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Isolation and Screening of Secondary Metabolites

Isolation of Macromolecules

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Isolation and Screening of Secondary Metabolites

  1. 1. © Angel L. Salaman. PhD ISOLATION AND SCREENING OF SECONDARY METABOLITES Angel L. Salaman, PhD angelsalaman@yahoo.com
  2. 2. INDUSTRIAL PRODUCTS AND THE MICROORGANISMS THAT MAKE THEM  Industrial microbiology  Uses microorganisms, typically grown on a large scale, to produce products or carry out chemical transformation  Originated with alcoholic fermentation processes  Later on, processes such as production of pharmaceuticals, food additives, enzymes, and chemicals were developed  Major organisms used are fungi and Streptomyces  Classic methods are used to select for high-yielding microbial variants
  3. 3. INDUSTRIAL PRODUCTS AND THE MICROORGANISMS THAT MAKE THEM  Properties of a useful industrial microbe include  Produces spores or can be easily inoculated  Grows rapidly on a large scale in inexpensive medium  Produces desired product quickly  Should not be pathogenic  Allow genetic manipulation
  4. 4. INDUSTRIAL PRODUCTS AND THE MICROORGANISMS THAT MAKE THEM  Microbial products of industrial interest include  Microbial cells  Enzymes  Antibiotics, steroids, alkaloids  Food additives  Commodity chemicals  Inexpensive chemicals produced in bulk  Include ethanol, citric acid, and many others
  5. 5. PRODUCTION AND SCALE  Primary metabolite  Produced during exponential growth  Example: alcohol  Secondary metabolite  Produced during stationary phase
  6. 6. PRODUCTION AND SCALE  Secondary metabolites  Not essential for growth  Formation depends on growth conditions  Produced as a group of related compounds  Often significantly overproduced  Often produced by spore-forming microbes during sporulation
  7. 7. © Angel L. Salaman. PhD FIGURE 15.1 Primary metabolite Secondary metabolite Alcohol Penicillin Cells Sugar Cells Sugar Time Time Alcohol,sugar,orcellnumber Penicillin,sugar,orcellnumber
  8. 8. PRODUCTION AND SCALE  Secondary metabolites are often large organic molecules that require a large number of specific enzymatic steps for production  Synthesis of tetracycline requires at least 72 separate enzymatic steps  Starting materials arise from major biosynthetic pathways
  9. 9. PRODUCTION AND SCALE  Fermentor is where the microbiology process takes place  Any large-scale reaction is referred to as a fermentation  Most are aerobic processes  Fermentors vary in size from 5 to 500,000 liters  Aerobic and anaerobic fermentors  Large-scale fermentors are almost always stainless steel  Impellers and spargers supply oxygen
  10. 10. © Angel L. Salaman. PhD FIGURE 15.2A
  11. 11. © Angel L. Salaman. PhD FIGURE 15.2B Steam Sterile seal Motor pH pH controller Acid–base reservoir and pump Viewing port Filter Exhaust Impeller (mixing) Cooling jacket External cooling water in External cooling water out Culture broth Steam in Valve Harvest Sparger (high- pressure air for aeration) Sterile air
  12. 12. © Angel L. Salaman. PhD FIGURE 15.2C
  13. 13. © Angel L. Salaman. PhD FIGURE 15.3
  14. 14. ANTIBIOTICS: ISOLATION, YIELD, AND PURIFICATION  Antibiotics  Compounds that kill or inhibit the growth of other microbes  Typically secondary metabolites  Most antibiotics in clinical use are produced by filamentous fungi or actinomycetes  Still discovered by laboratory screening Microbes are obtained from nature in pure culture  Assayed for products that inhibit growth of test bacteria
  15. 15. © Angel L. Salaman. PhD FIGURE 15.4A I. Isolation Sterile glass spreader Colonies of Streptomyces species Nonproducing organisms Zones of growth inhibition Producing organisms Spread a soil dilution on a plate of selective medium Incubation Overlay with an indicator organism Incubate
  16. 16. ANTIBIOTICS: ISOLATION, YIELD, AND PURIFICATION  Cross-streak method  Used to test new microbial isolates for antibiotic production  Most isolates produce known antibiotics  Most antibiotics fail toxicity and therapeutic tests in animals  Time and cost of developing a new antibiotic is approximately 15 years and $1 billion  Involves clinical trials and U.S. FDA approval  Antibiotic purification and extraction often involves elaborate methods
