An efficient blue-white screening based gene inactivation system for Streptomyces.

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Streptomyces species are crucial for producing bioactive secondary metabolites and exhibit complex morphological differentiation.
• Classical genetic manipulation in Streptomyces relies on laborious antibiotic resistance marker screening for double-crossover mutants.
• The absence of a suitable chromogenic reporter has hindered the development of simplified color-based screening systems for Streptomyces.

Purpose of the Study:

• To establish a novel blue reporter system for Streptomyces to facilitate gene inactivation.
• To develop a time-saving gene inactivation system for Streptomyces utilizing simple blue-white screening.
• To demonstrate the utility of the reporter system for gene functional analysis in Streptomyces.

Main Methods:

• Mining of an indigoidine synthetase gene (idgS) from Streptomyces lavendulae CGMCC 4.1386 to create a blue reporter.
• Construction of Streptomyces suicide and temperature-sensitive plasmids incorporating the idgS reporter cassette.
• Application of the idgS-based system for gene inactivation and subsequent blue-white screening to differentiate mutants.

Main Results:

• Successfully established a functional blue reporter system (idgS) for Streptomyces.
• Developed and validated a time-saving gene inactivation system based on blue-white screening.
• Demonstrated efficient differentiation between single-crossover (blue) and double-crossover (white) mutants.

Conclusions:

• The idgS-based blue reporter system offers a simple and efficient method for gene inactivation in Streptomyces.
• This system significantly reduces the time and effort required for screening double-crossover mutants.
• Application of the system revealed that afsA-y negatively controls paulomycin production in Streptomyces sp. YN86.