Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro) Biosynthetic Pathway inStreptomycessp. US24 Strain

Abstract

We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro) diketopiperazine (DKP). To identify the structural genes responsible for the synthesis of this DKP derivative, a PCR amplification (696 bp) was carried out using the Streptomyces sp. US24 genomic DNA as template and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases (NRPS). The detection of DKP derivative biosynthetic pathway of the Streptomyces sp. US24 strain was then achieved by gene disruption via homologous recombination using a suicide vector derived from the conjugative plasmid pSET152 and containing the PCR product. Chromatography analysis, biological tests and spectroscopic studies of supernatant cultures of the wild-type Streptomyces sp. US24 strain and three mutants obtained by this gene targeting disruption approach showed that the amplified DNA fragment is required for Cyclo (L-Phe, L-Pro) biosynthesis in Streptomyces sp. US24 strain. This DKP derivative seems to be produced either directly via a nonribosomal pathway or as a side product in the course of nonribosomal synthesis of a longer peptide.

New Chlorinated 2,5-Diketopiperazines from Marine-Derived Bacteria Isolated from Sediments of the Eastern Mediterranean Sea

“…The organic extracts of five marine-derived bacterial strains, specifically Bacillus endophyticus BI0327, Streptomyces albidoflavus BI0383, Nocardiopsis aegyptia BI0618, Streptomyces smyrnaeus BI0918, and Bacillus subtilis BI0980, isolated from marine sediments collected in the Aegean and Ionian seas, were subjected to repetitive chromatographic fractionations to yield three new natural products, namely cis-cyclo(Pro-3-chloro-Tyr) (15), trans-cyclo(Pro-3-chloro-Tyr) (16), and cis-cyclo(3-chloro-Tyr-Ile) (31), and 29 previously reported metabolites, which were identified as cyclo(Pro-Gly) (1) [24,25], cis-cyclo(Pro-Ala) (2) [26,27], cis-cyclo(Pro-Val) (3) [28][29][30], cis-cyclo(Pro-Leu) (4) [25,29,31,32], trans-cyclo(Pro-Leu) (5) [31,33], cis-cyclo(trans-4-Hyp-Leu) (6) [34], trans-cyclo(cis-4-Hyp-Leu) (7) [35], cis-cyclo(Pro-Ile) (8) [28,31,36], trans-cyclo(Pro-Ile) (9) [36,37], cis-cyclo(Pro-OMet) (10) [38], cis-cyclo(Pro-Phe) (11) [28,30,31,39,40], trans-cyclo(Pro-Phe) (12) [33,36,40], cis-cyclo(trans-4-Hyp-Phe) (13) [36,41], cis-cyclo(Pro-Tyr) (14) [42,…”

“…The origin of DKPs has been questioned and it has been proposed that they might even be chemical degradation products. However, sterile media do not contain DKPs [6][7][8][9] and specific bacterial genes encode their biosynthesis [10][11][12].…”

Diketopiperazines from Marine Organisms

“…Although the number of newly isolated naturally occurring DKPs has increased during the last few years, the biosynthetic pathways of these molecules remain largely unexplored. Generally, DKP derivatives seem to be produced by three different ways [94]. The first one is nonribosomal pathway, and product formation takes place under the catalytic control of a large multimodular enzyme complex, termed NRP synthetase (NRPS).…”

Untargeted Metabolomics of Streptomyces Species Isolated from Soils of Nepal

“…These cyclic peptides are also used in quorum-sensing signaling, inhibiting microtubule polymerization, and treating ischemic brain injury [107]. Diketopiperazines are believed to be undesired by-products during oligopeptides synthesis, or products of a nonribosomal pathway [108,109]. Actinomycin D and X2 are chemotherapeutic agents used to treat Wilm's kidney tumors, rhabdomyosarcoma, breast cancer, and trophoblastic tumors by inhibiting the transcription processes and RNA activity in cancer cells [110,111].…”