Total Biosynthesis for Milligram-Scale Production of Etoposide Intermediates in a Plant Chassis

Abstract

Etoposide is a plant-derived drug used clinically to treat several forms of cancer. Recent shortages of etoposide demonstrate the need for a more dependable production method to replace the semisynthetic method currently in place, which relies on extraction of a precursor natural product from Himalayan mayapple. Here we report milligram-scale production of (-)-deoxypodophyllotoxin, a late-stage biosynthetic precursor to the etoposide aglycone, using an engineered biosynthetic pathway in tobacco. Our strategy relies on engineering the supply of coniferyl alcohol, an endogenous tobacco metabolite and monolignol precursor to the etoposide aglycone. We show that transient expression of 16 genes, encoding both coniferyl alcohol and main etoposide aglycone pathway enzymes from mayapple, in tobacco leaves results in the accumulation of up to 4.3 mg/g dry plant weight (-)-deoxypodophyllotoxin, and enables isolation of high-purity (-)-deoxypodophyllotoxin after chromatography at levels up to 0.71 mg/g dry plant weight. Our work reveals that long (>10 step) pathways can be efficiently transferred from difficult-to-cultivate medicinal plants to a tobacco plant production chassis, and demonstrates mg-scale total biosynthesis for access to valuable precursors of the chemotherapeutic etoposide.

Figure 1.

Impact of CA infiltration and DIR expression on DPT production in planta according to LC-MS of extracts from Agro.- infiltrated tobacco. Gray bar height indicates mean (error bars show ± SD); individual biological replicates are represented as dots. Colors indicate absence or presence of CA infiltration (−CA or +CA, respectively).

Figure 2.

Impact of individual CA pathway enzyme expression on DPT yield in planta according to LC-MS of extracts from Agro.- infiltrated tobacco. Gray bar height indicates mean (error bars show ± SD); individual biological replicates are represented as dots. Asterisks indicate a significant difference between sets, p < 0.05. Gray-shaded boxes indicate enzymes expressed via Agro.-infiltration, with numbers showing the amount of the GFP-expressing strain used relative to the other strains.

Figure 3.

EA and (−)-morelensin production. (A) LC-MS extracted ion chromatograms (EIC) at 401 m/z, [EA + H]⁺. Green asterisk indicates EA; other peaks are likely derived from in-source fragmentation of EA glycosides. Both traces are from experiments where noted genes were expressed in tobacco via Agro.-infiltration. (B) Engineered pathway for (−)-morelensin production. Dotted arrows depict omitted enzymes. (C) (−)-Morelensin production with exogenous addition of varying CA levels or co-infiltration with CA pathway (PW)-expressing Agro. strains. Gray bar height indicates mean (error bars show ± SD); individual biological replicates are represented as dots. Asterisks indicate a significant difference between sets, p < 0.05.

Scheme 1.. Engineered Biosynthetic Pathway for Production of EA and Late-Stage Pathway Intermediates^a

^aArrows represent previously characterized biosynthetic genes. The coniferyl alcohol (CA) pathway is shared by all vascular plants, including tobacco, while the (−)-deoxypodophyllotoxin (DPT) and etoposide aglycone (EA) genes are less widespread (see Supporting Information). The latter five DPT enzymes and EA cytochromes P450 are thought to be more specific to EA production and are not found in tobacco.