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Enzyme Engineering
An authoritative and up-to-date discussion of enzyme engineering and its applications
In Enzyme Engineering: Selective Catalysts for Applications in Biotechnology, Organic Chemistry, and Life Science, a team of distinguished researchers deliver a robust treatment of enzyme engineering and its applications in various fields such as biotechnology, life science, and synthesis. The book begins with an introduction to different protein engineering techniques, covers topics like gene mutagenesis methods for directed evolution and rational enzyme design. It includes industrial case studies of enzyme engineering with a focus on selectivity and activity.
The authors also discuss new and innovative areas in the field, involving machine learning and artificial intelligence. It offers several insightful perspectives on the future of this work.
Readers will also find:
- A thorough introduction to directed evolution and rational design as protein engineering techniques
- Comprehensive explorations of screening and selection techniques, gene mutagenesis methods in directed evolution, and guidelines for applying gene mutagenesis in organic chemistry, pharmaceutical applications, and biotechnology
- Practical discussions of protein engineering of enzyme robustness relevant to organic and pharmaceutical chemistry
- Treatments of artificial enzymes as promiscuous catalysts
- Various lessons learned from semi-rational and rational directed evolution
A transdisciplinary treatise, Enzyme Engineering: Selective Catalysts for Applications in Biotechnology, Organic Chemistry, and Life Science is perfect for protein engineers, theoreticians, organic, and pharmaceutical chemists as well as transition metal researchers in catalysis and biotechnologists.
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Veröffentlichungsjahr: 2023
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Table of Contents
Cover
Title Page
Copyright
Preface
About the Authors
1 Introduction to Directed Evolution and Rational Design as Protein Engineering Techniques
1.1 Methods and Aims of Directed Enzyme Evolution
1.2 History of Directed Enzyme Evolution
1.3 Methods and Aims of Rational Design of Enzymes
References
2 Screening and Selection Techniques
2.1 Introductory Remarks
2.2 Screening Methods
2.3 Selection Methods
2.4 Conclusions and Perspectives
References
3 Gene Mutagenesis Methods in Directed Evolution and Rational Enzyme Design
3.1 Introductory Remarks
3.2 Directed Evolution Approaches
3.3 Diverse Approaches to Rational Enzyme Design
3.4 Merging Semi‐rational Directed Evolution and Rational Enzyme Design by Focused Rational Iterative Site‐Specific Mutagenesis (FRISM)
3.5 Conclusions and Perspectives
References
4 Guidelines for Applying Gene Mutagenesis Methods in Organic Chemistry, Pharmaceutical Applications, and Biotechnology
4.1 Some General Tips
4.2 Rare Cases of Comparative Directed Evolution Studies
4.3 Choosing the Best Strategy When Applying Saturation Mutagenesis
4.4 Techno‐economical Analysis of Saturation Mutagenesis Strategies
4.5 Generating Mutant Libraries by Combinatorial Solid‐Phase Gene Synthesis: The Future of Directed Evolution?
4.6 Fusing Directed Evolution and Rational Design: New Examples of Focused Rational Iterative Site‐Specific Mutagenesis (FRISM)
References
5 Tables of Selected Examples of Directed Evolution and Rational Design of Enzymes with Emphasis on Stereo‐ and Regio‐selectivity, Substrate Scope and/or Activity
5.1 Introductory Explanations
References
6 Protein Engineering of Enzyme Robustness Relevant to Organic and Pharmaceutical Chemistry and Applications in Biotechnology
6.1 Introductory Remarks
6.2 Rational Design of Enzyme Thermostability and Resistance to Hostile Organic Solvents
6.3 Ancestral and Consensus Approaches and Their Structure‐Guided Extensions
6.4 Further Computationally Guided Methods for Protein Thermostabilization
6.5 Directed Evolution of Enzyme Thermostability and Resistance to Hostile Organic Solvents
6.6 Application of epPCR and DNA Shuffling
6.7 Saturation Mutagenesis in the B‐FIT Approach
6.8 Iterative Saturation Mutagenesis (ISM) at Protein–Protein Interfacial Sites for Multimeric Enzymes
6.9 Conclusions and Perspectives
References
7 Artificial Enzymes as Promiscuous Catalysts in Organic and Pharmaceutical Chemistry
7.1 Introductory Background Information
7.2 Applying Protein Engineering for Tuning the Catalytic Profile of Promiscuous Enzymes
7.3 Applying Protein Engineering to P450 Monooxygenases for Manipulating Activity and Stereoselectivity of Promiscuous Transformations
7.4 Conclusions and Perspectives
References
8 Learning Lessons from Protein Engineering
8.1 Introductory Remarks
8.2 Additive Versus Nonadditive Mutational Effects in Fitness Landscapes Revealed by Partial or Complete Deconvolution
8.3 Unexplored Chiral Fleeting Intermediates and Their Role in Protein Engineering
8.4 Case Studies Featuring Mechanistic, Structural, and/or Computational Analyses of the Source of Evolved Stereo‐ and/or Regioselectivity
8.5 Conclusions and Suggestions for Further Theoretical Work
References
9 Perspectives for Future Work
9.1 Introductory Remarks
9.2 Extending Applications in Organic and Pharmaceutical Chemistry
9.3 Extending Applications in Biotechnology
9.4 Patent Issues
9.5 Final Comments
References
Index
End User License Agreement
List of Tables
Chapter 3
Table 3.1 Theoretical number of variants in a library obtained for a protei...
Table 3.2 Oversampling necessary for 95% library coverage as a function of ...
Table 3.3 Comparison of PCR‐based SM library with the on‐chip gene synthesi...
Chapter 4
Table 4.1 Conversion of cyclohexanone to stereoisomeric cyclohexane‐1,2‐dio...
Table 4.2 Statistical consequences as a function of grouping single CAST re...
Table 4.3 Choice of codon degeneracies at each position in the 441–444 loop...
Table 4.4 Combinatorial use of amino acids as building blocks employed in s...
Table 4.5 Quick quality control and
Q
‐values.
Table 4.6 Summary of P450‐BM3 sequencing results obtained from 96 single co...
Chapter 5
Table 5.1 Selected directed evolution studies of enzymes, many for enhanced...
Table 5.2 Selected studies of rational enzyme design (more publications are...
Chapter 6
Table 6.1 Specific activity,
T
50
, and 1‐propanol stability of WT and mutant...
Table 6.2 Summary of additional typical B‐FIT studies of enzymes for enhanc...
Chapter 7
Table 7.1 Summary of the directed evolution of the Kemp eliminase KE70.
Chapter 8
Table 8.1 Results of MD calculations.
Table 8.2 Stereoselective reduction of Baylis–Hillman adducts catalyzed by ...
Table 8.3 Computed activation barriers and reaction energies
a)
for rate‐det...
Table 8.4 Calculated energies (kcal mol
−1
) of the optimized structure...
Table 8.5 Catalytic profiles of WT RasADH and of selected single point muta...
Chapter 9
Table 9.1 Selected examples of therapeutic drugs, their access being possib...
Table 9.2 Classification of enzymes by the Enzyme Commission (EC).
List of Illustrations
Chapter 1
Scheme 1.1 The basic steps in the directed evolution of enzymes.
