BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 TABLE OF CONTENTS INTRODUCTION..................................................................................................................... 2 GENETIC ASPECTS OF ANTIBIOTICS ...................................................................... 5 ANTIBIOTIC PRODUCTION AND REGULATION ................................................ 7 DISCOVERY OF NEW ANTIBIOTICS........................................................................ 12 CONCLUSION AND PERSPECTIVES......................................................................... 13 REFERENCES......................................................................................................................... 14 1 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 Introduction Antibiotics have been used for more than fifty years, and they are the cornerstone of infectious disease treatment. In addition, these bioactive compounds have been applied to many other therapeutic purposes, for example, their use as cytotoxic agents, as disease-control agents in veterinary medicine and plant pathology, as food preservatives and as animal growth promoters. [1], [2] What is an antibiotic? Vuillemin (1889), in introducing the term `antibiosis` said, “..one creature destroys the life of another to perserve its own; the first is completely active, the second completely passive. The conception is so simple that no one has ever thought of giving it a name...In order to simplify words we will call it antibiosis...!” [3] The term ´antibiotic´ was coined by the discoverer of streptomycin, Selman A. Waksman (1945), who described an antibiotic as “a chemical substance of microbial origin that possesses antibiotic powers”. Thirty six years later, in the Webster´s Third International Dictionary (1981), an antibiotic was mentioned as “a substance produced by a micro-organism (as a bacterium or a fungus) and in dilute solution having the capacity to inhibit the growth of or kill another microorganism (such as a disease germ)”. Today, in Brock´s textbook of microbiology, antibiotic is defined as “a chemical agent produced by one organism that is harmful to other organisms”. [1], [3] Finally, antibiotics are products of secondary metabolism with an incidental action in minimal concentration on growth processes and they are the chemical interface between microbes and the rest of the world. [4],[5] Penicillin, discovered by Fleming in 1928, was isolated and clinically tested in the early 1940s by Chain and his group at Oxford, but to the surprise of many it was not the first known antibiotic. Mycophenolic acid had already been isolated from a penicillium by Gosio in 1896 in his studies of pellagra. [3], [6] In the 1940s, Waksman started systematic screening programmes in the search for antibiotics produced by actinomycetes. The discovery of streptomycin in 1943, isolated from Streptomyces griseus, became a milestone and opened the floodgates for the study of the production of antibiotics by actinomycetes, thus new compounds were isolated in rapid succession. [3], [6] 2 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 In the early 1960s, the isolation of 6-aminopenicillanic acid (6-APA) opened the way for semisynthetic antibiotics, followed by intensified search for new antibiotics and extensive investigation of the biosynthesis, chemistry, pharmacology and modes of action of resistance modification to known antibiotics. [3], [6] It is significant in the development of antibiotics to mention the rapid increase over a short period of time. In 1955 only 500 antibiotics had been known twenty years later, in 1975, this number was already 500. Today, more than 13000 antibiotic natural products are known. The total number of semi-synthetic derivatives of natural antibiotics is close to 100000. [5] Todays antibiotic research is characterized by studies of antibiotics as secondary metabolites. [3] Most of the antibiotics are produced by bacteria (especially by actinomycetes) and microscopic fungi. The antibiotic, which are produced by eubacteria, are mostly peptidic compounds. [3] Antibiotics can be classified in several ways. The most common method classifies them according to their chemical structure. As antibiotics sharing the same or similar chemical structure will generally show similar patterns of antibacterial activity, effectiveness, toxicity and allergic potential. [7] An open chemical classification system of antibiotics was proposed by Berdy (1974). This system consists of a primary and secondary classification, methods of classification and the appropriate code system. [6] In the primary classification, antibiotics are arranged into families according to the following nine principal constituents: sugar, macrocyclic lactone ring, quinone skeleton, amino acid, nitrogen-containing heterocyclic system, oxygen-containing heterocyclic system, alicyclic skeleton, aromatic skeleton and aliphatic skeleton. [6] The secondary system organizes antibiotics, within the families, into subgroups according to their individual characteristics like constituents, the size of the molecule, variants of similar identical skeletons, antibiotics of practical importance, the type of linkage or quality of chromophore, similarities of antibiotics and biological activity. [3] 3 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 In the code system of Berdy´s chemical classification, individual