Yi-Qiang Cheng

Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis

Type I polyketide synthases (PKSs) are multifunctional enzymes that are organized into modules, each of which minimally contains a β-ketoacyl synthase, an acyltransferase (AT), and an acyl carrier protein. Here we report that the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 consists of two PKS genes, lnmI and lnmJ, that encode six PKS modules, none of which contain the cognate AT domain. The only AT activity identified within the lnm gene cluster is a discrete AT protein encoded by lnmG. Inactivation of lnmG, lnmI, or lnmJ in vivo abolished LNM biosynthesis. Biochemical characterization of LnmG in vitro showed that it efficiently and specifically loaded malonyl CoA to all six PKS modules. These findings unveiled a previously unknown PKS architecture that is characterized by a discrete, iteratively acting AT protein that loads the extender units in trans to “AT-less” multifunctional type I PKS proteins for polyketide biosynthesis. This PKS structure provides opportunities for PKS engineering as exemplified by overexpressing lnmG to improve LNM production.

Identification and Localization of the Gene Cluster Encoding Biosynthesis of the Antitumor Macrolactam Leinamycin in Streptomyces atroolivaceus S-140

Leinamycin (LNM), produced by Streptomyces atroolivaceus, is a thiazole-containing hybrid peptide-polyketide natural product structurally characterized with an unprecedented 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a 18-member macrolactam ring. LNM exhibits a broad spectrum of antimicrobial and antitumor activities, most significantly against tumors that are resistant to clinically important anticancer drugs, resulting from its DNA cleavage activity in the presence of a reducing agent. Using a PCR approach to clone a thiazole-forming nonribosomal peptide synthetase (NRPS) as a probe, we localized a 172-kb DNA region from S. atroolivaceus S-140 that harbors the lnm biosynthetic gene cluster. Sequence analysis of 11-kb DNA revealed three genes, lnmG, lnmH, and lnmI, and the deduced product of lnmI is characterized by domains characteristic to both NRPS and polyketide synthase (PKS). The involvement of the cloned gene cluster in LNM biosynthesis was confirmed by disrupting the lnmI gene to generate non-LNM-producing mutants and by characterizing LnmI as a hybrid NRPS-PKS megasynthetase, the NRPS module of which specifies for L-Cys and catalyzes thiazole formation. These results have now set the stage for full investigations of LNM biosynthesis and for generation of novel LNM analogs by combinatorial biosynthesis.

Genomics-Guided Discovery of Thailanstatins A, B and C as Pre-mRNA Splicing Inhibitors and Antiproliferative Agents from Burkholderia thailandensis MSMB43

Mining the genome sequence of Burkholderia thailandensis MSMB43 revealed a cryptic biosynthetic gene cluster resembling that of FR901464 (4), a prototype spliceosome inhibitor produced by Pseudomonas sp. 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. thailandensis MSMB43. Thailanstatins are proposed to be biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase pathway. They differ from 4 by lacking an unstable hydroxyl group and by having an extra carboxyl moiety; those differences endow thailanstatins with a significantly greater stability than 4 as tested in phosphate buffer at pH 7.4. In vitro assays 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.

Chain Initiation in the Leinamycin-producing Hybrid Nonribosomal Peptide/Polyketide Synthetase from Streptomyces atroolivaceus S-140 DISCRETE, MONOFUNCTIONAL ADENYLATION ENZYME AND PEPTIDYL CARRIER PROTEIN THAT DIRECTLY LOAD d-ALANINE

Nonribosomal peptide natural products are biosynthesized from amino acid precursors by nonribosomal peptide synthetases (NRPSs), which are organized into modules. For a typical NRPS initiation module, an adenylation (A) domain activates an amino acid and installs it onto a peptidyl carrier protein (PCP) domain as a thioester; an elongation module, which has a condensation (C) domain located between every consecutive pair of A and PCP domains, catalyzes the formation of the peptide bond between the upstream aminoacyl/peptidyl-S-PCP and the free amino group of the downstream aminoacyl-S-PCP. d-Amino acid constituents in peptide natural products usually arise from the l-enantiomers through the action of integral epimerization (E) domains of an NRPS. The biosynthetic gene cluster for leinamycin, a hybrid nonribosomal peptide/polyketide containing a d-alanine moiety, does not encode a typical NRPS initiation module with the expected A-PCP-E domains; instead, it has only an A protein (LnmQ) and a PCP (LnmP), both of which are encoded by separate genes. Here we show the results of biochemical experiments as follows: (i) we demonstrate that LnmQ directly activates d-alanine as d-alaninyl-AMP and installs it onto LnmP to generate a d-alaninyl-S-PCP intermediate; (ii) we confirm that aminoacylation of LnmP by LnmQ in trans is the result of specific communication between the separate A and PCP proteins; and (iii) we reveal that leinamycin production can be improved by supplementation of exogenous d-alanine in the fermentation broth of Streptomyces atroolivaceous S-140. These findings unveil an unprecedented NRPS initiation module structure that is characterized by a discrete d-alanine-specific A protein and a PCP.

Isolation and characterization of spliceostatin B, a new analogue of FR901464, from Pseudomonas sp. No. 2663

Isolation and characterization of spliceostatin B, a new analogue of FR901464, from Pseudomonas sp. No. 2663

Abstract 2257: Thailanstatins: New pre-mRNA splicing inhibitors and potent antiproliferative agents discovered from Burkholderia Thailandensis MSMB43.

Natural products have been traditionally sought from actinomycetes, filamentous fungi and medicinal plants. Gram-negative bacterial species, such as Burkholderia, Chromobacterium, Lysobacter, Pseudomonas and Xenorhabdus, however, have recently captured attention as new sources of diverse bioactive natural products. Mining the genome sequence of B. thailandensis MSMB43 revealed a cryptic biosynthetic gene cluster highly resembling that of FR901,464, a prototype pre-mRNA splicing inhibitor produced by Pseudomonas sp. No. 2663. Transcriptioal analysis identified a cultivation condition in which a representive gene of the cryptic gene cluster is adequately expressed. Consequently, three new compounds, thailanstatins A, B and C, were isolated from the fermentation broth of B. thailandensis MSMB43 through natural product chemistry. Thailanstatins belong to the FR901,464-family of microbial products biosynthesized by a hybrid polyketide synthease-nonribosomal peptide synthetase pathway. They have an oveall structural similarity with FR901,464, but feature an extra acetic acid unit which endows the compouds with a significantly higher stability than FR901,464 under physiologically relevant conditions. In vitro assays showed that thailanstatins inhibit pre-mRNA splicing as potently as FR901,464, with half-maximal inhibitory concentrations in the single to sub μM range, causing pre-mRNA to accumulate and preventing the production of mRNA and splicing intermediates. In vitro 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 (Table 1). This work provides new chemical entities for research and development, and validates the Burkholderia species as an exciting new source of bioactive natural products. Citation Format: Xiangyang Liu, Sreya Biswas, Michael G. Berg, Christopher M. Antapli, Feng Xie, Qi Wang, Man-Cheng Tang, Gong-Li Tang, Lixin Zhang, Gideon Dreyfuss, Yi-Qiang Cheng. Thailanstatins: New pre-mRNA splicing inhibitors and potent antiproliferative agents discovered from Burkholderia Thailandensis MSMB43. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2257. doi:10.1158/1538-7445.AM2013-2257