Scientists exploit ZouA to amplify gene cluster between replication sites.

Scientists report on the development of a targeted gene amplification system for increasing production of antibiotics in Streptomyces coelicolor, and potentially in other bacterial species. The site-specific recombination system developed by a team based in Canada, Japan, and the U.K., comprises three genetic elements previously shown to be required for DNA amplification in another related species, Streptomyces kanamyceticus. They found that when inserted in S. coelicolor, these same elements led to amplification of the gene cluster responsible for biosynthesis of the polyketide antibiotic actinorhodin, and hence a 20-fold increase in antibiotic production. 

Encouragingly, they found that the same recombination system also functioned in E. coli, suggesting that it may represent a viable tool for effecting targeted amplification in a range of different bacteria. Reporting in PNAS, the team claims the same system could at least be used “readily and immediately to increase levels of antibiotic synthesis in many actinomycetes.”

The University of British Columbia’s Charles J. Thompson, Ph.D., and Takeshi Murakami, Ph.D., along with John Innes Centre researcher Mervyn J. Bibb, Ph.D., and colleagues, describe their work in a paper titled “A system for the targeted amplification of bacterial gene clusters multiplies antibiotic yield in Streptomyces coelicolor.”

About half of all agriculturally and pharmaceutically important compounds, including the majority of antibiotics, are produced in actinomycetes (most belonging to the Streptomyces genus) or fungi, the researchers report. The products are in the main secondary metaboliltes: when the genes are expressed, intermediates of primary metabolism are redirected to alternative pathways, generating antibiotics and other useful compounds. In S. coelicolor, for example, a cluster of 22 genes (act) is responsible for biosynthesis of the polyketide antibiotic actinorhodin.

Unfortunately, work to improve yield is time consuming, and generally involves years of repeated cycles of random mutagenesis and screening to stumble on more efficient strains, the authors continue. Independent studies have, however, shown that production in some antibiotic-overproducing bacterial strains is linked with amplification of the antibiotic biosynthetic gene clusters. These findings have been confirmed by researchers at the John Innes Centre in the U.K, who studied a kanamycin-overproducing strain of S. kanamyceticus that carries 36 tandem copies of a 145-kb DNA sequence housing the kanamycin biosynthetic gene cluster.

These observations raise the question of whether it is possible to harness the genetic elements that target and catalyze amplification to increase the number of antibiotic-related gene clusters in other producing organisms.

Although gene amplifications in Streptomyces species have been described, the enzymes and sequences required to generate and maintain them are not known, Drs. Thompson et al note. New insights were provided by a study published earlier this year by Drs. Murakami and Bibb, which found that DNA amplification in the overproducing S. kanamyceticus strain requires two recombination sites, RsA and RsB, and a putative relaxase, ZouA, which encodes a TraAlike protein with two domains that are homologous to those that mediate plasmid conjugation in many bacteria.

Building on this work, the researchers set out to see whether a genetic cassette comprising the same three elements could be introduced into S. coelicolor to target and amplify the organism’s actinorhodin biosynthetic gene cluster, and so increase antibiotic production. Regions of the S. kanamyceticus genome containing the three genetic elements were cloned into a cassette and targeted to directly flank the actinorhodin biosynthetic gene cluster in the S. coelicolor genome by homologous recombination.

The results were evident: in successfully engineered S. coelicolor strains (selected by antibiotic resistance), significant increases in actinorhodin production correlated with the presence of tandem repeats of the act gene cluster. In fact, a number of the engineered S. coelicolor strains carried 4–12 tandem copies of the act gene cluster, averaging nine repeats per genome. Importantly, the system only worked if the recombination sites directly abutted the act cluster. If one of the recombination sites was located just 60kb away, amplification occurred only very rarely.

Given the successful results, the researchers went on to test whether the same system could catalyze recombination in a basically unrelated organism, E. coli. They tested the platform in both both recA or recA+ strains of the bacterium to see if the process worked and, if so, whether it still depended on this universal bacterial enzyme RecA. Encouragingly, the RsA/ReB/ZouA system enabled recombination to occur in E. coli independent of RecA, although the authors stress further studies will need to be carried out to verify that recombination was related to gene amplification.

Nevertheless, they conclude, “ZouA-mediated DNA amplification promises to be a valuable tool for increasing the activities of commercially important biosynthetic, degradative, and photosynthetic pathways in a wide variety of organisms.”

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