(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization IN International Bureau (10) International Publication Number (43) International Publication Date 1 April 2010 (01.04.2010) PCT W O 2010/037119 Al

(51) International Patent Classification: (81) Designated States (unless otherwise indicated,for every C12N 9/88 (2006.01) C12R 1/46 (2006.01) kind of nationalprotection available): AE, AG, AL, AM, C12P 7/16 (2006.01) C12R 1/225 (2006.01) AG, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21)CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, EG, ES, Fl, GB, GD, GE, GH, GM, GT, PCT/US2009/058843PCT/S200/0584 FIN,DZ, HR,EC, HU,EE, 11), IL, IN, IS, JP, KE, KG, KM, KN, KP, (22) International Filing Date: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, 29 September 2009 (29.09.2009) ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, GM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, (25) Filing Language: English SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, (26) Publication Language: English TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 6 1/100,809 29 September 2008 (29.09.2008) us kind of regionalprotection available>: ARIPO (BW, GH, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, (71) Applicant (for all designated States except US>: BUTA- ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, NLALX 1fm ADVANCED BIOFUELS LLC [US/US]; 200 TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, Powder Mill Road, Wilmington, DE 19803 (US). ES, Fl, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (725) Inventors;uan TR)G,GAPIMC, MK, MT,(BF, NL,BJ, CF,NO, CG, PL, C PT, , CM, RO, GA, SE, GN, SI, GQ,SK, GW,SM,

James [US/US]; 115 Fairfax Boulevard, Wilmington, TZ, UA, UG, S, UZ, V N A , Delaware 19803 (US). SUH, Wonchul [KR/US]; 8 Salina Published: Court, Piersons Ridge, Hockessin, Delaware 19707 (US). with internationalsearch report (Art. 21(3 (74) Agent: LHULIER, Christine, M.; E. 1. du Pont de with sequence listing part of description (Rule 5.2(a Nemours and Company, Legal Patent Records Center, 4417 Lancaster Pike, Wilmington, Delaware 19805 (US).

Jae-U/S] 1yaraxBuead f W112imington

(54) Title: ENHANCED IRON-SULFUR CLUSTER FORMATION FOR INCREASED DIHYDROXY-ACID DEHYDRATASE ACTIVITY IN LACTIC ACID BACTERIA (8)DsgaeCtts(nesohrieidctd o vr

feoO.s~C'sfD -- \ = eA suU RF

FIG. 1lA

FIG. 1B (57) Abstract: Lactic acid bacteria expressing dihydroxyacid dehydratase polypeptides with increased specific activity are dis closed. The lactic acid bacteria comprise recombinant genes encoding iron-sulfr cluster forming proteins. WO 2010/037119 PCT/US2009/058843

TITLE ENHANCED IRON-SULFUR CLUSTER FORMATION FOR INCREASED DIHYDROXY-ACID DEHYDRATASE ACTIVITY IN LACTIC ACID 5 BACTERIA CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to and claims the benefit of priority of U.S. Provisional Application No. 61/100,809, filed September 29, 2008, the entirety of which is herein incorporated by reference. 10 FIELD OF THE INVENTION The invention relates to the field of microbiology. More specifically, lactic acid bacteria are disclosed expressing high levels of dihydroxy-acid dehydratase activity in the presence of introduced iron-sulfur cluster forming proteins. 15 BACKGROUND OF THE INVENTION Dihydroxy-acid dehydratase (DHAD), also called acetohydroxy acid dehydratase, catalyzes the conversion of 2,3-dihydroxyisovalerate to a ketoisovalerate and of 2,3-dihydroxymethylvalerate to a ketomethylvalerate. The DHAD enzyme requires binding of an iron-sulfur 20 (Fe-S) cluster for activity, is classified as E.C. 4.2.1.9, and is part of naturally occurring biosynthetic pathways producing valine, isoleucine, leucine and pantothenic acid (vitamin B5). DHAD catalyzed conversion of 2,3-dihydroxyisovalerate to a-ketoisovalerate is also a common step in the multiple isobutanol biosynthetic pathways that are disclosed in commonly 25 owned and co-pending US Patent Pub No. US 20070092957 Al. Disclosed therein is engineering of recombinant microorganisms for production of isobutanol. Isobutanol is useful as a fuel additive, whose availability may reduce the demand for petrochemical fuels.High levels of DHAD activity are desired for increased production of products from 30 biosynthetic pathways that include this enzyme activity, including for enhanced microbial production of branched chain amino acids, pantothenic acid, and isobutanol, however since DHAD enzymes are Fe-S

1 WO 2010/037119 PCT/US2009/058843

cluster requiring they must be expressed in a host having the genetic machinery to produce Fe-S proteins. [2Fe-2S] 2+ and [4Fe-4S] 2+ clusters can form spontaneously in vitro (Malkin and Rabinowitz (1966) Biochem. Biophys. Res. Comm. 23: 5 822-827). However, likely due to the toxic nature of both free Fe(II) and sulfide, biogenesis systems have evolved to form Fe-S clusters and insert them into their target apoproteins in vivo. The biogenesis of iron sulfur clusters is not completely understood but is known generally to include liberation of sulfur from the amino acid cysteine by a cysteine desulfurase 10 enzyme, combination of the sulfur with Fe(II) on a scaffold protein, and transfer of the formed Fe-S clusters, frequently in a chaperone-dependent manner, to the proteins and enzymes that require them. The Isc, Suf and Nif operons have been found to encode proteins involved in Fe-S cluster formation in different bacteria (Johnson et al. Annu. Rev. Biochem. 15 74:247-281 (2005)). Lactic acid bacteria are well characterized and are used commercially in a number of industrial processes. Although it is known that some lactic acid bacteria possess Fe-S cluster requiring enzymes (Liu et al., Journal of Biological Chemistry (2000), 275(17), 12367-12373) and 20 therefore posses the genetic machinery to produce Fe-S clusters, little is known about the ability of lactic acid bacteria to insert Fe-S clusters into heterologous enzymes, and little is known about the facility with which Fe S cluster forming proteins can be expressed in lactic acid bacteria. To obtain high levels of product in a lactic acid bacteria from a 25 biosynthetic pathway including DHAD activity, high expression of DHAD activity is desired. The activity of the Fe-S requiring DHAD enzyme in a host cell may be limited by the availability of Fe-S cluster in the cell. There remains a need therefore to engineer a lactic acid bacteria, which is a good industrial host, to provide sufficient levels of Fe-S cluster forming 30 proteins to accommodate the expression of Fe-S requiring proteins such as DHAD.

2 WO 2010/037119 PCT/US2009/058843

SUMMARY OF THE INVENTION Provided herein are lactic acid bacterial cells comprising a functional dihydroxy-acid dehydratase polypeptide and at least one recombinant genetic expression element encoding iron-sulfur cluster 5 forming proteins. In some embodiments, the functional dihydroxy-acid dehydratase polypeptide is encoded by a nucleic acid molecule that is heterologous to the bacteria. In some embodiments, the functional dihydroxyacid dehydratase polypeptide is a [2Fe-2S] 2+ dihydroxy-acid dehydratase, while in other embodiments, the functional dihydroxyacid 10 dehydratase polypeptide is a [4Fe-4S] 2+ dihydroxy-acid dehydratase. In one embodiment, the dihydroxyacid dehydratase polypeptide has an amino acid sequence that matches the Profile HMM of Table 7 with an E value of < 10-5 wherein the polypeptide additionally comprises all three conserved cysteines, corresponding to positions 56, 129, and 201 in the 15 amino acids sequences of the Streptococcus mutans DHAD enzyme corresponding to SEQ ID NO:168. In one embodiment, the dihydroxyacid dehydratase polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:310, SEQ ID NO:298 , SEQ ID NO:168, SEQ ID No:164, SEQ ID NO:346, SEQ ID NO:344, SEQ ID NO:232, and 20 SEQ ID NO:230. In some embodiments, the recombinant genetic expression element encoding iron-sulfur cluster forming proteins contains coding regions of an operon selected from the group consisting of Isc, Suf and Nif operons. In some embodiments, the Suf operon comprises at least one 25 coding region selected from the group consisting of SufC, Suf D, Suf S, SufU, Suf B, SufA and yseH, and in some embodiments, the Isc operon comprises at least one coding region selected from the group consisting of IscS, IscU, IscA, IscX, HscA, HscB, and Fdx. In some embodiments the Nif operon comprises at least one coding region selected from the 30 group consisting of NifS and NifU. In some embodiments, the Suf operon has the nucleotide sequence selected from the group consisting of SEQ ID NO:881 and SEQ ID NO:589. In some embodiments, the Suf operon is derived from Lactococcus lactis and comprises at least one coding region

3 WO 2010/037119 PCT/US2009/058843

encoding a polypeptide having an amino acid sequenced selected from the group consisting of SEQ ID NO: 598 (SufC), SEQ ID NO: 604 (SufD), SEQ ID NO: 610 (SufB), and SEQ ID NO: 618 (YseH). In some embodiments, the Suf operon is derived from Lactoabcillus plantarum and 5 comprises at least one coding region encoding a polypeptide having an amino acid sequenced selected from the group consisting of SEQ ID NO: 596 (SufC), SEQ ID NO: 602 (SufD), SEQ ID NO: 624 (SufS), SEQ ID NO: 620 (SufU) and SEQ ID NO: 608 (SufB). In some embodiments, the Isc operon is derived from E. Coli and comprises at least one coding 10 region encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 528 (IscS), SEQ ID NO: 530 (IscU), SEQ ID NO: 532 s(IscA), SEQ ID NO:534 (HscB), SEQ ID NO: 536 (hscA), SEQ ID NO: 538 (Fdx), and SEQ ID NO: 540 (IscX). In some embodiments the Nif operon is derived from Wolinella succinogenes and 15 comprises at least one coding region encoding a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 542 (NifS) and SEQ ID NO: 544 (NifU). In some embodiments, the lactic acid bacterial cell provided herein is a member of a genus selected from the group consisting of 20 Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus. In some embodiments, the bacteria produces isobutanol, and in some embodiments, the bacteria comprises an isobutanol biosynthetic pathway. In some embodiments, the isobutanol biosynthetic pathway comprises genes encoding acetolactate synthase, acetohydroxy 25 acid isomeroreductase, dihydroxy-acid dehydratase, branched-chain a keto acid decarboxylase, and branched-chain alcohol dehydrogenase. Also provided herein is a method for increasing the activity of a heterologous dihydroxyacid dehydratase polypeptide in a lactic acid bacterial cell comprising: a) providing a lactic acid bacterial cell 30 comprising: 1) a nucleic acid molecule encoding a heterologous dihydroxyacid dehydratase polypeptide; 2) a recombinant genetic expression element encoding iron-sulfur cluster forming proteins, wherein the proteins are expressed; and b) growing the lactic acid bacterial cell of

4 WO 2010/037119 PCT/US2009/058843

(a) under conditions whereby the dihydroxy-acid dehydratase polypeptide is expressed in functional form having a specific activity greater than the same dihydroxy-acid dehydratase polypeptide expressed in the same bacterial cell lacking the recombinant genetic expression element 5 encoding iron-sulfur cluster forming proteins. In one embodiment, the specific activity of the expressed dihydroxyacid dehydratase polypeptide is at least about two fold greater than the specific activity of the same dihydroxyacid dehydratase polypeptide expressed in the same bacteria lacking the recombinant genetic expression element encoding iron-sulfur 10 cluster forming proteins. Also provided herein is a method of making isobutanol comprising providing a lactic acid bacterial cell disclosed herein and growing said cell under conditions wherein isobutanol is produced. BRIEF DESCRIPTION OF THE FIGURES AND 15 SEQUENCE DESCRIPTIONS The invention can be more fully understood from the following detailed description, figure, and the accompanying sequence descriptions, which form a part of this application. Figure 1 shows a schematic drawing of the coding regions in the 20 Suf operon from Lactobacillus plantarum as well as the adjacent coding regions feoA and ORF (A), and the portion of the Suf operon that was deleted in Example 1 (B). Figure 2 shows a schematic drawing of the coding regions in the Suf operon from Lactococcus lactis, with each coding region named by the 25 designation from the publicly available genomic sequence and the corresponding coding region identified by sequence homology. No homologous protein is identified for the hypothetical protiein. Figure 3 shows a schematic drawing of the coding regions in the Suf operon from E. coli. 30 Figure 4 shows a schematic drawing of the coding regions of the Isc operon from E. coli, and the adjacent iscR gene.

5 WO 2010/037119 PCT/US2009/058843

Figure 5 shows a schematic drawing of the coding regions of the Nif operon from Wolinella succinogenes, with the bounding ORF1 and ORF2. Figure 6 shows biosynthetic pathways for biosynthesis of 5 isobutanol. Table 7 is a table of the Profile HMM for dihydroxy-acid dehydratases based on enzymes with assayed function prepared as described in Example 1. Table 8 is submitted herewith electronically and is incorporated herein by reference. 10 The following sequences conform with 37 C.F.R. 1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures - the Sequence Rules") and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT 15 (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

20 Table 1. SEQ ID NOs of representative bacterial [2Fe-2S] 2+ DHAD proteins and encoding sequences Organism of derivation S E Q I D N O: S E Q I D N O: Nucleic acid Peptide Mycobacterium sp. MCS 1 2 Mycobacterium gilvum PYR-GCK 3 4 Mycobacterium smegmatis str. MC2 155 5 6 Mycobacterium vanbaalenii PYR-1 7 8 Nocardia farcinica IFM 10152 9 10 Rhodococcus sp. RHA1 11 12 Mycobacterium ulcerans Agy99 13 14 Mycobacterium avium subsp. 15 15 16 paratuberculosis K-10 Mycobacterium tuberculosis H37Ra 17 18

6 WO 2010/037119 PCT/US2009/058843

Mycobacterium leprae TN * 19 20 Kineococcus radiotolerans SRS30216 21 22 Janibacter sp. HTCC2649 23 24 Nocardioides sp. JS614 25 26 Renibacterium salmoninarum ATCC 27 28 33209 Arthrobacter aurescens TC1 29 30 Leifsonia xyli subsp. xyli str. CTCB07 31 32 marine actinobacterium PHSC20C1 33 34 Clavibacter michiganensis subsp. 35 36 michiganensis NCPPB 382 Saccharopolyspora erythraea NRRL 37 38 2338 Acidothermus cellulolyticus 11 B 39 40 Corynebacterium efficiens YS-314 41 42 Brevibacterium linens BL2 43 44 Tropheryma whipplei TW08/27 45 46 Methylobacterium extorquens PA1 47 48 Methylobacterium nodulans ORS 2060 49 50 Rhodopseudomonas palustris BisB5 51 52 Rhodopseudomonas palustris BisB18 53 54 Bradyrhizobium sp. ORS278 55 56 Bradyrhizobium japonicum USDA 110 57 58 Fulvimarina pelagi HTCC2506 59 60 Aurantimonas sp. S185-9A1 61 62 Hoeflea phototrophica DFL-43 63 64 Mesorhizobium loti MAFF303099 65 66 Mesorhizobium sp. BNC1 67 68 Parvibaculum lavamentivorans DS-1 69 70 Loktanella vestfoldensis SKA53 71 72 Roseobacter sp. CCS2 73 74 Dinoroseobacter shibae DFL 12 75 76

7 WO 2010/037119 PCT/US2009/058843

Roseovarius nubinhibens ISM 77 78 Sagittula stellata E-37 79 80 Roseobacter sp. AzwK-3b 81 82 Roseovarius sp. TM1035 83 84 Oceanicola batsensis HTCC2597 85 86 Oceanicola granulosus HTCC2516 87 88 Rhodobacterales bacterium HTCC2150 89 90 Paracoccus denitrificans PD1222 91 92 Oceanibulbus indolifex HEL-45 93 94 Sulfitobacter sp. EE-36 95 96 Roseobacter denitrificans OCh 114 97 98 Jannaschia sp. CCS1 99 100 Caulobacter sp. K31 101 102 Candidatus Pelagibacter ubique 103 104 HTCC1 062 Erythrobacter litoralis HTCC2594 105 106 Erythrobacter sp. NAP1 107 108 Comamonas testosterone KF-1 109 110 Sphingomonas wittichii RW1 111 112 Burkholderia xenovorans LB400 113 114 Burkholderia phytofirmans PsJN 115 116 Bordetella petrii DSM 12804 117 118 Bordetella bronchiseptica RB50 119 120 Bradyrhizobium sp. ORS278 121 122 Bradyrhizobium sp. BTAi1 123 124 Bradhyrhizobium japonicum 125 126 Sphingomonas wittichii RW1 127 128 Rhodobacterales bacterium HTCC2654 129 130 Solibacter usitatus Ellin6076 131 132 Roseiflexus sp. RS-1 133 134 Rubrobacter xylanophilus DSM 9941 135 136 Salinispora tropica CNB-440 137 138

8 WO 2010/037119 PCT/US2009/058843

Acidobacteria bacterium Ellin345 139 140 Thermus thermophilus HB27 141 142 Maricaulis maris MCS10 143 144 Parvularcula bermudensis HTCC2503 145 146 Oceanicaulis alexandrii HTCC2633 147 148 Plesiocystis pacifica SIR-1 149 150 Bacillus sp. NRRL B-14911 151 152 Oceanobacillus iheyensis HTE831 153 154 Staphylococcus saprophyticus subsp. 155 156 saprophyticus ATCC 15305 Bacillus selenitireducens MLS10 157 158 Streptococcus pneumoniae SP6-BS73 159 160 Streptococcus sanguinis SK36 161 162 Streptococcus thermophilus LMG 18311 163 164 Streptococcus suis 89/1591 165 166 Streptococcus mutans UA159 167 168 Leptospira borgpetersenii serovar 169 170 Hardjo-bovis L550 Candidatus Vesicomyosocius okutanii 171 172 HA Candidatus Ruthia magnifica str. Cm 173 174 (Calyptogena magnifica) Methylococcus capsulatus str. Bath 175 176 uncultured marine bacterium 177 178 EB80_02D08 uncultured marine gamma 179 179 180 proteobacterium EBAC31AO8 uncultured marine gamma 181 181 182 proteobacterium EBAC20EO9 uncultured gamma proteobacterium 183 183 184 eBACHOT4E07 Alcanivorax borkumensis SK2 185 186

9 WO 2010/037119 PCT/US2009/058843

Chromohalobacter salexigens DSM 3043 187 188 Marinobacter algicola DG893 189 190 Marinobacter aquaeolei VT8 191 192 Marinobacter sp. ELB17 193 194 Pseudoalteromonas haloplanktis 195 196 TAC125 Acinetobacter sp. ADP1 197 198 Opitutaceae bacterium TAV2 199 200 Flavobacterium sp. MED217 201 202 Cellulophaga sp. MED134 203 204 Kordia algicida OT-1 205 206 Flavobacteriales bacterium ALC-1 207 208 Psychroflexus torquis ATCC 700755 209 210 Flavobacteriales bacterium HTCC2170 211 212 unidentified eubacterium SCB49 213 214 Gramella forsetii KT0803 215 216 Robiginitalea biformata HTCC2501 217 218 Tenacibaculum sp. MED152 219 220 Polaribacter irgensii 23-P 221 222 Pedobacter sp. BAL39 223 224 Flavobacteria bacterium BAL38 225 226 Flavobacterium psychrophilum JIPO2/86 227 228 Flavobacterium johnsoniae UW1 01 229 230 Lactococcus lactis subsp. cremoris SK 1 231 232 Psychromonas ingrahamii 37 233 234 Microscilla marina ATCC 23134 235 236 Cytophaga hutchinsonii ATCC 33406 237 238 Rhodopirellula baltica SH 1 239 240 Blastopirellula marina DSM 3645 241 242 Planctomyces maris DSM 8797 243 244 Algoriphagus sp. PR1 245 246 Candidatus Sulcia muelleri str. Hc 247 248

10 WO 2010/037119 PCT/US2009/058843

(Homalodisca coagulata) Candidatus Carsonella ruddii PV 249 250 Synechococcus sp. RS9916 251 252 Synechococcus sp. WH 7803 253 254 Synechococcus sp. CC9311 255 256 Synechococcus sp. CC9605 257 258 Synechococcus sp. WH 8102 259 260 Synechococcus sp. BL107 261 262 Synechococcus sp. RCC307 263 264 Synechococcus sp. RS9917 265 266 Synechococcus sp. WH 5701 267 268 Prochlorococcus marinus str. MIT 9313 269 270 Prochlorococcus marinus str. NATL2A 271 272 Prochlorococcus marinus str. MIT 9215 273 274 Prochlorococcus marinus str. AS9601 275 276 Prochlorococcus marinus str. MIT 9515 277 278 Prochlorococcus marinus subsp. pastoris 279 279 280 str. CCMP1986 Prochlorococcus marinus str. MIT 9211 281 282 Prochlorococcus marinus subsp. marinus 283 284 str. CCMP1375 Nodularia spumigena CCY9414 285 286 Nostoc punctiforme PCC 73102 287 288 Nostoc sp. PCC 7120 289 290 Trichodesmium erythraeum IMS101 291 292 Acaryochloris marina MBIC11017 293 294 Lyngbya sp. PCC 8106 295 296 Synechocystis sp. PCC 6803 297 298 Cyanothece sp. CCY0110 299 300 Thermosynechococcus elongatus BP-1 301 302 Synechococcus sp. JA-2-3B'a(2-13) 303 304 Gloeobacter violaceus PCC 7421 305 306

11 WO 2010/037119 PCT/US2009/058843

Nitrosomonas eutropha C91 307 308 Nitrosomonas europaea ATCC 19718 309 310 Nitrosospira multiformis ATCC 25196 311 312 Chloroflexus aggregans DSM 9485 313 314 Leptospirillum sp. Group II UBA 315 316 Leptospirillum sp. Group II UBA 317 318 Halorhodospira halophila SL1 319 320 Nitrococcus mobilis Nb-231 321 322 Alkalilimnicola ehrlichei MLHE-1 323 324 Deinococcus geothermalis DSM 11300 325 326 Polynucleobacter sp. QLW-P1 DMWA-1 327 328 Polynucleobacter necessarius STIR1 329 330 Azoarcus sp. EbN1 331 332 Burkholderia phymatum STM815 333 334 Burkholderia xenovorans LB400 335 336 Burkholderia multivorans ATCC 17616 337 338 Burkholderia cenocepacia PC184 339 340 Burkholderia mallei GB8 horse 4 341 342 Ralstonia eutropha JMP134 343 344 Ralstonia metallidurans CH34 345 346 Ralstonia solanacearum UW551 347 348 Ralstonia pickettii 12J 349 350 Limnobacter sp. MED105 351 352 Herminiimonas arsenicoxydans 353 354 Bordetella parapertussis 355 356 Bordetella petrii DSM 12804 357 358 Polaromonas sp. JS666 359 360 Polaromonas naphthalenivorans CJ2 361 362 Rhodoferax ferrireducens Ti 18 363 364 Verminephrobacter eiseniae EF01-2 365 366 Acidovorax sp. JS42 367 368 Delftia acidovorans SPH-1 369 370

12 WO 2010/037119 PCT/US2009/058843

Methylibium petroleiphilum PM1 371 372 gamma proteobacterium KT 71 373 374 Tremblaya princeps 375 376 Blastopirellula marina DSM 3645 377 378 Planctomyces maris DSM 8797 379 380 Microcystis aeruginosa PCC 7806 381 382 Salinibacter ruber DSM 13855 383 384 Methylobacterium chloromethanicum 385 386

Table 2. SEQ ID NOs of representative fungal and plant [2Fe-2S] 2+ DHAD proteins and encoding sequences SEQ ID NO: SEQ ID NO: Description Nucleic acid Peptide Schizosaccharomyces pombe ILV3 387 388 Saccharomyces cerevisiae ILV3 389 390 Kluyveromyces lactis ILV3 391 392 Candida albicans SC5314 ILV3 393 394 Pichia stipitis CBS 6054 ILV3 395 396 Yarrowia lipolytica ILV3 397 398 Candida galbrata CBS 138 ILV3 399 400 Chlamydomonas reinhardtii 401 402 Ostreococcus lucimarinus CCE9901 403 404 Vitis vinifera 405 406 (Unnamed protein product: CAO71581.1) Vitis vinifera 407 408 (Hypothetical protein: CAN67446.1) Arabidopsis thaliana 409 410 Oryza sativa (indica cultivar-group) 411 412 Physcomitrella patens subsp. patens 413 414 Chaetomium globosum CBS 148.51 415 416 Neurospora crassa OR74A 417 418

13 WO 2010/037119 PCT/US2009/058843

Magnaporthe grisea 70-15 419 420 Gibberella zeae PH-1 421 422 Aspergillus niger 423 424 Neosartorya fischeri NRRL 181 425 426 (XP_001266525.1) Neosartorya fischeri NRRL 181 427 428 (XP_001262996.1) Aspergillus niger 429 430 (hypothetical protein An03g04520) Aspergillus niger 431 432 (Hypothetical protein An14g03280) Aspergillus terreus NIH2624 433 434 Aspergillus clavatus NRRL 1 435 436 Aspergillus nidulans FGSC A4 437 438 Aspergillus oryzae 439 440 Ajellomyces capsulatus NAmi 441 442 Coccidioides immitis RS 443 444 Botryotinia fuckeliana B05.1 0 445 446 Phaeosphaeria nodorum SN15 447 448 Pichia guilliermondii ATCC 6260 449 450 Debaryomyces hansenii CBS767 451 452 Lodderomyces elongisporus NRRL 453 454 YB-4239 Vanderwaltozyma polyspora DSM 455 456 70294 Ashbya gossypii ATCC 10895 457 458 Laccaria bicolor S238N-H82 459 460 Coprinopsis cinerea okayama7#1 30 461 462 Cryptococcus neoformans var. 463 464 neoformans JEC21 Ustilago maydis 521 465 466 Malassezia globosa CBS 7966 467 468

14 WO 2010/037119 PCT/US2009/058843

Aspergillus clavatus NRRL 1 469 470 Neosartorya fischeri NRRL 181 471 472 (Putative) Aspergillus oryzae 473 474 Aspergillus niger (hypothetical 475 476 protein An18g04160) Aspergillus terreus NIH2624 477 478 Coccidioides immitis RS (hypothetical 479 480 protein CIMG_04591) Paracoccidioides brasiliensis 481 482 Phaeosphaeria nodorum SN15 483 484 Gibberella zeae PH-1 485 486 Neurospora crassa OR74A 487 488 Coprinopsis cinerea okayama 7#130 489 490 Laccaria bicolor S238N-H82 491 492 Ustilago maydis 521 493 494

Table 3. SEQ ID NOs of representative [4Fe-4S] 2+ DHAD proteins and encoding sequences Organism SEQ ID NO: SEQ ID NO: Nucleic acid Peptide Escherichia coli str. K-1 2 substr. 495 496 MG1655 Bacillus subtilis subsp. subtilis str. 168 497 498 Agrobacterium tumefaciens str. C58 499 500 Burkholderia cenocepacia MCO-3 501 502 Psychrobacter cryohalolentis K5 503 504 Psychromonas sp. CNPT3 505 506 Deinococcus radiodurans R1 507 508 Wolinella succinogenes DSM 1740 509 510 Zymomonas mobilis subsp. mobilis 511 512

15 WO 2010/037119 PCT/US2009/058843

ZM4 Clostridium acetobutylicum ATCC 824 513 514 Clostridium beijerinckii NCIMB 8052 515 516 Pseudomonas fluorescens Pf-5 517 518 Methanococcus maripaludis C7 519 520 Methanococcus aeolicus Nankai-3 521 522 Vibrio fischeri A TCC 700601 (ES 114) 523 524 Shewanella oneidensis MR-I ATCC 525 526 700550

Table 4. SEQ ID NOs of representative Suf operon Fe-S cluster forming proteins and encoding sequences. Organism and gene name SEQ ID SEQ ID NO: nucleic NO: amino acid acid Lactoabcillus plantarum sufC 595 596 Lactococcus lactis sufC 597 598 Escherichia coli sufC 599 600 Lactoabcillus plantarum sufD 601 602 Lactococcus lactis sufD 603 604 Escherichia coli sufD 605 606 Lactoabcillus plantarum sufB 607 608 Lactococcus lactis sufB 609 610 Escherichia coli sufB 611 612 Escherichia coli sufA 613 614 Escherichia coli sufE 615 616 Lactococcus lactis yseH 617 618 Lactoabcillus plantarum sufU 619 620 Lactococcus lactis sufU 621 622 Lactoabcillus plantarum, sufS 623 624 Lactobacillus reuteri, sufS 625 626 Lactobacillus fermentum, sufS 627 628

16 WO 2010/037119 PCT/US2009/058843

Enterococcus faecalis, sufS 629 630 Lactobacillus faecium DO ,sufS 631 632 Lactobacillus sakei subsp. sakei 23K, putative 633 634 sufS Carnobacterium sp. AT7, sufS 635 636 Streptococcus mutans UA159, sufS 637 638 Streptococcus suis 05ZYH33, sufS 639 640 Streptococcus sanguinis SK36, sufS 641 642 Leuconostoc mesenteroides subsp. 643 644 mesenteroides ATCC 8293, sufS

Streptococcus thermophilus LMG 18311, sufS 645 646

Lactococcus lactis subsp. cremoris SKI 1, 647 648 sufS hypothetical protein LACR_1972 Bacillus sp. B14905, sufS 649 650 00018 Streptococcus infantarius subsp. infantarius ATCC BAA-102, sufS hypothetical 651 652 protein STRINF Lactobacillus helveticus CNRZ32, sufS 653 654 Streptococcus pneumoniae CGSP14, sufS 655 656 Geobacillus sp. WCH70, sufS 657 658 Leuconostoc citreum KM20, sufS 659 660 Listeria monocytogenes EGD-e, sufS 661 662 hypothetical protein Imo2413 Lactobacillusjohnsonii NCC 533, sufS 663 664 Bacillus sp. SG-1, sufS 665 666 Bacillus clausii KSM-K16, sufS 667 668 Bacillus pumilus SAFR-032, sufS 669 670 Geobacillus kaustophilus HTA426, sufS 671 672 Bacillus selenitireducens MLS10, sufS 673 674 Streptococcus pyogenes MGAS10750, sufS 675 676 Bacillus sp. NRRL B-14911, sufS 677 678 Paenibacillus larvae subsp. larvae BRL- 679 680 230010, sufS Bacillus subtilis subsp. subtilis str. 168, sufS 681 682

17 WO 2010/037119 PCT/US2009/058843

Bacillus licheniformis A TCC 14580, sufS 683 684 Oceanobacillus iheyensis HTE831, sufS 685 686 Bacillus coagulans 36D1, sufS 687 688 Staphylococcus aureus subsp. aureus Mu50, 689 690 sufS Staphylococcus saprophyticus subsp. 691 692 saprophyticus ATCC 15305, putative sufS Paenibacillus sp. JDR-2, sufS 693 694 Lactobacillus salivarius UCC 118, sufS 695 696 Exiguobacterium sibiricum 255-15, sufS 697 698 Exiguobacterium sp. ATIb, sufS 699 700 Rubrobacterxylanophilus DSM 9941, sufS 701 702 Clostridium acetobutylicum A TCC 824, sufS 703 704 Clostridium beijerinckii NCIMB 8052, sufS 705 706 Clostridium kluyveri DSM 555, sufS 707 708 Lactobacillus casei ATCC 334, sufS 709 710 Thermoanaerobacter pseudethanolicus A TCC 711 712 33223, sufS Symbiobacterium thermophilum lAM 14863, 713 714 sufS Thermoanaerobacter tengcongensis MB4, 715 716 sufS Verrucomicrobium spinosum DSM 4136, sufS 717 718 Oenococcus oeni PSU-1, sufS 719 720 Mariprofundus ferrooxydans PV-1, sufS 721 722 Opitutus terrae PB90-1, sufS 723 724 Nitrosococcus oceani A TCC 19707, sufS 725 726 Lactobacillus delbrueckii subsp. bulgaricus 727 728 ATCC 11842, sufS Escherichia coli str. K-12 substr. MG1655, 729 730 sufS (PLP-dependent) Rhodoferax ferrireducens TI 18, sufS 731 732 Thermus thermophilus HB27, sufS 733 734 Streptomyces avermitilis MA-4680, sufS 735 736 Clostridium sp. L2-50, sufS protein CLOL25002464 737 738

Coprococcus eutactus ATCC 27759, sufS 739 740

18 WO 2010/037119 PCT/US2009/058843

hypothetical protein COPEUT_00639 Thermobifida fusca YX, sufS 741 742 Acidothermus cellulolyticus I IB, sufS 743 744 Methylococcus capsulatus str. Bath, sufS 745 746 Thauera sp. MZ1T, sufS 747 748 Streptomyces coelicolor A3(2), sufS 749 750 Solibacter usitatus Ellin6076, sufS 751 752 Coxiella burnetii RSA 493, sufS 753 754 Petrotoga mobilis SJ95, sufS 755 756 Synechocystis sp. PCC 6803, sufS 757 758 Ralstonia eutropha H16, sufS 759 760 Thermotoga maritima MSB8 sufS 761 762 Gloeobacter violaceus PCC 7421, sufS 763 764 Nitrococcus mobilis Nb-231, sufS 765 766 Pediococcus pentosaceus ATCC 25745, sufS 767 768 Streptomyces griseus subsp. griseus NBRC 769 770 13350, sufS Nitrosospira multiformis ATCC 25196, sufS 771 772 Frankia sp. EANipec, sufS 773 774 Propionibacterium acnes KPA 171202, 775 776 putative sufS Rhodococcus sp. RHAI, sufS 777 778 Alkalilimnicola ehrlichei MLHE-1, sufS 779 780 Anaeromyxobactersp. Fw109-5, sufS 781 782 Anaeromyxobacter sp. K, sufS 783 784 Mycobacterium abscessus, sufS 785 786 Lentisphaera araneosa HTCC2155, sufS 787 788 Saccharopolyspora erythraea NRRL 2338, 789 790 sufS Acidiphilium cryptum JF-5, sufS 791 792 Nocardia farcinica IFM 10152, sufS 793 794 Nocardioides sp. JS614, sufS 795 796 Corynebacterium urealyticum DSM 7109, sufS 797 798 Legionella pneumophila subsp. pneumophila 799 800 str. Philadelphia 1, sufS

19 WO 2010/037119 PCT/US2009/058843

Mycobacterium marinum M, sufS 801 802 Psychromonas ingrahamii 37, sufS 803 804 Corynebacterium efficiens YS-314, sufS 805 806 Corynebacterium jeikeium K411, putative sufS 807 808 Leptospira borgpetersenii serovar Hardjo- 809 810 bovis L550, sufS Mycobacterium vanbaalenii PYR-1, sufS 811 812 Mycobacterium gilvum PYR-GCK, sufS 813 814 Mycobacterium tuberculosis H37Rv, sufS 815 816 Janibacter sp. HTCC2649, sufS 817 818 Salinispora arenicola CNS-205, sufS 819 820 Polaromonas sp. JS666, sufS 821 822 Nitrosomonas eutropha C91, sufS 823 824 Mycobacterium sp. MCS, sufS 825 826 Frankia alni ACN14a, sufS 827 828 Salinispora tropica CNB-440, sufS 829 830 Nitrosomonas europaea ATCC 19718, sufS 831 832 Leptospira interrogans serovar Copenhageni 833 834 str. Fiocruz L1-130, sufS Mycobacterium avium subsp. paratuberculosis 835 836 K-10, sufS hypothetical protein MAP1 190 Thermotoga maritima MSB8, sufS 837 838 Pectobacterium atrosepticum SCRI1043, sufS 839 840 Corynebacterium glutamicum ATCC 13032, 841 842 sufS Clavibacter michiganensis subsp. 843 844 michiganensis NCPPB 382, putative sufS Frankia sp. CcI3, sufS 845 846 Gluconacetobacter diazotrophicus PAI 5, 847 848 putative sufS Candidates Pelagibacter ubique HTCC1062, 849 850 sufS Kineococcus radiotolerans SRS30216, sufS 851 852 Finegoldia magna ATCC 29328, sufS 853 854 Collinsella aerofaciens ATCC 25986, sufS 855 856 hypothetical protein COLAER_01633 Peptostreptococcus micros ATCC 33270, 857 858 hypothetical protein PEPMIC_00951

20 WO 2010/037119 PCT/US2009/058843

Arthrobacter chlorophenolicus A6, sufS 859 860 Granulibacter bethesdensis CGDNIHI, sufS 861 862 Arthrobacter sp. FB24, sufS 863 864 Thermosipho melanesiensis B1429, sufS 865 866 Renibacterium salmoninarum ATCC 33209, 867 868 sufS Leifsonia xyli subsp. xyli str. CTCB07, sufS 869 870 Acholeplasma laidlawii PG-8A, sufS 871 872 Brevibacterium linens BL2, sufS 873 874 Corynebacterium diphtheriae NCTC 13129, 875 876 sufS Bifidobacterium animals subsp. lactis HN019, 877 878 sufS Burkholderia thailandensis MSMB43, sufS 879 880 Annotations in public databases may have a different protein indicated for some of the SufS proteins above. Annotation as Class V aminotransferase refers to the same protein as cysteine desulfurase.

