Hyper-inducible expression system for streptomycetes

Abstract

Streptomycetes produce useful enzymes and a wide variety of secondary metabolites with potent biological activities (e.g., antibiotics, immunosuppressors, pesticides, etc.). Despite their importance in the pharmaceutical and agrochemical fields, there have been no reports for practical expression systems in streptomycetes. Here, we developed a "P(nitA)-NitR" system for regulatory gene expression in streptomycetes based on the expression mechanism of Rhodococcus rhodochrous J1 nitrilase, which is highly induced by an inexpensive and safe inducer, epsilon-caprolactam. Heterologous protein expression experiments demonstrated that the system allowed suppressed basal expression and hyper-inducible expression, yielding target protein levels of as high as approximately 40% of all soluble protein. Furthermore, the system functioned in important streptomycete strains. Thus, the P(nitA)-NitR system should be a powerful tool for improving the productivity of various useful products in streptomycetes.

Fig. 1.

Construction of a set of plasmids and nitrilase expression in each S. lividans TK24 transformant. (a) Genetic organization of the nitrilase expression system of R. rhodochrous J1. For clarity, only the restriction sites of the nitrilase expression system discussed in the text are shown. Various trimmed fragments are diagrammatically shown below the restriction map, and the construction procedures are described in Materials and Methods.(b) Nitrilase activity in cell-free extracts of S. lividans TK24 transformants containing each plasmid (pSH10–50 and pIJ487). Each transformant was cultured in the presence or absence of ε-caprolactam. ND, not detected at a lower limit of detection of 5 nmol/min per mg of protein. (c) Coomassie-stained SDS/PAGE showing nitrilase formation in S. lividans TK24 transformants. Lanes M were loaded with the following molecular mass standards: phosphorylase (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and soybean trypsin inhibitor (20 kDa). +, 20 μg of cell-free extracts of S. lividans TK24 transformants cultured in the medium supplemented with ε-caprolactam (0.1%, wt/vol); –,20 μg of cell-free extracts of S. lividans TK24 transformants cultured in the absence of ε-caprolactam. Arrows indicate the band corresponding to the nitrilase.

Fig. 2.

Hyper-inducible expression system. (a) Schematic model of the P_nitA-NitR system. NitR interacts with ε-caprolactam (inducer), and the activated form of NitR causes the production of a large amount of the target protein. To suppress transcriptional read-through from upstream of P_nitA, fd-ter is located just upstream of each P_nitA. (b) Physical structure of plasmid pSH19. Unique restriction endonuclease sites in the multicloning-site (MCS) of the plasmid are indicated in bold type.

Fig. 3.

Expression analysis of the P_nitA-NitR system by SDS/PAGE. +,20 μgof cell-free extracts of each transformant cultured in the medium supplemented with ε-caprolactam (0.1%, wt/vol); –, 20 μg of cell-free extracts of each transformant cultured in the absence of ε-caprolactam. Arrows indicate the band corresponding to each expressed heterologous protein. (a) Expression analysis of the P_nitA-NitR system in S. lividans TK24. In each induced culture, an enormous amount of the target protein was formed. Specific activity of each expressed enzyme is shown below the SDS/PAGE panel. (b) Hyper-production of heterologous proteins in S. coelicolor A3(2), S. avermitilis K139, and S. griseus IFO13350.