Hyaluronan Molecular Weight Is Controlled by UDP-N-acetylglucosamine Concentration in Streptococcus zooepidemicus

Abstract

The molecular weight of hyaluronan is important for its rheological and biological function. The molecular mechanisms underlying chain termination and hence molecular weight control remain poorly understood, not only for hyaluronan synthases but also for other ␤-polysaccharide synthases, e.g. cellulose, chitin, and 1,3-betaglucan synthases. In this work, we manipulated metabolite concentrations in the hyaluronan pathway by overexpressing the five genes of the hyaluronan synthesis operon in Streptococcus equi subsp. zooepidemicus. Overexpression of genes involved in UDP-glucuronic acid biosynthesis decreased molecular weight, whereas overexpression of genes involved in UDP-N-acetylglucosamine biosynthesis increased molecular weight. The highest molecular mass observed was at 3.4 ؎ 0.1 MDa twice that observed in the wild-type strain, 1.8 ؎ 0.1 MDa. The data indicate that (a) high molecular weight is achieved when an appropriate balance of UDP-N-acetylglucosamine and UDP-glucuronic acid is achieved, (b) UDP-N-acetylglucosamine exerts the dominant effect on molecular weight, and (c) the wild-type strain has suboptimal levels of UDP-Nacetylglucosamine. Consistent herewith molecular weight correlated strongly ( ‫؍‬ 0.84, p ‫؍‬ 3 ؋ 10 ؊5 ) with the concentration of UDP-N-acetylglucosamine. Data presented in this paper represent the first model for hyaluronan molecular weight control based on the concentration of activated sugar precursors. These results can be used to engineer strains producing high molecular weight hyaluronan and may provide insight into similar polymerization mechanisms in other polysaccharides.

Influence of competing metabolic processes on the molecular weight of hyaluronic acid synthesized by Streptococcus zooepidemicus

“…This is then activated by an uridyltransferase to form the HA precursor. The finding that UDP-N-acetylglucosamine is limiting the synthesis of high chain molecular weight has been shown in a recent study [26]. Studies have also claimed that the depletion of UDP-N-acetylglucosamine could be responsible for inhibiting HA synthesis process [42].…”

“…In a recent study on Lactococcus lactis housing the cloned szhasA and szhasB gene, an inverse relationship between the ratio of HasA enzyme and UDP-glucose concentration to the molecular weight of HA was observed [25]. A very recent study on HA molecular weight control in the native Streptococcus zooepidemicus has been reported, where it was observed that the unavailability of precursor UDP-Nacetylglucosamine, is the critical factor determining the molecular weight of the polymer [26]. However, it still remains to be seen if the various fermentation conditions yielding different molecular weight HA, presented in literature consistently explain this finding.…”

Heterologous Production of Hyaluronic Acid in an ε-Poly- l -Lysine Producer, Streptomyces albulus

“…Nielsen and coworkers reported that overexpression of genes involved in the precursor sugar pathway led to the production of high-molecular-weight HA (11). In addition, a fermentation study has demonstrated that enhanced ATP levels brought by changing fermentation parameters enhanced the production and improved molecular weight of HA, probably by increased fluxes of the sugar precursor pathways (12).…”

Temporal Dynamics of the Saccharopolyspora erythraea Phosphoproteome

“…The cell dry weight was quantified by filtering 15 ml of broth using 22-m nylon membranes (Millipore, Bedford, MA) and drying the samples at 60°C for 8 h. The concentration of glucose was determined via high-performance liquid chromatography (Agilent 1200 HPLC system) (as described in Ref. 34) using a Phenomenex Rezex RHM Monosaccharide H ϩ column (300 ϫ 7.8 mm; 8 m) and detected by Refractive index. Erythromycin production was determined via LC-MS using a Dionex UltiMate 3000 liquid chromatography system (Dionex, Sunnyvale, CA) coupled with an AB Sciex 4000 QTRAP mass spectrometer (AB Sciex, Ontario, Canada) as described in Ref.…”