Engineering the substrate scope of the Fe(II) dependent halogenase WelO15

Selective halogenation is an important reaction for late-stage functionalisation of drug-like molecules. Performing halogenations under mild conditions using sodium chloride as the chlorine source has great potential for sustainable catalysis. The discovery of non-heme iron (NHI) and 2-oxoglutarate dependent halogenases, acting directly on a small organic molecule and not on acyl-carrier bound substrates,[1,2] has eliminated a major drawback of know NHI-halogenases. Hence, these enzymes represent attractive starting points for developing biocatalytic routs for selective, aliphatic chlorination, a paramount challenge in organic synthesis. The wild-types have a narrow natural substrate-scope and are unexplored for biocatalytic applications.[3] After solving the crystal structure of WelO15 from Westiella intricata, we used directed evolution to redesign the active site using a small-but-smart amino acid alphabet, thereby limiting the screening effort to a HPLC compatible throughput. New variants were found, able to chlorinate novel synthesized non-natural substrates. This study represents a first step towards milder, selective chlorination using biocatalysis. Please click Additional Files below to see the full abstract

Exporling the Evolvability of Old Yellow Enzymes for Organic Synthesis

In the presented thesis, guidelines for the evolution of flavin-dependent ene reductases, an in-dustrially important catalyst class, are reported. In the first part of this thesis it should be tested if mining the existing knowledge of the Old Yellow Enzyme family (OYE), obtained from directed evolution studies, may allow guided traversing through the sequence space and thereby shortcutting biocatalyst development. Iden-tified hotspot positions of YqjM from Bacillus subtilis, i.e. C26D/I69T and C26G for improvement of activity and stereoselectivity, respectively, were transferred to seven OYE scaffolds. The new-ly created variants were tested with three compounds revealing more stereocomplementary OYE pairs with potent turnover frequencies (up to 660 h-1) and excellent stereoselectivities (up to >99%). Although systematic prediction of absolute enantioselectivity still remains for OYE variants, ‘scaffold sampling’ was confirmed as a fast engineering method for this family allow-ing access to new, potent biocatalysts for organic synthesis. In the second part of this thesis the development and characterisation of an engineered panel of ene reductases (ERs) from Thermus scotoductus SA-01 (TsER) is reported, that combines control over facial selectivity in the reduction of electron deficient carbon-carbon double bonds with thermostability (up to 70 °C), organic solvent tolerance (up to 40% (v/v)) and a broad substrate scope (23 compounds, three of them new). The panel shows excellent enantiomeric excess (ee) and yields during gram scale synthesis (3.8 g). Exquisite turnover frequencies (TOF) up to 40 000 h-1 are achieved, which are comparable to rates in hetero- and homogeneous metal cata-lysed hydrogenations. Efforts to rationalize the stereocomplementarity are reported, using the obtained crystal structure of TsER C25D/I67T and in silico docking studies. Our holistic charac-terisation, together with the preparative scale reactions, shows that these engineered ERs are truly practical catalysts for preparative organic synthesis. In the third section the aforementioned panel of TsER variants was screened for bulkier sub-strate classes and further mutation sites were identified over semi-rational design for the suc-cessful biotransformation of coumarin-like structures. Thereby chemoselective variants with either hydrogenation or evidence for acid/base catalysis in the active site of TsER have been discovered. In general there is a great interest in using these highly selective trans-hydrogenation catalysts in the late stage synthesis of complex organic molecules

Directed Evolution of an Feᴵᴵ-Dependent Halogenase for Asymmetric C(sp³)-H Chlorination

By using structure-guided directed evolution, the substrate scope of the FeII and a‑ketoglutarate dependent halogenase Wi‑WelO15 from Westiella intricata HT-29-1 was engineered to enable chemo-, regio- and diastereoselective chlorination of unactivated C(sp3)-H bonds using NaCl as chlorine source. While FeII dependent enzymes are often oxygen sensitive, variants of this halogenase could be screened in lysates under aerobic conditions. The new biocatalysts offer a sustainable approach for mild, late-stage chlorination on mg-scale of non-natural hapalindoles containing a ketone instead of an isonitrile functionality, thereby unlocking them for preparative biocatalysis.<br /

Bioorthogonal Enzymatic Activation of Caged Compounds

Engineered cytochrome P450 monooxygenase variants are reported as highly active and selective catalysts for the bioorthogonal uncaging of propargylic and benzylic ether protected substrates, including uncaging in living E. coli. observed selectivity is supported by induced-fit docking and molecular dynamics simulations. This proof-of-principle study points towards the utility of bioorthogonal enzyme/protecting group pairs for applications in the life sciences

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and β-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s

Speeding up Directed Evolution: Combining the Advantages of Solid-Phase Combinatorial Gene Synthesis with Statistically Guided Reduction of Screening Effort

Efficient and economic methods in directed evolution at the protein, metabolic, and genome level are needed for biocatalyst development and the success of synthetic biology. In contrast to random strategies, semirational approaches such as saturation mutagenesis explore the sequence space in a focused manner. Although several combinatorial libraries based on saturation mutagenesis have been reported using solid-phase gene synthesis, direct comparison with traditional PCR-based methods is currently lacking. In this work, we compare combinatorial protein libraries created in-house via PCR versus those generated by commercial solid-phase gene synthesis. Using descriptive statistics and probabilistic distributions on amino acid occurrence frequencies, the quality of the libraries was assessed and compared, revealing that the outsourced libraries are characterized by less bias and outliers than the PCR-based ones. Afterward, we screened all libraries following a traditional algorithm for almost complete library coverage and compared this approach with an emergent statistical concept suggesting screening a lower portion of the protein sequence space. Upon analyzing the biocatalytic landscapes and best hits of all combinatorial libraries, we show that the screening effort could have been reduced in all cases by more than 50%, while still finding at least one of the best mutants

P450-Catalyzed Regio- and Diastereoselective Steroid Hydroxylation: Efficient Directed Evolution Enabled by Mutability Landscaping

Cytochrome P450 monooxygenases play a crucial role in the biosynthesis of many natural products and in the human metabolism of numerous pharmaceuticals. This has inspired synthetic organic and medicinal chemists to exploit them as catalysts in regio- and stereoselective CH-activating oxidation of structurally simple and complex organic compounds such as steroids. However, levels of regio- and stereoselectivity as well as activity are not routinely high enough for real applications. Protein engineering using rational design or directed evolution has helped in many respects, but simultaneous engineering of multiple catalytic traits such as activity, regioselectivity, and stereoselectivity, while overcoming trade-offs and diminishing returns, remains a challenge. Here we show that the exploitation of information derived from mutability landscapes and molecular dynamics simulations for rationally designing iterative saturation mutagenesis constitutes a viable directed evolution strategy. This combined approach is illustrated by the evolution of P450_BM3 mutants which enable nearly perfect regio- and diastereoselective hydroxylation of five different steroids specifically at the C16-position with unusually high activity, while avoiding activity–selectivity trade-offs as well as keeping the screening effort relatively low. The C16 alcohols are of practical interest as components of biologically active glucocorticoids

Moisture variability over the Indo-Pacific region and its influence on the Indian summer monsoon rainfall

The Indo-Pacific Ocean (i.e. region between 30E and 150E) has been experiencing a spread warming since the 1950s. At the same time the large-scale summer monsoon rainfall over India and the moisture over the East Africa/Arabian Sea are both decreasing. In this study we intend to investigate how the decrease of moisture over the East Africa/Arabian Sea is related with the Indo-Pacific Ocean warming and how this could affect the variability of the Indian summer monsoon rainfall. We performed the analysis for the period 1951-2012 based on observed precipitation, sea surface temperature and atmospheric reanalysis products and we verified the robustness of the result by comparing different datasets. The decreasing trend of moisture over the East Africa/Arabian Sea coincides with an increasing trend of moisture over the western Pacific region. This is accompanied by the strengthening (weakening) of the upward motion over the western Pacific (East Africa/Arabian Sea) that, consequently, contributes in strengthening the western Pacific-Indian Ocean Walker circulation. Associated with it, the low-level westerlies are weakening over the peninsular India, thus contributing to the reduction of moisture transport towards India. Therefore, rainfall has decreased over the Western Ghats and central-east India. Differently from previous decades, since 2003 moisture over the East Africa/Arabian Sea started to increase and this is accompanied by the strengthening of convection due to increased warming of sea surface temperature over the western Arabian Sea. Despite this moisture increase over the Arabian sea, we found that moisture transport is still weakening over the Indian landmass in the very recent decade and still contributing to the decreased precipitation over the northeast India and southern part of the Western Ghats