Ola Pasternak
Biography: Ola started her undergraduate education at Queen’s University, where she completed a Bachelor of Science (honours) in Biochemistry in 2016 followed by a Bachelor of Arts in Classical Studies in 2018. In the fall of 2016, she joined the lab of Dr. David Zechel (Department of Chemistry at Queen’s University) for graduate school to study natural product biosynthesis. During her Ph.D., she spent a semester on exchange in the lab of Professor Andreas Bechthold in Freiburg, Germany, expanding her knowledge of genetic manipulation in Streptomyces. She recently (December, 2022) defended her Ph.D. thesis, which focused on elucidating the biosynthetic pathway of the fluoronucleoside, nucleocidin. When not in the lab, she is likely enjoying the outdoors or planning her next travel destination.
Title of Presentation: Towards sulfamate assembly in nucleocidin, and the search for related nucleoside natural products
Abstract: Nucleocidin is a natural product analog of adenosine that is produced by a limited number of Streptomyces strains. The molecule is notable for having 4’-fluorine and 5’-O-sulfamate groups, which are rarely seen in nature. In contrast, both substituents are widely used in pharmaceuticals. This includes 4’-fluorouridine, which was recently shown to have potential as a COVID-19 therapeutic. The biosynthetic route to the sulfamate and fluorine groups in nucleocidin are unknown. Within the nucleocidin biosynthetic gene cluster of Streptomyces calvus, five genes are proposed to be involved in sulfamate formation, including two sulfatases, nucI and nucG, two sulfotransferases, nucK and nucO and an amidinotransferase, nucN. Through recent gene inactivation experiments in the closely related strain and nucleocidin producer, Streptomyces virens, we show that nucI, nucG, and nucJ, the latter encoding a radical SAM/Fe-S dependent enzyme, are essential for sulfamate biosynthesis. We hypothesize that these genes may also be used as ‘marker genes’ toward identifying new sulfamate containing natural products and nucleosides. Through genome mining, a hybrid nucleocidin gene cluster encoding a new glucosylated tubercidin derivative was found in Streptomyces sp. AVP053U2. The glycosyltransferase, AvpGT, involved in biosynthesis is active with several purine and UDP-hexose substrates. Recent results towards nucleocidin biosynthesis, characterization of related derivatives and the kinetic parameters of AvpGT will be presented.
Connor Lorne Hodgins
Biography: My name is Connor Hodgins and I recently completed my PhD at the University of Calgary under the supervision of Dae-Kyun Ro. My interests are focused on developing gene-editing platforms in plants not only as a genetic tool to better understand plant physiology but also to produce unique cultivars with improved agronomic traits. While working in the Ro lab I have been successful in utilizing CRISPR-Cas9 gene-editing to study specialized metabolism in lettuce and peas. In lettuce two critical genes in the NR biosynthetic pathway were successfully knocked-out. Additionally, cis-prenyltransferase (CPT) lettuce mutants were used as a platform for the study of CPT genes from a variety of NR producing plants to gain insight into the mechanisms that control NR biosynthesis. The flavour of peas was improved by knocking-out the gene beta-amyrin synthase (BAS). BAS produces saponins which give peas an unpleasant bitter and astringent flavour. The saponin content in the bas mutant peas was reduced by 99% resulting in a pea with an improved flavour.
Title of Presentation: New insights into natural rubber biosynthesis from rubber deficient lettuce mutants expressing goldenrod and guayule cis-prenyltransferase
Abstract: Natural rubber (NR) is an industrially essential biopolymer characterized by its high Mw (≥ 1 million Da) and uniform cis-1,4-polyisoprene structure. Modern synthetic rubbers are still inadequate at perfectly replicating the physical properties of NR, making NR an irreplaceable resource in the manufacturing of hundreds of industrial and medical products, most notably pneumatic tires. Brazilian rubber tree (Hevea brasiliensis) plantations in Southeast Asia are the source of 90% of NR worldwide. Therefore, the limited geographic range and genetic diversity of the rubber tree pose existential threats to the global NR supply. The development of biotech solutions to this threat require a detailed understanding of NR biosynthesis. To date, the only necessary components identified for NR biosynthesis are, cis-prenyltransferase (CPT) and CPT binding protein (CBP). In NR producing plants these proteins form a complex on specialized rubber particle organelles in which CPT catalyzes the addition of isopentyl pyrophosphate (IPP) subunits to an allylic primer molecule. Lettuce (Lactuca sativa) is an ideal model to study NR as it produces high quality, long NR polymers (Mw ≥ 1 million Da). In this work, a CRISPR-Cas9 gene-editing platform was optimized in lettuce hairy roots and then implemented to produce lscpt3 mutant lines. These mutants were deficient in NR with no developmental or physiological abnormalities. Heterologous CPTs from guayule (Parthenium argentatum) and goldenrod (Solidago canadensis) were then expressed in the lettuce mutant background. In this talk, the qualitative and quantitative properties of NR in these mutant and transgenic lines will be discussed.
Connor L. Hodgins1,†, Moonhyuk Kwon1,2,†, Eman M. Salama1, Kayla R. Dias1, Aalap Parikh1, Ashlyn V. Mackey3, Karizza F. Catenza3, John C. Vederas3, and Dae -Kyun Ro1
1Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, 11 AB, T2N1N4, Canada
2Division of Applied Life Science (BK21 Four), ABC -RLRC, PMBBRC, Gyeongsang National 13 University, Jinju, 52828, Republic of Korea
3Department of Chemistry, University of Alberta, Edmonton, AB, T6G2G2, Canada
†These authors contributed equally to this work