Despite the advances in synthetic biology, the construction of efficient and balanced artificial pathways remains challenging and a general bottleneck in bacterial strain development. Although translational tuning can be applied for balancing consecutive catalytic steps at protein expression level, the potential remains underexplored in cyanobacterial engineering. To complement existing modular cloning systems for this purpose the objective here was to simplify the construct assembly procedure to make translational tuning more accessible for expression optimization in cyanobacteria.

This study describes the design and use of a one-pot DNA construct assembly system (CyanoConstruct) for the generation transformation-ready multi-gene expression plasmids in a single Golden Gate reaction. This approach allows the user to select the ribosome binding site element (RBS) for each target gene, thus serving as a tool for independently modulating the translation efficiency of the individual overexpressed enzymes. For easy adaptation, a custom online tool (www.cyanoconstruct.com) guides the sequence design of new compatible parts and the assembly of constructs from user-specified parts in silico. We demonstrate the use of the system by assembling different two-gene and three-gene expression constructs from parts selected specifically for optimal performance in Synechocystis sp PCC 6803; the constructs functioned as intended in vivo and showed different pathway fluxes construed by alternative RBS combinations. The efficiency and specificity of the assembly were shown to be high, enabling the generation of the final expression plasmids from the library parts in one assembly cycle.

CyanoConstruct offers a simple strategy for building bacterial operon-based expression constructs, specifically facilitating the use of modular cloning systems for RBS optimization in routine cyanobacterial engineering. With the help of the web-based design tool that also serves as a sequence repository, the part library described in this work (https://www.addgene.org/browse/article/28263931/) can be easily expanded with user-specified sequences. By increasing the throughput for generating pathway variants with different translational patterns, the system is expected to advance the design of more efficient strains with higher flux to the desired end-product, thereby contributing to the development of next-generation biotechnologies.