5 Table 5. SEQ ID NOs of representative Isc and Nif operon Fe-S cluster forming proteins and encoding sequences Organism and gene name SEQ ID NO: SEQ ID nucleic acid NO: amino acid Escherichia coli iscS 527 528 Escherichia coli iscU 529 530 Escherichia coli iscA 531 532 Escherichia coli hscB 533 534 Escherichia coli hscA 535 536 Escherichia coli fdx 537 538 Escherichia coli iscX 539 540 Wolinella succinogenes nifS 541 542 Wolinella succinogenes nifU 543 544

21 WO 2010/037119 PCT/US2009/058843

Table 6. SEQ ID NOs of additional proteins and encoding sequences Description SEQ ID NO: SEQ ID NO: Encoding seq protein Vibrio cholerae KARI 545 546 Pseudomonas aeruginosa PAO1 551 552 KARI Pseudomonas fluorescens PF5 547 548 KARI Achromobacter xylosoxidans 549 550 butanol dehydrogenase sadB

SEQ ID NOs:554 - 570, 572, 573, 575, 576, 578 - 588, 592 and 593 are nucleotide sequences of primers used in the Examples. 5 SEQ ID NOs:553, 571, 574, 577 and 594 are nucleotide sequences of vectors used in the Examples. SEQ ID NO:589 is the nucleotide sequence of the Suf operon from Lactobacillus plantarum PN0512. SEQ ID NO:590 is the nucleotide sequence of a ribosome binding 10 sequence used in the Examples. SEQ ID NO:591 is the nucleotide sequence of the promoter region of the ldhL1 gene from Lactobacillus plantarum PN0512. SEQ ID NO:881 is the nucleotide sequence of the Suf operon from Lactococcus lactis subsp lactis NCDO2118. 15 DETAILED DESCRIPTION OF THE INVENTION The present invention solves the stated problem by providing recombinant lactic acid bacterial cells that express DHAD and that express at least one recombinant genetic element encoding Fe-S cluster forming proteins. These cells have increased DHAD activity as compared 20 to DHAD activity in cells without the recombinant genetic element. In these cells, products synthesized by a pathway that includes DHAD activity may be increased, including amino acids valine, leucine and isoleucine, vitamin B5, and isobutanol. The amino acids and vitamin B5 may be used as

22 WO 2010/037119 PCT/US2009/058843

nutritional supplements, and isobutanol may be used as a fuel additive to reduce demand for petrochemicals. The following abbreviations and definitions will be used for the interpretation of the specification and the claims. 5 As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains" or "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those 10 elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not 15 present), A is false (or not present) and B is true (or present), and both A and B are true (or present). Also, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. 20 Therefore "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular. The term "invention" or "present invention" as used herein is a non limiting term and is not intended to refer to any single embodiment of the 25 particular invention but encompasses all possible embodiments as described in the specification and the claims. As used herein, the term "about" modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring 30 and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the

23 WO 2010/037119 PCT/US2009/058843

like. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term "about", the claims include equivalents to the quantities. In one embodiment, the term "about" 5 means within 10% of the reported numerical value, preferably within 5% of the reported numerical value The term "isobutanol biosynthetic pathway" refers to an enzyme pathway to produce isobutanol from pyruvate. The term "afacultative anaerobe" refers to a microorganism that 10 can grow in both aerobic and anaerobic environments. The term "carbon substrate" or "fermentable carbon substrate" refers to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, 15 and one-carbon substrates or mixtures thereof. The term "gene" refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5' non-coding sequences) and following (3' non coding sequences) the coding sequence. "Native gene" refers to a gene 20 as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and 25 coding sequences derived from the same source, but arranged in a manner different than that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign gene" or "heterologous gene" refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene 30 transfer. "Heterologous gene" includes a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding region that is a portion of a chimeric

24 WO 2010/037119 PCT/US2009/058843

gene including non-native regulatory regions that is reintroduced into the native host. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. 5 The term "recombinant genetic expression element" refers to a nucleic acid fragment that expresses one or more specific proteins, including regulatory sequences preceding (5' non-coding sequences) and following (3'termination sequences) coding sequences for the proteins. A chimeric gene is a recombinant genetic expression element. The coding 10 regions of an operon may form a recombinant genetic expression element, along with an operably linked promoter and termination region. As used herein the term "coding region" refers to a DNA sequence that codes for a specific amino acid sequence. "Suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non 15 coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector 20 binding site and stem-loop structure. The term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be 25 composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological 30 conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". It is further recognized that since in most cases the exact boundaries of

25 WO 2010/037119 PCT/US2009/058843

regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is 5 affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. 10 The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. The term "overexpression" ", as used herein, refers to expression 15 that is higher than endogenous expression of the same or related gene. A heterologous gene is overexpressed if its expression is higher than that of a comparable endogenous gene. As used herein the term "transformation" refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable 20 inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms. The terms "plasmid" and "vector" as used herein, refer to an extra chromosomal element often carrying genes which are not part of the 25 central metabolism of the cell, and usually in the form of circular double stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide 30 sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

26 WO 2010/037119 PCT/US2009/058843

As used herein the term "codon degeneracy" refers to the nature in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in 5 usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell. The term "codon-optimized" as it refers to genes or coding regions 10 of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. As used herein, an "isolated nucleic acid fragment" or "isolated 15 nucleic acid molecule" will be used interchangeably and will mean a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA. 20 A nucleic acid fragment is "hybridizable" to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well 25 known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloninq: A Laboratory Manual, 2 ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the "stringency" of 30 the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms).

27 WO 2010/037119 PCT/US2009/058843

Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45 0C for 30 min, and then repeated twice with 0.2X SSC, 0.5% 5 SDS at 50 0C for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X

SSC, 0.5% SDS was increased to 60 OC. Another preferred set of highly stringent conditions uses two final washes in 0.1X SSC, 0.1% SDS at 65

10 0C. An additional set of stringent conditions include hybridization at 0.1X SSC, 0.1% SDS, 65 0C and washes with 2X SSC, 0.1% SDS followed by 0.1X SSC, 0.1% SDS, for example. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the 15 hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of 20 nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations 25 with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a 30 hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that

28 WO 2010/037119 PCT/US2009/058843

the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe. A "substantial portion" of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a 5 polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more 10 contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent 15 methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a 20 nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding particular proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a 25 substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above. The term "complementary" is used to describe the relationship 30 between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.

29 WO 2010/037119 PCT/US2009/058843

The term "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness 5 between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Bioloqy (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputinq: 10 Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Bioloqy (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) 15 Stockton: NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed 20 using the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment of the sequences is performed using the "Clustal method of alignment" which encompasses several varieties of the algorithm including the "Clustal V method of alignment" corresponding to the alignment method labeled 25 Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Apple. Biosci., 8:189-191 (1992)) and found in the MegAlign TM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. 30 Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 30 WO 2010/037119 PCT/US2009/058843

and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program. Additionally the "Clustal W method of alignment" is available and corresponds to the 5 alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Apple. Biosci. 8:189-191(1992)) and found in the MegAlign TM v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH 10 PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB ). After alignment of the sequences using the Clustal W program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program. 15 It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer 20 percentage from 55% to 100% may be useful in describing the present invention, such as 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable 25 nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids. 30 The term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. "Sequence analysis software" may be commercially available or independently developed. Typical sequence

31 WO 2010/037119 PCT/US2009/058843

analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. 5 Madison, WI); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, MI); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, NY). Within the context of this application it will be understood 10 that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters that originally load with the software when first initialized. 15 Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Man iatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) (hereinafter "Maniatis"); and by Silhavy, T. J., Bennan, M. L. and 20 Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). The invention provides recombinant lactic acid bacterial cells 25 expressing a functional dihydroxy-acid dehydratase polypeptide where the lactic acid bacterial cell is also expressing at least one recombinant genetic element encoding iron-sulfur cluster forming proteins. It has been discovered that the co-expression of a dihydroxy-acid dehydratase polypeptide with a recombinant genetic expression element encoding iron 30 sulfur cluster forming proteins results in increased specific activity of dihydroxy-acid dehydratase. Specific activity is based on concentration of total soluble protein in a crude cell extract. Lactic Acid Bacterial cells

32 WO 2010/037119 PCT/US2009/058843

Lactic acid bacteria (LAB) which may be used as hosts in the present disclosure include, but are not limited to, Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus.These and any LAB cells that are amenable to genetic 5 manipulation may be modified as disclosed herein for increased DHAD activity. Expression of DHAD activity In the disclosed LAB cells, DHAD activity may be provided by natural expression of an endogenous DHAD protein, by expression of an 10 introduced heterologous DHAD gene, or both. For example, cells of Lactococcus, Streptococcus, and Leuconostoc.have endogenous genes encoding DHAD, and may have this endogenous activity enhanced by introduction of a heterologous DHAD encoding gene. DHAD genes are not known in cells of Lactobacillus, Pediococcus, and Oenococcus, which 15 then are engineered for DHAD expression through introduction of a heterologous DHAD encoding gene. Any gene encoding a DHAD enzyme may be used to provide expression of DHAD activity in a LAB cell. DHAD, also called acetohydroxy acid dehydratase, catalyzes the conversion of 2,3 20 dihydroxyisovalerate to a-ketoisovalerate and of 2,3 dihydroxymethylvalerate to a-ketomethylvalerate and is classified as E.C. 4.2.1.9. Coding sequences for DHADs that may be used herein may be derived from bacterial, fungal, or plant sources. DHADs that may be used may have a [4Fe-4S] 2+ cluster or a [2Fe-2S] 2+ cluster bound by the 25 apoprotein. Tables 1, 2, and 3 list SEQ ID NOs for coding regions and proteins of representative DHADs that may be used in the present invention. Proteins with at least about 95% identity to those listed sequences have been omitted for simplification, but it is understood that the omitted proteins with at least about 95% sequence identity to any of 30 the proteins listed in Tables 1, 2, and 3 and having DHAD activity may be used as disclosed herein. Additional DHAD proteins and their encoding sequences may be identified by BLAST searching of public databases, as well known to one skilled in the art. Typically BLAST (described above)

33 WO 2010/037119 PCT/US2009/058843

searching of publicly available databases with known DHAD sequences, such as those provided herein, is used to identify DHADs and their encoding sequences that may be expressed in the present cells. For example, DHAD proteins having amino acid sequence identities of at least 5 about 80-85%, 85%- 90%, 90%- 95% or 98% sequence identity to any of the DHAD proteins of Table 1 may be expressed in the present cells. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. 10 Additional [2Fe-2S] 2+ DHADs may be identified using the analysis described in commonly owned and co-pending US Patent Application 61/100792, which is herein incorporated by reference. The analysis is as follows: A Profile Hidden Markov Model (HMM) was prepared based on amino acid sequences of eight functionally verified DHADs. These DHA 15 Ds are from Nitrosomonas europaea (DNA SEQ ID NO:309; protein SEQ ID NO:31 0), Synechocystis sp. PCC6803 (DNA SEQ ID:297; protein SEQ ID NO:298 ), Streptococcus mutans (DNA SEQ ID NO:167; protein SEQ ID NO:168), Streptococcus thermophilus (DNA SEQ ID NO:163; SEQ ID No:164), Ralstonia metallidurans (DNA SEQ ID NO:345; protein SEQ ID 20 NO:346 ), Ralstonia eutropha (DNA SEQ ID NO:343; protein SEQ ID NO:344), and Lactococcus lactis (DNA SEQ ID NO:231; protein SEQ ID NO:232). In addition the DHAD from Flavobacteriumjohnsoniae (DNA SEQ ID NO:229; protein SEQ ID NO:230) was found to have dihydroxy acid dehydratase activity when expressed in E. coli and was used in 25 making the Profile. The Profile HMM is prepared using the HMMER software package (The theory behind profile HMMs is described in R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Bioloqical sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998; Krogh et al., 1994; J. Mol. Biol. 235:1501-1531), 30 following the user guide which is available from HMMER (Janelia Farm Research Campus, Ashburn, VA). The output of the HMMER software program is a Profile Hidden Markov Model (HMM) that characterizes the input sequences. The Profile HMM prepared for the eight DHAD proteins

34 WO 2010/037119 PCT/US2009/058843

is given in Table 7. Any protein that matches the Profile HMM with an E value of < 10-5 is a DHAD related protein, which includes [4Fe-4S] 2+ DHADs, [2Fe-2S] 2+ DHADs, arabonate dehydratases, and phosphogluconate dehydratases. Sequences matching the Profile HMM 5 are then analyzed for the presence of the three conserved cysteines, corresponding to positions 56, 129, and 201 in the Streptococcus mutans DHAD. The presence of all three conserved cysteines is characteristic of

proteins having a [2Fe-2S] 2+cluster. Proteins having the three conserved cysteines include arabonate dehydratases and [2Fe-2S] 2+ DHADs. The 10 [2Fe-2S] 2+ DHADs may be distinguished from the arabonate dehydratases by analyzing for signature conserved amino acids found to be present in the [2Fe-2S] 2+ DHADs or in the arabonate dehydratases at positions corresponding to the following positions in the Streptococcus mutans DHAD amino acid sequence. These signature amino acids are in 15 [2Fe-2S] 2+ DHADs or in arabonate dehydratases, respectively, at the following positions (with greater than 90% occurance): 88 asparagine vs glutamic acid; 113 not conserved vs glutamic acid; 142 arginine or asparagine vs not conserved; 165: not conserved vs glycine; 208 asparagine vs not conserved; 454 leucine vs not conserved; 477 20 phenylalanine or tyrosine vs not conserved; and 487 glycine vs not conserved. Additionally, the sequences of DHAD coding regions provided herein may be used to identify other homologs in nature. For example each of the DHAD encoding nucleic acid fragments described herein may 25 be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1.) methods of nucleic acid hybridization; 2.) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid 30 amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Patent 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392

35 WO 2010/037119 PCT/US2009/058843

(1992)]; and 3.) methods of library construction and screening by complementation. For example, genes encoding similar proteins or polypeptides to the DHAD encoding genes provided herein could be isolated directly by 5 using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired organism using methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the disclosed nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, 10 supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full 15 length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments by hybridization under conditions of appropriate stringency. Typically, in PCR-type amplification techniques, the primers have 20 different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, "The use of oligonucleotides as specific 25 hybridization probes in the Diagnosis of Genetic Disorders", in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50, IRL: Herndon, VA; and Rychlik, W., In Methods in Molecular Bioloy, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, NJ). 30 Generally two short segments of the described sequences may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned

36 WO 2010/037119 PCT/US2009/058843

nucleic acid fragments wherein the sequence of one primer is derived from the described nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding microbial genes. 5 Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers 10 oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (e.g., BRL, Gaithersburg, MD), specific 3' or 5' cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)). 15 Alternatively, the provided DHAD encoding sequences may be employed as hybridization reagents for the identification of homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest, and a specific hybridization method. Probes are typically single-stranded 20 nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are "hybridizable" to the nucleic acid sequence to be detected. The probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. 25 Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired 30 with the proper complementary base. Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the

37 WO 2010/037119 PCT/US2009/058843

presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe 5 or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and 10 stringent hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide and cesium trifluoroacetate, 15 among others. Typically, the chaotropic agent will be present at a final concentration of about 3 M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v). Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic 20 solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCI, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), 25 polyvinyl pyrrolidone (about 250-500 kdal) and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine. Other additives may also be included, 30 such as volume exclusion agents that include a variety of polar water soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).

38 WO 2010/037119 PCT/US2009/058843

Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non denaturing conditions. A primary component of a sandwich-type assay is 5 a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence. LAB cells are genetically modified for expression of DHAD activity using methods well known to one skilled in the art. Expression of DHAD is 10 generally achieved by transforming suitable LAB host cells with a sequence encoding a DHAD protein. Typically the coding sequence is part of a chimeric gene used for transformation, which includes a promoter operably linked to the coding sequence as well as a ribosome binding site and a termination control region. The coding region may be from the host 15 cell for transformation and combined with regulatory sequences that are not native to the natural gene encoding DHAD. Alternatively, the coding region may be from another host cell. Codons may be optimized for expression based on codon usage in the selected host, as is known to one skilled in the art. Vectors useful for 20 the transformation of a variety of host cells are common and described in the literature. Typically the vector contains a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. In addition, suitable vectors may comprise a promoter region which harbors transcriptional initiation controls and a transcriptional 25 termination control region, between which a coding region DNA fragment may be inserted, to provide expression of the inserted coding region. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the specific 30 species chosen as a production host. Initiation control regions or promoters, which are useful to drive expression of a DHAD coding region in LAB are familiar to those skilled in the art. Some examples include the amy, apr, and npr promoters; nisA

39 WO 2010/037119 PCT/US2009/058843

promoter (useful for expression Gram-positive bacteria (Eichenbaum et al. Apple. Environ. Microbiol. 64(8):2763-2769 (1998)); and the synthetic P11 promoter (useful for expression in Lactobacillus plantarum, Rud et al., Microbiology 152:1011-1019 (2006)). In addition, the ldhLland fabZ1 5 promoters of L plantarum are useful for expression of chimeric genes in LAB. The fabZ1 promoter directs transcription of an operon with the first gene, fabZ1, encoding (3R)-hydroxymyristoyl-[acyl carrier protein] dehydratase. Termination control regions may also be derived from various 10 genes, typically from genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included. Vectors useful in LAB include vectors having two origins of replication and one or two selectable markers which allow for replication 15 and selection in both Escherichia coli and LAB. Examples are pFP996(SEQ ID NO:565) and pDM1 (SEQ ID NO:563), which are useful in L. plantarum.and other LAB. Many plasmids and vectors used in the transformation of Bacillus subtilis and Streptococcus may be used generally for LAB. Non-limiting examples of suitable vectors include 20 pAMD1 and derivatives thereof (Renault et al., Gene 183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231 (1993)); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al. Apple. Environ. Microbiol. 62:1481 1486 (1996)); pMG1, a conjugative plasmid (Tanimoto et al., J. Bacteriol. 184:5800-5804 (2002)); pNZ9520 (Kleerebezem et al., Appl. Environ. 25 Microbiol. 63:4581-4584 (1997)); pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67:1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38:1899-1903 (1994)). Several plasmids from Lactobacillus plantarum have also been reported (e.g., van Kranenburg et al. Apple. Environ. Microbiol. 2005 Mar; 71(3): 1223-1230). 30 Vectors may be introduced into a host cell using methods known in the art, such as electroporation (Cruz-Rodz et al. Molecular Genetics and Genomics 224:1252-154 (1990), Bringel, et al. Apple. Microbiol. Biotechnol. 33: 664-670 (1990), Alegre et al., FEMS Microbiology letters 241:73-77

40 WO 2010/037119 PCT/US2009/058843

(2004)), and conjugation (Shrago et al., Appl. Environ. Microbiol. 52:574 576 (1986)). A chimeric DHAD gene can also be integrated into the chromosome of LAB using integration vectors (Hols et al., Appl. Environ. Microbiol. 60:1401-1403 (1990), Jang et al., Micro. Lett. 24:191-195 5 (2003)). Fe-S cluster forming proteins Disclosed herein are recombinant LAB cells that express DHAD and are engineered for expression of proteins involved in formation of Fe S clusters. Two or more proteins are involved in several systems that are 10 known to form Fe-S clusters, which may include proteins that acquire iron and sulfur, assemble Fe-S clusters, and transfer Fe-S clusters to apoproteins. The DHAD protein requires either a [2Fe-2S] 2+ cluster or a [4Fe-4S] 2+ cluster to be active, depending on the specific DHAD. Applicants have found that increasing the expression of Fe-S cluster 15 forming proteins effectively increased the activity of DHAD in LAB cells. Expression of any set of proteins for Fe-S cluster formation may be used to increase DHAD activity in LAB cells. There are three known groups of Fe-S cluster forming proteins. These proteins are encoded by three types of operons: the Suf operon, the Isc operon, and the Nif 20 operon. The putative Suf operons of Lactococcus lactis and of Lactobacillus plantarum were identified by applicants by the presence of coding regions with sequence homologies to suf coding regions from other organisms. Disclosed herein is the first demonstration that expression of the set of 25 genes including putative sufC, putative sufD, putative sufS, putative sufU, and putative sufB of L. plantarum affect function of an Fe-S protein. Similarly, disclosed herein is the first demonstration that expression of the set of genes including putative sufC, putative sufD, putative sufS, yseH (encoding hypothetical protein), putative nifU, and putative sufB of L. lactis 30 affect function of an Fe-S protein. Applicants have shown in Example 3 herein, that expression of the identified Lactococcus lactis suf operon in a Lactobacillus plantarum strain with the endogenous suf operon deleted allowed expression of activity of an introduced DHAD while there as no

41 WO 2010/037119 PCT/US2009/058843

DHAD acitvity in the Lactobacillus plantarum deletion strain with no Lactococcus lactis suf operon. Applicants have shown in Example 5 herein, that increased expression of the identified endogenous Lactobacillus plantarum suf operon provided increased activity of an 5 expressed DHAD. The Suf operons of L. plantarum and L. lactis are shown in Figures 1 and 2, respectively. SufS is a cysteine desulfurase which provides the sulfur for the cluster, and SufU is a scaffold protein that acts as a sulfur and iron acceptor. Functions of SufC and SufD are not established, 10 though SufC has ATPase activity, Suf B has cysteine desulfurase activator activity and a SufBCD complex has similarity to components of ATP binding cassette transporter proteins. The E. coli Suf operon, shown in Figure 3, includes SufE, another cysteine desulfurase activator. In addition, SufU is not present and is replaced by a different scaffold 15 protein, SufA. Thus there is some variation in the set of Fe-S cluster forming proteins that is included in a Suf operon depending on the source organism. Any set of Fe-S cluster forming Suf operon proteins may be expressed in the LAB cells disclosed herein. Representative examples of these proteins and their coding regions, with SEQ ID NOs, are given in 20 Table 4. Typically a set of coding regions that is used in preparing the LAB cells disclosed herein is derived from a single operon. However, coding regions for proteins that have high sequence identities may be interchanged for one another in a set of Fe-S cluster forming proteins. For example, the SufS protein from L. lactis may be used together with the 25 SufC, SufD, SufU, and SufB proteins of Lactobacillus reuteri whose SufS has 74% identity, or of Lactobacillus fermentum whose SufS has 72% identity, each to the SufS of L lactis. Also the SufS of L. plantarum may be interchanged. Though it has 62% identity with SufS of L. lactis, considering conservative amino acid changes the similarity is 80%. One 30 skilled in the art will recognize that generally proteins with sequence identities of at least about 70%, 75%, 80%, 85%, 90%, 95% or greater may be substituted for each other in a set of Fe-S cluster forming proteins. With similarities of about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or

42 WO 2010/037119 PCT/US2009/058843

greater, Suf proteins may be interchangeable if the amino acid changes are conservative for a final similarity of 70%, 75%, 80%, 85%, 90%, 95% or greater based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and 5 Gonnet 250 series of protein weight matrix over the full length of the protein sequence. Proteins of a Suf operon derived from a wide variety of LAB and other related bacteria may be used in the LAB cells disclosed herein. The SufS proteins and coding regions representing the Fe-S cluster forming 10 protein operons of a variety of organisms that may be used herein are given in Table 4. Each of the sufS coding regions given in Table 4 is a part of a Suf operon. One skilled in the art can readily use the sufS coding region or protein sequence of an organism that is given in Table 4 as a sequence probe to identify the entire Suf operon from that organism in 15 publicly available sequence databases. Each individual suf gene coding region may be identified using BLAST sequence analysis of individual coding or protein sequences, as described above, to identify the corresponding coding sequence from a desired organism. The suf gene sequences given in Table 4, for example, may be used as the gene probe 20 sequences. Alternatively, annotations present in publicly available databases may be used to identify suf genes. Fe-S cluster forming proteins may also be found in an Isc operon. The Isc operon of E. coli, for example, includes coding regions for the proteins IscS, IscU, IscA, HscB, HscA, Fdx and IscX, whose sequences 25 are listed in Table 5, with SEQ ID NOs, and operon diagram is shown in Figure 4. Expression of the operon is negatively regulated by IscR, encoded by an adjacent sequence (Figure 4) but that is not part of the Fe S cluster forming set of proteins in the Isc operon. IscS is a cysteine desulfurase that transfers sulfur to the scaffold protein IscU. IscA binds 30 iron and provides iron to IscU. HscA, also called Hsc66, is a chaperone (member of the Hsp70 protein family) whose ATPase activity is stimulated by IscU in the presence of the co-chaperone HscB, also called Hsc20. FdX is a ferredoxin which may function as an intermediate site for Fe-S

43 WO 2010/037119 PCT/US2009/058843

cluster assembly. IscX interacts with IscS, but may not be necessary for Fe-S cluster formation. Any Isc operon Fe-S cluster forming proteins may be used in the LAB cells disclosed herein. Fe-S cluster forming proteins may also be found in a Nif operon. 5 The Nif operon of Wolinella succinogenes, for example, includes coding regions for the proteins NifS and NifU, whose sequences are listed in Table 5 and operon diagram is shown in Figure 5. NifS is a cysteine desulfurase and NifU is a scaffold protein. Any Nif operon Fe-S cluster forming proteins may be used in the LAB cells disclosed herein. 10 A set of Fe-S cluster forming proteins, as described above, may be expressed in LAB cells as one recombinant genetic expression element that includes the coding regions as they are present in their natural operon, operably linked to a promoter and 3' termination sequence. Alternatively, a set of Fe-S cluster forming proteins may be expressed in 15 more than one operon or each as an individual chimeric gene, all of which are called recombinant genetic expression elements. One skilled in the art can readily choose and implement any of these methods of expressing two or more proteins that are a set of Fe-S cluster forming proteins. An additional approach to increase the expression of Fe-S cluster 20 forming proteins comprises replacing or augmenting the promoter of an endogenous gene whose product is known or predicted to be involved in Fe-S cluster assembly or the promoter for an operon containing genes whose products are known or predicted to be involved in Fe-S cluster assembly. The endogenous promoter may be replaced by a high 25 expression promoter or augmented by additional copies of the native promoter or a non-native promoter. Suitable promoters and methods are well known in the art. Promoters, termination control regions, and vectors used for expressing Fe-S cluster forming proteins as recombinant genetic 30 expression elements that are individual chimeric genes or one or multiple operons in LAB cells are the same as described above for expression of DHAD coding regions. Isobutanol and other products

44 WO 2010/037119 PCT/US2009/058843

Isobutanol and any other product made from a biosynthetic pathway including DHAD activity may be produced with greater effectiveness in a LAB cell disclosed herein having a functional dihydroxy acid dehydratase polypeptide and at least one recombinant genetic 5 expression element encoding iron-sulfur cluster forming proteins. Such products include, but are not limited to valine, isoleucine, leucine, pantothenic acid (vitamin B5), 2-methyl-1 -butanol, 3-methyl-1 -butanol, and isobutanol. For example, biosynthesis of valine includes steps of acetolactate 10 conversion to 2,3-dihydroxy-isovalerate by acetohydroxyacid reductoisomerase (ilvC), conversion of 2,3-dihydroxy-isovalerate to a ketoisovalerate (also called 2-keto-isovalerate) by dihydroxy-acid dehydratase (ilvD), and conversion of a-ketoisovalerate to valine by branched-chain amino acid aminotransferase (ilvE). Biosynthesis of 15 leucine includes the same steps to a-ketoisovalerate, followed by conversion of a-ketoisovalerate to leucine by enzymes encoded by leuA (2-isopropylmalaate synthase), leuCD (isopropylmalate isomerase), leuB (3-isopropylmalate dehydrogenase), and tyrB/ ilvE (aromatic amino acid transaminase). Biosynthesis of pantothenate includes the same steps to 20 a-ketoisovalerate, followed by conversion of a-ketoisovalerate to pantothenate by enzymes encoded by panB (3-methyl-2-oxobutanoate hydroxymethyltransferase), panE (2-dehydropantoate reductae), and panC (pantoate-beta-alanine ligase). Engineering expression of enzymes for enhanced production of pantothenic acid in microorganisms is 25 described in US 6177264. Increased conversion of 2,3-dihydroxy isovalerate to a-ketoisovalerate will increase flow in these pathways, particularly if one or more additional enzymes of a pathway is overexpressed. Thus it is desired for production of, for example, valine, leucine, or pantothenate to use an engineered LAB cell disclosed herein. 30 The a-ketoisovalerate product of DHAD is an intermediate in isobutanol biosynthetic pathways disclosed in commonly owned and co pending US Patent Publication 20070092957 Al, which is herein incorporated by reference. A diagram of the disclosed isobutanol 45 WO 2010/037119 PCT/US2009/058843

biosynthetic pathways is provided in Figure 6. Production of isobutanol in a strain disclosed herein benefits from increased DHAD activity. As described in US 20070092957 Al, steps in an example isobutanol biosynthetic pathway include conversion of: 5 - pyruvate to acetolactate (Fig. 6 pathway step a), as catalyzed for example by acetolactate synthase, - acetolactate to 2,3-dihydroxyisovalerate (Fig. 6 pathway step b) as catalyzed for example by acetohydroxy acid isomeroreductase; - 2,3-dihydroxyisovalerate to a-ketoisovalerate (Fig. 6 pathway step c) as 10 catalyzed for example by acetohydroxy acid dehydratase, also called dihydroxy-acid dehydratase (DHAD); - a-ketoisovalerate to isobutyraldehyde (Fig. 6 pathway step d) as catalyzed for example by branched-chain a-keto acid decarboxylase ;and - isobutyraldehyde to isobutanol (Fig. 6 pathway step e) as catalyzed for 15 example by branched-chain alcohol dehydrogenase. The substrate to product conversions and enzymes involved in these reactions, and for steps f, g, h, I, j, and k of alternative pathways shown in Figure 6, are described in US 20070092957 Al. Genes that may be used for expression of the pathway step 20 enzymes named above other than the DHADs disclosed herein, as well as those for two additional isobutanol pathways, are described in US 20070092957 Al, and additional genes that may be used can be identified by one skilled in the art through bioinformatics or experimentally as described above. The preferred use in all three pathways of ketol-acid 25 reductoisomerase (KARI) enzymes with particularly high activities is disclosed in commonly owned and co-pending US Patent Pub No. 20080261230. Examples of high activity KARIs disclosed therein are those from Vibrio cholerae (DNA: SEQ ID NO:545; protein SEQ ID NO:546), Pseudomonas aeruginosa PAO1, (DNA: SEQ ID NO:551; 30 protein SEQ ID NO:552), and Pseudomonas fluorescens PF5 (DNA: SEQ ID NO:547; protein SEQ ID NO:548). Additionally described in US 20070092957 Al are construction of chimeric genes and genetic engineering of bacteria for isobutanol

46 WO 2010/037119 PCT/US2009/058843

production using the disclosed biosynthetic pathways. Expression of these enzymes in LAB is as described above for expression of DHADs. Growth for production Recombinant LAB cells disclosed herein may be used for 5 fermentation production of isobutanol and other products as follows. The recombinant cells are grown in fermentation media which contains suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, or mixtures of monosaccharides, including C5 sugars such as xylose and arabinose, 10 oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Although it is contemplated that all of the above mentioned carbon 15 substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose. Sucrose may be derived from renewable sugar sources such as sugar cane, sugar beets, cassava, sweet sorghum, and mixtures thereof. Glucose and dextrose may be derived from renewable grain sources through 20 saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, and mixtures thereof. In addition, fermentable sugars may be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in co-owned and co-pending U.S. Patent 25 Application Publication No. 2007/0031918A1, which is herein incorporated by reference. Biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, 30 such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not

47 WO 2010/037119 PCT/US2009/058843

limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, 5 grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof. 10 In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for isobutanol production. 15 Typically cells are grown at a temperature in the range of about 25 0C to about 40 0C in an appropriate medium. Suitable growth media are common commercially prepared media such as Bacto Lactobacilli MRS broth or Agar (Difco), Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM) broth. Other defined or synthetic growth 20 media may also be used, and the appropriate medium for growth of the particular bacterial strain will be known by one skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2':3'-monophosphate, may also be incorporated into the fermentation 25 medium. Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the initial condition. Fermentations may be performed under aerobic or anaerobic conditions, where anaerobic or microaerobic conditions are preferred. 30 Isobutanol, or other product, may be produced using a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the

48 WO 2010/037119 PCT/US2009/058843

fermentation. A variation on the standard batch system is the fed-batch system. Fed-batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. 5 Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Batch and fed-batch fermentations are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second 10 Edition (1989) Sinauer Associates, Inc., Sunderland, MA., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated by reference. Isobutanol, or other product, may also be produced using continuous fermentation methods. Continuous fermentation is an open 15 system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of 20 one factor or any number of factors that affect cell growth or end product concentration. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra. 25 It is contemplated that the production of isobutanol, or other product, may be practiced using batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for isobutanol 30 production. Methods for Isobutanol Isolation from the Fermentation Medium Bioproduced isobutanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see for

49 WO 2010/037119 PCT/US2009/058843

example, Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process. Biochem. 27:61-75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the isobutanol may 5 be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation. EXAMPLES The present invention is further defined in the following Examples. 10 It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes 15 and modifications of the invention to adapt it to various uses and conditions. The meaning of abbreviations used is as follows: "min" means minute(s), "h" means hour(s), "sec' means second(s), "pl" means microliter(s), "ml" means milliliter(s), "L" means liter(s), "nm" means 20 nanometer(s), "mm" means millimeter(s), "cm" means centimeter(s), "tm" means micrometer(s), "mM" means millimolar, "M" means molar, "mmol" means millimole(s), "pmole" means micromole(s), "g"means gram(s), "pg" means microgram(s), "mg" means milligram(s), "rpm" means revolutions per minute, "w/v" means weight/volume, "OD" means optical density, and 25 "OD600" means optical density measured at a wavelength of 600 nm. GENERAL METHODS: Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory 30 Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,

50 WO 2010/037119 PCT/US2009/058843

1984, and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987. Materials and methods suitable for the maintenance and growth of bacterial cultures are also well known in the art. Techniques suitable for 5 use in the following Examples may be found in Manual of Methods for General Bacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds., American Society for Microbiology, Washington, DC., 1994, or by Thomas D. Brock in Biotechnology: A Textbook of Industrial 10 Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA, 1989. All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, WI), BD Diagnostic Systems (Sparks, MD), Life Technologies (Rockville, MD), or Sigma Chemical Company (St. Louis, MO), unless 15 otherwise specified. Example 1 Lactobacillus plantarum PN0512 suf operon deletion The purpose of this example is to describe the deletion of the suf operon in Lactobacillus plantarum PN0512 (ATCC strain # PTA-7727) to 20 create the Lactobacillus plantarum strain PN0512Asuf. This operon contains genes whose products are predicted to be involved in Fe-S cluster assembly. The coding regions of the operon were identified by sequence homology to suf gene coding regions that are present in publicly available sequence databases. 25 The deletion was constructed by a two-step homologous recombination procedure to yield an unmarked deletion using methods previously described (Ferain et al., 1994, J. Bact. 176:596). The procedure utilized a shuttle vector, pFP996 (SEQ ID NO:553). It can replicate in both E. coli and gram-postive bacteria. It contains the origins of replication 30 from pBR322 (nucleotides #2628 to 5323) and pE194 (nucleotides #43 to 2627). pE194 is a small plasmid isolated originally from a gram positive bacterium, Staphylococcus aureus (Horinouchi and Weisblum J. Bacteriol. (1982) 150(2):804-814). In pFP996, the multiple cloning sites (nucleotides

51 WO 2010/037119 PCT/US2009/058843

#1 to 50) contain restriction sites for EcoRI, BgIII, Xhol, Smal, Clal, KpnI, and Hindill. There are two antibiotic resistance markers; one is for resistance to ampicillin and the other for resistance to erythromycin. For selection purposes, ampicillin was used for transformation in E. coli and 5 erythromycin was used for selection in L. plantarum. Two segments of DNA containing sequences upstream and downstream of the intended deletion were cloned into the plasmid to provide the regions of homology for two genetic crossovers. The initial single crossover integrated the plasmid into the chromosome. The second crossover event yielded either 10 the wild type sequence or the intended gene deletion. The recombination plasmid was constructed using standard molecular biology methods known in the art. All restriction and modifying enzymes and Phusion High-Fidelity PCR Master Mix were purchased from New England Biolabs (Ipswich, MA). DNA fragments were purified with 15 Qiaquick PCR Purification Kit (Qiagen Inc., Valencia, CA). Plasmid DNA was prepared with QiAprep Spin Miniprep Kit (Qiagen Inc., Valencia, CA). L. plantarum PN0512 genomic DNA was prepared with MasterPure DNA Purification Kit (Epicentre, Madison, WI). Oligoucleotides were synthesized by Sigma-Genosys (Woodlands, TX). The vector construct 20 was confirmed by DNA sequencing. The homologous DNA arms were designed such that the deletion would encompass the majority of the first gene through the 5' end of the last gene in the operon, which is shown in Figure 1 A. The deleted sequence, as shown in Figure 1B, started at 94 base pairs into the sufC 25 coding sequence through 215 base pairs of the sufB coding sequence. The homologous arms cloned into the plasmid were approximately 1100 (left arm) and 1200 (right arm) base pairs long separated by 12 base pairs (Xhol and Xmal restriction sites). The suf operon left homologous arm was amplified from L. plantarum PN0512 genomic DNA with primers oBP97 30 (SEQ ID NO:554), containing an EcoRI site, and oBP98 (SEQ ID NO:555), containing an Xhol site using Phusion High-Fidelity PCR Master Mix. The suf operon right homologous arm was amplified from L. plantarum PN0512 genomic DNA with primers oBP1 01 (SEQ ID NO:556), containing

52 WO 2010/037119 PCT/US2009/058843

an Xmal site and oBP102 (SEQ ID NO:557), containing a KpnI site using Phusion High-Fidelity PCR Master Mix. The suf operon left homologous arm was digested with EcoRI and Xhol and the suf operon right homologous arm was digested with Xmal and Kpnl. The two homologous 5 arms were ligated with T4 DNA Ligase into the corresponding restriction sites of pFP996, after digestion with the appropriate restriction enzymes, to generate the vector pFP996-suf-arms. Deletion of the suf operon was obtained by transforming Lactobacillus plantarum PN0512 with pFP996-suf-arms. 5 ml of 10 Lactobacilli MRS medium (7406, Accumedia, Neogen Corporation, Lansing, MI) containing 1% glycine (G8898, Sigma-Aldrich, St. Louis, MO) was inoculated with PN0512 and grown overnight at 300C. 100 ml MRS medium with 1% glycine was inoculated with overnight culture to an OD600 of 0.1 and grown to an OD600 of 0.7 at 300C. Cells were 15 harvested at 3700xg for 8 min at 4 0C, washed with 100 ml cold 1 mM

MgCl 2 (M8266, Sigma-Aldrich, St. Louis, MO), centrifuged at 3700xg for 8 min at 4 0C, washed with 100 ml cold 30% PEG-1 000 (81188, Sigma Aldrich, St. Louis, MO), recentrifuged at 3700xg for 20 min at 4 0C, then resuspended in 1 ml cold 30% PEG-1 000. 60 pl of cells were mixed with 20 -100 ng of plasmid DNA in a cold 1 mm gap electroporation cuvette and electroporated in a BioRad Gene Pulser (Hercules, CA) at 1.7 kV, 25 pF, and 400 0. Cells were resuspended in 1 ml MRS medium containing 500

mM sucrose (S9378, Sigma-Aldrich, St. Louis, MO) and 100 mM MgCl2, incubated at 300C for 2 hrs, and then plated on MRS medium plates 25 containing 2 pg/ml of erythromycin (E5389, Sigma-Aldrich, St. Louis, MO). Transformants were screened by PCR using plasmid specific primers oBP42 (SEQ ID ON:558) and oBP57 (SEQ ID NO:559). Transformants were grown at 300C in Lactobacilli MRS medium with erythromycin (3 pg/ml) for approximately 10 generations and then at 370C 30 for approximately 45 generations by serial inoculations in Lactobacilli MRS medium. The cultures were plated on Lactobacilli MRS medium with erythromycin (1 pg/ml).The isolates were screened by colony PCR for a single crossover with chromosomal specific primer oBP1 25 [SEQ ID No.

