CyanoConstruct: a Golden Gate DNA assembly platform for translationally optimized engineered metabolic pathways in Cyanobacteria.

Abstract

Recruiting cyanobacteria as cell factories for bio-production provides a sustainable route for many products such as biofuels, etc. However, rational engineering of cyanobacteria is impeded by the unpredictability of heterologous gene expression level which is tuned by swapping variants of DNA parts in the target DNA construct. Ribosome-binding site (RBS) modulates heterologous gene expression by determining the translation rate and efficiency. Nonetheless, the unavailability of cloning methods tailored for RBS switching in the target DNA constructs hampers the optimization efforts at the translational level in cyanobacterial hosts. This work presents a user-friendly Golden Gate-based cloning method, CyanoConstruct, designed specifically to assemble multi-gene operons with various RBSs in a one-step reaction. CyanoConstruct was capable of assembling RBS-varied three-gene operons for ethanol pathway from nine fragments with 10% efficiency as opposed to 100% efficiency for a two-gene construct. The functionality of the generated three-gene constructs was verified in Synechocystis sp. PCC 6803 by qualitative detection of ethanol using gas chromatography. A detected misassembled construct, generated by misannealed overhangs, was responsible for the undermined efficiency of the three-gene assembly; which was improved by introducing fragments at two sequential time points into the assembly reaction to minimize the overhang misannealing. Overall, CyanoConstruct is an efficient and straightforward cloning method, uniquely designed to readily construct RBS-varied multi-gene operon to advance RBS-based optimization of heterologous gene expression in engineered cyanobacteria.