厭氧菌所產生的纖維素酵素複合體（Cellulosomes）被認為是自然界中最好的纖維素分解機器，因此科學家一直持續嘗試在工業酵母菌中建構人工纖維素酵素複合體。由於纖維素酵素複合體的基因數量龐大且複雜，因此非常具有挑戰性。我們以先進的合成生物學技術去設計與合成Clostridium thermocellum的骨架蛋白（CipA）與錨定蛋白（OlpB）成功克服此困難，經過改造的益生菌酵母菌（Kluyveromyces marxianus）可生產一系列來自真菌的複合纖維素分解酶，包括內切葡聚醣酶（TrEgIII），外切葡聚醣酶（CBHII），β-葡萄糖苷酶（NpaBGS）和纖維素酶增強劑（TaLPMO和MtCDH）。共軛焦顯微鏡的結果證實細胞表面有OlpB-ScGPI的表現，且透過螢光活化細胞分類計分析顯示幾乎有81％的酵母細胞表面具有OlpB-ScGPI。同時我們也進一步利用非變性聚丙烯酰胺凝膠電泳與質譜儀分析的結果驗證了纖維素酵素複合體蛋白確實形成複合體。相較於前人以質體構築的纖維素酵素複合體的研究只能容納12種酵素，本實驗透過重組染色體的基因工程可生產容納多達63種酵素，是目前已知構築纖維素酵素複合體中最多的。更特別的是，與其他改造後的CipA相比，本實驗所使用的CipA2B9C （具有兩個纖維素連接模塊，CBM）所釋放的還原糖量明顯提昇，藉此結果也證實了纖維素結合結構模塊的數量對纖維素酵素複合體的重要性。經過改造的酵母菌可有效分解纖維素，並分別從微晶纖維素（Avicel）和磷酸膨脹纖維素（PASC）中生產3.09 g / L和8.61 g / L的乙醇，其生產量高於前人所構建的酵母菌纖維素酵素複合體。

Cellulosomes from anaerobic bacteria are considered nature’s finest cellulolytic machinery. Thus, constructing a cellulosome in an industrial yeast has long been a goal pursued by scientists. However, it remains highly challenging due to the size and complexity of cellulosomal genes. Here, we overcame the difficulties by designing and synthesizing the Clostridium thermocellum scaffoldin gene (CipA) and the anchoring protein gene (OlpB) using advanced synthetic biology techniques. The engineered Kluyveromyces marxianus, a probiotic yeast, secreted a cocktail of dockerin fused fungal cellulases, including an endoglucanase (TrEgIII), exoglucanase (CBHII), β-glucosidase (NpaBGS) and cellulase boosters (TaLPMO and MtCDH). The confocal microscopy results confirmed the cell surface display of OlpB-ScGPI and Fluorescence-Activated Cell Sorting analysis results revealed that almost 81% of yeast cells displayed OlpB-ScGPI. We have also demonstrated the cellulosome complex formation using purified and crude cellulosomal proteins. Native PAGE and mass spectrometric analysis further confirmed the cellulosome complex formation. Our engineered cellulosome can accommodate up to 63 enzymes, whereas the largest engineered cellulosome reported thus far could accommodate only 12 enzymes and was expressed by a plasmid instead of chromosomal integration. Interestingly, CipA 2B9C (with two cellulose binding module, CBM) released significantly higher quantities of reducing sugars compared with other CipA variants, thus confirming the importance of cohesin numbers and CBM domain on cellulosome complex. The engineered yeast host efficiently degraded cellulosic substrates and released 3.09 g/L and 8.61 g/L of ethanol from avicel and phosphoric acid swollen cellulose (PASC), respectively, which are higher than any previously constructed yeast cellulosome.

