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Mining the genome sequence of Burkholderia thailandensisMSMB43 revealed a cryptic biosynthetic gene cluster resembling that of FR901464 (4), a prototype spliceosome inhibitor produced by Pseudomonassp. No. 2663. Transcriptional analysis revealed a cultivation condition in which a regulatory gene of the cryptic gene cluster is adequately expressed. Consequently, three new compounds, named thailanstatins A (1), B (2), and C (3), were isolated from the fermentation broth of B. thailandensisMSMB43. Thailanstatins are proposed to be biosynthesized by a hybrid polyketide synthase–nonribosomal peptide synthetase pathway. They differ from 4by lacking an unstable hydroxyl group and by having an extra carboxyl moiety; those differences endow thailanstatins with a significantly greater stability than 4as tested in phosphate buffer at pH 7.4. In vitroassays showed that thailanstatins inhibit pre-mRNA splicing as potently as 4, with half-maximal inhibitory concentrations in the single to sub-μM range. Cell culture assays indicated that thailanstatins also possess potent antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the single nM range. This work provides new chemical entities for research and development and new structure–activity information for chemical optimization of related spliceosome inhibitors.

Histone deacetylase (HDAC) inhibitors have emerged as a new class of anticancer drugs, with one synthetic compound, SAHA (vorinostat, Zolinza; 1), and one natural product, FK228 (depsipeptide, romidepsin, Istodax; 2), approved by FDA for clinical use. Our studies of FK228 biosynthesis in Chromobacterium violaceumno. 968 led to the identification of a cryptic biosynthetic gene cluster in the genome of Burkholderia thailandensisE264. Genome mining and genetic manipulation of this gene cluster further led to the discovery of two new products, thailandepsin A (6) and thailandepsin B (7). HDAC inhibition assays showed that thailandepsins have selective inhibition profiles different from that of FK228, with comparable inhibitory activities to those of FK228 toward human HDAC1, HDAC2, HDAC3, HDAC6, HDAC7, and HDAC9 but weaker inhibitory activities than FK228 toward HDAC4 and HDAC8, the latter of which could be beneficial. NCI-60 anticancer screening assays showed that thailandepsins possess broad-spectrum antiproliferative activities with GI50for over 90% of the tested cell lines at low nanomolar concentrations and potent cytotoxic activities toward certain types of cell lines, particularly for those derived from colon, melanoma, ovarian, and renal cancers. Thailandepsins thus represent new naturally produced HDAC inhibitors that are promising for anticancer drug development.

It's only natural: The biosynthetic pathway of himastatin from Streptomyces himastatinicusfeaturing unusual domain organizations is elucidated by genome scanning, generation of in‐frame gene deletion mutants, and complementation experiments. Three cytochrome P450s that catalyze novel post‐tailoring oxidative reactions (see picture) are characterized.

Der Biosyntheseweg von Himastatinaus Streptomyces himastatinicuswurde durch Genomanalyse, die Erzeugung von Gendeletionsmutanten und Komplementierungsexperimente aufgeklärt. Drei Cytochrome P450, die neuartige postmodifizierende Oxidationen katalysieren (siehe Bild), werden charakterisiert.

A fundamental feature of modular polyketide synthases (PKSs) is the highly predictable relationship between the domain order and the chemical functional groups of resultant polyketide products. Sequence analysis and biochemical characterization of the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 has revealed a gene, lnmJ, that encodes five PKS modules but with six acyl carrier protein (ACP) domains. The LnmJ PKS module 6 contains two ACP domains, ACP6-1 and ACP6-2, separated by a C-methyltransferase domain. Site-directed mutagenesis experiments were carried out with each of these ACPs to test alternative mechanisms proposed for their role in polyketide chain elongation. The in vivo results revealed a new type of polyketide chain “skipping” mechanism, in which either ACP is sufficient for LNM biosynthesis. Biochemical characterization in vitro showed that both ACPs can be loaded with a malonate extender unit by the LnmG acyl transferase; however, ACP6-2 appears to be preferred because the loading efficiency is about 5-fold that of ACP6-1. The results are consistent with ACP6-2 being used for the initial chain elongation step wth ACP6-1 being involved in the ensuing C-methylation process. These findings provide new insights into the polyketide chain skipping mechanism for modular PKSs.

The cyclic tetrapeptide HC-toxin is an essential virulence determinant for the plant pathogenic fungusCochliobolus carbonumand an inhibitor of histone deacetylase. The major form of HC-toxin contains thed-isomers of Ala and Pro. The non-ribosomal peptide synthetase that synthesizes HC-toxin has only one epimerizing domain for conversion of l-Pro to d-Pro; the source ofd-Ala has remained unknown. Here we present the cloning and characterization of a new gene involved in HC-toxin biosynthesis,TOXG. TOXGis present only in HC-toxin-producing (Tox2+) isolates of C. carbonum. TOXGis able to support d-Ala-independent growth of a strain ofEscherichia colidefective in d-Ala synthesis. A C. carbonumstrain with both of its copies ofTOXGmutated grows normally in culture, and although it no longer makes the three forms of HC-toxin that containd-Ala, it still makes a minor form of HC-toxin that contains Gly in place of d-Ala. The addition ofd-Ala to the culture medium restores production of thed-Ala-containing forms of HC-toxin by the toxGmutant. The toxGmutant has only partially reduced virulence. It is concluded that TOXGencodes an alanine racemase whose function is to synthesize d-Ala for incorporation into HC-toxin.