Ying Zhuo

High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections

The high mortality rate of immunocompromised patients with fungal infections and the limited availability of highly efficacious and safe agents demand the development of new antifungal therapeutics. To rapidly discover such agents, we developed a high-throughput synergy screening (HTSS) strategy for novel microbial natural products. Specifically, a microbial natural product library was screened for hits that synergize the effect of a low dosage of ketoconazole (KTC) that alone shows little detectable fungicidal activity. Through screening of ≈20,000 microbial extracts, 12 hits were identified with broad-spectrum antifungal activity. Seven of them showed little cytotoxicity against human hepatoma cells. Fractionation of the active extracts revealed beauvericin (BEA) as the most potent component, because it dramatically synergized KTC activity against diverse fungal pathogens by a checkerboard assay. Significantly, in our immunocompromised mouse model, combinations of BEA (0.5 mg/kg) and KTC (0.5 mg/kg) prolonged survival of the host infected with Candida parapsilosis and reduced fungal colony counts in animal organs including kidneys, lungs, and brains. Such an effect was not achieved even with the high dose of 50 mg/kg KTC. These data support synergism between BEA and KTC and thereby a prospective strategy for antifungal therapy.

Medium optimization for the production of avermectin B1a by Streptomyces avermitilis 14-12A using response surface methodology

Response surface methodology was employed to optimize the composition of medium for the production of avermectin B1a by Streptomyces avermitilis 14-12A in shaker flask cultivation. Corn starch and yeast extract were found to have significant effects on avermectin B1a production by the Plackett-Burman design. The steepest ascent method was used to access the optimal region of the medium composition, followed by an application of response surface. The analysis revealed that the optimum values of the tested variables were 149.57 g/l corn starch and 8.92 g/l yeast extract. A production of 5128 mg/l, which was in agreement with the prediction, was observed in verification experiment. In comparison to the production of original level (3528 mg/l), 1.45-fold increase had been obtained.

Reverse biological engineering of hrdB to enhance the production of avermectins in an industrial strain of Streptomyces avermitilis

Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are widely used in the field of animal health, agriculture, and human health. Here we have adopted a practical approach to successfully improve avermectin production in an industrial overproducer. Transcriptional levels of the wild-type strain and industrial overproducer in production cultures were monitored using microarray analysis. The avermectin biosynthetic genes, especially the pathway-specific regulatory gene, aveR, were up-regulated in the high-producing strain. The upstream promoter region of aveR was predicted and proved to be directly recognized by σhrdB in vitro. A mutant library of hrdB gene was constructed by error-prone PCR and selected by high-throughput screening. As a result of evolved hrdB expressed in the modified avermectin high-producing strain, 6.38 g/L of avermectin B1a was produced with over 50% yield improvement, in which the transcription level of aveR was significantly increased. The relevant residues were identified to center in the conserved regions. Engineering of the hrdB gene can not only elicit the overexpression of aveR but also allows for simultaneous transcription of many other genes. The results indicate that manipulating the key genes revealed by reverse engineering can effectively improve the yield of the target metabolites, providing a route to optimize production in these complex regulatory systems.

ContigScape: a Cytoscape plugin facilitating microbial genome gap closing

BackgroundWith the emergence of next-generation sequencing, the availability of prokaryotic genome sequences is expanding rapidly. A total of 5,276 genomes have been released since 2008, yet only 1,692 genomes were complete. The final phase of microbial genome sequencing, particularly gap closing, is frequently the rate-limiting step either because of complex genomic structures that cause sequence bias even with high genomic coverage, or the presence of repeat sequences that may cause gaps in assembly.ResultsWe have developed a Cytoscape plugin to facilitate gap closing for high-throughput sequencing data from microbial genomes. This plugin is capable of interactively displaying the relationships among genomic contigs derived from various sequencing formats. The sequence contigs of plasmids and special repeats (IS elements, ribosomal RNAs, terminal repeats, etc.) can be displayed as well.ConclusionsDisplaying relationships between contigs using graphs in Cytoscape rather than tables provides a more straightforward visual representation. This will facilitate a faster and more precise determination of the linkages among contigs and greatly improve the efficiency of gap closing.

