Development of an antibiotic marker-free platform for heterologous protein production in Streptomyces

Abstract

BackgroundThe 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.ResultsIn 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.ConclusionsThe 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.Graphical abstractAntibiotic marker-free platform for protein expression in Streptomyces. The antitoxin gene present in the expression plasmid counteracts the effect of the toxin gene in the genome. In absence of the expression plasmid, the toxin causes cell death ensuring that only plasmid-containing cells persist.

Characterization of Two Toxin-Antitoxin Systems in Deep-Sea Streptomyces sp. SCSIO 02999

“…by laboratory methods [41,42]. It has been suggested that TAs YoeB/YefM from S. lividans is a powerful tool for producing other proteins of interest in Streptomyces without the use of antibiotics in the production step [43,44]. Thus, TAs are expected to be a valuable tool for the mass production of metabolites in Streptomyces .…”

Bacillus and Streptomyces spp. as hosts for production of industrially relevant enzymes

“…But then again, the usage of antibiotics in industrial production of enzymes should be maximally minimized, not just for reduction in production costs, but also because of the emergence of antibiotic-resistant strains and complexed legal requirements. The expression platform without antibiotic resistance genes was developed and tested for production of two hydrolytic enzymes in S. lividans (Sevillano et al 2017 ). In this system, the toxin gene was integrated into the S. lividans genome, while the expression plasmid carried the antitoxin gene, which served to inactivate the toxin.…”

Streptomycetes: Attractive Hosts for Recombinant Protein Production

“…The more recent pIJ86 carries apramycin resistance marker ( aprR ) and it is a conjugative vector used for the strong constitutive expression of proteins under erythromycin promoter ( ermE ∗ promoter) from Saccharopolyspora erythraea . Recent works ( Sevillano et al, 2013 , 2017 ) described new replicative high copy number ‘marker-free’ systems, which allowed the production of high levels of proteins without using antibiotics as selection markers. One example is based on the presence of a toxin gene localized in the genome and of an anti-toxin gene located on the expression plasmid of the yefM/yoeBsl operon from S. lividans ( Sevillano et al, 2013 ).…”