[국내논문]Metagenomic SMRT Sequencing-Based Exploration of Novel Lignocellulose-Degrading Capability in Wood Detritus from Torreya nucifera in Bija Forest on Jeju Island원문보기
Lignocellulose, composed mostly of cellulose, hemicellulose, and lignin generated through secondary growth of woody plant, is considered as promising resources for biofuel. In order to use lignocellulose as a biofuel, biodegradation besides high-cost chemical treatments were applied, but knowledge o...
Lignocellulose, composed mostly of cellulose, hemicellulose, and lignin generated through secondary growth of woody plant, is considered as promising resources for biofuel. In order to use lignocellulose as a biofuel, biodegradation besides high-cost chemical treatments were applied, but knowledge on the decomposition of lignocellulose occurring in a natural environment is insufficient. We analyzed the 16S rRNA gene and metagenome to understand how the lignocellulose is decomposed naturally in decayed Torreya nucifera (L) of Bija forest (Bijarim) in Gotjawal, an ecologically distinct environment. A total of 464,360 reads were obtained from 16S rRNA gene sequencing, representing diverse phyla; Proteobacteria (51%), Bacteroidetes (11%) and Actinobacteria (10%). The metagenome analysis using single molecules real-time sequencing revealed that the assembled contigs determined originated from Proteobacteria (58%) and Actinobacteria (10.3%). Carbohydrate Active enZYmes (CAZy)- and Protein families (Pfam)-based analysis showed that Proteobacteria was involved in degrading whole lignocellulose, and Actinobacteria played a role only in a part of hemicellulose degradation. Combining these results, it suggested that Proteobacteria and Actinobacteria had selective biodegradation potential for different lignocellulose substrates. Thus, it is considered that understanding of the systemic microbial degradation pathways may be a useful strategy for recycle of lignocellulosic biomass, and the microbial enzymes in Bija forest can be useful natural resources in industrial processes.
Lignocellulose, composed mostly of cellulose, hemicellulose, and lignin generated through secondary growth of woody plant, is considered as promising resources for biofuel. In order to use lignocellulose as a biofuel, biodegradation besides high-cost chemical treatments were applied, but knowledge on the decomposition of lignocellulose occurring in a natural environment is insufficient. We analyzed the 16S rRNA gene and metagenome to understand how the lignocellulose is decomposed naturally in decayed Torreya nucifera (L) of Bija forest (Bijarim) in Gotjawal, an ecologically distinct environment. A total of 464,360 reads were obtained from 16S rRNA gene sequencing, representing diverse phyla; Proteobacteria (51%), Bacteroidetes (11%) and Actinobacteria (10%). The metagenome analysis using single molecules real-time sequencing revealed that the assembled contigs determined originated from Proteobacteria (58%) and Actinobacteria (10.3%). Carbohydrate Active enZYmes (CAZy)- and Protein families (Pfam)-based analysis showed that Proteobacteria was involved in degrading whole lignocellulose, and Actinobacteria played a role only in a part of hemicellulose degradation. Combining these results, it suggested that Proteobacteria and Actinobacteria had selective biodegradation potential for different lignocellulose substrates. Thus, it is considered that understanding of the systemic microbial degradation pathways may be a useful strategy for recycle of lignocellulosic biomass, and the microbial enzymes in Bija forest can be useful natural resources in industrial processes.
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제안 방법
Extracted DNA was used for PCR and 16S rRNA gene sequencing. The PCR amplification was carried out using the primer set V4-V5 region of the 16S rRNA gene (forward: CCA GCA GC[T,C] GCG GT[G,A] A.; reverse: CCG TCA ATT C.T TT[G,A] AGT). In the first PCR, thermal cycle conditions were 95oC for 3 min, followed by 33 cycles of 30 sec denaturation at 95oC, 30 sec at 55oC for annealing, and 1 min at 72oC for extension, and a final extension was performed at 72oC for 5 min.
Then, the product was barcoded using the Nextera XT Index Kit v2 (Illumina, USA) and amplified for 8 cycles. The second PCR product was purified using AMPure XP beads and the quality of the enriched libraries was evaluated using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, US). The libraries were sequenced at Macrogen (Korea) by means of the Illumina MiSeq platform, and the generated 16S rRNA gene sequences were processed and analyzed with QIIME [16, 17].
