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(1)Graduate School of Advanced Science and Engineering, Waseda University. 博士論文概要 Doctor Thesis Synopsis 論. 文. 題. 目. Thesis Theme. Screening of xylose isomerase metabolic enzymes through metagenome approach for bioethanol production 申請者 (Applicant Name). Dini. NURDIANI. ヌルディアニ. ディニ. (Major in Life Science and Medical Bioscience, Research on Biomolecular Engineering). December, 2014.

(2) Lignocellulosic biomass is a renewable and energy-rich resource that can be utilised for bioethanol production. Complete conversion of all available sugars in lignocellulosic biomass hydrolysates is required for cost effective and sustainable industrial bioethanol production. In lignocellulase biomass, hemicellulose, largely comprised of the pentose sugars D-xylose and L-arabinose, is a major component where it comprises 25-35% of total biomass composition. Furthermore, xylose in particular is generally the most abundant pentose which comprises up to 25% of the total sugar content in some hydrolysates and it is the second only to glucose in natural abundance of all monosaccharides. Therefore, the conversion of xylose becomes significantly important for the efficient conversion of lignocellulose to ethanol. Saccharomyces cerevisiae is an organism of choice for bioethanol production since it could efficiently produce ethanol from hexose. However it lacks the ability to utilize xylose but instead utilizes and ferments its isomer D-xylulose. Bacterial xylose isomerase (XI) can directly convert xylose to xylulose and for over the years several explorations have been attempted to introduce xylA, the only gene known to encode for XI, from naturally pentose-utilizing bacteria and fungi in S. cerevisiae by heterologous expression. XIs are widespread among prokaryotic microorganisms and the phylogenetic analysis of known xylA sequences gives a good indication of its diversity. Based on amino acid similarity, bacterial XIs are classified into two groups which differ by approximately an additional 50 amino acid residues at the N terminus of group II XIs. So far, few bacterial XIs have been reported to be functionally expressed in S. cerevisiae. Nevertheless, it remains insufficient for S. cerevisiae recombined with known xylA genes to efficiently produce bioethanol. Thus, the exploration of bacterial xylA is required in order to obtain diversity for efficient screening in S. cerevisiae. Soil as a complex environment is a major resource for diverse microbes. Diversity of the microbial community in soil is possibly considered as the highest prokaryotic diversity in comparison to any other natural environment. Since only 1-10% species in the soil are estimated cultivable, recently metagenomics has appeared as one useful approach to circumvent this problem and may provide access to obtain novel useful genetic resources from environmental samples. As diverse microbes inhabit soil environments that contain lignocellulosic biomass, soil metagenome could become one promising approach to explore potential bacterial xylA genes for modifying the yeast metabolic pathway. The goal of this study was to screen for novel and highly active xylA genes from soil metagenomes by heterologous expression in S. cerevisiae. Two strategies that are generally used in metagenome screening are activity-based and sequence-based screening. Both activity- and sequence-based screening have advantages and disadvantages respectively, and they have been applied successfully to discover biocatalysts from metagenome. Differ from activity-based screening that requires heterologous expression of the genes encoded within metagenome clones and high-throughput function assays for clone identification, sequence-based screening is not dependent on the expression of cloned genes in the heterologous host. In this work, a sequence-based screening approach was performed to obtain novel xylA genes from soil metagenome. Sequence-based screening is an efficient method for isolating novel proteins based on homology searches with different level of similarity from the previously known genes. The study consists of three main parts, first was investigation of microbial and xylose isomerase genes diversity in soil metagenomes,. Ｎｏ.1.

(3) second was isolation of xylA full length genes from soil metagenome and their functional expression in S. cerevisiae, and third was the establishment of a high-throughput technique by using chimeric xylA genes for efficient xylose degradation in S. cerevisiae from soil metagenome. This thesis consists of five chapters. The first chapter is a general overview and introduction of metagenome-based screening for isolating novel metabolic enzymes and utilization of C5 metabolic enzymes for yeast metabolic engineering to produce bioethanol. It leads to an understanding of the background, objectives and significance of this study. This chapter reviews about how metagenomics has emerged as a powerful approach for mining novel biocatalysts through sequence- or activity-based screening and the gene-mining based on the construction and screening of complex libraries derived from the metagenome provides opportunities to fully explore and exploit the enormous genetic and metabolic diversity of microorganisms. On the other hand, this chapter also reviews the attempts, challenges and current reports of the introduction of novel microbial sequences that expressed C5 metabolic enzymes in recombinant S. cerevisiae strains for efficient industrial bioethanol production. Chapter 2 presents the diversity analysis of bacterial xylA from soil metagenome. Three soil metagenomes which differ by plant vegetation were investigated. Pyrosequencing-based analysis of 16S rRNA and xylA genes were performed to measure the microbial community and functional gene diversity. To amplify internal xylA sequences from soil metagenome, two degenerate primer sets were newly designed based on known amino acid xylA sequences reported in database. The internal sequences of the xylA genes were successfully amplified by using two degenerate primer sets and the attained sequence reads comprised of 158,555 sequences from triplicate metagenomic DNA. Sequences showing 90% amino acid identity to currently known xylA genes were calssified to 1,127 bacterial phylotypes. The phylotype coverage of xylA genes were estimated to be within the range of 84.0-92.7%. The difference of xylA diversity and compositions strongly correlated with those of 16S rRNA genes within the soil samples, also assessed by amplicon pyrosequencing. Chapter 3 describes the isolation of xylA full length genes from soil metagenome and their functional expression in S. cerevisiae. The xylA full length genes were retrieved by employing the pre-amplified inverse polymerase chain reaction (PAI-PCR) method. Thus far, the standard inverse polymerase chain reaction (IPCR) technique for isolation of full length genes from metagenomes have been employed but have limitations when target genes are of low copy number. Pre-amplified inverse PCR method (PAI-PCR) is an inverse PCR method that was developed to amplify desired nucleotide sequences from environmental DNA. This method enriches the target DNA sequences by rolling circle amplification with a site specific primer containing locked nucleic acids (LNAs). Based on the internal xylA sequences amplified by two degenerate primer sets, inverse PCR primer sets have been designed from selected partial xylA sequences which were chosen based on the phylogenetic clades. PAI-PCR was performed to amplify flanking sequence of target xylA sequences from soil metagenome. As a result, six target xylA genes were successfully amplified in which four putative full length xylA genes were successfully isolated and cloned which have identity 73, 81, 62, and 67% to the XI gene of Candidatus koribacter, bacterium Ellin, Opitutus terrae, and Mesorhizobium sp., respectively. Subsequently, the four. Ｎｏ.2.

