Industrial production of beta-lactam antibiotics.

The industrial production of beta-lactam antibiotics by fermentation over the past 50 years is one of the outstanding examples of biotechnology. Today, the beta-lactam antibiotics, particularly penicillins and cephalosporins, represent the world's major biotechnology products with worldwide dosage form sales of approximately 15 billion US dollars or approximately 65% of the total world market for antibiotics. Over the past five decades, major improvements in the productivity of the producer organisms, Penicillium chrysogenum and Acremonium chrysogenum (syn. Cephalosporium acremonium) and improved fermentation technology have culminated in enhanced productivity and substantial cost reduction. Major fermentation producers are now estimated to record harvest titers of 40-50 g/l for penicillin and 20-25 g/l for cephalosporin C. Recovery yields for penicillin G or penicillin V are now >90%. Chemical and enzymatic hydrolysis process technology for 6-aminopenicillanic acid or 7-aminocephalosporanic acid is also highly efficient (approximately 80-90%) with new enzyme technology leading to major cost reductions over the past decade. Europe remains the dominant manufacturing area for both penicillins and cephalosporins. However, due to ever increasing labor, energy and raw material costs, more bulk manufacturing is moving to the Far East, with China, Korea and India becoming major production countries with dosage form filling becoming more dominant in Puerto Rico and in Ireland.

The beta-lactam antibiotics: past, present, and future.

The discovery and development of the beta-lactam antibiotics are among the most powerful and successful achievements of modern science and technology. Since Fleming's accidental discovery of the penicillin-producing mold, seventy years of steady progress has followed, and today the beta-lactam group of compounds are the most successful example of natural product application and chemotherapy. Following on the heels of penicillin production by Penicillium chrysogenum came the discoveries of cephalosporin formation by Cephalosporium acremonium, cephamycin, clavam and carbapenem production by actinomycetes, and monocyclic beta-lactam production by actinomycetes and unicellular bacteria. Each one of these groups has yielded medically-useful products and has contributed to the reduction of pain and suffering of people throughout the world. Research on the microbiology, biochemistry, genetics and chemistry of these compounds have continued up to the present with major contributions being made by both individual and collaborative groups from industry and academia. The discovery of penicillin not only led to the era of the wonder drugs but provided the most important antibiotics available to medicine. Continued efforts have resulted in the improvement of these compounds with respect to potency, breadth of spectrum, activity against resistant pathogens, stability and pharmacokinetic properties. On the research front, major advances are being made on structural and regulatory biosynthetic genes and metabolic engineering of the pathways involved. New semisynthetic compounds especially those designed to combat resistance development are being examined in the clinic, and unusual non-antibiotic activities of these compounds are being pursued. Although seventy years of age, the beta-lactams are not yet ready for retirement.

Results of radial and astigmatic keratotomy by beginning refractive surgeons.

BACKGROUND: There is little information available on the results of radial and astigmatic keratotomy surgery that is performed by beginning refractive surgeons. METHODS: A retrospective review of all refractive keratotomies performed by Corneal Fellows in the University of California, Los Angeles, Department of Ophthalmology between October 1985 and October 1991 was performed. Data from all eyes with at least 3 months of follow-up were analyzed. Visual acuity, refractive error, and complication rates were compared with published case series. RESULTS: The mean preoperative spherical equivalent for the 79 eyes analyzed was -3.97 diopters (D) (range, -0.75 to -8.50 D). The mean postoperative spherical equivalent was -0.44 D (range, +1.50 to -3.88 D). The postoperative spherical equivalent was within 1.00 D of emmetropia in 85% of eyes, and uncorrected visual acuity was 20/40 or better in 94% of eyes. There were no vision-threatening complications. No patient lost more than one line of best-corrected visual acuity. CONCLUSION: Radial and astigmatic keratotomies that are performed by beginning refractive surgeons in a supervised setting can be safe and effective procedures with results comparable with those obtained by experienced refractive surgeons.

Decreased production of para-hydroxypenicillin V in penicillin V fermentations.

Penicillin V (phenoxymethyl penicillin) is produced by industrial strains of Penicillium chrysogenum in the presence of phenoxyacetic acid (POAc), a side-chain precursor for the penicillin V molecule. The wild-type strain of P. chrysogenum produces an undesirable penicillin byproduct, para-hydroxypenicillin V (p-OH penicillin V), in addition to penicillin V, via para-hydroxylation of POAc and subsequent incorporation of the p-OH phenoxyacetic acid into the penicillin molecule. Most of the p-OH penicillin V is produced late in cycle when the POAc concentration in the medium is nearly depleted. The level of p-OH penicillin V produced by the control strain ranges up to 10-15% of the total penicillins produced. 3-Phenoxypropionic acid and p-bromophenylacetic acid partially inhibit the formation of p-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 of p-OH penicillin V. Multi-step mutation and screening, starting with the wild-type strain, have culminated in isolation of mutants which produce p-OH penicillin V as 1% of the total penicillins with no adverse effect on penicillin V productivity.

