Page 253 - PC2019 Program & Proceedings

PLANT CANADA 2019

P79. Recombinant protein expression in plants: The key influence of basic growth conditions
Shang, L.; M. Goulet; D. Michaud
Université Laval

Plants show practical advantages for the production of clinically valuable recombinant proteins, including
low upstream production costs, convenient large-scale cultivation and low risks of pathogenic or toxin
contamination. From a biotechnological standpoint, the yield of a plant-made protein depends on the
amount of recombinant protein per biomass unit, and on the biomass per plant or plant culture area.
Several strategies have been proposed to improve recombinant protein yield per plant biomass unit,
mostly relying on molecular tools and procedures for increased transgene expression or protein stability in
planta. By comparison, little attention has been paid to the influence of common cultural practices such as
supplemental lighting, atmospheric CO2 enrichment or ammonium nutrition in ‘molecular pharming’
contexts. For instance, extensive work has been done over the years to define optimal growth conditions
for leafy crops like lettuce or tobacco but the best cultural conditions for these same plants used as
bioreactors may differ, since the ultimate target is recombinant protein yield in leaf tissue, not maximal
leaf biomass or increased endogenous protein content. We here show the importance of carefully defining
cultural practices that are well suited to the particular constraints and goals pursued in recombinant
protein production settings. We also show how certain cultural practices can be harnessed to generate
recombinant protein yield gains on a whole-plant basis, notably via an impact on host plant architecture.

Dominique Michaud (dominique.michaud@fsaa.ulaval.ca)

P80. Identification of candidate cinnamyl alcohol dehydrogenases in Tabernanthe iboga root
*
McDonald, K. ; M. Kapasi; J. Stout
University of Manitoba

Vinblastine and vincristine are compounds used as chemotherapy drugs and as precursors to semi-
synthetic alternatives. These compounds are produced in low quantities (0.002% dry weight) by
Catharanthus roseus (rosy periwinkle). The low accumulation of these compounds contributes to their
high cost and places a strain on the supply chain of these drugs. Catharanthine is a precursor to
vinblastine and vincristine biosynthesis in rosy periwinkle.

Catharanthine is an intermediate in the biosynthesis of the iboga alkaloid ibogaine in Tabernanthe iboga
(iboga). Iboga accumulates large quantities of iboga alkaloids (4% dry weight) in its root bark. It is
hypothesized that a reductase belonging to the cinnamyl alcohol dehydrogenase (CAD) family is
responsible for committing catharanthine to the ibogaine biosynthetic pathway in iboga.

To identify the enzyme involved in this committal step, we generated an iboga root transcriptome from
plants confirmed to be producing ibogaine. A list of candidate reductase genes was generated by
searching this transcriptome for sequences homologous to CAD proteins.

This search revealed seven candidate genes which will be cloned, expressed in a heterologous system, and
purified. Purified protein will be used to determine in vitro function/substrate affinity of these candidate
genes. Once the reductase is identified, RNAi will be used to knock down reductase expression in iboga
hairy root cultures from which tissue will be harvested and analyzed for the accumulation of
catharanthine.

Kyle McDonald (ketchupkyle@gmail.com)

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