Assembly of Plant Enzymes in E. coli for the Production of the Valuable (-)-Podophyllotoxin Precursor (-)-Pluviatolide.

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).

Laccase Encapsulation in ZIF-8 Metal-Organic Framework Shows Stability Enhancement and Substrate Selectivity.

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%.

Insights into the functional properties of the marneral oxidase CYP71A16 from Arabidopsis thaliana.

The Arabidopsis thaliana gene encoding CYP71A16 is part of the gene cluster for the biosynthesis and modification of the triterpenoid marneral. Previous investigations of A. thaliana have revealed that CYP71A16 catalyzes marneral oxidation, while it also can accept marnerol as substrate. The aim of the present study was to investigate functional properties of CYP71A16 in vitro. For this purpose, heterologous expression of a N-terminally modified version of CYP71A16 was established in Escherichia coli, which yielded up to 50mgL-1 recombinant enzyme. The enzyme was purified and activity was reconstituted in vitro with different redox partners. A heterologous bacterial redox partner system consisting of the flavodoxin YkuN from Bacillus subtilis and the flavodoxin reductase Fpr from E. coli clearly outperformed the cytochrome P450 reductase ATR2 from A. thaliana in supporting the CYP71A16-mediated hydroxylation of marnerol. Substrate binding experiments with CYP71A16 revealed a dissociation constant KD of 225µM for marnerol. CYP71A16 catalyzed the hydroxylation of marnerol to 23-hydroxymarnerol with a KM of 142µM and a kcat of 3.9min-1. Furthermore, GC/MS analysis revealed an as of yet unidentified overoxidation product of this in vitro reaction. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Three-steps in one-pot: whole-cell biocatalytic synthesis of enantiopure (+)- and (-)-pinoresinol via kinetic resolution.

BACKGROUND: Pinoresinol 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. RESULTS: The 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%. CONCLUSION: A 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.

Expanding the laccase-toolbox: a laccase from Corynebacterium glutamicum with phenol coupling and cuprous oxidase activity.

Laccases are oxidases with potential for application in biotechnology. Up to now only fungal laccases have been applied in technical processes, although bacterial laccases are generally easier to handle and more stable at alkaline pH values and elevated temperatures. To increase the toolbox of bacterial laccases and to broaden our knowledge about them, new enzymes have to be characterized. Within this study, we describe the new bacterial laccase CgL1 from Corynebacterium glutamicum. CgL1 was found to oxidize typical laccase substrates like 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), syringaldazine and 2,6-dimethoxyphenol. The enzyme also demonstrates cuprous oxidase activity. Furthermore, CgL1 is active for several hours at temperatures up to 60°C and at alkaline pH, as well as stable in different organic solvents. This makes CgL1 a potential candidate for technical applications. In addition, CgL1 was found to catalyze the CC/CO coupling of several phenolic compounds which can serve as precursors for the synthesis of natural products like antibiotics and phytohormones. This activity and product distribution were influenced by pH value and mediators used.