亚硝基胍消除链霉菌FR-008的线性质粒

【目的】利用亚硝基胍(NTG)消除链霉菌FR-008线性质粒以简化其基因组,获得背景清晰的菌株,作为抗生素异源生物合成的底盘细胞。【方法】NTG溶液处理链霉菌FR-008孢子悬液,从存活的诱变株中筛选砷敏感的突变株,再通过脉冲场凝胶电泳(PFGE)检测线性质粒是否被消除;用生测实验定性检测各个线性质粒消除突变株杀念菌素合成的能力,最后通过HPLC定量比较突变株和野生型产生杀念菌素的差异。【结果】从103个诱变株中筛选到3株砷敏感的突变株(10#、59#、115#)。PFGE检测发现它们均丢失了大线性质粒p SSFR1,此外,42#突变株的小线性质粒p SSFR2被消除,在此基础上,第二轮NTG诱变获得了双质粒消除的突变株。大线性质粒p SSFR1消除率约为3%,小线性质粒p SSFR2消除率约为1%。发酵结果显示:10#、115#突变株杀念菌素有效组分III产量分别提高了40%和30%。【结论】首次发现NTG是一种有效消除链霉菌线性质粒的诱变剂,2株大线性质粒消除的突变株杀念菌素的产量得到提高。此方法可以用来消除特定链霉菌菌株中的巨型线性质粒以高效简化其基因组,因而是一种有效的抗生素遗传育种的方法。

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

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.