Scope of biotechnology


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Scope of biotechnology

  3. 3. One can be a good biologist without necessarily knowing muchabout microorganisms, but one cannot be a good microbiologistwithout a fair basic knowledge of biology!Stanier, R. Y., Doudoroff, M.
  4. 4. This is an era of explosive growth of analysis and manipulation ofmicrobial genomes as well as of invention of many new, creativeways in which both microorganisms and their genetic endowmentare utilized.The umbrella of microbial biotechnology covers many scientificactivities, ranging fromproduction of recombinant human hormonesmicrobial insecticidesmineral leachingbioremediation of toxic wastes etc.The purpose of this course is to convey the impact, theextraordinary breadth of applications, and the multidisciplinarynature of this technology. The common denominator to thesubjects discussed is that in all instances, prokaryotes or fungiprovide the indispensable component.
  5. 5. Microorganisms are important for many reasons such asThey produce things that are of value to usLarge material like proteins, carbohydrates, nucleicacids and even cells.Small molecules like primary metabolites that areessential for vegetative growth and secondarymetabolites (non essential).Why Microorganisms?
  6. 6. Microorganisms produce an array of metabolites but inminute quantity.Regulatory mechanisms that keep a check on overproduction of metabolites.However,Industrial biotechnologist seek for such wasteful strainthat will over produce a certain compound which can beisolated and marketed.After desired strain has been found a developmentprogram is initiated to improve titers by modification ofcultural conditions by mutations or by recombinantDNA technology.Metabolite production
  7. 7. The main reason to use a microorganisms over plant andanimal for synthesis of a compound is that microorganismscan be manipulated to increase the production to even1000 fold for small metabolites.
  8. 8. Primary metabolitesThese are small molecules produced by living cells; theyare intermediates or end products of the pathway ofintermediary metabolism such asBuilding blocks for essential macromolecules, or areconverted into coenzymes.TRADITIONAL MICROBIALBIOTECHNOLOGY
  9. 9. Alcohols (ethanol)Amino acids (monosodium glutamate, lysine, threonine,phenyl alanine, tryptophan)Flavour nucleotides (5’- guanylic acid, 5’- inosinic acid)Organic acid (acetic,propionic, succinic, fumaric and lactic )Polyols (glycerol, mannitol, xylitol etc.)Polysaccharides (xanthan, gellen)Sugars (fructose, ribose, sorbose)Vitamins (Ribo flavin B2, cyanocobalamin (B12), biotin)Examples of primary metabolites used in food and feedindustries are
  10. 10. MutantsAuxotrophic mutants‘‘A mutant strain of microorganism that will proliferateonly when the medium is supplemented with some specificsubstance not required by wild-type organisms ’’Amino acid production in which regulatory mechanismis bypassed by auxotrophic mutants by partially starvingthem for a requirement.DA B CEC - objective productEnzyme 2Enzyme 1
  11. 11. In parent strain enzyme 1 is subjected by cumulativefeed back regulation by end product D and E.A mutant is obtained that lacks enzyme 3.D must be supplied in the medium.If D is supplied in growth limiting concentrations,commulative feed back mechanism is broken and C isover produced.Example: Inosine 5- mono-phospahte (IMP production)
  12. 12. Produce mutants that are resistant to toxic analog of ametabolite i.e. antimetabolite.Due to feed back mechanism the presence of primarymetabolite inhibits over production of itself.Analog mimics the metabolite in chemical and structuralproperties.Strains are first grown at different conc, of an analog.Those isolates that are resistant to an analog can overproduce the metabolite.Examples, amino acid, vitamins and antibioticproduction.Resistance to toxic metabolite
