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Microbial Biotechnology
What are microbes?
Microbes are small single-celled organisms
Either free-living or in colonies
They can belong to any of the three domains
Eubacteria
(Bacteria)
Archaebacteria
(Archaea)
Eukaryota
(Eukaryotes)
Three Domains
Archea
Ancient domain, but only recently identified
Through DNA analysis they were determined to differ significantly from eubacteria
Found predominantly in extreme environments (Extremophiles)
Thermophiles 50- 110°C
Psychrophiles 0- 20°C
Alkaliphiles pH>9
Halophiles 3- 20% salt
Methanogens use H2 + CO2 to produce CH4
Eubacteria
Gram-negative and gram-positive prokaryotes
Either autotrophs or heterotrophs
Can be aerobic or anaerobic
Mesophiles
Examples:
E. coli
Lactobacillus
Agrobacterium
Staphylococcus
Eukaryotes
Predominately yeasts/molds, protists, algae
Sac shaped cells that form sexual spores
Examples:
Sacchromyces
Penicillium
Aspergillus
Pichia
Commercial Uses of Microbes
Products

Bioconversion/Biocatalysis

Agriculture

Bioremediation

Oil/Mineral Recovery
Fermentation is a process for the production of useful products through mass culture of single-cells
The end products or the various intermediate products (metabolites) are siphoned off & purified for commercial use
stirred tank reactor
Fermenter or Bioreactor
http://www.wavebiotech.com/products/wave_bioreactor/system500/index.html
http://www.pharmaceutical-technology.com/projects/lonza/lonza1.html
15 000L Fermenter
1000L Disposable Bag
Enzyme: chymosin - the enzyme used
to curdle milk products
Hormone: bST - bovine somatotropin; used to increase milk production
Examples of bacterially-expressed proteins:
1928: Alexander Fleming discovered the first antibiotic.
He observed that Penicillium fungus made an antibiotic, penicillin, that killed S. aureus.
1940s: Penicillin was tested clinically and mass produced.
Original Penicillium moulds produced less than 10 units of penicillin per ml of fermentation broth (1943)

By 1955 Penicillium strains produced 8000 units/ml

Mutation with UV, mustard gas, and X-Ray, strain selection / culture improvement
Is this GMO?
How Are Microbes Modified?
Artifical Selection

Recombiant DNA

Metabolic Engineering
Transgenic microbes are created when cDNAs for the protein product are cloned into expression vectors
Human genes inserted into E. coli
Genes from extremophiles are moved to mesophiles
Due to the ease in culturing of mesophiles
Mesophiles also have 5 to 10x higher growth rates
Recombinant DNA Microbes
Enzyme/Drug
Metabolic Engineering, manipulation of pathways within an organism to optimize the production of a compound
Done by turning off particular genes, either through mutation or deletion
Products are also gained by altering the microbe’s environment
The microbe is forced to produce alanine at higher than normal amounts
Carotenoid production in E.coli cells
Fermentation Products
Enzymes
Enzymes, the most common product produced by microbes
Overall value of industrial enzymes is about $2.0 billion1
They are found in many household items that you would never think to have a biotechnology component
Enzymes
1 2004 data
Enzyme Name GE Organism Use (examples)
-acetolactate bacteria Removes bitter substances decarboxylase from beer
 -amylase bacteria Converts starch to simple sugar
Catalase fungi Reduces food deterioration
Chymosin bacteria or fungi Clots casein to make cheese
-glucanase bacteria Improves beer filtration
Glucose isomerase bacteria Converts glucose to fructose
Glucose oxidase fungi Reduces food deterioration
Lipase fungi Oil and fat modification
Maltogenic amylase bacteria Slows staling of breads
Pectinesterase fungi Improves fruit juice clarity
Protease bacteria Improves bread dough structure
xylanase (hemicellulase) bacteria or fungi Enhances rising of bread dough
http://www.geo-pie.cornell.edu/crops/enzymes.html
Detergent Enzymes
Detergents are the largest application of industrial enzymes
Traditionally these are lipolases, proteases & amylases
A recent innovation is the addition of mannanase
This enzyme aids in removing stains containing guar gum
These enzymes are engineered to improve stability in the presence of detergent, alkaline pH, and cold water
Subtilisin, a protease used in laundry detergents
The recombinant protein was engineered to remain active in the presence of bleach
Bleach caused the oxidation of one amino acid (methionine) and the enzyme lost 90% of its activity
By replacing this amino acid with alanine, the engineered enzyme was no longer sensitive to oxidation
Directed evolution is the most recent tool utilized in the creation of new and better enzymes (& other proteins)
http://www.rsc.org/chemistryworld/Issues/2004/July/rational.asp
Subtilisin normally functions in aqueous solution
Mutations were introduced randomly throughout the structure of the enzyme
Only 0.1–1% of the mutations were beneficial, but…
Activity in 60% dimethylformamide was improved 256-fold
doi:10.1016/S1367-5931(02)00396-4
 
