Technological watch

Directed evolution of enzymes did not attract the attention of organic chemists until the control of stereoselectivity was successfully achieved 25?years ago. Traditionally, semi?rational directed evolution based on focused saturation mutagenesis at sites lining the binding pocket and rational enzyme design were considered to be two different approaches, especially when evolving stereoselectivity. However, because the former has become more and more rational, the two approaches have merged with the development of focused rational iterative site?specific mutagenesis (FRISM).This review outlines recent developments in protein engineering of stereo? and regioselective enzymes, which are of prime interest in organic and pharmaceutical chemistry as well as biotechnology. The widespread application of enzymes was hampered for decades due to limited enantio?, diastereo? and regioselectivity, which was the reason why most organic chemists were not interested in biocatalysis. This attitude began to change with the advent of semi?rational directed evolution methods based on focused saturation mutagenesis at sites lining the binding pocket. Screening constitutes the labor?intensive step (bottleneck), which is the reason why various research groups are continuing to develop techniques for the generation of small and smart mutant libraries. Rational enzyme design, traditionally an alternative to directed evolution, provides small collections of mutants which require minimal screening. This approach first focused on thermostabilization, and did not enter the field of stereoselectivity until later. Computational guides such as the Rosetta algorithms, HotSpot Wizard metric, and machine learning (ML) contribute significantly to decision making. The newest advancements show that semi?rational directed evolution such as CAST/ISM and rational enzyme design no longer develop on separate tracks, instead, they have started to merge. Indeed, researchers utilizing the two approaches have learned from each other. Today, the toolbox of organic chemists includes enzymes, primarily because the possibility of controlling stereoselectivity by protein engineering has ensured reliability when facing synthetic challenges. This review was also written with the hope that undergraduate and graduate education will include enzymes more so than in the past.