An In Vitro and In Vivo Study of Broad-Range Phosphopantetheinyl Transferases for Heterologous Expression of Cyanobacterial Natural Products.

Phosphopantetheinyl transferases catalyze the post-translational modification of carrier proteins involved in both primary and secondary metabolism. The functional expression of polyketide synthases and nonribosomal peptide synthetases requires the activation of all carrier protein domains by phosphopantetheinyl transferases. Here we describe the characterization of five bacterial phosphopantetheinyl transferases by their substrate specificity and catalytic efficiency of four cyanobacterial carrier proteins. Comparative in vitro phosphopantetheinylation analysis showed Sfp possesses the highest catalytic efficiency over various carrier proteins. In vivo coexpression of phosphopantetheinyl transferases with carrier proteins revealed a broad range substrate specificity of phosphopantetheinyl transferases; all studied phosphopantetheinyl transferases were capable of converting apo- carrier proteins, sourced from diverse biosynthetic enzymes, to their active holo form. Phosphopantetheinyl transferase coexpression with the hybrid nonribosomal peptide synthetases/polyketide synthases responsible for microcystin biosynthesis confirmed that the higher in vitro activity of Sfp translated in vivo to a higher yield of production.