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Microbial Products. Primary Metabolites: log phase, use nutrients fast, produce PM Secondary Metabolites: depletion of nutrients, growth retards, produce SM. Primary Metabolites: Vitamins. Vitamins : cannot be synthesized by higher organisms
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Primary Metabolites: log phase, use nutrients fast, produce PM
Secondary Metabolites: depletion of nutrients, growth retards, produce SM
Vitamins: cannot be synthesized by higher organisms
But microorganisms are capable of synthesizing (gut)
b- carotene (provitamin A)
Ergosterol (vitamin D)
b-carotene (provitamin A)
Poly unsaturated Fatty acids (PUFA; vitamin F)
Docosahexaenoic acid (DHA)
Arachidonic acid (ARA)
Riboflavin (vitamin B2)
Cobalamin (vitamin B12)
L-Ascorbic acid (Vitamin C)
R-Pantothenic acid (vitamin B5)
D-Biotin (vitamin H or B7)
Vitamin B1 (Thiamine)
Vitamin B6 (pyridoxol)
Ergosterol (vitamin D)
4 Pyrrole units
C63 H88 CoN14 O14P
Chemical syn not feasible
Genera known to produce vit B12
Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium, Flavobacterium, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus and Xanthomonas
Sanofi-Aventis (FRENCH) use genetically engineered versions to produce vit B12 under specialized conditions from Propionibacterium since they have no endotoxins or exotoxins
P. denitrificansalso used after strain modification; mutant more efficient than wild type
pH 7.5 +
Cobinamide production and accumulation
Combined with cobinamide
To yield 2ppm of cobalamin
Acidification of culture
To 2-3pH/ 100oC
Filter to remove cell debris
Used as feed additive
Betaine: sugar beet molasses
5,6-dimethylbenzimidazole is added and gets incorporated to form 5’-deoxyadenosylcobalamin
During the 7-day fermentation run, adenosylcobalamin is predominantly secreted from the biomass and accumulates in the fermentation broth in milligram amounts.
The down- stream steps comprise filtration, cyanide treatment, chromatography, extraction, and crystallization yielding vitamin B12 in high purity.
If to be used for treatment further purification (95-98% Purity)
Pseudomonas denitrificans: strain improvements resulted in increase in yeild
From 0.6mg/L to 60mg/L
Glucose : common carbon
Alcohols (methanol, ethanol, isopropanol)
Hydrocarbons(alkanes, decane, hexadecane)
With methanol 42mg/L was obtained using Methanosarcinabarkeri
7,8-dimethyl-10- (D-19-ribityl) isoalloxazine
Participates in O-R reactions
Flavin is ring moiety with yellow colour to oxidized form
genes encoding the riboflavin biosynthetic enzymes are well conserved among bacteria and fungi
Processed food is often fortified by the use of riboflavin as a colorant or vitamin supplement.
The main application (70%) of commercial riboflavin is in animal feed, since productive livestock, especially poultry and pigs, show growth retardation and diarrhea in case of riboflavin deficiency.
According to a report by SRIC, a consulting company in Menlo Park (California), in 2005 the need for industrially produced riboflavin was estimated at 6500–7000 tons per year.
50% by biotransformation
20% production by Chemical synthesis
Acetone butanol fermentation
C. butylicum riboflavin as
Bacillus subtillis (genetically modified)
Phase I use of glucose, accumulation of pyr, pH acidic, growth stops, no Riboflv
Phase II decrpyr, incr in ammonia, alkalinity incr, prod of Riboflv in form of FAD and FMN
Phase III autolysis, cell disruption, release of free FAD, FMN and riboflv
Carbon sources: glucose, acetate, methanol, aliphatic hydrocarbons
Major riboflavin producers are DSM Nutritional Products (Switzerland) and Hubei Guangji(Hubei Province, China), both using genetically engineered B. subtilisproduction strains, and BASF (first in Germany but now in South Korea), employing genetically engineered A. gossypii.
Precursor for its chemical synthesis can be obtained by biological methods
feed applications of L-ascorbic acid account for only 10%, whereas the main uses are in the
pharmaceutical industry (50%),
food (25%), and
Pharmaceutical applications include stimulation of collagen synthesis (especially cosmetic products) and high antioxidant capacity, used for the reported health benefits in the prevention of flu, heart diseases, and cancer, as well as an antidote for poisoning.
