Construction of non-invasively constitutive expression vectors using a metagenome-derived promoter for soluble expression of proteins.

Expression of soluble and functional proteins has been one of the critical challenges to many aspects of synthetic biology, metabolic and protein engineering. Among the current methods for expression of target proteins, constitutive expression systems offer several advantages over inducible systems, which require a chemical or physical inducer. In a previous study, a G196 DNA fragment containing constitutive promoters was mined from the soil metagenome and evaluated for the expression of target proteins in the functional and soluble state. In this study, we further improved this system by constructing a series of constitutive expression vectors, pCEM (using the CEM promoter trimmed from G196), pCEMT (incorporating rrnB T1 and T2 terminator into the downstream region of MCS in pCEM) and pRCEMT (grafting the cis-acting region of pCEMT into a low-copy-number plasmid). Subsequently, genes encoding GFPuv, esterase 1767 and β-glucosidase were subcloned into the resulting vectors, and their expression level and solubility were compared with those of IPTG-inducible vector systems pQE30 and pTrc99A. The extent of homogeneity and the ratio of the soluble fraction in the pRCEMT vector were relatively higher, without any delay of growth rate, than that of the pQE30 or pTrc99A. These results indicate that new expression vectors with moderate constitutive function could more easily lead to a homogenous population of cells expressing target proteins than those with conventionally inducible promoters.