A metagenomic approach for the identification and cloning of an endoglucanase from rice straw compost.
Over the years, culturable cellulase-producing microorganisms have been isolated from a variety of sources and genes of cellulolytic enzymes have been cloned. Then again, the "great plate count anomaly" phenomenon necessitates a culture-independent metagenomic approach for the isolation of cellulolytic genes from microorganisms in their natural environment. We have constructed a metagenomic library derived from rice straw composts. Of 2739 clones screened, a lambda clone carrying a 12kb genomic fragment was able to yield a clear zone on an agar plate containing carboxymethyl cellulose (CMC). A 4.7kb subclone, generated by restriction enzyme digestion, was found to harbor a GH12 cellulase gene, RSC-EG1, encoding 464 amino acids along with two other ORFs. The identified cellulolytic gene showed more than 70% similarity on the amino acid level with cellulase from Micromonospora aurantiaca and Thermobispora sp. Interestingly, RSC-EG1 contains a stretch of approximately 86 amino acids not present in either of these close relatives. Our results demonstrated that RSC-EG1, stable over a wide temperature and pH range, is a novel endoglucanase, and provided the first example of metagenomics approach to isolate cellulolytic gene from rice straw composts.
Biotechnology Center in Southern Taiwan, Academia Sinica, No. 59, Xiraya Blvd., Xinshi Dist., Tainan 74145, Taiwan, ROC.
Yi-Fang
YF
Yeh
Samuel Chia-yu
SC
Chang
Hsion-Wen
HW
Kuo
Chi-Gong
CG
Tong
Su-May
SM
Yu
Tuan-Hua David
TH
Ho
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D056804:Biofuels; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D056186:Metagenomics / Q000379:methods*; D008847:Micromonospora / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology / Q000235:genetics*; D018547:Plant Stems / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012987:Soil; D013696:Temperature
D001426:Bacterial Proteins; D056804:Biofuels; D018744:DNA, Plant; D011994:Recombinant Proteins; D012987:Soil; D002482:Cellulose; D002480:Cellulase