Module 8: Microbial Biotechnology (Applications) Flashcards
Red Biotechnology:
2 main uses
1) Secondary metabolite therapeutics
2) Human Protein therapeutics
What are some types of drugs that are based upon secondary metabolites (list them) (3)
1) Antibiotics
2) Statins
3) Artemissin (antimalarial)
What were the 3 first antibiotics?
1) Penicillin
2) Streptomycin
3) Actinomycin
What is the main source of streptomycin + actinomycin?
Actinomycetes
–> MOST ABUNDANTLY found in Streptomyces (genus) species
Actinomycetes
A group of bacteria that form saprophytic filaments in soil
What are some secondary metabolites of Streptomyces species?
1) Antibiotics
2) Antifungals
3) Anti-cancer agents
4) Immunosuppressants
Statins
Secondary metabolites produced by different fungi that inhibit cholesterol synthesis
What do statins act upon?
HMG-CoA-Reductase
–> They COMPETITIVELY inhibit this enzyme that is needed for the synthesis of cholesterol
In what microbe were statins first discovered
Penicillium fungus (strain citrinium)
Artemissin
An antimalarial secondary metabolite produced by the Artemissia annua plant!
What are some examples of human proteins that can be produced within microbes?
1) Type I interferons –> Antiviral + antitumor
2) Anti-coagulation Factor XIIIA (13A) –> Haemophilia treatment
3) Epidermal growth factor –> burn and organ damage treatment
What is insulin?
A protein hormone produced by the pancreas that regulates blood sugar
What is the process of producing insulin in E.coli?
1) 2 vectors used; one has A subunit and other has B subunit
–> in BOTH, the A and B subunit genes are fused to a LacZ gene tag sequence
2) Both vectors introduced into E. coli cultures
3) LacZ (B-gal) tagged insulin is replicated
4) Lyse cells
5) Isolate the A and B chains from all cell materials using affinity tag purification (affinity chromatography)
6) Cleave the A + B subunits from the LacZ peptide (B-gal.)
7) Combine A + B chains to form functional insulin
What is an additional step that might be needed at the end of E.coli insulin production?
Cleavage of an endotoxin that E.coli adds to the protein hormone
Biorefinery
the process of converting BIOMASS into a # of proteins
Biorefinery is modeled after __________ BUT differs in that it is _________________
Modeled after oil refineries
BUT instead is RENEWABLE
Feedstock
Biomass serving as the starting materials for biorefinery
What can serve as feedstock?
1) Crops specifically grown to be feedstock
2) Agricultural and forestry waste
3) Methane in biogas
What is the starting material that microbes actually act upon?
Cellulose/Hemicellulose
–> Microbes won’t act on the feedstock itself; must be broken down first to at least cellulose BUT usually needs to be broken down FURTHER
Cellulose is hard to digest because of…
B-1,4-linkages
Before microbes can act on feedstock, what might need to occur first?
BREAKDOWN
–> Breakdown of feedstock —> cellulose
+ in many cases:
Cellulose —> Simpler sugars
How can cellulose be pre-degraded?
1) Physically –> heat + alkaline treatment
2) Enzymatically –> Glycoside Hydrolases (Ex: cellulase)
the biorefinery process
Sawdust + Straw —> Cellulose (Not microbe)
cellulose —–> 1) Bioplastics, 2) Fuels, 3) Chemical feedstock (MICROBE ACTION)
Optional:
chemical feedstock –> Fine chemicals (solvents)
What products occur as a result of biorefinery?
1) Materials = Bioplastics
2) Fuels = Ethanol, acetone, butanol
3) Chemical feedstock = succinic acid, acetic acid
Biofuels
Fuels produced via biomass conversion
What were the original users of biofuels?
1) Original diesel engines == used biodiesel from vegetable oil
2) Ford Model T == used ethanol from corn starch
How is ethanol produced?
Produced by fermentation of sugars by yeast during the exponential phase under ANAEROBIC conditions
(a primary metabolite)
What is the issue with ethanol production by yeast?
What additional step is needed?
