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Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations.
14 July 2014
Zhao J, Kardashliev T, Joelle
Biotechnol Bioeng 2014
Diversity generation by random mutagenesis is often the first key step in directed evolution...
Bacillus gibsonii alkaline protease
1 November 2012
Martinez R, Jakob F, Tu R, Sie
Biotechnol Bioeng. 2013 Mar
Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting...
Review: To get what we aim for - progress in diversity generation methods
5 June 2013
Ruff AJ, Dennig A, Schwaneberg
FEBS J., 280 (2013), 2961-2978
Protein re-engineering by directed evolution has become a standard approach for tailoring enzymes...
Reengineered glucose oxidase for amperometric glucose determination in diabetes analytics
20 June 2013
Arango Gutierrez E, Mundhada H
Biosensors and Bioelectronics
Glucose oxidase is an oxidoreductase exhibiting a high β-d-glucose specificity and high stability...

Directed Evolution

Directed evolution
Steps in a Directed Evolution experiment: diversity generation, cloning & expression, screening and variant characterization

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 [8], lipases [9, 10], esterases [11, 12], epoxide hydrolases [13], dehalogenases [14, 15], aldolases [16] 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 [17] and [18].

More, recently, there is a trend in evolving enzymes that degrade biomass to biofuel as also described in [19] where a cellulase from a metagenomic library was evolved for activity in ionic liquids. In addition to evolving enzymes for speci c reactions in extreme conditions, directed evolution is useful to engineer therapeutic proteins such as monoclonal antibodies [20], adnectins [21], anticalins [22] and other protein scaffolds [23]. Furthermore, recently, a recombinase that can effciently and speciffcally modify DNA sequences was evolved for the help of enhanced gene therapies and genetic studies [24]. Other red biotech applications of directed evolution were reported in for vaccines [25] and for predicting antibiotic resistance [26]. 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 [31], 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 de nes rational design. This is how the positions that are important for the property of interest are identi ed 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 [32], thermostability [33], antibody affnity [34], protein expression level [35] 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 [36]. They shuffled two naturally occurring biphenyl dioxygenases that diff er 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 [37] and a 'Kemp eliminase' [38]. 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' [39].


1 -Otagiri M, Ui S, Takusagawa Y, Ohtsuki T, Kurisu G, Kusunoki M (2010) Structural basis for chiral substrate recognition by two 2,3-butanediol dehydrogenases FEBS Lett. 584, p219-23.

2 -Rutten L, Ribot C, Trejo-Aguilar B, Wsten HA, de Vries RP (2009) A single amino acid change (Y318F) in the L-arabitol dehydrogenase (LadA) from Aspergillus niger results in a signi cant increase in affinity for D-sorbitol BMC Microbiol. 9, p166.

3 -Sawayama AM, Chen MM, Kulanthaivel P, Kuo MS, Hemmerle H, Arnold FH (2009) A panel of cytochrome P450 BM3 variants to produce drug metabolites and diversify lead compounds Chemistry 15, p11723-9.

4 -Li HM, Mei LH, Urlacher VB, Schmid RD (2008) Cytochrome P450 BM-3 evolved by random and saturation mutagenesis as an effective indole-hydroxylating catalyst Appl Biochem Biotechnol. 144, p27-36.

5 -Nazor J, Dannenmann S, Adjei RO, Fordjour YB, Ghampson IT, Blanusa M, Roccatano D, Schwaneberg U (2008) Laboratory evolution of P450 BM3 for mediated electron transfer yielding an activity-improved and reductase-independent variant Protein Eng Des Sel.21, p29-35.

6 -Rehdorf J, Mihovilovic MD, Bornscheuer UT (2010) Exploiting the Regioselectivity of Baeyer-Villiger Monooxygenases for the Formation of beta-Amino Acids and beta-Amino Alcohols Angew. Chem. Int. Ed. 49

7 -Wu S, Acevedo JP, Reetz MT (2010) Induced allostery in the directed evolution of an enantioselective Baeyer-Villiger monooxygenase Proc Natl Acad Sci U S A 107, p2775-80.

8 -Wang NC, Lee CY(2007) Enhanced transaminase activity of a bifunctional L-aspartate 4-decarboxylase Biochem Biophys Res Commun. 356, p368-73

9 -Engstrom K, Nyhlen J, Sandstrom AG, Backvall JE (2010) Directed Evolution of an Enantioselective Lipase with Broad Substrate Scope for Hydrolysis of alpha-Substituted Esters J Am Chem Soc. [Epub ahead of print]

10 -Colin DY, Deprez-Beauclair P, Silva N, Infantes L, Kerfelec B (2010) Modification of pancreatic lipase properties by directed molecular evolution Protein Eng Des Sel. 23, p365-73.

11 -Chakiath C, Lyons MJ, Kozak RE, Laufer CS (2009) Thermal Stabilization of Erwinia chrysanthemi pectin methylesterase a for application in a sugar beet pulp biorefinery Appl Environ Microbiol. 75, p7343-9.

12 -Hawwa R, Larsen SD, Ratia K, Mesecar AD (2009) Structure-based and random mutagenesis approaches increase the organophosphate-degrading activity of a phosphotriesterase homologue from Deinococcus radiodurans J Mol Biol. 393, p36-57.

13 -Kahakeaw D, Reetz MT (2008) A cell-based adrenaline assay for automated high-throughput activity screening of epoxide hydrolases Chem Asian J. 3, p233-8.

