Microbial Metabolites -

Development of New Metabolite

Dr. Muhammad Sajjad Khattak

Stages of Microbial Growth
 Microbial Metabolites
 Primary metabolite
 Produced during exponential growth (trophophase)
 Example: Alcohols, aminoacids, vitamins
 Secondary metabolite
 Produced during stationary phase (idiophase)
 Not essential for growth and reproduction
 Formation depends on growth conditions
 Produced as a group of related compounds
 Often large organic molecules that require a large number of
specific enzymatic steps for production and significantly
overproduced
 Often produced by spore-forming microbes during
sporulation
 Example: Citric acid, Antibiotics (Synthesis of tetracycline
requires at least 72 separate enzymatic steps)
 Cell number / Biomass

Time
Primary metabolite

Sugar
Cells

Alcohol

Alcohol (Product), sugar (Substrate)

Cell number / Biomass

Sugar

Cells

Penicillin

Time
Primary vs Secondary Metabolites

Secondary metabolite

Penicillin (Product), sugar (Substrate)

 New Metabolites – Development and Production
 Interdisciplinary and requires input of:
 Microbiology – isolation and identification of
microorganisms, strain preservation, biological activity,
fermentation.
 Biochemistry – analytical procedures for biologically active
molecules and their purification.
 Chemistry – synthesis of substrates and inhibitors.
 Bioinformatics – literature search and computerized
databases and software
 Engineering – development of technical equipment
 New Metabolites – Development and Production

 Biochemical capabilities of microorganisms are vast.

 Development and production of new metabolite is done by:
 Screening – to obtain completely new class of compounds
 Chemical modification – known microbial substances
 Biotransformation – using microbe or enzymatic reaction
 Interspecific protoplast fusion – recombining genetic
information of closely related strains to produce new
products e.g. antibiotics
 Gene cloning – gene transfer between unrelated strains to
produce known substances e.g. enzymes
 Screening for New Metabolites
 No universal screening method and depends on selection of
appropriate
 Strain
 Tests i.e. method of detection
 Todays focus is on:
 Chemotherapeutically active products in the areas of:
 Antibiotic resistant strains
 Tumors
 Viruses
 Enzyme inhibitors
 Pharmacologically active substances e.g. hormones
 Better starter cultures in food industry and industrial enzymes
 Organisms capable of degrading hazardous and persistent chemicals
 Strain isolation in Screening
 Location of sample i.e. inoculum source
 For new metabolites – extreme and unusual environmental
conditions
High altitude, cold habitats, sea water, deep sea, hot geysers, deserts,
petroleum fields
 Strain isolation in Screening
 Enrichment procedure
 Testing the initial isolates for biological activity i.e. sample is diluted
directly onto selective (test) agar plates and only those colonies
showing activity are isolated.
 Antibiotics – inhibition zone on an antibiotic plate
 Proteases – Clear zone on substrate plate (Casein)
 Amylases – Clear zone on substrate plate (Starch)
 Lipases – Clear zone on substrate plate (Oil emulsion/Tributyrin)
 Test Systems Used
 Success of screening procedure also depend on development of
intelligent tests to eliminate known / undesirable metabolites and
to recognize the new compounds with required properties. For
Example
 Screening for inhibitors of bacterial cell wall (Have low toxicity)
 Culture filtrates of 10,000 strains (bacteria, actinomycetes and
fungi) – Discovery of Azureomycin
 Phase I – metabolites that inhibit growth of Bacillus subtilis (having
cell wall) but didn’t inhibit Acholeplasma laidlawii (no cell wall)
 Test Systems Used
 Phase II – compounds that inhibit the synthesis of meso-
diaminopimelic acid (m-DAP), derivative of lysine and component
of bacterial cell wall, but didn’t inhibit the incorporation of leucine
(protein synthesis)

Gram negative bacteria and Gram positive cocci

Gram positive bacilli

 Phase III – substances with molecular weight of < 1000 Da

(larger the size of molecule, more undesirable side effects)
 Test Systems Used
 Screening of β-lactamase inhibitors
 Penicillin, an antibiotic containing β-lactam ring
 Penicillin resistance in bacteria producing β-lactamase

 Inhibitors of β-lactamases prove to be useful in permitting penicillin

therapy against resistant microorganisms
 Culture supernatants placed on agar plates containing β-lactam
antibiotic and a microorganism producing β-lactamase
 Most common are Clavulanic acid and Sulbactam
 Chemical modification of known metabolite
 Aspirin – Sodium salt of acetylsalicylic acid
 Salicylic acid – active ingredient from bark of Willow tree and used for
pain relief. BUT
 Pure salicylic acid causes sever digestive problems such as bleeding,
diarrhea.
 Salicylic acid is mixed with acetic anhydride in the presence of conc.
H3PO4.

 Because of insolubility, the bioactivity of acetylsalicylic acid is limited.

Therefore its solubility is increased by converting it to its sodium salt.
 Chemical modification of known metabolite

 Penicillin – antibacterial drug, discovered in 1928 by A. Fleming.

 It saved more lives than any other pharmaceutical drug.
 Distinctive feature is the presence of β-lactam ring (responsible for
antibacterial activity)
 Binds to the enzyme transpeptidase, which is required for
bacterial cell wall synthesis and irreversibly inhibits it.
 Certain bacteria developed antibiotic resistance by producing
penicillinase, which deactivates the antibiotic.

 Resulted in Multidrug resistance (MDR) – a combination of many

antibiotics is required to overcome the infection
 To overcome this modifications were done in the side chain (R) to
make semi-synthetic penicillins
Chemical modification of known metabolite

Natural

Penicillinase
resistant

Extended
spectrum