Mycotoxins, drugs and other extrolites produced by species in Penicillium subgenus Penicillium

This article focuses on the recent world-wide advances of the biotechnology of the increasingly important fed-batch (or semi-batch) cultivation technique used in microbial processes. The history and characteristics of the fed-batch technique in microbial reactions are reviewed and examples of those fed-batch operations are cited which have greatly increased the productivity of microbial conversions in comparison to conventional batch operation; the various fed-batch techniques will be classified according to the mode of nutrient-feeding. Theoretical mathematical models are compared with experimental results. Fed-batch cultures with automatic feedback control are discussed in detail. Cultivation of high microbial cell concentrations, repeated fed-batch operations, and the start-up of microbial processes are described and, finally, some future prospects of fed-batch techniques in microbial processes are discussed.

Fed-batch techniques in microbial processes

The discovery and development of the β-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 β-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 β-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 β-lactams are not yet ready for retirement.

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

SUMMARY: The rates of utilization of energy-yielding substrates (glucose and oxygen) by Penicillium chrysogenum in glucose-limited chemostat cultures were resolved into requirements or ‘rations’ for growth and maintenance. The maintenance ration of glucose was almost all oxidized to carbon dioxide. Over the growth rate range 0.023--0.075 hr-1 only vegetative growth occurred; although the filamentous growth form predominated, the occurrence of pellets and swollen organisms increased with growth rate. At growth rates of 0.014 hr-1 and below, conidiation occurred and was maximal at a specific growth rate of 0.009 hr-1 (average doubling time 78 hr). After growth in chemostat culture the organism could be maintained in a non-growing state by supplying only the maintenance ration of glucose (0.022 g. glucose g. mycelial dry wt/hr). When growth in the chemostat was suddenly stopped by stopping the glucose feed, the mould autolyzed; autolysis was prevented by supplying the maintenance ration of glucose. When the glucose feed rate in chemostat cultures was decreased to the maintenance ration, mycelial differentiation occurred. Differentiation involved increased hyphal vacuolation, a decreased degree of oxidation of glucose, breakdown and resynthesis of nucleic acids and conidiation. The rates at which these changes occurred were inversely related to the growth rate prior to stopping growth. For maximum conidia formation there was an optimal glucose feed rate about 0.038 g. glucose/g. mycelial dry wt/hr, that is 1.7 x maintenance ration. The maintenance ration of glucose was shown to play a role in preventing autolysis and facilitating conidiation. Electron microscope studies showed that there was no change in the observed ultrastructure of cells (except degree of vacuolation) with change in specific growth rate from near the maximum to zero. The septa of the hyphae were found to be plugged.

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The Influence of Maintenance Energy and Growth Rate on the Metabolic Activity, Morphology and Conidiation of Penicillium chrysogenum

Many Fungi have a well-developed secondary metabolism. The diversity of fungal species and the diversification of biosynthetic gene clusters underscores a nearly limitless potential for metabolic variation and an untapped resource for drug discovery and synthetic biology. Much of the ecological success of the filamentous fungi in colonizing the planet is owed to their ability to deploy their secondary metabolites in concert with their penetrative and absorptive mode of life. Fungal secondary metabolites exhibit biological activities that have been developed into life-saving medicines and agrochemicals. Toxic metabolites, known as mycotoxins, contaminate human and livestock food and indoor environments. Secondary metabolites are determinants of fungal diseases of humans, animals, and plants. Secondary metabolites exhibit a staggering variation in chemical structures and biological activities, yet their biosynthetic pathways share a number of key characteristics. The genes encoding cooperative steps of a biosynthetic pathway tend to be located contiguously on the chromosome in coregulated gene clusters. Advances in genome sequencing, computational tools, and analytical chemistry are enabling the rapid connection of gene clusters with their metabolic products. At least three fungal drug precursors, penicillin K and V, mycophenolic acid, and pleuromutilin, have been produced by synthetic reconstruction and expression of respective gene clusters in heterologous hosts. This review summarizes general aspects of fungal secondary metabolism and recent developments in our understanding of how and why fungi make secondary metabolites, how these molecules are produced, and how their biosynthetic genes are distributed across the Fungi. The breadth of fungal secondary metabolite diversity is highlighted by recent information on the biosynthesis of important fungus-derived metabolites that have contributed to human health and agriculture and that have negatively impacted crops, food distribution, and human environments.

Biologically Active Secondary Metabolites from the Fungi

The production of penicillin by the surface culture of Penicillium notatum is the foundation upon which a large new industry has been built. Although cultivation of the mold in submerged culture now appears to be more economical and more practical from an industrial standpoint, surface cultures were utilized to produce the penicillin that first effected the remarkable clinical cures which indicated that the large investment of time and money made during the past three years would be justified. Previous investigators of penicillin production have not been successful in developing a highly productive medium. Fleming (1929), the discoverer of the drug, refers only to the use of a \"nutrient broth,\" whereas Clutterbuck, Lovell, and Raistrick (1932) used a modified Czapek-Dox medium. Although the yields of penicillin obtained by this group and by Fleming were not evaluated in terms of the Oxford unit adopted later, they were undoubtedly very low, at least when compared with present standards. Abraham et al. (1941), using the same modified Czapek-Dox medium with the addition of small amounts of a crude yeast extract, obtained only 2 to 6 Oxford units per ml. This paper will report on a few of more than five hundred experiments which have led to an increase in the yield of penicillin from the range of 2 to 6 Oxford units per ml to as much as 160 to 220 Oxford units per ml. This increase in yield has been achieved primarily by the proper selection of organisms and nutrients, including the use of corn steep liquor, the use of lactose as the principal carbohydrate, and the addition of nutrients during the course of the fermentation.3