Problem = Even the best yeast can only tolerate ethanol up to 15%
== Distillation step is needed to produce more concentrated ethanol
Distillation
A method for increasing the concentration of ethanol by evaporating ethanol out of the culture mix and condensing it back into a liquid
Distillation steps
1) Culture mix is heated
2) Produced ethanol is evaporated
3) Evaporated ethanol condenses around cooling coils
4) Liquid ethanol is collected
What determines the cost of ethanol production?
The price of the feedstock utilized
What are the most common feedstocks used for ethanol production?
Cornstarch + Sugarcane (juice)
What are the different methods of ethanol production from different feedstocks?
Cellulose Based Feedstock: (Still in development)
1) PRETREATMENT == to release cellulose
2) Enzymatic breakdown by glycoside hydrolases
3) Fermentation by bacteria or yeast == ethanol
Cornstarch;
1) Enzymatic breakdown by amylase
2) Fermentation by bacteria or yeast == ethanol
Sugarcane Juice:
1) Fermentation by bacteria or yeast == ethanol
What are the potential drawbacks of using sugarcane and corn starch as feedstock for ethanol?
1) Will increase demand for corn starch and sugar cane == drive prices up for food products that use these materials
2) Increased requirements for LAND and SYNTHETIC FERTILIZERS = could be bad for the environment
What is a proposed alternative to using sugarcane + cornstarch to produce ethanol?
What’s the problem with this alternative?
The use of waste biomass feedstock
Problem = composed of lignocellulose which microbes cannot break down
Waste Biomass Feedstock
Waste products from agriculture and forestry operations
Lignocellulose
A mix of polymeric cellulose, hemicellulose, and lignin
What is a potential solution to lignocellulose resistance to breakdown by microbes?
Pre-treatment! == Free the cellulose + hemicellulose from the lignocellulose so that microbes can act on it
What are potential methods of pretreatment for lignocellulose?
1) Cellulase breakdown
2) Acid Treatment
3) Physical breakdown
Bioplastics
Biodegradable plastics; mainly consists of PHAs
Plastics
Versatile polymers with a wide range of physical characteristics
Problem with plastics
Resistant to biodegradation == accumulate in the environment
What is PHA (and what does it stand for)?
(+ PHB?)
PHA = Polyhydroxyalkanoates
–> Natural polyester polymer that serves as carbon storage within bacteria AND has characteristics like fossil-fuel derived polyester
PHB = Polyhydroxybutyrate (1st PHA discovered + most simple PHA)
In what microbe was the first PHA discovered?
Bacillus megaterium
(1st discovered = PHB)
PHAs in natural life cycle:
In what conditions are PHAs synthesized and broken down?
Synthesis = Carbon is ABUNDANT
Breakdown = Carbon is SCARCE
On an industrial level, what conditions support high production of PHAs?
1) HIGH CARBON
2) LOW Nitrogen + Phosphorus
Why is low nitrogen and phosphorus used to increase industrial yield of PHAs?
Because N + P are necessary for growth SO without them, the cells are more likely to undergo carbon storage into PHA rather than carbon incorporation to grow!
–> Essentially directs the carbon usage to PHA production
PHAs exhibit a range of physical features, how can we alter their characteristics?
1) By altering the chain length of individual monomers
2) By altering the # of monomers making up the polymer
In PHAs: The greater the chain length, what changes about the features?
Greater chain length = greater elasticity + lower melting point
Copolymers of PHAs
PHAs with > 1 TYPE of monomers
What are 3 enzymes essential to PHA formation?
1) Ketothiolase
2) Acetoacetyl-CoA Reductase
3) PHA synthase
How many intermediates exist between sugars and PHA formation?
THREE
1) Acetyl-CoA
2) Acetoacetyl-CoA
3) 3-hydroxyalkanoate-CoA
Ketothiolase
Enzyme that converts Acetyl-CoA to Acetoacetyl-CoA
What are the key enzymes of PHA synthesis and degradation?
