Supplementary Material for: Accessing Carboxylesterase Diversity from Termite Hindgut Symbionts through Metagenomics

A shotgun metagenomic library was constructed from termite hindgut symbionts and subsequently screened for esterase activities. A total of 68 recombinant clones conferring esterolytic phenotypes were identified, of which the 14 most active were subcloned and sequenced. The nucleotide lengths of the esterase-encoding open reading frames (ORFs) ranged from 783 to 2,592 bp and encoded proteins with predicted molecular masses of between 28.8 and 97.5 kDa. The highest identity scores in the GenBank database, from a global amino acid alignment ranged from 39 to 83%. The identified ORFs revealed the presence of the G-X-S-X-D, G-D-S-X, and S-X-X-K sequence motifs that have been reported to harbour a catalytic serine residue in other previously reported esterase primary structures. Five of the ORFs (EstT5, EstT7, EstT9, EstT10, and EstT12) could not be classified into any of the original eight esterase families. One of the ORFs (EstT9) showed a unique primary structure consisting of an amidohydrolase-esterase fusion. Six of the 14 esterase-encoding genes were recombinantly expressed in <i>Escherichia coli</i> and the purified enzymes exhibited temperature optima of between 40–50°C. Substrate-profiling studies revealed that the characterised enzymes were ‘true’ carboxylesterases based on their preferences for short to medium chain length <i>p</i>-nitrophenyl ester substrates. This study has demonstrated a successful application of a metagenomic approach in accessing novel esterase-encoding genes from the gut of termites that could otherwise have been missed by classical culture enrichment approaches.