Shotgun Screening for Investigating the Effective Factors for Producing Heparosan

             Glycosaminoglycans, linear mucopolysaccharides, such as hyaluronate, chondroitin sulfate, and heparosan, have become a focus of research as useful compounds for producing cosmetics and pharmaceuticals. Heparosan produced by Escherichia coli K5 strain, a pathogenic bacteria,  as a capsular polysaccharide has been reported to be converted into a sulfated polysaccharide with anti-inflammatory activity. The biosynthesis and efflux system of heparosan in E. coli have been elucidated; however, there is little information about factors that effectively increase heparosan production. In the present study, we developed a heparosan producer strain from non-pathogenic bacteria, and isolated novel factors for increasing heparosan production.

              Heparosan production was studied by introducing the capsule biosynthetic genes of the E. coli K5 strain (called Region 2) into E. coli B strain. However, heparosan accumulation in the B strain with introduced region 2 was lower than that in the wild-type E. coli K5 strain. Enhanced expression of the rfaH gene, an activator of heparosan efflux, effectively increased heparosan production. However, little is known about other factors. Therefore, the purpose of the present study was to investigate possible genes for an increased production of heparosan using shotgun cloning.

              The study was designed as follows. E. coli BL21 (DE3), with introduced Region 2 and enhanced rfaH gene expression, was used as the recipient strain. The fragments of E. coli K5 genomic DNA (~4 kb) were randomly ligated into the pSTV28 vector and transformed into the recipient strain. We performed screening for novel factors increasing heparosan production using a shotgun cloning approach with the entire genome of the E. coli K5 strain. Transformants with increased heparosan production were selected, and the introduced genes were evaluated.

              Using this screening approach, 30 genes involved in the carbohydrate metabolism, efflux pump, and stress tolerance were identified from the library of 1,698 clones. Possible functions of the genes and mechanisms of the increase of heparosan production will be discussed.