2010 12 12 2 RITE 1
C6 C5 RITE Sustainable Industry C2 C3 C4 2
1 take-off food non-food DuPont C 6 2013 Advanced fuels 3
in ( kl/y) POET Broin 2011 2011 C5C6 9.5 Novozymes Z. mobilis Vercipia (Verenium/BP) 2010 2012 2 13.6 BP (Verenium) 2009 2009 2 0.6 E.coli KO11 K. oxytoca BW34 E.coli KO11 K. oxytoca BW34 Mascoma 2009 2012 CBP 7.6 Genencor, Yeast Themophilic anaerobe Bluefire Ethanol Inc. 2009 2012 C5C6 7.2 Yeast Abengoa 2011 2012 2 4.4 Novozymes, Genencor, Dyadic, DSM DuPont Danisco Cellulosic Ethanol 2012 2010 C5C6 0.1 Genencor Z. mobilis American Energy Enterprises 2010 mid 2010 C5C6 5.7 Yeast Pacific ethanol 2009 4Q, 2010 2 1.0 Novozymes Yeast Themophilic anaerobe KL Energy / Western Biomass Energy 2007 2008 C6 0.6 Novozymes Yeast 4
UP Land Usage CSR 5
2 $ 230 Billion in 2030 IT C 6 6
Green-chemical s market:$230billion by 2030 When bio-fuel gives you lemons, make green-chemicals at first. From BiofuelsDigest, partially modified 7
A Novel and Highly Efficient Bioprocess RITE Bioprocess 8
RITE RITE High productivity No energy loss for growth Simple system 9
RITE strain Corynebacterium glutamicum Under oxygen deprivation - Growth-arrested - Maintains main metabolic capabilities 10
Engineering of Hyper-producing Corynebacteria Sugars Gene introduction using compatible vectors Metabolite gene A I gene B II gene C III gene D IV Expression plasmids genome Selection of useful gene Optimization of gene expression level Identification of the best combination Products Chromosomal integration 11
Glucose PEP EM pathway DHAP NAD + NADH G-6-P F-6-P F-1,6-P 2 GAP PGP PTS PYR NADP + P8),15),16),20) GAPDH P15),12),17),19),23),21),22) NADPH 6-PGlucono -lactone PP pathway 6-PGluconate Rib5P GAP Xylose NADP + Xylose xyla Pentose transporter Xylulose xylb NADPH Ribu5P P3),2),4) Arabinose 5),6) Arabinose araa Ribulose arab Ribulose-5P arad Xlu5P Sed-7-P P4),3) PEP PYR F-6-P F-6-P GAP Ery-4-P P9) L-Alanine D-Lactic acid P14),11),13),14) P14),9) P13),P14),P16),P17), P18),P19),P20),7) P1),P2),1) P10),P11),P12), P14),8),10) L-Lactic acid Ethanol Isopropanol Succinic acid Branched-chain P9) amino acids(l-valine ) P5) Isobutanol P6),P7),18) Butanol Patent application / registration by RITE P1) WO2009028582 P2) WO2009131040 P3) PCT/JP2009/060637 P4) JP2009050236(A) P5) JP2009083668 P6) JP2009039031 P7) JP2009183259 P8) JP2007295809 P9) JP2007043947 P10) EP1647594A1 P11) WO2005010182A1 P12) US7368268 P13) JP4294373 P14) JP3869788 P15) JP4171265 P16) EP1291428A1 P17) US7598063 P18) CN1436240 P19) IN209524 P20) JP2002510689 Publication by RITE 1) Appl. Microbiol. Biotechnol. 77:1219-1224. 2008. 2) Appl. Environ. Microbiol. 72:3418-3428. 2006. 3) Appl. Microbiol. Biotechnol. 77:1053-1062. 2008. 4) Appl. Microbiol. Biotechnol. 81:691-699. 2008. 5) Appl. Environ. Microbiol. 75:3419-3429. 2009. 6) Appl. Microbiol. Biotechnol. 85:105-115. 2009. 7) J. Mol. Microbiol. Biotechnol. 8:243-254. 2004. 8) J. Mol. Microbiol. Biotechnol. 7:182-196. 2004. 9) Appl. Microbiol. Biotechnol. 68:475-480. 2005. 10) Appl. Microbiol. Biotechnol. 81:459-464. 2008. 11) Appl. Microbiol. Biotechnol. 78:449 454. 2008. 12) Microbiology 155:3652-3660. 2009. 13) J. Bacteriol. 191:4251-4258. 2009. 14) Appl. Microbiol. Biotechnol. 83:315-327. 2009. 15) J. Bacteriol. 191:968-977. 2009. 16) Appl. Microbiol. Biotechnol. 81:291-301. 2008. 17) Appl. Microbiol. Biotechnol. 78:309-318. 2008. 18) J. Mol. Microbiol. Biotechnol. 15:16-30. 2008. 19) Microbiology 154:264-274. 2008. 20) J. Mol. Microbiol. Biotechnol. 8:91-103. 2004. 21) Biochem. Biophys. Res. Commun. 289:1307-1313. 2001. 22) J. Biosci. Bioeng. 92:502-517. 2001. 23) Microbiology 149:1569-1580. 2003. 12
