Discoveries and improvements of microbes and enzymes for lignocellulosic ethanol production

Transcription

1 Discoveries and improvements of microbes and enzymes for lignocellulosic ethanol production Tuan-hua David Ho Department of Biology Washington University St. Louis Sumay Yu Institute of Molecular Biology Academia Sinica Taipei

2 How much energy is stored in cellulose? Cellulose is made of glucose and it is the most abundant biopolymer on earth The Taiwan case: 1.5 M tons of rice straws are routinely burned by farmers every year One ton of rice straws can be converted to 250 liters of ethanol Sufficient to produce E3 for all cars in Taiwan

3 Basic procedure of lignocellulosic ethanol production Seeds Photosynthesis CO 2 Energy Carbon Neutral Raw material Pretreatment Saccharification (Hydrolysis) Fermentation Ethanol Cellulose Hemi-cellulose Lignin 6C Sugars 5C Sugars Ethanol Butanol 6C 5C

4 Key enzymes involved in lignocellulosic conversion Endo-glucanase Exo-glucanase β-glucosidase.g-g-g-g-g-g-g-g-g-g-g-g.. G-G G Hemi-cellulases Laccase Peroxidase

5 Where to discover new enzymes/microbes? Wood or straw composts Wood or grass eating insects Gut tissues of grass eating animals Wood rotting fungi (AVRDC)

6 A highly active and thermostable endo-glucanase from the thermophilic bacterium Geobacillus Isolation of Geobacillus from rice straw compost and cloning of endo-glucanase gene, CelA 1st Geobacillus endo-glucanase gene cloned 10 X more active than commercial Trichoderma reesei endo-glucanase Stable and active over a broad temperature range Original bacterial colony Cloning and exp of Cel A Activity doubled in 2 mm MnSO 4 Paper submitted and patent filed Expression of CelA in E. coli CelA active and stable up to 70ºC Relative activity (%) ( ) Optimal temp. 20 ( ) Thermostability (6 hr) Temp ( o C)

7 Metagenomics Approach Isolation of RSC-EG1 by screening λphage library constructed from rice straw compost 120 Optimum Temperature Fresh compost samples 100 DNA extraction Subcloning of RSC-EG1 ty(%) Relative activit Metagenomics 4 kb 20 CMCase Temperature Sequencing revealed a novel endo-glucanase 120 Optimum ph Expression and purification from E.coli 100 Cloning & expression 49kDa Relative ac ctivity(%) mM sodium acetate buffer (ph ) 100mM sodium phosphate buffer (ph ) 100mM Tris-HCl buffer (ph ) Enzyme characterization Optimal temp is 70 Optimal ph is ph

8 Novel cellulase genes from white spotted longicorn beetle Anoplophora malasiaca Cloning and sequence homology of newly isolated AmEGases Gene GHF Type of cellulase Identidy(%) AmEG 1 45 Endo-β-1,4-glucanase 75.7 AmEG 2 5 Endo-β-1,4-glucanase 80.4 AmEG 3 5 Endo-β-1,4-glucanase 58.3 AmEG 4 5 Endo-β-1,4-glucanase 56.3 AmEG 5 5 Endo-β-1,4-glucanase 55.8 AmEG 6 48(9) Cellobiohydrolase (exo-glucanase) 10X higher yield of AmEG1 in whole Silkworm body than in body fluid 65 6 longicorn cellulase genes cloned so far All AmEGs appear to be eukaryotic type An efficient silkworm expression system developed for production of AmEGs 10X higher yield of AmEGs in whole silkworm body than in body fluid AmEG1 enhances efficiency of commercial Trichoderma reesei enzymes AmEG1 has an optimal ph at 4 Activity assay of AmEG 1 from silkworm body fluid with T. reesei (CMC by DNS) KD Body Body fluid Reducing sugar (mg/ml) AmEG 1 AmEG 1+T.reesei T.reesei T.reesei+N ovo 188

9 Filter paper p digestion (3 hr at 50ºC) Cellulases of Xylariaceae fungi Fungal culture medium 1 2 H 2 O Xylariaceae secretes 2 endoglucanases, 1 exo- glucanase and 1 β-glucosidase Four cellulase-related genes have been isolated from Xylariaceae 110 kda 72 kda 55 kda 43 kda M M kda kda Cip2 [Hypocrea jecorina] Identity: 48%, Positive: 64% CMC Zymogram SDS-PAGE -glucosidase 1-1 Cellulase [Melanocarpus albomyces] Identity: 64%, Positive: 77% 1-2 Cellulose 1,4-beta-cellobiosidase [Acremonium thermophilum] Identity: 57%, Positive: 71% 3 Endoglucanase [Talaromyces emersonii] Identity: 62%, Positive: 78% (Exo-glucanase)

10 Highly active laccase of Rigidoporus microporus A Taiwanese fungus R. microporus secreting high level laccase Lcc35 laccase is a novel and abundant secretory enzyme in R. microporus. >3 X more active than commercial laccases Paper to be submitted and patent filed Laccase is the single most abundant protein secreted by R. microporus Strain ABTS assay (U/mg) SGZ assay (U/mg) Lcc35 Rigidoporus sp (U/mg) 1700 (U/mg) Laccase (Fluka co.) Trametes versicolor 1300 (U/mg) 500 (U/mg) Laccase (patent Thielavia i nkat/mg application US2006/ A1) arenaria nkat/mg or or 29 (U/mg)* 61 (U/mg)* Laccase (Novozymes)** (U/mg)** (U/mg)** *16.67 nkat = 1U ** Laccase provided by 永豐餘

11 Fast growing and highly productive R. microporus mutants Fast growing mutants of R. microporus 25 Basal medium 20 H8 H5 WT H7 Laccase activity (U/ml) H6 mutant WT H Time (day) R. microporus mutant H6 grows twice as fast as wild type H6 mutant produces more than twice as much laccase as wild type H6 mutant culture does not have viscous metabolites in medium

12 Dramatic structural changes on rice straws after pretreatment with specific fungi Non-treated Treated with strain 40-3 for 3 weeks Treated with Treated with strain 40-5 strain 001 for 3 weeks for 6 weeks

13 Challenges ahead Low cost collection of raw materials Lowering energy input Lowering cost of enzymes and microbes More efficient i fermentation ti Nonezymatic conversion (pyrolysis)

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16 Output Papers -- 2 submitted (Ho, Yu and Tong) -- 2 under preparation (each for Ho/Yu/Tong and Chao) Patents -- 3 filed (Ho, Yu and Tong; 2 US and 1 ROC) -- 1 to be applied (Chao) Licensing agreements -- 2 under preparation for Ho,Yu and Tong (bio-pretreatment; fungal cellulase l production process) Collaborations -- INER (bioconversion) -- Scandinavian Biofuel (bio-pretreatment; bioconversion) -- Yuen-Foong Yu 永豐餘 (paper pulping; brightening)

17 Development of fungal enzyme formulations tested in INER pilot plant Fusarium proliferatum (F.P) cellulases Glucose yield (per g cellulosic materials) 24h 48h 72h Cellulast 1.5L (commercial enzymes) Cellulase-F.P (this project) Relat tive activit ty (%) FPase activity 核研所 assessment of our enzyme formulations *Local strain isolated at AVRDC, Taiwan * Has a full set of cellulases to hydrolyze rice straws *Crude enzyme preparation converts 50% of pretreated rice straws to glucose *Crude preparations can be produced using low-cost feedstocks such as rice straws *Some enzyme activities ities display synergistic effect with commercial enzymes

18 Development of bacterial enzyme formulations with synergistic effect Relativ ve activity (% %) Avicelase 100% 106% 115% 111% Glucanases from Thermobifida fusca (T.F) 125% 0% 1:0 1:1 1:2 1:4 1:10 βgl Weight ratio (crude T.F enzyme/ βgl) Up to 25% enhancement of T.F avicelase activity with supplement of β-glucosidase (βgl) 100 Relative e activity (% %) CMCase FPase 1 : 0 1 : : : 1 1 : 2 1 : 4 1 : 10 1 : 20 1 : 60 Molar ratio (E4/ βgl) Up to 70~100% enhancement of CMCase and FPase activities by combining purified E4 cellulase (with both endo- and exo- act) with T.F-β-glucosidase (βgl)

19 Discovery of highly active fungal β-glucosidases Fungi from Dr. YM Ju (IPMB) D2 D3 PC E9 β-glucosidas se (U/ml) Screening for β-glucosidase producing fungi A1 A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6 C1 C2 C3 C4 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 E1 E2 E3 E4 E5 E6 E7 E9 E10 E11 E13 E15 E16 PC β-glucosidase zymogram D2 D3 PC E9 N188 6 fungi with excellent β-glucosidase activity D2 is most promising in hydrolyzing cellobiose to glucose (Min -1 ) (Min -1 )