Penicillin production by glucose-derepressed mutants of Penicillium chrysogenum.

Wild-type strains of Penicillium chrysogenum produce lower penicillin V titers in media containing excess glucose. Two mutant strains were isolated and shown to produce normal penicillin V titers in the presence of excess glucose. These strains, designated as glucose-repression insensitive (GRI) mutants, produced higher penicillin V titers than the wild-type strain in media containing lactose as the main carbohydrate source. In lactose-based media, the production of penicillin V was depressed to a much lesser extent by in-cycle additions of glucose with the GRI mutants when compared to the wild-type strain. In short-term biosynthesis experiments using washed cells in a medium containing glucose as the sole carbon source, the GRI mutants produced penicillin V at a faster rate than the wild-type strain. In fed-batch fermentations in 14-liter fermentors, where glucose was fed continuously and pH controlled, both GRI mutants produced more than 10% higher penicillin V titers than the wild-type strain. These results suggest that isolation of GRI mutants is an effective way to select for higher producing strains and that the synthesis of penicillin synthesizing enzymes in GRI mutants may be less repressed by glucose than in wild-type strains.

Four-incision radial keratotomy for low to moderate myopia.

The first 112 consecutive eyes (75 patients) to undergo four-incision radial keratotomy (RK) performed by four surgeons were evaluated retrospectively. The preoperative myopia (spherical equivalent) ranged from -1.12 to -7.00 D (95% between -1.12 and -4.25 D). The preoperative uncorrected visual acuity was 20/100 or worse in 95% of the eyes. A six-month follow-up was achieved in 95% of the eyes and 45% were followed over one year. Following four incision RK, 79% of the eyes achieved 20/40 or better vision and 82% were corrected to within 1 D of emmetropia. Following repeat RK in 15 eyes (13%), 82% had uncorrected visual acuity of 20/40 or better, and 90% were corrected to within 1 D of emmetropia. Only 3.5% of the eyes were overcorrected by more than 1 D and only one eye had induced astigmatism greater than 1 D. For eyes with preoperative myopia up to -4.25 D over 90% achieved uncorrected visual acuity of 20/40 or better and were within 1 D of emmetropia following primary four incision RK and a second four incisions in 13 eyes (12%). Only 3.5% of the eyes in this group were overcorrected by more than 1 D. There were no serious complications.

Genetic manipulation of antibiotic-producing microorganisms.

The application of directed selection techniques and genetic engineering methods for manipulation of antibiotic-producing microorganisms is generating a new era in industrial microbiology. Modern methods, based on advances in the knowledge of the biosynthetic pathways and regulatory mechanisms involved in the induction and repression of genes involved in antibiotic synthesis, provide a means of increasing antibiotic activity. Hence, recombinant DNA and protoplast fusion methods are used to alter the genetics of antibiotic producers in a semirandom fashion for the development of novel hybrid antibiotics. Directed mutation and selection, protoplast fusion, and both semirandom and specific recombinant DNA methods are examples of alternative procedures for manipulating the biosynthetic pathways of microorganisms for strain improvement and for new hybrid antibiotic synthesis.

UCLA clinical trial of radial keratotomy. Preliminary report.

A clinical trail of radial keratotomy ws begun under a strict research protocol at the Jules Stein Eye Institute in November 1979. The results for the first 52 eyes undergoing radial keratotomy are reported three months after surgery. Preoperatively uncorrected visual acuity was less than 20/200 in all 52 eyes. and postoperative visual acuity was less than 20/200 in 11 eyes (21%). Three months postoperatively, uncorrected visual acuity was 20/40 or better in 27 eyes (52%) and 20/20 or better in 13 eyes (25%). Postoperatively the best-corrected visual acuity decreased in 10 eyes (20%), but maximum decrease in any eye was one line of snellen letters. Preoperatively mean refractive error was -4.9 (/+-2.2) diopters, and postoperatively the mean decrease in myopia was 3.4 (/+-2.2) diopters. Postoperatively 13 eyes (25%) had a hyperopic refractive error of +0.25 to +3.25 diopters, but all of these eyes were able to accommodate and obtain 20/20 visual acuity without glasses. Postoperatively there was no statistically significant change in axial length, anterior chamber depth, or scleral rigidity, but there was a mean corneal endothelial cell loss of 10% (P = 0.0002). Decrease in myopia achieved by radial keratotomy did not correlate with the steepness of corneal curvature, corneal diameter, or scleral rigidity. Three months after surgery, significant symptoms of glare were present in 10 eyes (20%0, and annoying variable visual acuity was noted in five eyes (10%).

