Biological activities and metabolomic profiles of extracts from the marine sediment bacterium Nocardiopsis alba DP1B cultivated in different media.

The soil bacterium DP1B was isolated from a marine sediment collected off the coast of Randayan Island, Kalimantan Barat, Indonesia and identified based on 16S rDNA as Nocardiopsis alba. The bacterium was cultivated in seven different media (A1, ISP1, ISP2, ISP4, PDB, PC-1, and SCB) with three different solvents [distilled water, 5 % NaCl solution, artificial seawater (ASW)] combinations, shaken at 200 rpm, 30 °C, for 7 days. The culture broths were extracted with ethyl acetate and each extract was tested for its antimicrobial activity and brine shrimp lethality, and the chemical diversity was assessed using thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The result showed that almost all extracts showed antibacterial but not antifungal activity, whereas their brine shrimp toxicity levels vary from high to low. The best medium/solvent combinations for antibacterial activity and toxicity were PC-1 (in either distilled water, 5% NaCl solution, or ASW) and SCB in ASW. Different chemical diversity profiles were observed on TLC, GC-MS, and LC-MS/MS. Extracts from the PC-1 cultures seem to contain a significant number of cyclic dipeptides, whereas those from the SCB cultures contain sesquiterpenes, indicating that media and solvent compositions can affect the secondary metabolite profiles of DP1B. In addition, untargeted metabolomic analyses using LC-MS/MS showed many molecular ions that did not match with those in the Global Natural Products Social Molecular Networking (GNPS) database, suggesting that DP1B has great potential as a source of new natural products.

Genome features and secondary metabolite potential of the marine symbiont Streptomyces sp. RS2.

Streptomyces sp. RS2 was isolated from an unidentified sponge collected around Randayan Island, Indonesia. The genome of Streptomyces sp. RS2 consists of a linear chromosome of 9,391,717 base pairs with 71.9% of G + C content, 8270 protein-coding genes, as well as 18 rRNA and 85 tRNA loci. Twenty-eight putative secondary metabolites biosynthetic gene clusters (BGCs) were identified in the genome sequence. Nine of them have 100% similarity to BGCs for albaflavenone, α-lipomycin, coelibactin, coelichelin, ectoine, geosmin, germicidin, hopene, and lanthionine (SapB). The remaining 19 BGCs have low (< 50%) or moderate (50-80%) similarity to other known secondary metabolite BGCs. Biological activity assays of extracts from 21 different cultures of the RS2 strain showed that SCB ASW was the best medium for the production of antimicrobial and cytotoxic compounds. Streptomyces sp. RS2 has great potential to be a producer of novel secondary metabolites, particularly those with antimicrobial and antitumor activities.

The Effects of Garlic and Red Chilli Pepper Powder on Physicochemical, Microbiological, and Sensory Properties of Cincalok.

Cincalok, a traditional fermented shrimp, is prepared by mixing rebon shrimps (Acetes sp) with coarse salt and granulated sugar in a certain ratio. This research was aimed at studying the effect of adding garlic and red chilli pepper powder on the physicochemical, microbiological, and sensory properties of cincalok. Cincalok was made to be three recipes, namely, original cincalok, A, consists of 2 kg of rebon shrimp, 400 g of granulated sugar, and 100 g of coarse salt; B (A ingredients plus 20 g of red chilli pepper powder); and C (A ingredients plus 20 g of garlic powder). Sensory analysis was conducted on recipe A, and the colour was observed by the naked eye on days 0, 2, 4, 6, 8, 13, 18, 23, 28, 33, 43, 60, 90, 120, 150, and 180. According to the highest criterion score on sensory results, the panellists chose day 6 as the best fermentation for recipe A. The colour of recipe A started changing from pink to a light brown colour on the surface on day 28. Therefore, the physicochemical, microbiological, and sensory properties of each recipe were analyzed for 28 days. Overall, the water, ash, and fat content; titratable acid (TA); total volatile base nitrogen (TVBN); and amino acid nitrogen (AAN) showed insignificant differences (p > 0.05) among the recipes during 28 days of the same observation. The crude protein, pH, and free fatty acid (FFA) of recipe C were significantly different (p < 0.05) from recipes A and B. All recipes contained the total count of mesophilic anaerobic bacteria (TMABs) and the lactic acid bacteria (LABs) except Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, and Enterobacteriaceae for all observation times. The highest criterion score for consumer acceptability was awarded for recipe C followed by recipes B and A. The addition of garlic and red chilli pepper powder affected the physicochemical, microbiological, and sensory properties of cincalok.

