Table 2.

Factors to consider in plant-based expression of plant cell wall degrading enzymes.

Category	Exemplary factors associated with a common strategy
Expression type	Selection between stable transformation in a transgenic plant and transient expression in a wildtype plant
Host plant	Composition of biomass Ease of DNA cloning Productivity of a plant crop Selection between monocot and dicot plants
Gene	Development of fusion protein Function and properties of enzyme for example, Thermal stability Subcellular protein localization using transit peptide Synthetic gene design for example, Codon/UTR optimization Use of plant cell wall modifying enzyme
Expression vector	Development of viral expression system for transient gene expression Easy selection markers for stably transformed plants Optimal promoter RBS optimization for example, Kozak's context sequence for monocot Selection between constitutive and inducible systems
Functional analysis	Change of enzyme stability with time Cleavage of holoenzyme for example, Truncated enzyme Comparison of plant produced enzyme with E. coli produced enzyme Effect of plant crude extract on enzyme stability Expression of multiple enzymes Microscopic analysis of protein localization Physical properties for example, Activity optima, thermal stability Posttranslational modification for example, Glycosylation Purification process
Phenotype analysis of transgenic plant	Compositional and structural change in transgenic plant Deleterious phenotype change for example, Fertile, pale leaves Location of protein accumulation for example, High level accumulation in leaf or stem New beneficial traits for example, Resistance to pathogens
Biomass hydrolysis	Application of enzyme cocktails Composition and property of cellulosic biomass Induction of in-planta hydrolysis Optimal combination of enzymes Resistance of enzymes to pretreatment process