Hyaluronic acid production is enhanced by the additional co-expression of UDP-glucose pyrophosphorylase in Lactococcus lactis

Abstract

Hyaluronic acid (HA) production was metabolically engineered in Lactococcus lactis by introducing the HA synthetic machinery from the has operon of the pathogenic bacterium Streptococcus zooepidemicus. This study shows that the insertion of uridine diphosphate (UDP)-glucose pyrophosphorylase (hasC) gene in addition to the HA synthase (hasA) and UDP-glucose dehydrogenase (hasB) genes has a significant impact on increasing HA production. The recombinant L. lactis NZ9000 strain transformed with the plasmid pSJR2 (co-expressing hasA and hasB genes only) produced a maximum of 107 mg/l HA in static flask experiments with varying initial glucose concentrations, while the corresponding experiments with the transformant SJR3 (co-expressing hasA, hasB, and hasC genes) gave a maximum yield of 234 mg/l HA. The plasmid cloned with the insertion of the full has operon comprising of five different genes (hasA, hasB, hasC, hasD, and hasE) exhibited structural instability. The HA yield was further enhanced in batch bioreactor experiments with controlled pH and aeration, and a maximum of 1.8 g/l HA was produced by the SJR3 culture.

Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor

“…As expected, the highest production of heparosan was reached by the co-expression of KfiA, KfiB, KfiC and KfiD. The similar phenomenon was also observed for the production of hyaluronic acid by recombinant Lactococcus lactis, where it was found that the insertion of uridine diphosphate-glucose pyrophosphorylase gene in addition to the HA synthase and UDP-glucose dehydrogenase genes can significantly increase HA production (Prasad et al, 2010).…”

Microbial production of hyaluronic acid: current state, challenges, and perspectives

“…The engineered strain produced 0.5 g/L HA in shaker flask and 2.0-3.8 g/L HA in a fed-batch culture process in a 1-L bioreactor [45]. L. lactis was engineered by introducing the HA synthetic machinery from the has operon of S. zooepidemicus , and it was found that the insertion of uridine diphosphate-glucose pyrophosphorylase ( hasC ) gene in addition to the HA synthase ( hasA ) and UDP-glucose dehydrogenase ( has B ) genes can significantly increase HA production [46]. The recombinant L. lactis NZ9000 strain transformed with the plasmid pSJR3 (co-expressing hasA , hasB , and hasC genes) gave a maximum of 1.8 g/L HA in a 2.4-L batch bioreactor [46].…”

“…L. lactis was engineered by introducing the HA synthetic machinery from the has operon of S. zooepidemicus , and it was found that the insertion of uridine diphosphate-glucose pyrophosphorylase ( hasC ) gene in addition to the HA synthase ( hasA ) and UDP-glucose dehydrogenase ( has B ) genes can significantly increase HA production [46]. The recombinant L. lactis NZ9000 strain transformed with the plasmid pSJR3 (co-expressing hasA , hasB , and hasC genes) gave a maximum of 1.8 g/L HA in a 2.4-L batch bioreactor [46]. The hasA gene from S. zooepidemicus was expressed in B. subtilis for the production of HA, and it was found that the production of UDP-glucuronic acid is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene is sufficient for high-level production of HA in B. subtilis [8].…”

Bacterial Polysaccharides: Production and Applications in Cosmetic Industry

“…Izawa et al (2010) reported that Streptococcus thermophilus YIT 2084, a GRAS strain isolated from a dairy food product, was able to produce HA from milk, although at a concentration lower than that achieved by S. equi. Recently, great effort has been put on the development of heterologous systems by transforming bacterial species that normally do not produce HA (e.g., Escherichia coli, Lactococcus lactis, or Bacillus subtilis) into HA producers (Mao et al 2009;Prasad et al 2010;Yamada and Kawasaki 2005;Yu and Stephanopoulos 2008;Widner et al 2005). However, there are restrictions to the use of recombinant strains in some countries and the production achieved by heterologous HA-producer systems, as well as the polymer's molecular weight, was considerably lower than those obtained by wild-type streptococcal fermentation.…”