L2/3

Question 1

What are 4 benefits of altering chemical structure?

Answer 1

Improved bioactivity
Counter resistance
allow easier administration
reduce S/E
IP considerations

Question 2

How can you generate new antibiotics by manipulating m/o?

Answer 2

Alter chemical structures

Question 3

What are 4 ways of altering chemical structure?

Answer 3

semi-synthesis
precursor-directed biosynthesis
OSMAC
genetic manipulation

Question 4

What happens when you remove the aromatic group on penicillin G?

Answer 4

gives handle on structure to add different side chains -> semi-synthetic ring

Question 5

What do cultivation parameters influence?

Answer 5

Secondary metabolites
manipulate nutritional/environmental factors

Question 6

Whats an example of changing cultivation parameters?

Answer 6

Aspergilus ochraceus orginally knwon to produce aspinonine -> changed parameters -> produced 15 additional compounds

Question 7

What does changing the parameters do?

Answer 7

Activation of cryptic/orphan pathways (changes genes)

Question 8

What is precursor-directed biosynthesis?

Answer 8

Modified biosynthetic precursor added to antibiotic-producing strain -> modified antibiotic with modified bioactivity

Question 9

Whats an example of a modified antibiotic with modified bioactivity?

Answer 9

Rumbrin produced by fungus Auxarthron umbrinum

Question 10

What is a characteristic of Rumbrin?

Answer 10

Cytotoxic -> depends on halogen

Question 11

What was the starting point of Rumbrin?

Answer 11

pyrrole-2-carboxylic acid

Question 12

Define IC50?

Answer 12

50% of cell inhibition - wants to lower IC50

Question 13

What is mutasynthesis?

Answer 13

Generate mutant deficient in key step

Question 14

What does mutasynthesis result in?

Answer 14

reduces competition between natural & unnatural precursors

Question 15

What is salinispora tropica?

Answer 15

Marine bacterium that produces salinosporamide A

Question 16

What is the 1st step in the biosynthesis of salinosporamide A?

Answer 16

Chlorination

Question 17

What is the activity of salinosporamide A determined by?

Answer 17

C-13 substituent - non-chlorinated salinosporamide B not as active

Question 18

How was fluoro-salinosporamide made?

Answer 18

Mutasynthesis by knocking out SalL -> added 5-fluoro-5-deoxyadenosine

Question 19

How can molecular biology techniqies to modify enzyme-catalysed steps?

Answer 19

Remove genes/enzymes
add genes/enzymes
combinatorial biosynthesis

Question 20

What does metabolic engineering require?

Answer 20

Requires that the biosynthetic gene cluster be known -> expressed in heterologous host

Question 21

What’s an example of new aminocourmarin antibiotics?

Answer 21

heterlogus expresssion in S. coelicolor ->knocked out novO -> co express clo-hal

Question 22

What is the role of novO?

Answer 22

catalyses methylation

Question 23

What is the MoA of clo-hal?

Answer 23

codes for halogenation

Question 24

What are biobricks?

Answer 24

create novel biological systems with various ‘parts’ taken from other organisms

Question 25

What is the outcome of synthetic metabolic pathways?

Answer 25

Convert cheap substrates to high value products e.g syn-bio vanillin from E. coli

Question 26

What is artemisinin?

Answer 26

Anti-malarial drug from sweet wormwood (low productivity)

Question 27

How is semi-synthetic artemisinin made?

Answer 27

Engineer yeast to produce artemisinin acid

Question 28

What are 5 steps leading to artemisinic acid?

Answer 28

Genes for amorphadiene production genes for oxidation optimisation fermentation chemical synthesis

Question 29

Why was porcine insulin not the best?

Answer 29

One aa difference (B30 thr- ala) caused immunological problems

Question 30

Why was human insulin difficult to acquire 60 yrs ago?

Answer 30

Difficult/expensive - available in small amounts

Question 31

How was recombinant insulin created?

Answer 31

Synthetic oligonucleotides coded for A & B chain -> E. coli -> fusion proteins -> chemical treatment

Question 32

Whats another way to create recombinant insulin

Answer 32

Using mRNA where splicing has already occured

Question 33

What are 4 therapeutic products?

Answer 33

Blood products hormones immune modulators therapeutic enzymes

Question 34

What is biocatalysis?

Answer 34

Engineer bacteria to produce high levels of the useful enzyme eg. penicillin acylase

Question 35

What is an example of steroid biotransformation?

Answer 35

Hydrocortisone is produced chemoenzymatically

Question 36

How is hyrodcortisone produced?

Answer 36

Progesterone chemically synthesised by plant sterols Regiospecific hydroxylation achieved using fungus Rhizopus nigricans

Question 37

What is an example of chiral resolution?

Answer 37

Formation of S-2-chloropropionic acid from racemate

Question 38

What is racemate?

Answer 38

Valuanle synthon in manufacture of pharmaceuticals

Question 39

How is S-2-chloropropionic acid produced?

Answer 39

Pseudomonas sp. with stereoselective dehalogenase -> degrades R enantiomer

Question 40

What does the production of sitagliptin involve?

Answer 40

TransaminaseW

Question 41

What is sitagliptin?

Answer 41

anti-diabetic drug

Question 42

What are the types of biocatalyst?

Answer 42

Whole cell biocatalyst or purified enzyme

Question 43

What are considerations for which biocatalyst?

Answer 43

product recovery enzyme stability substrate/product toxicity co-factor requirement cost of enzyme purification through conversion of desired product

Question 44

What are 3 ways of strain improvement?

Answer 44

Industrial production culturing mutagenesis

Question 45

What are 2 levels of feedback inhibition/repression?

Answer 45

Inhibits enzyme activity inhibits gene expression

Question 46

What are 3 ways of mutagenesis?

Answer 46

random - exposure to mutagen site-directed mutagenesis - change any base pair in a specific gene gene disruption - use restriction sites to insert another DNA fragment

Question 47

What is a restriction site?

Answer 47

sequence at where a restriction enzyme will cut a piece of DNA

Question 48

What is the kanamycin cassette?

Answer 48

Contains a gene that will confer resistance to Kanamycin

Question 49

What does CRISPR-Cas stand for?

Answer 49

Clustered regularly interspaced short palindromic repeats

Question 50

What are CRISPR regions?

Answer 50

region of bacterial genome where memory of previous viral infection resides

Question 51

What does CRISPR allow?

Answer 51

Quickly destroy invading viral DNA if it has been 'immunised'

Question 52

What activity does Cas have?

Answer 52

Endonuclease activity & is responsible for adding new spacers

Question 53

What do guide RNAs sequences do?

Answer 53

Complementary to sequence of gene where the Cas9 will cut

Question 54

What is the PAM sequence?

Answer 54

NGG downstream of cut site & is recognised by Cas9

Question 55

What does non-homologous end joining do?

Answer 55

Insert base pairs causing frameshift of gene -> no longer functional

Question 56

What does homology directed repair allow?

Answer 56

New gene with homologous upstream/downstream sequences to be introduced