Primary Metabolites: log phase, use nutrients fast, produce PM Secondary Metabolites: depletion of nutrients, growth r

1. Microbial Products Primary Metabolites: log phase, use nutrients fast, produce PM Secondary Metabolites: depletion of nutrients, growth retards, produce SM

2. Primary Metabolites: Vitamins Vitamins: cannot be synthesized by higher organisms But microorganisms are capable of synthesizing (gut) Thiamine Riboflavin Pyridoxine Folic acid Pantothenic acid Biotin Vitamin B12 Ascorbic acid b- 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

3. Vitamins Fat soluble Water soluble Carotenoids b-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)

4. 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

5. Pyrrole nitrogen 4 Pyrrole units C63 H88 CoN14 O14P cobinamide • 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 6 2 1 4 3 5 5,6-dimethyl benzimindazole nucleotide

6. Commercial production 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 20mg/L • Most commonly used for industrial production are • Streptomyces griesus • Pseudomonas denitrificans (aerobic) • Salmonella typhimuriu (anaerobic) • PropionibacteriumshermaniiGRAS 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. denitrificansalso used after strain modification; mutant more efficient than wild type

7. Commercial production • Produced in continuous culture with 2 fermenters in series • Addition of 5,6-dimethylbenzimidazol (0.1%) Glucose Corn steep Betaine (5%) Cobalt (5ppm) pH 7.5 + Anaerobic 70h Aerobic 50h Propionibacterium freudenreichii Cobinamide production and accumulation Nucleotide synthesized Combined with cobinamide To yield 2ppm of cobalamin KCN added Acidification of culture To 2-3pH/ 100oC Filter to remove cell debris CYNACOBALAMIN 80% purity Used as feed additive Filtrate Betaine: sugar beet molasses

8. Commercial production ANAEROBIC PHASE 2-4 DAYS 5-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)

9. Commercial production 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

10. 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

11. Isoalloxazine ring Isoalloxazine ring H H Ribitol FAD E101 FMN E101a genes encoding the riboflavin biosynthetic enzymes are well conserved among bacteria and fungi

12. INDUSTRIAL USE 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.

13. Commercial production Glucose 50% by biotransformation usingBacillus pumulis D-ribose 20% production by Chemical synthesis Riboflavin 1/3rdproduction by direct fermentation Acetone butanol fermentation Clostridium acetobutylicum C. butylicum riboflavin as by product Ashbyagossypii Candida famata Bacillus subtillis (genetically modified)

14. Commercial production 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.

15. Ascorbic acid or Vitamin C • Used in collagen biosynthesis, protects against nitrosamines, free radicals • Deficiency causes scurvy 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 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.

16. Submerged bioreactor fermentation Erwiniasp. Acetobacter sp. Gluconobacter sp. 2,5-diketogluconic acid 2-keto L-gluconic acid L-ASCORBIC ACID D-glucose (200g) Glucuronic acid Gluconolactone L-Gluconolactone L-ASCORBIC ACID 2,5-diketogluconic acid reductase Corynebacterium sp. D-sorbitol sorbitol dehydrogenase L-Sorbose chemical oxidation 2 keto L gulonic acid Enol formof 2 keto L gulonic acid acid treatment L-ASCORBIC ACID (100g) Bacillus megaterium Acetobacterxylinum, A,suboxydans L-Gluconolactone dehydrogenase Cloning of gene 2,5-diketogluconic acid Reductaseof Corynebacterium into Erwiniaherbicola Reichstein Grussner synthesis

17. 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.

18. Commercial production • Microbial fermentation • Blakesleatrispora (high yeild; 7g/L) • Phycomycesblakesleeanus • Choanephoracucurbitarum 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 stimulators 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.

19. 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.

20. 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. I. Terminal end products lactic acid (pyruvate, alcohol) Propionic acid II. Incomplete oxidation of sugars citric acid (glucose) Itaconic acid Gluconic acid Glycolysis Krebs cycle III. Dehydrogenation of alcohol with O2 acetic acid Manufactured on large scale as pure products or as salts

21. CITRIC ACID: industrial uses Flavoring agent 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 Chemical industry Antifoam Treatment of textiles Metal industry, pure metals +citrate (chelating agent) Pharmaceutical industry Trisodium citrate (blood preservative) Preservation of ointments and cosmetics Source of iron Acidifyer Flavoring Chelating agent Primary metabolite Present in all organisms Detergent cleaning industry Replace polyphosphates

22. Commercial Production Aspergillusniger clavatus Pencilliumluteum Strains that can tolerate high sugar and low pH with reduced synthesis of undesirable by products (oxalic acid, isocitric acid, gluconic acid) Glucose MEDIUM CYTOPLASM Pyruvate Glucose Pyruvate Pyruvate Acetyl CoA Pyr carboxylase CO2 OXA Malate MITOCHONDRIA Malate FumarateSuccinyl CoA OXA citric acid a-KG PyrDehy- drogenase CO2 Citrate synthase 100g sucrose --- 112g any citric acid or 123g citric acid-1hydrate

23. 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 concincr 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

24. Citric acid production Surface fermentation submerged fermentation Solid liquid Stirred Airlift Bioreactor bioreactor N alkanes (C9-C23) can also be used to produce citric acid; can result in excess production of isocitric acid

25. ACETIC ACID: industrial uses

26. ACETIC ACID Incomplete oxidation of ethanol Vinegar is prepared from alcoholic liquids since ceturies NAD+ NADH +H+ NADP+NADP +H+ CH3 CH2OH---- CH3CHO-------- CH3CH(OH)2 ------- CH3COOH Ethanol acetaldehyde acetaldehyde hydrate acetic acid Alcohol dehydrogenase Acetaldehyde dehydrogenase 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. Clostridium thermoaceticum

27. 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.

