Engineering S. equi subsp. zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest

Abstract

Glycosaminoglycans, such as hyaluronic acid and chondroitin sulphate, are not only more and more required as main ingredients in cosmeceutical and nutraceutical preparations, but also as active principles in medical devices and pharmaceutical products. However, while biotechnological production of hyaluronic acid is industrially established through fermentation of Streptococcus spp. and recently Bacillus subtilis, biotechnological chondroitin is not yet on the market. A non-hemolytic and hyaluronidase negative S. equi subsp. zooepidemicus mutant strain was engineered in this work by the addition of two E. coli K4 genes, namely kfoA and kfoC, involved in the biosynthesis of chondroitin-like polysaccharide. Chondroitin is the precursor of chondroitin sulphate, a nutraceutical present on the market as anti-arthritic drug, that is lately being studied for its intrinsic bioactivity. In small scale bioreactor batch experiments the production of about 1.46 ± 0.38 g/L hyaluronic acid and 300 ± 28 mg/L of chondroitin with an average molecular weight of 1750 and 25 kDa, respectively, was demonstrated, providing an approach to the concurrent production of both biopolymers in a single fermentation.

Keywords: Chondroitin; Co-production; Hyaluronic acid; Streptococcus equi subsp. zooepidemicus.

Fig. 1

HA biosynthesis pathways in Streptococcus. The pathway was modified by the introduction of two foreign genes (indicated in red), kfoA coding for an epimerase and kfoC coding for chondroitin polymerase leading to the production of chondroitin in S. equi subsp. zooepidemicus

Fig. 2

SEC-TDA chromatogram of intracellular chondroitin purified by ethanol precipitation, anion exchange and desalting chromatographies from S. equi subsp. zooepidemicus-pNZ8148kfoAkfoC. RI signal (red), viscometer signal (blue), right angle light scattering (green) and low angle light scattering (black)

Fig. 3

¹H and DEPT-HSQC NMR (600 MHz, 298 K, D₂O, acetone as internal standard) spectra of chondroitin polysaccharide from S. equi subsp. zooepidemicus-pNZ8148kfoAkfoC [chemical structure and numbering of chondroitin is shown in the inset; in the parenthesis below signal attributions are indicated the ¹H and ¹³C (in italic) chemical shift values]

Fig. 4

Average Mw and Mw/Mn obtained for HA and BC produced during batch fermentations on 3 L bioreactors