研究生: |
陳品臻 Pin-Chen Chen |
---|---|
論文名稱: |
建構可感測環境中多醣生物質之枯草芽孢桿菌 Construction of Bacillus subtilis for Sensing Environmental Polysaccharide Biomass |
指導教授: |
蔡伸隆
Shen-Long Tsai |
口試委員: |
李振綱
Cheng-Kang Lee 王勝仕 Sheng-Shih Wang 蔡伸隆 Shen-Long Tsai |
學位類別: |
碩士 Master |
系所名稱: |
工程學院 - 化學工程系 Department of Chemical Engineering |
論文出版年: | 2023 |
畢業學年度: | 111 |
語文別: | 中文 |
論文頁數: | 109 |
中文關鍵詞: | 木質纖維素生物質 、生物感測器 、西格瑪因子 、枯草芽孢桿菌 |
外文關鍵詞: | lignocellulosic biomass, biosensor, sigma factor, Bacillus subtilis |
相關次數: | 點閱:356 下載:0 |
分享至: |
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
隨著世界人口的成長,發展可替代能源以減低對化石能源的需求已是人類文明對未來發展的共識,生質能源以農業廢棄物作為原料,在生產能源的同時還可避免過去燃燒、掩埋帶來的汙染。以生物轉化法進行生物質的水解具有反應條件溫和與易於操作等優點,僅需要投入可大量分泌生物質水解酶之菌株於反應槽中即可,然而若環境中無相關底物存在,菌體仍會持續表達非必須基因,最終能量耗盡而導致死亡。為改善上述缺點,本研究設計了兩種系統,在環境中存在不同多醣生物質時,可分別轉錄不同報告基因,以期達到感測之目的,在確認感測效果正常後,將此報告基因替換為水解酶基因,可使其在生物轉化法應用上更具實用性。系統一以糖類誘導型啟動子作為設計元件,當環境中存在木質纖維素時,將會被持續少量分泌至胞外的纖維素水解酶與半纖維素水解酶水解,而後各自產物糖類將誘導不同啟動子開啟下游螢光蛋白轉錄,目前已達成對木聚醣之感測,對纖維素的感測亦完成了系統的建立,並持續調整以提升偵測效果;而另一系統參考Clostridium thermocellum在艱困環境下發展出的生存策略,利用σI2 轉錄因子與 RsgI2跨膜蛋白之間的交互作用力,使其可在環境中存在纖維素時,釋放轉錄因子並表達相關啟動子下游之綠螢光蛋白以達成感測之目的,先前的研究已建構了具相關基因之質體與確立排列調控方式,並逐一確認元件的效果,本研究專注於跨膜蛋白的正確表達與感測能力。
The global population growth leads to a consensus in human that the development of alternative energy to reduce reliance on fossil fuels is necessary. Biomass energy, which uses agricultural waste as raw material, has the advantage of generating energy and reducing pollution. Biocatalytic biomass hydrolysis provides mild reaction conditions and eazy operation. However, no matter there are any substrates in the environment, bacteria will continue expressing non-essential genes, ultimately depleting their own energy. To address these limitations, two systems were designed in this study, enable the transcription of different reporter genes in the presence of various polysaccharide biomass.
System 1 employs sugar-inducible promoters as design elements. Hydrolitic enzyme are continuously secreted in small quantities. Once lignocellulosic is present in the environment, they will be spontaneous hydrolysis. Subsequently, the sugar products induce different promoters, initiating the transcription of downstream fluorescent proteins. The sensing of xylan has been successfully achieved, and the establishment of a cellulose sensing system has been completed, with ongoing adjustments to enhance the detection effectiveness. The second system take the survival strategy of Clostridium thermocellum. Utilizes the interaction between the σI2 transcription factor and the RsgI2 transmembrane protein to release the transcription factor in the presence of cellulose. Previous research has constructed plasmids containing relevant genes, established the arrangement and regulation methods and confirmed the effects of individual components. This study focuses on ensuring the proper expression and sensing capabilities of transmembrane proteins.
1.Saha BC. Hemicellulose bioconversion. Journal of Industrial Microbiology & Biotechnology. May 2003;30(5):279-291.
2.Lee M-H, Jeon HS, Kim SH, et al. Lignin-based barrier restricts pathogens to the infection site and confers resistance in plants. The EMBO Journal. 2019;38(23):e101948.
3.Broda M, Yelle DJ, Serwańska K. Bioethanol Production from Lignocellulosic Biomass—Challenges and Solutions. Molecules. 2022;27(24):8717.
4.Yu Y, Wu H. Significant Differences in the Hydrolysis Behavior of Amorphous and Crystalline Portions within Microcrystalline Cellulose in Hot-Compressed Water. Industrial & Engineering Chemistry Research. 2010;49(8):3902-3909.
5.Tomme P, Boraston A, McLean B, et al. Characterization and affinity applications of cellulose-binding domains. J Chromatogr B Biomed Sci Appl. Sep 11 1998;715(1):283-296.
6.Menon V, Rao M. Trends in bioconversion of lignocellulose: Biofuels, platform chemicals & biorefinery concept. Progress in Energy and Combustion Science. Aug 2012;38(4):522-550.
