title: Genetic and biochemical characterization of genes involved in hyaluronic acid synthesis in Streptococcus zooepidemicus.
abstract: The biosynthetic pathway for hyaluronic acid (HA) has been proposed; however, a thorough genetic and functional analysis is required to further elucidate the roles of genes involved in HA production. Previously, we developed a markerless gene-deletion system for Streptococcus zooepidemicus and confirmed that hasA is essential for HA synthesis. Here, we constructed a comprehensive set of deletion mutants and investigated the roles of ten additional predicted genes in the HA synthetic pathway. Phenotypic assays revealed that all ten genes play a role in cell growth and/or HA synthesis. As expected, the deletion of hasA or hasB abolished HA production with little effect on growth, while the deletion of genes that are also required for peptidoglycan biosynthesis (hasE, glmM, and glmS) significantly reduced cell growth and HA production. Either of the glmU homologues (hasD and gcaD) was sufficient for optimal growth and the mucoid phenotype, while no double mutant could be isolated. Of the two UDP-glucose pyrophosphorylase (UGPase) paralogues, the operon-encoded hasC1 was responsible for 65 % of the activity, while hasC2 was responsible for the remaining 35 %. The deletion of hasC1 had no effect on cell growth and caused only a moderate decrease in the UDP-glucose level and HA production. The deletion of both hasC1 and hasC2 resulted in a severe growth defect and negligible UDP-glucose accumulation, HA production, and pyrophosphorylase activity. Of the two phosphoglucomutase paralogues, pgm1 and pgm2, the former is responsible for around 10 % of activity, while the latter is responsible for 90 %. The deletion of pgm1 showed no apparent effect on HA synthesis and growth, while the deletion of pgm2 resulted in the abolishment of HA synthesis and a significantly slower growth. These results should guide the metabolic engineering of S. zooepidemicus to improve HA productivity and quality.
authors.affiliation: MOE Key Laboratory of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, 300457, Tianjin, China.|National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science & Technology, 300457, Tianjin, China.
authors.forename: Yali
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: MOE Key Laboratory of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, 300457, Tianjin, China.
authors.forename: Kailai
authors.initials: K
authors.lastname: Luo
authors.affiliation: MOE Key Laboratory of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, 300457, Tianjin, China.
authors.forename: Qiushuang
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: MOE Key Laboratory of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, 300457, Tianjin, China.
authors.forename: Zhengliang
authors.initials: Z
authors.lastname: Qi
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, 4072, Australia.
authors.forename: Lars Keld
authors.initials: LK
authors.lastname: Nielsen
authors.affiliation: MOE Key Laboratory of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, 300457, Tianjin, China. liuhao@tust.edu.cn.|National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science & Technology, 300457, Tianjin, China. liuhao@tust.edu.cn.
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D053898:Biosynthetic Pathways; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D009876:Operon; D018502:Streptococcus equi / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006820:Hyaluronic Acid
keywords: Gene function; Hyaluronic acid; Metabolic engineering; Streptococcus zooepidemicus