NP discovery Flashcards

Question

What are the two broad strategies for waking up silent gene clusters?

Answer 1

Non-genetic methods and targetted genetic methods

Answer 2

Ribosome engineering Chemical elicitors Co-cultuvation Growth media screen

Answer 3

``` Deletion of repression Overexpression of activators Phosphomimics Manipulation of regulators Promoter engineering ```

Answer 4

Take a strep species and plate onto streptomycin or rifamycin plates Target the ribosome or RNA polymerase Cause spontaneous point mutations which alter the transcription and translation prolifes of some strainsvia the alarmone ppGpp to switch on gene clusters It tricks the bacteria into thinking it's starving which activates stress and therefroe biosyn clusters

Answer 5

Hosaka did this and idenified a new antibiotic called piperidmycin A to F where D was the best They use a strep species

Answer 6

Not high through-put and only works 1% of the time

Answer 7

HDAC removes the acetylation on histones causing the DNA to be wrapped around the histone Inhibitors of HDAC prevent this keeping the DNA unwrapped so it can be transcribed Whole series of HDAC inhibitors e.g. sodium buterate which can be put into the culture and switch on pathways. This was done in streptomyces nd psuedocardia where it showed increased anti-fungal activity Moore's group screened a whole range of HDAS inhibits looking for the increased production of RED or ACT pigments in bacteria

Answer 8

Sub-lethal doses of antibiotics can act as inducers of secondary metabolism This was done in Burkholderia bacteria where they fused the promoter of a cryptic gene cluster to LacZ and screen 640 known bioactive compounds They found 4 antibiotics which activated the reporter Trimethoprim antibiotic was a global activator of at least 5 biosynthetic pathways

Answer 9

DasR is a pleiotropic repressor which normally sits on a gene and represses it. However when GlcNAc is present it falls off the promoter and you get transcription of the genes it was repressing DasR normally represses cluster-situated regulators of actinorhodin and undecylprodios in biosyn. Tested the sprinkling of GlcNac where it works for some strain but not others highlighting the complexity of the pathways

Answer 10

Took strep coelicolor under conditions where actinorodin (pigmented) is not produced and screened high through-put librabry of synthetic compounds >30,000 Found 19 enhanced the yield of actinorhodin One called Arc2 is similar to triclosan which inhibits FabI Cannot completely inhibit FabI as the cells would die as need fatty acids biosynthesis but partially inhibition is thought to free up CoAs for PKS

Answer 11

Strep lividans does not normally produce undecylprodigiosin but direct physical contact with mycolic acid producing bacteria stimulates the production Interaction-mediated secondary metabolism

Answer 12

Seeing chemical mass changes from isolation to co-cultivation e.g. amycolatopsis sp and strep coelicolor for the battle for iron

Answer 13

Imperial college won last year with ecolibruim 3 systems using quorum sensing Allows the bacteria to detect it's own population density and the others in culture If it is growing too much an inhibitory protein is expressed to restore the ratios in culture

Answer 14

The ScbR2 repressor of the cpk gene cluster was deleted which revealed a novel antibacterial activity in strep coelicolor cpk is a type 1 PKS ScbR2 directly repressors the cpkO pathway specific activator

Answer 15

Strep ambofeciens has numerous biosyn clusters including a giant type 1 PKS but is not expressed in the lab Constitutive expression of a regulatory gene within the cluster by cloning the activator into a plasmid led to the identification of new compounds 4x 51 membered glycosylated macrolides called stambomycins Promising antiproliferants Database searches idenified numberous gene clusters containg LAL regulators so could be used on other clusters They have characterised the product and came up with a scheme on how it is made

Answer 16

2 component system are made up of a sensor histidine kinase which sits in the membrane and sensors something and auto-P itself The 2nd component had a response regulator which the P-SHK P's the response regulator allowing it to act on its target genes There is a highly conserved two-component system in strep species called AfsQ1 and 2 Problem is that AfsQ2 is only active when it is P by the AfsQ2 and the signal for this is unknown A point mutation was made in AfsQ2 which mimicked P and allowed it to bind They cloned this into a vector and put it in various Strep species which lead to the identification of a lasso peptide with antibacterial activity against lipid 2

Answer 17

Silent 'indigo' biosyn gene cluster in strep albus switched on using recombineering of a strong promotor upstream of the NRPS gene Another example from his lab where there was consistent failure of heterologous production of antimycins using strep coelicolor and promotr engineering alleviated the requirement for FscR1 regulator allowing the production

Answer 18

Use to vary chemical conditions with lead to the isolation of a new lasso peptide called capistrum Non-targetted approach with is cheap, easy and assessable

NP discovery Flashcards

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