Study And Application Of Phosphopantetheinyl Transferases From Industrial Streptomyces

Many secondary metabolites from Streptomyces have been widely used in the field of medicine, food, and agriculture because of their biological activities. Most of them are biosynthesized by polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). Phosphopantetheinyl transferases (PPTases) are essential to the activities of PKSs, NRPSs, fatty acid synthases (FASs) by converting acyl carrier proteins (ACPs) in PKSs, peptidyl carrier proteins (PCPs) in NRPSs, and ACPs in FASs from inactive apo-forms into active holo-forms. PPTases can be classified into group I PPTases, group II PPTases, and group III PPTases according to their structures.First we characterized the functions of PPTases from four bacterial. An industrial natamycin (NTM) producing strain, Streptomyces chattanoogensis L10, was identified to contain two PPTases. Their functions were characterized by both in vitro assay and in vivo assay. A strain with higher yield of NTM and faster production of NTM was also constructed by the engineering of phosphopantetheinylation network in S. chattanoogensis L10. An industrial tacrolimus (FK506) producing strain, S. tsukubaensis YN06, contains the most PPTases (five PPTases) among the genomic DNA sequenced species. In vitro characterization of these five PPTases showed that all of them had the phosphopantetheinylation activities to ACPs or PCPs; however, the substrate specificities of them to ACPs or PCPs were different. Unlike most bacterial contain at least one group I PPTase and at least one group II PPTase, Haemophilus influenza and Synechocystis sp. PCC6803 harbor merely one group II PPTases, Hppt and Sppt, respectively. In vitro characterization of Hppt and Sppt showed that both of them phosphopantetheinylated not only an ACP in a type I PKS involved in secondary metabolism, but also an ACP in a FAS involved in primary metabolism.Second we studied the structure-activity relationship of group II PPTases. Magnesium ion is essential to PPTase activity. Both in vitro and in vivo characterization of point mutations of SchPPT and Hppt indicated that the first and the third magnesium binding residues were essential to their activities; however, the second magnesium binding residues were important but not essential to their activities. Regarding the effect of N terminus and C terminus in group II PPTases to their substrate specificities, in vitro characterization of SchPPT suggested that its N terminus was essential to its activity; its C terminus probably controlled its substrate specificity.Finally we analyzed the evolution of group II PPTases. All known group II PPTases could be classified into three-magnesium-binding-residue-PPTase subgroup (containing Asp-Glu-Glu triad) and two-magnesium-binding-residue-PPTase subgroup (containing Asp-Xxx-Glu triad). The second residues in the above two triads were consverved in some species. Both phylogenetic tree and gene synteny analyses suggested that the animal group II PPTases may originate from one common ancestor; the plant two-magnesium-binding-residue-PPTases may originate from one common ancestor; the plant three-magnesium-binding-residue-PPTases may derive from horizontal gene transfer from prokaryotes.In summary, we characterized the functions of nine PPTases, studied the structure-activity relationship of group II PPTases, and evolutionary analyzed group II PPTases. Our results here not only expand our understanding to PPTases but also may help people to improve the activities and the substrate specificities of PPTases, engineer the phosphopantetheinylation network, and increase the yields of target natural products synthesized by PKSs and NRPSs.