Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsBoth, (−)−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.
Highlights
The aryltetralin lignan (−)−podophyllotoxin is a potent antiviral and anti-neoplastic compound that is mainly found in Podophyllum plant species
In two previous studies we have reported on efficient multi-enzyme cascades in E. colistarting from coniferyl alcohol or (+)−pinoresinol to yield (−)−matairesinol 1 [25, 26]
Screening of E. coli strains to express OMT3, OMT1, CYP71CU1 and 2‐ODD Following the genome mining efforts which led to their identification [23] the heterologous expression in E. coli of the individual genes of the methyltransferases OMT3, OMT1 and the dioxygenase 2-oxoglutarate/Fe(II)-dependent dioxygenase (2-ODD) have been previously reported in literature [23, 27]
Summary
The aryltetralin lignan (−)−podophyllotoxin is a potent antiviral and anti-neoplastic compound that is mainly found in Podophyllum plant species. 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 endangered by overexploitation and environmental damage. Conventional isolation of (−)−podophyllotoxin from natural sources like Podophyllum and Sinopodophyllum plants and other related Berberidaceace species [8, 9] has become environmentally unbearable due to unregulated plant uprooting within the few regions where these plants can be cultivated [10] To overcome these limitations, various organometallic chemical catalysts have been successfully combined with single or multiple enzymatic steps, such approaches often involve the usage of expensive/toxic reagents and harsh reaction conditions [11, 12]. Either alone or combined in multi-enzyme cascades, in vitro or in vivo, biocatalysts generally allow to achieve reactions under mild, biologically compatible reaction conditions, with minor waste production and lower reagents toxicity in comparison to chemical catalysts [13,14,15]
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