  17. 17. © Angel L. Salaman. PhD FIGURE 15.4B II. Testing Activity Spectrum Streak antibiotic producer across one side of plate Incubate to permit growth and antibiotic production Cross-streak with test organisms Incubate to permit test organisms to grow Antibiotic diffuses into agar Streptomyces cell mass Growth of test organism Inhibition zones where sensitive test organisms did not grow
  18. 18. INDUSTRIAL PRODUCTION OF PENICILLINS AND TETRACYCLINES  Penicillins are β-lactam antibiotics  Natural and biosynthetic penicillins  Semisynthetic penicillins  Broad spectrum of activity  Penicillin production is typical of a secondary metabolite  Production only begins after near-exhaustion of carbon source  High levels of glucose repress penicillin production
  19. 19. © Angel L. Salaman. PhD FIGURE 15.6 Glucose feeding Nitrogen feeding Cells Lactose Ammonia Penicillin Fermentation time (h) Biomass(g/liter),carbohydrate, ammonia,penicillin(g/liter×10) 100 90 80 70 60 50 40 30 20 10 0 20 40 60 80 100 120 140
  20. 20. INDUSTRIAL PRODUCTION OF PENICILLINS AND TETRACYCLINES  Biosynthesis of tetracycline has a large number of enzymatic steps  More than 72 intermediates  More than 300 genes involved!  Complex biosynthetic regulation
  21. 21. VITAMINS AND AMINO ACIDS  Production of vitamins is second only to antibiotics in terms of total pharmaceutical sales  Vitamin B12 produced exclusively by microorganisms  Deficiency results in pernicious anemia  Cobalt is present in B12  Riboflavin can also be produced by microbes
  22. 22. VITAMINS AND AMINO ACIDS  Amino acids  Used as feed additives in the food industry  Used as nutritional supplements in nutraceutical industry  Used as starting materials in the chemical industry  Examples include  Glutamic acid (MSG)  Aspartic acid and phenylalanine (aspartame [NutraSweet])  Lysine (food additives)
  23. 23. ENZYMES AS INDUSTRIAL PRODUCTS  Exoenzymes  Enzymes that are excreted into the medium instead of being held within the cell; they are extracellular  Can digest insoluble polymers such as cellulose, protein, and starch  Enzymes are useful as industrial catalysts  Produce only one stereoisomer  High substrate specificity
  24. 24. © Angel L. Salaman. PhD FIGURE 15.10 Starch oligosaccharides Time (h) Percentenzyme activityremaining Pullulanase 90°C 100°C 110°C 110°C plus Ca2+ 100 10 1 1 2 3 4 Pullulanase is used predominantly in conjunction with other enzymes that break down starch (glucoamylase). It is produced as an extracellular, cell surface- anchored lipoprotein by Gram-negative bacteria of the genus Klebsiella. Type I pullulanases specifically attack α-1,6 linkages, while type II pullulanases are also able to hydrolyse α-1,4 linkages. It is also produced by some other bacteria and archaea. Pullulanase is used as a processing aid in grain processing biotechnology (production of ethanol and sweeteners).
  25. 25. III. ALCOHOLIC BEVERAGES AND BIOFUELS  Wine  Brewing and Distilling  Biofuels
  26. 26. WINE  Most wine is made from grapes  Wine fermentation occurs in fermentors ranging in size from 200 to 200,000 liters  Fermentors are made of oak, cement, glass-lined steel, or stone  White wine is made from white grapes or red grapes that have had their skin removed  Red wine is aged for months or years  White wine is often sold without aging
  27. 27. © Angel L. Salaman. PhD FIGURE 15.12B
  28. 28. © Angel L. Salaman. PhD FIGURE 15.12C
  29. 29. © Angel L. Salaman. PhD FIGURE 15.12D
  30. 30. © Angel L. Salaman. PhD FIGURE 15.13 Stems removed Grapes crushed Must Juice sits in contact with skins for 16–24 h Press Yeast White wine Red wine Pomace (discard) Yeast Fermentation vat 10–15 days Aging 5 months Racking Clarifying agents Filtration Bottling Stems removed Grapes crushed Must Fermentation vat 3 weeks (pulp is not removed) Press Pomace (discard) Aging in barrels Racking Transfer to clean barrels 3 times per year Clarifying agents 2 years Settling tank Filtration Bottling: Age in bottles 6 months or more Sodium metabisulfite : It is used as a disinfectant, antioxidant and preservative agent
  31. 31. BREWING AND DISTILLING  Brewing is the term used to describe the manufacture of alcoholic beverages from malted grains. Yeast is used to produce beer  Two main types of brewery yeast strains  Top fermenting — Ale is a type of beer brewed from malted barley using a warm-fermentation with a strain of brewers' yeast.The yeast will ferment the beer quickly, giving it a sweet, full bodied and fruity taste.  Bottom fermenting — Lager (German: storage) is a type of beer that is fermented and conditioned at low temperatures.
