BT4: Module BE421

Lecturer: Donal OShea

Primary Versus Secondary Metabolites of Microorganisms

Autors: Jemma Keegan & Emma OLeary

Table of Contents
Section Page Abstract..1 Introduction1 Primary Metabolites...3 Secondary Metabolites...5 Antibiotic Production..7 Concluding Remarks..8 References..9

1.0 Abstract
The objectives of this essay are to describe and explain the differences between primary and secondary metabolites of microorganisms, to describe their key role in the development of the biotechnology industry, to outline the importance of antibiotic production and to describe some possible future applications of metabolites.

2.0 Introduction
The metabolism is defined as the sum of all the biochemical reactions carried out by an organism. Primary metabolic pathways converge too few end products while secondary metabolic pathways diverge to many products. Primary requires the cell to use nutrients in its surroundings such as low molecular weight compounds for cellular activity. There are three potential pathways for primary metabolism: the Embden Meyerhof-Parnas Pathway (EMP), the Entner-Dourdorof pathway and the hexose monophosphate (HMP) pathway. The EMP pathway produces two molecules of pyruvate via triose phosphate intermediates. This pathway occurs most widely in animal, plant, fungal, yeast and bacterial cells. Many microorganisms however use this pathway solely for glucose utilization. During primary metabolism hexoses such as glucose are converted to single cell protein (SCP) by yeasts and fungi. This is generally done by using a combination of EMP and HMP pathways, followed by the TCA cycle and respiration. Yeasts from the Sachcharomyces species produce alcohol as cells grow during the log phase (during trophophase) using an anaerobic primary metabolic pathway. This accounts for most of the alcohol found in nature and is widely used in the fermentation industry to produce beer, wine and spirits. In the citric acid fermentation process involving Aspergillus Niger, hexoses are converted via the EMP pathway, to pyruvate and acetyl Co-A that condenses with oxaloacetate to form citrate in the first step of the TCA cycle. Ethanol, lactic acid and acetic acid were the first commercial products of the fermentation industry. Several of these products have applications as alternative energy sources, for example alcohol has been used to

produce a cheaper alternative to petrol in developing countries such as Brazil and in Europe between World Wars I and II. Secondary metabolism synthesises new compounds. Secondary metabolites are not vital to the cells survival itself but are more so for that of the entire organism. Relatively few microbial types produce the majority of secondary metabolites. Secondary metabolites are produced when the cell is not operating under optimum conditions e.g. when primary nutrient source is depleted. Secondary metabolites are synthesized for a finite period by cells that are no longer undergoing balanced growth (22). A single microbial type can produce very different metabolites. Streptomyces griseus and Bacillus Subtillus each produce more than fifty different antibiotics. Most secondary metabolites are produced by families as closely related compounds. The chemical structure and their activities cover a wide range of possibilities, including antibiotics, ergot alkaloids, naphtalenes, nucleosides, peptides, phenazines, quinolines, terpenoids and some complex growth factors. The production of economically important metabolites such as antibiotics by microbial fermentation is one of the major activities of the bioprocess industry. Secondary metabolites such as penicillin are produced during the stationary phase (idiophase) of cell growth. Most of the knowledge concerning secondary metabolism comes from the study of commercially important microorganisms. There are some similarities between the pathways that produce primary and secondary metabolites, namely that the product of one reaction is the substrate for the next and the first reaction in each case is the rate-limiting step. Also the regulation of secondary metabolic pathways is interrelated in complex ways to primary metabolic regulation.

Primary Metabolites
Fermentation products of primary metabolism such as ethanol, acetic acid, and lactic acid were the first commercial products of the fermentation industry. These industrial revelations were soon followed by citric acid production along with other products of fungal origin. Due to the high product yield and the low reproducibility costs, major interest has been shown in the respective markets. Production of cell constituents i.e. lipids, vitamins, polysaccharides as well as intermediates in the synthesis of cell constituents such as amino acids and nucleotides are also of great economic importance in present-day industry. The effectiveness of yeasts along with other microorganisms as sources of the B-group vitamins has been recognized for more than 50 years and like products of catabolic primary metabolism e.g. ethanol, citric acid etc. are of great commercial importance. Citric acid is an organic acid that is of major economic use in todays industry. It is a very important commercial product and is widely used in the food and beverage industries as a food additive. A simple diagram outlining citric acid production is given below in Figure 1. The present annual global requirement is nearly 4 lakh MTons that is produced almost entirely by fermentation of molasses sugar with selected strains of Aspergillus niger. (www.fgsc.net/asilommar/citric.html) In addition to the beverage and food industry, citric acid is used in effervescent powders as well as being used in boiler and metal cleaning. Citric acid was Pfizers most popular product for decades. The process was vastly improved in 1917 when a Pfizer employee, Dr. James Currie began fermentation experiments in order to develop the sugar under conversion in acid citrate (SUCIAC) process. This began to outperform conventional citric acid production methods. Deep tank fermentation using molasses was also developed and aspects of this process subsequently contributed to the design of penicillin production. Factors effecting citric acid production vary considerable and depend predominantly on the strain of A. niger used. Other factors that effect citric acid production include the type of raw material fermented, the amount of methyl alcohol present, the substrates initial moisture content as well as the fermentation time and temperature. Much research has been conducted over the years in order to increase the yield of citric acid production. Much of the research carried out is in the area of improving the strains of A. niger used in

