title: Chromosomal integration of hyaluronic acid synthesis (has) genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis.
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.
authors.affiliation: Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, India.
authors.forename: Rothangmawi Victoria
authors.initials: RV
authors.lastname: Hmar
authors.forename: Shashi Bala
authors.initials: SB
authors.lastname: Prasad
authors.forename: Guhan
authors.initials: G
authors.lastname: Jayaraman
authors.forename: Kadathur B
authors.initials: KB
authors.lastname: Ramachandran
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D014453:Glucuronosyltransferase / Q000235:genetics* / Q000378:metabolism; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018502:Streptococcus equi / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006820:Hyaluronic Acid; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases
keywords: Genomic integration; Hyaluronic acid; Lactococcus lactis; Metabolic engineering; Molecular weight