Whole-cell screening of oxidative enzymes using genetically encoded sensors

Biocatalysis is increasingly used for synthetic purposes in the chemical and especially the pharmaceutical industry. Enzyme discovery and optimization which is frequently needed to improve biocatalytic performance rely on high-throughput methods for activity determination. These methods should ideally be generic and applicable to entire enzyme families. Hydrogen peroxide (H2O2) is a product of several biocatalytic oxidations and its formation can serve as a proxy for oxidative activity. We designed a genetically encoded sensor for activity measurement of oxidative biocatalysts via the amount of intracellularly-formed H2O2. A key component of the sensor is an H2O2-sensitive transcriptional regulator, OxyR, which is used to control the expression levels of fluorescent proteins. We employed the OxyR sensor to monitor the oxidation of glycerol to glyceraldehyde and of toluene to o-cresol catalysed by recombinant E. coli expressing an alcohol oxidase and a P450 monooxygenase, respectively. In case of the P450 BM3-catalysed reaction, we additionally monitored o-cresol formation via a second genetically encoded sensor based on the phenol-sensitive transcriptional activator, DmpR, and an orthogonal fluorescent reporter protein. Single round screens of mutant libraries by flow cytometry or by visual inspection of colonies on agar plates yielded significantly improved oxidase and oxygenase variants thus exemplifying the suitability of the sensor system to accurately assess whole-cell oxidations in a high-throughput manner.ISSN:2041-6520ISSN:2041-653

Directed Evolution of DNA Polymerases for Advancement of the SeSaM Mutagenesis Method and Biotransformations with P450 BM3 Monooxygenase

Directed evolution is a powerful algorithm to tailor proteins to needs and requirements in industry. Error-prone PCR (epPCR) based methods are the “golden standard” in random mutagenesis due to their robustness and simplicity. Despite their wide use in protein engineering experiments, epPCR methods are limited in their ability to generate highly diverse mutant libraries. This is due to 4 reasons – 1) the redundancy of the genetic code, 2) the low mutagenic frequency and lack of subsequent nucleotide exchanges in a standard epPCR library, 3) the tendency of polymerases to introduce mutations preferentially in certain DNA sequence contexts and 4) the innate transition bias of DNA polymerases leading to conservative amino acid exchanges. Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method that has been developed to overcome the aforementioned limitations. SeSaM complements the mutagenic spectrum of epPCR by introducing transversions and consecutive nucleotide substitutions. Indeed, SeSaM libraries enriched in consecutive nucleotide subsitutions and transversion mutations were reported; however the frequency of occurrence of such mutations remains low. In particular, the fraction of consecutive transversion mutations accounted for a meager 4.6 %. The increase of the fraction of consecutive transversion mutations is critical in order to advance the SeSaM technology and access previously unattainable sequence space. This can be achieved by the use of DNA polymerases adapted to the requirements of the SeSaM method. The biggest challenge is posed in SeSaM step 3 in which the employed DNA polymerase must be able to elongate consecutive transversion mismatches formed between degenerate base analogs in the primer strand and standard nucleobases in the template strand. The most feasible solution to this problem is to engineer a DNA polymerase capable of efficient consecutive transversion mismatch elongation. The latter has been the main objective pursued in CHAPTER I of this doctoral thesis. The work towards fulfilling this goal included identification from genetic databases of 4 potential candidates from the Y-family of DNA polymerases (exclusively involved in translesion DNA synthesis), followed by expression and preliminary biochemical characterization of the putative polymerases, and, finally, selection of one candidate (Dpo4 from Sulfolobus solfataricus) for directed evolution. The protein engineering work comprised development of a novel high-throughput screening system for non-processive DNA polymerases, screening and identification of Dpo4 variants capable of consecutive mismatch elongation. Finally, the most promising polymerase mutant was used in the preparation of a model SeSaM library. Direct comparison to data generated using an earlier version of the SeSaM protocol indicated a marked improvement in frequency of consecutive transversion mutations in the libraries prepared with the identified Dpo4 polymerase mutant. The identified polymerase variant enabled a significant advancement in consecutive transversion generation and, consequently, of the SeSaM random mutagenesis method. In CHAPTER II of this dissertation, P450 BM3 monooxygenase was studied in the context of regioselective biotransformations of benzenes with isolated enzyme (P450 Project I) and as a whole cell catalyst (P450 Project II). P450 BM3 is a promising biocatalyst which enables challenging chemical reactions, e.g., insertion of an oxygen atom into a non-activated C-H bond. In P450 Project I, P450 BM3 in purified form was employed in the synthesis of mono- and di-hydroxylated products from six monosubstituted benzene substrates. A P450 BM3 mutant (P450 BM3 M2 (R47S/Y51W/A330F) proved to be promiscuous and highly regioselective (95 % - 99 %) hydroxylation catalyst achieving good overall yields (up to 50 % with benzenes and ≥90 % with phenols). The developed process showed promising potential for sustainable synthesis on a semi-preparative scale of valuable chemical precursors, i.e., phenols and hydroquinones, even prior to bioprocess optimizations. Nevertheless, productivity, especially with non-halogenated substrates, would need to be further improved. Especially, whole cell cofactor regeneration systems have to be developed in order to bring this attractive synthesis route closer to industrial exploitation. The issue of cofactor regeneration served as a stimulus to initiate follow-up project dealing with whole cell catalysis with P450 BM3. The requirement for expensive reduced cofactor (NAD(P)H) in equimolar amounts is a major disadvantage of P450 BM3 preventing its wider use in organic synthesis. The NAD(P)H dependency of P450 BM3 necessitates the use of whole cells in preparative synthesis in order to achieve cost-efficient cofactor regeneration. However, the semi-permeable nature of the outer membrane of industrially relevant bacteria such as E. coli limits their use as whole cell systems. The issue of substrate permeability in whole-cell biocatalysts has been addressed in this thesis by co-expressing a large passive diffusion channel, FhuA Δ1-160, in the outer membrane of E. coli. The influence of the channel protein on P450 BM3-catalyzed conversions of 2 monosubstituted benzenes has been investigated in P450 Project II. Preliminary experiments indicated that the co-expression in the outer E. coli membrane of FhuA Δ1-160 had a positive effect, possibly of global nature, on the mass transfer in whole cell biotransformations (up to 10-fold relative increase of product titers). The presented drawback of whole-cell biocatalysis has not been extensively addressed to date, and with these preliminary and promising first results, we hope to entice further interest in the topic.In summary, this doctoral thesis addresses three questions from the related fields of protein engineering and biocatalysis: 1) Advancement of diversity generation methods in directed protein evolution; 2) Establishing biocatalytic process for oxy-functionalization of generally unreactive aromatic C-H bonds; 3) Developing a strategy to improve the mass transfer across the outer membrane of E. coli in whole cell oxy-functionalization of aromatic compounds