14 -Pavlova M, Klvana M, Prokop Z, Chaloupkova R, Banas P, Otyepka M, Wade RC, Tsuda M, Nagata Y, Damborsky J (2009) Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate Nat Chem Biol. 5, p727-33.

15 -Pavlova M, Klvana M, Jesenska A, Prokop Z, Konecna H, Sato T, Tsuda M, Nagata Y, Damborsk J.(2007) The identi cation of catalytic pentad in the haloalkane dehalogenase DhmA from Mycobacterium avium N85: reaction mechanism and molecular evolution J Struct Biol. 157, p384-92.

16 -Wolterink-van Loo S, Siemerink MA, Perrakis G, Kaper T, Kengen SW, van der Oost J (2009) Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase Archaea. 2, p233-9.

17 -Hudlicky T, Reed JW (2009) Applications of biotransformations and biocatalysis to complexity generation in organic synthesis Chem Soc Rev. 38, p3117-32.

18 -Patel, RN (2008) Synthesis of chiral pharmaceutical intermediates by biocatalysis Coordination Chemistry Reviews 252(5-7), p659-701.

19 -Pottkamper J, Barthen P, Ilmberger N, Schwaneberg U, Schenk A, Schulte M, Ignatiev N and Streit WR (2009) Applying metagenomics for the identi cation of bacterial cellulases that are stable in ionic liquids Green Chem. 11, p957-65

20 -Isaacs JD.(2009) Antibody engineering to develop new antirheumatic therapies Arthritis Res Ther. 11(3), p225.

21 -Dineen SP, Sullivan LA, Beck AW, Miller AF, Carbon JG, Mamluk R, Wong H, Brekken RA (2008) The Adnectin CT-322 is a novel VEGF receptor 2 inhibitor that decreases tumor burden in an orthotopic mouse model of pancreatic cancer BMC Cancer. 8, p352.

22 -Skerra A (2008) Alternative binding proteins: anticalins - harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities FEBS J. 275(11), p2677-83.

23 -Gebauer M, Skerra A (2009) Engineered protein scaffolds as next-generation antibody therapeutics Curr Opin Chem Biol. 13(3), p245-55.

24 -Gersbach CA, Gaj T, Gordley RM, Barbas CF 3rd (2010) Directed evolution of recombinase specifficity by split gene reassembly Nucleic Acids Res. [Epub ahead of print]

25 - Locher, CP, Soong, NW, Whalen, RG, Punnonen, J (2004). Development of novel vaccines using DNA shuffling and screening strategies. Curr Opin Mol Ther, 6, p34-39.

26 - Orencia MC, Yoon JS, Ness JE, Stemmer WP, Stevens RC (2001) Predicting the emergence of antibiotic resistance by directed evolution and structural analysis Nat Struct Biol, 8, p238-42.

27 - Wong, TS, Zhurina, D, Schwaneberg, U (2005) The diversity challenge in directed protein evolution. Comb Chem High Throughput Screen, 9, p271-88.

28 - Goddard, JP, Reymond, JL (2004) Enzyme assays for high-throughput screening. Curr Opin Biotech, 15, p314-322.

29 - Goddard, JP, Reymond, JL (2004) Recent advances in enzyme assays. Trends Biotechnol, 22, p363-370.

30 - Reymond, JL (2004) Spectrophotometric enzyme assays for high-throughput screening. Food Technol Biotech, 42, p265-269.

31 -Arnold FH (1998) When blind is better: Protein design by evolution Nature Biotechnology 16, p617-8

32 -Otten LG, Hollmann F, Arends IW (2010) Enzyme engineering for enantioselectivity: from trial-and-error to rational design? Trends Biotechnol. 28(1), p46-54.

33 -Gribenko AV, Patel MM, Liu J, McCallum SA, Wang C, Makhatadze GI (2009) Rational stabilization of enzymes by computational redesign of surface charge charge interactions Proc Natl Acad Sci U S A 106(8), p2601-6.

34 -Wei S, Mizaiko B (2007) Recent advances on noncovalent molecular imprints for affinity separations J Sep Sci. 30(11), p1794-805.

35 -Welch M, Govindarajan S, Ness JE, Villalobos A, Gurney A, Minshull J, Gustafsson C (2009) Design parameters to control synthetic gene expression in Escherichia coli PLoS One. 4(9), e7002.

36 -Kumamaru T, Suenaga H, Mitsuoka M, Watanabe T, Furukawa K (1998) Enhanced degradation of polychlorinated biphenyls by directed evolution of biphenyl dioxygenase Nat Biotechnol. 16(7), p663-6.

37 -Jiang L, Altho EA, Clemente FR, Doyle L, Rthlisberger D, Zanghellini A, Gallaher JL, Betker JL, Tanaka F, Barbas CF 3rd, Hilvert D, Houk KN, Stoddard BL, Baker D (2008) De novo computational design of retro-aldol enzymes Science. 319(5868), p1387-91.

38 -Rothlisberger D, Khersonsky O,Wollacott AM, Jiang L, DeChancie J, Betker J, Gallaher JL, Althoff EA, Zanghellini A, Dym O, Albeck S, Houk KN, Tawfik DS, Baker D (2008) Kemp elimination catalysts by computational enzyme design Nature. 453(7192), p190-5.

39 -Arnold F (1998) Chemical and Engineering News, April 27 p. 39.