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Item Open Access Biotechnologische Herstellung von Octan- und 8-Hydroxyoctansäure mit Escherichia coli(2016) Kirtz, Marko; Hauer, Bernhard (Prof. Dr.)In den letzten 20 Jahren rückt die Entwicklung biotechnologischer Prozesse zur Produktion von Diesel- und Benzinersatzstoffen immer mehr in den Fokus von Forschung und öffentlichem Interesse. Durch die Verknappung der Erdölressourcen[1], die negativen Auswirkungen der extensiven Nutzung dieser Ressourcen auf die Umwelt und nicht zuletzt das stetig wachsende Umweltbewusstsein der Bevölkerung in Industrieländern, wurden Prozesse gefordert, die auf regenerierbaren Ressourcen basieren, somit den permanent in Richtung Atmosphäre gerichteten CO2-Fluss eingrenzen und zudem die Kostenexplosion für Kraftstoffe eindämmen. Jedoch ist nicht nur der weltweite Transportsektor von Erdöl und seinen weiterverarbeiteten Produkten abhängig, auch die produzierende chemische Industrie bezieht einen Großteil ihrer Grundstoffe aus dem „schwarzen Gold“[2,3,4,5]. Hier gilt es, effektive alternative Prozesse zu entwickeln, die zum Großziel einer „grünen Chemie“ beitragen und von der Abhängigkeit von Erdöl losgelöst sind. Diese Arbeit hatte das Ziel durch die Realisierung eines mikrobiellen Produktionsprozesses von mittelkettigen Fett- und ω-Hydroxyfettsäuren einen Beitrag zum Ziel der sog. „grünen Chemie“ zu leisten. Die wirtschaftliche und damit einhergehend umweltschutztechnische Relevanz von Fettsäuren und ihren terminal hydroxylierten Derivaten spiegelt sich in der großen Vielfalt an Produkten, deren Grundbausteine sie bilden, wider. Zum Einsatz kam hier das Bakterium Escherichia coli, für welches bereits in vergangenen Studien Strategien entwickelt worden waren, um beispielsweise (Hemi-)Cellulosen für den Aufbau von eigener Biomasse verwerten zu können[6]. Daher ist es für einen Ansatz, der auf regenerierbaren Ressourcen beruhen soll, bestens geeignet. Um mit diesem Bakterium in einem Produktionsprozess Fettsäuren zu generieren, musste seine native Fettsäurebiosynthese zu einer Überproduktion der gewünschten Fettsäure angeregt werden. Dazu wurde die pflanzliche Thioesterase FatB2 aus Cuphea hookeriana[7] in einer β-oxidationsdefizienten Zelle überexprimiert, um sowohl die Fettsäuresynthese zu deregulieren als auch gleichzeitig die Länge des Produktes zu bestimmen. Nach einer N-terminalen Fusion des Enzyms mit dem Thioredoxin-1-Anhang trxA und einigen Optimierungen des Produktionsprozesses, konnten mit diesem System in 24 h etwa 330 mg/L der für die Zellen toxischen Fettsäure Octansäure in einem Bioreaktor-fed batch-Verfahren hergestellt werden. Um aus dieser Fettsäure ihr ω-hydroxyliertes Derivat zu formen, wurde in den Produktionsstamm zusätzlich ein Monooxygenase-Fusionsprotein eingebracht, das bereits aus früheren Arbeiten des ITBs hervorging[8,9]. Das Fusionsprotein von CYP153A aus Marinobacter aquaeolei mit der Reduktasedomäne CPR von CYP102A1 aus Bacillus megaterium ist in der Lage sich selbst mit Reduktionsäquivalenten zu versorgen ohne dabei auf die Hilfe einer zusätzlichen Reduktase angewiesen zu sein. Die in dieser Arbeit verwendete Mutante G307A ist zudem gegenüber Octansäure um das 20-fache aktiver als es die Wildtypform ist[9]. Diese Enzymmutante wurde in vier unterschiedlichen Ansätzen mit dem octansäureproduzierenden Bakterienstamm kombiniert. So konnte letztlich in einem Einzellsystem mit paralleler Expression der Thioesterase sowie der Monooxygenase in 20 h ein 8-Hydroxyoctansäure-Titer von etwa 230 µM erreicht werden, was umgerechnet 40 mg/L entspricht. Nachdem der erfolgreiche konzeptionelle Beweis für dieses Produktionssystem erbracht war, sollte ein ungerichtetes Mutageneseverfahren angewendet werden, um die Möglichkeit das Endprodukt nach Wunsch in seiner Länge variieren zu können, zu erhalten. Das Verfahren sollte gentechnisch veränderte Thioesterase-Varianten generieren, die verglichen zum eingesetzten Wildtypenzym eine erhöhte Aktivität gegen kürzere oder längere Fettsäuren als Octansäure aufwiesen. Für ein hierzu benötigtes high throughput screening-Verfahren sollten im Rahmen dieser Arbeit die Grundlagen erarbeitet werden. Getestet wurden dabei verschiedene Methoden des screenings sowohl auf Agarplatten und in Flüssigmedium, so beispielsweise die extrazelluläre Präsentation des Enzyms auf Hefezelloberflächen[10], als auch die Möglichkeit eines screenings mittels aufgereinigtem Enzym.Item Open Access Studien zum Einsatz von enzymatischen Lewis- und