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

Abstract

Streptomyces is studied intensively for its outstanding ability to produce bioactive secondary metabolites and for its complicated morphological differentiation process. A classical genetic manipulation system for Streptomyces has been developed and widely used in the community for a long time, using antibiotic resistance markers to select for double-crossover mutants. The screening process is always laborious and time-consuming. However, the lack of a suitable chromogenic reporter for Streptomyces has limited the use of color-based screening system to simplify the selection process for double-crossover mutants. In this study, a blue reporter system for Streptomyces has been established by mining an indigoidine synthetase gene (idgS) from Streptomyces lavendulae CGMCC 4.1386, leading to the development of a time-saving gene inactivation system for Streptomyces by simple blue-white screening. A series of Streptomyces suicide and temperature-sensitive plasmids containing the idgS reporter cassette were constructed and used successfully to inactivate genes in Streptomyces, allowing a simple and efficient screening method to differentiate the colonies for double-crossover (white) and single-crossover (blue) mutants. Inactivation of the putative γ-butyrolactone synthase gene afsA-y via the idgS-based blue-white screening method revealed that the paulomycin production is negatively controlled by afsA-y in Streptomyces sp. YN86.

Utilization of a reporter system based on the blue pigment indigoidine biosynthetic gene bpsA for detection of promoter activity and deletion of genes in Streptomyces

“…Our simple system, on the other hand, could be suitable for the deletion of individual genes or larger DNA regions even in poorly genetically characterized Streptomyces strains that lack an order cosmid library or a known genomic sequence. During the preparation of this manuscript, a similar inactivation system was reported using the homologous bpsA gene from S. lavendulae (Li et al, 2015).…”

Development of a novel compound microbial agent for degradation of kitchen waste

“…However, Aspergillus flavus produces aflatoxin, a compound potentially harmful to the environment and human health 20 and Streptomyces somaliensis produces several secondary metabolites (i.e., streptomycin), which inhibit the growth of other microorganisms 21 . These two species were excluded from further study.…”

Engineering microbial hosts for production of bacterial natural products

“…Traditionally, this process is inefficient and time consuming due to low homologous recombination (HR) efficiency and the multiple replica plating steps needed to distinguish single-crossover (antibiotic-resistant) from the much less abundant double-crossover (antibiotic-sensitive) mutants. Chromogenic reporter genes such as xylE, 33 gusA, 32 and idgS 35 facilitate the workflow by allowing visual identification of the second crossover event without colony replication. The counterselectable marker cytosine deaminase CodA, which converts 5-fluorocytosine (5FC) to the highly toxic 5-fluorouracil (5FU), has been used to select for double-crossover mutants 36 and plasmid curing 37 without the need for further genetic modifications.…”