Genome mining and metabolic engineering for bioactive natural product production in Streptomyces

Zhijie Yang*

*Corresponding author for this work

Research output: Book/ReportPh.D. thesis

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Abstract

Streptomyces, a genus of Gram-positive bacteria widely distributed in natural environments, has garnered considerable attention due to its diverse metabolic pathways and the production of bioactive compounds. Among these, the secondary metabolites produced by Streptomyces exhibit substantial comprehensive pharmacological and industrial potential with significant applications in medical, agricultural, and biotechnological domains. To unravel the biosynthetic potential of Streptomyces, recent research efforts have shifted towards the elucidation and analysis of its genomic content. Employing high-throughput sequencing technologies and bioinformatics analysis, numerous putative biosynthetic gene clusters (BGCs) have been successfully unveiled within Streptomyces genomes, laying the foundation for a deeper understanding of the pathways responsible for the production of natural products. In addressing the gaps in our understanding of Streptomyces biosynthetic pathways, genome mining has emerged as a pivotal research approach. In-depth analysis of the Streptomyces genome enables the precise localization and identification of genes involved in biosynthesis, thereby providing crucial support for subsequent endeavors. Furthermore, the integration of metabolic engineering strategies, particularly the integration of CRISPR-based genome editing, offers a means to optimize metabolic pathways, enhance the production of target compounds, and establish effective technical platforms for the sustainable production of Streptomyces-derived natural products.

This thesis primarily unveils the discovery and biosynthesis mechanisms underlying two novel types of natural products: plant growth-promoting pteridic acids and antifungal spirolactone, derived from Streptomyces iranensis. We found that the culture broth of S. iranensis could significantly help plants relieve abiotic stresses such as salinity, osmotic, and drought stresses. Metabolomics, activity-guided chemical isolation, and structure elucidation led to the discovery of the bioactive ingredients, pteridic acids H and F. Pteridic acids also exhibited significant efficacy in enhancing plant stress resistance during Arabidopsis seedling experiments. Transcriptome analysis revealed that pteridic acids treatment not only upregulated the expression of plant photosynthesis and auxin biosynthesis-related genes but also activated various transcription factors associated with plant stress resistance under salt stress condition. The BGC and biosynthesis pathway of pteridic acids was determined by in silico analysis and in vivo CRISPR base editing. Phylogenetic and comparative genome mining indicated that the streptomycetes containing pteridic acids BGC were evolutionarily conserved and globally distributed (Section 2.1 and Paper 1). Genome mining of S. iranensis revealed an unprecedented PKS-type BGC located in the terminal region of its linear chromosome. CRISPR-BEST mutation, combined with HR-LC-MS analysis, followed up by large-scale fermentation and purification led to the identification of a novel class of macrolide spirolactones A-C. Spirolactones showcased the structure of a rare β-lactone moiety, a [6,6]-spiroketal ring, and an unusual extender unit in structure. Spirolactone A exhibits potent anti-Aspergillus activity and proteomic analysis shows it may act by affecting fungal cell wall integrity (Section 2.2 and Paper 2). To further improve the production of pteridic acids and spirolactones, a comprehensive metabolic strategy was employed such as regulatory and transportation genes manipulation, precursors enhancement, promoter engineering, and side pathway elimination (Section 2.3). In addition to the application of the CRISPR-based gene editing toolbox in the aforementioned studies, we employed it in the other two non-model Streptomyces strains to elucidate the BGCs associated with diverse microbial natural products (Section 2.4 and Paper 3-4).

In conclusion, this PhD study uncovered novel natural products, plant growth-promoting pteridic acids, and antifungal spirolactone, from S. iranensis. Pteridic acids exhibited significant efficacy in enhancing plant stress resistance, while spirolactone demonstrated potent anti-Aspergillus activity. Genomics, metabolomics, and CRISPR-based gene editing were employed to elucidate their biosynthetic pathways. This work also involved optimizing the production of pteridic acids and spirolactone through metabolic engineering. These findings significantly advance our understanding of ecological roles and therapeutic potential of Streptomyces secondary metabolites, presenting promising applications in the fields of sustainable agriculture and medicine.
Original languageEnglish
Place of PublicationKgs. Lyngby, Denmark
PublisherDTU Bioengineering
Number of pages260
Publication statusPublished - 2024

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