Biosynthetic Gene Cluster for the Polyenoyltetramic Acid α-Lipomycin

Abstract

The gram-positive bacterium Streptomyces aureofaciens Tü117 produces the acyclic polyene antibiotic ␣-lipomycin. The entire biosynthetic gene cluster (lip gene cluster) was cloned and characterized. DNA sequence analysis of a 74-kb region revealed the presence of 28 complete open reading frames (ORFs), 22 of them belonging to the biosynthetic gene cluster. Central to the cluster is a polyketide synthase locus that encodes an eight-module system comprised of four multifunctional proteins. In addition, one ORF shows homology to those for nonribosomal peptide synthetases, indicating that ␣-lipomycin belongs to the classification of hybrid peptide-polyketide natural products. Furthermore, the lip cluster includes genes responsible for the formation and attachment of D-digitoxose as well as ORFs that resemble those for putative regulatory and export functions. We generated biosynthetic mutants by insertional gene inactivation. By analysis of culture extracts of these mutants, we could prove that, indeed, the genes involved in the biosynthesis of lipomycin had been cloned, and additionally we gained insight into an unusual biosynthesis pathway.

Common biosynthetic origins for polycyclic tetramate macrolactams from phylogenetically diverse bacteria

“…We expected the same to be true for the frontalamides. These expectations were further bolstered by the observation that bacterial tetramic-acid polyketides such as α-lipomycin are produced by modular PKS and NRPS enzymology, requiring multiple PKS enzymes (23). However, genome searches in the vicinity of ftd clusters revealed no juxtaposed PKS genes and left unresolved the issue of how the remainder of the frontalamide/HSAF-type backbone is biosynthesized.…”

Insights into polyketide biosynthesis gained from repurposing antibiotic-producing polyketide synthases to produce fuels and chemicals

“…Hence we turned to the lipomycin PKS, which was proposed to use isobutyryl-CoA to initiate α-lipomycin biosynthesis, and whose AT-ACP loading didomain is linked to module 1 to form the first polypeptide (LipPks1) of the PKS complex ( Figure 1a). 8 In our hands, linkage of lipPKS1 to the erythromycin PKS thioesterase (TE) gave rise to soluble, active protein (LipPks1+TE) in E. coli. One of the key findings in our analysis was an unexpected substrate profile of the loading AT domain of the lipomycin PKS (Figure 1b).…”

“…One of the key findings in our analysis was an unexpected substrate profile of the loading AT domain of the lipomycin PKS (Figure 1b). 8,9 AT domains of type I PKSs are the primary determinants of building block specificity in polyketide biosynthesis. Previously, in vitro kinetic studies of the loading AT domain of the erythromycin PKS revealed its specificity for propionyl-CoA, although acetyl-and butyryl-CoA were also accepted as substrates with about 40-fold lower k cat /K M .…”

“…This was not anticipated because an α-lipomycin analog that would use 2-methylbutyryl-CoA as the starter had not been reported in the native producer, Streptomyces aureofaciens Tü117. 8 Nonetheless, the in vitro kinetic parameters suggested that an analog of α-lipomycin that used 2-methylbutyryl-CoA to start the biosynthesis should be produced in the native host if the intracellular concentration of 2-methylbutyryl-CoA were raised to a level sufficient to enable incorporation. To achieve this, we added isoleucine, the precursor of 2-methylbutyryl-CoA, to the S. aureofaciens Tü117 culture and subsequently identified the predicted novel analog 21-methyl-α-lipomycin (Figure 1a), which exhibited similar in vitro antibiotic activity to lipomycin.…”

Bio-based production of fuels and industrial chemicals by repurposing antibiotic-producing type I modular polyketide synthases: opportunities and challenges

“…Hence, we turned to the lipomycin PKS, whose loading acyltransferase (AT) domain shares 50% amino acid similarity with the avermectin loading AT. 9 Unlike the avermectin PKS, the first protein of the lipomycin PKS (LipPks1) contains only the loading didomain and module 1, and linkage of LipPks1 to the erythromycin PKS TE domain Figure 2 Biosynthesis of erythromycin (a) and tiacumicin (b). Schemes for the step-wise biosynthesis of the polyketide backbones of 6-deoxyerythronolide B (6-dEB) and tiacumicinone showing the structure of the intermediate tethered to the acyl carrier protein (ACP) after all reactions by the modular domains are complete.…”