Curing linear plasmids by nitrosoguanidine in Streptomyces sp. FR-008

Abstract:

[Objective] Streptomyces sp. FR-008 was treated with nitrosoguanidine (NTG) to screen mutant with reduced genome in size and without any effect on its growth rate. The resultant mutants were measured on their potentiality to be further developed into chassis cells for heterologous production of secondary metabolites. [Methods] Treatment of the spore suspension of Streptomyces sp. FR-008 by NTG was immediately followed by screening of mutants that are sensitive to arsenite, indicative of loss of large linear plasmid. Pulsed-Field Gel Electrophoresis (PFGE) analysis of 103 strains that survived NTG treatment was used to detect the presence or loss of linear plasmids. Bioassay and HPLC analysis were used to compare the yield of candicidin component III for four mutants with that of the wild type strain. [Results] From 103 strains, 4 independent mutants named 10#, 42#, 59# and 115# with one linear plasmid cured were obtained, and the second-round NTG mutagenesis cured both two linear plasmids in XYS1. Fermentation analysis revealed that the yield of candicidin component III in 10# and 115# increased by 40% and 30%, respectively. [Conclusion] It is the first report that NTG is an effective reagent to cure the stable linear plasmids in Streptomyces, two strains from which the large linear plasmid were cured with enhanced yields of candicidin were obtained. NTG treatment can be used to cure the linear plasmids in some Streptomyces to efficiently reduce its genome, and therefore an effective approach in genetic breeding of antibiotic producer.

Key words:Streptomyces sp. FR-008, plasmids curing, linear plasmids, NTG, PFGE, arsenic resistance, reduced-genome