Antibacterial Potential of Secondary Metabolites from Indonesian Marine Bacterial Symbionts.

Indonesian marine environments are known to house diverse organisms. However, the potential for bacteria from these environments as a source of antibacterial agents has not been widely studied. This study aims to explore the antibacterial potential of secondary metabolites produced by bacterial symbionts from sponges and corals collected in the Indonesian waters. Extracts of 12 bacterial isolates from sponges or corals were prepared by cultivating the bacteria under a number of different media conditions and using agar well diffusion assays to test for antibacterial activity. In addition, the morphology, physiology, and biochemical characteristics and 16S rRNA sequence of each isolate were used to determine their taxonomic classification. All tested bacterial isolates were able to produce secondary metabolites with various levels of antibacterial activity depending on medium composition and culture conditions. Two of the bacteria (RS3 and RC4) showed strong antibacterial activities against both Gram-negative and Gram-positive bacteria. A number of isolates (RS1, RS3, and RC2) were co-cultured with mycolic acid-containing bacteria, Mycobacterium smegmatis or Rhodococcus sp. However, no improvements in their antibacterial activity were observed. All of the 12 bacteria tested were identified as Streptomyces spp. LC-MS analysis of EtOAc extracts from the most active strains RS3 and RC4 revealed the presence of a number of dactinomycin analogues and potentially new secondary metabolites. Symbiotic Streptomyces spp. from sponges and corals of the Indonesian marine environments have great potential as a source of broad-spectrum antibacterial agents.

3-Ketoacyl-ACP synthase (KAS) III homologues and their roles in natural product biosynthesis.

The 3-ketoacyl-ACP synthase (KAS) III proteins are one of the most abundant enzymes in nature, as they are involved in the biosynthesis of fatty acids and natural products. KAS III enzymes catalyse a carbon-carbon bond formation reaction that involves the α-carbon of a thioester and the carbonyl carbon of another thioester. In addition to the typical KAS III enzymes involved in fatty acid and polyketide biosynthesis, there are proteins homologous to KAS III enzymes that catalyse reactions that are different from that of the traditional KAS III enzymes. Those include enzymes that are responsible for a head-to-head condensation reaction, the formation of acetoacetyl-CoA in mevalonate biosynthesis, tailoring processes via C-O bond formation or esterification, as well as amide formation. This review article highlights the diverse reactions catalysed by this class of enzymes and their role in natural product biosynthesis.

Strobilurin biosynthesis in Basidiomycete fungi.

Strobilurins from fungi are the inspiration for the creation of the ß-methoxyacrylate class of agricultural fungicides. However, molecular details of the biosynthesis of strobilurins have remained cryptic. Here we report the sequence of genomes of two fungi that produce strobilurins and show that each contains a biosynthetic gene cluster, which encodes a highly reducing polyketide synthase with very unusual C-terminal hydrolase and methyltransferase domains. Expression of stpks1 in Aspergillus oryzae leads to the production of prestrobilurin A when the fermentation is supplemented with a benzoyl coenzyme A (CoA) analogue. This enables the discovery of a previously unobserved route to benzoyl CoA. Reconstruction of the gene cluster in A. oryzae leads to the formation of prestrobilurin A, and addition of the gene str9 encoding an FAD-dependent oxygenase leads to the key oxidative rearrangement responsible for the creation of the ß-methoxyacrylate toxophore. Finally, two methyltransferases are required to complete the synthesis.

Investigations into the biosynthesis of the antifungal strobilurins.

The strobilurins are important antifungal metabolites isolated from a number of basidiomycetes and have been valuable leads for the development of commercially important fungicides. Isotopic labelling studies with early and advanced intermediates confirm for the first time that they are produced via a linear tetraketide, primed with the rare benzoate starter unit, itself derived from phenylalanine via cinnamate. Isolation of a novel biphenyl metabolite, pseudostrobilurin B, provides evidence for the involvement of an epoxide in the key rearrangement to form the ß-methoxyacrylate moiety essential for biological activity. Formation of two bolineol related metabolites, strobilurins Y and Z, also probably involves epoxide intermediates. Time course studies indicate a likely biosynthetic pathway from strobilurin A, with the simplest non-subsubstituted benzoate ring, to strobilurin G with a complex dioxepin terpenoid-derived substituent. Precursor-directed biosynthetic studies allow production of a number of novel ring-halogenated analogues as well as a new pyridyl strobilurin. These studies also provide evidence for a non-linear biosynthetic relationship between strobilurin A and strobilurin B.