Better enzymes through Directed Evolution.
Fungal-derived unspecific peroxygenases (UPOs) have emerged over the past two decades as highly efficient biocatalysts for the oxyfunctionalization of carbon, sulfur, and nitrogen. However, while their excellent activity and stereoselectivity make the enzymes attractive for future industrial syntheses, their low regio- and chemoselectivity leads to product mixtures, which compromises their utility. To date, this reduced utility of UPOs for large-scale product syntheses has been remedied by smart substrate selection. The development of UPOs that catalyze selective hydroxylations in a substrate-independent manner would greatly increase their applicability as biocatalysts and is therefore the focus of many scientific efforts.
IPB researchers now succeeded in optimizing the enzyme MthUPO from the thermophilic fungus Myceliophthora thermophila for chemical and regioselective transformations. For this purpose, they used the Golden Mutagenesis technique to insert targeted mutations at nine different amino acid positions of the enzyme. The resulting mutant library was transformed into Saccharomyces cerivisiae and subsequently screened for variants showing good oxyfunctionalization activities with high chemo- and regioselectivities. This screen of over 5300 transformants was realized using split-GFP colorimetric assays. It led to MthUPO variants catalyzing both regioselective aromatic oxidations and chemoselective benzylic hydroxylations with 2-methylnaphthalene and other naphthalene derivatives. Among the promising enzyme candidates were triple and quadruple mutants with up to 16 times higher catalytic efficiency. Using computer modeling methods, the Halle scientists were able to show that this protein engineering procedure had led to significant active site rearrangements in some of the resulting enzyme variants.
This impressive effort towards Directed Evolution was only made possible by the previously developed high-throughput yeast expression system, which also originated in the laboratory of the Halle enzyme experts. The study paves the way for a broader application of UPOs in enzyme cascades and in organic chemistry.