Table of Contents
Acknowledgements i
中文摘要 iii
Abstract iv
List of Tables vii
List of Figures viii
List of Appendix x
1. Introduction 1
1.1. Lignocellulosic biomasses as an alternative 1
1.2. Enzyme synergism: a key factor 1
1.3. ‘Cellulosome’: a superior enzyme complex 2
1.4. Cellulosome on yeast 5
1.5. Cellulolytic biorefinery concept 6
1.6. Consolidated bioprocessing (CBP): an ideal approach 6
1.7. Kluyveromyces marxianus: a potential host for CBP 7
1.8. Synthetic biology tool for K. marxianus 7
2. Materials and Methods 9
2.1. Strains and Media 9
2.2. Cellulosomal construct design and synthesis 9
2.3. Yeast transformation and clone screening 10
2.4. Expression and purification of cellulosomal proteins 11
2.5. Cellulosome complex assembly and purification 12
2.6. Immunofluorescence microscopy and FACS 13
2.7. Cellulosome assembly on cell surface 13
2.8. Real-time quantitative PCR 14
2.9. Enzyme assays 14
2.10. Fermentation and ethanol production 15
2.11. Homology modeling of dockerin-fused enzymes 15
2.12. Statistical analysis 15
3. Results 16
3.1. Design and synthesis of cellulosomal scaffoldins 16
3.2. To study the cellulolytic synergism with cellulase boosters 18
3.3. Conversion of free cellulases into cellulosomal mode 22
3.4. Chromosomal integration of cellulosomal genes 27
3.5. Demonstration of cell-surface display using immunofluorescence analysis 30
3.6. Confirmation of cellulosome complex assembly 34
3.7. Constructing enzyme hosts 40
3.8. Boosting effects of cellulase boosters 44
3.9. Effects of cohesin and CBM numbers on cellulosome efficiency 49
3.10. Demonstration of consolidated bioprocessing 51
3.11. Future direction: Designing designer Scaffoldin 54
3.12. Chromosomal integration of Designer cellulosomal genes 57
4. Discussion 59
5. References 65
6. Appendixes 73

Abdel-Banat, B.M.A., Nonklang, S., Hoshida, H., and Akada, R. (2010). Random and targeted gene integrations through the control of non-homologous end joining in the yeast Kluyveromyces marxianus. Yeast 27(1), 29-39. doi: doi:10.1002/yea.1729.
Arantes, V., and Saddler, J.N. (2010). Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis. Biotechnology for Biofuels 3(1), 4. doi: 10.1186/1754-6834-3-4.
Arfi, Y., Shamshoum, M., Rogachev, I., Peleg, Y., and Bayer, E.A. (2014). Integration of bacterial lytic polysaccharide monooxygenases into designer cellulosomes promotes enhanced cellulose degradation. Proc Natl Acad Sci U S A 111(25), 9109-9114. doi: 10.1073/pnas.1404148111.
Bayer, E.A., Morag, E., and Lamed, R. (1994). The cellulosome--a treasure-trove for biotechnology. Trends Biotechnol 12(9), 379-386. doi: 10.1016/0167-7799(94)90039-6.
Bayer, E.A., Setter, E., and Lamed, R. (1985). Organization and distribution of the cellulosome in Clostridium thermocellum. Journal of Bacteriology 163(2), 552-559.
Boder, E.T., and Wittrup, K.D. (1997). Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15(6), 553-557. doi: 10.1038/nbt0697-553.
Boraston, A.B., Bolam, D.N., Gilbert, H.J., and Davies, G.J. (2004). Carbohydrate-binding modules: fine tuning polysaccharide recognition. Biochem J 382. doi: 10.1042/bj20040892.
Caspi, J., Barak, Y., Haimovitz, R., Irwin, D., Lamed, R., Wilson, D.B., et al. (2009). Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode. Applied and Environmental Microbiology 75(23), 7335-7342. doi: 10.1128/aem.01241-09.
Chang, J.-J., Ho, C.-Y., Ho, F.-J., Tsai, T.-Y., Ke, H.-M., Wang, C.H.-T., et al. (2012). PGASO: A synthetic biology tool for engineering a cellulolytic yeast. Biotechnology for Biofuels 5(1), 53. doi: 10.1186/1754-6834-5-53.
Chang, J.-J., Ho, F.-J., Ho, C.-Y., Wu, Y.-C., Hou, Y.-H., Huang, C.-C., et al. (2013). Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production. Biotechnology for Biofuels 6(1), 19. doi: 10.1186/1754-6834-6-19.
