Engineered Biosynthesis of Nonribosomal Lipopeptides with Modified Fatty Acid Side Chains

Abstract

The biological properties of the calcium-dependent antibiotics (CDAs), daptomycin and related nonribosomal lipopeptides, depend to a large extent on the nature of the N-terminal fatty acid moiety. It is suggested that the chain length of the unusually short (C6) 2,3-epoxyhexanoyl fatty acid moiety of CDA is determined by the specificity of the KAS-II enzyme encoded by fabF3 in the CDA biosynthetic gene cluster. Indeed, deletion of the downstream gene hxcO results in three new lipopeptides, all of which possess hexanoyl side chains (hCDAs). This confirms that HxcO functions as a hexanoyl-CoA or -ACP oxidase. The absence of additional CDA products with longer fatty acid groups further suggests that the CDA lipid chain is biosynthesized on a single ACP and is then transferred directly from this ACP to the first CDA peptide synthetase (CdaPS1). Interestingly, the hexanoyl-containing CDAs retain antibiotic activity. To further modulate the biological properties of CDA by introducing alternative fatty acid groups, a mutasynthesis approach was developed. This involved mutating the key active site Ser residue of the CdaPS1, module 1 PCP domain to Ala, which prevents subsequent phosphopantetheinylation. In the absence of the natural module 1 PCP tethered intermediate, it is possible to effect incorporation of different N-acyl-L-serinyl N-acetylcysteamine (NAC) thioester analogues, leading to CDA products with pentanoyl as well as hexanoyl side chains.

Biosynthesis and biosynthetic engineering of calcium-dependent lipopeptide antibiotics

“…Subsequently, Kopp et al [14] were able to confirm that. HxcO and HcmO do catalyze desaturation and epoxidation of the hexanoyl-ACP precursor, which was in agreement with our earlier study [13]. Following this, we developed a mutasynthesis approach, for modifying the CDA lipid moiety.…”

“…In the case of the ∆hxcO mutant, we were able to isolate several new CDAs, which were shown by tan- dem MS experiments to be lipopeptides with hexanoyl fatty acid side chains (Fig. 7, hCDA3b, 4a, and 4b) [13]. In addition, we overproduced HxcO and HcmO and showed that these enzymes were unable to process a putative hexanoyl-CoA precursor.…”

“…In the absence of such a gene or enzyme, we resorted to classical labeling experiments to determine the stereochemical course of Trp dehydrogenation during CDA biosynthesis [11]. In these experiments, synthetic stereospecifically deuterated Trp precursors, including doubly labeled (2'S,3'R)-[3-2 H 1 , 13 C 1 ]-tryptophan 10, were fed to the Trp-auxotrophic strain of S. coelicolor WH101. The use of the auxotroph enabled the fate of the 2 H labels during CDA biosynthesis to be determined by MS and NMR.…”

“…5). hxcO and hcmO encode proteins that are similar to FAD-dependent oxidase and monooxygenase enzymes, respectively, which are predicted to catalyze the desaturation and epoxidation of the CDA hexanoyl fatty acid precursor [4,13]. To explore this, we deleted the hxcO and hcmO genes from the S. coelicolor chromosome.…”

“…This blocks phosphopantetheinylation of the PCP domain and the subsequent accumulation of intermediates on module 1 of the mutant NRPS (∆PCP1). In the absence of the natural intermediate it was possible to affect incorporation of different exogenous N-acyl-L-serinyl N-acetylcysteamine (NAC) analogs (16,17), in the ∆PCP1 strain leading to new lipopeptides with different fatty acid side chains [13]. In addition to generating hexanoyl-CDAs, we showed that feeding Npentanoyl-L-serinyl N-acetylcysteamine (16) gave rise to a new CDA with a pentanoyl side chain (pCDA4b).…”