Enzymes for Feed
Enzymes are used in animal feed to breakdown cellulose (cellulase)

New use of enzymes (phytases) which breakdown phytic acid
This allows better utilization of plant phosphorus stores
Allowing bone-meal to be removed from feeds
The latest generation of phytases are from fungus and have been engineered to survive high temperatures used during food processing
65% of poultry and 10% of swine feeds contain enzymes
Where do the genes for these enzymes come from?
Nature is still an important source (Gene Prospecting)
~<1% of the microbes have been grown in pure cultures
But what if you cannot find the enzyme you want?
You engineer it…

In the 1980’s rational protein engineering was introduced as a way of optimizing enzymes
Recombinant Drugs
Besides antibiotics which are derived from microorganisms
Protein medicines are produced by inserting human genes into microbes
1982, FDA approves the first recombinant protein drug, human insulin produced by E. coli developed by Genentech
Today there are >75 recombinant protein drugs approved by the FDA with 100s more being studied
Currently the global market for recombinant protein drugs is $47.4 billion1
(2006)
Product Microbe Purpose
Insulin E. coli Diabetes treatment
Interleukin-2 E. coli Cancer/immune system stimulant
EGF E. coli wound healing
Interferons E. coli/yeast Cancer/virus treatments
Prourokinase E.coli/yeast Anticoagulant/heart attacks
CSF E. coli/yeast Immune stimulant
Taxol E. coli ovarian cancer
Other Products From Microbes
Fuels, Plastics, Medications
Ethanol Production
Produced via anaerobic fermentation by yeast
Corn starch is hydrolyzed to glucose monomers
Plastics
Polyhydroxyalkanoate (PHA) is a polymer made by some microbes as a way of storing carbon
Up to 80% of the microbe’s biomass is plastic
PHA is sold to make shampoo bottles in Germany, and disposable razors in Japan
The microbe Pseudomonas putida converts styrene to PHA
http://www2.unil.ch/ibpv/WWWPoirier/images/Fig1.jpg
http://www2.unil.ch/ibpv/WWWPoirier/images/Fig2.jpg
Bioconversion
Utilization of microbes to modify a compound
Useful when multi-step chemical synthesis is expensive or inefficient
Often microbial conversion is combined with traditional chemistry to reduce the steps necessary
The most common use of bioconversion is in the synthesis of steroids such as hormones & corticosteroids
starting product
End products
Microbes and Agriculture
Frost damages many crops such as citrus trees & strawberries

