Biosynthesis of Hyaluronic acid polymer: Dissecting the role of sub structural elements of hyaluronan synthase.

Hyaluronic acid (HA) based biomaterials have several biomedical applications. HA biosynthesis is catalysed by hyaluronan synthase (HAS). The unavailability of 3-D structure of HAS and gaps in molecular understanding of HA biosynthesis process pose challenges in rational engineering of HAS to control HA molecular weight and titer. Using in-silico approaches integrated with mutation studies, we define a dictionary of sub-structural elements (SSE) of the Class I Streptococcal HAS (SeHAS) to guide rational engineering. Our study identifies 9 SSE in HAS and elucidates their role in substrate and polymer binding and polymer biosynthesis. Molecular modelling and docking assessment indicate a single binding site for two UDP-substrates implying conformationally-driven alternating substrate specificities for this class of enzymes. This is the first report hypothesizing the involvement of sites from SSE5 in polymer binding. Mutation at these sites influence HA production, indicating a tight coupling of polymer binding and synthase functions. Mutation studies show dispensable role of Lys-139 in substrate binding and a key role of Gln-248 and Thr-283 in HA biosynthesis. Based on the functional architecture in SeHAS, we propose a plausible three-step polymer extension model from its reducing end. Together, these results open new avenues for rational engineering of Class I HAS to study and regulate its functional properties and enhanced understanding of glycosyltransferases and processive enzymes.

Chromosomal integration of hyaluronic acid synthesis (has) genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis.

Microbial production of hyaluronic acid (HA) is an attractive substitute for extraction of this biopolymer from animal tissues. Natural producers such as Streptococcus zooepidemicus are potential pathogens; therefore, production of HA by recombinant bacteria that are generally recognized as safe (GRAS) organisms is a viable alternative that is being extensively explored. However, plasmid-based expression systems for HA production by recombinant bacteria have the inherent disadvantage of reduced productivity because of plasmid instability. To overcome this problem, the HA synthesis genes (hasA-hasB and hasA-hasB-hasC) from has-operon of S. zooepidemicus were integrated into the chromosome of Lactococcus lactis by site-directed, double-homologous recombination developing strains VRJ2AB and VRJ3ABC. The chromosomal integration stabilized the genes and obviated the instability observed in plasmid-expressed recombinant strains. The genome-integrated strains produced higher molecular weight (3.5-4 million Dalton [MDa]) HA compared to the plasmid-expressed strains (2 MDa). High molecular weight HA was produced when the intracellular concentration of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-glucuronic acid (UDP-GlcUA) was almost equal and hasA to hasB ratio was low. This work suggests an optimal approach to obtain high molecular weight HA in recombinant strains.

Transcription analysis of hyaluronan biosynthesis genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactis.

The has operon genes in the hyaluronan (HA) producer, Streptococcus zooepidemicus, encode for some of the critical enzymes in the HA biosynthetic pathway. Heterologous expression of different combinations of multiple has genes has resulted in increasing HA production to varying degrees in different recombinant strains. In this work, a recombinant Lactococcus lactis strain (SJR6) was constructed, with insertion of three has operon genes (hasABD) from S. zooepidemicus. The SJR6 strain was found to be a better HA producer than two previously constructed recombinant L. lactis strains (SJR2 and SJR3), containing hasAB and hasABC genes, respectively, but exhibited lower HA production than the native HA producer S. zooepidemicus. To understand the differences in HA yield between the various strains, transcriptions of the HA biosynthesis genes (has genes and their homologues) were compared at different phases of exponential growth of the L. lactis and S. zooepidemicus cultures. The mRNA levels of all the heterologous has genes were expectedly far higher than their corresponding homologues in the L. lactis strains. The relative mRNA level of the hasB-homologue, viz. ugd (encoding UDP-glucose dehydrogenase), was found to be much lower than that of other homologues, corroborating earlier reports which indicate tight transcriptional regulation of the ugd gene in L. lactis. Interestingly, all the has gene homologues were found to be up-regulated in all the recombinant L. lactis strains, when compared with the corresponding genes in the untransformed strain, L. lactis NZ9000. A transcription analysis of S. zooepidemicus cultures revealed that the has operon was down-regulated in the mid-exponential growth phase in comparison to the early- and late-exponential growth phases. The transcription analyses in this study have provided insights for the design of recombinant strains with higher HA productivity.

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

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.