Showing papers by "Arnold L. Demain published in 1975"

Microbiological studies on the formation of gramicidin S synthetases

Abstract: Rapid and extensive growth of Bacillus brevis ATCC 9999 was obtained in a complex medium containing yeast extract and peptone. Gramicidin S (GS) production in this medium reached 2.5 g/liter and 0.25 g/g dry cell weight. GS synthetase I production was also high in this complex medium. Chemically defined media were also developed for this strain. In a glycerol-ammonium sulfate-Tris-salts medium, the culture grew about 40% as well (rate and extent) as in complex medium. Although GS production was low (0.23 g GS/liter), peak specific activity of GS synthetase I was as high as on complex medium. Nutritional experiments showed that growth was stimulated by glutamine, methionine, proline, arginine, and histidine. Addition of these amino acids almost doubled the rate and extent of growth and GS production on a volumetric basis. However the increase in GS was due merely to the increased cell density; GS synthetase I specific activity was in fact decreased by the supplement. Complex medium is better than defined medium for GS and GS synthetase production due to increased cell density and a slower rate of synthetase disappearance.

Mutational biosynthesis of a new antibiotic, streptomutin A, by an idiotroph of Streptomyces griseus.

Abstract: Microorganisms producing antibiotics have been genetically converted by earlier workers to mutants which cannot produce antibiotic without supplementation with a moiety of the antibiotic. These antibiotics include neomycin, kanamycin, paromomycin, butirosin, sisomicin, ribostamycin and novobiocin. Success has not been reported for organisms producing guanidinocyclitol antibiotics such as streptomycin. We mutagenized conidia of the streptomycin-producing Streptomyces griseus strain 7-455F3 with nitrosoguanidine at pH 7.0. Non-producers of streptomycin were visually selected by the agar-plug technique using Bacillus subtilis. We successfully isolated mutant MIT-A5 which produces no streptomycin unless streptidine is added to the agar medium. The streptidine-dependent phenotype was confirmed in submerged culture in flasks. Attempts to produce new antibiotics by feeding aminocyclitols to mutant MIT-A5 failed. However a new antibiotic (streptomutin A) was produced by supplementation with the guanidinocyclitol, 2-deoxystreptidine. We propose the term "mutational biosynthesis" for the production of new metabolites by the use of mutants blocked in the biosynthetic pathway to the secondary metabolite. We further propose the term "idiotroph" to properly describe such mutants.

Production of Cephalosporin C by Single and Double Sulfur Auxotrophic Mutants of Cephalosporium acremonium

Abstract: An early blocked sulfur amino acid auxotroph, Cephalosporium acremonium mutant 274-1 (which could be satisfied by methionine or cysteine), utilized organic sulfur compounds for cephalosporin C production in the following order of decreasing effectiveness; methionine > cystathionine > cysteine, despite the fact that cysteine is considered to be the immediate precursor of the antibiotic. When a genetic block was added to mutant 274-1 in the transsulfuration pathway from cysteine to methionine, the double mutant 11-8 (which grows on methonine but not cysteine) failed to produce cephalosporin C from cysteine even though enough methionine was added to support normal growth. Addition of the non-sulfur analogue, norleucine, resulted in antibiotic production from cysteine in the double mutant. These facts support the hypothesis that methionine stimulation of cephalosporin C production is due to a role of methionine other than that of sulfur donation.

Stimulation of cephalosporin production by methionine peptides in a mutant blocked in reverse transsulfuration.

Abstract: The previously reported inability of methionine to stimulate cephalosporin C production in a cysteine auxotroph is due to cysteine interference with methionine uptake. In such a cse, "illicit transport" of alanylmethionine can be used to demonstrate the efficacy of methionine in such mutants blocked in the path from methionine to cysteine. This result supports the hypothesis that the stimulatory activity of methionine is not due to its ability to donate sulfur to the caphalosporin C molecule.

[52] Mannosidostreptomycin hydrolase

Abstract: Publisher Summary This chapter discusses the assay procedure and properties of mannosidostreptomycin hydrolase. Mannosidostreptomycin hydrolase is an enzyme that transforms mannosidostreptomycin to streptomycin by the hydrolytic removal of the mannose moiety. The two antibiotics are made concurrently during the course of fermentation by strains of Streptomyces griseus. The hydrolase is an inducible enzyme whose formation is subject to catabolite repression so that it is usually synthesized late in fermentation when the repressible carbon source is near depletion. The α-D-mannosidase enzyme is most conveniently assayed by use of the chromogenic substrate p-nitrophenyl-α-D-mannopyranoside. The p-nitrophenol liberated is determined spectrophotometrically at 400 nm. Streptomyces mannosidase hydrolyzes phenyl-α-D-mannopyranoside, mannosidostreptomycin, mannosidodihydrostreptomyein in addition to p-nitrophenyl-α-D-mannopyranoside. The Streptomyces enzyme is bound to the cell until lysis occurs. The cell-bound enzyme can be extracted into water, but its release is inhibited by sodium chloride, phosphate, or Tris.

Derivatives of streptomycin and method of producing streptomycin derivatives by mutational biosynthesis

Abstract: A method of making new derivatives of streptomycin by mutational biosynthesis. A mutant of Streptomyces griseus makes streptomycin production dependent on streptidine addition. A new antibiotic, deoxystreptomycin, is disclosed.