ANS/PLSS 433: Microbiology Applications

Microbial Synthesis of Commercial Products:

I. Protein Pharmaceuticals

	1. Recombinant Human Insulin, Genentech, 1978
		A. Bovine/Porcine Insulin used previously
		B. Many diabetics had side effects (allergies, etc.)
	2. bST approved for use in dairy cattle, 1994
		A. Controversial
		B. Health Issues
		 	a. bST milk is safe for human consumption
		C. FDA approval pending for use of pST/bST in meat animals
			a. Increase lean:fat ratio
			b. Reproductive advantages--increase ovulation?
	3. The "genes" for over 300 different proteins have been cloned 
		that may have human therapeutic significance.
	4. Isolation of cDNAs
		A. Easy for abundant proteins 
			a. Insulin makes up 70% of Islet of Langerhans 
				mRNAs
			b. Screen DNA Libraries from Site of Synthesis 
				(Gland, Organ, Cell type, Tumors, etc.)
		B. Harder for low expressed proteins and proteins with 
			unknown sites of synthesis
			a. Interferons
				i.	Reverse transcribe size fractioned 
					mRNAs from leukocytes and cloned 
					into pBR322
				ii.	6,000 clones divided into pools
				iii.	Crude IFN mRNA was used to                                                                               hybridize 
					to cDNA clone pools
				iv.	Hybridized mRNA was isolated and 
					translated in a cell-free protein 
					synthesis system (Rabbit 
					Reticulocyte)
				v.	Protein products tested for 
					antiviral activity.
				vi.	Positive pools further subgrouped 
					until specific cDNA found.				
	5. Many other proteins, including enzymes are made this way.
	6. Remember Post-transcriptional Modification & Optimizing Gene 
		Expression		 

II. Restriction Endonucleases

	1. Restriction Enzymes are a commercial product of microbes.
	2. In microbes, they serve as a protection system against DNA 
		viruses and other microorganisms.
	3. Grow under a wide range of conditions--Costly!!
	4. Clone R.E. genes into E. coli host
		A. Must also clone genes that protect against self
			-DNA degradation
			a. Usually methylation genes

III. Small Biological Molecules

	1. Recombinant DNA technology in microorganisms can be used to 
		modify metabolic pathways
	2. Goal:  Use Microorganism instead of chemical synthesis 
	3. Examples:

		L-Ascorbic Acid (Vitamin C)

		A. Synthesized from Glucose
			a. Currently:  	Fermentation + a number of 
					chemical steps--Expensive!!!!
			b. One Wild-type microbe doesn't have all the 	
				enzymatic steps.
		B. Combine microbes
			a. Tandem fermentation causes one microbe to 	
				wash-out
			b. Sequential Fermentation--Also Costly
		C. Genetic Engineering
			a. Clone all needed genes into one organism
				--Cornebacterium gene into Erwinia

		Indigo (Dye)
 
		A. Serendipity discovery in Pseudomonas genes cloned 
			into E.coli
			a. E.coli had genes for enzyme to convert 
				tryptophan to indole.
			b. Two different Psuedomonas had genes that utilize 
				either naptholene or xylene as 
				carbons sources.
			c. These enzymes also convert Indole to Indoxyl 
				which oxidizes to Indigo
		B. Bioreactor Designed--Solid support of bacteria

		Amino Acids 

		A. Building blocks of protein
		B. Uses in food industry, agriculture, medicine, and 	
			chemical industries
			a. $3 billion industry--500 thousand tons.
		C. Synthesis
			a. Protein Hydrosylates--Expensive
			b. Corynebacterium or Brevi bacterium
				--Mutant strains
				--Genetic Engineering--gram positive makes 
					difficult (few vectors)

		Others: Vitamins, Steroids, Cyclosporin, Mevinolin
			
IV. Antibiotics

	1. $5 billion industry
		A. $100 million in animal feed 
			a. Disease prevention
			b. Growth Promoters
		B. 6,000 Discovered since Penicillin, 1920s
			a. 100-200 new discovered each year
		C. Antibiotic resistance makes new discoveries more 
			important
			a. 1-2% of discoveries are added to disease
				-fighting arsenal	
		D. Most made from Streptomyces
			a. Gram Positive, Soil Bacteria
		E. Other Bacteria and Fungi also used
	2. Impact of recombinant DNA technology
		A. Development of structurally unique antibiotics
			a. Hybrids
		B. Genetic manipulation to increase yields
				--lower costs.
			
V. Biopolymers

	1. Large, multi-unit macromolecules
 		A. Used in Food Processing, Manufacturing, 
			& Pharmaceuticals
	2. Can be derived from Microorganisms, Animals and Plants.
	3. Genetic Engineering
		A. New Biopolymers
		B. Replace synthetic with biological equivalents
		C. Modify existing Biopolymers to enhance properties
		D. Increase yields
	4. Examples:
		A. Xanthene Gum
			a. Xanthomonas Campestris
			b. Engineered to grow on Whey (Cheese making 
				by-product)
		B. Melanin
			a. Streptomyces antibioticus 
			b. Two genes involved cloned into E.coli
		C. Byssal Adhesives
			a. Water proof adhesive from Blue Mussel
			b. Gene cloned into Yeast
			c. Cross-linking (Oxidation) inhibited by 
				Ascorbic acid
		D. Rubber
			a. Plant rubber polymerase cloned into microbes
		E. Others:
			a. Gellan--Solidification of food products
			b. Emulsan--Cleaning oil spills
			c. Pullulan--Food coating
			d. Dextrans--Blood Expander

Commercial Uses of Microbes: Bioremediation and Biomass

	Bioremediation--A process that uses living organisms to remove 
		contaminants, pollutants, or unwanted substances form 
		soil and water.

