English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Beating Bias in the Directed Evolution of Proteins: Combining High-Fidelity on-Chip Solid-Phase Gene Synthesis with Efficient Gene Assembly for Combinatorial Library Construction

Li, A., Acevedo-Rocha, C. G., Sun, Z., Cox, T., Xu, J. L., & Reetz, M. T. (2018). Beating Bias in the Directed Evolution of Proteins: Combining High-Fidelity on-Chip Solid-Phase Gene Synthesis with Efficient Gene Assembly for Combinatorial Library Construction. Chembiochem, 19(3), 221-228. doi:10.1002/cbic.201700540.

Item is

Basic

show hide
Genre: Journal Article

Files

show Files

Locators

show

Creators

show
hide
 Creators:
Li, Aitao1, 2, 3, Author              
Acevedo-Rocha, Carlos G.4, Author
Sun, Zhoutong5, Author
Cox, Tony6, Author
Xu, Jia Lucy6, Author
Reetz, Manfred T.1, 2, Author              
Affiliations:
1Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445588              
2Department of Chemistry, Philipps-Universität Marburg, 35032 Marburg (Germany), ou_persistent22              
3Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources Hubei Key Laboratory of Industrial Biotechnology College of Life Sciences, Hubei University, 368 Youyi Road, Wuchang Wuhan, 430062 (P.R.China), ou_persistent22              
4Biosyntia ApS, 2100 Copenhagen (Denmark), ou_persistent22              
5Tianjin Institute of Industrial Biotechnology Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308 (P.R. China), ou_persistent22              
6Twist Bioscience, 455 Mission Bay Boulevard South, San Francisco, CA 94158 (USA, ou_persistent22              

Content

show
hide
Free keywords: directed evolution; enzymes; polymerase chain reaction; saturation mutagensis; solid-phase synthesis
 Abstract: Saturation mutagenesis (SM) constitutes a widely used technique in the directed evolution of selective enzymes as catalysts in organic chemistry and in the manipulation of metabolic paths and genomes, but the quality of the libraries is far from optimal due to the inherent amino acid bias. Herein, it is shown how this fundamental problem can be solved by applying high-fidelity solid-phase chemical gene synthesis on silicon chips followed by efficient gene assembly. Limonene epoxide hydrolase was chosen as the catalyst in the model desymmetrization of cyclohexene oxide with the stereoselective formation of (R,R)- and (S,S)-cyclohexane-1,2-diol. A traditional combinatorial PCR-based SM library, produced by simultaneous randomization at several residues by using a reduced amino acid alphabet, and the respective synthetic library were constructed and compared. Statistical analysis at the DNA level with massive sequencing demonstrates that, in the synthetic approach, 97% of the theoretically possible DNA mutants are formed, whereas the traditional SM library contained only about 50%. Screening at the protein level also showed the superiority of the synthetic library; many highly (R,R)- and (S,S)-selective variants being discovered are not found in the traditional SM library. With the prices of synthetic genes decreasing, this approach may point the way to future directed evolution.

Details

show
hide
Language(s): eng - English
 Dates: 2017-10-062017-11-242018-02-02
 Publication Status: Published online
 Pages: 8
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1002/cbic.201700540
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: Chembiochem
  Other : Chembiochem
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: Weinheim, Germany : Wiley-VCH
Pages: - Volume / Issue: 19 (3) Sequence Number: - Start / End Page: 221 - 228 Identifier: ISSN: 1439-4227
CoNE: https://pure.mpg.de/cone/journals/resource/110978984568897