The food and beverage industry predominantly exploits the antioxidant capacity of L-ascorbic acid to extend durability, prevent discoloration, and to protect flavor and nutrient contents of their products.
2-keto L-gluconic acid
2 keto L gulonic acid
2 keto L gulonic acid
L-ASCORBIC ACID (100g)
Cloning of gene
Reductaseof Corynebacterium into Erwiniaherbicola
Reichstein Grussner synthesis
Provitamin A -----> Vitamin A (intestine)
Carotenoids Used as food colorants and animal feed supplements for poultry and aquaculture, carotenoids play an increasing role in cosmetic and pharmaceutical applications due to their antioxidant properties.
The pigments are often regarded as the driving force of the nutraceutical boom, since they not only exhibit significant anticarcinogenic activities but also promote ocular health, can improve immune response, and prevent chronic degenerative diseases.
Submerged Fermentation process
Corn starch, soyabean meal, b-ionone, antioxidants
Trisporic acid: act as microbial sex hormone, improves yield
b-Ionone: incrb-carotene syn by incr enzyme activity
Purified deodorized kerosene increases solubility of hydrophobic substrates
Recovery: b- carotene rich mycelium used as feed additive
Mycelium is dehydrated by methanol, extracted in methylene chloride and crystallized which is 70-85% pure
DSM Nutritional Products (Switzerland) and BASF (Germany) dominate the market with their chemical synthesis processes, but Chinese competitors are catching up.
Halophilicgreen microalgae Dunaliellasalina. It accumulates the pigments in oil glo- bules in the chloroplast interthylakoid spaces, protecting them against photoinhibition and photodestruction.
Excessive pigment formation in D. salinais achieved by numerous stress factors like high temperature, lack of nitrogen and phosphate but excess of carbon, high light intensity, and high salt concentration, the latter two having the highest impact.
Dried D. salinabiomass for sale contains 10–16% carotenoids, mainly b-carotene. In addition crystalline material obtained after extraction with edible oil is also sold.
Organic acids are produced by through metabolisms of carbohydrates. They accumulate in the broth of the fermenter from where they are separated and purified.
I. Terminal end products lactic acid
(pyruvate, alcohol) Propionic acid
II. Incomplete oxidation of sugars citric acid
(glucose) Itaconic acid
III. Dehydrogenation of alcohol with O2 acetic acid
Manufactured on large scale as pure products or as salts
In food and beverages
Jams, candies, deserts, frozen fruits, soft drinks, wine
Antioxidants and preservative
Agent for stabilization of
Fats, oil or ascorbic acid
Stabilizer for cheese preparation
Treatment of textiles
Metal industry, pure metals +citrate (chelating agent)
Trisodium citrate (blood preservative)
Preservation of ointments and cosmetics
Source of iron
Present in all organisms
Detergent cleaning industry
Strains that can tolerate high sugar and low pH with reduced synthesis of undesirable by products (oxalic acid, isocitric acid, gluconic acid)
Malate FumarateSuccinyl CoA
100g sucrose --- 112g any citric acid or 123g citric acid-1hydrate
High sugar concincr uptake and production of citric acid
Surface fermentation submerged fermentation
N alkanes (C9-C23) can also be used to produce citric acid; can result in excess production of isocitric acid
Incomplete oxidation of ethanol
Vinegar is prepared from alcoholic liquids since ceturies
NAD+ NADH +H+
CH3 CH2OH---- CH3CHO-------- CH3CH(OH)2 ------- CH3COOH
Ethanol acetaldehyde acetaldehyde hydrate acetic acid
Gluconobacter, Acetobacterwith acid tolerant A. aceti
One molecule of ethanol one molecule of acetic acid is produced
12% acetic acid from 12% alcohol
It is an obligate anaerobe, Gram-positive, spore-forming, rod-shaped, thermophilic organism with an optimum growth temperature of 55–60 o C and optimum pH of 6.6–6.8.
VINEGAR: 4% by volume acetic acid with alcohol, salts, sugars and esters
flauoring agent in sauces and ketchups, preservative also
Wine, malt, whey (surface or submerged fermentation process)
Surface: trickling generator; fermentale material sprayed over surface, trickle thro shavings contaning acetic acid producing bacteria; 30oC (upper) and 35oC (lower). Produced in 3 days.
