A. Primary Metabolites: log phase, use nutrients fast, produce PM

B. Secondary Metabolites: depletion of nutrients, growth retards, produce SM

Microbial Products

Primary Metabolites: Vitamins

Vitamins: cannot be synthesized by higher organismsBut microorganisms are capable of synthesizing (gut)

ThiamineRiboflavinPyridoxineFolic acidPantothenic acidBiotinVitamin B12Ascorbic acidb- carotene (provitamin A)Ergosterol (vitamin D)

Studies reveal vitamin deficiencies Reported beneficial health effects Growing vitamin market demand (cost

effective) Genetically engineered MO as

alternatives to chemical synthesis

Vitamins

Fat soluble Water soluble

Carotenoidsb-carotene (provitamin A)Astaxanthin

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)Folic acid

Ergosterol (vitamin D)

Vitamin B12 or Cyanocobalamin

• Water soluble vitamin ; complex sructure• Has role in functioning of brain and nervous system, formation

of blood• Contains rare element cobalt

• Deficiency causes pernicious anemia which is an causes low Hb, less RBCs

• Pernicious anemia: autoimmune disorder, parietal cells (stomach) responsible for secreting intrinsic factor are destroyed. Intrinsic factor is crucial for the normal absorption of B12, so a lack of intrinsic factor, as seen in pernicious anemia, causes a deficiency of Vitamin B12

• dietary reference intake for an adult ranges from 2 to 3 µg per day

• used in treating cyanide poisoning, prevents brain atrophy in Alzheimer’s patients

• COMMON INGREDIENT IN ENERGY DRINKS

cobinamide

nucleotide

• Corrin ring• Deep red colour due to corrin

ring • Central Co atom• Coordination state 6• 4 of 6 coord sites have pyrrole

ring• 5 has dimethylbenzimidazole

group• 6 is center of reactivity,

variab;e• CN, OH, Me, 5-deoxyadenosyl

for 4 types of B12

4 Pyrrole unitsPyrrole nitrogen

5,6-dimethyl benzimindazole

C63 H88 CoN14 O14P

1 2

34

5

6

Commercial production

Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium, Flavobacterium, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus and Xanthomonas

Genera known to produce vit B12

Most commonly used for industrial production are Streptomyces griesusPseudomonas denitrificans (aerobic)Salmonella typhimuriu (anaerobic)Propionibacterium shermanii GRAS by FDA

(anaerobic) (Generally Regarded As Safe)

Sanofi-Aventis (FRENCH) use genetically engineered versions to produce vit B12 under specialized conditions from Propionibacterium since they have no endotoxins or exotoxins

P. denitrificans also used after strain modification; mutant more efficient than wild type

20mg/L

Chemical syn not feasible

Commercial production• Produced in continuous culture with 2 fermenters in series

Anaerobic70h

Aerobic 50h

GlucoseCorn steep Betaine (5%)Cobalt (5ppm)pH 7.5 +Propionibacterium freudenreichii

Cobinamide production and accumulation

Nucleotide synthesizedCombined with cobinamideTo yield 2ppm of cobalamin

Acidification of cultureTo 2-3pH/ 100oCFilter to remove cell debris

Filtrate

KCN added

CYNACOBALAMIN80% purityUsed as feed additive

Addition of 5,6-dimethylbenzimidazol (0.1%)

Betaine: sugar beet molasses

Commercial production

ANAEROBIC PHASE

2-4 DAYS5-deoxyadenosylcobinamide produced

AEROBIC PHASE

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)

Commercial production

Pseudomonas denitrificans: strain improvements resulted in increase in yeildFrom 0.6mg/L to 60mg/L

Glucose : common carbon

Alcohols (methanol, ethanol, isopropanol)Hydrocarbons(alkanes, decane, hexadecane)

With methanol 42mg/L was obtained using Methanosarcina barkeri

Riboflavin (Rf) or Vitamin B2

• Water soluble• Essential for growth and reproduction; key role in energy metabolism, ketone

bodies, fats, CHO and protein metabolism• Deficiency leads to cheliosis (fissures around mouth), glossitis (purple tounge)

and dermatitis• Required in coenzymes FAD (flavin adenine dinucleotide) and FMN (flavin

mononucleotide)• Used as an orange-red food colour additive, designated in Europe as E101

7,8-dimethyl-10- (D-19-ribityl) isoalloxazine

Participates in O-R reactions

Flavin is ring moiety with yellow colour to oxidized form

FADE101

FMNE101a

Isoalloxazine ring Isoalloxazine ring

Ribitol

H

H

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.