53 WO 2010/037119 PCT/US2009/058843

560] and plasmid specific primer oBP42 (SEQ ID NO:558), and chromosomal specific primer oBP127 9SEQ ID NO:561) and plasmid specific primer oBP57 (SEQ ID NO:559). Subsequently, single crossover integrants were grown at 370C for 5 approximately 44 generations by serial inoculations in Lactobacilli MRS medium. The cultures were plated on MRS medium. Colonies were patched to MRS plates and grown at 370C. The isolates were then patched onto MRS medium with erythromycin (1 pg/ml). Erythromycin sensitive isolates were screened by (colony) PCR for the presence of a 10 wild-type or deletion second crossover using chromosomal specific primers oBP1 25 (SEQ ID NO:560) and oBP1 27 (SEQ ID NO:561). A wild type sequence yielded a 6400 bp product and a deletion sequence yielded a 2500 bp product. The deletion was confirmed by sequencing the PCR product while the absence of plasmid was tested by colony PCR using 15 plasmid specific primers oBP42 (SEQ ID NO:558) and oBP57 (SEQ ID NO:559).

Example 2 Construction of plasmid pDM1-ilvD(L. lactis)-suf(L. lactis) 20 The purpose of this example is to describe cloning of the ilvD coding region (SEQ ID NO:231) and suf operon (SEQ ID NO:881) from Lactococcus lactis subsp lactis NCDO2118 (NCIMB 702118) [Godon et al., J. Bacteriol. (1992) 174:6580-6589]. The Lactococcus lactis suf operon comprises ysfB (sufC), ysfB (sufC), ysfA (sufD), ysel (sufS), yseH 25 (hypothetical protein), nifU, and yseF (sufB) genes as diagrammed in Figure 2) A shuttle vector pDM1 (SEQ ID NO:571) was used for cloning and expression of the ilvD coding region and suf operon from Lactococcus lactis subsp lactis NCDO2118 (NCIMB 702118) in Lactobacillus plantarum 30 PN0512 (ATCC PTA-7727). Plasmid pDM1 contains a minimal pLF1 replicon (-0.7 Kbp) and pemK-peml toxin-antitoxin(TA) from Lactobacillus plantarum ATCC1 4917 plasmid pLF1, a P1 5A replicon from pACYC1 84, chloramphenicol resistance marker for selection in both E. coli and L.

54 WO 2010/037119 PCT/US2009/058843

plantarum, and P30 synthetic promoter [Rud et al., Microbiology (2006) 152:1011-1019]. Plasmid pLF1 (C.-F. Lin et al., GenBank accession no. AF508808) is closely related to plasmid p256 [Servig et al., Microbiology (2005) 151:421-431], whose copy number was estimated to be -5-10 5 copies per chromosome for L. plantarum NC7. A P30 synthetic promoter was derived from L. plantarum rRNA promoters that are known to be among the strongest promoters in lactic acid bacteria (LAB) [Rud et al., Microbiology (2005) 152:1011-1019]. The L. lactis suf operon (6,108 bp) was PCR-amplified from 10 genomic DNA of L. lactis subsp lactis NCDO2118 (NCIMB 702118) with T sufLI(Notl) (SEQ ID NO:572) and B-sufLI(Spel) (SEQ ID NO:573) primers. L. lactis subsp lactis NCDO2118 genomic DNA was prepared with a Puregene Gentra Kit (QIAGEN, CA). The resulting suf PCR fragment containing ysfB (sufC), ysfB (sufC), ysfA (sufD), ysel (sufS), yseH, nifU, 15 and yseF (sufB) coding regions was digested with Notl and Spel, and the 6.1 Kbp suf operon fragment was gel-purified. A cloning plasmid pTnCm (SEQ ID NO:574) was digested with Notl and Spel, and ligated with 6.1Kbp suf operon fragment. pTnCm contains a pE194 replicon, pBR322 replicon, ampicillin resistance marker for selection in E. coli, and 20 chloramphenicol resistance marker for selection in L. plantarum. The ligation mixture was transformed into the E. coli Top1 0 strain (Invitrogen, CA), and spread on LB plates containing 100 pg/ml ampicillin for selection. Positive clones were screened by Xhol digestion, giving two fragments with an expected size of 5,136bp and 8,413bp. The correct plasmid was 25 named pTnCm-suf(L. lactis). The L. lactis ilvD coding region was PCR-amplified from genomic DNA of L. lactis subsp lactis NCDO2118 (NCIMB 702118) with T ilvDLI(BamHI) (SEQ ID NO:575) and B-ilvDLI(NotlBamHI) (SEQ ID NO:576) primers. The resulting ilvD PCR fragment was digested with 30 BamHI and Noti, This 1.7Kbp ilvD coding region fragment was gel purified, and ligated into BamHI and Notl sites of plasmid pAMAC8-Papha (SEQ ID NO:577), which contained the Papha promoter from the pJH1 plasmid of Enterococcus faecalis (Trieu-Cuot, P. & Courvalin, P. Gene

55 WO 2010/037119 PCT/US2009/058843

(1983) 23:331-341). pAMAC8 carries a pAMp1 replication origin (Renault et al., Gene 183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231 (1993)), P15A replicon from pACYC184, chloramphenicol resistance gene from Staphylococcus aureusplasmid pC194 for selection in L. plantarum, 5 and ampicillin gene for selection in E. coli. As a result of the ligation, pAMAC8-Papha-ilvD (L.lactis) was generated. Plasmid pAMAC8-Papha ilvD(L. lactis) was then digested with Xhol and Noti, and the 2,147 bp Papha-ilvDLI fragment was gel-purified. The 2,147 bp Papha-ilvDLI fragment was ligated into Sall and Notl sites of pTnCm-suf(L. lactis). The 10 ligation mixture was transformed into E. coli Topl0 cells (Invitrogen, CA), which were spread on LB plates containing 100 pg/ml ampicillin for selection. Positive clones were screened by BamHI and Notl digestion, giving two fragments with an expected size of 13,934 bp and 1,742 bp. The correct plasmid was named pTnCm-Papha-ilvD(L. lactis)-suf(L. 15 lactis). The ilvD(L. lactis)-suf(L. lactis) cassette (SEQ ID NO:594) was isolated from pTnCm-Papha-ilvD(L. lactis)-suf(L. actis). Plasmid pTnCm Papha-ilvD(L. lactis)-suf(L. lactis) was digested with Spel, treated with Klenow fragment of DNA polymerase to make blunt ends, and then 20 digested with BamHI. The 7.9 Kbp ilvD(L. lactis)-suf(L. lactis) cassette (SEQ ID NO: 594) was gel-purified, and ligated into BamHI and Smal sites of pDM1 to clone the ilvD(L. lactis)-suf(L. lactis) cassette under the control of the P30 promoter in pDM1. The ligation mixture was transformed into E. coli Topl0 cells (Invitrogen, CA), and spread on LB 25 plates containing 25 pg/ml chloramphenicol for selection. Positive clones were screened by ApaLl digestion, giving two fragments with an expected size of 8,040bp and 3,562bp. The correct plasmid was named pDM1 ilvD(L. lactis)-suf(L. lactis). The sequence of the ilvD(L. lactis)-suf(L. lactis) cassette in pDM1-ilvD(L. lactis)-suf(L. lactis) was confirmed with sequence 30 primers, DL11(R) (SEQ ID NO:578), DLI2 (SEQ ID NO:579), DLI3 (SEQ ID NO:580), Suf1 (SEQ ID NO:581), Suf2 (SEQ ID NO:582), Suf3 (SEQ ID NO:583), Suf4 (SEQ ID NO:584), Suf5 (SEQ ID NO:585), Suf6 (SEQ ID NO:586), Suf7 (SEQ ID NO:587), and Suf8 (SEQ ID NO:588).

56 WO 2010/037119 PCT/US2009/058843

Plasmid pDM1-ilvD(L.lactis)-suf(L.lactis) was digested with ApaLl and Noti, treated with Klenow fragment of DNA polymerase to make blunt ends, and the 5.3 Kbp fragment containing pDM1-ilvD(L.lactis) was gel purified. The gel-purified pDM1-ilvDLI fragment was self-ligated to create 5 pDM1-ilvD(L.lactis). Positive clones were screened by Sall digestion, giving one fragment with an expected size of 5,262 bp.

Example 3 Recombinant co-expression of Lactococcus lactis suf operon with 10 Lactococcus lactis ilvD restores DHAD activity in Lactobacillus plantarum PN0512Asuf strain The purpose of this example is to describe co-expression of the Lactococcus lactis ilvD coding region and Lactococcus lactis suf operon in the Lactobacillus plantarum PN0512 Asuf strain. Construction of 15 Lactobacillus plantarum PN0512 Asuf operon deletion mutant and that of plasmids pDM1-ilvD(L. lactis)-suf(L. lactis) and pDM1-ilvD(L. lactis) are described in examples 1 and 2, respectively. L. plantarum PN0512 was transformed with plasmid pDM1-ilvD(L. lactis)-suf(L. lactis) or pDM1-ilvD(L. lactis) by electroporation. Electro 20 competent cells were prepared by the following procedure. 5 ml of Lactobacilli MRS medium containing 1% glycine was inoculated with PN0512 cells and grown overnight at 300C. 100 ml MRS medium with 1% glycine was inoculated with the overnight culture to an OD600 = 0.1 and grown to an OD600 = 0.7 at 300C. Cells were harvested at 3700xg for 8

25 min at 4 C, washed with 100 ml cold 1 mM MgCl 2, centrifuged at 3700xg for 8 min at 4 0C, washed with 100 ml cold 30% PEG-1 000 (81188, Sigma Aldrich, St. Louis, MO), recentrifuged at 3700xg for 20 min at 4 0C, and then resuspended in 1 ml cold 30% PEG-1000. 60 pl of electro-competent cells were mixed with -100 ng plasmid DNA in a cold 1 mm gap 30 electroporation cuvette and electroporated in a BioRad Gene Pulser (Hercules, CA) at 1.7 kV, 25 pF, and 400 Q. Cells were resuspended in 1

ml MRS medium containing 500 mM sucrose and 100 mM MgCl 2,

57 WO 2010/037119 PCT/US2009/058843

incubated at 300C for 2 hrs, and then plated on MRS medium plates containing 10 pg/ml of chloramphenicol for selection. Lactobacilli MRS medium (7406, Accumedia, Neogen Corporation, Lansing, MI)) was inoculated with L. plantarum PN0512 Asuf 5 transformants carrying pDM1 -ilvD(L.lactis)-suf(L.lactis) or pDM 1 ilvD(L.lactis) and grown overnight at 300C. 120 ml MRS medium with 40 pM ferric citrate (F3388, Sigma-Aldrich, St. Louis, MO) , 0.5 mM L cysteine (30089, Sigma-Aldrich, St. Louis, MO), and 10 pg/ml chloramphenicol was inoculated with overnight culture to an OD600 of 0.1 10 and grown to an OD600 of 2-3 anaerobically at 30 C in a 50 ml conical tube. Cultures were centrifuged at 3700xg for 10 min at 4 C, the pellets

washed with 50 mM potassium phosphate buffer pH 6.2 (6.2 g/L KH2PO4

and 1.2 g/L K2HPO 4) and re-centrifuged. Pellets were frozen and stored at -80 0C until assayed for DHAD activity. Cell extract samples were assayed 15 for DHAD activity using a dinitrophenylhydrazine based method as follows. Enzymatic activity of the crude extract was assayed at 37 0C as follows. Cells to be assayed for DHAD were suspended in 2-5 volumes of 50 mM

Tris, 10 mM MgSO 4, pH 8.0 (TM8) buffer, then broken by sonication at 0 0C. The crude extract from the broken cells was centrifuged to pellet the 20 cell debris. The supernatants were removed and stored on ice until assayed (initial assay was within 2 hrs of breaking the cells). It was found that the DHADs assayed herein were stable in crude extracts kept on ice for a few hours. The activity was also preserved when small samples were 0 frozen in liquid N2 and stored at -80 C. 25 The supernatants were assayed using the reagent 2,4-dinitrophenyl hydrazine as described in Flint and Emptage (J. Biol. Chem. (1988) 263: 3558-64). When the activity was so high that it became necessary to dilute the crude extract to obtain an accurate assay, the dilution was done in 5 mg/ml BSA in TM8. 30 Protein assays were performed using the Piece Better Bradford reagent (cat # 23238) using BSA as a standard. Dilutions for protein assays were made in TM8 buffer when necessary.

58 WO 2010/037119 PCT/US2009/058843

The DHAD activity results are given in Table 8. Plasmid expression of the L. lactis ilvD coding region showed 0.004 pmol min-1 mg- 1 DHAD activity in L. plantarum PN0512. Plasmid expression of the L. lactis ilvD coding region, however, showed no DHAD activity in L. plantarum PN0512 5 Asuf. Co-expression in L.plantarum PN0512 Asuf of L. lactis suf operon with L. lactis ilvD from pDM1-ilvD(L.lactis)-suf(L.lactis) restored the DHAD activity to 0.004 pmol min-1 mg-1. The data indicate that either L. plantarum native suf operon or L. lactis suf operon is involved in Fe-S cluster biogenesis for DHAD activity in L. plantarum PN0512. 10 Table 8. DHAD activity in L. plantarum PN0512 Asuf. Strain/Plasmid Specific Activity (pmol min-1 mg-) L. plantarum PN0512 / pDM1-ilvD(L.lactis) 0.004 L. plantarum PN0512 Asuf / pDM1-ilvD(L.lactis) 0.000 L. plantarum PN0512 Asuf / pDM 1-ilvD(L.lactis)-suf(L.lactis) 0.004

Example 4 Construction of plasmids for co-expression of Lactococcus lactis ilvD and 15 the Lactobacillus iplantarum PN0512 suf oDeron. The purpose of this example is to describe the construction of plasmids used for the co-expression of Lactococcus lactis llvD(SEQ ID NO:231) and the Lactobacillus plantarum PN0512 suf operon (SEQ ID NO:589). A shuttle vector pDM1 (SEQ ID NO:571), described in Example 20 2, was used for cloning and expression of the ilvD coding region from Lactococcus lactis subsp lactis NCDO2118 (NCIMB 702118) [Godon et al., J. Bacteriol. (1992) 174:6580-6589] and the suf operon from Lactobacillus plantarum PN0512. Plasmids were constructed using standard molecular biology 25 methods known in the art. All restriction and modifying enzymes and Phusion High-Fidelity PCR Master Mix were purchased from New England Biolabs (Ipswich, MA). DNA fragments were purified with Qiaquick PCR Purification Kit (Qiagen Inc., Valencia, CA). Plasmid DNA was prepared

59 WO 2010/037119 PCT/US2009/058843

with QiAprep Spin Miniprep Kit (Qiagen Inc., Valencia, CA). L. plantarum PN0512 genomic DNA was prepared with MasterPure DNA Purification Kit (Epicentre, Madison, WI). Oligoucleotides were synthesized by Sigma Genosys (Woodlands, TX). All vector constructs were confirmed by DNA 5 sequencing. Vector pDM1 was modified by deleting nucleotides 3281-3646 spanning the lacZ region which were replaced with a multi cloning site. Primers oBP120 [SEQ ID NO:562], containing an Xhol site, and oBP182 [SEQ ID NO:563], containing Drdl, Pstl, Hindill, and BamHI sites, were 10 used to amplify the P30 promoter from pDM1 with Phusion High-Fidelity PCR Master Mix. The resulting PCR product and pDM1 vector were digested with Xhol and Drdl, which drops out lacZ and P30. The PCR product and the large fragment of the pDM1 digestion were ligated to yield vector pDM20 in which the P30 promoter was reinserted, bounded by 15 Xhol and DrdI restriction sites. The ilvD coding region (SEQ ID NO:231) from Lactococcus lactis and a ribosome binding sequence (SEQ ID NO:590) were cloned into pDM20 to create vector pDM20-ilvD(L. lactis). Primers oBP190 (SEQ ID NO:564), containing a BamHI site and ribosome binding sequence, and 20 oBP192 (SEQ ID NO:565), containing a Pstl site, were used to amplify the ilvD coding region from pDM1-ilvD(L. lactis) with Phusion High-Fidelity PCR Master Mix. Construction of pDM1-ilvD (L.lactis) is described in Example 2. The resulting PCR product and pDM20 were ligated after digestion with BamHI and Pstl to yield vector pDM20-ilvD(L.lactis) in which 25 the ilvD coding region is expressed from the P30 promoter. The promoter region of the IdhL1 gene (SEQ ID NO:591) from Lactobacillus plantarum PN0512 with a multi cloning site and the suf operon containing sufC, sufD, sufS, sufU, and sufB (SEQ ID NO:589) from Lactobacillus plantarum PN0512 were cloned into pDM20-ilvD(LI) by two 30 consecutive steps to create vector pDM20-ilvD(LI)-PldhL1-suf(Lp)]. sufC was preceded by a ribosome binding sequence (SEQ ID NO:590). Primers AA178 (SEQ ID N0567), containing Drdl, Sall and AfIIl sites, and AA179 (SEQ ID NO:568), containing Drdl, Pmel, Sac, AvrII, Pac, Kasi, and Not I

60 WO 2010/037119 PCT/US2009/058843

sites, were used to amplify the IdhL1 promoter from L. plantarum PN0512 genomic DNA using Phusion High-Fidelity PCR Master Mix. The resulting PCR product and pDM20-ilvD(LI) were ligated after digestion with Drdl. Clones were screened by PCR for inserts that were in the same 5 orientation as the ilvD coding region using primers AA178 (SEQ ID NO:567) and AA177 (SEQ ID NO:566). A clone that had the correctly oriented insert was named pDM20-ilvD(LI)-PldhLl. Primers oBP211 (SEQ ID NO:569), containing a Notl site and ribosome binding sequence, and oBP195 (SEQ ID NO:570), containing a Pac site, were used to amplify 10 the suf operon from L. plantarum PN0512 genomic DNA using Phusion High-Fidelity PCR Master Mix. The resulting PCR product and pDM20 ilvD(LI)-PldhLl were ligated after digestion with Notl and Pac to yield vector pDM20-ilvD(LI)-PldhLl -suf(Lp), where the suf operon is expressed from the ldhL1 promoter. 15 Example 5 Increased DHAD activity with co-expression of Lactococcus lactis ilvD and the Lactobacillus plantarum PN0512 suf operon in a wild-type PN0512 strain background 20 The purpose of this example is to demonstrate the effect of co expression of the Lactobacillus plantarum PN0512 suf operon, containing the Fe-S cluster assembly genes, with Lactococcus lactis ilvD on DHAD activity in wild-type Lactobacillus plantarum PN0512. Lactobacillus plantarum PN0512 was transformed separately with 25 vectors pDM20-ilvD(LI) and pDM20-ilvD(LI)-PldhL1-suf(Lp). Lactobacillus plantarum PN0512 was transformed as in Example 1, except transformants were selected for on MRS medium plates containing 10 pg/ml of chloramphenicol (C0378, Sigma-Aldrich, St. Louis, MO). Strains PN0512/pDM20-ilvD(LI) and PN0512/pDM20-ilvD(LI)-PldhL1 -suf(Lp) were 30 grown overnight in Lactobacilli MRS medium (7406, Accumedia, Neogen Corporation, Lansing, MI) with 10 pg/ml chloramphenicol (C0378, Sigma Aldrich, St. Louis, MO) at 300C. 120 ml of MRS medium supplemented with 100 mM MOPS (M1254, Sigma-Aldrich, St. Louis, MO), 40 pM ferric

61 WO 2010/037119 PCT/US2009/058843

citrate (F3388, Sigma-Aldrich, St. Louis, MO), 0.5 mM L-cysteine (30089, Sigma-Aldrich, St. Louis, MO), and 10 pg/ml chloramphenicol adjusted to pH 7.5 with KOH was inoculated with overnight culture to an OD600 -0.05-0.1 in a 125 ml screw cap flask. The cultures were placed in an 5 anaerobic chamber (Coy Laboratories Inc., Grass Lake, MI) for 1 hour with the caps loose or the cultures were inoculated in the anaerobic chamber using medium which had been stored in the anaerobic chamber. The caps on the flask were sealed tight and the cultures were incubated at 37 C until reaching an OD600 - 1.0-2.0. Cultures were centrifuged at 3700xg 10 for 10 min at 4 0C. Pellets were washed with 50 mM potassium phosphate

buffer pH 6.2 (6.2 g/L KH2PO4 (P5379, Sigma-Aldrich, St. Louis, MO) and

1.2 g/L K2HPO 4 (P8281, Sigma-Aldrich, St. Louis, MO)) and re centrifuged. Pellets were frozen and stored at -80 C until assayed for DHAD activity. 15 Samples were assayed for DHAD activity using a dinitrophenylhydrazine based method as described in Example 3. The DHAD activity results are given in Table 9. The presence of the overexpressed suf operon led to a two-fold increase in DHAD activity in the PN0512 strain background . 20 Table 9. Co-expression of ilvD and the suf operon in wild-type Lactobacillus plantarum PN0512. DHAD activity in pmoles KIVA/min/mg total protein. Data represent the average of two independent experiments. Strain DHAD Activity PN0512/pDM20-ilvD(LI) 0.022 PN0512/pDM20-ilvD(LI)-PldhLl-suf(Lp) 0.051

25 Example 6 (Prophetic) Construction of plasmid for co-expression of Bacillus subtilis ilvD and the Lactococcus lactis suf operon The purpose of this example is to describe how to clone the ilvD 30 coding region (SEQ ID NO:497) from Bacillus subtilis 168 (ATCC 23857) and suf operon (SEQ ID NO:881) from Lactococcus lactis subsp lactis 62 WO 2010/037119 PCT/US2009/058843

NCDO2118 (NCIMB 702118) [Godon et al., J. Bacteriol. (1992) 174:6580 6589] into pDM1. Plasmid pDM1-ilvD(B. subtilis)-suf(L.lactis) is constructed by swapping the ilvD(L. lactis) coding region of pDM1-ilvD(B. subtilis) 5 suf(L.lactis) with a B. subtilis ilvD coding region. The B. subtilis ilvD coding region including a ribosomal binding site (RBS) is PCR-amplified from genomic DNA of Bacillus subtilis 168 with primers T-ilvDBs(BamHI) (SEQ ID NO:592) and B-ilvDBs(Notl) (SEQ ID NO:593). Bacillus subtilis 168 genomic DNA is prepared with a Puregene Gentra Kit (QIAGEN, CA). The 10 B. subtilis ilvD PCR product is digested with BamHI and Noti, and the 1.7kbp B. subtilis ilvD fragment is gel-purified. Plasmid pDM1 ilvD(L.lactis)-suf(L.lactis) is digested with BamHI and Noti, and 9.8 kbp fragment containing pDM1-suf(L.lactis) is gel-purified. The construction of pDM1-ilvD(L.lactis)-suf(L.lactis) is described in Example 2. The resulting 15 9.8 kbp pDM1-suf(L.lactis) fragment is ligated with the 1.7 kbp B. subtilis ilvD fragment. The ligation mixture is transformed into E. coli Top1O strain (Invitrogen, CA), and spread on LB plates containing 25 ptg/ml chloramphenicol for selection. Positive clones are screened by colony PCR with primers T-ilvDBs(BamHI) and B-ilvDBs(Notl), giving a PCR 20 product with an expected size of 1.7kbp. The positive plasmid is named as pDM1-ilvD(B. subtilis)-suf(L.lactis). Plasmid pDM1-ilvD(B. subtilis) suf(L.lactis) is digested with ApaLl and Noti, treated with Klenow fragment of DNA polymerase to make blunt ends, and then the 5.3 Kbp fragment containing pDM1-ilvD(B. subtilis) is gel-purified. The gel-purified fragment 25 is self-ligated to create pDM1-ilvD(B. subtilis). Positive clones are screened by Sall digestion, giving one fragment with an expected size of 5.3 kbp.

Example 7 (Prophetic) 30 Co-expression of Bacillus subtilis lvD with Lactococcus lactis suf operon in Lactobacillus plantarum PN0512

63 WO 2010/037119 PCT/US2009/058843

The purpose of this example is to describe how to express Bacillus subtilis llvD with Lactococcus lactis suf operon in Lactobacillus plantarum PN0512. L. plantarum PN0512 is transformed with plasmid pDM1-ilvD(B. 5 subtilis)-suf(L.lactis) or pDM1-ilvD(B. subtilis) by electrophoration. Preparation of electro-competent cells and electro-transformation are performed as described in Example 1. L. plantarum PN0512 transformants carrying pDM1-ilvD(B. subtilis) suf(L.lactis) or pDM1-ilvD(B. subtilis) are grown overnight in Lactobacilli 10 MRS medium at 300C. 120 ml of MRS medium supplemented with 40 pM ferric citrate, 0.5 mM L-cysteine, and 10 pg/ml chloramphenicol is inoculated with overnight culture to an OD600 = 0.1 in a 50 ml conical tube for each overnight sample. Cultures are anaerobically incubated at 30 C until reaching an OD600 of 2-3. Cultures are centrifuged at 3700xg for 10 15 min at 4 C. Pellets are washed with 50 mM potassium phosphate buffer

pH 6.2 (6.2 g/L KH2PO4 and 1.2 g/L K2HPO 4) and re-centrifuged. Pellets are frozen and stored at -80 C until assayed for DHAD activity. Cell extract samples are assayed for DHAD activity using a dinitrophenylhydrazine based method as in Example 3. In preferred 20 embodiments, DHAD activity is higher in the cells transformed with pDM1 ilvD(B. subtilis)-suf(L.lactis) than in those transformed with pDM1-ilvD(B. subtilis).

64 WO 2010/037119 PCT/US2009/058843

C 0)

E E~ C) . o( ) l0 ( 0 N ) -D c~~~I E- a em 0v[)v v0 0)0 CE a) a) a00 10S 0 ~ 1~ '~~ N

E C N C? x0) MO f0 O 10 00 0) (0

m C? Cr I? r' 0 In >

E m I'! C)' N'C 1 0. '

-0 0) (0)0) Go00) 00 ) 100C) NO ) 0) 0) C') V) 10 101 N-b 101 .10 10 00 CC) 0 Or) I In 0? C' N -l li~ 0)

E1 aN1T70 76 (D r- 101 N ) 01 N NO 1?00 O () 00) 1D0) 00) 0 0) 0) m 0 E E L)0~I E E000 w

01 010 010 N04 V 01 V-1 101 1')(L m m c ;E m10o102E o T w

2CC 4 E) C0n m ' 0

C151 a cmm M mQ co a 1 ' ) C

0) Cn 0)0I. Ni Ln 0 O ( ) mL oL 0t E Zlin t-~ C,0 I,, LO) 1N 0N O N (D N ON E~ ', :s E0 '-N 20 S 0 C N N 1N 1N 0)

N) - -W o NO 1to0 -0 100 (100 co 0 00 N, li) C0o D1~ . DJO toN PN Oq N 1')N O)N ED N 10N N, C CN -N ON (0n r D 9 a- 0111 C- - m

1 -0 U)o E 0Z a)~ N0)~'m ' Nf 6 S tI 2a53 &i ) z) 'o 2nC 'E to m m N O 'o a) ~ ~ l3 01I G a) 0 1in m~ 1 00 00 0 70 10 O N

E10 r 2,>N DU, DU 0 05 n EliE 0 1 -N Ul N ON N N N 0)

NE a E d)' (D A m' 2 2 m=X A C1 CD 't( D ,( LO0 m t )( N 0N NON *ON C4 ON ON 100N iN 1

E a 10

N Nor~ ~~'10) 10 0)0 0)) 0) 0'2' N .- ' , 0( 1 1', N V1)N N 10N 1')

0 -x0 0 x '1 0'1 00 INr '--1 m''10 1

00 0 A 1 (Dc

M - N N 1T0 1) 10 10 10 m 10V ) 10 10 0 -) C0 m

E2 10I. 10 z2 10 10 10 1 - C0 NC)1 'n01 N to U) N mIL *1')10 '(0 *

OPO C N CN - ! N N N N 04 N CN 104 CC 10( * 7 r 1 r 10 vL -0 NF -0 N 101' 100' No' 101' 001') NO' 001 10 003 N N m 00 a 00 0 m000 000 IT00 000 100 N NO 10C C N LON C n10N 010N 'T0 L? 10 C-40 L91 10? -4 OD1 -o1 -- 71 ~9 9 '9 (9II7 E E E 0 v 10 A C zr OD m0 G0 1 '0) 00)0) 1o0)0) I *0) 100 5 0)0 00)05 'S'D a)-N N*vci N N N 0* V 0* CVN-) C ) ) 0 (n 0 . '0 10 N N0'- 1C - C') N ' 0)r 1 ,2 B C 3 10 In C,' E'7 17~ 0 , , '1 , 7 AL 0-aC -T -' --- N E 10 0 L--

O2o' < w Wj 0 U) X Z i< 0U ZDX Z x Z z- N, 'I . U)I , N, 1 WO 2010/037119 PCT/US2009/058843

Ii) aCO N 0 It in ')U - - - - N N N N N N

0) C') m )0 r0D mO 0) 10) '-0 C) m m0 W mDO DO m00 00 NO). N t-N 0)N O4 N NCN -N N O -N WN vN C N vN N-N C* ' N' N' N .- ' N N' N' N'

03) ?2(O) 0) 0) (O ) (DO)M 0)0) 2(O) NO)m C) () .0) (OD NO), 00)) C* '-' N' 0 N' N' U) N? Sco C')'C C') I 21

0) ()) D) '0)f)0) 00) C) C 'F ) 00m C) 0) 0) 0) -) (D 0 (D V(D N (D CD ((D (ID v (D Cl)CD CF)(D f-0 CD (D C) (a

N- T en CN t2N ON- ON T -- V) C NC- N ON- N-N ON ? 0

- O (D 0. co) D OD 0r 'CO. "C. - LO - (C). sn

- r N-. C) N (1 Or N-r (Dr 0- 0, (D r-m0)

0f) NO) rO r) NO) W) UO) 00) 00) NO) 0)0W ) W) 0)W C') ( C'1) 0 V) C) ODCm NC1) OC') 0Cl) ) v NC C) (9CV) 9 ~ - 4 - C,)

(0 (DH - (D 77 D O)I N7 7I 7')6 q( (D( C'7 7FrI -D COC 01) .0) ()201) I () -0) NO) NO ) CV)a) ODOC) V'2) 0)0) m NO) 0)0)D 0) 0 N O 0) IT ID U) ID ( 9) N N .- Cm 7 7

M ) L ('2 to NW fW (OW qW 0( OW 90 NW O)( 7 7 q- )U nI (D v N vC ' 00 vD to *~I I (9r U)

7 (DT Tv~ 77 -u' 0)T NT N-' '' N-7 V) 0 't (D 0) ('20) 0)0) (DO0) NO0) (DO) 00 00) (0O) (DO) NO ) COO) C')0) C') m-' OC'm (DC' mDCm) OC) ( C') (C') N-C'f) C')) (1 C')c O),' (C) N - - r C') N NLr

(D N-(D a)( LO to n U lW (D o U NW C'O ( m( N( (DWO N- (DO ON N N O )NP CN CON N-N- C,'N- q r N NN O- 0 - I-P N IT(N ON O)N ON ON C') (DLON (ON n(N C'N ?4 U C m ' 'D' m' C)

14 N N-N O N LW N m')N 'N 'N N -N (D N V)N N-4 C-N r C l 1 I r r r r S I ' r N N

CON

(D~0 LO(('w w-C(DC0( O) OD ~ O COO co(NC (

0 ')O 00j coO (DO (DO (DO -O (DO NO- N COO 00?

(D N-9 N- N.D N- I D 0. LO N- "*' N-'-w q0 (N N N (ON CO0 4 N0N C')N CN M) N -N W)N ITN ON 0CN

NF I- I- I

NW( 0 00 W(0 M D(OD (O wDWD 0N(D 00 (D (D 00 0O C0 I OD N (D N CO (OD o N- N - 0 N 0NN to)2N N- ON Nr ,N CD N (DN 0) ?N N-FN

m~ r- C r . -rVrr m r tr C' - fr .- '

91 ? N? C? IC '2 ?C

V D C' ) CO I 0) CO ) CO (D Na ) 0 CO) ITa9)v () v m CO 9q0 r ) N- ) Op' ITM

'6W OC'R W -6C')( C')(Dr (D'( m-( 0 I) )0 rc)( OC')(D w OC') C) D NC') 0 (C.) N- NN C'N. N-N4 NO . vN. N- ,9 C')n .- 0 O )rl m ~ ONC)1, l (n9c' (0Jv) v) c)'n .9 ) .19 N9)

00 1 00 0) 00 000 OD 00 COD Va r) ! Cc'O WOO ) N c~OD FO WO) OD 0)0 0)0'Lo 'N- .- 'N .-'N N'? .?-'N- 9 L r'N - .'NL? r N ' - C)'- N

C7 I I). IDr ' (Drc ' r

7 I 7 77 ' ') '0 9.7 mr WT N-.- Or') mD w O0 LOr

0 R q N >- 0 v 0

0)- r I ' -1 , r .* I - S' WO 2010/037119 PCT/US2009/058843

rwc 0 in V N ' 0 n ( N N el m ' ' ' ' ' '

NO QC 0C) NCD (00 m (00) c(00) tNC) cl NO m0 m0 Vf) ) ON CNN V)N .N NN -I ON ON v-( (O N v W)N N I N -1 N'1 C,) N C -' N' LN'c C) N C N 0 C' 'i

0) Q)0) w00) (0 0) m m I) .0 NO ) .0) m)0 (I) m ;0) W00) V)N C C)N NN C-4 t0N ( tC ')NC (V N a? CN 'CN CN N' N '' N N C'

0)M V 0) (0) (0 v .0) U)0) NC0) 00 C) NO C) 00) C) 9) IT 0) Nto 0( t -o .- D .(D L2( cc T t 0) (O 0(0 (1)( ( 0(m0 N0 (

r--0.C)- 0- cr). (Y) - (0. - 0' 0). v N

0)) 0) 0) C) 0) NC0) NC0) 100) (0)0) .0 O C 0) C0 (0(0 N(0 W( V) ( (0(w 0( V-( N(0W)( I O0( V(0(0L .- 5 n U.)C') 0OC') (C'C) N C') L1CV) rC') C') 0) C') C)) 0 CCC') l

0 0) 0)0 CD 0 .- 0 00) 0 (0) (1) () M) V-C .0) (1) m - (00 ) 0( (0 r to N N, N -- IN')[NN N

LC 0( 4 N(0 (0 U') LI (0) (0(0 (-(0 (0( (0( 0) L0 m ) N(0C 0) N 1 (0N 0 N 0 C') N 0? ' . N - CI C Iv

00 (0 r)0) (00() CD0 NO ) NO ) (00 ) NC) (0 C) () NC) C'0) 01'm NC 'C ) (1) C') mOC IT .Cl) (C) W0' M0' PNC') DC) l N N C' N -l C) N N I

(0(0 m( C) ( ) (0( to0 (0( 00 U) U (0(0 .- ( N LO 0(0 m) (O 0N P-N Nl 1 l C)r lr- 0) (N cON 0N ,,N N CN ON (? N CN (N 04N -N (ON1 CNN C) N (ON r N -N - C') 7 ) - .

() N NN 04 vN IT CN4 N (ON w C404N .1 0 o N CN C NN N - N N ~ CN (ON g 1ON 0N ON 0O (O- c)N 'N cr)N N-N N N I( .- I (N I 0 I - I

(0 Q 0)10 0)1W .(0 W01 (0( N0 (0 )0 ((0 N0 0

CI I I I I I7 C

NO 0) NO ) C) NO 0 0 (0 (0 m1)0 (0- 0.- 0). m'. (1). (0. NCNO N. (0 .- (. N ON CvN. (ON N I? N CN In N ( N ON (N NON .-

r N N o N 0 0 N 0 o N wC)t

CNN (O N (ON .N IT N .N (O N N -I.N ITN .N NN

(0W( 0)0( m(( (00 M 00 -T E (00 . 0 W 0(0o (0 W)0( N(01 N(O( 0)0o (0 ND0 N . N 00 N C1 1 W N O OD0 NO WO NO (0O W0O Ln~ ~ ~ V)L?2~ C' C C,

T 0 F) 77)0 (0'N 0-6 ( ) 77 0) 0)) W0) C. .- )- U71m0) D 0)0) ~ 0 0 0 to0) 0 0 0)0 N'0)O 0 10 .- 0 O N (DO 00) 0 00) 0 N1-- 00m0 00)0

Lon ON0. NO. (0 on(0 (0(0 N t, N(. W 0m co. N 0.- NO. - w0 NC). C C. I(,-I? C OC?).- (C. NC CC). (O ). C?). CC. N C') DC.

