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