Directed Evolution of Enantioselective Enzymes

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Directed Evolution of Enantioselective Enzymes
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Reetz, Manfred T.
Jerabek, Paul
Hegemann, Julian
Authmann, Andreas
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Paul Jerabek, Julian Hegemann, Andreas Authmann
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Prof. Reetz (MPI Mühlheim) talks about the synthesis of enantionselective enzymes through the means of Directed Evolution.
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while the other 2 the a you In a demand what
made me into our research area which is far away from synthetic organic chemistry you may know that have worked in an hour and done research in synthetic organic chemistry developing methods for for decades so some people have asked me what made you go into this completely different area which involves molecular biology and homology and so on what I thought about this question and it turns out that the roots can be traced actually too not OK when I came from 1
2 this university In 1980 my colleagues cannot Hoffman suggested that we offer to the that project Himika German chemical Society a course on serious selective synthesis methods in asymmetric at
palaces and story committed reactions so I thought That's a nice idea and I said yes I do it and we asked another colleague now progressive guys at that time that such a bunch that university it also participate and we divided the subjects and Professor guys was responsible for enzymes as catalysts and synthetic organic chemistry and we offered this course every 2 years for a whole decade and we present as are the speakers as the 3 speakers during the whole course was always a one-week courses 8 hours
a day so I learned a lot myself and not just the participants which were students investor people so long from all over Europe as was really 1 of the best the German put causes there is a sharp budget gimmick so I learned something about insects and I had previously no knowledge of that subject whatsoever so I was sensitive and then in 1994 I read a paper by chance in nature entitled DNA shuffling written by a molecular biologist at the name of mission so I was curious operated I didn't really understand the details but it was clear this has to do with directed at motion and he was interested in and have the havoc resistance beta-lactam aces so
long so I started to read the literature more on not directed evolution and the government agreed pay the seminal paper by
France's Arnold at Caltech written and appeared in 1993 she went through several cycles of mutagenesis In order to increase the stability of a protein so what does this actually means what is directed evolution that's the 2nd question I have 1 moment list here and everybody knows what evolution in nature is it's a continuous cycle of our mutagenesis Jean mutagenesis selection genes Genesis selection it's a powerful driving force in nature and nothing can be understood in biology in the absence of hot evolution and it has been the dream of entomologists and revolutionary power researchers to simulate this process in the laboratory in other words the perform Oct evolution in the test tube and this is what she it this is what I know it performed and we read this at a very early stage and then this posed a question can we harness this powerful force In the evolution put it in the test In order to
control a parameter which admittedly is not trivial namely asymmetric catalysis in Nantou selectively so let me begin with a the are cartoon here you see Our new approach to asymmetric catalysis directed evolution of selective enzymes and on the rights upper part you see our circle it symbolizes the wild-type enzyme in other words the enzyme that occurs in nature it has poor selected poor enter selectivity in the reaction that you or we may be interested in it so we take the gene the square to the left I which encodes bets are enzyme and subjected to Beijing mutagenesis methods and there are a number of these methods available which were developed in the 1980's and 1990's even to this day and so on such things as error-prone
polymerase chain reaction shotgun method the most popular method used to this day
then we have DNA shuffling which is a recombinant method mentioned it's already watched them a gene or 2 genes you slice them and semantically into pieces and you reassemble them so this is simulating sexual evolution let me now show you some details of what is actually done in the laboratory once you perform 1 of those methods on some gene which encodes an enzyme
of interest to you you have it and they test to here and then you transfer that this collection of mutated genes into a bacterial hosts such as E. coli life you played out on actor plates on many other plates here symbolically the 1st plates and after a while you see the colony's growing each coming from a single cell producing a mutant you collect them you you harvest and you give them food and a few good you put them into individually into the wells of Michael tighter plates and then you had suddenly have hundreds and thousands of Little factories producing of potentially the man selective instead so we used some of these methods such as error-prone PCR and also DNA shuffling in proof of principle studies using light basis and were able to increase the Ananta selected the team to a notable decrease if you do something completely new for the 1st time it doesn't matter how efficient it is sold we were not concerned about our practical ramifications on the efficiency of what we're actually doing no made challenge in directed evolution is to develop methods which allow you to probe protein sequence space efficiently and what do I mean by that consider for example enzyme composed of 300 amino acids if you introduce 1 mutation 1 point mutations randomly everywhere at every position and remember the 20 building blocks 20 different amino acids you can calculate their about 7 thousand different mutants are possible if you introduce 2 mutations simultaneously this jumps up to about 15 million and 3 30 billion which are impossible 2 screens and remember how are you going to screen even a thousand or 5 thousand samples for an engineering purity so this was 1 challenge to develop high-throughput methods for determination I would not go into any details that the other intellectual challenge is what I just addressed namely methodology development we published our proof of principle paper others joined
