I. Degradation of organic pollutants Genetic engineering to enhance bioremediation II. Plant biomass Utilization of sugars, starch, cellulose and lignin
I. Degradation of organic pollutants
Genetic engineering to enhance bioremediation
II. Plant biomass
Utilization of sugars, starch, cellulose and lignin
I. Bioremediation.
Chemical Pollutants 1. Heavy metals: mercury, lead, copper 2. Radionuclides: uranium, cesium 3. Nutrients: N and P from animal waste and fertilizer 4. Organics: pesticides, petroleum, chlorinated solvents
Chemical Pollutants
1. Heavy metals: mercury, lead, copper
2. Radionuclides: uranium, cesium
3. Nutrients: N and P from animal waste and fertilizer
4. Organics: pesticides, petroleum, chlorinated solvents
The Carbon Cycle, Microorganisms and Xenobiotic Pollutants
Global carbon cycle:
Naturally-occurring organic compounds are: 1. Biosynthesized from inorganic CO2 and H2O Mainly through photosynthesis by plants and bacteria (primary producers) 2. Usually metabolically transformed into various other organic compounds by consumer organisms 3. Eventually mineralized back to CO2 and H2O by catabolic reactions Mainly bacteria and fungi
Naturally-occurring organic compounds are:
1. Biosynthesized from inorganic CO2 and H2O Mainly through photosynthesis by plants and bacteria (primary producers) 2. Usually metabolically transformed into various other organic compounds by consumer organisms 3. Eventually mineralized back to CO2 and H2O by catabolic reactions Mainly bacteria and fungi
1. Biosynthesized from inorganic CO2 and H2O
Mainly through photosynthesis by plants and bacteria (primary producers)
2. Usually metabolically transformed into various other organic compounds by consumer organisms
3. Eventually mineralized back to CO2 and H2O by catabolic reactions
Mainly bacteria and fungi
Most are man-made via chemical synthesis --don't occur naturally Often designed to be chemically stable and may persist in the environment for decades Ex. Chlorinated organics: DDT (insecticide) and PCBs (polychlorinated biphenyls --electrical insulators and other uses) Toxic to humans and wildlife "Microbial infallibility": A hypothesis that microorganisms can mineralize any naturally-occurring organic compound (i.e. biochemically-produced compounds) Chemical bonds produced by enzyme-catalyzed biosynthetic reactions should be able to be broken by enzyme-catalyzed catabolic reactions. Can we extend the principle of microbial infallibility to xenobiotic compounds which are not biochemically produced? Some xenobiotic compounds can be cooxidized by degradative enzymes of pathways of naturally occurring compounds. Ex. Some PCBs are partially degraded via the degradation pathway for biphenyl Ex. Degradation of trichloroethylene (TCE) by a reaction catalyzed by toluene dioxygenase
Most are man-made via chemical synthesis --don't occur naturally
Often designed to be chemically stable and may persist in the environment for decades
Ex. Chlorinated organics: DDT (insecticide) and PCBs (polychlorinated biphenyls --electrical insulators and other uses) Toxic to humans and wildlife
Ex. Chlorinated organics: DDT (insecticide) and PCBs (polychlorinated biphenyls --electrical insulators and other uses)
Toxic to humans and wildlife
"Microbial infallibility": A hypothesis that microorganisms can mineralize any naturally-occurring organic compound (i.e. biochemically-produced compounds)
Chemical bonds produced by enzyme-catalyzed biosynthetic reactions should be able to be broken by enzyme-catalyzed catabolic reactions.
Can we extend the principle of microbial infallibility to xenobiotic compounds which are not biochemically produced?
Some xenobiotic compounds can be cooxidized by degradative enzymes of pathways of naturally occurring compounds.
Ex. Some PCBs are partially degraded via the degradation pathway for biphenyl Ex. Degradation of trichloroethylene (TCE) by a reaction catalyzed by toluene dioxygenase
Ex. Some PCBs are partially degraded via the degradation pathway for biphenyl
Ex. Degradation of trichloroethylene (TCE) by a reaction catalyzed by toluene dioxygenase
Benefits of Bioremediation
1. Cheaper than physical removal, incineration or chemical treatments. 2. Less disruptive to the environment. 3. Public acceptance.