Brønsted-Säuren in Carbonyl-aktivierten organischen Reaktionen(2016) Bischoff, Jennifer N.; Hauer, Bernhard (Prof. Dr.)Item Open Access SiaA/D interconnects c-di-GMP and RsmA signaling to coordinate cellular aggregation of Pseudomonas aeruginosa in response to environmental conditions(2016) Colley, Brendan; Dederer, Verena; Carnell, Michael; Kjelleberg, Staffan; Rice, Scott A.; Klebensberger, JanoschPseudomonas aeruginosa has emerged as an important opportunistic human pathogen that is often highly resistant to eradication strategies, mediated in part by the formation of multicellular aggregates. Cellular aggregates may occur attached to a surface (biofilm), at the air-liquid interface (pellicle), or as suspended aggregates. Compared to surface attached communities, knowledge about the regulatory processes involved in the formation of suspended cell aggregates is still limited. We have recently described the SiaA/D signal transduction module that regulates macroscopic cell aggregation during growth with, or in the presence of the surfactant SDS. Targets for SiaA/D mediated regulation include the Psl polysaccharide, the CdrAB two-partner secretion system and the CupA fimbriae. While the global regulators c-di-GMP and RsmA are known to inversely coordinate cell aggregation and regulate the expression of several adhesins, their potential impact on the expression of the cupA operon remains unknown. Here, we investigated the function of SiaA (a putative ser/thr phosphatase) and SiaD (a di-guanylate cyclase) in cupA1 expression using transcriptional reporter fusions and qRT-PCR. These studies revealed a novel interaction between the RsmA posttranscriptional regulatory system and SiaA/D mediated macroscopic aggregation. The RsmA/rsmY/Z system was found to affect macroscopic aggregate formation in the presence of surfactant by impacting the stability of the cupA1 mRNA transcript and we reveal that RsmA directly binds to the cupA1 leader sequence in vitro. We further identified that transcription of the RsmA antagonist rsmZ is controlled in a SiaA/D dependent manner during growth with SDS. Finally, we found that the siaD transcript is also under regulatory control of RsmA and that overproduction of RsmA or the deletion of siaD results in decreased cellular cyclic di-guanosine monophosphate (c-di-GMP) levels quantified by a transcriptional reporter, demonstrating that SiaA/D connects c-di-GMP and RsmA/rsmY/Z signaling to reciprocally regulate cell aggregation in response to environmental conditions.Item Open Access Enzymatic asymmetric dihydroxylation of alkenes(2016) Gally, Christine; Hauer, Bernhard (Prof. Dr.)The introduction of chirality into C=C double bonds is of special interest in organic synthesis. In particular, the catalytic asymmetric dihydroxylation (AD) of alkenes has attracted considerable attention due to the facile transformation of the chiral diol products into valuable derivatives. By chemical means, the metal-catalyzed AD of olefins provides both stereo- and regiospecific cis-diol moieties. Next to their toxicity, however, these metal catalysts can also lead to byproduct formation as a result of oxidative fission. In nature, Rieske non-heme iron oxygenases (ROs) represent promising biocatalysts for this reaction since they are the only enzymes known to catalyze the stereoselective formation of vicinal cis-diols in one step. ROs are key enzymes in the degradation of aromatic hydrocarbons and can target a wide variety of different arenes. Despite their broad substrate scope, limited data is available for the conversion of unnatural substrates by this class of enzymes. To explore their potential for alkene oxidation, three ROs were tested for the oxyfunctionalization of a set of structurally diverse olefins including linear and cyclic arene-substituted alkenes, cycloalkenes as well as several terpenes. Naphthalene- (NDO), benzene- (BDO) and cumene dioxygenases (CDO) from different Pseudomonas strains where selected as they are amongst the RO enzymes that have already been reported to catalyze the oxidation of a small number of olefins. The majority of compounds from the selected substrate panel could be converted by NDO, BDO or CDO and products were either isolated and identified by NMR analysis or using the authentic standards. Dependent on the substrate, allylic monohydroxylation was found in addition to the corresponding diol products, a reaction which is chemically still most reliably achieved by the use of SeO2 in stoichiometric amounts. However, having