Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG

Abstract

Cystargolides are natural products originally isolated from Kitasatospora cystarginea NRRL B16505 as inhibitors of the proteasome. They are composed of a dipeptide backbone linked to a β-lactone warhead. Recently, we identified the cystargolide biosynthetic gene cluster, but systematic genetic analyses had not been carried out because of the lack of a heterologous expression system. Here, we report the discovery of a homologous cystargolide biosynthetic pathway in Streptomyces durhamensis NRRL-B3309 by genome mining. The gene cluster was cloned via transformation-associated recombination and heterologously expressed in Streptomyces coelicolor M512. We demonstrate that it contains all genes necessary for the production of cystargolide A and B. Single gene deletion experiments reveal that only five of the eight genes from the initially proposed gene cluster are essential for cystargolide synthesis. Additional insights into the cystargolide pathway could be obtained from in vitro assays with CysG and chemical complementation of the respective gene knockout. This could be further supported by the in vitro investigation of the CysG homolog BelI from the belactosin biosynthetic gene cluster. Thereby, we confirm that CysG and BelI catalyze a cryptic SAM-dependent transfer of a methyl group that is critical for the construction of the cystargolide and belactosin β-lactone warheads.

Keywords: biosynthesis; gene knockout; heterologous expression; inhibitor; lactone; natural product; proteasome.

Figure 1

Chemical structure of cystargolides and belactosins.

Figure 2

The cystargolide biosynthetic gene cluster from K. cystarginea NRRL-B16505 (top), S. durhamensis NRRL-B3309 (middle), and the belactosin biosynthetic gene cluster from Streptomyces sp. UCK 14 (bottom). The individual genes are colored according to their putative biosynthetic function. Homologous genes in the cystargolide and belactosin BGCs are indicated.

Figure 3

LC-MS analysis of culture crude extracts from the heterologous host strain S. coelicolor M512 containing the cystargolide BGC from S. durhamensis NRRL-B3309 and its knockout variants compared to S. coelicolor M512 wild type. The retention time of cystargolide B: t = 9.5 min, cystargolide A: t = 10.2 min. A, total ion chromatograms (TIC), cystargolide peaks are highlighted. B, extracted ion chromatograms (EIC) of cystargolide B m/z 357.2 [M + H]⁺ and cystargolide A m/z 371.2 [M + H]⁺, the peaks are normalized to the highest intensity.

Figure 4

LC-MS analysis of the in vitro enzyme activity of CysG and BelI.A, in vitro methylation of 3-isopropyl malate (3-IPM) by the S-adenosyl methionine (SAM)-dependent methyltransferase CysG. Total ion chromatograms with highlighted signals for 3-IPM (substrate) and Met 3-IPM (3-IPM-1-methyl ester, product). B, in vitro methylation of 3-sec-butylmalate (3-sBM) by the S-adenosyl methionine (SAM)-dependent methyltransferase BelI. Total ion chromatograms with highlighted signals for 3-sBM (substrate) and methylated 3-sBM (product).