INDUSTRIAL USE

Commercial production

Glucose

50% by biotransformationusing Bacillus pumulis

D-ribose

20% production by Chemical synthesis

Riboflavin

1/3rd production by direct fermentation

Acetone butanol fermentationClostridium acetobutylicumC. butylicum riboflavin as

by product

Ashbya gossypiiCandida famataBacillus subtillis (genetically modified)

Major riboflavin producers are DSM Nutritional Products (Switzerland) and Hubei Guangji (Hubei Province, China), both using genetically engineered B. subtilis production strains, and BASF (first in Germany but now in South Korea), employing genetically engineered A. gossypii.

Commercial production

Phase I use of glucose, accumulation of pyr, pH acidic, growth stops, no Riboflv

Phase II decr pyr, 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

Ascorbic acid or Vitamin C

Precursor for its chemical synthesis can be obtained by biological methods

• Used in collagen biosynthesis, protects against nitrosamines, free radicals• Deficiency causes scurvy

feed applications of L-ascorbic acid account for only 10%, whereas the main uses are in the pharmaceutical industry (50%), food (25%), and beverages (15%).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.

D-glucose (200g)

Submerged bioreactor fermentation

D-sorbitol

sorbitol dehydrogenase

L-Sorbosechemical oxidation

2 keto L gulonic acid

Enol form of 2 keto L gulonic acid

acid treatment

L-ASCORBIC ACID (100g)

Acetobacter xylinum, A,suboxydans

Glucuronic acid

Gluconolactone

L-Gluconolactone

L-ASCORBIC ACID

L-Gluconolactonedehydrogenase

Reichstein Grussner synthesis

Erwinia sp.Acetobacter sp.Gluconobacter sp.

2,5-diketogluconic acid

2-keto L-gluconic acid

L-ASCORBIC ACID

Corynebacterium sp.

2,5-diketogluconic acidreductase

2,5-diketogluconic acidReductase of Corynebacterium into Erwinia herbicola

Cloning of gene

Bacillus megaterium

b- carotene or provitamin A

Provitamin A -----> Vitamin A (intestine)

• Fat soluble• Deficiency leads to night blindness• Best source is liver and whole milk also coloured fruits and vegetables

• Isoprene derivatives• Tetraterpenoids with eight isoprene residues• 400 naturally occurring carotenoids: b-carotene, a-carotene, d-carotene,

lycopene, zeaxanthin

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.

Commercial production Microbial fermentationBlakeslea trispora (high yeild; 7g/L)Phycomyces blakesleeanusChoanephora cucurbitarumSubmerged Fermentation process

Corn starch, soyabean meal, b-ionone, antioxidants

DSM Nutritional Products (Switzerland) and BASF (Germany) dominate the market with their chemical synthesis processes, but Chinese competitors are catching up.

Trisporic acid: act as microbial sex hormone, improves yieldb-Ionone: incr b-carotene syn by incr enzyme activityPurified deodorized kerosene increases solubility of hydrophobic substrates

Recovery: b- carotene rich mycelium used as feed additiveMycelium is dehydrated by methanol, extracted in methylene chloride and crystallized which is 70-85% pure

stimulators

Halophilic green microalgae Dunaliella salina. It accumulates the pigments in oil glo- bules in the chloroplast interthylakoid spaces, protecting them against photoinhibition and photodestruction.

Excessive pigment formation in D. salina is 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. salina biomass for sale contains 10–16% carotenoids, mainly b-carotene. In addition crystalline material obtained after extraction with edible oil is also sold.

