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 Z*0|- 9°0|- ZSyns/19SÁOgns/VJSÁ · Ogns/gJSK Patent Application Publication Apr. 1, 2010 Sheet 3 of 4 US 2010/0081182 A1 |7°0|- 9°0|-! Patent Application Publication US 2010/0081182 A1 Z O US 2010/0081182 A1 Apr. 1, 2010 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.