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LECTURE PRESENTATIONS For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark Chapter 15 Commercial Products and Biotechnology Lectures by John Zamora Middle Tennessee State University © 2012 Pearson Education, Inc. I. Putting Microorganisms to Work • 15.1 Industrial Products and the Microorganisms That Make Them • 15.2 Production and Scale © 2012 Pearson Education, Inc. 15.1 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 highyielding microbial variants © 2012 Pearson Education, Inc. 15.1 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 – Amenable to genetic manipulation © 2012 Pearson Education, Inc. 15.1 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 © 2012 Pearson Education, Inc. 15.2 Production and Scale • Primary metabolite – Produced during exponential growth – Example: alcohol • Secondary metabolite – Produced during stationary phase © 2012 Pearson Education, Inc. 15.2 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 © 2012 Pearson Education, Inc. Primary metabolite Cells Alcohol Sugar Penicillin, sugar, or cell number Alcohol, sugar, or cell number Figure 15.1 Secondary metabolite Sugar Cells Penicillin Time © 2012 Pearson Education, Inc. Time 15.2 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 © 2012 Pearson Education, Inc. 15.2 Production and Scale • Fermentor is where the microbiology process takes place (Figure 15.2a and b) • 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 (Figure 15.2c) © 2012 Pearson Education, Inc. Figure 15.2a © 2012 Pearson Education, Inc. Figure 15.2b Motor pH Steam Sterile seal pH controller Acid–base reservoir and pump Viewing port Filter Exhaust Impeller (mixing) External cooling water out Cooling jacket Culture broth External cooling water in Sparger (highpressure air for aeration) Steam in Sterile air Valve Harvest © 2012 Pearson Education, Inc. Figure 15.2c © 2012 Pearson Education, Inc. 15.2 Production and Scale • Industrial Fermentors – Closely monitored during production run – Growth and product formation must be measured – Environmental factors must be controlled and altered as needed • Including temperature, pH, cell mass, nutrients, and product concentration – Data on the process must be obtained in real time © 2012 Pearson Education, Inc. 15.2 Production and Scale • Scale-up – The transfer of a process from a small laboratory scale to large-scale commercial equipment – Major task of the biochemical engineer • Requires knowledge of the biology of producing organism and the physics of fermentor design and operation – Many challenges in scale-up arise from aeration and mixing • Flask laboratory fermentor pilot plant commercial fermentor (Figure 15.3) © 2012 Pearson Education, Inc. Figure 15.3 © 2012 Pearson Education, Inc. II. Drugs, Other Chemicals, and Enzymes • 15.3 Antibiotics: Isolation, Yield, and Purification • 15.4 Industrial Production of Penicillins and Tetracyclines • 15.5 Vitamins and Amino Acids • 15.6 Enzymes as Industrial Products © 2012 Pearson Education, Inc. 15.3 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 (Figure 15.4a) • Microbes are obtained from nature in pure culture • Assayed for products that inhibit growth of test Animation: Isolation and Screening bacteria of Antibiotic Producers © 2012 Pearson Education, Inc. Figure 15.4a I. Isolation Spread a soil dilution on a plate of selective medium Sterile glass spreader Incubation Colonies of Streptomyces species Nonproducing organisms Zones of growth inhibition Producing organisms © 2012 Pearson Education, Inc. Overlay with an indicator organism Incubate 15.3 Antibiotics: Isolation, Yield, and Purification • Cross-streak method (Figure 15.4b) – 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 © 2012 Pearson Education, Inc. Figure 15.4b II. Testing Activity Spectrum Streak antibiotic producer across