Primary Metabolites: Organic Acids

Organic acids are produced by through metabolisms of carbohydrates. They accumulate in the broth of the fermenter from where they are separated and purified.

GlycolysisKrebs cycle

I. Terminal end productslactic acid

(pyruvate, alcohol)Propionic acid

II. Incomplete oxidation of sugars citric acid(glucose)

Itaconic acid

Gluconic acidIII. Dehydrogenation of alcohol with O2 acetic acid

Manufactured on large scale as pure products or as salts

CITRIC ACID: industrial uses

Flavoring agentIn food and beverages

Jams, candies, deserts, frozen fruits, soft drinks, wine

Antioxidants and preservative

Chemical industryAntifoamTreatment of textilesMetal industry, pure metals +citrate (chelating agent)

Pharmaceutical industryTrisodium citrate (blood preservative)Preservation of ointments and cosmeticsSource of iron

Agent for stabilization of Fats, oil or ascorbic acidStabilizer for cheese preparation

Detergent cleaning industryReplace polyphosphates

AcidifyerFlavoringChelating agentPrimary metabolitePresent in all organisms

Aspergillus nigerA. clavatusPencillium luteum

Commercial ProductionStrains that can tolerate high sugar and low pH with reduced

synthesis of undesirable by products (oxalic acid, isocitric acid, gluconic acid)

Glucose

Pyruvate

Pyruvate

Acetyl CoACO2

CO2

Pyruvate

OXA

Malate

MITOCHONDRIAMalate Fumarate

Succinyl CoA

OXA

citric acid

a-KG

CYTOPLASM

Glucose MEDIUM

Pyr carboxylase

Pyr Dehy-drogenase Citrate

synthase

100g sucrose --- 112g any citric acid or 123g citric acid-1hydrate

Factors for regulation CARBOHYDRATE SOURCE: sugar should be 12-25%

Molasses (sugar cane or sugar beet) Starch (potato) Date syrup Cotton waste Banana extract Sweet potato pulp Brewery waste Pineapple waste

High sugar conc incr uptake and production of citric acid TRACE METALS:

Mn2+, Fe3+, Zn2+ incr yield Mn2+ incr glycolysis Fe3+ is a cofator for enzymes like aconitase

pH: incr yield when pH below 2.5, production of oxalic acid and gluconic acid is suppressed and risk of contamination is minimal

DISSOLVED O2: high O2, sparging or incr aeration can affect if interrupted

NITROGEN SOURCE: addition of ammonium stimulates overproduction, molasses is good source of nitrogen

Citric acid production

Surface fermentationsubmerged fermentation

Solidliquid

Stirred AirliftBioreactorbioreactor

N alkanes (C9-C23) can also be used to produce citric acid; can result in excess production of isocitric acid

ACETIC ACID: industrial uses

ACETIC ACID

Vinegar is prepared from alcoholic liquids since ceturies

CH3 CH2OH---- CH3CHO-------- CH3CH(OH)2 ------- CH3COOHEthanol acetaldehyde acetaldehyde hydrate acetic acid

NAD+NADH

+H+

NADP+ NADP +H+

Alcoholdehydrogenase Acetaldehyde dehydrogenase

Gluconobacter, Acetobacter with acid tolerant A. aceti

Incomplete oxidation of ethanol

One molecule of ethanol one molecule of acetic acid is produced12% 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

optimumpH of6.6–6.8.

Clostridium thermoaceticum

VINEGAR: 4% by volume acetic acid with alcohol, salts, sugars and esters

flauoring agent in sauces and ketchups, preservative alsoWine, 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 Acetobacterium woodii. 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 smallquantity 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

1mol

2moles

2moles

1mol

1molCODH

LACTIC ACID: industrial uses

Technical grade

20-50%

Ester manufactureTextile industry

Food grade>80%

Food additive (sour flour and dough)

Pharmaceutical grade

>90%

Intestinal treatment(metal ion lactates)

Glucose

G3P NAD+

NADH +H+1,3-biphosphoglycerate

G3P dehy-drogenase Pyruvate

Lactic acid

LDH(Lactate dehydrogenase)

