Cyanobacteria are attractive hosts for sustainable biotechnology due to their ability to perform oxygenic photosynthesis and directly convert light energy and carbon dioxide into valuable compounds. Their photosynthetic electron transport chain offers a unique opportunity to drive heterologous enzymatic reactions using light. In this work, strategies were developed to enhance the performance of plant cytochrome P450 monooxygenases (P450s) in the model cyanobacterium Synechocystis sp. PCC 6803 by improving enzyme targeting to the thylakoid membrane, where photosynthetic electron flow occurs. Fusion constructs of the plant P450 CYP79A1 with the native thylakoid protein PetC1 were designed to optimize membrane localization and electron transfer. All fusion variants showed enhanced thylakoid association and retained catalytic activity, with the highest activity achieved by replacing the native P450 transmembrane domain with that of PetC1. In parallel, cyanobacteria were engineered as photoautotrophic platforms for natural pigment production. Introduction of the betalain biosynthesis pathway, combined with increased aromatic amino acid supply, enabled betaxanthin production up to 15 mg L -1 using only light and CO₂, representing the first demonstration of betaxanthin synthesis in cyanobacteria. For ketocarotenoid biosynthesis, expression of a β-carotene ketolase from Chlamydomonas reinhardtii enabled astaxanthin and canthaxanthin accumulation. Overall, this study demonstrates that rational P450 targeting enhances light-driven catalysis and establishes cyanobacteria as versatile, sustainable cell factories for the production of high-value natural pigments and secondary metabolites.
Sayali S. Hanamghar (Wed,) studied this question.