Nanoparticle intervention of microbial metabolism for drug discovery

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University of Newcastle
Natural products have been a rich source of inspiration for a diverse range of medical drugs with widely different chemical structures (e.g., terpenoids, alkaloids and phenolic compounds) and various biological activities. Microbes can produce a wide range of natural compounds, known as secondary metabolites. Increasingly, it has been recognized that microbes have an extensive range of gene clusters coding for secondary metabolites in which only some are transcribed, and the others remain silent and are not expressed in normal cultivation conditions. In the past years, great progress has been made in characterizing the gene clusters and understanding the biosynthetic pathways of secondary metabolites. Genetic engineering approaches have also been developed to manipulate secondary metabolism to enhance production of known secondary metabolites or discovery of new natural products. However, genetic engineering approach is time-consuming and often requires multiple cloning experiments and culture generations to achieve a suitable microbial strain. Recently, it has been demonstrated that NPs can be used as an elicitor to modulate microbial metabolic processes. Therefore, NPs intervention strategies for optimizing culture conditions affecting bacterial metabolism can be extremely useful for future drug discovery and development. Our preliminary research showed that the use of gold nanoparticles (AuNPs) under a specific cultivation method may activate silent biosynthetic pathways of a bacterial strain obtained from a potato common scab, where a novel gene cluster is activated or an existing gene cluster is enhanced to produce bioactive secondary metabolite. Subsequently, this bacterial strain was identified as Priestia megaterium (P. megaterium). Genome sequencing, bioinformatics approaches were applied to explore the potential secondary metabolites that might be produced by this bacterium. The culture of these bacteria was then intervened with different concentrations of AuNP to investigate if this intervention could promote the production of certain secondary metabolites, which were characterized and assessed for their anti-cancer effects on MDA-MB-231 breast cells using an MTT assay. Th genes that could potentially be associated with the production of these secondary metabolites were then assessed by RT-PCR to investigate the effects of AuNPs on gene expression associated with the metabolic pathways.
Our research has been arranged into five chapters: Chapter 1: Background and Literature Review This chapter includes the general introduction of the discovery of bioactive natural products from different source of microbes that possess biological activities. Three major classes of secondary metabolites have been described. The chapter has also discussed the biosynthetic pathways of these secondary metabolites and the possible of modulation of microbial secondary metabolism, by nanoparticles It finally leads to the development of the research hypothesis and objectives for which this thesis aims address. Chapter 2: Identification of the bacterial isolate from potato crops in Saudi Arabia This chapter is intended to provide detailed information about the identification and genomic study of strain P. megaterium, which was isolated from a potato common scab crop located in Saudi Arabia. Based on the data obtained from DNA sequencing and the use of bioinformatics tools, the verification of assembly, alignment, and annotation of whole genome sequence was carried out, contributing to the identification of genes involved in metabolite biosynthesis, prediction of the structures of metabolites as well as determination of similarities and differences across organisms through comparative genomic analysis. Chapter 3: Nanoparticles induced biosynthesis of secondary metabolites in Priestia megaterium This chapter provides proof-of-concept for AuNPs intervention on P. megaterium metabolism for the enhanced biosynthesis of secondary metabolite. This chapter studied the effect of various concentrations of AuNPs during the culture period, and the profile of secondary metabolite production of P. megaterium with or without the presence of AuNPs were evaluated using HPLC and a suite of mass spectrum techniques. Finally, the enhanced metabolites were isolated and investigated for their effect on the viability of MDA-MB-231 breast cell Chapter 4: Identification, isolation, and transcriptome analysis of naringenin derivatives from nanoparticles-intervention of Priestia megaterium metabolism This chapter describes the isolation and characterization of the enhanced naringenin derivatives due to AnNP-intervention of Priestia megaterium metabolism. Bioinformatics and transcriptome analysis tools have been then used to examine the biosynthetic pathway of naringenins for which showed significant enhancement in their transcriptomic responses in the presence of AuNPs. Chapter 5: Research summary and future directions This chapter provides a comprehensive summary of our new findings and anticipates the presence of additional biosynthetic gene clusters (BGCs) involved in the production of alkaloid secondary metabolites within the P. megaterium genomic draft. The isolation of secondary metabolites from P. megaterium highlights the potential existence of compounds originating from indole alkaloid BGCs. Our goal is to delve into the implications of these discoveries and offer recommendations for future research.
Drug discovery, Common scab, potato crops, genomic draft, secondary metabolites, Priestia megaterium, biosynthetic gene clusters, Gold nanoparticles., genetic manipulation