These notes are from the master degree course of Pharmaceutical Biotechnology (University of Insubria - Italy).
These course focuses on the research and exploitation of naturally synthesized secondary metabolites for the design of novel antibiotics that can cope with the uprising antibiotic res...
Pharmaceutical Biotechnology means a lot of things:
- Diagnostics procedures
- Production of recombinant proteins
- Vaccines
This course will be related to the natural products (secondary metabolites) produced by microbes.
Microbes’ production of small molecules: ethanol, butanol, biofuels, organic acids, amino acids,
vitamins, nucleotide.
Primary metabolites:
- Amino acids are an important group of molecules produced by microbes and some of them are used
in pharmaceutical or cosmetic field, but they are primary metabolites
- Vitamins
Secondary metabolites:
They are produced during the secondary metabolism (antibiotics, statins, cyclosporine and other
immunosuppressive agents, antitumor agents…). These molecules are also called natural products.
Other important metabolites are (industrial enzymes, DNA proteins RNA vaccines) but they are
recombinant proteins and for this reason they are NOT considered natural products.
When we talk about microorganism, we refer not only to bacteria, but
also to archaea and some Eukaryotes (fungi, algae, and protozoa).
The microbial diversity can produce a lot of valuable products:
- Biomass (yeast for bread)
- Products from anaerobic metabolism
- Products from incomplete oxidations
- Products from secondary metabolism
- Enzymes
- Polysaccharides
- (Heterologous proteins)
WHAT ARE SECONDARY METABOLITES?
Secondary metabolites are also produced by plants.
Bu’Lock - 1961 (plant biologist) introduces the term “secondary metabolites” also referring to microbes:
he defined “secondary metabolites” as all the molecules that are not essential for cell life and are not
found in every growing cell.
The evolution has driven the selection of primary metabolites in a very strict way: amino acids are always
the same from eukaryotic to mammalian, plants, and microbial cells.
Some cells produce secondary metabolites, some produce no secondary metabolites, and some others
produce a huge chemical diversity of secondary metabolisms. That’s because the secondary metabolism
is not conserved as the primary one, is more variable and it’s not essential.
Growth and duplication necessitate the production of biomass which is made up by 99% of polymers
(chains of primary metabolites) such as proteins, fatty acids, polysaccharides, nucleotide.
SM are useful when the cell is living together with other cells; they are essential if we focus the attention
on the environment that surrounds the cell.
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,ex: antibiosis (the most common example, but not the only one) refers to microbes that produce
antibiotics to defend themselves.
SM are essential to compete for the nutrient sources and for ecological needs, to find a way to survive in
the environment (e.g., penicillin). Diversity is generated by evolution just to fit different situations to
which microbes or plants are subjected in their lives.
Secondary metabolites were for a long time defined for the low molecular weight (<3000 Da): Waksman
and Fleming were the first who discovered SM and they called antibiotics (first class of SM discovered)
“low molecular wight molecules”. That’s a misleading definition… amino acids and ethanol has a lower
mw than SM.
At their time this definition could make sense because they were working on enzymes (Fleming was
working on the lysozyme) which are way heavier than SM.
SM are produced generally by polymerization of few primary metabolites, or they derive form primary
metabolites.
Sigle low-weight molecules → polymerization of more low-weight molecules * → SM
*(Penicillium is derivative of polymerization of 3 amino acids)
The single molecules that are polymerized in different composition create a huge chemical. Secondary
metabolites are not conserved among cells of different species, genera, family (contrary to the primary
metabolites).
From a pre-genomic era point of view, SM are produced only by few microorganisms and some
filamentous microorganisms, such as actinomycetes (actinobacteria order; prokaryotic) and ascomycota
(fungi; eukaryotic), are particularly gifted.
From a post-genomic era point of view (a lot of genomes were sequenced), SM are formed not only by
few microorganisms.
Streptomyces coelicolor is a popular example of actinomycetes,
they grow “as fungi” but they are procaryotic).
SM are produced by secondary metabolism which is common in complex organisms which passes
through several steps during their development (differentiation). The onset of morphological
differentiation usually coincides with the production of secondary metabolites.
Apparently, SM are produced by groups which are not taxonomically correlated (plants, filamentous
actinomycetes, filamentous fungi). However, their common trait is sessility and a specific differentiation
cell cycle. Actinomycetes (procaryotic) and fungi (eucaryotic) have a quite similar growth; the first one
produces ifes with a smaller diameter in respect to the second one.
Ifes are a group of linear cellular chains that form a mycelium (pluricellular structure). A group of
vegetative ifes in the soil constitute the vegetative mycelium, which differentiate (when nutrients
are limited and there is a competition with surrounding microorganisms) producing the aerial
mycelium. This mycelium produces spores, allowing the microorganism to start a new life in
another place with optimal conditions.
