Abstract
Hyaluronic acid (HA) has many applications in the pharmaceutical and cosmetic industries. Streptococcus zooepidemicus is naturally producing HA with high yield. There have been many attempts to produce HA in GRS strains but none have been able to produce HA as much as S. zooepidemicus. This study was undertaken to prove the dependence of S. zooepidemicus life on Glutamate produced by Glutamine fructose 6-phosphate transaminase (GFAT) related to the HA pathway. The presence of Glutamate synthase (GOGAT) in S. zooepidemicus was investigated by in-silico, genome annotation, and enzymatic assay methods, respectively. Recombinant expression of GOGAT was investigated. The deletion of GFAT was done by the homologous recombination method. Recombinant expression of GOGAT and deletion of GFAT were performed simultaneously. In these last three steps, growth rate and HA production were determined. Not only the absence of GOGAT but also the presence of GFAT as the only enzyme-producing glutamate was observed. Recombinant expression of GOGAT led to alack of HA production along with any change in the bacterial growth rate. A lack of bacterial growth in the glutamate-free medium was observed. Also, no HA production was seen in the glutamate-supplemented medium. The almost natural growth rate of bacteria, as well as no HA production, was shown. Overall, the S.zooepidemicus genome lacks GOGAT leading to a natural driving force to increase HA production. Knocking out of GOGAT in recombinant HA-producing strains like those coli and B. subtilis aimed to increase HA production would be welcomed.