"a F' '7 "F C'T. U;I. C) Iq -7 (NR. W' I -T 'V1. I I.. - 0 Cl) It 3 co

N 110) 0C) V 0) W V a) C) 0) C) 0) (M 0) q 0 ) P) 'T 0) V)'I- (D m) 0~0 IT v10) C') (0 (0 (0) c aU

(0 ()V0 (0 V V 0 (0 (0) (0 N Co o M ' N ' 0 OD m N (0 ( 0 0 (0 )l " N L 0 - N .- N N - . o N .- N C

(0 (0 N (0 - (0 C!N (0 .- 0 n0 L9 r,?~ C 7 C ' I l' ' ? rl ? 19 9? (? 04 ? C?

7 0) mo OO .- D C)OO '20 m10 0)0 00 00w D0 C)00 VOO(

a I I I C) I * I I N i

Io m CI I I

M I C) I I N I I N Il ." N N I N I N N . N C') WO 2010/037119 PCT/US2009/058843

NO) NO (D o ) C) O) co ) NOD NO CO) Na) ODO) NO) CDa . N4 v 0 O)v V I LO -1 '-V C 0V Ni .v N NNi ON 'ON CON (ON N4 N 'ON (ON O)N ON 0- (ON

N'=,NT; ' C .* It

* C I? I? IC C? C

va CO) O) Na) Wa) Wa0) Ca ) Na C) Wa V) NO) * N' C? NO C? (2, C' C?' ('' -, (

Wa) 0)O v (DO) (Ma OCD Va) 0) ) Ca) Na wOO )O ) 20 COW 0(0 (0(0Q CW Q 04W ) U) 0 COW OW m O (D( 0( 0'I 0 II

N 0 - - C? N N

N ('( )0 L0( (0D W 0C(V0 10( C) 0U) CO(0 co0 0(0 N0) CD

C,,

0O m0O NO) O)O (0O (DOLnt 0) O) O 7) OO wNO) CO ) (0aU) ( V Vn 00q v q w vCOv n C3' N . N v ~ IT CO CO N ) .- N CO N 0- C C? CI C.

Nv a)VNV v0 rl (DM .- LV v (a IT 0o0 3 na C N)(Y 0 W CO (D0) (3) 0 (3) Nn CD

00 l) mC) NOC) T O) Va) (0O) ma COO) CO) Ca co Ia() C',m

C? C I C? II

N NNo l f ON CON (ON LI N O)N c0N to VO) N 'ON N0 NN (Din aLI NF CN l 11 CO aCO N N co N' 2t

(1) .N CN ON (O N (DN NN CM N ON CON 'ON .- N NNi ( O

I

-(0~~(CO COWNW O)C- OW N OW N v0

,D C) C If1

CO -0 (0I0?.0 N CO? C? 'C? C) (0 )

CO. (0. CO.- a). O0. CO, 0.- N (0 ( 0. (0.- CN T N O)N (O N (O9N N 04N CN (DN WN NN- CON

N 0 CO A CON (DCl Cl l Nq N -l N .i CO C.- 1

WN) tN CON,) ON NN) CON (O ONJ .- 1N O

0 - W N(a)0 O)WW C)O) (W 9 VW COWD M O)o( VNMWW C O)(M0 0(0(0 3) 0 (n0 VO.C 0 . ( CO r O -C ( 0.)C t.-CrOV CO.C N.-C (0.C N-C N-C N-C

,Na) N)- ..- (900) (0). 00V, COO) NOU.O' O- N) C) CO I N 1 ? 1 ? a) OD I? V 'I N '? N C If C? C? I? I? CI

7;7 ( ( C ( r0 '.( 0-0 V( CO.-( NQI) N 0.(-T N777 'O7 COW v (0 v(. N7- w) UW v00 .- (00. CO

V ( D 0 ) N (3) q OO) O)VO q M ) to V ( ~) O I a) to a ) ' v a) O ITa) 00'a

I I

(nCO0 - CO(O NOD 0 C( n C) C(n CO(.V)O0C(1)oVO(00V)0 OCOW0Nm()L) f I 1 -f U 9 C 9 C) m 0 VO ( O C' (9 ( CO1 N4 (9) CO 9 (

C)O CC WO WoO WOO NC. C-COC 00 -OOO 0

(00.- (0 . (D(. 0 (0.- NCO N. . . . O( 0( 0)0. 0(0(W.0 N'C -' - N 9' C'N N'? N N N'N N'9 -? N ''9N T -'N

CD 0) 0 D N 0) (D ( CON V (0 ( (D) - NC Ca COCC Oa '. O, O a V' Vaa V ' Va WO 2010/037119 PCT/US2009/058843

o1 m v W' co (0 co. m1 0 - N La 0n an ) V) W) W) U) ccU 0 0(

CNa 0) CD 0 W 10 0) C) w(D C-) ' 0) 0)0 00 N-D (00 0 cli v -, (')~ IT q IT v (D~ IT 4 '4I 0 C' ODc ~ W )C G)N N-NC 0) 'Tn ~C j~ ' N * (N C, I

OD4 0)q ca7 'F VJ 7) N0 04I 1-4 (04 IT 4 04) vCT'T (00 Q W)0) V')) Q 0 ) N ) (0) (1)) 0) Q (c00) (00CD (0) (0 0) U) wa~ Ca) NN C') .' C') N' N' '' N' C'4 ' C')N CI ? I Ct C? CH

40 0) C)0) 10) 0 1, 0)0(A N a) -) 00) 0) O a0 0) N ID0 w'( to( to0(0 (D w N(0 M (( C'(D m(0 10( D0

N . - N N C? C CN

GoL) toU N-U) 0U) N) CU) 00 001 w01 10 0)1 Nr

N C') (N N ' '' I N0

N-) ra)0)) 0 0) 40 ) (D0) 0q0) 0IT (00 G )(00) .a a '0 N- 0)) (D LO - ) O P I 0L 0 L .- 04 Uf Y L D) NOit (0) (O 0 00cq M V') N - 'q -l -' Nvm c) pc C') N r n 0

N C) (10 L 0) NU) 0) 0)10 ( D1 (010 go0 CD1 CD (10 0() CA

'Go (04 w o (nV N ) LO 0 (0 LO to 04 ('1 104 04 0O ('4 N- (0 v 0 T v 0 ( N N- m 0 IT N I

N4 w0 N 0) 94C) 4 N4 (4 0 04 10, N

-MC) 0a 7-' 7)' -IF' a)C') 01) -qC' 01a IT) m0a C'C' C') C ') C') m- m - '- 9) m- C Nm ( C') mCl) N

-U) 0)1 to t W 1 N-U) (01 0'U CoV n O U) N10V C)UW) w01 .O U) C')CN N- N1 -Ncl N- N N(4N N-N 0N '-N (N NIJN (N (D0N N C) C'? N N rl 'm - '

N- NO (00 -s N-C 0) 0 '0 (00 10 00 (00 '0 0)0Q N ('I N 0) N (o0N '-N toN C') 0 C1N NNR ('0 ('N N CV (9 N

'-(0 N-U (0( 0( N-U N(((0 C) )0 00 ((D0 X 0( (0 (00 ( 10( 4(0 0)( (D)( 4(T (0( 'I( w( '( C)

U)O C'' v1' (D. ' U)O 1. N' O0' 0'- N- ' (0 0) N ( N 00C14 NN N U)4 N-N U) L N N ( NN '-N N M'

-' C') N - a' (D ,(D to( Cl) (0 N-U 4 to0 (0 05 (0 NED 0(0o (ON(0( C% 0o(WN .- N C)N -N 4 ID0N N N U) N 'N N4N C) N~ (DN

Ni(D00 m to 00 4(o00 w w0(D N(0 D ED00 1O0(0(0 N(D0(O N-(0(0c N-(0 00 w00( w 00 N- r ta 0 11 CRr TFt ' -N L N N- C' N

CD 0 10 m0 N-C')N-~OC)NN- D0 00)D0 .- 0) 0 0C)N)0 NN-mN 0O 0 U)oN~~~0) N0,m'CN o)'N N'C'NN00 wCN100 0N oC)N (00N-w N C')Z( ') a an a'~~~~ - a , ar a - l 1a a' a C- a .- C9 a, a

,NO-U '-'-U N-U -U) (a ) C.U '' 1 0'-U N-U N-U) 00U 'U

N40 04) N-40 C-V) -V40 (0 V ') 40 Nv0 04q0)n I40 100V) q 40 (04 C0 lv0 ) V 9 o Q mv 0 vI ) cl ) c Tmt '7C

M C'0 0'o (0a1o0l)0 C4mC) 0 ) 10 n00 m'CI 0 0 mC'0 ; 0 m 4av 0n0 q 4C0 CbM 0 It)1 V)C1 in01 0 MC')U 0 4mU 0) 0 Ln -1 KC)U m'C'U to0en1 N-)U 0M10 1 0NC1,pr- NN'- 1 'ON' '- ON'- 0N' ON .- N' 4L c)n ? 91r C ')4 P n '-TC,) 91' UN - -N-N'- a? '91 Op9 Op'9 1 7 ) ' 1 ~ 1 '

0)00 00L0 3 0 .0 100 m00 00 a00 0 0 m C)0 U) VU -) -)- V) 4U NUr NU)?( (10 1010 ? 010. U)U) LU

0) (D m)00 w0)( 00 0 ( N N0)(0 0a( PN-0)( a) 0)( (0a)(CD N0( M )(0

-, 7 0 -0 IT 0 Z U) (0 N 0 0 0 ' ' 4U 0 N 4'a a4'' 4a 4 a a 4aa U) a 10aaUOU. ~ a ) a Ia a 4 WO 2010/037119 PCT/US2009/058843

4' (D UO a) t-M vm0 0 ) 0 - N 0 C C D C ( D CD - NI'

IT0) Q m00 co0) NO) r-0) rO) (00 Ita '0) 00)0 NO ) (0) (00) C' O No ON ON NN C'N CN qO NN ON O N NN

0) Cl) (7 DO) rO) NO)) n 0) 0J) NO ) 'mO (D) 'O 0) '

(O 0 C) 0) COO)D Co0 '0) ( DO O) (3 (D C)0 0) 'O

(D CIT C' LO - D' C' DO (0 C ) 0)0 at') (')

- - N 9 r N

a-( D ( NJ (0( 77 6 C"D (0 0(0 m)( r( NC0 m0( COC mD Q t , O L ) (DC') c i (DC' Co'U -D m 9m Cn') (Cv m C' (DC' m A' NC, co D') ac ' 0

NO 0a) 0) 0) w-C (0m 0)0) c (PO) (D (DOC) 0)D 0 m)0m N A O (0 - ( (D LO

NCD ' C0 O OWLo) t r-( W(0 (o( 00 to D 'J) O( (0 Lo 0) C' N N 'C' 0) O 0 r N 7 C') 2 CN N -

N 0) ra) mm 00) 00 Na ) NNO 00) aC) 00 0) 0 0) vO cD' mD' (C') ":rC' C') NC' m CV) m C' ) (DC') m (D' (C') CC') 'OC'

04 N ) N N C') a- C' N

N N CON CON ON Nl ?N O)NC N NNl C- NN N N (ON NCIN 'ON oN ( N C14 N N rNN NN r, " N - 'N N 'N NN O NO (DO0 N- 0 V') C') 0 NO 'O (D0D NO0 NO (OD0 a- NN ON ON m)N (O)N Lo0N 3) N (ON (O N IqN a1) NN N3 NN CD ON C) D C')N O)NIVr C) NN Nr 0)

(( 0 0 N NW ( NW CW C')( 19 OW OIOW (

r O ~ ~ 0 00 (0 N O '0 'JO COO 00 00NO (O

m- Or E Dr r ( (Dr a- ) C .4 Nr ) Or Dr (N N N go4 ON WN 4 (ON N ND.*N NN N aN N N N N N CN C') C14')

O) o OW) CD( 'O (D 0E OWD- o IT( )( (D0 r NW NW (D ( IT0 ID wW D C - N N ON O N NN O1 r N (0N N N (ON (DN 0)N aN WN N W(9 NQ 9 (09 '9 (9 W9 r(9 w'( ('1(9 0( '( 0(9

w NW0 00 0 00-O C(DOD CD(0(0 E G N 't CDW ID (0(O t ((0c N (D( D (OW V W( NON ON, CrO C')O0 rON ( WON NO ~0 r VO) N COO (DO ar O C) N V) NC-4 Na- 'r' r ' r ' 'r' 0 r- ' -C)

m0~ 777~ CDE) O0) 0)) ( ) N a N0) 00) 0) (D)' W0)r 'OOj N0)O (00) C'c0 O)O 0a)C) 0 0() 0 )0O 0 m)0 OD00)0 0)09)0 a) a 0 OD0)0 N-0)0

NCr rO 0' 2~ r 0 N0 (DO C? - ~- N Inr C- N IN- NIr 0 N ' 1 a - r

IT D ao 10) N ) 11 O)q0 a)0 1-O .1O aO NCD ~ N -1C N~0 (0 D ( 0 0 - (0 N ( - 0 r (0CD t, Nq0 a (9 ( 0 OD IT

rM No ( m 0))0 m0 00(cl) 0 00(l0 M0( m )00 0)(0 00 '70 n0 OC 0(0 ra-)0C 0)N UNr ) N rN n NN N0V)r CDlr ONm NM ~ C-(N [ C' 0) [(9 -9 r N9 00) N - (9 e4 C' C(9

a? 'N r'? TN a N N4'?N N'N N' r'N N' r N T' r'

O) N0) CDD ) (D)0 N W C0W 0)D N)O 0O (OW N NO) C O aY aq a an C ao a 1Dr a- a) a (D r a mr N N aq *1c r a Or D a ~ C a- ID aN'(~

I JC) V)'n I~c 0 N ( 0) C0 a) 0 r N 0 4 C 0 a~~a a a C a O aa C a CN0 a a a a WO 2010/037119 PCT/US2009/058843

On 0 N o CD 0 N v w f ' N N N P N oo 0 0 o co o 0o

N, 9! 0) 00 00 W0 m0 .m 0 0 ) NO mOND C) 00 m0 w- D 0)c -m 0D 00 ON v N N.v N v DNq L I-N ON .N nN v N

N D0 n (D 0) 00 a0 00 G 0( D 0 D0 0 v .-0 00 0) Nm 0? 00 00 LO0 0 00 0 0 00 -O 00 00 00C Ir

coC)0 00 NOM 00 00 0) NO 00 00 C-OC) NO 00 D OD w 0 oC :)w w V0 00 00 (0 0 to (D 00 00 00 00 m0 00 .- 0 NO 00 C 00oC 00 1? 00 N'~ 0 co ~ ~ ~w~ ~C I?* I? ' N -S I N

- N- 0 0- . .- N9. N N'-~~~~; N-C ~

00 004 00 0) .0) NO to ;0N00 00 0

O0 0 00 0D O0 OD 00 00 Lo C) 0*M0 00 OD M 0) w 0 c r2 O ro 00ILf 0C' U) 00) NO 00 V0

m 7) 00 00 .- 0 00 00 oQ C* 00 NO 0) 00 ) CD 00 0 r ( 0 N v 0 v go 0 N N ? 0 N N7 - N - ~

Il RI IT t- 11 co 0) vv wT

N0 0 00 V No CIO 0mN 0 00 00

0) N ) 0r ) ) 0) 0) 0 M N(3 0 0 N ODG

N3 r- (1 - r )- 04 - P) 11 V - N U , c N N 0CDr

04 U) N - N N N 0 N N2. I? C? IIS

ON 0N -N ' N O N N N vN 4 00N ON ON ON 0 N -' 0 r- v 0 1-N I N I?

N' 00 - 00 No 00 (0 0o0 0o ' 0 00 00 00 00

N O Lr - N 0 N IT rN r-C4 0

t0 0oto0 00 0n 00 0 ,w ,w 0 00 0 0M ID 00 -0 00 N O 0 0c 0 0 0 qN t zN ."0 00 00 00 c N 0T 00N S N O 0 v~ N c? I?~ IV ~ wI !g N'9

.0 (0 00 t, wO00 N 00 00 00 O0 t0 0C.--O 0 00 - (D E.- NOD 0- 0 co co 0. 0.D Go MC O 0OC - N -Fr N U -F N2 1 0 N C r qC Nr.Fr " N or

0 )0 0 )0 0)0 0 0 C 00 NOT NO 0 0M0 0 0 ON NN NNCN ON ON pN mON) ')f .r-n CD9ON ON)ON 0(9C') 0(9C)( N( ( (9 ( 9 (

"o- A'- 0.-0 .- - Nm- o t2 'n - 0 ,DS Lon) .- 0 D 0, IZ !9-- - r IV No .- - - 0 . C?0.-CC C? IO' NO LO' 00 N0.I? - 0 - 0- 0 0 00 00.

N N a ? .- a C4 .- 17 N r ' a? a? N T

m O- N'0 0-m0 .O- m .- '.0 00 ) C 0 NCr 00 0 OrD M .mc0 w0 - '-0. NO 00. NM.- NO. N NO NO' 00- NO. C1 00C C' N 1a C? a?

0 0 (9) N 0 0 0 ( 0 0 C. (9 0 rl 0 O O N

0 000 00 l0 NO 00a)0 000 000 000 .0 O0000 0 0 0 0 -00 NOO 00 NT C.- L9 O 000 00 00

7 9 ( cr m9 0-( m9 N 6 ( N) ( N) Q CN go CD1 ( P 'm (9 0) (9 N (

- NO 00' 0------00 - -N - N--- 0--- 0- 0.

C,) 0c? 0 CC 0~~~- N I TO,0 0 N 0 WO 2010/037119 PCT/US2009/058843

co 0m 0 C4 m to l N co m)

co m 0 0) 0 0) 0DC) 0 NM C 0) 0)m 0 W )

ON '-N ON ON vN N v NN vN ON vIN NNv C? C? (0 N C C? C') '2 N' C'

(0) m (0o0) C00 Go0 m (0m (D0) (0 .O (0 '0) 0-m 0 m (N 0 O (ON N? N (OenL N CN NN VON C ('-Nr (D N C? VON ON 0 N C? C? CI C I T

(n0) NO (0D (00 NOc m 0 (D0) to00) 0O (n 00 FF0 v0 0(0 0 ( OW to0 cow w 0 W W( OW 0 -0 (0(TO (( 0 (0 0C) m ') NC' ( C C) C C' OC' C') (C') (C') C ') D C? N' I ' C~' ' T' IT' 7' 7

(0 0 (00~ IT0 (0 0 L).O ( ) 00) NO 4 NO OC) 'C) OC' NC' C'C) .C) (f) O') C' O' C) ((

LO) 0)()0 n0 n )1 m0 N C') Nlm 7 '-N LO Ln0,V w C',L n mL ) wt )L m ( NW V0 NW N N 00 0(0 m 0( NW 0(0) 0(0 C')O (0) 00 ) 0) NO (0 NO '0 C') 00 N

On N) "CC'm 0 r n N N n CD 'co -n NDC t N~ N N Cl C) Nv'

m Iq m -I co IT u ) "

0 ' IC' ~NN C 0 ' - N

(D N N ND C'In m w V) N aC) C')a ( N CD C')

(00 00 NO (0 00 m 0 m - (0 00 C)0,O 0

N LO Nn C') U- ) C' ) C') NO C'O)o n *~~C M NN .N O N Cl N CNN (ON CNN N '- mN N

W~ N (0 N t') 'N (O '-N 0)C4 )N 0 N 04N C)N (O NN4 C')C1 - . N N l N F- C') -, V') C) N -C

NOC N (0 O ' w00 C) 0 (00 LN O (00 CD O0( ON r)C') (O0- ON (ON .N NN ON0N Of

o00 00 ( ( -( 0 (00 NW0 (00 (0(0 v(0 V(0

)0 No O 1 00 NO 10 N0 00 U0 (00 00 "O 10

N'- 00 N- O) N'-D( (D OD(0 Go( 0' CD ) N(D IT- ON (0N N )N N M')N (DN 'I)N 0 N ON ~N C4 ON Nr 9 D N NrU C') N c 9 L N , C'R N C'P)C T r,( N I f 4 1 (n I f I

vr0(D0 (0(00 O DD DC O m 0 w w(0 (0( '-'DC C(0 OD 0 (D0 -() 0 '(D0 , (ON ON ONl Y 0 (ON O)C n Tor )N (ON (O N 0N N- 1 I

O NN (0N OO) V O N0) CD 0 ~0N ( 0)ON V WON WONo 4 CON M C'N (

(O)C')N P 0C')N r (0IC')N 1 OC')Nr. 0 C')N Nr C')N P )vC')N 1r NC')N W, oC')N r C') Onf C')N N, -C')Nr NCN ~H N C') C'I?, ' r '

N O (D O V') W~ W9 0 (Om O W) 0 O, C., IMO .-u O N

'- 0 v0 (0 0 N) (0 0 C; V0 N7Q Nv 0 m ( 0" m N 0) VM 04 'T) 0 T (0 N OP N 1? N OP C') NP V I? '2 C) w C'? 1 - N c? ;'

p m'C3 (0O r O 0 CD00 0 C') c 0 w') VC) o C) vmo c')Cm)0 Nf'C)O NC0 q m o N1 N N N Op 1 N Op If N N ) '1 C) 0 S9 N ~C

00( ')0 0)0 (00 WO O O 0 0 0 0 00 00( 00

000 rOC( NO 0 00 000 (00 C000 0cC,00 000 (C' O (000 00

to - i 09 v CDtn - v, CD) N C') 'I (0 ( ( ? '

OD0) ,. c 03~ to s 0,,C 0~ m , m . v 0,. C 0'') 00,, 0 0)' 0)' WO 2010/037119 PCT/US2009/058843

w -7 c N 0) C' l) 4 (In I0- U 0) 0 -0) L N a W 0 v (D 01"T NI 0 0 IT -1 v

(00 ' N N4 C)O NO (0 00 NO (0 0) (0 co C4 IT N4 04 (4 0 4 N4 (04 N4 (0 (4 N'~C I 4' N' N ' C)' N ' C)' m' N' N

44O (04 (D4 'C) 0(4 IT 4 4 4 4 (4 0

N' N o C) N' N'v C C)' N' N) r'' ') C) N'

to00 (0) 410) co0 NOli (00 co) (0) 00 2 ') 00(3 N )m (0) N~jW q. o C 0(D to0 (') t O t (0( -(0 C) (0)-( (D (D 0)

IT I II?

(0oo In (r 0r 0r rW (D0'- v

W)' 0C) 0) 0) C 0) NCD M ') NC (D0) 4C' ) OC') 0'(C) Cto -' ' ) N8 - C) w' to C) C' '

(0) (00 W NO 0)0 r LO 40) 00) (00 ('(0 U 0 4 (0 00 V (0 C') No ND 0l 0) C) M 4 N m 01 M NV 0 0 (0 V * C') N C N N C' C) VN IN C')

0 67 0(0 -9( 6 T0( C- T N(0 T7'( (D( (0o ('0 (0 (( C P- 0 It ( - 0 4- 0 4N CC? I) 'l N. N I?)N NI?

N C'O C't) 0 L o) Nltf C' ) Nl C') ED) N) ID) N CW

ON 0 UNWN') NNC 1 ') C-N OND 9) 9N ON ON WN 0N NoC f C M CO') ITC') L'- a l 1 N C) N n Nl 'l C') C l) g

N? 40 C'(' NO C'0 NO (0 0 C') 00 NO 00 ?

r- (0N (N0Nr - fC' (' (0N r.. m') 1, 1, N (0N NN) C)0 9) N ON4 NlN O N OmN ONC1 9 N N )N m- -Ni ONCl C Cj I

(0(0 7(0 NW- 7(0 NW7 T''( 0(0 F')6 N-( 00 NW0 (1

N) U)0 oN ON C4 ITN (0)N O3N W(04 nN O N (ON4 r (1N NN4( (0)

- ') C) N C') NY W' C') No -C'

C? C? C C? C

ON ON CD 4N (0N ()N ON N N N ON G9 N ON 0

* C C I? C? C IC

0(o( 00 NW r(0 t vc M(( 00( (00( 4(0o (0(0 ((( 4(D( W00( ('0 N NO)N OO WON NONcl (0N a)N NO r-O ON NON WON aO -1 - ' C) - 9 0) N (9 N C ) ' N '- 9 '- C'

400 c OO M0 0 O T 00 Nf DOD (0 400 0D()Go L 00 00 0 400 00 cc0 N O 0, (0C C 3 CD) V7 '71

c00' 0(' N' N o NWF- NW'N l v ,n-M-1 (0- C'). 0 WW) 00 I00- (

7 N7 ''7 (''7 C'7 N ' )'7 '7 , 7 '7 ('7 C'' '''7

qN4)0 040 .4ico0 '-40 440 D C 04 (0v4 0 4 0 C)0 m0 N 44 0 co()0 C)C ) ( V r-0 l 0 0) ( N C (0 (0 rC) (0C4) co 4 o (0- 4 0 C')-r -) ifC)F oC' (0N ( N ( C' I?) C ? CI N I'

ID )( L2(( C 40 C')')( (C')(I0 Lon,)( C'C''( In, ')( Lon, ( C') ( 'C)( coC' 0o NN N' C'N WM' WN. IN C'N0 1? 1 ?N' 1'N - C)r (N' '

'7 9) 9 77 9) C) 9 )N )~ ) ~ C'? ) IT ) C' 9r ) q 9)C) v

CD 00 0 IT0 V MO (0V 0 NO NO (0) Nv NOW r v NO (0 ' 0(0? 0)0 (00' (0(' ( P0(op op I?'" 0? C N( C)' N()' 0?0 F,0 0(0? N( '-'N? C-' I? ' ' N N ' ' 'N '' C ') 'N CN I?

00 (00 c l0 NO 400 (0(0 NoW (0)(0 NV)o '(0 v W M O NO 0 00(w l0 W (04' 1-4'-C 0 4'- l C 0to 04- '-4r W4' N4- (0' 04 - (9' to 4' 4)m 0'-' (0r ' I ( 0- 4I 0'-' N 9 l (0' 4 'n (0 . ('('7 N)- N 'ON MC N- N N ' N' C'T C?' N' N7 IT' WJ Co 7-7 U-7 U-7 -C U- U R- U 0 0- 77 N, C) 4 U 0 C (0 c)4 ( m0 (D N3 ( o0 a0 ! (D0 U)0 w0 (D0 N0 '2 w)c)N0 0 WO 2010/037119 PCT/US2009/058843

V) I'. m DM ) m 0 0 a v C') m'0r- Q c

0) C 0) tNO 0) C')0 vOO (-O C0 Va) 040 NC) NN N mN m') CON 0)0 V C'N -N TO 04 ON' CC) N * N C') C'' N N C') N

C) () (0 -) C)) (0 0) N-09) 0 ) 0) t- r-) a) CoO) ) vN N N-N .N C or O CN 1, ON 'IN N-N N C (ON vN C' C ' C'' N N'CC' C)'C? N ' V)' I')

I'T0 0) CO N0) M)0M N0 0) )l (0) 0) a)0C) 0) (D)M0 ) C'0) OD W0( ('2( 00 W OD CO v W (D v0( W 0( 00W"tt N t D(

a)N- N4 C' a?

00 NW 0) 0) 0) V) (0) 0) q'0 V0) 03)0) 00 ) 000) (00) r-C' LO' COC) ITC') .C ) (0C'L) 0 L) L) C') .C' r) N ' (0C' .- C' m 'q '- No-)0?V 9 ' C') C') ) N N CO'l ) cl C')

(0) WVO) U)00) 0) NO) MO ('0 00 NO) (0 COO N-O (00 C' m CM) ND C' N) N C 'CN

V .V C? C? 04VCI C')I I WV N 'I

N-) U) COO) )N-W) V)20 U0) r) V'2) 0O N-) VO mL) ,0) 00) .C) q )' IT' N-C' C') CO' 0) CC' CCo (00 0)0 )' 'q0 C') C' N C) N .- N N

c-)c) ) C'D N) 0) C') .- ) 0) V-

0N NN- CO- N VN- V N- (DN (ON- 0)N m -N N- CON-

f N- 0.t C) C. N.- N.- 0)4- (0- N- LO N N- (N C N co (0. CN O CO (O CO N N C) N- .- i N ND N~fC') C' N N C ') Nr N a-

C'N VN .- ,ci l )N N-N ON N 4 0 (N VD N OfN N-Nci C1N .- N

GoW 0(0( 0W ) W (( 0 .CW NOW(D IW ( (0(0 (DWt0 (0 (D NCW NWC WO- N N'TONO- ION 0 ON W77 N- .- ' CN-rC W N ) -C')N COONC'. ') COONN C) .- C') COON-- ' 0.C' V--C)) 04 N - --~ ~ ~- ~- C------C? - - -

N0). 00 00.- 0) 0 r 0' 0 ' go 0)- O.- (0)c 00' V -4 NOF N ' N ' C- 'D C' 'o Nl V) ' N ' N-

to vW COWD NWE NU (0m w-0. 10 . V(0 in. N-W (0 0 OC. O).0 ~ C'(D D W-'.'. NV WO'. (0 . N) . 04C). C')' IC'T - NCN. COC')l0

C')V OCN 'I NO 0C) N-N) OC') NO Go') NC') QC2 NC) 0OO -' (90 VV0 ) TVO (0) VV) ')O COV) C)V),) 0 O NV0 O VO)O (y CO N 0 N O VU IT I?( ( 0 ' ) ( 0 C

C 0 )C' CO) (DC'Q)0 O 0 00 V c ')( C' a NC) V WCO .- C'W(W COC')CW N-Cto O 0)C 0 0)C'C to 'N NI ) (N N- ooN. 0N. (ON. N ON CN. N . C'N0 CL ) Cn ) N 9) 9 9 004C , 9) 0 ) m M) V ) Cl ) ' 9)

0( o (4 0 . c ')(0 VCD O)CO. N-)Q - C') 0 )CO r c .CDr 0(0. NCO. m 0 (.- a N-O0 CO N'N m-N C''N ''N N'N- N'N N'C.-') N- .- N. .'N C''-

M0. )' V CV W. ' - W V -V 'V V.~~~~~~~~ -) C'V. (0.

-~~I 0 '-- W CO) t O N- OD 0 0 to Nw C' VCI O C? C, 0 w w- w ?- C? 9- WO 2010/037119 PCT/US2009/058843

M' ' ' ' ' N N N Nq N m'

C O) ND 00 N) NO) 00m 00 l .0) C40 04 N4r w 't4 04 m4 00z) 04 94 04 04 C') 04 44

4'm0) 0 m 0 NQ 0'a 0 co N' N'm N' 0D 0 CD~ 0D c4 04w (D to 04 04 4 '-T w. ON 4N N VN 0N-N C?~ v- ON p- -n rN ON 0 0' N' N' N ' 0' 0 '

* I I I I

00 0D 0) 40 0) - ) M0 00 0) N- NO 0 0 40

0'- 0- ' V)0. 0 0- N-'to- .-. C-') C ) Lfr Do

No .- w w- NV to CDN

00 0 r) No 00) 00 00) 00 (D cc)0 N4 ND r-00 ' - N

00~~~C 40 C? - 00 '00 00 NON N-O 00mtn a 00 L V 00 00 00 O LO-O 00 NO m00 IT N-v 0 0 N- 0t Nt. , N- q , 0 . 0 0 0 - 0 N 0 0 N 0 N

0 0 0 0 0 4 N- 0 m N- N C?? I?C? C

N N N 0 D N to4 0 N 0l C? C? I I? C?

00 00 .O 00 00 N- 00 00 '0 7077 0 00 0 - N 0 - 00 N 0 N

LONci ON ODN ONI ON O4(N O N ON OI DN a)4DN O)N ON l .0) - - - 0 '- 0 N 0 C? II'

00) N0 00 ) 00 40 00 0 0 00 '0 - 00

T Nl N o 0 N 0 Nl 0

ON ON ON Cll N-I ON N-N ON T N LaON F4 N N 00(D U 'V-D L) o V N' ( 'D V' 'V (D ('V coV N0 000 00 0 N 0 0 NO 0 , 40, N-O N- 000 N-0 - ~ C N N.N - N N OC '

0 V ' 0D c I 0 co N (D NO L D r Dc - V N 'o N- ' o r- V N 0 CD 0 ' Do F NvrOP. 0 N O, 0- N-- Tr O. 0'o'' 0 A0 0.- r- w.- t, C,' 40.a,. 00 N2 '- ' 00wr- 00' m ~vo n t 00. 00-C'-D -D 00' 00- - - a-0 - 00- 00O M -O 00'

0 0 0 0 0 0 0 0) 0 m 0 .1 N 'V N 'VCl 'D co 0 0 MV NM CN 0? 0?

0 0 00 00 00 0 r0 0 000 . 0 1 r 0 000 000 000) C' V C? ' 7 ' ' V?' 'V ' V C? ' ~ V ~ 'V C V f ' C?

0001 0 00 c 00 000v T N G 00 N00T0 !2 00 00 00 0'100 400

0' O r NOM 00 m o w0' q N 40'- m~ en N .

r 0-' N-' 0 - M44'- co gNC4 N 04' 04' 04' N C4r 04'-c

0l ag 0 - o0C l D L 0 co 00co 0c (AO 0 -n 0 C4 0 C 0 N- 0 I 0) 0 -~~~~a NN N N N N r~~I - ' r.. 1 WO 2010/037119 PCT/US2009/058843

G Go ) m) a, N) v CD N o0 om mm

(00 0?0) NO C tO 0 'O (OC N ) (00 (OC ITO NDO C-04 (00) 'iC

m) )D co mN N 0 N (MN N NN m N PN m r -Ncl N' N' '-o0i 00N' C ' '' N (.IJC')S 'V ~ C ,) C') C? WV W V V N V V () ;0) NO0-) NOm M N '00 c '-D) N)0) v(0) m00) 00 NDmmO ) C') C ' N C ' C ' )' m U) w-C C')O .- 0 C 0 m0 cO) M O 0)0 to W o co' 0 0 Om CvC? (0( 0 ' D C C 0 C ? (OW 'T 'o I? m) W C? (OWc?( N 'C), rN I I 0) N CD

C O N (O C)' 0N NNO)* 0N 0)N C1N to N Lfl NN 0.0)P 1r -~ a?(O N2 N N C) N- N' N

0D CDO VC) C) N ) 0)0 (D (Cl) 0) C- -q) C)O 0o

(00 NC) VCL c L ( ) m W) wtO w ) (00 00) LO 0 C)n C) 00) (9 N 0 C)1 V(m 0 N n' 0 (0 V) m ' N N C') C)N C'I'? C

m C) C) mC) N ND0 C 0 0 m N 0) N V C)

N, No0t l ) Nn Cnu ) (D U) to ND COwLn L ') N

C) N C) NN) V )C C) N

coW m0( CO (0 co( W(m0 (OW m i ND OWC (DW m OW D NW V NN WN NN (NC)Cl r)N (O ON N a NN ) NN C V) 'N N o l Cl) EDC)

to0 r (0 N 0)0 NO I) (0 C ' 0C0 LO N' 0N (ON CN 4 )N (N N N (ON n N C)N ON CA ) N (ON U IC CONNn C) CN

N'0 C o l C' 0 N 0 0) (ON VNl ( N V 04 N N N 9) N') C)' (0 N.4 P Nf

N (OW NW NW4 (OW C) 0)W O)W NW NW (OW

0 NO0 0 0 C Vq (00 o )0 C) mt (O - LON N p NN (O O N (ON C)N NN (DN - (ON N C N 0 r) N N' C) C N C) C) OW N W (( CWON C)VN C(0 C(0 1W (O COW VN N N (ON O)N (O C W) WD O c y N ( O r V (W ( ( N CW OqV NI ItCN Wo N CON w N WWON ( oUON ( DW( W4ooNv) W 0 WO NO WO4ITN NO WON C 4 (o C ) W W CONWDW) D ( D 0 ) N.C WrC V.C) P.C 01-C (OrC NrC 9C 0. N C)) NC) (-C .- N -2 N 9- C)-)-.CN ) N W '

T I I I.)' (0 ) I If0) N0) WO)- IO ) mO) I - )'-1 N C- I )I 0)(00 P(0OD)0 00 NC) VC) 0)0) N(oc0)C O DCD 0 O) 0 (000 N ) (00CDO O

' C)j ' C) ' m 0 r ' '0 N) '~ N) ' C ' 040 N C) ') tOr NSm ). C)WV'Z (O mO(N m-( (-C 0r enW-0) Nr ' en N'r C)'. o C) 'l - V'r (M 'lr 'nr C)'r '0 V)P r NCV C) C'C) C)V W ) CV W )V ((V (C C)') 0C) NC) C) VVC VV) 0VO O~) NO) )V ) NC V) N co rV) NVO M (0O N Col ~ co" N0 C,' C? 9 . ) ) ' C ' ? I? I? C, 0? N0 C? (9

0 v~ C) 0 C) N C) CD NT a0V ( ) W N 0) V 0 N,0 v-( v O WO WO O OWCO0WW 0( O WOWNOr

't o 0C 00 m0 ('O m too WOO CDOO 0 c )00 anO 0 )vco0 w n0 m CW C)OO N'W-4 p 'N N' N' NN 'N N'N NN 'N N' N0.-' .'N r

NV' -7r N5 UO O) V' ) 0)V cV - NV' OV0M W

ODW N 0 0 0 ' N O C) 0 V 0)00V) DC C) Cf) C ) C) C ,0 ) C) CO0 c) C)0 C) cp ? qI C f N I OD 1? 0 L? L? L? r - 7 I ' WO 2010/037119 PCT/US2009/058843

Ch 11 N M) U) wP c' ( )

00) 0 0) co0 4Dv0) w00) m)0m 00) W)0) M 14 N0 NO4 0 D0) ) 1N N- C') ON ON co v0 0Ct' wv N N-N NN N v N v 0 4)17 0

a) 0) m 0) r, 0) 0)D N0) (00 o ) N0 00Q ) v40) (00) to0 m 0 N a)l (0? N-N IflN NNC- CN N NN 'T w0 (N w- ( c I?' - C) C)' N'N C) C') C')

'-0) N0) 0 0) N-0M (D0 m (c00) (00C) 00 ) '0) 40) v40) 0 0) 0D '-C C) co w coC(D (D .4w (0D NC' to' CC CC) (D' N-' to4 E * ' ' r 'r C') C') * 'T' C?