Chang, J.-J., Lin, Y.-J., Lay, C.-H., Thia, C., Wu, Y.-C., Hou, Y.-H., et al. (2018a). Constructing a cellulosic yeast host with an efficient cellulase cocktail. Biotechnology and Bioengineering 115(3), 751-761. doi: doi:10.1002/bit.26507.
Chang, J.J., Anandharaj, M., Ho, C.Y., Tsuge, K., Tsai, T.Y., Ke, H.M., et al. (2018b). Biomimetic strategy for constructing Clostridium thermocellum cellulosomal operons in Bacillus subtilis. Biotechnol Biofuels 11, 157. doi: 10.1186/s13068-018-1151-7.
Chang, J.J., Thia, C., Lin, H.Y., Liu, H.L., Ho, F.J., Wu, J.T., et al. (2015). Integrating an algal beta-carotene hydroxylase gene into a designed carotenoid-biosynthesis pathway increases carotenoid production in yeast. Bioresour Technol 184, 2-8. doi: 10.1016/j.biortech.2014.11.097.
Chen, H.-L., Chen, Y.-C., Lu, M.-Y.J., Chang, J.-J., Wang, H.-T.C., Ke, H.-M., et al. (2012). A highly efficient β-glucosidase from the buffalo rumen fungus Neocallimastix patriciarum W5. Biotechnology for Biofuels 5(1), 24. doi: 10.1186/1754-6834-5-24.
Christopher, L.P., Yao, B., and Ji, Y. (2014). Lignin Biodegradation with Laccase-Mediator Systems. Frontiers in Energy Research 2(12). doi: 10.3389/fenrg.2014.00012.
Colussi, P.A., and Taron, C.H. (2005). Kluyveromyces lactis LAC4 promoter variants that lack function in bacteria but retain full function in K. lactis. Appl Environ Microbiol 71(11), 7092-7098. doi: 10.1128/AEM.71.11.7092-7098.2005.
Currie, D.H., Herring, C.D., Guss, A.M., Olson, D.G., Hogsett, D.A., and Lynd, L.R. (2013). Functional heterologous expression of an engineered full length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum. Biotechnology for Biofuels 6(1), 32. doi: 10.1186/1754-6834-6-32.
Demain, A.L., Newcomb, M., and Wu, J.H. (2005). Cellulase, clostridia, and ethanol. Microbiol Mol Biol Rev 69(1), 124-154. doi: 10.1128/MMBR.69.1.124-154.2005.
Dien, B.S., Cotta, M.A., and Jeffries, T.W. (2003). Bacteria engineered for fuel ethanol production: current status. Applied Microbiology and Biotechnology 63(3), 258-266. doi: 10.1007/s00253-003-1444-y.
Dimarogona, M., Topakas, E., and Christakopoulos, P. (2012). Cellulose degradation by oxidative enzymes. Comput Struct Biotechnol J 2, e201209015. doi: 10.5936/csbj.201209015.
Fan, L.-H., Zhang, Z.-J., Mei, S., Lu, Y.-Y., Li, M., Wang, Z.-Y., et al. (2016a). Engineering yeast with bifunctional minicellulosome and cellodextrin pathway for co-utilization of cellulose-mixed sugars. Biotechnology for Biofuels 9(1), 137. doi: 10.1186/s13068-016-0554-6.
Fan, L.H., Zhang, Z.J., Mei, S., Lu, Y.Y., Li, M., Wang, Z.Y., et al. (2016b). Engineering yeast with bifunctional minicellulosome and cellodextrin pathway for co-utilization of cellulose-mixed sugars. Biotechnol Biofuels 9, 137. doi: 10.1186/s13068-016-0554-6.
Fan, L.H., Zhang, Z.J., Yu, X.Y., Xue, Y.X., and Tan, T.W. (2012). Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production. Proc Natl Acad Sci U S A 109(33), 13260-13265. doi: 10.1073/pnas.1209856109.
