Quantification, regulation and production of 5-aminolevulinic acid by green fluorescent protein in recombinant Escherichia coli

Abstract

5-Aminolevulinic acid (5-ALA) is an unnatural amino acid and has been approved as a biodegradable, non-toxic pesticide and herbicide with applications in sustainable agriculture. 5-ALA can also be applied for cancer targeting via tumor localization and photodynamic therapy. Herein, we developed a feasible quantification, regulation and production method of 5-ALA in Escherichia coli is based on the chimera of 5-ALA synthetase from Rhodobacter sphaeroides (RshemA) and super-fold green fluorescent protein (sfGFP) under the control of dual promoters/double plasmids. 5-ALA production based on quantification with the reporter sfGFP was unsuccessfully for the RshemA-sfGFP fusion protein owing to a steric hindrance effect, but was effective using dual constitutive promoters (i.e., J23100 and PLacI) for RshemA and sfGFP independently. Moreover, a simple quantification method based on the linear relationship between 5-ALA concentration and the change in sfGFP intensity was calculated with the Hill equation according to the results of dual plasmids which composed of RshemA-threonine/homoserine exporter (RhtA) and the sensing plasmid pSU-T7-sfGFP. Compared with the conventional detection method for 5-ALA using Ehrlich's reagent, our proposed method is advantages in effectiveness, real-time detection, and outstanding sensitivity. Finally, the highest yield of 5-ALA was obtained in E.coli D2TT strain, reaching 2.46g/L of 5-ALA produced in a 2.5-L baffle flask fermentation. Hence, this approach shows strong potential for improving 5-ALA production with appropriate regulation and detection based on the fluorescent signal.