This kind of cell lifestyle and the ability to produce SM are shared by actinomycetes and fungi,
both generally living in the soil.
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,Plants present these common traits too: sessility, SM production, seeds spreading to colonize other
places. Plants produce antibiotics, toxins, pigments to attract insects and more. This kind of molecules
are also used as a way of communication.
HOW SECONDARY METABOLITES ARE PRODUCED?
Both in nature and in lab, SM are produced by microorganisms when
nutrients are running out (starvation induction) and the cells stop
growing. In lab SM are produced by fermentation of microorganisms
in liquid cultures.
SM production is extremely dependent on the cultural conditions and
generally confined to the stationary phase of growth = idiophase.
On the other hand, primary metabolism is common to the exponential
growth phase = trophophase.
Another peculiar aspect is that most of the secondary metabolites are produced as a group of closely
related structures (and not as a single molecules) → the enzymes involved in their production are very
flexible and can create a range of similar products.
When Penicillium produces penicillin, it does not produce only penicillin G (the most abundant
one) but also a small number of similar penicillins. This flexibility does not exist in primary
metabolism.
Different SM nomenclatures
- Natural products: called like this by industries and companies.
- Secondary metabolites (Bu’Lock definition – 1961)
- Small bioactive molecules (Julian Davis definition - 2000s): low-mw organic compounds with an
extraordinary diversity of molecular structures and activities produced by living organisms (the
Parvome).
- Specialized metabolites (Dubrovnik Summer School definition 2012): small bioactive molecules
made by defined, specialized and regulated biosynthetic pathways and involved in highly specific
interactions with cellular targets.
ANTIBIOTICS
This was the first class of SM discovered (discovered by Waksman and Fleming → streptomycin).
At their time they call them magic bullets because they can precisely and effectively treat some diseases.
Now we know that microbes can became resistant to antibiotics, and, for this reason, new molecules
must be found. At that time, 2 definitions of antibiotics were given:
- “A chemical substance of microbial origin that possesses antimicrobial activities”
- “Low molecular weight chemical substances produced by microorganisms, which at low
concentrations inhibit the growth of other microorganisms”
There are two concepts in these definitions that are important to understand the role of antibiotics:
1. They are produced by microorganisms = they are natural products;
To date, antibiotics can belong to 3 classes:
- natural
- semisynthetic derivatives (the natural molecule is chemically modified)
- fully synthetic molecules (quinolones, fluoroquinolones)
2. Low concentrations of antibiotics are enough to kill specific cells (specificity of action); other
molecules also can kill cells (like ethanol) but at higher concentrations. The specificity of action and
the low concentrations gave them the name “magic bullets”.
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, Actinomycin: is active on bacteria and cell (cytotoxic). So, it cannot be considered as an antibiotic.
Today is used as an anti-cancer molecule.
WAKSMAN PLATFORM
In addition to the discovery of streptomycin Waksman and his group starts what is called the “Waksman
platform” (1940).
It’s a biological activity guided screening aimed at discovering other specialized metabolites. The
group starts to screen the microbial diversity to find new antibiotics. This approach is still used today by
all big pharma to find new specialized metabolites.
Before this innovation, with plants, the molecules were extracted form plant tissue and then tested to
find which of them were active (this was a chemistry-based approach).
This approach consists in:
1. Systematically collect, enrich, and isolate soil microorganisms, such as actinomycetes and fungi
(collect samples and isolate strains)
2. Growing them in axenic cultures (pure liquid culture)
3. Testing the culture broths for their ability to inhibit the growth of pathogens (collecting culture broth
and test it)
4. Recovering the active substances produced (if the broth was active, then try to find which
component gave activity/was active)
To sum up: prepare a huge number of strains, isolate them in axenic cultures, grow them in liquid
culture, test the broths and focus only on those that had microbial activity; then try to understand
whether that microbial activity was interesting; eventually the chemistry of the molecule can be studied
(studying the structure).
In the last century, this system was upgraded. Not only soil is
screened, but also water and plants. The isolated microbes were
obtained thanks to a selective media (→ pure cultures) from these
sources. After the fermentation a library or a strain collection is
obtained.
Library = a collection of broth samples.
At the same time, it is also important to collect strains.
Most of the antibiotics we are using today were discovered during the
Golden Age (1940-1950) from soil actinomycetes and fungi by
classical screening.
ANTIBIOTICS CLASSES
When we talk about antibiotics classes, we refer to chemistry groups that have a specific cellular target
and are produced by some microorganisms; to describe an antibiotic class lots of information are
needed (chemical structure, mechanism of action, microbial producer, antimicrobial spectrum, cellular
target…)
ex:
Penicillin
Microbial producer: Penicillium notatum (ascomycota)
Chemical structure = beta-lactam
MOA = inhibition of peptidoglycan synthesis
Target = transpeptidase
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