Plant biotin synthase

#This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding biotin synthase in plants and seeds.

#Biotin is an essential component for all living organisms even though many, including humans, cannot synthesize biotin and are dependent upon its uptake from their environment or diet (Eisenberg (1973) Adv Enzymol 38:317-372, Pai (1975) J Bacteriol 121:1-8). Biotin serves as a cofactor that covalently binds to carboxylases and facilitates the transfer of carboxyl groups during enzymatic reactions involving carboxylation, decarboxylation and transcarboxylation (Dakshinamurti and Bhagavan, eds., (1985) “Biotin”, Ann NY Acad Sci 447:1-441; Knowles (1989) Ann Rev Biochem 58:195-221).

#Biotin biosynthesis has been extensively studied in microorganisms, using biotin auxotrophic mutants to characterize the pathway. The biosynthesis of biotin involves four enzymatic steps in all microorganisms that starts with the precursor pimeloyl-CoA. The final step in this pathway involves the addition of sulfur to desthiobiotin to form biotin. The enzyme responsible for this reaction is known as biotin synthase and is encoded by the bioB gene (Birch et al. (1995) J Biol Chem 270:19158-19165).

#The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing which form a part of this application.

#FIG. 1 shows a comparison of the amino acid sequences of the barley (SEQ ID NO: 18), corn (SEQ ID NOs:20, 22, and 24), prickly poppy (SEQ ID NO:26), soybean (SEQ ID NOs:28 and 30), and wheat (SEQ ID NO:32) biotin synthase polypeptides to the enzymes from Arabidopsis thaliana (SEQ ID NO:33), fission yeast (Schizosaccharomyces pombe, SEQ ID NO:34), and yeast (Saccharomyces cerevisiae, SEQ ID NO:35). The conserved iron binding consensus sequence (GXCXEDCXYCXQ) is highlighted in italics and underlined (SEQ ID NO:36).

#FIG. 2 shows a comparison of the sequences from nucleotides 301-441 of clone cdt2c.pk002.c17:fis (SEQ ID NO:19) and the comparable region (nucleotides 253-492) of clone cho1c.pk009.j14:fis (SEQ ID NO:21). The SEQ ID NO:19 sequence has a 99 nucleotide “deletion” from this region with respect to the SEQ ID NO:21 sequence. This region encompasses the conserved iron binding sequence noted in FIG. 1. The “deleted” sequence shown in SEQ ID NO 21 has consensus intron border sequences (GT . . . AG) and the two sequences may represent alternative splice products of the same precursor.

#Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. 1 TABLE 1 Biotin Synthase SEQ ID NO: Protein Clone Designation (Nucleotide) (Amino Acid) barley bsh1.pk0005.d10 1 2 [Hordeum vulgare] maize [Zea mays] cdt2c.pk002.c17 3 4 maize [Zea mays] cho1c.pk009.j14 5 6 maize [Zea mays] Contig of: cca.pk0012.g11 7 8 cco1n.pk069.f1 p0004.cb1hi70r p0041.crtax65r p0094.cssth33r p0094.cssth33ra prickly poppy pps1c.pk008.m8 9 10 [Argemone mexicana] soybean Contig of: [Glycine max] sah1c.pk001.b19 11 12 sfl1.pk128.m2 sgc5c.pk001.j23 sgs2c.pk003.p6 sr1.pk0026.d1 src2c.pk025.k23 ssm.pk0072.h10 soybean sls2c.pk010.124 13 14 [Glycine max] wheat-common wr1.pk0104.b6 15 16 [Triticum aestivum] barley bsh1.pk0005.d10 17 18 [Hordeum vulgare] maize [Zea mays] cdt2c.pk002.c17 19 20 maize [Zea mays] cho1c.pk009.j14 21 22 maize [Zea mays] cca.pk0012.g11:fis 23 24 prickly poppy pps1c.pk008.m8:fis 25 26 [Argemone mexicana] soybean sgc5c.pk001.j23:fis 27 28 [Glycine max] soybean Contig of: [Glycine max] sls1c.pk015.d12 29 30 sls2c.pk010.124:fis wheat-common wr1.pk0104.b6:fis 31 32 [Triticum aestivum]

#The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the requirements of 37 C.F.R. §1.822.

#In the context of this disclosure, a number of terms shall be utilized. The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid sequence,” and “nucleic acid fragment”/“isolated nucleic acid fragment” are used interchangeably. These terms encompass nucleotide sequences and the like. A polynucleotide may be an RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolated polynucleotide of the present invention may include at least 30 contiguous nucleotides, preferably at least 40 contiguous nucleotides, most preferably at least 60 contiguous nucleotides derived from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31, or the complement of such sequences.