groups of antibiotics received four-element code numbers. The first element of the code number shows the family, the second elements indicates the subfamily. The third element indicates the group and the fourth gives the type. [3] The role of antibiotic in the metabolism is not completely understood. Several proposed functions for antibiotics are no longer accepted, whereas other ideas are still under consideration. For example, antibiotics as evolutionary relics, waste products of cellular metabolism, reserve food materials, spore coat components, or breakdown products derived from cellular macromolecules are all currently being considered. [6] There is remarkable significance in killing or inhibiting the growth of other organisms, common among soil saprophytes. Also the important function of antibiotics in cellular differentiation, for instance, in the transition from vegetative cells to spores or from spores to vegetative cells, is a remarkable function to note. It is inconceivable that the complex multienzyme reactions sequences of antibiotic biosynthesis have been retained in nature without some beneficial effect on survival. [6] 4 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 Genetic Aspects of Antibiotics The synthesis of antibiotics by micro-organisms is not a random phenomenon. It is, rather, the logical consequence of the expression of genetic information coded in the DNA, which must have a beneficial survival effect for the producer strain. [8] The over-production of huge amounts of penicillin by industrial strains of Penicillium chrysogenum is more difficult to understand. It is obviously a wasteful process for the producer strain because more energy is spend in precursors for synthesizing and excreting penicillin than in synthesizing DNA, protein and other macromolecules. [8] The goal of industrial microbiologists is to exploit the microbial strain in order to get more, higher quality and cheaper antibiotics for the welfare of humanity. [8] The expression of the genetic information for antibiotics, located in the chromosome, the extra chromosomal DNA elements, or in both, is controlled at the transcriptional, translational and post-translational levels by a series of intracellular effectors that exert regulatory signals in response to the environmental conditions. This genetic information for the synthesis of secondary metabolites might be amplified by increasing the number of gene copies in the cell as this will generally lead to an increase in the antibiotic produced, assuming that the system controlling gene expression does not limit expression of the duplicated genes. [8] The availability of a genetic recombination system is useful in constructing a genetic map of the culture. The model for investigations is the map of streptomyces coelicolor. Most Streptomyces chromosomal DNA molecules are about 8-Mb long, with terminal-inverted repeats. This size is unusually large for a bacterium, and they also have a higher G - C content (more than 70%) than nearly all other organisms. Streptomyces strains produce many kinds of secondary metabolites, including antibiotics and bioactive compounds, because of having many gene clusters, which encode enzymes for many secondary metabolic pathways. [10] Additional, plasmids have been found in many antibiotic producers, for instance, actinomycetes contain plasmids, or extra chromosomal DNA. In S. coelicolor, the linear 356kb plasmid is a sex factor (SCP 1), which can be transferred to other species of Streptomyces. An example of this is antibiotic synthetase (methylenomycin A) that is coded by plasmid genes. [9] The method, which consists of attaching a fragment of DNA that codes for antibiotic synthesis to a plasmid that replicates independently, is a very useful tool for gene amplification. Transferring 5 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 the ability to produce an antibiotic, to a microbial species different of the natural producer, may be convenient for several reasons: firstly, it would be adequate to use fast-growing organisms, secondly, it would be convenient to transfer the antibiotic-production ability to biochemically and genetically well-known micro-organisms and lastly, a new host might be chosen in which membrane permeability would be easily altered, facilitating excretion. [8] 6 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 Antibiotic Production and Regulation Wild type antibiotic-producing cells appear to be strictly regulated. Compared to the industrial mutants, they do not waste large amounts of metabolites. Industrial microbiologists are interested in eliminating these regulatory mechanisms in order to obtain strains which are even more strongly deregulated. [8] Antibiotics exert feed-back regulations on their own formation, either by repressing one or more antibiotic synthetases or by interfering with their own formation by inhibiting an enzyme involved in their biosynthesis. A second type of feed-back regulation is involved in a branched pathway leading to a primary and secondary metabolite. Negative feed-back regulation of an early common enzyme by the primary end product might be expected to diminish antibiotic production; for instance, lysine interferes with penicillin and cephalosporin biosynthesis by feed-back inhibition and repression of homocitrate synthase. The use of different carbon sources for production of a given antibiotic results in different rates and extents of production. The earliest recognition of a negative effect of glucose on a secondary process involved benzylpenicillin production. Glucose was excellent for the growth of Penicillium chrysogenum but poor for penicillin production. Lactose showed the opposite consequences. This demonstrates why disaccharides are better carbon sources than glucose.