Biol 443

Define respiration.

*Catabolic* reactions producing ATP, in which organic or inorganic compounds are primary electron donors, and organic or inorganic compounds are ultimate electron acceptors.

What might be the roles of an R&D microbiologist in fermentation?

- selection of suitable organism - selection of suitable media - determination of environmental conditions (i.e. temperature, aeration) to allow maximum growth of the organism - improvement of performance by genetic manipulation or media reconstitution

What are some properties of an environment that could affect microbial growth rates?

- temperature, water activity, pH, redox environment, available nutrients

What could be the 'products' of modern day biotechnology?

- the cells themselves - products made by the cells - the cells (& their enzymes) may be used to biotransform a specific substance to the desried product (i.e. they are used as the reaction catalysts).

What does white biotechnology consist of?

- Designing an organism to produce a useful chemical. - enzymes as industrial catalysts. * A white biotech process tends to be less resource-intensive than a traditional process for an industrial product*.

Bacillus is considered a traditional "workhorse" in fermentation. What specific features make it useful?

- Gram-positive bacterium - makes extracellular enzymes --> these would be released into the supernatant - makes Antibiotics and insecticides (Bacillus thuringensins; Bt toxin)

What are some differences in prok. and euk. regarding transcription and translation?

- In prokaryotes, there is no temporal (time and space) separation - polycistronic mRNAs and operons in prok. - mRNA in prokaryotes has a short half life to allow them to adjust to environment quickly - introns in euk. and mRNA processing - mRNA half life of eukaryotes varies

What are the major players in industrial biotechnology?

- bacteria, yeasts, filamentous fungi (major players) - animal cell cultures (used for monoclonal Ab production) - plant cell cultures (plant flavonoids, alkaloids for use in foods, cheical production)

E. coli is considered a "workhorse" in fermentation. What specific features make it useful?

- because it can be used for many purposes - recombinant proteins - sorting and shuttling genes

What are secondary metabolites NOT used for/not essential for?

- biosynthesis - reproduction - energy production/metabolism - survival (at least under lab conditions)

What features do we want the organisms used in fermentation/biotechnology to have?

- cheap to grow - easy to grow - produce product of interest in a commercially viable amount

Describe how the glycolytic pathway was manipulated to yield more glycerol in WW1.

- knew glucose could be converted to ethanol and CO2 with a little bit of glycerol production on the side - in the pathway (conducted in Saccharomyces cerevisiae) glucose is eventually converted into 2 Acetaldehyde molecules, which are then converted to alcohol and CO2 (also converting NADH to NAD+ during this step) - the addition of bisulphite blocks this conversion of acetaldehyde to alchohol, resulting in a build -up of NADH in the cell - cell tries to fix the redox balance by rerouting the carbon flow to glycerol production, where NAD+ is also regenerated - a large amount of glycerol is produced and 2 ATP

Describe the general process of how semi-synthetic penicillins could be made.

- mass culture P. chrysogenum to produce PenG - mass culture suitable bacteria to produce pencillin acylase --> recover and purify enzyme - at Ph 7.5-8.5, convert PenG to 6-APA - run reverse reaction in acidic conditions with a suitable acyl donor to produce semi-synthetic penicillins

What are some examples of process control that occurs during fermentation?

- monitor for absence/presence of contaminants - quality control to ensure uniformity - ensure proper custody of cultures (culture maintenance) - improvement of process performance

Describe how an inducible enzyme system works.

- you have a regulatory gene that codes for a repressor protein (can be 1000s bp upstream) - under normal conditions, the repressor is bound to the operator, which interferes with the binding of the polymerase to the promoter --> transciption of downstream genes does not occur. - in the presence of the inducer, the inducer will bind to the repressor protein, changing its shape so that it can no longer bind to the operator--> transcription of downstream genes occurs.

What can be said about the relationship between organism complexity and their doubling time?

As complexity increases, so does doubling time (generally).

What are the traditional "workhorses" of fermentation?

Bacillus, Aspergillus, Saccharomyces sp.

Why is it important that filamentous fungi grow from tips?

Because tips are the metabolically active part and therefore, you may want to keep the tips short to maintain productivity (while conserving SA).

Why is biotechnology useful and describe how it has changed since the term/idea was first introduced.

Biotechnology could provide solutions to societal crises like food & energy shortages. In the beginning, fermentation did not involve the molecular manipulation of organisms at all; was simply using microorganisms to perform processes (i.e. beverage modification). Now it often involves molecular techniques to genetically pioneer new microbes for a specific use.

