Mining the biomass deconstructing capabilities of rice yellow stem borer symbionts

Abstract

Background: Efficient deconstruction of lignocellulosic biomass into simple sugars in an economically viable manner is a prerequisite for its global acceptance as a feedstock in bioethanol production. This is achieved in nature by suites of enzymes with the capability of efficiently depolymerizing all the components of lignocellulose. Here, we provide detailed insight into the repertoire of enzymes produced by microorganisms enriched from the gut of the crop pathogen rice yellow stem borer (Scirpophaga incertulas).

Results: A microbial community was enriched from the gut of the rice yellow stem borer for enhanced rice straw degradation by sub-culturing every 10 days, for 1 year, in minimal medium with rice straw as the main carbon source. The enriched culture demonstrated high cellulolytic and xylanolytic activity in the culture supernatant. Metatranscriptomic and metaexoproteomic analysis revealed a large array of enzymes potentially involved in rice straw deconstruction. The consortium was found to encode genes ascribed to all five classes of carbohydrate-active enzymes (GHs, GTs, CEs, PLs, and AAs), including carbohydrate-binding modules (CBMs), categorized in the carbohydrate-active enzymes (CAZy) database. The GHs were the most abundant class of CAZymes. Predicted enzymes from these CAZy classes have the potential to digest each cell-wall components of rice straw, i.e., cellulose, hemicellulose, pectin, callose, and lignin. Several identified CAZy proteins appeared novel, having an unknown or hypothetical catalytic counterpart with a known class of CBM. To validate the findings, one of the identified enzymes that belong to the GH10 family was functionally characterized. The enzyme expressed in E. coli efficiently hydrolyzed beechwood xylan, and pretreated and untreated rice straw.

Conclusions: This is the first report describing the enrichment of lignocellulose degrading bacteria from the gut of the rice yellow stem borer to deconstruct rice straw, identifying a plethora of enzymes secreted by the microbial community when growing on rice straw as a carbon source. These enzymes could be important candidates for biorefineries to overcome the current bottlenecks in biomass processing.

Keywords: Carbohydrate-active enzymes; GH10 family; Gut consortium; Metaexoproteome; Microbial diversity; Rice yellow stem borer; Targeted enrichment; Xylanase.

Fig. 1

Rice yellow stem borer gut microbial community structure at the level of Phylum. Relative abundance of phylum in the a gut consortium and in the c enriched consortium. b Total number of Operational Taxonomy Unit (OTU) in the gut consortium and in the enriched consortium

Fig. 2

Rice yellow stem borer gut microbial community structure at the level of genus. Relative abundance of genus in the a gut consortium and in the c enriched consortium. Top 20 genera in terms of their unique OTUs detected in the b gut consortium and in the d enriched consortium

Fig. 3

Evaluation of different culture conditions for biomass degrading enzyme production. Cultures were grown under various conditions, and secretory proteins (a) and cell bound protein extract (b) were evaluated for release of glucose and xylose using CMC and xylan as substrates, respectively. Data in a and b represent mean ± SD. TSB Tryptic Soya Broth, YE yeast extract

Fig. 4

Enrichment of rice straw deconstructing YSB gut microbial community and assessment of available enzymes and biomass degrading ability. a The microbial community was passaged for 1 year on the rice straw containing medium and analyzed for various features. b Reduction in rice straw weight after incubation with either enriched consortium or original symbionts; c CMCase activity shown by the supernatant and cell bound protein fraction of YSB gut consortium on plate containing CMC and trypan blue dye; d CMCase and xylanase assay of YSB gut consortium proteins on zymogram; e Morphologically different colonies grown as a result of plating on YEB agar plate

Fig. 5

Venn diagram showing the proportion of CAZy assignments observed exclusively in the Bound Fraction, Supernatant or in both fractions

Fig. 6

Dynamics of changes in different classes of CAZy families upon cultivation on rice straw for 20 days. Hierarchical clustering of CAZy family proteins detected at 4th, 7th, 13th and 20th day of cultivation in the bound (a) and supernatant (b) fractions. c Comparison of the expression level of various CAZy classes in the 30 high expressed contigs at each timepoints

Fig. 7

Annotation, expression and characterization of xylanase from the enriched consortium derived from rice stem borer gut. a Schematic representation of various modules present in the xylanase polypeptide; SP signal peptide, GH10 glycoside hydrolase of family 10, CBM2 carbohydrate-binding modules of family 2. b Cloning of xylanase ORF without the SP in the expression vector pET30a at the NdeI and HindIII restriction sites to derive the expression of xylanase with the help of T7 promoter. c Xylanase protein purification. Lane1, uninduced total cellular protein; lane 2, Induced total cellulase protein and Lane 3, Purified xylanase protein after metal affinity chromatography. d Optimal temperature and e optimal pH for activity of xylanase