Abstract
BackgroundGenetic tools including constitutive and inducible promoters have been developed over the last few decades for strain engineering in Streptomyces. Inducible promoters are useful for controlling gene expression, however only a limited number are applicable to Streptomyces. The aim of this study is to develop a controllable protein expression system based on an inducible promoter using sugar inducer, which has not yet been widely applied in Streptomyces.ResultsTo determine a candidate promoter, inducible protein expression was first examined in Streptomyces avermitilis MA-4680 using various carbon sources. Xylose isomerase (xylA) promoter derived from xylose (xyl) operon was selected due to strong expression of xylose isomerase (XylA) in the presence of d-xylose. Next, a xylose-inducible protein expression system was constructed by investigating heterologous protein expression (chitobiase as a model protein) driven by the xylA promoter in Streptomyces lividans. Chitobiase activity was detected at high levels in S. lividans strain harboring an expression vector with xylA promoter (pXC), under both xylose-induced and non-induced conditions. Thus, S. avermitilis xylR gene, which encodes a putative repressor of xyl operon, was introduced into constructed vectors in order to control protein expression by d-xylose. Among strains constructed in the study, XCPR strain harboring pXCPR vector exhibited strict regulation of protein expression. Chitobiase activity in the XCPR strain was observed to be 24 times higher under xylose-induced conditions than that under non-induced conditions.ConclusionIn this study, a strictly regulated protein expression system was developed based on a xylose-induced system. As far as we could ascertain, this is the first report of engineered inducible protein expression in Streptomyces by means of a xylose-induced system. This system might be applicable for controllable expression of toxic products or in the field of synthetic biology using Streptomyces strains.
Highlights
Genetic tools including constitutive and inducible promoters have been developed over the last few decades for strain engineering in Streptomyces
The detected protein was identified as putative xylose isomerase (XylA, SAV_7182) by MALDI-TOF–MS analysis after the purification of cell extracts and culture supernatants
These results suggest that the expression of the Xylose isomerase (xylA) gene was induced in S. avermitilis by the addition of d-xylose
Summary
Inducible promoters are useful for controlling gene expression, only a limited number are applicable to Streptomyces. Streptomyces strains are aerobic, gram-positive, mycelia-forming soil bacteria with high G+C content DNA. These strains have produced many useful compounds such as secondary metabolites [1, 2]. Streptomyces strains enable the secretion of various hydrolytic enzymes into culture medium; they are useful for native and heterologous protein production as a secretion from culture medium [3, 4]. This secretion property might be employed to eliminate endotoxin contamination to simplify purification processes, and to fold proteins precisely [5, 6]. Only a limited number of inducible promoters, such as PtipA and PnitA, have been developed for Streptomyces [9,10,11]
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