Margit Winkler

Monooxygenases as biocatalysts: Classification, mechanistic aspects and biotechnological applications

Monooxygenases are enzymes that catalyze the insertion of a single oxygen atom from O(2) into an organic substrate. In order to carry out this type of reaction, these enzymes need to activate molecular oxygen to overcome its spin-forbidden reaction with the organic substrate. In most cases, monooxygenases utilize (in)organic cofactors to transfer electrons to molecular oxygen for its activation. Monooxygenases typically are highly chemo-, regio-, and/or enantioselective, making them attractive biocatalysts. In this review, an exclusive overview of known monooxygenases is presented, based on the type of cofactor that these enzymes require. This includes not only the cytochrome P450 and flavin-dependent monooxygenases, but also enzymes that utilize pterin, metal ions (copper or iron) or no cofactor at all. As most of these monooxygenases require nicotinamide coenzymes as electron donors, also an overview of current methods for coenzyme regeneration is given. This latter overview is of relevance for the biotechnological applications of these oxidative enzymes.

Carbon source dependent promoters in yeasts.

Budding yeasts are important expression hosts for the production of recombinant proteins.The choice of the right promoter is a crucial point for efficient gene expression, as most regulations take place at the transcriptional level. A wide and constantly increasing range of inducible, derepressed and constitutive promoters have been applied for gene expression in yeasts in the past; their different behaviours were a reflection of the different needs of individual processes.Within this review we summarize the majority of the large available set of carbon source dependent promoters for protein expression in yeasts, either induced or derepressed by the particular carbon source provided. We examined the most common derepressed promoters for Saccharomyces cerevisiae and other yeasts, and described carbon source inducible promoters and promoters induced by non-sugar carbon sources. A special focus is given to promoters that are activated as soon as glucose is depleted, since such promoters can be very effective and offer an uncomplicated and scalable cultivation procedure.

Biocatalytic reduction of carboxylic acids

An increasing demand for non‐petroleum‐based products is envisaged in the near future. Carboxylic acids such as citric acid, succinic acid, fatty acids, and many others are available in abundance from renewable resources and they could serve as economic precursors for bio‐based products such as polymers, aldehyde building blocks, and alcohols. However, we are confronted with the problem that carboxylic acid reduction requires a high level of energy for activation due to the carboxylates thermodynamic stability. Catalytic processes are scarce and often their chemoselectivity is insufficient. This review points at bio‐alternatives: currently known enzyme classes and organisms that catalyze the reduction of carboxylic acids are summarized. Two totally distinct biocatalyst lines have evolved to catalyze the same reaction: aldehyde oxidoreductases from anaerobic bacteria and archea, and carboxylate reductases from aerobic sources such as bacteria, fungi, and plants. The majority of these enzymes remain to be identified and isolated from their natural background in order to evaluate their potential as industrial biocatalysts.

Fluoro amino acids: A rarity in nature, yet a prospect for protein engineering

Fluoro amino acids are highly valuable compounds constantly gaining relevance in diverse fields of the biosciences as well as in the pharmaceutical industry. The value of these compounds can be attributed to the properties of the extremely electronegative fluorine atom. This atom forms a highly polarized bond of extraordinary strength with carbon. The formation of the fluorine–carbon bond is challenging: its chemical synthesis demands harsh reaction conditions and to date only one class of enzyme has been found capable of introducing the fluoride ion into an organic compound. Most of these fluorinating enzymes participate in the biosynthesis of 4‐fluoro‐L‐threonine, the only fluoro amino acid of natural origin discovered so far. Despite their scarcity in nature, fluoro amino acids are valuable tools to fluorinate proteins. The fluoro protein variants often show improved stability and folding as well as altered activity and fluorescence characteristics. This review details the biosynthesis of 4‐fluoro‐L‐threonine with a special focus on the fluorinating enzymes. Moreover, we elaborate on the application of fluoro amino acids as building blocks for fluorinated protein variants. Insight into different techniques to incorporate fluoro amino acids into proteins is also provided. We highlight prospects and the current relevance of fluoro amino acids as a tool to engineer proteins with novel traits.

Biocatalytic Characterization of Human FMO5: Unearthing Baeyer–Villiger Reactions in Humans

Flavin-containing mono-oxygenases are known as potent drug-metabolizing enzymes, providing complementary functions to the well-investigated cytochrome P450 mono-oxygenases. While human FMO isoforms are typically involved in the oxidation of soft nucleophiles, the biocatalytic activity of human FMO5 (along its physiological role) has long remained unexplored. In this study, we demonstrate the atypical in vitro activity of human FMO5 as a Baeyer-Villiger mono-oxygenase on a broad range of substrates, revealing the first example to date of a human protein catalyzing such reactions. The isolated and purified protein was active on diverse carbonyl compounds, whereas soft nucleophiles were mostly non- or poorly reactive. The absence of the typical characteristic sequence motifs sets human FMO5 apart from all characterized Baeyer-Villiger mono-oxygenases so far. These findings open new perspectives in human oxidative metabolism.

