Rieske non-heme dioxygenases: Versatile biocatalysts for the generation of vicinal Cis- Diols

Vicinal cis-diols are a common motif in many biologically active natural products and important intermediates in the flavor and fragrance as well as pharmaceutical industries. Most of the chemical approaches for cis-dihydroxylations are based on transition-metal catalysts like osmium and manganese, which are expensive and toxic. Hence, the enzyme-catalyzed asymmetric dihydroxylation is a powerful tool to overcome the obstacles encountered using such chemical approaches. With the Rieske non-heme dioxygenases (ROs) a promising biocatalytic alternative to generate very selectively vicinal cis-diols is at our disposal. Also known as the non-heme analog to P450 monooxygenases1, these enzymes are multicomponent systems, which only need molecular oxygen for the vicinal cis-dihydroxylation (s. Figure 1). Please click Additional Files below to see the full abstract

Generation of new imine reducing enzymes - expansion of the imine reductase sequence space

The synthesis of chiral molecules by asymmetric hydrogenation applying rhodium and ruthenium-based metal catalysts and molecular H2 as hydride donor is an important and established technology in the pharmaceutical and fine-chemical industries.1 The tremendous progress in enzyme discovery, enzyme engineering and process development made in recent years enable to extend these technologies by biocatalytic alternatives. Many reductase enzymes catalyzing the stereocontrolled addition of hydrogen from NAD(P)H to α,ß-unsaturated carbonyl compounds, cyclic/acyclic imines and aldehydes/ketones are described.2 The latest discovered enzymes in this field are imine reductases (IREDs), catalyzing the reduction of various C=N bonds.3 Recently the structural similarity of IREDs to ß-hydroxyacid dehydrogenases (ßHADs) was investigated4, suggesting a common ancestry to C=O reducing enzymes. Please click Additional Files below to see the full abstract

H-2-driven biotransformation of n-octane to 1-octanol by a recombinant Pseudomonas putida strain co-synthesizing an O-2-tolerant hydrogenase and a P450 monooxygenase

An in vivo biotransformation system is presented that affords the hydroxylation of n-octane to 1-octanol on the basis of NADH-dependent CYP153A monooxygenase and NAD(+)-reducing hydrogenase heterologously synthesized in a bacterial host. The hydrogenase sustains H-2-driven NADH cofactor regeneration even in the presence of O-2, the co-substrate of monooxygenase.DFG, EXC 314, Unifying Concepts in CatalysisEC/FP7/297503/EU/Modular beads for regeneration of bio-cofactors in enzyme-catalysed synthesis/HydRege

Synthesis of N-heterocycles from diamines via H2-driven NADPH recycling in the presence of O2

Herein, we report an enzymatic cascade involving an oxidase, an imine reductase and a hydrogenase for the H2-driven synthesis of N-heterocycles. Variants of putrescine oxidase from Rhodococcus erythropolis with improved activity were identified. Substituted pyrrolidines and piperidines were obtained with up to 97% product formation in a one-pot reaction directly from the corresponding diamine substrates. The formation of up to 93% ee gave insights into the specificity and selectivity of the putrescine oxidase.DFG, 53182490, EXC 314: Unifying Concepts in CatalysisDFG, 284111627, H2-basierende Kaskaden für die Biosynthese von N-HeterocyclenTU Berlin, Open-Access-Mittel - 201

Biocatalyst Screening with a Twist: Application of Oxygen Sensors Integrated in Microchannels for Screening Whole Cell Biocatalyst Variants

Selective oxidative functionalization of molecules is a highly relevant and often demanding reaction in organic chemistry. The use of biocatalysts allows the stereo- and regioselective introduction of oxygen molecules in organic compounds at milder conditions and avoids the use of complex group-protection schemes and toxic compounds usually applied in conventional organic chemistry. The identification of enzymes with the adequate properties for the target reaction and/or substrate requires better and faster screening strategies. In this manuscript, a microchannel with integrated oxygen sensors was applied to the screening of wild-type and site-directed mutated variants of naphthalene dioxygenase (NDO) from Pseudomonas sp. NICB 9816-4. The oxygen sensors were used to measure the oxygen consumption rate of several variants during the conversion of styrene to 1-phenylethanediol. The oxygen consumption rate allowed the distinguishing of endogenous respiration of the cell host from the oxygen consumed in the reaction. Furthermore, it was possible to identify the higher activity and different reaction rate of two variants, relative to the wild-type NDO. The meander microchannel with integrated oxygen sensors can therefore be used as a simple and fast screening platform for the selection of dioxygenase mutants, in terms of their ability to convert styrene, and potentially in terms of substrate specificity

Semi-pilot scale-up of a continuous packed-bed bioreactor system developed for the lipase-catalyzed production of pseudo-ceramides

Ceramides are sphingolipid compounds that are very attractive as active components in both the pharmaceutical and the cosmetic industries. In this study, the synthesis of 1-O,3-N-diacyl 3-amino-1,2-propanediol-type pseudo-ceramides was developed at the semi-pilot scale, starting from a two-step continuous enzymatic process with immobilized Candida antarctica lipase B (Novozym® 435) in a packed-bed bioreactor, previously optimized at the laboratory scale. This process involved the selective N-acylation of 3-amino-1,2-propanediol (step 1), followed by the selective O-acylation of the N-acyl 3-amino-1,2-propanediol synthesized in the first step, with various fatty acids as acyl donors, to produce N,O-diacyl 3-amino-1,2-propanediol-type pseudo-ceramides (step 2). Under partially optimized operating conditions, high synthesis yields and production rates were obtained, within the ranges 76–92% and 3.7–4.6 g h−1 (step 1), or 23–36% and 1–1.4 g h−1 (step 2), respectively, depending on the fatty acids used as acyl donors. The overall synthesis yields varied from 20 to 33%: the best yield was obtained using palmitic acid and lauric acid as first and second acyl donors, respectively. Together with the high production rates also obtained with these acyl donors, this confirms that this two-step process has great potential for the production of differently functionalized 1-O,3-N-diacyl 3-amino-1,2-propanediol-type pseudo-ceramides on an industrial scale

Inverting the stereoselectivity of an NADH-dependent imine reductase variant

Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the (R)-selective IRED from Myxococcus stipitatus (NADH-IRED-Ms) yielding a NADH-dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and (S)-selectivity in asymmetric reductions has yet been reported. Herein, we applied semi-rational enzyme engineering to switch the selectivity of NADH-IRED-Ms. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2- methylpyrroline compared to the wild-type and afforded the (S)- amine product with >99% conversion and 91% enantiomeric excess. We also report the crystal-structures of the NADPH-dependent (R)- IRED-Ms wild-type enzyme and the NADH-dependent NADH-IREDMs variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant

Powering Artificial Enzymatic Cascades with Electrical Energy

We have developed a scalable platform that employs electrolysis for an in vitro synthetic enzymatic cascade in a continuous flow reactor. Both H2 and O2 were produced by electrolysis and transferred through a gas‐permeable membrane into the flow system. The membrane enabled the separation of the electrolyte from the biocatalysts in the flow system, where H2 and O2 served as electron mediators for the biocatalysts. We demonstrate the production of methylated N‐heterocycles from diamines with up to 99 % product formation as well as excellent regioselective labeling with stable isotopes. Our platform can be applied for a broad panel of oxidoreductases to exploit electrical energy for the synthesis of fine chemicals.DFG, 284111627, H2-basierende Kaskaden für die Biosynthese von N-HeterocyclenTU Berlin, Open-Access-Mittel – 2020DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"DFG, 390677874, EXC 2033: RESOLV (Ruhr Explores Solvation