Integrative gene cloning and expression system for Streptomyces sp. US 24 and Streptomyces sp. TN 58 bioactive molecule producing strains

Abstract

Streptomyces sp. US 24 and Streptomyces sp. TN 58, two strains producing interesting bioactive molecules, were successfully transformed using E. coli ET12567 (pUZ8002), as a conjugal donor, carrying the integrative plasmid pSET152. For the Streptomyces sp. US 24 strain, two copies of this plasmid were tandemly integrated in the chromosome, whereas for Streptomyces sp. TN 58, the integration was in single copy at the attB site. Plasmid pSET152 was inherited every time for all analysed Streptomyces sp. US 24 and Streptomyces sp. TN 58 exconjugants under nonselective conditions. The growth, morphological differentiation, and active molecules production of all studied pSET152 integrated exconjugants were identical to those of wild type strains. Consequently, conjugal transfer using pSET152 integration system is a suitable means of genes transfer and expression for both studied strains. To validate the above gene transfer system, the glucose isomerase gene (xylA) from Streptomyces sp. SK was expressed in strain Streptomyces sp. TN 58. Obtained results indicated that heterologous glucose isomerase could be expressed and folded effectively. Glucose isomerase activity of the constructed TN 58 recombinant strain is of about eighteenfold higher than that of the Streptomyces sp. SK strain. Such results are certainly of importance due to the potential use of improved strains in biotechnological process for the production of high-fructose syrup from starch.

Figure 1

(a) Southern blot of Streptomyces sp. US24 BamHI-digested total DNA hybridised with ³²P-labeled pSET152 plasmid. Lane 1, untransformed wild type Streptomyces sp. US24; lanes 2–9, the eight studied exconjugants; lane 10, BamHI-digested pSET152 plasmid DNA (5.7 Kb); lane 11, the 1 kb ladder used as DNA marker. (b) Schematic representation of tandemly insertion of the pSET152 into the Streptomyces sp. US24 attB site. Chromosomal DNA hybridising is represented by thin lines and pSET152 by thick line. The integrase gene (int), and the apramycin resistance gene [aac3(IV)] from pSET152 attP, attL, and attR sites are also shown.

Figure 2

(a) Southern blot of Streptomyces sp. TN 58 BamHI-digested total DNA hybridised with ³²P-labeled pSET152 plasmid. Lanes 1–8, the eight studied exconjugants; lane 9, untransformed wild type Streptomyces sp. TN 58; lane 10, BamHI-digested pSET152 plasmid DNA (5.7 Kb); lane 11, the 1 kb ladder used as DNA marker. (b) Schematic representation of the integration of plasmid pSET152 in the chromosome of Streptomyces sp. TN 58 by site-specific recombination.

Figure 3

Alignment of the attB region of Streptomyces sp. US 24 and TN 58 strains. Solid boxes, identical sequences, underlined sequence, attB site.

Figure 4

Natural variation of ΦC31 attB sites in various species of Streptomyces. (a) Alignment of attB nucleotide sequence of Streptomyces sp. US 24 strain (S. US 24) and Streptomyces sp. TN 58 (S. TN 58) with attB sequences of S. hygroscopicus NRRL5491 (S. hygro); S. avermitilis MA-4680 (S. aver); S. longisporoflavus 83E6 (S. longi); S. coelicolor (S. coel); S. clavugerus (S. clav); S. cinnamonensis (S. cinna); S. aureofaciens (S. aureo); S. lividans 66 TK64 (S. livid); S. griseus ATCC 12475 (S. grise); S. ambofaciens (S. ambof). Dark boxes, identical sequences; black shading sequence at which crossover occurs (TT) for the natural attB and attP sites. (b); Sequence of the attP site in φC31.

Figure 5

Antibacterial activities in solid media against M. luteus of the Streptomyces sp. US 24 strain wild type “WT US24” (a1) and one corresponding exconjugant US24/pSET152 “Ex US24” (a2) and Streptomyces TN 58 strain wild type “WT TN 58” (b1) and one corresponding exconjugant TN 58/pSET152 “Ex TN 58” (b2).