Heterologous expression of cellobiohydrolase II (Cel6A) in maize endosperm

Abstract

The technology of converting lignocellulose to biofuels has advanced swiftly over the past few years, and enzymes are a significant constituent of this technology. In this regard, cost effective production of cellulases has been the focus of research for many years. One approach to reach cost targets of these enzymes involves the use of plants as bio-factories. The application of this technology to plant biomass conversion for biofuels and biobased products has the potential for significantly lowering the cost of these products due to lower enzyme production costs. Cel6A, one of the two cellobiohydrolases (CBH II) produced by Hypocrea jecorina, is an exoglucanase that cleaves primarily cellobiose units from the non-reducing end of cellulose microfibrils. In this work we describe the expression of Cel6A in maize endosperm as part of the process to lower the cost of this dominant enzyme for the bioconversion process. The enzyme is active on microcrystalline cellulose as exponential microbial growth was observed in the mixture of cellulose, cellulases, yeast and Cel6A, Cel7A (endoglucanase), and Cel5A (cellobiohydrolase I) expressed in maize seeds. We quantify the amount accumulated and the activity of the enzyme. Cel6A expressed in maize endosperm was purified to homogeneity and verified using peptide mass finger printing.

The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse

“…Interestingly, the magnitude of the enhancement was different for each pretreatment (3 to 36% glucan conversion), and there was a positive correlation (R 2  = 0.76) between the lignin content in the residue and the magnitude of the enhancement. Protein extracts from non-transgenic corn seed, shoot, and stem have been shown to enhance the performance of commercial cellulase mixtures on a pure cellulosic substrate [51,52] with a crystallinity index between 53% and 91%, depending on the method used to determine crystallinity [43]. In contrast to these studies, the non-transgenic extracts generated in the present study did not contain appreciable MULase activity (Figure 1) or the ability to hydrolyse carboxymethylcellulose (data not shown), the substrates were pretreated lignocellulosic biomass, and glucan conversions were, with the exception of a single experimental condition, above 10%.…”

The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse

“…Interestingly, the magnitude of the enhancement was different for each pretreatment (3 to 36% glucan conversion), and there was a positive correlation (R 2 = 0.76) between the lignin content in the residue and the magnitude of the enhancement. Protein extracts from non-transgenic corn seed, shoot, and stem have been shown to enhance the performance of commercial cellulase mixtures on a pure cellulosic substrate [51,52] with a crystallinity index between 53% and 91%, depending on the method used to determine crystallinity [43]. In contrast to these studies, the nontransgenic extracts generated in the present study did not contain appreciable MULase activity (Figure 1) or the ability to hydrolyse carboxymethylcellulose (data not shown), the substrates were pretreated lignocellulosic biomass, and glucan conversions were, with the exception of a single experimental condition, above 10%.…”

Plant Molecular Farming: Much More than Medicines

“…For transgenic plants and transient expression platforms, the environment of the plant can have a substantial impact; for example, differences in temperature of only 1-2 • C can change the yields of a recombinant protein by up to 15% (30). The combination of good construct design, optimal genetic background, and a supportive environment has achieved extraordinary yields of up to 10.6% of the total soluble protein (TSP) for human serum albumin expressed in rice seeds (31), 30% TSP for industrial enzymes expressed in maize seeds (32), 36% TSP for a murine antibody expressed in Arabidopsis (33), and more than 70% TSP for proteins expressed in tobacco chloroplasts (34).…”