NP discovery Flashcards
What are NP?
Secondary metabolites produced by a microorganism or plant. The soil is filled with actinomycete bacteria e.g. streptomyces which produce the most
Draw the life cycle of a filamentous bacteria
See diagram
When did Flemming discover Penicillin
Late 1920s
What has Penicillin been derived into?
Amoxicillin
What did Waksman discover?
Streptomycin
What is Streptomycin used to treat?
1st treatment for tuberculosis
Draw the Waksman platform
See diagram
When did the innovation gap start?
from the 1960s
What was the innovation gap?
When pharma pulled out due to rediscovery problems and the Waksman platform takes a lot of time and money. Started focusing on synthetic chemistry but this can lack complexity and chemical diversity
What is the NP renaissance?
Advancements in genome-mining, bioprospecting, bioengineering and activation of silent gene clusters
Types of genome sequencing platforms (short or long reads)
454 sequencing and Illumina (short reads)
PacBio (Longer reads)
Oxford nanopore - beta testing but allows real time
Tools for annotating the genome
BLAST
RAST
EMBL-EBI
What bioinformatics tool can be used to help find gene clusters?
antiSMASH
Draw antiSMASH pathway
See diagram
Genome mining- Antimycin/neoantimycin
Strep albus from leaf cutting ants was isolated and bioactivity aginst fungus
Grew up and separated on normal phase column into fractions
Bioassay of fractions showed two peaks
LCMS showed was antimycin and candicidin
No-one knew how antimycin was made
454 sequencing of genome and identified the biosyn gene cluster
The core of it looks like threonine suggest NRPS pathway so search for a threonine adenylation domain
Antimycin is produced by a NRPS/PKS hybrid
Mutations were made and the pathway solved
A lot of interest as inhibit BCL-XL which is overexpressed in cancers
Neoantimycin downregulates BiP - known about for a while
Did not know the pathway but similar in structure to antimycin - seq the producer and the pathway was identified (not using antiSMASH)
Strep orinocci couldn’t be genetically modified therefore was cloned into heterologous host - albus and the pathway was idenified
Which bacteria produced Antimycin/candicin
Strep albus
Which bacteria produced neoantimycin
Strep orinocci
Genome mining examples
Antimycin/neomycin
Nystatin P1
Surugamide
Anthracimycin
Genome mining - Nystatin P1 example
Also from leaf-cutting ants
Non-strep species called psuedocardia which is rare and hard to isolate
Took a known closely related gene cluster and aligned the contigs from 454 sequencing draft genome
Found an extra gene
LCMS showed it was not nystatin A1 and tandem MS showed it had an extra mannose sugar which was attached onto the 1st one
This was encoded by the extra gene NypY which belongs to the same glycosyltransferase family as NypD1 but only has 41% sequence idenitity
Sugars are essential for anti-fungal activity
Species of Nystatin P1
Psuedocardia
Genome mining - Surugamide
Mass spec molecular networking suggest a surugamide was present
Surugamide was idenified 4 years ago as an inhibit of cathepsin B but also suggested to have antibacterial activity
Structure is an octapeptide so looked for 8 adenylation domains with antiSMASH
Found cluster 15 had too many so carried out deletions
Deletion of SurA or SurB revealed there was actually two biosynthetic gene clusters i.e. a cluster within a cluster
Genome mining - Anthracimycin
Anthracimycin is a potent antibacterial against MRSA and VRE
Unsual for a macrolide antibiotic as has a tricyclic ring system with no sugars
Structure suggest made by PKS
The producer was sequenced and antiSMASH identified a suitable PKS. The cluster was cloned and a PAC library was made and it was put into a heterologous host
What did the connecting of molecules to genes allow?
A modified ‘Waksman’ platform to be used
Dereplication by genomics and genome-guided chemical characterisation
Which genome sequence revealed silent pathways and how many?
Strep coelicolor
~30 biosynthetic pathways but only 4 expressed in the lab
What are the two broad strategies for waking up silent gene clusters?
Non-genetic methods and targetted genetic methods
What are the 4 non-genetic methods
Ribosome engineering
Chemical elicitors
Co-cultuvation
Growth media screen
What are the 5 genetic methods
Deletion of repression Overexpression of activators Phosphomimics Manipulation of regulators Promoter engineering
Ribosome engineering principle
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
Ribosome engineering example
Hosaka did this and idenified a new antibiotic called piperidmycin A to F where D was the best
They use a strep species
Problems with ribosome engineering
Not high through-put and only works 1% of the time
Chemical elicitors HDAC principle and example
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
Chemical elicitors sub-lethal doses example
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
Chemical elicitors pleiotropic repressors example
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
Craneys group chemical elicitors example
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
Co-cultivation example
Strep lividans does not normally produce undecylprodigiosin but direct physical contact with mycolic acid producing bacteria stimulates the production
Interaction-mediated secondary metabolism
What can imaging MS be used for?
Seeing chemical mass changes from isolation to co-cultivation e.g. amycolatopsis sp and strep coelicolor for the battle for iron
co-culivation iGem
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
Genetic method - deletion of repressors example (cdk)
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
Genetic methods - overexpression of activators
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
Genetic methods - phosphomimics
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
Genetic methods - promoter exchange example
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
Non-targetted OSMAC approach
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
