Metabolic engineering of Lactococcus lactis to produce medical-grade Hyaluronic aci
Hyaluronic acid (HA) is vital for normal growth and development of vertebrate which makes it as a commercially valuable polysaccharide in pharmaceutical and cosmetics industries. HA is synthesized by polymerization of two nucleotide sugars UDP-glucuronic acid and UDP-N-acetylglucosamine by the enzyme HA synthase (hasA). Two major genes required for the formation of HA monomers are hasB (UDP-glucose dehydrogenase) and hasD (glucosamine--phosphate acetyl transferase) respectively. Streptococcus zooepidemicus, the native HA producer contains has-operon genes (has ABCDE) which code for the critical enzymes of the HA biosynthetic pathway. Streptococcal species are being used for industrial scale production of HA, however, the presence of endotoxins and pathogenic factors renders it unsuitable for medical purposes. Hence, this demands for metabolic engineering of Generally Recognized As Safe (GRAS) microbes to produce HA. The production of HA thus requires a strain that has been proved safe, and at the same time capable of supporting the expression of genes in the HA pathway. Lactococcus lactis is found to fulfill these requirements as it has GRAS status and belongs to the same bacterial family as Streptococcus, sharing common metabolic pathways. The genome sequence analysis of L. lactis revealed presence of all the homologous genes present in HA pathway, except hyaluronan synthase gene (hasA). Increased HA production through heterologous expression of different combinations of has-genes has been reported in different recombinant microorganisms. However, the productivity and molecular weight of HA are influenced by the metabolic fluxes (precursor availability and proportions) in the two branches of the HA biosynthetic pathway. Thus, HA yield and molecular weight can be improved by enhancing the metabolic fluxes in the HA pathways and by achieving equimolar ratio of HA precursors. Our research aim at metabolic engineering of L.lactis for medical-grade HA production. We have engineered L.lactis for HA production by cloning HA pathway genes from Streptococcus in L.lactis. Over expression of HA pathway genes will increase the flux towards HA production.
Abstract: 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.
Pub.: 22 Jul '14, Pinned: 31 Jul '17
Abstract: The potential advantages of hyaluronic acid (HA) production by metabolically-engineered Lactococcus lactis is constrained by the lower molecular weight and yield of HA obtained in these strains, compared to natural producers. Earlier studies have correlated lower HA yield with excessive lactate production in L. lactis cultures ( Chauhan et al., 2014). In the present study, a three-fold increase was observed in the amount as well as molecular weight of HA produced by recombinant ldh-mutant L. lactis strains. The diversion from lactate production in the ldh-mutant strains resulted in excess ethanol and acetoin production and higher NAD+/NADH ratio in these cultures. The initial NAD+/NADH ratio showed a positive correlation with HA molecular weight as well as with the HA-precursor ratio (UDP-GlcUA/UDP-GlcNAc). The influence of NAD+/NADH ratio on regulation of the concerned metabolic pathways was assessed by transcriptional analysis of key genes having putative binding sites of the NADH-binding transcriptional factor, Rex.
Pub.: 22 Jan '16, Pinned: 31 Jul '17
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.
Pub.: 29 Oct '09, Pinned: 31 Jul '17