Koroleva, A. | Search bioRxiv

bioRxiv2023

Physical interactions trigger Streptomyces to prey on yeast using natural products and lytic enzymes

Microbial predators obtain energy from killing other living cells. In this study, we present compelling evidence demonstrating that widely distributed Streptomyces soil bacteria, typically not considered as predators, possess the ability to detect and prey on Saccharomyces cerevisiae. Using fluorescence microscopy, we observed that predation is initiated by physical contact between Streptomyces lavendulae YAKB-15 and yeast cells. Comparative transcriptomics data indicated that the interaction triggered the production of numerous lytic enzymes to digest all major components of the yeast cell wall. The production of various glucanases, mannosidases and chitinases was confirmed by proteomics and enzymatic activity measurements. In order to destabilise the yeast cell membrane and assimilate yeast, Streptomyces lavendulae YAKB-15 induced production of cell-associated antifungal polyenes, namely pentamycin and filipin III, and cholesterol oxidase ChoD. In response, yeast downregulated protein synthesis and attempted to enter a quiescence-like state. We show that yeast predation is a common phenomenon in Streptomyces, including well-characterized strains such as Streptomyces peucetius ATCC 27952, where the interaction led to production of 14-hydroxyisochainin. Finally, gene inactivation studies lead us to propose a multidirectional assault model harbouring numerous redundancies that are not dependant on any single individual factor. Our results provide insights into the ecological role of Streptomyces and highlight the utilization of predation as a mechanism to elicit the production of bioactive natural products for drug discovery. Significance StatementSoil is a rich environment for microbes, where they compete for space and resources. Streptomyces bacteria are well-known for their ability to synthesize natural products, particularly antibiotics, that are used in chemical defense against competing microbes. Here we show that Streptomyces are, in fact, predatory bacteria. Upon encountering yeast cells, Streptomyces initiate the production of numerous enzymes that digest the cell wall of yeast. In addition, the interaction triggers the production of natural products that destabilize the yeast cell membrane. Collectively these actions lead to the death of yeast cells and release of cellular building blocks that Streptomyces can use as nutrients. The work fundamentally shifts the paradigm of how Streptomyces are perceived within the soil microbiome ecosystem.

microbiology↗

bioRxiv2022

Single cell mutant selection for metabolic engineering of actinomycetes

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l-1 in a second round of mutant selection. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g-1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.

bioengineering↗