Industrial Microbiology

Question 1

What is the definition of an antibiotic?

Answer 1

Compounds of natural, semi-synthetic, or synthetic origin which inhibit growth of microorganisms without significant toxicity to the host

Question 2

What do all penicillins have as part of their chemical structure?

Answer 2

Beta Lactam Ring (4 membered ring/square)

Question 3

Why do antibiotics exhibit selectivity?

Answer 3

Due to the separation (by many years) between eukaryotic and bacterial life forms = different cell organization, biochemical pathways, and protein/RNA structures…

Question 4

What needs to be true about the target of an antibiotic in order to ensure selectivity? [2 - one or other]

Answer 4

Target can be present only in bacteria but not eukaryotic host (eg: B-lactams vs. cell wall)

OR

Target in bacteria is different from the homologous target in host (bacterial vs eurkaryotic ribosome)

Question 5

What are antibiotics used for naturally?

Why are many antibiotics omnipotent and inhibit growth of a wide range of microorganisms?

Answer 5

Defending vs other microorganisms

Microorganisms share common evolutionary origins and thus share biochemical targets

Question 6

What are some of the possible targets of antibiotics?

Answer 6

+ folate biosynthesis (sulfonamides)

Question 7

Is the bacterial cell wall essential for survival? What’s its purpose?

What makes it a good target for antibiotics?

Answer 7

Yes - it protects bacteria from changes in osmotic pressure

It is biochemically and structurally different from cell envelope of eukaryotes - as it is unique to bacteria, it makes for a great target

Question 8

What is the bacterial cell wall made up from?

What are the major components of this material?

Answer 8

Cross-linked peptidoglycan

Alternating N-acetylgucosamine and N-acetylmuramic acid (sugars) cross-linked by 3-5 amino acids between N-acetylmuramic acids.

Question 9

What is the name of the enzyme responsible for cross-linking peptidoglycan strands? When does it undertake this process? What does it lead to?

What type of antibiotic targets this enzyme? What does it do? What type of bond does it form with the enzyme? What is the result?

Answer 9

Transpeptidase (active during normal growth, leads to cell wall strength)

B-lactam antibiotics (penicillins) - it inhbits the enzyme (and forms a covalent bond - “suicide substrate” and irreversible), preventing cross-linking, and leads to cell wall weakness (and will rupture, killing the cell, under osmotic pressure).

Question 10

Why do B-lactam antibiotics have the effect they have (weakening cell wall and leading to rupture)?

Answer 10

The B-lactam ring of the antibiotic is similar to the peptide bond connecting two D-alanine resides of the peptidoglycan precursor. Transpeptidase (cross-linking enzyme) recognizes the antibiotic as its substrate, and forms a covalent bond with it, thus irreversibly inactivating the enzyme and failing to create cross-links

Question 11

Who discovered antibiotics?

Who developed methods for growing and purifying penicillin (making it into a drug)?

What did Mary Hunt discover that made commercial production of penicillin possible? What was the original issue?

Answer 11

Alexander Fleming

Howard Florey and Ernst Chain

P. chrysogenum (penicillin notatum didn’t produce enough to make it viable)

Question 12

Bacterial-produced antibiotics…

Which one produces over two thirds of clinically useful antibiotics of natural origin? Examples?

Clavulanic acid is produced by S. clavuligerus. What is it used in combo with? Why?

Answer 12

Streptomyces (neomycin, cypermycin, grisemycin, bottromycins, chloramphenicol)

Used w/beta lactams to block/weaken bacterial resistance mechanisms caused by beta-lactamase enzyme

Question 13

Most antibiotics are termed _________ metabolites. What does this mean?

Considering this, what are the types of culture used to produce them?

Answer 13

Secondary - produced in large quanities only towards the end of batch growth (end of exponential growth phase)

Fed batch or chemostat (continuous nutrition)

Question 14

B-lactam group (four atom cyclic amide) is the _____________ of all B-lactam antibiotics. Give the names of some of the B-lactam antibiotics. [4]

Answer 14

Pharmacophore. Penicillins, carbapenems, cephalosporins, monobactams

Question 15

What can cause a sensitive bacteria to become resistant to an antibiotic? [2]

What are some of the ways that this can happen?

Answer 15

Spontaneous mutation or acquisition of a gene (horizontal transfer)

[see picture]

Question 16

What is the major cause of resistance to penicillins? What does it do?

What drug can help deal with this when used alongside penicillin?

Answer 16

Acquisition of beta-lactamase gene - it cleaves the beta-lactam ring.

Clavulanic acid

Question 17

What are the three basic steps of using genetics to create a drug from a microorganism

Answer 17

1. Find appropriate microorganism
2. Transform gene -> enzyme expressed, metabolite synthesized
3. Utilize whatever you have created as a therapy. Muahaha.

Question 18

How is human insulin created nowadays?

What other advantage does this have over natural insulin?

Answer 18

In yeast or E Coli. Genes to create human insulin were inserted into these microbes - polypeptide chains A and B manufactured separately, and then disulfide bonded afterwards

Recombinant insulin can be modified (eg: fast/slow acting)

[NB: possibly interesting - disulfide bonds need oxidising conditions… in cell cytoplasm it is a reducing environment, due to glutathione (sp?), which is why most proteins with disulfide bonds are extracellular]

Question 19

What is the effect of Hep B?

How was the hep B vaccine created?

