Tailored molecular weight hyaluronic acid production by engineered Lactococcus lactis

Hyaluronic acid (HA) is a glycosaminoglycan with a wide range of biological functions that depend on its molecular weight (MW). Recently, there has been an increasing interest in producing HA at particular MWs for various cosmetic and biomedical applications. HA is traditionally produced by extraction or microbial fermentation, which is then subjected to chemical or enzymatic treatments to customize the MW. On the other hand, direct microbial synthesis at desired MWs has considerable advantages over conventional techniques. The present study introduces a combinatorial approach using four critical variables which influence the molecular weight of HA (MWHA): (1) Expression of HA synthases from different Streptococcus species (S. parauberis, S. uberis, S. zooepidemicus, and S. pyogenes) which intrinsically produce different MWHA; (2) Supply of HA precursors by varying heterlogous gene expression in the HA-precursor pathways (hasAB vs. hasABE); (3) Re-routing of metabolic fluxes by deletion of the lactate dehydrogenase (ldh) gene; and (4) Varying the initial glucose concentration in batch fermentation. Recombinant Lactococcus lactis strains expressing HA synthase genes taken from diverse Streptococcal sp. were found to produce varying MWHA under otherwise identical genetic and bioreactor conditions. The HA synthases sourced from S. uberis and S. parauberis synthesized higher MWHA, whereas those from S. pyogenes produced lower MWHA. In silico analysis of the HA synthase sequences indicated that differences in the transmembrane regions among the various isoforms are the probable cause of variations in MWHA. Compared to their wild-type counterparts, ldh-knockout L. lactis strains showed a noticeable increase in MWHA due to a substantial increase in HA precursor levels. Further, the co-expression of hasE in addition to hasAB, considerably increased MWHA due to a better balance of the intracellular HA-precursor ratios. This multiplexing approach, involving simultaneous manipulation of the above factors, allowed us to produce HA with tailored MWHA over a broad range from 0.2 to 2.6 MDa. Our technology eliminates the need for enzymatic desizing or post-processing of HA to achieve the desired MWHA. In summary, this multiplexing approach enables one-pot synthesis of desired MWHA, opening up new avenues for producing customized HA.