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Cascade reactions are not only of interest to chemists and biotechnologists, but also to life in general, because every metabolic reaction resembles a cascade reaction. This principle of substrate/intermediate channeling was only adapted by scientists. That way especially one-pot reactions became very attractive as for this no isolation of intermediates is necessary. Furthermore, unstable or toxic intermediates are only produced in low amounts and directly transformed in situ. In this PhD thesis two previously established cascade reactions were subject of further optimization. In the first part, a cascade reaction established in a DFG-funded project (Bo1862/6-1)in cooperation with the Vienna Technical University (Austria) for the production of chiral lactones was further optimized and extended. Therefore, on the one hand the genes encoding the needed enzymes were cloned for co-expression into a single plasmid in different arrangements to be expressed in pseudo-operon mode, with the aim to lower the metabolic burden of the cascade host cell. One out of the welve created constructs showed a reasonable activity of 15.3 ± 1.2 U · gCDW-1. On the other hand, this cascade reaction was aimed to be extended by the use of a hydroxylating enzyme to enable the use of limonene as renewable and chiral precursor for the proposed production of chiral polymers. Therefore, the feasibility of cytochrome P450-monooxygenases was studied. These turned out to be not applicable due to their bad regioselectivity for the hydroxylation of limonene or due to the difficulties of activity reconstitution. As alternative system for an initial hydroxylation step the use of a Rhodococcus equi strain, which was isolated from Cellulosimicrobium cellulans EB-8-4 and which is capable of very regioselective limonene-hydroxylation, was investigated. Therefore, the dioxygenase cluster responsible for the desired reaction was identified and especially the recombinant expression in a suitable host (Pseudomonas putida S12) was further studied. The results from these experiments revealed that the recombinant expression needs to be further optimized to enable the use of the recombinant dioxygenase in combination with the other enzymes for cascade reactions. The third part of this PhD thesis dealt with the immobilization of an established cascade reaction for the synthesis of poly-[caprolactone] precursors. Therefore, the use of a rotating bed reactor (RBR) was investigated. Preliminary studies using single enzymes involved in the desired cascade reaction demonstrated the general feasibility of this reactor concept. Especially the reusability of the catalysts was highly improved, because the catalytic particles were protected very effectively from mechanical forces within the voids of the reactor. For further work-flow optimization the immobilization was transformed into an in situ process by the application of a gas-shear device, which leads to decreased capsule size and thereby to increased mass transfer inside the particles. The developed methods were applied for encapsulation of the cells containing the enzymes needed for the reaction. After additional improvement of the reaction parameters a conversion of 93% (based on substrate depletion) was reached using catalysts produced by the established encapsulation procedure. In summary, the described cascade reactions were successfully optimized by either co-expression, extension applying a dioxygenase or immobilization. Furthermore, the general feasibility of an RBR was demonstrated.
Entdeckung und Design promiskuitiver Acyltransferase‐Aktivität in Carboxylesterasen der Familie VIII
(2021)
In 2010, the identification of 17 novel (R)-ATAs represented a breakthrough for the biocatalytic asymmetric synthesis of chiral amines, because only one (R)-ATA was described before. These novel ATAs were identified in a bioinformatic approach by studying the substrate acceptance of BCATs and DATAs to deduce the unknown substrate coordination of (R)-ATAs. Article I describes an alternative approach for the identification of (R)-ATA activity by reengineering the substrate- recognition site of α-AATs. While the engineering of the eBCAT led to the formation of an initial (R)-amine acceptance only, the (R)-ATA activity was successfully introduced in the DATA scaffold. These results demonstrate the transformation of an α-AAT in a moderately active (R)-ATA for the first time and highlight the evolutionary relationship between α-AATs and ATAs. Despite the availability of different ATAs nowadays, their substrate spectrum is limited due to the natural composition of their active sites. Several protein-engineering studies showed the widening of the substrate spectrum and the acceptance of bulky substrates by screening large mutant libraries to identify beneficial variants. In Article II, we developed an in silico engineering approach for amine transaminases to improve the conversion of bulky substrates and to reduce the number of variants to be tested in the laboratory. The resulting double-mutants of the (S)-ATA from C. violaceum displayed a >200-fold improved activity towards the bulky benchmark substrate. These variants expand the available biocatalytic toolbox for the synthesis of bulky amines, and the developed framework paves the way for rational protein-engineering protocols.
