(12) Patent Application Publication (10) Pub. No.: US 2010/0081182 A1 PAUL Et Al

US 20100081182A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0081182 A1 PAUL et al. (43) Pub. Date: Apr. 1, 2010

(54) ENHANCED IRON-SULFUR CLUSTER (22) Filed: Sep. 29, 2009 FORMATION FOR INCREASED DHYDROXY-ACID DEHYDRATASE Related U.S. Application Data ACTIVITY IN LACTIC ACID BACTERA (60) Provisional application No. 61/100,809, filed on Sep. (75) Inventors: BRIAN JAMES PAUL, 29, 2008. Wilmington, DE (US); Wonchul Suh, Hockessin, DE (US) Publication Classification Correspondence Address: (51) Int. Cl. E DUPONT DE NEMOURS AND COMPANY CI2P 7/16 (2006.01) LEGAL PATENT RECORDS CENTER CI2N I/2 (2006.01) BARLEY MILL PLAZA 25/1122B, 4417 LAN CASTER PIKE (52) U.S. Cl...... 435/160; 435/252.3 WILMINGTON, DE 19805 (US) (57) ABSTRACT (73) Assignee: BUTAMAX(TM) ADVANCED BIOFUELS LLC, Wilmington, DE Lactic acid bacteria expressing dihydroxyacid dehydratase (US) polypeptides with increased specific activity are disclosed. The lactic acid bacteria comprise recombinant genes encod (21) Appl. No.: 12/569,103 ing iron-sulfur cluster forming proteins. Patent Application Publication Apr. 1, 2010 Sheet 1 of 4 US 2010/0081182 A1

Patent Application Publication Apr. 1, 2010 Sheet 2 of 4 US 2010/0081182 A1

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Patent Application Publication US 2010/0081182 A1

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ENHANCED IRON-SULFUR CLUSTER little is known about the ability of lactic acid bacteria to insert FORMATION FOR INCREASED Fe—S clusters into heterologous enzymes, and little is known DHYDROXY-ACID DEHYDRATASE about the facility with which Fe—S cluster forming proteins ACTIVITY IN LACTIC ACID BACTERIA can be expressed in lactic acid bacteria. 0006 To obtain high levels of product in a lactic acid CROSS-REFERENCE TO RELATED bacteria from a biosynthetic pathway including DHAD activ APPLICATIONS ity, high expression of DHAD activity is desired. The activity of the Fe—S requiring DHAD enzyme in a host cell may be 0001. This application is related to and claims the benefit limited by the availability of Fe—S cluster in the cell. There of priority of U.S. Provisional Application No. 61/100,809, remains a need therefore to engineer a lactic acid bacteria, filed Sep. 29, 2008, the entirety of which is herein incorpo which is a good industrial host, to provide sufficient levels of rated by reference. Fe—S cluster forming proteins to accommodate the expres FIELD OF THE INVENTION sion of Fe—S requiring proteins such as DHAD. 0002 The invention relates to the field of microbiology. SUMMARY OF THE INVENTION More specifically, lactic acid bacteria are disclosed express 0007 Provided herein are lactic acid bacterial cells com ing high levels of dihydroxy-acid dehydratase activity in the prising a functional dihydroxy-acid dehydratase polypeptide presence of introduced iron-sulfur cluster forming proteins. and at least one recombinant genetic expression element encoding iron-sulfur cluster forming proteins. In some BACKGROUND OF THE INVENTION embodiments, the functional dihydroxy-acid dehydratase 0003 Dihydroxy-acid dehydratase (DHAD), also called polypeptide is encoded by a nucleic acid molecule that is acetohydroxy acid dehydratase, catalyzes the conversion of heterologous to the bacteria. In some embodiments, the func 2,3-dihydroxyisovalerate to C.-ketoisovalerate and of 2,3-di tional dihydroxyacid dehydratase polypeptide is a 2Fe-2S hydroxymethylvalerate to O-ketomethylvalerate. The DHAD 2+ dihydroxy-acid dehydratase, while in other embodiments, enzyme requires binding of an iron-sulfur (Fe-S) cluster for the functional dihydroxyacid dehydratase polypeptide is a activity, is classified as E.C. 4.2.1.9, and is part of naturally 4Fe-4S2+ dihydroxy-acid dehydratase. occurring biosynthetic pathways producing Valine, isoleu 0008. In one embodiment, the dihydroxyacid dehydratase cine, leucine and pantothenic acid (vitamin B5). DHAD cata polypeptide has an amino acid sequence that matches the lyzed conversion of 2,3-dihydroxyisovalerate to O-ketoisov Profile HMM of Table 7 with an Evalue of <10 wherein the alerate is also a common step in the multiple isobutanol polypeptide additionally comprises all three conserved cys biosynthetic pathways that are disclosed in commonly owned teines, corresponding to positions 56, 129, and 201 in the and co-pending US Patent Pub No. US 20070092957 A1. amino acids sequences of the Streptococcus mutans DHAD Disclosed therein is engineering