Scheme 1.2 Pedigree of
ebgA
alleles in evolved strains [10a]. Strain 1B1 car...
Scheme 1.3 First example of directed evolution of thermostability of an enzy...
Scheme 1.4 Mixed oligonucleotide mutagenesis of the gene MATa1 from
Saccharo
...
Scheme 1.5 Catalytic efficiency of WT subtilisin E and variant PC3 as cataly...
Scheme 1.6 Steps in the recombinant technique of splicing by overlap extensi...
Scheme 1.7 DNA shuffling starting from a single gene encoding a given enzyme...
Scheme 1.8 Concept of directed evolution of stereoselective enzymes with (
R)
Scheme 1.9 Hydrolytic kinetic resolution of
rac
‐
1
catalyzed by the lipase fr...
Scheme 1.10 First example of directed evolution of a stereoselective enzyme....
Scheme 1.11 First example of focused saturation mutagenesis at sites next to...
Scheme 1.12 Illustration of ISM. (a) Sites chosen for ISM; (b) 2‐, 3‐, and 4...
Scheme 1.13 Timeline of enzyme‐directed evolution.
Chapter 2
Scheme 2.1 Screening versus selection in directed evolution.
Figure 2.1 (a) Time course of the lipase‐catalyzed hydrolysis of two enantio...
Figure 2.2 On‐plate pretest for lipase activity based on halos that form upo...
Scheme 2.2 Periodate‐coupled fluorogenic assay designed for assessing the en...
Scheme 2.3 An enzyme‐coupled assay for assessing the activity of lipases or ...
Scheme 2.4 Utilization of isotopically labeled pseudo‐enantiomers in high‐th...
Scheme 2.5 First example of a medium‐throughput GC unit used in the directed...
Scheme 2.6 Developing a desensitized niacin repressive biosensor and its app...
Scheme 2.7 (a) Kinetic resolution of NAcetyl‐DL‐amino acid using
D
‐Acylase a...
Scheme 2.8 Genetic selection in the directed evolution of enantioselective e...
Scheme 2.9 A growth‐based selection method based on pro‐antibiotic substrate...
Scheme 2.10 A genetic selection system for directed evolution of enantiosele...
Scheme 2.11 A selection system utilizing a pseudo‐racemate (
S
)‐
1
/(
R
)‐
4
in th...
Scheme 2.12 Hydrolytic kinetic resolution of
rac
‐
6
directed by LipA.
Scheme 2.13 Compounds used in devising a selection system for the evolution ...
Scheme 2.14 Dual selection system for evolving enantioselectivity of a
Bacil
...
Scheme 2.15 Radical polymerization of
L
‐ or
D
‐tyrosine and Alexa‐488 derivat...
Scheme 2.16 EstA‐catalyzed hydrolytic kinetic resolution and used in FACS‐ba...
Scheme 2.17 Coupling reactions ensuring covalent attachment of tyramide spec...
Scheme 2.18 Two distinct labeled enantiomers of 2‐MDA tyramide ester substra...
Figure 2.3 FACS‐based high‐throughput screening for evolving stereoselectivi...
Scheme 2.19 The esterase PFE‐directed hydrolytic kinetic resolution toward t...
Scheme 2.20 Schematic representation of gene selection in compartmentalized ...
Figure 2.4 Deep mutational scanning‐assisted ultrahigh‐throughput sequence‐f...
Chapter 3
Scheme 3.1 Illustration of epPCR. Source: Adapted from Ref. [7].
Scheme 3.2 Mutational bias of epPCR in the case of the lipase from
Bacillus
...
Figure 3.1 epPCR‐directed evolution of prodigiosin ligase PigC with the aim ...
Scheme 3.3 Illustration of random insertion/deletion (RID) mutagenesis for t...
Figure 3.2 Comparison of the crystal structure of PAMO (1W4X) (left) and the...
Figure 3.3 The general procedure of transposon‐based mutagenesis approach (T...
Figure 3.4 TRIAD‐guided directed evolution of a bifunctional enzyme.
Scheme 3.4 Illustration of saturation mutagenesis based on the QuikChange™ (...
Scheme 3.5 General illustration of megaprimer PCR.
Scheme 3.6 Steps in site‐directed mutagenesis by overlap extension PCR which...
Scheme 3.7 Efficient method for saturation mutagenesis useful for cases of d...
Scheme 3.8 (a) Illustration of canonical SSM library construction using the ...
Scheme 3.9 Illustration of the CAST/ISM strategy. (a) Rationally chosen site...
Figure 3.5 Library coverage calculated for NNK codon degeneracy at sites con...
Figure 3.6 Library coverage calculated for NDT degeneracy at sites consistin...
Figure 3.7 Correlation between oversampling factor
O
f
and percent library cov...
Figure 3.8 Probabilities of “full coverage” and of discovering at least one ...
Figure 3.9 Patrick–Firth versus Nov statistical metrics. There is a relation...
Figure 3.10 Screening effort required for different randomization schemes re...
Figure 3.11 Distribution of nucleotide bases in the randomized residue Leu42...
Figure 3.12 Searching sequence space by single‐gene shuffling versus family ...
Figure 3.13 Workflow of the directed evolution of the enzyme PvGmGSTUG via D...
Scheme 3.10 A schematic example of biased mutation‐assembling, assuming a ba...
Scheme 3.11 General concept of ADO with two strategies for the linking of fr...
Scheme 3.12 Schematic overview of CALB engineering by circular permutation a...
Figure 3.14 Model reaction and library design for comparing traditional PCR‐...
Figure 3.15 Combinatorial mutant library fabrication via the Twist system....
Figure 3.16 LEH‐catalyzed hydrolytic desymmetrization of prochiral cyclohexe...
Figure 3.17 Comparison of the Twist‐based library (blue) and the conversiona...
Figure 3.18 Percentage distribution of different residues at positions 78, 8...
Scheme 3.13 SCHEMA disruption based upon a contact matrix representing inter...
Scheme 3.14 Formal representation of ProSAR.
Scheme 3.15 Steps when applying ASRA to directed evolution.
Figure 3.19 Optimal reordering of the
E
‐value enantioselectivity landscapes ...
Figure 3.20 Workflow of the Innov'SAR method. (a) A protein sequence is enco...
Figure 3.21 Ranking of the enantioselectivity
E
‐values for the 512 possible ...
Figure 3.22 Schematic illustration of the machine‐learning‐guided mutagenesi...
Figure 3.23 Enzymatic Diels–Alder reaction achieved by Rosetta‐based rationa...
Figure 3.24 RosettaRemodel effective for a variety of protein backbone manip...
Figure 3.25 Conversion of penicillin G and N into cephalosporins catalyzed b...
Figure 3.26 Adapted illustration of Focused Rational Iterative Site‐specific...
Figure 3.27 Stereoconvergent transacylation between 2‐phenylpropionic acid
p
Figure 3.28 Focused Rational Iterative Site‐specific Mutagenesis (FRISM) app...
Chapter 4
Scheme 4.1 Utility of computationally designed mutants of ω‐transaminase fro...
Scheme 4.2 Reaction mechanism for the transamination of acetophenone to enan...
