De novo transcriptome assembly of the midgut glands of theherbivorous land crab Chiromantes haematocheir, andidentification of laccase genes involved in lignin degradation
Katsuhide MIYAKE
Department of Environmental Technology, Faculty of Science and Technology,
Meijo University
Research objective
Many land crabs are herbivores. Chiromantes haematocheir and C. dehaani are
herbivorous land crabs that live in Japanese maritime forests, where they often eat decayedwood and fallen or green leaves (Fig. 1).
Therefore, herbivorous land crabs are thought todegrade plant biomass, and in particularlignocellulose. The latter consists of three major
components: cellulose, hemicellulose, lignin,
and tannins. We recently reported guaiacol
oxidase and cellulase activities in the midgut glands of the land crabs C. haematocheir and C.
dehaani (Miyake et al. 2019). Since guaiacol oxidation is probably involved in lignin
degradation, these land crabs seem to endogenously express lignin degrading enzymes.
Detailed genetic information is required to study the molecular basis of the degradation ofcomplex plant molecules such as lignin. However, the lack of genomic and transcriptomic
resources for land crabs strongly limits molecular studies. RNA sequencing (RNA-seq) is a
powerful tool that is widely used to profile the genomes of non-model species. In particular,
de novo sequencing and assembly of the transcriptome represent the first step for theidentification of genes involved in enzymatic activities in non-model animal species. In thisstudy, we sequenced and assembled the transcriptome of C. haematocheir midgut glands.
Genes encoding putative enzymes involved in the degradation of biopolymers, such as lignin
and cellulose, were identified, and their expression was further characterized. Here we report
the analysis of a laccase gene, which showed the highest expression level in the midgut glandsof this land crab species. The properties of the corresponding laccase protein and its role inplant biomass degradation were studied. To assess whether this laccase could oxidize ligninrelated substrates, we expressed this enzyme in Escherichia coli, purified it, and examined itsactivity.

Methods
Total RNA was purified from 30 mg of midgut glands using an RNeasy Plus Mini kit(Qiagen) according to the manufacturer’s instructions. RNA sequencing was performed by
Hokkaido System Science Co., Ltd. (Sapporo, Japan). The libraries were subjected to 100-bp
paired-end sequencing on an Illumina HiSeq 2500 instrument (Hokkaido System Science Co.,Ltd).
To investigate laccase activity, we used 2,6-dimethoxyphenol (2,6-DMP) as a substrate.
The reaction was initiated by mixing 100 µL of enzyme solution with 900 µL of a 4-mM
substrate solution. To estimate the oxidation rate of 2,6-DMP to coerulignone at 25 °C,absorbance was measured at 470 nm every minute for 5 min using a UV-VIS
Spectrophotometer UV-1280 (Shimadzu, Kyoto, Japan).
Laccase was produced as a C-terminal His6-tagged enzyme. Transformant clones werecultured in 100 mL of LB medium supplemented with 100 µg/mL ampicillin at 37 °C. Whenthe cultures reached an A600 of 0.6, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and
0.5 mM CuSO4 were added, and incubation was continued for 4 h. Cells were then collectedby centrifugation at 8000 ×g for 10 min at 4 °C. Laccase was purified using a HisTALONTM
gravity column purification kit (Clontech Laboratories, Inc., Mountain View, CA, USA).
Results
Land crabs were rearedwith plant or non-plant diets
to study the effect of diet on
laccase activity. After the
preparation of crude extracts
fromdigestiveorgans,enzymeactivity
wasmeasured. As shown in Fig.
2, significant laccase activity
against2,6-DMPwasdetected only in land crabs
fed with Zelkova leaves (plant diet). Laccase activity was mainly detected in midgut glandsamples, although several cardiac stomach samples also showed significant activity.
Nevertheless, laccase activity in the stomach was consistently lower than that in midgut glands.
These results suggest that laccase is synthesized within the midgut glands and secreted into
the digestive fluid of gut organs such as the cardiac stomach.
Based on de novo assembly and annotation of RNA-seq reads, 22 contigs were identified

as laccase gene fragments. These were then consolidated into 11 different genes, among which
the expression of TRINITY_DN24131 (24131) accounted for more than 99% of the totallaccase expression. BlastP analysis against the latest version of the database showed that theamino acid sequence of the 24131-laccase was highly similar to that of the predicted laccase
proteins of Portunus trituberculatus (swimming crab) (75.47%), Callinectes sapidus (bluecrab) (74.6%), and Penaeus vannamei (Pacific white shrimp) (67.65%). The 24131-laccase
consists of 613 amino acids, and exhibits a deduced molecular mass of 67.708 kDa and an
estimated pI of 4.17. The search for a conserved domain based on BlastP analysis revealed aCu-binding domain at the C terminus of this protein. This domain was similar to thecupredoxin domains of insect laccases, ascorbate oxidase, and multicopper oxidase. Theselaccase-like proteins harbor signal sequences in their N-terminal regions, indicating that they
are secretory enzymes.
We attempted to verify the function of the 24131-laccase by introducing its cDNA into thepETBlue-2 expression vector. This enzyme was produced as a His6-tagged protein in E. coli
BL21 (DE3) and purified using a His6-tag-binding resin. After purification of His6-tagged
24131-laccase, we analyzed its enzymatic activity by performing oxidation assays using 4 mM
2,6-DMP. Notably, the purified 24131-laccase exhibited significant oxidation activity against
2,6-DMP. As expected, the addition of CuSO4 to the culture medium was found to be essentialfor laccase activity.
Conclusion
The present study is the first to report the laccase activity of land crabs. Nevertheless, it isstill unclear whether crab laccases are involved in the assimilation of lignin. We speculate thatcrabs may use laccases to remove barriers or deterrents, such as lignin or tannins, in order toutilize carbohydrates. In possible future biotechnological applications, these enzymes may be
useful for the conversion of lignin structures, which may improve the extraction ofcarbohydrates such as cellulose and hemicellulose. For further applications of these putative
lignin degrading enzymes, it is pivotal to improve the production of 24131-laccase and identify
a guaiacol oxidase.
References
１） Miyake, K., Ura, K., Chida, S., Ueda, Y., Baba, Y., Kusube, T., and Yanai, S. (2019)
Guaiacol oxidation activity of herbivorous land crabs, Chiromantes haematocheir andChiromantes dehaani. J. Biosci. Bioeng. 128: 316-322.
２） Miyake, K. and Baba Y. (2021) De novo transcriptome assembly of the midgut glandsof herbivorous land crabs, Chiromantes haematocheir, and identification of laccase
genes involved in lignin degradation. J. Comp. Physiol. B in press.