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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...


Screening: choosing the best

Screening is an important component part of an evolution project. Once the diversity is generated at the gene level, the library is cloned and expressed in organisms that will be used for the selection of improved variants (see Directed evolution). Nature also selects for the evolved species letting survive only the fittest i.e. having the needed properties for survival. Nature is able to do so because the link between the genotype and the phenotype is preserved in a living organism. Building on nature's success, protein engineers try to also link the genotype and phenotype for selection.
In order to cover the entire protein space, one needs high throughput screening methods that will allow reliable sampling as much as possible in a short time. Frances Arnold said in a 1999 review [1] that: 'You get what you screen for'. What she meant was that, unfortunately, there is no universal screening technique, and each enzyme evolved has to have its own screening assay.
In the past, only 'toothpicks and logic' were enough for screening, as the Nobel Prize winner Sydney Brenner said during his Nobel Prize speech [2]. However, since Brenner did his work for determining genetic regulation during development and programmed cell death, a lot of screening assays were developed.
In order to develop a good screening assay, it has to be taken into consideration the limitations in the library size screened by each type of assay. For example, colorimetric or fluorometric methods lead to 103 - 105 clones screened per experiment [3] while solid phase screening on agar plates, filter papers or membranes can increase the number of clones screened in one experiment to 104 - 106 [4, 5]. Recently, fluorescent activating cell sorting (FACS) based methods were developed for screening > 107 clones/hour [6].
When reviewing the recent developments on the absorbance based methods, surface enhanced resonance Raman scattering (SERRS) is was found worth of being mentioned. Using this method, benzotriazoles are sensitively detected as products of enzymatic cleavage of corresponding esters [7]. Using the same method, protease activity was measured in [8]. Alternatively to developing a new detection method, classical colorimetric methods can be coupled to the target product in order to quantify its formation rate. In this way, recently [9], the evolution of a hydroxynitrile lyase was done using a colorimetric assay to quantify the generated cyanide. In a similar manner, in the group of Bornscheuer, an enzymatic cascade reaction for the detection of the release of acetate was developed for the screen of hydrolases [10, 11].
Next, while studying recent fluorogenic based assays recent reports, the introduction of FACS for detection of activity at the cell level was acknowledged as a major development in this area. Using FACS, enantioselectivity of horseradish peroxidase (HRP) was the property screened for in a library of cells displaying HRP on the surface [12, 13]. Enantiomers of tyrosinol were covalently attached to red- and blue-fluorescent dyes and subsequently oxidized by the surface HRP variants. The resulting radicals labeled the cells that were sorted according to enantioselectivity of respective HRP variant using FACS. Several other enzymes that were evolved by FACS screening of single cells: proteases [5, 14], aminoacyl-tRNA synthetases [15, 16], recombinases [17], glutathione-S transferases [18, 19], glycosyltransferases [20], esterases [21], and glucose oxidase in the Schwaneberg group (unpublished results).
In the case of the abovementioned examples, the substrate of the respective enzymes could permeate the cells, being available to the cytosolic enzyme, while the product was entrapped in the cell. However, when this is not the case, and the product freely di uses out of the cell, water in oil emulsions were the solution found to screen the needed enzymes using FACS.
At the next level, the genes are entrapped in water in oil emulsions and in vitro transcriptiontranslation is carried out. In the latter case, in addition to the translation machinery, the enzyme substrate is also encapsulated in the droplet. Enzyme activity is reported by the substrate becoming fluorescent.
Furthermore, in the case of compartmentalised self-replication (CSR), water in oil emulsions are used to entrap the cells in which a library of polymerase gene was cloned. The polymerase that has improved activity will have more copies of its own gene. This will lead to an enrichment of the library as described for Taq DNA polymerase in the group of Holliger [22]. The latter authors also suggest that the CSR system can be used for the directed evolution of enzymes involved in DNA replication or gene expression such as polymerases, ligases, helicases, etc This statement is proven in 2004 by evolving a restriction endonuclease using CSR [23].
The next generation of screening assays started to be developed for microfluidic devices in which up to 10000 highly monodisperse aqueous droplets per second are generated in a continuous oil phase. However, most of the work published in this area is proof-of-principle work. This is why there is more work to be done to integrate the existing physical and biological unit operations in such a way that there is reproducibility when larger number of experiments are performed [24].
Our company has experience with developing screening assays for different classes of enzymes. Through various collaborative projects, our high throughput screening systems were successfully validated. Amongst our expertise we can mention: colorimetric and fluorometric multiple well plate screening, solid plate screening (qualitative pre-screen for improving hit rate before a quantitative re-screen of the positive hits), CSR (emulsion screening for enzymes involved in DNA replication or gene expression) etc. Moreover, we can also use HPLC, GC or TLC for low to medium throughput screening or enzyme characterization. All this is possible because SeSaM-Biotech has access to a fully equipped lab.