Figure 4.1 Key residues lining the large and small binding pockets of ω‐TA P...
Figure 4.2 Ketone substrates (left half) and (
S
)‐configurated chiral amines ...
Scheme 4.3 The conventional procedure of synthesizing hydroquinone (HQ) from...
Figure 4.3 The flexibility analysis of GNCA and TEM‐1 β‐lactamase. (a) The d...
Figure 4.4 The substituting residues selected by flexibility profiles. (a) N...
Scheme 4.4 Two choices when attempting to optimize thermostability and activ...
Scheme 4.5 Preferred approach for the simultaneous optimization of two catal...
Scheme 4.6 The strategy of
in vitro
coevolution (substrate walking) for engi...
Scheme 4.7 Model reactions in the directed evolution of a fucosidase from a ...
Scheme 4.8 DNA shuffling process used in the directed evolution of a fucosid...
Figure 4.5 Structure of BGAL active site [32] used as a guide in designing s...
Figure 4.6 Selected BGAL variants resulting from saturation mutagenesis at a...
Scheme 4.9 Model reaction used in the directed evolution of the enzyme PAL....
Scheme 4.10 Alternating saturation mutagenesis at different positions with e...
Scheme 4.11 Extended CMCM in the evolution of an (
S
)‐selective variant X in ...
Scheme 4.12 Summary of all comparative studies of PAL as a catalyst in the h...
Figure 4.7 Schematic representation of amino acid residues considered for sa...
Scheme 4.13 Two different approaches to the use of reduced amino acid alphab...
Scheme 4.14 Biocatalytic route to sitagliptin phosphate using a transaminase...
Scheme 4.15 Model compound (
11
) used in substrate walking based on
in vitro
...
Scheme 4.16 Model hydrolytic kinetic resolution of the glycidyl ether
rac
‐
15
Figure 4.8 CAST sites A–E [21] of the epoxide hydrolase from
Aspergillus nig
...
Figure 4.9 Complete experimental exploration of a 24‐pathway ISM system invo...
Figure 4.10 Fitness pathway landscape featuring the 24 trajectories leading ...
Figure 4.11 Free energy profiles of the 24 ISM pathways in the directed evol...
Figure 4.12 Toward universal blood using ISM [57]. (a) Carbohydrate antigeni...
Figure 4.13 Evolutionary pathways of Sp3GH98 based on iterative saturation m...
Figure 4.14 Schematic illustration of a unique mechanism to generate H antig...
Scheme 4.17 Oxidative kinetic resolution catalyzed by PAMO mutants.
Scheme 4.18 Multiple Sequence Alignment (MSA) of eight BVMOs (441–444 loop i...
Scheme 4.19 PAMO‐catalyzed oxidative kinetic resolution of 2‐alkyl substitut...
Scheme 4.20 Substrates investigated in the CAST‐based directed evolution of ...
Figure 4.15 Binding pocket of CALA showing tetrahedral intermediate with sub...
Scheme 4.21 Hydrolytic desymmetrization of meso‐epoxides catalyzed by LEH an...
Figure 4.16 (a) All theoretically possible reductive products of ethyl secon...
Figure 4.17 Large randomization site defined by 10 amino acid positions (gre...
Scheme 4.22 Primer design and library construction using valine as the sole ...
Figure 4.18 Best hits discovered in a mutant library created by a single sat...
Scheme 4.23 Application of best variants of alcohol dehydrogenase TbSADH as ...
Figure 4.19 Total cost as a function of screening cost, when randomizing a s...
Figure 4.20 Cost space partitioned into regions according to the optimal ran...
Figure 4.21 How to proceed when choosing solid‐phase gene for mutant library...
Figure 4.22 Engineering the regioselectivity of UGT74AC2 guided by Focused R...
Chapter 6
Figure 6.1 Overlay of the eightfold mutated TLP‐ste variant. The substitutio...
Figure 6.2 Residual activity and stability determined for TLP‐ste at 80 °C (...
Figure 6.3 Temperature melt measuring (a) and activity half‐life measurement...
Figure 6.4 (a) van der Waals representation of sites A23 and I140 in WT yCD ...
Figure 6.5 (a) Overlay of the 51 mutations in the designed variant. (b) Rela...
Figure 6.6 (a) Representations of the salt bridges in BSLA WT. (b) OSs resis...
Figure 6.7 The thermal resistance assay of BSLA WT and its variants.
Scheme 6.1 The procedure of Proteus to predict beneficial mutations. (a) Usi...
Figure 6.8 FRESCO strategy for protein thermostabilization.
Figure 6.9 Positions of 12 stabilizing mutations as revealed by the crystal ...
Figure 6.10 Workflow of the FireProt method. Individual steps involved in th...
Figure 6.11 Prediction of the candidate residues for mutagenesis.
Figure 6.12 Schematic representation of VisualCNA. (a) Illustration of the t...
Figure 6.13 Divide and combine approach to protein thermostabilization featu...
Figure 6.14 Catalytic performance of a variant of the feruloyl esterase from...
Figure 6.15 Workflow in the directed evolution of an improved xylanase.
Figure 6.16 NMR spectra recorded for native and thermally treated
15
N‐labele...
Figure 6.17 Results of limited ISM exploration starting from the best mutant...
Scheme 6.2 The workflow of ACS strategy for improving enzyme stability.
Figure 6.18 (a) Thermostability assay of Lip1 mutants. (b) Evaluation of the...
Figure 6.19 Thermostabilization of PcDTE following application of ISM [131]....
Chapter 7
Scheme 7.1 Diels–Alder reaction of azachalcones
2
with
3
leading to endo‐prod...
Scheme 7.2 Systematization for generating artificial metalloenzymes, origina...
Scheme 7.3 Whitesides system comprises a biotinylated achiral diphosphine/Rh...
Scheme 7.4 Chemical modification of tHisF mutant Cys9Ala/Asp11Cys by means o...
Scheme 7.5 Model Diels–Alder cycloaddition used in Rosetta‐design.
Scheme 7.6 Model reaction used in the directed evolution of the promiscuous ...
Figure 7.1 Model of the biotinylated diphosphine‐Rh‐complex in streptavidin ...
Scheme 7.7 Directed evolution of stereoselectivity of a promiscuous enzyme b...
Scheme 7.8 Enantioselective ketone reductions using the streptavidin platfor...
Figure 7.2 Iterative Saturation Mutagenesis (ISM) in the directed evolution ...
Figure 7.3 X‐ray crystal structure of [η
6
‐(benzene)RuCl(Biot‐
p
‐
L
)]⊂S112K Sav...
Scheme 7.9 Protein engineering of a HAMase based on dual gold activation of ...
Scheme 7.10 Directed evolution of a HAMase in an iterative fashion.
Scheme 7.11 Representation of a unique artificial metalloenzyme (ARM). (a) D...
Scheme 7.12 New artificial metalloenzymes based on the LmrR scaffold in whic...
Scheme 7.13 Protein‐catalyzed Kemp elimination
10
→
11
.
Figure 7.4 (a) The KE07 design, showing the TIM barrel scaffold of HisF (PDB...
Scheme 7.14 P450‐catalyzed insertion of nitrenes into nonactivated C—H bonds...
