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TERM PAPER OF

“MICROBIAL PHYSIOLOGY AND METABOLISM”

SUBMITTED BY:

NAME: SHASHI SHARMA

COURSE: M.Sc. (MICROBIOLOGY)

PROGRAM CODE: 2403

SECTION: RP8003

ROLL NO: B15

REG. NO: 11006142

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Table of Contents:

1. INTRODUCTION………………………………………………………..22. Industrial importance……………………………………………………2-3

2.1 PENICILLIN AND CEPHALOSPORIN BIOSYNTHESIS………3-42.2 IMPROVEMENT OF β-LACTAM ANTIBIOTICS INDUSTRIAL

PRODUCTION………………………………………………………4-72.3 Molecular improvement of penicillin and cephalosporin production…82.4 Industrial application…………………………………………………..9

3. Discovery of Penicillium notatum…………………………………………104. Further development of penicillin…………………………………………115. The Nobel Prize and the continuing importance of penicillin…………..126. Recent researches……………………………………………………………12-147. Market Value of Penicillium Notatum……………………………………..158. Future Prospective……………………………………………………………159. References……………………………………………………………………..16

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1. INTRODUCTION

Here stated is the introduction for penicillin notatum which makes clear the reason for selecting this microorganism for this term paper .Species of Penicillium are ubiquitous saprobes, whose numerous conidia are easily distributed through the atmosphere and are common in soils. In soil analyses, using dilution plate techniques, Penicillium species are detected with high frequency (Domsch et al., 1993). However, very little is known of interactions between Penicillium species and other soil fungi, or even on plan growth. Penicillium species generally occurr at greater soil depths than species of other genera, and have low concentrations in rhizosphere soils (Domsch et al., 1993). Some species of Penicillium are well known for their activities to produce antibiotics (e.g. Penicillin), and therefore Penicillium sp. is one of the best researched genera, with regard to biochemistry. All strains of Penicillium so far tested are able to solubilize metaphosphates and utilize them as phosphorus sources (Picci, 1965). Many species have been shown to contain mycoviruses (Bozarth, 1972). There are some reports that Penicillium species can suppress root pathogens; Penicillium chrysogenum has been reported to be able to control Verticillium wilt of tomato, when roots are dipped in a spore suspension before planting (Dutta, 1981). Penicillium notatum has also been reported to inhibit and reduce the number of rust pustules in wheat caused by Puccinia graminis f. sp. tritid (Mishra and Tiwari, 1976). Little is also known about plant growth stimulants produced by Penicillium spp. The objective of this study was therefore to investigate whether P. notatum (KMITL 99) can promote plant growth of Chinese mustard (Brassica campestris var. chinensis), Chinese radish (Raphanus sativas var. longipinnatus) and cucumber (Cucumis sativus). The optimum concentration of spore suspensions for promotion of plant growth was also investigated.

2. Industrial importance:

In industrial fermentations, Penicillium chrysogenum uses sulfate as the source of sulfur for the biosynthesis of penicillin. By a PCR-based approach, two genes, sutA and sutB, whose encoded products belong to the SulP superfamily of sulfate permeases were isolated. Transformation of a sulfate uptake-negative sB3 mutant of Aspergillus nidulans with the sutB gene completely restored sulfate uptake activity. The sutA gene did not complement theA. nidulans sB3 mutation, even when expressed under control of the sutBpromoter. Expression of both sutA and sutB in P. chrysogenum is induced by growth under sulfur starvation conditions. However, sutA is expressed to a much lower level than is sutB. Disruption of sutB resulted in a loss of sulfate uptake ability. Overall, the results show that SutB is the major sulfate permease involved in sulfate uptake by P. chrysogenum.

