Single cell mutant selection for metabolic engineering of actinomycetes

Abstract

<p><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/actinobacteria" title="Learn more about Actinomycetes from ScienceDirect's AI-generated Topic Pages">Actinomycetes</a> 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 <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/metabolic-engineering" title="Learn more about metabolic engineering from ScienceDirect's AI-generated Topic Pages">metabolic engineering</a> tool based on random <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/mutagenesis" title="Learn more about mutagenesis from ScienceDirect's AI-generated Topic Pages">mutagenesis</a>, 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. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/cholesterol-oxidase" title="Learn more about cholesterol oxidase from ScienceDirect's AI-generated Topic Pages">cholesterol oxidase</a> ChoD producer <em><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/streptomyces-lavendulae" title="Learn more about Streptomyces lavendulae from ScienceDirect's AI-generated Topic Pages">Streptomyces lavendulae</a></em> 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. 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 <em><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/amycolatopsis" title="Learn more about Amycolatopsis from ScienceDirect's AI-generated Topic Pages">Amycolatopsis</a></em><em>.</em> 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. 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.<br></p>