[8],[9] In the last few years, a new regulatory mechanism, similar to carbon catabolite regulation, which controls utilization of nitrogen sources by the cell has been discovered. [8] In addition, induction is clearly involved in the control of biosynthesis of special (secondary) metabolism. One example is the tryptophan induction of enzymes involved in ergot alkaloid synthesis by Claviceps. Dimethylallyl-tryptophan synthase is induced by tryptophan analogues and the inhibition occurs by phosphate. [8] Finally, the concentration of phosphate has an effect on the biosynthesis of many antibiotics and secondary metabolites. Antibiotics are synthesized only at concentrations of inorganic phosphate sub optimal for growth. The phosphate effect seems to be exerted at two different expression levels. First, phosphate represses the formation of candicidin synthases during the growth phase and second, phosphate inhibits the activity of the same enzymes once they have been formed. [8] 7 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 Fermentation The growth phases of bacteria in batch culture was investigated by Robert Koch (1843-1910) and Nägeli (1877). They did this by determining the amount of acid produced in a culture, whilst others (e.g. Buchner et al., 1887) used the plating-out method of Koch in quantitative manners. Lane-Claypon (1909) and later Slator (1917) did much to develop the phases of growth in batch cultures of micro-organisms. [11] In batch cultures containing nutritionally rich media, high levels of antibiotics are usually produced only after most of the cellular growth has already occurred. The logarithmic phase of growth is called the "trophophase", however, the production phase is termed the "idiophase". [12] In the growth phase there is extensive metabolism where the microbes rapidly replicate their cell components as a prerequisite to growth and cell division. The stationary phase of growth represented virtually total metabolic inactivity on part of micro-organisms. The separation between trophophase and idiophase is unclear in filamentous micro-organisms (actinomycetes and fungi). [11] Dry weight is a poor criterion of true growth because cellular mass consists of true structural material required for cell replication (cell walls, membranes, cytoplasmic organelles, ribosomes, nuclei, etc.) and assimilatory reserve materials, such as polyols, lipids, polyphosphates, and nonstructural carbohydrates. Nonreplicatory growth usually results from the accumulation of reserve materials, which may account for up to 50 to 60% of the dry weight at the end of a fermentation. The best parameter with which to measure true replicatory growth is the increase in deoxyribonucleic acid. This is true because cell growth often can be clearly dissociated from antibiotic production. Other parameters that often indicate the end of the replicatory growth phase are a drop in respiratory activity and a decrease in the ribonucleic acid synthesis rate. [12] In many antibiotic fermentations, trophophase-idiophase dynamics occur in complex media capable of supporting rapid growth, but the two phases overlap in defined media supporting slow growth. Depletion of a nutritional growth-limiting components arrests growth and initiates idiolite biosynthesis. In defined medium supporting only slow growth, some nutritional factors may be growth limiting from the very start of cultivation, thus favoring antibiotic production while slow growth is still occurring. In this case, the development and control of fermentation processes have increasingly attracted the attention of biochemical engineers and microbiologists. 8 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 In most antibiotic fermentations, more can be gained by optimizing control of the environment in which the culture grows than by improving unit operations. That is why building up and controlling a fermentation process requires the integration of both engineering and microbiological knowledge. [12], [13] Generally, antibiotics are produced in batch fermentations or in feed-type processes and the aerated and agitated vessel is the standard type. The batch fermentation has the advantage of simplicity because only the physical parameters have to be optimised during the scale. Air lift fermenters and reactors with special mechanical agitation systems have been designed for the above reason. [13] The feed rate, which might lead to higher yields and to greater reproducibility, has to be considered in every optimization. There are three methods for controlling the feed rate. First, the feed is predetermined and can be constant or variable. Second, in a weakly buffered medium, the pH can be kept constant by adding a controlled amount of substrate. Finally, the feed can be controlled by a function of the dissolved oxygen concentration. Which controlling system to chose depends on the organism and on the equipment available. [13] In the laboratory a careful standardization of flask form and size, filling volume and flask cover are important, as are the control of throw and