Submerged: stainless steel, aerated using suction pump, production is 10X higher
Clostridium thermoaceticum(from horse manure) is also able to utilize five-carbon sugars:
2C5H10O5 --- 5CH3COOH
A variety of substrates, including fructose, xylose, lactate, formate, and pyruvate, have been used as carbon sources in an effort to lower substrate costs. This factor is also important if cellulosic renewable resources are to be used as raw materials.
Typical acidogenic bacteria are Clostridium aceticum, C. thermoaceticum, Clostridium formicoaceticum, and Acetobacteriumwoodii. Many can also reduce carbon dioxide and other one-carbon compounds to acetate.
These enzymes are metalloproteins; for example, CODH contains nickel, iron, and sulfur; FDH contains iron, selenium, tungsten, and a small
quantity of molybdenum; and the corrinoid enzyme (vitamin B12 compound) contains cobalt. C. thermoaceticum does not have any specific amino acid requirement; nicotinic acid is the sole essential vitamin
(metal ion lactates)
Food additive (sour flour and dough)
2 isomeric forms L(+) and D(-) and as racemic mixture DL-lactic acid
First isolated from milk
Toady produced microbial
Other than lactate products only lactate as product
L. delbrueckii Glucose
L.helvetii Whey (lactose)
L.lactis ------- Maltose
L.amylophilus -------- Starch
L.pentosus ------ Sulfite waste liquor
Mostly one isomer is produced
1mol of glucose gives 2 moles of lactic acid; L lactic acid is predominantly produced
Fermentation broth (12-15% glucose, N2, PO4, salts micronutrients)
pH 5.5-6.5/temp 45-50oC/75h
Heat to dissolve Ca lactate
Addition of H2SO4
(removal of Ca SO4)
Filter and concentrate
Addtion of Hexacyanoferrant
(removes heavy metal)
Purification (Ion exchange)
Used in stainless steel manufacturing, leather (can remove rust and calcareous deposits)
Food additive for breverages
Used in Ca and Fe therapy
Na gluconate used in sequestering agent in detergets
Desizing polyester or polyamide fabric
Manufacture of frost and cracking resistant concrete
Bacteria: Gluconobacter, Acetobacter, Pseudomonas, Vibrio
Fungi: Aspergillus, Penicillium, Gliocladium
High conc of glucose and pH above 4
H2O2 antagonist for other micro-organisms
Submerged fermentation process
Use glucose from corn
28-30oC for 24h
Incr supply of O2 enhances yield
Used in plastic industry, paper industry
Manufacturing of adhesives
Cis-aconitic acid undergoes decarboxylation
Itaconic acid Oxidase
(-) By Ca to incr yield
Control growth of wide range of unrelated organisms
Control growth of selected number of organisms
Streptomyces,eg. Tetracyclin, actinomycin D,
Antimicrobial agents for chemotherapy
Antitumour antibioticseg. Actinomycin D and mitomycin D
Food preservative antibiotics eg in canning (chlortetracycline) or fish or meat preservation (pimarcin, nisin)
Antibiotics in animal feed and veterinary medicine egenduracidin, tylosin and hygromycinB, theostrepton, salinomycin
Control of plant diseases egblasticidin, teranactin, polyoxin
CELL WALL SYNTHESIS
DNA DIRECTED RNA POLYMERASE
Selective toxicity: concept, Paul Ehrlich
URACIL 5-FLOUROURACIL (Uracil analog)
THYMINE 5-BROMOURACIL (thymine analog)
Addition of F or Br does not alter the shape but changes chemical properties such that the compound does not function in the cell metabolism, thereby blocking the nucleic acid synthesis.
These analogs are used in treatment of viral and fungal infections and many of these occur as mutagens.
