Systematic Plasmid Engineering for Targeted Carotenoid Synthesis in Bacteria

Carotenoids are natural pigments known for their vibrant colors and roles in various biological functions, such as antioxidant activity, photosynthesis, and radiation tolerance. The microbial production of carotenoids through heterologous expression of biosynthetic genes on engineered plasmid has facilitated the exploration of these functions. However, the complexity of creating carotenoid pathway plasmids has limited the number of available constructs, despite the vast diversity of carotenoids found in nature. This study aims to overcome these challenges by developing a platform for the systematic creation of plasmids that efficiently produce specific carotenoids in bacteria. A novel strategy, called Slot Assignment Cloning (SA-Clo), which applies the modular cloning (MoClo) method, was introduced. Using this strategy, plasmids for 17 carotenoids were constructed, including lycopene, β-carotene, zeaxanthin, canthaxanthin, astaxanthin, phytoene, δ-carotene, ε-carotene, α-carotene, violaxanthin, isorenieratene, several C 30 carotenoids, and non-natural C 50 -carotenoids. These plasmids enable the targeted and specific production of carotenoids, streamlining future studies on lesser-known carotenoids and their applications.