  13. 13. Outward permeability i.e. how much conc. per litre.example., sodium glutamate an amino acidAnnual production is 1.2 billion employing different bacterialike Corneybacterium and Brevebacterium.From sugur conc. of 100g/ lit. have been achieved.Glutamic AcidOver production of glutamic acid is inhibited by feed backmechanisim.It is only regulated by change in conformity of cell membraneby biotin limitation process (biotin auxotrophs) that result inphospholipid deficient cell membrane.Efflux is carried out by special system through a carrier thatis dependent on membrane potential.Fermentation
  14. 14. In E.coli Threonine, lysine and methionine produced bya tight system of 3 enzymes through feed backmechanism. Naturally this does not lead tooverproduction of any amino acid at commercial level.Commercially C. glutamicum is used for commercialproduction of lysine. Homoserine dehydrogenase isremoved genetically and threonine and methionine areprovided in limited amount in the media.Assignment diagrammatic representation of L-lysineproduction in both E.coli and mutant strain.Lysine
  15. 15. No feed back repression of aspartate kinase occurs inlysine over producers.The first and second enzyme in lysine production areneither repressed or inhibited by lysine conc.L- lysine decarboxylase is absent in over producersWorld market for amino acidsL-glutamate US dollar 915 millionL-lysine US dollar 450 millionL-phenylalanine US dollar 198 millionL-aspartate US dollar 43 millionDifference between lysine overproducers and E.coli
  16. 16. Recombinant technology along with mutations andselection procedures have led to production of aminoacids to these levels g/lL-Threonine 100L-Isoleucine 40L- leucine 34L-valine 31 etc.Recombinant DNA technology
  17. 17. Riboflavin (vitamin B2)Over producer are two yeast Eremothecium ashbyii andAshbya gossypii (20g/l)Candidia and recombinant Bacillus subtilis strains haveimproved yeild by 30g/l.Vitamins
  18. 18. Produced by Propionibacterium shermani andPseudomonas denitrificansP. shermanii fermentation first step is under anaerobicconditions without addition of 5,6 benzimidazoleresulting in inhibition of B12 and accumulation ofintermediate cobinamide.Later under aerobic conditions precursor is added andB12 is synthesized.With Pseudomonas denitrificans fermentation entireprocess takes place under low oxygen content.Production level is 150mg/l and world market value of71 million US dollar.Vitamin B12
  19. 19. Strains of Serratia marcescens after recombinationhave produced yield of biotin upto 600mg/l.FungiMainly used for the production of organic acids e.g. 1 billionpounds of citric acid (CA) produced per year of 1.4 million USdollar market value.Produced by Embden-Meyerhof pathway and the first step ofTCA cycleControl of production is by inhibition of phosphofructokinase bycitric acid.Commercially Aspergillus niger is used for CA production in ironand manganese deficient media.High level of CA is also associated with high intracellular conc. offructose 2-6, bisphosphate, an activator of glycolysis.Assignment : diagrammatic representation of CA production.Biotin
  20. 20. Other factors effecting CA productionHigh CA production by Inhibition off isocitratedehydrogenase by CA.Low pH (1.7-2.0), inhibits glucose oxidase that wouldnormally produce gluconic acid. After 4 -5 days 80%sugar is converted to CA with titers of 100g/l.Higher pH values (3.0) leads to production of oxalicacid and gluconic acid instead of CA.CA from hydrocarbons using Candida yeast havereported to yield 150-170% of CA and titre upto 225g/l.