citric acid production using mutations. One such mutant of strain 72/4 was produced by X-ray and ultraviolet irradiation, which clearly gave better yields than that of the parent (18). Recently, research carried out in the Institute of Food and Radiation Biology in Bangladesh has found several high citric acid producing strains of Aspergillus niger (14). Using molasses as its substrate, these strains were found to yield 3-5 times more citric acid than that of its parent strain (CA16), thus being more resourceful than the parent form

Figure 1. A basic outline of citric acid cycle Chemical riboflavin production, successfully used for decades, is in the course of being replaced by microbial processes. This promises to save half the costs, reduce waste and energy requirements, and use renewable resources like sugar or plant oil (15). Vitamin B12 is found in practically all animal tissues and originates either from organisms within the animals own digestive tract of from ingestion of animal food. Human beings are completely dependent on dietary vitamins as they are unable to utilize any of the intestinally synthesized vitamins. It therefore seems apparent that the only primary source of the vitamin B12 in nature is the metabolic activity of microorganisms. It is synthesized by a wide range of bacteria and streptomycetes, however not so much by yeasts and fungi. Three microorganisms are currently in use for industrial riboflavin production, which naturally overproduces the vitamin: the hemiascomycetes Ashbya 4

gossypii, a filamentous fungus, and Candida famata, yeast. Riboflavin production is also possible using the gram-positive Bacillus subtilis, however the deregulation of purine synthesis is required. Riboflavin production is identifiable among all three organisms by the growth of yellow coloured colonies. This is of great importance during the screening of improved mutants. Antimetabolites like itaconate, which inhibits the isocitrate lyase in A. gossypii, tubercidin, which inhibits purine biosynthesis in C. famata, or roseoflavin, a structural analogue of riboflavin used for B. subtilis, have been applied successfully for mutant selections (18). These mutations greatly improve the production yield of the vitamin riboflavin, in comparison to the parent strain. Nucleotides are used in the preparation of poly and oligonucleotides as well as being of potential nutritional and medical interest. However the greatest interest in nucleotides lies in the fact that they have the ability to enhance the flavour of foods. Yeast extract is extensively used as a flavouring agent in the food industry and is widely available either in powder or paste form. After autolysis and partial hydrolysis of RNA, ribonucleotides such as 5-monophpsphate (GMP) and inosine 5-monophosphate (IMP) may be extracted from the biomass.(Ref) Flavour enhancement is a property of these purine ribonucleosides as well as the ribonucleoside, xanthylic acid (XMP). These food enhancers are responsible for meaty flavours found in foods and are available on the market worldwide. These products are of major importance in the food industry and currently international trade surpasses US $1.1 billion per year (23).

Secondary Metabolites
Antibiotics were first defined as a chemical compound produced by a microorganism, which has the capacity to inhibit the growth of and even destroy bacteria and microorganisms in dilute solutions (A. Waksman 1942). Antibiotic production includes 160 different products and has a total annual worldwide market of $23 billion. Sir Alexander Fleming first discovered the antibiotic properties of the mould Penicillin notatum in 1929 at St. Marys hospital in London, when he noticed that Penicillin notatum destroyed a staphylococcus bacterium in culture. Penicillin is bactericidal to a

