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.
Highlights
Bacteria included in the genus Streptomyces exhibit several attractive characteristics that make them adequate hosts for the heterologous expression of proteins
The S. lividans-ΔTA strain was transformed with a temperaturesensitive plasmid containing the antitoxin gene (Figure 1 step 2)
No protein production was observed in the control strains transformed with the same plasmids. These results indicated that the absence of antibiotic in the culture medium was responsible for the loss of the expression plasmids in the control strains that did not harbour the toxin in their genomes
Summary
Bacteria included in the genus Streptomyces exhibit several attractive characteristics that make them adequate hosts for the heterologous expression of proteins. Streptomyces has the following advantages over other systems: 1) the formation of inclusion bodies has not been described in the literature; 2) it is a wellsuited host for the expression of very GC-rich genes without codon adaptation [12]; 3) it has high protein secretion efficiency, which makes it feasible to obtain the protein of interest in the culture supernatant, facilitating the protein folding and downstream procedures of extraction and purification [11]; 4) the species used to express proteins (see below) display a relatively low level of endogenous extracellular proteolytic activity in comparison with other hosts such as Bacillus, where deletion of up to seven extracellular proteases had to be done to improve protein production [13,14]. Most of them are summarized in two reviews [1,11]
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