Scheme 7.15 Promiscuous reactivity of P450‐BM3.
Scheme 7.16 Protein engineering of P411‐C10 in the synthesis of internal cyc...
Scheme 7.17 (a) Site‐directed mutagenesis of sperm whale myoglobin on the ke...
Scheme 7.18 Intra‐ and inter‐molecular promiscuous reactions of mutants of I...
Scheme 7.19 Rational enzyme design based on a combination of semi‐rational d...
Figure 7.5 An artificial olefin metathease based on anchoring a Grubbs–Hovey...
Scheme 7.20 Representation of hemin association with catalytic self‐assembli...
Chapter 8
Figure 8.1 Changes in transition‐state stabilization energies for the multip...
Scheme 8.1 Model reaction catalyzed by the lipase PAL.
Figure 8.2 Best ISM pathway B → A leading to the triple mutant 1B2 (Leu162As...
Scheme 8.2 Mechanism of lipase‐catalyzed hydrolysis of esters.
Figure 8.3 Comparison of the oxyanions with bound (
S
)‐substrate at the catal...
Scheme 8.3 Systematization of additive and nonadditive mutational effects in...
Figure 8.4 Fitness pathway landscape showing the 24 pathways leading from WT...
Figure 8.5 Fitness pathway landscape in the frontal view of Figure 8.4 of al...
Scheme 8.4 Concept of fleeting chiral intermediates. (a) General representat...
Figure 8.6 Directed evolution of the esterase from
Archaeoglobus fulgidus
(A...
Figure 8.7 Structural basis for reversed enantioselectivity of AFEST. (a) Do...
Scheme 8.5 Hydrolytic kinetic resolution of
rac
‐
1
catalyzed by the epoxide h...
Scheme 8.6 Mechanism of the epoxide hydrolase ANEH.
Figure 8.8 Kinetic analysis of variant LW202 as catalyst in separate reactio...
Scheme 8.7 Definition of the distance
d
in the rate‐determining step of the ...
Figure 8.9 Interpretation of crystal structures of WT ANEH and evolved varia...
Scheme 8.8 Binding modes in the active site of ene‐reductases. (a) Tradition...
Figure 8.10 Location of substrate 2‐hydroxymethylcyclopentenone in OYE1 muta...
Figure 8.11 Schematic representation of the role of OYE1 variants characteri...
Figure 8.12 Superposition of CmOYE structures in the absence (green) and pre...
Scheme 8.9 Two‐step biocatalytic conversion of ketoisophorone to (4
R
,6
R
)‐act...
Figure 8.13 Computed transition state geometries of the lowest energy pathwa...
Scheme 8.10 Mechanism of P450 monooxygenases. Intermediate
VII
is catalytica...
Scheme 8.11 Ideal pose of a substrate for smooth oxidative hydroxylation ini...
Figure 8.14 The substrate binding cavity of P450 BM3 harboring cyclododecane...
Scheme 8.12 P450‐BM3 catalyzed oxidative hydroxylation of testosterone.
Figure 8.15 Computed pose of testosterone (
6
) explaining 2ß‐selectivity (mut...
Scheme 8.13 Secondary structural elements determining regioselectivity and a...
Scheme 8.14 Computed preferred poses of testosterone in binding pockets of P...
Scheme 8.15 Mechanism of Baeyer–Villiger reactions using synthetic reagents ...
Scheme 8.16 Model TmCHMO‐catalyzed reaction used in two BVMO studies.
Figure 8.16 Results of deconvoluting the best TmCHMO mutant LGY3‐D‐E1 (L145G...
Figure 8.17 Computationally‐optimized structures of the transition states in...
Scheme 8.17 Reductive desymmetrization of 1,3‐cyclodiketones. (a) Model star...
Scheme 8.18 ISM campaign of RasADH. Enzyme activity of WT is taken as 1.0, a...
Figure 8.18 X‐ray structures of WT RasADH and variant F12. (a) Overlay of WT...
Figure 8.19 Conformational population analyses of the two most important com...
Figure 8.20 Molecular dynamics analysis of variant F12. (a) Distribution of ...
Chapter 9
Scheme 9.1 Terminal hydroxylation of medium‐chain fatty acids to ω‐hydroxy f...
Figure 9.1 Illustration of the bio‐based circular plastic economy in the fut...
Figure 9.2 Computer‐guided design of a phenylalanine aminomutase for the β‐t...
Guide
Cover
Table of Contents
Title Page
Copyright
Preface
About the Authors
Begin Reading
Index
End User License Agreement
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Manfred T. Reetz, Zhoutong Sun, and Ge Qu
Enzyme Engineering
Selective Catalysts for Applications in Biotechnology, Organic Chemistry, and Life Science
Authors
Dr. Manfred T. ReetzMax‐Planck‐Institut für KohlenforschungMülheim an der RuhrGermany
Prof. Zhoutong SunTianjin Institute of Industrial BiotechnologyChinese Academy of SciencesChina
Prof. Ge QuTianjin Institute of Industrial BiotechnologyChinese Academy of SciencesChina
Cover Images: © Zhoutong Sun, Manfred T. Reetz, and Ge Qu; © theasis/Getty Images
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Preface
The term directed evolution is relevant in two very different research areas: (i) the genetic manipulation of functional RNAs as pioneered by Sol Spiegelmann in 1967 in a Darwinian manner and continued by a number of present‐day experts for potential applications, inter alia, as aptamers; and (ii) the genetic manipulation of DNA with the aim to engineer the catalytic profiles of enzymes as catalysts in organic and pharmaceutical chemistry and in biotechnology. Both approaches concern the age‐old dream of imitating evolution in Nature as it has developed for billions of years, but of course not wanting to wait for such long periods! This monograph is a comprehensive treatise on the second research area.
A number of books have appeared on the directed evolution of enzymes (also called laboratory evolution), generally published by editors who asked scientists of their choice to write specific chapters on certain special topics. A different publishing approach is to write a monograph by a single author, specifically in the field of directed evolution. This would enable a more critical and comparative view of the different mutagenesis approaches and strategies practiced in this exciting research field. It was first presented in the Wiley‐VCH book by Manfred T. Reetz, titled Directed Evolution of Selective Enzymes: Catalysts for Organic Chemistry (2016). The limitation of this seminal monograph is the fact that the alternative to directed evolution, namely rational enzyme design as a protein engineering technique, was not at all highlighted. During the last six years, the methodology development of directed evolution has continued to produce notable advances, as have the number of applications in synthetic organic chemistry, natural products synthesis, and pharmaceutical chemistry. The time for a second edition of the Reetz monograph seemed to be ripe. However, the editors of Wiley‐VCH made a better and more challenging suggestion, namely to produce a unique monograph which covers both approaches to protein engineering with the evolution of selective catalysts. For this transdisciplinary task, two additional authors were willing to participate, a microbiologist with experience in applications of enzymes in chemistry and biotechnology and a bioinformatician with expertise in the computational design of proteins. Thus, readers may find the languages of chemistry and biology meet to talk about protein engineering in this book.
