Heterologous complementation in bacteria for functional analysis of genes encoding carotenoid biosynthetic enzymes

Carotenoids represent a large class of isoprenoid pigments found in nature. These compounds are synthesized in non-photosynthetic and photosynthetic organisms and have a multitude of functions related to photosynthesis, protection against biotic and abiotic stress, and signaling in development and with other organisms. Thus, manipulation of carotenoid content can influence plant growth, development, and stress tolerance. In mammals, provitamin A and nonprovitamin A carotenoids are important for health. Mammals must obtain carotenoids in the diet, because of the inability to produce carotenoids de novo. Due to these important functions, carotenoids have been a major target for crop biofortification in the last decades. The plant kingdom is a great source of genetic material that encodes a wide range of variant enzymes for carotenoid synthesis. Thus, efficient systems to validate the functionality of structural genes are required. For this purpose, heterologous complementation in E. coli is a widely used in vivo platform to test functionality of carotenogenic enzymes of different origins such as bacteria, yeast, algae and plants. Here we describe the methodology for applying the E. coli heterologous platform to determine functionality of carotenoid enzymes, using the examples of phytoene synthase (PSY) and lycopene β-cyclase (LCYB) from apple and carrot.