Targeting the Substrate Binding Site of E. coli Nitrile Reductase QueF by Modeling, Substrate and Enzyme Engineering

Nitrile reductase QueF catalyzes the reduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ0) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ1) in the biosynthetic pathway of the hypermodified nucleoside queuosine. It is the only enzyme known to catalyze a reduction of a nitrile to its corresponding primary amine and could therefore expand the toolbox of biocatalytic reactions of nitriles. To evaluate this new oxidoreductase for application in biocatalytic reactions, investigation of its substrate scope is prerequisite. We report here an investigation of the active site binding properties and the substrate scope of nitrile reductase QueF from Escherichia coli. Screenings with simple nitrile structures revealed high substrate specificity. Consequently, binding interactions of the substrate to the active site were identified based on a new homology model of E. coli QueF and modeled complex structures of the natural and non-natural substrates. Various structural analogues of the natural substrate preQ0 were synthesized and screened with wild-type QueF from E. coli and several active site mutants. Two amino acid residues Cys190 and Asp197 were shown to play an essential role in the catalytic mechanism. Three non-natural substrates were identified and compared to the natural substrate regarding their specific activities by using wild-type and mutant nitrile reductase.

Whole‐Cell Carboxylate Reduction for the Synthesis of 3‐Hydroxytyrosol

3‐Hydroxytyrosol (3‐HT) is a phenolic antioxidant that has a number of beneficial effects on human health and is a valuable building block in the synthesis of various pharmaceuticals. Herein, we report a new method for the production of 3‐HT through reduction of 3,4‐dihydroxyphenylacetic acid. The reduction was performed in whole Escherichia coli BL21 (DE3) cells overexpressing carboxylic acid reductase from Nocardia and phosphopantetheinyl transferase from E. coli. An endogenous E. coli aldehyde reducing activity turned out to be highly efficient for further reduction of the aldehyde intermediate to the desired alcohol. The influence of different buffer components, cofactors, and cofactor recycling systems was investigated. A very economic combination of glucose, citrate, and air proved sufficient for recycling of the essential cofactors ATP and NAD(P)H. Selected crucial parameters were then further optimized within a “design of experiments” approach. Finally, first preparative‐scale bioreductions resulted in pure 3‐HT.

Mini-Review: Recent Developments in Hydroxynitrile Lyases for Industrial Biotechnology

Hydroxynitrile lyases (HNLs) catalyze the cleavage as well as the formation of cyanohydrins. The latter reaction is valuable for the stereoselective C-C bond formation by condensation of HCN with carbonyl compounds. The resulting cyanohydrins serve as versatile building blocks for a broad range of chemical and enzymatic follow-up reactions. A significant number of (R)- and (S)-selective HNLs are known today and the number is still increasing. HNLs not only exhibit varying substrate scope but also differ in sequence and structure. Tailor-made enzymes for large-scale manufacturing of cyanohydrins with improved yield and enantiomeric excess are very interesting targets, which is reflected in a solid number of patents. This review will complement and extend our recent review with a strong focus on applications of HNLs for the synthesis of highly functionalized, chiral compounds with newest literature, recent and current patent literature.

Trametes versicolor carboxylate reductase uncovered

The first carboxylate reductase from Trametes versicolor was identified, cloned, and expressed in Escherichia coli. The enzyme reduces aromatic acids such as benzoic acid and derivatives, cinnamic acid, and 3-phenylpropanoic acid, but also aliphatic acids such as octanoic acid are reduced.Graphical abstract

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

Abstract The enzymatic reduction of carboxylic acids is in its infancy with only a handful of biocatalysts available to this end. We have increased the spectrum of carboxylate‐reducing enzymes (CARs) with the sequence of a fungal CAR from Neurospora crassa OR74A (NcCAR). NcCAR was efficiently expressed in E. coli using an autoinduction protocol at low temperature. It was purified and characterized in vitro, revealing a broad substrate acceptance, a pH optimum at pH 5.5–6.0, a T m of 45 °C and inhibition by the co‐product pyrophosphate which can be alleviated by the addition of pyrophosphatase. The synthetic utility of NcCAR was demonstrated in a whole‐cell biotransformation using the Escherichia coli K‐12 MG1655 RARE strain in order to suppress overreduction to undesired alcohol. The fragrance compound piperonal was prepared from piperonylic acid (30 mM) on gram scale in 92 % isolated yield in >98% purity. This corresponds to a productivity of 1.5 g/L/h.