Aminoacyl chain translocation catalysed by a type II thioesterase domain in an unusual non-ribosomal peptide synthetase

Non-Ribosomal Peptide Synthetases (NRPSs) assemble a diverse range of natural products with important applications in both medicine and agriculture. They consist of several multienzyme subunits that must interact with each other in a highly controlled manner to facilitate efficient chain transfer, thus ensuring biosynthetic fidelity. Several mechanisms for chain transfer are known for NRPSs, promoting structural diversity. Herein, we report the first biochemically characterized example of a type II thioesterase (TEII) domain capable of catalysing aminoacyl chain transfer between thiolation (T) domains on two separate NRPS subunits responsible for installation of a dehydrobutyrine moiety. Biochemical dissection of this process reveals the central role of the TEII-catalysed chain translocation event and expands the enzymatic scope of TEII domains beyond canonical (amino)acyl chain hydrolysis. The apparent co-evolution of the TEII domain with the NRPS subunits highlights a unique feature of this enzymatic cassette, which will undoubtedly find utility in biosynthetic engineering efforts.

Bioactive compounds derived from Streptomyces sp. SA32: antibacterial activity, chemical profile, and their related genes

Microbes which are resistant to drugs and antibiotics as well as multi-drug resistant (MDR) microbes have developed due to the improper use of antibiotics and led to explore the microbial isolates as the sources of new antibiotics or those with highly effective and stabile attack. Streptomyces sp. SA32 was selected to inhibit the growth of MDR bacteria Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Enterobacter cloaceae, and Enterococcus sp. E. coli was sensitive to crude extracts of Streptomyces sp. SA32 at the concentration of 19 g.mL-1 with moderate strength against E. cloacae. The bioactive compounds analyzed using thin layer chromatography and phytochemical methods showed that the spot with Rf 0.63 and 0.68 was polyketide compound and that with Rf 0.74 was flavonoid compound. The bioautography assay on the TLC plate confirmed the absence of MDR bacterial growth on polyketide and flavonoid spots. The synthesis of antibacterial compounds was also confirmed by the successful analysis on both non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene sequences.

Antifungal activity, identification and biosynthetic potential analysis of fungi against Rhizoctonia cerealis

Purpose Wheat sheath blight mainly infected by Rhizoctonia cerealis is one of the soil-borne fungal diseases of wheat worldwide and prevalent in major wheat growing areas in China at present. This study aimed to evaluate the antifungal activity of 163 endophytic fungi on R. cerealis. Antifungal strains were identified and their biosynthetic potential was analysed. Methods The antifungal activity of the strains was evaluated via dual-culture antagonism assay. The antifungal strains were identified on the basis of morphological characteristics and internal transcribed spacer gene sequencing. The polyketide synthases (PKSs) and nonribosomal peptide synthetase (NRPS) genes in antifungal strains were detected via specific amplification of chromosomal DNA. Result Twelve out of 163 fungal strains, including seven strains with matrix competition and five strains with antibiosis, were obtained. The twelve antifungal strains belonged to four genera: Alternaria, Ascochyta, Botryosphaeria, and Talaromyces. The inhibition rate of the seven strains with matrix competition was greater than 50%, with that of Botryosphaeria dothidea S2-33 being the highest at 84.6%. The inhibition zone of Talaromyces assiutensis R-03 amongst the five strains with antibiosis was the widest at up to 7 mm. Among the twelve antifungal strains, the strain S2-16 contained all the genes tested, five B. dothidea strains contained PKS-II and NRPS genes, two Alternaria alternata strains only contained PKS-II gene and the remaining four strains did not contain any. Conclusion Results demonstrated twelve potential strains for the biocontrol of wheat sheath blight. In particular, T. assiutensis R-03 was determined as a promising agent. The active substances secreted by antifungal strains may be produced by other biosynthetic pathways.

Isolation, Selection and Antimicrobial Activity of Actinomycetes from Mangrove Soil of Can Gio Forest, Hochiminh City, Vietnam

The Can Gio Mangrove is a Biosphere Reserve of UNESCO since 2000 and it is also a well-known example of “mangrove afforestation and reforestation area’’. A total of 63 actinomycetes were isolated from 25 samples of 9 different sites in mangrove forest soil Can Gio, HoChiMinh city, Vietnam. Almost their colonies have round-shaped; milky, white clear and yellow, entire or loabate margin; diameter size of these colonies varied from 0.2 to 3.0 mm. Twenty-nine of 63 tested isolates could produce antimicrobial active metabolites inhibiting at least one of the tested pathogens and 9 isolates were selected for 16S rDNA sequencing. The result showed that genus Streptomyces (8 isolates) and other genera including 1 isolates were Gram-negative bacteria (Stenotrophomonas). The antimicrobial activity and the amplifying genes coding for polyketide synthetase (PKS) and nonribosomal peptide synthetase (NRPS) showed that 8 strains had broad-spectrum antimicrobial activity, mainly against gram-positive bacteria as Bacillus cereus and Staphylococcus aureus.

Analyzing the biosynthetic potential of antimicrobial-producing actinobacteria originating from Indonesia

We investigated the biosynthetic potential of soil-associated actinobacteria originating from Indonesia, identified as Streptomyces luridus and as Streptomyces luteosporeus. Antimicrobial assays indicated inhibitory activity by both strains against the pathogen Pseudomonas aeruginosa, with S. luteosporeus particularly inhibiting the growth of Bacillus subtilis. PCR-amplification, cloning, and sequencing of ketosynthase (KS) domains of type I modular polyketide (PKS-I) and adenylation (AD) domains of non-ribosomal peptide synthetase (NRPS) indicated the diversity of KS and AD domains derived from both Indonesian Streptomyces. Further phylogenetic analysis showed that KS domains from the subclass cis-AT PKS can be classified as being a part of a loading module or an extension module, along with their predicted substrate specificity. The results suggest that both strains are a potential source of novel biosynthetic pathways. This genetic analysis approach can be used as a fast guide to obtain insight into natural product biosynthetic gene diversity in microorganisms.