one side of plate Incubate to permit growth and antibiotic production Antibiotic diffuses into agar Streptomyces cell mass Cross-streak with test organisms Incubate to permit test organisms to grow Growth of test organism Inhibition zones where sensitive test organisms did not grow © 2012 Pearson Education, Inc. 15.4 Industrial Production of Penicillins and Tetracyclines • Penicillins are -lactam antibiotics – Natural and biosynthetic penicillins (Figure 15.5) – Semisynthetic penicillins • Broad spectrum of activity • Penicillin production is typical of a secondary metabolite – Production only begins after near-exhaustion of carbon source (Figure 15.6) – High levels of glucose repress penicillin production © 2012 Pearson Education, Inc. Figure 15.5 Add precursor I Biosynthetic penicillin I Add precursor II Biosynthetic penicillin II Penicillin fermentation Add precursor III Chemical or enzymatic treatment of penicillin G Biosynthetic penicillin III Natural penicillins (for example, penicillin G) 6-Aminopenicillanic acid Add side chains chemically Semisynthetic penicillins (for example, ampicillin, amoxycillin, methicillin) © 2012 Pearson Education, Inc. Figure 15.6 Biomass (g/liter), carbohydrate, ammonia, penicillin (g/liter 10) Glucose feeding 100 Nitrogen feeding 90 80 Penicillin 70 60 50 40 30 Cells 20 Lactose 10 Ammonia 0 20 40 60 80 100 120 140 Fermentation time (h) © 2012 Pearson Education, Inc. 15.4 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 (Figure 15.7) © 2012 Pearson Education, Inc. Figure 15.7 Inoculum (spores on agar slant or in sterile soil) Agar plates Medium 2% Meat extract; 0.05% asparagine; 1% glucose; 0.5% K2HPO4; 1.3% agar Growth in optimal medium Spores as inoculum Shake flask 2% Corn steep liquor; 3% sucrose; 0.5% CaCO3 24 h Prefermentor Medium mimics production medium Same as for shake culture 19–24 h pH 5.2–6.2 Fermentor 60–65 h pH 5.8–6.0 1% Sucrose; 1% corn steep liquor; 0.2% (NH4)2HPO4; 0.1% CaCO3; 0.025% MgSO4 0.005% ZnSO4 0.00033% and each of CuSO4, MnCl2 Production medium, no glucose, low phosphate Antibiotic purification from broth after cell removal Chlortetracycline © 2012 Pearson Education, Inc. 15.5 Vitamins and Amino Acids • Production of vitamins is second only to antibiotics in terms of total pharmaceutical sales – Vitamin B12 produced exclusively by microorganisms (Figure 15.8a) • Deficiency results in pernicious anemia • Cobalt is present in B12 – Riboflavin can also be produced by microbes (Figure 15.8b) © 2012 Pearson Education, Inc. Figure 15.8a B12 © 2012 Pearson Education, Inc. Figure 15.8b Flavin ring Riboflavin © 2012 Pearson Education, Inc. 15.5 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; Figure 15.9) © 2012 Pearson Education, Inc. Figure 15.9 Methionine Threonine Isoleucine ATP Aspartate Aspartyl-P Aspartokinase Feedback inhibition Aspartate semialdehyde Diaminopimelate Lysine AEC: Lysine: © 2012 Pearson Education, Inc. 15.6 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 © 2012 Pearson Education, Inc. 15.6 Enzymes as Industrial Products • Enzymes are produced from fungi and bacteria – Bacterial proteases are used in laundry detergents (can also contain amylases, lipases, and reductases) • Isolated from alkaliphilic bacteria • Amylases and glucoamylases are also commercially important – Produce high-fructose syrup © 2012 Pearson Education, Inc. 15.6 Enzymes as Industrial Products • Extremozymes – Enzymes that function at some environmental extreme (e.g., pH or temperature; Figure 15.10) – Produced by extremophiles © 2012 Pearson Education, Inc. Figure 15.10 Percent enzyme activity remaining 100 10 Starch Pullulanase oligosaccharides 90°C 100°C 110°C 110°C plus Ca2 1 1 2 Time (h) © 2012 Pearson Education, Inc. 3 4 15.6 Enzymes as Industrial Products • Immobilized enzymes are attached to a solid surface – Used in the starch processing industry • Three ways to immobilize an enzyme (Figure 15.11) – Bonding of enzyme to a carrier – Cross-linking of enzyme molecules – Enzyme inclusion © 2012 Pearson Education, Inc. Figure 15.11 Carrier-bound enzyme Enzyme inclusion in fibrous polymers © 2012 Pearson Education, Inc. Cross-linked enzyme Enzyme inclusion in microcapsules III. Alcoholic Beverages and Biofuels • 15.7 Wine • 15.8 Brewing and Distilling • 15.9 Biofuels © 2012 Pearson Education, Inc. 15.10 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, glasslined steel, or stone (Figure 15.12b, c, and d) • White wine is made from white grapes or red grapes that have had their skin removed (Figure 15.13) • Red wine is aged for months or years • White wine is often sold without aging © 2012 Pearson Education, Inc. Figure 15.12b © 2012 Pearson Education, Inc. Figure 15.12c © 2012 Pearson Education, Inc. Figure 15.12d © 2012 Pearson Education, Inc. Figure 15.13 Stems removed Grapes crushed Stems removed Grapes crushed Must Must Yeast Juice sits in contact with skins for 16–24 h Fermentation vat 3 weeks (pulp is not removed) Press Press Pomace (discard) Yeast Fermentation vat 10–15 days Pomace (discard) Aging in barrels Racking Aging 5 months Racking Transfer to clean barrels 3 times per year 2 years Clarifying agents Settling tank Clarifying agents Filtration Filtration Bottling Bottling: Age in bottles 6 months or more White wine © 2012 Pearson Education, Inc. Red wine 15.8 Brewing and Distilling • Brewing is the term used to describe the manufacture of alcoholic beverages from malted grains (Figure 15.14) • Yeast is used to produce beer • Two main types of brewery yeast strains – Top fermenting — ales – Bottom fermenting — lagers © 2012 Pearson Education, Inc. Figure 15.14 © 2012 Pearson Education, Inc. 15.8 Brewing and Distilling • Distilled alcoholic beverages are made by heating previously fermented liquid to a temperature that volatilizes most of the alcohol (Figure 15.16) – 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 © 2012 Pearson Education, Inc. Figure 15.16 © 2012 Pearson Education, Inc. 15.9 Biofuels • Ethanol Biofuels – Ethanol is a major industrial commodity chemical – Over 60 billion liters of alcohol are produced yearly from the fermentation of feedstocks (Figure 15.17a and b) – Gasohol and E-85 • Petroleum Biofuels – Production of butanol – Synthesis of petroleum from green algae (Figure 15.17c) © 2012 Pearson Education, Inc. Figure 15.17 © 2012 Pearson Education, Inc. IV. Products from Genetically Engineered Microorganisms • 15.10 Expressing Mammalian Genes in Bacteria • 15.11 Production of Genetically Engineered Somatotropin • 15.12 Other Mammalian Proteins and Products • 15.13 Genetically Engineered Vaccines • 15.14 Mining Genomes • 15.15 Engineering Metabolic Pathways © 2012 Pearson Education, Inc. 15.10 Expressing Mammalian Genes in Bacteria • Biotechnology – Use of living organisms for industrial or commercial applications • Genetically modified organism (GMO) – An organism whose genome has been altered • Genetic engineering allows expression of eukaryotic genes in prokaryotes (e.g., insulin) • This is achieved by – Cloning the gene via mRNA (Figure 15.18) – Finding the gene via the protein (Figure 15.19) © 2012 Pearson Education, Inc. Figure 15.18 Poly(A) tail mRNA Addition of primer Oligo dT primer Reverse transcription to form single-stranded cDNA cDNA Hairpin loop Removal of RNA with alkali DNA polymerase I to form doublestranded cDNA Nuclease Single-strand-specific nuclease Double-stranded cDNA Clone © 2012 Pearson Education, Inc. Figure 15.19 Protein Possible mRNA codons DNA oligonucleotides (possible probes) and so on Preferred DNA sequence (based on the organism’s codon bias) © 2012 Pearson Education, Inc. 15.10 Expressing Mammalian Genes in Bacteria • Protein synthesis in a foreign host is subject to other problems – Degradation