Systematics-guided bioprospecting for bioactive microbial natural products

Advances in the taxonomic characterization of microorganisms have accelerated the rate at which new producers of natural products can be understood in relation to known organisms. Yet for many reasons, chemical efforts to characterize new compounds from new microbes have not kept pace with taxonomic advances. That there exists an ever-widening gap between the biological versus chemical characterization of new microorganisms creates tremendous opportunity for the discovery of novel natural products through the calculated selection and study of organisms from unique, untapped, ecological niches. A systematics-guided bioprospecting, including the construction of high quality libraries of marine microbes and their crude extracts, investigation of bioactive compounds, and increasing the active compounds by precision engineering, has become an efficient approach to drive drug leads discovery. This review outlines the recent advances in these issues and shares our experiences on anti-infectious drug discovery and improvement of avermectins production as well.

Synthetic biology of avermectin for production improvement and structure diversification

Natural products are still key sources of current clinical drugs and innovative therapeutic agents. Since wild‐type microorganisms only produce natural products in very small quantities, yields of production strains need to be improved by breaking down the precise genetic and biochemical circuitry. Herein, we use avermectins as an example of production improvement and chemical structure diversification by synthetic biology. Avermectins are macrocyclic lactones produced by Streptomyces avermitilis and are well known and widely used for antiparasitic therapy. Given the importance of this molecule and its derivatives, many efforts and strategies were employed to improve avermectin production and generate new active analogues. This review describes the current status of synthetic strategies successfully applied for developing natural‐product‐producing strains and discusses future prospects for the application of enhanced avermectin production.

Engineering of a genome-reduced host: practical application of synthetic biology in the overproduction of desired secondary metabolites

Synthetic biology aims to design and build new biological systems with desirable properties, providing the foundation for the biosynthesis of secondary metabolites. The most prominent representation of synthetic biology has been used in microbial engineering by recombinant DNA technology. However, there are advantages of using a deleted host, and therefore an increasing number of biotechnology studies follow similar strategies to dissect cellular networks and construct genomereduced microbes. This review will give an overview of the strategies used for constructing and engineering reduced-genome factories by synthetic biology to improve production of secondary metabolites.

Magnetic Field Is the Dominant Factor to Induce the Response of Streptomyces avermitilis in Altered Gravity Simulated by Diamagnetic Levitation

Background Diamagnetic levitation is a technique that uses a strong, spatially varying magnetic field to simulate an altered gravity environment, as in space. In this study, using Streptomyces avermitilis as the test organism, we investigate whether changes in magnetic field and altered gravity induce changes in morphology and secondary metabolism. We find that a strong magnetic field (12T) inhibit the morphological development of S. avermitilis in solid culture, and increase the production of secondary metabolites. Methodology/Principal Findings S. avermitilis on solid medium was levitated at 0 g*, 1 g* and 2 g* in an altered gravity environment simulated by diamagnetic levitation and under a strong magnetic field, denoted by the asterix. The morphology was obtained by electromicroscopy. The production of the secondary metabolite, avermectin, was determined by OD245 nm. The results showed that diamagnetic levitation could induce a physiological response in S. avermitilis. The difference between 1 g* and the control group grown without the strong magnetic field (1 g), showed that the magnetic field was a more dominant factor influencing changes in morphology and secondary metabolite production, than altered gravity. Conclusion/Significance We have discovered that magnetic field, rather than altered gravity, is the dominant factor in altered gravity simulated by diamagnetic levitation, therefore care should to be taken in the interpretation of results when using diamagnetic levitation as a technique to simulate altered gravity. Hence, these results are significant, and timely to researchers considering the use of diamagnetic levitation to explore effects of weightlessness on living organisms and on physical phenomena.

Assessing the Potential of an Induced-Mutation Strategy for Avermectin Overproducers

ABSTRACT Mutant libraries of avermectin-producing Streptomyces avermitilis strains were constructed by different mutagenesis strategies. A metric was applied to assess the mutation spectrum by calculating the distribution of average phenotypic distance of each population. The results showed for the first time that a microgravity environment could introduce larger phenotype distribution and diversity than UV and N-methyl-N-nitro-N-nitrosoguanidine (NTG) could.

Revised Genome Sequence of Burkholderia thailandensis MSMB43 with Improved Annotation

There is growing interest in discovery of novel bioactive natural products from Burkholderia thailandensis. Here we report a significantly improved genome sequence and reannotation of Burkholderia thailandensis MSMB43, which will facilitate the discovery of new natural products through genome mining and studies of the metabolic versatility of this bacterium.