A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units

Abstract

Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of a, b-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemicallysynthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual b-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.

Structural and functional comparison of Saccharomonospora azurea strains in terms of primycin producing ability

“…Nevertheless, these efforts revealed the presence of unusual butlymalonyl-CoA, pentlymalonyl-CoA or hexylmalonyl-CoA substrate specificity in module 18. Similar AT domain characteristics were reported in case of stambomycin, thailandi, neoansamycin, antimycin and cinnabaramide biosynthesis (Greule et al 2016;Li et al 2015;Rachid et al 2011;Ray et al 2016). These findings were further supported by R 2 side chain variability of primycin molecules (Fig.…”

Searching for Glycosylated Natural Products in Actinomycetes and Identification of Novel Macrolactams and Angucyclines

“…In fact, all the PKSs encoded by cluster 21 of CS227 (except Orf20 and Orf21) and those of the stambomycin cluster of Streptomyces ambofaciens ATCC23877 are very similar both in amino acid sequence (percentage of identity ranging between 55 and 80; Supplementary Table 9 ) and domain architecture (they share the same predicted modules and domains) The main differences concerning PKSs of both clusters relay in PKS Orf20 that shares the first two modules with SAMR466 but has four additional modules and in PKS Orf21, composed by four modules, that has not an ortholog in the stambomycin biosynthesis cluster. Furthermore, acyltransferase domain analysis using antiSMASH and PRISM software ( Skinnider et al, 2016 ) predicts that 23 × malonyl-CoA, 9 × methylmalonyl-CoA and one ethylmalonyl-CoA units might participate in the biosynthesis of the final compound in CS227, whilst in stambomycin biosynthesis the PKSs introduce 16 × malonyl-CoA, 8 × methylmalonyl-CoA and one unusual alkylmalonyl-CoA unit ( Laureti et al, 2011 ; Ray et al, 2016 ). In addition, the proposed functions for the enzymes involved in the biosynthesis of the sugar attached to the polyketide deduced from cluster 21 of CS227 indicates that it might be a 3-amino-2,6-DOH, in contrast to the 3-amino-6-DOH β-mycaminose of stambomycins.…”

Acyltransferases as Tools for Polyketide Synthase Engineering

“…An alternative strategy to the construction of modified biocatalysts for the provision of unusual polyketide precursors is the screening and identification of independent pathways or novel enzymes, directly delivering the exotic substrates. In 2016 Ray et al described a CCRC (crotonyl-CoA reductase/carboxylase)-independent mechanism for assembly of unusual PKS precursors, where an acyl-CoA carboxylase (YCC, biotin-dependent enzyme) directly carboxylates medium chain acyl-CoA thioesters to alkylmalonyl-CoA [ 98 ].…”

“…Researchers aiming at AT-based PKS engineering will most probably continue to use the established and approved strategies and methodology. Those include in general, the expanding of the pool of precursors [ 97 , 98 , 190 ] for the ATs, the modification of the AT enzymes under the consideration of the obtained structural and mechanistic insights, AT-domain swapping and cross-complementation of inactivated ATs with ATs from other modules/pathways, and site-directed mutagenesis to exchange the substrate specificity of the AT. Specifically, the information existing in databases [ 191 , 192 ] is incredibly helpful, not only for direct sequence alignments and phylogenetic analysis [ 193 ], but also for the development of advanced bioinformatics tools supporting the AT-targeted PKS engineering [ 194 ].…”