4-Methylproline Guided Natural Product Discovery: Co-Occurrence of 4-Hydroxy- and 4-Methylprolines in Nostoweipeptins and Nostopeptolides

4-methylproline (4-mPro) is a rare nonproteinogenic amino acid produced by cyanobacteria through the action of a zinc-dependent long-chain dehydrogenase and a Delta(1)-pyrroline-5-carboxylic acid (P5C) reductase homologue. Here, we used the presence of 4-mPro biosynthetic genes to discover new bioactive compounds from cyanobacteria. Eight biosynthetic gene clusters containing the 4-mPro biosynthetic genes nosE and nosF were found from publicly available cyanobacteria genomes, showing that 4-mPro is a good marker to discover previously unknown nonribosomal peptides. A combination of polymerase chain reaction (PCR) and liquid chromatographymass spectroscopy (LC-MS) methods was used to screen 116 cyanobacteria strains from 8 genera. The 4-mPro biosynthetic genes were detected in 30 of the 116 cyanobacteria strains, 12 which were confirmed to produce 4-mPro by amino acid analysis. Species from the genus Nostoc were responsible for 80% of the positive results. Altogether, 11 new nonribosomal cyclic peptides, nostoweipeptin W1W7 and nostopeptolide L1L4, were identified from Nostoc sp. XPORK 5A and Nostoc sp. UK2aImI, respectively, and their chemical structure was elucidated. Interestingly, screening with 4-mPro genes resulted in the detection of peptides that do not contain just one 4-mPro but also 4-hydroxylproline (nostopeptolides) and, in case of nostoweipeptins, two 4-mPros and two 4-hydroxyprolines. Peptides from both groups inhibit microcystin-induced apoptosis of hepatocytes HEK293. The cell experiments indicated that these cyclic peptides inhibit the uptake of microcystin by blocking the organic anion-transporters OATP1B1/B3. This study enriches the drug library of microcystin antitoxin.