Frank Schulz

Light-Driven Biocatalytic Oxidation and Reduction Reactions: Scope and Limitations

The quest for practical regeneration concepts for nicotinamide‐dependent oxidoreductases continues. Recently we proposed the use of visible light to promote the direct reductive regeneration of a flavin‐dependent monooxygenase. With this enzyme (PAMO‐P3) light‐driven enantioselective Baeyer–Villiger oxidations were performed. In spite of the significant reduction in the complexity achieved, catalytic performance of the novel approach did not meet the requirements for an efficient biocatalytic oxygenation system. Driven by this ultimate goal, we further investigated the limiting factors of our particular system. We discovered that oxidative uncoupling of the flavin‐regeneration reaction from enzymatic O2‐activation accounts for the futile consumption of approximately 95 % of the reducing equivalents provided by the sacrificial electron donor, EDTA. Furthermore, it was found that the apparent turnover frequency (TOF) for PAMO‐P3 in the present setup is approximately two orders of magnitude lower than in conventional setups that use NADPH as reductant. This finding was traced to sluggish electron transfer kinetics that arose from an impeded interaction between PAMO‐P3‐bound FAD and the reducing catalyst. The limiting factors and potential approaches for their circumvention are discussed. Furthermore, we broadened the light‐driven regeneration approach to the class of flavin‐dependent reductases. By using the Old Yellow Enzyme homologue YqjM as a model system, a significantly higher catalytic turnover for the enzyme catalyst was achieved, which we assign to a higher accessibility of the prosthetic group as well as to the absence of oxidative uncoupling.

Enzyme-Directed Mutasynthesis: A Combined Experimental and Theoretical Approach to Substrate Recognition of a Polyketide Synthase

Acyltransferase domains control the extender unit recognition in Polyketide Synthases (PKS) and thereby the side-chain diversity of the resulting natural products. The enzyme engineering strategy presented here allows the alteration of the acyltransferase substrate profile to enable an engineered biosynthesis of natural product derivatives through the incorporation of a synthetic malonic acid thioester. Experimental sequence-function correlations combined with computational modeling revealed the origins of substrate recognition in these PKS domains and enabled a targeted mutagenesis. We show how a single point mutation was able to direct the incorporation of a malonic acid building block with a non-native functional group into erythromycin. This approach, introduced here as enzyme-directed mutasynthesis, opens a new field of possibilities beyond the state of the art for the combination of organic chemistry and biosynthesis toward natural product analogues.

Towards practical biocatalytic Baeyer-Villiger reactions: applying a thermostable enzyme in the gram-scale synthesis of optically-active lactones in a two-liquid-phase system

Baeyer-Villiger monooxygenases (BVMOs) are extremely promising catalysts useful for enantioselective oxidation reactions of ketones, but organic chemists have not used them widely due to several reasons. These include instability of the enzymes in the case of in vitro and even in vivo systems, reactant/product inhibition, problems with upscaling and the necessity of using specialized equipment. The present study shows that the thermally stable phenylacetone monooxygenase (PAMO) and recently engineered mutants can be used as a practical catalysts for enantioselective Baeyer-Villiger oxidations of several ketones on a preparative scale under in vitro conditions. For this purpose several parameters such as buffer composition, the nature of the solvent system and the co-factor regeneration system were optimized. Overall a fairly versatile and efficient catalytic system for enantioselective laboratory scale BV-oxidations of ketones was developed, which can easily be applied even by those organic chemists who are not well versed in the use of enzymes.

Deazaflavins as mediators in light-driven cytochrome P450 catalyzed hydroxylations

A light-driven deazaflavin-dependent direct enzyme regeneration system has been developed for a P450-BM3 catalyzed CH-activating hydroxylation, thereby avoiding the need for the expensive NADPH cofactor.

Minimally Invasive Mutagenesis Gives Rise to a Biosynthetic Polyketide Library

Not in the public domain: Site-directed mutagenesis of megasynthases was the key to the generation of a library of polyketides in bacteria. Redox derivatizations are used to change the bioactivity profile of the compounds.