Fonseca, G.G., Heinzle, E., Wittmann, C., and Gombert, A.K. (2008). The yeast Kluyveromyces marxianus and its biotechnological potential. Applied Microbiology and Biotechnology 79(3), 339-354. doi: 10.1007/s00253-008-1458-6.
Gai, S.A., and Wittrup, K.D. (2007). Yeast surface display for protein engineering and characterization. Curr Opin Struct Biol 17(4), 467-473. doi: 10.1016/j.sbi.2007.08.012.
Ghose, T.K. (1987). "Measurement of cellulase activities", in: Pure and Applied Chemistry.).
Gilmore, S.P., Henske, J.K., and O''Malley, M.A. (2015). Driving biomass breakdown through engineered cellulosomes. Bioengineered 6(4), 204-208. doi: 10.1080/21655979.2015.1060379.
Gold, N.D., and Martin, V.J.J. (2007). Global View of the Clostridium thermocellum Cellulosome Revealed by Quantitative Proteomic Analysis. Journal of Bacteriology 189(19), 6787-6795. doi: 10.1128/jb.00882-07.
Goldstein, M.A., Takagi, M., Hashida, S., Shoseyov, O., Doi, R.H., and Segel, I.H. (1993). Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A. J Bacteriol 175(18), 5762-5768.
Goyal, G., Tsai, S.L., Madan, B., DaSilva, N.A., and Chen, W. (2011). Simultaneous cell growth and ethanol production from cellulose by an engineered yeast consortium displaying a functional mini-cellulosome. Microb Cell Fact 10, 89. doi: 10.1186/1475-2859-10-89.
Harris, P.V., Welner, D., McFarland, K.C., Re, E., Navarro Poulsen, J.-C., Brown, K., et al. (2010). Stimulation of Lignocellulosic Biomass Hydrolysis by Proteins of Glycoside Hydrolase Family 61: Structure and Function of a Large, Enigmatic Family. Biochemistry 49(15), 3305-3316. doi: 10.1021/bi100009p.
Ho, C.-Y., Chang, J.-J., Lee, S.-C., Chin, T.-Y., Shih, M.-C., Li, W.-H., et al. (2012). Development of cellulosic ethanol production process via co-culturing of artificial cellulosomal Bacillus and kefir yeast. Applied Energy 100, 27-32. doi: https://doi.org/10.1016/j.apenergy.2012.03.016.
Horn, S.J., Vaaje-Kolstad, G., Westereng, B., and Eijsink, V. (2012). Novel enzymes for the degradation of cellulose. Biotechnology for Biofuels 5(1), 45. doi: 10.1186/1754-6834-5-45.
Huang, G.L., Anderson, T.D., and Clubb, R.T. (2014). Engineering microbial surfaces to degrade lignocellulosic biomass. Bioengineered 5(2), 96-106. doi: 10.4161/bioe.27461.
Källberg, M., Wang, H., Wang, S., Peng, J., Wang, Z., Lu, H., et al. (2012). Template-based protein structure modeling using the RaptorX web server. Nature Protocols 7, 1511. doi: 10.1038/nprot.2012.085.
Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N., and Sternberg, M.J.E. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols 10, 845. doi: 10.1038/nprot.2015.053.
Kondo, A., and Ueda, M. (2004). Yeast cell-surface display--applications of molecular display. Applied Microbiology and Biotechnology 64(1), 28-40. doi: 10.1007/s00253-003-1492-3.
Kricka, W., Fitzpatrick, J., and Bond, U. (2014). Metabolic engineering of yeasts by heterologous enzyme production for degradation of cellulose and hemicellulose from biomass: a perspective. Frontiers in Microbiology 5(174). doi: 10.3389/fmicb.2014.00174.
Kruus, K., Lua, A.C., Demain, A.L., and Wu, J.H. (1995). The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome. Proceedings of the National Academy of Sciences 92(20), 9254-9258. doi: 10.1073/pnas.92.20.9254.
Kumar, P., Barrett, D.M., Delwiche, M.J., and Stroeve, P. (2009). Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production. Industrial & Engineering Chemistry Research 48(8), 3713-3729. doi: 10.1021/ie801542g.
