Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles

The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min−1; R255L: 1168 ± 163 min−1), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation

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)

Advances in directed monooxygenase evolution : from diversity generation and flow cytometry screening to tailor-made monooxygenases

Directed Evolution became a powerful tool for proteins engineers to generate tailor-made biocatalyst. Directed protein evolution consist of the following three consecutive main steps, which are performed in iterative cycles; Step 1 the gene diversity generation, Step 2 the screening for improved variants and Step 3 the isolation of gene encoding for improved proteins. In this thesis, methodological advancements in the two key steps of the directed evolution, the diversity generation (SeSaM method) and high throughput screening (flow cytometer based screening technology) were performed and complemented by the evolution of P450 BM3 for alternative cofactor systems. Step 1 consisting of generation of high quality mutant libraries was advanced by developing an improved SeSaM protocol in which a universal base complementary to the P-base was employed to generate more diverse libraries. Step 2 was advanced by generating a whole cell high throughput screening assay for monooxygenases allowing to efficiently sample the diversity generated in Step1. Subsequently, the advanced protocols were applied to evolve P450 BM3 for alternative cofactor systems in iterative rounds (Step 3). These developed methods (flow cytometer assay for P450 BM3 and advanced SeSaM method) are of main interest for the researchers focusing on protein engineering. In Chapter I of the thesis, the application of the R-base as universal base complementary to P-base increase the amino acid sequence space that can be covered with the SeSaM method. SeSaM-P/R allows for the first time to introduce at all four nucleotides transversion mutations which are homogeneously distributed over the targeted gene and which mutate in an ideal case each nucleotide of a gene. Second approach to advance the SeSaM method has been undertaken via a modified base at the 3’ position of the sugar backbone. Through a chemical cleavable block the number of added bases was aimed to be controlled. In Chapter II of the thesis, the first flow cytometer based ultra-high throughput screening platform that can be applied in directed evolution campaigns of P450 monooxygenases was developed. A whole cell high throughput screening assay for P450 BM3 based on a coumarin derivative (7-Benzoxy-3-Carboxy-Coumarin Ethyl ester (BCCE)) was established. Furthermore, the presented screening platform is not only restricted to P450 BM3, the reported BCCE screening system will very likely be applicable to all P450 monooxygenases that can be expressed in E. coli and catalyze an O-dealkylation of coumarin derivatives (e.g. human CYP3A4). However, the flow cytometer-based ultra-high throughput screening assay for directed evolution of P450 BM3 towards improved mediated electron transfer with Zn-dust could not be developed. The inhibitory effect of the Zn-dust on the microbial grow was pre-dominent and excluded the use of the double emulsion technology for the evolution of P450 BM3 for mediated electron transfer (MET). In Chapter III, the advanced methods developed in Chapter I and II were employed to generate P450 BM3 libraries to evolve the catalyst towards alternative cofactor systems. P450 monooxygenases have a remarkable catalytic capability of inserting one atom from molecular oxygen into inert CH bonds. This unique oxygen chemistry makes the P450s very attractive for organic synthesis in the chemical and pharmaceutical industries. The evolution of P450 BM3 for improved alternative cofactor systems allows a step forward to liberate P450 BM3 from the requirement of expensive NADPH addition as cofactor which restricts the industrial use. The enzyme was evolved by generating random mutant libraries with the SeSaM method and by epPCR. The libraries were enriched with the flow cytometer screening system and subsequent screening in MTP format for improved mediated electron transfer. P450 BM3 M3 could further be engineered towards increased activity in presence of the alternative cofactor system Zn/Co(III)-sep. Indeed, new hot spots which were not reported before as relevant for the mediated electron transfer, could be identified. The second investigated alternative cofactor system, the direct electron transfer was explored by directed evolution to enhance the activity of the monooxygenase P450 BM3 in presence of a conducting polymer. Focus mutagenesis as well as random approach were performed, but no P450 BM3 variants with improved activity, an increased electron transfer rate or new hot spots for the direct electron transfer could be identified. Subsequently, collaborative P450 BM3 projects are summarized: the monooxygenase mutein database (muteinDB) was supported in which muteins altering substrate specificity are collected, the evolution of P450 BM3 for substrate specificity was contributed and P450 BM3 muteins with surface modifications for electrochemical characterization were generated

Advances in directed monooxygenase evolution : from diversity generation and flow cytometry screening to tailor-made monooxygenases