FROM BACTERIA, FUNGI
LESS THAN 1% OF 1000S OF ANTIBIOTICS ARE USEFUL BECAUSE OF TOXICITY OR LACK OF UPTAKE BY HOST CELLS
Natural antibiotics can be artificially modified to enhance their efficacy then they are semi-synthetic antibiotics
Broad spectrum antibiotics: effective against both gram +ve and gram-ve
Narrow may also be beneficial to target specific group of bacteria eg. Vancomycin: narrow spectrum effective for gram positive pencillin resistant Staphylococcus, Bacillus, Clostridium
Targets for antibiotics maybe
ribosomes (Cm and Str for Bacteria and Cyclohexamide for eukarya), Cell wall, cytoplasmic membrane, lipid biosynthesis, enzymes, DNA replication and transcription elements
Protein synthesis, Transcription (RNA poly, RNA elongation etc)
B-LACTAMS (b-lactam ring)
Produced by Prokaryotes
AMINOGLYCOSIDES (amino sugars with glycosidic linkage)
MACROLIDES (lactone ring bonded to sugars)
PEPTIDE ANTIBIOTICS (Daptomycin, (Streptomyces)
Pencillin G and V (natural)
Pencillin G first clinically useful antibiotic
For Gram positive bacteria
Slight modification in N-acyl groups results in semi synthetic penicillin which is able to act on gram negative bacteria (goes past outer membrane) to act on cell wall
MANY BACTERIA HAVE BETA LACTAMASE HENCE THOSE BACTERIA ARE PENCILLIN RESISTANT
EG.Oxacillin and Methicillin beta lactamase resistant semi synthetic antibiotics
MECHANISM OF ACTION
Natural penicillin: i.e. V and G are effective against several gram positive bacteria
They are effective against b-lactamase producing MO (enz which can hydrolyze penicillins)
Eg. Staphylococcus aureus
Production of penicillin is used: 45% (human), 15% (animal health) and 45% for production of semi synthetic penicillin
P. notatum, P.chrysogenum and its mutant strain which is a high yeilding strain (Q176)
Genetically engineered strains for improved pencillin production are being used now
UDP tansfers NAG to bactoprenol-NAM peptapeptide. For pentaglycine use special glycyl-tRNA moc but not ribosomes
UDP deriv of NAM and NAG are synthesized
Transport of completed NAM-NAG-pepntapeptide across membrane
Sequentially aa are added to UDP-NAM to form NAM -pentapeptide
ATP is used, no tRNA or ribosomes involved in peptide bond formation
Transfer of UDP-NAM-pentapeptideto bactoprenol PO4
Bactoprenol carrier moves back across membrane by losing one PO4 for a new cycle
Attached to growing end of PG chain and incr by one repeat unit
Bactoprenol is a 55 carbon alcohol and linked to NAM by pyrophosphate
In S. aureus pepntapeptide has L-lys and in
E. coli DAP
Final step is TRANSPEPTIDATION which creates peptide cross links between PG chains. The enzyme removes terminal D-alanine as cross link is formed
The b-lactam group of antibiotics includes an enormous diversity of natural and semi-synthetic compounds that inhibit several enzymes associated with the final step of peptidoglycan synthesis.
All of this enormous family are derived from a b-lactam structure: a four-membered ring in which the b-lactam bond resembles a peptide bond. The multitude of chemical modifications based on this four-membered ring permits the astonishing array of antibacterial and pharmacological properties within this valuable family of antibiotics.
Clinically useful families of b-lactam compounds include the penicillins, cephalosporins, monobactams and carbapenems. Many new variants on the b-lactam theme are currently being explored. Certain b-lactams have limited use directly as therapeutic agents, but may be used in combination with other b-lactams to act as b-lactamase inhibitors.
Co-amoxyclav, for example is a combination of amoxycillin and the b-lactamase inhibitor clavulanic acid. During cross-linking of the peptidoglycan polymer, one D-alanine residue is cleaved from the peptidoglycan precursor and this reaction is prevented by b-lactam drugs.
More recent studies have shown that the activity of this class of drugs is more complicated and involves other processes as well as preventing cross-linking of peptidoglycan.
An increasing number of bacteria are penicillin resistant. Penicillinase-resistant penicillins such as methicillin, nafcillin, and oxacillin are frequently employed against these bacterial pathogens.
Although penicillins are the least toxic of the antibiotics, about 1 to 5% of the adults in the United States are allergic to them. Occasionally a person will die of a violent allergic re- sponse; therefore patients should be questioned about penicillin allergies before treatment is begun.
Cephalosporium: Cephalosporin C
Dihydrothiazine ring (6 member)
Same mode of action with broader spectrum than penicillins
Resistant to b-lactamases
Hence used to treat infections which are penicillin resistant
Used to treat Nesseria gonorrhea (STD)
Most cephalosporins (including cephalothin, cefoxitin, ceftri- axone, and cefoperazone) are administered parenterally.
Cefoperazoneis resistant to destruction by b-lactamases and effective against many gram-negative bacteria, including Pseudomonas aeruginosa.
Cephalexineand cefixime are given orally rather than by injection.
