Glycans (or carbohydrates) are the most abundant biomolecules on the planet, but we are far from elucidating their role in the design and regulation of biological systems spanning from the organismal to protein-level. The Chundawat Lab is focused on the study and manipulation of glycans and its interactions with other biomolecules in cellular systems with molecular-scale precision to address open-problems in diverse areas ranging from the design of more efficacious glycosylated drug molecules to engineering robust glycan-active enzymes for cheaper biofuels production. Our lab takes a carbohydrate or glycan-centric approach to solve interdisciplinary problems in the areas of bioenergy, healthcare, and advanced biomaterials engineering. We have expertise in developing a variety of analytical techniques for design and characterization of carbohydrate-active enzymes/proteins, as well as advanced bioprocessing and characterization of glycans/glycoconjugates. Specifically, our lab develops and applies protein and glycan engineering, bioprocessing, and biophysical techniques to address waste upcycling, accessible healthcare, and biophysics of life related research problems, as briefly outlined below.
- Advanced Biomanufacturing of Glycosylated Biopharmaceuticals: There has been an emergent need to develop advanced and continuous manufacturing bioprocesses for production of low cost but high-quality biotherapeutics like mRNA, monoclonal antibodies (mAbs), and CAR-T cells. Chundawat lab is developing and applying tools for real-time monitoring of glycans to understand cellular glycosylation states for enabling advanced biomanufacturing of glycosylated biotherapeutics. We are developing novel Process Analytical Technology (PAT) methods to autonomously monitor critical quality attributes (CQAs) of biotherapeutics like post-translational N-linked glycosylation of proteins and RNA produced by mammalian cells. Our group has developed an automated analytical workflow to continuously monitor mammalian cell cultures producing glycosylated biotherapeutics, called the ‘N-GLYcanyzer’, capable of cell free drug sampling/capture from batch/perfusion bioreactors, rapid enzymatic deglycosylation to release N-linked glycans, chemical derivatization/cleanup of released N-glycans, and detailed glycosylation composition profiling using online UHPLC/MS.
- Glycoengineering for Biomedical, Biomaterials, and Biotechnology Applications: Carbohydrate-active enzymes (or CAZymes) are a broad category of enzymes and functional proteins/domains that can synthesize, degrade, modify and/or recognize glycans and glycoconjugates. However, we have a poor understanding of the structure-function relationships of CAZymes that can be used to tweak enzyme specificity towards desired acceptor or donor sugar groups during biocatalysis. Our group is specifically working with glycosynthases, transglycosidases, and glycosyl transferases to tailor these enzymes for various biocatalysis applications. We are developing novel directed evolution techniques for CAZymes to synthesize bespoke human milk oligosaccharides for biomedical and prebiotics related applications. We are interested in engineering CAZymes to synthesize/modify biomedicine relevant glycans or glycoconjugates (e.g., N-linked glycosylation for designer mAbs synthesis).
- Advanced Biophysical Characterization of Carbohydrate-Active Enzymes & Cellular Systems: Chundawat group is broadly exploring the structure-function relationships relevant to binding interactions of multivalent glycans and glycan-binding proteins (GBP) and CAZymes using advanced single-molecule force spectroscopy and other structural characterization techniques (e.g., fluorescence microscopy). For example, we have applied force spectroscopy and super-resolution microscopy to probe and track the biomechanical steps of processive CAZymes as well as living cells involved in glycan polysaccharides synthesis and hydrolysis with sub-nanometer and millisecond resolution. We are developing single-molecule force spectroscopy and other imaging methods to characterize multivalent binding interactions of CAZymes/GBP to glycans to gain mechanistic insights into how cells and biomolecules sense, synthesize or degrade/modify glycans. Lastly, we are designing novel CAZymes (guided by computational protein design and insights from novel advanced characterization techniques) that are more efficient in catalytic turnover of glycan substrates.
- Biotechnology Toolkit Development for Waste Materials Upcycling to Value-Added Bioproducts: Advanced biofuels from inedible plant/algal biomass or waste plastics will allow addressing the impending energy, climate-change, and related socio-economic challenges facing humanity. Our lab has interests in developing integrated upstream-downstream biochemical conversion processes for enabling sustainable bioproducts synthesis such as biofuels, biochemicals, and biologics. Plant/algal biomass (i.e., cell walls) is composed mostly of insoluble glycan (~60-70%) and phenolic (~15-30%) polymers. However, plant and algal polysaccharides are 'recalcitrant' to extraction/conversion to soluble sugars and/or phenolic intermediates. Our lab has focused on overcoming this recalcitrance and developing a biochemical conversion platform to facilitate enzymatic hydrolysis of polysaccharides to sugars and fermentation to bioproducts using engineered CAZymes & microbial strains using advanced biomanufacturing methods (e.g., consolidated bioprocessing).