Richard Paul Elander

Stimulation of the conversion of penicillin N to cephalosporin by ascorbic acid, α-ketoglutarate, and ferrous ions in cell-free extracts of strains of Cephalosporiumacremonium

Abstract A cell-free system of the cephalosporin C fungus, Cephalosporium acremonium , has been described which conversts penicillin N to a cephalosporin-like material which was biologically-active upon treatment with penicillinase with subsequent loss of activity when treated with cephalosporinase. We have confirmed this conversion independently in our laboratories and have shown that the activity was lost upon boiling, indicating the reaction is enzymatic in nature. Furthermore, we have shown that the production of the cephalosporin-like material was stimulated by the addition of ascorbic acid (3.8 μM) and ferrous ions (0.075 μM) to the reaction mixture, components which are co-factors in monooxygenase and dioxygenase reactions.

Enzymatic hydrolysis of penicillin V to 6-aminopenicillanic acid byFusariumoxysporum

SummaryA strain ofFusariumoxysporum was identified as having an intracellular penicillin V acylase activity (penicillin V amidohydrolase EC 3.5.1.11). Activity was induced by phenoxyacetic acid and had a good tolerance for high substrate and product concentrations. Washed cells could be used repeatedly for the complete hydrolysis of 5% penicillin V solutions. The enzyme was partially purified and concentrated from disrupted cells by fractional precipitation with water miscible solvents.

Decreased production ofpara-hydroxypenicillin V in penicillin V fermentations

SummaryPenicillin V (phenoxymethyl penicillin) is produced by industrial strains ofPenicillium chrysogenum in the presence of phenoxyacetic acid (POAc), a side-chain precursor for the penicillin V molecule. The wild-type strain ofP. chrysogenum produces an undesirable penicillin byproduct,para-hydroxypenicillin V (p-OH penicillin V), in addition to penicillin V, viapara-hydroxylation of POAc and subsequent incorporation of thep-OH phenoxyacetic acid into the penicillin molecule. Most of thep-OH penicillin V is produced late in cycle when the POAc concentration in the medium is nearly depleted. The level ofp-OH penicillin V produced by the control strain ranges up to 10–15% of the total penicillins produced. 3-Phenoxypropionic acid andp-bromophenylacetic acid partially inhibit the formation ofp-OH penicillin V with a minimal effect on penicillin V productivity. Mutants deficient in their ability to hydroxylate POAc were found to produce lower levels ofp-OH penicillin V. Multi-step mutation and screening, starting with the wild-type strain, have culminated in isolation of mutants which producep-OH penicillin V as 1% of the total penicillins with no adverse effect on penicillin V productivity.

Biotechnology: Present and Future Roles in the Pharmaceutical Industry

AbstractBiotechnology is the integration of a number of scientific disciplines including microbiology, genetics, biochemistry and chemical engineering. It uses living organisms, or systems or products from these organisms to make or modify useful products. New biotechnology comprises genetic engineering, protoplast fusion and monoclonal antibody techniques, powerful new “tools” designed to generate efficient bioprocesses and products for the pharmaceutical industry. The following areas of biotechnology are highlighted: human insulin, interferons and other growth factors, neuroactive peptides, blood products, antibiotics, enzymes, monoclonal antibodies, vaccines and oncogenes.

STRAIN IMPROVEMENT PROGRAMS IN ANTIBIOTIC-PRODUCING MICROORGANISMS - PRESENT AND FUTURE STRATEGIES

ABSTRACT Directed selection procedures using rational prescreening methodology based on known genetic and biochemical mechanisms have been reported to be successful for many established fermentation organisms and now have largely replaced the empirical mutation and selection procedures. The following techniques have been successful for a variety of producer organisms: (1) direct selection of colonies following plate overlay bioassay; (2) use of analogue-resistant mutants which overproduce rate-limiting biosynthetic intermediates; (3) isolation of mutants resistant to metallic ions which complex antibiotic molecules; (4) selection of auxotrophs followed by reversion to prototrophy; (5) selection of mutants resistant to toxic precursor molecules or to toxic levels of end-product; (6) isolation of desirable morphological types; (7) use of stable recombinant strains and (8) isolation of strains following exposure to agents inducing higher ploidy or compounds known to eliminate chromosomal aberrations. The recent rapid advances in protoplast fusion and recombinant DNA technology now allow the microbial geneticist to perform gene transfer and subsequent amplification in organisms in which the genetics and biochemistry are much better understood, i.e., Escherichia coli , Bacillus subtilis , Streptomyces coelicolor , Saccharomyces cerevisiae , etc. The new technologies will culminate in the generation of new highly productive recombinant strains capable of producing novel useful metabolites.