7.Sandhya J, Renuka R. Microbial Cellulases: An Overview and Applications. In: Alejandro Rodríguez P, María EEM, eds. Cellulose. Rijeka: IntechOpen; 2019:Ch. 5.
8.Anoop Kumar V, Suresh Chandra Kurup R, Snishamol C, Nagendra Prabhu G. Role of Cellulases in Food, Feed, and Beverage Industries. In: Parameswaran B, Varjani S, Raveendran S, eds. Green Bio-processes: Enzymes in Industrial Food Processing. Singapore: Springer Singapore.
9.Moreira LRS, Filho EXF. Insights into the mechanism of enzymatic hydrolysis of xylan. Applied Microbiology and Biotechnology. 2016/06/01 2016;100(12):5205-5214.
10.Hoffmam ZB, Zanphorlin LM, Cota J, et al. Xylan-specific carbohydrate-binding module belonging to family 6 enhances the catalytic performance of a GH11 endo-xylanase. New Biotechnology. Jun 2016;33(4):467-472.
11.Roncero MI. Genes controlling xylan utilization by Bacillus subtilis. Journal of Bacteriology. 1983;156(1):257-263.
12.Sunna A, Antranikian G. Xylanolytic enzymes from fungi and bacteria. Crit Rev Biotechnol. 1997;17(1):39-67.
13.Borukhov S, Nudler E. RNA polymerase holoenzyme: structure, function and biological implications. Current Opinion in Microbiology. 2003;6(2):93-100.
14.Winkelman JT, Gourse RL. Open complex DNA scrunching: A key to transcription start site selection and promoter escape. Bioessays. Feb 2017;39(2).
15.Ferrari E, Henner DJ, Perego M, Hoch JA. Transcription of Bacillus subtilis subtilisin and expression of subtilisin in sporulation mutants. J Bacteriol. Jan 1988;170(1):289-295.
16.Jordan S, Junker A, Helmann JD, Mascher T. Regulation of LiaRS-dependent gene expression in bacillus subtilis: identification of inhibitor proteins, regulator binding sites, and target genes of a conserved cell envelope stress-sensing two-component system. J Bacteriol. Jul 2006;188(14):5153-5166.
17.Bartosiak-Jentys J, Hussein AH, Lewis CJ, Leak DJ. Modular system for assessment of glycosyl hydrolase secretion in Geobacillus thermoglucosidasius. Microbiology. 2013;159(Pt_7):1267-1275.
18.Singh KD, Schmalisch MH, Stülke J, Görke B. Carbon catabolite repression in Bacillus subtilis: quantitative analysis of repression exerted by different carbon sources. J Bacteriol. Nov 2008;190(21):7275-7284.
19.Yaniv O, Fichman G, Borovok I, et al. Fine-structural variance of family 3 carbohydrate-binding modules as extracellular biomass-sensing components of Clostridium thermocellum anti-σI factors. Acta Crystallogr D Biol Crystallogr. Feb 2014;70(Pt 2):522-534.
20.Chang J-J, Anandharaj M, Ho C-Y, et al. Biomimetic strategy for constructing Clostridium thermocellum cellulosomal operons in Bacillus subtilis. Biotechnology for Biofuels. 2018;11(1):157.
21.Ichikawa S, Ito D, Asaoka S, et al. The expression of alternative sigma-I7 factor induces the transcription of cellulosomal genes in the cellulolytic bacterium Clostridium thermocellum. Enzyme and Microbial Technology. May 2022;156:8.
22.Muñoz-Gutiérrez I, Ortiz de Ora L, Rozman Grinberg I, et al. Decoding Biomass-Sensing Regulons of Clostridium thermocellum Alternative Sigma-I Factors in a Heterologous Bacillus subtilis Host System. PLoS One. 2016;11(1):e0146316.
23.Wei Z, Chen C, Liu Y-J, et al. Alternative σI/anti-σI factors represent a unique form of bacterial σ/anti-σ complex. Nucleic Acids Research. 2019;47(11):5988-5997.
24.Grinberg IR, Yaniv O, de Ora LO, et al. Distinctive ligand-binding specificities of tandem PA14 biomass-sensory elements from Clostridium thermocellum and Clostridium clariflavum. Proteins. Nov 2019;87(11):917-930.
25.Mahoney BJ, Takayesu A, Zhou A, Cascio D, Clubb RT. The structure of the Clostridium thermocellum RsgI9 ectodomain provides insight into the mechanism of biomass sensing. Proteins. Jul 2022;90(7):1457-1467.
26.Kahel-Raifer H, Jindou S, Bahari L, et al. The unique set of putative membrane-associated anti-Sigma factors in Clostridium thermocellum suggests a novel extracellular carbohydrate-sensing mechanism involved in gene regulation. Fems Microbiology Letters. Jul 2010;308(1):84-93.
27.Chow N, Wu JHD. Chapter 10 - The Cellulosome: A Supramolecular Assembly of Microbial Biomass-Degrading Enzymes. In: Brahmachari G, ed. Biotechnology of Microbial Enzymes: Academic Press; 2017:243-266.
