Evolution of the C(30) Carotenoid Synthase CrtM for Function in a C(40) Pathway

The C(30) carotene synthase CrtM from Staphylococcus aureus and the C(40) carotene synthase CrtB from Erwinia uredovora were swapped into their respective foreign C(40) and C(30) biosynthetic pathways (heterologously expressed in Escherichia coli) and evaluated for function. Each displayed negligible ability to synthesize the natural carotenoid product of the other. After one round of mutagenesis and screening, we isolated 116 variants of CrtM able to synthesize C(40) carotenoids. In contrast, we failed to find a single variant of CrtB with detectable C(30) activity. Subsequent analysis revealed that the best CrtM mutants performed comparably to CrtB in an in vivo C(40) pathway. These mutants showed significant variation in performance in their original C(30) pathway, indicating the emergence of enzymes with broadened substrate specificity as well as those with shifted specificity. We discovered that Phe 26 alone determines the specificity of CrtM. The plasticity of CrtM with respect to its substrate and product range highlights the potential for creating further new carotenoid backbone structures.