of recombinant microorgan enzyme corresponding to SEQ ID NO:168. In one embodi isms for production of isobutanol. Isobutanol is useful as a ment, the dihydroxyacid dehydratase polypeptide has an fuel additive, whose availability may reduce the demand for amino acid sequence selected from the group consisting of petrochemical fuels. High levels of DHAD activity are SEQID NO:310, SEQID NO:298, SEQIDNO:168, SEQID desired for increased production of products from biosyn No: 164, SEQID NO:346, SEQID NO:344, SEQID NO:232, thetic pathways that include this enzyme activity, including and SEQID NO:230. for enhanced microbial production of branched chain amino 0009. In some embodiments, the recombinant genetic acids, pantothenic acid, and isobutanol, however since expression element encoding iron-sulfur cluster forming pro DHAD enzymes are Fe—S cluster requiring they must be teins contains coding regions of an operon selected from the expressed in a host having the genetic machinery to produce group consisting of Isc, Sufand Nifoperons. In some embodi Fe—S proteins. ments, the Suf operon comprises at least one coding region 0004 2Fe-2S]2+ and 4Fe-4S2+ clusters can form spon selected from the group consisting of Sufc. Suf D, SufS. taneously in vitro (Malkin and Rabinowitz (1966) Biochem. SufU, SufB, SufA and yseH, and in some embodiments, the Biophys. Res. Comm. 23: 822-827). However, likely due to Isc operon comprises at least one coding region selected from the toxic nature of both free Fe(II) and sulfide, biogenesis the group consisting of IscS, IscU, IscA, Iscx, HscA, HscB. systems have evolved to form Fe—S clusters and insert them and Fdx. In some embodiments the Nif operon comprises at into their target apoproteins in vivo. The biogenesis of iron least one coding region selected from the group consisting of sulfur clusters is not completely understood but is known NifS and Niflu. In some embodiments, the Suf operon has the generally to include liberation of sulfur from the amino acid nucleotide sequence selected from the group consisting of cysteine by a cysteine desulfurase enzyme, combination of SEQID NO:881 and SEQID NO:589. In some embodiments, the sulfur with Fe(II) on a scaffold protein, and transfer of the the Suf operon is derived from Lactococcus lactis and com formed Fe—S clusters, frequently in a chaperone-dependent prises at least one coding region encoding a polypeptide manner, to the proteins and enzymes that require them. The having an amino acid sequenced selected from the group Isc, Suf and Nif operons have been found to encode proteins consisting of SEQ ID NO. 598 (SufC), SEQ ID NO: 604 involved in Fe—S cluster formation in different bacteria (Sufi)), SEQ ID NO: 610 (SufB), and SEQ ID NO: 618 (Johnson et al. Annu. Rev. Biochem. 74:247-281 (2005)). (YseH). In some embodiments, the Suf operon is derived 0005 Lactic acid bacteria are well characterized and are from Lactoabcillus plantarum and comprises at least one used commercially in a number of industrial processes. coding region encoding a polypeptide having an amino acid Although it is known that some lactic acid bacteria possess sequenced selected from the group consisting of SEQID NO: Fe—S cluster requiring enzymes (Liu et al., Journal of Bio 596 (Suf(c), SEQ ID NO: 602 (Sufi)), SEQ ID NO: 624 logical Chemistry (2000), 275(17), 12367-12373) and there (SufS), SEQ ID NO: 620 (SufU) and SEQ ID NO: 608 fore posses the genetic machinery to produce Fe—S clusters, (SufB). In some embodiments, the Isc operon is derived from US 2010/0081182 A1 Apr. 1, 2010

E. Coli and comprises at least one coding region encoding a 0016 FIG. 3 shows a schematic drawing of the coding polypeptide having an amino acid sequence selected from the regions in the Suf operon from E. coli. group consisting of SEQID NO: 528 (IscS), SEQID NO:530 0017 FIG. 4 shows a schematic drawing of the coding (IscU), SEQ ID NO: 532 s(IscA), SEQID NO:534 (HscB), regions of the Isc operon from E. coli, and the adjacent iscP SEQID NO:536 (hscA), SEQIDNO:538 (Fdx), and SEQID gene. NO: 540 (Isc)x). In some embodiments the Nif operon is 0018 FIG. 5 shows a schematic drawing of the coding derived from Wolinella succinogenes and comprises at least regions of the Nif operon from Wolinella succinogenes, with one coding region encoding a polypeptide having an amino the bounding ORF1 and ORF2. acid sequence selected from the group consisting of: SEQID 0019 FIG. 6 shows biosynthetic pathways for biosynthe NO: 542 (NifS) and SEQID NO: 544 (Nifu). sis of isobutanol. 