- -17 7 7 NF7 N-7 7) 4 - 6 Nt r- MC') N No CC) N 0 '- ') N 77 CN N0 (' N'

C'0 ~0) (0 0) Nj0) C')) N040) to00) 40 (00 0)) m)0 00) v (0( LN 0)( (0O COU)L 0) 0 m')0 c0( '-( C'O ( n0( Lo( 0) -C) co) N-C -C) m 0I C) (0O m (I W( (0C' (OC' w-' M 2 I IS I CS C

N-0 N0) 00) 0)0) w0 CA 0 m0) (0 v0 m -0 P0 Q4P. N C') C ( C'? C'? N-? N C) N- N- NC N N ~~ ~ ~~~' ') N N C' ' ) N C

C)U 4(O v 0( U)( Go( 4O(0In N ) m)( m ( (0 (0( LO( co(0D O U (04v N4 (0 (04 v- '-4 v- w0 0v4 0 C N ( NM 0 C C' 0 - N- N 0

C) (3 -N0 0C C') N V N CY) C'M ) C) C) 0 P NA 9) I~ S n r04 I N4 N4 NC4 [N - )4 04 0 N4 4 N 0

NV (00 ) 0)0 NOLo0 ) 0 LD 0) 0 40) N-0 ) (00) (00 (00 N N)C N -C (0(0EDN N 0)C) N0 C)C N vC) N-C C)C C)C NC (0t L N N 0 *N L) N IT C) C) N N C4 C) 04 C' W)-N C) S I? I? C? CC I? I?

a) L0N I ci 0 Nl ON 4 LoN 4N cl (aN CN ON CN CN (0N (0 G t, C) Dr N C) Op C))1 N N C ) C) 9 C) N C? N I III

N-- 0- N 'N-c N 1-N- (0N (N- NN- NN O- ( N

C) N 00 )0 (00 (0 NC4 C 00 C)m00 C (0) 4N NNm N 0 -N 4 N ON N-N (0N C1N (Y)N 'N

14 N C 4C w(T0(0 t N0 (0( w-( (0( NID N-(r0 N- (D C0 '-(0 w-( 0( V N-N N0 C) N~ NNl N N N-N N-N~ -N ONJ (0 (0 0 WN (0 '-o5 (9 eq 909 v9 99) 0Cj(9 N 9 i

* I N C M C *IC

NON-, v CN CON-r w 0N (0C N 0CN CN- 0 ON .- N coCwN w 0N N-ON (0 CD) C)N 2 C C) .- N 2 . N- 9 - q -

N m O IT0) 0 N-0)a c N0)o 00) 0 v0)0 00 00 0)0 (0O0M0 40)0400 O4)0 C) C)C-' N N ' ' C ) ' ) , ' ) C') ' C-) r, C)- ' '- C

4 0 1 -. t- D l f M l .-- ( .- - MN1r0 (0U (0( C)(0 (( V0( m0( m'0 ( N0 N . ( ' N I? 0. C?0.4(c?00- ((. 00 ( ' C(. )0-

C? C? I?) C C) N C M C) ? ) ' N ' C

m79 Nq 0) 0 v) 0) IT 0)' 91 ) N' 91 ) C' v9) 'q (D (I9) N79) C14 9) (7

a?0 NC N C N-C 400 p 0 N 0p)T 0?0 1N C ( 0 )0 0)0 N-4 CO D

n V)(0 (o0 0 .- 0)(0 (0)( M 0( 40)(0 77)( NU( V0( 77)( -6 )( (00C)(0 (0I' (0. 0N. (0' (0 04' )O (0 N0 m If (NiC~l n U) (NI (O NI N') I N

C) N 0 O IU11 C0111NI NI( I0 4 C0 N C ') M 4 N (0

4O 4 0 0 of N 0 v im 0 4i 0 w o (0 0 m0 t--0 I( W ) - 0 W) a) %n N '-9 m' '? L?~~ L? J L.. L9J - LO - W W WO 2010/037119 PCT/US2009/058843

m 00 m m C) 0) CD m N C 0 ) - m 0 to vD ND IT N D VD CD IT N T T N v m

a) (,0) c0) NO) CD 0) m (a6m CDO ('O m -0 LO w ) v40) 0) NO0) 4 6)4 w ? (. C4 43 (64 CN4 N4 (0D N4 mN4C' C (n 4 04 wNl C* C? C') I? CI T I

m' .- C m' m4 m) (4 CA4 (04 N- 0 0 v 44mm 0 N4C" - 0 -4 to LNo) t (O COO 00 00o ) ED 00w (0 0 (N) to 6 4OD CDO C? 0 N' N' C) N0 4' N' 0' N N' N N' '

0 n (0 (0 00 (0D NC' (0 0 00 N) ') .- O LA

N,4 'o ND NI - N

0) N) lAO0) 6)) (06o ) co0 IT6 ( 0 m6 06 O) 0) 6)6)D tnC' (OC' LA, NC' m0 )W LO C')C') mO' CC'I) NC' TW) w W) (0 NC)

(0 c N c0 .(Y OD) L Y)0 C')( 00 .CO 0:) CO 0 (0(0 (00 Q)V (op0 V(

a)0)) 4q0) NO) C) 0) (D0 ) NO0) ) 0) 06) co0) NO)m 0) C') 0)N, C'w' LO

LA NLA) 0L (OL 0 0 U) LA c (( (OLO (00 LO 0(D tn ( LAL M M ) LA v (04 v 0 a) (04 v- v0 IT ' v ' 04 04 m IT (0IT C'o LA NP . L N N - N 0 N N

C'D0) v 9) L) ND C' l ) C)C ) ) 0 ) 0 7 0' C'0)C' )

0) (0l) 4) (0Y ) (06Cl C) 0) 00 C) LAO m (00C) (6) O6) ) O)

Ii ' 'I'

L 0( 00 0(0 0 (LO 0 In LA .LA w 00 COL (LA 0(o m -L 0(0C) N NN wO I N ON LAN Cr N r N f ) N LA N N NN NN ) ON N4 0-N (9N O N N L AN o N ON LN -N ON '- N (D0N

N 2 -a NO LAO NO (00 NO NO NOf NO N7 O (0 ( 0 0 0N .- N N (o N -N (ON NN LAN 0 NCJN ON OD Q) N 0NN LN ODN I N N ON 4N NN ON N NN N (ON

(0 -( 0(0o N(0 (( 'TO 6( NCO 0 ) ( 0 )( LA( V(0 LAC o

N Nt LAN ON .N N ON )N N ON (N LAN (0 n ' o 0 0 0 N e 0o NgoC)

(0 40 N( (O (0 .0 0(0 Lo( F )( 0ID 6)O N(2 N. NNi N (N L n 6 N 0N CN q LN CLN (ON LN (ONC

* C4* C ? N C ?

to( I C 0(0 N (0(0D W00( 6C( V(0( to 0 0 (0w LA(0(0 0 ( w0( 4(D ON r-O 40i N CON LON 0 o .- ITN CON NO N L)N c l wN IT0N4 N

4 - N 0 N. r- - 0 11 'o a '1a) 0 'l 0 N C')

N) R"- NO . (0)o 6) )- "- 6) O). N ) - 6) - NO v NOA V0 . 0 )- NO)

6)o )0 a) (0)0 v m)0 0-) 0 --) 06) o00 .-) NO0 ( 0 6 )0 ))

' ' o w ' I'

04004- N0 0 0 (00 (004 002 (00 404 t2 00 4 00 c (9 46) .- 4) L46 CN,) M40 40) N4 N O M46 ) N4) (06 06) N CN 0 LA C0 C0 (0i ) W) 0 N Iq ') ') ' ' 1 0C'()N ' ( NN a C '0 1 ' a) MI 0 P (0 0 (w - ( ' 0 . 0 (

(9 9 9 C? C?9C 9 (9 C 9 9 N 9 ( ' 9

0( 40( NO( 00( o0(0 6 0 0 (0 00m(0 mA( (0 N( NO(0 0 00 NOO 00n n 0 0)00m 6)0 000 (000 0)0to 0 (00 0 m 0 NOmO 0m NO 6)0 C-4 CLA. N VA) CL.4 .L. .- 0. 64 )N- 0L r 0(N 0 ) COLA. C 0)L 2 4. 0(0

0)( 06C )0 0)C moo 0 N60 (0C 4)0 4(D a C 00 N0( N-6)0 0 N. 09 (00? (0. 6)4. 44 SN4.- (04 U )4 4D4.- ( 97- 9,7. (04?

C. N2 (0f U6v 0 LA LA 7 A LA 7.a(0 0 N 7 N WO 2010/037119 PCT/US2009/058843

V) M U V. 00 m 0 - C4d 0 0D (a I. 0 0 co

(0) NO)i 00 NO)D LO ) O) 0D)0) LAO m 0 LAO)D CNO )0) (Nr - (IN (ON v - ON ('T(N,1 -q ON V ON -q q IN OM 'qN~ (4)'0N N** A4) C N' C?

NN 0N ON ON ON NA NNmN mO NA ON NJ

( 0(0 rO r (0 NO 0 0)0 0(0 N o NO LAO

N' N- (C, N

00m ) 0 0 N 00 0 -C40 0*0 00 NOm ( CD N~Q - a) N24 (,o I N , N , N

N N

CO' N . 0 N)~ ~ 1 a) 0 ' 7 Iq Q

0) 0) o0) - 0) (00 M I c) 00 0P) NO) 00mcm C0) LAO) 00) (00L m)c) 0) o 0O (00 C) (00r OV N O (4 L) c O 0U 0) 10 00 No N9 C' r 0D Nl W- (Y CCo v) N' N 00) (4)

0l CD 0 ) (a) 0 . a) 0- m 0)

I I inI$ 0)0 00 Lo 00V No (0 00 NO 0l 0 LAO NO .e N NN ON N ON LA NN LN r-N .- O N

I -a ItI 0- IT 0' N'

0(V 00) 0(0 M) 0000 NO ( (0 ND o 0) 0

Caa? C? I I

0.- La V ) LA 4) c N mt N. to' (). N- N4) N -N O1mN (n , N LAN P, 14N RN I N LAN W N (N N .- C'4 a, o '- N N (4) (4N C,) N I

(0 0 0 0 0 1( ) 0 (0 0 -O (4) 0 ON N ON LA O CN c N0) OlN 0 N0('I ON C-N (N

19) (D (9) ED049)o a DC) 09 00 )0 0~~ ~C(00 '0I ;!O '-(0 w( 0(0 N(0 N0 0 000

-- ) 1'O 0- c ) Ao r4 CD N.I '(a) Nm a 1 0 w-Lca) 0-' N (4 o ( D. - N- N C - ). - ' N - a - N-) 0,'I0. ) 00)00) 't). LA). 00) N NO) NO).N0'

In ) 10) I0) ID) M0)0 to 0 NW) 0)0 m ID0D)0 10) 100 '00 ON 4N (N m04N LO N N ON WO cm (DN LAN 0O mAO CA NN

a a ( I C) a - N I. I S~~I (a) aI I L.a! a a a -&aa -A (0L AL v-0 c )- w .L 0wL NL (0L 0L co L LA Nt-L (00- NO- F . NON 0 0 0). 1 - 0) N 0'(0- N0.- )0' q0 a2 ' 2 t ) 2m0L)Ll t N a Na Q()1 - . ' -6 Ta - v' -a 7 7 -6 a ( 0 w() m() o )0 m 1or0a G 0)1 C, m)0 a,1 No 4O NC0) 'v'( )0 '0 1) .'09 0 o) 5 0 v0 (D0 0 10 n 0) 00 1) 01) N0 (0) 0, 0- 0 fr 0 a V9 0 0 0 a 02 c ) N 02 N 0r (A V) N ' If 0

7 N ao aD aT aO aa a) 7 4 v)0 C.) 0 ( ) 0 4 0 . O o 040 (0o 0 0 0 m' O wC4) IT 9) Ln9) '7 ) 9) a 9) 7' N) C) N~ Cl a N ) IT m ) M ) C7 -0 000 000 D CPI a 0) MOD 00 (00 NIt mO 00 (0 0 0)0

OC) 0 v0)m 0 )0)-60 N0)( 0 0)0m 0q .-) NO 0 ) m N0) 0 m O) 0) 0)0C 01. tN V)o - Nn (01'V- (0I) U)~ 01'01 m1 U). N'o.

0) - N 0n to L 0 N 0 0 0) 0 co m N4 N N N 0l N N) N 0 0 N N NT a a ' -a a a . a a a ' a a a a a a ' a a - a a a WO 2010/037119 PCT/US2009/058843

V, co.(0 p0 0 in toI F0U C

(0 (q0 m0) 9) 0) v) 0) 0)Itm 03 0o)C 0) 0 m )

(n0 v (00v ) v0)0)I v0 v00 v00 IT) v0 v00 00IT 0)0 v 0o N c-N C') 00 (0N qN (N rNN Na C. N' 1 C N' - V N N

00) c (0 (0) N 0) w00) 0 0) N0) m0 00) 0 00) N 0) GoN .- N wA NNC C)N -N LN 0) N r- , (N N'C N' N' N N' N' (0 'a '' ' N' N

CD0 N0 00C) .0 0) CD0 0D r 'CD 0) 9 () 0 0) C)) (0 I-- 0( ('(D Co (a Nco (D( ( 0( 0oto 0 0(0 Cl) D N (D NC') () 'T ? a ) (0( (0oa 0?a LA) C0(a (0Da 0(a (o0?

IT v m 0 (o a II

00) v00) (00) (0 0) (0 4 N0) V')) N0C) m 0) cn 0) (0 0) 00 (co) C? V 0(am C00 mNPC' ' ) (2' v')

N0) (0)0) N0 () .0) LA) m N0 00) C'0) V) 0 0) (0) m (,0 0 ) LA N 0) 0 N 0 ? () V) ' -~~~' ."r . '

LA .-a NLin (LA IT( (03L .LO ) (nU) 0(0o I-LAo (0 V' ) C A N A a' 0 C,) N NN

L0((0 (00 (00m0) w00Nf) LV0 (D 0)0) C040

q0a L 0( (0(a U U ) (-( (0( OC' W0) (00 (00 L C) (( NC' N , C') t, ND N .- ) - C') Nf l

NC-- -qN C4 LN C'N N LN LN (0N N VLAN NNj

1- (0 (00 (00 C'O (0 (0 0 .0 NO NO LAO 0 0 NO. a) coCN oN ( 0N A (4VN (0N V) N C1N C0N NN4 LAN C14 0)N C1 (N N 't LA - NN N NNCN (N - 0

C)r- (D OD NO ( 'D t-N m v ao a a ao a Da- a a) al 'T 0) a

'( o a( (0 0( 0 ('-( (0 'D( (0 i0 N(0 ( (0

0- N- co 0. N- IT 0 C'- m0 0- -- ( (0 0 'N (N M N c? N (-N C ')N mA C4N

-7 U C' N w 0)( OU N (D nCN( r D C

(N N A NN CVN C1 AiN IT0N V0 C') LAN (0N ONci * 0)N0 N? 09 (9 t9, co T F )

Nw( O (c0C(co0(0 D w00( .(( N0 0 P(0L w 00( co0( toO0t(o (00c L (00 N(0(0o (00( tON .- O (00 0)TOlc o N NON r (OD 00 r-O m0( .0N ( N (0O N

NDC' . 0C' (P Im 0 (M0.CD) . Cm 0 ItC) N.-C) 0 -C') N.-C) 0 ~ C' NC)Oma N M . a M N V)t- MP )r V OMt t0r FI a a r.- t r 0C

()0 0(00 0)0) 03)0 0t- Do 0 !90) (0)0 (00) 00)0l L9) ON ' (a) - a ', ' C') ?

(057.v N W 77Lw 7 - T - (0 0 (0(0T N( 7 70 (0 (0("q. - ) 'T U 7 7 ( 0 q

to a0 OD(IT00) N(00 ) (000 M M ) N-(00) N IT0) 0(V00) M00( V (M v0) 0(0 v 0Q00O

0 0 0 (0 0) (0 m 0 0 ' l ) C)C N ( Itn 0 (0o (0 (oC)0 0( N a O 0 V ag W a m a a co to U) a l n L N N ) r r a n()L

NN ( MN - - N - N N- a)N NC4-a (N N 4N N -N' NN0.-l NN. 79P,?I T T P 0 N 0? LA

(0 0 .0 4 NO 0 NO M 000 (00 0 00F 0 00 00 NO 0 a-) (0 0 A NLA. oLA' Ln0. 0L' ( - rL) .A- LAOO(0(0 L - (LA.

.- (0 OD 0. ( -1 (-0 - (0D 0)CD (0 nc 0)(0 (00 N0 '- (00 - N 0.

w, in q. ) V 0 - C') ( 0) (0 N( 0) (0 30 LA *LA 10 70 ( 70 ) 70 7A -2 a9 a - a a . a a - a a . WO 2010/037119 PCT/US2009/058843

10 M 0 - N to CD r- 0) m

0 )0 0 0 0 0 0 0 0 0 0 - - N N4 N N N N N N N N N

() -) 00 NO m 20 Itm O) N4a) N) 00 m )0 N) NO) m

r- C c 12I CP Pi C.-C in C ~ CJ C i

0O 0) 0) , mO 0 NO) NO Pm coO) wD0) (D0a) CD a) (DOD I q- i (CJ ViN CN 0) ON CN No) C? qN CN (D N CO

-~ N M Mii Oi .C' f f Ci ON (N N CON 0)1' CONlm 00) t)C) I C) ( ) g 0)1' vN ON ON M- ON fir C.4 o N' o - Nr()( 0 D v( Or CD' O O ). (1- (r 1 D 0' w 0 0 nI CO. fir Q?) N. 0)? 02 T~ 0).- 0r If t I IT If v

I? CI C7 CI

0) 00 '0) ) al) )) 00) 0 0) N) 0D0) m 0) mN0) N) N ) 0 R 0 ) ) 0) C O C f ) NN0

flU) .CO) M OCO M C NU) M C 01 ODC 01 N OCOC) 0 c 00 C

NO) coO) -10) 0)) N) (35 o ) w 0) COO) 0)0) (0)0) NCOY ) N 0 01 D Nf ) coi rDf Nfl Of N rl ODf Nf ii fii f N4 N N N (2'? N? ' - C' C

NCOc 101 COCO MC rCO 0) vC OODU) n 0C r OCO 00 C' ( *~~1 0' N fi 0 i . i f i f

NY f ) 1 ) a j NO CO N) 00 COO NO CO ) 2 0) 0 NO N1 ' o C'4 (2) NN m o) CN 0N CO) N NN) l liN M - co tN CON -N) (-N Cl l

NN CY o N CN CO 0N IT N CN O)N N)N 1,9N CO(N NN N m N 0

tC N 21 101 01 0)1 N)1 10(0C NN N fI CO1 NC NN 0)q

a CP CIO.,

fiC 0(0101 10N NCO ). '-( mC010 0)1 -0) OD9 0)1 (01 m)1 1019 C* ' C? IT ?

1010 NCOO 100) NC0 10 NO N(00 N00 1010 1010 NO0 N10 CO4 fiN fON CO N COO N O N NON ) CON CON CON 0)O COO)N N

* N - 1- N N !r- i - N v - ~ - N f - C) r f 100).- NW) C0 r O) w CODO) ID) CO)' NO) ED) fi . NOD. NO). CO(C P Ofi C N fN vN ( CfN 0C')N P N NCN NI N Cl4 NC4N Nl Ni N N, (9 toND (v fi r, (' (9 T f t9 P- Q?

w (D O N (D co oG N NC 0C c 10.- co CO 00 )U 10.-CDN o 00 1C NCO to-C OrC CO U.) -,- Ir CO, 0))1 f- - 10(01-1 N0 COO.n ! - vr - N r No (2 - N

I I c - - I

0) 0 m0) 0 Nm 0 C0) O0 0 ) 0 OD m 0 IT0 N0 OM0 (D a) v m "N 0) 0 m 19,1 C') N- mf t9 m9r N19.-O m

IT00) C OfiC VfC OO NIC NfC C)O OO C1,00 NC)1 ODiC N ODiC fi O ' NN' fiN. NN- M. CiN, flr fiN. NN. 0 )?Nr CON. 7N' 1 t 0) I! 0 f CO -' Cr C 9 vi C'; 0

N100 0 vO 0) vOT0) -0)0 D V 01 0 0) NOV 000 0010) 100TQ 0) 0 001 10

NO NOO NO mO -0 mO NOO '(0w0) 0)0 '00 0)0 0)0

fN NN -' N Q N NN rN '-N N'N C4N 4 C4N NN N' N, c

0r - T0 2 -") r .;r- N ' C OO- 10 - 1 ' 2 - 01

10 (e - 'U w0 N 0 w S0) 0 0 L 0 00O 00 N 6 fiz 'R 2 a) 0 C,2 0)) 0) 0) 0 ~ 00 N 0 N r u co r a 0 'C -1 0 ( 0 u N) WO 2010/037119 PCT/US2009/058843

N Nm N N N N N N N 0N 04

m) (D0 0) 00) 00) m00 (00 M 00 NO (0m0) N0 In Q) (0) m 0 C O0 (0 N 0 ~ w 0i C m) (0C ( N ;- (00 ;N TN ON C ON OCN C) . ?12cON

M0 M (0 (00) 00 D o M (D0) 'l) 0 0) (0D NO) 0 V0) 0CO 00C)

(DO4 NO f00 CD v0 m 0) 10 NO) (0 N 0 0)m w(C0(0U CD (00 w ( o 0( w( R 0om(0 rN(0 W0(0 0 (D VC 1 w ( Q( II w1 NI (0o N i CI v' . C? S I IIr

'- 1- C' (' ' 0- N- C4 N' C'- 0' '- '' (0) ~~~ C, V- N ~r N '- '

0) 0O) M0 (0) 0) (0) NO NOi 00 u (0 IT0 0) COO) C, CN C, IC, C, NC, '0 CC W(0V(m 0 OC C' m ( wC (o0C(C0

C, '-N '- 0 N N r N

0) (00 Q 0 LOD 0 0 0 V0) NO (00) NO m 1M 0) NO (00 M 00) C ) (0 N N (0 C- (0

C, Nn NO N coND VN C, n LNn ' NO o LN) nC4L N v IT 0 v 0 IT v I l

(0 N N N ( .0 '- I 0 N ) M0 (0 N C

a' 10 10 C' c

0 q NO 00 t L LO0 (00 00 n (0 L0O 1 0 )0 (0 PV) O n)

(0 10 '-( (0( (0( '-( 00 (0(0 c (( to( N(0 '- 00 N N N (N Oli N 'n i ( CN N N CN ON CN IN

N6 NF N- w N5 -6 '- - - -6 0

C) i N NNi '-N CO N CN 'N wCN O N 00cl N N O)N LnC-N aN N

0( C 00D(0 (( (0 (01(( ( 00 (( -0 CO( (0N( (0 (00 (00 (O(( T 00 (( t-(NO -(0 0(0

w N0 C, N) N) C, N

I I C? C S

N C' N 0 IN l)N O-N ON CON (00 CN (0N ON4 ON C C C C? C? C9 1C? I?? 19 1 C? C '?

(0 1(0( 0 0 to0( mNto w 00 0 (00( (D(( N(0 t(00 0 0 C,00 (0 ON CON CO (N CON mON COO O N ION NO vi CON 0ON 0)0N N, w - ND t, N9 CD N9 '- N - N- N 0 - N-?

t (000 N)EDODO (D0OD 0)0 0) 00 D v 0 (00 (0 00 OD w 00m IT( c0) a)0 D f 00 (00N q(0 (0(0 C (0NU O0 N 0 NL, r m0N CD(0 0 0 N 0(0 0C, OC) NC, ! N C, ' 0 .-0-2c)-c 7 N ' '-'' ,

0(0' CC .0 C,. .0 .0 I0C O)IC .' 0(0 - CC, 0( ((

() 0 N M0 N c () 0 010 0)0 0 0 (0M0) 0 (1 0 0)0 01Q0 U010) 0 0 (01 q (0) C, (0 ) 0 C? N C?m P7 m t1 (0 m- (0 (0 r1 ( 2m0 0 N2 m

N M '-N' NN' 0' ON' NN- ( N- IN'- u ON' P- O ' (0 ' (0 M? ' C) M ?~ ? ' C? N I? V) I? ~ C? L? ? C? ? C?

'N 7' '-' 'N N' -' -N 'N 'N ''N '-N C0'

v m'- v 0) 0 v 0 '- vI N1 0 V 0 m I 0 1 I' 9) v0 11 tm 0I 0' m 1I 0 0 ( ' - ' 9 'F N ' N ' NT ' 9 ' 0 ' W ' N '

Z C/? Cl - WI

'0 (D 0 7U0 ( Q0 Om (0 ND m ( oC0 n 0 F -6 i7 0V) F)0 N m

06 066 0a 06- 10O 0 OD w- 7 To-7 N ' N 0 N N 0 ' N 0 0 ND , N m N r, N *n N v N 0N0 WO 2010/037119 PCT/US2009/058843

W) f 0 r- 00 0 0 - N ( N N N N N Nl N ( ' ' 0 ( N N Nm N N N 04 NM cm N N N

Q00 N C) IT0) ) 0) N) m NOl ) 00C) (0 0 NO)b (0 0 0) O(00)

(0 C4 (0 04 44 N0 4 0 0) go4 0 D (0

.) (D00) N G) C) (0)0) NC) (0) ; 0) IT0) 40) 40) C0) .- CN N 0N N C')N - C0CN (CN N- C N

(D) IT0) r- ) () co m00 '0) ) ( (00 v0 r040 0)

(oN 0N (NDO (D N ) ( N ON 'N ( N Cn -N N: N Nt N (3- 00 I . a) l [ I)

NO0) v40) M)0) m)0) (00 T 0)0) C)) ( 0 (00D L) NOl) (1)0) CD00) 0UV) C)( Lo 0 v L U) 0) 11) 0) 11) m U DV) (0(0f 0( o to( P-U) N U NC' ) ) N m v C') (0 .) V) )- C C) LN mc

0( N)( C 00 -0 00 (( (0(0 VC 4(0 ((

go0 m O)9 0) 0) 0 0) IT0) (0) (00)D 00C) (o0 (0) to00) 0Y)0) 4 N 0 N) (0C) n C T r I I I "? '?

W) 1 w 't 0g0 0 It c0 '- 0 c N (0- N N (0 NN N

C') D0 C) N0 Lo) N Nv .0 0(m m

In0(0 0 (0 mL m)( Lo( tN-U C')m0 e(0 mCto 0 'U) 0o m( W') O N f))N L)(N ) N -N -N 'oN N (m0 oN (I)04 5) N (ON M0 C,)

N 74 NO (0 0 0 NO (0 V0 0 (0 00 0 0)0 0 a) (Q04 .N NN- (ON 11 0 (N (O N O N co0N (oN v N 0)NC '-N l 4N~~C' NNNN ON C)N N a0 N 0N N N

00 r- C) (D'- (D' N.' (0' (0- ( N- (0D N N' In (D go No C') Nl 04 N I?

7 I 71 0)( ( (0( c 40 0 0 C3O o( (0 (D(0 4(0 0)O 0( 00 N (O N 0)N ( N 0 N cN N N NN a) (0 0)N M CN cm co (D co) (9) V9 to C Dc D 39 D ) ( 9) 0) (D C

N ) (ON (0ON m (0N NON (0N 0N (0O N NO C-4 m N OONC~ (0ON C C0N N 0 N - - N

-t0'- N )- (00 0 0)'t r 00. ~0 - CD0) 00)' 40- 00' 0) (0 O~~~~~~~ (00) "-) (0) 000) -) )) 0 00) N0 0)) 4)

V0 4-m( ( 0 (o m 0 IT m(0 N.m0 O.0 0 -(M0 M0.-0 0 M 0 N M-( 0 0,C D-o

CoU ' 17 '' '?' 0 77 ' C' '7 W

(0 ( (0 (0 N 0 0 (0 N ; ) ( ) ( 0 ( ( N 44 (0 - ( 0 (0 ) NO( 'C' C7 I' C ' N 1? 7 ' C' 7 ' CN

9) 1-) 9) N P' M9 OF99 T W ) T v T 9 '? 9 N 9

N N'N N' '-'N -' (N'N -' '-'N -N 'N 'N -N . (0 (O)0 v F)( o 0 ( coino m0 (D () a( 1 co )0 U0)(0) N0) M0 NC)) ( D NV)(0 OO) 0 00) 0 N'4 N' N'4 0 NI NT I 9l) - I) I INN I N'

9 ? f C? C. C CO CO 0

N ( 0) 0 N - N (0 4 0 N ( I? - N N N N N N

N' (n cN I- N. CD N Nm NI N. N Ni I N. 0 N, I N WO 2010/037119 PCT/US2009/058843

10 In -. 0 ) V) ~ ' 10 W NN N N N N N N N N N C

0 0) U0) N NOm 1 ) to0) (00 m ) 100C) 100 0) s-) 10

(D0 M 1(00) m00) co0) (0 0) 0 0) CID) 0 0 0M )0C) 10D Na 00)

00- 0)1 N10 co0 N(0 C')(tn0 Nb C- C 01 0)1 "(1 01 I4 ') 1

100 00) O) C) (0) N)0 '0) U) 0) &-C0) (0) a(0) 00 (D0'T) '1 N) q N, N

s-U() 101 (D1 co1 10 co 10 101 1(D 0)1 101(0 -0 101(D NGo M 00 N"Om ) C? ) 100 100C) m00 0I? 'T0 C? 0 (04 C? 2 C

N N710 -5 1 7 N -7 010t N0 w)0 (00 100 1) 10) ('- 100 100 ;00 10 (0) L00

s IDN105 M mO 10 00 00 0 0) - 00)- 00 100 '0 ON NLO 'JN f-.1- V) C U N r 0)NLr c) s-N ' N 0) Nv' Lo N~ NN 0 r N ' 'I (

10( 01 101 00 (D10 (D-0 )00 s-0 N 0)D 0)0 N 010 Ia

OD ) 010O (010r 10 '- 04 U) 0 10(0 1yL 00 010 L )0 o 101 N0)I rv ( I Tv L T 1 L~ D N9. 80s-9 0~ 09. 109 R91

T O s-OL 100 100 m - 100 'JO NO NOwwW 0 0 s- (o00 s- (0n NN s-N N sN N)N 'N 1N N N (

-~~( N00() 0 0 q

1-0N U) N (0 N NN) N ODN LN ON 04 N s-N ON 0014 N 0N

(0-1 Js1 0s0 ws1 Os- D OD-1 '5-1 (0 0 U (s-1 NsD1 Ns1 1s OD N- N s- rl 'T s- 'q s- I T1 CI I? 1 C

10)- 00) 10). N0)s s-m-'0s N0)s0)- N)s 0)- 'O.

M00 CV ODN 101- s-1DN 0N N0 N co N U)1 N U)1N 1N ~1N c)CN U) 'g . ' ' N ? ' 0 N- ' (D 1? 0)Ns-s '

V) toOD 00 C0-OD Ns-(0 D -1 10s-o1co 0 Ins-D M rs-U) O s- Ns-D (00s-DUO ) 'OD 0) C 1,0 1. o-0 N U) s- 1010to 0 10s- N1s 01' 0)0. s-0s (D' (10s (sg0') - r ' N N - -1 0 - N2 ' - 10 's- s- N ' s- C) ~

I*- U) (3 (0 M 0 0 ) t 1M0 N) M0 0 0 0 ) N 1,M0 M) 10 (D M 0 N 10 ( 0

' Z c) ' r t2 N ) r ' '9) N s- 9) m r -)P nr 2 np Em

(100 00 1000 NM0 1000 000 1000 100 100 )0 0

(10 C)0)1 C0)1 Cl)1 s0)U 00)1 N0U 00 1? 00)1 9 1?01 101 C?)1

v3 co 0 0 V cwC)I N) 0 N? 10 ' 0 10 N? 1 0 0 .

N 10, P L C0 13c0 ( 1 0 1 0 1oci0 1 0 0 1 o0 l N DV - N n N to,' N. ' N ' N N GO N N. C-N4N WO 2010/037119 PT/US2009/058843

W) U) to UO wO In Lo LO W ) W) N N N N N04 N Of N N N Nq

*0 M-0 r-.0 CD ) 00m) C'0 0 0) (a) (o00D 0 0) t r- ) 0 ) C 0 g C N NV tN tov 'V wD NV NV (V ITV V (V WV V~V N NN '-N coN Ca) m NO N NO NC? () -N N - a . N' N I m (a) C. NTNI a aV twoI~ 7 IT rI V ) V WV VV aV C)V N - V to V CD4 9)) (n0 0D to) 0) 00) 0) (0) V V)0 Ca) m0 00) CD m

I I C? a ? T T

V 0) (w to0) c(00) t,0). m 0) N m Ca)0 c(0) cn 0) 0)0 (D (a0) ( 00 ( 0 (D 00 ( (0 V (0( 030 ( to0 V( 0 Tw 00 w 0 0

. C) 0 (. 0.- N. 0. . .~ 9 . 0

-- D Go (0t(m 0 m0( V( CD coCD 0 (0 0-) ( 0o(0 L (-0) N00 w-( 0(0 a- C) 0 00 0 to) -n LO C

N) N 0) V) N0U) VO) V-0 V0) (00 m~0 V))

P0 DC) D0 N (0 (D (0 ND 0 m Cm N N ND 'T aD Q N m' Wa

C' V)- m ) Va a) N1 (a)D0 (aO No U-) Lo to C') N- a-

0- IT NV (V 0a0 IT ( N aV 0) 0) V 0V

00) P N0D O C) V 0 C)O 0) 0) 00)m (00() N0 m m C) N ) 0)C) (0') (0) (DCV) 0(a) cn C') C() m N 0a OC,N4 (C) C)' Nom m 0' I C') NV' 1 N (a) N C (

V'( N(0 V(0 N-( VW t(0 (0o in0 (0( V(0 0( 0) cO N N-N 0 N N N- (ON (-- PN (0N r- ON- r0- mN- NN N(N N V ON w0 1N -Ni N N C14 - N I C') N NO 04j* CN I? N

Nc .- 0 (0 (00 (0 NO (0 NO VO (0 (0 (- V L C) (ON C-N N) N- (0 -N) NN (N (N (N N N V14 N CNN ()N N (N OlN (O N NO (ON N0N 0

( )0 0)0 0)0 c 0)0 0 0 aC (0 (0 .0 (0 0. ( D N'0- V- a. 0 0a '. 0- 0 N N'. 0 ( 0 N ( O CDN 'OO N C' ) N O N) N - N

N( V (00 0 0 (0(0 C', 0)0 00 '-(0 (0( to( N(0 04N ON (0N O N 1rN VN N ON ) 0N tON ONN N

0(0( to( 0(0 o (00 0 (D(( .(0( N(0 ( 0(D( ()(0 ED (0 0( (ot IT N (a) .00N VN - a-OsN (0ON .- N NO- ON- NN N-ON NN- ) 0N- (?~rl a- a a N) aR N -- 0- (N

(D) D0) 0 0) a Ca)0)O(00) ~ ~ (0) ~ 0))' w0) 0) .00.O ~ ~ ~ OWN (O N NCo N (0 o 1a)N co w N- (O DN N-0 (ON- 0)N - ol a N ao ) (0)N a- N ao N aO a a - I-C'

NC)- N2~- 0 (0).- TCD0 (S NC'. V '. (() 6 C)~ N-Cma Nm . m (o(). 0 W. 0(D.

MV m)V r. 0m) N-V) N m-V Nm0 0)V0 (0V0 C')V0 a rnV) VVpr (0V0 0 (0 7 (0 (D (0 t V (0) N- (C0 ( 0 ( ) Ca ) ( ( 0 0 (0 ' 0 a~NC - Na N a a . N() a N a . '

C?aN C?C ( 'T 9l ) C? 9 ' T ) C, ~ 9) N 9) '

C')O 70 (00 (00 0)0 W0 v0 0)0 V-O (000 (000 C' V(0V C'3(0 q30. 0). V(. (0D. (00 0(. (0) (0 m0-I - O(a 00 - N -cr a m'. a 0 N. 0( 0)N'- a N00 ~ N0( 0)0 0( a t, .1(D N V( ( 0 C T0) 0 (0 0) )( 0( C)v a ' ( 0. V a V.- a (. ) (1)00.- a N - a

) l N 0a 00 v) 0 N-) (0 00 m (0 U) a-r f) ( I N(0 (1 (0U - V V) Vl Vl v 0 co V N a Na a NI Na) V ar Na m Na Na aNa~ a a N N a N a WO 2010/037119 PCT/US2009/058843

w w w w' 0 C w w N N N N N N N N N N em N N

a) v 0) q Q O C) NO ) COO) .0) COD CD O 'Ot COO co0 nm coOm ) mOO c 0 Ic w S IT 0) IT U)I oI ). o o CC CO o C?C '0 COC COCO0 COC .-C OCO CCO C4O CC CO

a) ) C )) v00) CO) COO COO cOO COO COO L ) 0a Im V) COwO) N' CV) N' N'C N c C') N' N' C') in C) N' LO C C?I S I I? C?

