Production of multifunctional chimaeric enzymes in plants: A promising approach for degrading plant cell wall from within

Multifunctional chimaeric hydrolases can be created by covalently linking heterologous catalytic and functional domains in a single polypeptide. Previously, we have generated a number of chimaeric lignocellulosic hydrolases that contain two to five modules [. Biotechnol Bioeng (2009) 102: 1045; Appl Environ Microbiol (2009) 75: 1754]. These chimaeras closely resemble the parental enzymes in kinetics and other enzymatic properties, and some exhibit improved synergy in degrading natural substrates when compared to mixtures of parental enzymes. In addition to the applications in fermentative enzyme production, the chimaeric genes can be used in the construction of a single plant transformation binary vector carrying several genes that encode a complete set of lignocellulosic hydrolase activities. The advantages of this approach include ease in vector construction and transformation, as well as downstream plant analysis and breeding. The hydrolases sequestered in biomass feedstock can potentially assist enzymatic pretreatment and sugar conversion. Here, we report the gene expression and functional characterization of a chimaeric hemicellulase in transgenic tobacco plants. T1 transgenic plants produced up to 19-mg active enzymes per gram of total-soluble leaf proteins. The results demonstrate the feasibility of producing multifunctional lignocellulosic hydrolases in plants. Key considerations in the design, construction and plant expression of the chimaeric genes are discussed.