0010. In some embodiments, the lactic acid bacterial cell (0020 Table 7 is a table of the Profile HMM for dihydroxy provided herein is a member of a genus selected from the acid dehydratases based on enzymes with assayed function group consisting of Lactococcus, Lactobacillus, Leuconos prepared as described in Example 1. Table 8 is submitted toc, Oenococcus, Pediococcus, and Streptococcus. In some herewith electronically and is incorporated herein by refer embodiments, the bacteria produces isobutanol, and in some ence. The following sequences conform with 37 C.F.R. embodiments, the bacteria comprises an isobutanol biosyn 1.821-1.825 (“Requirements for Patent Applications Con thetic pathway. In some embodiments, the isobutanol biosyn taining Nucleotide Sequences and/or Amino Acid Sequence thetic pathway comprises genes encoding acetolactate Syn Disclosures—the Sequence Rules”) and are consistent with thase, acetohydroxy acid isomeroreductase, dihydroxy-acid World Intellectual Property Organization (WIPO) Standard dehydratase, branched-chain C-keto acid decarboxylase, and ST.25 (1998) and the sequence listing requirements of the branched-chain alcohol dehydrogenase. EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and 0011. Also provided herein is a method for increasing the Annex C of the Administrative Instructions). The symbols activity of a heterologous dihydroxyacid dehydratase and format used for nucleotide and amino acid sequence data polypeptide in a lactic acid bacterial cell comprising: a) pro comply with the rules set forth in 37 C.F.R. S1.822. viding a lactic acid bacterial cell comprising: 1) a nucleic acid molecule encoding a heterologous dihydroxyacid dehy TABLE 1 dratase polypeptide; 2) a recombinant genetic expression ele SEQID NOS of representative bacterial 2Fe-2S2 + ment encoding iron-sulfur cluster forming proteins, wherein DHAD proteins and encoding sequences the proteins are expressed; and b) growing the lactic acid bacterial cell of (a) under conditions whereby the dihydroxy SEQID NO: SEQID NO: acid dehydratase polypeptide is expressed in functional form Organism of derivation Nucleic acid Peptide having a specific activity greater than the same dihydroxy Mycobacterium sp. MCS 1 2 acid dehydratase polypeptide expressed in the same bacterial Mycobacterium gilvium PYR-GCK 3 4 cell lacking the recombinant genetic expression element Mycobacterium smegmatis str. MC2 155 5 6 Mycobacterium vanbaalenii PYR-1 7 8 encoding iron-sulfur cluster forming proteins. In one embodi Nocardia farcinica IFM 10152 9 10 ment, the specific activity of the expressed dihydroxyacid Rhodococcus sp. RHA1 11 12 dehydratase polypeptide is at least about two fold greater than Mycobacterium ulcerans Agy.99 13 14 the specific activity of the same dihydroxyacid dehydratase Mycobacterium avium subsp. 15 16 paratuberculosis K-10 polypeptide expressed in the same bacteria lacking the Mycobacterium tuberculosis H37Ra 17 18 recombinant genetic expression element encoding iron-sul Mycobacterium leprae TN* 19 2O fur cluster forming proteins. Kineococcus radiotoierans SRS30216 21 22 Janibacter sp. HTCC2649 23 24 0012. Also provided herein is a method of making isobu Nocardioides sp. JS614 25 26 tanol comprising providing a lactic acid bacterial cell dis Renibacterium salmoninarum ATCC 33209 27 28 closed herein and growing said cell under conditions wherein Arthrobacier attrescens TC1 29 30 isobutanol is produced. Leifsonia xyli Subsp. xvii str. CTCB07 31 32 marine actinobacterium PHSC20C1 33 34 Clavibacter michiganensis subsp. 35 36 BRIEF DESCRIPTION OF THE FIGURES AND michiganensis NCPPB 382 SEQUENCE DESCRIPTIONS Saccharopolyspora erythraea NRRL 2338 37 38 Acidothermus cellulolyticus 11B 39 40 Corynebacterium efficiens YS-314 41 42 0013 The invention can be more fully understood from Brevibacterium inens BL2 43 44 the following detailed description, figure, and the accompa Tropheryma whippiei TWO8/27 45 46 nying sequence descriptions, which form a part of this appli Methyliobacterium extorquens PA1 47 48 cation. Methyliobacterium nodulans ORS 2060 49 50 Rhodopseudomonas palustris Bisb5 51 52 0014 FIG. 1 shows a schematic drawing of the coding Rhodopseudomonas palustris Bisb18 53 S4 regions in the Suf operon from Lactobacillus plantarum as Bradyrhizobium sp. ORS278 55 56 well as the adjacent coding regions feo A and ORF (A), and Bradyrhizobium japonicum USDA 110 57 58 the portion of the Suf operon that was deleted in Example 1 Fulvimarina pelagi HTCC2506 59 60 Aurantimonas sp. SI85-9A1 61 62 (B). Hoeflea phototrophica DFL-43 63 64 0015 FIG. 2 shows a schematic drawing of the coding Mesorhizobium ioii MAFF303099 65 66 Mesorhizobium sp. BNC1 67 68 regions in the Suf operon from Lactococcus lactis, with each Parvibaculum iavanentivorans DS-1 69 70 coding region named by the designation from the publicly Loktanella vestfoldensis SKA53 71 72 available genomic sequence and the corresponding coding Roseobacter sp. CCS2 73 74 region identified by sequence homology. No homologous Dinoroseobaciershibae DFL 12 75 76 protein is identified for the hypothetical protiein. US 2010/0081182 A1 Apr. 1, 2010

