Riboswitches are structured RNA motifs that can directly bind specific metabolites. The binding of metabolites further regulates downstream metabolism eliminating the need for any regulatory proteins. We searched for novel bacterial vitamin B1 binding riboswitches in the metagenome of sun-dried saline soil. Soil microbial metagenomes were studied using NGS analysis. A total of approximately 50 Gb of the sequence data was obtained by Hi-seq and 454 GS FLX sequencing, and these sequences were subjected to riboswitch search. Hi-seq generated 614 contigs showing similarity to riboswitches, while 454-based sequencing generated 383 similar contigs. We matched whole metagenome contigs to local BLAST databases constructed using 91 previously known bacterial vitamin B1 thiamine pyrophosphate (TPP)-box motifs, and 11 SAM S-box motifs. Repetitive BLAST comparisons to local BLAST databases with nucleotide sequences from NGS identified 14 novel TPP-box motifs, and 7 S-box motifs respectively from the metagenome contigs. Further, RNA secondary structure analysis with public databases Rfam, and RibEx using these 21 riboswitch candidates revealed one contig, D8PYI to possess the most probable TPP-box structure. We constructed intragenic synthetic riboswitches to investigate whether the TPP-box motif region in D8PYI could harness gene expression in the presence of TPP. Construction of biosensors containing 100~400 bp fragments of D8PYI contigs, and in vivo imaging using the biosensors displayed TPP-specific changes in the expression of a green fluorescence protein reporter. In this regard, the adaptation of in silico riboswitch screening from environmental metagenomes could provide biosensors for detection of specific metabolites.

A carboxymethyl cellulase gene, cel5B, was cloned, sequenced, and expressed in Escherichia coli. pRCS20 in E. coli was identified from metagenomic cosmid library of cow rumen for cellulase activity on a carboxymethyl cellulose agar plates. Cosmid clone (RCS20) was partially digested with Sau3AI, ligated into BamHI site of pBluescript II SK+ vector, and transformed into E. coli DH5α. The insert DNA of 1.3 kb was obtained, designated cel5B, which has the activity of hydrolyzation of CMC. The cel5B gene had an open reading frame (ORF) of 1,059 bp encoding 352 amino acids with a signal peptide of 48 amino acids and the conserved region, VIYEIYNEPL, belongs to the glycosyl hydrolase family 5. The molecular mass of Cel5B protein expressed from E. coli DH5α exhibited to be about 34 kDa by CMC-SDS-PAGE. The optimal pH was 8.0, and the optimal temperature was about 50℃ for its enzymatic activity.

국내 하수처리장의 바이오가스화 시설은 낮은 농축율로 인한 저농도 유입수, 공정 흐름별 운영기술에 대한 노하우 부족 및 관리 미숙으로 인하여 혐기소화 효율이 미국 등 선진국 대비 약 54.2 %에 불과한 실정이다. 환경부는 바이오가스화 효율을 증진하고 하수슬러지를 바이오매스로 활용하기 위하여 2010년부터 전국적으로 에너지자립화 사업을 추진하였다. 특히 혐기소화조가 미설치된 하수처리장을 대상으로 음식물류폐기물과 병합소화시 연계 하수처리장의 유입 수질 등을 사전에 검토한 후 신규 소화조를 건설을 진행하고 있다. 하수슬러지 단독 및 병합처리 시설의 혐기소화조 내부 유출액에 대한 최우점 미생물 종을 분석하고 바이오가스화 시설의 주요 운전인자(유기물부하율, 메탄가스 발생량, C/N 비 등) 간의 정준상관분석을 실시하였다. 하수슬러지 단독 및 병합처리 시설의 상관관계 분포가 뚜렷이 구분되었으며, 메탄가스 생성율은 음폐수의 영향으로 VFAs, C/N비, VS/TS 등과 비례관계를 보임과 동시에 Mesotoga, Methanosaeta 미생물 군이 우점하는 경향을 보였다. 또한 하수슬러지 단독처리 바이오가스화 시설의 혐기소화조에서는 Copro-thermobacter, Methanosarcina 등의 미생물 분포가 높게 나타났다.