Single cell mutant selection for metabolic engineering of actinomycetes

Area of Science:

• Microbial Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Actinomycetes are crucial for producing pharmaceuticals and industrial enzymes, but wild-type strains need extensive development for industrial use.
• Current methods for strain improvement are laborious and time-consuming, hindering efficient development for industrial applications.

Purpose of the Study:

• To develop a generally applicable reporter-guided metabolic engineering tool for actinomycetes.
• To enable rapid identification and isolation of high-performing mutant strains using single-cell sorting.
• To enhance the yield of industrial enzymes and activate silent metabolic pathways for drug discovery.

Main Methods:

• Development of a fluorescence-activated cell sorting (FACS) methodology for single-cell screening of mutant libraries.
• Application of random mutagenesis and selective pressure to generate diverse mutant populations.
• Iterative rounds of screening and selection to achieve significant yield improvements.

Main Results:

• Achieved a 5-fold yield improvement for cholesterol oxidase (ChoD) in *Streptomyces lavendulae*, reaching 20.4 U/g after three rounds.
• Successfully activated a silent mutaxanthene metabolic pathway in *Amycolatopsis*, increasing yields 9-fold to 99 mg/L in two rounds.
• Demonstrated the ability to screen millions of mutants in a single-cell format, bypassing traditional medium optimization.

Conclusions:

• The developed reporter-guided metabolic engineering tool is broadly applicable for actinomycetes.
• This technology accelerates the development of industrial strains for enzyme and natural product production.
• Enables efficient activation of silent metabolic pathways for novel drug discovery.