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Background: The industrial use of enzymes produced by microorganisms is continuously growing due to the need for sustainable solutions. Nevertheless, many of the plasmids used for recombinant production of proteins in bacteria are based on the use of antibiotic resistance genes as selection markers. The safety concerns and legal requirements surrounding the increased use of antibiotic resistance genes have made the development of new antibiotic-free approaches essential. Results: In this work, a system completely free of antibiotic resistance genes and useful for the production of high yields of proteins in Streptomyces is described. This system is based on the separation of the two components of the yefM/yoeBsl (antitoxin/toxin) operon; the toxin (yoeBsl) gene, responsible for host death, is integrated into the genome and the antitoxin gene (yefMsl), which inactivates the toxin, is located in the expression plasmid. To develop this system, the toxin gene was integrated into the genome of a strain lacking the complete operon, and the antibiotic resistance gene integrated along with the toxin was eliminated by Cre recombinase to generate a final host strain free of any antibiotic resistance marker. In the same way, the antibiotic resistance gene from the final expression plasmid was removed by Dre recombinase. The usefulness of this system was analysed by checking the production of two hydrolases from different Streptomyces. Production of both proteins, with potential industrial use, was high and stable over time after strain storage and after serial subcultures. These results support the robustness and stability of the positive selection system developed. Conclusions: The total absence of antibiotic resistance genes makes this system a powerful tool for using Streptomyces as a host to produce proteins at the industrial level. This work is the first Streptomyces antibiotic marker-free system to be described. [ABSTRACT FROM AUTHOR]

Background: Bacteria included in the genus Streptomyces exhibit several attractive characteristics that make them adequate hosts for the heterologous expression of proteins. One of them is that some of its species have a high secretion capacity and hence the protein of interest could be released to the culture supernatant, facilitating downstream processing. To date, all the expression vectors described for these bacteria contain antibiotic resistance genes as selection markers. However, the use of antibiotics to produce proteins at industrial level is currently becoming more restricted owing to the possibility of contamination of the final product. In this report, we describe the use of the S. lividans yefM/yoeBsl toxin-antitoxin system to develop a stable plasmid expression system. Results: In order to use the yefM/yoeBsl system to stabilize expression plasmids in Streptomyces, a S. lividans mutant strain that contained only the toxin gene (yoeBsl) in its genome and the antitoxin gene (yefMsl) located in a temperature-sensitive plasmid was constructed and used as host. This strain was transformed with an expression plasmid harbouring both the antitoxin gene and the gene encoding the protein of interest. Thus, after elimination of the temperature-sensitive plasmid, only cells with the expression plasmid were able to survive. On using this system, two proteins - an α-amylase from S. griseus and a xylanase from S. halstedii - were overproduced without the addition of antibiotic to the culture medium. The production of both proteins was high, even after long incubations (8 days), and after serial subcultures, confirming the stability of the plasmids without antibiotic selection. Conclusions: This is the first report that describes the use of a toxin-antitoxin system to maintain high -copy plasmids in Streptomyces. This finding could be a valuable tool for using Streptomyces as a host to produce proteins at the industrial and pharmaceutical levels without the use of antibiotics in the production step. [ABSTRACT FROM AUTHOR]

Phosphate metabolism regulates most of the life processes of microorganisms. In the present work we obtained and studied a Streptomyces lividans ppk/pstS double mutant, which lacks polyphosphate kinase ( PPK) and the high-affinity phosphate-binding protein ( Pst S), impairing at the same time the intracellular storage of polyphosphate and the intake of new inorganic phosphate from a phosphate-limited medium, respectively. In some of the aspects analyzed, the ppk/pst S double mutant was more similar to the wt strain than was the single pst S mutant. The double mutant was thus able to grow in phosphate-limited media, whereas the pst S mutant required the addition of 1 mM phosphate under the assay conditions used. The double mutant was able to incorporate more than one fourth of the inorganic phosphate incorporated by the wt strain, whereas phosphate incorporation was almost completely impaired in the pst S mutant. Noteworthy, under phosphate limitation conditions, the double ppk/pst S mutant showed a higher production of the endogenous antibiotic actinorhodin and the heterologous antitumor 8-demethyl-tetracenomycin (up to 10-fold with respect to the wt strain), opening new possibilities for the use of this strain in the heterologous expression of antibiotic pathways. [ABSTRACT FROM AUTHOR]

Summary Interaction of the predatory myxobacterium Myxococcus xanthus with the non-motile, antibiotic producer Streptomyces coelicolor was examined using a variety of experimental approaches. Myxococcus xanthus cells prey on S. coelicolor, forming streams of ordered cells that lyse the S. coelicolor hyphae in the contact area between the two colonies. The interaction increases actinorhodin production by S. coelicolor up to 20-fold and triggers aerial mycelium production. Other bacteria are also able to induce these processes in S. coelicolor though to a lesser extent. These studies offer new clues about the expression of genes that remain silent or are expressed at low level in axenic cultures and open the possibility of overproducing compounds of biotechnological interest by using potent inducers synthesized by other bacteria. [ABSTRACT FROM AUTHOR]