By studying unconventional transaminase substrates, we explored the potential of the available in- house transaminase toolbox in Articles III, IV, V, and VI. In Article III, we showed the transamination of a β-keto ester, leading to the synthesis of β-phenylalanine. The described cascade in Article IV enables the synthesis of amino carbohydrates. In addition, Article V describes an enzymatic cascade for the synthesis of amino fatty acids, which was extended in Article VI to obtain fatty amines.
The findings of this thesis clearly contribute to the understanding of the substrate scope and specificity of amine transaminases and expand the application of this versatile biocatalyst beyond classical ketone substrates.
In this thesis, two novel assay systems had been developed, which allow a fast and easy screening for amine transaminase activity as well as the characterization of the amino donor and acceptor specificity of a given amine transaminase. The assays overcome some limitations of previously described assays but of course have some limitations themselves. The relatively low wavelength of 245 nm, at which the production of acetophenone is detected with the spectrophotometric assay, limits the amount of protein/crude extract that can be applied, which eventually results in a decreased sensitivity at higher enzyme loads due to an increased initial absorbance. Otherwise, this assay can be used very easily for the investigation of the amino acceptor specificity and both pH and temperature dependencies of amine transaminases. The conductometric assay is – by its very nature – limited to low-conducting buffers, a neutral pH and constant temperatures. In summary, the assays complement one another very well and the complete characterization of the most important enzyme properties can be accomplished quickly. Furthermore, we developed and applied a novel in silico search strategy for the identification of (R)-selective amine transaminases in sequence databases. Structural information of probably related proteins was used for rational protein design to predict key amino acid substitutions that indicate the desired activity. We subsequently searched protein databases for proteins already carrying these mutations instead of constructing the corresponding mutants in the laboratory. This methodology exploits the fact that naturally evolved proteins have undergone selection over millions of years, which has resulted in highly optimized catalysts. Using this in silico approach, we have discovered 17 (R)-selective amine transaminases. In theory, this strategy can be applied to other enzyme classes and fold types as well and for this reason constitutes a new concept for the identification of desired enzymes. Finally, we applied the seven most promising candidates of the identified proteins to asymmetric synthesis of various optical pure amines with (R)-configuration starting from the corresponding ketones. We used a lactate dehydrogenase/glucose dehydrogenase system for the necessary shift of the thermodynamic equilibrium. For all ketones at least one enzyme was found that allowed complete conversion to the corresponding chiral amine with excellent optical purities >99% ee. Bearing in mind that until last year there was only one (R)-selective amine transaminase commercially available and two microorganisms with the corresponding activity described, the identification of numerous enzymes is a breakthrough in asymmetric synthesis of chiral amines.
In this thesis several methods of protein engineering were applied to explore and increase enantioselectivity and thermostability of certain carboxylesterases and to better understand the relationship between sequence, structure and function. For example, we were able to confirm the observed conservation of motifs like GX/GGGX and GXSXG, which was reported earlier. Yet, even more details were revealed and some were designated in numbers. However, the numbers may vary when even more sequences will be available, but the trend should remain the same. The power of the ABHDB lies in the information available throughout the very diverse and quite large superfamily. Structural equal positions can be easily compared and analysed regarding mutations, correlated mutations, prevalence etc., and visualization is simplified through direct output with YASARA software. The ABHDB was the first structural alignment of such a large number of known enzymes of the alpha/beta-hydrolase fold superfamily. With methods of rational protein engineering we were able to show that there is little flexibility of the GGG(A)X motif for the eukaryotic enzyme PLE 1 and the natural motif appears to be a good solution for high activity and enantioselectivity of PLE 1 in the conversion of tertiary alcohol esters. In a focused directed evolution approach, we were able to identify variants of BsteE with moderate, but significantly increased enantioselectivity in the kinetic resolution of tetrahydrofuran-3-yl acetate, and hence, were able to proof that the concept of ‘small but smart’ libraries is an efficient way to find improved mutants, while the screening effort was reduced. Moreover, we were able to show that the domain exchange enhanced the thermostability of BsubE, while expression level and activity were maintained or increased, respectively. Despite the great achievements and possibilities at present, we are not yet in the position to directly modify the gene to alter the structure in a complete predictable fashion to improve functional properties as imagined by Ulmer (1983). Nevertheless, substantial changes can be targeted and as demonstrated in this work, several broadly applicable methods are at hand. Furthermore, bioinformatics tools play an essential role in planning of experiments, analysis and interpretation.