rotational speed of the shaker and the broth temperature on the different shelves. In long lasting antibiotic fermentations, small variations of one of the parameters are determined. [13] After optimising the control parameters so that the product formation will be at its maximum, the production will be scaled-up from flask to the fermenter. [14] There are many important variables which are measured and controlled at all times. First, the composition of the fermentation medium is important. Influencing factors are the carbon sources and the nitrogen sources. For example, highest oxytetracycline production was obtained on media containing starch or maltose compared to lowered antibiotic production, which was found on glucose media. [14] An optimum biosynthesis production requires the presence of both organic and mineral nitrogen sources in the medium. Streptomyces aureofaciens utilizes predominantly ammonium nitrogen. [14] Besides the composition of the fermentation medium, some crucial cultivation conditions are aeration, temperature, effect of pH value, effect of inoculum and technological problems, growth 9 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 and production, morphology of the cells, rheology of the culture, effect of orthophosphate, stimulators and inhibitors of production as well as Chlorination/Methylation and the effect of antibiotics on producing strains. [13],[14] In particular, the mechanical agitation receives a lot of attention. It is important to facilitate transfer of oxygen, nutrients and heat. For example, the cellular morphology of Penicillium chrysogenum is influenced by the agitation intensity. On one hand, at reduced agitation the cells grew as hyphae, while on the other and, more intensive agitation produced short, branched mycelium. Additional, agitation determines the formation and the stability of pellets. It is incidental that the morphology of the cells and the formation of pellets control the rheology of the culture broth. Fig. 1 attempts to summarize the complex interactions of all the possible parameters. [13] Fig.1: Schematic presentation of the interplay between the organism and its environment [13] The first antibiotic of the tetracycline series, chlortetracycline, produced by the actinomycete, Streptomyces aureofaciens, was described by Duggar (1948). The chemical structure consists of a naphtacene ring. After the discovery of tetracyclines, they began to be produced on an industrial scale. For this reason, results of suitable fermentation technological procedures were obtained in various laboratories in the period 1948-1957. [2], [14] Fermentation tanks for tetracycline production, with a working volume of 100-150m³, must provide for sufficient oxygen transfer (0.4-0.8 µmol/liter per minute) because the minimum supply with oxygen is of particular importance. The strains for industrial fermentations are held either at freeze dried or at liquid nitrogen temperatures. The cultivation takes place at 29°C and pH 5,8-6,0. 10 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 The production strains utilize sucrose, starch, or technical glucose. Calcium carbonate binds the formed antibiotic. Inorganic salts and a controlled level of phosphates are also influencing factors for a successful fermentation. Chloride ions serve as precursors. Antifoaming agents are animal or vegetable lipids. The growth rate is regulated by the selection of C and N sources in broth, additionally by the technical possibility of oxygen transfer. [15] The chlortetracycline synthesis is a complex metabolic pathway, which starts with the formation of malonamoyl CoA and anthracene synthase. The glycolysis is suppressed by using the inhibitor benzylthiocyanate whereas, the pentose phosphate cycle is increased. The concentration of the second last enzyme, anhydrotetracycline oxygenase, is proportional to the rate of antibiotic sythesis; stimulated by benzylthiocyanate and repressed by phosphate. Besides penicillin, tetracyclines are the most frequently applied antibiotics in human medicine in the United States, and it is used in technical and veterinary practice as well. [2],[ 15] 11 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 Discovery of new Antibiotics Because of the microbial formation of resistance to antibiotics, there is a continuing and cyclical demand on new antibiotics. [16] In general, the screening process for a new bioactive microbial metabolite begins with the isolation of the producing species from its natural habitat. They mostly belong to the actinomycetes or to streptomyces sp..[16] In the first step the species are cultivated by fermentation for one year. After the recognition of a new microbial metabolite, it is evaluated in detail, including animal tests, toxicology, pharmacokinetics, scale-up and structure determination, that all together takes 2-3 years to complete. This step is followed by the clinical or field trial. Specific properties are investigated with the help of isolation and separation techniques, especially automated chromatographic methods. Criteria for the characterization are the taxonomy, culture morphology, biochemistry etc. [5] The essential pathways of the bacterial fatty acid biosynthesis and the peptidoglycan layer offer are very interesting fundamentals for new antibiotic research. As for instance, daptomycin (Cubicin), which biosynthetic gene cluster is related to acyldepsipeptidolactones, is a