scholarly journals Improvement of alkaliphily of thermostable GH family 10 xylanase from Thermotoga maritima by directed evolution

2010 ◽  
Vol 9 (1) ◽  
pp. 15-18
Author(s):  
W Tsukimura ◽  
K Watanabe ◽  
C Morokuma ◽  
R Yatsunami ◽  
T Fukui ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Thermotoga Maritima ◽  
Family 10 Xylanase

scholarly journals Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Gordon Rix ◽  
Ella J. Watkins-Dulaney ◽  
Patrick J. Almhjell ◽  
Christina E. Boville ◽  
Frances H. Arnold ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Thermotoga Maritima ◽  
Enzyme Engineering ◽  
Tryptophan Synthase ◽  
Β Subunit ◽  
Evolutionary Processes ◽  
Separate Species ◽  
Continuous Evolution ◽  
Primary Activity ◽  
Natural Diversity

Abstract Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB’s primary activity of synthesizing l-tryptophan from indole and l-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions.

scholarly journals Directed Evolution of a Hyperthermophilic Endoglucanase Cel12B from Thermotoga maritima

BioResources ◽  
2014 ◽  
Vol 9 (2) ◽  
Author(s):  
Hao Shi ◽  
Lili Wang ◽  
Xun Li ◽  
Liangliang Wang ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Thermotoga Maritima

scholarly journals Scalable, continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

2020 ◽  
Author(s):  
Gordon Rix ◽  
Ella J. Watkins-Dulaney ◽  
Patrick J. Almhjell ◽  
Christina E. Boville ◽  
Frances H. Arnold ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Thermotoga Maritima ◽  
Enzyme Engineering ◽  
Tryptophan Synthase ◽  
Β Subunit ◽  
Evolutionary Processes ◽  
Separate Species ◽  
Continuous Evolution ◽  
Primary Activity ◽  
Natural Diversity

AbstractEnzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolved the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only onTmTrpB’s primary activity (synthesizing L-tryptophan from indole and L-serine). We find that the resulting sequence-diverseTmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of new biomolecular functions.

Exemplar Abstract for Thermotoga maritima Huber et al. 1986.

2003 ◽  
Author(s):  
Charles Thomas Parker ◽  
Dorothea Taylor ◽  
George M Garrity
Keyword(s):  
Thermotoga Maritima

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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