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4 Publications visible to you, out of a total of 4
Winning the numbers game in enzyme evolution - fast screening methods for improved biotechnology proteins.

Abstract (Expand)

The booming demand for environmentally benign industrial processes relies on the ability to quickly find or engineer a biocatalyst suitable to ideal process conditions. Both metagenomic approaches and … directed evolution involve the screening of huge libraries of protein variants, which can only be managed reasonably by flexible platforms for (ultra)high-throughput profiling against the desired criteria. Here, we review the most recent additions toward a growing toolbox of versatile assays using fluorescence, absorbance and mass spectrometry readouts. While conventional solution based high-throughput screening in microtiter plate formats is still important, the implementation of novel screening protocols for microfluidic cell or droplet sorting systems supports technological advances for ultra-high-frequency screening that now can dramatically reduce the timescale of engineering projects. We discuss practical issues of scope, scalability, sensitivity and stereoselectivity for the improvement of biotechnologically relevant enzymes from different classes.

Authors: Yuriy V Sheludko, Wolf-Dieter Fessner

Date Published: 29th Jun 2020

Publication Type: Journal

Convergent in situ Generation of Both Transketolase Substrates via Transaminase and Aldolase Reactions for Sequential One‐Pot, Three‐Step Cascade Synthesis of Ketoses

Abstract

Not specified

Authors: Marion Lorillière, Christine Guérard‐Hélaine, Thierry Gefflaut, Wolf‐Dieter Fessner, Pere Clapés, Franck Charmantray, Laurence Hecquet

Date Published: 12th Dec 2019

Publication Type: Journal

Aldolase-Catalyzed Asymmetric Synthesis of N-Heterocycles by Addition of Simple Aliphatic Nucleophiles to Aminoaldehydes

Abstract (Expand)

Nitrogen heterocycles are structural motifs found in many bioactive natural products and of utmost importance in pharmaceutical drug development. In this work, a stereoselective synthesis of functionalized … N‐heterocycles was accomplished in two steps, comprising the biocatalytic aldol addition of ethanal and simple aliphatic ketones such as propanone, butanone, 3‐pentanone, cyclobutanone, and cyclopentanone to N‐Cbz‐protected aminoaldehydes using engineered variants of d‐fructose‐6‐phosphate aldolase from Escherichia coli (FSA) or 2‐deoxy‐d‐ribose‐5‐phosphate aldolase from Thermotoga maritima (DERATma) as catalysts. FSA catalyzed most of the additions of ketones while DERATma was restricted to ethanal and propanone. Subsequent treatment with hydrogen in the presence of palladium over charcoal, yielded low‐level oxygenated N‐heterocyclic derivatives of piperidine, pyrrolidine and N‐bicyclic structures bearing fused cyclobutane and cyclopentane rings, with stereoselectivities of 96–98 ee and 97:3 dr in isolated yields ranging from 35 to 79%.

Authors: Raquel Roldán, Karel Hernández, Jesús Joglar, Jordi Bujons, Teodor Parella, Wolf-Dieter Fessner, Pere Clapés

Date Published: 6th Jun 2019

Publication Type: Not specified

Second-Generation Engineering of a Thermostable Transketolase (TK Gst ) for Aliphatic Aldehyde Acceptors with Either Improved or Reversed Stereoselectivity

Abstract (Expand)

The transketolase from Geobacillus stearothermophilus (TKGst) is a thermostable enzyme with notable high activity and stability at elevated temperatures, but it accepts non‐α‐hydroxylated aldehydes … only with low efficiency. Here we report a protein engineering study of TKGst based on double‐site saturation mutagenesis either at Leu191 or at Phe435 in combination with Asp470; these are the residues responsible for substrate binding in the active site. Screening of the mutagenesis libraries resulted in several positive variants with activity towards propanal up to 7.4 times higher than that of the wild type. Variants F435L/D470E and L191V/D470I exhibited improved (73 % ee, 3S) and inverted (74 % ee, 3R) stereoselectivity, respectively, for propanal. L191V, L382F/E, F435L, and D470/D470I were concluded to be positive mutations at Leu191, Leu382, Phe435, and Asp470 both for activity and for stereoselectivity improvement. These results should benefit further engineering of TKGst for various applications in asymmetric carboligation.

Authors: Chaoqiang Zhou, Thangavelu Saravanan, Marion Lorillière, Dongzhi Wei, Franck Charmantray, Laurence Hecquet, Wolf-Dieter Fessner, Dong Yi

Date Published: 2nd Mar 2017

Publication Type: Not specified

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