scholarly journals Combination of computational prescreening and experimental library construction can accelerate enzyme optimization by directed evolution

2005 ◽  
Vol 18 (11) ◽  
pp. 509-514 ◽  
Author(s):  
Susanne Aileen Funke ◽  
Nikolaj Otte ◽  
Thorsten Eggert ◽  
Marco Bocola ◽  
Karl-Erich Jaeger ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Library Construction ◽  
Enzyme Optimization

A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences

2007 ◽  
Vol 388 (12) ◽  
pp. 1291-1300 ◽  
Author(s):  
Ai-Sheng Xiong ◽  
Ri-He Peng ◽  
Jing Zhuang ◽  
Jin-Ge Liu ◽  
Feng Gao ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Dna Sequences ◽  
Active Sites ◽  
Rational Design ◽  
Specific Activity ◽  
Dna Shuffling ◽  
Gene Encoding ◽  
Library Construction ◽  
Key Sites

Abstract Directed evolution in vitro is a powerful molecular tool for the creation of new biological phenotypes. It is unclear whether it is more efficient to mutate an enzyme randomly or to mutate just the active sites or key sites. In this study, the strategy of a semi-rational design of directed evolution combined with whole sequence and sites was developed. The 1553 bp gene encoding the thermostable β-galactosidase of Pyrococcus woesei was chemically synthesized and optimized for G+C content and mRNA secondary structures. The synthesized gene product was used as a template or as a wild-type control. On the basis of the first round of DNA shuffling, library construction and screening, one mutant of YH6754 was isolated with higher activity. Eight potential key sites were deduced from the sequence of the shuffled gene, and 16 degenerate oligonucleotides were designed according to those eight amino acids. Two variants of YG6765 and YG8252 were screened in the second part of DNA shuffling, library construction and screening. For comparison, one mutant of YH8757 was screened through the same routine rounds of directed evolution with YH6754 as template. The purified β-galactosidase from YH8757 exhibited a lower specific activity at 25°C than those purified from mutated YG6755 and YG8252.

Solid-Phase Gene Synthesis for Mutant Library Construction: The Future of Directed Evolution?

ChemBioChem ◽  
2018 ◽  
Vol 19 (19) ◽  
pp. 2023-2032 ◽  
Author(s):  
Aitao Li ◽  
Zhoutong Sun ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Solid Phase ◽  
Gene Synthesis ◽  
Mutant Library ◽  
Library Construction ◽  
The Future

scholarly journals Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

2003 ◽  
Vol 69 (8) ◽  
pp. 4448-4454 ◽  
Author(s):  
So-Young Kim ◽  
Jung-Hoon Sohn ◽  
Jung-Hoon Bae ◽  
Yu-Ryang Pyun ◽  
Michael O. Agaphonov ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Gene Dosage ◽  
Hot Spot ◽  
Hansenula Polymorpha ◽  
Autonomously Replicating Sequence ◽  
Library Construction ◽  
In Vivo Recombination ◽  
Recombination System ◽  
Selection Of

ABSTRACT A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

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