Na) GNO ) O O)) 0 a) cn 0) NO)n a) Ca 1,O) CDO 0 ) N NO) C o (0 co coC (DC o) c0 U 00 0(, t Co t o )C wOC WO w wD w C?C ED Ln ' N' ? rm CN N' N'C, l

N' N ! - ') N N

O) F) COO) NO1) GoO0) Vl) m COO ) COO) MlO OO) coO 0 -) N4CLwCDO) NC .-C COU WOC IU) LO C NC COC N'O Q~t WC U) LON C') v N W) U) O N Lo

a) .--O O)O .- ) ) VO) ) C OO) V)0 co CO) O CO) CO, CO, C, ;IN vO f Nl ? C? NI C

N mC)OD ( m 0) )N9 C) 9) N) W) N .- m LO

mi L N 4L No to -0 nI N 0 a') C) N No Nl )

fl) qOC m -CO CO .- C .- N C CU) COC COC .-U flmwU D ) mCO

NO NO NO NO C N On NO COO n Nl P, NO COO fl CON CON COc V) N .-N CO N O) ) ON CON fiN

N U)CO N) .- N .N N N C.4-N N .- N CN O) N mCN N I-N CO COW 0(0 O)C NCO w-C COC NCO NiC NOC OO O g '~C '0 .C' 'I'? .

f. U). N C'o . CO. N)m- CO. Om w O. 0i. O) O fi0 ' C) f N co- NNfIT N * C C C CC C ITC

O 0C;O m CO 0 0 ) VCO fCO V) CC CO fiC -CO OCO COO CON O N O N N ON fN C N C a N NN CO N CO

(ON NO ON CON COO ON mCON CON O)N CON r-CO Ln 0 COO flN CD Ni . 'T E? - f - N9 . N9 r3 'D ' - N r- - - N9

CO 0)- l). OO- C)- C? O)- Ca O' COO' NO) CO)IT0)-

OD - CD LOl (D Cl)(fiN D00 V CO 7i OD 0 iN OD fi Nfl CDc ( o flNo c OC'C 0.-fiN .) . ' N- ' N- CO C ) '

t-C~ flo. COw. . - CO )- C OCO.? - 0iC COC CO- )C . CO O. CO O. O)).

OCO oC c4m o cn ) CoLo) NM O NCO) 0 ' ) CO) D) O m o Cola) oCma NrOa) N O coOO C N C' O Fr CO N) CO 0) CO -, C) o 0 CO ) CO M 0 e ( ) C C ) co CO ) CO

CO m CO fi Cl m- ;i r, Mf 9 O N C O D CO n N O C OO O C O O C COOC CO O C CO C O C O O CO C 1CO 0O C 00? '00 OC 00 WO 100 N c NO COC C InOCOC0

CO. .- O. COO CO- COO.- COC- OCO- OCO- C. COO- OCN OC- fU 'N~~~~~~~~~~N'V-NNN.' N' -N-NNNNN'N-NC)

C DC ' 0)W) 0).- N- v) N- N-' q-. 'D 0v0 wICf i-'0) N CO ' - 0.' CO

COn CO C N C ) 01 -N fi CO C

, Cn o mV . CO) V O CO CO CO CO mmT Ul a ) CO4 CO) CO CO mn N N. N. a N O l N t N N. N..4 N -' r N. N,.-i N "t WO 2010/037119 PCT/US2009/058843

N 0) v LO w V 10 0 0 .- N m) P, fl fl h. N N I. P. w OD 00 go 4 N~ N1 N4 N1 N- N N4 N4 N4 N N

00) 00C) w0 0" 0 ) v C) 00) m co 00) to0 m O NO) m00)m NN C ') OnC N N? LON .- N ON Cc C N NN NNi ONC' C CI C? CI CI II

7 77 00)T T0 0) T 0 67 -0 v) 'UV ' 00) v 0 w v v In m 0' 0 0) N' Ni NmN'4 N) w' m 0) C)D

NOl ) )) m 0 L m m 0 m0 00)m 00m NO m 0 v ~ O CD0 0D 00 V )0 'TO NO 0 00 N 00 00 2

0 _) Q 0.w- a,~. 0 N N. 0 0). 0 .r

77 .- K W -F ) -F K 77 N

'-0 0 0) N) 'nO 'n 0 00 ' 0), 0 N 0 0 00 0) 0 O 0 ' ) 00 0 C) NOC) 0)0 0D0l ) N~l0) 00)C N0 ) 0) C 00v) 0) 0) 0)0) 'CC'D C'I I ) r ) C' N .- . - 1 N CN 0)

OD ~ 0 0 0) m N 0 m 00 0 0 NO 00 00CD r, 0)m 0 0 0o w 0 0 v 0l) Nl Nl N C ? H I? I? N I C? I?

LO 0 0)O l)0L 00 V m ) 00 0cot 0 0 w00 ) 0 0 m0)O N ) N v 0) IT 0 IT r 0 C) 0 v- N N0 v N- 0) E ) N- N Nr) ) C? IC I I C? C

C') wm C"0 v)0 m)0 N0 0 w0) 0)0) 0 0 m )0 W0 m 0w m m V)) N N N) r) n 0 N l C~ N I n Wn N m 'l CI C I? C? CI

Nl O 0W NO C14 00 LO 0 n L) 0 0)0 W) 00 00Lf 0l)0t N )N N4N ON ON N 5 tN-O r- 0NN N. NN v N N N N N WON ON o N O)N ON M 0N l) N O)N C0) N 0) C0 (0 C, N NC) 0,.) 7

7 F- ON -N '(N -N NN O O5N - ON N6 ODrlN O N ON 0N0N r-)N )N ON -N N N N

N-.- N. N.- W)- 0) 0 0 .- 0. 0. 0q L- N) 0o N? C') N ) 0 C)NN

C? C *CCC

NN N ON ON ON N- ON O N ON O4[)N ON ODN

I? I? C C C C? C C

N 00 00 0 00f) 00 000 00D ED W 00 NO TDOO ( 'T (D N '0ON v)N NO)N C)ON NO N PON MC-4 .0ON C)0N OON ON NONC1 9.0 'r0) C4-0 9*' 0) 0'0 N.EpC') Q?0 .0 )0 O.-0 U? -0 N.0 - N . -~ ~~ ~~------~~------)

0W 0 0. NO- W 00W.W 0. 0 0GO 00.- ) D 00.- NO- N - 00.I t 'T0 r_ 00 NO C400 000 00 0 NO a)0) 000-wF % 'D0 '00 '000 r P N- 0 N N-2N 0N N0V)N ON 0)0mN- M0N C,0) -) N0N N N 0) (n, -n l mN C' ' 0 0) ' 777 '? . 7? 1 1 ;-1 . ? I ? I C

O '-0 CNM0 v 9) 0. 0 Q0m0 LO m 0 N (D a 0m 0 0Oa)0 a)0 IT D0 v0 NOn.- ! 0(0. m). m00 '0( 00.- NO- '0- 0- NOm NO - 00-mr,

NC04 00 0 ~ C) NN0 0 N N) OD)0

' 0 ) ' 0)4f N v09 F- W v I r W7) 0) N, ' N m IT' V~ ) C. v)

0 0)0 T p - 0) 00 ' t T0) 0v0) 00vm)0 00) M0) 0V) '0D ) N T '*C40) 0) 9 00) '00 N0) 0)) (0P) 0) 0)0 0 0 0)0 0 0 0 0 T))

0 n 000 N0 00 0 0m0 vC)0 0 n0 n0 OC .- 0 00cn0 000) 0 00 0 )006 0 LMmin Wm03.-C f to0. co0 m)0 in 00Y.- t E to0 00.-V Sc 00V 0 0C' 0. L 0.n

O~~~C N O (00 000 000?0'0 0 00 - 0 '0 '0 NoO

- N ' - 0) 04 N N- W W0 - 1 N r N- 'r NN ' N'

0 -0 N 0) 0 0 000 r 0 ) 0 N0 0) 0~II II~II~I

r4OD G 0 T 0 D 0 Nt ND Nm No r INI N II II N N,. N. N, .. N. N., 0 12 M 2 WO 2010/037119 PCT/US2009/058843

c go 00 c 00 C ) a 0 N N C4 N N N4 N N4 N N N N

wCm) ) m mO ; a) N) m ) N m 00 Ca a)O m ) 00 m Wa)

C) m ' C') N' N' C1' ro' N'0 C C') N' C')'

Ca) CaO ) c'o) m ca ) C') 0)0() M-0 C ) 00 WO) 00) Va) CN4 N N C) C L CN - C')N C') I? ( C4 N 0 N VN 'tC N1 C')'~~C C') C') N? N' N - ' C'' N '' N

NO ) Na ) va0) Ca) coa) C) CD ) Ca ) C 0) Iqa) C')) C Ma) 0c 0(D IT v-C to 0C cc (3) CC( f ( N c CC NrCO

m) ) N V 0N CN -T) NN NN ON NN O C')~L N- N C') - -

('() Va ) 0a -) C')) 0) 0) 0) Va) Ca) a) Ca N ) IDC') NC'r) CC'L) 0C') N) NC'W) C'0' CCO () N C') 00mNC') n N C ) NC' LOC'4 CS) ) NC' ((C')(

C) OO0 ) C) 0) Coa) Va)m Na0) 0D Na ) Va) Wa)0) aT ) Ca) N C C W C C C C? N )

ND N) NO Nf 0 5') o ) N1 Lo U) NO C'O ) It) No L - 0L

N N N V C3 0 N r- N a) N, N N N N N C') N C) N

C7 N aV 'Cq NV CV 7 N 7 Cq C') NV T V N 0C0) ()a) C)a) NVO) C0) Ca () Ca ) C)) V Ta) NO() v a) C0a) (0C') m)' 0)C') N C' C) C' CC') CC' V D Tc') C') v En N C') NCN C' C') C') V) Nq C) NN

N IN r P f)1 1 0 l t, m [rN r l, co

cN O1 N C4 N N N W NN CN4 ( CN CN 04( WN4 C'CN 0N N V C vi C? C?') N N

N~~~~1 NO aC NO N C . O ( C 00 NOU ') CN VoN V NN c) V0 N WoN CN W N wCN4 -N CD -Ni C40 mCC C NC CCC C OC CC NC C

CC CO CO (C COD N C C ')O CC 00 CC V NN. C?) ?N CN 04 CN CN CN C'N O? CC

ND N NI N C') ND N C) C' C') w'

CC C') CC .Ci CC C CN) I? I?0 CC CW

0)CN VN C4(DN ('(N 0)(N C)N NN N IqN CN Vl tN CN 0 (D~ U C? j N(Y) W(9 CjC (')C v-o~ 1? gC9 19 0 90Q D C* I? - N C)

CU) Nf- ' C'(D' 0 - N v -') rv- CJ -' Nv--C') 0 N '-') av- NC ') C'N). -' ND - N C - V c)F 2 I !M N - V ------N c) N 0) - N r- - m ,

N )- Va)- C')) Va) M0 Ca), Ca- C Ca)- C04 7 Ca)- Ca).

N04 )Oa CO) 0 0 )0 0m0 C3 OO) 0 V0)o C)O N CDO )O Oa)co N0) C~o 0)m - r N) 0 -r N ' M')r OMP C) ' N) Pr n 0 ' 0P CO ) r )n

Nv-C C CrC 0I- NIC N- I I~ C C'I- I )-I. NI

MC) CV N') a)C'S CC') OC') VC') NC) C) 'C) C) C VVO C0a OOa) coa a)Va V ;;Oa rV.- -W0 I'-aV ) NR !9 m CCCM ) C C - C N ) I C N0 C C? C N ' C ' C Nl I - N? No '?

C) CCC VoC0 7u 0)W a)5 CO o C)C NO NOW NOW VOWf N(1 NOC WOO COO V)C OC - 0 WO NOCv N C)OC T NC NOC'I COO ' NC CCCD -'N CC-N'N ~ ~ ~''N ~ ~ -'N0 CC2 N'N C'' N) . IC-C--'N O v '-V' C Vto- -'N CC-2 N N ' - C- VN' v

.- 0 NV(C 1 Cr) C ). nV- NS - 'V M'C WM C ')vCv N- 'F - a? av ? C '? N? 'p C '? '). G? C? or' V.

=7 7J F-J' '67i J I 6 C, 0 0) 0 C 0( N C 0 N C N! C' a)10 P 0 00 , 0 0

N N C- C) C, CC C C W C C) C Nl 'E NY ND , NW (% ' N ' Iq( Go WO 2010/037119 PCT/US2009/058843

r, co fn 0 .- In w KO wD N N ) m 0 0 0 0 0 0. 0. 0 0

m)0 (00) 0 CDO 0m COO 0)D .0) It) 0 v m N 0) m0 00 WV q qV OD V to v OW vO ITW COW IV 0 rNC CN mN NN CN C? CN ON CONt i CN oN ON

TV Cv !gW CO 7V 7O N v WV W W OW IT) NOci) NO0) .O) m C(O) 0 0) 00) ) COO) CO) 0) NO) C') O NN .- NN CN 0)N rN CONI CON N NP NN N 0 0' (4 0 ' N N C? - 10 C'N

NO) ; ) COO0) COO CO) NO0) OO) 00) w COO COO) COO0) N COO O OCO Om COW CO CW COW N'W WCO COW NW CO T , N' ' - I - ', C) C) N', N'

0.- N' N. N O- C). .. O-0. O. W O ~~( (I)0 ND( 0 C) NN

coO0) NO ) 0 o0) 00 O) 0) CO) 0) 0 MO 0) 0 0) COO0) M' O )O oLr COCO OW O)W CW O)CO n D .- W NW No O) Ln 0)W L W COL) COO 0) 0 VC) NOC 9 CO 04 CO V)0 MO v C.) CO C? CICC I £ I

C) 0) 0) M 0) 0O M-O NO) 0-) 0) C O) 0P) CD TO) coO) N N ' 0N N N 0 0D I C I N I . I

No Lo 0 o r- CD LD a) -o U N-0 0 0 Lo N N r - N ' N to No IT Lo -t It Nm c IT Iq r T I

COO) 0)0 NO 00) COO MD D0 COO) v m 0)0) NO .- O COO m O ) Co N )M M c M i 0 N CD- N N 0 09 r- -) c 1 CI C? CI CI CI I

OW OW) OW) 'CO COW NW COW NW) NW -CO ) In CO OD)N 0N NNj Nl N O)N CON ON Nl)t 4N NNr CON 7)N .N V 'tN CON ON CON 'N L CN CN IN N~ ?N CON NN CON4 C

I ) N~ COO- NO6 NO CO7OSO 0 CO 0 CO 0 a) 0) O) N CO)N NN 4 0N CO N vN ON .N CN ON CO(N N4 CON~ NN -N CON CON CON ONPN CO 0)N O)N NN I - 00C * I - ?- I:

COW NW COW -CO 77CCOW NW NW OWu CO 7 C70 VW

N N ' 0 (D Nn N N U N

* *. C C S C CC

CON~ CO L)N - -N CO O N V 0CN C

goO(N NoN ON WON CON CON ON .- N NON 0)ON OON OON N1

COO) CO NO)-R wO) 000 N)). MO. 0- ) CO) QO) NO) r CO) CO) O ON NN CO COF NO O N C -O N CON O)O N)O VON IT F Ic - N -2 '7 0 L - !2 N - 0 w C - N 0C-

cy C, )a 0 0)-C NaC N DCO 0.- O D N.W 0 .C C O.CO0-0) 1)0 0-O 0-CO V.C 0.-C COm

N ; qg - N Lo M - N (N to M c - M VD

OW ;)O N V (N) W NV W04OW COO WO ) NOV 0W COF 9 v ' N O VO )O V) OVO NWO) OVO) IO ;: O I?0 ;- O NV ) O Nl C O CO - CO C) CO 0) 0) CO VC - CO C CO -0 O C

N N7 -T 07 -c N TU OP - - ' - 2

ON. ON NN. ON- CNN -N- CON ON- ON NN CO ON.- 'T V ? w NC O C C O V O CO 0 C) O N CO M CGO 0

'-0 WO WOO 0)0cCD0 WOO .- O N O WOO WO 0007 N 00 CO O)O. O)O NW- N W-o 0W. .- CO O)W NW. LO C U3 NW. COW CO 'N 'N NN "t LN N'N N-' '' -' ' 'N N' -?

M0) O)W COO )C VO)C NO)C 0) CDO)W wO)C O0C COO)C OO)C OO)C

N N~ - - I - -N '

0 0 J o U 0) 0) 0 ) ) ) 0) 0) C) 000 N. ' N N-. N NI 1 N. ' N . I'* 0 WO 2010/037119 PCT/US2009/058843

D D 0 a) N ) 04l 0) 0) 0 0 - Co N- r0 - - -% N (0 0 ( I? C4) . ' C') .0 (0 C ? C'

M) C11O) (00 NO m *0) NO) a m NO m NV) m ) (Dm o)a 0 0D) (N N( ON NN O)N '- N vNl N N 0CN N(N CON O *' Iv I C? I I

) 0D) mO 0O -() CN) O)W 00 00 DO O) 1 ' t0o( (D 0 0 O) NW (D 00 W ( M0 N (D 0(to C)( W C'(n )' A0( (D' (C') ODC C? 0C) GoC' V-) (Dc) NC')

N14 04~ 0N CN CO C') 4 N C')N ON191 CN

(. CI) (0'- -- ~ - .- 10- N' 0.- O9r C)

0)) 00) CDO 00 0 F) ')O m0 0) c) NO) (C) (NO ~ 0n(0U (n C4Ln Ln( (o 0 (N0 cc( (0o Go( to( co( (0( C') C) 00 o)( m'C' M D' !9C' MC' m D' 0D' 0C' (D9' M(DwCo

ND 0 -) - C') 0 3 W0 ) - C' D ) (3 N- MCo ) (N0 CI W C? N CIC I I

0) 0 C'4)0) NO) 00 UD) 0)0) to) 0) ) NOU) 00)V) (00) 0 D 00 0 C'N NN 1 N0 ( 0 0) Nq C) - 0 N (

0) 0) (0) NO) IT0) W C) (NO ) ODO (00) Iq m 0 0) (00C) C) m C') (Do' m (to') (C) (NC') (aCV) C') cl ' mD' V ) C) (C) N0 O

C') C' C') 04 - N l (N r- r 0 N C') 1, N

N 0NN (N N N O N O)N N CN (N coN ' N 0 N v0 I (NC C') If '- C?

N~ ~~~~~~ 0 0 O N C'O (O (O '0 N 00 (0 N 0 al) N Q) N Lo N~~ CN NN- 0i N CON (D N C0N C)N O~ N NlN C)N 0 BN ~ D - N (0N (0N O)N ON1 'N ( O 0) I

V.' 0) 0)' ''f 0 W~ Ow N N 0I (NIL .

I I IT C O (00 (O 0 O - CO NF C) (iO 0)0 N '- N C')( C N N n (N w NI CN GoN Nm N C'N coN N N C' N N C') - ' N 7 ') C')

N(0D Lr (D 0 W0 0 0 W-( O( Nw r0( Nw N t C) 0 (0 r- c ON N C') N 00 N NN C ON ~ r l N (N 0N 00N CN m)N P CON N(

00' .- r) C) (0C' C.( O (0Vto O w0-' (C') N.-C' 0) C')'-C'a 0.- oC' (0 0 - , 0N - N F IV' - CNFN - C) (D ' l

Can)N (00N C')N 0 CN C' N NC' C')N mc (0') aC) C') C) NC) A C')N 0C)N I 7 C)r ,V c' 0c' )r c) (N m ( N N I) N' Ir C' F.vC)r

0 1 11 : 1 ? 1 01 C? CI T I 9 T II

COO 03 ov0) N VO) w *qO) 00~O ( O) 0 N0) C, T0 0 N M 0 ) I- mO 0)0 v N I c) W'IT

I C' C? I I 0 )

0 (00 co )'O .( (0 ' 0 CD) 0(.10 F0CM)0 0)0 0 (0 N'0 N m0 cr) N'- 0o Cp CON' NN'2 O C N ' ~N 0 N - 0N ON' TO' N-N' CON 0 T If C' "9 -T - 9) C' 9 C') C ) 'r 9 ? T' ) 1 9)

00~ 000 ;-0 (0 0 F NO N-000 N C (00 (0 O 0 ~~(co ~~ 000 0(0' F)0' (00- 00-r(w N0 - )0- F (' CO(0- 0(0? 7 )(o 'N '-'?11N N'N L?'N N N '? - ' N ('N T' -'N - , - (' ('N N'

(D I 0)r 10.- N0 0 )'- I m)~ w moo r , C ' I) '0'. 7 (0 v co TIn V- ' a) mN I) T N V N I Il mI 9: I v 0

(0~ (0 ( 0 0F 0 - N ( C

(onC') IM . (0 I 1(01 Il ( 'i (, 01 (01 (0. -1 -0 0 *(1 WO 2010/037119 PCT/US2009/058843

- 0 ' ~ Il D . co 0 0 C4 N N N N N N ~ C4 N m (n M m

v ) V 0) 0 0 (D 0 oC 0 0 0 CD 0 0 C)

00 CO NO 00) . CO COO CO N0 C O a) 00) C N N' N I?

- C) ) 00) m Q w) NOi NO 0 00 m 0 CD V0 m O Q ON ON ( CN N N? tiN ON ON ON NIN, CNN IN

0 0' 0 0 N' '' N' N' M N' D IT ) tom 0 0 ci in I I? C' m

OW) NW tao O COrC CO CO I'-W COW COW O ti') 00 0 VD) COO ,C' 0 NO M- O DC) IN'0

N. 0).- C 0. m . COm COD N.0- 0 CO. m . c- 0) )

tiC' COC' DN O1' WC'0' VC) N -, t)tiC' 0 COOLLN 01'' ' N - N I?

mm N O ' C N No 4m 00 ')m O N0 C'LO 0 . 0 N

' LN M~ to No to N LO to CO N 0 . C) N 0 N GO N- .- .- ')

0 N NO COO COO CO CONN O . O

NO 00)( C') C') 00 NO M01'04) 00 ) NO COO VO cO

ON NN CNN CONm NN CN N CN CN CO RV O NI ON ON CON ON toIN ti4N -N ON Ol(DN CN ON r -I I NC)0 0 -' ON VNJ WIa)N COI CO N 0~~~ -)N NN CN 0N 0N CO4 N N ON

II .- N6 'V N ' 7

1 NN

ON N1 ON CO V 4 )N ITiN1 L IN P CON QCON O N ON 0 CON D1N .- ) O N N m N 0V.

'N i DN .I ON CON ON ON ON ON ODN CO

a0) o 9 N9) 09 r) 0O9 OD 9) OD9

OWC N (0 It CO CO COCO tOC INCOC (DCO 0C CCO NCC N O N-OC N COO NON CO ON NOC-N WN NO N CO COO COO O

(00 WO WOO NOD (D0 WOO DOD 0OD NOD O CND (OD W VO DO(D COO C4N CO4 0 0ONC N 1 O )ON 4 D OON NO MO C O N OO OO4)N CON

.- FOI- -! 00IO- NO -0F CO0 rO. NO. COF COO. NO.-w M- l'- 2

c CO C C C C0' C 0 O C 0 CO O CON COO - - . C 0 C 0 CO ' 77 7 '7 m0 nm n0)0 N 000 WO 0OD DODLr A r NO 000 NO aO 000 0 O ~o . 0 WW OO VW ' DCq0O OC O CO DW~ ~ ~NW ~ DW ~ NW ~NOW ~ ~9 ~ - ~ON N-O.~ LVI. N- O. NN-CO-N-O-O. 9)~~I 0 C'CN9) 9 ' )C' )N 9 9 '9 9 CO~m O 0 O O O 0WVC OC2O 0 O

0 04W COCl NOW VOWIO NOW NOC WO OCO W W -

' N V N N' 0' - ) M 't 'v

C. CO >

0C) N CO 0 qV Rl ) 0 . m 0 )k)0 COlCl O~ D 0l () O 0~ (1) LO MN MmLO 0~ M * U)' M0O WO 2010/037119 '~PCT/US2009/058843

in qD La- w00

.0) NOM N) 0 O v CC 0) 11m 0) N-C) wOO O 0 D CD 00( m I 0 Nv C NC 0IT co I r, C r-v N cI V)' vO CNI N V mOlN CN cl CN CNt O N ONC ( CN ON OCN CN N C N CN IC f N'CN'N C# N 7~ CI CI CII

N -tCOv WO v W'O 00 "CVq~~N- 0 NOi COO ( CO CO 0) ) m 0) r- COO m 0 0 0) 0 0 0)0) ( CON CO ? ( N C U) C 0 ON CN ON ON ON N0 CN m C N' C') C') C') C') N' C') N' I?) N' N

vCOO C) In CO r- m COOa CO "O v ~0) cca) COOa C'C' CC' OC? COC' 0C) CC) LlC) 1? C)O ' C)

CN -N- 77N ON- ON- ON- N-N vON CO- - ON- O

N- 0 0- N- N N- In. 00. C- DO N? N C'C N .

COO 00 00 NO NO 004 CO - 0 m0 co) C)7OO 7 C' ) MO' OC) COC'r L) .co CO CC) COC' COC' N C') N-C') D N-C) N N. NoC) c l C )m V) l N1 (D N l C ') NC')

N C'r 0 0) C' )C' a) ' 1 C;) C') ' N) 9) c') C' )

LOCO O COCO go )C .t COC C') u) CO7 FC N to COCO CoCO O N- N- CO N- CO C) O No ID 04 N N C') C'I?) C C' NI?. 'o' C') C'

77777 C1 7'TI m

0 C')C'0) C)C) COC') C') N-C C')') COC') C') 0 COC3) N-C N-C') C' ) ) C) C') C) X) .C V) C' ) C' N C o '- C')

NN N CNr- C')N ON- m N :rN- CO N - I 'N N m I'N C')N NN C')N V)N N 'N 19N N N CON O N CNN O N O N C I? I? ? C

Wu '00 NO No5 C' CO COO CO 00 00 N-C 0 -' 0 0C)N -1 N - N m')N CO N 04CN -N CODN C') N N C')N PCON-v1- o N

C'DN N- CN- CON 0N 0)N ON- U)N CD t D ON- ON- 'N- 'N-l Q

CO9 0C Co C' C OC9 COC'C NC N-C C') C OCOITC'

N-C V)0 VC) C 0 C OO 0o0 nO 0 0 0 0 N C') C') ) C C') ' C' C') N P,) N C')l r C') CI I? I? C? C? C

CO N CN CN N- N NN CO4 N ON 04 N ON ON 'Oi N 'ON IT~ 17 .I. V I - I (o9 N-9 t_ 't0 - 0)P- ;O~ C,9 r - c -9)0

CON CON 2 m - o ( l - O- CON vON CO- COON N-ON COON COON CON

N 0-C) 0 C'm 0-C' 0 ) N-3) 0 N C) O-C' ) M.C COU ( 0 ) C) NC') 0 0 ) 0 -. C' C')o N V C) - ' - IfC' C) C)p C' C) ' ------

NO C? 0 M 'C0 1?O 1?C CON C? N C ?0 C)0 0C0 0

C')'7 C'7 O 'q C6 1 ' 'C')' 'T 7' 7 ' C)' C)'

CC CC O. C')? . NCO. I?'O. 'ONO 0ITOO NC. Cr O~- O

CoCO0 OC m o C' OClO'O CCO' 0 a,) 0 OCO'0 'CO' m0 CC) 0 CoC' C'0)CO'0 CCO O~0 CO CO CO C O C co CO CO9CO - C CO P C' 'C 'CO 9) 17 9) ' N N ' . 'I

00COC 0 a)CO '00 0 C? NC0 a O100 000 OCO 0 CO OO .- C 0 .C')2 w o CON CON CON CO. '9N N-N N-N CON L9~ -N- NN-t 0 N N

CO CO CO CO CO CO 'I C C CO (D 9) Lf 9D)C N 00

OC.- COO' CCO COO' COO- '0O OC OI? 0C OCO 7 0O 'CO. - - -'N N- ''N - N - -'- -'N -'- - N'N- - - N' - - O oC 0CO O O N- C CO COnC NC .- C O 0O O OO OOO O

N- CO 0 N CO M m CO, C N C No N) N CO CO ) CO CO C' CO CO C WO 2010/037119 PCT/US2009/058843

le w 4 ) in No W UV Lo to to V n e en In in In In) m

Q0 WO (0) V) CD :Va) m-O NO NCD -a) ma (DO ItO (D t m0 0 D D NVi V0 U)vVrC1 1 U V 'o m 0V o V CD v) I ~(V CN NC) ( CN CN C? NN1 C4C1 CNN (ON NN N (o CcNiN I~ , C?IIS

VV VV (DV (DV (97 N0 0D DV (V N V m) 0) tom NO4) to) NO1 ) 0) 0) w(m m s-a) NO Va w tO 0A) ( ON w) (ON N NN toN mO wN NN N" C? 0N (ON

N CD N4 (D ( M in0 w0( 0, 0 0(0 W 0 .- 0 tow O( (W 0 W (0

N 0o N9 Nn ') N to - to

0O -) DOC) gDo0) ODO) *O )O)O) to) NO) NO) toO) 0O in Nto VOt WVVtf o Nto O) o toto C)t 'ato P)(DL) D c ' C4V VNt (Dt 0)t I

N( VT0 (( T0( Ow 0(05 -5( N( V(0 TO tow77 0 (DO0) VO NO) .O) 0) ) NO) NO ) to) mDO (DO C3 0 ) toO ) (D N - to) N to to - to N to N

NV0 O)V NV (04 o V tV 44 OV D I? V

N0 U) 0) D- (0t o DL DV N. ) 0 N) - 0)

mV V 0DVI toV ( NtN V ) O) V DV IT

Nn N tD0 oC o t o t o to -l to N to r C* I? I? I? I C

F ) f, N(o 0(Lo l N0 0)~ In Ow 0 Lo' tow m( Lo 'TU) (ONDD O)N O)N DN (rDN tN (0N toN r- N to NN ( N 4 OND to4co o C4 C4 0 N N (0 00 N (0 NO . N (00 N0 0D N oO

a) f C4 CJ 4 ON (DN V)N 'N 'aN C1 N NN O)N O - (O4LN N

ID toO 0D 4 w DO 0 QO 1 0 (DO C7D 40) O N.1 r- r- (D ( -. 0- . (D (D (0- N? Nto N? to NCto N t

( -N -N ON oN .- ()N toN (3)N ODN C -N D (0N O)N .- )N

N((D v(OCD D((D -H~( O)(0(R D 0 N((D (D(( (0( O) (D( O)O( (00( N(D O) DN NO NON (DO (0N C)ON 4O O)DN rc O 0D4 CDD 04N tON NOt Nto svt IV ? -t? o to-to N-t q-t O' 0 C? I ITI II

04c t oD NO)O (O) N)(D c o 0 D V (000 coWOD (000 N)WO DO)D CO 0 ) N Nto2 N01,toN P w - nFr Nt) Fto Vto O o 1-to Vto 1-o totoN OUto (toN - 2 ' - '2 to N 2 ' ' ' to ' '0 - Sf C 7 C5 C? N vf .? ?

UD-' M0.-t0 (0.-( 0 0 N-0 It 0 V M 0 U3CM0 r- M0 N(D D0 C4oM0 V'(M V N(D. C' ' N(D V0D CO)Dr (0(' r 0 . N(D ,m - V(D C 0(D 1r m0( N(" 0nmr m V tol t. t- 't- s-o- V)otts -o- Oto ( o- t 0o ~ O

(ID, too c (0toV CUD,4 NtV 0o V (toV Nto toto Ot V IDto 0 C'? 'C ?' N Cf' C? 'I?

toD (0to0 mV Nto mOo No totoD m N C Vto (Oo NoO VoD atoD nt N) ) c? (? -? 9 S 9 - (9 - (9 V2 T9 ODJ (9 Sf9 Vf G?

0( N ( O)a a00(0 L(OOmD 0 )( )( 0 q0 C)0 00( m0 NM( NO('0 NV ( C"0 )0 (000NCl f 1m (000 toOO 0 0 (n00 NCD O)M to) (000 NOD NOD NOD N,()L)Ln c .- N -' -' N .- N N'N ''N -'N to' N'N to-4 t'N

VW ( NO D O)OM0 NO)( 00O( (0O)0 VOD0 V)0 V) 0 N )0 to)( 0 0),o)(0 V

0)) 02 N) to In IT V0 N m I to ' , t ' o ' to I t ' ' to I o to ' ' to ' to WO 2010/037119 PCT/US2009/058843

( D C U) In wD C D C D C D

0 0 ) 0 om mm oa L ( CO a m 0 m 0P 0)

V 0 co -0 04 ('T Nt ) v OO to0 00) 00 It0 C0'r t T 0) 0 LO T ) ~ CN N 1 1N -N ;-N ON C')C ON C N ON CON CI I

G 70 -T CDV -It Uzi F') N- N-V 7D (DV Iq V a to0 a0 0) 00 Oa)00) 00) M00) O ) 03)00) D00) m00 00 ON ON DN CON ON NNC ( CN 'T O V N C N CNN NN ON

0 0) m0 NOa 0 0) .0) COO0) 0)0() (00Y) I VO) 0Q IT) Q 0) r- 0) 0 (0 (0 (D N C (0 0(0 (0(0 0(0 0(D co( N C VO (( co t C I? I? N , ,)

c-0). (0' V.N-j 0F- N-CD N r, 0 co 9 - t ~ - 00 0)'

(t0) 0) NO 00 V0) m 00 ) NO 140 0 ) 0) NO 0 0) m O 'r 0 0( 0 OD( (0 0(0L 0(0 (0 LO ( '-O ) t nO 011 0(0 m 0 i

C3 ) NO 00 I Ol NO M U) (0 vO ' CO, ) (00 N U (0 N- 0o 0 (D N- 0w

P0 0 N- m- N- N N O - 0 - N

(0V) NO VO NO OO (00 Lo0 00) (00 mO 00 n '0 N Nq ) O - nN 0 N N0

q - N 0 N1 . '-T N! v 0I C? I? C

O N V N VNi Cl)(5 N N 0 N CN C N O O N N- CN N-N CN '- CON ON- C?-V- NN

w0( W0( V) ( 0(0 V N( L N -COV ( '-0 C OCO (0 N-C (0( '( C N P-( (0 (00 OC N CO N ( c(o(

NN CN ON4 N ON N-N n- N ON '-( N C N N ON ON 0? N N 0 C' 0 .

