Functional expression of the Cre recombinase in actinomycetes

Abstract

Site-specific recombinases revolutionized "in vivo" genetic engineering because they can catalyze precise excisions, integrations, inversions, or translocations of DNA between their distinct recognition target sites. We have constructed a synthetic gene encoding Cre recombinase with the GC content 67.7% optimized for expression in high-GC bacteria and demonstrated this gene to be functional in Streptomyces lividans. Using the synthetic cre(a) gene, we have removed an apramycin resistance gene flanked by loxP sites from the chromosome of S. lividans with 100% efficiency. Sequencing of the chromosomal DNA part showed that excision of the apramycin cassette by Cre recombinase was specific.

The ROK Family Regulator Rok7B7 Pleiotropically Affects Xylose Utilization, Carbon Catabolite Repression, and Antibiotic Production in Streptomyces coelicolor

“…This was then used to insert the apramycin (Apr) resistance cassette aacC4 flanked by loxP sites, using engineered XbaI sites. The presence of the loxP recognition sites allows the efficient removal of the apramycin resistance cassette following the introduction of plasmid pUWLcre (expressing the Cre recombinase) (30,31). We constructed the knockout plasmids pGAM8 to pGAM14 for the single gene replacement of SCO0794, SCO1060, SCO2846, SCO6008, SCO6566, SCO6600, SCO7543, respectively.…”

Direct Involvement of the Master Nitrogen Metabolism Regulator GlnR in Antibiotic Biosynthesis in Streptomyces

“…The apramycin-resistant and kanamycin-sensitive exconjugants were selected, and the glnRsavdisruption mutant (⌬glnRsav:aac(3)IV) was verified by colony PCR with primers JglnRsav-fw/rv. Finally, the apramycin resistance gene acc(3)IV in the ⌬glnRsav:aac(3)IV mutant was deleted by introducing the plasmid pUWLCRE containing the synthetic cre(a) gene (30), which resulted in the ⌬glnRsav mutant. pUWLCRE was removed by three continuous passages on MS agar medium without antibiotics.…”

Is PhoR–PhoP partner fidelity strict? PhoR is required for the activation of the pho regulon in Streptomyces coelicolor

“…To remove the resistance marker, pUWLCRE, containing a synthetic cre recombinase (Fedoryshyn et al 2008), was transformed into E. coli ET12567 (pUZ8002) and 125 conjugated into the above S. coelicolor strain containing the apramycin cassette flanked by the loxP sites instead of the phoR sequence. Around 15% of the colonies obtained were apramycin sensitive and after two rounds of plating on non-selective (without thiostrepton) medium, pUWLCRE was also lost.…”

“…The recent availability of molecular tools to facilitate the construction of non-polar deletion mutants in Streptomyces (Gust et al 2003;Fedoryshyn et al 2008;Fernández-Martínez et al 2011) has allowed us to construct and characterize for the first time a S. coelicolor ∆phoR mutant strain in which phoP is transcribed from the native phoRP 95 promoter, a key step in order to assess the fidelity of the PhoR-PhoP system in this organism. In this work, we demonstrate that the expression of genes of the pho regulon is not induced in the absence of PhoR thus pointing towards the specificity of the phosphorylation of PhoP by its cognate partner PhoR in S. coelicolor.…”