number of gram-positive bacteria and acts by inhibiting transpeptidation thus preventing new cells from forming walls. It belongs to the beta-lactam family of antibiotics. A team at Oxford University further proved penicillins value as a drug by developing methods of growth, extraction and purification. During World war two research was moved to the USA where large-scale growth of the mould began. Firstly penicillin moulds were grown in small shallow containers on nutrient broth. Methods of growth were improved by using deep fermentation tanks with continuous sterile air supply and corn steep liquor as a source of nutrients. In 1943 a cantaloupe mould, P. Chysogenum was found to produce twice the amount of penicillin than P. notatum. Since then researchers continued to find higher yielding penicillin moulds and have also improved yields further by exposing moulds to x-rays and UV light. The first type of penicillin produced was Penicillin G, which had to be administered to patients parenterally because it is broken down by stomach acid. Penicillin V was later formulated so that it could be taken orally, unfortunately it was less active than Penicillin G. The enhancement of antibiotic industrial yield has been achieved through traditional strain improvement programs based on random mutation and screening. Recombinant DNA techniques have existed since the 1970s and involve the introduction of DNA fragments into host cells using a vector (a plasmid or phage) that contains a selection marker. The DNA fragments are integrated into the host genome or autonomously replicated as a plasmid. Transformants are then screened for improved characteristics. The pharmaceutical company Eli Lilly was responsible for the first recombinant DNA improvement of an antibiotic producing microorganism. excreted by the organism. Transformation of C . acremonium 394-4 caused an increase in the amount of antibiotic cephalosporin C Cephalosporins are beta-lactam compounds that are structurally and pharmacoligically related to penicillins. Cephalosporins resist hydrolysis by enzymes referred to as penecillinases, which are secreted by a number of bacteria. They are now one of the most widely prescribed antibiotics and are very effective for the treatment of hospital-acquired infections. Actinomycetes are aerobic spore forming bacteria that originate from soil. A large number of antibiotics are produced by actinomycetes and in particular Streptomyces.

They resemble a fungal mycelium in form, but have thinner filaments. These filaments are formed when cells divide to form long chains of up to 50 cells. Actinomyces griseus was first isolated from soil in the Andes, this bacterium produced a substance that killed many bacteria unaffected by penicillin, including Tuburculosis bacillus. The antibiotic was named streptomycin. However tubercle bacilli soon became resistant to streptomycin and it has since been replaced by para-amino-salicylic acid (PAS). Stretomycetes are still very important bacterial producers of antibiotics and cytostatics. Due to the emerging resistance of bacteria to common antibiotics, new technologies such as combitatorial biosynthesis are being used for the production of novel metabolites using streptomycetes. metabolites. Ordinarily Actinomycetes use the EMP pathway to metabolise glucose because this pathway is a more efficient one than the ED pathway. However, one actinomycete, Nonomureae in Mycobacterium smegmatis, has been found to preferentially use the ED pathway (6). This bacterium is now used to produce a novel commercial antibiotic for the treatment of multiresistant gram-positive bacteria. The secondary metabolites of the fungi including Drechslera, Trichoderma, Aspergillus and Curvularia have the ability to produce green dyes / anthraquinones.. These dyes are from natural sources and do not cause the pollution to the environment associated with chemical dyes. This technology involves the use of a combination of genes from different biosynthetic pathways to produce modified

Antibiotic Production
The production of penicillin G or V usually begins with lyophilized spores of P. chrosogenum. Using these spores, a sterile agar slant is inoculated, sporulation and growth occur under controlled conditions. Spores are harvested from mature slants and a spore suspension is used to inoculate a shake flask containing vegetative medium. A typical vegetative medium will contain an organic nitrogen source (corn-steep liquor) and a sufficient concentration of fermentable carbohydrate (2% sucrose or glucose). Log 7

phase growth is required in this stage and a minimum doubling time of six hours is usually required. The purpose of this inoculum development stage is to increase the concentration of the fungal mycelium necessary for a high volumetric rate of penicillin synthesis. Addition of lactose in combination with limited nitrogen availability stimulates a greater accumulation of penicillin because of the slower hydrolysis rate of the disaccharide. Slow continuous feeding of culture with glucose also produces the same result. In order to produce particular penicillin a specific precursor can be added to the medium, for example phenyl acetic acid is added to maximize the production of penicillin G. The pH is kept neutral by adding sterile alkali, which stabilizes the newly synthesized penicillin. The fermentation usually takes 6-7 days; the fungal mycelium is then separated from the broth and processed by adsorption, precipitation and crystallization to yield the final product. The fermentation of penicillin G or V is carried out in a liquid culture in volumes of 0,000 to 200,000 litres. This aerobic process has a volumetric oxygen uptake of 0.4 to 0.8mmole/L/min (Sylvester and Coghill, 1954: Perlman, 1970).