1. Cheaper than physical removal, incineration or chemical treatments.
2. Less disruptive to the environment.
3. Public acceptance.
Problems that Need to be Solved
1. Bioavailability. Low aqueous solubility of hydrophobic pollutants, sorption to sediments. Limits availability of compound and uptake by cell 2. Toxicity of pollutants to microorganisms. 3. Mixed wastes. --several different microorganisms usually needed. a. Most microorganisms have degradative pathways for a few related compounds b. Substrate specificity of most enzymes is narrow 4. Predation. Protozoa graze on microbial degraders lowering their numbers. 5. Environmental factors. Not optimal for rapid degradation. -Temperature, pH, oxygen tension, nutrients 6. Competition. Utilization of nutrients, oxygen and space by other microorganisms. 7. Growth. Often not supported by pollutant. Microorganisms preferentially use other compounds in environment and ignore the pollutant 8. Metabolic regulation. Degradative pathways may not be induced by the pollutant. 9. Incomplete degradation. Dead-end metabolites (which may also be toxic) may be produced. 10. Government regulations. Some of the above problems could be solved using genetically engineered microorganisms (GEMS) but they usually can't be released into the environment.
1. Bioavailability. Low aqueous solubility of hydrophobic pollutants, sorption to sediments.
Limits availability of compound and uptake by cell
2. Toxicity of pollutants to microorganisms.
3. Mixed wastes. --several different microorganisms usually needed.
a. Most microorganisms have degradative pathways for a few related compounds b. Substrate specificity of most enzymes is narrow
a. Most microorganisms have degradative pathways for a few related compounds
b. Substrate specificity of most enzymes is narrow
4. Predation. Protozoa graze on microbial degraders lowering their numbers.
5. Environmental factors. Not optimal for rapid degradation.
-Temperature, pH, oxygen tension, nutrients
6. Competition. Utilization of nutrients, oxygen and space by other microorganisms.
7. Growth. Often not supported by pollutant.
Microorganisms preferentially use other compounds in environment and ignore the pollutant
8. Metabolic regulation. Degradative pathways may not be induced by the pollutant.
9. Incomplete degradation. Dead-end metabolites (which may also be toxic) may be produced.
10. Government regulations. Some of the above problems could be solved using genetically engineered microorganisms (GEMS) but they usually can't be released into the environment.
Increasing the Range of Substrates Degraded by a Microorganism
(Related to problem 3 a. above) See Fig. 13.5 Some nautrally occurring plasmids carry genes that encode enzymes that catalyze degradation of toxic organic pollutants Ex. aromatic solvents: benzene, toluene, xylenes Transfer several different catabolic plasmids to one organism via conjugation. "Superbug" was created for remediation of oil spills. 1st genetically engineered organism to be patented United States Patent No. 4,259,444 Inventor: Ananda Chakrabarty
(Related to problem 3 a. above)
See Fig. 13.5
Some nautrally occurring plasmids carry genes that encode enzymes that catalyze degradation of toxic organic pollutants
Ex. aromatic solvents: benzene, toluene, xylenes
Transfer several different catabolic plasmids to one organism via conjugation.
"Superbug" was created for remediation of oil spills. 1st genetically engineered organism to be patented United States Patent No. 4,259,444 Inventor: Ananda Chakrabarty
"Superbug" was created for remediation of oil spills.
1st genetically engineered organism to be patented United States Patent No. 4,259,444 Inventor: Ananda Chakrabarty
1st genetically engineered organism to be patented
United States Patent No. 4,259,444 Inventor: Ananda Chakrabarty
Engineering a Psychrotroph for Low-Temperature Growth on Toluate
(Related to problem 5 above) Many polluted aquatic environments (and soil in winter) have temperatures between 0-20oC. Mesophile: an organism with an optimum growth temperature of 20-45oC Psychrotroph: a mesophile that can grow at low temperature. Psychrophile: has an optimum growth temperature below 20oC.
(Related to problem 5 above)
Many polluted aquatic environments (and soil in winter) have temperatures between 0-20oC.
Mesophile: an organism with an optimum growth temperature of 20-45oC Psychrotroph: a mesophile that can grow at low temperature. Psychrophile: has an optimum growth temperature below 20oC.
Bacterial Strain
Strain Q5T grows on toluate at low temperatures.
