Biosynthesis of Alterochromide Natural Products in Pseudoalteromonas piscicida JCM 20779

Abstract

The alterochromides are a family of structurally related lipopeptides (many of which are brominated) produced by several species of Pseudoalteromonas including Pseudoalteromonas piscicida, Pseudoalteromonas flavipulchra, and Pseudoalteromonas maricaloris. Previous investigations identified the alt locus in P. piscicida as the biosynthetic gene cluster responsible for production of alterochomides and particularly the gene product of altN as being responsible for the bromination. AltN corresponds to a flavin-dependent halogenase that appears to selectively generate brominated alterochromides. It is not, however, known the timing of the bromination within the biosynthesis of alterochromides. However, it was hypothesized that AltN would carry out the bromination reaction once coumaric acid has been covalently linked to an acyl carrier protein towards the beginning of the fatty acid synthesis. The objective of this thesis is to determine at which step of the biosynthesis procedure does AltN act to brominate the natural product moiety by conducting in vitro enzymatic assays to reconstruct the brominated natural product moiety. Enzymes native to the P. piscicida JCM 20779 biosynthetic pathway, AltA, AltC, and AltN were successfully heterologously expressed and purified. The carrier protein AltB, however, could not be successfully expressed. Accessory enzymes including the 4′-phosphopantetheinyl transferase Sfp, NAD(P)H-dependent FMN reductase SsuE, and NAD-dependent phosphite dehydrogenase PtdH were successfully heterologously expressed and purified. Enzymatic activities of AltA were examined using its native substrate L-tyrosine, and the kinetic parameters of AltA were determined using steady state kinetic experiments. Substrate promiscuity tests were further conducted, and results showed that AltA was highly selective towards L-tyrosine compared to L-, D-phenylalanine, L-, D-tryptophan, and L-, D- histidine.