The present monograph with the title, Enzyme Engineering: Selective Catalysts for Applications in Biotechnology, Organic Chemistry, and Life Science, brings together three experts who have written a comprehensive treatise on both approaches to protein engineering, namely directed evolution and rational enzyme design. We believe that it is ideally suited for master's and doctoral students in university courses, but also for postdocs and other advanced scientists at different research institutions and those already working in industry. The first chapter introduces the basic concepts, goals, and mutagenesis methods as well as the areas of practical applications. Thereafter, several chapters follow in which the complexity of the material is gradually increased, with further details and information on effective mutagenesis strategies being offered. Importantly, the differences, limitations, and advantages of the different approaches are critically analyzed.
The monograph also contains chapters representing special topics, which are nevertheless important. These include information on how to evolve protein robustness under operating conditions as an alternative to traditional enzyme immobilization, developing promiscuous enzymes, as for example artificial metalloenzymes, and learning lessons from directed evolution, which reveals hitherto unnoticed mechanistic intricacies, thereby enabling further effective methodology developments, which include machine learning. The final chapter constitutes a concise resume, offers ideas for future research and challenging applications in organic and pharmaceutical chemistry and biotechnology, and gives tips on patent problems. The importance of ethical issues is likewise stressed.
The reader will discover in several chapters how semi‐rational directed evolution and rational enzyme design have merged, specifically by focusing mutations at sites lining the binding pocket. The graphic on the front cover is a succinct reminder of this strategy. It features the key as a symbol of the substrate entering the binding pocket. The optimal shape at the active site for ensuring high stereo‐ and/or regioselectivity can be achieved by protein engineering.
March 2023
Manfred T. Reetz, Zhoutong Sun, and Ge QuMax‐Planck‐Institut für KohlenforschungMülheim an der RuhrGermany
Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesChina
About the Authors
Manfred T. Reetz (born 1943) obtained his doctoral degree in synthetic organic chemistry in 1969 under the direction of Ulrich Schöllkopf at Göttingen University in Germany followed by a postdoctoral stay with Reinhard W. Hoffmann at Marburg University. He then performed independent research in various institutions in Germany and the USA, including directorship of the Max‐Planck‐Institut für Kohlenforschung in Mülheim for two decades, where he pioneered the concept of directed evolution of stereo‐ and regio‐selective enzymes as catalysts in organic and pharmaceutical chemistry. After his first retirement in 2011, he continued research at Marburg University and at the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, and returned to Mülheim in 2019.
Zhoutong Sun obtained his doctoral degree in microbiology with Prof. Sheng Yang at the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, in 2012. He then moved to Nanyang Technological University in Singapore as a research fellow in metabolic engineering. One year later, he joined the Reetz group as a postdoc at MPI für Kohlenforschung and Marburg University in directed evolution and biocatalysis. In 2016, he became a full professor at the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. His research interests are in enzyme engineering, metabolic engineering, and synthetic biology.
Ge Qu received his PhD in bioinformatics from Adam Mickiewicz University in Poland in 2016. Currently, he is working as an associate professor at the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, in the group of Professor Zhoutong Sun. His major research interests are the discovery and computational engineering of enzymes that have potential as industrial biocatalysts.
1Introduction to Directed Evolution and Rational Design as Protein Engineering Techniques
1.1 Methods and Aims of Directed Enzyme Evolution
For decades organic chemists have viewed enzymes as possible catalysts in their toolbox of synthetic methods with a great deal of skepticism, traditional textbooks hardly mentioning them. Indeed, even modern monographs on organic chemistry provide little information on the importance of these biocatalysts [1]. On the other hand, textbooks on enzymes in organic chemistry and biotechnology have interested mainly specialists [2], not organic chemists. Apart from psychological reasons, the actual limitations of enzymes were known to everyone, which are as follows:
Insufficient robustness under operating conditions.
Insufficient activity.
Narrow substrate scope.
Insufficient or wrong stereoselectivity.
Insufficient or wrong regioselectivity.
Sometimes product inhibition.
In addition, organic chemists were not trained to handle enzymes and therefore preferred to be content with the use and development of their own synthetic homogeneous and heterogeneous catalysts, knowing that enzymes cannot catalyze many or most of the important reaction types that dominate modern synthesis. Today all of these problems can be addressed and generally solved by applying directed evolution, as summarized in a 2016 monograph [3] authored by Manfred T. Reetz, and in more recent reviews [4]. It mimics Darwinian evolution as it occurs in nature, but it does not constitute real natural evolution. The process consists of several steps (Scheme 1.1), beginning with mutagenesis of the gene encoding the enzyme of interest. The library of mutated genes is then inserted into a bacterial or yeast host, such as Escherichia coli or Pichia pastoris, respectively, which is plated out on agar plates. After a growth period, single colonies appear, each originating from a single cell, which now begins to express the respective protein variants. This ensures the required linkage between phenotype and genotype. Multiple copies of transformants as well as wild type (WT) appear, which unfortunately decreases the quality of libraries and increases the screening effort. Colony harvesting must be performed carefully because cross contamination leads to the formation of inseparable mixtures of mutants with concomitant misinterpretations. The colonies are picked by a robotic colony picker (or manually using toothpicks) and placed individually in the wells of 96‐ or 384‐format microtiter plates, which contain nutrient broth. Portions of each well content are then placed in the respective wells of another microtiter plate where the screening (Chapter 2) for a given catalytic property ensues. In some (fortunate) cases, a sufficiently improved variant (hit) is identified in such an initial library. If this does not happen, which proves to be the case most often, then the gene of the best variant is extracted and used as a template in the next cycle of mutagenesis/expression/screening (Scheme 1.1). This exerts “evolutionary pressure,” the underlying characteristic of directed evolution.
Scheme 1.1 The basic steps in the directed evolution of enzymes.
If a library in a recursive mode fails to harbor an improved mutant/variant, the Darwinian process ends abruptly at a local minimum on the fitness landscape. Fortunately, researchers have developed ways to escape from such local minima (“dead ends”) (Chapter 4).
Directed evolution is thus an alternative to so‐called “rational design” in which the researcher utilizes structural, mechanistic, and sequence information, possibly flanked by computational aids, to perform site‐directed mutagenesis at a given position in a protein (Section 1.3). Needed for this alternative is the molecular biological technique of site‐specific mutagenesis with exchange of an amino acid at a specific position in a protein by one of the other 19 canonical amino acids, as established by Michael Smith in the late 1970s [5a], which led to the Nobel Prize [5b]. The method is based on designed synthetic oligonucleotides and has been used extensively by Fersht [6] and numerous other groups in the study of enzyme mechanisms. Application of the Smith technique in rational enzyme design has been shown to be successful mainly when aiming to increase protein robustness (Section 1.3 and Chapter 6). However, when aiming for enhanced or reversed enantioselectivity, diastereoselectivity, and/or regioselectivity, rational design is much more difficult, in which case directed evolution is generally the preferred strategy [3, 4].
