evSeq: Cost-Effective Amplicon Sequencing of Every Variant in a Protein Library DOI
Bruce J. Wittmann, Kadina E. Johnston, Patrick J. Almhjell

et al.

ACS Synthetic Biology, Journal Year: 2022, Volume and Issue: 11(3), P. 1313 - 1324

Published: Feb. 17, 2022

Widespread availability of protein sequence-fitness data would revolutionize both our biochemical understanding proteins and ability to engineer them. Unfortunately, even though thousands variants are generated evaluated for fitness during a typical engineering campaign, most never sequenced, leaving wealth potential information untapped. Primarily, this is because sequencing unnecessary many strategies; the added cost effort thus unjustified. It also results from fact that, lower-cost strategies have been developed, they often require at least some access experience with or computational resources, which can be barriers access. Here, we present every variant (evSeq), method collection tools/standardized components variable region within gene produced campaign cents per variant. evSeq was designed democratize low-cost engineers and, indeed, anyone interested in biological systems. Execution its wet-lab component simple, requires no perform, relies only on resources services typically available biology labs, slots neatly into existing workflows. Analysis likewise made simple by accompanying software (found github.com/fhalab/evSeq, documentation fhalab.github.io/evSeq), run personal laptop accessible users experience. Low-cost easy-to-use, makes extensive practical.

Language: Английский

The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes DOI
Ge Qu, Aitao Li, Carlos G. Acevedo‐Rocha

et al.

Angewandte Chemie International Edition, Journal Year: 2019, Volume and Issue: 59(32), P. 13204 - 13231

Published: July 3, 2019

Directed evolution of stereo-, regio-, and chemoselective enzymes constitutes a unique way to generate biocatalysts for synthetically interesting transformations in organic chemistry biotechnology. In order this protein engineering technique be efficient, fast, reliable, also relevance synthetic chemistry, methodology development was still is necessary. Following description early key contributions, review focuses on recent developments. It includes optimization molecular biological methods gene mutagenesis the design efficient strategies their application, resulting notable reduction screening effort (bottleneck directed evolution). When aiming laboratory selectivity activity, second-generation versions Combinatorial Active-Site Saturation Test (CAST) Iterative Mutagenesis (ISM), both involving saturation (SM) at sites lining binding pocket, have emerged as preferred approaches, aided by silico such machine learning. The recently proposed Focused Rational Site-specific (FRISM) fusion rational evolution. On-chip solid-phase chemical synthesis rapid library construction enhances quality notably eliminating undesired amino acid bias, future evolution?

Language: Английский

Citations

401
Machine Learning in Enzyme Engineering DOI Creative Commons
Stanislav Mazurenko, Zbyněk Prokop, Jir̆ı́ Damborský

et al.

ACS Catalysis, Journal Year: 2019, Volume and Issue: 10(2), P. 1210 - 1223

Published: Dec. 13, 2019

Enzyme engineering plays a central role in developing efficient biocatalysts for biotechnology, biomedicine, and life sciences. Apart from classical rational design directed evolution approaches, machine learning methods have been increasingly applied to find patterns data that help predict protein structures, improve enzyme stability, solubility, function, substrate specificity, guide design. In this Perspective, we analyze the state of art databases used training validating predictors engineering. We discuss current limitations challenges which community is facing recent advancements experimental theoretical potential address those challenges. also present our view on possible future directions applications biocatalysts.

Language: Английский

Citations

343
Power of Biocatalysis for Organic Synthesis DOI Creative Commons
Christoph K. Winkler, Joerg H. Schrittwieser, Wolfgang Kroutil

et al.

ACS Central Science, Journal Year: 2021, Volume and Issue: 7(1), P. 55 - 71

Published: Jan. 14, 2021

Biocatalysis, using defined enzymes for organic transformations, has become a common tool in synthesis, which is also frequently applied industry. The generally high activity and outstanding stereo-, regio-, chemoselectivity observed many biotransformations are the result of precise control reaction active site biocatalyst. This achieved by exact positioning reagents relative to each other fine-tuned 3D environment, specific activating interactions between protein, subtle movements catalyst. Enzyme engineering enables one adapt catalyst desired process. A well-filled biocatalytic toolbox ready be used various reactions. Providing nonnatural conditions evolving biocatalysts play with myriad options creating novel transformations thereby opening new, short pathways target molecules. Combining several pot perform reactions concurrently increases efficiency biocatalysis even further.