  32. 32. © Angel L. Salaman. PhD FIGURE 15.14
  33. 33. BREWING AND DISTILLING  Distilled alcoholic beverages are made by heating previously fermented liquid to a temperature that volatilizes most of the alcohol  Whiskey, rum, brandy, vodka, gin  >50,000,000,000 liters of ethanol are produced yearly for industrial purposes  Used as an industrial solvent and gasoline supplement
  34. 34. BIOFUELS  Ethanol Biofuels  Ethanol is a major industrial commodity chemical  Over 60 billion liters of alcohol are produced yearly from the fermentation of feed stocks  Gasohol and E-85  Petroleum Biofuels  Production of butanol  Synthesis of petroleum from green algae
  35. 35. © Angel L. Salaman. PhD FIGURE 15.17
  36. 36. BOTOX® (ALLERGAN) MYOBLOC® (SOLSTICE NEUROSCIENCES, INC), DYSPORT® (BIOPHARM LIMITED), OR XEOMIN® (MERZ PHARMA GMBH & CO.)  BOTOX COSMETIC® (onabotulinumtoxinA for injection) is a sterile, vacuum-dried form of purified botulinum neurotoxin type A complex, (AB5-type exotoxin) produced by the bacterium Bordetella pertussis, which causes whooping cough produced from a culture of the Hall strain of Clostridium botulinum grown in a medium containing N-Z amine, glucose and yeast extract.  Pertussis toxin (PT) is a proteBOTOX® Cosmetic is a prescription medicine that is injected into muscles and used to improve the look of moderate to severe frown lines between the eyebrows (glabellar lines) in people 18 to 65 years of age for a short period of time (temporary).
  38. 38. PRODUCTS FROM GENETICALLY ENGINEERED MICROORGANISMS  Expressing Mammalian Genes in Bacteria  Ex. Production of Genetically Engineered Somatotropin  Other Mammalian Proteins and Products  Genetically Engineered Vaccines  Mining Genomes  Engineered Metabolic Pathways
  39. 39.  Successful genetic engineering depends not only on being able to carry out molecular cloning but also on knowledge of replication, transcription, translation, and the regulatory aspects that control all of these processes.
  40. 40. HOSTS FOR CLONING VECTORS  The choice of a cloning host depends on the final application. In many cases, the host can be a prokaryote, but in others it is essential that the host be a eukaryote.  Any host must be able to take up DNA, and there are a variety of techniques by which this can be accomplished, both natural and artificial.
  41. 41. Nucleic acid gun for transfection of certain eukaryotic cells.
  42. 42. FINDING THE RIGHT CLONE  Special procedures are needed to detect the foreign gene in the cloning host
  43. 43.  If the gene is expressed, the presence of the foreign protein itself, as detected either by its activity or by reaction with specific antibodies, is evidence that the gene is present. However, if the gene is not expressed, its presence can be detected with a nucleic acid probe.
  44. 44. SHUTTLE VECTORS  allow cloned DNA to be moved between unrelated organisms. A shuttle vector is a cloning vector that can stably replicate in two different organisms.
  45. 45. SPECIALIZED VECTORS  Many cloned genes are not expressed efficiently in a new host. Expression vectors have been developed for both prokaryotic and eukaryotic hosts  These vectors contain genes that will increase the level of transcription of the cloned gene and make its transcription subject to specific regulation. Signals to improve the efficiency of translation may also be present in the expression vector.