Biological Containment of Genetically Engineered Microorganisms
(Not in text, related to problem 10 above) Desirable for GEMS released into the environment to self destruct when no longer needed Presence or absence of a pollutant could regulate expression of a lethal (suicide) gene In presence of pollutant cells grow and degrade the pollutant Absence of pollutant signals the cell to commit suicide Killing is effected by a regulatory cascade that responds to presence or absence of a pollutant 1. Regulatory cassette: controls expression of the killing gene xylS is a positive regulatory protein of the Pm promoter 3-methylbenzoate is the effector Pm promoter controls expression of lacI (negative regulatory protein)
(Not in text, related to problem 10 above)
Desirable for GEMS released into the environment to self destruct when no longer needed
Presence or absence of a pollutant could regulate expression of a lethal (suicide) gene
In presence of pollutant cells grow and degrade the pollutant Absence of pollutant signals the cell to commit suicide
In presence of pollutant cells grow and degrade the pollutant
Absence of pollutant signals the cell to commit suicide
Killing is effected by a regulatory cascade that responds to presence or absence of a pollutant
1. Regulatory cassette: controls expression of the killing gene xylS is a positive regulatory protein of the Pm promoter 3-methylbenzoate is the effector Pm promoter controls expression of lacI (negative regulatory protein)
1. Regulatory cassette: controls expression of the killing gene
xylS is a positive regulatory protein of the Pm promoter 3-methylbenzoate is the effector Pm promoter controls expression of lacI (negative regulatory protein)
xylS is a positive regulatory protein of the Pm promoter
3-methylbenzoate is the effector
Pm promoter controls expression of lacI (negative regulatory protein)
2. Killing cassette: expression of the suicide gene kills the host cell Located on host cell chromosome for stability Plac promoter controls expression of the suicide gene gef the suicide gene Gef protein inserts into the cell membrane Membrane potential collapses, ATP not made, cell dies
2. Killing cassette: expression of the suicide gene kills the host cell
Located on host cell chromosome for stability Plac promoter controls expression of the suicide gene gef the suicide gene Gef protein inserts into the cell membrane Membrane potential collapses, ATP not made, cell dies
Located on host cell chromosome for stability
Plac promoter controls expression of the suicide gene
gef the suicide gene
Gef protein inserts into the cell membrane Membrane potential collapses, ATP not made, cell dies
Gef protein inserts into the cell membrane
Membrane potential collapses, ATP not made, cell dies
II. Biomass
Organic matter produced primarily by photosynthesis. A renewable resource.
Ex. Starch and lignocellulose from trees and agricultural crops Waste biomass paper and crop wastes Current uses involve conversion of starch and cellulose to: Ethanol for fuel and alcoholic beverages Sweeteners for processed foods
Ex.
Starch and lignocellulose from trees and agricultural crops
Waste biomass paper and crop wastes
Current uses involve conversion of starch and cellulose to:
Ethanol for fuel and alcoholic beverages Sweeteners for processed foods
Ethanol for fuel and alcoholic beverages
Sweeteners for processed foods
Plant storage polymer, abundant in grain (corn)
Composed of: 1. Amylose --linear glucose chains 2. Amylopectin --branched glucose chains
Composed of: 1. Amylose --linear glucose chains
2. Amylopectin --branched glucose chains
1. First, starch is converted to glucose by enzymes
Enzymes that convert starch to glucose a-amylase and b-amylase glucoamylase
Enzymes that convert starch to glucose
a-amylase and b-amylase glucoamylase
a-amylase and b-amylase
glucoamylase
2.. Glucose may then be converted to:
Furctose (sweetner) by glucose isomerase or Ethanol (fuel) by microbial fermentation
Furctose (sweetner) by glucose isomerase or
Ethanol (fuel) by microbial fermentation
Fig. 13.13 1. a-Amylase and b-amylase hydrolyze amylose and amylopectin to: glucose, maltose and limit dextrin (short branched chains) 2. Glucoamylase converts limit dextrins to glucose
Fig. 13.13
1. a-Amylase and b-amylase hydrolyze amylose and amylopectin to:
glucose, maltose and limit dextrin (short branched chains)
2. Glucoamylase converts limit dextrins to glucose
Fig. 13.14 1. Milled grain is converted to gelatinized starch by steam 2. a-amylase used to liquify the gelatinized starch, at 50-60OC and pH ~6.5 3. Glucoamylase completes conversion to glucose at pH ~4.5 4. Saccharified starch is converted to fructose by glucose isomerase at pH~7.5 or is fermented to ethanol by yeast Enzymes are expensive and contribute significantly to cost of starch conversion 30% of cost of all enzymes used for industrial processes are for starch conversion Each enzyme-catalyzed step requires different conditions of temperature and pH, adding to production costs
Fig. 13.14
1. Milled grain is converted to gelatinized starch by steam