Filamentous fungi are saprophytic organisms secreting a wide array and high level of proteins involved in the breakdownand recycling of complex polymers from both plants andanimal

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tissues. These characteristics and the existence of a well-established technology for large-scale fermentation of these organisms advanced their industrial application in secretion of heterologous proteins. Species of Aspergillus and Trichoderma have been extensively used as model organisms for diverse transformation and expression systems. Although Penicillium chrysogenum is of significant industrial importance and has the “generally recognized as safe” status of the U.S. Food and Drug Administration, only preliminary attempts have been made to utilize this fungus as a host for homologous and heterologous protein production and secretion. One of the major reasons might be that in contrast to Aspergillus and Trichoderma, no suitable promoter system for P. chrysogenum has been available so far.

2.1 PENICILLIN AND CEPHALOSPORIN BIOSYNTHESIS

Once some physiological characteristics of both P. chrysogenum and A. chrysogenum were known, the nextstudies were focused on the biosynthesis of penicillin andcephalosporin C. It was observed that these and some othermicroorganisms use the same pathway for the production ofdiverse β-lactam antibiotics (Fig. 2). For penicillin andcephalosporin, the pathway begins with non-ribosomal condensationof three leading amino acids: L-α-aminoadipicacid, L-cysteine, and L-valine to produce the tripeptide δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (or its abbreviation,ACV) (Arnstein and Morris, 1960). During the secondstep of the pathway, tripeptide cycling takes place to formisopenicillin N (Fawcett et al., 1976), the first intermediary of the pathway with antibiotic activity. At this point, thepathway diverges for the two microorganisms: in the case ofPenicillium (and the other penicillin-producing fungi), the lateral chain of the aminoadipyl is exchanged by a hydrophobiclateral chain (Jayatilake et al., 1981), whereas inAcremonium, isopenicillin N is converted into penicillin Nby means of a two-enzymes system: an acyl CoA synthetaseand an acyl CoA racemase (Ullán et al., 2002a); althoughthe final transformation into penicillin N also requires hydrolysisof the CoA thioester, by means of differentthioesterases (Knihinicki et al., 1991). In the case of cephalosporin’sbiosynthetic pathway, penicillin N is transformedsubsequently into deacetoxycephalosporin C, expandingthe thiazolidinic ring of five atoms into adihydrothiazonic ring of six (typical of cephalosporins andcephamicins). The enzyme responsible for this step, calledDAOC synthase/DAC hydroxylase, is also in charge of hydroxylatingdeacetoxicephalosporin C leading to deacetylcephalosporinC formation (Samson et al., 1987). The laststep in cephalosporin biosynthesis is the acetylation ofdeacetylcephalosporin C into cephalosporin C (Fujisawa etal., 1973 and 1975).

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Figure. Biosynthetic pathways for penicillin and cephalosporin.

2.2IMPROVEMENT OF β-LACTAM ANTIBIOTICS INDUSTRIAL PRODUCTION

Application of classical and recombinant DNA technique has been very important in the increased production of penicillin and cephalosporin as well as the production of suitable intermediates to obtain more potent and low-cost semi-synthetic antibiotics.Classical improvement of penicillin and cephalosporin production The low production of the original P. chrysogenum and A. chrysogenum strains forced the potential genetic exploitation of both microorganisms, aimed at generating commercial superproducer strains of β-lactam antibiotics. From a classical improvement approach, where mutant agents are used at random and high producing mutants are subsequently selected,

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Figure 3. A. Gene organization of penicillin biosynthesis genes in Penicilliumchrysogenum, sizes estimated from Fierro et al. (2006). B. Geneorganization of cephalosporin biosynthesis genes, modified from Schmittet al. (2004).

two types of mutant agents have been traditionally employed: physical (ultraviolet radiation, gamma rays or Xrays), and chemical (ethyl methanosulphonate, N-methyl-N’-nitro-N-nitrosoguanidine, nitrogenated mustards, or nitrousacid).

