Abstract
Cyanobacteria produce structurally and functionally diverse polyketides, nonribosomal peptides and their hybrids. Sfp-type phosphopantetheinyl transferases (PPTases) are essential to the production of these compounds via functionalizing carrier proteins (CPs) of biosynthetic megaenzymes. However, cyanobacterial Sfp-type PPTases remain poorly characterized, posing a significant barrier to the exploitation of cyanobacteria for biotechnological and biomedical applications. Herein, we describe the detailed characterization of multiple cyanobacterial Sfp-type PPTases that were rationally selected. Biochemical characterization of these enzymes along with the prototypic enzyme Sfp from Bacillus subtilis demonstrated their varying specificities toward 11 recombinant CPs of different types of biosynthetic pathways from cyanobacterial and Streptomyces strains. Kinetic analysis further indicated that PPTases possess the higher binding affinity and catalytic efficiency toward their cognate CPs in comparison with noncognate substrates. Moreover, when chromosomally replacing the native PPTase gene of Synechocystis sp. PCC6803, two selected cyanobacterial PPTases and Sfp supported the growth of resulted mutants. Cell lysates of the cyanobacterial mutants further functionalized recombinant CP substrates. Collectively, these studies reveal the versatile catalysis of selected cyanobacterial PPTases and provide new tools to synthesize cyanobacterial natural products using in vitro and in vivo synthetic biology approaches.
Highlights
More than 1,000 structurally diverse natural products have been isolated from cyanobacterial species over the past decades[1,2]
Sfp-type phosphopantetheinyl transferases (PPTases) can further be subdivided into the NRP synthetases (NRPSs)-preferred F/ KES and PK synthases (PKSs)-favored W/KEA groups based on their sequences[22]
These results identify cyanobacterial PPTases with the catalytic proficiency and efficiency in activating carrier proteins (CPs) from diverse natural product biosynthetic pathways and lay a solid foundation to harness the biosynthetic potential of cyanobacteria via synthetic biology approaches
Summary
More than 1,000 structurally diverse natural products have been isolated from cyanobacterial species over the past decades[1,2]. Bioinformatic analysis of over 140 cyanobacterial genomes available in the NCBI database reveals at least three NRP synthetases (NRPSs) per genome on average, while some genomes contain more than forty natural product gene clusters[8,9] These results illustrate the untapped potential of cyanobacteria as a source of new chemicals. These results identify cyanobacterial PPTases with the catalytic proficiency and efficiency in activating CPs from diverse natural product biosynthetic pathways and lay a solid foundation to harness the biosynthetic potential of cyanobacteria via synthetic biology approaches
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