us industrial power companies also use this method to create new catalysts for asymmetric catalysis but we were not happy with the with the methods so let me know show you how to beat the so-called numbers problems and directed the pollution how to make small libraries high-quality libraries with less efforts that is the challenge
and our answer is shown on the slide here we call it iterative saturation mutagenesis isn't you 1st make a decision regarding sites in the enzyme where you want a randomized so saturation
mutagenesis is a method that I have not introduced you it yet but it is as follows you can choose for example 1 position anywhere in the
enzyme you define it and introduce randomly all 20 ProTour Janik amino acids there and you get a library of all that 20 if you saturate a randomized as people 2 positions I'm instantaneously it's 20 the power of 2 forms and so on there are 3 amino acid positions sites would be 8 thousand so you need a decision of where to go random so it's knowledge-driven mechanism driven structures driven and we have
developed criteria with which you can choose the right appropriate positions where to randomized but let's say and I will show you in a minute what those criteria are let's say you know have analyzed your system and there are 4 sites ABC days and just to make it less abstract let's say a and B are sites composed of 2 amino acid positions and C and D 3 amino acids positions in see on the slide that in the case of 3 as I said there are 8 thousand mutants it doesn't mean that you choose to harvest the 1st 8 thousand bacterial colonies and you have all of us there's a statistical arguments and statistics according to which you have to do so-called oversampling so you have to harvest and many many many more if you insist on really screening all of those 8 thousand for example but if you look at the scheme it looks a little complicated we make 8 we make for
libraries screen them for an his electability and put the winner but as you can see in each case a B C and D. then the interactivity comes into play we have changed the catalysts the enzyme structurally and we take a gene that encodes this mutant enzymes and then visit the other sites In the case of a B C and D and that will continue until we have I visited all 4 sites and then a converge so when we set up this year we did not dream how successful it would be this is the most efficient way to do directed evolution now let's look at the criteria we have to make the decisive but choice where 2 mice so
it is a combination of let's say rational design and randomization in that shown on the next flight we call this combinatorial active-site saturation test cast that surmise acronym casting for substrates gold and images selected at end you simply look at the binding pocket and see which amino acid residues airlines this finding pocket those are our ABC NBC and then we perform best systematically in the sense of it creativity according to the scheme that I showed you on the last slide now let's briefly look at our 1st example and that's shown on the next slide here this concerns the a so-called kinetic resolution
overestimate in this case and the Parkside and b we use in the Parkside hydrolase so it's arrested 1 1 mixture of of R S and we only won 1 of the tumors react to the dial and that would leave after 50 per cent conversion the others starting material untouched and those can then be separated so we performed the cast an analysis based on the X-ray structure and we came up with 6 sites ABC D E and F each composed of 2 or 3 amino acid positions on the right side you can see how a cartoon Oro which picture these half past 6 sites so the students performed the muted saturation mutagenesis 6 times got 6 libraries and the best hits came out of Liberia beat and this was then used as a starting point for people call it a template to visit another side so the question is where should you go if you remember the dendritic scheme was somewhat complicated today we know it really doesn't matter which pact will you you take but on the next slide you see the results we have about the wild type this is the selectively the fact of the relative rate of 1 with respect to the other indenture mayor and you
see In the best came out of the sails of selective factor of 14 men and the student visited C D E F and G and we came up with the mutant W 2 old 2 which has a selectively factor of 115 and that we only have to look at 20 thousand reactions which happens to be the same number that we had already screened using the old strategies error-prone PCR and older study but the results there were very very poor we could only double in year we have are multiplied by a factor of 20 at 25 an interesting question concerns the problem of identifying the reasons for enhanced inanities selected so we just recently published a paper concerning the questions investors specific cases and it's a long story you can read it in the journal American Chemical Society 2000 9 just a few months ago but I only want to show you 1 little thing that is very important namely the X-ray structure of the wild type the starting in
inside and the X-ray structure of the involved in Nantou selective ones if you look at the 2 X-ray structures there is essentially identical


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