Metabolic pathway optimization through fusion with self-assembling amphipathic peptides

Abstract

Fusion with self-assembling amphipathic peptides (SAPs) could improve enzyme stability and activity, as well as enhancing protein expression. In this study, we proposed a metabolic regulation strategy based on an iterative SAP fusion with key enzymes in the pathway. First, an SAP, S1nv10 (ANANARARANANARAR), was separately fused to the N-terminus of metabolite enzymes. To achieve diversity in the enzyme activities, a library of the fused SAPs that varied in hydrophobicity, length, and net charge was generated by a multiprimer PCR procedure and the DATEL-assembly method. After simultaneous modification of three enzymes (CrtE, CrtY, and CrtI) using the SAP library, an Escherichia coli strain with a 3.91-fold increase in β-carotene production was isolated. As indicated by western blotting and catalytic property analysis, SAP fusion induced increases in both enzyme expression (CrtY and CrtE) and enzymatic activities (CrtY). The effectiveness of the strategy was further confirmed by the metabolic regulation of eriodictyol and (2S)-naringenin, the yields of which were increased by 111.3 % and 186.3 % in E. coli, respectively. These results indicate that fusing SAPs to the N-terminus of enzymes in metabolic flux is an efficient strategy for metabolic engineering.