Biotechnology for Biofuels | |
Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions | |
Shara D McClendon2  Tanveer Batth2  Christopher J Petzold2  Paul D Adams2  Blake A Simmons3  Steven W Singer1  | |
[1] Deconstruction Division, Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA, 94608, USA | |
[2] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA | |
[3] Biomass Science and Conversion Technology Department, Sandia National Laboratories, Livermore, CA, USA | |
关键词: Switchgrass (Panicum virgatum); 1-ethyl-3-methylimidazolium acetate; Ionic liquid; Ammonia fiber expansion; Fungal secretome; Polysaccharide monooxygenases; GH 61; Thielavia terrestris; Thermoascus aurantiacus; | |
Others : 798254 DOI : 10.1186/1754-6834-5-54 |
|
received in 2012-06-06, accepted in 2012-07-13, 发布年份 2012 | |
![]() |
【 摘 要 】
Background
Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents.
Results
Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of ionic liquid pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails.
Conclusions
T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or genetic modifications. Therefore, T. aurantiacus provides an excellent platform to develop a thermophilic fungal system for enzyme production for the conversion of biomass to biofuels.
【 授权许可】
2012 McClendon et al.; licensee BioMed Central Ltd.
【 预 览 】
Files | Size | Format | View |
---|---|---|---|
20140706112900617.pdf | 1970KB | ![]() |
|
Figure 5. | 80KB | Image | ![]() |
Figure 4. | 74KB | Image | ![]() |
Figure 3. | 68KB | Image | ![]() |
Figure 2. | 76KB | Image | ![]() |
Figure 1. | 91KB | Image | ![]() |
【 图 表 】
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
【 参考文献 】
- [1]Berka RM, Grigoriev IV, Otillar R, Salamov A, Grimwood J, Reid I, Ishmael N, John T, Darmond C, Moisan M-C, et al.: Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotech 2011, 29(10):922-927.
- [2]Maheshwari R, Bharadwaj G, Bhat MK: Thermophilic Fungi: Their Physiology and Enzymes. Microbiol Mol Biol Rev 2000, 64(3):461-488.
- [3]Brienzo M, Arantes V, Milagres AMF: Enzymology of the thermophilic ascomycetous fungus Thermoascus aurantiacus. Fungal Biology Reviews 2008, 22(3–4):120-130.
- [4]Bhat MK, Parry NJ, Kalogiannis S, Beever DE, Owen E: Thermostable Cellulase and Xylanase from Thermoascus aurantiacus and Their Potential Industrial Applications. In Glycosyl Hydrolases for Biomass Conversion vol. 769. Washington D C: American Chemical Society; 2000:204-221.
- [5]Waters DM, Murray PG, Ryan LA, Arendt EK, Tuohy MG: Talaromyces emersonii Thermostable Enzyme Systems and Their Applications in Wheat Baking Systems. Journal of Agricultural and Food Chemistry 2010, 58(12):7415-7422.
- [6]Viikari L, Alapuranen M, Puranen T, Vehmaanperä J, Siika-aho M: Biofuels: Advances in Biochemical Engineering/Biotechnology. In Thermostable Enzymes in Lignocellulose Hydrolysis Biofuels. vol. 108 edition. Edited by Olsson L. Berlin/Heidelberg: Springer; 2007:121-145.
- [7]Li C, Knierim B, Manisseri C, Arora R, Scheller HV, Auer M, Vogel KP, Simmons BA, Singh S: Comparison of dilute acid and ionic liquid pretreatment of switchgrass: Biomass recalcitrance, delignification and enzymatic saccharification. Bioresource Technology 2010, 101(13):4900-4906.
- [8]Petkovic M, Ferguson J, Bohn A, Trindade J, Martins I, Carvalho MB, Leitao MC, Rodrigues C, Garcia H, Ferreira R, et al.: Exploring fungal activity in the presence of ionic liquids. Green Chemistry 2009, 11(6):889-894.
- [9]Singer SW, Reddy AP, Gladden JM, Guo H, Hazen TC, Simmons BA, VanderGheynst JS: Enrichment, isolation and characterization of fungi tolerant to 1-ethyl-3-methylimidazolium acetate. Journal of Applied Microbiology 2011, 110(4):1023-1031.
- [10]Liang C, Xue Y, Fioroni M, Rodríguez-Ropero F, Zhou C, Schwaneberg U, Ma Y: Cloning and characterization of a thermostable and halo-tolerant endoglucanase from Thermoanaerobacter tengcongensis MB4. Applied Microbiology and Biotechnology 2011, 89(2):315-326.
