Computational protocol: Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to improve the production of (2S,5S)-hexanediol at moderate temperatures

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Protocol publication

[…] Plasmid DNA was isolated from 5-ml cultures by using the QIAprep spin miniprep kit (Qiagen) and the sequence of the different clones was determined by sequence analysis of both DNA strands (BaseClear). Amino acid substitutions were deduced from identified mutations in the DNA sequence of the adhA gene. A structural model of AdhA was constructed by homology modeling using the crystal structures of the NADP+-dependent clavulanic acid dehydrogenase from Streptomyces clavuligerus as a template (PDB-ID: 2JAP and 2JAH) (MacKenzie et al. ). The primary sequence of this enzyme aligns with the complete AdhA sequence while sharing 30% sequence identity. The aligned sequences were modeled using Swiss Model (Schwede et al. ), an automated comparative protein-modeling server. The validity of the model was checked by WhatCheck (Hooft et al. ). The model was compared to several homologous short-chain ADH structures, i.e., to the sepiapterin reductase of Chlorobium tepidum (26% identity; CT0609; PDB-ID 2BD0) (Supangat et al. ), the gluconate-5-dehydrogenase of Thermotoga maritima (22% identity; TM0441; PDB-ID 1VL8), and the human 17-β-hydroxysteroid dehydrogenase (23% identity, PDB-ID 1A27) (Breton et al. ; Mazza et al. ). […]

Pipeline specifications

Software tools SWISS-MODEL, WHAT_CHECK
Application Protein structure analysis
Chemicals Ethanol, NADP