LACTIC ACID

2 isomeric forms L(+) and D(-) and as racemic mixture DL-lactic acidFirst isolated from milkToady produced microbial

HeterofermentationHomofermentation

Other than lactate products only lactate as product

Lactobacillus

L. delbrueckii Glucose

L. leichmanni

L. bulgaricusL.helvetiiWhey (lactose)

L.lactis ------- MaltoseL.amylophilus -------- StarchL.pentosus ------ Sulfite waste liquor

Mostly one isomer is produced

LACTIC ACID: production process

Fermentation broth (12-15% glucose, N2, PO4, salts micronutrients)

pH 5.5-6.5/temp 45-50oC/75hHeat to dissolve Ca lactate

Addition of H2SO4(removal of Ca SO4)

Filter and concentrate

Addtion of Hexacyanoferrant(removes heavy metal)

Purification (Ion exchange)

Concentration

Lactic acid

1mol of glucose gives 2 moles of lactic acid; L lactic acid is predominantly produced

GLUCONIC ACID: Applications

1. Used in stainless steel manufacturing, leather (can remove rust and calcareous deposits)

2. Food additive for breverages3. Used in Ca and Fe therapy4. Na gluconate used in sequestering agent in detergets5. Desizing polyester or polyamide fabric6. Manufacture of frost and cracking resistant concrete

Bacteria: Gluconobacter, Acetobacter, Pseudomonas, VibrioFungi: Aspergillus, Penicillium, Gliocladium

D-Glucose D-gluconolactone Gluconic Acid

PQQ PQQH2Glucose

dehydrogenase

PQQ: pyrroliquinoline quinonecoenzyme

Bacteria

Fungi FAD FADH2

O2H2O2

Glucose oxidase

Catalase

Lactonase

H2O

fungi

intracellular extracellular

ExtracellularInducible

High conc of glucose and pH above 4H2O2 antagonist for other micro-organisms

Submerged fermentation processUse glucose from cornH 4.5-6.528-30oC for 24hIncr supply of O2 enhances yield

ITACNIC ACID: Applications

1. Used in plastic industry, paper industry2. Manufacturing of adhesives

Aspergillus itoconicus and A.terreus

Cis-aconitic acid undergoes decarboxylation

Itaconic acid Itatartaric acid

(-) By Ca to incr yield

Itaconic acid Oxidase

SECONDARY METABOLITES

ANTIBIOTICS

BROAD SPECTRUM NARROW SPECTRUM

Control growth of wide range of unrelated organismsTet, Cm

Control growth of selected number of organismsPen, Str

Streptomyces,eg. Tetracyclin, actinomycin D,

ANTIBIOTICS: applications

1. Antimicrobial agents for chemotherapy2. Antitumour antibiotics eg. Actinomycin D and mitomycin D3. Food preservative antibiotics eg in canning (chlortetracycline) or fish or

meat preservation (pimarcin, nisin)4. Antibiotics in animal feed and veterinary medicine eg enduracidin,

tylosin and hygromycin B, theostrepton, salinomycin5. Control of plant diseases eg blasticidin, teranactin, polyoxin6. Molecular biology

MODE OF ACTION OF ANTIBIOTICS

DNA

RNARIBOSOMES

PABA

DHF

THF

CELL WALL SYNTHESISDNA GYRASE

RNA ELONGATION

DNA DIRECTED RNA POLYMERASE

PROTEIN SYNTHESIS(50S INHIBITORS)

PROTEIN SYNTHESIS(30S INHIBITORS)

PROTEIN SYNTHESIS(tRNA)

LIPID BIOSYNTHESIS

CYTOPLASMIC MEMBRANE STRUCTURE AND FUNCTION

SYTHETIC ANTIBIOTICS

Selective toxicity: concept, Paul Ehrlich

1. GROWTH FACTOR ANALOGS:structurally similar to a growth factor required in a micro-

organism; small differences of analogs in authentic growth factor prevent analog to function in the cell.

