (Fig. 5b). When GnK was co-expressed along with CBT-1 and CAP in S. cerevisiae, cellobionic acid consumption was observed in aerobic conditions (Fig. 5c). These results suggest that gluconokinase activity in S. cerevisiae was limiting the cellobionic acid pathway. Notably, even though YDR248C has gluconokinase activity in vitro and is annotated to be a cytoplasmic protein [18], its pattern of co-expression suggests that it is more likely to be a mitochondrially targeted protein (SGD co-expression analysis, [19]).Fig. 5


Cellobionic acid utilization: from Neurospora crassa to Saccharomyces cerevisiae.

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

Reconstituted cellobionic acid utilization pathway in S. cerevisiae.a Aerobic cellobionic acid consumption of S. cerevisiae expressing CBT-1 and CAP. b In vitro assays of the purified gluconokinases from N. crassa (NCU07626, GnK) and S. cerevisiae (YDR248C). c Aerobic cellobionic acid consumption of S. cerevisiae expressing CBT-1, CAP and GnK. d Anaerobic cellobionic acid consumption of S. cerevisiae expressing CBT-1, CAP and GnK, providing cellobionic acid and glucose or xylose as an additional carbon source.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Reconstituted cellobionic acid utilization pathway in S. cerevisiae.a Aerobic cellobionic acid consumption of S. cerevisiae expressing CBT-1 and CAP. b In vitro assays of the purified gluconokinases from N. crassa (NCU07626, GnK) and S. cerevisiae (YDR248C). c Aerobic cellobionic acid consumption of S. cerevisiae expressing CBT-1, CAP and GnK. d Anaerobic cellobionic acid consumption of S. cerevisiae expressing CBT-1, CAP and GnK, providing cellobionic acid and glucose or xylose as an additional carbon source.
Mentions: With the identification and characterization of the cellobionic acid transporter (CBT-1) and the intracellular cellobionic acid phosphorylase (CAP), we aimed to transfer the cellobionic acid pathway from N. crassa to the industrial yeast S. cerevisiae. However, S. cerevisiae expressing CBT-1 and CAP did not consume cellobionic acid (Fig. 5a). Since the activity assays of the CBT-1 and CAP revealed that they were both functionally expressed in S. cerevisiae, the failure to consume cellobionic acid indicated that there are likely other components in the cellobionic acid utilization pathway missing in S. cerevisiae. Whereas the glucose 1-phosphate released by CAP is likely consumed directly by conversion to glucose 6-phosphate by phosphoglucomutase (Pgm2 or Pgm1 in yeast), we hypothesized that the activity of the putative endogenous S. cerevisiae gluconokinase (YDR248C) responsible for converting gluconic acid to 6-phosphogluconate was limited, resulting in the failure of the cellobionic acid consumption pathway to function. To test this hypothesis, gluconokinases from S. cerevisiae (YDR248C) and N. crassa (NCU07626) were purified and tested for activity in vitro. In comparison to YDR248C, N. crassa gluconokinase (GnK, hereafter) was capable of converting more gluconic acid to 6-phosphogluconate at all enzyme concentrations tested (Fig. 5b). When GnK was co-expressed along with CBT-1 and CAP in S. cerevisiae, cellobionic acid consumption was observed in aerobic conditions (Fig. 5c). These results suggest that gluconokinase activity in S. cerevisiae was limiting the cellobionic acid pathway. Notably, even though YDR248C has gluconokinase activity in vitro and is annotated to be a cytoplasmic protein [18], its pattern of co-expression suggests that it is more likely to be a mitochondrially targeted protein (SGD co-expression analysis, [19]).Fig. 5

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