Mandana Gruber-Khadjawi

Application of Designed Enzymes in Organic Synthesis

Application of Designed Enzymes in Organic Synthesis Gernot A. Strohmeier, Harald Pichler, Oliver May, and Mandana Gruber-Khadjawi* Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz, Austria Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, A-8010 Graz, Austria DSM—Innovative Synthesis BV, Geleen, P.O. Box 18, 6160 MD Geleen, The Netherlands

Biocatalytic Friedel–Crafts Alkylation Using Non‐natural Cofactors

The formation of C C bonds is a central aspect of synthetic organic chemistry. However, in biocatalysis only few enzymes capable to perform this reaction are known, among which aldolases, transketolases, and hydroxynitril lyases have been investigated thoroughly. Some have even found their way into industrial applications. Friedel–Crafts alkylation is a classic organic reaction of great importance. However, in particular for large scale application, this transformation is ecologically very critical and regiospecific monoalkylation is difficult to achieve. Therefore, an environmentally friendly and selective alternative would be highly desirable. In nature methyl groups are selectively introduced into reactive aromatic rings by methyltransferases (Mtases), in particular with S-adenosyl-lmethionine (SAM) as the cofactor. Furthermore, enzyme-catalyzed reactions are important for access to isoprenoids. Also, prenylation of aromatic rings has been performed. For phenylalanine ammonia lyases a Friedel–Crafts-type mechanism has been proposed. Recently, it has been shown that besides the methyl group other alkyl, alkenyl, and alkinyl groups can be introduced into S-adenosyl-l-homocystein. These modified cofactors of transferases were used for a sequence-specific alkylation of DNA. Having cofactor and modified cofactors in hand, we investigated the possibility of alkylation of aromatic substrates, thus transferring the biosynthesis into the laboratory (Scheme 1). Aminocoumarins are antibiotics produced by some Streptomyces species and are targets for the methyl transfer from the natural cofactor SAM. The Mtase A and B are involved in the biosynthesis of the antibiotics coumermycin A1 [7] (produced by Streptomyces rishiriensis) and novobiocin (produced by Streptomyces spheroides ; Scheme 1). SAM analogues were synthesized by modifying the strategy published by Klimašauskas, Weinhold, and co-workers. S-Adenosyl-l-homocysteine (SAH) was alkylated by seven different alkyl bromides using formic acid as the solvent and AgOTf as a Lewis acid activator and catalyst. We observed quantitative conversion in less than 2 days (average reaction time 24 h; Table 1). The chemical synthesis of SAM analogues results in approximately 1:1 diastereomeric mixtures at the sulfonium center. In the first screenings the diastereomers were separated by preparative HPLC and used as cofactors for the alkylation of coumarin compound 3a. Both epimers were accepted by the enzymes NovO and CouO Scheme 1. C-Mtases involved in the biosynthesis of the antibiotics coumermycin A1 in Streptomyces rishiriensis and Novobiocin in Streptomyces spheroides.

Biocatalytic Methods for CC Bond Formation

Carbon‐carbon bond formation is among the most challenging transformations in the organic synthetic chemistry. Enzymes capable to perform this reaction are of great interest. The enzymes for stereoselective CC bond formations have been investigated very intensively during the last two decades. New recombinant DNA technologies have paved the way for improved catalysts and broaden the application scope of the already known enzymes and reactions. On the other side new discoveries have brought more enzyme players in the arena of CC bond formation reactions. Novel enzymatic CC bond formation reactions have been applied, implying the most important benefit of biocatalysis, namely the high selectivity.

Asymmetric Retro-Henry Reaction Catalyzed by Hydroxynitrile Lyase from Hevea brasiliensis

Hydroxynitrile lyase from Hevea brasiliensis (HbHNL) is a promiscuous biocatalyst that, besides the native cyanohydrin reaction, also catalyzes the asymmetric Henry reaction yielding (S)‐β‐nitroalcohols with high enantiomeric excess. Since the Henry reaction is reversible, the enzyme can be also utilized for the production (R)‐enantiomers by means of resolution of racemic β‐nitroalcohols. Herein the biocatalytic retro‐Henry reaction is studied using the cleavage of 2‐nitro‐1‐phenylethanol as a model system. The main problem that prevents high levels of conversion or high ee values during the cleavage of the β‐nitroalcohol is the formation of benzaldehyde, which is known to be a strong enzyme inhibitor. The product inhibition is overcome by performing the biocatalytic retro‐Henry reaction in the presence of HCN, which reacts in situ with benzaldehyde and converts it to the less‐inhibitive mandelonitrile. By using such a reaction cascade, it was possible to conduct the resolution practically to completion (95 % ee, 49 % conversion). Furthermore, the catalyst productivity achieved during the resolution was ten times higher than that in the HbHNL‐catalyzed synthesis of (S)‐2‐nitro‐1‐phenylethanol by condensation of benzaldehyde and nitromethane.

