Stable expression plasmids for Streptomyces based on a toxin-antitoxin system

Abstract

BackgroundBacteria 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.ResultsIn 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.ConclusionsThis 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.

Antibiotic-Free Selection in Biotherapeutics: Now and Forever

“…Interestingly, separated components toxin/antitoxin stabilization systems have been developed for protein production in Streptomces lividans , using YoeBsl gene on the chromosome and YefMsl gene on the plasmid [ 50 ].…”

Synthetic Biology of Antibiotic Production

“…These systems, which are plasmid-free and do not rely on antibiotic selection, are also important for the transition of synthetic biology from the research laboratory to the production environment. This is also the case for stable expression plasmid based on toxin-antitoxin systems, which have been available for some time but have only recently been developed for the important antibiotics producers of the genus Streptomyces [122]. While antibiotics production by synthetic biology will, for the foreseeable future, be restricted to contained reactor systems, its widespread application will nonetheless benefit from considering the introduction of engineered intrinsic biocontainment mechanisms, as reviewed by Moe-Behrens et al [123].…”

BtoxDB: A comprehensive database of protein structural data on toxin–antitoxin systems

“…TA-system components have been used to enhance clonal selection and protein expression in living bacterial cells [17][18][19]. Furthermore, because TA systems are able to repress growth or kill cells and are widely present in bacterial genomes, they are considered as potential targets for the development of new antibacterial drugs [3,20,21].…”