Abstract
Streptomyces are one of the most important industrial microorganisms for the production of proteins and small-molecule drugs. Previously reported flow cytometry-based screening methods can only screen spores or protoplasts released from mycelium, which do not represent the filamentous stationary phase Streptomyces used in industrial cultivation. Here we show a droplet-based microfluidic platform to facilitate more relevant, reliable and rapid screening of Streptomyces mycelium, and achieved an enrichment ratio of up to 334.2. Using this platform, we rapidly characterized a series of native and heterologous constitutive promoters in Streptomyces lividans 66 in droplets, and efficiently screened out a set of engineered promoter variants with desired strengths from two synthetic promoter libraries. We also successfully screened out several hyperproducers of cellulases from a random S. lividans 66 mutant library, which had 69.2–111.4% greater cellulase production than the wild type. Our method provides a fast, simple, and powerful solution for the industrial engineering and screening of Streptomyces in more industry-relevant conditions.
Highlights
Streptomyces are one of the most important industrial microorganisms for the production of proteins and small-molecule drugs
In a previous study based on Fluorescence-activated cell sorting (FACS) and the green fluorescence protein (GFP) reporter system, a quantitative method was developed to characterize synthetic promoters and ribosomal binding site (RBS) strength in protoplasts released from the mycelium of Streptomyces[9]
We first optimized elution conditions to obtain a suspension of monodispersed S. lividans 66 spores
Summary
Streptomyces are one of the most important industrial microorganisms for the production of proteins and small-molecule drugs. Reported flow cytometry-based screening methods can only screen spores or protoplasts released from mycelium, which do not represent the filamentous stationary phase Streptomyces used in industrial cultivation. We show a droplet-based microfluidic platform to facilitate more relevant, reliable and rapid screening of Streptomyces mycelium, and achieved an enrichment ratio of up to 334.2. Using this platform, we rapidly characterized a series of native and heterologous constitutive promoters in Streptomyces lividans 66 in droplets, and efficiently screened out a set of engineered promoter variants with desired strengths from two synthetic promoter libraries. The most obvious drawback of these flow cytometry-based Streptomyces-screening methods is that neither protoplasts nor spores represent the filamentous cells that are used in industrial fermentation environments, which usually produce target metabolites in the stationary phase.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