Association of 6-oxo-piperidine-2-carboxylic acid with penicillin V. Production on Penicillium chrysogenum fermentations.

Analysis of a 13C NMR spectrum of a concentrated broth from Penicillium chrysogenum fermentation revealed the presence of penicillin V and 6-oxo-piperidine-2-carboxylic acid(1) as the principal constituents. The latter lactam, identical to an authentic sample prepared by the cyclization of alpha-aminoadipic acid was present to the extent of 28 mol% of penicillin V. The lactam isolated form the broth was nearly racemic, having a slight excess of the L-isomer. This isolation provides further evidence regarding the biosynthetic precursors of the hydrophobic penicillins.

Biochemical properties of haploid and diploid strains of Penicillium chrysogenum.

An intensive parasexual genetics program in which industrial strains of Penicillium chrysogenum were used culminated in the isolation of a number of heterozygous diploid strains. The diploid clones were selected from heterokaryons formed from matings between mutant strains having complementary biochemical and conidial color markers. Several diploid cultures were compared with their haploid wild-type parents and other distantly related production strains on the basis of a variety of cultural and physiological criteria. The diploid strains characteristically produced conidia of larger volume and higher deoxyribonucleic acid content. Some were vigorous with respect to growth rate and onset and degree of conidiation. One diploid strain (WC-9) had a 46% greater oxygen uptake rate and oxidized glucose at a 57% greater rate than its haploid parent (M-2). It also produced 33% higher concentrations of beta-galactosidase, 66% more alkaline protease, and 53% more glucose oxidase than the M-2 haploid parent. The selection of rare stable diploid mold cultures through the use of parasexual genetics offers a unique approach to the direct selection of mutants with potential for increased enzyme formation.

Metabolism of tryptophan by Pseudomonas aureofaciens. 3. Production of substituted pyrrolnitrins from tryptophan analogues.

Exogenous tryptophan is metabolized by Pseudomonas aureofaciens to yield pyrrolnitrin [3-chloro-4-(2'-nitro-3'-chlorophenyl)-pyrrole], an antifungal agent. The ability of this culture to metabolize tryptophan analogues in a similar manner was investigated by addition of the appropriate compound to the fermentation. Tryptophan precursors and metabolites or nonphenyl-substituted tryptophans had little effect on pyrrolnitrin biosynthesis, but simple derivatives of indole inhibited the production of pyrrolnitrin. Tryptophans substituted at the 4 position decreased pyrrolnitrin production and were converted into the corresponding substituted indoles. Tryptophans substituted at the 5, 6, and 7 position with fluorine or at the 5 and 7 position with methyl yielded new pyrrolnitrin derivatives. Substitution of larger groups (such as chloro, bromo, trifluoromethyl, and methoxy) at these positions led to the formation of the intermediate, amino pyrrolnitrin [3-chloro-4-(2'-amino-3'-chlorophenyl)-pyrrole], with the appropriate new substituent. The trifluoromethyl group at the 6 position of tryptophan prevented chlorination at the 3 position of pyrrolnitrin.

Metabolism of tryptophans by Pseudomonas aureofaciens. VI. Production of pyrrolnitrin by selected Pseudomonas species.

Twenty-nine strains of Pseudomonas, classified as P. fluorescens biotype D or E or as P. multivorans, were examined for the production of pyrrolnitrin, an antifungal agent synthesized in P. aureofaciens. Eight strains were shown to produce pyrrolnitrin in shake-flask fermentation. Four cultures were from the multivorans taxon, and the remaining four were members of the fluorescens group. The antifungal agent produced in these strains was isolated and shown to be pyrrolnitrin by comparison with an authentic sample. The strains differed markedly with respect to the amount of pyrrolnitrin produced and in their utilization of exogenous tryptophan. Secondary metabolites, not related to pyrrolnitrin, were also examined and compared with those synthesized in P. aureofaciens. Marked differences were noted in both phenazine pigments and phenolic metabolites. The results of the study suggest that the production of pyrrolnitrin may be widespread in selected taxonomic groups of Pseudomonas.