Directed evolution of the monooxygenase P450 BM3 toward aromatic hydroxylations

The direct and selective hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a major challenge in organic chemistry. The monooxygenase P450 BM3 from Bacillus megaterium is known to catalyze the CH-activating functionalization of benzene with atmospheric dioxygen. The main objective of this thesis project has been the engineering of P450 BM3 toward the aromatic hydroxylation of pseudocumene to produce trimethylhydroquinone (TMHQ). The aromatic TMHQ is a key building block in vitamin E synthesis. The capability of P450 BM3 to catalyze the direct aromatic hydroxylation of pseudocumene to TMHQ was validated and a first biocatalytic route to TMHQ in a one-pot reaction was established. P450 BM3 variants were investigated for their side product formation and selectivity. TMHQ concentrations up to 0.19 g L- 1 were obtained with P450 BM3 M3 (R47S, Y51W, A330F, I401M). This P450 BM3 variant was subsequently used as a starting variant in protein engineering campaigns. Identification of P450 BM3 variants with improved TMHQ formation in large mutant libraries required an effective screening strategy. Therefore, a hydroquinone (HQ) specific screening assay based on the interaction of 4-nitrophenylacetonitrile (NpCN) with HQ under alkaline conditions was established. In the 96-well plate format, a low detection limit (5 μM), broad linear detection range (5 to 250 μM) and low standard deviation (10-14%) was obtained. The NpCN assay was successfully applied for screening of random mutagenesis and semi-rational designed libraries. The P450 BM3 variant with the highest TMHQ formation obtained in protein engineering campaigns was P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P). P450 BM3 AW2 had a 70-fold improved TMHQ formation when compared to the wildtype (WT) and a 1.8-fold improved TMHQ formation compared to P450 BM3 M3. Until now, the industrial application of P450s is restricted to whole-cell systems which bring along several benefits e.g., cofactor regeneration and process stability. Whole-cell catalysts co-expressing P450 BM3 variants and alcohol dehydrogenases (ADHs) in an E. coli host were evaluated to produce cyclooctanone. Furthermore, solvent-resistant Pseudomonas putida strains were explored as hosts for whole-cell conversions as well as the co-expression of FhuAΔ1-160 for improved substrate uptake was studied

A hydroquinone-specific screening system for directed P450 evolution

The direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 μM) and a broad linear detection range (5 to 250 μM) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M). © 2018, The Author(s)

Dataset to "A hydroquinone-specific screening system for directed P450 evolution"

In direct evolution, an effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. In the manuscript “A hydroquinone specific screening system for directed enzyme evolution” (A. M. Weingartner, D. F. Sauer, G. V. Dhoke, M. D. Davari, A. J. Ruff, and U. Schwaneberg, Appl Microbiol Biotechnol. 2018; 102(22): 9657–9667. doi: 10.1007/s00253-018-9328-3), we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-Nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. Here the dataset to the above mentioned manuscript is reported. Source Data of the NMR spectra (Fig1 and FigS1) are given as well as source data in Excel format to display the spectra of the hydroquinones (FigS3), the linear detection range of the assay of different hydroquinones (FigS6) or of TMHQ (Fig2) and screening data of the SSM library on position 330 with the NpCN assay (FigS7). In the description of dataset a summary of the collected source data is given

Dataset to "An Enzymatic Route to α‐Tocopherol Synthons: Aromatic Hydroxylation of Pseudocumene and Mesitylene with P450 BM3"

Aromatic hydroxylation of pseudocumene and mesitylene with P450 BM3 yields key phenolic building blocks for a-tocopherol synthesis. Site-saturation mutagenesis generated a new P450 BM3 mutant, named “variant M3” (R47S, Y51W, A330F, I401M), with significantly increased coupling efficiency (3- to 8-fold) and activity (75- to 230-fold) for the conversion of pseudocumene and mesitylene. Here the dataset to “An Enzymatic Route to α‐Tocopherol Synthons: Aromatic Hydroxylation of Pseudocumene and Mesitylene with P450 BM3” (Alexander Dennig, Alexandra Maria Weingartner, Tsvetan Kardashliev, Christina Andrea Müller, Erika Tassano, Martin Schürmann, Anna Joëlle Ruff, Ulrich Schwaneberg, Chemistry. 2017 Dec 19;23(71):17981-17991., doi: 10.1002/chem.201703647) is given. This study provides an enzymatic route to key phenolic synthons for a-tocopherols and the first catalytic and mechanistic insights into direct aromatic hydroxylation and dearomatization of trimethylbenzenes with O2.Source data of the performed substrate docking, and mechanistic considerations are given. Docking of mesitylene into the active site of P450 BM3 WT (source data to Fig1) and the homology model of P450 BM3 variant M3, as well as Docking of pseudocumene into the active site of P450 BM3 WT (1BU7) (source data to Fig2) and P450 BM3 variant M3 (R47S, Y51W, A330F, I401M) (source data to Fig3) are submitted. In the description of dataset a summary of the docking source data is given

Versatile Gene-Specific Sequence Tags for Arabidopsis Functional Genomics: Transcript Profiling and Reverse Genetics Applications

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics