Commercial Products and Biotechnology

Commercial Products and Biotechnology
Chapter 15 Commercial Products and Biotechnology

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 high-yielding microbial variants © 2012 Pearson Education, Inc.

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.

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 Secondary 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 Secondary metabolite Alcohol Penicillin Cells
Figure 15.1 Primary metabolite Secondary metabolite Cells Alcohol Alcohol, sugar, or cell number Penicillin, sugar, or cell number Sugar Cells Sugar Figure 15.1 Contrast between production of primary and secondary metabolites. Penicillin Time Time © 2012 Pearson Education, Inc.

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 15.2b Figure 15.2 Fermentors. © 2012 Pearson Education, Inc.
Motor Steam pH pH controller Acid–base reservoir and pump Sterile seal Viewing port Filter Exhaust Impeller (mixing) External cooling water out Cooling jacket External cooling water in Culture broth Figure 15.2 Fermentors. Sparger (high- pressure air for aeration) Steam in Sterile air Valve Harvest © 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 Figure 15.3 Research and production fermentors.
© 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
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 bacteria Animation: Isolation and Screening of Antibiotic Producers © 2012 Pearson Education, Inc.

Figure 15.4a I. Isolation © 2012 Pearson Education, Inc.
Spread a soil dilution on a plate of selective medium Sterile glass spreader Incubation Colonies of Streptomyces species Overlay with an indicator organism Nonproducing organisms Incubate Zones of growth inhibition Producing organisms © 2012 Pearson Education, Inc.

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 Figure 15.4 Isolation and screening of antibiotic producers. 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.

Penicillin fermentation
Figure 15.5 Add precursor I Penicillin fermentation Biosynthetic penicillin I Chemical or enzymatic Add precursor II Add precursor III treatment of penicillin G Biosynthetic penicillin II 6-Aminopenicillanic acid Biosynthetic penicillin III Add side chains chemically Natural penicillins Figure 15.5 Industrial production of penicillins. (for example, penicillin G) Semisynthetic penicillins (for example, ampicillin, amoxycillin, methicillin) © 2012 Pearson Education, Inc.

Penicillin Glucose feeding Nitrogen feeding 100 90 80 70
Figure 15.6 Glucose feeding Nitrogen feeding 100 90 80 Penicillin 70 Biomass (g/liter), carbohydrate, ammonia, penicillin (g/liter  10) 60 50 40 Figure 15.6 Kinetics of the penicillin fermentation with Penicillium chrysogenum. Cells 30 20 Lactose 10 Ammonia 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.

Medium Figure 15.7 Chlortetracycline
Inoculum (spores on agar slant or in sterile soil) Medium Growth in optimal medium 2% Meat extract; 0.05% asparagine; 1% glucose; 0.5% K2HPO4; 1.3% agar Agar plates Spores as inoculum 2% Corn steep liquor; 3% sucrose; 0.5% CaCO3 Shake flask Medium mimics production medium 24 h Prefermentor Same as for shake culture 19–24 h pH 5.2–6.2 1% Sucrose; 1% corn steep liquor; 0.2% (NH4)2HPO4; 0.1% CaCO3; 0.025% MgSO % ZnSO % and each of CuSO4, MnCl2 Fermentor Production medium, no glucose, low phosphate 60–65 h pH 5.8–6.0 Figure 15.7 Production scheme for chlortetracycline using Streptomyces aureofaciens. 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.

Riboflavin Flavin ring Figure 15.8b
Figure 15.8 Vitamins produced by microorganisms on an industrial scale. Riboflavin © 2012 Pearson Education, Inc.

15.5 Vitamins and 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.

ATP Lysine Aspartate Feedback inhibition AEC: Lysine:
Figure 15.9 Methionine Threonine Isoleucine ATP Aspartate Aspartyl-P Aspartate semialdehyde Aspartokinase Feedback inhibition Diaminopimelate Figure 15.9 Industrial production of lysine using Corynebacterium glutamicum. 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.

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.

Percent enzyme activity remaining
Figure 15.10 100 Percent enzyme activity remaining 10 Pullulanase Starch oligosaccharides 90°C 100°C 110°C 110°C plus Ca2 Figure Examples of extremozymes, enzymes which function under environmentally extreme conditions. 1 1 2 3 4 Time (h) © 2012 Pearson Education, Inc.

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.

