A Priming Cassette Generates Hydroxylated Acyl Starter Units in Mupirocin and Thiomarinol Biosynthesis

Abstract

Mupirocin, a commercially available antibiotic produced by Pseudomonas fluorescens and thiomarinol, isolated from the marine bacterium Pseudoalteromonas sp. SANK 73390, both consist of a polyketide-derived monic acid homologue esterified with either 9-hydroxynonanoic acid (mupirocin, 9HN) or 8-hydroxyoctanoic acid (thiomarinol, 8HO). The mechanisms of formation of these deceptively simple 9HN and 8HO fatty acid moieties in mup and tml respectively remain unresolved. To define starter unit generation the purified mupirocin proteins MupQ, MupS and MacpD and their thiomarinol equivalents (TmlQ, TmlS and TacpD) have been expressed and shown to convert malonylcoenzyme A (CoA) and succinyl-CoA to 3-hydroxypropionoyl (3-HP) or 4-hydroxybutyryl (4-HB) fatty acid starter units respectively via the MupQ/TmlQ catalysed generation of an unusual bis-CoA/acyl carrier protein (ACP) thioester, followed by MupS/TmlS catalysed reduction. Mix and match experiments show MupQ/TmlQ to be highly selective for the correct CoA, MacpD/TacpD were interchangeable but an alternate transacting ACPs from the mupirocin pathway (MacpA/TacpA) or a heterologous ACP (BatA) were non-functional. MupS and TmlS selectivity was more varied and these reductases differed in their substrate and ACP selectivity. The solution structure of MacpD determined by NMR revealed a C-terminal extension with partial helical character that has been shown to be important for maintaining high titres of mupirocin. We generated a truncated MacpD construct, MacpD_T, which lacks this C-terminal extension but retains an ability to generate 3-HP with MupS and MupQ, suggesting further downstream roles in protein-protein interactions for this region of the ACP.

Food‐Poisoning Bacteria Employ a Citrate Synthase and a Type II NRPS To Synthesize Bolaamphiphilic Lipopeptide Antibiotics**

“…The activated tricarboxylic fatty acid would then be transferred onto the type II NRPS to form the acyl‐β‐Ala subunit by the second KS III. A similar cooperation of two KS III and an ATP‐dependent ligase has been implicated in the biosynthesis of unusual fatty acids in mupirocin and thiomarinol biosynthesis [20] . Finally, a desaturase would introduce the enamide double bond.…”

“…A similar cooperation of two KS III and an ATP-dependent ligase has been implicated in the biosynthesis of unusual fatty acids in mupirocin and thiomarinol biosynthesis. [20] Finally, a desaturase would introduce the enamide double bond. We confirmed this model by targeted gene inactivation of bolC, bolN and bolB (Figure 3 C).…”

“…In conclusion, by genome mining of infamous food-spoiling bacteria we discovered a new family of antibiotics that are an important addition to the metabolic inventory of diverse, ecology-and healthrelevant bacteria of the genus Burkholderia. [20] The bolagladins feature an unprecedented lipopeptide modification, a tricarboxylic fatty acid that creates a natural bolaamphiphile. We unveiled the key role of a citrate synthase in the bol pathway and provide an evolutionary model for the recruitment of a biocatalyst from the Krebs cycle.…”

Recent highlights of biosynthetic studies on marine natural products

“…The FAS starter units are generated by the class I adenylate forming enzyme TmlQ, the reductase TmlS and the ACP TacpD. 109 The NRPS produces holothin, which is incorporated into marinolic acid by the ligase TmlU and the acetyltransferase HolE, yielding thiomarinol A. 110 Work by Murphy and colleagues could finally show that the 8-hydroxyoctanoic acid side-chain originates from 4-hydroxy- Fig.…”

Food‐Poisoning Bacteria Employ a Citrate Synthase and a Type II NRPS To Synthesize Bolaamphiphilic Lipopeptide Antibiotics**

“…In conclusion, by genome mining of infamous food‐spoiling bacteria we discovered a new family of antibiotics that are an important addition to the metabolic inventory of diverse, ecology‐ and health‐relevant bacteria of the genus Burkholderia [20] . The bolagladins feature an unprecedented lipopeptide modification, a tricarboxylic fatty acid that creates a natural bolaamphiphile.…”