28. 1mol 2moles 2moles 1mol 1mol CODH 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

29. LACTIC ACID: industrial uses Pharmaceutical grade >90% Technical grade 20-50% Food grade >80% Intestinal treatment (metal ion lactates) Food additive (sour flour and dough) Ester manufacture Textile industry Glucose G3P NAD+ NADH +H+ 1,3-biphosphoglycerate Lactic acid LDH (Lactate dehydrogenase) G3P dehy- drogenase Pyruvate

30. LACTIC ACID 2 isomeric forms L(+) and D(-) and as racemic mixture DL-lactic acid First isolated from milk Toady produced microbial HeterofermentationHomofermentation Other than lactate products only lactate as product Lactobacillus L. delbrueckii Glucose L. leichmanni L. bulgaricus L.helvetii Whey (lactose) L.lactis ------- Maltose L.amylophilus -------- Starch L.pentosus ------ Sulfite waste liquor Mostly one isomer is produced

31. LACTIC ACID: production process 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) Concentration Lactic acid

32. GLUCONIC ACID: Applications 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

33. intracellular extracellular PQQH2 Bacteria PQQ Glucose dehydrogenase H2O D-gluconolactone Gluconic Acid D-Glucose Lactonase Glucose oxidase fungi Extracellular Inducible FAD FADH2 Fungi Catalase PQQ: pyrroliquinolinequinone coenzyme H2O2 O2 High conc of glucose and pH above 4 H2O2 antagonist for other micro-organisms Submerged fermentation process Use glucose from corn H 4.5-6.5 28-30oC for 24h Incr supply of O2 enhances yield

34. ITACNIC ACID: Applications Aspergillusitoconicusand A.terreus Used in plastic industry, paper industry Manufacturing of adhesives Cis-aconitic acid undergoes decarboxylation Itaconic acid Oxidase Itaconic acid Itatartaric acid (-) By Ca to incr yield

35. SECONDARY METABOLITES ANTIBIOTICS BROAD SPECTRUM NARROW SPECTRUM Control growth of wide range of unrelated organisms Tet, Cm Control growth of selected number of organisms Pen, Str Streptomyces,eg. Tetracyclin, actinomycin D,

36. ANTIBIOTICS: applications 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 Molecular biology

37. MODE OF ACTION OF ANTIBIOTICS RNA ELONGATION DNA GYRASE CELL WALL SYNTHESIS DNA DIRECTED RNA POLYMERASE DNA PROTEIN SYNTHESIS (50S INHIBITORS) PROTEIN SYNTHESIS (30S INHIBITORS) PROTEIN SYNTHESIS (tRNA) THF RNA RIBOSOMES DHF CYTOPLASMIC MEMBRANE STRUCTURE AND FUNCTION LIPID BIOSYNTHESIS PABA

38. SYTHETIC ANTIBIOTICS Selective toxicity: concept, Paul Ehrlich • 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. • 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 • 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.

39. NUCLEIC ACID BASE ANALOGS URACIL 5-FLOUROURACIL (Uracil analog) PHENYLALANINE p-FLOUROPHENYLALANINE 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. • QUINOLONES: • Antibacterial compounds interfere with bacterial DNA gyrase, prevent supercoiling (packaging of DNA) egFlouroquinolones 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.

40. New generation Flouroquinolnes Ouinolones

41. NATURALLY OCCURING ANTIBIOTICS 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)

42. Produced By Fungi B-LACTAMS (b-lactam ring) Penicillin Cephalosporins Produced by Prokaryotes AMINOGLYCOSIDES (amino sugars with glycosidic linkage) MACROLIDES (lactone ring bonded to sugars) TETRACYLINES (Streptomyces) PEPTIDE ANTIBIOTICS (Daptomycin, (Streptomyces) PLATENSIMYSIN (Streptomyces)

46. Beta Lactam Antibiotics PENICILLINS, CEPHALOSPORINS, MONOBACTAMS AND CARBAPENEMS

47. PENCILLIN--------b-LACTAM ANTIBIOTIC Pencillin G and V (natural) Penicilliumchrysogenum Alexander Fleming Pencillin G first clinically useful antibiotic For Gram positive bacteria Used for Pneumococcal Streptococcal infections 6-AMINOPENICILLIANIC ACID

48. 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 RESISTANT EG.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

49. 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

50. 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 LIPID I LIPID II 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