Abstract
BackgroundPlant-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.ResultsHere, 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.ConclusionWe 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.
Highlights
Plant-produced biomass-degrading enzymes are promising tools for the processing of lignocellulose to fermentable sugars
Cloning TrCel5A and the generation of transgenic tobacco plants The cDNA encoding TrCel5A was introduced into two different plant expression vectors, one under the control of the constitutive double Cauliflower mosaic virus (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)
Both constructs were introduced into tobacco (Nicotiana tabacum SR1) leaf by Agrobacterium-mediated transformation
Summary
Plant-produced biomass-degrading enzymes are promising tools for the processing of lignocellulose to fermentable sugars. Plants can express specific recombinant proteins [1,2,3], so the heterologous production of lignocellulolytic enzymes in plants could help to process biomass more efficiently [4,5,6]. It is necessary to consider the potential impact of heterologous cellulases on normal plant growth and development. This is relevant when using strong promoters like the Cauliflower mosaic virus (CaMV) 35S promoter derived from dicotyledons [12], the tissue specific maize embryopreferred globulin-1 promoter [13] or the artificial Mac promoter [14], based on the Escherichia coli maltose and lactose operons [15]
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