  21. 21. Ethyl alcohol is a Primary metabolite produced byfermentation of sugars.Saccharomyces cerevicae for fermentation of hexoses.Kluyveromyces fragilis or Candida for lactose and pentoses.Under optimum conditions 10-12% of alcohol by volume canbe produced.After this conc. ethanol intolerance inhibit furtherconversion.With special yeast 20% yield can be achieved by afterseveral month and years of fermentation.Fermentation by bacteria include Zymomonas andClostridium thermocellum .Recombinant E.coli has given 43% (v/v) of yield.Alcohols
  22. 22. Microbes have enzyme systems for almost every type ofreaction.In bioconversion a compound is converted to anothercompound that is structurally related to the initialcompound i.e. stereospecific.Bioconversions yield 90-100% conversion.Bioconverting-organisms
  23. 23. Very important from health and nutrition point of view.Includes antibiotics, other medicinals, toxins,biopesticides and other plant and animal growth factors.Produced by restricted group of microbes and chemicalsmixtures formed as closely related member of achemical family.Secondary metabolites
  24. 24. AntibioticsThe best known group of secondary metabolitesIn 1996 over 160 antibiotics are known to be produced and worldmarket value of US 23 billion dollar.targets include DNA replication (actinomycin, bleomycin andgriseofulvin)transcription (rifamycin)translation by 70-S ribosomes (chloramphenicol, tetracycline,lincomycin, erythromycin and streptomycin)transcription by 80-S ribosomes (cyclohexamide)transcription by 70- and 80-S ribosomes (puromycin and fusidicacid)cell wall synthesis (cycloserine, bacitracin, penicillin, cephalosporinand vancomycin)cell membranes (surfactants including: polymyxin andamphotericin; channel forming ionophores, such as lineargramicidin; and mobile carrier ionophores, such as monensin).
  25. 25. The search for new antibiotics continues, in order to:combatevolving pathogens,natural resistancebacteria and fungi,previously susceptible microbes that have developedresistanceimprove pharmacological propertiescombat tumorsviruses and parasitesand discover safer more potent and broad spectrumcompounds.Antibiotics are used not only for chemotherapy in humanand veterinary medicine, but also for growth promotion infarm animals and for the protection of plants
  26. 26. In nature, secondary metabolites are important to theorganisms that produce them, functioning as:(1) Sex hormones(2) Ionophores(3) competitive weapons against other bacteria, fungi,amoebae, insects and plants(4) agents of symbiosis(5) effectors of differentiation.Non-antibiotic agents
  27. 27. Despite the testing of thousands of syntheticcompounds, only a few promising structures werefound.As new lead compounds became more andmore difficult to find, microbial broths filled the voidand microbial products increased in importance in thetherapy of non-microbial diseases.polyethers: monensin, lasalocid and salinomycin dominatethe coccidiostat marketThese are also chief growth promoters in use for ruminantanimals.avermectins, another group of Streptomycete products with amarket of more than US$1 billion per year, have high activityagainst helminths and arthropods.
  28. 28. Inhibitors such as statins, including lovastatin (also knownas mevinolin) and pravastatin: fungal products that are usedas cholesterol-lowering agents in humans and animals.Lovastatin in its hydroxy acid form, is a potent competitiveinhibitor of 3-hydroxy-3-methylglutaryl-coenzyme Areductase from liver.Other wellknown enzyme inhibitors include:Clavulanic acid, a penicillinase-inhibitor that protectspenicillin from inactivation by resistant pathogensAcarbose, a natural inhibitor of intestinal glucosidase, whichis produced by an actinomycete of the genus Actinoplanes.Acarbose decreases hyperglycemia and triglyceride synthesisin adipose tissue, the liver and the intestinal wall of patientssuffering from diabetes, obesity and type IV hyperlipidemia.
  29. 29. In commercial or near-commercial use are biopesticides, includingBiofungicides e.g. kasugamycin, polyoxins,Bioinsecticides e.g. nikkomycin, spinosynsBioherbicides such as bialaphosAntihelminthics e.g. avermectinCoccidiostats, ruminant-growth promoters (monensin, lasalocid,salinomycin)plant-growth regulators (gibberellins),immunosuppressants for organ transplants (cyclosporin A, FK-506,rapamycin)Anabolic agents in farm animals (zearelanone)Uterocontractants (ergotalkaloids)Antitumor agents (doxorubicin, daunorubicin, mitomycin,bleomycin).Biopesticides
  30. 30. Tropophase and idiophaseIn batch culture, most secondary metabolite processeshave a distinct growth phase (trophophase) followed by aproduction phase (idiophase). In other fermentations, the twophases overlap; the timing depends on1. The nutritional environment presented to the culture2. The growth rate or both.A delay in antibiotic production until after trophophase helpsthe producing organism because the microbe is sometimessensitive to its own antibiotic during growth.Resistance mechanisms that develop in producingmicroorganisms include:1. enzymatic modification of the antibiotic,2. alteration of the cellular target of the antibiotic and3. decreased uptake of the excreted antibiotic.
