Application of Dual Promoter Expression System for the Enhanced Heparosan Production in Bacillus megaterium

Heparosan, a capsular polysaccharide synthesized by certain pathogenic bacteria, is a promising precursor for heparin production. Heparosan production is catalyzed by the formation of KfiC-KfiA complex and the subsequent action of KfiC and KfiA proteins. Polycistronic expression of kfiC and kfiA in Bacillus megaterium yielded an unbalanced expression of KfiC and KfiA proteins resulted in decreased heparosan production. In this study, dual promoter plasmid system was constructed to increase the expression levels of KfiC and KfiA proteins. Dual promoter plasmid system along with UDP-glucuronic acid pathway overexpression (CADuet-DB) increased the heparosan production to 203 mg/L in shake flask experiments. Batch fermentation of strain CADuet-DB under controlled conditions yielded a maximum heparosan concentration of 627 mg/L, which is 59% higher than strain CA-DB. A modified logistic model is applied to describe the kinetics of heparosan production and biomass growth. Fed batch fermentation resulted in 3-fold enhancement in heparosan concentration (1.96 g/L), compared to batch fermentation. Nuclear magnetic resonance analysis revealed that heparosan from strain CADuet-DB was similar to Escherichia coli K5 heparosan. These results suggested that dual promoter expression system is a promising alternative to polycistronic expression system to produce heparosan in B. megaterium.