TABLE 1-continued TABLE 1-continued SEQID NOS of representative bacterial 2Fe-2S2 + SEQID NOS of representative bacterial 2Fe-2S2 + DHAD proteins and encoding sequences DHAD proteins and encoding sequences SEQID NO: SEQID NO: SEQID NO: SEQID NO: Organism of derivation Nucleic acid Peptide Organism of derivation Nucleic acid Peptide Roseovarius nubinhibens ISM 77 78 Kordia algicida OT-1 205 2O6 Sagittula stellata E-37 79 8O Flavobacteriaies bacterium ALC-1 2O7 208 Roseobacter sp. AZwK-3b 81 82 Psychroflexus torquis ATCC 700755 209 210 Roseovarius sp. TM1035 83 84 Flavobacteriales bacterium HTCC2170 211 212 Oceanicola haiSensis HTCC2597 85 86 unidentified eubacterium SCB49 213 214 Oceanicola granulosus HTCC2516 87 88 Gramella forseti KTO803 215 216 Rhodobacieraies bacterium HTCC2150 89 90 Robiginitalea biformata HTCC2501 217 218 Paracoccus denitrificans PD1222 91 92 Tenacibaculum sp. MED152 219 220 Oceanibulbus indolifex HEL-45 93 94 Polaribacteringensii 23-P 221 222 Sulfitobacter sp. EE-36 95 96 Pedobacter sp. BAL39 223 224 Roseobacter denitrificans OCh 114 97 98 Flavobacteria bacterium BAL38 225 226 Jannaschia sp. CCS1 99 OO Flavobacterium psychrophilum JIPO2/86 227 228 Caulobacter sp. K31 O1 O2 Flavobacterium johnsoniae UW 101 229 230 Candidatus Pelagibacter ubique HTCC1062 O3 O4 Lactococcus lactis subsp. cremoris SK11 231 232 Erythrobacter litoralis HTCC2594 05 O6 Psychromonas ingrahamii 37 233 234 Erythrobacter sp. NAP1 O7 O8 Microscilia marina ATCC 23134 235 236 Connaimonastestosterone KF-1 09 10 Cytophaga hutchinsoni ATCC 33406 237 238 Sphingomonas wittichii RW1 11 12 Rhodopinellula baitica SH 1 239 240 Burkhoideria xenovorans LB400 13 14 Blastopirellula marina DSM 3645 241 242 Burkholderia phytofirmans PsyN 15 16 Planctomyces maris DSM 8797 243 244 Bordetella petri DSM 12804 17 18 Algoriphagus sp. PR1 245 246 Bordeteila bronchiseptica RB50 19 2O Candidatus Suicia mieieri str. He 247 248 Bradyrhizobium sp. ORS278 21 22 (Homalodisca coagulata) Bradyrhizobium sp. BTAi1 23 24 CandidatusCarsoneiia ruddi PV 249 250 Bradhyrhizobium japonicum 25 26 Synechococcus sp. RS9916 251 252 Sphingomonas wittichii RW1 27 28 Synechococcus sp. WH 7803 253 254 Rhodobacieraies bacterium HTCC2654 29 30 Synechococcus sp. CC9311 255 256 Soibacter usitatus Ellinó076 31 32 Synechococcus sp. CC9605 257 258 Roseiflexus sp. RS-1 33 34 Synechococcus sp. WH 8102 259 260 Rubrobacter xylanophilus DSM 9941 35 36 Synechococcus sp. BL107 261 262 Salinispora tropica CNB-440 37 38 Synechococcus sp. RCC307 263 264 Acidobacteria bacterium Ellin345 39 40 Synechococcus sp. RS9917 26S 266 Thermus thermophilus HB27 41 42 Synechococcus sp. WH 5701 267 268 Maricantis maris MCS10 43 44 Prochiorococcus marinus str. MIT 9313 269 270 Parviliarctia bermudensis HTCC2503 45 46 Prochiorococcus marinus str. NATL2A 271 272 Oceanicatiis alexandri HTCC2633 47 48 Prochiorococcus marinus str. MIT9215 273 274 Plesiocystis pacifica SIR 49 50 Prochiorococcus marinus str. AS9601 275 276 Bacilius sp. NRRLB-14911 51 52 Prochiorococcus marinus str. MIT 9515 277 278 Oceanobacilius iheyensis HTE831 53 S4 Prochlorococcus marinus subsp. pastoris 279 28O Staphylococci is saprophyticus Subsp. 55 56 Str. CCMP1986 Saprophyticus ATCC 15305 Prochiorococcus marinus str. MIT9211 281 282 Bacilius seleniiireducens MLS10 57 58 Prochlorococcus marinus subsp. marinus 283 284 Streptococcus pneumoniae SP6-BS73 59 60 Str. CCMP1375 Streptococcus sanguinis SK36 61 62 Nodularia spumigena CCY9414 285 286 Streptococcus thermophilus LMG 18311 63 64 Nostoc punctiforme PCC 73102 287 288 Streptococcus suis 89,159 65 66 Nostoc sp. PCC 7120 289 290 Streptococcus mutans UA159 67 68 Trichodesmium erythraeum IMS101 291 292 Leptospira borg.petersenii serovar 69 70 Acaryochloris marina MBIC11017 293 294 Hardjo-bovis L550 Lyngbya sp. PCC 8106 295 296 Candidatus Vesicomyosocius Okutani HA 71 72 Synechocystis sp. PCC 6803 297 298 Candidatus Ruthia magnifica