(4) putative xylA full length genes isolated were introduced into a yeast expression vector, pULD1, that enables the heterologous proteins to be displayed on the surface of S. cerevisiae. The pULD1 expression vector harbors the secretion signal sequence of glucoamylase gene from Rhizopus oryzae, the 3’-half region of the α-agglutinin gene as the cell wall anchoring domain and a FLAG tag. The vectors harboring the putative xylA full length genes were successfully established, transformed into S. cerevisiae and the expression of the genes were confirmed by immunofluorescence labeling. Fluorescing S. cerevisiae transformant cells were observed by using fluorescence microscopy and flow cytometric analysis. From this analysis, three out of the four putative xylA full length genes which have similarity to the xylA gene of C. Koribacter, O. terrae, and Mesorhizobium sp. were expressed on the S. cerevisiae cells, while the remaining full length gene which had similarity to the B. ellin xylA was not expressed. The investigation was conducted along with known xylA genes of Piromyces and Escherichia coli which had previously been reported as active or inactive in S. cerevisiae, respectively. Chapter 4 presents a different attempt to obtain novel active xylA genes from soil metagenome by constructing chimeric xylA genes for establishment of a high-throughput technique for efficient xylose degradation in S. cerevisiae from soil metagenome. In this screening system, both sequence- and activity-based screenings were used simultaneously to obtain active xylose isomerase enzyme in S. cerevisiae. Partial xylA sequences were introduced from metagenome samples amplified by uniquely designed degenerate primers to replace targeted regions of the Piromyces xylA within a yeast expression vector, pRS436GA-PiXI-opt via in vivo homologous recombination. Three S. cerevisiae recombinant strains harbored chimeric xylA were able to grow on xylose media. These chimeric genes contain metagenome xylA inserts that show homology to the Niastella koreensis, Pedobacter heparinus and uncultured bacterium with sequence similarity of 91, 95, and 87 %, respectively. Furthermore, upon fermentation of the yeast strains harboring these chimeric xylA genes, the strain harboring the xylA fragment with similarity to Niastella koreensis showed significant activity in bioethanol production comparable to the Pyromices XI. Chapter 5 presents the conclusions of this thesis. First, diverse xylA genes were successfully amplified from soil metagenomes and based on this analysis four putative full length xylA genes have been successfully retrieved. However, functionality of these genes were not successfully confirmed. Simultaneously, an efficient screening system was developed by constructing chimeric xylA sequences using the Pyromices xylA as a backbone, where functional chimeric XIs were successfully attained showing xylose degradation and ethanol production in S. cerevisiae. In conclusion, I believe that the efforts in this thesis have allowed further exploitation of bacterial xylA from the highly genetically diverse soil metagenome for efficient bioethanol production in S. cerevisiae from xylose.. Ｎｏ.3.

(5) No.1. 早稲田大学. 博士（工学）. 学位申請. 研究業績書. (List of research achievements for application of doctorate (Dr. of Engineering), Waseda University). 氏名. Nurdiani, Dini. 印（As of February, 2015）. 種類別 (By Type) Academic paper. 題名、発表・発行掲載誌名、発表・発行年月、連名者（申請者含む） (theme, journal name, date & year of publication, name of authors inc. yourself). 1. D. Nurdiani, M. Ito, T. Maruyama, T. Terahara, T. Mori, S. Ugawa, H. Takeyama Analysis of the bacterial xylose isomerase gene diversity with gene targeted metagenomics. J. Biosci. Bioeng. (in printing). Lecture 1. D. Nurdiani, Y. Okamura, T. Terahara, N. Takehiro, H. Takeyama Efficient screening of xylose isomerase genes from soil metagenome for bioethanol production. International Union of Microbiological Societies 2011 Congress, September 6-16, 2011. 2. D. Nurdiani, Y. Hamamoto, T. Mori, K. Kuroda, M. Ueda, H. Takeyama Efficient screening of xylose isomerase genes from soil metagenome for bioethanol production German-Japanese 2nd Joint Symposium for Diamond Researchers on Sustainable Life Science Innovation and Biomedical Research, February, 2012. 3. D. Nurdiani, Y. Okamura, T. Terahara, N. Takehiro, H. Takeyama Molecular diversity of xylose isomerase for bioethanol production 第 4 回バイオ関連化学シンポジウム, September, 2010.

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博士論文概要