Improving the Properties of Bacterial R-selective Hydroxynitrile Lyases for Industrial Applications

Hydroxynitrile lyases (HNLs) catalyse the reversible cleavage of cyanohydrins to carbonyl compounds and HCN. The recent discovery of bacterial HNLs with a cupin fold gave rise to a new promising class of these enzymes. They are interesting candidates for the synthesis of cyanohydrins on an industrial scale owing to their high expression levels in Escherichia coli. The activity and enantioselectivity of the manganese‐dependent HNL from Granulicella tundricola (GtHNL) were significantly improved by site‐saturation mutagenesis of active site amino acids. The combination of beneficial mutations resulted in a variant with 490‐fold higher specific activity in comparison to the wild‐type enzyme. More importantly, GtHNL‐A40H/V42T/Q110H is a highly competitive alternative for the synthesis of chiral cyanohydrins, such as 2‐chlorobenzaldehyde cyanohydrin, (R)‐2‐hydroxy‐4‐phenylbutyronitrile, and (R)‐2‐hydroxy‐4‐phenyl‐3‐butene nitrile, which serve as intermediates for the synthesis of pharmaceuticals.

Discovery of a novel (R)-selective bacterial hydroxynitrile lyase from Acidobacterium capsulatum

Hydroxynitrile lyases (HNLs) are powerful carbon–carbon bond forming enzymes. The reverse of their natural reaction – the stereoselective addition of hydrogen cyanide (HCN) to carbonyls – yields chiral cyanohydrins, versatile building blocks for the pharmaceutical and chemical industry. Recently, bacterial HNLs have been discovered, which represent a completely new type: HNLs with a cupin fold. Due to various benefits of cupins (e.g. high yield recombinant expression in Escherichia coli), the class of cupin HNLs provides a new source for interesting, powerful hydroxynitrile lyases in the ongoing search for HNLs with improved activity, enantioselectivity, stability and substrate scope. In this study, database mining revealed a novel cupin HNL from Acidobacterium capsulatum ATCC 51196 (AcHNL), which was able to catalyse the (R)-selective synthesis of mandelonitrile with significantly better conversion (97%) and enantioselectivity (96.7%) than other cupin HNLs.

High-level biosynthesis of norleucine in E. coli for the economic labeling of proteins.

The residue-specific labeling of proteins with non-canonical amino acids (ncAA) is well established in shake flask cultures. A key aspect for the transfer of the methodology to larger scales for biotechnological applications is the cost of the supplemented ncAAs. Therefore, we established a scalable bioprocess using an engineered host strain for the biosynthesis of the methionine analog norleucine at titers appropriate for the efficient and economic labeling of proteins. To enhance the biosynthesis of norleucine, which is a side-product of the branched chain amino acid pathway, we deleted all three acetolactate synthase isoforms of the methionine auxotrophic Escherichia coli expression strain B834(DE3). Additionally, we overexpressed leuABCD to boost the biosynthesis of norleucine. We systematically analyzed the production of norleucine under the conditions for its residue-specific incorporation in bioreactor cultures that had a 30-fold higher cell density than shake flask cultures. Under optimized conditions, 5g/L norleucine was biosynthesized. This titer is two times higher than the standard supplementation with norleucine of a culture with comparable cell density. We expect that our metabolically engineered strain for the improved biosynthesis of norleucine in combination with the proposed bioprocess will facilitate the efficient residue-specific labeling of proteins at a reasonable price in scales beyond the shake flask.

Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily

Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.

Methyltransferases: Green Catalysts for Friedel–Crafts Alkylations

A set of S‐adenosyl‐l‐methionine (SAM) dependent methyltransferases has been characterized as versatile catalysts for the enzymatic Friedel–Crafts (alkylation) reaction. Although the substrate specificity of the enzymes range from high (in the case of SfmM2, SacF, and ORF19) to moderate (in the case of NovO and CouO), the cofactor spectrum is broad. Modified cofactors decorated with alkyl groups other than methyl were used for biocatalytic Friedel–Crafts alkylation, and conversions up to 99 % were achieved. In contrast to the classical chemical reaction the biotransformation is very selective and environmentally compatible.

CHEMOENZYMATIC METHODS FOR THE PREPARATION OF OPTICALLY ACTIVE CYCLIC POLYAZIDO ALCOHOLS FROM EASILY AVAILABLE ACHIRAL STARTING MATERIALS

Abstract Optically active 2,5-diazido-1,4-cyclohexanediol, 4,6-diazido-1,3-cyclohexanediol and 2,5-diazido-1,4-cyclohexanediol as precursors for polyfunctional cyclic amino alcohols were prepared by enzymatic hydrolysis of the respective butyrates with lipases from Candida rugosa (CRL), Pseudomonas cepacia (PCL), and Geotrichum candidum (GCL). The enantiomeric excesses obtained were very high.