Enzymes are nature's way of catalyzing chemical reactions. Directed evolution is a collection of protocols through which natural enzymes are evolved in the laboratory towards non-natural properties. In the last 2 decades, using directed evolution, industry built up on nature's success to develop clean ways to get to the products needed in modern world. Since the 1989 Exxon Valdez oil spill in Alaska was solved by bacteria degrading oil, mankind has turned to biotechnological methods such as directed evolution to answer many worldwide problems such as pollution due to waste coming from the industry, fossil fuel shortage, diseases or even poverty.
Many reports can be found in recent literature about using enzymes for industrial or pharmacological processes. In organic synthesis, enzymes are used because of their ability to have increased chemoselectivity, enantioselectivity and regioselectivity. Furthermore, they do their job at low temperatures, neutral pH, while no harsh chemicals are needed. Some of the enzymes protein engineers have evolved for such chemical reactions are alcohol dehydrogenases [1, 2], oxygenases such as P450 monooxygenases [3-5] or Baeyer-Villiger monooxygenases [6, 7], transaminases , lipases [9, 10], esterases [11, 12], epoxide hydrolases , dehalogenases [14, 15], aldolases  etc. The uses of the classes of enzymes enumerated above both in the biosynthesis of chemical compounds for white biotech and new chemical entities for red biotech are reviewed in  and .
More, recently, there is a trend in evolving enzymes that degrade biomass to biofuel as also described in  where a cellulase from a metagenomic library was evolved for activity in ionic liquids. In addition to evolving enzymes for specic reactions in extreme conditions, directed evolution is useful to engineer therapeutic proteins such as monoclonal antibodies , adnectins , anticalins  and other protein scaffolds . Furthermore, recently, a recombinase that can effciently and speciffcally modify DNA sequences was evolved for the help of enhanced gene therapies and genetic studies . Other red biotech applications of directed evolution were reported in for vaccines  and for predicting antibiotic resistance . A directed protein evolution project includes two main parts (left figure): generating diverse mutant libraries [25, 27] and screening for the improved protein variants [28-30].
In a 1998 review , Frances Arnold, a pioneer of protein engineering, was comparing directed evolution with rational design by naming the first one the work of a blind watchmaker while the latter one was the development of an algorithm to copy the work of the watchmaker for new molecules. In the same article, Frances Arnold described how rational design has evolved once genome databanks were lled, and we expanded our understanding of how proteins have evolved and their intricate familial relationships. As a consequence, the protein engineers that need to redesign enzymes for new properties started looking for patterns that would explain the way natural evolution works. The collection of these patterns that are applied to fast forward through natural evolution are what denes rational design. This is how the positions that are important for the property of interest are identied in silico. Afterwards, these positions are targeted by either site-saturation or site-directed mutagenesis in the lab. This is how diversity is generated. However, the diversity is limited to those mutants that have more chances in presenting the desired features. As a comparison, this method is reducing the search of a needle in the haystack. Once diversity on the gene level is generated, all the other steps of a directed evolution project are undergone as described in the top left figure.
Rational design can be used to change enantioselectivity , thermostability , antibody affnity , protein expression level  etc.
Both directed evolution and rational design have advantages over each other. This makes them complementary. Directed evolution might lead to unpredicted results such as the case of Kumamaru and coworkers . They shuffled two naturally occurring biphenyl dioxygenases that differ at less than 5% of their amino acids to create enzymes with new substrate speciffcities.
On the other hand, by rational design, it was recently possible to design de novo a retro-aldolase  and a 'Kemp eliminase' . However, in the case of the last two, directed evolution was needed in order to further optimise the novel enzymes. This is why, good practice would recommend to combine the two for better results.
This is what we also do here at SeSaM-Biotech by offering though our evolution packages both directed evolution and rational design (see Evolution services).
All in all, if directed evolution and rational design knowledge is used at their maximum potential, we are not anymore limited by our ignorance in terms of novel enzymatic activities, but just by our imagination, as Frances Arnold was saying in 1998 on the podium of 'Challenges for chemistry in the 21st Century' .
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