Experimental models for production, selection, and employmentof P. chrysogenum mutant strains, with highpenicillin yields, have been developed since the middle ofthe last century. The starting point was the isolation of theWisconsin Q-176 strain, although the subsequent mutationexperiments were not achieved directly with thisstrain, but rather with its descendants, obtained after analyzingthousands of spontaneous mutant strains (consideringtheir capacity to produce the antibiotic). Reductionsin sporulation and even in fungal growth are also desirablecharacteristics (Elander and Espenshade, 1976); itshould not be forgotten that β-lactam antibiotics are secondarymetabolites and, therefore, they are not linked tomicrobial growth. An example of this is the NRRL-1951strain, in which a decrease of up to 60% in growthand sporulation was obtained, along with a six-fold increasein antibiotic production.Regarding Acremonium chrysogenum, a program to improve the low cephalosporin C titers produced by theBrotzu strain began in the middle of the last century. Mutagenesi of this strain was useful to isolate a mutant strain called M8650, which became the progenitor strain fomany programs of industrial improvement (Elander an Aoki, 1982). Fig. 5 shows the first A. chrysogenum improved mutants production chart, obtained after treatment with UV rays as mutant agent. As an example, an improved mutant strain developed by the American company Eli Lilly & Co., called CW19, produced three time more antibiotics than the Brotzu strain and, under

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favorablefermentation conditions; it could produce up to 15 times more antibiotic than the progenitor strain (Elander and Espenshade, 1976).

For both Penicillium and Acremonium, as importan as mutagenesis per se, is the rational selection of mutant to find superproducer strains (mainly in largescal strain improvement programs). To accomplish this, different strategies, chemical compounds, and even protein activities have been employed as indicators o the presence of improved mutants (Queener et al., 1975 Trilli et al., 1978; Chang and Elander, 1979). Perhaps, one of the most used strategies, due to its low cost, Inthe isolation of mutants grown in smaller and more compact colonies on well-defined chemical media. The good correlation between the presence of improved mutants and a progressive reduction in the diameter anvegetative development of colonies was confirmed later (Vialta et al., 1997) Although highly productive strains have been obtained through the above mentioned methods, further improvement is needed, specifically in the titer achieved at the end of fermentation and the stability of strains. Industrial strains are very unstable, because mutagenic events are random and do not necessarily affect only the genes involved in antibiotic synthesis. In practice, industrial strains mutate constantly, and re-isolation of the best performing strains is conducted routinely, since prolonged storage of high producing strains can occasionally result in the loss of their productivity or, at least, part of it (Barber et al., 2004). Molecular biology techniques helped to know in detail the nature of changes occurring during the random mutagenic processes (either when increased production is achieved or when lower yields are obtained) and to specifically locate such changes in the microorganism’s genome. In this way, it has been proven that high penicillin-producer strains possess the biosynthetic “cluster” amplified in tandem repeats (several repeated copies, one after the other) (Fierro et al., 1995). Instability of industrial strains can be explained by a higher probability of recombinogenic events, implying these iterative sequences.

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Figure: Penicillin production improvement: a classical approach (from Elander, 1967).

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Figur: First A. chrysogenum improved mutants production chart (fromElander et al., 1976).

2.3 Molecular improvement of penicillin and cephalosporin production:

Molecular biology represents an advanced tool in the genetic improvement of industrial producer strains. These techniques allow for controlled modifications of the microorganisms’ genome and establishment of causeeffect relationships between any introduced alteration and the observed result (which is harder to analyze in mutations induced by the classic methods). Thanks to this discipline, the changes introduced at all levels of the biosynthetic pathway (incorporation of precursor compounds, assembly of intermediary products of the pathway, and secretion into the medium) can be followed. Likewise, the effects of introducing factors affecting the process globally can be observed and compounds that formerly could not be produced by the microorganism can be obtained.

