Abstract
Kirromycin is the main product of the soil-dwelling Streptomyces collinus Tü 365. The elucidation of the biosynthetic pathway revealed that the antibiotic is synthesised via a unique combination of trans-/cis-AT type I polyketide synthases and non-ribosomal peptide synthetases (PKS I/NRPS). This was the first example of an assembly line integrating the three biosynthetic principles in one pathway. However, information about other enzymes involved in kirromycin biosynthesis remained scarce. In this study, genes encoding tailoring enzymes KirM, KirHVI, KirOI, and KirOII, and the putative crotonyl-CoA reductase/carboxylase KirN were deleted, complemented, and the emerged products analysed by HPLC-HRMS and MS/MS. Derivatives were identified in mutants ΔkirM, ΔkirHVI, ΔkirOI, and ΔkirOII. The products of ΔkirOI, ΔkirOII, and kirHVI were subjected to 2D-NMR for structure elucidation. Our results enabled functional assignment of those enzymes, demonstrating their involvement in kirromycin tailoring. In the ΔkirN mutant, the production of kirromycin was significantly decreased. The obtained data enabled us to clarify the putative roles of the studied enzymes, ultimately allowing us to fill many of the missing gaps in the biosynthesis of the complex antibiotic. Furthermore, this collection of mutants can serve as a toolbox for generation of new kirromycins.
Highlights
Polyketides are synthesised by megaenzyme complexes termed modular polyketide synthases (PKSs), which catalyse the controlled initiation and elongation of simple carboxylic acid monomers into polyketide chains
The genes kirM, kirHVI, kirOI, kirOII, kirHIV, kirHV, and kirN are located in the kirromycin gene cluster and a potential function of their gene products was postulated based on sequence analysis using the sequence aligner DIAMOND19 against the MIBiG database (Table 1)
With 31% aa identity and 55% similarity, the hydroxylase Fum3p involved in C-5 hydroxylation in fumonisin biosynthesis in Gibberella fujikuroi[21] was the best hit for KirHVI
Summary
Polyketides are synthesised by megaenzyme complexes termed modular polyketide synthases (PKSs), which catalyse the controlled initiation and elongation of simple carboxylic acid monomers into polyketide chains. The linear molecule is composed of three internal ring structures: a pyridone ring, a central tetrahydrofuran (THF) group, and a sugar-like moiety, referred as goldinonic acid[4] (Fig. 1A) These three kirromycin moieties are directly involved in the binding of the antibiotic kirromycin to the elongation factor (EF) Tu in prokaryotes[5,6]. The underlying reactions leading to the THF ring closure, the introduction of hydroxyl groups at C-16 and C-30, methylation of the oxygen at O-42, and the formation of the double bond between C-5 and C-6 in the pyridone ring have remained elusive These enzymatic reactions cannot be explained from the enzymatic domains encoded within the PKS I/NRPS complex, and it was speculated that those product modifications are achieved through tailoring reactions
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