Synthesis = PHA synthase
Degradation = PHA depolymerase
PHA synthesis:
Sugar Excess vs Sugar NOT in excess
Sugar Excess = 4 acetyl-coA produced == promotes the PHA pathway
Sugar NOT in excess = 2 acetyl-coA produced = promotes Kreb Cycle
What microbes accumulate a lot of PHA?
CYANOBACTERIA
Transgenic PHA Plants
Plants containing bacterial PHA synthase genes
Transgenic Plants
Plants containing DNA transferred from a different type of organism
What are the main goals with transgenic PHA plants?
1) Produce PHAs as a COPRODUCT of agricultural production
2) Localize PHA production to specific regions of plants (stems, roots, etc.)
Biocatalyst
Enzyme that facilitates an industrial chemical reaction
Why are biocatalysts preferred over chemical catalysts? (3)
1) Greater specificity
2) Greater efficiency
3) BIODEGRADABLE
Two examples of biocatalyst uses:
1) High Fructose Corn Syrup production
2) Laundry detergents
What is the process of producing HFCS?
1) Corn starch converted to corn syrup by AMYLASE
2) Corn syrup isomerized to HFCS via GLUCOSE ISOMERASE
In HFCS production, from what microbes do the two main enzymes originate from?
AMYLASE = From Bacillus species
GLUCOSE ISOMERASE = From Streptomyces species
What is the largest industry for microbial produced enzymes?
LAUNDRY DETERGENT
Why do food manufacturers fortify foods? (3)
1) Nutritive compounds were lost during food processing
2) Nutritive composition is off balance
3) Development of FUNCTIONAL FOODS
What are functional foods?
(Example?)
= Foods that provide a specific function other than basic nutritional value
Ex: Breads + pastas fortified w/ FOLIC ACID (for proper fetal development)
Common Names for B12 and B2
B12 = Cobalamin
B2 = Riboflavin
What organisms NEED B12?
ANIMALS
(plants do not need + therefore do not make it)
How do we get B12?
1) Diet
2) Microbes in our gut produce it
What illness does B12 deficiency cause?
Pernicious Anemia
Differences in commercial production of B2 and B12
(WHY?)
B12 = Mainly microbial production (due to very complicated chemical structure)
B2 = Mainly chemical production (due to simpler chemical structure BUT can be produced by microbes too!)
What microbes predominantly produce B12?
Pseudomonas
What are the application of microbial produced AAs? (5)
1) Food Industry
2) Supplement for Animal Feed
3) Production of Synthetic Chemicals
4) Nutritional supplements
What is the advantage of producing AAs in microbes?
STEREOSPECIFICITY
–> All AAs produced by microbes are the L-ISOMER
(we can use the L-isomer!)
What are the most abundantly produced AAs using microbes? (give their percentages)
1) (L)-Glutamic Acid (51%)
2) (DL)- Methionine (22%)
3) (L) -Lysine (22.6%)
What is glutamic acid used for?
Flavor enhancer
What are methionine and lysine used for?
Animal feed supplement
What is the natural synthesis pathway + regulatory process of LYSINE?
Aspartate —-Aspartate Kinase—–> Lysine
–> When lysine content is high it inhibits aspartate kinase by ALLOSTERIC inhibition!
What reactant and enzyme is needed for lysine production?
Reactant = Aspartate
Enzyme = Aspartate Kinase
What constitutes a good microbial strain for industrial production of Lysine?
A strain that has an Aspartate Kinase with:
1) Functional ACTIVE SITE (= Lysine can be produced)
2) Dysfunctional ALLOSTERIC SITE (= no negative feedback!)
== No shutdown of the production system!
A. tumefaciens causes ____________ disease mainly in _____________ plants
Causes CROWN GALL DISEASE
–> Mainly in DICOT plants
How does TDNA of pTi move from A. tumefaciens to plant cell?
Conjugation
When TDNA of A. tumefaciens is expressed in plant cells, what is produced?
1) OPINES = act as nutrient source for A. tumefaciens to live within the tumors!
2) Phytohormones (cytokines + auxins) = cell proliferation is triggered to cause tumor formation
What genes does TDNA of pTi naturally contain?
