title: Construction of efficient Streptococcus zooepidemicus strains for hyaluoronic acid production based on identification of key genes involved in sucrose metabolism.
abstract: Biosynthesis of polysaccharide hyaluoronic acid (HA) by Streptococcus zooepidemicus is a carbon-intensive process. The carbon flux and factor(s) restricting HA yield were not well understood. Here, we investigated the function of genes involved in sucrose metabolism and identified targets limiting HA yield, which were exploited to construct efficient S. zooepidemicus strains for HA production. The sucrose uptake was addressed by deletion of scrA and scrB, which encodes sucrose-PTS permease and sucrose-6-phosphate hydrolase, respectively. We found that scrB was essential for the growth of S. zooepidemicus and HA biosynthesis, and accumulation of sucrose-6-phosphate was toxic. ΔscrB could not grow in THY-sucrose medium, while ΔscrA and ΔscrAΔscrB showed negligible growth defects. Overexpression of scrA significantly reduced biomass and HA production, while overexpression of scrB resulted in 26% increase of biomass and 30% increase of HA yield. We revealed that fructose-6-phosphate for HA biosynthesis mainly originates from glucose-6-phosphate. Deletion of scrK, a gene encoding hexokinase, led to 11% reduction of biomass and 12% decrease of HA yield, while deletion of hasE, a gene encoding phosphoglucoisomerase, resulted in the abolishment of HA biosynthesis and a significantly slow growth. We found that HA biosynthesis could be improved by directing carbon flux to fructose-6-phosphate. Deletion of fruA encoding the EII of fructose-PTS and fruK encoding phosphofructokinase showed no apparent effect on cell growth, but resulted in 22 and 27% increase of HA yield, respectively. Finally, a strain with 55% increase of HA was constructed by overexpression of scrB in ΔfruK. These results provide a solid foundation for further metabolic engineering of S. zooepidemicus for highly efficient HA production.
authors.affiliation: MOE Key Lab of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China.
authors.forename: Xuzhen
authors.initials: X
authors.lastname: Zhang
authors.affiliation: MOE Key Lab of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China.
authors.forename: Man
authors.initials: M
authors.lastname: Wang
authors.affiliation: MOE Key Lab of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China.
authors.forename: Tuanjie
authors.initials: T
authors.lastname: Li
authors.affiliation: MOE Key Lab of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Fu
authors.affiliation: MOE Key Lab of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Cao
authors.affiliation: MOE Key Lab of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China. liuhao@tust.edu.cn.
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
keywords: Hyaluoronic acid; Metabolic engineering; Streptococcus zooepidemicus