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Chapter 32 : Enzyme Engineering by Directed Evolution

Author: Manfred T. Reetz
Content Type: Reference
Publication Year: 2010

Category: Applied and Industrial Microbiology

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Abstract:

This chapter treats exclusively directed evolution of proteins, the primary focus being on recent methodology developments. During the past 25 years, numerous gene mutagenesis methods have been developed. It is difficult even for experts to make the optimal choice, because comparative studies focusing on relative efficiency are rare. A few select studies in directed evolution are reviewed in the chapter, because they allow conclusions regarding the relative merits of different mutagenesis methods and strategies. In a landmark study, single-gene DNA shuffling was applied to turn β-galactosidase (BGAL) into a β-fucosidase, thereby changing the substrate scope. The enzyme hydrolyzes β-galactosyl linkages such as the β (1,4)-linkage in lactose. In principle, epPCR and DNA shuffling are independent of structural data, whereas saturation mutagenesis generally needs such knowledge to make a decision regarding the randomization sites. Alternatively, saturation mutagenesis can be applied systematically at every single amino acid position, as was reported in the thermostabilization of a xylanase. Subsequent to the first directed evolution study regarding the enantioselectivity of enzymes, which involved four cycles of epPCR in the hydrolytic kinetic resolution of a chiral ester catalyzed by a lipase, numerous academic and industrial studies have appeared that contribute to the generalization of this new approach to asymmetric catalysis. Subsequent investigations addressed the reasons for the apparent efficacy of iterative combinatorial active-site saturation testing as a form of iterative saturation mutagenesis (ISM).

Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32

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Enzyme Engineering by Directed Evolution
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Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32
/content/10.1128/9781555816827.ch32.ch32fig01
FIGURE 1
Individual steps in directed evolution.
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Image of FIGURE 1
FIGURE 1

Individual steps in directed evolution.

Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32
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FIGURE 2
Genetic selection system for laboratory evolution of enantioselectivity in a kinetic resolution ( ).
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Image of FIGURE 2
FIGURE 2

Genetic selection system for laboratory evolution of enantioselectivity in a kinetic resolution ( ).

Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32
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/content/10.1128/9781555816827.ch32.ch32fig03
FIGURE 3
Schematic illustration of QuikChange (Stratagene) ( ).
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Image of FIGURE 3
FIGURE 3

Schematic illustration of QuikChange (Stratagene) ( ).

Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32
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/content/10.1128/9781555816827.ch32.ch32fig04
FIGURE 4
Scheme illustrating the improved method for PCR-based saturation mutagenesis useful in the case of difficult-to-amplify templates ( ). The gene is represented by the dotted section, the vector backbone is shown in light gray, and the formed megaprimer in black. In the first stage of the PCR both the mutagenic primer (positions randomized represented by a white square) and the antiprimer (or another mutagenic primer, shown to the right) anneal to the template, and the amplified sequence is used as a megaprimer in the second stage. Finally, the template plasmids are digested by use of DpnI and the resulting library is transformed in bacteria. The scheme on the left illustrates the three possible options in the choice of the megaprimer size for a single-site randomization experiment. The scheme to the right represents an experiment with two sites simultaneously randomized.
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10.1128/9781555816827/f0470-01.gif
Image of FIGURE 4
FIGURE 4

Scheme illustrating the improved method for PCR-based saturation mutagenesis useful in the case of difficult-to-amplify templates ( ). The gene is represented by the dotted section, the vector backbone is shown in light gray, and the formed megaprimer in black. In the first stage of the PCR both the mutagenic primer (positions randomized represented by a white square) and the antiprimer (or another mutagenic primer, shown to the right) anneal to the template, and the amplified sequence is used as a megaprimer in the second stage. Finally, the template plasmids are digested by use of DpnI and the resulting library is transformed in bacteria. The scheme on the left illustrates the three possible options in the choice of the megaprimer size for a single-site randomization experiment. The scheme to the right represents an experiment with two sites simultaneously randomized.

Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32
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FIGURE 5
Library coverage calculated for NNK codon degeneracy at sites composed of one, two, three, four, and five amino acid positions (aa, amino acids) ( ).
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Image of FIGURE 5
FIGURE 5

Library coverage calculated for NNK codon degeneracy at sites composed of one, two, three, four, and five amino acid positions (aa, amino acids) ( ).

Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32
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/content/10.1128/9781555816827.ch32.ch32fig06
FIGURE 6
Library coverage calculated for NDT degeneracy at sites composed of one, two, three, four, and five amino acid positions (aa, amino acids) ( ).
10.1128/9781555816827/f0472-01_thmb.gif
10.1128/9781555816827/f0472-01.gif
Image of FIGURE 6
FIGURE 6

Library coverage calculated for NDT degeneracy at sites composed of one, two, three, four, and five amino acid positions (aa, amino acids) ( ).

Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32
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FIGURE 7
ISM using four sites, A, B, C, and D, each site in a given upward pathway in the fitness landscape being visited only once ( ).
10.1128/9781555816827/f0472-02_thmb.gif
10.1128/9781555816827/f0472-02.gif
Image of FIGURE 7
FIGURE 7

ISM using four sites, A, B, C, and D, each site in a given upward pathway in the fitness landscape being visited only once ( ).

Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32
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FIGURE 8
Scheme illustrating DNA shuffling for the case in which the parental genes originate from the WT by some sort of mutagenesis.
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10.1128/9781555816827/f0473-01.gif
Image of FIGURE 8
FIGURE 8

Scheme illustrating DNA shuffling for the case in which the parental genes originate from the WT by some sort of mutagenesis.

Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32
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/content/10.1128/9781555816827.ch32.ch32fig09
FIGURE 9
Thermostability diagram in the B-FIT-based evolution of Lip A ( ).
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10.1128/9781555816827/f0476-01.gif
Image of FIGURE 9
FIGURE 9

Thermostability diagram in the B-FIT-based evolution of Lip A ( ).

Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32
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/content/10.1128/9781555816827.ch32.ch32fig010
FIGURE 10
Iterative CASTing in the enhancement of enantioselectivity of the hydrolytic kinetic resolution of glycidyl phenyl ether catalyzed by ANEH variants ( ).
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10.1128/9781555816827/f0477-01.gif
Image of FIGURE 10
FIGURE 10

Iterative CASTing in the enhancement of enantioselectivity of the hydrolytic kinetic resolution of glycidyl phenyl ether catalyzed by ANEH variants ( ).

Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32
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Tables

Enzyme Engineering by Directed Evolution
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Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32
/content/10.1128/9781555816827.ch32.ch32tab01
TABLE 1
Oversampling necessary for 95% coverage as a function of NNK and NDT codon degeneracy assuming the absence of amino acid bias (44)
Generic image for table
TABLE 1

Oversampling necessary for 95% coverage as a function of NNK and NDT codon degeneracy assuming the absence of amino acid bias (44)

Citation: Reetz M. 2010. Enzyme Engineering by Directed Evolution, p 466-479. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch32

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