English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Speeding up Directed Evolution: Combining the Advantages of Solid-Phase Combinatorial Gene Synthesis with Statistically Guided Reduction of Screening Effort

Höbenreich, S., Zilly, F., Acevedo-Rocha, C. G., Zilly, M., & Reetz, M. T. (2015). Speeding up Directed Evolution: Combining the Advantages of Solid-Phase Combinatorial Gene Synthesis with Statistically Guided Reduction of Screening Effort. ACS Synthetic Biology, 317-331. doi:10.1021/sb5002399.

Item is

Files

show Files

Locators

show

Creators

show
hide
 Creators:
Höbenreich, Sabrina1, 2, Author              
Zilly, Felipe1, 2, Author              
Acevedo-Rocha, Carlos G.1, 2, Author              
Zilly, Matias3, Author
Reetz, Manfred T.1, 2, Author              
Affiliations:
1Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445588              
2Philipps-Universität Marburg, Fachbereich Chemie, ou_persistent22              
3Universität Duisburg Essen, Fakultät für Physik, Lotharstraße 1, 47048 Duisburg, Germany, ou_persistent22              

Content

show
hide
Free keywords: protein engineering; metabolic engineering; genome engineering; screening effort; biocatalysis; cytochrome P450
 Abstract: Efficient and economic methods in directed evolution at the protein, metabolic, and genome level are needed for biocatalyst development and the success of synthetic biology. In contrast to random strategies, semirational approaches such as saturation mutagenesis explore the sequence space in a focused manner. Although several combinatorial libraries based on saturation mutagenesis have been reported using solid-phase gene synthesis, direct comparison with traditional PCR-based methods is currently lacking. In this work, we compare combinatorial protein libraries created in-house via PCR versus those generated by commercial solid-phase gene synthesis. Using descriptive statistics and probabilistic distributions on amino acid occurrence frequencies, the quality of the libraries was assessed and compared, revealing that the outsourced libraries are characterized by less bias and outliers than the PCR-based ones. Afterward, we screened all libraries following a traditional algorithm for almost complete library coverage and compared this approach with an emergent statistical concept suggesting screening a lower portion of the protein sequence space. Upon analyzing the biocatalytic landscapes and best hits of all combinatorial libraries, we show that the screening effort could have been reduced in all cases by more than 50%, while still finding at least one of the best mutants.

Details

show
hide
Language(s): eng - English
 Dates: 2014-06-122015-01
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/sb5002399
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: ACS Synthetic Biology
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: -
Pages: - Volume / Issue: - Sequence Number: - Start / End Page: 317 - 331 Identifier: -