Biosynthesis of brazzein using the bacterial cell-free protein synthesis system

Abstract

Feasibility of producing brazzein plant sweetener by the cell-free biosynthesis procedure as an alternative to the classical submerged fermentation method was assessed in the present investigation. Chimeric RNA polymerase of T7 bacteriophage, S30-cell extract of Escherichia coli and multicopy plasmid vector pET42mut with the inserted brazzein gene were included into the protein synthesis. The completed research resulted in the first successful demonstration of the sweet protein biosynthesis in the cell-free system. The volumetric brazzein yield under optimized process conditions was 2 mg/ml of the reaction mixture, exceeding 57 times the maximum values that had been achieved in the previous studies applying whole-cell expression systems.