Construction of the soil metagenomic library from the agricultural soil and discovery of the novel biocatalysts

• Main Authors: Wen-Han Yu, 郁文涵
• Other Authors: Chia-Yin Lee
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/38921573501607245423

碩士 === 國立臺灣大學 === 農業化學研究所 === 92 === Environmental microorganisms hold the greatest abundant resource applied to medicine, food and industry. But the source of natural-products in microbiota is constrained because the majority of the microbial species in the biosphere cannot be cultured in the laboratory; these sources must be discovered by direct-cloning method and applied further by construction of metagenomic library. A large number of different methods have been published for extraction of total microbial soil DNA. But DNA fragments were sheared seriously due to physical disruption. We have developed newer method to extract DNA from the agricultural soils and obtain high molecular weight and pure DNA (100~1000kb). To access these uncultured genetic materials, we have used bacterial artificial chromosome (BAC) vector to construct 12,000-clones libraries of microbiota genomes. The average insert size of BAC library we prepared is about 60 kb. The phylogenetic analysis of 16S rRNA and 18S rRNA gene sequences from the soil metagenomic library approximately belonged to the undescribed microbes, and revealed the divergent microbial phyla such as Proteobacteria, Nitrospina, High G+C Gram positive, Sporomusa, Ascomycota, and Basidiomycota. N-acylamino acid racemase (NAAAR) is an enzyme that specifically catalyzes the racemization of N-acetyl-D, L-amino acids but not general amino acids and was applied to the production of optically active amino acids. The BAC library was screened by colony PCR and seminested PCR for sequences similar to the high conserved region of NAAAR and related homologous proteins. Four positive recombinant clones (pBAB1 to pBAB4) of 12,000 clones were obtained by double PCR screening. Analysis the cell extracts of four positive clones with different substrates and cofactors, the NAAAR activity in pBAB3 and pBAB4 were two folds higher than the negative control E.coli/BAC. All of related naaar genes were identified during subcloning and sequencing of the inserts of pBAB1 to pBAB4. After analysis of BLAST, ORF finder, and CD-search programs, the deduced gene functions showed similarities to homocysteine methyltransferase (pBAB1), Hypothetical protein (pBAB2), Uncharacterization protein (pBAB3), sensor kinase (pBAB4). But the sequences homologous to naaar gene cannot be found. Therefore, it is possible that the pBAB3 and pBAB4 utilize the substrates (N-acetyl-D-methionine, N-acetyl-D-phenylalanine) in a way with the unknown functions. Based on these data, we provide a strong platform for searching the novel-type enzymes.