str. Cm 73 74 Cyanothece sp. CCYO110 299 300 (Calyptogena magnifica) Thermosynechococcus elongatus BP-1 301 3O2 Methylococci is capsulatus str. Bath 75 76 Synechococcus sp. JA-2-3B'a(2-13) 303 304 uncultured marine bacterium EB80 02D08 77 78 Gioeobacter violiaceus PCC 7421 305 306 uncultured marine gamma 79 8O Nitrosomonas eutropha C91 307 3O8 proteobacterium EBAC31A08 Nitrosomonas europaea ATCC 19718 309 310 uncultured marine gamma 81 82 Nitrosospira multiformis ATCC 25196 311 312 proteobacterium EBAC20E09 Chloroflexus aggregains DSM 94.85 313 314 uncultured gamma proteobacterium 83 84 Leptospirilium sp. Group II UBA 315 316 eBACHOT4E07 Leptospirilium sp. Group II UBA 317 3.18 Alcanivorax borkumensis SK2 85 86 Halorhodospira halophila SL1 319 320 Chromohalobacter salexigens DSM 3043 87 88 Nitrococcus mobilis Nb-231 321 322 Marinobacter algicola DG893 89 90 Aikailimnicola ehrlichei MLHE-1 323 324 Marinobacter aquaeolei VT8 91 92 Deinococcus geothermalis DSM 11300 325 326 Marinobacter sp. ELB17 93 94 Polynucleobacter sp. QLW-P1 DMWA-1 327 328 Pseudoalteromonas haloplanktis TAC125 95 96 Polynucleobacter necessarius STIR1 329 330 Acinetobacter sp. ADP1 97 98 Azoarcus sp. EbN1 331 332 Opituitaceae bacterium. TAV2 99 200 Burkholderia phymatum STM815 333 334 Flavobacterium sp. MED217 2O1 2O2 Burkhoideria xenovorans LB400 335 336 Cellulophaga sp. MED134 2O3 204 Burkhoideria multivorans ATCC 17616 337 338 US 2010/0081182 A1 Apr. 1, 2010 4

TABLE 1-continued TABLE 2-continued SEQID NOS of representative bacterial 2Fe-2S2 + SEQID NOS of representative fungal and plant 2Fe-2S2 + DHAD proteins and encoding sequences DHAD proteins and encoding sequences SEQID NO: SEQID NO: SEQID NO: SEQID NO: Organism of derivation Nucleic acid Peptide Description Nucleic acid Peptide Burkholderia cenocepacia PC184 339 340 Coccidioides immitis RS 443 444 Burkhoideria maiei GB8 horse 4 341 342 Botryotinia fuckeiana B05.10 445 446 Ralstonia eutropha JMP134 343 344 Phaeosphaeria nodorum SN15 447 448 Raistonia metailidurans CH34 345 346 Pichia guilliermondii ATCC 6260 449 450 Raistonia Soianacearum UW551 347 348 Debaryomyces hansenii CBS767 451 452 Ralstonia picketti 12J 349 350 Lodderomyces elongisporus NRRLYB-4239 453 454 Limnobacter sp. MED105 351 352 Vanderwaltozyma polyspora DSM 70294 455 456 Herminimonas arsenicoxydans 353 3S4 Ashbya gossypii ATCC 10895 457 458 Bordeteila parapertissis 355 356 Laccaria bicolor S238N-H82 459 460 Bordetella petri DSM 12804 357 358 Coprinopsis cinerea okayama7#130 461 462 Polaromonas sp. JS666 359 360 Cryptococci is neoformans var. 463 464 Polaromonas naphthalenivorans CJ2 361 362 neoformans JEC21 Rhodoferax ferrireducens T118 363 364 Ustilago maydis 521 465 466 Verminephrobacter eiseniae EFO1-2 365 366 Malassezia globosa CBS 7966 467 468 Acidovorax sp. JS42 367 368 Aspergillus clavatus NRRL 1 469 470 Delfia acidovorans SPH-1 369 370 Neosartorya fischeri NRRL 181 (Putative) 471 472 Methylibium petroleiphilum PM1 371 372 Aspergillus Oryzae 473 474 gamma proteobacterium KT71 373 374 Aspergillus niger (hypothetical 475 476 Tremblaya princeps 375 376 protein An 18g.041 60) Blastopirellula marina DSM 3645 377 378 Aspergillus terreus NIH2624 477 478 Coccidioides immitis RS (hypothetical 479 480 Planctomyces maris DSM 8797 379 380 protein CIMG 04591) Microcystis aeruginosa PCC 7806 381 382 Paracoccidioides brasiliensis 481 482 Sainihacier rather DSM 13855 383 384 Phaeosphaeria nodorum SN15 483 484 Methyliobacterium chloromethanicum 385 386 Gibbereia zeae PH-1 485 486 Neurospora crassa OR74A 487 488 Coprinopsis cinerea okayama 7#130 489 490 Laccaria bicolor S238N-H82 491 492 TABLE 2 Ustilago maydis 521 493 494 SEQID NOS of representative fungal and plant 2Fe-2S2 + DHAD proteins and encoding sequences SEQID NO: SEQID NO: TABLE 3 Description Nucleic acid Peptide SEQID NOS of representative 4Fe–4S 2+ Schizosaccharomyces pombe ILV3 387 388 DHAD proteins and encoding sequences Saccharomyces cerevisiae ILV3 389 390 Kluyveromyces lactis ILV3 391 392 SEQID NO: SEQID NO: Candida albicans SCS314 ILV3 393 394 Organism Nucleic acid Peptide Pichia stipitis CBS 6054 ILV3 395 396 Yarrowia lipolytica ILV3 397 398 Escherichia coistr. K-12 substr. MG1655 495 496 Candida gallbrata CBS 138 ILV3 399 400 Bacilius subtilis Subsp. subtilis str. 168 497 498 Chlamydomonas reinhardtii 4O1 402 Agrobacterium tumefaciens str. C58 499 500 Osteococcus lucinarinus CCE9901 403 404 Burkholderia cenocepacia MCO-3 5O1 502 Vitis vinifera 40S 406 Psychrobacter cryohaiolentis K5 503 SO4 (Unnamed protein product: CAO71581.1) Psychromonas sp. CNPT3 505 SO6 Vitis vinifera 407 408 Deinococcus radiodurans R1 507 SO8 (Hypothetical protein: CAN67446.1) Wolinella succinogenes DSM 1740 509 510 Arabidopsis thaliana 409 410 Zymomonas mobilis Subsp. mobilis ZM4 511 512 Oryza Saiva (indica cultivar-group) 411 412 Clostridium acetobutyllicum ATCC 824 513 S14 Physcomitrella patens Subsp. patens 413 414 Clostridium beijerinckii NCIMB 8052 515 S16 Chaetomium globosum CBS 148.51 415 416 Pseudomonas fluorescens Pf-5 517 S18 Neurospora crassa OR74A 417 418 Methanococcus maripaludis C7 519 52O Magnaporthe grisea 70-15 419 420 Meihanococcusaeoicus Nankai-3 521 522 Gibbereia zeae PH-1 421 422 Vibrio fischeri ATCC 700601 (ES114) 523 524 Aspergilius niger 423 424 Shewaneia Oneidensis MR-1ATCC 700550 525 526 Neosartorya fischeri NRRL 181 425 426 (XP 001266525.1) Neosartorya fischeri NRRL 181 427 428 (XP 001262996.1) TABLE 4 Aspergilius niger 429 430 (hypothetical protein An()3g04520) SEQID NOS of representative Suf operon Fe—S cluster Aspergilius niger 431 432 forming proteins and encoding Sequences. (Hypothetical protein An14g03280) Aspergillus terreus NIH2624 433 434 SEQID NO: SEQID NO: Aspergillus clavatus NRRL 1 435 436 Organism and gene name nucleic acid amino acid Aspergillus nidulans FGSC A4 437 438 Aspergilius Oryzae 439 440 Lactoabcillus plantarum sufc 595 596 Aiellomyces capsulatus NAm1 441 442 Lactococci is lactis SufC 597 598 US 2010/0081182 A1 Apr. 1, 2010

TABLE 4-continued TABLE 4-continued SEQID NOS of representative Suf operon Fe–S cluster SEQID NOS of representative Suf operon Fe—S cluster forming proteins and encoding sequences. forming proteins and encoding sequences. SEQID NO: SEQID NO: SEQID NO: SEQID NO: Organism and gene name nucleic acid amino acid Organism and gene name nucleic acid amino acid Escherichia Coi sufC 599 600 ATCC 33223, sufS Lactoabcillus plantarum suf) 6O1 6O2 Symbiobacterium thermophilum IAM 14863, 713 714 Lactococcus iactis suf) 603 604 SufS Escherichia Coi suf) 60S 606 Thermoanaerobacter tengcongensis MB4, 715 716 Lactoabcillus plantarum sufB 607 608 SufS Lactococcus iactis suf3 609 610 Verrucomicrobium spinosum DSM 4136, 717 718 Escherichia Coi sufB 611 612 SufS Escherichia Coi sufA 613 614 Oenococcus oeni PSU-1, SufS 719 720 Escherichia Coi sufE 615 616 Mariprofundus ferrooxydans PV-1, SufS 721 722 Lactococci is lactis ySeH 617 618 Opituitus terrae PB90-1, SufS 723 724 Lactoabcillus plantarum sufU 619 62O Nitrosococcus oceani ATCC 19707, SufS 725 726 Lactococci is lactis Sufly 621 622 Lactobacillus delbrueckii subsp. 727 728 Lactoabcilius plantarum, SufS 623 624 bulgaricus ATCC 11842, SufS Lactobacilius reuteri, SufS 625 626 Escherichia coistr. K-12 substr. 729 730 Lactobacilius fermentum, SufS 627 628 MG1655, sufS (PLP-dependent) Enterococcus faecalis, SufS 629 630 Rhodoferax ferrireducens T118, SufS 731 732 Lactobacilius faecium DO, SufS 631 632 Thermus thermophilus HB27, SufS 733 734 Lactobacilius Sakei Subsp. Sakei 23K, 633 634 Streptomyces avermitilis MA-4680, SufS 735 736 putative sufS Clostridium sp. L2-50, SufS protein 737 738 Carnobacterium sp. AT7, SufS 635 636 CLOL250 02464 Streptococcus mutans UA159, SufS 637 638 Coprococcus eutacitus ATCC 27759, SufS 739 740 Streptococcus suis 05ZYH33, SufS 639 640 hypothetical protein COPEUT OO639 Streptococcus sanguinis SK36, SufS 641 642 Thermobifida fisca YX, sufS 741 742 Leticonostoc mesenteroides Subsp. 643 644 Acidothermus cellulolyticus 11B, sufS 743 744 mesenteroides ATCC 8293, SufS Methylococci is capsulatus str. Bath, 745 746 Streptococcus thermophilus LMG 18311, 645 646 SufS SufS Thauera sp. MZ1T, sufS 747 748 Lactococcus lactis subsp. cremoris SK11, 647 648 Streptomyces coelicolor A3(2), SufS 749 750 SufS hypothetical protein LACR 1972 Soibacter usitatus Ellinó076, SufS 751 752 Bacilius sp. B14905, SufS 649 6SO Coxielia burnetii RSA 493, SufS 753 754 00018 Streptococcus infantarius Subsp. 