been evolved for the dihydroxylation of aromatic compounds, wild type ROs displayed low conversions (< 50%) and modest stereoselectivities (≤ 80% ee/de) for several of the tested olefins. To overcome these limitations, changes in the active site topology of RO catalysts were introduced. A single targeted point mutation that was identified based on sequence and structural comparisons with other members of the RO family proved to be sufficient to generate BDO and CDO variants displaying remarkable changes in regio- and stereoselectivity for various substrates. In particular biotransformations with CDO M232A gave excellent stereoselectivities (≥ 95% ee/de) and good activities (> 90%) also for linear alkenes, which have been reported to be challenging substrates for RO-catalyzed oxyfunctionalizations. Site-saturation mutagenesis at position 232 in CDO revealed a correlation between the steric demand of the amino acid side chain and its influence on regio- and/ or stereoselectivities for styrene and indene. While the wild type enzyme almost exclusively catalyzed the dihydroxylation of the aromatic ring, the regioselectivity was shifted with decreasing side chain size to the terminal vinyl group of styrene, yielding up to 96% of the alkene-1,2-diol. For cis-1,2-indandiol formation, enantiocomplementary enzymes could be generated, a fact further highlighting the importance of position 232 for the engineering of ROs. Moreover, site-saturation mutagenesis of additional residues in the substrate binding pocket of CDO (F278, I288, I336 and F378) identified further positions having an influence on selectivity and product formation for alkene oxidation. To proof the applicability of ROs for organic synthesis, semi-preparative scale biotransformations (70 mg) of selected substrates were performed with CDO M232A. Without further optimization of the reaction set-up, products were successfully isolated in > 30% yield. In addition, up-scaling of (R)-limonene hydroxylation to 4 L in a bioreactor with growing cells gave final isolated product titers of 0.4 g L-1 even though substrate volatility and product toxicity diminished the yield. In conclusion, these examples demonstrated that a single point mutation was sufficient to transform CDO wild type into an efficient catalyst, furthermore constituting the first example of the rational engineering of CDO and BDO enzymes for the oxyfunctionalization of a broad range of alkenes.Item Open Access Metabolismus nicht-physiologischer Substrate in Mikroorganismen(2016) Reznicek, Ondrej; Hauer, Bernhard (Prof. Dr.)Item Open Access Asparaginsäure-vermittelte enzymatische Reaktionen(2016) Kühnel, Lisa C.; Hauer, Bernhard (Prof. Dr.)Die vorliegende Arbeit befasst sich mit der Untersuchung der Promiskuität zweier Enzyme aus unterschiedlichen Proteinklassen, die allerdings auf eine gemeinsame, in der Biochemie sehr häufige Reaktion, die Säure-(Base)-Reaktion, zurückgreifen. Beide Enzyme verwenden die Asparaginsäure als katalytisch aktive Aminosäure, wobei Haloalkan Dehalogenasen aus der alpha/beta-Hydrolase-Familie, die deprotonierte Form der Asparaginsäure als nukleophiles Aspartat verwenden, wohingegen die Squalen-Hopen-Zyklasen die protonierte Asparaginsäure als Brønsted-Säure nutzen. Das Substratspektrum der Dehalogenase LinB aus Sphingobium japonicum, das sonst nur halogenierte Alkane umfasst, sollte um alternative Substrate erweitert werden. Es konnte erfolgreich demonstriert werden, dass LinB eine eindeutige Hydrolyseaktivität gegenüber organischen Thiocyanaten und Isothiocyanaten aufweist, was auf erstmals beschriebene promiskuitive Eigenschaften der LinB schließen lässt. Es konnte gezeigt werden, dass Substrate von unterschiedlicher Größe toleriert werden, um dabei Alkohole und Amine zu generieren. Mittels rationalem Design konnten Varianten erzeugt werden, die eine deutliche Verbesserung gegenüber der Bildung von Benzylamin demonstrierten. Zur Untersuchung der Art der Promiskuität, wurden sogenannte knockout-Mutanten der katalytischen Triade der LinB erstellt und für Studien bezüglich des Katalysemechanismus herangezogen. In der Tat konnte über die einfache Substratpromiskuität hinaus, katalytische Promiskuität der LinB in Bezug auf die organischen Isothiocyanate gezeigt werden. Anhand der Squalen-Hopen-Zyklase aus Alicyclobacillus acidocaldarius (AacSHC) wurden Untersuchungen bezüglich intermolekular katalysierter Reaktionen vorgenommen. Diese Fähigkeit der Etherbildung ist zwar bei der nativen AacSHC anhand des Substrates Farnesol