Answer 19

Affects liver. Sickness w/vomiting, jaundice, tiredness, cirrhosis, liver cancer

Took gene for protein shell of virus, put it into baker’s yeast via a plasmid… the yeast will then express these proteins (antigens), which can be harvested and used to create the vaccine

Question 20

Artemisinin & Malaria

What is synthetic/systems biology? Why was this difficult in the above case?

Answer 20

Trangenically creating not just one gene but a whole pathway (there were many steps in the sequence, and they not easy to locate)

Question 21

Ethanol is produced by _____ (scientific name: ___________ __________). It takes _______ and converts it into what?

Answer 21

yeast, Saccharomyces cerevisiae, glucose

2 molecules of carbon dioxide, and 2 molecules of ethanol

Question 22

Glucose is _______ during glycolysis (when NADH is produced). During ____________, pyruvate is converted to ___________ by the enzyme ________ _____________, which is then reduced to _______ by the enzyme _______ ____________.

Answer 22

oxidised, fermentation, acetaldehyde, pyruvate decarboxylase, ethanol, alcohol dehydrogenase

Question 23

What enzymes are overexpressed in yeast?

Why might they produce ethanol (evolutionarily)?

Answer 23

Pyruvate decarboxylase, alcohol dehydrogenase

To kill other microbial life forms (and they have enzymes that convert ethanol and allow it to re-enter the TCA cycle, and eventually produce energy)

Question 24

What foodstuffs can be converted into ethanol?

Why is this not actually environmentally friendly (at least when it comes to production of fuel)?

Answer 24

Sugar rich crops (sugar cane, sugar beet, potato, cassava, etc)

It is only a partially renewable resource. Sunlight might fix the energy into sugars, but ethanol produced is only 10%, and it needs energy input to distill it into a form that can be used for fuel.

Question 25

What does auxotrophic mean?

Which vitamin are only bacteria capable of making?

What do you get if you don’t have the above vitamin?

So how do we get it?

Answer 25

No natural synthesis of necessary organic compound (eg: vitamin)

B12

Pernicious anaemia (not enough red blood cells produced)

By eating animals that have eaten the bacteria - their tissues are full of stuff (eg: cows, sheep, fish, etc)

Question 26

What is vitamin B12 needed for in humans? [2]

Answer 26

Metabolism of propionyl-CoA (derived from degradation of amino acids) and conversion of homocysteine to methionine by methionine synthase

Question 27

What can cause malabsorption of vitamin B12?

What are the symptoms?

Answer 27

Leaky/inflamed gut, atrophic gastritis/hypochlorhydria (low stomach acid), pernicious anemia (autoimmune), medications (especially proton pump inhibitors and other acid-suppressing drugs), and alcohol

Extreme tiredness/fatigue, lack of energy, shortness of breath, faintness, headache, tinnitus, lack of appetite, and those ones more specific to Vit B12 deficiency (yellowing skin, loss of sensation, reduction in pain sensation, walking problems, vision problems, mood changes, symptoms of dementia)

Question 28

A _____-____ _____-_______ pathway is used in bacteria to produce B12. What co-factor is needed?

Which amino acid is used as a precursor? What else is needed? Where are these produced?

Which bacterial species are used in industrial B12 fermentation? How have they been improved?

Answer 28

multi-gene, multi-enzyme, cobalt co-factor

Glycine and succinyl-CoA (from the TCA cycle)

Pseudomonas and nocardia species (with yields improved by random UV mutagenesis and by genetic engineering)

Question 29

How many amino acids are “essential”? What does this mean? Can you remember what they are?

Which one (if added to cow/chicken feed) makes them grow big? What aminos are normal feed (soya) protein lacking?

What is also used in poultry feed?

Answer 29

Needed in the diet (10): Leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, arginine

Lysine (lysine, methionine, threonine, tryptophan)

DL-Methionine (produced by chemical synthesis)

Question 30

Monosodium glutamate is a type of modified _____ ____ (_______ ____). What effect does it have on flavour?

What bacteria is used in its production? Give some info about it.

How much of the above (bracketed) precursor does it make naturally? After genetic engineering?

Answer 30

Amino acid (Glutamic acid) - intensifies meaty/savoury flavour

Corynebacteria glutamicum (gram positive, facultatively anaerobic, heterotrophic, irregular rods in V formation, non-pathogenic, lives in soil)

10g/L -> 100g/L

Question 31

MSG precursor is glutamic acid. What is needed to make it? Which TCA cycle intermediate is needed to make that?

Answer 31

Glutamate (Alpha-Ketoglutarate)

Question 32

Which microorganism is used to create acetone? What else can be made at the same time? And so what is the process called?

Who created the process? Why was it important in WW1? What was used to make it because maize couldn’t be provided?

Answer 32

Clostridium acetobutylicum (ABE Process: Acetone Butanol Ethanol Fermentation Process)

Chaim Weizmann (acetone used to make cordite for shells) - used horse chesnuts for a while..

Question 33

Single Cell Protein: what is the famous example?

How is it grown?

Disadvantages?

[Image: waste products that can be used to grow SCPs on]

Answer 33

Quorn

Give microorganism carbon, nitrogen and sulfur source. Then let it grow, and eventually harvest it.

SCP has lower protein:nucleic acid ratio than meat. If consumed, this produces more uric acid -> gout and kidney stones

Question 34

Single Cell Protein: What is quorn made from?

How is it grown?

Answer 34

Fusarium venenatum (filamentous funghi)

Through continuous culture process - glucose and ammonium sulphate used as medium, plus vitamins and minerals

Question 35

NB: Revise pic…

Answer 35