CO U *C to 0 0(40 (0(0 -0 NCO N-C OW NC NC -- '-N ITCN CD N N V)CN ON4 ON (DN O)N CD N ODN ON rN

C- N r -l co' r 0r U5 N'

0 ' (0 . 0 (000 0ID 00 (D0 go) N-O) NOD .00 NN 0

0 ' .0 'C 0N V' - ' 0 ' . '

(0'- N(' I(~ (0- 0(0-w C OC (0(. -4 ((N0 V) NO 0(' 040 (0(0 ? N O- 00' N 0' N -.- NO- N0 ). - - - N N'' ' N

'- - 0''!- N- -* or f - DC 0 L v' - .0'- 0''L - 0

0 NC D CO 0 ( D)a C ) w (0 U) ( (0 ( 0 (0 O N CD CD( t 00 N(0 o C ) c 0 - (9 No

00 0 00O 0 00 00 00 00 N 0 cm0 14mO O m0 000 (D N-O ~o N' r - ON'r- ON'- ) , Mc N-N NN' CO- (0N 'N' ON7 m-N ON' Nn N ' CON V

DO 0)00 000 N-DO 000 N -. 00 -0O O 00(O 0 N - O 0 (0'-~U COO COO' to0 040 ;O !P0 co( NO- 0 -

w( (0o( NO tOCO (00( 00( M-) w (00 '0 00(0 r 00( (o00( 0 0( V' 0 'r--' V' O'-V- V' Ot' 't Ot' ) . NV

0 N [ -NO ( 0 O(0( C,,(0 [ ID 0N N 0,. M [ I (D v~ V,)0 0 q T . 0., 'T 0, *l ol WO 2010/037119 PCT/US2009/058843

N- N- N N . N P. N N- P 0 ~ 0 M ~ 0 m* M

(0) 0) 0) m0 C) 0 ) CD ) N) 0C m m0) 00) M m -N~r ON (N (N NN 00 ( N -N N a-N N, C? 0 V N N £ (N I IV N

COOC) 00G) 0 0) 020) OD00) (0) N-C G-) ) N-C) '-C) 9-)

v 0) N- C) NC) co00) NC 0 (to0) -T) Q0 (A) 0)0)M (00) 0 0) N-D C N ( 0 (0 0 (D NW -W LAW ( W C(D vW N-CO 5 LW

M I? I2 I? I TC

V OD N - N r- Do0

0) N- C) 0)) )) CDC 00 ) (00 ) LAC() m0 m V0) IT0) NC ) V )C) 0 ) NLA) LO NA (00 VOC' U) NC' V 0 r) '-') L n m pm uI aL? N '- ) ODV f 0 C '4m -) ( vC' mD

(O ) (0 0) m 0) 00) r40 (00) (D0a) 0C) -t0) NO () N-C0) 00 () o C') 4 C - (D0 N o LA 0

4 in LO ' ) 0 a- wL

It0I N LA4 (4 04 e (04 It (04 IT 4 -q -q4 (04T U

00m) 00 m00 N-C) LnC 00)0 0) 0) (0) 0V)0) N- CD C) 00) N l - l m N m C' m 0 ( v)00C') C') m

NN-L) o r LAN- 0N 4N- N- 0)N ON- AN N-N CO G ) ) N- W 0NL0 - ON ON l LAN .N 4N ON '-N -N COrl N IAN 1 l

C4 ON 4N .- N ON N- N AN (N 4N NN C r) N N-N (O N

(3) "6 -- ,o - 6 76 - -a 5 a a as

0CW V (04 C1W 4W) N O OWC4 1)N 00N C 0)1 CO r- C)W CD0 N(W (0 4~(q (OW 0)W (N LAW 0O OW 0(0 (0(0 N-C

(00 w' 00 N-C COO 0) (0 m) 0 .- O NC 0) -~C' a9 ' '

ON ,q O N CN N CN N '-C4 N ON (N ON

(0 -C( T 0( 0 L WO C) 0 '- 0 UCO( C(0( Q? O (00 C)W( (00( 4W(0

N 0)a-c') n(Dc LA0 NDrM' ) E-D- (0.- ) NCD 000 0(DO')0 0.C) (0.C' WaC' VN'-Om (O N-C) * n N 0 r- a D N a 0- aT N N a N- C' .- 0' -l N) r

- ) 0' '-0)4 WCa F0)' (00).- (0)a -). mC 0)0' N C. 0)D. (00)'- 1 N- LC)N N-C)N C'- 400 CC'N NON CON L - c ) 0N to'N- W N- 4 a~ aI a , I a N a N I aI

- Cl N-M(0r a( 1 ! MA0 Pr 0 (0(0'-? C N(Oa r (0(0.- 0(0L 0 - -0 W(0a 0)(m0.-2M - N-')0 - ~ r4 . 00 NO- NO NO- N-r (0'- 0'- N-O

6, (00 0)0 00 C04 004 00 -0 00 a-O 004 wO4 ( l N (0 4~ (0 Nm 0 ' ( - ( ) ( 0 0 1 ( N (0C?( (0 ~ ~ ~ ~ ~ ~ ~ ? . I ? Ie C- a a: a? a? aC

mLA (0( 0v00 00 (0 0 0 0( 40( (00( v 0)N' C ')vLmNA (0) v-L v0 9) C q

V) n9N!9 0 ; L) mL O mL(o ( rM N 9Nu 0 0 c N 0 (0

02 000 W0 OFM F cC 0 00(0 (000 000 000 (00 0)00 400 (00 0

-a '&(0U K770'm 00- (00 0- N(0 0))LA C)0 N0 N-a.- C (- I 1 ,FU N '(7CYo T(' (0~ ~ ~ ~ ~ ~ ~~~- N0-- 0-) (0)LC0N) CWN) A) 0) )O 00WC)W

ID ID, I 1- w1(MNN MI a c l A I a a WO 2010/037119 PCT/US2009/058843

NO w ) ( 1,. 00 CD 0 NC' 10 w co 0 0 0 w 00 M m m

NO) .O m-O 0)0 CD0 (0 o (0) NO) m NOi) NO) '00 m A t- C'J 0) .-4 (04 (0? .4 N4 (0! .-4 ON ON NN ON O O 4 N CN (O N

w(0 a-) .0) COO0) NO m (0o0) (00 LAOn ) 00 (00 NO)4 NO)

Q-O 0) (0o0) 20) CD) LAO) N.) W ) v O) v0 0 0) 0 (D IT0 ((w0 '-( w 0 .- 2 N0 0 (D (0 0 (D N to( 0(D I? (n~ -l 1? ? .- 0(Na C?~ C) I? OL

KO '-N 7-1 4N 0)!' LA 7 'tN 77N 7- 77 L!. LA, 0,- N,- 0 (0- N.- I.DL. 0),- 01), 0.- LA

-O) NOL) WNO CW) ,-0) - ) (0) to 00t) 0)0) LO0 (M0 'tm O

0 T( -) LA) NO M (9C) 03C) 00) V0) (00 NO) No ) m)0 w 0) 0) 0 m )c 0) 4 (D m) m0 (, 0t a C ND 04 m- (0 04 0D m 4 r- NDr4) ) 0) m m0 N~~r LONr0

NO ) NO) NO o oo mU) N (0) U NO) LOn O (v00 ) 00 (0NNO 0 O 0 (00 00 N0 (00 0~ ~~ .- 0 - -q

NLA NOi 0) L N Y 1()0 (0 0LA (0) Itm ,LA) LI 0) N O) ( 0) 0( 0 0CN ON ON NN C1 nNV N NN CNN (ON 0'N c)cNN 0 2c

to (00 O (00 00 4) A V0 (00 NO (0LO N U) NA 0)N Nj ON ON NN ON ON '-N LAN ()N f)N (o(0(4 o LN - N S 4 E CN )N C 04 0)N ON N0 LN AN CNN C4

LDCN ONcl 4 O N O N 'N CON N 0) N CN CJ N ( 0N 0) Ii 9 N N) ,- N N 0' 0, C4r n1 or 1

0C o0 (0v 0( N( (( 0(0 0( ( 0(0 0(0 0 (0

N- (D o- - -D 0 0 v 0 N

LO)'- (0N NO)' VO)' 0' 4. OD , (0)N N). 0) 00), 0

400 0)0 0 (0D (D0) 0N 0 (0o) 00) o0) 5)0) 00 000w-) NN 'tN 'tN 0 (N toN NON mN 'tN N N CON mtO NO

(o ' 0(0. Lo - (00. (D0 (D D E 0-(0. 0(D0 0)0,-c O O (0(0, N(D0O' ((OD. a (00 CE (00 N00 011 RN )0. 00. N00, NO.- J ! O0 D '0.N0- (0D 00 V) N LA) . .- 0ED U, N (1 M - NI . N. N . 7 r

0)D 0.-40) 0 N4) 0 40C) 0 0) (O40) 0 4 0 40) cmo I N00 04) 040) N0

N- ( 0 0 Nm 0 0 ( 0 ( 0 0 C (0 (0 (0 co ( 0 0 Nw (

(00 v 0) (00v(0 0- ) v0)00 (0 00( 00( 0 I 0 0 ( 00T 00TCD ( 10 NN. (Oo .0 O . CN N. OpN. NNp ONNN) 0N. aN LANp NNO C? .) 9)4 OP 0? OP Ir9 7 ) )' 9 ? 9 9 9)'9

00(0 00( 7 70 0m (0000 04 .o0 00mo NOWm 0 0()0 0)0( 0 0(- M0 000 V 00 400 '-0 NC) 000 ) ) V MW 0)0 0 NOD L 0 )00 (00 (00M)0 N(D)0C.00 0IO (00- 0(C40 N '- ,' NO- (,- V NCLA.- L)NO.- CLA I- 0 (4, N 0- 0(0' '-N NF NN -- ' '? p N NN - 'N N N op N NIN IF' a? 7 N

000 3 t0)0 000 N)0 > N0)0 .)0) 0 (0 0)F N )0 F 40)0 )Go0 .- cO)0 0 (0. N . N, 0 . F U, (04- 0 .- L 4 0)4. (04.- 04. W) U) C, I? I? L?rz N, N?

70 I 0 )- 0 It 0

(0c Nm0 0 0 Go N004 ( ( O (n £I 0* ,n IE- 01 1,0 01 -1 1,1 0 I~ I I MI 0 -I0 WO 2010/037119 PCT/US2009/058843

V nw M. (0 N) m n

N0 0 (Nma) 0) m 0 0) t 0)m 0) C4a a.,a') IT m' Iq c T C T )- ,

Na ) 00) NN) 0) 0) 00m () 00 v00 m (00 ) 0 (0 NW 0 Cq N NN C ( 0N CN 0)NC CN O'N (0C * N ' CN ON (ON (3 N' N' N? '!' I?(0' N' I?' C?

a) D)C) 00) v00 M 00 0 m W0) v00) C.-0) N00 M (00) 00) NW 0)

(00 ( CD v ) (D tN (D 0 0 0 V (00C) ( 0 (D0 (D0 (D0 (00 (D 0 000 ( (0 (0(0 (0( 0(0 0? m)( (0(2 0( (00 00?00 ( C' C2( C? ? 0(0 (0( 0( (OC' mc') (00

N' 00 -I (0 N .- - DD - ( 00

rh 00N r. 'N 0) N 00N (DN 0) -- .N- -- Np N N N N ' N

(0m (0 4 NMMW) ' 0) m0) (0 (00) c (0 m0M 0)0w NW m 00) ( L0( (n( 0( (0 IN( W (0 W (0m5 N (0 0)( In 0 N-( Nm (, (, C-N N , '- N N

to) 00 'F N 00) W00 (0) 7 '0) N0 77 (00 7 (0 N- M0 (D 0 N) Nt(0 M0 co- co r0 toN ( ' 0 N(

0)( (0O U ) r n c ) O0 ( D LO( IT( (0( L-( Nm W (0D(V N V V') Go (D( t 0( OD( V( 0oI(vv 0 (0( v ( co v 0)( IT ( IT( V0( V0v)O (0 N - l 04 N V N- Nl - N s

(0 (C)0) U) (0) N ( 0 N(

(00 0)) (0) (00) (53) 0)0) (0) M )0) (0) M (0)0) IT00) (3)0 V (00 L m(n0 ~(1 (Y0(Y)0 0) N-cl C') C') .- 0 00 C 00 V (0 V( V V m0 (0 o N (0co. N (0 co (0

N4hr- (0 - ON NI 4- ON (N cor o N r- (0N cor- N r 01' mI' ( WN N-N m)N 0 N N C N N mN (N 'N -N n N C4 N

(0 (0 (04 V (0 a- 0 0 0 0 -- (70 N

) - N (N 0N -N C0 (0' N (0 (N .- N NN C1 N ON 0 N-PN (0 N N r - N' V'I N1 N ' rI. N T3 L9 (0r Ir l

' I

(0 0 )0 0 0 N0 00 00 00U) (00 V)0w V)0 (0 N.to- r (0 VQT ( - (0- (. 0 (.- (0. (0- N. N T0 - 0 ITN (0N - (0N N - 0) ()D ( N

(0) 'T N (0 ND (0 N' 0 q (0 N ( (0

0N (0N N (ON CN(0Cj MN rN (0 N 04N (N (0 N (0N ON 0 V

NO 00O EON (0O (DO (0 N ) (DO VO ID O (0 N (DO (0 ( C14 C1 co ( N - 0 . N D C1 CV -n (0 -~ WN . 0 C4 -qN

0)). (0) 00. N-). a)- 0). (0o. (09. r- -) - . (09 OW1 (?0

(0 t c D N o 040 N r (0 -Oco L (0 co m (D 'o -)to

O.-( (- 0 0 - (00 (0to0 N (0 -0 .-. 0) (0-0 .. (0 (( 0 N0 N' N-0. 0(0. 0)0. (0(0 (00 0(0- 0(0- N(. C?(0 It(- 0 (0 0(. N

(0(0) 000) NV0 NV) ((00 OV N00 VV 00 ) N - N-a (0V2 0)? (0C00 0 0 0 N ( .i0( ( ( ? ~ ( ? ~ C

(0 (0 ' - 'N N ' 0 m' 0 (0(0 o0 m (0 N-(00 ((M00 (0(0D0 -(00 a0)0 M 0(0 (00 00 0 (0 ( N(0( (0(0( (00( 0(0( (0,( m0( (00( 10( N00 0(0( (00( (0(0(cl)r 0 V '! C '! C ' ? ? C ' C! C ' ?' ? - C? ? ' C?? C?

D0( N-t( (00( 007 70( NO( (007 (0 0 00( IDD 000 000 (000 1- NO (00 (0 Go00 (0 0 -0 (00 CD 0 (00 000 N

to0) 0 -0(3 ) W( VM 0m)T( I m-)( '0 Vm '0(D V N7 T0)m I 'W 0) (0 ) N I )0 0) (0 (00(0.- ((. 0. NU. No. NV? NV' F0. 0 v VV' N Vo (0

C) 0 -6 N) v( v 0 T m (0 N0 0 0 (f)C0 0 0) 0D (04 (r ) 0 0) 0 ) 0) a) 0, o) 0) 0 ' 2 N 04 'l CC " i0 0 ' f ' f (0 ' (0) ' 0 ' ' ( ' ( 0 ' 0 ' ' ( WO 2010/037119 PCT/US2009/058843

0) go00) N-O) co0m NO 0) 0 T ) N m 0)0 m) N) 0o0) NO)

0) 0) '0) mO (0) ) It0 a) 0 to m0 m0 N ) (DO0) C (0N toNC ON 0N NNi (ON C4N (ON tN C') ONt !

-T.O 00 V) -T O D (0)0 (O) VO 0(00) 0) N77 0)0) CO) m0 co( .(D N( 0(0 (0 ( ( 0o(0 NN(D (D CD( (0 (D 0(D S -1 .- C00 00 C) C ')cnC') 0 a0 C'C' V0 -C' OC' C) N'I N' N'I? I T

N (DN ON N r CT) ON (N (DNON OGo LO (0- O.- ( (Or 0. ~ 0 0 ~ N- ( (0. C'N4')- O. (0 . (. D C'). N'- 0.- 0 C' 0

LO 0O VOo to 0 n 0 00V ) N O - ) - DO ) V C)) m')V) W)0U) 0m0L) C') (0O C ')' mDnC'() 0OC' (C' NC' co C') NCm' ) CC) NC')00C N C) C' N

(0 L (D N(D m( C 0 0w C) ID (D0(D 0(0 to ( T 0( T 0( OT O ) NO) (D0l0) NO C) 11() a0 '0 (00 .0) NO ) NO) 0 ) a NO() (0 N Nt N C') (0 V0N N

0 0 N(4 N N C')

0) C') w0 V) N(0oV) .- O (00 0o 0) 00 ) 00 (00 C'v D' (D C' C' (O' vA' OC' ( c o) qC' m' VD' vO' CWC' (D IS.)

N( -N vN O VN N N C) N wNN VN(N

N I C) .- ' - ' C'

(Dn O V) r-tn) o N 0 c -t0 (00 (DO N VO (1O VO) (0tn 0 (DO VoN N N N N N (O (Om l 0 1 N -N IT C') ON C1N - ON N o- (N t- N -Ni NN N ON (OLc4 N C')N to-N 'Nl C N V)

Ol 76 0 0 VO N '0 (00 00 NO 0 00 a) NO C'V)-7 d) N NN (ON N ON - VN(~ (N ON NN (l r N -N C O N ONG C') ) N C'4 - NN C') r

7U( 0 (0 70( V( 0to .(D 70 00 (0( (0(-s0 -(0 'a( N( tN (N No oN ( ,0 (ON C'N w 0 NN (ON ON (AN C? I(~ 1 .I N

(0(0r. 0(( W'(( (0 N (O ND 0 0(0( N0(0 (0( N(( N((-F(( 00 ON NO N O N NON a)ON (DN No N OON WO1 N (JONC (DO 04 C') l') NC ') 0-' .C) 0.C) (. r') O O '.O N.-' - C

0)0 L 000 N0) Nto ) 0D 0 0 0 0 OD 000 O000 (0)0 (00 G) V o N OC'N NON NCN m('N (C')14 N C'N (co N U)N O CC') qC'N N' c')N 0 N' ', N9 (9 '! C'). NO (9 - ' 9 C') T r .- N (

0.(0 co O ( m'.( O.-( (D.( No-W (0 0 (0 0-55 t,-( C')-( (DD( (D ?r - a0 (0 N- (0t0. 3 t - N 0 O 0 (0 N(O P 00m N (D C')(N wN(0 0- N r N)- 00.- OC') L?~ .- ) (DC. c?') 00. 7.5 (D ') O' I .. I 5 I I C'

CD 0 (Dm 0 40 0) N D 0 (qCD0 0 ) (0 (D (0 v (0 NO m 0 ( (00 ) D00 ) (0 (0C'

No v 0o N ~04 '' N W - C" 'O 'I ' W

N- N ON. NN- ON. (0 ON. ON-ON NNTN. IN? ?CN? 1 ? C ? I ONC)C

0) 0)m Wv m J) N0) (00 (0 (0WTm (0m(0iI

(0 O (D .- 0(L)0 (0( V )( 0 C-0 o r 0( N"t en0( c ) N0 N m( 0 0 0 mO N V)N Of~- 0.- (0.-', '1 Or (0.-'nN 0

-r Z o;. 0. N o 0 M a 0 (0 N u(077 7 7-a- 0 c N0 NW0 o 0 0; 0 0 0 c 0 .' 0 0 0 0) V 00. . V ! mooVoo 00V.00 . ~ ' V.' V. ~~1 WO 2010/037119 PCT/US2009/058843

0 0 .- N en) 4) If N Go m) N N N N N N N N '

0-) 00a 0 C) - 0) G) 0) m () ) LO0 m-0 O-D 0) (0)

N' C C')' .- N' No No C') V-' .- ' V' N S i I Ir P CV

c00) 0 0) v0m Nl- 0 ) C'0) (00 ) P,.m co00) m0 -T0) (0 0) p)0 v- 4N CNNC 1 IN W-N (CN ON (ON Lo C N ON N C

.0) M')a) 00C) CC)0) co00) 0 0) (D00) N4OY N 0) N0) 0) ) (0) (N' (D) co w C0 C ') 0 (D NI (D -j w- 0 co t o w

C'? (. C? 0)- C (0 0C? N -' C C') I? C9CC 0

00 - N L') - N - D'

NO r0)C'C,0) (0 00 N00) ' 4) 0))., N0) (D C) co M0 a 9) C) (o0) ) Nr N O NoL C') W C' f) O r Lo Nn 'r a) ')

'l ) ITC') (0 (0 N ) c4l (0C) CD N- ( C - ' N NC') C') ',

0) .- i0) q40) (N40 ) 0) (009) (0)0) NDC) (00 0MN0) v(0) 0) 03) CV" 0() NCD w0a G-C' ITa W( ) V-' () .- ')D0 C')C N N 1?) 9' N N - N C'?

w0(O 00 0(0O V0( 0(0n N0 (0( -04 0(0 N-( Nm (0( FU) U3W (0 0)N -v -cN -SN 0 C ')N q'N - N- co 1 N- N- -ON-q ) 0O N N N N CNN 4N (N N N (N ( CNY a ' - -a '

W N)O N-N N-N ON 0) N a) N (1) oNl (0N9 0N 0N 0) C ( N -m -ON V) - (ON- ON V0N ON- '-N -CN N

(0( N-W 0( N( - 0 4 CV N- (0(0 NW4 (( ( N 4w

-N N CN 4N ON ON N) -NNNN

C V c 14 C4L) N W') N .- N OD N' (DN N az a- Ca a?a a a co rS ar 11 U) rV () 'W (( (0 N00( W C) 00 C'W C)O ((

C')N~~~c 4N O N N .N 4N CN ON C)N (O (09 4( LO( 0( C(D (0(9 (0( I? 09 -9 N(

v Z; '- N ; .- C') r I- al ,- a ; N a p c a 2al cl a C a 1 N L

0 (00 N0(DO (D 0 ) (00D ) (D 0) 0D)0 ( 00) 70) 0to (0) to-co

4. ( 0)' (0 (.U -- 0 4) N -- 0 .( N '.( N0.-(" ON.-U N N-.-( N)( N'. C''. (NI t? a. va C? I C'). C''-?- '

OD 44D O 40 W-0 (DOD) co () go 0) N-400) 8 '40 D () N40t)' ( D DO to N 03 N- ((0 0 ( 0 '- 0 C F0 - ( 0 0 ( ( N (9 o t, 14 (0 0 a2 a N a2 al Cl) a C" C') -7 6 'N _ 7 M a6- (0' a,; a a N 0 ) m '0 (0Cm)O 0 o 0m OC) (0 '0 w') ) 0 00 (O 0 0 )Cm)O 0 a)0 U3 0)Oa 0)) N 0 NC')'t N -( m)')L 0)Cr ( 0(0( (0C2( m '.'m) m 0 m-C')( (0 N-C)( 0 (0(0 0 m C')( CN 'N' N O ' C) ' - (0N-ON NN'- -N. ON. (0 .- ~ .- 9 a -C(

C ') C9 M (9 M (9 2c ? a (0 a N? 099 . a? 0 a01 1c ,

IT 7,77- 0 7v 0. (00U N-7' 4(0. 77 (- 'T (0.- 00 0(v 7 0( W7037 N 'T m' T -T'N N'TN) 0 -. 'T N'D C')'N- ' -' -(0'Nv -- ' C -' -- N

V) o Da ( l 0 Ci) Li 0 NC') v M 0 LO ND -() W L 0 0) M LO 4jr 2 'e Nt N N!;; a ~ aT a C a a C? a 4 a a V a a 4 I WO 2010/037119 PCT/US2009/058843

' V le V V

w 0) (00) (0) m m ) m)0 m O m O 11-0 (00 CD0 NoOtC) 00

C? N) Nn r r1 '' N? M' r,

(DO0) 90) m)0) go 0) 0) 0) VO) v 0) -0) CD00) (0 O) W 0) (0)Q (EN VN 0N 0N NN Go NN IO n 0) N N-.N ( N N-N N' 0) N ' N' N N N i N - ) 0

T)0 00) It)Q V) 0) 0) V0) D 0 09 N ) (00) 770 7O)06 0 r( Q( g (9 a w)9 P'-(w w( Q( (D( (0 ( N( N9 0a0 ' C? 92 cN'I C? C7' C? C?9

a)- (D'- O- - ) ON - (N N- '4h

0-0 90 N0) 0D)0) N 0) 0) 0) (00 CD)0) (0)0) N-0 M))

W) (0) 90) w (0 0) (0 Nn N-W LO 0 ) 0 N U V) ( ' N M) C' I l - ' 0l l) . 0) N

m0) l 0) WV OV (D 9 t N-V (0) NV) (3) m V VV N ) 0 0f)

(00)n N 0) 90) 0)0w) (00 '-0D) (0 1 (00) (00) N-) 0) 00L)

0)) 00) N - 00) (000 C ) 0) 0 (0)0) N-0) (0) OD0 0))V)0) N m 00) OD C) .- -l N M V) '- C' 04) ,m TF 1 ) N r 9 I? C9 2 9 99

1-( N( N1 N(0 '-(0 '-( N(0 (0(7 0 W 0( m( 010 N(V NN-. C)N VN- OTrl N N (0 P, N X N- N NN O1 O2N rN N (ON (04 ON N '-N N-N N (ONl)C 4 9 - ) 9 - c9 ~

Cl N N- N -N N N ON (4IDN N N vN Ol N )N '- 0'N4

l N' C 00~ NN- 0N N- 'N GoN (ON NN ON-(

N-(0 N0 N90U V)( (00 N-m 00 009 '-0 N-( V

N) (DO (0 (0 )9 0)0 00 '-1- ) )0 10) NN oN N-N -N (N VTN l) NV (ON 'N U? C4 O) N 0N

*1 ND (D ) U 9C) ND ( Y)( f 0) toO-D o m t N N N MN, ~ N N Cl 0) N l CI V C Sl Ln N-)(0 ."O 0( 0) 9 '0 0( v9( (0( 0)0 (00 '(0 0

N N N 9 (9 co1?I9 C

(00000 0 0)00 0900 (0) t (0) N -0 0)() 0 ( 0(( D0 (0- NO9( CO- MOO 0 - NON L? V ( - (0N (00 F0N r-O 00N t .- o) . 9 - - 9' 'T-2 N - - - V0 0- '0-) . I) C - r . 0)

N-a)O 0 C)00 0)a) 0 (00M)0 -0) 0 N 0) 0 N00 0)0)0 (0 0)0 N0)O0 V 0)0 V0 )0

SI c I I I I 1 17 I I m I I

r-4-N)' 0' u.)) ;))' 0)). 10 . 0 0) ' 00) ; N- 2. 10) . Nc -0 N I? C) C? N- C? 0 NC? NI C - C? 0 N 0- N? 119

777V0)) N-0v 70V 0)0)V 00)V (0V '0)V 777) O0)V 0))7N N-Vq0() n)V0) N- v0) (0) v v0) ED v0) 0) vVIT0) N- vO) 00 m 4I N 0 9)Vm

(D ('S0 10 m(0 0) c) 0 (0 0) 0 M 0 t (0 0 Ono 0) m (0 M- 0 0 0 0

L90n)0 L2) 0 m r (00)0L 00)0 -) 00M) C 00) 0))0 0) V) M!p 0 00pQ)0 (0)( -40( O00 N -L?)00 (00)9 N))( (00)1 C0)9 4 C0C4DD10 C- 0))1 0C)9 0)0D)AN

(0 N (o04 V 0 M 0 N 0 N 0) 0 O 0 M 0 M 0 0) 0 0r) CA N 9 ? 9 2 T 9 N 9 p 9 9 . N I

VO N 0)0 NOO U 0)0 N 57 (00 .00 (0 00)00 )00 (0

0- - . 0- 0) .- 0 N-r v 'T 0)' tf 0). N-Q Vr-~~~ - - -~ - - - -!

( 0 ! t L t0 - i . 0 ) 1) - I , 1 V o0( WO 2010/037119 PCT/US2009/058843

U) I Li O )in

NOo mO NO) (D A) w(D0 0 0) (N C) v 0) NO N) N C) 41 N NN (O UN Ni vN vN vN ON v CN vN toI0 l v ciI N7'I I' )C ? C N' N' LA' LA' 0 "j C '' C '4 C

LAO OD0M NO m LUAO) 0)0) m 0)0 ) (00M 0 0) 0O w00 m w0) 0D

mO 00) ma C) m 00) -0 r-d C)) N) (D 0) v 0) -T (D N OD c (W (q ( 0 (D OD 0 0) (D 0(0 8')0 N 0 LW N( 0( 03 NL 00 C? U. LC? C') ? ( (1) C) IDL 0OC? 0 ') Ln C? OC' SA

.- 0 [ID NVI C)' 0 ~ ' -. Na' N.- 0 -N C - 0

N' N C') C') C4 -0 N '' NN0

[0NW N( )0 (( 00 (( 0 CAW NW N) )W

(0) Nan) LAO NO) m Da) Wa) 0)OO ) N 0 0) m0 0) NO) N) 0) i N 0 (,U0 0 V0 m0 N 0 C' N C') N C C ( ) C' ) - m C') ( C') C')C) CC)') C')

O)7 NW 777 V0( 7)L NT 7'L -6( 0))OU) ( (D ' ( C) )l ') (0 (D M C' oA (D ~ (0l ED V~' ~ ~ ~ ~ ) a ) N N Nr N C' mN ) r ) 0 ) ( .0 N N 0 C'to N LO ) C) -n In -l C' Co'ODL) CD C'o) c' ) C'

C') Dm (3 ) U) c N m P') C' O) N C') m C'0 ) C) N

OW) m0 (0(0 NWm -L N0( V(0 OLAco () -U ) NWV (1) 0(0 m - 1

1, cir N N 0a) C Ca)q N LA C') N - a) N LA NN 0N N 0 (4 N N M- N N UN m N -l)N (

NW7- -6( 7W N6 -( W6 O)W -W- 00 ( -W .- W

(00 NO 0)0 NO 0 .O C) )0 . 'O LO C) ( NNl (0 N IN (ivN to N 0) N CO N O)1 (0N N0N )N 0N

NW LA(w A (0 W N ')0 -0 -0 0( - ON~a w) ON w0 vN.N N N N NN (0N

NCO( N0 mW( N00 (0(00 0(0( ,(( N(( V)0( NWO N( OW ( WO N 0 rON (0O 'V O NO WO14 (9 N WO NON WON WON W O N WO-1. O V)()N

C') ~ ~ ~ ~ ~ ~ ~ L - - C) - L- - - - - . ' - L A -' -'.- C'

N 07)0)0 L (0) mA) N0) (D 0) Na)O COO (O0 N(D to) t0O o) v0') (C')N OCN OC'NI OC'N L 'N [I A N (DC')N cl 0C')N P, N to'N NC14 0

C' W aD P N D CC o MC0') aD W aa N C ) co aD W 0 - aD 0 A a W ' a- N000

0(0 COW' m'(0 0 O). I,0 NWP'to-?P, V N W (00' C'W' NWD N(0. (0 ' NM - .- . N a - I ma a.- NaY) aC a CO C~~ , - aq

()0 0l) (0 IV 0 N - (0 V 0 9) (0 09 N (0 (0) 0 ( 0 (Om0 (l0 (0 N ) 0 ) ~ cm m nP oC') N a C') 1, CD) 'o r Y , N n a , ) r CD) a P r p , ) C') rr

L - C NN C?. NN. NN-? C)N'- 0 . -9 LA - CAN' ;N- ON;- LA .

0 C v) (D 9v ) 0 T) n 09) 0) r-9T) N79v (D 9. 9) ItM(0v 79r ) N v9) C

NOW (00? LA?( (00( N W (00 OW 00 (00(0 00( 00(0 (0

n0 V LAO 0 c)0 'T 30 r N 0 WOO )00 0)0CO0 N 00 l)0 (000 C 00 ) 0 ( LAW.- 0L.- (0tW-Om mA ' 0 (0P 0) mr * NW) (0LmUA 4() f (1) A' 0W W . C L. aI 'V1)N -N N C,' C'-' N N'N NN I N I NN N N')N N

) .- a LA- a) 0 )) a LA- N- aq N - a C- a D LA-a 0 0

C a 0' ? r D C? C?

(3 (0 )(D N0- N LA m) v CO mA N (0 0A LA ITAr LoW q aaa~ ~ ~ ~~ ~ aa~ ~ ~a~ ~ ~aa~ ~ ~ 'a~~ u 7aa~ WO 2010/037119 PCT/US2009/058843

In in in

) 0 t CD '0 CO)Q M) NO) NO) CO COO) t. 0) 0C) '30 COO v C-4 ,t ,2It Iz I ,2

'3q C) c O)' O C C C' C' N C mO CO' 04 C

CO) .0). IT0 '0 C) Co) Q -O (M (0OO) .- O m 0) 0)0 0C N C O ONC - q3 N * n C CN CN N CN CON C NO CNm N NNq N -

a)0 aO) I~) N) CO O) NO 3 C O ) )COO) CNC) NC) 0) 0)1 CO) OC( C)C 0)1m0( NCD 0 (D CD COW (D C CW (D C CO CO N-O C') -~ ON NC N- 1')vCO ~ C C? CO CO) C? OC1- CO 2 T C? L? I? CI ? I

CO.- CO (DO C) N- CO. (D. vO'. CO' CO' CO. N. nO CO. 0 0- ). in.- N- LI) CO- CO- i O N N N N N C'? m

C) MOO) N ) 0) 00 ) 00) 0) .C) CO) ) Nd0 ) VOO CO .CO COC N q Ln O (0V COW CO to M CO .- C COV N-C V) co C)C M T mO CON C)M MW C) m N-C m O CO mO N N 'o N N Nr - C

0) MOO C)0 m COO ) Nl.CD NC ) .- 0 0) MOO) CO) a) Nd 0)0 Nl F! CO N ) 0 CO 0 4 N NC N N N N N C1 CC')O O CO CO ICO CC C O N(N COO CW OC -O Ce) LO N2L N- 0D 0 0 f -1 0 'O N) N (1 O L O ND LO CD)

I SI I - NN nO - C' N nON n a)N N-N ON- ON N L NN0 dN N N C m 0 M N d) CON ON CN Nt 0O NN CO CO NN N CO '-N CON v) m CON N . - . N~ ~ ; CO C?-N C

00V)V) NO VO '30 COO NO o- '-0 0O CO 00 mO N ) CO,N a N CNN .- N 0,N N-N CON aN CON NN r, l VN IN N, (1 N NY14 LN CN O N 0)NN NN N N I 1N ON4 N CON ON4 ' N CO' N' CNn - ' ' N

CO C'

CO7CO6O O, C 0 00 7CO "60 COO COO COOa (D N )N .N N 04 NI N N-V ?N N- O14 N 0N CN CON N-N0 CO N CO CO - N t-N.C N 0 I* * I C I 9) 0) 09 ( 19 )1).9 CO9 NC 9

C~C,

m- CO-O 0- O 0-O I' N-?C ND . cO ) 0C W. C ) COC OC CO0 - -f N -0 -q . CO4 - C ------

.- O) CON NC) NOO C O CO M) 100 00) '3) CO0 4 00N) 0 04 N 'D N

CON N 1) ld 0IC) N IT ) ) 0 N33O CO N v N 01 0) 04C N~d ~

' N CO, CO '- N CO 'I ' N'N

CO CO C OON C 9)) 7 77 C4 9) 9) 17 ' ) 0 ) ) CN0

0D0 0)0 0 ) 0 00 00 0 OO D)0 CO NO WOO M O COO

0) IC WCC I ))C .-d)C W-) C)C Wd)O 7fWdC O) OC O

Go 7. w JCj CS CO N - CO 00) 0 00 C4 N2 CO 'o(3 o 0 ;O CO C COCC C C O C CO , CO CO O WO 2010/037119 PCT/US2009/058843

PNC ) r m ci0 v0 C140 0) U ) Wl m ' m ( V 0)

NO)V (N) NT) V NO v W) NO)v 0l)0) X0 RNO ~0) (O ('' C' U N' v C') N")' ('' ') C') '0) I CN OD N N 0( N CN .- N (NN TN T)N

coO aO U) 0) COO 0 v0m (Do) 0)) 0 )mQ 0) CrO ) N mNO V 0) wN CN (4 N CN CN CON to( c! ( toN 'ON v( ON

00 T0 N0) V 0 w 0) v to U) 0) N ') CO) OO (00, 0(D 0to 0 0 v0( w ( Nw w 0( ND 0 w (D C D)O '( C? CO' NC'V) 0CC) OF ? 0 mC') mOC' m ) V) 0)C' OC') N C') UD'm

IDn (' O CO- WD CDr N' 0 (0- (0 - N r N N ' rN N

'0o V)0 CO 0)0 NO ) V)0)O (00) 0-0) ID00) Lo ) 0)0') L'O A')V) oo C) C9C') wD' m C' 0D' (DC' gom C'w ) rDC) 0 w' (C') mO' C) CN N. N N N ' ' ' * I ? I

NW m m)C C')( ) 0 0) )COm 0 (D( M ( NW m'C NW L O N, - l N C' N C' 0 CD LO

C') (') C') o in U I P- C') OD C'O N) C'0 0 o c L) C') o C',( 11 ( V 0 COWC ~ NW O IT) (D( 0W IT W OW VNWv 0 ID N- co N 0N(0 Co 0) C') C') C') C C' ( N '

(O) O) m co ) UD) 0 ITm 0) (D m')O) 'O) (NO) NO0) NO)3 O) (c')0 C') C') NCaC)' D' ') C) O ' ) C') m C') N ' C'M' C') N o C') M N (N N a' C'

NW 0( NOlW W COW O W W 0) NO(D On r ) M (') m O) N CN (DU)N CNN Wc lN NN p F CON 0N. (NN N C 1N N N N P') 0 CN CON )N C)(N 'N N-N NN NN0 CN N N C'V' ) - C') ' . C') C')

0) O)N r N C) N C4 a) N rNJ 1 N O N CON 00 N (ONcl NC' CN CN CN CN N CC-4 1rI C)N Ln NN.4P 0

(0( (DC 00 rCO OW rW NW NW COW OW 0)O (0( N 0 NW 'D W rW 0( (D C CO N0(0

ON ) N ONl O) N CNN O)N ON CN N CoN g CN N 0C C? CC

t( -W N '-C w0( NW 0 NW Cto( 0( -(ED 'It o CN CO1(DN 0N CN w(0N CON ON N PN wNC U 04 (9 N RCN

co S *C C $ C * C *

CD C wCO0 N co0 0(0 N 0D (D( w -(D 00 N (0OD O (0OD 0( o( ((( (0(0n 0 c N(W 0 c (0(o(0 N - wO ON 0 )O COON (DON COO NO C')O NO COO r"0t0('O1. N MOON -2 C'm ' C) r C ') r ' - ' o - - N N C )V) 2

- ~ ~ ~ ~ ~ c N0r N) (0. N)- 70) N7 ) COT '0 )) 0) 0) 0'

O 040)O 'O 00 '00) 0 o o V Da 0))0 v00m0 co00)0 m0) 1 m0 moo) N 0)0 '))O r C' r r V ' gC') ', I- C')r r C') 'r C (N ( w m N C C m 0 r C '' CV m C 1 C

(0~~~~ C'7- C-,(r W0r 77N-( Wr o~(O.- 0r(COW (D t2 NC) C)'C'')r C') ) L 9 CC(on 0C'CD 0 NCO) LO OD') NC- C') 00 C'' - r ? VI )C p 0' CN' - ( C I- C?'r1 C

0) WV 0) 7~0 -7 0 V ~ O V 6NVto ) N'O0Cl V v) 'T0 coc v 7 -y 07) 7 7)~0 (DCO) 7 77 ) I- mO CO4 V (D0 0) 9) IT 0) - V C) -T a) 9) 0 ' D 0 IT0) C) v 0 C ( 0) (0 NVa C' ? C 7 op 2NC C) I? I? C? I? C C

(0 CO) OC')W L C')t(D2 0 r) E c)V CO mOO OCOW .C(f)t C)C) OIv C'()( OCOW 0y CO OW)L c E')) l) CO c ' ) (0C C? (0O C') 0 N C C') P4 N ' CP IT C ? C I? C C?9 9) C')C op CI I? N op IT op I C0N q C?'

