Combinatorial assembly platform enabling engineering of genetically stable metabolic pathways in cyanobacteria

Area of Science:

• Synthetic biology
• Metabolic engineering
• Microbial biotechnology

Background:

• Cyanobacteria are promising biocatalysts for CO2 capture and conversion due to their photosynthetic efficiency.
• Genetic instability and low productivity hinder the practical application of engineered cyanobacteria.
• Developing stable and efficient microbial cell factories is crucial for sustainable bioproduction.

Purpose of the Study:

• To develop a massively parallel approach for engineering cyanobacteria.
• To create libraries of synthetic promoters and RBSs for Synechocystis sp. PCC 6803.
• To assemble and screen millions of metabolic pathway variants for improved productivity and stability.

Main Methods:

• Generation and characterization of synthetic promoter and RBS libraries.
• Assembly of a sparse combinatorial library of metabolic pathway constructs.
• High-throughput screening of engineered cyanobacteria under photoautotrophic conditions.

Main Results:

• Despite initial observations of genetic instability in some variants, 80% of randomly selected constructs accumulated lycopene from atmospheric CO2.
• The developed platform enabled the production of a target terpenoid in a single combinatorial round without iterative optimization.
• The study demonstrated a method to overcome genetic instability in engineered cyanobacteria.

Conclusions:

• Large-scale parallel metabolic engineering offers a novel platform for developing stable cyanobacterial biocatalysts.
• This approach facilitates sustainable, light-driven production of valuable products directly from CO2.
• The findings pave the way for utilizing cyanobacteria as efficient and stable microbial cell factories, avoiding fossil carbon reliance.