A total of 217,558 reads ranging from 5,202 to 61,553 bp in length were obtained and subsequently assembled by the hierarchical genomeassembly process (HGAP) into 204 contigs with a contig N50 of 15,225 bp. The mean coverage of the assembled sequences was 45.5-fold, and this coverage was relatively low, but accurate analysis was possible by providing the long reads to reduce the error through assembly. The metagenome dataset statistics are summarized in Table 1.
대상 데이터
Taxonomic classification of contigs was performed by Edge Perl scripts [26] using Burrows-Wheeler Aligner (BWA) [27] mapping to National Center for Biotechnology Information (NCBI) RefSeq [26-28]. The data reported in this paper are available at the SRA database with the accession number of SRR5230003.
성능/효과
Both results revealed the dominance of Proteobacteria and Actinobacteria in Bija’s wood detritus, and two orders (Xanthomonadales and Rhizobiales) dominated.
As results of the sequencing, a total of 366,621 pairend reads were merged into 94,290 sequences. From all the 16S rRNA sequences, 16,283 OTUs were obtained, and 15,277 OTUs were obtained finally after removing the ones from Chloroplast and Mitochondria. A total of 99.
From all the 16S rRNA sequences, 16,283 OTUs were obtained, and 15,277 OTUs were obtained finally after removing the ones from Chloroplast and Mitochondria. A total of 99.9% of OTUs represented the kingdom Bacteria, 0.003% corresponded to Archaea, and the remaining were unclassified. Three major phyla were Proteobacteria (48.
The most abundant genus was unclassified Xanthomonadaceae, followed by unclassified Sinobacteraceae and unclassified Chitinophagaceae. OTUs assigned to unclassified Xanthomonadaceae (most closely related to genus Mizugakiibacter and Povalibacter by SeqMatch) were the most dominant (7.2%) (Fig. 2).
The contigs have been classified using BWA mapping to NCBI RefSeq. Only two phyla, Proteobacteria (57.8%) and Actinobacteria (10.3%), were observed, and the most abundant orders were Xanthomonadales (39.2%), Burkholderiales (7.8%), and Solirubrobacterales (4.4%) (Table 2).
Overall, a higher abundance of members of Proteobacteria was detected, and among them, Xanthomonadales was the largest bacterial order, which had remarkable abundance in entire lignocellulose degradation in Bija’s detritus (Table 4).
A total of 65 proteins were found for these five modules (Table 2). Our results indicated that some GHs (GH5, 9, and 10) and CBM35 were included in M1, consensus lignocellulose degradation modules, whereas GH16 and some CBMs (CBM32, 47, and 13) were affiliated with M2, which are xylan-binding and xyloglucan degradation modules. In addition, PF00759, PF00331, and PF13472 were assigned to M1, whereas PF13229, PF13472, and PF13524 were assigned to the M3 module (pectin degradation modules).
3). It was confirmed that CAZymes related to lignocellulose degradation are proportional to the number of total metagenomes. Xanthomonadales (Gammaproteobacteria), Burkholderiales (Betaproteobacteria), and Solirubrobacterales (Actinobacteria) were widely distributed in the Bija metagenome, and many of the lignocellulose-degrading enzymes were assigned to the ORFs of these orders.
The main findings of this study confirmed that the dominant members—Burkholderiales (Proteobacteria), Xanthomonadales (Proteobacteria), and Solirubrobacterales (Actinobacteria)—play an important role in lignocellulose decomposition, as shown in other studies [40, 41].
Two orders showed large distribution in both results commonly; we investigated 16S rRNA gene analysis at the genus level. It was supposed that the most abundant genus was Mucilaginibacter of Xanthomonadales (RDP SeqMatch results), which was also found in the OTU analysis the most frequently, and the second largest genus of Xanthomonadales was Povalibacter. Among the Burkholderiales members, the most abundant genus was considered to be Burkholderia, which accounted for 0.
It was supposed that the most abundant genus was Mucilaginibacter of Xanthomonadales (RDP SeqMatch results), which was also found in the OTU analysis the most frequently, and the second largest genus of Xanthomonadales was Povalibacter. Among the Burkholderiales members, the most abundant genus was considered to be Burkholderia, which accounted for 0.9% in the OTU analysis, and the greatest number of Solirubrobacterales was Conexibacter.
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