建構土壤多源基因庫與開發新型酵素

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

郁文涵(Wen-Han Yu)

指導教授 ： 李佳音

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10100

摘要

近年來，不論是醫藥、食品、工業等方面對於生物資源的需求與日俱增，而地球上所蘊藏最富饒自然資源的所在莫過於環境中的微生物，然而由於人工培養的限制，以致於我們對於絕大部分的微生物仍所知有限，其大部分無法人工培養 (culture-independent)之微生物的遺傳物質，弁鄋[異度也較單一微生物基因體高的多。為獲得這些遺傳資訊，須以直接萃取土壤 DNA之方式(Direct-cloning) 建構土壤多源基因庫。本研究室在台灣南部芒果園採樣土壤，為直接地得取完整的土壤微生物之DNA，本研究室發展出一套萃取土壤DNA技術平台，直接從土壤中獲取高分子量的DNA 片段 (100~1000 kb)，在三種不同土壤中總共建構12,000轉殖株的Bacterial artificial chromosome (BAC) 基因庫，經過限制酶截切分析後，平均每個轉殖株所攜帶的DNA 片段約為50~60 kb。進一步分析其16S rDNA 與18S rDNA 的序列，95% 的轉殖株所攜帶的DNA 皆為無法人工培養的微生物。分析生物系統演化樹，發現大部分的芒果園土壤之細菌相主要屬於 Proteobacteria、 Nitrospina、 High G+C Gram positive 、Sporomusa；真菌方面主要為Ascomycota和 Basidiomycota。醯基胺基酸消旋酶 (N-acylamino acid racemase) 可與醯基胺基酸水解酶 (aminoacylase) 配合生產D型或L型胺基酸，但目前已發表的少數幾個胺基酸消旋酶其活性及基質專一性仍未達理想。故我們設計PCR 引子利用兩次PCR 的方式篩選醯基胺基酸消旋酶，從12,000株轉殖株中篩選到4株帶有PCR 產物。為了進一步證明基因弁遄A我們直接抽其BAC 選殖株的粗酵素液，配合胺基酸氧化酶法進行醯基胺基酸消旋酶的活性檢測，經由提供不同的基質與輔酶發現僅有pBAB3 與pBAB4 其活性明顯比對照組高。再由次選殖方式將完整基因組找到並定序，由BLAST 比對及保守區域的搜尋，推測此4株選殖株分別帶有homocysteine methyltransferase (pBAB1), hypothetical protein (pBAB2), Uncharacterization protein (pBAB3), sensor kinase (pBAB4)，其相似度分別為30 %, 47 %, 42 % ,31 %，推測其轉殖株帶有的基因組可能以未知的方式轉換所提供的基質(N-acetyl-D-methionine, N-acetyl-D-phenylalanine) 形成L-methionine 與L-phenylalanine。也證明本研究室所建構之基因庫可作為篩選新型酵素的平台。

關鍵字

土壤多源基因庫 ； 菌相演化樹 ； 醯基胺基酸消旋酶

並列摘要

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.

並列關鍵字

BAC vector ； phylogenetic tree ； N-acetylamino acid racemase ； soil metagenomic library

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