Assembly and Absolute Configuration of Short‐Lived Polyketides from Burkholderia thailandensis

Abstract

Complex polyketides from bacteria comprise a broad range of macrolides, polyenes, and polyethers, many of which play eminent roles for the development of therapeutics. Typically, these compounds feature multiple chiral centers that render them challenging benchmarks for organic synthesis. In light of this, it is impressive to learn how swiftly complex polyketides are assembled in microorganisms, utilizing only simple building blocks such as activated acyl and malonyl units. The types, number, and processing of these basic blocks are programmed by the architecture of modular polyketide synthases (PKS) which are encoded in the microbial genomes. One most remarkable discovery was that the order of catalytic domains involved in chain elongation and side-chain processing is typically colinear with the final product. This principle has not only opened the door to rationally engineering polyketides, but also enabled forecasting PKS architectures from polyketide structures and vice versa. However, through recent full genome sequencing projects it has become more and more obvious that most of the genes coding for biosynthetic assembly lines remain silent under standard laboratory conditions. Furthermore, PKS expression and polyketide formation may only occur transiently, thus suggesting that the metabolites function as temporary signals. This remarkable case has been observed for Burkholderia thailandensis, a bacterium that serves as a model for the pathogenic relatives B. mallei and B. pseudomallei. Mining the genome of B. thailandensis revealed a giant trans-acyltransferase (trans-AT) PKS gene cluster, and a bioinformatic structure prediction aided in the discovery of a hitherto unknown polyketide, thailandamide A (1, Scheme 1A). However, the absolute configuration of the labile polyene 1 and the structure of a short-lived congener (thailandamide B; 2 ; see Figure 2 for structure), have remained elusive. Yet this very structural information would serve as a stepping-stone to clarify the biological function of this enigmatic, quorum-sensing-regulated metabolite. Herein we report a multidisciplinary approach involving bioinformatics, chemical degradation, and precursor-directed biosynthesis to rigorously determine the absolute configuration of thailandamide A. On the basis of the stereochemical assignments, in silico predictions, and mutational pathway dissection, we also unveil the structure of the immediate, thermolabile PKS product thailandamide B. Thailandamide features six chiral centers, at C2, C3, C13, C22, C24, and C29. We initiated the stereochemical analysis by chemical degradation and derivatization. For the isolation of preparative amounts of thailandamide A (1) we employed a genetically engineered strain, the quorum sensing mutant B. thailandensis DPthaA, which produces thailandamide constitutively. From an upscaled fermentation (10 L) of the mutant we succeeded in isolating sufficient amounts of Scheme 1. Structure of thailandamide A (1) and experiments revealing the absolute configuration of 1. A) Degradation and derivatization. a) O3, 78 8C; b) 35% H2O2, AcOH, 60 8C; c) 6m HCl, 105 8C; d) S-PGME, PyBOP; e) l-FDAA; f) (S,R)-PGME, PyBOP. B) Synthesis of deoxythailandamide. g) B. thailandensis DPthaA, precursor-directed biosynthesis; h) (S,R)-MTPA chloride, pyridine. B. Dd values in ppm.