  31. 31. Directed biosynthesisThe manipulation of the culture media in any developmentprogram often involves the testing of hundreds of additives aspossible limiting precursors of the desired product.Occasionally, a precursor that increases production of thesecondary metabolite is found.The precursor may also direct the fermentation towards theformation of one specific desirable product: this is known asdirected biosynthesis.Examples of directed biosynthesis include the use of1. Phenylacetic acid in the fermentation of benzylpenicillin2. Specific amino acids in the production of actinomycins andtyrocidins.Stimulatory precursors include:1. Methionine, as an inducer in cephalosporin C formation2. Valine, in tylosin production3. Tryptophan for ergot-alkaloid production.
  32. 32. In many fermentations, however, precursors show noactivity because their syntheses are not rate-limiting.In screening of additives often revealed dramaticeffects, both stimulatory and inhibitory, of non-precursor molecules on the production of secondarymetabolites.These effects are usually due to the interaction ofthese compounds with the regulatory mechanismsexisting in the fermentation organism.Antibiotic biosynthesis ends via the decay ofantibiotic synthetases or because of feedbackinhibition and repression of these enzymes.
  33. 33. Because the regulatory mechanisms are geneticallydetermined, mutations have had a major effect on theproduction of secondary metabolites. Indeed, it is thechief factor responsible for the 100–1000-fold increasesobtained in the production of antibiotics from their initialdiscovery to the present time.These tremendous increases in fermentation productivityand the resulting decreases in costs have come aboutmainly by random mutagenesis and screening for higher-producing microbial strains.Mutation has also served to:(1) shift the proportion of metabolites produced in afermentation broth to a more favorable distribution;(2) elucidate the pathways of secondary metabolism; and(3) yield new compounds.
  34. 34. Modern biotechnology is now over 25 years old.In addition to recombinant DNA technology, modern microbialbiotechnology encompassesfermentation,microbial physiology,high-throughput screening for novel metabolites,strain improvement,bioreactor designDownstream processing,cell immobilization (enzyme engineering),cell fusion,metabolic engineering,in vitro mutagenesis (proteinengineering)directed evolution of enzymes (applied molecular evolution).Modern microbial biotechnology
  35. 35. With the revolutionary exploitation of microbial genetic discoveries in the1970s, 1980s and 1990s.The major microbial hosts for production of recombinant proteins areE. coli,B. subtilis,S. cerevisiae,Pichia pastoris,Hansenula polymorphaAspergillus niger.The use of recombinant microorganisms provided the techniques andexperience necessary for the successful application of higherorganisms, such asmammalian and insect cell culture,transgenic animals and plants as hosts for the production of glycosylatedrecombinant proteins.Recombinant microorganisms
  36. 36. The progress in biotechnology has been truly remarkable. Withinfour years of the discovery of recombinant DNA technology,genetically engineered bacteria were making human insulin andhuman growth hormone.This led to an explosion of investment activity in new companies,mainly dedicated to innovation via genetic approaches.Newer companies entered the scene in various niches such asbiochemical engineeringdownstream processing.Today, biotechnology in the USA is represented by some1300 companies with revenues of US$19.6 billion, of which salesrepresent US$13.4 billion and approximately 153 000 employeesProgress
  37. 37. CanadaThe number of biotechnology companies reached 282 in1998,employing 10 000 workers and with revenues ofapproximately US$1.1 billion.Japanbiotechnology sales were approximately US$10 billion,mainly byestablished pharmaceutical, food and beverage companies.Europeanbiotechnology moved rapidly in the 1990s, after years oflagging behind and, in 1998, 1178 biotechnology companiesexisted with 45 000 employees, and revenues of US$3.7billion.