Directed Evolution of DNA Polymerases for Advancement of the SeSaM Mutagenesis Method and Biotransformations with P450 BM3 Monooxygenase

Directed evolution is a powerful algorithm to tailor proteins to needs and requirements in industry. Error-prone PCR (epPCR) based methods are the “golden standard” in random mutagenesis due to their robustness and simplicity. Despite their wide use in protein engineering experiments, epPCR methods are limited in their ability to generate highly diverse mutant libraries. This is due to 4 reasons – 1) the redundancy of the genetic code, 2) the low mutagenic frequency and lack of subsequent nucleotide exchanges in a standard epPCR library, 3) the tendency of polymerases to introduce mutations preferentially in certain DNA sequence contexts and 4) the innate transition bias of DNA polymerases leading to conservative amino acid exchanges. Sequence Saturation Mutagenesis (SeSaM) is a random mutagenesis method that has been developed to overcome the aforementioned limitations. SeSaM complements the mutagenic spectrum of epPCR by introducing transversions and consecutive nucleotide substitutions. Indeed, SeSaM libraries enriched in consecutive nucleotide subsitutions and transversion mutations were reported; however the frequency of occurrence of such mutations remains low. In particular, the fraction of consecutive transversion mutations accounted for a meager 4.6 %. The increase of the fraction of consecutive transversion mutations is critical in order to advance the SeSaM technology and access previously unattainable sequence space. This can be achieved by the use of DNA polymerases adapted to the requirements of the SeSaM method. The biggest challenge is posed in SeSaM step 3 in which the employed DNA polymerase must be able to elongate consecutive transversion mismatches formed between degenerate base analogs in the primer strand and standard nucleobases in the template strand. The most feasible solution to this problem is to engineer a DNA polymerase capable of efficient consecutive transversion mismatch elongation. The latter has been the main objective pursued in CHAPTER I of this doctoral thesis. The work towards fulfilling this goal included identification from genetic databases of 4 potential candidates from the Y-family of DNA polymerases (exclusively involved in translesion DNA synthesis), followed by expression and preliminary biochemical characterization of the putative polymerases, and, finally, selection of one candidate (Dpo4 from Sulfolobus solfataricus) for directed evolution. The protein engineering work comprised development of a novel high-throughput screening system for non-processive DNA polymerases, screening and identification of Dpo4 variants capable of consecutive mismatch elongation. Finally, the most promising polymerase mutant was used in the preparation of a model SeSaM library. Direct comparison to data generated using an earlier version of the SeSaM protocol indicated a marked improvement in frequency of consecutive transversion mutations in the libraries prepared with the identified Dpo4 polymerase mutant. The identified polymerase variant enabled a significant advancement in consecutive transversion generation and, consequently, of the SeSaM random mutagenesis method. In CHAPTER II of this dissertation, P450 BM3 monooxygenase was studied in the context of regioselective biotransformations of benzenes with isolated enzyme (P450 Project I) and as a whole cell catalyst (P450 Project II). P450 BM3 is a promising biocatalyst which enables challenging chemical reactions, e.g., insertion of an oxygen atom into a non-activated C-H bond. In P450 Project I, P450 BM3 in purified form was employed in the synthesis of mono- and di-hydroxylated products from six monosubstituted benzene substrates. A P450 BM3 mutant (P450 BM3 M2 (R47S/Y51W/A330F) proved to be promiscuous and highly regioselective (95 % - 99 %) hydroxylation catalyst achieving good overall yields (up to 50 % with benzenes and ≥90 % with phenols). The developed process showed promising potential for sustainable synthesis on a semi-preparative scale of valuable chemical precursors, i.e., phenols and hydroquinones, even prior to bioprocess optimizations. Nevertheless, productivity, especially with non-halogenated substrates, would need to be further improved. Especially, whole cell cofactor regeneration systems have to be developed in order to bring this attractive synthesis route closer to industrial exploitation. The issue of cofactor regeneration served as a stimulus to initiate follow-up project dealing with whole cell catalysis with P450 BM3. The requirement for expensive reduced cofactor (NAD(P)H) in equimolar amounts is a major disadvantage of P450 BM3 preventing its wider use in organic synthesis. The NAD(P)H dependency of P450 BM3 necessitates the use of whole cells in preparative synthesis in order to achieve cost-efficient cofactor regeneration. However, the semi-permeable nature of the outer membrane of industrially relevant bacteria such as E. coli limits their use as whole cell systems. The issue of substrate permeability in whole-cell biocatalysts has been addressed in this thesis by co-expressing a large passive diffusion channel, FhuA Δ1-160, in the outer membrane of E. coli. The influence of the channel protein on P450 BM3-catalyzed conversions of 2 monosubstituted benzenes has been investigated in P450 Project II. Preliminary experiments indicated that the co-expression in the outer E. coli membrane of FhuA Δ1-160 had a positive effect, possibly of global nature, on the mass transfer in whole cell biotransformations (up to 10-fold relative increase of product titers). The presented drawback of whole-cell biocatalysis has not been extensively addressed to date, and with these preliminary and promising first results, we hope to entice further interest in the topic.In summary, this doctoral thesis addresses three questions from the related fields of protein engineering and biocatalysis: 1) Advancement of diversity generation methods in directed protein evolution; 2) Establishing biocatalytic process for oxy-functionalization of generally unreactive aromatic C-H bonds; 3) Developing a strategy to improve the mass transfer across the outer membrane of E. coli in whole cell oxy-functionalization of aromatic compounds

