Undecylprodigiosin

Marine Streptomyces species are underexplored for their pigment molecules and genes. In this study, we report the genome of the undecylprodigiosin biosynthesizing gene cluster carrying Streptomyces sp. strain BSE6.1, displaying antioxidant, antimicrobial, and staining properties. This Gram-positive obligate aerobic bacterium was isolated from the coastal sediment of the Andaman and Nicobar Islands, India. Pink to reddish pigmented colonies with whitish powdery spores on both agar and broth media are the important morphological characteristics of this bacterium. Growth tolerance to NaCl concentrations was 2 to 7%. The assembled genome of Streptomyces sp. BSE6.1 contains one linear chromosome 8.02 Mb in length with 7157 protein-coding genes, 82 tRNAs, 3 rRNAs and at least 11 gene clusters related to the synthesis of various secondary metabolites, including undecylprodigiosin. This strain carries type I, type II, and type III polyketide synthases (PKS) genes. Type I PKS gene cluster is involved in the biosynthesis of red pigment undecylprodigiosin of BSE6.1, similar to the one found in the S. coelicolor A3(2). This red pigment was reported to have various applications in the food and pharmaceutical industries. The genome of Streptomyces sp. BSE6.1 was submitted to NCBI with a BioProject ID of PRJNA514840 (Sequence Read Archive ID: SRR10849367 and Genome accession ID: CP085300).

Genetic manipulation of the undecylprodigiosin-producing strains and engineered culture medium approaches were applied as the most economical induction strategy for improving production. The hyper-producing recombinant strain ALAA-R20 was obtained after applying protoplast fusion strategy between the potent producer marine endophytic strains Streptomyces sp. ESRAA-10 (P1) and Streptomyces sp. ESRAA-31 (P2) of Dendronephthya hemprichi. Recombinant strain ALAA-R20 produced undecylprodigiosin yield higher than its parental strains ESRAA-10 and ESRAA-31 by 82.45% and 105.52% under submerged fermentation using modified R2YE medium. In order to reduce the costs of producing undecylprodigiosin, a solid-state fermentation (SSF) was applied. Scaled-up of optimized SSF parameters consisting of groundnut oil cake (GOC) sized to 3 mm, initial moisture content 80% with a mixture of dairy mill and fruit processing wastewaters (1:1), pH 7.0, inoculum size equal to 3 × 105 spores/g dry substrate (gds), incubation temperature 30 °C, and 7-day incubation period yielded the highest yield of 181.78 mg/gds of undecylprodigiosin by the recombinant strain Streptomyces sp. ALAA-R20. Extraction and purification of the pigment using the chromatographic techniques as well as mass spectral analysis exhibited maximum absorbance at 539 nm which is physiological property of the undecylprodigiosin. Undecylprodigiosin was stable over a wide temperature ranged from - 20 to 35 °C even after storage for 6 months. The maximum yield and stability of pigment was obtained at the acidic pH (acidified methanol, pH 4.0). Undecylprodigiosin obtained from the recombinant strain Streptomyces sp. ALAA-R20 demonstrated strong antimicrobial activity against all multidrug-resistant bacterial and fungal strains tested with minimum inhibitory, minimum bactericidal, and minimum fungicidal concentrations ranged between 0.5 and 4.0, 0.5 to 4.0, and 1.0 to 8.0 μg/mL, respectively. It also showed complete inhibition of cancer cells; HCT-116, HepG-2, MCF-7 and A-549 at 5, 8, 4, and 7 μM with IC50 equal to 2.0, 4.7, 1.2, and 2.8 μM, respectively.

Streptomyces are efficient chemists with a capacity to generate diverse and potent chemical scaffolds. The secondary metabolism of these soil-dwelling prokaryotes is stimulated upon interaction with other microbes in their complex ecosystem. We observed such an interaction when a Streptomyces isolate was cultivated in a media supplemented with dead yeast cells. Whole-genome analysis revealed that Streptomyces sp. MBK6 harbors the red cluster that is cryptic under normal environmental conditions. An interactive culture of MBK6 with dead yeast triggered the production of the red pigments metacycloprodigiosin and undecylprodigiosin. Streptomyces sp. MBK6 scavenges dead-yeast cells and preferentially grows in aggregates of sequestered yeasts within its mycelial network. We identified that the activation depends on the cluster-situated regulator, mbkZ, which may act as a cross-regulator. Cloning of this master regulator mbkZ in S. coelicolor with a constitutive promoter and promoter-deprived conditions generated different production levels of the red pigments. These surprising results were further validated by DNA-protein binding assays. The presence of the red cluster in Streptomyces sp. MBK6 provides a vivid example of horizontal gene transfer of an entire metabolic pathway followed by differential adaptation to a new environment through mutations in the receiver domain of the key regulatory protein MbkZ.