The use of genetic engineering techniques has enabled new approaches to the development of higher antibiotic- producing strains. Once the genes responsible for the biosynthesis of antibiotics had been cloned (or, at least, some of them), the first assays to improve the productive strains were based on the idea that if production depended upon the expression of given genes, any change capable of positively affecting that expression would be translated into increased production. The simplest way to increase production at these initial stages, applying recombinant DNA techniques, consisted in increasing the genetic dose (the increase of the number of gene copies encoding a determined proteinwithin the microorganism, a copy “without secondary effects” of the amplifying process observed in classical improvement processes). The development of better fungal transformation techniques contributed to molecular improvement; these transformation methods were based on modifications developed for the bread yeast, Saccharomyces cerevisiae (Hinnen et al., 1978). These methods consist of some basic steps: protoplasts production from mycelium, and their transformation and regeneration. Through these techniques, it was possible to observe the effect induced by integration of several structural genes in the fungal genome on antibiotic production.

The first successful application, in the field of β-lactam antibiotics, with increased doses of relevant genes was achieved with the cefEF gene in Acremonium chrysogenum (Skatrud et al., 1989). Over-expression of the gene that encodes the expandase/hydroxylase enzyme produced an increase in this activity, a decrease in the amount of accumulated penicillin N, and an increase in cephalosporin C production in the modified strain. Even better results were obtained when over-expressing the cefG gene, which encodes the deacetylcephalosporin C acetyltransferase, the last enzyme in the biosynthesis pathway of cephalosporin C (Gutierrez et al., 1997). Regarding the genes of penicillin biosynthesis, overexpression of the pcbAB gene in Aspergillus nidulans has led to 30 times higher yields than normally obtained (Kennedy and Turner, 1996).Introduction of additional copies of the pcbC genein the P. chrysogenum strain Wisconsin 54-1255 didnot produce any remarkable increase in penicillin Gproduction, although it slightly accelerated its

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biosynthesis between 30 and 80 hours of fermentation (Barredo, 1990). This author explained these results by stating that probably the activity of isopenicillin N synthase did not constitute a “bottle neck” in penicillin production and the increase observed for this enzymatic activity would not be enough to favor biosynthesis of this compound. In any case, utilization of an integrative plasmid in this transformation suggested caution in interpreting the results. Introduction of the last gene of the penicillin pathway, i.e., penDE gene of P. chrysogenum (Fernández, 1997) and of A. nidulans (Fernández- Cañón and Peñalva, 1995; Montenegro, 1996) did not lead to satisfactory results. Good results were obtained when a DNA fragment, containing pcbC and penDE genes of P. chrysogenum (Veenstra et al., 1991) was used in the transformation. Penicillin G production mean values obtained with 26 transformants of P.chrysogenum strain Wisconsin 54-1255, in which the

DNA fragment had been integrated, became significantly higher than the yields of those transformants where only the plasmid without insert had been integrated. The two transformants with the highest production reached a 40% increase as compared with the control. The homogeneous amplification of both genes (and therefore their joint expression) would seem necessary to get an improvement in penicillin production (at least in the strain studied). This hypothesis was confirmed after introducing the complete cluster for penicillin biosynthesis (pcbAB, pcbC, and penDE genes), although this was performed in a low-producing laboratory strain(Theilgaard et al., 2001). Molecular strategies, different from those involving an increase in biosynthesis gene doses, have also been developed. One of them, of great interest, involves semi-synthetic cephalosporins (the most useful ones from the medical point of view). These antibiotics are normally produced from 7-aminocephalosporic acid (7-ACA) or from 7-aminodeacetoxycephalosporanic acid (7-ADCA) (Demain and Elander, 1999), both coming from CPC. Traditionally, the production processes for these compounds involved complex, expensive, and environmentally unfriendly chemical processes. However, through the application of genetic engineering techniques, it has been possible to produce 7-ACA and 7-ADCA directly through fermentation processes, using new metabolic pathways created in both Penicillium and Acremonium (Velasco et al., 2000).

