Bài giảng Nhập môn công nghệ sinh học - Microbial Biotechnology

or deletion 
Products are also gained by altering the microbe’s environment 
Metabolically Engineered Cell 
Cheap Carbon Source 
Industrial Chemicals 
Chemical Precursors 
Biopolymers 
Vitamins 
Amino Acids 
Glucose 	 NH 4 + 
Pyruvate 
L-lactate 
D-Alanine 
L-Alanine 
100% 
L-Alanine 
NAD + 
NADH 
NADH 
NAD + 
L-LDH 
AlaOH 
NADH NAD+ 
Racemase 
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 billion 1 
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 
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) 
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 billion 1 
(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 
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 
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 CO 2 and H 2 0 
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 
6000yards 3 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 
Cu 2 S not soluble	 	 CuSO 4 is soluble 
Commercial Bioleaching Tanks