Production of high-molecular weight hyaluronic acid (HA) is a significant challenge in metabolically-engineered bacterial systems. Hyaluronic acid is a linear polymer made up of alternating units of β-(1-4)-glucuronic acid and β-(1-3)-N-acetylglucosamine, whose precursors are synthesized in parallel branches of the HA biosynthetic pathway. This study investigates the effect of metabolic flux distribution and redox balance on HA yield and molecular weight obtained from metabolically-engineered Lactococcus lactis cultures. The hyaluronic acid biosynthetic pathway was engineered in L. lactis using a combination of genes obtained from the has-operon of Streptococcus zooepidemicus. It was observed that an optimal combination of three has-genes is required for substantial HA production. Two different sets of heterologous has-genes (hasABC and hasABD) were expressed in nisin-inducible recombinant strain L. lactis NZ9000 (Prasad et al. 2010, 2012). This allowed for independent variation of fluxes in the two precursor pathways. The yield and molecular weight of HA obtained from L. lactis NZ9000 cultures was lower than HA produced by S. zooepidemicus cultures. It was observed that HA yield from glucose substrate correlates inversely with lactate yield and HA molecular weight obtained varies inversely with glucose uptake rate (Sanghe et al., 2011). Hence, the same combination of has-genes were expressed in ldh-mutant strain L. lactis NZ9020 (Bongers et al.,
2003) procured from NIZO (Netherlands) in order to study the effect of flux diversion from lactate production to HA and other metabolites. A dramatic improvement in HA yield and molecular weight was observed in recombinant cultures of L. lactis NZ9020 (ldh-mutant) compared to recombinant L. lactis NZ9000 cultures under similar conditions. The L. lactis NZ9020 cultures also produced substantial levels of ethanol, acetoin and formate, which were not observed in L. lactis NZ9000 cultures. The production of these metabolites also resulted in a significant shift in the redox balance, which may contribute to enhancement of fluxes in the biosynthetic pathway of the HA precursors, viz. UDP-glucuronic acid and UDP-N- acetylglucosamine. This study will investigate the effect of flux diversion from lactate towards formation of more reduced metabolites and the concomitant effect of flux variations in HA pathway. The study will also correlate the effect of HA-precursor concentrations on the
molecular weight of HA produced. Finally, we will investigate the effect of NAD+/NADH ratios
on metabolic flux regulation in the pathways leading to HA production and other metabolites in the engineered L. lactis cultures. The insights obtained from these studies will be used to further
re-engineer the metabolic pathways as well as to develop bioprocess strategies for high-MW HA
production by recombinant L. lactis cultures.

References:

Bongers R.S., Hoefnagel M.H.N., Starrenburg M.J.C., Siemerink M.A.J., Arends J.G.A., Hugenholtz J., Kleerebezem M. (2003) â??IS981-Mediated Adaptive Evolution Recovers Lactate Production by ldhB Transcription Activation in a Lactate Dehydrogenase-Deficient Strain of Lactococcus lactisâ?, Journal of Bacteriology, 185, 4499-4507
Prasad S.B., Jayaraman G., Ramachandran K.B. (2010) â??Hyaluronic acid production is enhanced by the additional co-expression of UDP-glucose pyrophosphorylase in Lactococcus lactisâ?, Appl Microbiol Biotechnol, 86, 273â??283
Prasad S.B., Ramachandran K.B., Jayaraman G. (2012) â??Transcription analysis of hyaluronan biosynthesis genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactisâ?, Applied Microbiology and Biotechnology, 94, 1593-1607

Sanghe N., Prasad S.B., Ramachandran K.B., Jayaraman G. (2011), â??Hyaluronic acid production in metabolically engineered Lactococcus lactis NZ9000: Bioreactor studies and flux balance analysisâ? (Oral Presentation), 1st European Congress of Applied Biotechnology, Sept 25-29,

2011, Berlin, Germany