Amplified DNA in Streptomyces fradiae.

A spontaneous mutant of Streptomyces fradiae contained an amplifiable unit of DNA with a sequence length of approximately 10.5 kilobases that was amplified to approximately 500 copies per chromosome. The amplified DNA appears to be cryptic. SalI fragments of the amplified DNA were cloned into Escherichia coli to construct a restriction map and characterize the amplified DNA. The amplified DNA contained tandem repeats of the amplifiable unit of DNA. The unit had an average base composition of 71% guanine plus cytosine, similar to the chromosomal DNA of Streptomyces species. At least a portion of the amplifiable unit of DNA was present at a low copy number in the wild-type strain. The phenotype of amplified DNA was designated Ads1SF for amplified DNA sequence 1 in S. fradiae.     

Efficient plasmid transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts.

A procedure for efficient transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts with plasmid DNA was developed. Transformation frequencies with S. fradiae protoplasts were strongly influenced by the temperatures for cell growth, protoplast formation, and protoplast regeneration. Transformation frequencies for both species were also influenced by the culture age before protoplast formation, the source and concentration of polyethylene glycol, the transformation-inducing agent, the concentration of protoplasts used in the transformation procedure, and the number of protoplasts added to regeneration plates. Transformation frequencies were substantially higher for both species when calf thymus DNA and protamine sulfate were added to the transformation mix. With S. fradiae, transformation frequencies were much lower with plasmid DNA prepared from other species than with the same plasmids prepared from S. fradiae, suggesting that S. fradiae expresses restriction and modification. With the modified transformation procedures using DNA prepared from homologous hosts, S. ambofaciens and S. fradiae are now transformed routinely at frequencies of 10(6) to 10(7) transformants per micrograms of plasmid DNA.     

Aspartate aminotransferase and tylosin biosynthesis in Streptomyces fradiae.

Aspartate aminotransferase as well as valine dehydrogenase and threonine dehydratase was required for the biosynthesis of tylosin in Streptomyces fradiae NRRL 2702. The biosynthesis of these enzymes and tylosin production were repressed by high concentrations of ammonium ions. The change in specific tylosin production rates in batch cultures with different initial concentrations of ammonium ions showed patterns similar to those of the specific production rates of aspartate aminotransferase, valine dehydrogenase, and threonine dehydratase. Aspartate aminotransferase has been purified by acetone precipitation, DEAE-cellulose, hydroxyapatite, and preparative electrophoresis chromatographies. The purified enzyme (120 kDa) consisted of two subunits identical in molecular mass (54 kDa) and showed homogeneity, giving one band with a pI of 4.2 upon preparative isoelectric focusing. The enzyme was specific for L-aspartate in the forward reaction; the Km values were determined to be 2.7 mM for L-aspartate, 0.7 mM for 2-oxyglutarate, 12.8 mM for L-glutamate, and 0.15 mM for oxaloacetate. The enzyme was somewhat thermostable, having a maximum activity at 55 degrees C, and had a broad pH optimum that ranged from 5.5 to 8.0. The mode of action was a ping-pong-bi-bi mechanism.     

Protease production by immobilized mycelia of Streptomyces fradiae

Streptomyces fradiaewas immobilized in polyacrylamide gel prepared from 5% total acrylamide (90% acrylamide and 10%N,N′-methylenebisacrylamide). Production of protease by the immobilized mycelia was attempted in a batch system. A dilute medium containing 0.5% starch, 0.5% meat extract, and 0.05% yeast extract was employed. The reusability of the immobilized and washed mycelia was examined. The activity of protease production by washed mycelia was rapidly decreased with increasing use cycles. The activity of the immobilized mycelia increased gradually, and reached a maximum after ten use cycles. Then, the activity gradually decreased with increasing reaction cycles. This might be caused by destruction of the gels. On the other hand, the sterilization of the surface of the immobilized mycelia was effective for elongation of the lifetime. As a result, the half-life of protease production by the sterilized immobilized mycelia was about 30 days. The rate of protease production by immobilized mycelia was 12,000 U/ml/hr. This value was four times higher than that by submerged culture.     

Proteinases of Streptomyces fradiae. II. Specificity.

It has been shown that extracellular proteinases synthesized by a keratinolytic strain of S. fradiae are characterized by diversified activity in the decomposition of both proteins and synthetic substrates. Among the six proteinases isolated, apart from the ones dominating and having relatively low specificity, there are two enzymes characterized by narrow catalytic abilities--extremely similar to those of trypsin. These proteinases intensively degraded all the trypsin substrates studied, but they were inactive or showed slight activity toward others. They were also highly sensitive to such specific inhibitors of trypsin as TLCK, SBTI and TIO.     