651 652 Petrotoga mobilis SJ95, SufS 755 756 infantarius ATCC BAA-102, SufS Synechocystis sp. PCC 6803, SufS 757 758 hypothetical protein STRINF Ralstonia eutropha H16, SufS 759 760 Lactobacilius helveticus CNRZ32, SufS 653 654 Thermotoga maritima MSB8 SufS 761 762 Streptococcus pneumoniae CGSP14, SufS 655 656 Gioeobacter violiaceus PCC 7421, SufS 763 764 Geobacillus sp. WCH70, sufS 657 658 Nitrococcus mobilis Nb-231, SufS 765 766 Leuconostoc Citreum KM20, SufS 659 660 Pediococcus pentosaceus ATCC 25745, 767 768 Listeria monocytogenes EGD-e, SufS 661 662 SufS hypothetical protein Imo2413 Streptomyces grisetts Subsp. grisetts 769 770 Lactobacilius Johnsonii NCC 533, SufS 663 664 NBRC 13350, sufS Bacilius sp. SG-1, SufS 665 666 Nitrosospira multiformis ATCC 25196, 771 772 Bacilius clausii KSM-K16, SufS 667 668 SufS Bacilius pumilus SAFR-032, SufS 669 670 Frankia sp. EAN1 pec, SufS 773 774 Geobacilius kaustophilus HTA426, SufS 671 672 Propionibacterium acnes KPA171202, 775 776 Bacilius selenitireducens MLS10, SufS 673 674 putative sufS Streptococcus pyogenes MGAS10750, SufS 675 676 Rhodococcus sp. RHA1, SufS 777 778 Bacilius sp. NRRL B-14911, SufS 677 678 Aikailimnicoia ehrlichei MLHE-1, 779 780 Paenibacilius larvae subsp. larvae BRL- 679 68O SufS 230010, sufS Anaeromyxobacter sp. Fw 109-5, SufS 781 782 Bacilius subtilis Subsp. subtilis str. 681 682 Anaeromyxobacter sp. K, SufS 783 784 168, sufS Mycobacterium abscessus, sufS 785 786 Bacilius licheniformis ATCC 14580, SufS 683 684 Lentisphaera araneosa HTCC2155, SufS 787 788 Oceanobacilius iheyensis HTE831, SufS 685 686 Saccharopolyspora erythraea NRRL 2338, 789 790 Bacilius coagulans 36D1, SufS 687 688 SufS Staphylococcusatiretts Subsp. aureus MuSO, 689 690 Acidiphilium cryptum JF-5, SufS 791 792 SufS Nocardia farcinica IFM 10152, sufS 793 794 Staphylococci is saprophyticus Subsp. 691 692 Nocardioides sp. JS614, SufS 795 796 Saprophyticus ATCC 15305, putative sufS Corynebacterium urealyticum DSM 7109, 797 798 Paenibacilius sp. JDR-2, SufS 693 694 SufS Lactobacilius Saivarius UCC118, SufS 695 696 Legionella pneumophia Subsp. 799 800 Exiguobacterium sibiricum 255-15, SufS 697 698 pneumophila str. Philadelphia 1, SufS Exiguobacterium sp. AT1b, SufS 699 700 Mycobacterium marinum M, SufS 8O1 8O2 Rubrobacter xylanophilus DSM 9941, SufS 701 702 Psychromonas ingrahamii 37, SufS 803 804 Clostridium acetobutyllicum ATCC 824, 703 704 Corynebacterium efficiens YS-314, SufS 805 806 SufS Corynebacterium jeikeium K411, 807 808 Clostridium beijerinckii NCIMB 8052, 705 706 putative sufS SufS Leptospira borg.petersenii serovar 809 810 Clostridium kluyveri DSM 555, sufS 707 708 Hardjobovis L550, SufS Lactobacilius casei ATCC 334, SufS 709 710 Mycobacterium vanbaalenii PYR-1, SufS 811 812 Thermoanaerobacter pseudethanolicus 711 712 Mycobacterium gilvium PYR-GCK, SufS 813 814 US 2010/0081182 A1 Apr. 1, 2010

TABLE 4-continued TABLE 5-continued SEQID NOS of representative Suf operon Fe—S cluster SEQID NOS of representative Isc and Nif operon Fe—S cluster forming proteins and encoding sequences. forming proteins and encoding sequences SEQID NO: SEQID NO: SEQID NO: SEQID NO: Organism and gene name nucleic acid amino acid Organism and gene name nucleic acid amino acid Mycobacterium tuberculosis H37RV, SufS 815 816 Escherichia coifix 537 538 Janibacter sp. HTCC2649, SufS 817 818 Escherichia coi iscX 539 S4O Salinispora arenicola CNS-205, SufS 819 820 Wolinella succinogenes nifS 541 S42 Polaromonas sp. JS666, SufS 821 822 Wolinella succinogenes niftJ 543 544 Nitrosomonas eutropha C91, SufS 823 824 Mycobacterium sp. MCS, SufS 825 826 Frankia aimi ACN14a, sufS 827 828 Salinispora tropica CNB-440, SufS 829 830 TABLE 6 Nitrosomonas europaea ATCC 19718, SufS 831 832 Leptospira interrogans Serovar 833 834 Copenhagenistr. Fiocruz L1-130, SufS SEQ ID NOS of additional proteins and encoding sequences Mycobacterium avium subsp. 835 836 SEQ ID NO: SEQID NO: paratuberculosis K-10, SufS Description Encoding seq. protein hypothetical protein MAP1190 Thermotoga maritima MSB8, SufS 837 838 Vibrio choierae KARI 545 S46 Pectobacterium atrosepticum SCRI1043, 839 840 Pseudomonas aeruginosa PAO1 KARI 551 552 SufS Pseudomonas fluorescens PF5 KARI 547 S48 Corynebacterium glutamicum ATCC 13032, 841 842 Achromobacter xylosoxidans S49 550 SufS butanol dehydrogenase sadB Clavibacter michiganensis subsp. 843 844 michiganensis NCPPB 382, putative SufS Frankia sp. Cel3, SufS 845 846 Gluconacetobacter diazotrophicus PAI5, 847 848 0021 SEQIDNOs:554-570,572,573,575,576,578-588, putative sufS 592 and 593 are nucleotide sequences of primers used in the Candidatus Pelagibacter ubique 849 850 Examples. HTCC1062, sufS 0022 SEQID NOs:553,571,574, 577 and 594 are nucle Kineococcus radiotolerans SRS30216, 851 852 SufS otide sequences of vectors used in the Examples. Finegoidia magna ATCC 29328, SufS 853 854 (0023 SEQ ID NO:589 is the nucleotide sequence of the Collinseila aerofaciens ATCC 25986, 855 856 Suf operon from Lactobacillus plantarum PNO512. SufS hypothetical protein COLAER 01633 Peptostreptococcus micros ATCC 33270, 857 858 (0024 SEQ ID NO:590 is the nucleotide sequence of a hypothetical