Hyaluronan (HA) is a commercially valuable biopolymer with numerous biomedical applications. The molecular weight of HA (MWHA) determines its suitability for different applications. Commercial production of HA majorly depends on extraction from rooster comb and fermentation of the native HA producer, Streptococcus zooepidemicus, a known pathogen. However, these sources have risks of cross-viral and endotoxin contamination, respectively. A safer route for HA production is through microbial fermentation of recombinant GRAS (Generally Recognized As Safe) organisms. However, the titer and MWHA produced from these organisms are lower than the native producer. Literature reports are not sufficiently available to understand the factors influencing HA titer and MWHA and devise strategies to improve HA production or modulate MWHA in recombinant cultures.

In this work, Lactococcus lactis - a Gram-positive bacterium, has been chosen as a host for pathway engineering. This organism was chosen due to its GRAS status, sufficient genetic tools for gene manipulation, and fermentation ease. In this present study, the recombinant L. lactis GJP5 (has ABE) and L. lactis GJP6 (hasABpgmA) were constructed in L. lactis NZ9000 using nisin-inducible vector pNZ8148. By comparing with L. lactis GJP2 (has AB) strain, the effect of genes hasE and pgmA on HA production and MWHA was evaluated in two different process conditions- static and shake flask experiments. Overexpression of the genes hasE and pgmA improved HA titer and MWHA.

The effect of glucose concentration on MWHA during fermentation has not been studied. In this particular study, batch fermentation was carried out at two different initial glucose concentrations (10g/l and 30g/l) for three recombinants (GJP2, GJP5 and GJP6), and the MWHA profile was measured. All these strains confirmed the positive correlation of MWHA with initial glucose concentration. However, the MWHA decreases during batch fermentation from a peak value attained in the initial phase of the process. A glucostat strategy was successfully used to control glucose concentration and the MWHA. This strategy was validated to achieve constant MWHA across three recombinant strains and two different initial glucose concentrations. By manipulating a combination of genetic (coexpression of different HA biosynthetic genes) and process (glucose concentration), constant- MWHA was produced in a range of 0.4-1.4 MDa.

Aeration has been shown to improve HA titer in recombinants. As the respiratory capacity of the L. lactis can be induced by heme addition, this strategy can be exploited to enhance recombinant HA production. Respiration improves the intracellular level of two cofactors, acetyl-CoA and NAD+, critical in the biosynthesis of the HA precursors UDP-GlcNAc and UDP-GlcUA biosynthesis, respectively. This study reports a HA titer of 4.6 g/l in hemin-supplemented cultures, which is much higher than literature reports on HA production with recombinant L. lactis strains.
Publications:
1) P. Jeeva*, S. Shanmuga Doss*, V. Sundaram, G. Jayaraman (2019). Production of controlled molecular weight hyaluronic acid by glucostat strategy using recombinant Lactococcus lactis cultures. Appl Microbiol Biotechnol 103:4363–4375. DOI: 10.1007/s00253-019-09769-0.

2) S. Schulte, S. Shanmuga Doss, P. Jeeva, M. Ananth, G. Jayaraman and L.M. Blank (2019). Exploiting the diversity of streptococcal hyaluronan synthases for the production of molecular weight–tailored hyaluronan. Appl Microbiol Biotechnol 103:7567–7581. DOI: 10.1007/s00253-019-10023-w