Efficient synthesis of 2,6-bis(hydroxymethyl)pyridine using whole-cell biocatalysis

We demonstrate a novel one-pot biocatalytic process for the preparation of a versatile chemical intermediate, 2,6-bis(hydroxymethyl)pyridine, from naturally-occurring 2,6-lutidine using recombinant microbial whole cells as a catalysts. After scale up, the bioconversion enabled titers exceeding 12 g L-1 with a space-time yield of 0.8 g L-1 h(-1). This biocatalytic route offers a simpler and more sustainable alternative to multistep organic synthesis protocols.ISSN:1463-9262ISSN:1463-927

Directed Evolution of a Surface-Displayed Artificial Allylic Deallylase Relying on a GFP Reporter Protein

Artificial metalloenzymes (ArMs) combine characteristics of both homogeneous catalysts and enzymes. Merging abiotic and biotic features allows for the implementation of new-to-nature reactions in living organisms. Here, we present the directed evolution of an artificial metalloenzyme based on; Escherichia coli; surface-displayed streptavidin (Sav; SD; hereafter). Through the binding of a ruthenium-pianostool cofactor to Sav; SD; , an artificial allylic deallylase (ADAse hereafter) is assembled, which displays catalytic activity toward the deprotection of alloc-protected 3-hydroxyaniline. The uncaged aminophenol acts as a gene switch and triggers the overexpression of a fluorescent green fluorescent protein (GFP) reporter protein. This straightforward readout of ADAse activity allowed the simultaneous saturation mutagenesis of two amino acid residues in Sav near the ruthenium cofactor, expediting the screening of 2762 individual clones. A 1.7-fold increase of; in vivo; activity was observed for Sav; SD; S112T-K121G compared to the wild-type Sav; SD; (wt-Sav; SD; ). Finally, the best performing Sav isoforms were purified and tested; in vitro; (Sav; PP; hereafter). For Sav; PP; S112M-K121A, a total turnover number of 372 was achieved, corresponding to a 5.9-fold increase vs wt-Sav; PP; . To analyze the marked difference in activity observed between the surface-displayed and purified ArMs, the oligomeric state of Sav; SD; was determined. For this purpose, crosslinking experiments of; E. coli; cells overexpressing Sav; SD; were carried out, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The data suggest that Sav; SD; is most likely displayed as a monomer on the surface of; E. coli; . We hypothesize that the difference between the; in vivo; and; in vitro; screening results may reflect the difference in the oligomeric state of Sav; SD; vs soluble Sav; PP; (monomeric vs tetrameric). Accordingly, care should be applied when evolving oligomeric proteins using; E. coli; surface display

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