2.4 Industrial application: PenPenicillium chrysogenum is a mold that is widely distributed in nature, and is often found living on foods and in indoor environments. It was previously known as Penicillium notatum.[1] It has rarely been reported as a cause of human disease. It is the source of several β-lactam antibiotics, most significantly penicillin. Other secondary metabolites of P. chrysogenum include various different penicillins, roquefortine C, meleagrin, chrysogine, xanthocillins, secalonic acids, sorrentanone, sorbicillin, and PR-toxin.Like the many other species of the genus Penicillium, P. chrysogenum reproduces by forming dry chains of spores (or conidia) from brush-shaped conidiophores. The conidia are typically carried by air currents to new colonisation sites. In P. chrysogenum the conidia are blue to blue-green, and the mold sometimes exudes a yellow pigment. However, P. chrysogenum cannot be

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identified based on colour alone. Observations of morphology and microscopic features are needed to confirm its identity. The airborne spores of P. chrysogenum are important human allergens. Vacuolar and alkaline serine proteases have been implicated as the major allergenic proteins. P. chrysogenum has been used industrially to produce penicillin and xanthocillin X, to treat pulp mill waste, and to produce the enzymes polyamine oxidase, phosphor gluconate dehydrogenase, and glucose oxidase vicillium chrysogenum is a mold that is widely distributed in nature, and is often found living on foods and in indoor environments. It was previously known as Penicillium notatum. It has rarely been reported as a cause of human disease. It is the source of several β-lactam antibiotics, most significantly penicillin. Other secondary metabolites of P. chrysogenum include various different penicillins, roquefortine C, meleagrin, chrysogine, xanthocillins, secalonic acids, sorrentanone, sorbicillin, and PR-toxin.Like the many other species of the genus Penicillium, P. chrysogenum reproduces by forming dry chains of spores (or conidia) from brush-shaped conidiophores. The conidia are typically carried by air currents to new colonisation sites. In P. chrysogenum the conidia are blue to blue-green, and the mold sometimes exudes a yellow pigment. However, P. chrysogenum cannot be identified based on colour alone. Observations of morphology and microscopic features are needed to confirm its identity. The airborne spores of P. chrysogenum are important human allergens. Vacuolar and alkaline serine proteases have been implicated as the major allergenic proteins. P. chrysogenum has been used industrially to produce penicillin and xanthocillin X, to treat pulp mill waste, and to produce the enzymes polyamine oxidase, phospho-gluconate dehydrogenase, and glucose oxidase.

3. Discovery of Penicillium notatum:

Penicillium chrysogenum (also known as Penicillium notatum) is the source for penicillin, the first antibiotic. Penicillin works against gram-positive bacteria, such as Staphylococcus and Pneumococcus by disrupting bacterial cell wall synthesis-- crosslinking of the peptidoglycan polymers is prevented by inhibition of the enzyme transpeptidaase, causing the malformed cells walls to take on excess water, which causes them to burst (cell lysis).The name Penicillium comes from the resemblance of the conidiophore of the fungus to a paintbrush-- penicillus is the Latin word for paintbrush. Penicillium is a member of the deuteromycetes, fungi with no known sexual state. Some species of Penicillium have an additional sexual state in the Ascomycota in the Eurotiales. New research suggests penicillin is becoming obsolete, and antibiotic resistance could lead to a "major health crisis" unless governments act to promote research into new drugs.

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4. Further development of penicillin in Great Britain and the United States: "Penicillin would undoubtedly still have remained a fairly unknown substance, interesting to the bacteriologist but of no great practical importance, if it had not been taken up at the Pathological Institute at the venerable University of Oxford." -- Professor G. Liljestrand, introducing the winners of the 1945 Nobel Prize for Medicine.