Since when has wine-making been practiced?

10 000 BC

Define batch culture.

A closed system (that may be aerated for aerobic organisms) which is provided with a limited amount of medium.

What is penicillin acylase?

An enzyme found in bacteria that is able to cleave Penicillin G (the natural form) to convert it into 6-APA and a benzyl ester, thus inactivating it (note, it may inactivate it by cleaving the square ring, but not sure). Note: this reaction occurs at an alkaline pH.

Are repressive enzyme systems typically found in anabolic or catabolic systems?

Anabolic (i.e. biosynthetic pathways)

What are two ways that proteins can be secreted? What is an advantage of this?

Co-translationally or post-translationally. Note: the protein usually folds *after* secretion. An advantage of this is that it may simplify product recovery, if the product you want is secreted into the media.

What is an example of post-translational control?

Covalent modifications that alter the protein conformation (i.e. addition of AMP, ADP, phosphorylation, methylation, glycosylation). Note, if the protein is an enzyme, these types of covalent modifications will modulate its activity (can activate or inhibit)

Define fermentation (basic definition).

Catabolic reactions producing ATP, in which *organic* compounds serve as both the primary electron donor & the ultimate electron acceptor. ATP is produced by substrate-level phosphorylation.

Are indicible enzyme systems typically used in anabolism or catabolism?

Catabolism.

What did most scientists believe was responsible for fermentation around Pasteur's time?

Chemical processes (rather than biological ones).

Give an example of a thermophile.

Geobacillus stearothermophilus

Why are the structural difference between Gram-positive and Gram-negative bacteria significant?

In Gram-negative bacteria, molecules can get trapped in the periplasmic space. Therefore, an enzyme that is extracellular in a Gram-positives may be periplasmic in Gram-negatives.

How can the production of penicillin acylase by bacteria be manipulated to form semi-synthetic penicillin?

In alkaine conditions, this enzyme cleaves natural penicillin to produce 6-APA and a benzyl ester. In acidic conditions, the reaction is reversed. Therefore, this enzyme could be added to a flask containing 6-APA and a suitable acyl donor (does not have to be the benzyl ester) to produce semi-synthetic penicillin. This allows you to change the functional group while maintaining the square ring found on 6-APA (that makes the antibiotic functional).

Why is it important to understand how organisms regulate their metabolisms?

In fermentation, the flux of a substrate through the various anabolic and catabolic pathways is influenced by a variety of regulatory mechanisms. We need to understand this to optimize desired product formation (i.e. need to know how it wokrs before being able to manipulate it). i.e. if your desired product is halfway down the metabolic pathway, you'd want to be able to stop the pathway at that particular point.

What is a difference in prok. and euk. regarding control of metabolism?

In prok. this is *mostly* at the level of transcription. In euk. we see controls at levels of transcription, translation and mRNA processing.

True or false: Yeast is able to grow on and metabolize lactose.

False: it can grow on, but not metabolize it.

What are some differences between fermentation and respiration with respect to the reaction itself?

Fermentation uses only organic compounds as the electron donor and acceptor; there is no electron transport chain so less ATP is produced.

What was Koch's contribution to fermentation? (1881)

Development of pure cultures.

What are isoenzymes?

Different enzymes that catalyze the same reaction.

Give an example of a mesophile.

E. coli

What is the significance of phages that are able to infect Lactococcus lactis?

Lactococcus lactis is important in dairy fermentation so these phages cause lots of problems for the dairy industry.

What are the four stages of bacterial growth?

Lag phase, log phase, deceleration phase, stationary phase. (growth plotted against time).

In Eukaryotes where are proteins made? Where do covalent modifications occur?

Made by ribosomes on the rough ER, modified in the Golgi.

What is the purpose of secondary metabolites to humans?

Many are of commercial interest (i.e. antibiotics).

Are the repressive and inducible systems described in here examples of positive or negative controls?

Negative controls because when the repressor is bound, you don't get transcription. In positive control, binding of allosteric proteins increases transcription.

Do archaea play an important role in biotechnology and fermentation?

No, so far

What was Pasteur's contribution to fermentation? (1856-7)

Observed that yeast cells covert sugar to ethanol and CO2 in the presence of air.

Other than glycerol, what is another example of an early attempt at biological engineering?

