Biocatalysis has become an important aspect of modern organic synthesis, both in academia and across the chemical and pharmaceutical industries. Its success has been largely due to a rapid expansion of the range of chemical reactions accessible, made possible by advanced tools for enzyme discovery coupled with high-throughput laboratory evolution techniques for biocatalyst optimization. A wide range of tailor-made enzymes with high efficiencies and selectivities can now be produced quickly and on a gram to kilogram scale, with dedicated databases and search tools aimed at making these biocatalysts accessible to a broader scientific community. This Primer discusses the current state-of-the-art methodology in the field, including route design, enzyme discovery, protein engineering and the implementation of biocatalysis in industry. We highlight recent advances, such as de novo design and directed evolution, and discuss parameters that make a good reproducible biocatalytic process for industry. The general concepts will be illustrated by recent examples of applications in academia and industry, including the development of multistep enzyme cascades.

No abstract

No abstract

The synthesis of substituted d-phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one-pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high-throughput solid-phase screening method has also been developed to identify PALs with higher rates of formation of non-natural d-phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the d-configured product. Furthermore, the system was extended to the preparation of those l-phenylalanines which are obtained with a low ee value using PAL amination.

The combination of computational design and directed evolution could offer a general strategy to create enzymes with new functions. To date, this approach has delivered enzymes for a handful of model reactions. Here we show that new catalytic mechanisms can be engineered into proteins to accelerate more challenging chemical transformations. Evolutionary optimization of a primitive design afforded an efficient and enantioselective enzyme (BH32.14) for the Morita-Baylis-Hillman (MBH) reaction. BH32.14 is suitable for preparative scale transformations, accepts a broad range of aldehyde and enone coupling partners, and is able to promote selective mono-functionalizations of dialdehydes. Crystallographic, biochemical and computational studies reveal that BH32.14 operates via a sophisticated catalytic mechanism comprising a His23 nucleophile paired with a judiciously positioned Arg124. This catalytic arginine shuttles between conformational states to stabilize multiple oxyanion intermediates and serves as a genetically encoded surrogate of privileged bidentate hydrogen bonding catalysts (e.g. thioureas). This study demonstrates that elaborate catalytic devices can be built from scratch to promote demanding multi-step processes not observed in Nature.