bekannt, allerdings nur in geringem Umfang. So konnte mittels Varianten aus einer fokussierten Mutantenbibliothek eine 10-fache Steigerung der Produktbildung erzielt werden. Ferner konnten unter Verwendung nicht-natürlicher Substrate und unterschiedlicher Nukleophile neue, nicht-literaturbekannte intermolekular gebildete Produkte dargestellt werden. Durch die Wahl des Nukleophils und dessen Konzentration gelang es, Produktverhältnisse eindeutig zu Gunsten der intermolekular gebildeten Produkte zu verschieben.Item Open Access Development of highly efficient CYP153A-catalysed terminal hydroxylation of fatty acids(2016) Notonier, Sandra; Hauer, Bernhard (Prof.Dr.)Terminally hydroxylated fatty acids (omega-OHFA) are of great interest to industry: in the area of high end polymers, in fine chemicals, in the cosmetic and fragrance industry. The activation and oxidation of C-H bonds, is however still chemically challenging. It requires rough conditions, high pressure, high temperature and the processes still suffer from poor selectivity. Alternatively, the functionalization of carbon atoms can be achieved by using versatile cytochrome P450 monooxygenases with high regio- and stereoselectivity (P450s or CYPs). These heme containing proteins can be found in many organisms, they are involved in a diverse range of reactions and they are able to catalyse the conversion of a large panel of substrates. CYP153A from Marinobacter aquaeolei (CYP153AM.aq.) constitutes a promising catalyst for the oxidation of non-activated carbon atoms due to its high regioselectivity in the hydroxylation of different small to medium chain alkanes, fatty acids and primary alcohols as well as its efficient expression in Escherichia. coli at high yields. For bacterial whole-cell applications, the enzyme was further engineered as a protein chimera by fusing the reductase domain of P450BM3, from Bacillus megaterium, to the heme domain of CYP153AM.aq. (CYP153AM.aq.-CPRBM3). The mutant G307A (position located in the binding pocket) was further identified as improved variant towards medium chain-length fatty acids regarding activity, regioselectivity and coupling efficiency. To perform efficient whole-cell biotransformations and therefore to increase the yield of bioconversion of fatty acids into omega-OHFA, the system requires the identification of present bottlenecks. The approaches to characterize and to optimise the up-scaling process involved testing different feeding strategies, evaluation of substrate/product inhibition, transport limitations estimation, cofactor availability evaluation and biocatalyst stability investigations. Such limitations often associated to the P450 biocatalysts and/or related to whole-cell biotransformations were evaluated in this study, using the fusion constructs CYP153AM.aq.-CPRBM3 towards the model substrate dodecanoic acid (C12:0) in vitro and in vivo. Strategies to overcome existing bottlenecks regarding low activity involved the generation of mutant libraries to screen and to characterize improved versions of the current chimera biocatalyst. In parallel to the mutant libraries generation, a whole-cell colorimetric assay was also established.Item Open Access Characterization and application of novel imine reductases(2016) Scheller, Philipp; Hauer, Bernhard (Prof. Dr.)Chiral amines are an ubiquitously distributed class of bioactive compounds, what turns them into preferred scaffolds for pharmaceuticals. The high chemical and enantiomerical purities required for such an application are ideally suited for biocatalysis as enzymatic methods routinely display high specificities. The established methods for chiral amine synthesis with lipases, ω-transaminases and amine oxidases, however have considerable limitations regarding their access to pharmaceutically relevant chiral secondary and tertiary amines. Recently the new enzyme class of imine reductases (IREDs) was described, offering an attractive extension to the currently used techniques as the preparation of imines by chemical methods in organic solvents is a well established and widely applicable method. As the number of IREDs known initially was limited to only three enzymes, this project started with a database search for the discovery of novel enzymes. For the first time it was shown that the IRED family is much larger than assumed and over 350 novel, putative IREDs were identified. A sequence analysis of the database members revealed (R)- type and (S)- type superfamilies and led after an update to the identification