The effect of manipulating glucuronic acid biosynthetic pathway in Bacillus subtilis strain on hyaluronic acid production

Abstract

Abstract Hyaluronic acid (HA), composed of glucuronic acid (GlcUA) and N-acetyl glucoseamine (GlcNAc), is a versatile biopolymer with high commercial value and innumerous physiological roles and pharmaceutical applications. The hasA gene is responsible for HA biosynthesis by Streptococcusstrain as a natural producer. The hasB and hasC genes are also responsible for GlcUA precursor biosynthesis. In the present study, S. equisimilis hasA gene; B. subtilis tuaD and gtaB genes for GlcUA precursorsenhancement, and vgb gene coding bacterial hemoglobin as an oxygen provider were used to engineer the B. subtilis strainfor HA production. RBSHA (hasA), RBSHA2 (hasA/tuaD/gtaB), and RBSHA3 (hasA/tuaD/gtaB/vgb) strains were developed and confirmed through genotype and phenotype analysis. After HA production and purification, FTIR spectroscopy confirmed the HA structures produced by the strains. HA assay showed the highest HA titer for RBSHA3 (2.1 ± 0.18 mg/ml) and then RBSHA2 (1.9 ± 0.03 mg/ml), and RBSHA (0.6 ± 0.14 mg/ml). Statistical analysis indicated there is no significant difference in HA titer between RBSHA2 and RBSHA3 strains (p-value > 0.05), however, these strains produced HA approximately 4-fold higher than that of RBSHA strain. Agarose gel electrophoresis showed the same molecular weight (< 30 kDa) of produced HA. Dynamic light scattering (DLS) revealed all HA polymers had a relatively low polydispersity index (PDI < 0.5). Our study demonstrates the successful GlcUA biosynthetic pathway engineering strategy in improving HA yield by recombinant B.subtilis, metabolically-robust, and industrially potential strain.