Heterologous Reconstitution of Ikarugamycin Biosynthesis in E. coli

Abstract

Polycyclic tetramate macrolactams (PTMs) are a family of biomedically promising natural products with challenging molecular frameworks. Despite these interesting properties, so far only relatively little is known about the biosynthetic origin of PTMs, in particular concerning the mechanism by which their ring systems are formed. Herein we present the first insights into these processes by using the biosynthesis of ikarugamycin as an example. This has been facilitated by the first heterologous expression of a PTM biosynthetic gene cluster in Escherichia coli. With this approach it will not only become possible to mechanistically investigate already known PTM biosynthetic pathways in more detail in the future, but also to interrogate cryptic PTM biosynthetic pathways chemically and biochemically.

Iterative Assembly of Two Separate Polyketide Chains by the Same Single‐Module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

“…Furthermore, PTM-type gene clusters have been identified from numerous bacterial genomes, [5,6] and the biochemical investigation of this system has only started recently. [3,7,8,28] Here, our studies of HSAF provide direct evidence for this iterative biosynthetic mechanism for bacterial polyketide-peptide natural products. The evidence comes from both the in vivo approach -heterologous production of HSAF analogs and the polyene tetramate intermediate in the Gram-positive bacterial hosts with a clean background, and the in vitro approach -expression of the ~200 kDa PKS and the ~150 kDa NRPS separately in E. coli, purification of the giant enzymes, and reconstitution of the biosynthetic activity.…”

“…[1][2][3][4] This suggests that the same PKS module would act not only iteratively, but also separately, in order to link the two hexaketide chains with the NRPS-activated ornithine to form the characteristic PTM scaffold. Recently, the Gulder group reported heterologous expression of the ikarugamycin (4) biosynthetic gene cluster in E. coli, [7] and the Zhang group reported the enzymatic mechanism for formation of the inner 5-memebered ring and demonstrated the polyketide origin of the ikarugamycin skeleton. [8] Ikarugamycin is a Streptomyces-derived PTM which has a 5,6,5-tricyclic system (Figure 1).…”

“…[3,5,6,23] The determining factor for this timing is not known, but probably related to the redox enzymes that are proposed to cooperate with the PKS-NRPS in forming the PTM scaffold. [7,8,15,23] The transfer of the second hexaketide chain leads to a NRPS-bound polyene-ornithinepolyene intermediate. Finally, the intermediate is released from the NRPS by formation of the tetramate moiety, which is through the attack of the nucleophilic alpha carbon of the second hexaketide at the thioester carbonyl carbon of L-ornithine.…”

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…[190] One thing to note is that they can act together with modular type I PKSs, forming hybrid NRPS-PKS. Several successes in the nonribosomal peptides category include the production of echinomycin, [191] D-Phe-Pro-diketopiperazine, [192] yersiniabactin, [193] ikarugamycin, [194] and antimycins [195] in E. coli.…”

Microbial biodiscovery: Exploring venomous animal associated microbes as sources of new chemical diversity

“…Many PTMs described are reported to exhibit antifungal activity while other exhibit cytotoxic and nematocidal activity. [196][197][198][199][200][201][202][203] PTMs biosynthetic genes have been expressed in E.coli 211 and in vitro production of individual functional PKS and NRPS enzymes has been achieved. …”