Page 234 - PC2019 Program & Proceedings

PLANT CANADA 2019

TOPIC 4: Biochemistry, Metabolism, Photosynthesis
(Posters P41-P68)

P41. A central role for polyprenol reductase in plant dolichol biosynthesis
*
Van Gelder, K. ; L. Virta; T. Akhtar
University of Guelph)

Dolichol is an essential polyisoprenoid within the endoplasmic reticulum of all eukaryotes. It serves as a
membrane bound anchor onto which N-glycans are assembled prior to being transferred to nascent
polypeptides that enter the secretory pathway. Historically, it has been posited that the ‘rate-limiting’ step
in protein N-glycosylation, a process that affects the efficacy of approximately one fifth of the entire
eukaryotic proteome, is determined by the extent to which dolichol accumulates. Therefore, this study
aimed to enhance dolichol accumulation by manipulating the enzymes involved in its biosynthesis using
an established Nicotiana benthamiana platform. Co-expression of a Solanum lycopersicum (tomato) cis-
prenyltransferase and its cognate partner protein that catalyze the antepenultimate step in dolichol
biosynthesis led to a 400-fold increase in the levels of long-chain polyprenols but resulted in only modest
increases in dolichol accumulation. However, when combined with a newly characterized tomato
polyprenol reductase, dolichol biosynthesis was enhanced by approximately 20-fold. We provide further
evidence that in the aquatic macrophyte, Lemna gibba, dolichol is derived exclusively from the mevalonic
acid (MVA) pathway with little participation from the evolutionary co-adopted non-MVA
pathway. Taken together these results indicate that to effectively enhance the in planta accumulation of
dolichol, coordinated synthesis and reduction of polyprenol to dolichol, is strictly required.

Kristen Van Gelder (kvangeld@uoguelph.ca)

P42. Cytochrome P450 and O-methyltransferase catalyze the final steps in the biosynthesis of the
anti-addictive alkaloid ibogaine from Tabernanthe iboga
Farrow, S.; M. Kamileen; S. O'Connor
The John Innes Centre

Monoterpenoid indole alkaloids are a large and structurally diverse class of metabolites restricted to a
limited number of plant families in the order Gentianales. Tabernanthe iboga is native to western
equatorial Africa and has been used in traditional medicine for centuries. Howard Lotsof is credited with
bringing iboga to the attention of modern medicine through his discovery that iboga can alleviate opioid
withdrawal symptoms. Since this observation, iboga has been investigated for its use in general addiction
management. We were interested in elucidating ibogaine biosynthesis to understand the unique reaction
steps en route to ibogaine. Furthermore, because ibogaine is currently sourced from plant material, these
will help improve the ibogaine supply chain through synthetic biology approaches. We used next-
generation sequencing to generate the first iboga transcriptome and leveraged homology-guided gene
discovery to identify the penultimate hydroxylase and final O-methyltransferase steps in ibogaine
biosynthesis, herein named ibogamine 10-hydroxylase (I10H) and noribogaine-10-O-methyltransferase
(N10OMT). Heterologous expression in S.cerevisiae (I10H) or E.coli (N10OMT) and incubation with
putative precursors, along with HPLC–MS analysis, confirmed the predicted activities of both enzymes.
Moreover, high expression levels of their transcripts were detected in ibogaine-accumulating plant tissues.
These discoveries coupled with our publicly available iboga transcriptome will contribute to additional
gene discovery efforts and could lead to the stabilization of the global ibogaine supply chain and its
development as a treatment for addiction.

Scott Farrow (scott.farrow@jic.ac.uk)

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