Concluding Remarks
Industrially important primary microorganisms are continually being improved to optimize product yield and substrate utilization in order to minimize the cost of production. Primary products of microbial metabolism have made a significant contribution to the food and beverage industries. Primary metabolites have been used to produce petroleum derived products as well as ethanol for liquid fuels (gasahol). Considering the current trends in oil prices, microorganisms that have the ability to produce such products will no doubt be exploited to their full potential. In the not too distant future biomass energy could become a major contributor to the Earths energy requirements as petroleum resources run out.

The introduction of antibiotics revolutionised the treatment of infectious disease in humans. Antibiotics are used in large quantities in animal farming to prevent infection as well as to treat diseases. Smaller doses are added to animal feed to promote growth. Fruits and vegetables are also treated for bacterial infections using antibiotics such as 8

streptomycin and oxytetracycline. Owing to this widespread use, antibiotics have been found in liquid waste at animal feedlots and have spread into many surface and groundwater supplies. Residues of antibiotics have also been detected in sewage treatment plants and raw water resources in many European countries. The prescence of antibiotics has upset the delicate balance of microorganisms in the environment by depletion of microorganisms susceptible to antibiotics and providing favourable environments for the proliferation of resistant strains (11). Three EU projects were undertaken in 2003 to assess the presence and effects of antibiotics in the aquatic environment and in soils: the ERAVMIS, PEPHARMAWATER and POSEIDON projects. These projects also propose solutions to this problem, such as the removal of antibiotics from wastewater by ozonation and sunlight. Bioresearch Italia began a project to isolate previously unexploited microorganisms from actinomycetes and uncommon filamentous fungi in 2002. This project may provide some insight into methods of combating the increasing number of antibiotic resistant strains of bacteria. References 1. Davies, J., 1999, In praise of antibiotics, American Society for Microbiology (News), Vol.65,Issue 5. 2. Diez, B. et al, 1997, Recombinant microorganisms for industrial production of antibiotics, Biotechnology and Bioengineering, Vol.55, No.1. 3. Donidio, S. Monciardini, P., Microbial technologies for the discovery of novel bioactive metabolites, 2002, Jornal of Biotechnology, Vol. 99, Issue 3 pp. 187198 4 . Doull.J.L., Vining L.C.,1990, Physiology of antibiotic production in actinomycetes and some underlying control mechanisms, Biotechnology Advances, pp141-158 5. Drew & Wallis, Secondary Metabolism and Differentiation in Fungi, Chapter 2 6. Gottsshalk G, 1986, Bacterial Metabolism 7 . Gunnarrson, N., Bruheim, P., Nielson, J., 2004, Glucose metabolism in the antibiotic producing actinomycee Nonomuraea sp. ATTC 339727, Biotechnology and Bioengineering, Vol. 88, No.5, pp652-663

8. http://aem.asm.org/cgi/content/full/68/4/1914 9. http://bio.aiche.org/pdf/Articles/KEYSTON1.pdf 10. http://template.bio.warwick.ac.uk/staff/dhodgson/strep/secondary.html 11. http://www.cumc.columbia.edu/news/reviews/archives 12. http://www.ejbiotechnology.info/content/vol5/issue2/full/5/bip/ 13. http://www.ejbiotechnology.info/content/vol5/issue3/full/3/ 14. http://www.eng.auburn.edu/~wfgale/usda_course/pasteur_pagel.htm 15. http://www.fgsc.net/asilomar/citric.html 16. http://www.lilly.com/about/milestones.html 17. http://www.msu.edu/course/mmg/301/lec 18. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list _uids=10855708&dopt=Abstract 19. http://www.socgenmicrobial.org.uk/pubs/micro_today/pdf/050204.pdf 2 0 .James, L.V., Rubbo, S.D. and Gardner, J.F. (1956). Journal of General Microbiology. 14. 228 21. Krishna, C., 19xx, Solid state fermentation systems - an overview, Journal of Applied Microbiology, Vol. 25 Issues 1-2, pp1-30 22. Naturwissenschaften,1980, Vol. 67, Issue 12, pp.582-587. 23. R villion, J.P.P., Brandelli, A. and Ayub, M.A.Z. (2000), Production of yeast extracts from whey for food use, market and technical considerations. Cincia Technol. Alim., 20, 246-249 2 4 .Rodrigues, K.F.,Hosse, M., Werner, C., 2000, Antimicrobial activities of secondary metabolites produced by endophytic fungi from Spondias mombin, Journal of Basic Microbiology, Vol. 40, Issue 4 25. Rose, A.H., 1979, Economic Microbiology, Vol. 3, Academic Press

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