protein PEPMIC 00951 ribosome binding sequence used in the Examples. Arthrobacter chlorophenolicus A6, SufS 859 860 (0025 SEQ ID NO:591 is the nucleotide sequence of the Grantibacter beihesdensis CGDNIH1, 861 862 promoter region of the IdhL1 gene from Lactobacillus plan SufS tartin PNO512. Arthrobacter sp. FB24, SufS 863 864 Thermosipho melanesiensis BI429, SufS 865 866 (0026 SEQ ID NO:881 is the nucleotide sequence of the Renibacterium salmoninarum ATCC 33209, 867 868 Sufoperon from Lactococcus lactis subsp. lactis NCDO2118. SufS Leifsonia xyli Subsp. xvii str. CTCB07, 869 870 DETAILED DESCRIPTION OF THE INVENTION SufS Acholeplasma laidlawi PG-8A, SufS 871 872 0027. The present invention solves the stated problem by Brevibacterium inens BL2, SufS 873 874 Corynebacterium diphtheriae NCTC 13129, 875 876 providing recombinant lactic acid bacterial cells that express SufS DHAD and that express at least one recombinant genetic Bifidobacterium animalis subsp. lactis 877 878 element encoding Fe—S cluster forming proteins. These cells HNO19, sufS have increased DHAD activity as compared to DHAD activ Burkhoideria thailandensis MSMB43, 879 880 ity in cells without the recombinant genetic element. In these SufS cells, products synthesized by a pathway that includes DHAD Annotations in public databases may have a different protein indicated for activity may be increased, including amino acids valine, leu some of the SufS proteins above. Annotation as Class V aminotransferase cine and isoleucine, Vitamin B5, and isobutanol. The amino refers to the same protein as cysteine desulfurase. acids and vitamin B5 may be used as nutritional Supplements, and isobutanol may be used as a fuel additive to reduce demand for petrochemicals. TABLE 5 0028. The following abbreviations and definitions will be SEQID NOS of representative Isc and Nif operon Fe—S cluster used for the interpretation of the specification and the claims. forming proteins and encoding sequences 0029. As used herein, the terms “comprises.” “compris SEQID NO: SEQID NO: ing,” “includes.” “including,” “has.” “having.” “contains' or Organism and gene name nucleic acid amino acid “containing, or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, Escherichia coi iscS 527 528 Escherichia coi iscO 529 530 a mixture, process, method, article, or apparatus that com Escherichia coi iscA 531 532 prises a list of elements is not necessarily limited to only those Escherichia coihscB 533 534 elements but may include other elements not expressly listed Escherichia coihscA 535 536 or inherent to Such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the US 2010/0081182 A1 Apr. 1, 2010

contrary, “or refers to an inclusive or and not to an exclusive native gene. For example, a heterologous gene may include a or. For example, a condition A or B is satisfied by any one of native coding region that is a portion of a chimeric gene the following: A is true (or present) and B is false (or not including non-native regulatory regions that is reintroduced present), A is false (or not present) and B is true (or present), into the native host. Foreign genes can comprise native genes and both A and B are true (or present). inserted into a non-native organism, or chimeric genes. A 0030. Also, the indefinite articles “a” and “an preceding “transgene' is a gene that has been introduced into the an element or component of the invention are intended to be genome by a transformation procedure. nonrestrictive regarding the number of instances (i.e. occur rences) of the element or component. Therefore “a” or “an 0037. The term “recombinant genetic expression element' should be read to include one or at least one, and the singular refers to a nucleic acid fragment that expresses one or more word form of the element or component also includes the specific proteins, including regulatory sequences preceding plural unless the number is obviously meant to be singular. (5' non-coding sequences) and following (3' termination 0031. The term “invention' or “present invention' as used sequences) coding sequences for the proteins. A chimeric herein is a non-limiting term and is not intended to refer to any gene is a recombinant genetic expression element. The cod single embodiment of the particular invention but encom ing regions of an operon may form a recombinant genetic passes all possible embodiments as described in the specifi expression element, along with an operably linked promoter cation and the claims. and termination region. 0032. As used herein, the term “