Identification and heterologous expression of an NRPS biosynthetic gene cluster responsible for the production of the pyrazinones Ichizinone A, B and C

Abstract

Pyrazinones are a growing family of microbial NRPS-derived natural products showing interesting biological activities. These compounds are characterized by the presence of either a di- or trisubstituted heterocyclic, nonaromatic 2(1 H)-pyrazinone core in their structure. The most commonly occurring disubstituted pyrazinone natural products are synthesized through a dipeptide intermediate, which is further cyclized to yield the pyrazinone moiety. Trisubstituted pyrazinones are seldom found in natural products, with JBIR56 and JBIR57, isolated from marine Streptomyces, being notable examples. In contrast to the simply organized disubstituted pyrazinones, JBIR56 and JBIR57 are syn-thesized as tetrapeptides with unnatural beta-amino acid residue involved in the for-mation of the pyrazinone moiety. Despite interesting structural features, biosynthetic routes leading to the production of these compounds have not been reported yet. Here we report the discovery of new members of trisubstituted pyrazinone family- tetrapeptides ichizinones A-C in Streptomyces sp. LV45-129. Through sequence analysis and heterologous expression, a biosynthetic gene cluster encoding ichizinone production was identified. Based on gene annotation and sequence homology, a biosynthetic model was suggested. The presented results provide insights into the biosynthesis of rare trisubstituted pyrazinone natural products.

Keywords: Streptomyces; Actinobacteria; Biosynthesis; Gene cluster; Heterologous expression; NRPS; Pyrazinone.

Fig. 1

Representatives of the pyrazinone family of natural products: 1–2(1 H)-pyrazinone core structure (1), 2– leuvalin (2), 3– tyrvalin (3), 4– argvalin (4), 5– sorazinone B (5), 6– butrepyrazinone (6), 7– JBIR-56 (7), 8– JBIR-57 (8). The disubstituted pyrazinone natural products are enclosed within a solid-line rectangle, while the trisubstituted pyrazinone natural products are enclosed within a dashed-line rectangle

Fig. 2

LC-MS detection of ichizinones. A– Base peak chromatogram of crude extract from S. sp. LV45-129. Peaks corresponding to ichizinones A (9), B (10), and C (11) are marked with the numbers 9, 10 and 11, respectively. B, C, and D– Mass spectra of the peaks corresponding to ichizinones A (9), B (10), and C (11), respectively

Fig. 3

¹³C/¹⁵N HMBC () and COSY (s) key correlations of ichizinone A

Fig. 4

Structures of the isolated compounds: ichizinone A (9), ichizinone B (10), ichizinone C (11)

Fig. 5

LC-MS analysis of ichizinone production by the heterologous host S. albus E514. Extracted ion chromatograms (409.3 ± 0.5 Da, 423.3 ± 0.5 Da, 485.3 ± 0.5 Da) of crude extracts from S. sp. LV45-129, S. albus E514, and S. albus Del14 are shown. Peaks corresponding to ichizinones A (9), B (10), and C (11) are marked with the numbers 9, 10, and 11, respectively

Fig. 6

The chromosomal fragment of S. sp. LV45-129 containing the ichizonone biosynthetic gene cluster

Fig. 7

LC-MS analysis of ichizinone production in S. albus strains harboring E514 cosmids with inactivated biosynthetic genes. Extracted ion chromatograms (409.3 ± 0.5 Da, 423.3 ± 0.5 Da, 485.3 ± 0.5 Da) of crude extracts from S. albus E514, S. albus E514 ΔichH, S. albus E514 ΔichI, and S. albus E514 ΔichJ are shown. Peaks corresponding to ichizinones A (9), B (10), and C (11) are marked with the numbers 9, 10, and 11, respectively

Fig. 8

Proposed biosynthetic pathway of ichizinones. A– Assembly of ichizinones by linear NRPS. B– Biosynthesis of beta-amino acid precursors of ichizinones