of IRED specific sequence motifs. These criteria allowed to define this new enzyme family on a sequence level and discriminate them from the closest related homologues. Based on the biochemical information about the three published IREDs and a conservation analysis of the database members, three new enzymes from Streptosporangium roseum DSM43021, Streptomyces turgidiscabies and Paenibacillus elgii were selected for characterization. The enzymes were shown to encode for functional IREDs with much higher activity than the previously known IREDs. By site directed mutagenesis the mechanism of the IREDs was probed and the importance of a conserved Tyr for catalysis of an (S)- type IRED shown, while the crucial role of the proposed Asp residue for catalysis in the (R)- type IREDs was questioned. The characterization of the new IREDs revealed their pH optima and confirmed the suspected dimerization. The thermostability of the IREDs was investigated and the selected (S)- type IRED identified as the most stable enzyme known to date. Further the activity in the presence of water miscible organic solvents was tested and high tolerance versus MeOH found. In biotransformations all IREDs showed high activity and a broad panel of cyclic imines was fully converted to piperidines and tetrahydroisoquinolines with enantioselectivities up to 99% ee. With purified IREDs kinetic constants for these substrates were recorded and their substrate preference investigated. This indicated a preference of the (S)- type IRED for more bulky substrates, compared to the (R)- type IREDs. After optimization of the reaction conditions, with purified IREDs also high activities and chemo- as well as enantioselectivities for very labile exocyclic imines were detected. The possibility to effectively reduce already low levels of such imines led to the application of one (R)- type IRED for the generation of novel C-N bonds by reductive aminations. The established methodology revealed the crucial influence, conditions that favor imine formation (high molar excess of the amine nucleophile and high pH) display on the conversion rates. Under optimized conditions, different carbonyls could efficiently be transformed with a variety of amines in the aqueous buffer system with moderate to good conversions into primary and secondary (chiral) amines with very high selectivities (ee up to 98%). Finally, an application of IREDs in cascade reactions to produce saturated N-heterocyclic compounds was envisioned. A microbial putrescine oxidase (PuO) was chosen to selectively oxidize polyamines to aminoaldehydes, thereby triggering their spontaneous cyclization to an imine. To target a broad range of heterocycles, PuO was characterized with a range of unnatural polyamines. The results indicated a narrow substrate scope and low activity for these compounds. To enhance the activity for such substrates directed evolution of PuO with epPCR was performed and led to the identification of a Glu residue, representing a hotspot for mutagenesis. This residue aligns to one of the multiple channels that lead into the deeply buried active site of PuO and it is located in the second shell around the active site. By site-saturation mutagenesis further mutants in the active site and this channel were generated and many mutants with smaller amino acids demonstrated the influence of this hotspot position to increase the activity of the enzyme. The best mutant exhibited considerably increased activities (up to 25-fold) for unnatural polyamines and also for natural polyamines the substrate spectrum was strongly shifted from putrescine towards longer polyamines like spermidine which is now transformed with a 10-fold increased catalytic efficiency (kcat/KM). The combination of both enzymes in purified form as well as in whole cells enabled the production of heterocyclic amines relying on the consecutive transformation of the substrate by both enzymes. While with the whole cell system only low amounts of the N-heterocyclic compounds were produced, the utilization of purified enzymes led in case of all three IREDs to high conversions of different polyamines into pyrrolines and piperidines.Item Open Access Stereoselektive enzymatische Brønsted-Säure-Katalyse unter Verwendung einer Squalen-Hopen Cyclase(2016) Bastian, Silke A.; Hauer, Bernhard (Prof. Dr.)Item Open Access Novel route to vanillin - an enzyme-catalyzed multi-step cascade synthesis(2016) Klaus, Tobias; Hauer, Bernhard (Prof. Dr.)The selective hydroxylation of aromatic compounds