Thesis Defence: Integrated approaches to study the camptothecin biosynthetic pathway in Camptotheca accuminata

Matthew McConnachie, supervised by Dr. Thu-Thuy Dang, will defend their thesis titled “Integrated approaches to study the camptothecin biosynthetic pathway in Camptotheca accuminata” in partial fulfillment of the requirements for the degree of Master of Science in Chemistry.

An abstract for Matthew McConnachie’s thesis is included below.

Defences are open to all members of the campus community as well as the general public. Registration is not required for in-person defences.

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ABSTRACT

Over 40 years of research have been spent investigating the camptothecin biosynthetic pathway in Camptotheca accuminata, yet it remains elusive. While there has been a large amount of work done in the last decade to generate new genomic, transcriptomic and metabolomic resources, there are still major gaps in the knowledge such as the localization of the tissue responsible for the biosynthesis of camptothecin and the missing steps in the biosynthetic pathway. To fill this gap, isotopically labelled precursors were fed to six tissues from C. accuminata, which were then analyzed using metabolomics. It was found that stem tissue was best able to produce downstream products in the CPT biosynthetic pathway. To identify candidate genes encoding enzymes involved in the camptothecin biosynthetic pathway, a novel bioinformatics workflow was developed, which utilized a new machine learning algorithm CLEAN to generate new functional annotations of the C. accuminata genome. This annotation was then combined with orthology data generated using another machine learning algorithm, TOGA, from Nyssa sinensis and Davidia involucrate, two non-CPT producing plants closely related to C. accuminata in the family
Nyssaceae to identify biosynthetic candidates highly expressed in stem tissues. To access costprohibitive and non-commercially available substrates, a workflow was developed to isolate gramscale quantities of secologanin and secologanic acid from Lonicera tatarica, and 20-(S)-deoxycamptothecin was produced semi-synthetically from camptothecin using a Marko-Lam deoxygenation. A new O-methyltransferase capable of methylating both 10 and 11-hydroxycamptothecin, as well as an array of flavonoids, was identified using correlation analysis of transcriptomic data. Finally, new activity of the previously discovered camptothecin-10-hydroxylase was identified when screened against 20-(S)-deoxycamptothecin putatively producing 10-hydroxy-20-(S)-deoxycamptothecin.