  46. 46. REPORTER GENES  are incorporated into vectors because they encode proteins that are readily detected. These genes can be used to signal the presence or absence of a particular genetic element or its location. They can also be fused to other genes or to the promoter of other genes so that expression can be studied
  47. 47. EXPRESSION OF MAMMALIAN GENES IN BACTERIA  It is possible to achieve very high levels of expression of mammalian genes in prokaryotes. However, the expressed gene must be free of introns.
  48. 48. • This can be accomplished by using reverse transcriptase to synthesize cDNA from the mature mRNA encoding the protein of interest.
  49. 49.  One can also use the amino acid sequence of a protein to design and synthesize an oligonucleotide probe that encodes it. This process is in effect reverse translation.
  50. 50. • Fusion proteins are often used to stabilize or solubilize the cloned protein.
  51. 51. © Angel L. Salaman. PhD Production of Insulin: The Beginnings of Commercial Biotechnology PRACTICAL APPLICATIONS OF GENETIC ENGINEERING
  52. 52. • The first human protein made commercially using engineered bacteria was human insulin, but many other hormones and human proteins are now being produced. In addition, many recombinant vaccines have been produced.
  53. 53. OTHER MAMMALIAN PROTEINS AND PRODUCTS  Many human proteins that were formerly extremely expensive to produce because they were found in human tissues only in small amounts can now be made in large amounts from the cloned gene in a suitable expression system.
  54. 54. ANAKINRA® (AMGEN LTD)  Anakinra (Kineret) is an interleukin-1 (IL-1) receptor antagonist. Anakinra blocks the biologic activity of naturally occurring IL-1, including inflammation and cartilage degradation associated with rheumatoid arthritis, by competitively inhibiting the binding of IL-1 to the Interleukin-1 type receptor, which is expressed in many tissues and organs. IL-1 is produced in response to inflammatory stimuli and mediates various physiologic responses, including inflammatory and immunologic reactions. IL-1 additionally stimulates bone reabsorption and induces tissue damage like cartilage degradation as a result of loss of proteoglycans.  In patients with rheumatoid arthritis the natural IL-1 receptor antagonist is not found in effective concentrations in synovium and synovial fluid to counteract the elevated IL-1 concentrations in these patients.  It is produced by recombinant DNA technology using an E coli bacterial expression system.
  55. 55. ENBREL® (AMGEN LTD)  ENBREL treats moderate to severe rheumatoid arthritis, adult chronic moderate to severe plaque psoriasis in patients who are candidates for systemic therapy or phototherapy, psoriatic arthritis, ankylosing spondylitis, and moderately to severely active polyarticular juvenile idiopathic arthritis.  Enbrel (etanercept) is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. The Fc component of etanercept contains the CH2 domain, the CH3 domain and hinge region, but not the CH1 domain of IgG1. Etanercept is produced by recombinant DNA technology in a Chinese hamster ovary (CHO) mammalian cell expression system. It consists of 934 amino acids and has an apparent molecular weight of approximately 150 kilodaltons.
  56. 56. NEUPOGEN™ (AMGEN LTD)  Neupogen (filgrastim) is a form of a protein that stimulates the growth of white blood cells in your body. White blood cells help your body fight against infection.  Filgrastim is a granulocyte colony-stimulating factor (G- CSF) analog used to stimulate the proliferation and differentiation of granulocytes. It is produced by recombinant DNA technology. The gene for human granulocyte colony-stimulating factor is inserted into the genetic material of Escherichia coli. The G-CSF then produced by E. coli is different from G-CSF naturally made in humans.  Neupogen is used to treat neutropenia, a lack of certain white blood cells caused by cancer, bone marrow transplant, receiving chemotherapy, or by other conditions.
  57. 57. GENETICALLY ENGINEERED VACCINES  Many recombinant vaccines have been produced. These include live recombinant, vector, subunit, and DNA vaccines
  58. 58. • Lists some genetically engineered vaccines.
  59. 59. •Production of recombinant vaccinia virus and its use as a recombinant vaccine.
  60. 60. GENETIC ENGINEERING IN ANIMAL AND HUMAN GENETICS  Genetic engineering can be used to develop transgenic organisms capable of producing proteins of pharmaceutical value  The techniques of genetic engineering are also applied to identifying individuals using DNA fingerprinting  One of the great hopes of genetic engineering is gene therapy, in which functional copies of a gene can be supplied to an individual to treat human genetic disease.