Degradation of feather keratin by culture filtrates of Streptomyces fradiae.

Treatment of milled defatted capon feathers with filtrates of Steptomyces fradiae ATCC 10745 that had been concentrated 20 times showed that the most keratin degradation in 0.5h occurred at pH 8.0 and 60degrees C. If assay time was extended, however, adecrease in reaction rate occurred and appeared to result from keratinase instability at elevated temperatures. Exposure of commercial feather meal to unconcentrated culturefiltrates for 15 h produced greatest degradation at 50 degrees C.     

Improvement of expression level of keratinase Sfp2 from Streptomyces fradiae by site-directed mutagenesis of its N-terminal pro-sequence

The keratinase Sfp2, produced by Streptomyces fradiae var. k11, is a serine alkaline protease first synthesized as pre-pro-mature precursor, of which the N-terminal propeptide must be autocatalytically cleaved on the C-terminal of P1 amino acid to produce mature enzyme. Single amino acid substitutions were introduced at positions ?1 and ?2 to improve the expression level of mature Sfp2. The specific activity of L(?1)F mutant (48935 U/mg) was nine times that of wild-type Sfp2, whereas the mutants L(?1)D, L(?1)G, L(?1)H, K(?2)E, and K(?2)L had 2–52 % of the specific activity of wild-type. The yield of mature Sfp2 of L(?1)F mutant was estimated to be 800 μg/mg total protein and 112 mg/l culture supernatant, nine and twice that of wild-type, respectively. The L(?1)F mutant exhibited similar enzymatic properties to wild-type.     

Alanine dehydrogenase from Streptomyces fradiae. Purification and properties

Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.     

Proteinases of Streptomyces fradiae. I. Preliminary characterization and purification

A keratinolytic strain of S. fradiae has been shown to synthesize a complex of extracellular proteinases degrading native keratin proteins, elastin and collagen as well as some globular proteins. These enzymes are characterized by basic optimal pH and are inactivated by pheynlmethylsulfonyl fluoride (PMSF). Using preparative polyacrylamide gel electrophoresis, ion-exchange chromatography and affinity chromatography, 6 fractions of active protein of diversified proteolytic activity have been distinguished in the preparation studied.     

Effects of Minerals on Neomycin Production by Streptomyces fradiae

A study was made on the mineral requirements of Streptomyces fradiae strain 3535 for neomycin production. It was observed that optimal levels of the elements Ca, Fe, and Zn per milliliter of a synthetic medium for neomycin production were 10.8, 1.0, and 0.115 μg, respectively. K₂HPO₄ was required at a concentration of 0.1% for maximal yield of neomycin, whereas NaCl and the metals Mn and Cu were without any effect. High doses of Zn (0.23 μg/ml or above) caused destruction of neomycin after the fifth day of fermentation.     

Transposition mutagenesis in Streptomyces fradiae: identification of a neutral site for the stable insertion of DNA by transposon exchange

We explored transposition in Streptomyces fradiae (Sf) as a means to insert a second copy of the tylF gene to improve tylosin (Ty) production. Transposons Tn5096 and Tn5099 transposed relatively randomly in Sf, and many of the insertions caused no deleterious effects on Ty production yields. Tn5098, a derivative of Tn5096 containing tylF and tylJ genes, recombined into the chromosome into the tyl gene cluster and transposition was not observed. However, following the tagging of a neutral site (NS) by Tn5099 transposition, tylF was effectively inserted into the NS by homologous recombination (transposon exchange). Recombinants obtained by transposon exchange produced higher yields of Ty.     

The tylosin-biosynthetic genes of Streptomyces fradiae

The tylosin-biosynthetic (tyl) gene cluster occupies about 1% of the genome of Streptomyces fradiae and includes at least 43 open reading frames. In addition to structural genes required for tylosin production, the tylcluster contains three resistance determinants and several regulatory genes. Tylosin production is evidently controlled by pathway-specific and pleiotropic regulators with the likely involvement of -butyrolactone signalling factors. Accumulation of the polyketide aglycone is controlled by glycosylated macrolides and optimal performance of the complex polyketide synthase enzyme requires the activity of an editing thioesterase.     

Two new extracellular serine proteases from Streptomyces fradiae.

1. Two new extracellular serine proteases have been purified to homogeneity from Streptomyces fradiae. 2. On amino acid sequencing, striking homology is observed between the first enzyme and Streptomyces griseus Protease A, and the second enzyme and S. griseus trypsin. 3. The sequence information shows for the first time that structurally and enzymatically related serine proteases are extracellularly expressed by different Streptomycetes. 4. Differential keratinolytic substrate specificity among these two microbes are probable due to a difference in disulfide reduction capacity.