Penicillin production - was first produced by Penicillium notatum in very small amounts - exposed the WT strain to various mutagens to try and manipulate it - eventually developed commercial strain, Penicillium chrysogenum - increased production from 120 inernational units (IU) to 2580 IU

What commercial strain of mold is used to make penicillin? What was the original WT strain?

Penicillium chrysogenum; Penicillium notatum.

What do antibacterial substances target in bacteria?

Peptidoglycan, since this is unique to bacteria.

Give an example of a psychrophile.

Polaromonas vacuolata (typcally below 15 degrees C)

What was Buchner's contribution to fermentation? (1897)

Prepared cell-free extracts that were capable of sugar fermentation; this showed that it was a number of different steps.

What is the purpose of secondary metabolites *to the microorganism*?

Presumably confer some advantage in the natural environment. Note that their purpose varies and some are unknown.

What are the precursors of secondary metabolites?

Primary metabolites or derivatives of primary metabolites (i.e. organic acids, amino acids, sugar derivatives, purines, pyrimidines etc.)

Describe how the cumulative pathway is able to exert post-translational control.

In this pathway, one enzyme catalyzes the conversion of A --> B. This enzyme has three regulatory sites and the binding effects are cumulative. So if one inhibitor (from downstream of the pathway) binds the pathway is slowed down a little. Binding of another shuts it down a little more etc.

Describe how the concerted pathway is able to exert post-translational control.

In this pathway, you have one enzyme that catalyzes the conversion of A --> B. This enzyme has three regulatory sites and all three have to be occupied to inhibit it.

What influences microbial growth rate?

Inherent physiological properties and the environment.

What was necessary to allow mass production of penicillin?

Introduction of submerged culture, as penicillin grows in liquid far more efficiently (compared to on dry agar plates exposed to air).

What is the basic process of endocytosis?

Invagination of the plasma membrane --> fusion with a lysozome --> enzymes (from lysosome) degrade contents that were in the vesicle.

Name the three pathways of post-translational control.

Isoenzymes, concerted, cumulative.

Are inducible and repressive enzyme systems examples of transcriptional or translational control?

Transcriptional.

What is active transport?

Transport of molecules across the plasma membrane *against their concentration gradient* that requires specific membrane proteins and ATP.

What is facilitated diffusion?

Transport of molecules across the plasma membrane that requires specific membrane proteins and a concentration gradient (no ATP).

Who first discovered the concept of 'enzymes'?

Traube (1887) --> theorized that fermentation was conducted by protein-like substances that were unchanged at the end of the reaction.

True or false? When t = td (doubling time), X = 2*Xo Where X is the cell concentration and Xo is the initial cell concentration.

True.

True or false: secondary metabolites tend to be produced as families of related compounds (i.e. set of very similar antibiotic molecules).

True. Note that not all of these related compounds may be useful to us, so you must also consider how to manipulate conditions to promote production of the useful product.

True or false: there is still cell division occurring during stationary phase.

True: although the total cell number is not changing, cell division is still occurring. Cell division = cell death, so no net changes in cell number.

What can viruses be used for in the biotechnology industry?

Vaccine production.

Describe how an repressive enzyme system works.

When the compound-of-interest is present in the media: - the compound acts as a co-repressor. It binds to the apo-repressor (produced by the regulatory gene) and the combination of these two makes a functional repressor protein that binds to the operator, preventing transcription (which we don't want, since the compound is present in the media) - when the corepressor from the media is all used up, there is nothing bound to the apo-repressor, so the repressor is not made and nothing binds to operator --> transciption occurs.

What is post-translational control?

When the protein/enzyme has been made and the question becomes whether or not it is active.

What does the Arrhenius equation: µ = Ae^(-EaRT) give?

The growth rate based on temperature.

Taking into account the bacterial growth curve, what equation gives the cell concentration at a specific time? What equation gives the doubling time?

dx = μx - αx dt where x is the cell concentration, μ is the specific growth rate and α is the rate of lysis or endogenous metabolism. td = ln2 μ

What 'colour' of biotechnology is fermentation biotechnology?

White.

Describe how post-translational control via isoenzymes works.

You have several isoenzymes that catalyze the conversion of A --> B. Each of these are inhibited by a different product downstream of the pathway. If there is too much of this product, it will bind to its respective isoenzyme and inhibit it, reducing the amount of A --> B conversion.

Which scientist began the study of fermentation?

van Leeuwenhoek --> observed yeast cells (1680).

