Non-restricting And Methylation Deficient Escherichia Coli Strains For Bacterial Secondary Metabolism Research

Actinomycetes are important antibiotic producers. In the research of secondary metabolism, biosynthesis gene clusters are first cloned into E. coli strains, then transferred into actinomycetes to make gene disruption or heterologous expression by another E. coli strain. There is no such strain which can finish these two tasks at the same time. So we have constructed two non-restricting and methylation deficient Escherichia coli strains, JTU006 and JTU007, by successively deleting the DNA methylation genes dcm and dam from the widely used cloning host DH10B.JTU006 and JTU007 were tested as donors for the conjugative transfer of a plasmid containing the 39 kb actinorhodin biosynthesis gene cluster to Streptomyces lividans, Streptomyces coelicolor and three predecessors of high yielding antibiotic producer strains Streptomyces avermitilis, Streptomyces clavuligerus and Saccharopolyspora erythraea. The Dcm- Dam- strain JTU007 transferred DNA into S. coelicolor A(3)2 derivatives at high frequency. Surprisingly, neither dcm deletion nor dam deletion was required for efficient conjugation to S. avermitilis. For S. clavuligerus and Sac. erythraea, the Dcm- Dam~+ strain JTU006 gave the highest conjugation efficiency, over 100-fold and 10-fold higher than JTU007, respectively. Actinorhodin production was detected in exconjugants of all 5 actinomycetes.To demonstrate the usefulness of E. coli JTU007 for gene cloning, we constructed a comprehensive Streptomyces toxytricini cosmid library, and transferred it using high-throughput conjugation to the methyl-restricting S. coelicolor. One of the clones produced a brown pigment. Melanin biosynthesis related melC operon was found on the corresponding cosmid by sequencing. JTU007 was better than E. coli ET12567 because it gave higher transformation and cosmid infection frequencies. The cosmid infection frequency of JTU006 was between that of DH10B and JTU007. JTU006 successfully transferred cosmids to methyl-restriction strain S. coelicolor, but the conjugation frequency was lower than that of JTU007.JTU006 and JTU007 are also more useful than ET12567 because they do not restrict methylated DNA in primary cloning, and they are useful for exploring the methyl-specific restriction of potential Streptomyces and other expression hosts.