	Biomass--The cell mass produced by a population of living 
		organisms (from food and agricultural industries)

I. Microbial Degradation of Xenobiotics

	1. Pseudomonas species can breakdown synthetic aromatic compounds
		A. Herbicides, pesticides, refrigerants, solvents, 
			PCPs, etc.
		B. Broken down to catechols or protocatechuates
		C. Catechols or protecatechuates processed:
			a. To acetyl CoA and succinate ortho-cleavage
			b. To pyruvate and acetaldehyde meta-cleavage
	2. Other microbes are being genetically engineered to combine 
		different biodegradative pathways.
		A. Oil-eating bacterium
			a. Contains three plasmids containing genes that 
				degrade Camphor, Octane, Xylene, 
				& Naphthalene
			b. First patent of microorganism, 1980

II. Utilization of Starch & Sugar

	1. Starch--Polysaccarides (Amylose & Amylopectic)
		A. Amylases & Glucoamylases breakdown to sugar
		B. Sugar can then undergo microbial fermentation into 
			commercially important products
			a. Ethanol--Yeast
			b. Acetone--Clostridium
			c. Butanol--Clostridium
		
	2. Ethanol & Fructose from Starch 
		A. Uses of Ethanol
			a. Commodity Chemical
			b. Fuel
			c. Beverage Industry
				--20 billion gallons of beer are 
					produced in the world annually
		B. Uses of Fructose--Syrup		
		C. Steps in Ethanol & Fructose production
			a. Steam Gelatinization of Milled Grain
			b. Liquification with Amylase
			c. Saccharification with Glucoamylase
			d. Yeast fermentation to get alcohol
				or Glucose isomerase to get high
				fructose syrup (Corn Syrup)
		D. The use of Amylase, Glucoamylase and Glucose Isomerase 
			make up 30% of costs of all enzymes used in 
			industrial processes.
		E. Glucoamylases and other enzymes are also used to 
			reduce carbohydrates (limit dextrins) in 
			the production of dry and lite beers
		F. Biotech. methods under consideration to improve 						Fructose & Alcohol Production
			a. Overproduction of Enzymes in microbes that 
				utilize an inexpensive substrate
			b. Develop amylase that works at higher temp.
				--Would make liquification more efficient 
					and save energy.
			c, Alter amylase and glucoamylase so that they 
				would work under same temp. 
				and pH conditions.
			d. Find or engineer an enzyme that would degrade 
				raw starch, thus deleting the 
				gelatinization step.
			e. Develop a fermentation organism that could 
				synthesize and secrete glucoamylase.

III. Utilization of Lignocellulose

	1. Lignin, hemicellulose, & cellulose
		A. Combine in terrestrial plants in different degrees 
	2. Classes of lignocellulose
		A. Primary cellulosics--plants harvested for cellulosic 
			content (cotton), structural use (timber), 
			or feed value (hay)
		B. Agricultural waste cellulosics remaining after harvest 
			or processing.
			--Straw, stovers, rice hulls, 
				sugar cane bagasse, manures, 
				& timber residues.
		C. Municipal waste cellulosics--Paper waste products
	3. Three polymers can be separated by strong acid or base
	4. Ease of degradation:   cellulose > hemicellulose > lignin 	
	5. A large number of bacteria and fungi can degrade cellulose
		A. Several enzymes collectively known as cellulase
			a. Endoglucanase
			b. Exoglucanase
			c. Cellobiohydrolase
			d. B-Glucosidase
		B. Efforts are underway to incorporate cellulase genes 
			into yeast as a use of cellulose for alcohol 
			production
	6. Efforts are also underway to engineer microbes to utilize 
		hemicellulose and lignin

IV. Biomass--Production of whole cells
	
	1. Bioremediation 
	2. Inoculants
		A. Penicillium roquefortii--blue cheese flavor
		B. Rhizobium sp.--N2 fixation
	3. Insecticides
		A. Bacillus thuringiensis--Caterpillars, etc
	4. Starter Cultures
		A. Lactobaccilus sp.--Cheese, Yogurt, Sour Cream, etc.
		B. Yeasts--Bread, Beer, Wine, etc.
	5. Single Cell Protein
		A. Human or animal protein supplement
			a. Also contains fats, CHOs, nucleic acids, 
				vitamins, & minerals.
		B. Bacteria, Yeasts, Fungi, Algae, actinomycetes
		C. Use a variety of substrates
			a. CO2, Whey, Petroleum Hydrocarbons,
				 Cellulosic wastes, Methane
		D. Drawbacks:
			a. High nucleic acid hazardous to humans
			b. Possible Toxicity--Heavy Metals, Mycotoxins
			c. Microbes are digested slowly--indigestion, 
				allergies, etc.
			d. SCP more expensive than other sources of protein 
				(e.g. soybean meal)
	6. Bacterial Vaccines
		A. Killed bacterial cells make up many vaccines
			a. Cholera, whooping cough, plague, typhoid, etc.
		B. Attenuated
			a. TB
	7. Others
		A. Pseudomonas syringae
			a. Prevent Frost Damage
			b. Make Artificial Snow