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After Fleming's paper was published, some work continued by Clutterbuck et al. in Pennsylvania, but Fleming's penicillin work went otherwise largely ignored, even by Fleming. At the 1939 International Microbiological Congress in New York when Charles Thom asked Fleming what had become of his penicillin, Fleming replied, "I forgot about that some years ago."As World War II approached, the search for new drugs to combat bacterial infections of battle wounds became a primary focus of research, especially at Oxford university, where Howard Florey and colleagues E.P. Abraham, Boris Chain, Norman Heatley, and Florey's wife Mary, who was a physician.Penicillin was the prime candidate for an antibacterial antibiotic for the following reasons:• Penicillin kills gram-positive bacteria very well. On the plate to the left, P. chrysogenum (a descendent of Fleming's original strain) was inoculated into the center of a lawn of Staphylococcus aureus. Note the clear zone of inhibition of growth of S. aureus.• Penicillin causes no ill effects in humans and other animals, except for allergies in about 10% of humans. (We now know that penicillin allergies are usually caused by its binding to serum proteins, causing an IgE-mediated inflammation.)However there were also obstacles to be overcome:• Penicillin was unstable, especially at low and high pH.• Penicillin was produced in small quantities by even the most prolific cultures.• Penicillium grows well only in surface culture.Florey and his colleagues at Oxford recognized the importance of Fleming's discovery and set out to solve these problems. They had already treated some patients with promising results. However the number of flasks they had to use to grow enough of the fungus to produce enough penicillin to treat even a single patient was prohibitively high. The first vessels they tried were some old-style bedpans, with a large surface area, a lid, and a side-arm for inoculation and

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withdrawal. It's always been unclear to me why a bedpan should have a side-arm-- maybe that's why they don't make them that way anymore. In fact by the time they discovered this as an ideal culture vessel they had already been replaced by more modern bedpans, and no more were available.About that time the bombing of Britain made large scale production of penicillin there unlikely. Legend has it the researchers dusted the insides of their coats with spores of the fungus--in case something happened to destroy the lab. at least the fungus would survive. Florey and Heatley came to the United States in 1941 to try to interest some pharmaceutical companies in producing penicillin. They were not greeted with open arms, since penicillin production was in its very early stages of development. In particular, the production of penicillin was very low (4 units/ml where 1 unit=0.6micrograms), and the companies did not envision huge profits from such a small production. The National Academy of Sciences was consulted and sent them to Charles Thom, noted Penicillium expert (and coauthor of the monograph of Penicillium with Ken Raper). Thom sent them to the new Fermentation Division of the newly created Northern Regional Research Laboratory (NRRL) in Peoria, Illinois. It was there that most of the work on the industrialization of penicillin production was coordinated, with significant work by Ken Raper, and Drs. Moyer and Coghill.By Christmas 1941, production was up to 40 units/ml due to modification of the culture medium. The main problem was that no derivative of the Fleming strain was ever found to produce penicillin in submerged culture or shaken flasks. The researchers surmised that they could find a strain of P. chrysogenumthat would grow well in submerged culture. They asked everyone throughout the world to send in samples, moldy fruit,grains, and vegetables. Air Force and other military personnel were instructed to scoop up soil from exotic locations and have them sent to Peoria. The Peoria researchers even employed a young woman to scour the markets in Peoria for produce bearing blue-green molds. She became known as "Moldy Mary." However, after all that it was a moldy cantaloupe brought in by a Peoria housewife that proved to be the bonanza strain of Penicillium chrysogenum. In submerged culture the initial isolate produced 70-80 units/ml. By isolating single uninucleate conidia they found a mutant that yielded 250 units/ml.

Once word got around about this new wonder drug, home penicillin treatments were all the rage. Gauze floating on a nutrient broth grew mold, and the gauze was applied directly with a bandage to superficial wounds. Of course not all molds that people could grow were the right species of Penicillium. Moreover, even if the isolate is the correct species, not all strains even produce penicillin. Unfortunately this home remedy did not work.Demands for penicillin were "unbelievable," so the War Production Board set up projects at other sites, including the University of Wisconsin-Madison, where J.F. Stauffer and Myron Backus tested thousands of ultraviolet light-induced mutations. (Incidentally, Ken Raper eventually became a professor of Botany and Bacteriology at UW-Madison, where I met him several times as a young graduate student. I even sat between him and Folke Skoog [discoverer of the plant hormone cytokinin] at a Badger football game!). Due to careful selection of mutants Backus and Stauffer were able to increase production from 250 to 900 units/ml. Additional