Predicted incorporation of non-native substrates by a polyketide synthase yields bioactive natural product derivatives

The polyether ionophore monensin is biosynthesized by a polyketide synthase that delivers a mixture of monensins A and B by the incorporation of ethyl‐ or methyl‐malonyl‐CoA at its fifth module. Here we present the first computational model of the fifth acyltransferase domain (AT5mon) of this polyketide synthase, thus affording an investigation of the basis of the relaxed specificity in AT5mon, insights into the activation for the nucleophilic attack on the substrate, and prediction of the incorporation of synthetic malonic acid building blocks by this enzyme. Our predictions are supported by experimental studies, including the isolation of a predicted derivative of the monensin precursor premonensin. The incorporation of non‐native building blocks was found to alter the ratio of premonensins A and B. The bioactivity of the natural product derivatives was investigated and revealed binding to prenyl‐binding protein. We thus show the potential of engineered biosynthetic polyketides as a source of ligands for biological macromolecules.

Substrate Flexibility of a Mutated Acyltransferase Domain and Implications for Polyketide Biosynthesis.

Polyketides are natural products frequently used for the treatment of various diseases, but their structural complexity hinders efficient derivatization. In this context, we recently introduced enzyme-directed mutasynthesis to incorporate non-native extender units into the biosynthesis of erythromycin. Modeling and mutagenesis studies led to the discovery of a variant of an acyltransferase domain in the erythromycin polyketide synthase capable of accepting a propargylated substrate. Here, we extend molecular rationalization of enzyme-substrate interactions through modeling, to investigate the incorporation of substrates with different degrees of saturation of the malonic acid side chain. This allowed the engineered biosynthesis of new erythromycin derivatives and the introduction of additional mutations into the AT domain for a further shift of the enzymes substrate scope. Our approach yields non-native polyketide structures with functional groups that will simplify future derivatization approaches, and provides a blueprint for the engineering of AT domains to achieve efficient polyketide synthase diversification.

Quantification of N-acetylcysteamine activated methylmalonate incorporation into polyketide biosynthesis

Summary Polyketides are biosynthesized through consecutive decarboxylative Claisen condensations between a carboxylic acid and differently substituted malonic acid thioesters, both tethered to the giant polyketide synthase enzymes. Individual malonic acid derivatives are typically required to be activated as coenzyme A-thioesters prior to their enzyme-catalyzed transfer onto the polyketide synthase. Control over the selection of malonic acid building blocks promises great potential for the experimental alteration of polyketide structure and bioactivity. One requirement for this endeavor is the supplementation of the bacterial polyketide fermentation system with tailored synthetic thioester-activated malonates. The membrane permeable N-acetylcysteamine has been proposed as a coenzyme A-mimic for this purpose. Here, the incorporation efficiency into different polyketides of N-acetylcysteamine activated methylmalonate is studied and quantified, showing a surprisingly high and transferable activity of these polyketide synthase substrate analogues in vivo.

Heterologous fermentation of a diterpene from Alternaria brassisicola

A variety of different applications render terpenes and terpenoids attractive research targets. A promising but so far insufficiently explored family of terpenoids are the fusicoccanes that comprise a characteristic 5-8-5 fused tricyclic ring system. Besides herbicidal effects, these compounds also show apoptotic and anti-tumour effects on mammalian cells. The access to fusicoccanes from natural sources is scarce. Recently, we introduced a metabolically engineered Saccharomyces cerevisiae strain to enable the heterologous fermentation of the shared fusicoccane–diterpenoid precursor, fusicocca-2,10(14)-diene. Here, we show experiments towards the identification of bottlenecks in this process. The suppression of biosynthetic by-products via medium optimisation was found to be an important aspect. In addition, the fermentation process seems to be improved under oxygen limitation conditions. Under fed-batch conditions, the fermentation yield was reproducibly increased to approximately 20 mg/L. Furthermore, the impact of the properties of the terpene synthase on the fermentation yield is discussed, and the preliminary studies on the engineering of this key enzyme are presented.

Stereochemical assignment of fusiccocadiene from NMR shielding constants and vibrational circular dichroism spectroscopy

The absolute configuration (AC) of the common precursor of the fusicoccane family of terpenoids, fusicocca-2,10(14)-diene (FCdiene), had only been deduced by a lengthy total synthesis, or indirectly from crystal structures of fusicoccin A. However, in particular the AC determinations based on downstream products of the terpene synthase intrinsically overlook potential epimerization reactions. In this contribution, we confirm the relative stereochemistry of FCdiene by comparison of experimental and predicted 13 C-NMR chemical shifts, and finally determine the absolute configuration from an analysis of its infrared and vibrational circular dichroism spectra.