Bioproduction Optimization and Characterization of Hyaluronic Acid in Escherichia coli and Nicotiana benthamiana

Hyaluronic Acid (HA) is a polymer widely used in biomedical and pharmaceutical applications, with its biological and physical properties strongly dependent on molecular weight. Conventional production methods, including animal tissue extraction and Streptococcus fermentation, yield high-molecular weight HA and require additional processing to obtain low-molecular weight polymers. Additionally, these methods carry ethical and toxicity concerns, respectively. This study investigates alternative strategies for the controlled biosynthesis of HA using both microbial and plant-based expression systems. Recombinant Escherichia coli strains were engineered to express human hyaluronic acid synthase 2 (hHAS2) with and without the co-expression of hyaluronidase and were evaluated based on total HA yield and molecular weight. The engineered strains successfully produced HA with exceptionally low molecular weights (2.3–3.6 kDa) and narrow polydispersity index, indicating a highly uniform product compared to chemically degraded HA. Contrary to expectations, co-expression of hyaluronidase did not further reduce polymer size, likely due to enzyme interference or substrate limitations at low molecular weights. Optimization of culture conditions revealed that magnesium supplementation significantly enhanced HA production, increasing yield by up to 256%. Additionally, while longer incubation increased total yield, production rates declined over time, suggesting that multiple short cultivation cycles may be more efficient than extended fermentation. To assess biocompatibility, purified HA was tested using dauer Caenorhabditis elegans, showing no detectable toxicity. This confirms the suitability of the recombinant product for biological applications. In parallel, plant-based HA production was investigated using Agrobacterium-mediated gene transfer to transform Nicotiana benthamiana with hHAS2. To enhance precursor availability, N. benthamiana was further engineered to co-express glmS and glmU, which encode key enzymes involved in HA biosynthesis. Co-expression of these genes resulted in a substantial increase in HA yield, reaching up to 6.82 g/kg wet weight, a 40-fold improvement compared to previously reported plant-based systems. Overall, this study demonstrates the combination of E. coli and human hyaluronic acid synthase 2 as an effective system to produce uniform low-molecular weight HA, while N. benthamiana offers an improved system for an improved high yield production.

Department dc:contributor.department

Biology

Year dc:date.issued

2026

Author dc:creator

• Gavrin, Elliot

Advisor dc:contributor.supervisor

• Lefebvre, Daniel

• Hyaluronic Acid
• Bioprocessing
• Nicotiana
• E. coli
• Genetic Engineering
• Synthetic Biology

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1974/36368

OAI identifier oai:identifier

oai:queensu.scholaris.ca:1974/36368