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).

Two new sesquiterpenes, innatifolone A (1) and pinnatifolone B (2), along with 6 known compounds, furostan (3), isocalamendiol (4), pluviatolide (5), (8S,8'R,9S)-cubebin (6), 2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dimethoxybenzyl) tetrahydrofuran (7), and methyl 3-acetoxy-12-oleanen-28-oate (8), were isolated from Mongolian herb Syringa pinnatifolia.

Microbial synthesis of monolignols and lignans from simple substrates is a promising alternative to plant extraction. Bottlenecks and byproduct formation during heterologous production require targeted metabolomics tools for pathway optimization. In contrast to available fractional methods, we established a comprehensive targeted metabolomics method. It enables the quantification of 17 extra- and intracellular metabolites of the monolignol and lignan pathway, ranging from amino acids to pluviatolide. Several cell disruption methods were compared. Hot water extraction was best suited regarding monolignol and lignan stability as well as extraction efficacy. The method was applied to compare enzymes for alleviating bottlenecks during heterologous monolignol and lignan production in E. coli. Variants of tyrosine ammonia-lyase had a considerable influence on titers of subsequent metabolites. The choice of multicopper oxidase greatly affected the accumulation of lignans. Metabolite titers were monitored during batch fermentation of either monolignol or lignan-producing recombinant E. coli strains, demonstrating the dynamic accumulation of metabolites. The new method enables efficient time-resolved targeted metabolomics of monolignol- and lignan-producing E. coli. It facilitates bottleneck identification and byproduct quantification, making it a valuable tool for further pathway engineering studies. This method will benefit the bioprocess development of biotransformation or fermentation approaches for microbial lignan production.