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Genomic and Metagenomic Analyses of a Wood-Feeding Cockroach, Cryptocercus Punctulatus
Department of Entomology
As readily available fossil fuels are depleted, attention has turned to lignocelluloses to convert solar energy into thermochemical energy. Lignocellulose is a naturally occurring complex of plant-derived materials that includes the hydrophilic sugar polymers cellulose and hemicellulose, and the hydrophobic organic glue lignin. However, to release the solar energy stored in plants through photosynthesis, an array of enzymes referred to as lignocellulases are required to disassociate a matrix of cellulose, hemicelluloses, pectins, lignin, and glycosidic linkages. Similar to lower termites, wood-feeding C. punctulatus and their gut symbionts have co-evolved into a suite of specialized enzymes that synergistically break down lignocelluloses.
While the pressing needs for bioenergy and biomass conversion have redirected termite research to focus more on the lignocellulose degradation, research on Cryptocercus has been primarily focused on evolutionary relationships with Isoptera (termites), and the development of sociality. There is virtually no information available on lignocellulose digestion and degradation in wood-feeding cockroaches. To fill this knowledge gap and to enrich the existing gene pool of lignocellulolytic enzymes, I propose to sequence 1) a cDNA library synthesized from the entire digestive tract without symbionts, and 2) a gDNA library extracted from hindgut microbial community of the wood-feeding cockroach using a high throughput 454 pyrosequencing platform.
The potential impacts of the proposed project are that
1) a complete genome-metagenome-wide survey of lignocellulases will add novel information regarding the enzymatic conversion of lignocellulosic biomass;
2) information gained from the wood-feeding cockroach may help to identify a subset of conserved lignocellulolytic enzymes throughout C. punctulatus "C R. flavipes lineage; the distribution of lignocellulase genes among different insects has yet to be explored in great detail. However, given the fact that Cryptocercus and lower termites share many flagellate symbionts such as oxymonadid and hypermastigid in their hindguts, it is germane to survey the homogeneity of lignocellulases across Dictyoptera with the basal C. punctulatus as a reference using a comparative genomic approach. Generally, evolutionarily conserved lignocellulases bear more biological meaning in the processes of lignocellulosic biomass conversion and may shed light on the core enzymes of the lignocellulose digestion machinery;
3) Cryptocercus maintains more diverse gut flagellate species than any existing termite species, and provides an invaluable resource for novel lignocellulases; and
4) it will help us to elucidate the operating mechanism underlying the host-symbiont lignocellulose digestion systems.
The role that endogenous and symbiotic lignocellulases play in lignocellulose digestion has remained a point of conjecture. The proposed sequencing will shed light on the genome organization and interactions (synergistically or independently) of the existing dual lignocellulase system in both C. punctulatus and R. flavipes.
2010 Project Description
Dictyopterans including termites and woodroaches by far are the most efficient lignocellulose-digesting bioreactors on this planet, and have intrigued scientists for decades because of their unique capability of breaking down seemingly recalcitrant lignocelluloses. In the first year of this Hatch project, we initiated several sequencing projects to inventory a comprehensive set of genes encoding lignocellulases in the wood-feeding C. punctulatus.
1. Transcriptomic analysis of C. punctulatus digestive tract Tissue-specific transcriptomes were sequenced at the University of Arizona Genetics Core. Salivary gland, foregut, midgut, and hindgut, respectively, from 30 adult roaches were dissected and pooled for the total RNA extraction. Emulsion PCR (emPCR) and 454 Pyrosequencing were carried out according to Roche GS FLX Titanium protocols. Each woodroach tissue accounted for 1/8 of a sequencing plate, and the average total reads and total bases from these digestive tract tissues was 165,120 and 60,819,523, respectively. A total number of Isotigs from 4 tissue-based libraries are 1730, 4498, 3152, and 4560, respectively.
2. Metatranscriptomic analysis of hindgut microbiota in C. punctulatus Pyrosequencing of the woodroach hindgut metatranscriptome were performed by the Advanced Genetic Technologies Center (AGTC) at the University of Kentucky. Digestive tract from seven C. punctulatus adults was removed, and the luminal contents from hindgut were collected carefully. The total numbers of Reads and Bases from a half plate run are 1,010,243 and 228,975,825, respectively. A total number of Isotigs from symbiont librarys is 11,584. In comparison to the lower termite, R. flavipes, symbiotic lignocellulolytic enzymes in the woodroach hindgut microbiota (170) is significantly more abundant and diverse.
3. Metaproteomic profiling of hindgut microbiota in C. punctulatus The luminal contents collected from a total of nine C. punctulatus adults were used for metaproteomics analyses. Total protein was extracted directly from the luminal contents, and then it was subjected to the trypsin digestion, and followed by peptide identification using MALDI-TOF/TOF mass spectrometry. A total of 694 and 565 proteins were identified from C. punctulatus hindgut microbiota using peptide libraries from R. flavipes symbionts and guts, respectively. Among them, 194 and 169 sequences were unclassified. Based on the GO term molecular function, 79 peptide sequences were putatively classified as glycoside hydrolases in which majority of them belonged to GHF-7,-8, -2, -3, -5, and -18 (66 derived from the symbiont library and 13 came from the gut Library, respectively). Hindgut microbiota from C. punctulatus and R. flavipes apparently shared some lignocellulolytic enzymes (e.g. GHF-7), however the low degree of overlapping GHFs in the preliminary metaproteomics study supported the contention that C. punctulatus is a rich reservoir for novel lignocellulases.
This project has considerable potential economic impacts in biomass conversion and process applications. Because of their unique biology and physiology, wood-feeding Dictyopterans provide a rich reservoir for novel lignocellulases, especially the enzymes associated with biomass pretreatments. The enzymatic pretreatment is a critical step to improve the efficiency of current biomass conversion process.
Sun, J. and X. Zhou. 2010. Utilization of lignocellulose-feeding Insects for viable biofuels: an emerging and promising area of entomological science. In: T. X. Liu and L. Kang (eds), Recent advances in entomological research: from molecular biology to pest management. High Education Press, Beijing, China. In press.