- [11]Gladden JM, Allgaier M, Miller CS, Hazen TC, VanderGheynst JS, Hugenholtz P, Simmons BA, Singer SW: Glycoside Hydrolase Activities of Thermophilic Bacterial Consortia Adapted to Switchgrass. Applied and Environmental Microbiology 2011, 77(16):5804-5812.
- [12]Graham JE, Clark ME, Nadler DC, Huffer S, Chokhawala HA, Rowland SE, Blanch HW, Clark DS, Robb FT: Identification and characterization of a multidomain hyperthermophilic cellulase from an archaeal enrichment. Nat Commun 2011, 2:375.
- [13]Rosgaard L, Pedersen S, Cherry JR, Harris P, Meyer AS: Efficiency of New Fungal Cellulase Systems in Boosting Enzymatic Degradation of Barley Straw Lignocellulose. Biotechnology Progress 2006, 22(2):493-498.
- [14]Harris PV, Welner D, McFarland KC, Re E, Navarro Poulsen JC, Brown K, Salbo R, Ding H, Vlasenko E, Merino S, et al.: Stimulation of Lignocellulosic Biomass Hydrolysis by Proteins of Glycoside Hydrolase Family 61: Structure and Function of a Large, Enigmatic Family. Biochemistry 2010, 49(15):3305-3316.
- [15]Langston JA, Shaghasi T, Abbate E, Xu F, Vlasenko E, Sweeney MD: Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proceedings of the National Academy of Sciences of the United States 2011, 108(37):15079-84.
- [16]Walker JM: The Bicinchoninic Acid (BCA) Assay for Protein Quantitation. In The Protein Protocols Handbook. Edited by Walker JM. New Jersey: Humana Press; 2002:11-14.
- [17]Li C, Cheng G, Balan V, Kent MS, Ong M, Chundawat SPS, Sousa L, Melnichenko YB, Dale BE, Simmons BA, et al.: Influence of physico-chemical changes on enzymatic digestibility of ionic liquid and AFEX pretreated corn stover. Bioresource Technology 2011, 102(13):6928-6936.
- [18]Chundawat SPS, Vismeh R, Sharma LN, Humpula JF, Sousa LD, Chambliss CK, Jones AD, Balan V, Dale BE: Multifaceted characterization of cell wall decomposition products formed during ammonia fiber expansion (AFEX) and dilute acid based pretreatments. Bioresource Technology 2010, 101(21):8429-8438.
- [19]Gao D, Chundawat SP, Krishnan C, Balan V, Dale BE: Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover. Bioresource Technology 2010, 101(8):2770-2781.
- [20]Cannella D, Hsieh CW, Felby C, Jorgensen H: Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content. Biotechnology for Biofuels 2012, 5(1):26. BioMed Central Full Text
- [21]Banerjee G, Car S, Scott-Craig JS, Borrusch MS, Bongers M, Walton JD: Synthetic multi-component enzyme mixtures for deconstruction of lignocellulosic biomass. Bioresource Technology 2010, 101(23):9097-9105.
- [22]Le Crom S, Schackwitz W, Pennacchio L, Magnuson JK, Culley DE, Collett JR, Martin J, Druzhinina IS, Mathis H, Monot F, et al.: Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing. Proceedings of the National Academy of Sciences of the United States of America 2009, 106(38):16151-16156.
- [23]Pore RS, Larsh HW: First occurrence of Thermoascus aurantiacus from animal and human sources. Mycologia 1967, 59(5):927.
- [24]Bals B, Rogers C, Jin M, Balan V, Dale B: Evaluation of ammonia fibre expansion (AFEX) pretreatment for enzymatic hydrolysis of switchgrass harvested in different seasons and locations. Biotechnology for Biofuels 2010, 3(1):1. BioMed Central Full Text
- [25]Torget R, Werdene P, Himmel M, Grohmann K: Dilute acid pretreatment of short rotation woody and herbaceous crops. Applied Biochemistry and Biotechnology 1990, 24–25(1):115-126.
- [26]Brown RE, Jarvis KL, Hyland KJ: Protein measurement using bicinchoninic acid: elimination of interfering substances. Analytical Biochemistry 1989, 180(1):136-139.
- [27]Xiao Z, Storms R, Tsang A: Microplate-based carboxymethylcellulose assay for endoglucanase activity. Analytical Biochemistry 2005, 342(1):176-178.
- [28]Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M: In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protocols 2007, 1(6):2856-2860.
- [29]Grigoriev IV, Nordberg H, Shabalov I, Aerts A, Cantor M, Goodstein D, Kuo A, Minovitsky S, Nikitin R, Ohm RA, et al.: The Genome Portal of the Department of Energy Joint Genome Institute. Nucleic Acids Research 2012, 40(D1):D26-32.