1 20012005 NEDO PL JBA) G SPL: G SPL: G SPL: L RITE 2 1
2006.12.16. SPL 3 ATP 4 2
3 5 1400kb1000kb524kb 510kb, 200kb, 2Thr 2 GH30Phytase7 5 6 Minimum genome factory 10
Nature (2002) 420:350 Synthetic Genomics Hydrogen production from water, Ethanol and Butanol from cellulose best 7 MGF Best Best Synthetic genome MGF 8 4
Predictable system Simple genome only with defined genes Designed and activated metabolism Predictable system MGFMinimum Minimum Genome Factory 9 4320 75 10 5
6 11 30% MGF-01 12
Mbp 20 100 PTS SKIN (sigk) 1.5 (wpra) MGB874 1/4 1/4 DNA 28 13 MGF JBA psp3-isp6- ppp53- ppp16- ppp22- sxa2 ght5 pmr1 gmn2 isp6 400kb hgh 300500kb MGF Cre-loxP Latour 100kb 14 7
MGF 15 OGABOrdered Gene Assembly in Bacillus subtilis MGF OGAB 16 8
9 17 B. subtilis 168 100300 kda5 pps pps 38.4 kb degq 0.6 kb sfp 1.0 kb Glu-D-Orn-Tyr-D-Thr- Glu-D-Ala D-Tyr-Gln- Pro Ileβ-OH FA- - - 18 49 50 51 52 53 54 55 56 57 58 60 62 65 67 69 71 59 61 63 64 66 68 70 72 74 76 78 80 83 85 88 73 75 77 79 81 82 86 89 92 94 96 84 87 90 91 93 95 97 98 99 100 101 102 103 104 105 106 108 110 113 115 117 119 107 109 111 112 114 116 118 120 122 124 126 128 131 133 136 121 123 125 127 129 130 134 137 140 142 144 132 135 138 139 141 143 BEST8824 0 178 173 degq sfp pps 1 2 3 4 5 6 7 8 9 10 12 14 17 19 21 23 11 13 15 16 18 20 22 24 26 28 30 32 35 37 40 25 27 29 31 33 34 38 41 44 46 48 36 39 42 43 45 47 144 144
Pantoea ananatis E Y I B Z E B I Y Z Z E B I Y Y I Z Y E Z B E I B B I Y Z E 19 144degQpps 5 5 5 PR, CI5 20 10
.. 21 MGF PCR-mediated Chromosome Splitting : PCS 22 11
M1 M2 M3 Chr. I (230 kb) Chr. XV (1,091 kb) Chr. II (813 kb) Chr. III (315 kb) Chr. XI (666 kb) Chr. XIV (784 kb) 16 596 kb 287 ORFs Chr. VIII (563 kb) Chr. XIII (924 kb) Chr. XVI (948 kb) Chr. X (745 kb) Chr. IV (1,522 kb) Chr. XI (666 kb) Chr. VI (270 kb) Chr. V (574 kb) 8 285 kb 135 ORFs Chr. V (575 kb) Chr. X (745 kb) Chr. IX (440 kb) 11 376 kb 198 ORFs Chr. XIV (784 kb) 23 24 12
Glycerol (g/l) 2.5 YPD 2 1.5 1 0.5 10 8 6 4 GPD1 2 +GPD1 50 40 30 20 10 0 WT Percoll 0 WT #7 0 WT #7 25 2.5 2 1.5 1 0.5 0 5209 1150 1157 1211 1 0.8 0.6 0.4 0.2 0 5209 1150 1157 1211 26 13
MGF ade2 ade3 YAC-AWA (12 kb) TRP1/ARS1 CEN4 Telomere ADE3 Telomere ade2 ade3 ADE3 DNA ade2ade3yac-awa ade2ade3 Loss rate ( /generation) 0.06 0.05 0.04 0.03 0.02 0.01 0 YEp13 YEp13- YEp13- ZDS1 27 MGF 28 14
Research Institute of Innovative Technology for the Earth RITE 1H13H17 H18 C. glutamicum R (3,314,179bp) 2990 ORF DNA Cre/loxP Cysteine synthase Transketolase Enolase Elongation factor Tu (2-phosphoglycerate dehydratase) Fumarase 75 kda Chaperonin GroEL 50 kda (HSP60 family) 35 kda Phosphoserine 30 kda aminotransferase 25 kda Myo-inositol inositol-1-phosphate synthase 15 kda L-2,3-Butandiol dehydrogenase 10 kda Inorganic pyrophosphatase 10 kda Chaperonin (Protein Cpn10) Peptidyl-prolyl prolyl cis-trans isomerase (rotamase) Glyceraldehyde-3-phosphate dehydrogenase Acetyl-CoA hydrolase Pyruvate kinase Ribosome-associated protein Y Superoxidedismutase Putative fructose-1,6 1,6- bisphosphatase Fructose-bisphosphate aldolase ABC-type transporter, ATPase component Phosphoglycerate mutase Translation elongation factor Ts Nucleoside diphosphate Malate dehydrogenase kinase Triosephosphate isomerase 29 C. glutamicum R (3,314,179bp) 2990 ORF Blue: Tn5-based mini transposon Red: mini-tn31831 185 650 445 320 300 370 720 C. glutamicum R (2990 ORFs) 30 15
Cre recombinase DNA loxp loxp C. glutamicum R genome 3,314,179 bp Ant Transposase 1294bp Transposase (765bp,254AA) DR (3bp) 1453bp Transposase (1311bp,436AA) DR (8bp) 1150bp Transposase DR (2bp)(1035bp,344AA) 1453bp Transposase (1311bp,436AA) DR (8bp) DR (3bp) DR (8bp) DR (2bp) DR (8bp) 31 lox71 1001000 Cre/ mutant lox lox66 lox71 Km r hemf heme hema hemb hemc hemd Cre recombinase hemf heme hema hemb hemchemd loxp LE+RE lox 32 16
5 6 33 o C, 48 h 33 3 genome Corynebacterium glutamicum R genome Genome manipulation C. glutamicum R DNA 5 Superior cells 34 17
Appl. Environ. Microbiol. 71:407-416. 2005. Microbiology 151:501-508. 2005. Appl. Microbiol. Biotechnol. 67225-233. 2005. Appl. Environ. Microbiol. 71:3369-3372. 2005. J. Mol. Microbiol. Biotechnol. 8:243-254 2005. Appl. Environ. Microbiol. 71:7633-7642. 2005 (Review). Appl. Environ. Microbiol. 71:8472-8480. 2005. Appl. Microbiol. Biotechnol. 69151-161. 2005. Appl. Environ. Microbiol. 72:3750-3755. 2006. Appl. Microbiol. Biotechnol. 2006. (in press). Microbiology 2006. (in press). 2005-65649 DNAPCT/JP2005/16269 17 14 2 C. glutamicum R AEM 35 (OGAB) 36 18
ATP 37 MGF DGF Designed GenomeRefined genomeintelligent Genome 38 19