A. SULFA DRUGS: specifically inhibit bacteria (streptococcal infections) eg. SULFANILAMIDE: is an analog of PABA (p-aminobenzoic acid) which is part of folic acid and nucleic acid precursor. Combination: sulfamethoxazole and trimethoprim; disadvantages and advantages

B. ISONIAZID: important growth factor with narrow spectrum only against Mycobacterium. It interferes with synthesis of mycolic acids, a cell wall component. It is an analog of nicotinamide (vitamin). Single most effective drug against tuberculosis.

2. NUCLEIC ACID BASE ANALOGS

URACIL 5-FLOUROURACIL (Uracil analog)PHENYLALANINE p-FLOUROPHENYLALANINETHYMINE 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.

3. QUINOLONES:Antibacterial compounds interfere with bacterial DNA gyrase, prevent supercoiling (packaging of DNA) eg Flouroquinolones like ciprofloxin (UTI, anthrax). B. anthracis maybe resistant to pencillin. These are effective in both G+ve and G-ve bacteria since DNA gyrase is present in all.Also used in beef and poultry for prevention and treatment of respiratory diseases.

Ouinolones

New generation Flouroquinolnes

NATURALLY OCCURING ANTIBIOTICS

FROM BACTERIA, FUNGILESS THAN 1% OF 1000S OF ANTIBIOTICS ARE USEFUL BECAUSE OF TOXICITY OR LACK OF UPTAKE BY HOST CELLSNatural 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-veNarrow 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 mayberibosomes (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)

Produced By Fungi

B-LACTAMS (b-lactam ring)PenicillinCephalosporins

Produced by Prokaryotes

AMINOGLYCOSIDES (amino sugars with glycosidic linkage)MACROLIDES (lactone ring bonded to sugars)TETRACYLINES (Streptomyces)PEPTIDE ANTIBIOTICS (Daptomycin, (Streptomyces)PLATENSIMYSIN (Streptomyces)

1. PENICILLINS, 2. CEPHALOSPORINS, 3. MONOBACTAMS AND 4. CARBAPENEMS

Beta Lactam Antibiotics

PENCILLIN--------b-LACTAM ANTIBIOTIC

Pencillin G and V (natural)Penicillium chrysogenumAlexander Fleming

Used for PneumococcalStreptococcal infections

Pencillin G first clinically useful antibioticFor Gram positive bacteria

6-AMINOPENICILLIANIC ACID

Ampicillin, carbencillin

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 RESISTANTEG. Oxacillin and Methicillin beta lactamase resistant semi synthetic antibiotics

MECHANISM OF ACTION

• Pencillins block cell wall synthesis: transpeptidation (cross linking 2 glycan peptide chains)

• Transpeptidases bind to pencillin hence they are called PENCILLIN BINDING PROTEINS (PBP)

• Newly synthesized bacterial wall is no longer cross linked and has poor strength

• PBP also stimulates release of AUTOLYSINS (ENZYMES TO DIGEST CELL WALL)• Osmotic pressure differences cause lysis

• VANCOMYCIN: does not bind PBPs but D-alanyl- Dalanine peptide to block transpeptidation

• BECAUSE OF SELECTIVE PROCESS B-LACTAMS DO NOT AFFTECT HOST CELLS AND MECHANISM IS UNIQUE TO BACTERIA

MECHANISM OF ACTION

Natural penicillin: i.e. V and G are effective against several gram positive bacteriaThey 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 deriv of NAM and NAG are synthesizedSequentially aa are added to UDP-NAM to form NAM -pentapeptideATP is used, no tRNA or ribosomes involved in peptide bond formation

Transfer of UDP-NAM-pentapeptideto bactoprenol PO4

UDP tansfers NAG to bactoprenol-NAM peptapeptide. For pentaglycine use special glycyl-tRNA moc but not ribosomes Transport of completed NAM-

NAG-pepntapeptide across membrane

Attached to growing end of PG chain and incr by one repeat unit

Bactoprenol carrier moves back across membrane by losing one PO4 for a new cycle

LIPID I LIPID II

UDP glucose

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.