Directed Evolution became a powerful tool for proteins engineers to generate tailor-made biocatalyst. Directed protein evolution consist of the following three consecutive main steps, which are performed in iterative cycles; Step 1 the gene diversity generation, Step 2 the screening for improved variants and Step 3 the isolation of gene encoding for improved proteins. In this thesis, methodological advancements in the two key steps of the directed evolution, the diversity generation (SeSaM method) and high throughput screening (flow cytometer based screening technology) were performed and complemented by the evolution of P450 BM3 for alternative cofactor systems. Step 1 consisting of generation of high quality mutant libraries was advanced by developing an improved SeSaM protocol in which a universal base complementary to the P-base was employed to generate more diverse libraries. Step 2 was advanced by generating a whole cell high throughput screening assay for monooxygenases allowing to efficiently sample the diversity generated in Step1. Subsequently, the advanced protocols were applied to evolve P450 BM3 for alternative cofactor systems in iterative rounds (Step 3). These developed methods (flow cytometer assay for P450 BM3 and advanced SeSaM method) are of main interest for the researchers focusing on protein engineering. In Chapter I of the thesis, the application of the R-base as universal base complementary to P-base increase the amino acid sequence space that can be covered with the SeSaM method. SeSaM-P/R allows for the first time to introduce at all four nucleotides transversion mutations which are homogeneously distributed over the targeted gene and which mutate in an ideal case each nucleotide of a gene. Second approach to advance the SeSaM method has been undertaken via a modified base at the 3’ position of the sugar backbone. Through a chemical cleavable block the number of added bases was aimed to be controlled. In Chapter II of the thesis, the first flow cytometer based ultra-high throughput screening platform that can be applied in directed evolution campaigns of P450 monooxygenases was developed. A whole cell high throughput screening assay for P450 BM3 based on a coumarin derivative (7-Benzoxy-3-Carboxy-Coumarin Ethyl ester (BCCE)) was established. Furthermore, the presented screening platform is not only restricted to P450 BM3, the reported BCCE screening system will very likely be applicable to all P450 monooxygenases that can be expressed in E. coli and catalyze an O-dealkylation of coumarin derivatives (e.g. human CYP3A4). However, the flow cytometer-based ultra-high throughput screening assay for directed evolution of P450 BM3 towards improved mediated electron transfer with Zn-dust could not be developed. The inhibitory effect of the Zn-dust on the microbial grow was pre-dominent and excluded the use of the double emulsion technology for the evolution of P450 BM3 for mediated electron transfer (MET). In Chapter III, the advanced methods developed in Chapter I and II were employed to generate P450 BM3 libraries to evolve the catalyst towards alternative cofactor systems. P450 monooxygenases have a remarkable catalytic capability of inserting one atom from molecular oxygen into inert CH bonds. This unique oxygen chemistry makes the P450s very attractive for organic synthesis in the chemical and pharmaceutical industries. The evolution of P450 BM3 for improved alternative cofactor systems allows a step forward to liberate P450 BM3 from the requirement of expensive NADPH addition as cofactor which restricts the industrial use. The enzyme was evolved by generating random mutant libraries with the SeSaM method and by epPCR. The libraries were enriched with the flow cytometer screening system and subsequent screening in MTP format for improved mediated electron transfer. P450 BM3 M3 could further be engineered towards increased activity in presence of the alternative cofactor system Zn/Co(III)-sep. Indeed, new hot spots which were not reported before as relevant for the mediated electron transfer, could be identified. The second investigated alternative cofactor system, the direct electron transfer was explored by directed evolution to enhance the activity of the monooxygenase P450 BM3 in presence of a conducting polymer. Focus mutagenesis as well as random approach were performed, but no P450 BM3 variants with improved activity, an increased electron transfer rate or new hot spots for the direct electron transfer could be identified. Subsequently, collaborative P450 BM3 projects are summarized: the monooxygenase mutein database (muteinDB) was supported in which muteins altering substrate specificity are collected, the evolution of P450 BM3 for substrate specificity was contributed and P450 BM3 muteins with surface modifications for electrochemical characterization were generated

Dataset to A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3

The main challenge for the success of directed enzyme evolution campaigns is the availability of suitable high-throughput screening systems. Multiplex capillary electrophoresis with a universal UV-based product detection is a promising analytical tool, which allows to quantify product formation simultaneously in 96-capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. The platform allows a comprehensive view on enzyme activity through the detection of all products formed during the target reaction. In the manuscript A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3. (Gärtner, A., Ruff, A.J. & Schwaneberg, U., Sci Rep 9, 15479 (2019), doi:10.1038/s41598-019-52077-w), we report for the first time the usage of a MP-CE system for the screening of evolution libraries. After adaptation of the screening preparation and measurement conditions, the system was validated for the suitability as screening platform for P450 BM3 evolution. The oxidation of α-isophorone was selected as target reaction. The MP-CE platform was compared to the standard cofactor (NADPH) screening system and was found to be 3.5-fold more efficient in identification of beneficial. Here the dataset to the above mentioned manuscript is reported. Source Data of the figures are given in Origin and Excel format to display the CE-measurements (CE-chromatograms), the screening data (summary variants and 1st screening round) and how the data evaluation described in the MS (standard curves and standard deviation) was performed. In the description of dataset a summary of the collected source data is given