2. a-amylase used to liquify the gelatinized starch, at 50-60OC and pH ~6.5
3. Glucoamylase completes conversion to glucose at pH ~4.5
4. Saccharified starch is converted to fructose by glucose isomerase at pH~7.5 or is fermented to ethanol by yeast
Enzymes are expensive and contribute significantly to cost of starch conversion
30% of cost of all enzymes used for industrial processes are for starch conversion
Each enzyme-catalyzed step requires different conditions of temperature and pH, adding to production costs
1. Produce enzymes more cheaply by cloning genes and expressing in microbial strains that grow on cheap carbon sources Ex. Bacillus sp. grown on soy molasses, a waste product of soybean oil production
1. Produce enzymes more cheaply by cloning genes and expressing in microbial strains that grow on cheap carbon sources
Ex. Bacillus sp. grown on soy molasses, a waste product of soybean oil production
2. Use thermostable enzymes for faster conversion rates and reduced cooling costs -Clone genes from thermophilic microorganisms -Engineer enzymes already in use by directed mutagenesis -Introduce Cys for intramolecular disulfide bond formation -Change Asn and Gln to other aminoa acids to prevent high temp. deamidatiioon
2. Use thermostable enzymes for faster conversion rates and reduced cooling costs
-Clone genes from thermophilic microorganisms -Engineer enzymes already in use by directed mutagenesis -Introduce Cys for intramolecular disulfide bond formation -Change Asn and Gln to other aminoa acids to prevent high temp. deamidatiioon
-Clone genes from thermophilic microorganisms
-Engineer enzymes already in use by directed mutagenesis
-Introduce Cys for intramolecular disulfide bond formation
-Change Asn and Gln to other aminoa acids to prevent high temp. deamidatiioon
3. Engineer enzymes by mutagenesis to have similar pH optima -Eliminates need for adjusting pH for each enzyme
3. Engineer enzymes by mutagenesis to have similar pH optima
-Eliminates need for adjusting pH for each enzyme
4. Clone and introduce glucoamylase gene into yeast for ethanol production -Same organism synthesizes the enzyme and ferments glucose to alcohol -Eliminates one step in conversion of starch to ethanol
4. Clone and introduce glucoamylase gene into yeast for ethanol production
-Same organism synthesizes the enzyme and ferments glucose to alcohol -Eliminates one step in conversion of starch to ethanol
-Same organism synthesizes the enzyme and ferments glucose to alcohol
-Eliminates one step in conversion of starch to ethanol
Lowering Costs and Increasing the Efficiency of Conversion of Glucose to Fructose
Fructose is ~ 2.5 times sweeter than glucose and 2 times sweeter than sucrose (table sugar) Fructose is cheaper to use in processed foods because less is needed to achieve the same level of sweetness Also used to produce low calorie foods because less is used
Fructose is ~ 2.5 times sweeter than glucose and 2 times sweeter than sucrose (table sugar)
Fructose is cheaper to use in processed foods because less is needed to achieve the same level of sweetness Also used to produce low calorie foods because less is used
I. High level expression of a thermostable glucose isomerase
Use of high temperature increases rate of conversion of glucose to fructose
1. Gene encoding a thermostable isomerase was cloned from a thermophile (Thermus thermophilus).
2. Isomerase gene placed on multicopy plasmid to increase gene dosage.
3. Highest level of expression achieved in industrial strain (Bacillus brevis) with gene under control of strong Bacillus promoter and with a Bacillus ribosome binding site that promotes efficient translation.
See Table 13.5
II. Alter enzyme's substrate preference by site directed mutagenesis
See Table 13.6 Glucose isomerase actually favors xylose (w/ 5 carbons) over glucose (w/ 6 arbons) as a substrate Substrate specificity of the thermostable enzyme from Clostridium thermosulfurogenes was modified Two amino acids involved in substrate binding in the active site of the enzyme were identified Site-directed mutagenesis used to change them to amino acids that favored binding of glucose over xylose Wild-type enzyme: xylose converted 17 X more efficiently than glucose Double mutant: glucose converted 1.5 X more efficiently than xylose
See Table 13.6
Glucose isomerase actually favors xylose (w/ 5 carbons) over glucose (w/ 6 arbons) as a substrate
Substrate specificity of the thermostable enzyme from Clostridium thermosulfurogenes was modified Two amino acids involved in substrate binding in the active site of the enzyme were identified Site-directed mutagenesis used to change them to amino acids that favored binding of glucose over xylose Wild-type enzyme: xylose converted 17 X more efficiently than glucose Double mutant: glucose converted 1.5 X more efficiently than xylose
Substrate specificity of the thermostable enzyme from Clostridium thermosulfurogenes was modified
Two amino acids involved in substrate binding in the active site of the enzyme were identified