new antibiotic. It is approved for human use to treat bacterial interfections. Effective antibacterial targets are also natural products like cerulenin and thiolactomycin, inhibitors of the FabB/F ketosynthases, and the synthetic molecules triclosan and isoniazid target the enoyl-ACP reductase FabI. [16] New antibiotic research shows Platensimycin, produced by Streptomyces platensis, as a selective FabF inhibitor. Platensimycin interacts specifically with the acylenzyme intermediate of the targeting protein. It is selective for b-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. [17] Additional, there are glycopeptides, which block lipid-II-dependent peptidoglycan maturation steps. For example, dalbavancin, telavancin and oritavancin prevent the transpeptidase-mediated cross linking. [16] The discovery of new antibiotics is very important because bacterial infection will be always a serious threat to human lives. 12 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 Conclusion and Perspectives Searching for new antibiotics is becoming increasingly necessary. Antibiotics are useful in combating human, animal, and plant diseases. Micro-organisms provide a unique source of unexpected, helpful products and their natural products are the origin of most of the antibiotics on the market today. [6] Although antibiotics are becoming more necessary, there is an alarming deficiency of new antibiotics today. Furthermore, new antibiotic resistant bacterial strains seem to arise daily. The development of the genetic and regulatory mechanisms as well as controlling and optimisation of antibiotic synthesis is very important. [8] Besides antibiotics, there are “other” bioactive products including enzyme inhibitors, pharmacologically, immunologically, and physiologically active agents, mycotoxins, phytotoxins, herbicides etc., which are also isolated from micro-organisms. The investigation of interactions of micro-organisms via their secondary metabolites with other microbes, plant or invertebrates is of high value. 13 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 References [1] Julian Davies, 2006 Are antibiotics naturally antibiotics? Journal Industrial Microbiology Biotechnology 33: 496–499 [2] Ward, O.P., 1989 Fermentation Biotechnology: Principles, Processes and Products Pp. i-xii, 1-227, Prentice-Hall [3] Vladimir Betina, 1994 Bioactive secondary metabolite of micro-organisms Vol 30, Elsevier Science B.V., Amsterdam: 98-120 [4] Abraham, E.P. et al., 1978 Antibiotics and Other Secondary: Metabolites Biosynthesis and Production No. 5, Academic Press, London/ H. Zähner The Search of New Secondary Metabolism Institut of Biology II, Tübingen: 1-17 [5] M.E. Bushell and U. Gräfe, 1989 Bioactive metabolites from micro-organisms Vol 27, Publishers B.V., Amsterdam/ J. Berdy The Discovery of New Bioactive Microbial Metabolites: Screening and Identification Institut of Drug Research, Budapest: 3-25 [6] Erick J. Vandamme, 1984 Biotechnology of Industrial Antibiotics Vol 22, Marcel Dekker, Inc./ Antibiotic Search and Production: An Overview, Laboratory of General and Industrial Microbiology Ghent: 3-31 14 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” Nadine Kallweit (20288655) October 31th, 2007 [7] http://dermnetnz.org/treatments/antibiotics.html (last update: 26. December) [8] Abraham, E.P. et al., 1978 Antibiotics and Other Secondary: Metabolites Biosynthesis and Production No. 5, Academic Press, London/ J.-F. Martin Manipulation of Gene Expression in the Development of Antibiotic Production Department of Microbiology, Salamanca: 19-37 [9] Erick J. Vandamme, 1984 Biotechnology of Industrial Antibiotics Vol 22, Marcel Dekker, Inc./ A.L. Demain Biology of Antibiotic Formation Fermentation Microbiology Laboratory, Massachusetts: 33-42 [10] Satoshi Omura et al, 2001 Microbiology, Genome sequence of an industrial micro-organism Streptomyces avermitilis: Deducing the ability of producing secondary metabolites The Kitasato Institute for Life Sciences, Kitasato University, Tokyo [11] A.H. Rose, 1979 Secondary products of metabolism Vol 3, Academic Press, London/ Production and Industrial Importance of Secondary Products of Metabolism Zymology Laboratory, School of Biological Science, Bath: 2-34 [12] Juan F. Martin and Arnold Demain, l980 Control of Antibiotic Biosynthesis Microbiological review, Vol. 44, No. 2: p. 230-251 15 BIOL 443 Fermentation Biotechnology „Microbial production of antibiotics” [13] Nadine Kallweit (20288655) October 31th, 2007 Abraham, E.P. et al., 1978 Antibiotics and Other Secondary: Metabolites Biosynthesis and Production No. 5, Academic Press, London/ M.T. Küenzi Process Design and Control in Antibiotic Fermentations Basel: 39-56 [14] A.H. Rose, 1979 Secondary products of metabolism Vol 3, Academic Press, London/Z. Hostalek, M. Blumauerova and Z. Vanek Tetracycline Antiobiotics Czechoslovak Academy of Sciences, Praque: 293-354 [15] Erick J. Vandamme, 1984 Biotechnology of Industrial Antibiotics Vol 22, Marcel Dekker, Inc./ Miloslav Podojil et al The Tetracyclines: Properties, Biosynthesis, and Fermentation Institute of Microbiology, Praque: 259-279 [16] Jon Clardy, Michael A. Fischbach & Christopher T. Walsh, 2006 New antibiotics from bacterial natural products Nature Biotechnology, Vol 24, Number 12: 1541-1550 [17] Jun Wang et al, 2007 Platensimycin is a selective FabF inhibitor with potent antibiotic properties nature 04784, Vol 441: 358-361 16