Recombinant E. coli was used as a host to explore the biosynthesis of Hyaluronic acid (HA) in Gram-negative strain using the optimized medium. In addition, measurement of HA molecular weight produced by the recombinant strains in shake-flask and 2 L stirred tank bioreactor (batch and fed-batch cultivation) were compared too .In this study, the hasA and hasE genes expressed together in a plasmid in recombinant E. coli strains were successfully produced HA using glucose as a carbon source under aerobic condition. Production of 127 mg/L HA was observed in a batch fermentation process with the highest HA molecular weight (70056 Da) using strain sAE in 2 L stirred tank bioreactor with controlled DO at 30%. The fed-batch culture with constant feeding of glucose was favorable for cell growth and the highest OD600 achieved at 1.923. However, the HA biosynthesis and molecular weight in fed-batch culture were lower than that observed in the batch culture.
Aims: Hyaluronic acid (HA) is a high molecular weight polymer and a major component of mucoid capsule in bacteria and extracellular matrix (ECM) of vertebrate tissue. Due to its unique characteristics, HA is used extensively in medical and cosmetic field. However, because of the exotoxins production from animal tissues extraction and Streptococcus zooepidemicus, HA production by recombinant microorganisms has gained interest. The present study was aimed at cloning of hasA gene in Escherichia coli and optimization of the medium components for HA production. Methodology and results: A fragment of an approximate size of 1.5 kb that encodes the hyaluronan synthase (hasA) gene from S. zooepidemicus ATCC 39920 was amplified by PCR using hasA-specific primers. The hasA gene was ligated into the bacterial expression vector pLbADH and transformed into the expression host, Escherichia coli BL21 strain. Then, genetically engineered E. coli strain BL21 was used for the production of HA by fermentation using different glucose concentration (10-50 g/L) and different IPTG concentration (0.1, 0.5 and 1.0 mM) in shake flask culture. Amongst varying glucose concentrations, results showed that 50 g/L glucose with nutrient rich media containing nitrogen source was able to produce the highest HA concentration (0.115 ± 0.002 g/L). With addition of 1.0 mM IPTG, HA production reached a peak 0.532 ± 0.026 g/L which is around fivefold higher compared to without IPTG. Conclusion, significance and impact of study: The hasA gene was cloned from S. zooepidemicus and successfully expressed in recombinant E. coli BL21 cells. This low molecular weight HA is gaining more importance in medical and cosmetic application due to possess pronounced free radical scavenging and antioxidant activities.
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