by intracellular proteases – Toxicity to prokaryotic host – Formation of inclusion bodies • Fusion of a target protein with a carrier protein facilitates protein purification (Figure 15.20) © 2012 Pearson Education, Inc. Figure 15.20 Ptac Encodes Shine–Dalgarno lacI malE Encodes protease cleavage site Polylinker lacZ pBR322 origin M13 origin © 2012 Pearson Education, Inc. Ampicillin resistance 15.11 Production of Genetically Engineered Somatotropin • Insulin was the first human protein made commercially by genetic engineering • Somatotropin, a growth hormone, is another widely produced hormone (Figure 15.21) – Cloned as cDNA from the mRNA – Recombinant bovine somatotropin (rBST) is commonly used in the dairy industry; stimulates milk production in cows © 2012 Pearson Education, Inc. Figure 15.21 Bacterial promoter and RBS BST mRNA from cow Bovine somatotropin mRNA Expression vector Convert BST mRNA to cDNA using reverse transcriptase Inject rBST into cow to increase milk yield Bovine somatotropin cDNA rBST Transform into cells of Escherichia coli © 2012 Pearson Education, Inc. Commercial production 15.12 Other Mammalian Proteins and Products • Many mammalian proteins are produced by genetic engineering – These include hormones and proteins for blood clotting and other blood processes © 2012 Pearson Education, Inc. 15.13 Genetically Engineered Vaccines • Recombinant vaccines – Vector vaccine – Subunit vaccine – DNA vaccine • Polyvalent vaccine – A single vaccine that immunizes against two different diseases © 2012 Pearson Education, Inc. 15.13 Genetically Engineered Vaccines • Vector vaccine – Vaccine made by inserting genes from a pathogenic virus into a relatively harmless carrier virus (e.g., vaccinia virus; Figure 15.22) Animation: Production of Recombinant Vaccina Virus © 2012 Pearson Education, Inc. Figure 15.22 Cloning plasmid Foreign DNA Foreign DNA inserted into tdk gene Part of vaccinia tdk gene tdk Insert into host cell Wild-type vaccinia virus DNA Host cell with defective tdk gene Foreign DNA Recombinant vaccinia virus DNA © 2012 Pearson Education, Inc. Recombination Select with 5-bromo-dU 15.13 Genetically Engineered Vaccines • Subunit vaccines – Contain only a specific protein or proteins from a pathogenic organism (e.g., coat protein of a virus) – Preparation of a viral subunit vaccine • Fragmentation of viral DNA by restriction enzymes • Cloning of viral coat protein genes into a suitable vector • Provision of proper conditions for expression (promoter, reading frame, and ribosome-binding site) • Reinsertion and expression of the viral genes in a microbe © 2012 Pearson Education, Inc. 15.13 Genetically Engineered Vaccines • DNA vaccine (genetic vaccine) – Vaccine that uses the DNA of a pathogen to elicit an immune response – Defined fragments of genomic DNA or specific genes encoding immunogenic proteins are used • They are cloned into a plasmid or viral vector and delivered by injection © 2012 Pearson Education, Inc. 15.14 Mining Genomes • Gene mining – The process of isolating potentially useful novel genes from the environment without culturing the organism – To do so, DNA (or RNA) is directly isolated from the environment and cloned into appropriate expression vectors, and the library is screened for activities of interest (Figure 15.23) © 2012 Pearson Education, Inc. Figure 15.23 Collect DNA samples from different environments Construct gene library Transform host cells and plate on selective media Screen library for reactive colonies Large DNA inserts in BAC Plates of differential media Analyze and sequence positive clones © 2012 Pearson Education, Inc. Vector 15.15 Engineering Metabolic Pathways • Transgenic organism – An organism that contains a gene from another organism © 2012 Pearson Education, Inc. 15.15 Engineering Metabolic Pathways • The production of small metabolites by genetic