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Ethanol inducible expression of a mesophilic cellulase avoids adverse effects on plant development.

Klose H, Günl M, Usadel B, Fischer R, Commandeur U - Biotechnol Biofuels (2013)

Bottom Line: However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development.These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, Aachen, 52074, Germany. commandeur@molbiotech.rwth-aachen.de.

ABSTRACT

Background: Plant-produced biomass-degrading enzymes are promising tools for the processing of lignocellulose to fermentable sugars. A major limitation of in planta production is that high-level expression of such enzymes could potentially affect the structure and integrity of the plant cell wall and negatively influence plant growth and development.

Results: Here, we evaluate the impact on tobacco plant development of constitutive versus alcohol-inducible expression of the endoglucanase TrCel5A from the mesophilic fungus Trichoderma reesei. Using this system, we are able to demonstrate that constitutive expression of the enzyme, controlled by the doubled Cauliflower Mosaic Virus promoter, leads to lower cellulose content of the plant combined with severe effects on plant growth. However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.

Conclusion: We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development. These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

No MeSH data available.


Related in: MedlinePlus

Western blot of different transgenic lines constitutively expressing TrCel5A after SDS-PAGE (A) and zymography performed with SDS-PAGE containing 0.15% (w/v) CMC (B). Lanes contain 10 μg of plant total soluble protein. The recombinant enzyme was detected with a polyclonal α-cellulase antibody and an alkaline phosphatase conjugated goat-anti-rabbit secondary antibody. The zymogram control is performed with purified TrCel5A produced in Hansenula polymorpha.
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Figure 2: Western blot of different transgenic lines constitutively expressing TrCel5A after SDS-PAGE (A) and zymography performed with SDS-PAGE containing 0.15% (w/v) CMC (B). Lanes contain 10 μg of plant total soluble protein. The recombinant enzyme was detected with a polyclonal α-cellulase antibody and an alkaline phosphatase conjugated goat-anti-rabbit secondary antibody. The zymogram control is performed with purified TrCel5A produced in Hansenula polymorpha.

Mentions: Western blot analysis showed the recombinant protein had a molecular weight of approximately 35 kDa (Figure 2A), which is similar to the molecular weight of the native catalytic domain [35]. The truncated enzyme retained its activity (Figure 2B). TrCel5A expression levels in transgenic tobacco plants were determined by measuring the activity of total soluble protein (TSP) against the substrates azoCMC and 4-MUC. Plants with constitutive TrCel5A expression achieved a specific enzyme activity of up to 1.5 U mg-1 TSP for azoCMC and 35 nmol 4MU min-1 mg-1.


Ethanol inducible expression of a mesophilic cellulase avoids adverse effects on plant development.

Klose H, Günl M, Usadel B, Fischer R, Commandeur U - Biotechnol Biofuels (2013)

Western blot of different transgenic lines constitutively expressing TrCel5A after SDS-PAGE (A) and zymography performed with SDS-PAGE containing 0.15% (w/v) CMC (B). Lanes contain 10 μg of plant total soluble protein. The recombinant enzyme was detected with a polyclonal α-cellulase antibody and an alkaline phosphatase conjugated goat-anti-rabbit secondary antibody. The zymogram control is performed with purified TrCel5A produced in Hansenula polymorpha.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643885&req=5

Figure 2: Western blot of different transgenic lines constitutively expressing TrCel5A after SDS-PAGE (A) and zymography performed with SDS-PAGE containing 0.15% (w/v) CMC (B). Lanes contain 10 μg of plant total soluble protein. The recombinant enzyme was detected with a polyclonal α-cellulase antibody and an alkaline phosphatase conjugated goat-anti-rabbit secondary antibody. The zymogram control is performed with purified TrCel5A produced in Hansenula polymorpha.
Mentions: Western blot analysis showed the recombinant protein had a molecular weight of approximately 35 kDa (Figure 2A), which is similar to the molecular weight of the native catalytic domain [35]. The truncated enzyme retained its activity (Figure 2B). TrCel5A expression levels in transgenic tobacco plants were determined by measuring the activity of total soluble protein (TSP) against the substrates azoCMC and 4-MUC. Plants with constitutive TrCel5A expression achieved a specific enzyme activity of up to 1.5 U mg-1 TSP for azoCMC and 35 nmol 4MU min-1 mg-1.

Bottom Line: However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development.These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, Aachen, 52074, Germany. commandeur@molbiotech.rwth-aachen.de.

ABSTRACT

Background: Plant-produced biomass-degrading enzymes are promising tools for the processing of lignocellulose to fermentable sugars. A major limitation of in planta production is that high-level expression of such enzymes could potentially affect the structure and integrity of the plant cell wall and negatively influence plant growth and development.

Results: Here, we evaluate the impact on tobacco plant development of constitutive versus alcohol-inducible expression of the endoglucanase TrCel5A from the mesophilic fungus Trichoderma reesei. Using this system, we are able to demonstrate that constitutive expression of the enzyme, controlled by the doubled Cauliflower Mosaic Virus promoter, leads to lower cellulose content of the plant combined with severe effects on plant growth. However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.

Conclusion: We were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development. These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

No MeSH data available.


Related in: MedlinePlus