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Cell products > L2/3 > Flashcards

L2/3 Flashcards

1
Q

What are 4 benefits of altering chemical structure?

A

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

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2
Q

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

A

Alter chemical structures

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3
Q

What are 4 ways of altering chemical structure?

A

semi-synthesis
precursor-directed biosynthesis
OSMAC
genetic manipulation

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4
Q

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

A

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

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5
Q

What do cultivation parameters influence?

A

Secondary metabolites
manipulate nutritional/environmental factors

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6
Q

Whats an example of changing cultivation parameters?

A

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

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7
Q

What does changing the parameters do?

A

Activation of cryptic/orphan pathways (changes genes)

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8
Q

What is precursor-directed biosynthesis?

A

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

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9
Q

Whats an example of a modified antibiotic with modified bioactivity?

A

Rumbrin produced by fungus Auxarthron umbrinum

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10
Q

What is a characteristic of Rumbrin?

A

Cytotoxic -> depends on halogen

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11
Q

What was the starting point of Rumbrin?

A

pyrrole-2-carboxylic acid

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12
Q

Define IC50?

A

50% of cell inhibition - wants to lower IC50

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13
Q

What is mutasynthesis?

A

Generate mutant deficient in key step

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14
Q

What does mutasynthesis result in?

A

reduces competition between natural & unnatural precursors

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15
Q

What is salinispora tropica?

A

Marine bacterium that produces salinosporamide A

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16
Q

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

A

Chlorination

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17
Q

What is the activity of salinosporamide A determined by?

A

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

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18
Q

How was fluoro-salinosporamide made?

A

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

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19
Q

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

A

Remove genes/enzymes
add genes/enzymes
combinatorial biosynthesis

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20
Q

What does metabolic engineering require?

A

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

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21
Q

What’s an example of new aminocourmarin antibiotics?

A

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

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22
Q

What is the role of novO?

A

catalyses methylation

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23
Q

What is the MoA of clo-hal?

A

codes for halogenation

24
Q

What are biobricks?

A

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

25
Q

What is the outcome of synthetic metabolic pathways?

A

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

26
Q

What is artemisinin?

A

Anti-malarial drug from sweet wormwood (low productivity)

27
Q

How is semi-synthetic artemisinin made?

A

Engineer yeast to produce artemisinin acid

28
Q

What are 5 steps leading to artemisinic acid?

A

Genes for amorphadiene production
genes for oxidation
optimisation
fermentation
chemical synthesis

29
Q

Why was porcine insulin not the best?

A

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

30
Q

Why was human insulin difficult to acquire 60 yrs ago?

A

Difficult/expensive - available in small amounts

31
Q

How was recombinant insulin created?

A

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

32
Q

Whats another way to create recombinant insulin

A

Using mRNA where splicing has already occured

33
Q

What are 4 therapeutic products?

A

Blood products
hormones
immune modulators
therapeutic enzymes

34
Q

What is biocatalysis?

A

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

35
Q

What is an example of steroid biotransformation?

A

Hydrocortisone is produced chemoenzymatically

36
Q

How is hyrodcortisone produced?

A

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

37
Q

What is an example of chiral resolution?

A

Formation of S-2-chloropropionic acid from racemate

38
Q

What is racemate?

A

Valuanle synthon in manufacture of pharmaceuticals

39
Q

How is S-2-chloropropionic acid produced?

A

Pseudomonas sp. with stereoselective dehalogenase -> degrades R enantiomer

40
Q

What does the production of sitagliptin involve?

A

TransaminaseW

41
Q

What is sitagliptin?

A

anti-diabetic drug

42
Q

What are the types of biocatalyst?

A

Whole cell biocatalyst or purified enzyme

43
Q

What are considerations for which biocatalyst?

A

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

44
Q

What are 3 ways of strain improvement?

A

Industrial production
culturing
mutagenesis

45
Q

What are 2 levels of feedback inhibition/repression?

A

Inhibits enzyme activity
inhibits gene expression

46
Q

What are 3 ways of mutagenesis?

A

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

47
Q

What is a restriction site?

A

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

48
Q

What is the kanamycin cassette?

A

Contains a gene that will confer resistance to Kanamycin

49
Q

What does CRISPR-Cas stand for?

A

Clustered regularly interspaced short palindromic repeats

50
Q

What are CRISPR regions?

A

region of bacterial genome where memory of previous viral infection resides

51
Q

What does CRISPR allow?

A

Quickly destroy invading viral DNA if it has been ‘immunised’

52
Q

What activity does Cas have?

A

Endonuclease activity & is responsible for adding new spacers

53
Q

What do guide RNAs sequences do?

A

Complementary to sequence of gene where the Cas9 will cut

54
Q

What is the PAM sequence?

A

NGG downstream of cut site & is recognised by Cas9

55
Q

What does non-homologous end joining do?

A

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

56
Q

What does homology directed repair allow?

A

New gene with homologous upstream/downstream sequences to be introduced

57
Q
A

Cell products (12 decks)

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