engineering typically involves multiple genes that must be coordinately expressed • Pathway engineering – The process of assembling a new or improved biochemical pathway using genes from one or more organisms (e.g., indigo; Figure 15.24) © 2012 Pearson Education, Inc. Figure 15.24 Tryptophan Tryptophanase activity (already in E. coli) Indole Naphthalene oxygenase activity (from Pseudomonas) Dihydroxy-indole Spontaneous dehydration Indoxyl Spontaneous oxidation by O2 Indigo © 2012 Pearson Education, Inc. V. Transgenic Eukaryotes • 15.16 Genetic Engineering of Animals • 15.17 Gene Therapy in Humans • 15.18 Transgenic Plants in Agriculture © 2012 Pearson Education, Inc. 15.16 Genetic Engineering of Animals • Genetic engineering can be used to develop transgenic animals • Transgenic animals are useful for – Producing human proteins that require specific posttranslational modifications – Medical research – Improving livestock and other food animals for human consumption (Figure 15.25) © 2012 Pearson Education, Inc. Figure 15.25 © 2012 Pearson Education, Inc. 15.17 Gene Therapy in Humans • Gene therapy holds promise for tackling many human genetic diseases • Gene therapy: introduces a functional copy of a gene to treat a disease caused by a dysfunctional version of the gene • The use of recombinant DNA technology and conventional genetic studies allows for the localization of particular genetic defects to specific regions of the genome © 2012 Pearson Education, Inc. 15.18 Transgenic Plants in Agriculture • Plants can be genetically modified through several approaches, including – Electroporation – Particle gun methods – Use of plasmids from bacterium Agrobacterium tumefaciens • Many successes in plant genetic engineering; several transgenic plants are in agricultural production © 2012 Pearson Education, Inc. 15.18 Transgenic Plants in Agriculture • The plant pathogen Agrobacterium tumefaciens can be used to introduce DNA into plants (Figure 15.26) • A. tumefaciens contains the Ti plasmid, which is responsible for virulence • The Ti plasmid contains genes that mobilize DNA for transfer to the plant – The segment of the Ti plasmid that is transferred to the plant is called the T-DNA © 2012 Pearson Education, Inc. Figure 15.26 Mobilized region Foreign DNA Kanamycin resistance Spectinomycin resistance Chromosomes D-Ti Transfer to E. coli cells Transfer by conjugation Origin A. tumefaciens Origin E. coli Cloning vector Transfer to plant cells “Disarmed” Ti plasmid E. coli Nucleus A. tumefaciens Plant cell © 2012 Pearson Education, Inc. Grow transgenic plants from plant cells 15.18 Transgenic Plants in Agriculture • Tobacco was the first genetically modified (GM) plant to be grown commercially – 2005 estimate: >1 billion acres of agricultural land are used to grow GM crops • Several areas are targeted for genetic improvements in plants including herbicide, insect, and microbial disease resistance as well as improved product quality © 2012 Pearson Education, Inc. 15.18 Transgenic Plants in Agriculture • Plants are engineered to have herbicide resistance to protect them from herbicides applied to kill weeds (e.g., glyphosate; Figure 15.27) © 2012 Pearson Education, Inc. Figure 15.27 © 2012 Pearson Education, Inc. 15.18 Transgenic Plants in Agriculture • One of the most widely used approaches for genetically engineering insect resistance in plants involves the introduction of genes encoding the toxic protein of Bacillus thuringiensis (Bt toxin; Figure 15.28) © 2012 Pearson Education, Inc. Figure 15.28 © 2012 Pearson Education, Inc. 15.18 Transgenic Plants in Agriculture • Improving product quality is another target area of genetic engineering of plants – For example, spoilage delay • Transgenic plants can also be employed to produce human proteins for medical use – Examples: interferon, antibodies, vaccines © 2012 Pearson Education, Inc.