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mutagenesis on those strains resulted in a strain that could produce 2500 units/ml. Other universities became involved, including Stanford, Minnesota, and the Carnegie Institution in Cold Spring Harbor. It really became a group effort, with all groups making significant contributions to the development of penicillin. Industrial strains of P. chrysogenum now produce 50,000 units/ml, or about 30mg, significantly improved from the 4 units/ml starting point. Apparently, all the industrial strain used today are descendents of that single strain that came from the Peoria cantaloupe in 1943.

5. The Nobel Prize and the continuing importance of penicillin For their work, Sir Alexander Fleming, Ernst Boris Chain, and Sir Howard Walter Florey were awarded the 1945 Nobel Prize in Medicine "for the discovery of penicillin and its curative effect in various infectious diseases." You can read more about their Nobel Prize here.

6. Recent researches :Antibiotics such as penicillin have been key to the decline of infectious diseases over the last 60 years, but bacteria are becoming increasingly resistant to existing drugs. That means many antibiotics are no longer effective at combating common diseases, and a lack of research into new drugs means there is a dire shortage of alternatives, according to the report by London School of Economics and Political Science (LSE).Elias Mossialos, professor of health policy at LSE, led the research and has called for governments to do more to tackle the problem.He told CNN that penicillin is becoming obsolete in some developing countries, as well as in France, Spain and Romania, because of over-prescription by doctors and pharmacists. He said the emergence of "superbugs" such as methicillin-resistant staphylococcus aureus (MRSA) is causing the growing problem of hospital-acquired infections. "Antibiotic resistance is a much more important situation than swine flu and it will only get worse," he said. He said doctors are commonly misdiagnosing viral infections as bacterial infections, and then prescribing antibiotics to treat them, while in some countries pharmacists are selling antibiotics without a doctor's prescription. Excessive use of antibiotics encourages the emergence of resistant bacteria. Dr Kathleen Holloway of the World Health Organization (WHO) told CNN that antibiotic resistance is a global problem, with diseases including childhood pneumonia, dysentery and tuberculosis (TB) no longer responding to first-line antibiotics in some parts of the world. "We've got to a situation where there are no more drugs for certain conditions. There are some people with extreme drug-resistant TB and there are no drugs to treat them," Holloway said."Research and development of new antibiotics isn't keeping up with development of resistance. If we don't do something about it we'll end up with a situation where all the old drugs have resistance and we don't have any new ones." Mossialos said the lack of development of new antibiotics is largely because antibiotics don't earn pharmaceutical companies as much as other products.

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7. Market Value of Penicillium Notatum: Today, the use of penicillin and other antibiotics are common place. The various antibiotics are used to treat a number of what are now common diseases and to prevent the onset of infections when our skin, our first barrier to fight off disease, is somehow broken through a simple cut or a more serious wound. It is something that we all take for granted, today. However, many diseases and simple wounds that are so easily treated today because of the availability of antibiotics has not always been available. Antibiotics are a relatively recent discovery and the first practical one, penicillin, was not available until the early 1940s. Even the concept of using fungal products, such as penicillin, to produce medicine is a relatively new one. However, many folk remedies that have included fungi have long been utilized, but the incorporation of fungi into the remedy was inadvertent and not known. For example, over three thousand years ago, the Chinese had put moldy soybean curd on boils and other types of skin infections. Other cultures have placed warm earth, which contains molds and other fungi, as first aid in injuries. There was undoubtedly antibiotics in the soybean curd and earth that were placed on injuries. So, although the concept of antibodies is relatively recent, its use has been around for some time.The discovery of penicillin has often been described as a miracle drug, and that is exactly what it was. Prior to the discovery of penicillin, death could occur in what would seem, today, to be very trivial injuries and diseases. It could occur from minor wounds that became infected or from diseases such as Strep Throat, and venereal diseases such as syphilis and gonorrhea were a much more serious issue.

