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).

Background The aryltetralin lignan (−)−podophyllotoxin is a potent antiviral and anti-neoplastic compound that is mainly found in Podophyllum plant species. Over the years, the commercial demand for this compound rose notably because of the high clinical importance of its semi-synthetic chemotherapeutic derivatives etoposide and teniposide. To satisfy this demand, (−)−podophyllotoxin is conventionally isolated from the roots and rhizomes of Sinopodophyllum hexandrum, which can only grow in few regions and is now endangered by overexploitation and environmental damage. For these reasons, targeting the biosynthesis of (−)−podophyllotoxin precursors or analogues is fundamental for the development of novel, more sustainable supply routes. Results We recently established a four-step multi-enzyme cascade to convert (+)−pinoresinol into (−)−matairesinol in E. coli. Herein, a five-step multi-enzyme biotransformation of (−)−matairesinol to (−)−deoxypodophyllotoxin was proven effective with 98 % yield at a concentration of 78 mg/L. Furthermore, the extension of this cascade to a sixth step leading to (−)−epipodophyllotoxin was evaluated. To this end, seven enzymes were combined in the reconstituted pathway involving inter alia three plant cytochrome P450 monooxygenases, with two of them being functionally expressed in E. coli for the first time. Conclusions Both, (−)−deoxypodophyllotoxin and (−)−epipodophyllotoxin, are direct precursors to etoposide and teniposide. Thus, the reconstitution of biosynthetic reactions of Sinopodophyllum hexandrum as an effective multi-enzyme cascade in E. coli represents a solid step forward towards a more sustainable production of these essential pharmaceuticals.

Laccases are multi-copper oxidases that catalyze the oxidation of various electron-rich substrates with concomitant reduction of molecular oxygen to water. The multi-copper oxidase/laccase CueO of Escherichia coli is responsible for the oxidation of Cu + to the less harmful Cu 2 + in the periplasm. CueO has a relatively broad substrate spectrum as laccase, and its activity is enhanced by copper excess. The aim of this study was to trigger CueO activity in vivo for the use in biocatalysis. The addition of 5 mM CuSO 4 was proven effective in triggering CueO activity at need with minor toxic effects on E. coli cells. Cu-treated E. coli cells were able to convert several phenolic compounds to the corresponding dimers. Finally, the endogenous CueO activity was applied to a four-step cascade, in which coniferyl alcohol was converted to the valuable plant lignan (À )-matairesinol.

The evaluation of drug metabolites is compulsory during drug development. Since recently, bacterial cytochromes P450 and their mutated variants have attracted considerable interest as an alternative to hepatic P450s for the synthesis of human drug metabolites. Thus, straightforward screening approaches are required that enable rapid identification and evaluation of drug‐metabolizing bacterial P450s with different product selectivities. Herein, we report a two‐step screening method for discovery and characterization of new P450s from actinomycetes that enable oxidation of various drugs. In the first step, substrate profiling with three structurally different model drugs, ritonavir, testosterone, amitriptyline, allowed us to select CYP105D and CYP107Z from Streptomyces platensis DSM 40041 that accepted all model substrates and produced human‐like drug metabolites. In the second step, activity tests with an array of 25 structurally‐related molecules and derivatives of the three model compounds revealed a correlation between structural variations in the target drugs and the enzyme chemo‐ and regioselectivity.

Towards establishing a prospective industrial microbial lignan production process, we set up and investigated the biotransformation of coniferyl alcohol to secoisolariciresinol with recombinant Escherichia coli in a stirred-tank reactor (STR). Initially, we tested different cofactor concentrations and antifoam additions in shake flasks. Next, we designed an STR batch bioprocess and tested aeration rates, pH regulation, and substrate-feeding strategies. Targeted metabolomics of phenylpropanoids and lignans assisted the bioprocess development by monitoring the lignan pathway activity. We found that the copper concentration and the substrate-feeding strategy had considerable impact on lignan production. Furthermore, time-resolved monitoring of pathway metabolites revealed two maximal intracellular lignan concentrations, the first shortly after induction of gene expression and the second after the cells entered the stationary growth phase. During STR cultivation, a maximal intracellular titer of 130.4 mg L−1 secoisolariciresinol was achieved, corresponding to a yield coefficient of 26.4 mg g−1 and a space–time yield of 2.6 mg L−1 h−1. We report for the first time the in-depth evaluation of microbially produced lignans in a well-controlled STR bioprocess. Monitoring of the lignan pathway activity showed that lignan accumulation is highly dynamic during the cultivation and points towards the need for a more efficient coniferyl alcohol dimerization system for optimal microbial production conditions.