Rubén Agudo

Achieving Regio‐ and Enantioselectivity of P450‐Catalyzed Oxidative CH Activation of Small Functionalized Molecules by Structure‐Guided Directed Evolution

Directed evolution of the monooxygenase P450‐BM3 utilizing iterative saturation mutagenesis at and near the binding site enables a high degree of both regio‐ and enantioselectivity in the oxidative hydroxylation of cyclohexene‐1‐carboxylic acid methyl ester. Wild‐type P450‐BM3 is 84 % regioselective for the allylic 3‐position with 34 % enantioselectivity in favor of the R alcohol. Mutants enabling R selectivity (>95 % ee) or S selectivity (>95 % ee) were evolved, while reducing other oxidation products and thus maximizing regioselectivity to >93 %. Control of the substrate‐to‐enzyme ratio is necessary for obtaining optimal and reproducible enantioselectivities, an observation which is important in future protein engineering of these mono‐oxygenases. An E. coli strain capable of NADPH regeneration was also engineered, simplifying directed evolution of P450 enzymes in general. These synthetic results set the stage for subsequent stereoselective and stereospecific chemical transformations to form more complex compounds, thereby illustrating the viability of combining genetically altered enzymes as catalysts in organic chemistry with traditional chemical methods.

Designer cells for stereocomplementary de novo enzymatic cascade reactions based on laboratory evolution

Designer cells for a synthetic cascade reaction harnessing selective redox reactions were devised, featuring two successive regioselective P450-catalyzed CH-activating oxidations of 1-cyclohexene carboxylic acid methyl ester followed by stereoselective olefin-reduction catalysed by (R)- or (S)-selective mutants of an enoate reductase.

Cytochrome P450 Catalyzed Oxidative Hydroxylation of Achiral Organic Compounds with Simultaneous Creation of Two Chirality Centers in a Single C-H Activation Step

Regio- and stereoselective oxidative hydroxylation of achiral or chiral organic compounds mediated by synthetic reagents, catalysts, or enzymes generally leads to the formation of one new chiral center that appears in the respective enantiomeric or diastereomeric alcohols. By contrast, when subjecting appropriate achiral compounds to this type of C-H activation, the simultaneous creation of two chiral centers with a defined relative and absolute configuration may result, provided that control of the regio-, diastereo-, and enantioselectivity is ensured. The present study demonstrates that such control is possible by using wild type or mutant forms of the monooxygenase cytochrome P450 BM3 as catalysts in the oxidative hydroxylation of methylcyclohexane and seven other monosubstituted cyclohexane derivatives.

Directed evolution by using iterative saturation mutagenesis based on multiresidue sites.

Iterative saturation mutagenesis (ISM) in combination with reduced amino acid alphabets has been shown to be an efficient method for directed evolution. In order to minimize the screening effort, the number of residues in a given randomization site has thus far been restricted to two or three; this prevents oversampling from reaching astronomical numbers when 95 % library coverage is aimed for. In this study, ISM is applied for the first time by using randomization sites composed of five amino acid positions. The use of just two such sites (A and B) results in two different ISM pathways, A→B and B→A. A severely reduced amino acid alphabet (only five members) was employed for the building blocks—a minimal set of structurally representative amino acids. The Baeyer–Villiger monooxygenase PAMO was chosen as the enzyme for this proof‐of‐principle study. The test system employed tuning of activity and diastereoselectivity in the oxidation of 4‐(bromomethylidene)cyclohexanone, which is not accepted by wild‐type PAMO. Although only 8–9 % library coverage was ensured (as calculated by traditional statistics), notable activity and 99 % diastereoselectivity were obtained, thus indicating that such an ISM strategy is viable in protein engineering.

Directed evolution of stereoselective enzymes based on genetic selection as opposed to screening systems.

Directed evolution of stereoselective enzymes provides a means to generate useful biocatalysts for asymmetric transformations in organic chemistry and biotechnology. Almost all of the numerous examples reported in the literature utilize high-throughput screening systems based on suitable analytical techniques. Since the screening step is the bottleneck of the overall procedure, researchers have considered the use of genetic selection systems as an alternative to screening. In principle, selection would be the most elegant and efficient approach because it is based on growth advantage of host cells harboring stereoselective mutants, but devising such selection systems is very challenging. They must be designed so that the host organism profits from the presence of an enantioselective variant. Progress in this intriguing research area is summarized in this review, which also includes some examples of display systems designed for enantioselectivity as assayed by fluorescence-activated cell sorting (FACS). Although the combination of display systems and FACS is a powerful approach, we also envision innovative ideas combining metabolic engineering and genetic selection systems with protein directed evolution for the development of highly selective and efficient biocatalysts.

Biocatalytic Route to Chiral Acyloins: P450-Catalyzed Regio- and Enantioselective α-Hydroxylation of Ketones

P450-BM3 and mutants of this monooxygenase generated by directed evolution are excellent catalysts for the oxidative α-hydroxylation of ketones with formation of chiral acyloins with high regioselectivity (up to 99%) and enantioselectivity (up to 99% ee). This constitutes a new route to a class of chiral compounds that are useful intermediates in the synthesis of many kinds of biologically active compounds.