1) Opine synthesis genes
2) Phytohormone synthesis genes
What are the DNA components (gene regions) of the pTi?
1) TDNA
2) Opine catabolism genes
3) vir genes
What regions of pTi can be altered in agrobacterium-mediated plant transformation?
The opine + phytohormone synthesis genes of the TDNA can be replaced by genes of interest
What plants are NOT susceptible to Agrobacterium-Mediated Plant Transformation?
MONOCOTS
–> Mainly important agricultural crops like WHEAT, BARLEY, RICE
For plants not susceptible to Agrobacterium-Mediated Plant Transformation, what are other methods that can be utilized? (2)
1) BIOLISTICS = metal beads with desired DNA on them get FIRED into plant cells
2) PROTOPLAST TRANSFORMATION = cell wall removed before DNA is introduced
Agrobacterium-Mediated Plant Transformation process:
1) Leaf tissue discs taken from a given plant
2) Plant discs plated in medium with:
i. A. tumefaciens containing modified pTi (Recomb. TDNA)
ii. phenolic compounds (to support transfer of TDNA)
3) Add antibiotic to plates to kill A. tumefaciens
4) Add phytohormones to support plant growth
5) Collect the calli that are BLUE (= marker that demonstrates proper transformation of TDNA)
6) Plate calli on solid medium and allow shoots to form
7) Transplant shoots
8) Grow transgenic plant
What are two main applications of making transgenic plants?
Give crops:
1) Herbicide tolerance
2) Insect resistance
What is Round-Up
A broad range herbicide used by most farmers to kill weeds
What is the active ingredient in Round up?
Glyphosate
What does glyphosate do?
In PLANTS, glyphosate inhibits the production of aromatic AAs by inhibiting the EPSP synthase enzyme
What amino acids does glyphosate prevent the production of?
Aromatic AAs =
1) Tyrosine
2) Tryptophan
3) Phenylalanine
Why is glyphosate NOT toxic to humans?
Because we do not have the EPSP synthesis pathway for aromatic AAs
What is the issue with using glyphosate (roundup) in farming?
Doesn’t just kill weeds, will kill the crops too!
(Glyophosate is very efficiently absorbed by plant tissues!)
What is the solution to making crops resistant to glyphosate?
Making CP4-EPSPS transgenic plants!
–> Giving crops a form of EPSP synthase gene that produces EPSP synthase resistant to glyphosate action!
From what microbe is the glyphosate resistant EPSP synthase derived?
Soil bacterium CP4
What did the plasmid contain that gives plants glyphosate resistance?
1) 2 copies of the following unit:
promoter –> CTP (chloroplast transit peptide) fused to CP4-EPSPS gene
2) OriV
3) Two markers: one selectable marker + one screening marker
In the plasmid to make glyphosate resistant transgenic plants, what was the purpose of CTP?
CTP = chloroplast transit peptide
–> Fused to CP4-EPSPS gene so that when the EPSP synthase protein is produced, it has a CTP tag on it that marks it to be moved to the chloroplasts! (where the EPSP pathway accumulates!)
In what crops has glyphosate tolerance been initiated so far?
1) Corn
2) Cotton
3) Soybeans
4) Canola
What are the two main problems with insecticides?
1) Most are NEUROTOXINS
2) Most kill non-pest insects
What are CRY proteins?
(where are they found)
CRY proteins AKA Bt Toxin
= Found in Bacillus thuringiensis
Proteins that kill insects by permeabilizing their gut membranes!
What do insect resistant transgenic plants have? (what are they capable of?)
Bt toxin transgenic plants!
= have genes to produce CRY proteins SO when insects try to eat them, they die
Bt toxin killing process:
1) CRY proteins crystals ingested
2) Crystals solubilized into gut liquid
3) Proteases cleave the inactive CRY proteins producing an ACTIVE conformation
4) ACTIVE CRY proteins bind to receptors on surface of epithelial cells of digestive tract lining
5) Triggers pores to form = permeabilizes the membrane!
6) Water rushes into cells and they lyse
== Overall effect is that it kills the insect
