The phytoestrogen pinoresinol is a high‐value compound that has a protective effect against diverse health disorders, and thus is of interest for the pharmaceutical industry. Isolation of pinoresinol from plants suffers from low yields, and its chemical synthesis involves several work‐up steps. In this study we devised a novel two‐step one‐pot enzymatic cascade combining a vanillyl‐alcohol oxidase and a laccase for the production of pinoresinol from eugenol via the intermediate coniferyl alcohol. Along with the well‐characterized vanillyl‐alcohol oxidase from Penicillium simplicissimum used to catalyze the oxidation of eugenol, enzyme screening revealed three bacterial laccases that were appropriate for the synthesis of pinoresinol from coniferyl alcohol. The cascade was optimized regarding enzyme ratios, pH value, and the presence of organic solvents. Under optimized conditions, pinoresinol concentration achieved 4.4 mM (1.6 g l−1), and this compound was isolated and analyzed.

BackgroundPinoresinol is a high-value plant-derived lignan with multiple health supporting effects. Enantiomerically pure pinoresinol can be isolated from natural sources, but with low efficiency. Most chemical and biocatalytic approaches that have been described for the synthesis of pinoresinol furnish the racemic mixture. In this study we devised a three-step biocatalytic cascade for the production of enantiomerically pure pinoresinol from the cheap compound eugenol. Two consecutive oxidations of eugenol through vanillyl-alcohol oxidase and laccase are followed by kinetic resolution of racemic pinoresinol by enantiospecific pinoresinol reductases.ResultsThe addition of the enantiospecific pinoresinol reductase from Arabidopsis thaliana for kinetic resolution of (±)-pinoresinol to an in vitro cascade involving the vanillyl-alcohol oxidase from Penicillium simplicissimum and the bacterial laccase CgL1 from Corynebacterium glutamicum resulted in increasing ee values for (+)-pinoresinol; however, an ee value of 34 % was achieved in the best case. The ee value could be increased up to ≥99 % by applying Escherichia coli-based whole-cell biocatalysts. The optimized process operated in a one-pot “two-cell” sequential mode and yielded 876 µM (+)-pinoresinol with an ee value of 98 %. Switching the reductase to the enantiospecific pinoresinol lariciresinol reductase from Forsythia intermedia enabled the production of 610 µM (−)-pinoresinol with an ee value of 97 %.ConclusionA new approach for the synthesis of enantiomerically pure (+)- and (−)-pinoresinol is described that combines three biotransformation steps in one pot. By switching the reductase in the last step, the whole-cell biocatalysts can be directed to produce either (+)- or (−)-pinoresinol. The products of the reductases’ activity, (−)-lariciresinol and (−)-secoisolariciresinol, are valuable precursors that can also be applied for the synthesis of further lignans.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0472-0) contains supplementary material, which is available to authorized users.

CgL1 laccase from Corynebacterium glutamicum was encapsulated into the metal‐organic framework (MOF) ZIF‐8 which was synthesized in a rapid enzyme friendly aqueous synthesis, the fastest in situ encapsulation of laccases reported to date. The obtained enzyme/MOF, i. e. laccase@ZIF‐8 composite showed enhanced thermal (up to 70 °C) and chemical (N,N‐dimethylformamide) stability, resulting in a stable heterogenous catalyst, suitable for high temperature reactions in organic solvents. Furthermore, the defined structure of ZIF‐8 produced a size selective substrate specificity, so that substrates larger than the pore size were not accepted. Thereby, 2’‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) (ABTS) was used to verify that the enzyme is immobilized inside the MOF versus the outside surface. The enzyme@MOF composite was analyzed by atomic absorption spectroscopy (ASS) to precisely determine the enzyme loading to 2.1 wt%.

Lignans are plant secondary metabolites with a wide range of reported health-promoting bioactivities. Traditional routes toward these natural products involve, among others, the extraction from plant sources and chemical synthesis. However, the availability of the sources and the complex chemical structures of lignans often limit the feasibility of these approaches. In this work, we introduce a newly assembled biosynthetic route in E. coli for the efficient conversion of the common higher-lignan precursor (+)-pinoresinol to the noncommercially available (−)-pluviatolide via three intermediates. (−)-Pluviatolide is considered a crossroad compound in lignan biosynthesis, because the methylenedioxy bridge in its structure, resulting from the oxidation of (−)-matairesinol, channels the biosynthetic pathway toward the microtubule depolymerizer (−)-podophyllotoxin. This oxidation reaction is catalyzed with high regio- and enantioselectivity by a cytochrome P450 monooxygenase from Sinopodophyllum hexandrum (CYP719A23), which was expressed and optimized regarding redox partners in E. coli. Pinoresinol-lariciresinol reductase from Forsythia intermedia (FiPLR), secoisolariciresinol dehydrogenase from Podophyllum pleianthum (PpSDH), and CYP719A23 were coexpressed together with a suitable NADPH-dependent reductase to ensure P450 activity, allowing for four sequential biotransformations without intermediate isolation. By using an E. coli strain coexpressing the enzymes originating from four plants, (+)-pinoresinol was efficiently converted, allowing the isolation of enantiopure (−)-pluviatolide at a concentration of 137 mg/L (ee ≥99% with 76% isolated yield).

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