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

Abstract

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.

Biosynthesis, total synthesis, and pharmacological activities of aryltetralin-type lignan podophyllotoxin and its derivatives

“…This six-step strategy produced 8.3 mg L À1 (À)-epipodophyllotoxin with 10% conversion. 32 Besides enzyme engineering and pathway optimization in vitro, engineering the supply and recycling of cofactors, which participate in many intracellular metabolic processes and reactions, is also a feasible way to enhance PTOX production. In 2022, on the basis of pathway optimization in yeast S. cerevisiae, ) reached gram-level production in the fedbatch fermentation, which has a considerable production potential at present 33 (Fig.…”

“…Natural Product Reports (À)-PTOX (Scheme 4). Researchers rst used the oxidative enolate coupling methodology to couple oxazolidinone (30) with an ester (31) to form a single diastereomer dibenzylbutyrolactone (32) as the natural substrate of the DPS enzyme. Then, they used chaperones GroEL and GroES to optimize the soluble expression level of the DPS enzyme, leading 32 to completely transform into (À)-DPS (19).…”

“…For example, using the latestage biosynthetic precursor DPT has realized milligram-scale production, both in Escherichia coli and plant classis. 31,32 The upstream compounds caffeic acid (CaA) and ferulic acid (FA) were achieved in gram-level production (5.5 AE 0.2 g L À1 CaA, 3.8 AE 0.3 g L À1 FA) in microbial cell factories by engineering cofactor supply and recycling. 33 Moreover, the catalytic mechanism of pathway enzymes, such as dirigent proteins (DIRs), secoisolariciresinol dehydrogenase (SDH), and deoxypodophyllotoxin synthase (DPS) have been revealed, enhancing understanding of a growing number of biochemical transformations.…”

Chemistry and Biology of Podophyllotoxins: An Update

“…[37] In addition, Decembrino et al completely biosynthesized (À )-deoxypodophyllotoxin and (À )-epipodophyllotoxin in E. coli. [38] Moreover, Schultz et al also achieved milligram-level synthesis of (À )deoxypodophyllotoxin in tobacco. [39] The complete biosynthesis of PPT in plants was described in Scheme 10, phenylalanine (116) was deaminated by the action of phenylalanine ammonia lyase (PAL) to obtain cinnamic acid (117), followed by the formation of coniferaldehyde (118) catalyzed by seven enzymes, which was then converted to coniferyl alcohol (119) by the action of cinnamyl alcohol-dehydrogenase (CAD).…”

Production of beneficial lignans in heterologous host plants

“…Previous studies have suggested the potential capability of bacteria to produce lignans. Two sequential Escherichia coli lines, harboring individual metabolic modules of (-)-matairesinol to (-)-5′desmetyl-yatein, and (−)-5′desmetyl-yatein to (−)-deoxypodophyllotoxin, reportedly metabolized exogenously applied (-)-matairesinol to (-)-deoxypodophyllotoxin ( Decembrino et al., 2021 ). The human gut microbiome also metabolizes exogenously applied (+)-pinoresinol to various lignans ( Xie et al., 2003 ; Espín et al., 2017 ; Bess et al., 2020 ).…”