This thesis is about the establishment and the application of novel methods and tools that are re-lated to the most widely used enzyme class: hydrolases. It covers all fields from the identification to the application of these valuable enzymes with particular focus on lactonases, acylases and proteases. The activity assay introduced in Article I substantially extends the method toolbox for studies on lactonases and acylases that interfere with the bacterial cell-cell communication system. Article II describes a fully automatized robotic platform that represents the next-level tool for the high-throughput enzyme screening in the microtiter plate format. It was used, for instance, for the screening for improved porcine aminoacylase I variants. Diverse aspects of the protease-mediated hydrolysis of non-resistant proteins for the purification of resistant target proteins are highlighted in Article III.
Chiral amines represent high-value fine chemicals serving as key intermediate products in pharmaceutical, chemical and agrochemical industries. In the past decades, application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones emerged to an environmentally benign and economically attractive alternative to transition metal-catalyzed asymmetric synthesis to afford optically pure amines at industrial scale. However, the restricted substrate scope of wild-type transaminases prohibited the conversion of particularly sterically demanding substrates, making protein engineering indispensable. The following thesis covers elaboration of a novel assay for transaminases (Article I) and identification and development of transaminase variants in order to achieve biocatalytic preparation of a set of pharmaceutically relevant model amines, ideally in optically pure form for both stereoisomers, preferentially using asymmetric synthesis and most preferably using isopropylamine as cost-efficient amine donor co-substrate (Article II-IV). The aforementioned target amines and the corresponding precursor ketones (see Scheme 4.1) were conceived and provided by the company F. Hoffmann-La Roche to attain suitable biocatalysts for a variety of potential intermediates for active pharmaceutical ingredients. Protein engineering of the transaminase scaffolds investigated in this thesis comprised: Initial screening for suitable starting enzyme scaffolds, structure-guided rational design of these scaffolds to enable bulky planar substrate acceptance, elaboration of a sequence motif, verification of the motif and preparative-scale asymmetric synthesis reactions (Article II). For non-planar and structurally different target substrates, namely spatially bulky or bi-cyclic bridged substrates, the transaminase variants were specifically refined and a different evolutionary route had to be pursued (Article III and Article IV). These results (Article II) represent not only the first successful endeavor to engineer a PLP-fold type I amine transaminase (commonly denoted as (S)-selective) for the conversion of highly sterically demanding substrates, but also generally expanded the scope of available fold type I amine transaminases by enzymes having a novel and exceptionally broad substrate spectrum. Aside from structure-guided rational protein engineering, as well non-rational methods, such as site-specific saturation mutagenesis or directed evolution, were applied for protein-engineering. In order to do so for all of the target compounds, a novel high-throughput solid phase activity assay for transaminases that was actually developed during the master thesis, was refined and published (Article I). In the context of this thesis, the same assay principle was as well adapted for quantification of specific activities in liquid phase (Article III). A comparison of different methodologies for developing agar plate assays and a detailed step by step protocol of our transaminase assay are illustrated in a book chapter.
In this work, the regioselectivity of different Baeyer-Villiger monooxygenases (BVMOs) for the conversion of selected substrates was reversed or improved by protein engineering. These studies highlight the importance of substrate positioning for the regioselectivity and that the position of the substrate can be efficiently influenced by introducing proper mutations. It was shown that the beneficial mutations for all BVMOs were partly in corresponding positions. Additionally, the sulfoxidation activity and the stability of BVMOs were targeted and improved by applying protein engineering.
