Computational protocol: A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans

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Protocol publication

[…] The PUL database (PULDB, http://www.cazy.org/PULDB) [] was searched for PULs that contained at least two of the predicted xylan-active CAZyme families of interest, such as GH10, GH43, GH67, and GH115. From the obtained list of PULs, all proteins of unknown function were extracted and analyzed using Signal P4.1 []; those containing a predicted signal sequence for secretion were further analyzed based on the presence of Pfam domains, sequence length, and the type of CAZymes present on the corresponding PUL. Based on these criteria, FjoAcXE (PULDB ID: Fjoh_3879; GenBank ID: ABQ06890.1) was selected for recombinant protein expression. A structural model of FjoAcXE was built using PHYRE2.0 [] for the CBM-like domain, using PDB 2O14 as template, and Modeller 9.19 [] for the CE/catalytic domain using PDB 2O14 and PDB 1K7C []. The model was displayed with PyMOLv1.7.4.5 Edu (PyMOL Molecular Graphics System, Schrödinger, LLC). [...] The gene encoding FjoAcXE lacking the predicted signal sequence (residues 1–21) was codon optimized for expression in Escherichia coli K12 using IDT Codon Optimization Tool (http://www.idtdna.com/CodonOpt). Fifteen base pair extensions homologous to the p15TV-L vector (GenBank ID: EF456736.1) (T7, 5′-TTGTATTTCCAGGGC and T7term, 5′-CAAGCTTCGTCATCA) were added to each end of the sequence and the gene was synthesized as gBlock® gene fragments (Integrated DNA Technologies, Inc., Coralville, IA, USA). gBlock fragments were cloned into p15TV-L using the In-Fusion® HD EcoDry™ Cloning Kit (Clontech Laboratories, Inc., Palo Alto, CA, USA). The resulting plasmid was transformed into E. coli HST08 Stellar™ Competent Cells (Clontech Laboratories, Inc.), and the sequence was verified using DNA sequencing service at the Center of Applied Genomics at the SickKids Hospital (Toronto, ON, Canada). […]

Pipeline specifications

Software tools Codon Optimization Tool, Gblocks
Application Synthetic biology