from-natural-product-to-its-biosynthetic-gene-cluster-demonstration

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from <em>Streptomyces diastatochromogenes</em> T&#252;6028

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different ...

Albert-Ludwigs-Universität Freiburg, Germany

The antibiotic kirromycin is assembled by a hybrid modular polyketide synthases (PKSs)/nonribosomal peptide synthetases (NRPSs). Five of six PKSs of this complex assembly line do not have acyltransferase (AT) and have to recruit this activity from discrete AT enzymes. Here, we show that KirCI is a discrete AT which is involved in kirromycin production and displays a rarely found three-domain architecture (AT₁-AT₂-ER). We demonstrate that the second AT domain, KirCI-AT₂, but not KirCI-AT₁, is the malonyl-CoA-specific AT which utilizes this precursor for loading the acyl carrier proteins (ACPs) of the trans-AT PKS in vitro. In the kirromycin biosynthetic pathway, ACP5 is exclusively loaded with ethylmalonate by the enzyme KirCII and is not recognized as a substrate by KirCI. Interestingly, the excised KirCI-AT₂ can also transfer malonate to ACP5 and thus has a relaxed ACP-specificity compared to the entire KirCI protein. The ability of KirCI-AT₂ to load different ACPs provides opportunities for AT engineering as a potential strategy for polyketide diversification.

The AT₂ domain of KirCI loads malonyl extender units to the ACPs of the kirromycin PKS.

Minimum Information about a Biosynthetic Gene cluster.

Two new polyene macrolactone antibiotics, thailandins A, 1, and B, 2, were isolated from the fermentation broth of rhizosphere soil-associated Actinokineospora bangkokensis strain 44EHW(T). The new compounds from this strain were purified using semipreparative HPLC and Sephadex LH-20 gel filtration while following an antifungal activity guided fractionation. Their structures were elucidated through spectroscopic techniques including UV, HR-ESI-MS, and NMR. These compounds demonstrated broad spectrum antifungal activity against fungi causing anthracnose disease (Colletotrichum gloeosporioides DoA d0762, Colletotrichum gloeosporiodes DoA c1060, and Colletotrichum capsici DoA c1511) as well as pathogenic yeasts (Candida albicans MT 2013/1, Candida parasilopsis DKMU 434, and Cryptococcus neoformans MT 2013/2) with minimum inhibitory concentrations ranging between 16 and 32 μg/mL. This is the first report of polyene antibiotics produced by Actinokineospora species as bioactive compounds against anthracnose fungi and pathogenic yeast strains.

Thailandins A and B, New Polyene Macrolactone Compounds Isolated from Actinokineospora bangkokensis Strain 44EHW(T), Possessing Antifungal Activity against Anthracnose Fungi and Pathogenic Yeasts.

We report the draft genome sequence of Actinokineospora bangkokensis 44EHW(T), the producer of the antifungal polyene compounds, thailandins A and B. The sequence contains 7.45 Mb, 74.1% GC content and 35 putative gene clusters for the biosynthesis of secondary metabolites. There are three gene clusters encoding large polyketide synthases of type I. Annotation of the ORF functions and targeted gene disruption enabled us to identify the cluster for thailandin biosynthesis. We propose a plausible biosynthetic pathway for thailandin, where the unusual butylmalonyl-CoA extender unit is incorporated and results in an untypical side chain.

The Draft Genome Sequence of Actinokineospora bangkokensis 44EHW(T) Reveals the Biosynthetic Pathway of the Antifungal Thailandin Compounds with Unusual Butylmalonyl-CoA Extender Units.

Article	Year
The AT₂ domain of KirCI loads malonyl extender units to the ACPs of the kirromycin PKS. Chembiochem : a European journal of chemical biology\| PubMed ID: 23828654	2013
Minimum Information about a Biosynthetic Gene cluster. Nature chemical biology\| PubMed ID: 26284661	2015
Thailandins A and B, New Polyene Macrolactone Compounds Isolated from Actinokineospora bangkokensis Strain 44EHW(T), Possessing Antifungal Activity against Anthracnose Fungi and Pathogenic Y... Journal of agricultural and food chemistry\| PubMed ID: 27267862	2016
The Draft Genome Sequence of Actinokineospora bangkokensis 44EHW(T) Reveals the Biosynthetic Pathway of the Antifungal Thailandin Compounds with Unusual Butylmalonyl-CoA Extender Units. Molecules (Basel, Switzerland)\| PubMed ID: 27886115	2016

Anja Greule

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