Lambertz, C., Garvey, M., Klinger, J., Heesel, D., Klose, H., Fischer, R., et al. (2014). Challenges and advances in the heterologous expression of cellulolytic enzymes: a review. Biotechnol Biofuels 7(1), 135. doi: 10.1186/s13068-014-0135-5.
Lamed, R., Setter, E., and Bayer, E.A. (1983). Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum. J Bacteriol 156(2), 828-836.
Lee, M.H., Lin, J.J., Lin, Y.J., Chang, J.J., Ke, H.M., Fan, W.L., et al. (2018). Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain. Sci Rep 8(1), 7305. doi: 10.1038/s41598-018-25366-z.
Lemaire, M., Ohayon, H., Gounon, P., Fujino, T., and Béguin, P. (1995). OlpB, a new outer layer protein of Clostridium thermocellum, and binding of its S-layer-like domains to components of the cell envelope. Journal of Bacteriology 177(9), 2451-2459. doi: 10.1128/jb.177.9.2451-2459.1995.
Lertwattanasakul, N., Kosaka, T., Hosoyama, A., Suzuki, Y., Rodrussamee, N., Matsutani, M., et al. (2015). Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses. Biotechnology for Biofuels 8(1), 47. doi: 10.1186/s13068-015-0227-x.
Lertwattanasakul, N., Sootsuwan, K., Limtong, S., Thanonkeo, P., and Yamada, M. (2007). Comparison of the gene expression patterns of alcohol dehydrogenase isozymes in the thermotolerant yeast Kluyveromyces marxianus and their physiological functions. Biosci Biotechnol Biochem 71(5), 1170-1182. doi: 10.1271/bbb.60622.
Liang, Y., Si, T., Ang, E.L., and Zhao, H. (2014). Engineered pentafunctional minicellulosome for simultaneous saccharification and ethanol fermentation in Saccharomyces cerevisiae. Appl Environ Microbiol 80(21), 6677-6684. doi: 10.1128/AEM.02070-14.
Liu, Z., Ho, S.H., Sasaki, K., den Haan, R., Inokuma, K., Ogino, C., et al. (2016). Engineering of a novel cellulose-adherent cellulolytic Saccharomyces cerevisiae for cellulosic biofuel production. Sci Rep 6, 24550. doi: 10.1038/srep24550.
Livak, K.J., and Schmittgen, T.D. (2001). Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 25(4), 402-408. doi: https://doi.org/10.1006/meth.2001.1262.
Masran, R., Zanirun, Z., Bahrin, E.K., Ibrahim, M.F., Lai Yee, P., and Abd-Aziz, S. (2016). Harnessing the potential of ligninolytic enzymes for lignocellulosic biomass pretreatment. Appl Microbiol Biotechnol 100(12), 5231-5246. doi: 10.1007/s00253-016-7545-1.
Morais, S., Morag, E., Barak, Y., Goldman, D., Hadar, Y., Lamed, R., et al. (2012). Deconstruction of Lignocellulose into Soluble Sugars by Native and Designer Cellulosomes. mBio 3(6), e00508-00512-e00508-00512. doi: 10.1128/mBio.00508-12.
Morais, S., Shterzer, N., Grinberg, I.R., Mathiesen, G., Eijsink, V.G., Axelsson, L., et al. (2013). Establishment of a simple Lactobacillus plantarum cell consortium for cellulase-xylanase synergistic interactions. Appl Environ Microbiol 79(17), 5242-5249. doi: 10.1128/AEM.01211-13.
Morais, S., Shterzer, N., Lamed, R., Bayer, E.A., and Mizrahi, I. (2014). A combined cell-consortium approach for lignocellulose degradation by specialized Lactobacillus plantarum cells. Biotechnol Biofuels 7, 112. doi: 10.1186/1754-6834-7-112.
Oberortner, E., Cheng, J.-F., Hillson, N.J., and Deutsch, S. (2017). Streamlining the Design-to-Build Transition with Build-Optimization Software Tools. ACS Synthetic Biology 6(3), 485-496. doi: 10.1021/acssynbio.6b00200.
Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., et al. (2004). UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25(13), 1605-1612. doi: 10.1002/jcc.20084.
Phillips, C.M., Beeson, W.T., Cate, J.H., and Marletta, M.A. (2011). Cellobiose Dehydrogenase and a Copper-Dependent Polysaccharide Monooxygenase Potentiate Cellulose Degradation by Neurospora crassa. ACS Chemical Biology 6(12), 1399-1406. doi: 10.1021/cb200351y.
Read, J.D., Colussi, P.A., Ganatra, M.B., and Taron, C.H. (2007). Acetamide selection of Kluyveromyces lactis cells transformed with an integrative vector leads to high-frequency formation of multicopy strains. Appl Environ Microbiol 73(16), 5088-5096. doi: 10.1128/AEM.02253-06.
Schwarz, W. (2001). The cellulosome and cellulose degradation by anaerobic bacteria. Applied Microbiology and Biotechnology 56(5), 634-649. doi: 10.1007/s002530100710.
Sørensen, A., Lübeck, M., Lübeck, P., and Ahring, B. (2013). Fungal Beta-Glucosidases: A Bottleneck in Industrial Use of Lignocellulosic Materials. Biomolecules 3(3), 612.
Spencer, J.F., Ragout de Spencer, A.L., and Laluce, C. (2002). Non-conventional yeasts. Appl Microbiol Biotechnol 58(2), 147-156.
Stern, J., Morais, S., Ben-David, Y., Salama, R., Shamshoum, M., Lamed, R., et al. (2018). Assembly of Synthetic Functional Cellulosomal Structures onto the Cell Surface of Lactobacillus plantarum, a Potent Member of the Gut Microbiome. Appl Environ Microbiol 84(8). doi: 10.1128/AEM.00282-18.
Tang, H., Wang, J., Wang, S., Shen, Y., Petranovic, D., Hou, J., et al. (2018). Efficient yeast surface-display of novel complex synthetic cellulosomes. Microb Cell Fact 17(1), 122. doi: 10.1186/s12934-018-0971-2.
Tsai, S.L., DaSilva, N.A., and Chen, W. (2013). Functional display of complex cellulosomes on the yeast surface via adaptive assembly. ACS Synth Biol 2(1), 14-21. doi: 10.1021/sb300047u.
Tsai, S.L., Oh, J., Singh, S., Chen, R., and Chen, W. (2009). Functional Assembly of Minicellulosomes on the Saccharomyces cerevisiae Cell Surface for Cellulose Hydrolysis and Ethanol Production. Applied and Environmental Microbiology 75(19), 6087-6093. doi: 10.1128/aem.01538-09.
Vazana, Y., Barak, Y., Unger, T., Peleg, Y., Shamshoum, M., Ben-Yehezkel, T., et al. (2013). A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates. Biotechnology for Biofuels 6(1), 182. doi: 10.1186/1754-6834-6-182.
Wen, F., Sun, J., and Zhao, H. (2010). Yeast surface display of trifunctional minicellulosomes for simultaneous saccharification and fermentation of cellulose to ethanol. Appl Environ Microbiol 76(4), 1251-1260. doi: 10.1128/AEM.01687-09.
Wieczorek, A.S., and Martin, V.J. (2010). Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis. Microb Cell Fact 9, 69. doi: 10.1186/1475-2859-9-69.
Wieczorek, A.S., and Martin, V.J. (2012). Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis. Microb Cell Fact 11, 160. doi: 10.1186/1475-2859-11-160.
Yoshida, E., Hidaka, M., Fushinobu, S., Koyanagi, T., Minami, H., Tamaki, H., et al. (2010). Role of a PA14 domain in determining substrate specificity of a glycoside hydrolase family 3 β-glucosidase from Kluyveromyces marxianus. Biochemical Journal 431(1), 39-49. doi: 10.1042/bj20100351.
Zheng, J., and Rehmann, L. (2014). Extrusion pretreatment of lignocellulosic biomass: a review. Int J Mol Sci 15(10), 18967-18984. doi: 10.3390/ijms151018967.