1 -Schmidt-Dannert C, Arnold FH (1999) Directed evolution of industrial enzymes Trends Biotechnol. 17(4), p135-6.
2 -Link AJ, Jeong KJ, Georgiou G (2007) Beyond toothpicks: new methods for isolating mutant bacteria Nat Rev Microbiol. 5(9), p680-8.
3 -Cohen,N., Abramov, S., Dror, Y., and Freeman, A (2001) In vitro enzyme evolution: the screening challenge of isolating the one in a million Trends Biotechnol. 19, p507-510.
4 -Lin H, and Cornish VW (2002) Screening and selection methods for large-scale analysis of protein function Angew. Chem. Int. Ed Engl. 41, p4402-4425.
5 -Olsen M, Iverson B, and Georgiou G (2000) High-throughput screening of enzyme libraries Curr. Opin. Biotechnol. 11, p331-337.
6 -Aharoni A, Amitai G, Bernath K, Magdassi S, and Tawfik DS (2005) High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments Chem. Biol 12, p1281-9.
7 -Moore BD, Stevenson L, Watt A, Flitsch S, Turner NJ, Cassidy C, Graham D (2004) Rapid and ultra-sensitive determination of enzyme activities using surface-enhanced resonance Raman scattering Nat Biotechnol. 22(9), p1133-8.
8 -Ingram A, Byers L, Faulds K, Moore BD, Graham D (2008) SERRS-based enzymatic probes for the detection of protease activity J Am Chem Soc. 130(36), p11846-7.
9 -Andexer J, Guterl JK, Pohl M, Eggert T (2006) A high-throughput screening assay for hydroxynitrile lyase activity Chem Commun (Camb). 240, p4201-3.
10 -Bottcher D, Bornscheuer UT (2006) High-throughput screening of activity and enantioselectivity of esterases Nat Protoc. 1(5), p2340-3.
11 -Bartsch S, Kourist R, Bornscheuer UT (2008) Complete inversion of enantioselectivity towards acetylated tertiary alcohols by a double mutant of a Bacillus subtilis esterase Angew Chem Int Ed Engl. 47(8), p1508-11.
12 -Lipovsek D, Antipov E, Armstrong KA, Olsen MJ, Klibanov AM, Tidor B, Wittrup KD (2007) Selection of horseradish peroxidase variants with enhanced enantioselectivity by yeast surface display Chem Biol. 14(10), p1176-85.
13 -Antipov E, Cho AE, Wittrup KD, Klibanov AM (2008) Highly L and D enantioselective variants of horseradish peroxidase discovered by an ultrahigh-throughput selection method Proc Natl Acad Sci U S A. 105(46), p17694-9.
14 -Varadarajan N, Pogson M, Georgiou G, Iverson BL (2009) Proteases that can distinguish among di erent post-translational forms of tyrosine engineered using multicolor flow cytometry J Am Chem Soc. 131(50), p18186-90.
15 -Santoro SW, Wang L, Herberich B, King DS, Schultz PG (2002) An efficient system for the evolution of aminoacyl-tRNA synthetase specifficity Nat Biotechnol. 20(10), p1044-8.
16 -Liu W, Alfonta L, Mack AV, Schultz PG (2007) Structural basis for the recognition of para-benzoyl-L-phenylalanine by evolved aminoacyl-tRNA synthetases Angew Chem Int Ed Engl. 46(32), p6073-5.
17 -Santoro SW, Schultz PG (2003) Directed evolution of the substrate specificities of a site-specific recombinase and an aminoacyl-tRNA synthetase using fluorescence-activated cell sorting (FACS) Methods Mol Biol. 230, p291-312.
18 -Eklund BI, Edalat M, Stenberg G, Mannervik B (2002) Screening for recombinant glutathione transferases active with monochlorobimane Anal Biochem. 309(1), p102-8.
19 -Griswold KE, Aiyappan NS, Iverson BL, Georgiou G (2006) The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase J Mol Biol. 364(3), p400-10
20 -Aharoni A, Thieme K, Chiu CP, Buchini S, Lairson LL, Chen H, Strynadka NC,Wakarchuk WW, Withers SG (2006) High-throughput screening methodology for the directed evolution of glycosyltransferases Nat Methods. 3(8), p609-14.
21 -Becker S, Schmoldt HU, Adams TM, Wilhelm S, Kolmar H (2004) Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts Curr Opin Biotechnol. 15(4), p323-9.
22 -Ghadessy FJ, Ong JL, Holliger P (2001) Directed evolution of polymerase function by compartmentalized self-replication Proc Natl Acad Sci U S A. 98(8),p4552-7.
23 -Doi N, Kumadaki S, Oishi Y, Matsumura N, Yanagawa H (2004) In vitro selection of restriction endonucleases by in vitro compartmentalization Nucleic Acids Res. 32(12), e95.
24 -Schaerli Y, Hollfelder F (2009) The potential of microfluidic water-in-oil droplets in experimental biology Mol Biosyst. 5(12), p1392-404