Site-directed mutagenesis used to change them to amino acids that favored binding of glucose over xylose
Wild-type enzyme: xylose converted 17 X more efficiently than glucose Double mutant: glucose converted 1.5 X more efficiently than xylose
Wild-type enzyme: xylose converted 17 X more efficiently than glucose
Double mutant: glucose converted 1.5 X more efficiently than xylose
Lignocellulose Biomass
A potential source for production of fuel and industrial chemicals
I. Sources of Cellulose
1. Primary cellulose: cotton, wood, hay
2. Agricultural crop wastes: straw, corn stalks, rice hulls
3. Municipal waste: paper, cardboard
II. Composition of Lignocellulose
1. Cellulose. Most abundant polymer on earth
Homopolymer of glucose Fig. 13.24
3. Hemicellulose
Heteropolymer of hexoses and pentoses
2. Lignin
Heteropolymer of phenylpropane subunits Fig. 13.23
III. Commercial Utilization of Cellulose
Acid attacks equipment and produces unwanted by-porducts Acid must be neutralized, producing salt
Acid attacks equipment and produces unwanted by-porducts
Acid must be neutralized, producing salt
Actually is four different enzymes Hydrolytic activity is slow however
Actually is four different enzymes
Hydrolytic activity is slow however
Has high cost, price needs to be reduced
IV. Cellulase structure
Fig. 13.25 Components arranged as a multienzyme complex called a cellulosome Catalytic subunits are responsible for hydrolytic activity Cellulose-binding domain Scafolding proteins bind catalytic subunits to cellulose-binding domain Cellulosome is located on the exterior of the cell
Fig. 13.25
Components arranged as a multienzyme complex called a cellulosome
Catalytic subunits are responsible for hydrolytic activity Cellulose-binding domain Scafolding proteins bind catalytic subunits to cellulose-binding domain
Catalytic subunits are responsible for hydrolytic activity
Cellulose-binding domain
Scafolding proteins bind catalytic subunits to cellulose-binding domain
Cellulosome is located on the exterior of the cell
V. Genes encoding catalytic subunits have been cloned
Screening a DNA library for cloned cellulose genes
1. Endoglucanases (Screening for enzyme activity)
Plate library onto medium containing carboxymethyl cellulose After colonies develop, flood plate with Congo red Dye binds to intact CMC but not hydrolyzed CMC
Plate library onto medium containing carboxymethyl cellulose
After colonies develop, flood plate with Congo red
Dye binds to intact CMC but not hydrolyzed CMC
2. Exoglucanases (Screening by immunoassay)
Use a specific antibody that bind the target protein
3. b-Glucosidases
Screen for ability of recombinant host to grow on cellobiose as sole carbon source
VI. Engineering yeast to convert cellulose to ethanol
1. Endo- and exoglucanase genes cloned from Cellulomonas fimi
2. Fused to a yeast signal peptide sequence and placed under control of a S. cerevisiae promoter
3. Introduced into S. cerevisiae on a plasmid
VII. Waste biomass as a potential source of fuel
100 million tons waste paper/year in U.S 400 liters etahnol /ton of paper are possible Enough to replace 16% of gasoline used Agricultural wastes are also a potential source Bagasse from sugarcane, corn stovers, rice hulls and straw 1. Cellulase converts waste cellulose to glucose 2. Yeast ferments glucose to ethanol
100 million tons waste paper/year in U.S
400 liters etahnol /ton of paper are possible
Enough to replace 16% of gasoline used
Agricultural wastes are also a potential source
Bagasse from sugarcane, corn stovers, rice hulls and straw
1. Cellulase converts waste cellulose to glucose
2. Yeast ferments glucose to ethanol
Single Cell Protein (Microbial Biomass)
Protein produced by microorganisms (bacteria, yeast, fungi, algae)
Carbon sources for growth of microorganisms may include: Waste CO2 (greenhouse gas) , whey, cellulose (paper and agricultural) Petroleum alkanes or methane 60-80% of cell dry weight is protein Contains high levels of essential amino acids and vitamins However, may be poorly digestible or contain toxins Large scale production using the bacterium Methylophilus methylotrophus grown on methanol was not profitable, but may be in the future Genetic engineering may create improved strains that overexpress proteins containing higher levels of essential amino acids for animal feed or human consumption
Carbon sources for growth of microorganisms may include:
Waste CO2 (greenhouse gas) , whey, cellulose (paper and agricultural) Petroleum alkanes or methane
Waste CO2 (greenhouse gas) , whey, cellulose (paper and agricultural)
Petroleum alkanes or methane
60-80% of cell dry weight is protein
Contains high levels of essential amino acids and vitamins
However, may be poorly digestible or contain toxins
Large scale production using the bacterium Methylophilus methylotrophus grown on methanol was not profitable, but may be in the future
Genetic engineering may create improved strains that overexpress proteins containing higher levels of essential amino acids for animal feed or human consumption
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