Reconstructive procedures for impaired upper airway function: laryngeal respiration

The larynx is the "bottleneck" of the human airway. For this reason, the effects of stenosing laryngeal pathologies on the vital factor respiratory gas exchange are particularly critical.Internal stabilization is a prerequisite for recovery of the laryngeal respiratory function in severe forms of inspiratory collapse (laryngomalacia). Effective laser surgery techniques have been developed to this end in recent years.Glottis-dilating surgery in cases of bilateral vocal cord motion impairment is now moving in the direction of endoscopic laser cordotomy or cordectomy, whereas arytenoidectomy and open surgical procedures are now used only rarely due to higher secondary morbidity rates. In individual cases, in particular if functional recovery is expected, temporary laterofixation of a vocal cord using an endoscopic suturing technique can be a helpful approach.Extensive laryngeal defects can be covered by means of composite grafts with mucosal lining, a supporting skeleton and their own vascularization. Autologous transplantation of the larynx, with its complex surgical and immunological problems, has become a manageable procedure. The problems of post-transplantation reinnervation and risk assessment of immunosuppression-induced recurrence of the tumor are still under consideration.Reanimation of the bilaterally paralyzed larynx by means of neurorrhaphy (neurosuture), neural grafting and, more recently, functional electrostimulation (pacemaker) represents a challenge for the coming years. In most cases of paralysis of the recurrent laryngeal nerve, a part of the muscles is maintained by synkinetic reinnervation when therapy is carried out, which however also prevents effective vocal cord movement due to simultaneous activity of agonists and antagonists. Modulation of reinnervation by means of electrostimulation and modern genetic therapy approaches justify hopes of better outcomes in the future.Der Kehlkopf stellt hinsichtlich des Atemwegsquerschnittes den Flaschenhals des menschlichen Atemweges dar. Aus diesem Grund wirken sich stenosierende Kehlkopferkrankungen in besonderem Maße auf den lebenswichtigen Atemgasaustausch aus.Die Wiederherstellung der Atemfunktion des Larynx erfordert bei schweren Formen des inspiratorischen Kollapses (Laryngomalazie) die innere Stabilisierung. In den letzten Jahren sind hierzu wirksame Verfahren der laserchirurgischen Intervention entwickelt worden.Die Glottis erweiternde Chirurgie bei bilateraler Immobilität der Stimmlippen zeigt einen Trend zur endoskopischen laserchirurgischen Chordotomie bzw. Chordektomie, während die Arytänoidektomie und offen chirurgische Verfahren wegen stärkerer Sekundärmorbidität nur noch selten zum Einsatz kommen. Im Einzelfall, insbesondere bei absehbarer Funktionswiederkehr, kann die temporäre Laterofixation einer Stimmlippe durch eine endoskopische Nahttechnik hilfreich sein.Ausgedehnte Kehlkopfdefekte können mit Composite grafts, die über eine Schleimhautauskleidung, ein Stützskelett und eine eigene Gefäßversorgung verfügen, gedeckt werden. Die autologe Transplantation des Kehlkopfes ist mit ihren komplexen chirurgischen und immunologischen Problemen beherrschbar geworden. Probleme der Reinnervation nach Transplantation und der Abwägung des Risikos eines durch die Immunsuppression induzierten Tumorrezidivs sind noch Gegenstand weiterer Untersuchungen.Die Reanimation des bilateral gelähmten Kehlkopfes durch Nervennaht, durch Nerventransfer und neuerdings durch funktionelle Elektrostimulation (Pacemaker) stellt die Herausforderung für die nächsten Jahre dar. Bei der überwiegenden Zahl der Rekurrensparesen liegt zum Zeitpunkt der Therapie eine synkinetische Reinnervation vor, die einen Teil der Muskulatur erhält, aber durch gleichzeitige Aktivität von Agonisten und Antagonisten eine effektive Stimmlippenbewegung verhindert. Die Modulation der Reinnervation durch Elektrostimulation und moderne Ansätze der Gentherapie lassen ein zukünftig besseres Outcome erwarten