If a cell has a lot of L-glutamine, what will happen to the activity of Glutamine synthetase?

Its activity will go down --> AMPs will be added to inhibit the enzyme so that it does not produce L-glutamine.

What are some inherent physiological properties of a microorganism that would affect its growth?

Its complexity, its temperature growth range.

Describe the general secretory pathway.

1. The protein is synthesized with a signal peptide. 2. SecB chaperone proteins prevent the newly synthesized protein form folding as it makes its way to the membrane. 3. Other Sec proteins make a channel in the membrane. 4. Protein is secreted, the signal peptide is cleaved and the protein is able to fold properly.

How do yeast typically grow and divide?

By budding (asexual).

Where would you harvest organisms to get the maximum amount of a secondary metabolite?

Near end of stationary phase.

Knowing that ethanol is a primary metabolite, where would you harvest cells to get maximum ethanol production?

Near the end of exponential phase.

In respiration, is the electron donor oxidized or reduced?

Oxidized --> electron acceptor is reduced.

What implications come from the fact that animal cells don't have cell walls?

They are subject to osmotic stress and therefore, more finicky to grow (require isotonic media).

How do gasses get across plasma membranes?

They diffuse across.

Are yeast unicellular or multicellular?

Yeast are unicellular fungi.

Do Saccharomyces cerevisiae have GRAS status?

Yes

What is secondary metabolism?

"optional pathways" not required for growth and reproduction.

Describe the different colours of biotechnology (There are 4).

*green biotechnology*: applied to agricultural processes. *red biotechnology*: applied to medicine. *blue biotechnology*: applied to marine and aquatic systems (rarely used). *white biotechnology*: applied to industrial processes.

Saccharomyces is considered a traditional "workhorse" in fermentation. What specific features make it useful?

- eukaryotic organism (mold; used to make eukaryotic proteins that differ in their 'decorating i.e. methylation from prokaryotic proteins) - bioethanol - brewer's, baker's, distiller's yeast - potential for other fine and bulk chemicals

Aspergillus is considered a traditional "workhorse" in fermentation. What specific features make it useful?

- eukaryotic organism (mold; used to make eukaryotic proteins that differ in their 'decorating i.e. methylation from prokaryotic proteins) - citric acid - exoenzymes used in food industry biotransformations

What types of covalent modifications does Glutamine synthetase undergo?

Adenylation. This is 12-o-mer enzyme that can covalently bind to 12 AMPs. The more AMPs bound the less active it is. Note: this is a reversible process.

Why is it important that bacteria are haploid?

Any mutants will be expressed, so easier to study mutations. Note: bacteria are asexual reproduction only.

What is the definition of fermentation for a food microbiologist?

Any process involving the mass culture of microorganisms, either aerobic or anaerobic. The use of microorganisms to carry out enzyme-catalyzed transformations of organic matter.

What two components were considered major parts of a diet in Ancient Egypt (~4000 BC)? What does this show?

Bread and wine; shows how important fermentation is and how long it has been around.

What is an example of a very early attempt at biological engineering?

Glycerol production in WWI

What type of covalent modification is most often seen in mammalian proteins?

Glycosylation; Pichia yeast is able to glycosylate proteins in a mammalian-like manner.

What are most bacteria in fermentation Gram-positive or Gram-negative?

Gram-positive Actinobacteria or Proteobacteria

What is the difference between Gram-positive and Gram-negative bacteria?

Gram-positive bacteria have a thick peptidoglycan layer that acts like a weave so molecules can easily pass through. Gram-negative bacteria have a periplasmic space prior to a thin peptidoglycan layer, and an outer membrane. The outer membrane prevents things from being excreted.

Define primary metabolite and give an example.

One that is formed during the growth phase of the microorganism. i.e. ethanol produced by Saccharomyces cerevisiae during fermentation. *be able to draw how this growth curve looks in comparison to metabolite production curve (they are essentially the same thing, overlapped with one another)

Define secondary metabolite and give and example.

One that is formed near the end of the growth phase, near/in stationary phase. i.e. penicillin production by Penicillium chrysogenum *be able to draw how this growth curve looks in comparison to metabolite production curve (metabolite curve rises steeply near the end of the exponential phase and rises past the growth curve in stationary phase).

What is an example of an inducible enzyme system.

The lac operon; lactose is the inducer and the operon codes for protein products that will break down lactose.

Give an example of a hyperthermophile.

Thermococcus celer Pyrolobus fumarii