Structure– and sequence–function relationships in (S)-amine transaminases and related enzymes
(2015)
Chiral primary amines are valuable building blocks for many biologically active compounds. Environmentally friendlier alternatives to the classical methods for α-chiral primary amine synthesis are highly desired. A biocatalytic alternative that recently proved beneficial for industrial applications is asymmetric synthesis utilising (S)-selective amine transaminases (S-ATAs). These enzymes can be utilized to transaminate a prochiral ketone with an amino donor (e.g. isopropylamine), to achieve a chiral amine and a carbonyl product (e.g. acetone). However, for several potential applications protein engineering is required to fit (S)-ATAS to the demands of an industrial process. Since no (S)-ATA crystal structure required for understanding the substrate recognition and thus protein engineering was available, we first aimed at obtaining structural data. Instead of solving crystal structures ourselves, we took advantage of structural genomics projects and discovered, that the protein data bank (PDB) already contained crystal structures of four enzymes with unknown function that we hypothesised to possess (S)-ATA activity. After developing a screening method, the four enzymes could be characterized as ω-amino acid:pyruvate transaminases (ωAA:pyr TAs). (S)-amine conversion was suggested to be a ‘substrate-promiscuous’ activity of these enzymes, as it is pronounced differently in the four investigated ones. By comparing the active sites of the highly and poorly active (S)-ATAs, the residues that determine the ability of amine conversion in these enzymes were discovered. Furthermore, the mechanism for dual substrate recognition, the binding of both, carboxyl and bulky hydrophobic substrates in the same active site, could be elucidated with the crystal structures. A flexible arginine side chain is able to adopt various positions thus enabling carboxylate binding and by ‘flipping’ out of the active site, to create space for amine binding. Then, a limitation of these enzymes, the restricted substrate scope caused by a small binding pocket was addressed. First, a rational protein engineering approach was set up to create more space. The tested mutations, however, destroyed most of the activity for both regular and more bulky substrates. We thus learned that the structural requirements for (S)-ATA activity are more complex than initially anticipated and a semi-rational approach was applied to broaden the substrate scope. By systematic saturation of active site positions, substantially improved mutants for bulkier amine synthesis could be obtained. As this study highlighted a lack of understanding of (S)-ATA, the functional important residues in the enzymes belonging to the class III TA family were surveyed. This family is defined by common sequence and structure features and besides (S)-ATAs mainly comprises TAs of various substrate scopes but also a few phospholyases, racemases and decarboxylases. To enable the comparison of active site residues among them, a commercial bioinformatics tool was used to create a family wide structure-based alignment of around 13,000 sequences. Based on statistical analyses of this alignment, structural inspections and literature evaluation, active site residues crucial for certain specificities within this family have been identified. By investigating the ingenious active site designs that enable such a plethora of reactions, and by identifying sets of functional important residues termed ‘active site fingerprints’, the understanding of catalysis in this enzyme family could be broadened. Furthermore, these functional important residues can on the one hand be applied to predict the specificity of uncharacterised enzymes, if a fingerprint is matched. On the other hand, if no fingerprint is matched, they can help to discover yet unknown activities or mechanisms to achieve a known specificity. We exemplified the latter case by functionally characterising a Bacillus anthracis enzyme with the crystal structure 3N5M, whose substrate specificity was unknown and could not be predicted. The 3N5M enzyme was found to possess ωAA:pyr TA and (S)-ATA activity even though it lacks the above-mentioned ‘flipping’ arginine. Based on molecular dynamics simulations we were able to propose an alternative mechanism for dual substrate recognition in the B. anthracis ωAA:pyr TA. By these findings the understanding of the requirements for (S)-ATA activity could be further broadened and a functional knowledge gap within the class III TA family was closed. The active site residue composition in 3N5M is now connected to enzymatic function and may be applied for future specificity predictions.