Optimized Expression of a Hyperthermostable Endoglucanase from Pyrococcus horikoshii in Arabidopsis thaliana

Manufacturing microbial cellulase in plants is an attractive strategy for the cost-effective production of cellulosic ethanol, especially the expression of thermostable cellulase, which causes no negative effects on plant growth and development. The beta-1,4-endogenous cellulase from Pyrococcus horikoshii (EGPh) is considered one of the most promising glycosyl hydrolase in the biofuel and textile industry for its hyperthermostability and its capability to hydrolyze crystalline celluloses, which has been researched extensively during recent years. In this study, the coding sequence of EGPh was expressed in Arabidopsis thaliana under the control of a CaMV35S promoter after codon optimization, with the addition of a eukaryotic Kozak sequence. The expression of EGPh caused no deleterious effects to the growth and development of transgenic A. thaliana. The heterologous EGPh showed relatively high activities, up to 111.69 and 13.35 U.mg(-1) total soluble protein against soluble cellulose carboxymethyl cellulose (CMC) and insoluble microcrystalline cellulose (Avicel), respectively. The subcellular localization analysis showed that the EGPh protein was targeted to the plasma membrane and cell wall. Based on these results, it is proposed that EGPh is an ideal candidate for the commercial production of hyperthermostable endoglucanase using plants as biofactories.