Directed evolution of enzymes is not as straightforward as it may appear to be. The challenge in putting Scheme 1.1 into practice has to do with the vastness of protein sequence space. High structural diversity is easily achieved in random mutagenesis, but the experimenter is quickly confronted by the so‐called “numbers problem,” which in turn relates to the screening effort (bottleneck). When mutagenizing a given protein, the theoretical number of variants N is described by Eq. (1.1), which is based on the use of all 20 canonical amino acids as building blocks [3, 4]:
where M denotes the total number of amino acid substitutions per enzyme molecule and X is the total number of residues (size of protein in terms of amino acids). For example, when considering an enzyme composed of 300 amino acids, 5700 different mutants are possible if only one amino acid is exchanged randomly, 16 million if two substitutions occur simultaneously, and about 30 billion if three amino acids are substituted simultaneously.
Calculations of this kind pinpoint a dilemma that accompanies directed evolution to this day, namely how to probe the astronomically large protein sequence space efficiently. One strategy is to limit diversity to a point at which screening can be handled within a reasonable time, but excessive reduction of diversity reduction should be avoided because then the frequency of hits in a library begins to diminish dramatically. Finding the optimal compromise constitutes the primary issue of this monograph. A very different strategy is to develop selection systems rather than experimental platforms that require screening (Chapter 2). In a selection system, the host organism thrives and survives because it expresses a variant having the catalytic characteristics that the researcher wants to evolve. A third approach is based on the use of various types of display systems, which are sometimes called “selection systems,” although they are more related to screening. These issues are delineated in Chapter 2, which serves as a guide for choosing the appropriate system. Since it is extremely difficult to develop genuine selection systems or display platforms for directed evolution of stereo‐ and regioselective enzymes, researchers had to devise medium‐ and high‐throughput screening systems (Chapter 2).
1.2 History of Directed Enzyme Evolution
Readers of this monograph may not be interested in historical aspects, but certainly doctoral students and postdocs who want to learn directed evolution are advised to read this section carefully. Much can be learned by seeing in some detail how a given field developed, some contributions being interesting but perhaps lacking essential conclusions for further work, others actually opening a new research field by posting seminal publications. At the end of this section, the most important developments are summarized in a “timeline” scheme.
Scientists have strived for a long time to “reproduce” or mimic natural evolution in the laboratory. In 1965–1967 Spiegelman et al., performed a “Darwinian experiment with a self‐duplicating nucleic acid molecule” (RNA) outside a living cell [7]. It was believed that this mimics an early precellular evolutionary event. Later investigations showed that Spiegelman's RNA molecules were not truly self‐duplicating, but his contributions marked the beginning of a productive new research area on RNA evolution, fueled seminally by Szostak and Joyce [8]. Directed evolution at RNA level is a very different field of research with totally different goals, focusing on selection of RNA aptamers, selection of catalytic RNA molecules, or evolution of RNA polymerase ribozyme and ribozymes by continuous serial transfer. Directed evolution in this area has been reviewed [8b]. The term “directed evolution” in the area of protein engineering was used as early as 1972 by Francis and Hansche, describing an in vivo system involving an acid phosphatase in Saccharomyces cerevisiae[9]. In a population of 109 cells, spontaneous mutations in a defined environment were continuously screened over 1000 generations for their influence on efficiency and activity of the enzyme at pH 6. A single mutational event (M1) induced a 30% increase in the efficiency of orthophosphate metabolism. The second mutational event (M2 in the region of the structural gene) led to an adaptive shift in the pH optimum and in the enhancement of phosphatase activity by 60%. Finally, the third event (M3) induced cell clumping with no effect on orthophosphate metabolism [9].
In the 1970s, further contributions likewise describing in vivo directed evolution processes appeared sporadically. The contribution of Hall utilizing the classical microbiological technique of genetic complementation constitutes a prominent example [10]. In one of the earliest directed evolution projects in the Hall group, new functions for the ebgA (ebg = evolved ß‐galactosidase) were explored (Scheme 1.2) [10a]. Growth of different carbohydrates as the energy source was the underlying evolutionary principle. Wildtype (WT) ebgAo is an enzyme showing very little or no activity toward certain carbohydrates such as the natural sugar lactose. It was shown, inter alia, that for an E. coli strain with lac2 deletion to obtain the ability to utilize lactobionate as the carbon source, a series of mutations must be introduced in a particular order in the ebg genes. It was also found experimentally when growing cells on different carbon sources, that in some cases old enzyme functions remain unaffected or are actually improved.
Scheme 1.2 Pedigree of ebgA alleles in evolved strains [10a]. Strain 1B1 carries the wild‐type allele, ebgAO. Strains on line one have a single mutation in the ebgA gene; those in line two have two mutations in ebgA; those in line three have three mutations in ebgA. All strains are ebgR‐. Strains enclosed in rectangles were selected for growth on lactose; those enclosed in diamonds were selected for growth on lactulose; those in circles were selected for growth on lactobionate. This pedigree shows only the descent of the ebgA gene; i.e. strains SJ‐17, A2, 5A2, and D2 were not derived directly from IBI, but their ebgA alleles were derived directly from the ebgA allele carried in IBI.
Two decades later the technique was extended by Kim et al. [11a]. It may have inspired other groups to study and develop new evolution experiments, e.g. by Lenski et al., who investigated parallel changes in gene expression after 20 000 generations of evolution in bacteria [11b], and more recently by Liu et al., who implemented a novel technique for continuous evolution [11c], including a phage‐assisted embodiment [11d].
Although originally not specifically related to directed evolution, developments such as the Kunkel method of mutational specificity based on depurination [12a] deserves mention because it was used two decades later in mutant library design based on error‐prone rolling circle amplification (epRCA) [12b]. These and many other early developments inspired scientists to speculate about potential applications of directed evolution in biotechnology. As can be seen, these and other developments were suggesting that directed evolution could be generalized with the emergence of a general protein engineering method. Indeed, in 1984 Eigen and Gardiner summarized these intriguing perspectives by emphasizing the necessity of self‐replication in molecular in vitro evolution [13]. At the time the best self‐replication system for the laboratory utilized the replication of single‐stranded RNA by the replication enzyme of the coliphage Qf3. The logic of laboratory Darwinian evolution involving recursive cycles of gene mutagenesis, amplification, and selection was formulated. However, the generation of bacterial colonies on agar plates for ensuring the required genotype–phenotype relation, as employed later by essentially all directed evolution researchers, was not considered. It should be remembered that in the early 1980s the polymerase chain reaction (PCR) for high‐fidelity DNA amplification had not yet been developed. Following its announcement in the 1980s by Mullis [14], which won him the Nobel Prize, completely new perspectives emerged in many fields, including directed evolution.
Parallel to these developments, researchers began to experiment with different types of mutagenesis methods to generate mutant libraries which were subsequently screened or selected for an enzyme property, generally protein thermostability. Sometimes such methods were introduced without any real applications at the time of publication. Only a few early representative developments are highlighted here. In 1985 Matsumura and Aiba subjected kanamycin nucleotidyltransferase (cloned into a single‐stranded bacteriophage M13) to hydroxylamine‐induced chemical mutagenesis [15a]. It had been known for a long time that certain chemicals can induce mutations in genes. Following recloning of the mutagenized gene of the enzyme into the vector plasmid pTB922, the recombinant plasmid was employed to transform Bacillus stearothermophilus so that more stable variants could be identified by screening. About 12 out of 8000 transformants were suspected to harbor thermostabilized variants, the best one out of the 12 then being characterized as having a single‐point mutation and a stabilization of 6 °C. Unfortunately, a second cycle for further improvement was not reported. Several other early studies of T4 lysozyme using chemical mutagenesis appeared, summarized by Matthews in a 2010 review [15b]. Today it is accepted that the formation of improved enzymes in an initial mutant library does not (yet) constitute an evolutionary process and that at least one additional cycle of mutagenesis/expression/screening as shown in Scheme 1.1 is required before the term directed evolution applies.
