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(12) Patent Application Publication (10) Pub. No.: US 2016/0024534 A1 Anthony Et Al US 2016.0024534A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0024534 A1 Anthony et al. (43) Pub. Date: Jan. 28, 2016 (54) INCREASED HETEROLOGOUS FE-S Publication Classification ENZYME ACTIVITY IN YEAST (51) Int. Cl. (71) Applicant: Butamax Advanced Biofuels LLC, CI2P 7/40 (2006.01) Wilmington, DE (US) CI2P 7/16 (2006.01) CI2N 9/88 (2006.01) (72) Inventors: Larry Cameron Anthony, Aston, PA (52) U.S. Cl. (US); Lori Ann Maggio-Hall, CPC. CI2P 7/40 (2013.01); C12N 9/88 (2013.01); Wilmington, DE (US): Steven Cary CI2P 7/16 (2013.01); C12Y402/01009 Rothman, Princeton, NJ (US); (2013.01) Jean-Francois Tomb, Wilmington, DE (US) (57) ABSTRACT (21) Appl. No.: 14/873,956 Yeast strains were engineered that have increased activity of (22) Filed: Oct. 2, 2015 heterologous proteins that require binding of an Fe—S cluster for their activity. The yeast strains have reduced activity of an Related U.S. Application Data endogenous Fe—S protein. Activities of heterologous fungal or plant 2Fe-2S dihydroxy-acid dehydratases and Fe—S pro (62) Division of application No. 12/569,069, filed on Sep. panediol dehydratase reactivase were increased for increased 29, 2009, now Pat. No. 9,206,447. production of products made using biosynthetic pathways (60) Provisional application No. 61/100,801, filed on Sep. including these enzymes. Such as valine, isoleucine, leucine, 29, 2008, provisional application No. 61/100,806, pantothenic acid (vitamin B5), isobutanol, 2-butanone and filed on Sep. 29, 2008. 2-butanol. Patent Application Publication Jan. 28, 2016 Sheet 1 of 2 US 2016/0024534 A1 Patent Application Publication Jan. 28, 2016 Sheet 2 of 2 US 2016/0024534 A1 Noh so US 2016/0024534 A1 Jan. 28, 2016 INCREASED HETEROLOGOUS FE-S ing high functional expression of heterologous Fe—S cluster ENZYME ACTIVITY IN YEAST containing enzymes is problematic due to the Fe—S cluster requirement, which involves availability and proper loading CROSS REFERENCE TO RELATED of the cluster into the apo-protein. APPLICATIONS SUMMARY OF THE INVENTION 0001. This application is related to and claims the benefit of priority to U.S. Provisional Application Nos. 61/100,801 0006 Provided herein are recombinant yeast host cells filed Sep. 29, 2008 and 61/100,806 filed Sep. 29, 2008. The comprising at least one heterologous Fe—S cluster protein entirety of each is hereby incorporated by reference. wherein the yeast host has reduced expression of at least one endogenous Fe—S cluster protein. FIELD OF THE INVENTION 0007. The recombinant yeast cell may be grown under Suitable conditions for the production of products including 0002. The invention relates to the field of industrial micro isobutanol, 2-butanol and 2-butanone. biology and the expression of proteins that require an iron 0008. In one aspect, the recombinant yeast cell comprises sulfur cluster for activity. More specifically, expression of a disruption in the gene encoding the at least one endogenous heterologous Fe—S protein activity in yeast cells is improved Fe—S cluster protein. through specific host gene inactivation. 0009. In another aspect, the endogenous Fe—S cluster protein is selected from the group consisting of dihydroxy BACKGROUND OF THE INVENTION acid dehydratase, isopropylmalate dehydratase, Sulfite reduc 0003 Engineering of yeast for fermentative production of tase, glutamate dehyddrogenase, biotin Synthase, aconitase, commercial products is an active and growing field. Enzy homoaconitase, lipoate synthase, ferredoxin maturation, matic pathways engineered for biosynthesis of some products NADH ubiquinone oxidoreductase. Succinate dehydroge include enzymes that require binding of an iron-sulfur (Fe nase, ubiquinol-cytochrome-c reductase, ABC protein Rli1. S) cluster for activity. Dihydroxy-acid dehydratase (DHAD) NTPase Nbp35, and hydrogenase-like protein. is one example. DHAD is part of naturally occurring biosyn 0010. In another aspect, the yeast is selected from the thetic pathways producing Valine, isoleucine, leucine and group consisting of Saccharomyces, Schizosaccharomyces, pantothenic acid (vitamin B5). Increased expression of Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia. DHAD activity is desired for enhanced microbial production 0011. In another aspect, the endogenous Fe—S protein is of branched chain amino acids or of pantothenic acid. In expressed in the mitochondria, and in another embodiment, addition, DHAD catalyzed conversion of 2,3-dihydroxyisov the endogenous Fe—S clusterprotein has an activity selected alerate to C-ketoisovalerate is a common step in the multiple from the group consisting of dihydroxy-acid dehydratase isobutanol biosynthetic pathways that are disclosed in co and isopropylmalate dehydratase