Enzyme inclusion in fibrous polymers Enzyme inclusion in microcapsules
Figure 15.11 Carrier-bound enzyme Cross-linked enzyme Figure Procedures for the immobilization of enzymes. Enzyme inclusion in fibrous polymers Enzyme inclusion in microcapsules © 2012 Pearson Education, Inc.

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, glass-lined 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 Figure 15.12 Commercial wine making.

Figure 15.12c Figure 15.12 Commercial wine making.

Figure 15.12d Figure 15.12 Commercial wine making.

Figure 15.13 Figure 15.13 Wine production.
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 Yeast Pomace (discard) Pomace (discard) Fermentation vat 10–15 days Aging in barrels Figure Wine production. Racking Aging 5 months Transfer to clean barrels 3 times per year Clarifying agents 2 years Racking Settling tank Clarifying agents Filtration Filtration Bottling Bottling: Age in bottles 6 months or more White wine Red wine © 2012 Pearson Education, Inc.

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 Figure 15.14 Brewing beer in a large commercial brewery.

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 Figure 15.16 Distilled spirits.

15.9 Biofuels Ethanol Biofuels Petroleum 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.

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 Figure 15.18 Complementary DNA (cDNA).
Poly(A) tail mRNA Addition of primer Reverse transcription to form single-stranded cDNA Oligo dT primer cDNA Hairpin loop Removal of RNA with alkali DNA polymerase I to form double- stranded cDNA Nuclease Figure Complementary DNA (cDNA). Single-strand-specific nuclease Double-stranded cDNA Clone © 2012 Pearson Education, Inc.

DNA oligonucleotides (possible probes)
Figure 15.19 Protein Possible mRNA codons DNA oligonucleotides (possible probes) Figure Deducing the best sequence of an oligonucleotide probe from the amino acid sequence of a protein. 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.

Encodes Shine–Dalgarno
Figure 15.20 Ptac Encodes Shine–Dalgarno lacI malE Encodes protease cleavage site Polylinker Figure An expression vector for fusions. lacZ pBR322 origin Ampicillin resistance M13 origin © 2012 Pearson Education, Inc.

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.

rBST BST mRNA from cow Bacterial promoter and RBS
Figure 15.21 BST mRNA from cow Bacterial promoter and RBS Inject rBST into cow to increase milk yield Bovine somatotropin mRNA Expression vector Convert BST mRNA to cDNA using reverse transcriptase Bovine somatotropin cDNA Figure Cloning and expression of bovine somatotropin. rBST Transform into cells of Escherichia coli Commercial production © 2012 Pearson Education, Inc.

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.

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.

Recombinant vaccinia virus DNA
Figure 15.22 Cloning plasmid Foreign DNA Foreign DNA inserted into tdk gene Part of vaccinia tdk gene Insert into host cell tdk Wild-type vaccinia virus DNA Figure Production of recombinant vaccinia virus. Host cell with defective tdk gene Recombination Foreign DNA Select with 5-bromo-dU Recombinant vaccinia virus DNA © 2012 Pearson Education, Inc.

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.

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.

Analyze and sequence positive clones
Figure 15.23 Collect DNA samples from different environments Construct gene library Vector Large DNA inserts in BAC Transform host cells and plate on selective media Figure Metagenomic search for useful genes in the environment. Screen library for reactive colonies Plates of differential media Analyze and sequence positive clones © 2012 Pearson Education, Inc.

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.

Tryptophan Indole Dihydroxy-indole Indoxyl Indigo Figure 15.24
Tryptophanase activity (already in E. coli) Indole Naphthalene oxygenase activity (from Pseudomonas) Dihydroxy-indole Spontaneous dehydration Figure Engineered pathway for production of the dye indigo. 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 Figure 15.25 Transgenic animals.

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.

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 “Disarmed” Ti plasmid Transfer to plant cells Grow transgenic plants from plant cells Foreign DNA Chromosomes Kanamycin resistance Transfer to E. coli cells D-Ti Origin A. tumefaciens Transfer by conjugation Spectinomycin resistance Nucleus Origin E. coli Cloning vector E. coli A. tumefaciens Plant cell Figure Production of transgenic plants using a binary vector system in Agrobacterium tumefaciens. © 2012 Pearson Education, Inc.

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.

Figure 15.27 Figure 15.27 Transgenic plants: herbicide resistance.

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 Figure 15.28 Transgenic plants: insect resistance.

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.

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