Non‐heme iron halogenases are synthetically valuable biocatalysts that are capable of halogenating unactivated sp3‐hybridized carbon centers with high stereo‐ and regioselectivity. The reported substrate scope of these enzymes, however, is limited primarily to the natural substrates and their analogues. We engineered the halogenase WelO5* for chlorination of a martinelline‐derived fragment. Using structure‐guided evolution, a halogenase variant with a more than 290‐fold higher total turnover number and a 400‐fold higher apparent kcat compared to the wildtype enzyme was generated. Moreover, we identified key positions in the active site that allow direction of the halogen to different positions in the target substrate. This is the first example of enzyme engineering to expand the substrate scope of a non‐heme iron halogenase beyond the native indole‐alkaloid‐type substrates. The highly evolvable nature of WelO5* underscores the usefulness of this enzyme family for late‐stage halogenation.

Late-stage functionalization of natural products offers an elegant route to create novel entities in a relevant biological target space. In this context, enzymes capable of halogenating sp3 carbons with high stereo- and regiocontrol under benign conditions have attracted particular attention. Enabled by a combination of smart library design and machine learning, we engineer the iron/α-ketoglutarate dependent halogenase WelO5* for the late-stage functionalization of the complex and chemically difficult to derivatize macrolides soraphen A and C, potent anti-fungal agents. While the wild type enzyme WelO5* does not accept the macrolide substrates, our engineering strategy leads to active halogenase variants and improves upon their apparent kcat and total turnover number by more than 90-fold and 300-fold, respectively. Notably, our machine-learning guided engineering approach is capable of predicting more active variants and allows us to switch the regio-selectivity of the halogenases facilitating the targeted analysis of the derivatized macrolides’ structure-function activity in biological assays.

Many drug candidate molecules contain at least one chiral centre, and consequently, the development of biocatalytic strategies to complement existing metal‐ and organocatalytic approaches is of high interest. However, time is a critical factor in chemical process development, and thus, the introduction of biocatalytic steps, even if more suitable, is often prevented by the limited availability of off‐the‐shelf enzyme libraries. To expand the biocatalytic toolbox with additional ene reductases, we screened 19 bacterial strains for double bond reduction activity by using the model substrates cyclohexanone and carvone. Overall, we identified 47 genes coding for putative ene reductases. Remarkably, bioinformatic analysis of all genes and the biochemical characterization of four representative novel ene reductases led us to propose the existence of two new Old Yellow Enzyme subclasses, which we named OYE class III and class IV. Our results demonstrate that although, on a DNA level, each new OYE subclass features a distinct combination of sequence motifs previously known from the classical and the thermophilic‐like group, their substrate scope more closely resembles the latter subclass.

C–H functionalization is a chemically challenging but highly desirable transformation. 2-oxoglutarate-dependent oxygenases (2OGXs) are remarkably versatile biocatalysts for the activation of C–H bonds. In nature, they have been shown to accept both small and large molecules carrying out a plethora of reactions, including hydroxylations, demethylations, ring formations, rearrangements, desaturations, and halogenations, making them promising candidates for industrial manufacture. In this review, we describe the current status of 2OGX use in biocatalytic applications concentrating on 2OGX-catalyzed oxyfunctionalization of amino acids and synthesis of antibiotics. Looking forward, continued bioinformatic sourcing will help identify additional, practical useful members of this intriguing enzyme family, while enzyme engineering will pave the way to enhance 2OGX reactivity for non-native substrates.

Squalene-hopene cyclases (SHCs) have great potential for the industrial synthesis of enantiopure cyclic terpenoids.Alimitation of SHC catalysis has been the enzymes strict (S)-enantioselectivity at the stereocenter formed after the first cyclization step.T ogain enantio-complementary access to valuable monocyclic terpenoids,a nS HC-wild-type library including 18 novel homologs was set up.Apreviously not described SHC (AciSHC) was found to synthesizes mall amounts of monocyclic (R)-g-dihydroionone from (E/Z)geranylacetone.U sing enzyme and process optimization, the conversion to the desired product was increased to 79 %. Notably,a nalyzed AciSHC variants could finely differentiate between the geometric geranylacetone isomers:While the (Z)isomer yielded the desired monocyclic (R)-g-dihydroionone (> 99 %ee), the (E)-isomer was converted to the (S,S)-bicyclic ether (> 95 %e e). Applying the knowledge gained from the observed stereodivergent and enantioselective transformations to an additional SHC-substrate pair,a ccess to the complementary (S)-g-dihydroionone (> 99.9 %ee) could be obtained.