7-ACA: 7- aminocephalosporanic acid nucleus structure in all cephalosporins
G+ > G-
G+ < G-
Three members of the tetracycline family. Tetracycline lacks both of the groups that are shaded. Chlortetracycline (aureomycin) differs from tetracycline in having a chlorine atom (blue); doxycycline consists of tetracycline with an extra hydroxyl (purple).
20 diff species producing mix of tet
Str. aureus. S.flavus
S. rimosus, S. antibioticus
Antibiotics synthesized by successive condensation of small carboxylic acids
Like acetate, butyrate, propionate, malonate
High doses of tetracycline may result in nausea, diarrhea, yellowing of teeth in children, and damage to the liver and kidneys.
Streptomycin, kanamycin, neomycin, and tobramycin are synthesized by Streptomyces, whereas gentamicin comes from a related bacterium, Micromonosporapurpurea.
Known as reserve antibiotics as they develop resistance quickly
Streptomycin Streptomyces griesus
Neomycin B and C S.fradiae
Kanamycin A, B and C S.kanamyceticus
Hygromycin B S.hygroscopicus
Antibiotics with a large lactone ring (macrocyclic lactone ring)
Which consists of 12-, 14- and 16-membered lactone rings with 1-3 sugars linked by glycosidic bond
Effective agaist penicillin resistant MO, G+ org, inhibitb y binding to 50S ribosome
Clarithromycin (Erythromycin derv)
Used to treat stomach ulcers
Erythromycin : Streptomyces erythreus
14-membred connected to 2 sugars
Genetic modifications by polyketide synthesis
Polyene macrolides: lactone rings in range of 26-28
Eg. Nystatin, amphotericin
Actinomycetes are most common organisms which produce them
Erythromycin is a relatively broad-spectrum antibiotic effective against gram-positive bacteria, mycoplasmas, and a few gram-negative bacteria. It is used with patients allergic to penicillinsand in the treatment of whooping cough, diphtheria, diarrhea caused by Campylobacter, and pneumonia from Legionella or Mycoplasma infections.
Newer macrolides are now in use.
Clindamycinis effective against a variety of bacteria including staphylococci and anaerobes such as Bacteroides.
Azithromycinis particularly effective against Chlamydia trachomatis.
Chloroamphenicol, griesofluvin, novobiocin
Broad spectrum antibiotic against G+ and G- bacteria, rickettesia, chlamydia, actinomycetes
chloramphenicol binds to 23S rRNA on the 50S ribosomal subunit. It inhibits the peptidyltransferase and is bacteriostatic.
Streptomyces venezuelae and S.omiyanesis
This antibiotic has a very broad spectrum of activity but unfortunately is quite toxic. One may see allergic responses or neurotoxic reactions. The most common side effect is a temporary or permanent depression of bone marrow function, leading to aplastic anemia and a decreased number of blood leukocytes. Chloramphenicol is used only in life-threatening situations when no other drug is adequate.
Maybe attacks chitin biosynthesis hence acts as anti fungal antibiotic
Following a 40-year hiatus in discovering new classes of antibacterial compounds, three new classes of antibacterial antibiotics have been brought into clinical use:
Cyclic lipopeptides (Daptomycin), Glycylcyclines (tigecycline) and Oxazolidinones (Linezolid)
Daptomycin : Streptomycesroseosporususedtotreat MDR infections
Tigecycline: Tygacil® marketed by Wyeth usedtotreat MDR strainsof
Staphylococcusaureus and Acineotobacterbaumanii. Mechanismsimilartotetracycline.
Also shows suceptibilityto NDML (New Delhi metallo-b-lactamasemultidrugresistantEnterobacteriaceae)
NDML is an enzyme which makes bacteria resistant to broad range of b-lactam antibiotics.
This includes antibiotics of carbapenems for treatment of antibiotics resistant infections.
Termed as “SUPERBUGS” Such bacteria susceptible to polymixins and tigecyclines
Superinfection: Clostridium difficile, Candida albicans
Transformation, conjugation, transduction, ABC transporters
There has been some recent progress in developing new antibiotics that are effective against drug-resistant pathogens.
Two new drugs are fairly effective against vancomycin-resistant enterococci. Synercid is a mixture of the streptogramin antibiotics quinupristin and dalfopristinthat inhibits protein synthesis.
A second drug, linezolid (Zyvox), is the first drug in a new family of antibiotics, the oxazolidinones. It inhibits protein synthesis and is active against both vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus.