Improved Production of Mannanase by Streptomyces lividans

Replacement of the natural promoter of the (beta)-mannanase gene of Streptomyces lividans by lacp resulted in a 15-fold increase in enzyme production over that of the previously reported clone S. lividans IAF36, a clone carrying multiple copies of manA, and a 350-fold increase over that of the wild-type strain S. lividans 1326. In addition, the use of lacp in the shuttle vector pIAF199 allowed synthesis of the enzymes on carbon sources that did not contain mannan, such as xylan and whey, which offers interesting possibilities for industrial production of the enzyme.     

Effects of Metals on Streptomyces coelicolor Growth and Actinorhodin Production

Actinorhodin production by Streptomyces coelicolor was used as a model system to study the effects of metals on growth and polyketide synthesis in a streptomycete. Numerous metals were tested in cultures grown in liquid media. Mercury and cadmium were highly toxic, and copper, nickel, and lead were less so, but all tended to inhibit both growth and antibiotic synthesis to a similar extent. Unexpectedly, manganese, cobalt, zinc, and, to a lesser extent, chromium caused complex effects that in general resulted in some enhancement of growth yield but a reduction in antibiotic titers. These complex effects meant that cobalt, manganese, and zinc had lower 50% inhibitory concentrations for antibiotic yields compared with those for biomass. The physiologically active divalent cations calcium and magnesium were also tested. Calcium at high concentrations was particularly effective in reducing antibiotic titers and enhancing growth yields. By adding calcium at different phases of growth, it could be demonstrated that it was most effective in reducing the antibiotic yield when added during the early growth phase. Addition during the antibiotic-producing phase resulted in little reduction of final actinorhodin titers.     

Engineering of Primary Carbohydrate Metabolism for Increased Production of Actinorhodin in Streptomyces coelicolor

The objectives of the current studies were to determine the roles of key enzymes in central carbon metabolism in the context of increased production of antibiotics in Streptomyces coelicolor. Genes for glucose-6-phosphate dehydrogenase and phosphoglucomutase (Pgm) were deleted and those for the acetyl coenzyme A carboxylase (ACCase) were overexpressed. Under the conditions tested, glucose-6-phosphate dehydrogenase encoded by zwf2 plays a more important role than that encoded by zwf1 in determining the carbon flux to actinorhodin (Act), while the function of Pgm encoded by SCO7443 is not clearly understood. The pgm-deleted mutant unexpectedly produced abundant glycogen but was impaired in Act production, the exact reverse of what had been anticipated. Overexpression of the ACCase resulted in more rapid utilization of glucose and sharply increased the efficiency of its conversion to Act. From the current experiments, it is concluded that carbon storage metabolism plays a significant role in precursor supply for Act production and that manipulation of central carbohydrate metabolism can lead to an increased production of Act in S. coelicolor.     

Production of aminoglycosides in non-aminoglycoside producing Streptomyces lividans TK24

The pRBM4 cosmid, which harbors a putative cluster of genes spanning a 31.8-kb chromosomal region of the ribostamycin producer Streptomyces ribosidificus ATCC 21294, was heterologously expressed in Streptomyces lividans TK24. ESI-MS/MS, HPLC, and LC-ESI MS analyses showed that the transformation gave rise to ribostamycin production in various culture broths. This is the first report of heterologous aminoglycoside production.     

Streptavidin secretion by Streptomyces lividans strains

Recombinant strains of S. lividans capable of secreting streptavidin were isolated. Various constructions containing either streptavidin gene copies integrated within a chromosome or a streptavidin gene within the secretory vector were investigated. S. lividans SA2 containing a gene copy integrated within the chromosome had the maximum productivity amounting to 60-80 per cent of the S. avidinii productivity. New vectors for Streptomyces used in the study are described.     

Synthesis and secretion of hirudin by Streptomyces lividans

Summary To examine the secretory production of heterologous proteins by Streptomyces lividans, we fused the DNA encoding the signal peptide of the -amylase inhibitor tendamistat, derived from S. tendae with a synthetic gene encoding the thrombin inhibitor hirudin. The analysis of secretion by immunoblots revealed an efficient translocation of hirudin through the membrane, with no detectable immunoreaction among the cellular proteins. The secreted hirudin was stable in the shaking culture for about 6 days. A comparison of the hirudin secreted by S. lividans and recombinant reference hirudin from yeast by immunoblots and thrombin inhibition assays shows that hirudin from Streptomyces has a lower specific activity, which may be due to a different aminoterminal sequence or to inexact processing of the precursor.Offprint requests to: J. Engels     

Native and heterologous protein secretion by Streptomyces lividans

The secretion of the heterologous parathion phosphotriesterase in S. lividans using the Streptomyces -galactosidase signal sequence was further characterised using a pulse/chase system. Unsecreted cell-associated protein in both the precursor and signal-cleaved forms was observed when the protein was expressed from both low- and high-copy vectors. Fractionation of the cells followed by immunoprecipitation with phosphotriesterase antibody suggests that the precursor is membrane-bound while the signal cleaved form is present in the soluble fraction. Preliminary data on the processing of -amylase, a native Streptomyces protein, showed much more rapid processing and secretion, but nevertheless still revealed cell-associated, signal-cleaved protein.National Institutes of Health, Laboratory of Host Defence     

Synthesis of bovine growth hormone by Streptomyces lividans

Streptomyces lividans 66 was transformed with a plasmid containing the regulatory region of the Streptomyces fradiae aph gene and a structural gene that specifies bovine growth hormone (bGH). When grown in liquid culture the transformant contained a protein identical to authentic bGH, as judged by radioimmunoassay and immuno-blotting (Western analysis). The bGH was present in cells that had been in culture for up to four weeks but was not found in the medium. The strategy employed should be generally applicable to the expression of foreign genes in actinomycetes.     

Chitinolytic properties of Streptomyces lividans

Streptomyces lividans TK24, an established host for genetic and molecular studies in actinomycetes, is able to use chitin as sole carbon and nitrogen source. Extracellular chitinase and N-acetyl--d-glucosamidinase (chitobiase) activities were detected in liquid cultures. Chitinase production was inducible by chitin and its low molecular weight derivatives. Low levels of chitinase were also produced in the absence of chitin. Production of extracellular N-acetylglucosaminidase was correlated with the beginning of the stationary phase of growth and was independent of the presence of chitin. Beside highly N-acetylated chitin, supernatants of chitin-induced cultures were able to hydrolyse chitosans with a wide range of degrees of N-acetylation.Abbreviations MS minimal salts - GlcNAc N-acetyl--d-glucosamine - pNP-GlcNAc p-nitrophenyl-2-acetamido-2-deoxy--d-glucopyranoside - d.a. degree of N-acetylation - TLC thin-layer chromatography     

Random insertion of Tn4560 in Streptomyces lividans and Streptomyces avermitilis

Summary Pulsed-field gel electrophoresis (PFGE) was used to show that insertions of transposon Tn4560 in Streptomyces lividans 66 and the avermectin-producer S. avermitilis are randomly distributed. DNA from the latter strain suffered degradation during electrophoresis that could be avoided by modification of the buffer. Inverse PCR primers were developed for Tn4560.     

Twin-Arginine Translocation Pathway in Streptomyces lividans

The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans, a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in the S. lividans genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the S. lividans secretome. To demonstrate its functionality, a tatC deletion mutant was constructed. This mutation impaired the translocation of the Streptomyces antibioticus tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in Escherichia coli, could be translocated in wild-type S. lividans but not in the tatC mutant. In contrast, the secretion of the Sec-dependent S. lividans subtilisin inhibitor was not affected. This study therefore demonstrates that also in general in Streptomyces spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the E. coli TorA signal peptide can direct Tat-dependent transport in S. lividans.     

Peptidases affecting recombinant protein production by Streptomyces lividans

The influence of peptidases on human interleukin-3 (rhIL-3) production by a recombinant Streptomyces lividans strain was investigated. The bacterium produced several general peptidases and tripeptidyl peptidases compromising the authenticity of rhIL-3. The level of peptidases depended on growth morphology. Growing S. lividans as compact pellets successfully reduced peptidase activity. Maximum general peptidase activity in pellet culture was delayed after maximum rhIL-3 concentration was achieved. The activity of the tripeptidyl peptidase was product (rhIL-3) associated.     

Intraplasmid recombination in Streptomyces lividans 66

Summary An Escherichia coli-Streptomyces shuttle plasmid pIF132 containing two direct mel repeats was constructed. While pIF132 replicated relatively stably in E. coli (Rec⁺ or recA), its structure was unstable in S. lividans: recombination between the mel repeats resulted in a smaller plasmid, pIF138. Furthermore, pIF132 formed oligomers extensively in E. coli but not in S. lividans.