Ch. 13 Flashcards
Pathway engineering -
The assembly of new or improved biochemical pathways, using genes from one or more organisms
What may help convert waste plant-derived material, including paper, into fuel alcohol?
Pathway engineering
Biomass -
- the total mass of organisms in a given area or volume
- fuel that is developed from organic materials, a renewable and sustainable source of energy used to create electricity or other forms of power
Polysaccharides -
A carbohydrate whose molecules consist of a number of sugar molecules bonded together
Plant material contains ___.
Polysaccharides
Outline the basic steps from biomass to ethanol.
- plant material contains polysaccharides: starch and cellulose
- enzymes degrade starch and release glucose molecules
- the glycolytic pathway converts glucose to pyruvic acid, which is fermented into alcohol and carbon dioxide
Yeast -
A microscopic fungus consisting of single oval cells that reproduce by budding, and are capable of converting sugar into alcohol and carbon dioxide
Fermentation -
The chemical breakdown of a substance by bacteria, yeasts, or other microorganisms, typically involving effervescence and the giving off of heat
Yeast and fermentation
Yeast is used to ferment sugars derived from grain or grapes, which produces an alcoholic liquid- the basis of beer and wine, respectively. Distillation is then used to make concentrated liquors such as whisky and vodka.
Zymomonas and fermentation
Bacterium which ferments sugar from the sap of the agave plant to give a liquid known as pulque. Distillation converts this into tequila.
Gasohol -
- alcohol blended with gasoline
- works well in most internal combustion engines
Waste biomass -
- waste plant-derived material
- conversion of waste biomass to fuel alcohol would not only get rid of large amounts of waste but would also reduce gasoline consumption
Benefits and limits of yeast and Zymomonas in performing fermentation
- they make only alcohol during fermentation, whereas most microorganisms generate mixtures of fermentation products
- Zymomonas lives entirely on glucose and lacks the enzymes to break down other sugars. Yeast is almost as narrow in its growth requirements.
- Zymomonas grows faster than yeast and makes alcohol faster as well.
- Yeasts are more alcohol resistant and are therefore capable of accumulating higher concentrations of ethanol in the medium before growth is halted
Xylose -
- Five-carbon sugar that is a major component of various hemicellulose polysaccharides found in plant cell walls
Why is xylose an important molecule to use for fermentation?
Vast amounts of waste material from plants are available for possible biodegradation. Breakdown of the polysaccharide polymers would release large amounts of xylose.
How was Zymomonas developed to ferment xylose instead of glucose?
This has been done in two stages.
- First, the genes for metabolism of xylose itself must be introduced, because Zymomonas does not naturally use this sugar
- The xylA and xylB genes encode the enzymes xylose isomerase and xylulose kinase, respectively, which convert xylose to xylulose and then to xylulose 5-phosphate.
- The two genes are carried on shuttle plasmids and transformed into bacteria such as Zymomonas
Tkt -
Enzyme that converts pentose phosphates back into hexose phosphates
Tal -
Enzyme that converts pentose phosphates back into hexose phosphates
How are bacteria/yeasts engineered to start with a 5 carbon sugar and ultimately use a 6 carbon pathway ending in fermentation?
- Transketolase (tktA) converts two five-carbon (C5) sugar molecules into one three-carbon (C3) and one seven-carbon sugar (C7)
- Next, transaldolase (tal) convert these products into a four-carbon sugar and fructose 6-P, a six-carbon sugar
- Fructose 6-P is degraded by glycolysis into ethanol
- The four-carbon sugar (C4) and another pentose 5-P (C5) are converted by transketolase into a second six-carbon sugar (C6) and a three-carbon sugar (C3), which both feed into the glycolytic pathway to make ethanol
Starch -
- a storage polysaccharide found in many plants
- consists of long chains of glucose residues with other glucose chains branching off the main backbone
- the main chain has glucose residues linked by alpha-1,4 linkages, and the side chain starts with an alpha-1,6 linkage
Starch consists of a mixture of ___ and ___.
Amylose and amylopectin
What is used in the food and brewing industry and is mostly converted to glucose by using the purified enzymes alpha-amylase and glucoamylase, rather than microorganisms?