In light of this, the report by Hageman and coworkers in 1986, in which for the first time two mutagenesis/screening cycles were described, is indeed a hallmark event. In their efforts to enhance the thermostability of kanamycin nucleotidyltransferase by a true evolutionary process, they employed a mutator strain [16]. Mutator strains such as E. coli XL1‐red induce randomly mutagenized plasmid libraries because they contain defects in one or more of their DNA repair genes [17]. Basically, Hageman's seminal study consisted of cloning the gene that encodes the enzyme from a mesophilic organism, introducing the gene into an appropriate thermophile, and selecting for activity at the higher growth temperatures of the host organism (in this case B. stearothermophilus). The host organism is resistant to the antibiotic at 47 °C, but not at temperatures above 55 °C. Upon passing a shuttle plasmid through the E. coli mutD5 mutator strain and introduction into B. stearothermophilus, a point mutation that led to resistance to kanamycin at 63 °C was identified, namely Asp80Tyr. Using this as a template, the second round was performed under higher selection pressure at 70 °C, leading to the accumulation of mutation Thr130Lys, the respective double mutant Asp80Tyr/Thr130Lys showing even higher thermostability (Scheme 1.3) [16]. The Darwinian character of this approach to thermostabilization of proteinsis self‐evident, and opened the door to a new research area. Importantly, in a follow‐up study, it was demonstrated that the best mutant also shows higher resistance to hostile solvents such as aqueous dimethylformamide (DMF) solutions and even urea, relative to WT [18].
Scheme 1.3 First example of directed evolution of thermostability of an enzyme. Kanamycin nucleotidyltransferase (KNT) served as the enzyme and a mutator strain as the random mutagenesis technique in an iterative manner.
Source: Adapted from Liao et al. [16].
As noted above, the original site‐specific mutagenesis of Smith allows the specific exchange of any amino acid in a protein by any one of the other 19 canonical amino acids [5], but if all 19 mutants are needed, then 19 separate experiments are required. This could be expensive and time‐consuming. The generation of random mutations all at once in a given experiment at a single residue or at a defined multiresidue randomization site was not developed until later. Early on (during the period 1983–1988), several variations of cassette mutagenesis based on the use of “doped” synthetic oligodeoxynucleotides, which allows the combinatorial introduction of all 19 canonical amino acids at a given position, were developed [19]. These and similar studies were performed for different reasons, not all having to do with enzyme catalysis. The early study by Wells et al., based on saturation mutagenesis is highlighted here [20]. Saturation mutagenesis means focused randomization at a chosen site in the protein (Chapter 3). The Wells study constitutes a clever combination of rational design and directed evolution for the purpose of increasing the robustness of the serine protease subtilisin so that it shows enhanced resistance to chemical oxidation by H2O2 relative to WT [20]. At the time it was known that residue Met222 constitutes a site at which undesired oxidation occurs. Therefore, saturation mutagenesis was performed at this position, which led to several improved variants showing resistance to 1 M H2O2 as measured by the reaction of N‐succinyl‐L‐Ala‐L‐Ala‐L‐Pro‐L‐Phe‐p‐nitroanilide, including mutants Met222Ser, Met222Ala, and Met222Leu [20].
Cassette mutagenesis as a form of saturation mutagenesis was developed early on. As pointed out by Ner, Goodin, and Smith in 1988, a disadvantage of cassette mutagenesis is the fact that the synthetic oligodeoxynucleotides in form of a cassette have to be introduced between two restriction sites, one on either side of the to be randomized sequence [21a]. Since the restriction sites had to be generated by standard oligodeoxynucleotide mutagenesis, additional steps were necessary prior to the actual randomization procedure. Therefore, an improved version was developed using a combination of the known primer extension procedure [21a] and Kunkel's method of strand selection [12a]. The technique uses a mixed pool of oligodeoxynucleotides prepared by contaminating the monomeric nucleotides with low levels of the other three nucleotides so that the full‐length oligonucleotide contains on average one to two changes/molecule [21b]. It was employed in priming in vitro synthesis of the complementary strand of cloned DNA fragments in M13 or pEMBL vectors, the latter having been passed through the E. coli host. The method allows random point mutations as well as codon replacements. Scheme 1.4 illustrates the case of the MATa1 gene from Saccharomyces cerevisae[21b].
Scheme 1.4 Mixed oligonucleotide mutagenesis of the gene MATa1 from Saccharomyces cerevisae.
Source: Adapted from Zoller and Smith [21b].
Despite these advancements, further improvements with respect to generality, efficiency, and ease of performance were necessary. Indeed, these began to appear in the late 1980s. They included the generation of mutant libraries using spiked oligodeoxyribonucleotide primers according to Hermes et al. [22]. A particular important study was published by Pease and coworkers at the Mayo Clinic, which is based on the use of overlap extension polymerase chain reaction (OE‐PCR) for site‐specific mutagenesis and saturation mutagenesis [23a], see Chapter 3 for details. It has greatly influenced directed evolution [3, 4]. OE‐PCR can also be used in insertion/deletion mutagenesis [24].
In the 1980s, further interesting contributions appeared, including a publication by Loeb et al. [25]. Accordingly, ß‐lactamase mutants that render E. coli resistant to the antibiotic carbenicillin by replacing the DNA sequence corresponding to the active site with random nucleotide sequences can be evolved without exchanging the codon encoding catalytically active Ser70. The inserted oligonucleotide Phe66XXXSer70XXLys73 contains 15 base pairs of chemically synthesized random sequences that code for 2.5 million amino acid exchanges. It should be noted that ß‐lactamase is an ideal enzyme with which randomization‐based protein engineering can be performed because a simple and efficient selection system is available (Chapter 2).
Major advancements followed in the 1990s, these allowing saturation mutagenesis‐based simultaneous randomization at more than one residue site. Based on some of these developments, the so‐called QuikChange™ protocol for saturation mutagenesis emerged in 2002 [26], see Chapter 3 for details. Another important version of saturation mutagenesis is the “megaprimer” method of site‐specific mutagenesis introduced by Kamman et al. [27] and improved by Sarkar and Sommer [28]. The overall procedure is fairly straightforward and easy to perform, but it also has limitations as discussed in Chapter 3. Early developments of site‐directed mutagenesis, which can also be used for randomization, were summarized by Tao in 1992 [29].
In 1989 a landmark study was published by Leung, Chen, and Goeddel describing error‐prone polymerase chain reaction (epPCR) [30a], but it was not applied to enzymes until a few years later (see below). It relies on Taq polymerase or similar DNA polymerases, which lack proofreading ability (no removal of mismatched bases). To control the mutational rate, the reaction conditions need to be optimized by varying such parameters as the MgCl2 or MnCl2 concentrations and/or employing unbalanced nucleotide concentrations (see details in Section 3.3). The mutation rate can be controlled empirically by varying such parameters as the MgCl2 or MnCl2 concentrations and/or employing unbalanced amounts of nucleotides [30b].
