Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis

Abstract

Type I polyketide synthases (PKSs) are multifunctional enzymes that are organized into modules, each of which minimally contains a beta-ketoacyl synthase, an acyltransferase (AT), and an acyl carrier protein. Here we report that the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 consists of two PKS genes, lnmI and lnmJ, that encode six PKS modules, none of which contain the cognate AT domain. The only AT activity identified within the lnm gene cluster is a discrete AT protein encoded by lnmG. Inactivation of lnmG, lnmI, or lnmJ in vivo abolished LNM biosynthesis. Biochemical characterization of LnmG in vitro showed that it efficiently and specifically loaded malonyl CoA to all six PKS modules. These findings unveiled a previously unknown PKS architecture that is characterized by a discrete, iteratively acting AT protein that loads the extender units in trans to "AT-less" multifunctional type I PKS proteins for polyketide biosynthesis. This PKS structure provides opportunities for PKS engineering as exemplified by overexpressing lnmG to improve LNM production.

Figure 1

Hypothesis for LNM (1) biosynthesis and modular organization of the Lnm hybrid NRPS–PKS megasynthetase with the discrete LnmG AT enzyme loading the malonyl CoA extender to all six PKS modules. The structures in brackets are hypothetical. It is not known whether one or both ACPs in module 6 are loaded with the malonyl group in vivo, although LnmG prefers ACP6-2 in vitro. Cy, condensation/cyclization; DH, dehydratase; MT, methyl transferase; Ox, oxidation; ?, domain of unknown function; TE, thioesterase.

Figure 2

HPLC analysis of LNM production by S. atroolivaceus wild-type and recombinant strains. (A) LNM standard. (B) S-140. (C) SB3002 (ΔlnmI). (D) SB3003 (ΔlnmJ). (E) SB3004 (ΔlnmG). (F) SB3005 (SB3004 harboring the lnmG overexpression plasmid of pBS3032). (G) SB3006 (SB3004 harboring the lnmG overexpression plasmid of pBS3033). ♦, LNM; ▿, an unknown metabolite with production that is independent to LNM biosynthesis.

Figure 3

In vitro assays of LnmG-catalyzed loading of malonyl CoA to individual LnmIJ PKS ACP domains and the LnmJ-(DH-ACP-KR) tridomain protein. (A) Purified LnmG on 4–15% SDS/PAGE. Lane 1, molecular mass standards; lane 2, LnmG. (B) Purified LnmIJ ACPs and LnmP on 4–15% SDS/PAGE. Lane 1, molecular mass standards; lane 2, ACP3; lane 3, ACP4; lane 4, ACP5; lane 5, ACP6-1; lane 6, ACP6-2; lane 7, ACP7; lane 8, ACP8; lane 9, LnmP. The numbers after the ACPs refer to the PKS modules from which they are derived with 6-1 and 6-2 to indicate the first and second ACPs, respectively, for PKS module 6. (C) Incubation of holo-ACPs or PCP with [2-¹⁴C]malonyl CoA and LnmG as visualized on 4–15% SDS/PAGE (I) and by phosphorimaging (II). Lane 1, molecular mass standards; lanes 2–8; ACP3–ACP8; lane 9, LnmP. (D) Time course of LnmG-catalyzed loading of [2-¹⁴C]malonyl CoA to ACP3 as visualized on 4–15% SDS/PAGE (I) and by phosphorimaging (II). (E) HPLC analysis of LnmG-catalyzed loading of malonyl CoA to ACP3. I, a negative control in the absence of Svp; II, a negative control in the absence of LnmG; III, complete assay. ●, apo-ACP3; ▿, holo-ACP3; ♦, malonyl-S-ACP3. (F) Purified LnmJ-(DH-ACP-KR) on 9% SDS/PAGE. Lane 1, molecular mass standards; lane 2, LnmJ-(DH-ACP-KR). (G) Incubation of holo-LnmJ-(DH-ACP-KR) with [2-¹⁴C]malonyl CoA and LnmG as visualized on 9% SDS/PAGE (I) and by phosphorimaging (II). Lane 1, molecular mass standards; lane 2, a negative control in the absence of Svp; lanes 3–6, complete assay with incubation times of 2, 5, 15, and 60 min, respectively.

Figure 4

Phylogenetic analysis of LnmG AT and its homologs from other AT-less type I PKS clusters and their relationships to cognate ATs from type I PKS clusters. The LnmG AT and its homologs fall into distinct groups that differ from cognate ATs of type I PKSs. Multiple sequence alignment and phylogenetic analysis were performed by the GCG program. We predict that PksC/PksD/PksE-AT [for the unknown polyketide in Bacillus subtilis (33)], PedC/PedD [for pederin in a bacterial symbiont of Paederus beetles (30)], MmpIII-AT1/AT2 [for mupirocin in Pseudomonas fluorescens (GenBank accession no. AF318063)], FenF [for mycosubtilin in B. subtilis ATCC6633 (31)], and Mx-TaK [for Ta1 in Myxococcus xanthus (32)], acting in a mechanistic analogy to LnmG, load the malonyl CoA extender unit onto the AT-less PKS modules in trans for polyketide biosynthesis in these clusters. For cognate ATs from rifamycin (Rif), rapamycin (Rap), erythromycin (Ery), and epothilone (Epo) clusters, protein GenBank accession numbers are given after the protein names: Rif-AT4 and Rif-AT2, AF04570; Rap-AT1 and Rap-AT2, X86780; Ery-AT1, Q03131; Epo-AT3, AF217189. MCoA, malonyl CoA; mMCoA, methyl malonyl CoA.