Starch
Amylose -
- linear polymers
- chain lengths vary from 100 to 500,000 glucose residues
Amylopectin -
- branched polymers
- branches are due to alpha-1,6 bonds and they occur every 20 glucose residues along the polymer chain
- chain lengths vary up to 40 million glucose residues
Explain why or how the listed improvement may be made to starch degradation by genetic engineering.
More enzyme production
- recombinant organisms could be made that produce more enzyme
- would improve the breakdown of starch to sugars
Explain why or how the listed improvement may be made to starch degradation by genetic engineering.
More thermally stable enzymes
- the enzymes themselves could be engineered for better thermal stability or higher rates of reaction
- would improve the breakdown of starch to sugars
Explain why or how the listed improvement may be made to starch degradation by genetic engineering.
Glucoamylase into a yeast strain under a yeast promoter
It may be possible to engineer yeast strains that also express high enough levels of alpha-amylase to completely convert raw starch to ethanol
Make up of plant cell walls ?
The major components are cellulose, hemicellulose, and lignin.
How is glucose linked in cellulose?
Cellulose is a structural polymer of glucose residues joined by beta-1,4 linkage.
How is glucose linked in starch?
Starch and glycogen are storage materials also consisting solely of glucose, but with an alpha-1,4 linkage.
Could paper be converted to glucose?
Yes, because paper consists almost entirely of cellulose, this might potentially be converted to glucose.
Why would converting paper to glucose be a good idea and how would it be accomplished?
- because paper accounts for the largest fraction of the trash of industrial nations
- it would be accomplished by using cellulose-degrading microorganisms
What is the major challenge of converting paper to glucose?
The challenge is to break down cellulose, yielding glucose that can be turned into alcohol or other products
What can’t humans eat paper?
Most paper is treated with harmful chemicals
Endoglucanase -
Snips open the polymer chains in the middle
Cellobiohydrolase -
Cuts off molecules with 10 or more glucose units from the free ends
Exoglucanase -
Chops off units of two or three which are called cellobiose and cellotriose, respectively
Beta-glucosidase -
- also known as cellobiase
- converts cellobiose and cellotriose to glucose
What are some items to consider related to cellulose degradation?
- because cellulose is too big to enter the cell, the first three enzymes must be secreted and work outside
- such multistage procedures are inefficient
- cellobiose acts as a feedback inhibitor of cellulose degradation
- therefore, the end products of cellulose breakdown must be rapidly removed to allow continuous degradation of the starting materials
Ice nucleation factors -
- “seed” that helps in the formation of ice
- specialized proteins in bacteria
Role of pseudomonas syringae in ice formation
The seeding of ice crystals on and within plants is mostly due to proteins on the surface of bacteria, especially pseudomonas syringae and related species, which live on plants.
How does ice harm plants?
The ice crystals that form damage the plant tissues and disrupt the vessels that carry water and nutrients throughout the plant.
Role of inaZ in ice formation, and how mutations to this gene can prevent ice formation (supercooling)?
- cell surface proteins of P. syringae provide a nucleation point for ice
- the inaZ gene encodes an ice nucleating protein
- under freezing temperatures, wild-type P. syringae allow ice crystals to form, disrupting any plant tissues the bacteria are on or within
- if the inaZ gene is disrupted, the P. syringae mutant will not nuclear any ice crystals, allowing the water to super cool
How could inaZ be used in biotech applications? How could inaZ be used to display a protein on a surface of a cell, and what might be one benefit/application of this?
- one application is in the degradation of lignocellulose. Enzymes from cellulose-degrading bacteria have been displayed externally on both E. coli and Zymobacter (an ethanol producer) and shown to function correctly.
- another, quite different use is to bind virus particles in water or other fluids. This can be used both for detecting virus contamination and removing the viruses.
Antibiotics are produced through a biosynthetic pathway, why does this make their biosynthesis in the lab difficult?
- 20 or more steps
- each step has a different enzyme, encoded by its own gene
- many of these pathways are branched and/or interact with other metabolic pathways
Why is antibiotic development important?