Language: Английский

Citations

288
Recent trends in biocatalysis DOI Creative Commons
Dong Yi, Thomas Bayer, Christoffel P. S. Badenhorst

et al.

Chemical Society Reviews, Journal Year: 2021, Volume and Issue: 50(14), P. 8003 - 8049

Published: Jan. 1, 2021

Technological developments enable the discovery of novel enzymes, advancement enzyme cascade designs and pathway engineering, moving biocatalysis into an era technology integration, intelligent manufacturing enzymatic total synthesis.

Language: Английский

Citations

283
The Hitchhiker's guide to biocatalysis: recent advances in the use of enzymes in organic synthesis DOI Creative Commons
R. Ann Sheldon, Dean Brady, Moira L. Bode

et al.

Chemical Science, Journal Year: 2020, Volume and Issue: 11(10), P. 2587 - 2605

Published: Jan. 1, 2020

Enzymes are excellent catalysts that increasingly being used in industry and academia. This Perspective provides a general practical guide to enzymes their synthetic potential, primarily aimed at organic chemists.

Language: Английский

Citations

261
Advances in ultrahigh-throughput screening for directed enzyme evolution DOI
Ulrich Markel, Khalil Essani,

Volkan Besirlioglu

et al.

Chemical Society Reviews, Journal Year: 2019, Volume and Issue: 49(1), P. 233 - 262

Published: Dec. 9, 2019

This review summarizes how ultrahigh-throughput screening methods employ cells and biomimetic compartments to access the vast, unexplored diversity of biocatalysts with novel functions derived from directed evolution metagenomics libraries.

Language: Английский

Citations

222
Expanding the enzyme universe with genetically encoded unnatural amino acids DOI
Ivana Drienovská, Gérard Roelfes

Nature Catalysis, Journal Year: 2020, Volume and Issue: 3(3), P. 193 - 202

Published: Jan. 6, 2020

Language: Английский

Citations

196
Biocatalytic Reduction Reactions from a Chemist's Perspective DOI Creative Commons
Frank Hollmann, Diederik J. Opperman, Caroline E. Paul

et al.

Angewandte Chemie International Edition, Journal Year: 2020, Volume and Issue: 60(11), P. 5644 - 5665

Published: April 24, 2020

Abstract Reductions play a key role in organic synthesis, producing chiral products with new functionalities. Enzymes can catalyse such reactions exquisite stereo‐, regio‐ and chemoselectivity, leading the way to alternative shorter classical synthetic routes towards not only high‐added‐value compounds but also bulk chemicals. In this review we describe state‐of‐the‐art potential of enzymes that reductions, ranging from carbonyl, enone aromatic reductions reductive aminations.

Language: Английский

Citations

170
Informed training set design enables efficient machine learning-assisted directed protein evolution DOI Creative Commons
Bruce J. Wittmann, Yisong Yue, Frances H. Arnold

et al.

Cell Systems, Journal Year: 2021, Volume and Issue: 12(11), P. 1026 - 1045.e7

Published: Aug. 19, 2021

Language: Английский

Citations

161
Stereodivergent Protein Engineering of a Lipase To Access All Possible Stereoisomers of Chiral Esters with Two Stereocenters DOI
Jian Xu, Yixin Cen, Warispreet Singh

et al.

Journal of the American Chemical Society, Journal Year: 2019, Volume and Issue: 141(19), P. 7934 - 7945

Published: April 26, 2019

Enzymatic stereodivergent synthesis to access all possible product stereoisomers bearing multiple stereocenters is relatively undeveloped, although enzymes are being increasingly used in both academic and industrial areas. When two thus four stereoisomeric products involved, obtaining enzyme mutants for individually accessing would be ideal. Although significant success has been achieved directed evolution of general, engineering one into highly stereocomplementary variants the full complement remains a challenge. Using Candida antarctica lipase B (CALB) as model, we report protein this needed transesterification reactions between racemic acids alcohols organic solvents. By generating screening less than 25 each isomer, >90% selectivity model reaction. This difficult feat was accomplished by developing strategy dubbed "focused rational iterative site-specific mutagenesis" (FRISM) at sites lining enzyme's binding pocket. The accumulation single mutations mutagenesis using restricted set rationally chosen amino allows formation ultrasmall mutant libraries requiring minimal stereoselectivity. crystal structure CALB variants, flanked MD simulations, uncovered source selectivity.

Language: Английский

Citations

149