Penicillin VIII. Production of Penicillin in Surface Cultures

Penicillin was first produced by cultivation of the fungus Penicillium notatum Westling on the surface of a liquid nutrient, such as Czapek-Dox glucose medium. Such a method of cultivation is very laborious, costly, and time-consuming when practiced on a large scale. Huge numbers of bottles or pans must be washed and sterilized, relatively small volumes of nutrient media must be dispensed into individual containers, and each container with its allotment of medium must be sterilized and inoculated. The incubation period for such cultures is usually 6 to 12 days, and, at the conclusion of the fermentation, considerable hand labor is required to remove the penicillin-containing liquors from the numerous fermentation vessels and from the fungus mycelium. It was obvious that a more economical fermentation process would result from growing the mold submerged and uniformly distributed in vats or tanks such as are used in other fermentation industries. Submerged mold fermentation processes have been previously used for the production of gallic acid (Calmette, 1902), gluconic acid (Moyer et at., 1940), and lactic acid (Ward et al., 1938), and the adaptation of this method to the cultivation of P. notatum appeared to offer a means of decreasing the labor involved, of decreasing the fermentation time, and of increasing the penicillin yield. This paper deals with the selection of a strain of P. notatum Westling suitable for the production of penicillin in submerged culture, and with investigations of nutrient media and culture conditions as nearly optimal as possible for this procedure.3 (P. notatum NRRL 832 was originally selected and evaluated for submerged penicillin production by the senior author.)

Penicillin: IX. The Laboratory Scale Production of Penicillin in Submerged Cultures by Penicillium notatum Westling (NRRL 832).

As penicillin chemistry unfolded during the early days of production development, it soon became evident that there existed more than one compound having the pharmacological and bactericidal properties associated with the name \"penicillin.\" American workers had isolated a pure crystalline penicillin (G) which was characterized by the benzyl radical and which yielded phenylacetic acid as a degradative product, whereas the crystalline penicillin isolated by the English workers (penicillin F) contained the A-2-pentenyl radical and yielded no phenylacetic acid on degradation, A-3-hexenoic acid being obtained in its place (Committee on Medical Research, Office of Scientific Research and Development, Washington; and the Medical Research Council, London). It was of interest, therefore, to ascertain the effect of adding phenylacetic acid to the culture medium in or upon which the mold was grown. Two possibilities were anticipated, both based on the assumption that the mold could use the phenylacetic acid as a building stone for the penicillin molecule. If this supposition were true, it would follow that (a) yields would be increased if synthesis of phenylacetic acid by the mold were the bottleneck in its penicillin production, and (b) the penicillin G:penicillin F ratio would be increased. The experiments described below show that the total penicillin yield was increased in both surface and submerged cultures, but no marked change in the ratio of penicillin types could be demonstrated as being due to phenylacetic acid.'

Penicillin: X. The Effect of Phenylacetic Acid on Penicillin Production.

(Granted under the act of March 3, 1883, as' amended April 30, 1928; 370 0. G. 757) 1 This application is made under the act of March 3, 1883, as amended by the act of April 30, 1928, and the invention herein described, if pat ented, may be manufactured and used by or for the Government of the United States of America for governmental purposes without the payment to us of any royalty thereon. This invention relates to a new and useful method for producing penicillin by the cultiva tion of molds, whereby the yield of penicillin is substantially increased above that previously ob tained. Although the production of penicillin is now a commercial enterprise, the yield of this valuable antibiotic material per unit volume of employed medium is very low, despite attempts to improve the yield by making numerous variations in the usual constituents of the nutrient medium and in the operating conditions. Accordingly, any means by which substantial ‘increases in yield can be obtained are of great practical signi?cance. We have found that striking increases in peni cillin production are obtained when phenylacetic acid or potential sources of phenylacetate ion, such as salts of phenylacetic acid or esters of phenylacetic acid, are incorporated in the aque ous nutrient medium used for the cultivation of penicillin-producing molds. These compounds are effective at levels ranging from 0.005 to 0.5 gram per 100 ml. of nutrient medium; however, we have observed that concentrations within the range of 0.02 to 0.20 gram per 100 ml. are espe cially effective, The use of quantities of phenyl acetic acid or its salts or esters in excess of 0.20 gram per 100 ml. does not result in proportion ately increased yields of penicillin, and, accord ingly, we prefer to use concentrations of these compounds within the range 0.02 to 0.20 gram per

Andrew J. Moyer

Papers

Penicillin VIII. Production of Penicillin in Surface Cultures

Penicillin: IX. The Laboratory Scale Production of Penicillin in Submerged Cultures by Penicillium notatum Westling (NRRL 832).

Penicillin: X. The Effect of Phenylacetic Acid on Penicillin Production.

Method for production of increased yields of penicillin

The production and characterization of ultraviolet‐induced mutations in aspergillus terreus. iii. biochemical characteristics of the mutations