Cloning of a Microbispora bispora cellobiohydrolase gene in Streptomyces lividans

Abstract

The cellobiohydrolase II (CBHII) of Microbispora bispora, originally cloned in Escherichia coli, was subcloned into Streptomyces lividans using shuttle vectors pSKN 01 and pSKN 02. The enzyme was secreted from Streptomyces, whereas it was intracellular in E. coli. The yields of CBHII produced by S. fividans transformants were 15-20-fold higher than those produced by E. coli transformants. The optimal pH of M. bispora native cellobiohydrolase and the cloned enzyme from S. lividans is 6.5. The thermal and pH stability of CHBII produced in M. bispora, E. coli and S. lividans were compared. Enzyme produced in E. coli was inactivated more rapidly (k = 0.252 rain -1 at 90 ° C; 90% inactivation after 10rain vs. 0.119rain -1 for the others). CBHII was monitored following electrophoretic separation by reaction with a monoclonal antibody. The apparent molecular mass of the protein produced from the S. lividans clone was 93 kDa, the same as that of the native enzyme, but that of the enzyme produced in E. coil was smaller (82 kDa).

Purification and characterization of the major β‐1,4‐endoglucanase from Thermomonospora curvata

“…However, the similarity in the first 10 amino acid residues of the N-terminus sequences of the E G s from the two actinomycetes was only 60%. It should be noted that a high concentration of acidic amino acids (and hence a low PI) appears to be a general characteristic of other Thermomonospora exoenzymes including the major polygalacturonate lyase (Stutzenberger 1987) and endoxylanase (Stutzenberger and Bodine 1992) of T. curvata, as well as those of the related thermophilic actinomycete, Microbispora bispora (Hu et al 1992).…”

Streptomyces lividans groES, groEL1 and groEL2 genes

“…Among the streptomycetes, S. lividans is almost exclusively used as a host for the cloning and expression of exogenous DNA because of the lack of an endonuclease restriction system (Kieser & Hopwood, 1991). S. lividans is able to produce extracellular proteins in high amounts (Strickler et al, 1992) and glycosylate proteins (Hu et al, 1993), and has been broadly used for the production of proteins both of prokaryotic and eukaryotic origin. Successful overproduction of homologous and heterologous proteins might require a concomitant increase of production levels of cellular chaperones to properly stabilize the newly made polypeptides.…”

Studies of Streptomyces reticuli cel-1 (cellulase) gene expression in Streptomyces strains, Escherichia coli, and Bacillus subtilis

“…In contrast to our data, several genes from different streptomycetes and actinomycetes had been especially overexpressed in S. lividans 66: ␣-amylase from Streptomyces griseus (40), chitinase from S. olivaceoviridis (3), protein protease inhibitor from Streptomyces lividans and Streptomyces longisporus (37), and ␤-lactamase from Streptomyces albus (8) (for a review, see reference 1). Cellulases were also successfully overproduced in streptomycetes, i.e., cellobiohydrolase of Microbispora bispora (16); E1, E4, and E5 from Thermomonospora fusca (11,17); and an exoglucanase of C. fimi (22). Therefore, streptomycetes are generally useful for overproducing heterologous polypeptides if no specific regulatory system is required for their synthesis.…”