Showing papers by "Arnold L. Demain published in 1980"

Cell-free cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N.

Abstract: Cell-free cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N by lytic enzyme extracts of Cephalosporium acremonium M-0198 was stimulated by ferrous ions. The optimum concentration of FeSO4 was 80 microM. No additional stimulation was observed with ascorbate, adenosine 5'-triphosphate, or alpha-ketoglutarate, but Triton X-100 and sonication of the extracts increased activity. ZnSO4 was very inhibitory to enzyme activity; CuSO4 was somewhat less inhibitory, and the least effective of the three was MnCl2. The dimer of the tripeptide was converted to a penicillin that has the biological spectrum of isopenicillin N, and this reaction was also stimulated by FeSO4. We found that sonication can be used directly to prepare extracts with cyclization activity from mycelia, without preparing protoplast lysates. The kinetics of cyclase appearance and disappearance during fermentation were similar to those of ring-expansion activity, i.e., enzyme appeared and peaked 13 h after growth ceased and then disappeared.

Incorporation of 3H and 14C from (6 alpha-3H)penicillin N and (10-14C,6 alpha-3H) penicillin N into deacetoxycephalosporin C.

Abstract: 1. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium mutant M-0198, 3H and 14C were incorporated from singly- and doubly-labelled penicillin N into deacetoxycephalosporin C. 2. The deacetoxcephalosporin C obtained from the above feeding experiments was converted into two different crystalline derivatives, namely N-phthalimidodeacetoxycephalosporin C bisbenzhydryl ester and N-phthalimidodeacetoxycephalosporin C bisdicyclohexylamine salt and recrystallized to constant specific activity or constant ratio of specific activity. 3. That 3H is incorporated at C-7 in the biosynthesized deacetoxycephalosporin C was shown by the loss of radioactivity (95.2%) after methoxylating the derived N-phthalimidodeacetoxycephalosporin C bisbenzyhydryl ester. 4. Deacetoxycephalosporin C was also the product of the cell-free reaction conducted in the presence of ferrous ions and ascorbic acid, as shown by two-dimensional paper electrophoresis-chromatography; these additives appreciably improved the efficiency of conversion.

Microbial production of primary metabolites.

Abstract: Microbial production of primary metabolites contributes significantly to the quality of life. Through fermentation, microorganisms growing on inexpensive carbon sources can produce valuable products such as amino acids, nucleotides, organic acids, and vitamins which can be added to food to enhance its flavor or increase its nutritive values. The contribution of microorganisms will go well beyond the food industry with the renewed interest in solvent fermentations. Microorganisms have the potential to provide many petroleum-derived products as well as the ethanol necessary for liquid fuel. The role of primary metabolites and the microbes which produce them will certainly increase in importance.

Stimulation of cell-free ring expansion of penicillin N by sonication and Triton X-100

Abstract: Cell-free protoplast lysates of Cephalosporium acremonium convert penicillin N to deacetoxycephalosporin C by ring expansion. Filtration eliminates 90% of their activity, indicating that the enzyme activity is particulate. The surfactant Triton X-100 or sonication of the lysate stimulates protoplast lysate activity. Active extracts could be produced by merely sonicating intact mycelia.

The astonishing synthetic versatility of microorganisms

Abstract: The versatility of microbial biosynthesis is enormous. Today's products of the microbe vary from the simplicity of methane and acetic acid to the complexity of vitamin B12 and proteins. The importance of the fermentation industry results from three important characteristics of microorganisms -ci) a high ratio of surface area to volume, which facilitates the uptake of nutrients needed for high rates of metabolism and biosynthesis, (ii) the tremendous variety of reactions which microorganisms can carry out, and (iii) a facility to adapt to a large array of different environments, allowing a culture to be transplanted from nature to the laboratory, where it is abILe to grow on inexpensive carbon and nitrogen sources while producing valuable compounds.

Recent biosynthetic studies on beta-lactam antibiotics.

Abstract: Recent experimental observations on the cell-free formation of penicillins and cephalosporins in protoplast lysates from C. acremonium are described. Conversion of radiolabelled penicillin N into deacetoxycephalosporin C is described. A proposal is made about the general types of mechanism involved in these conversions.

Enrichment of Auxotrophic Mutants in Streptomyces griseus.

Abstract: A method for the isolation and enrichment of tryptophan auxotrophic mutants of the indolmycin-producing strain Streptomyces griseus ATCC 12648 was developed, using penicillin selection. With UV irradiation of 2.2 × 104 μW cm−2, a mutation rate to tryptophan auxotrophy of 5.6 × 10−4 was achieved. With 300 μg ml−1 of penicillin G, an enrichment of tryptophan auxotrophs of about 1,000-fold was attained. Approximately 40% of all survivors were tryptophan auxotrophs.

The New Biology: Opportunities for the Fermentation Industry

Abstract: Publisher Summary This chapter presents a brief overview of the new opportunities possible for the fermentation industry. The field of industrial fermentations is in a new phase of growth, propelled by revolutionary discoveries and applications of molecular genetics. Although mutation had been successfully applied to industrial processes since the 1940s, recombination had not. This new growth phase of the industry will feature the application of genetic recombination in many ways. Recombination is being carried out via protoplast fusion, plasmid and viral transfer, and recombinant DNA technology. Success has already been obtained in antibiotic strain-improvement and in the discovery of new antibiotic derivatives. The amplification of genes is another valuable technique that will be used by itself or together with recombination. New opportunities include the microbial production of mammalian polypeptides, antibodies, vaccines, and new processes for the production of primary metabolites, microbial enzymes, industrial chemicals, liquid fuel, and antibiotics. Applications in the agricultural area are also anticipated.