Mutasynthesis of novel prodiginines derived from the antibiotic prodigiosin by exploiting substrate specificity of H2MAP oxidases PigB and HapB

Abstract

The natural product, prodigiosin, caught researchers’ interest more than a century ago because of its bright red color. Today, interest in this tripyrrolic secondary metabolite remains strong due to its biological effects, including potent antibiotic activity against various Gram-positive bacteria. Further exploration of the potential of this class of molecules requires libraries of analogues. Yet, the total synthesis of prodigiosin-like compounds (prodiginines) proves challenging. This can be overcome by highjacking the bacterial biosynthetic machinery via mutasynthesis. Although a number of different bacteria produce prodigiosin, its biosynthetic pathway is well conserved. The final precursors, MAP and MBC, are always produced independently, before a terminal condensation reaction. Our work focuses on the last step of the formation of MAP: the oxidation of H2MAP. We were able to isolate and characterize HapB, the enzyme catalyzing this reaction in Hahella chejuensis. In addition, we showed that some modifications of alkyl substituents on the C2 and C3 positions of H2MAP did not alter HapB activity significantly. We then fed these analogues of H2MAP into Serratia ΔpigD, a mutant of Serratia ATCC sp.39006 which does not produce any endogenous H2MAP. As expected from the in vitro testing, chain elongation past two carbons on the C2 position could not be accepted whereas all substrates with a modification on the C3 position restored pigmentation, leading to the formation of eight novel prodiginines.