Directed evolution Flashcards
What is an example of why selection can have problems
Chorismate mutase
Describe the problems of selection using the chorismate mutase example
CM is involved in the conversion of chorismate to prephenate in the Shikimate pathway for Phr/Tyr.
Tried to generate a hexameric enzyme from the WT dimer by introducing linkers but the activity dropped.
They set up a growth assay as Phe/Tyr are essential so you need these to grow cells i.e. selection can be used.
Hexamer had a high Km so they carried out two round of random mutagenesis and selection in a CM deficient strain which generated a trimer which had better activity than the hexamer but lower activity than the WT.
Problems that the timer couldn’t be improved further because the cells can grow. This showed the system wasn’t sensitive enough.
They went on to develop a system where expression was reduced and turnover of CM was enchanced to allow for sensitive selection which produced an improved variant. They developed a system for more sensitive selection.
What is the first law of DE?
You get what you screen for. Therefore conditions in which you screen are important.
Describe the fungal peroxidase example
Wanted to develop a peroxidase better for use in washing machine detergents. Therefore used ER-PCR to mutate the WT gene and looked for improvements under mild ‘washing machine conditions’.
Library was tested in yeast so this limited the library size (transformation efficiency). Screen 64,000 variants where 2 showed improved residual activity (activity after x hours). Both of these contained the E239G mutation which is not near the active site so would not have found doing SDM mutagenesis.
Describe the cytochrome P450 monooxygenase example
They have relativity low turnover rates with unnatural substrates, low stability and need e- donating factors such as O2 and NADPH.
Task looked at evolving efficient P450 that acts on naphthalene and uses H2O2 instead of O2/NADPH.
Naturally uses camphor and very little activity with naphthalene. Did ER-PCR of P450-cam and screen 32,000 in a fluorescence-based screen. The majority were worse with no activity but a number were better than the WT where 3 variants were tested further. All contained E331K which showed it was important in switching the specificity but it was also an allosteric site.
Describe the APEX 2 example
Heme peroxidases are power tools in biotechnology. Horseradish peroxidase is widely used but it is limited as it is hard to express and you have to add it externally and we often want to explore inside cells.
APEX is another peroxidase which is small in size and doesn’t interact with cellular activity. APEX was engineered as a monomeric peroxidase reporter which was derived from dimeric pea.soy bean ascorbate peroxidases. APE lacks DS and Ca2+ binding sites and can be easily expressed in cells. It can be used for intracellular specific protein imaging and EM.
Problem with APEX is that it is not as efficient as you might like therefore DE was used to make APEX2 using a yeast display system and FACs. ER-PCR of APEX and displayed it on yeast surface. Treat the yeast cells with a substrate which if is acted on by the enzyme it labels the cell and you can use FACs to sort and extract the yeast cells depending on the amount of labelling and therefore the enzyme activity. Biotinylated the yeast surface.
In rounds 2 and 3 they increased the selective pressure by targeting heme.
Biotin-phenol was the substrate used with H2O2.
They identified 2 mutants - APEX2 and APEX-v/g which had a common mutation. All of the mutations were away from the active site.
APEX 2 is used in EM and light microscopy as reporters using DAB as a substrate. Also used for proteomic tagging in live cells with MS analysis.
Describe the enchanced thermostability of Fructose-1,6 biphosphate aldolase
Extra reading
The thermostability of Fructose 1,6 biphosphate was increased using DNA shuffling which created a variant 280x higher half-life at 53oC than either parent.
It also had enhanced activity it various polar and non-polar organic solvents showing an example where you got more than what you screen for.
Describe the savinase example
Savinase is a leading commercial Subtilisin which is based on a serine protease and is found in washing powders.
Segments of subtilisins were cloned by PCR from 25 natural Bacilli and cloned in context of a savinase gene - retained N/C terminals of the WT. Generated lots of constructs and shuffled these.
Used hierarchical screening method - first used milk as proteases can cleave the proteins in milk and you get a clearing. From this they grew the clones with activity in a 96-well plate and assayed the supernatant for protease activity using a caesin-derivative fluorescence which fluorescences when cleaved.
Then looked at multiple properties of temperature, pH and solvent stability which had been previously targetted by engineering separately. Used directed evolved family shuffling to see if you can combine and showed that the properties could be combined and the best thermostable variant had 32aa changes so would not have found this without family shuffling.
Often the best parent is not always obvious and it is not always the most sequence related.
What is the best codon to use in saturation mutagenesis?
NNK
What is a metagenomic search approach?
Isolate and sequence the genomes of non-culturable organisms and HTP screen for novel enzyme activities.
Based on spontaneous natural mutations.
Can reveal new activities for immediate use or for family shuffling.
4x soil samples allowed the isolation of 94 subtilisin sequences with 38 mutations. 52 could be expressed and showed functional differences.
Also search for (R) selective transaminases where computer searches gave >5000 seqs revealing 21 new sequences. 17 of these were amine transaminases with (R) selectivity. Can use shuffling to try and improve these.
Design of Kemp eliminase reaction
Kemp eliminase reaction is a well-studied reaction but no enzyme was available for this. It is a model system for a proton transfer from a carbon.
Based on principle of raising something to stabilize the TS then you can push it into catalysis.
TIM is the classic enzyme used as a scaffold. They designed and evolved an artificial enzyme for kemp elimination reaction. 17 rounds of mutagenesis and selection (ER-PCR and DNA shuffling).
Successfully made a good enzyme with high shape complementary to the TS. The alignment of the catalytic base had to be optimal to attack in the right position. Introduction of other groups allowed stabilization of developing negative charge and push towards generation.
Not de novo as used a scaffold to put the design AS into.
Example of a computational structure-based design
3DM is a commercial structure based sequence alignment and analysis tool. It identifies distribution of amino acids with a protein superfamily and analyses connectivity between residues based on protein structure alignments.
