scholarly journals A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

10.3791/4216 ◽  
2012 ◽  
Author(s):  
Ryan D. Heselpoth ◽  
Daniel C. Nelson
Keyword(s):  
Directed Evolution ◽  
Screening Method ◽  
Bacteriolytic Enzymes

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.

scholarly journals Directed evolution  improves the enzymatic synthesis of L-5-hydroxytryptophan by an  engineered tryptophan synthase

2021 ◽  
Author(s):  
Xu Lisheng ◽  
Tingting Li ◽  
Ziyue Huo ◽  
Qiong Chen ◽  
Qiuxia Xia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Enzymatic Synthesis ◽  
High Throughput Screening ◽  
Screening Method ◽  
Mutant Enzyme ◽  
Tryptophan Synthase ◽  
Effective Strategy ◽  
Reaction Conditions ◽  
Important Amino Acid

Abstract L-5-Hydroxytryptophan is an important amino acid that is widely used in food and medicine. In this study, L-5-hydroxytryptophan was synthesized by a modified tryptophan synthase. A direct evolution strategy was applied to engineer tryptophan synthase from Escherichia coli to improve the efficiency of L-5-hydroxytryptophan synthesis. Tryptophan synthase was modified by error-prone PCR. A high activity mutant enzyme (V231A/K382G) was obtained by a high-throughput screening method. The activity of mutant enzyme (V231A/K382G) is 3.79 times higher than that of its parent, and kcat/Km of the mutant enzyme (V231A/K382G) was 4.36 mM− 1∙s− 1. The mutant enzyme (V231A/K382G) reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 h, reaching a yield of L-5-hydroxytryptophan of 86.7%. Directed evolution is an effective strategy to increase the activity of tryptophan synthase.

scholarly journals Improvement of an Escherichia coli whole-cell biocatalyst for geranyl glucoside production using directed evolution

2021 ◽  
Author(s):  
Julian Ruediger ◽  
Wilfried Schwab
Keyword(s):  
Directed Evolution ◽  
Production Systems ◽  
Low Cost ◽  
Continuous Production ◽  
Expression System ◽  
Screening Method ◽  
Fold Increase ◽  
Small Scale ◽  
Biotechnological Production ◽  
Whole Cell

The biotechnological production of glycosides is an economically competitive manufacturing alternative to classical chemical synthesis. Through continuous production improvement, glycosides can now be used in low-cost products by various industries. However, many production systems still suffer from low yields. Directed evolution, coupled with a suitable screening method, can tackle this challenge. We generated glycosyltransferase mutants through error-prone-PCR and screened the library using a small-scale whole-cell biotransformation system. The screening of only 176 colonies yielded three putative candidates. Detailed investigations revealed that the reason for the increase in product titer was mainly due to different expression effects of the mutagenized genes rather than improved enzyme kinetics. In total, a 60-fold increase in product formation was achieved. Therefore, in addition to the quality of the mutant library, an efficient and stable expression system is crucial to achieve high concentrations of active enzyme and product, as formation of inclusion bodies and other inactive forms of the biocatalyst reduces productivity.

scholarly journals Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method

2019 ◽  
Vol 400 (3) ◽  
pp. 405-415
Author(s):  
Sebastian W. Meister ◽  
Natalie M. Hendrikse ◽  
John Löfblom
Keyword(s):  
Directed Evolution ◽  
Proteolytic Activity ◽  
Fluorescent Protein ◽  
Screening Method ◽  
Fold Increase ◽  
Label Free ◽  
Peptide Bonds ◽  
3C Protease ◽  
High Throughput Method ◽  
Protease Substrate

Abstract Proteases are crucial for regulating biological processes in organisms through hydrolysis of peptide bonds. Recombinant proteases have moreover become important tools in biotechnological, and biomedical research and as therapeutics. We have developed a label-free high-throughput method for quantitative assessment of proteolytic activity in Escherichia coli. The screening method is based on co-expression of a protease of interest and a reporter complex. This reporter consists of an aggregation-prone peptide fused to a fluorescent protein via a linker that contains the corresponding substrate sequence. Cleavage of the substrate rescues the fluorescent protein from aggregation, resulting in increased fluorescence that correlates to proteolytic activity, which can be monitored using flow cytometry. In one round of flow-cytometric cell sorting, we isolated an efficiently cleaved tobacco etch virus (TEV) substrate from a 1:100 000 background of non-cleavable sequences, with around 6000-fold enrichment. We then engineered the 3C protease from coxsackievirus B3 (CVB3 3Cpro) towards improved proteolytic activity on the substrate LEVLFQ↓GP. We isolated highly proteolytic active variants from a randomly mutated CVB3 3Cpro library with up to 4-fold increase in activity. The method enables simultaneous measurement of proteolytic activity and protease expression levels and can therefore be applied for protease substrate profiling, as well as directed evolution of proteases.

scholarly journals Ultra-High-Throughput Screening Method for the Directed Evolution of Glucose Oxidase

2014 ◽  
Vol 21 (3) ◽  
pp. 414-421 ◽  
Author(s):  
Raluca Ostafe ◽  
Radivoje Prodanovic ◽  
Jovana Nazor ◽  
Rainer Fischer
Keyword(s):  
Glucose Oxidase ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method
Sign in / Sign up

Export Citation Format

Share Document