When fruit freeze the ice crystals form

As the plants thaws they are effectively turned to mush
Frost damage to an orange leaf and fruit
Frost Damage
Some ice crystal nucleation is due to bacterial activity
Pseudomonas syringae promotes the development of ice at 0 to 2°C
If the bacteria are not present ice does not form until between –6 and –8°C
A strain of P. syringae called “ice minus” was developed
Plants were to be sprayed with the ice minus strain
This inhibits colonization by the “ice plus” (wild) strain
The EPA declared the new strain to be a pesticide
This made the review process lengthy and burdensome
The company thought it too expensive to pursue
However the “ice plus” strain has found a purpose…
Bacillus thuringiensis (Bt) is an aerobic spore-forming bacterium
During sporulation produces insecticidal crystal protein (ICP), a toxin (Cry)
The toxin brakes down quickly in the environment
They have no toxicity to humans & there is no withholding period on produce sprayed with Bt
Cry toxins vary in their toxicity and specificity
Microbial Pesticides
http://www.bioc.cam.ac.uk/~dje1/
Bioremediation
Bioremediation is reclaiming or cleaning of contaminated sites using microbes or other organisms
This entails the removal, degradation, or sequestering of pollutants &/or toxic wastes
http://www.cleanearthltd.com/en/contamination_cleanup/index.php
Bacteria are isolated based on their efficiency at digesting & converting the waste
The bacteria are tested for performance and safety
Bacteria are placed back in the waste environment in high concentrations
The bacteria grow & in the process digest & convert the waste into CO2 and H20
What can be cleaned up using bioremediation?
Oil spills
Waste water
Plastics
Chemicals (PCBs)
Toxic Metals
Oil/Wastewater Cleanup
Bioremediation




Bacteria degrade organic matter in sewage.




Bacteria degrade or detoxify pollutants such as oil and mercury
Microbes that digest hydrocarbons found throughout the environment
These naturally occurring microbes are utilized during a spill to clean shore lines
Fertilizer is added to supply the nutrients phosphorus and nitrogen
This was approach was used after the Exxon Valdez
Stimulated the natural rate of biodegradation by 2 to 5x
There have yet to be any other instances of this being used on a large-scale
Before After
Smaller scale cleanup is feasible
For 3 months nutrients and microbes were sprayed on this field
After 11 months the site was deemed clean
6000yards3 petroleum conc. Before 4000ppm After 100ppm
Before After
Treatment of domestic sewage or industrial waste
Utilizes aeration to oxygenate allowing aerobic microbes to digest solid waste
Wastewater
Plastic Degradation
140 million tons of plastics are produced each year
Traditional plastics are very stable and do not degrade
Some plastics have been shown to be biodegradable
Strains of bacteria have been isolated that breakdown:
Polyurethane
Polyvinyl alcohol
Nylon-66
The degradation pathways are currently under study
Chemicals
Polychlorinated biphenyls (PCBs)
PCBs have low water solubility, good insulating properties, high boiling points and resistance to chemicals
The largest uses for PCBs was in capacitors, transformers, & as plasticizers
1977, Monsanto (main producer) stops all PCB production
Millions of lbs of PCBs are still in place around the world
The stability properties that made PCBs so useful have allowed them to persist in the environment
Most people in industrialized countries have PCBs in their tissue
Microbes that dehalogenate PCBs have been isolated
This process is referred to as halorespiration
Involves the replacement of the Cl with an –OH
This process is multi-step with four enzymes required
These enzymes are now the target of protein engineering to optimize their performance
Heavy Metal Clean up
-Uranium processing has left contaminated groundwater sites across the United States and the world
-Traditional “pump-and-treat” methods take decades and expose workers to toxic levels of uranium
-Geobacter to convert soluble uranium to insoluble uraninite
-Uraninite stays put instead of mixing with water used for drinking or irrigation
-The microbes are encouraged to multiply by injecting acetate
In ~50 days, 70% of the uranium is converted into uraninite
Biomining
-Microbe assisted mining has gone on for millennia
-Early copper miners used microbes to leach copper from ore without even knowing it
-Low-grade ore and mine tailings are exploited biologically
-Sulfides of metals like zinc, copper, nickel, cobalt, iron, tungsten, lead are insoluble in water
-These sulfides are converted to sulfate which are soluble
-The sulfates leach out of the ore and are then extracted
Cu2S not soluble CuSO4 is soluble
Commercial Bioleaching Tanks