- the emergence of antibiotic-resistant bacteria, coupled with the scarcity or newly developed antibiotics, compromises a looming crisis in health care
- there is a shortage of new classes of antibiotics
Briefly describe the point of figure 13.14: biosynthesis of beta-lactam antibiotics
- when certain molds grow on agar originally covered with bacteria, a clear zone appears around the mold where no bacteria grew
- the clearing is due to release of antibiotics such as penicillin and cephalosporin C from the mold
PHA -
- group of related plastics (long chains of repeating subunits)
- polyhydroxyalkanoates (PHA) have repeated hydroxyacid subunits linked through their carboxyl and hydroxyl groups
Why were PHA pathway genes expressed in the chloroplast?
The genes were inserted into the chloroplast genome so that the enzymes are expressed only in the chloroplast
What material do PHA pathway genes added to the chloroplasts use as their starting material to synthesize PHA?
The enzymes use the newly synthesized organic matter from photosynthesis to create PHA
Genetic circuit -
- combinations of genes, promoters, enhancers, and repressors that control the output or expression of the final gene product
- includes the input, either UV light or mitomycin C that cause DNA damage, a regulatory circuit of two repressor genes, and an output module containing the biofilm gene (traA) controlled by a promoter that responds to the lambda cl repressor
Biosensory system -
A genetic circuit that is controlled by a specific environmental cue
On/off motif -
- has a repressor protein that prevents the transcription and translation of the output gene
- when the input signal is received, the repressor is released from the promoter and the output gene is made
Repressor protein -
Regulatory protein that prevents a gene from being transcribed
Input signal -
- what you put in to get a particular output gene
- particular pollutant
Promoter -
Region of DNA in front of a gene that binds RNA polymerase and so promotes gene expression
Output gene -
- caused by the input signal
- fluoresce green
Feed forward motif -
- a master regulator gene controls a variety of different genes (labeled 1-5)
- the input signal activates the promoter on the master regulator, which in turn activates genes 1-5
Regulatory feedback motif -
- the final gene product represses the entire pathway
- two genes are linked in tandem with separate promoters
- promoter 2 is expressed continually to make repressor protein
- this prevents the expression of promoter 1 unless an input signal is received
- then the repressor is released, and the output gene is expressed
How could you design a system to express GFP in the presence of a pollutant? Which type of genetic circuit would you use and why?
- want a particular pollutant (the input signal) to cause a cell to fluoresce green (the output)
- this can be engineered by making the presence of the pollutant turn on the gene for GFP
- we can use on/off motif
- the pollutant may bind to the repressor protein that controls the GFP gene
- if the pollutant is present, the repressor is released from the GFP gene and the cells fluoresce
Briefly describe the E. coli circuit described in figure 13.20. Use the following terms to guide you: input, regulatory circuit, output module.
Biofilm produced by E. coli in response to DNA damage
The genetic circuit includes the input, either UV light or mitomycin C that cause DNA damage, a regulatory circuit of two repressor genes, and an output module containing the biofilm gene controlled by a promoter that responds to the lambda cl repressor.
What is the role of a repressor protein in transcription? What depressors are used in figure 13.20?
Biofilm produced by E. coli in response to DNA damage
- prevents a gene from being transcribed
- cl repressor
What is XNA?
- X is the sugar/ring
- the pentose sugar of the nucleic acid may be replaced with several possible alternative ring structures
Can XNA bind to RNA or DNA?
Yes
Can XNA be used to transmit genetic information?
- it has proven possible to use some XNA polymers to carry and transmit genetic information
- this requires engineered DNA polymerases that are capable of using the XNAs as substrates
Is it possible to generate a self-replicating XNA system?
- replication of XNA involves conversion to the complementary strand of DNA, amplification of the DNA, and conversion back to XNA
- eventually, it may prove possible to create a self-replicating system based on XNA
Describe an application you might invent to use XNA.
Recreate DNA that is affected by cancer cells by using XNA.
What is quorum sensing?
Regulatory system used by bacteria to coordinate their response to population density
How does quorum sensing work in P. aeruginosa?
Allows them to cooperate for group activities such as biofilm formation
In figure 13.25, how does E. coli “detect” P. aeruginosa? What proteins are involved?
Pathogen sensing and killing system
Carries genes allowing both detection and killing of target strains of Pseudomonas
In figure 13.25: the E. coli cell accumulated pyocin, and lyses to release it. Why is this a good strategy, and what is the role of pyocin and E7?
After the E7 proteins attain the threshold concentration that causes the E. coli to lyse, the accumulated pyocin S5 is released into the environment to kill the P. aeruginosa.