The first applications of epPCR are due to Hawkins and Winter in 1992 [31], who reported in vitro selection and affinity maturation of antibodies from combinatorial libraries. The creation of large combinatorial libraries of antibodies was a new area of science at the time, as shown by Lerner and coworkers using different techniques, including the use of OE‐PCR [32]. It should be noted that epPCR suffers from various limitations, which are discussed in Chapter 3. To this day the technique continues to be employed, especially when X‐ray structural data of the protein are not available. A different but seldom used molecular biological random mutagenesis method was developed and applied in 1992/1993 by Zhang et al., to increase the thermostability of aspartase as a catalyst in the industrially important addition reaction of ammonia to fumarate with formation of L‐aspartic acid [33]. Unbalanced nucleotide amounts were used in a special way, but diversity is lower than in the case of epPCR [33b].
In 1993, Chen and Arnold published a paper describing the use of random mutagenesis in the quest to increase the robustness of the protease subtilisin E in aqueous medium containing a hostile organic solvent (DMF) [34]. This study is described here in detail, because at the Nobel Prize announcement on 3rd October 2018, it was highlighted as constituting the seminal turning point in directed evolution (see Nobel Prize Lecture by Frances H. Arnold [4b] and also her contribution in developing artificial metalloenzymes as highlighted in Chapter 7). The only difference in the 1993 study was the use of epPCR as the random mutagenesis method (although the term epPCR is not mentioned in the paper), and the fact that not thermostability was aimed for as Hageman et al. had already described [16], but resistance to DMF in an otherwise aqueous environment [34]. This paper does not involve any increase in the activity of the WT in the absence of a cosolvent, as Arnold et al. actually pointed out [34]. Thus, the aim was not to increase the activity of an enzyme[4b], which is sometimes misunderstood. The traditional aim in directed evolution to enhance enzyme activity is a different challenge, which was addressed much later and is still relevant today [3].
In the Arnold paper, the mutations of three variants obtained earlier by rational design were first combined with formation of the respective triple mutant Asp60Asn/Gln103Arg/Asn218Ser to which was added a fourth point mutation Asp97Gly, leading to variant Asp60Asn/Gln103Arg/Asn218Ser/Asp97Gly (“4M variant”) [34]. The HindIII/BamHI DNA fragment of 4M subtilisin E from residue 49 to the C‐terminus was then employed as the template for PCR‐based random mutagenesis (epPCR). Thus, this diverges a little from epPCR as originally developed by Leung, Chen, and Goeddel [30], which addresses the whole gene. The PCR conditions were modified so that the mutational frequency increased (including the use of MnCl2). An easy‐to‐perform prescreen for activity was developed using agar plates containing 1% casein, which upon hydrolysis forms a halo. The roughly identified active mutants were then sequenced and used as catalysts in the hydrolysis of N‐succinyl‐L‐Ala‐L‐Ala‐L‐Pro‐L‐Met‐p‐nitroanilide and N‐succinyl‐L‐Ala‐L‐Ala‐L‐Pro‐L‐Phe‐p‐nitroanilide. Upon going through three cycles of random mutagenesis, the final best hit (PC3) was identified as having a total of 10 point mutations. The catalytic efficiency of variant PC3 relative to WT subtilisin E in aqueous medium containing different amounts of DMF is shown in Scheme 1.5[34]. This study constitutes the second example of more than one cycle of mutagenesis/expression/screening in directed evolution [16].
Scheme 1.5 Catalytic efficiency of WT subtilisin E and variant PC3 as catalysts in the hydrolytic cleavage of N‐succinyl‐L‐Ala‐L‐Ala‐L‐Pro‐L‐Met‐p‐nitroanilide.
Source: Chen and Arnold [34].
In addition to epPCR and OE‐PCR, other important mutagenesis methods were developed in the late 1980s. A key study by Pease, Horton, and coworkers is a prime example [23b] (Scheme 1.6). It is an extension of their earlier work on OE‐PCR [23a]. Fragments from two genes that are to be recombined are first produced by separate PCR, the primers being designed so that the ends of the products feature complementary sequences (Scheme 1.6). Upon mixing, denaturing, and reannealing the PCR products, those strands that have matching sequences at their 3′ ends overlap and function as primers for each other. Extension of the overlap by a DNA polymerase leads to products in which the original sequences are spliced together. This recombinant technique for producing chimeric genes was called splicing by overlap extension (SOE), which also allows the introduction of random errors (mutations). The technique was illustrated using two different mouse class‐I major histocompatible genes. However, at the time it was not exploited by the biotechnology community active in directed evolution.
Scheme 1.6 Steps in the recombinant technique of splicing by overlap extension (SOE), illustrated here using two different genes.
Source: Horton et al. [23b]/With permission of Elsevier.
In 1994, a novel gene mutagenesis method called DNA shufflingwas reported by Stemmer[35a]. It is a process that simulates sexual evolution as it occurs in nature. In the original study, ß‐lactamase served as the enzyme, the selection system is based on the increased resistance to an antibiotic. DNA shuffling is illustrated here when starting with mutants of a given enzyme previously produced by some other mutagenesis technique (Scheme 1.7). Family shuffling, also introduced in 1994, is a variation that in many cases constitutes the superior approach [35b]. See Chapter 3 for a description of this seminal technique and other recombinant methods.
Scheme 1.7 DNA shuffling starting from a single gene encoding a given enzyme.
These landmark papers sparked a great deal of further research in the area of directed evolution in the 1990s. In many of the studies, recombinant and/or nonrecombinant methods were applied to shed light on the mechanism of enzymes, but usually, only initial mutant libraries were considered. To this day directed evolution is sometimes employed in the quest to study enzyme mechanisms rather than for the purpose of evolving altered enzymes for practical purposes. Contributions by Hilvert [4d, 36] and Benkovic et al. [37] are prominent examples, as are the studies of Hecht et al. concerning binary patterning [38]. In an informative overview by Lutz and Benkovic which appeared in 2002, many of these and other early developments in directed evolution including applications were assessed [39].
Phage display, invented by George P. Smith in 1985 [40], was originally not intended for directed evolution of enzymes, but it inspired Gregory Winter to apply it with the aim of developing antibodies as medicines [41]. These seminal developments led to the Chemistry Nobel Prize 2018[40c, 41b] for Smith and Winter, which they shared with Arnold [4b]. Despite the evolutive nature of such systems, application of phage display in directed evolution of stereo‐ and/or regioselective enzymes is problematic and has limitations [42]. Although flow cytometry had been invented decades ago, it was not combined with fluorescence‐activated cell sorter technology (FACS) for application in directed evolution until much later, especially relevant in vaccine development, as demonstrated by the early contributions of Georgiou and coworkers [43]. The water‐in‐oil emulsion technology, elegantly developed by Griffiths and Tawfik, is likewise a step forward[44]. All of these selection platforms, which are really screening techniques (Chapter 2
