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Transcriptome-Guided Identification of Carbohydrate Active Enzymes (CAZy) from the Christmas Island Red Crab, Gecarcoidea natalis and a Vote for the Inclusion of Transcriptome-Derived Crustacean CAZys in Comparative Studies

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Abstract

The Christmas Island red crab, Gecarcoidea natalis, is an herbivorous land crab that consumes mostly fallen leaf litter. In order to subsist, G. natalis would need to have developed specialised digestive enzymes capable of supplying significant amounts of metabolisable sugars from this diet. To gain insights into the carbohydrate metabolism of G. natalis, a transcriptome assembly was performed, with a specific focus on identifying transcripts coding for carbohydrate active enzyme (CAZy) using in silico approaches. Transcriptome sequencing of the midgut gland identified 70 CAZy-coding transcripts with varying expression values. At least three newly discovered putative GH9 endo-β-1,4-glucanase (“classic cellulase”) transcripts were highly expressed in the midgut gland in addition to the previously characterised GH9 and GH16 (β-1,3-glucanase) transcripts, and underscoring the utility of whole transcriptome in uncovering new CAZy-coding transcripts. A highly expressed transcript coding for GH5_10 previously missed by conventional screening of cellulase activity was inferred to be a novel endo-β-1,4-mannase in G. natalis with in silico support from homology modelling and amino acid alignment with other functionally validated GH5_10 proteins. Maximum likelihood tree reconstruction of the GH5_10 proteins demonstrates the phylogenetic affiliation of the G. natalis GH5_10 transcript to that of other decapods, supporting endogenous expression. Surprisingly, crustacean-derived GH5_10 transcripts were near absent in the current CAZy database and yet mining of the transcriptome shotgun assembly (TSA) recovered more than 100 crustacean GH5_10s in addition to several other biotechnological relevant CAZys, underscoring the unappreciated potential of the TSA database as a valuable resource for crustacean CAZys.

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Authors and Affiliations

  1. Centre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University, Geelong, VIC, 3220, Australia

    Han Ming Gan, Christopher Austin & Stuart Linton

  2. School of Science, Monash University Malaysia, Jalan Lagoon Selatan, 47500, Bandar Sunway, Selangor, Malaysia

    Han Ming Gan & Christopher Austin

  3. Genomics Facility, Tropical and Medicine Biology Platform, Monash University Malaysia, Jalan Lagoon Selatan, 47500, Bandar Sunway, Selangor, Malaysia

    Han Ming Gan & Christopher Austin

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  1. Han Ming Gan
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  2. Christopher Austin
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Correspondence to Stuart Linton.

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Figure S1

qPCR-based quantification of selected CAZy transcripts and single copy reference gene, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Mean Cq values were indicates on each barchart and error bars represent standard deviation of three technical replicates. (PNG 50 kb)

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Gan, H.M., Austin, C. & Linton, S. Transcriptome-Guided Identification of Carbohydrate Active Enzymes (CAZy) from the Christmas Island Red Crab, Gecarcoidea natalis and a Vote for the Inclusion of Transcriptome-Derived Crustacean CAZys in Comparative Studies. Mar Biotechnol 20, 654–665 (2018). https://doi.org/10.1007/s10126-018-9836-2

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