8. Future Prospective: After World War II, penicillin was the antibiotic widely used for the treatment of infections such as syphilis,pneumonia, streptococcal infections of the pharynx, scarletfever, diphtheria, bacterial meningitis, and septicemia.In 1945, Fleming, Florey, and Chain were awardedthe Nobel Prize of Medicine for this discovery. Years later,the anti-staphylococcus penicillins appeared, as wellas the wide spectrum ones, to be used also orally (knownas ampicillins and amoxicillins). After penicillin finding,a great step in antibiotics therapy was the discovery ofcephalosporin C, another β-lactam compound producedby Acremonium chrysogenum.The discovery of the active nucleus of cephalosporin C and the possibility of modifying its lateral chain allowedfor the development of new semi-synthetic compoundswith a greater antibacterial activity, especiallyefficient against β-lactam resistant microorganisms (replacingin many cases penicillin-based antibiotics). Developmentprograms for new semi-synthetic cephalosporinscontinue at different pharmaceutical companies, sonew generations of these antibiotics will be available inthe future. However, and due to the high prices of theseproducts, the new advances must be paralleled byprogresses in the molecular biology knowledge regardingCPC biosynthesis.Molecular biology and its practical counterpart (geneticengineering) have been incorporated into thestrain improvement programs of most pharmaceuticalcompanies and, together with a careful control of thefermentation processes, they are expected to becomemore efficient. It remains to be seen whether it is possibleto obtain cephalosporin

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production levels similar tothose of penicillin, although it has to be noted that thePenicillium improvement programs began before thoseof Acremonium.At our Laboratory of the Universidad Autónoma Metropolitana (Genetics Engineering and Secondary MetabolismGroup of the Biotechnology Department) we are workingunder this guideline, searching to increase production ofpenicillin G as well as cephalosporin C, by means of a strategyof a coordinated increase in the doses of those genesimplicated in the biosynthesis of these antibiotics.

9. References:

1. Samson RA, Hadlok R, Stolk AC (1977). "A taxonomic study of the Penicillium chrysogenum series". Antonie van Leeuwenhoek 43 (2): 169–175. doi:10.1007/BF00395671. PMID 413477.2. a b de Hoog GS, Guarro J, Gené J, Figueras F (2000), Atlas of Clinical Fungi - 2nd Edition, Centraalbureau voor Schimmelcultures (Utrecht)

3. Shen HD, Chou H, Tam MF, Chang CY, Lai HY, Wang SR (2003). "Molecular and immunological characterization of Pen ch 18, the vacuolar serine protease major allergen of Penicillium chrysogenum". Allergy 58 (10): 993–1002. doi:10.1034/j.1398-9995.2003.00107.x. PMID 14510716.

4. http://www.bioline.org.br/pdf?ej02009 5. Abraham, E. P. 1991. From penicillins to cephalosporins, pp 15-23. In H. Kleinklauf & H. von Döhren. 50 years of Penicillin Application. History and Trends. Technische University at Berlin. PUBLIC Ltd., Czech Republic.6. Elander, R. P. 1967. Enhanced penicillin biosynthesis in mutantand recombinant strains of Penicillium chrysogenum, pp. 403-423. In H. Stübbe (Ed). Induced mutations and their utilization.Academie-Verlag, Berlin.7. http://en.wikipedia.org/wiki/Penicillin 8. Elander, R. P. & M. A. Espenshade. 1976. The role of microbialgenetics, pp. 192-256. In B. M. Miller & W. Litsky (Eds).Industrial Microbiology. McGraw-Hill, New York.9. Carr, L. G, P. L. Skatrud, M. E. Scheetz 2nd, S. W. Queener &T. D. Ingolia. 1986. Cloning and expression of the isopenicillinN synthetase gene from Penicillium chrysogenum. Gene48:257-266.

10. http://tomvolkfungi.net/