Directed evolution of a 13-hydroperoxide lyase (CYP74B) for improved process performance

The performance of a 13-hydroperoxide lyase from guava, an enzyme of the CYP74 family, which is of interest for the industrial production of saturated and unsaturated C6-aldehydes and their derivatives, was improved by directed evolution. Four rounds of gene shuffling and random mutagenesis improved the functional expression in E. coli by offering a 15-fold higher product yield factor. The increased product yield factor relates to an improved total turnover number of the variant enzyme, which also showed higher solubility and increased heme content. Thermal stability was also dramatically improved even though there was no direct selection pressure applied for evolving this trait. A structure based sequence alignment with the recently solved allene oxide synthase of Arabidopsis thaliana showed that most amino acid alterations occurred on the surface of the protein, distant of the active site and often outside of secondary structures. These results demonstrate the power of directed evolution for improving a complex trait such as the total turnover number of a cytochrome P450, a critical parameter for process performance that is difficult to predict even with good structural information at hand.

► Improved catalytic performance of a 13-hydroperoxide lyase by directed evolution.
► Variant with increases in total turnover number, heme content and thermal stability.
► Most amino acid alterations were non-obvious, located on the surface of the protein.
► Significantly improved functional expression in a heterologous host.
► High volumetric yields with poorly water soluble substrate.