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

Abstract

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).

Synthesis of (−)−deoxypodophyllotoxin and (−)−epipodophyllotoxin via a multi-enzyme cascade in E. coli

“…Under the established optimized expression conditions for CYP71CU1, when co-expressed with OMT3 in the same E. coli cell, CYP71CU1 concentration achieved 240 ± 15 µg P450 /g cww which is 3-fold lower in comparison to the single expressed CYP71CU1 (Additional file 1 : Figure S10A). This is not surprising because in our previous work we obseved a significant reduction of the P450 concentration when multiple genes were co-expressed from the same expression vector [ 25 ].…”

“…Next, the activity of OMT3 was evaluated. In our previous work, the OMT3’s substrate (−)−pluviatolide 2 was successfully isolated after production in E. coli from (−)−matairesinol 1 by S. hexandrum CYP719A23 supported by the cytochrome P450 reductase ATR2 from Arabidopsis thaliana [ 25 ]. To perform the two-step biotransformation of (−)−matairesinol 1 to (−)−5’-desmethoxy-yatein 3 , CYP719A23 and ATR2 (plasmid pETDuet_atr2_cyp719a∆23oc) were co-expressed with OMT3 (plasmid pCDFDuet_omt3) (Additional file 1 : Table S3).…”

“…1 ). In two previous studies we have reported on efficient multi-enzyme cascades in E. coli starting from coniferyl alcohol or (+)−pinoresinol to yield (−)−matairesinol 1 [ 25 , 26 ]. In the subsequent steps of the S. hexandrum metabolic pathway, (−)−matairesinol 1 is first converted to (−)−pluviatolide 2 by the cytochrome P450 monooxygenase (P450 or CYP) CYP719A23 [ 23 ].…”

“…Individual compounds produced in the herein described multi-enzyme cascade are highlighted by a 1 to 7 numbering. Compounds 2 , 3 , 4 , and 7 are not commercially available; (−)−pluviatolide 2 was isolated and identified in our previous work [ 25 ] …”

Control of synthetic microbial consortia in time, space, and composition

“…Modern engineered biological systems are able to solve more complex problems than ever before. Bioproduction is increasingly able to synthesize valuable plant-based products, significantly reducing production time [1][2][3]. Novel gene circuit topologies continue to increase in complexity as our understanding of the genetic code and its regulation deepens [4,5].…”