B-lactamase

MRSAVRSA

CEPHALOSPORINS

cefatrioxone

B-lactam ring Dihydrothiazine ring (6 member)

Same mode of action with broader spectrum than penicillinsResistant to b-lactamasesHence used to treat infections which are penicillin resistantUsed to treat Nesseria gonorrhea (STD)

Cephalosporium: Cephalosporin C

Most cephalosporins (including cephalothin, cefoxitin, ceftri- axone, and cefoperazone) are administered parenterally. Cefoperazone is resistant to destruction by b-lactamases and effective against many gram-negative bacteria, including Pseudomonas aeruginosa. Cephalexine and cefixime are given orally rather than by injection.

7-ACA: 7- aminocephalosporanic acid nucleus structure in all cephalosporins

G+ > G- G+ = G-

G+ < G-

R1 R2

TETRACYCLINES

• Broad spectrum• Effective for G+ and G- (mycoplasmas, rickettesia, chlamydia)• Used for combatting stomach ulcer (Helicobacter pylori)• Inhibit protein synthesis by blocking binding of amino acyl tRNA to ribosome (A site)

BASIC STRUCTURE

• Napthacene ring• Chlortetracycline and oxytetracycline are most commonly used in human and

veterinary diseases and for preservation of meat, fish and poultry

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).

TETRACYCLINES Streptomyces aureofaciens20 diff species producing mix of tetGenetic modificationPolyketide synthesis

Antibiotics synthesized by successive condensation of small carboxylic acidsLike 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.

Str. aureus. S.flavusS. rimosus, S. antibioticus

AMINOGLYCOSIDESOligosaccharide antibiotics

Streptomycin, kanamycin, neomycin, and tobramycin are synthesized by Streptomyces, whereas gentamicin comes from a related bacterium, Micromonospora purpurea.

Known as reserve antibiotics as they develop resistance quickly

• Structurally all contain a cyclohexane ring and amino sugars bound by glycosidic linkages

• Bind to the 30S small ribosomal subunit and interfere with protein synthesis in at least two ways. They directly inhibit protein synthesis and also cause misreading of the genetic message carried by mRNA…prolonged use can cause kidney damage and hearing loss

AMINOGLYCOSIDES producing organisms

Streptomycin Streptomyces griesus

Neomycin B and C S.fradiae

Kanamycin A, B and C S.kanamyceticus

Hygromycin B S.hygroscopicus

Gentamycin Micromonospora purpurea

Sisimicin M.inyoensis

MACROLIDESAntibiotics 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

Erythromycin : Streptomyces erythreus14-membred connected to 2 sugarsGenetic modifications by polyketide synthesis

Clarithromycin (Erythromycin derv)Used to treat stomach ulcers

MACROLIDES

Polyene macrolides: lactone rings in range of 26-28Eg. 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 penicillins and in the treatment of whooping cough, diphtheria, diarrhea caused by Campylobacter, and pneumonia from Legionella or Mycoplasma infections.

Newer macrolides are now in use. Clindamycin is effective against a variety of bacteria including staphylococci and anaerobes such as Bacteroides. Azithromycin is particularly effective against Chlamydia trachomatis.

AROMATIC ANTIBIOTICS

CHLORAMPHENICOL

Aromatic rings in structureChloroamphenicol, 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 peptidyl transferase and is bacteriostatic.

Streptomyces venezuelae and S.omiyanesis

GRIESOFULVIN

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

Penicillium patulum

PEPTIDE ANTIBIOTICS

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 : Streptomyces roseosporus used to treat MDR infections

Tigecycline: Tygacil® marketed by Wyeth used to treat MDR strains of Staphylococcus aureus and Acineotobacter baumanii.

Mechanism similar to tetracycline.

Also shows suceptibility to NDML (New Delhi metallo-b-lactamase multidrug resistant Enterobacteriaceae)

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

MECHANISM OF DRUG RESISTANCE

PlasmidsR-PlasmidsSuperinfection: Clostridium difficile, Candida albicansTransformation, conjugation, transduction, ABC transportersPhage therapy

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 dalfopristin that 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.