(0 W =C 00(0 mo0 U7 7 C'a( 00( NOW 000 0 0 0(0 '0( Go 0(16 NOC NOW j O OO0)00 MM)0 0)00 000 000 Too (00 000 C') OO (00 Co O

CO W VC CO) ID '-0)C(O 9O)C CF)0C 7O) C')U (D N 0 -6)C O0)CO6 N (nCO C')C C~ N7 C (D 'i (Dr C) (D. D D Dr C ( r C rD C , (D C o- C

CC C C ? C C

- -J - - - - -. -D - CO to CO NN NN C (0 C O O ( q VP vC C .0~ -IT~ C C V0' Ct ' ''' ICC ,~ ,~ 0'' 1C -. WO 2010/037119 PCT/US2009/058843

(D0

to0) Nl o W W m0 0)0) to00) NO ) NOa) 0C3 00 D) N m t 0 v Ov 4CNI IN NN v C', ON CSJN 9T UN v - NN I lq coN

(0(0 0(0? (0(0 N0( C') C 00 C 0 (0( N(0 0(0 0(0 C1 . 0C(0

00) 0) ( 0 0 m co0) m00 M ) CD) 00) co0 ) 0) NO ) 0DO m m0) N0 (0' c'2 '2' 02 (0'C l C wC C) (0' (0' (' C? C? I? I'

(D0 (00 m (00) 0) 00)c c'O (0) (00 00 0)0 (00) (mV00)D to0 (0( 0( 0 co0 w- 0 w 0( w 0 c -O 0( to 0(0t V)' 0(0 0(1? (0(' (0( (0C 0( 070 C -' 0 00 C? (

GIN MN CNN (0- O. (ON T -N .- N TN ON. C-.

('2 N) N ; N : 't N N , N N N

(00 (D0m0) m)0) w00) 00) L (0 0 0)0 00 (00 to0D)D0D 0) N Nn (0 In C, N in CDt oW m. o - In 0 N ) 0 W

to0 Nm m 0( m 0( q 0( m) m) OD-( M0(0o 00 m m0

7. N6 N. 0 0 N3 N Lo NW F- N. 17 Nq 0 00 (0 r0 N0 1.,0 N ( m0 (0

N. 0 Q . OD ()q w 0 ( 0. N. 03() (0 In OD Nl N (0 ND (0 rr(000N Q0

7 V V IF IR V

00 .0) (00 (00) 00 () (0 0) (0 0) ( 00 3) L ) NO (00 m '20) ND (1 0 (0) (0 0 N (0 0- (0 (0) V(V'2 V C)

In( r(0 0( in ( m)1 (0( 0( 00 .- (0 V 00 0( ( N-. N NN. -. D O)N t-N NN.4r, N ON NN N N C4-N.F 0N O (N ON N1N o) N (0N (0 mN (0 N CON (0N N-NC

Nq- .- -a( 0 00 U (Cl 0 (0 " V l (N (0 w(0

n) N- 0N .- N ON ON 4 N N 'N cON P.N -N ON ( ON. tiN- NN r N. r-N N.N NN. NN. ONC4N N .

00 (D N(D (D 0 0)0 N N.( wO 0)0 NO f (0D0c

w- nO 0 ON ON w)n 0N 0- ON o)N O N1 N (0 N) 0 (0 N (0v a ( )cq N L (0 (0w

C * C) *(. C?

(00w tO(0( co( NO7-( ((90 CDU '-(0 0to w -o M N O N ON -NC -N 0) N C(0 CI o N N N wN ON

0)0 IDc .to00 N o c (0 OD OW 00 0( IT NOM(~ C0 N (D0OD (000 OD(0 00 NO (000 r Qc 0 ~ ~~~~~~~~~ (D1 D - D t !Nr ,O 0 N N

N0. O)- .D- (0)- (0. 00). N.). N-n' NO. (0- - Nto 0)0N C?0N N.0. (. I 0( . O(0N. N.0N (N. N(N 0)0N .( . -N

N~ ((0 ND M (0 )) m( - ~ 3 (D (0 V (~ ~o NC0 V(0

00. (0(0 'q m1' N C) - ) 0. (00 (00-C' P NO'-P , ' NO. 0( D. 0(' NO. (00(0.

0(0) 0(0 N(0) (0r(0) (0M(0) (000 N(0Ok) () N00) ' -0 N(o) (0)

'0 ' 7 7 7 (7 ' 7 -v 7 I=i

(0v( '-(0 (M N0(0 '-0( N0 (00( 0(0( 0(0(0 Y) cl N(0 m (V0 0(0(0 (00 N-N'- ON- 0?'- T. N W0N q N- (N- TN )' (0N T N'a

N.6 (000u 0)0F0 NM .0 ( ) M00 n 0)0 0q)00 N.O ( 00 T 00 0

NN 0N N-4 N'J (0' N ' N' N' '2 1 (0'I NN N IN V oN. 0fl00 7FHV 6 U-- 65IT 7 -670 wIVH N(0 ( 0 0 m 0 N0 ( 0 0) 0 ' R Ni 1IIIm50 II qD II uI0 N. N. N. N? N. N. (0 0 (0III(L9 w IIII0I9 (0C0 0 0 0 0'(, (0,, J!LLA!UN~ i!L!LLL WO 2010/037119 PCT/US2009/058843

cm M) ) (0 P, 00 0" (0 .N 0)~W 0) ) 0 ( W) W) W)0

r- Vp- vO C., Iq 00 Nf v0 r- v) o N v I I 1 ," (0I

w 0 N ) N U) 00) NO) 'O N V) 0M C (O OO) NN 1N

NO) O) CO) C,0F NO C -O O)O CO qO CO? to0 (00 w O ON CN C)N N 'N . N IT N '- ON C C)' C) C)' N' C) N ) ) CN )

COO (DmvO ) CO O U) 00 M COO) NO) NO m CM ) coO 0)0) VO) M C, IO V') U 0 0(0 (D (0 0(0D (D NC (0ED0 (D N to 0(0 0 to05') WC' OC? CC') CC') CC' OW C? wC' ( ' 0' DC' 0

NN ) N N CN N CN CNON N N C ?

I I If I? I?

NW NUO v)( N( NW C)0 (( W OU) NW 0( Go( C

NC CO OCO 00 ( CC n0( 0(4 V(0 .- C 0( 0 C'0 ') O) NO -) O) o)O COO) CO) 00 N) 4O N

Nq N C, A N ODC'CO co 0 NO M)( m0 M C) ( C)wM O') C'O 2 C) NO C'V V) N C' o) Nl NO U n 00 MV

rDC') (D N l C ) CO) M 1N co M NT M N D N' ' N C) C' CN? C') C')

0n0 00 M00 N0a) NO M'0 NO M0 m O 0qD 0 00) (00 (DN 0 C')C(D to'N ' U') W M0'VC) .C D') ND'C)' PC)'

Cn ) C) C' M n cr) C') N to V WN N N C'0 0 ) C')

MO CON NN CON NN ON ON C) C) CN N NN NN C') CON NN CON I? 0N N N C')

C') M ) C') q o c ) 0 N 0) M v V)) V' C) C'

N COO COO NO .- O C? 0 0 0 N ) 0 N

NI NN GN -N CO CN (0N CON v0 CONlr. NN -N -N .- U) ONj Ol N NN (0N (0 wci -N ON -N4 L(N COiN -N ONI NN N0 I? ? T C ' '1 ' ' C' C'IN ( NN

-6C 'aC 70 W(6 -O VUC 0(0 CO7 N F NCO 7(

N' N. (0. ., V V- (. N- N- 0- (0. ( CON rq O N ONC4~~~~ CN CN ONi ~LCON '-N toN .- .ON 0 C)( C) toN~ C)7 ) ) C

ON N O N C'N N (DN NN ON VNi ED - CN C

U2 f tf t*f 0t C ftD (D

M N (DC~j N N -l '- 04 ' '7 C) M t 0)

(000 ( 00) 77 O 770 0)) (00 NOD (00 N to '0 (00 to0 OW OWN NC) toN V) N OCN vCW ) N NW N -CN 0C) N) N

C) C ) ' C) '0 C) 'o co '4 ND ND cc 'o C)0 o0 V DO ncoc

N.0 N- C .- O.- V 0U N C% 1 I - (0 N .U P)-( N0-( (D- C OW- N OC) C 04.). NC- COW N VOC)q- N,FC) C) M DC). C). C)v

COV C O C - NpO UVO PO) 7 0O 7VO C)V CV C)?Q O O N1? N

CO am) CO CO CO m- 0 0 L ) CO N 0 (0 Cnm l Ma I) (0 co0)0 1 C) C) 'C 1 ) r7 C) N C) 0 nt Mp C') C) 'T M 'r M.- M' C') DWO 0C)O VC')O~ ~ ~ ~ ' C)CO OO N) W CCO CW )O N)0 r C)lC

tN( (C(0 )) NC)C4) NC( NC)( P)) Vc ( LOC( OD - (0 OC( 0 NN. NN.- C) NNCCN O' CON.- CON CN- CON CON WN? 0c Q q C) C? 79 C ( ( ( '( ( ~ 9 ?~ (9 7( 1? CI ( ~ ( . 9 C? ((

NO OD NV 0D WOO0 V 0 0Nqq0 0 000 D O 00 0 0 NO 0)0 a qQ Dv0 NV N( N W CO Q? 0- ( 0 - NC (0 ( (D W C00. 900. .

o-oc0(000 Cl (D 0( COC (00( VOC NOC .0(0 co ()0 (D a r (00(0 N co 0 1 COC 0 O Nf . 04' MV C O4O- - V CCO C')V4 CCO C)V Q O4 - O- 0 C? C? N? C)'9 ' N ? N ' C)' C' '?

CO N CO O) P 0 Nl C) rO (0 C7P N C O

CO T CMO CD (0 0) T 0) 0 0) Rw w0) w 0 0 0 0 CO. 7 O. n CO. Lro In CD v. v W) . O', V , WO 2010/037119 PCT/US2009/058843

U, CD r0) 0 C4 m in) St U)

o 0 0 0 0 - Z . -s %n La w) WO Ua U) WO LO to CO U)

0) s-m 0)0) OD00) N) (D )0) (009) 0)) m v C NO m 00 NOw O) 0 c 0) vO~ 0)S v~ (O:t s-S t I OT N C, Lo V o c~ vO~ SOt St LON V-N NN, r v s-N Nt NN U) NN N-N -N CN CN aN * - I? N* C) a NCN ) a I a aa

-5 v~ 77S v ~ 77 S CO7 LO5t Ot -q ~ 0 v N-S v O~ q ) 0 00)m N 0) m t) NCD m0) Vtm a)0a) 0) 0) 0) W(0m 0) .C) (aD N (ON (ON SN IN U)N1 NNC 0)N (ON w- 0)? NN (, 5*I(I) N* I (JN C'a a) I I I () CI CI aIa

to m)0 0) 0) sC)3 U)0) StO) co0 M OV) 0 0) (00) NO) 00 0 )0) (D0 ( v)1 S(D w0( 'TO w -( to ( V) OD0 (D IT S(D 0to ( 0 (D 9) 1?0 1?(' 1?0 C)0 0C?0 0)a .0) 0(a C? 0 I? 0 C? tC

LO I ) tos -a a)N0 N 11 aa Nn

CN 9 N .- a, N.

0) 0) St) StO v-C S) (00) 00 00 F)) 00 SO C UO (0(V0 0) w0( StWcU L 0V qU Ina Lo m-U LOU t-U LO u I-UI m) Vf) -co) (00 N-m 0)(' m(0m0?) 0) 0 (n00) In0) 00 V) 0 (0 0) N N I - 0

a) 0 0) NO StO) (00 (0) 0) 0) 0 )0) 0) 0) 0) ) 0) C) NO ) 00) Lf) c0 CD N- m) - Ur) N - 0 0 I Ig a II

(0 L (0( Ln( N-U Lo ( m V v ) w0(0 NUV) go( V )1 (0(0 aa)U (oO (D0L ,It m) v 0 (D 0 0 Go w0 0 0) N N14 N - C0

m)s0 N-0 m 0)) 0) c00 (0co) 00) Lo( (00 n-C m0 (0) m (Go)

:! N) N V) 0) f, .- N )

(0 00 N) 01 (( -) S( (0( LOU s0U N 0( (0m

C) N O N 0)N 0N q i 04 -4N N U)N (N 0-N (0)N N-N0 3) N ON C4

CO NW7 "F( NW c 0( c0( 'r( 0( NW (0 (D0 770 (0(DCC

N w N0 (O N w) tN wt )N (N 0 ( N 'D DN (N

0O N NDcl 0) .- v N N N c' N CN

CO a)( w VC 0(w N W OW 0( 0)10o (D (0(CD CD V)( s-C CN m0)N WN 0)N CIA vN (ON IT N IqN N O N .N NN0 0N

C l C * CC C C I (00 0(( ((0 0 (00 0(0w0 s-CO N(O (0w( w)( N-O N-0 c)(0 to ON- C.)O WON WON C-ON 0)0N (ON Ns-)NON M-N 0 Ns O 0N 00 .- ) 0)0 N.0 0.0 Ns0 Os-0 N.0 (0 ) -0) (0 1) .- C,) 0 N .- 0 0) P. a N N N -l ' co 0) .

0)0 0) 0)) (00) ND o -0)0o N NO N0)O 0)0)0 (0 00 N-M0O N0) NO al a' m) a, F- aC ) a in C)p 0 ) ar co V) N - a' q) a ' N a s a

(0. N- NW. NW. ;~s N , ;;0 St2 s IT0. wt0s ;O s v- . 0 as- s- as- s- -0)%4 s-IT N w.0) ;sa c)-)t s 0s w

(0 !2 (0 0U) ) St M 0 m c ( D P - 0) (0 N T (0) (0 N) C 0 Nq (0Cl 0v 0 0 a~1 T (D 0) NT)' 0 N ' 9 T a 2 a0)

mL 0 0(D)m0 M-)U S0 0 0 w m (0)U m)00 0)U .0 m s0)L c-)o o 0 N.- P Ns 0)N -Stcl Ips- N N.- s- 1 (0Ns N' 0N ON. (N. 0N- (N 9)0)9)~~ N )co ) N Co 0 t 9

00 00 'T0 C')0 0)0 WOD ( 00 N0 (000 0l)0a0 NO - 0 00 (o0 N -'N-'N -'N -'N s ' - N' Nz- - ' ''N '9

0))( 0))1 IT)( NOC N0)( (1)( a)O 0)(D O N ID (O St.-~~N 0)( s-,C 0)t- ))- ~s 0)Sts- (D tOt. (0. N0t CD t. 0)ts N-St. COt a a o a It w

- NJC U) C 0'N) N S ) ( C)N 0 0

0G) 0 0O in0 0 0 WO 2010/037119 PCT/US2009/058843

N ) ) 0 M N0 In) wt 0 I' - - N N N Nm Nm 4 N N N W) W) %a 0D UO W) ta w) w) wD W)

N ) NO) a) NO) NOc) 0) L r) (D M 00) 0)) 0) 00)l

NO) 7 0) (1))a) 0 0) () () 0) NO ) 0 M 0) r-O N c) Q .-0) NO coNI~ CN O -N -N C m C')" rN CON N') N

(O0) (DO0) C) c) NO0) 0 0) V)Oa) (D 0) 00 Q (D)) 0 0) NO ) TDOT (D V E 01' (D' N') (DC' N') NC) D(D' (D C )('N I ) (C) D DC')

- N? N ,-) N GoN l ID 10 D ) (D'0 Lo0 0 ) 0 O C,)Ot) ~ ) 0C) () 0)' (C) D' 0 (DO)q ' C' C ')'

0) U)) CN(D 0) -)0) NaD 0)0) 00) (0) 00) 03 .- (CD 0 -( 00) CO U) 0)W) LNO (DO) 0)) )) NW) NOW) 0)0) - 00) r-0O r., N , 0 V (D ) ?V O ) 0 Cl (D M N) m ) I?) C') . -) , C) C 0)~~~~~~0N

C 04 ND N3 t- - C) N N T !2 - ' No ) .- O0) 0) , 0COO) (DO )NO) NO0) .- 0) C4d 0M M D) O -0

C' C') 0 W) .- tv U- ) ) ITC'I)o n CD

N( Iq ( co( C')( ITD 0)0) v)0 C')( Nv C'D( 0)) NN (D 0N N 'N 'N 0N N C)N (D(DN N

mC') co n al -D C'C, C' V)CV) NC'

N O (O C) a0 Lo NLO m NO (D 0)0 NJ 'JO (DO N C') (D N N ON Om NN (D 0P N ' - DN C')00N ~N 0 ONv) N 0N NN -N )N ON V-N C'N (N

N T- 'F 0 7Fa , f

L (04 N 0) N N0 C 0)04 t)0 NN C-0 N0N)oC1 N 0) Q)C)04 LN900) N ) N ) '0 0 N0) )0) C',( 0)) 00

(D. N- )' CO- C)- - 0). 0.- 0) O ED, C1-( C')~C' N NC') 1 I

o) N ' to0 n -0 an C') N '0 0)) )0 N( 0)0 0)0 00) 0'0 N00) 0)) 00 !2N ON NN 0N ) N N N N (DN ITN N c N

N o '-O 0N rlON (DON C')N (DON C') N C)N Dto N NON P0N (DO N C')~ ~~~ (?') - C' W0-' V-'C) N-' ''C) N ) C' N0-a EDC' NC'

S al an -2 r- r) .N- CO . - N, .- rl . C) 2

-~~~~~~~~ NO) -D) 2, )0. ) NO' ')) M)) 0))- 00. 0))2C))

to (SU O(D0 an 0 C'(D o M N.(D CO 0 0 (D -03 ) N-(D) 0 to(D 0 -(D) - C9)) t 0). 00v m)0 1N, N0c(). N-0O ) 00t) N .)' (Drl 0) r vc '-0) N0 C)

r)O, O) 2O C' 0)1) C) O) 10 0 CID NO ) 0)N [1;D; 0D 04v) 0I 2m 100T wI 0) TI D c 0) 0 a) 0) 0) ) 0) 0q N 0) N 0)a N v 9 ' C') O 0 N 00 Ta a? o a I aOaNa Ca) C'? ' ' ?

0 c)cl) 0 )Cm)0 0) C) I 'C')O (D Cl0 w C')O C) C) 0 wCmQ O 0C'0 N C')0 m 0 aC' 0

a? N'0C NO0 .- 00 000 0)00 C004 NO0 -q0 0)04 a'O 0) N O0)

OC) 00 1) 0 0 OD 000 000 OD0 0)Co00 000 000 VQ0 -0 0)00 00) 00 T a)) 0)0)- 00)' N(D' N(D N L. C')D' V(' L? L? L I 0)T07)'- ' N ' N C') 'N ? 'Nr ' ' N .' - N - ' '

-CM OD N C'U) 0)L N 0T ) 0 [ N

ON 0) a a (a a 0 a a ( '' 0 ) a) a, ( . a 0 a' 0) 4 '

C-'-, WO 2010/037119 PCT/US2009/058843

M0 COO C) COO) 0 CD 020) 1, ) CO) 0) )0) v'0 CC) (D 0) cli coN n C c NN C N N CON N CNN V) '0N?

V)0 M ' N.M (''0) C0'4 M 90 0 m C) .'i0 02' M 0a'D 0)

COO) IT) M O ) NC) (1)0) COO) ) T 00) Q NM) CC M C0) NO) tO' ( O' (D(D (D C14~ (D ' N' C N' t ' In (D mN (D P(D'w c

COO) , '00) 0(0) NC () N a -) '0 0 0C O C 0

CN CN C0' N C ONN C C?* ON CON CN CN

7' 7f - 77 l -7 7a ~ N. J7 77 F, T- 0, (0F F- F LIN i N 00 N N

CNO M O) 0 MO) C) 0) 00) NC) co0 C) 0am am) '0) CO

U) 00 ) 0) (00L) '-C) N) V-) 12V) NO) O) Nw N) I ) o '0M c CO () tO 0' 0 C O) N, C.)O N. N' CO ' O ' m0N

CO N tO 020 C) 1- 0 ft Q tO 3) C 0) a (1 N 0)

NO) CCC) COC ) COO)L 0) 0)0o ) 00 020 COO COOn q ) NO) a2) U COW ODCCC' QC'NW N04 W W OW CW N

NWCY CW w -CO V)C COW C 0) a M c .CO LO m 00)2 4'0 OW a CON C 0( ) NN) 'N WN CON ClN CN CN .N c CN '-N () CON V

NNO 0)0 COO COO COO COO '00 NO NO CO D fI) O r COO LO (D r-N NN fN CON N 0N ON CN. P N NN 0N c0 r, 0(N ONi CN CON ON CNNC' CNN -N COaN ON .- N0

Ol 04 N a) a DaO N 0 a NNci a, N a N 0 NG II?

CO0W)W CW O O 0 CO 0)C '0 -C 0CO COW NW '-C CO W W CW C )W W N

OD0 CNO O 2 O 0 0)0 CO '0 (O N OD' N. CO N I CO' CO R N IT0- '' 0- N- 0' NN, tON N -N N N 0)NC~ CNN CON '-N C 4N CN CON

ON CO N 'N O N v N CN CO N sN CON tON O N C) N 1(29 r, 0 9 -) '9 09) 9 0)9 09 f') 9 c)9 09 a 9 9t f t 9 t 9

ODW0 (W0 OD w200 CO0 OO O w0 N0wW 0 (D00 CO 0(0 CI00 CO ' -O ON 0O ;)O WOD 0 N OD '00 No wO NO WO CON tO-W N-CO - wF OD-C9 N.- 0.-C 0- N.- 0 0 , - aN N v2 - N r, - ~

00)0 v C)0 '0) 0 COC)0 0 0)0 W) N 0)O 0)0M)0 00)0 m 00 m)) W)O NCN ON OW 0CN W WN N N )N CON CW W NW

a, CO aD . a N c aaa aC a0 a- OD al ' 0- '

N'-W~~~~~~ m21 CO7 CO-4C O- 0C OC 0C )C -O 0.C NWI?-O-' ' CO' Ct NW- C7t' CO'- CO O CW' NW- I? W'

N a- a- CO ws v a -a .- 'U '- N -' 7 - CO as- Nas Na,

(N' 0) (D -q m 0) M C) It M V a CO') IM 't'qt C (1) 0)'O IT O'0)IT0) N'00) CN ITM CO 02 N w2 0 02 0 2 - N C 2 0 9)) N a ~~~ca a Ta a N Taa. aT) v) 2 0

cN M ) CO M ) M9 0) a0 V) 0a aD 9) a M) C2 9) No a ar 9

'002 N0)0 0 0200 0)00 000 0200 NOW VOC M00 '000 RML l Nt ) L n o 0 nO

NO '00 WOO0 000 0)0 0 )0 0)00 N 0 -00 00 NlOO O 00 O)W COW' )0 0W- CC )W OW' OWn 0 ? 0 - NW- NWLO- GO2L a ~ CO N W N a - N NN Wa9N N -'

N a C M a N- a '0)- -0 6,6 a (0 COg - aCT0),.a 0 2 a a r, a N a Nn a N aa 0N ) a) N a) -) a a

L2 N N'0~ ~NCO tNC N 0) 0)O C'- O ()C) t CO a a CI O a O a a CO a O CO O a a C O a a C WO 2010/037119 PCT/US2009/058843

N 0 0) CD '. ~ 0) .- N m' ul Lo ) 4 in W to to W) ) W) W) U) ) v) m ) v0 m) N) m D 0 0) 0 in m ) U) m c 0

0)) 40) m0 Nv coO v0 v0 0) It 0) NO) (0 00) 0I 2C n C ON NCN 04N ONi 4 -N N CN NN C? S- 4 5 N l)S r 4 N C)'

0D m m) C') Q 0m) 00C) rO) (D0 m co m N) m )0 COOw0 ) 0 a)0

-0o (D) 1-) N) ' C' C) to) (00 00)j 40? C-) C? 0 N

t,( (D0N0 CIO) m ( .- 0) 1D-o 0 ) 0to m)0 CV(0 CD0 CND (-C' 0)0 C O) CC' CC' (D w ) w 0) NC' C'C' (C o 40 1-0 w -' r) Co C: pr N:1 04 V) m 't CI)c r C) C

Lo ) N N7 N .- N )G

0o))co0) N C' (n 0) 0' WO) m -' OD) w o C') m N O' C'C'C') m'-)m9vC C') 'w - - C') 4 - O NCo CO Lo( 4( 1-C NCO W00 00) (0o N)0 1-C 4(O

-0)D 0 0) to0 m ) m )0 1-0)N 00 m )0 1-CD r 00) 0)0) m)0) OD0M IT4C -( 0) w) (0 C(D- ) 19 v N

U)~~~~~' t o O I O C' C'O -nW r~~ No C'N7'

U' C'-4 ) ' ) CD) 4) ED'-i m' NoC N) om O 0 U )) c')U N)0 0) 0)) 40) m 0) 1-U) NU )l) '0C) 00)1) OD0) r 0N N CON C') C? )N 0 'N~~~C C'N 4N 9 ) 'N ON C') -O N C') WC'7' 3

- OD0 10 NO 0. 0 S0 C' 0) C? ) 10 )0 00 0 CO N (904N 0N NN N Cl 0)N 4N - C)' - 0 C? 0N CN 0 1 ' N 1 - '-1-N) N 1 - 0) - ) - ' '- 'C'C?1 I-?

NN C ( -O 0C 77CC')C 7C .0 0)0 00) 00

N -1 N 1N, 4 a)-N N0N CN C')N 0 N N CON [N- N

C' ' N 0) t ( N r 0) '

C C C C I? C ? C

)) NCO Cl 0) ) .- CO NCO C 0)O (20 C'( C -(

U, 0 C 4 C n CD 0 0 V C)O0 0(0 C) 0)C) ))0 0) 000 0C0) CCCO C)C' -(C ((0 ((0) C\1 0') - C' C - .- 0 r'.- Y 'r ' OD'C'4.C' N.0 U)-) 4-' ~ '0 )- ''C) )-) 4-) 1'C) .. '

' 04 N ' l C') 'N 0 N ' ['7~ '~ N ~ 'l 'O N 'a N NW- 0))' 0))'q 0)0)'-~ COo) 0))' 0))' 00)? 0)' 1 )- ) )0)' . 7' 1' N N' I' . I I.' If' I.'' I '' I- ' 'I N'' .?' If''

4 0) 1-(04o o o ODt 0)0N 00 W D) LI)0 0) O 40) (OD 0) G (0(D)O V0)4 (00 00 40 N I lI 040 1 -40 0 40 ) '240)o Rw - ) 04) 00) N40 f)0 (20 140

0)0 v 0) - 0) CO No Q ) ' 0 1- 0) N c) (M 9 O CD 9 N 05 9) '- 0) ) 0 )

0) N) In '7) ' ' 0) C- n O N

v)U Q -qom 11)m N 10)0T ) N0) r0) N0)q ( 00) v 0q0CF ) 'I)D0)v0) 00) v ))0 a-)0 00)0 m

0)' 0 - N '- 0 vNN 0 t CON- N) 9) ~~~~~f0 )N ODm0 ( 0) ON'-n 0 )N'- ON' C')N''to 0? C''l'9 )~'9 -) '9 ) '2 M) T ) 000 0)0 -0 '-00l) to -00lU) 0)00l) CD 04 r -1 -00 ) (1-V) n 0 )0) 0)0) t OC

0 04'- 0)9 CN 4N 0) C11 Cl C N .D -4 0-C-4 4M . CN4 04 - 0l)4 4N' 1-4NN

.0 0 7 0 0 O 0 ... U) CO 1 - 0 ) 0)ci 0 - ND00 E C') 0 4 0

o0) 0) 0 ) 0 4m4 0 v)4 ) U),, 0)11 0) n a 0)I 01' N). a,' IT'0, ), ~ 0 WO 2010/037119 PCT/US2009/058843

LO to co N- ( 0n N M LnU on CD U) W) CD CD wD wD C D

0) D C v ) D MD CD CD Co MD -q toM 0 M D CD M I)

co 170 C) 00)i q lz v-C Ma vOC M0 (0 00)I (0) IT v Nav q~~C WY NC(4 0 N ~ a) ) ~ O ~ r)i (N )D ON aN '-N ON (ON N N It ON ONN

-,; -a v U7I Tv vv 06v

a) 00) ~ 0 00) t-a M a) (0) v ) Na ) M0 M -C N0 C) to00) I? C') N'N C? ' ' 0 0

0) CD o0 00) N) WaM D0 ) 0)0 0 Na) '0) 00) M (00) IT0) ID w( 0) w O NW (w C (W V (D (OW q( . 0W OW 02 co (00 (o toN C' ) ( (D0 10 w 0 * N N I? N! N - I? - I? C? 1I CI C? Ili

N T (O 77N N- N-N O-6 rN0- N-N (ON (N NN vN .- ) Or 0- wO 0). WvO- N-v cv W - 4' 'v Wv- (0. NO v CO - Q- - - , .N

(0 M( 0) 0 M( NW (00 Nw 0 0) ( c(0 M(0M(V 0) 0 0D(C In (0 N NO U00 M0 W) L -- O ) 0 0 A) M 0 0)0OcnL Nl 0 C') 0 -q M N MN M0DM M

0 (00) L (0 It0 Na)l Na M 0) M 0a) 40) 00)M 00 Wa0) N- a) M A)0 0 N 0 N S N (0 N N * - .

00 0 4(0 MOf N-( V 0( (0( Ln inV 0 0to a( U(0 4(0 Wo 4q (0 IT 4 O vN4 T r, MI v) ) o 0v A4 (O V ;rI 1)N ' N ) N N 4 N N~~C N0 a , v N Nq N

4n 0)o M 4 M , D0) N (0 (M MO N-4 (0 0 D 0 N4 N4L M) 00 n 00) n t0) Mc Na) 0) Na)W9 0 0) (0 M0 Na (00)

QV OD 0 0)0 40 (D (00 LOC') 00 '-0 00 00 00 0oL0NW I 0- N - 0r N CN N N, Iv- N C4 0

(N 4N I-N ONN (N N N4 vN N N (ON NN ON M N N~~~~~~ NN- - - . Cn C' W)'

4) N O (N (O N to0N v N N M)N ) N O (N (o N N0

0 I-- PC

0 40 NO (0 N0 ) 0 0 (0 0 (DO 00 00' to O 0 ? If- N

(0 CW0( OW -W 00( 0 M 0 w(o c (OW 'O a)

N oN N C'N (N N (0N ON (00N (OCN 0)N to(N (OCN

(0(OD 0(00 ' ( 0 o n(D0 V) 0(D00 0)W( go (0O NWODW (0 00 t0((0 D ITto00 0 CO(O NWt N N-N 0)N ON - N NON-0 0)ON O O )N -N N ' O w0 N M0 N -0 N-v-C') ?0- (Oy MO FS Nr' Nr rO -l WrO - - - V !9 N -rV OD (1NN V

0)r ra). 0)0). (0T. -0) O0) O0)r 067)7 40) M 0)r ,q C')) Na)3 0) 0 M0))0 -q0M0 N0)O) 00)0 (0)D0 t- O 0 ()0)0 0))0 00)0 IT) 0)0 q M r N C't o coN M r v c' ' v- C' tr 1' ') P7 ') 1 ' 0C .

Ur r-~ 00 Nor4 ,; 00P CW (00. NOV (00. 00 V r vO 00 1%- 1?r N * C- M C? 0 r -? Cr- N w N'. 0'. 0'r N2 w- vT V )C

7F7~~9 v~ F9 77 '5 7 7nC)vC ) v(0C'

(0 ) 0 0 ED V) 0 toM ) 0 M D9)4 (0 co M0 - -r ITC4g oC4- 0C4V 0 N - D(' - vN rN- V- V N N O 0 C

0670 (0Z( N-O( w0( N0( T0( 7 W WO(0 -&a "77 - ZF70 w00( 0 0 0 O 0 0

00 (00 00 N-a NOO O C' 400, 0 gc NOC .- 00 000 U (0 NOD 0() 0 (Or0)(0 c-0v( 0 NO0-P (OqO O~ )~ 40 00- (Dt~( I?0-

0( ;;)( v0)W C W0W v-D) 'On) O0W N0)( ID )( M -a)W N-0) W W0)M07 40)

(0 () N, (0) (0 0 r N C00 (0( I?0t (D 7 (? (0 ( 0 * . ' ( , ' CH 1?~(0 71 V) S ( ~ ( ~ ' 0 0 0 WO 2010/037119 PCT/US2009/058843

La W) W) W) W

(0 ( 0 0 CD 0 IIW-q r) LO Ir cU)L

CD0 NO)0 (D -0 M 0 0 0 N 0N

C0 T 0jm0 1CD F 0) w

"ID w O). (D ' 0) r (D W(D I? ~ -Co 01NnC)

'a) VDO 00) to 0) m (01 wNU) (W 0)10o (0 CN N

NO ND FFT NO CN) NO)C c 0) (DO C')mO) CD

U7 m O' 4 ) N N q c 0 N ) v C

Ln- m0 11 0)1

v -57O 7 0~ LID Q a) 00 0

40 0 c Ol U)00l) nO 0 00 0 (,0 0

N C1 N C1 N)

O)CN N N O 0 A CV P- l )C

m

7 70)T

NO 0)0 DO .- O (D 0 w' N,- 0 N 4

N 04 N N 1 00

1o0U0 0C (D C r (D N

* ~ I I I I

O 00)0 10)0O NO)O woo0 0

I 2 - ~ o N NO

If CI) N-f 01 0-I) (

IT N'. 0'N'o (D-)I 4)' m F 0) 00 v) 0 V 0) 0IT0) N 0 - 0 to 0) 0 N 0

F U - -F7W

CNfN N01 [I EN0 0 N l 0N

0N OCD )( 00 0 NOI) If -? N-. (DOC CD?' ) if)N C

O- 0'N 'NLoN (

0 40 0 0O) N 00 0 4)- in

'T 0). -64 77- toN

N - * N L co 1 0 0 O - N 0 4 WO 2010/037119 PCT/US2009/058843

CLAIMS What is claimed is: 1. A lactic acid bacterial cell comprising a functional dihydroxy-acid dehydratase polypeptide and at least one recombinant genetic expression 5 element encoding iron-sulfur cluster forming proteins.

2. The lactic acid bacterial cell of Claim 1 wherein the functional dihydroxy-acid dehydratase polypeptide is encoded by a nucleic acid molecule that is heterologous to the bacteria. 10 3. The lactic acid bacterial cell of Claim 2 wherein the functional dihydroxyacid dehydratase polypeptide is a [2Fe-2S] 2+ dihydroxy-acid dehydratase.

15 4. The lactic acid bacterial cell of Claim 2 wherein the functional dihydroxyacid dehydratase polypeptide is a [4Fe-4S] 2+ dihydroxy-acid dehydratase.

5. The lactic acid bacterial cell of Claim 2 wherein the dihydroxyacid 20 dehydratase polypeptide has an amino acid sequence that matches the Profile HMM of table 7 with an E value of < 10-5 wherein the polypeptide additionally comprises all three conserved cysteines, corresponding to positions 56, 129, and 201 in the amino acids sequences of the Streptococcus mutans DHAD enzyme corresponding to SEQ ID NO:168. 25 6. The lactic acid bacterial cell of Claim 1 wherein the recombinant genetic expression element encoding iron-sulfur cluster forming proteins contains coding regions of an operon selected from the group consisting of Isc, Suf and Nif operons. 30 7. The lactic acid bacterial cell of Claim 6 wherein the Suf operon comprises at least one coding region selected from the group consisting of SufC, Suf D, Suf S, SufU, Suf B, SufA and yseH.

112 WO 2010/037119 PCT/US2009/058843

8. The lactic acid bacterial cell of Claim 6 wherein the Isc operon comprises at least one coding region selected from the group consisting of IscS, IscU, IscA, IscX, HscA, HscB, and Fdx. 5 9. The lactic acid bacterial cell of Claim 6 wherein the Nif operon comprises at least one coding region selected from the group consisting of NifS and NifU.

10 10. The lactic acid bacterial cell of Claim 7 wherein the Suf operon is derived from Lactococcus lactisor Lactobacillus plantarum.

11. The lactic acid bacterial cell of Claim 8 wherein the Isc operon is derived from E. Coli. 15 12. The lactic acid bacterial cell of Claim 9 wherein the Nif operon is derived from Wolinella succinogenes.

13. The lactic acid bacterial cell of Claim 1 wherein the bacteria is a 20 member of a genus selected from the group consisting of Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus.

14. The lactic acid bacterial cell of Claim 1 wherein the bacteria 25 produces isobutanol.

15. The lactic acid bacterial cell of Claim 1 comprising an isobutanol biosynthetic pathway.

30 16. The lactic acid bacterial cell of Claim 15 wherein the isobutanol biosynthetic pathway comprises genes encoding acetolactate synthase, acetohydroxy acid isomeroreductase, dihydroxy-acid dehydratase,

113 WO 2010/037119 PCT/US2009/058843

branched-chain a-keto acid decarboxylase, and branched-chain alcohol dehydrogenase.

17. A method for increasing the activity of a heterologous dihydroxyacid 5 dehydratase polypeptide in a lactic acid bacterial cell comprising: a) providing a lactic acid bacterial cell comprising: 1) a nucleic acid molecule encoding a heterologous dihydroxyacid dehydratase polypeptide; and 2) a recombinant genetic expression element encoding iron 10 sulfur cluster forming proteins, wherein the proteins are expressed; and b) growing the lactic acid bacterial cell of (a) under conditions whereby the dihydroxy-acid dehydratase polypeptide is expressed in functional form having a specific activity greater than the same 15 dihydroxy-acid dehydratase polypeptide expressed in the same bacterial cell lacking the recombinant genetic expression element encoding iron-sulfur cluster forming proteins.

18. The method of Claim 17 wherein the specific activity of the 20 dihydroxyacid dehydratase polypeptide expressed in functional form is at least about two fold greater than the specific activity of the same dihydroxyacid dehydratase polypeptide expressed in the same bacteria lacking the recombinant genetic expression element encoding iron-sulfur cluster forming proteins. 25 19. A method of making isobutanol comprising providing the lactic acid bacterial cell of Claim 15 and growing said cell under conditions wherein isobutanol is produced.

30

114