activity. pending US Patent Pub No. US 20070092957 A1. Disclosed 0012. In another aspect, the host cell is Saccharomyces therein is engineering of recombinant microorganisms for expressing a gene encoding a polypeptide having the amino production of isobutanol, which is useful as a fuel additive acid sequence as set forth in SEQID NO:114. and whose availability may reduce the demand for petro 0013. In some embodiments, the at least one heterologous chemical fuels. Fe—S clusterprotein is selected from the group consisting of 0004 Diol dehydratase provides an enzyme activity in a fungal 2Fe-2S dihydroxy-acid dehydratases and plant 2Fe-2S biosynthetic pathway for production of 2-butanone and 2-bu dihydroxy-acid dehydratases. In one embodiment, the heter tanol that is disclosed in co-pending US Patent Pub No. US ologous fungal or plant 2Fe-2S cluster dihydroxy-acid dehy 2007-0292927A1. Disclosed in US Patent Pub No. dratase is expressed in the cytosol. In one embodiment, the US20090155870 is abutanediol dehydratase that is useful for heterologous fungal or plant 2Fe-2S cluster dihydroxy-acid expression in this pathway due to its coenzyme B-12 inde dehydratase is a polypeptide having an amino acid sequence pendence. A diol dehydratase reactivase that is an Fe—S that matches the Profile HMM of table 9 with an E value of cluster protein required for activity of the B12-independent <10 wherein the polypeptide additionally comprises all butanediol dehydratase, is also disclosed in US Patent Pub three conserved cysteines, corresponding to positions 56, No. US20090155870.2-Butanone, also referred to as methyl 129, and 201 in the amino acids sequences of the Streptococ ethyl ketone (MEK), is a widely used solvent, extractant and cus mutans DHAD enzyme corresponding to SEQ ID activator of oxidative reactions, as well as a Substrate for NO:179. In one embodiment, the heterologous fungalor plant chemical synthesis of 2-butanol. 2-butanol is useful as a fuel 2Fe-2S cluster dihydroxy-acid dehydratase is a polypeptide additive, whose availability may reduce the demand for pet having an amino acid sequence that has at least about 95% rochemical fuels. sequence identity to an amino acid sequence selected from 0005 For improved production of compounds synthe the group consisting of SEQ ID NOs:46, 48, 50, 52, 54, 56. sized in pathways including an Fe—S cluster containing 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78,80, 82,84, 86, 88,90, enzyme, it is desirable to provide a host cell capable of 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, expressing high levels of this enzymatic activity in the pro 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, duction host of interest. Whereas a number of commercially 142, 144, 146, 148, 150 and 152. In one embodiment, the relevant bacteria and yeast can express activity of Fe—S heterologous fungal or plant 2Fe-2S cluster dihydroxy-acid cluster containing proteins, this activity is at levels far below dehydratase is a polypeptide having an amino acid sequence what is commercially useful for enhancing introduced bio that is at least about 90% identical to SEQID NO: 114 using synthetic pathways. Consequently a need exists for the dis the ClustalW method of alignment using the default param covery of host cells capable of expressing activity of Fe—S eters of GAP PENALTY=10, GAP LENGTH PENALTY=0. cluster containing proteins at levels high enough to enhance 1, and Gonnet 250 series of protein weight matrix over the full introduced pathways that have Fe—S requirements. Obtain length of the protein sequence. US 2016/0024534 A1 Jan. 28, 2016 0014. In another aspect, a method for the conversion of Disclosures—the Sequence Rules”) and are consistent with 2,3-dihydroxyisovalerate to O-ketoisovalerate is provided, World Intellectual Property Organization (WIPO) Standard said method comprising: ST.25 (1998) and the sequence listing requirements of the 00.15 a) providing (1) a recombinant yeast host cell com EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and prising at least one heterologous gene encoding a 2Fe-2S Annex C of the Administrative Instructions). The symbols dihydroxy-acid dehydratase wherein the recombinant yeast and format used for nucleotide and amino acid sequence data host cell has reduced activity of at least one endogenous comply with the rules set forth in 37 C.F.R. S1.822. Fe—S cluster protein; and (2) a source of 2,3-dihydroxyis ovalerate; and TABLE 1 0016 b) growing the recombinant host cell of (a) with said source of 2,3-dihydroxyisovalerate under conditions where Inactivation target Fe-S protein encoding genes the 2,3-dihydroxyisovalerate is converted by the host cell to SEQ ID NO: SEQID NO: C.-ketoisovalerate. Organism and gene Nucleic Acid Peptide 0017. In another aspect, a method for
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