NP Discovery (OSMAC & Genome Mining) Flashcards
Anti-microbial resistance
- antibiotic not effective anymore
- antimicrobial resistance could kill 1.9 million people a year by 2050
Golden age of antibiotics
after WWII, we discovered lots of natural products
phase between 60s and 90s where we had a surplus of antibiotics - if infection starts use drugs to treat it, but because of overuse, resistance began
challenges with new NPs
- hard to determine what to use it for
- hard to upscale into drug to use it
what three themes are the main challenges in NP discovery?
- time
- sources
- rediscovery & screening
Time
1. when was penicillin discovered and when was it developed into drug?
2. Artemisinin?
- antibiotic properties were discovered in 1928 BUT developed as a drug in 1946
- (Artemisia annua) antimalaria molecule was discovered a long time after the properties of the plant were known - The Beijing team tested 240,000 compounds against malaria, with no success; they discovered that artemisinin was damaged during the normal extraction process
Sources
1. Pacific yew tree (Taxus brevifolia)
2. more plants & microbes to discover
3. lab?
- taxol: anticancer from pacific yew tree
- naturally produced in low-quantity in the bark
- not sustainable - have to remove almost whole bark on one tree
- now produced by plant cell cultures - fungi 70,000 have been described
estimated 5.1M fungal species
outnumber plant 6 to 1 - can they be grown easily in the lab?
Rediscovery & Screening
1. limited microbes & plants
2. laboratory conditions
3. limited screening
- isolation of same organisms
rediscovery (same NPs) - hard to culture some species - may usually grow in particular environments
different from the natural environment -> few secondary metabolites are produced - we can’t test for all bioactivities
may be missing some cool applications
NP discovery process (traditional) (3 steps)
- isolate, identify & culture organisms
- chemically extract & characterise natural products
- bioactivity & toxicity screening
NP discovery process
Isolate, identify & culture organisms
1. animals or pasture
2. correlation?
3. microbes (and plants) have?
4. how does genome analysis occur?
5. what occurs after organism identified?
- lots of gut microbes have not been studied due to specific culture - environment needs no O2
- usually correlation for some adapted to grow in difficult environments that may be very good at producing bioactive molecule
- a huge biosynthetic potential: ecological observation, genome analysis
- huge amount of genome data - mining this resource to identify a biosynthetic gene cluster
- once organism identified -> lab -> grow them mainly on plates or greenhouses for plants; characterise them to ensure they are new, can they be grown and is the organism new?
NP discovery process
Isolate, identify & culture organisms
1. what is OSMAC?
2. Aspergillus flavus?
3. most microbes (and plants) have…?
4. ‘Ecological’ approach?
5. marine bacterium (Pseudoalteromonas luteoviolacea)?
6. OSMAC + GENOME Mining?
- OSMAC - One Strain Many Compounds
- Aspergillus flavus -> Aspergillus flavus on a petri dish
-> on a media (food) at a temp with light
-> on a different condition (e.g. media, light, temperature, pH…)
-> genome encodes for 56 BGCs, most of them are silent under laboratory conditions (cryptic)
- mould that contaminates crops - can change growth of organism by changing condition to produce different metabolites - need screening to allow production of different metabolites
- a huge biosynthetic potential: ecological observation, genome analysis
- co-culturing, different growth scaffold, mechanical stresses, light temp pH…
- marine bacterium (Pseudoalteromonas luteoviolacea) - isolated from sponges - new strain -> lab -> add fake sponges -> bacteria growing in fake environment and changing colour -> uses the fake structures to grow on and produce and develop to produce certain metabolites
- Aspergillus flavus -> encodes for 56 BGC’s, most of them are silent under laboratory conditions (cryptic) -> unknown clusters -> synthetic biology + genetic tools, New Natural Products
NP discovery process
Isolate, identify & culture organisms
1. how does genome mining occur?
2. what occurs after isolate, identify & culture organisms?
- genome sequence (cheap & many) -> predict a natural product BGCs (Biosynthetic Gene Clusters)
-> bring the organism to the Lab -> New Natural Products
-> synthetic biology + genetic tools - finally organism looks new and is growing and might produce something, next = extraction
NP discovery process
Chemically extract & characterise natural products
1. chemical extraction?
2. what is left after?
3. Penicillium fuscoglaucum (PF) culture?
- solvent extraction (solvent polarity), solid phase extraction (resins), cell disruption
- oil or microbial extract -> different compounds seen due to colour
- PF culture -> mass culture/metabolite production -> metabolite with ethyl acetate -> ethyl acetate evaporation and metabolite concentration
NP discovery process
Chemically extract & characterise natural products
1. analyse the extract (what is inside?)?
2. bioactivity (is it active?)?
- separation of the mixture into pure Natural Products (chromatography); detection of the components
- cell lines, microbes, proteins
NP discovery process
chemically extract & characterise Natural Products
Analyse the extract: Chromatography
1. what are the phases/components?
2. separated by?
3. molecule movements and separation?
4. if stationary phase is polar?
5. if stationary phase is non-polar?
- stationary phase (separate mixture), mobile phase (help separate by moving things), analytes (sample we want to purify)
- separated by their physical properties (interactions)
- molecules move at different speeds because interact more or less with stationary phase
polar - most polar molecules, e.g. sugar will stay longer - spending more time in stationary phase
non-polar - do not interact with phase as much - Stationary Phase is Polar (Silica) -> Mobile Phase is Non-Polar (Hexane)
- Stationary Phase is Non-Polar (C18) -> Mobile Phase is Polar (Aqueous)
NP discovery process
Chemically extract & characterise Natural Products
1. types of chromatography?
2. detection?
- Thin layer chromatography (TLC), flash column chromatography, high performance liquid chromatography (HPLC)
- sample -> HPLC -> mass spectrometry (MS), ultraviolet, other detectors
on graph each peak represents something detected
