Engineering a Controllable Targeted Protein Degradation System and a Derived OR-GATE-Type Inducible Gene Expression System in Synechococcus elongatus PCC 7942.

Abstract

Cyanobacteria are important model organisms for exploring the mechanisms of photosynthesis and are considered as promising microbial platforms for photosynthetic biomanufacturing. The development of efficient cyanobacteria cell factories requires efficient and convenient tools to dynamically regulate and manipulate target proteins, modules, and pathways. Targeted protein degradation is important to achieve rapid responses of cellular metabolic networks to artificial or environmental signals, and there are currently limited approaches to induce protein degradation in cyanobacteria. In this work, we developed an Escherichia coli sourced ssrA-tagging system in an important cyanobacteria strain, Synechococcus elongatus PCC 7942, to achieve inducible degradation of target proteins. A modified version of the E. coli ssrA tag (ssrADAS) proved to be immune to the native ClpXP system in Synechococcus elongatus PCC 7942, while induced expression of the E. coli sourced adaptor SspB and ClpXP resulted in effective degradation of the tagged proteins. Compared to the previously developed down-regulation approaches, the inducible ssrADAS-SspB-ClpXPEc system facilitated the smart and rapid degradation of target proteins in PCC7942 cells at different growth stages. Furthermore, when used to regulate the degradation of LacI, the repressor element of LacO-LacI transcription regulation system, an efficient and stringent inducible gene expression system was obtained based on an OR-GATE type genetic circuit design. The tools developed in this work expanded the cyanobacteria synthetic biology toolbox and will facilitate the success of future dynamic metabolic engineering.