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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

Schematic presentation of the plant expression cassettes for constitutive (A) and inducible (B) expression of TrCel5A. The CaMV promoter (P35SS) and terminator signal (pA35S) are indicated in light grey. The chimeric promoter (alcAmin35S) comprising the CaMV 35S minimal promoter (±31 to +5) fused to the upstream promoter sequences of alcA (Caddick et al., 1998) is shown in black. 5′-UTR of chalcone synthase (CHS), plant codon-optimized leader peptide (LPH) derived from the heavy chain of the murine mAb24, the gene of interest (trcel5A) and the His6 coding sequence (His6) are indicated in dark grey.
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Figure 1: Schematic presentation of the plant expression cassettes for constitutive (A) and inducible (B) expression of TrCel5A. The CaMV promoter (P35SS) and terminator signal (pA35S) are indicated in light grey. The chimeric promoter (alcAmin35S) comprising the CaMV 35S minimal promoter (±31 to +5) fused to the upstream promoter sequences of alcA (Caddick et al., 1998) is shown in black. 5′-UTR of chalcone synthase (CHS), plant codon-optimized leader peptide (LPH) derived from the heavy chain of the murine mAb24, the gene of interest (trcel5A) and the His6 coding sequence (His6) are indicated in dark grey.

Mentions: The cDNA encoding TrCel5A (without its native signal peptide sequence) was introduced into two different plant expression vectors, one under the control of the constitutive double CaMV 35SS promoter and the other under the control of the inducible alcAmin35S promoter [27]. Both constructs included a plant codon-optimized leader peptide, derived from the heavy chain of the murine monoclonal antibody mAb24 for secretion to the apoplast, and a C-terminal His6 tag for detection and purification (Figure 1). The functionality of the cloned constructs was verified by transient expression [33] followed by the detection of the active enzyme by Western blot and activity staining (Additional file 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)

Schematic presentation of the plant expression cassettes for constitutive (A) and inducible (B) expression of TrCel5A. The CaMV promoter (P35SS) and terminator signal (pA35S) are indicated in light grey. The chimeric promoter (alcAmin35S) comprising the CaMV 35S minimal promoter (±31 to +5) fused to the upstream promoter sequences of alcA (Caddick et al., 1998) is shown in black. 5′-UTR of chalcone synthase (CHS), plant codon-optimized leader peptide (LPH) derived from the heavy chain of the murine mAb24, the gene of interest (trcel5A) and the His6 coding sequence (His6) are indicated in dark grey.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic presentation of the plant expression cassettes for constitutive (A) and inducible (B) expression of TrCel5A. The CaMV promoter (P35SS) and terminator signal (pA35S) are indicated in light grey. The chimeric promoter (alcAmin35S) comprising the CaMV 35S minimal promoter (±31 to +5) fused to the upstream promoter sequences of alcA (Caddick et al., 1998) is shown in black. 5′-UTR of chalcone synthase (CHS), plant codon-optimized leader peptide (LPH) derived from the heavy chain of the murine mAb24, the gene of interest (trcel5A) and the His6 coding sequence (His6) are indicated in dark grey.
Mentions: The cDNA encoding TrCel5A (without its native signal peptide sequence) was introduced into two different plant expression vectors, one under the control of the constitutive double CaMV 35SS promoter and the other under the control of the inducible alcAmin35S promoter [27]. Both constructs included a plant codon-optimized leader peptide, derived from the heavy chain of the murine monoclonal antibody mAb24 for secretion to the apoplast, and a C-terminal His6 tag for detection and purification (Figure 1). The functionality of the cloned constructs was verified by transient expression [33] followed by the detection of the active enzyme by Western blot and activity staining (Additional file 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