Accessing Carboxylesterase Diversity from Termite Hindgut Symbionts through Metagenomics

Abstract

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 Escherichia coli 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 p-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.

Omic research in termites: an overview and a roadmap

“…At the time of writing this article, at least 12 prokaryotic metagenomes had been partially sequenced ( Table 1 ). Most metagenome publications have reported on lignocellulase identification from genome sequences of gut bacteria that selectively grew on lignocellulose media ( Liu et al, 2011 ; Mattéotti et al, 2011a , b , 2012 ; Nimchua et al, 2012 ; Rashamuse et al, 2012 , 2014 ; Wang et al, 2012 ). Another study used targeted xylanase screening from gut and ectosymbiotic fungi-associated bacteria of the higher termite Pseudacanthotermes militaris ( Bastien et al, 2013 ).…”

A novel metagenome-derived gene cluster from termite hindgut: Encoding phosphotransferase system components and high glucose tolerant glucosidase

“…The metagenomic approach has become an important tool to discover novel lignocellulases from hindgut-associated obligate symbionts of termites in the last decade, and several enzymes related to lignocellulose degradation were successfully identified from termites via metagenomic strategy. These enzymes included feruloyl esterases [27], carboxylesterase [28], hemicellulases [29], -glucosidases [30].…”

Application of termite hindgut metagenome derived carboxyl ester hydrolases in the modification of cephalosporin substrates

“…The construction and screening of a metagenomic library from termite hindgut symbionts have been described previously [12] . Briefly, following the extraction of a total High molecular weight community DNA from the P3 section of termite hindgut, a large insert DNA library was generated using the copy control fosmid library production kit (Epicentre Biotechnologies, WI, USA).…”

“…Briefly, following the extraction of a total High molecular weight community DNA from the P3 section of termite hindgut, a large insert DNA library was generated using the copy control fosmid library production kit (Epicentre Biotechnologies, WI, USA). The library was screened for clones displaying esterolytic activity by tributyrin hydrolysis on LB tributyrin agar plates [12] . Esterase genes (AxeA and AxeB) from the positive fosmids were identified by generating pUC19 subclone libraries from fosmids partially digested with Sau 3A1.…”

“…As a result, hindguts of termite species provide an ideal source for bio-prospecting for novel enzyme genes. In this study, we have screened a previously constructed termite hindgut metagenomic library [12] for esterases/cephalosporin C-deacetylases that can be applied in a chemo-enzymatic synthesis of deacetylated β -lactam molecules. The two identified carboxyl ester hydrolase encoding genes ( axe A and axe B) were recombinantly expressed in Escherichia coli and their deacetylating catalytic abilities against acetylated β -lactam substrates were demonstrated.…”