Cell-Free Metabolic Engineering of Carotenoid Biosynthesis

Engineering heterologous metabolic pathways is a time consuming endeavor, even in model microbial systems like E. coli and S. cerevisiae that have benefited from recent advances in genetic assembly and pathway optimization techniques. Cell-free metabolic engineering, or the use of genetically engineered biochemically active cell extracts for the optimization and synthesis of biochemical products, is an accompanying strategy that can reduce engineering times, explore biosynthetic pathways in diverse organisms and produce novel biochemicals. Isoprenoids are plant and microbial secondary metabolites of interest to metabolic engineers due to their varied ring structures and degrees of unsaturation. They have applications in pharmaceuticals, biocontrol agents and fuels. Carotenoids are a predominantly linear member of the isoprenoids that play important roles in membrane structure and resistance to oxidative stress in microbes; they have also been shown to be beneficial to human health. Here, we are examining the use of a cell free system for the development of a multi-step biosynthetic pathway for carotenoids. Crt I and Crt B from Pantoea sp. YR343 are being produced through cell-free protein synthesis. These protein products constitute the biosynthetic pathway for lycopene starting with the substrate geranylgreranyl pyrophosphate. Without the limitations of a cellular membrane and cell viability, gene variants and enzyme stoichiometry can be manipulated through the addition of heterologous DNA for increased product yield without repeated genetic engineering. Recent progress will be presented.