CH 2 O O PYR Glucose PEP EM pathway C O C CH 3 DHAP NAD + NADH 336.1 kj/mol G-6-P F-6-P F-1,6-P 2 GAP PGP PEP PTS GAPDH PYR NADP + NADPH 6-PGlucono -lactone HOCH 2 O 6-PGluconate Rib5P GAP F-6-P F-6-P GAP Xylose 793.7 kj/mol 662.9 kj/mol CO C O CH 2 DAHP PP pathway NADP + CO C O CH 2 CH HC HC CH 2 O CO 2 Xylose xyla Pentose transporter Xylulose xylb NADPH Ribu5P Arabinose Arabinose araa Ribulose arab Ribulose-5P arad Xlu5P Sed-7-P Ery-4-P 662.9 kj/mol CHO HC HC CH 2 O O HOCH 2 13
RITE 14
CH 2 O O PYR Glucose PEP EM pathway O C C CH 3 DHAP NAD + NADH 336.1 kj/mol G-6-P F-6-P F-1,6-P 2 GAP PGP PEP PTS GAPDH PYR NADP + NADPH 6-PGlucono -lactone HOCH 2 O 6-PGluconate Rib5P GAP F-6-P F-6-P GAP Xylose 793.7 kj/mol 662.9 kj/mol CO C O CH 2 DAHP Phenol PP pathway CO C O CH 2 CH HC HC CH 2 O NADP + CO 2 Xylose xyla Pentose transporter Xylulose xylb NADPH Ribu5P Arabinose Arabinose araa Ribulose arab Ribulose-5P arad Xlu5P Sed-7-P Ery-4-P CHO HC HC CH 2 O 662.9 kj/mol O HOCH 2 Phenol 32.9 kj/mol 15
Green Sustainable Chemistry 2008 2,000 5 16
- 100 100 66 84 83 RON 95 120 94 109 17
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Air 19
BSE 120 t 16 0.3 20
RITE C6 C5 STY Space/Time/Yield 21
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6 5 23
6 5 24
C6 C5 C6 C5 25
C6, C5 Biomass C5 Sugars Fermentation Solid/Liquid Separation Liquid Hemi-sugars Pretreatment & hemicellulose hydrolysis Solids Cellulose + Lignin Detoxification C-5 Fermentation Ethanol Cellulose Hydrolysis Glucose Fermentation Product Recovery Enzymatic Hydrolysis & Fermentation Lignin to Boilers 26
Sugar Metabolism Engineering Cellulose Cellobiose (C 6 -C 6 ) Adaptive mutant for cellobiose uptake ability 1) Glucose (C 6 ) RITE strain Hemicellulose Xylose (C 5 ) Chromosomal integration for xylose metabolic ability promoter promoter 2) xyla xylb xylose isomerase xylulokinase Arabinose (C 5 ) Chromosomal integration for arabinose metabolic ability arab araa arad L-arabinose isomerase L-ribulokinase L-ribulose-5-P-4-epimerase 1) Microbiology 149: 1569-80. 2003. 2) Appl. Environ. Microbiol. 72: 3418-28. 2006. 3) Appl. Microbiol. Biotechnol. 77: 1053-62. 2008. 27 3)
Simultaneous Utilization of Mixed Sugars Integration of a pentose transporter 200 200 Glucose Glucose Sugar mm 150 100 50 Xylose Arabinose Sugar mm 150 100 50 Xylose Arabinose 0 0 2 4 6 8 10 12 0 0 2 4 6 8 10 12 Time h Time h Appl. Microbiol. Biotechnol. 85: 105-115. 2009. 28
Corynebacterium glutamicum Under oxygen deprivation - Growth-arrested -Maintains main metabolic capabilities 29
Corynebacterium glutamicum Shift Corynebacterium 30
Phase Trends Biotechnol. (2008) 26:100-108 EU Appl. Environ. Microbiol. (2007) 73:2079-2084 2009 8 RITE 31
RITE - - 32
ETP2010: GHG 4 IEA Energy Technology Perspective 2010 33
ETP2010: IEA Energy Technology Perspective 2010 34 34
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