is one of the most challenging chemical reactions. As an alternative to traditional chemical catalysis, biocatalysis emerged during the past decades. Hence, in the present work, a number of biocatalysts was investigated with regard to the realization of a novel synthesis route to the valuable aromatic compound vanillin, starting from the simple low-cost aromatic substrate 3-methylanisole via the intermediate products 3-methoxybenzyl alcohol or 4-methylguaiacol and via vanillyl alcohol, as an example of consecutive enzyme-catalyzed oxidation reactions accomplished in a multi-enzymatic three-step cascade reaction. For this reason a preselected set of enzymes, namely the m-hydroxybenzoate hydroxylase MobA from Comamonas testosteroni GZ39 and the cytochrome P450 monooxygenases CYP116B3 from Rhodococcus ruber DSM 44319 and CYP102A1 from Bacillus megaterium ATCC 14581, was investigated towards the selective hydroxylation of the substrate 3-methylanisole. Beside the wild type enzymes, a variant of MobA, which was created by rational protein design, and an existing focused minimal mutant library of CYP102A1 were applied in initial biotransformation reactions, combined with an efficient cofactor recycling system. Though the wild type enzymes of CYP116B3 and CYP102A1 displayed only a basic level of activity towards 3-methylanisole, highly increased activity was detected for many of the CYP102A1 variants with a maximum of 59% total conversion for the double mutant F87V/A328L. With 3-methoxybenzyl alcohol and 4-methylguaiacol both intermediate compounds of the intended cascade synthesis were generated, though 4-methoxy-2-methylphenol was the main product in most of the reactions. However, none of the so far investigated variants accepted any of the intermediate compounds as substrate. As CYP116B3 was a good candidate for further protein engineering approaches, as a basic level of activity towards the substrate of interest was already present in the wild type enzyme, a focused mutant library of 20 single mutant variants of CYP116B3 was created based on literature, sequence and structure information in order to improve the enzymes activity and selectivity towards conversion of the substrate 3-methylanisole and in order to find variants for the conversion of 3-methoxybenzyl alcohol and/or 4-methylguaiacol. Therefore a homology model of the monooxygenase domain of CYP116B3 was generated. Though, compared to the wild type, variants with up to almost six time increased activity towards the model substrate 7-ethoxycoumarin were found, total activity towards 3-methylanisole was still much lower compared to the best CYP102A1 variants. In addition, none of the variants displayed appropriate conversion of the intermediate compounds 3-methoxybenzyl alcohol and 4-methylguaiacol. Moreover, additional mutation in the literature known amino acid position 437 of CYP102A1 variant F87V/A328L revealed no benefit towards conversion of any of the substrates, too. Molecular dynamics simulations of a CYP102A1 variant with 4-methylguaiacol as substrate revealed the bottleneck in the conversion of this compound. 4-Methylguaiacol was shown to be stabilized at the entrance of the substrate access channel by the polar amino acid residues R47 and Y51. Replacement of these residues by the hydrophobic residues leucine and phenylalanine, respectively, resulted in successful conversion of 4-methylguaiacol to vanillyl alcohol, the precursor of vanillin in the intended cascade synthesis. Though, as the yield of vanillyl alcohol synthesized from 4-methylguaiacol with CYP102A1 variants was rather low, a vanillyl alcohol oxidase from Penicillium simplicissimum and rationally designed variants thereof, described in literature, were investigated. As a result, not only vanillyl alcohol but also 4-methylguaiacol was converted in high yield to vanillin. Finally, a combination of the best 4-methylguaiacol producing variant, CYP102A1 variant A328L, with the best 4-methylguaiacol converting variant, VAO variant F454Y, in one reaction system both in vitro and in vivo yielded vanillin from 3-methylanisole with a maximal product formation of 2.0% and 1.1% vanillin, respectively. We demonstrated as a proof-of-principle the establishment of the proposed multi-enzymatic three-step cascade reaction pathway. Though further optimizations concerning increase of enzyme activity and improvement of enzyme selectivity are required, the above mentioned exemplary synthesis of vanillin illustrates the capability of biocatalysis.