28.Devkota SR, Kwon E, Ha SC, Chang HW, Kim DY. Structural insights into the regulation of Bacillus subtilis SigW activity by anti-sigma RsiW. PLOS ONE. 2017;12(3):e0174284.
29.Brunet YR, Habib C, Brogan AP, Artzi L, Rudner DZ. Intrinsically disordered protein regions are required for cell wall homeostasis in Bacillus subtilis. Genes Dev. Sep 1 2022;36(17-18):970-984.
30.Zhao H, Roistacher DM, Helmann JD. Deciphering the essentiality and function of the anti-σ(M) factors in Bacillus subtilis. Mol Microbiol. Aug 2019;112(2):482-497.
31.Zhang W, Yang M, Yang Y, Zhan J, Zhou Y, Zhao X. Optimal secretion of alkali-tolerant xylanase in Bacillus subtilis by signal peptide screening. Applied Microbiology and Biotechnology. 2016;100(20):8745-8756.
32.Ling Lin F, Zi Rong X, Wei Fen L, Jiang Bing S, Ping L, Chun Xia H. Protein secretion pathways in Bacillus subtilis: Implication for optimization of heterologous protein secretion. Biotechnology Advances. 2007;25(1):1-12.
33.Lan Thanh Bien T, Tsuji S, Tanaka K, Takenaka S, Yoshida K. Secretion of heterologous thermostable cellulases in Bacillus subtilis. J Gen Appl Microbiol. 2014;60(5):175-182.
34.Errington J. Regulation of endospore formation in Bacillus subtilis. Nature Reviews Microbiology. Nov 2003;1(2):117-126.
35.Kamionka A, Dahl MK. Bacillus subtilis contains a cyclodextrin-binding protein which is part of a putative ABC-transporter. Fems Microbiology Letters. Oct 2001;204(1):55-60.
36.Abdel-Fattah AF, Mahmoud DAR, Esawy MAT. Production of levansucrase from Bacillus subtilis NRC 33a and enzymic synthesis of levan and fructo-oligosaccharides. Current Microbiology. Dec 2005;51(6):402-407.
37.Jin JQ, Yin Y, Wang X, Wen JP. Metabolic engineering of Bacillus subtilis 168 for the utilization of arabinose to synthesize the antifungal lipopeptide fengycin. Biochemical Engineering Journal. Jul 2022;185:12.
38.Xu L, Lu Y, Cong YZ, et al. Polysaccharide produced by Bacillus subtilis using burdock oligofructose as carbon source. Carbohydrate Polymers. Feb 2019;206:811-819.
39.Liu JM, Xin XJ, Li CX, Xu JH, Bao J. Cloning of Thermostable Cellulase Genes of Clostridium thermocellum and Their Secretive Expression in Bacillus subtilis. Applied Biochemistry and Biotechnology. Feb 2012;166(3):652-662.
40.Aminov RI, Golovchenko NP, Ohmiya K. Expression of a celE gene from Clostridium thermocellum in Bacillus. Journal of Fermentation and Bioengineering. 1995;79(6):530-537.
41.Qureshy AF, Khan LA, Khanna S. Expression of Bacillus circulans Teri-42 xylanase gene in Bacillus subtilis. Enzyme and Microbial Technology. Aug 2000;27(3-5):227-233.
42.Gallardo O, Diaz P, Pastor FIJ. Cloning and production of Xylanase B from Paenibacillus barcinonensis in Bacillus subtilis hosts. Biocatalysis and Biotransformation. 2007;25(2-4):157-162.
43.Alponti JS, Fonseca Maldonado R, Ward RJ. Thermostabilization of Bacillus subtilis GH11 xylanase by surface charge engineering. International Journal of Biological Macromolecules. 2016;87:522-528.
44.Lin CC, Yan CJS, Kan SC, et al. Deciphering characteristics of the designer cellulosome from Bacillus subtilis WB800N via enzymatic analysis. Biochemical Engineering Journal. Jan 2017;117:147-155.
45.Brejc K, Sixma TK, Kitts PA, et al. Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc Natl Acad Sci U S A. Mar 18 1997;94(6):2306-2311.
46.Shaner NC, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnology. 2004;22(12):1567-1572.
47.Shen Y, Chen Y, Wu J, Shaner NC, Campbell RE. Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity. PLOS ONE. 2017;12(2):e0171257.
48.Marciniak BC, Pabijaniak M, de Jong A, et al. High- and low-affinity cre boxes for CcpA binding in Bacillus subtilis revealed by genome-wide analysis. BMC Genomics. 2012;13(1):401.
49.Yano K, Inoue H, Mori H, et al. Heterologous Expression of the Oceanobacillus iheyensis SigW and Its Anti-Protein RsiW in Bacillus subtilis. Bioscience Biotechnology and Biochemistry. May 2011;75(5):966-975.
50.Kaur J, Bachhawat AK. A modified Western blot protocol for enhanced sensitivity in the detection of a membrane protein. Analytical Biochemistry. 2009;384(2):348-349.