A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis

Abstract

The ultimate step in the formation of thebaine, a pentacyclic opiate alkaloid readily converted to the narcotic analgesics codeine and morphine in the opium poppy, has long been presumed to be a spontaneous reaction. We have detected and purified a novel enzyme from opium poppy latex that is capable of the efficient formation of thebaine from (7S)-salutaridinol 7-O-acetate at the expense of labile hydroxylated byproducts, which are preferentially produced by spontaneous allylic elimination. Remarkably, thebaine synthase (THS), a member of the pathogenesis-related 10 protein (PR10) superfamily, is encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes immediately upstream. THS is a missing component that is crucial to the development of fermentation-based opiate production and dramatically improves thebaine yield in engineered yeast.

The opium poppy genome and morphinan production

“…the recently discovered THEBAINE SYNTHASE (19) were identified on an unplaced scaffold in synteny with the BIA biosynthesis gene cluster on chromosome 11 ( Fig. 2A;…”

“…Comparison of released opium poppy assembly (accession number PRJNA435796) with: (A) the PacBio assembly, (B) the NRgene 10X assembly, (C) scaffold MH011344 from Chen et al (19). Fig.…”

A yeast platform for high-level synthesis of natural and unnatural tetrahydroisoquinoline alkaloids

“…Although our titers are commensurate with this benchmark, morphine is synthesized several enzymatic steps downstream of (S)-reticuline. The efficiency of these reactions within a high titer production host remains unknown, yet the recent discovery of dedicated thebaine and neopinone biosynthetic enzymes 41,42 primes our engineered yeast for production of opioid pharmaceuticals at scale.…”

The Energetic Viability of Δ1-Piperideine Dimerization in Lysine-derived Alkaloid Biosynthesis

“…However, ( S,S )-piperideine dimer was not reported as a plant metabolite, indicating that an enzyme could mediates the formation of ( R,R )-piperideine dimer or decompose ( S,S )-piperideine dimer selectively. In thebaine biosynthesis, thebaine synthase (THS) catalyzes the conversion of (7 S )-salutaridinol 7- O -acetate to thebaine, although this reaction spontaneously proceeds in aqueous conditions [ 42 ]. THS-catalyzed reaction can prevent the formation of hydroxylated byproducts, which is the benefit for regulating the bioactive product metabolism.…”

“…Therefore, we conclude that ( S,S )- or ( R,R )-piperideine dimers are spontaneously formed immediately after the conversion of -piperideine to 2-piperideine. However, we cannot rule out the possibility of enzymatic reaction, because nature uses an enzyme for the reaction that can spontaneously proceeds in aqueous conditions [ 42 ]. In addition, the detection of (+)-epilupinine suggests that ( R,S )-piperideine dimer formation may take place very slowly in plant, as the activation energy is just the threshold value for the spontaneous reaction.…”