The Kwan Lab
Centre for Structural and Functional Genomics
7141 Rue Sherbrooke Ouest
Montréal, Quebec H4B 1R6
F. Ifthiha Mohideen
Function and engineering of enzymes involved in the glycosylation of natural products
Many small-molecule natural products produced by plants and bacteria are decorated with sugar moieties that have a great significance upon their biological activity. Glycosyltransferases (GTs) are involved in the biosynthesis of these glycosides and many of these enzymes can assemble different sugar residues to different acceptor molecules which will produce novel glycosides with improved or altered bioactivity. To characterize, analyze, and engineer these enzymes we have developed a method for assaying GTs in a high-throughput fashion. Moreover, using synthetic biology tools, we are engineering an improved GT for the production of a high-value anthracycline anticancer drug which is conventionally produced semisynthetically. Towards this aim, using a novel in vitro enzymatic pathway we are producing modified sugar donor substrates for the enzymatic synthesis of anthracyclines.
Lan Huong Nguyen
Investigating glycosylated natural products biosynthesis and pathway engineering for novel valuable biomolecules with the overall goal of uncovering new drug candidates.
I completed my Ph.D. program focusing on the discovery and characterization of an enzyme involved in biosynthetic gene clusters for a range of bioactive small molecules, and pathway design and re-engineering toward combinatorial natural product biosynthesis. During my postdoctoral research, a major focus of mine is on the enzymes involved in the biosynthesis of glycosylated natural products. The main goals of this research are to use these enzymes as biocatalytic tools for synthesis, to understand their enzymatic mechanisms, and to engineer them to produce new to-nature biosynthetic compounds that have anticancer and antibiotic activity. My proposed research will involve approaches in both molecular biology and chemical biology.
Designer Biosensors for Engineered Metabolic Pathway Optimization
Synthetic biology techniques aimed at constructing artificial metabolic pathways in genetically modified microorganisms are emerging as important methods to produce biofuels, pharmaceuticals and value-added chemicals. To reach industrially relevant scales, challenges related to pathway bottlenecks and system optimization must be addressed. Since these are typically complex multi-enzyme pathways, screening methods for most intermediates and products are laborious and inefficient. In this project, we utilize transcription factor-based biosensors to develop high-throughput molecule detection tools. Additionally, we employ a semi-rational approach to engineer and expand the substrate specificity of our biosensors. Our “designer” biosensors will be utilized for improving the productivity of an engineered yeast strain by pathway dynamic control as well as using them as screening tools for the directed evolution of pathway enzymes.
Role of Fucosyltransferase Enzymes in Cancer
My project aims to develop a high-throughput assay to discover drugs for FUT8 inhibition. FUT8 is the only enzyme involved in core fucosylation and acts specifically on a branched core N-glycan structure attached to certain proteins. Screening of FUT8 inhibitors will be performed using a previously developed strategy involving synthetic, fluorogenically labeled oligosaccharides and specific glycosidase and glycosyltransferase enzymes.
Development of a high-throughput assay to detect decarboxylase activity
Many industrial products have now been shown to be naturally occurring in nature, and the goal of sustainable biotechnology is to produce these fine chemicals in an environmentally safe way. To reach this goal, our group employs protein engineering to improve the activity of the enzymes that make these products. My project focuses on the development of a high-throughput screen for decarboxylases- which make a variety of molecules that are of interest in biofuel, medicine and food industries. This screen allows us to engineer these enzymes semi-rationally and detect higher activity variants.
Peptide-based Drug Discovery for Mycobacterium tuberculosis
The aim of this research project is to identify potent inhibitory molecules that restrict cell wall biosynthesis by binding to UDP-Galactopyranose Mutase (UGM) and inhibiting its activity. Inhibitory molecules will be identified from a library of non-standard macrocyclic peptides and affibodies. These peptides/protein will be subjected to screening for their bioactivity by using mRNA display technique. mRNA display is a dynamic technique that can be used to screen trillions of molecules for their binding affinity to the target molecule. In this method, the macrocyclic peptides bound with puromycin are covalently linked to 3’ end of their corresponding mRNA sequence. The genotype coding sequence and the phenotype polypeptide sequence are covalently combined within the same molecule, the selected protein can be revealed by DNA sequencing after reverse transcription and PCR amplification. Therefore, mRNA display is a powerful tool. This will be used to read and amplify a peptide sequence after it has been functionally isolated from a library with high diversity. A specialized form of mRNA display utilizes in vitro translation machinery to synthesize natural-product like non-standard macrocyclic peptides. This technique is called Flexible In vitro Translation (FIT). mRNA display using the FIT system is called Randon non-standard Peptide Integrated Discovery (RaPID). The RaPID system will be used to isolate macrocyclic peptide ligands that bind to UGM. Once these, peptide/protein ligands are obtained we will develop an assay to test them for its inhibitory activity.
Chemoenzymatic Synthesis and Modification of Sialyl Lewis X
My project is the Synthesis and Modification of Sialyl Lewis X (sLeX) which enable it to attach to cell surface ex vivo. Stem cells are thought important in therapies for a variety of diseases. But stem cells’ efficiency of engraftment is low due to the lack of relevant adhesion molecules on their surface. Since some research reported that sLeX is important in mediating binding interactions between cells, we plan to increase cells homing ability by install sLeX on the cell surface.