  38. 38. The major thrust of recombinant DNA technology has been in the area of raremammalian peptides, such ashormones,growth factors,enzymes,antibodies andbiological response modifiers.Among those genetically engineered products that have been approved for usein the USA arehuman insulin,human growthhormone,erythropoietin,antihemophelia factor,granulocyte-colony stimulating factor,Granulocyte macrophage-colonystimulating factor,Epidermal growth factor and other growth factors, interleukin-2, -, - and -interferons, and bovine somatotropin.
  39. 39. VaccinesVaccine production is another important part of thenew technology.the first subunit vaccine on the market was that ofhepatitis B virus surface antigen produced in yeast.The great contribution made by recombinantvaccines is the elimination of the tragic problemsassociated with conventional vaccines.Through reversion of the attenuated pathogen, someindividuals receiving the conventional vaccine notonly failed to be protected, but also came down withthe disease.
  40. 40. Combinatorial biosynthesisMost microbial biosynthetic pathways are encoded byclustered genes, which facilitatesthe transfer of an entire pathway in a fungi, pathway genes are sometimes clustered,such as the penicillin genes in Penicillium or theaflatoxin genes in Aspergillus.For the discovery of new or modified secondaryproducts, recombinant DNA techniques are beingused to introduce genes for the synthesis of oneproduct into producers of other antibiotics or intonon-producing strains (combinatorial biosynthesis).
  41. 41. Enzyme productionThe production of enzyme by fermentation was an established businessbefore modern microbial biotechnology.However, recombinant DNA methodology was so perfectly suited to theimprovement of enzyme production technology that it was almostimmediately used by companies involved in manufacturing enzymes.Industrial enzymes have now reached an annual market of US$1.6 billion.Important enzymes are proteases,lipases,carbohydrases,recombinant chymosin for cheese manufacturerecombinant lipase for use in detergents.Recombinant therapeutic enzymes already have a market value of overUS$2 billion, being used forthromboses,gastrointestinalrheumatic disorders,metabolic diseases and cancer.plasminogen activator,human DNAase andCerozyme
  42. 42. AgricultureIndustrial microbiology through genetic engineering and its associateddisciplines has brought about a revolution in agriculture.Two bacteria have had a major influence:Agrobacterium tumefaciens, a bacterium that normally produces crown galltumors on dicotyledonous plants andBacillus thuringiensis, an insecticidal bacterium.The tumor-forming genes of A. tumefaciens are present on its tumor-inducing (Ti) plasmid, along with genes directing the plant to form opines(nutritional factors required by the bacterium that it cannot produce byitself).The Ti vector has been exceedingly valuable for introducing foreign genesinto dicotyledonous plants for production of transgenic plants.However, the Ti plasmid is not very successful for transferring genes intomonocotyledonous plants, a problem bypassed by, for example, thedevelopment of a particle acceleration gun, which shoots DNA-coated metalparticles into plant cells.The activity of the insecticidal bacterium, B. thuringiensis, is caused by itscrystal protein produced during sporulation. Crystals and spores have beenapplied to plants for many years to protect them against lepidopteraninsects. B. thuringiensis preparations are highly potent, approximately 300times more active on a molar basis than synthetic pyrethroids and 80 000times more active than organophosphate insecticides.
  43. 43. In the modern biotechnology era, plants resistant toinsects have been produced by expressing forms of theB. thuringiensis toxin gene in the plant.Recently developed bioinsecticides include insectviruses, such as baculoviruses, that are engineered toproduce arthropod toxins.Transgenic plants, resistant to herbicides, are alsoavailable, as are virus-resistant plants produced byexpressing viral-coat-protein genes in plants.Interestingly, chemical pesticides against plant viruseswere never available.
  44. 44. ConclusionAlthough most of the early promises of biotechnologyhave been achieved, major challenges remain. We must useour brains, technology, drive and dedication to solve theproblems ofevolving diseases (e.g. AIDS),established diseases (cancer and parasitic infection),Antibiotic resistance development andenvironmental pollution, by converting urban, industrialand agricultural wastes into resources such as liquid fuel.These efforts will require continued interaction betweendifferent disciplines, major support by governments andinternational agencies, as well as an understanding andsupportive public.