RESUMO
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 has main role in HA biosynthesis by Streptococcus strain as a natural producer. The hasB and hasC genes are also mediate GlcUA precursor biosynthesis. In the present study, S. equisimilis hasA gene; B. subtilis tuaD and gtaB genes for GlcUA precursors enhancement, and vgb gene coding bacterial hemoglobin as an oxygen provider were used to construct the B. subtilis strain for 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 produced HA structures. 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 by strains. Dynamic light scattering (DLS) revealed all HA polymers had a relatively low polydispersity index (PDI < 0.5). These findings demonstrate the successful GlcUA biosynthetic pathway engineering strategy in improving HA yield by recombinant B. subtilis, metabolically-robust, and industrially potential strain.
RESUMO
Hyaluronic Acid (HA) is a natural biopolymer that has important physiological and industrial applications due to its viscoelastic and hydrophilic characteristics. The responsible enzyme for HA production is Hyaluronan synthase (HAS). Although in vitro structure-function of intact HAS enzyme has been partly identified, there is no data on in vivo function of truncated HAS forms. In the current study, novel recombinant Bacillus subtilis strains harboring full length (RBSFA) and truncated forms of SeHAS (RBSTr4 and RBSTr3) were developed and HA production was studied in terms of titer, production rate and molecular weight (Mw). The maximum HA titer for RBSFA, RBSTr4 and RBSTr3 was 602 ± 16.6, 503 ± 19.4 and 728 ± 22.9 mg/L, respectively. Also, the HA production rate was 20.02, 15.90 and 24.42 mg/L.h-1, respectively. The findings revealed that RBSTr3 produced 121% and 137% more HA rather than RBSFA and RBSTr4, respectively. More interestingly, the HA Mw was about 60 kDa for all strains which is much smaller than those obtained in prior studies.
