Abstract
BACKGROUND: The methanol-regulated AOX1 promoter (P(AOX1)) is the most widely used promoter in the production of recombinant proteins in the methylotrophic yeast Pichia pastoris. However, as the tight regulation and methanol dependence of P(AOX1) restricts its application, it is necessary to develop a flexible induction system to avoid the problems of methanol without losing the advantages of P(AOX1). The availability of synthetic biology tools enables researchers to reprogram the cellular behaviour of P. pastoris to achieve this goal. RESULTS: The characteristics of P(AOX1) are highly related to the expression profile of methanol expression regulator 1 (Mxr1). In this study, we applied a biologically inspired strategy to reprogram regulatory networks in P. pastoris. A reprogrammed P. pastoris was constructed by inserting a synthetic positive feedback circuit of Mxr1 driven by a weak AOX2 promoter (P(AOX2)). This novel approach enhanced P(AOX1) efficiency by providing extra Mxr1 and generated switchable Mxr1 expression to allow P(AOX1) to be induced under glycerol starvation or carbon-free conditions. Additionally, the inhibitory effect of glycerol on P(AOX1) was retained because the synthetic circuit was not activated in response to glycerol. Using green fluorescent protein as a demonstration, this reprogrammed P. pastoris strain displayed stronger fluorescence intensity than non-reprogrammed cells under both methanol induction and glycerol starvation. Moreover, with single-chain variable fragment (scFv) as the model protein, increases in extracellular scFv productivity of 98 and 269% were observed in Mxr1-reprogrammed cells under methanol induction and glycerol starvation, respectively, compared to productivity in non-reprogrammed cells under methanol induction. CONCLUSIONS: We successfully demonstrate that the synthetic positive feedback circuit of Mxr1 enhances recombinant protein production efficiency in P. pastoris and create a methanol-free induction system to eliminate the potential risks of methanol.