AU2001274408A1 - Nucleic acid encoding a plant very long chain fatty acid biosynthetic enzyme - Google Patents

Description

NUCLEIC ACID ENCODING A PLANT VERY LONG CHAIN FATTY

AC-ID BIOSYNTHETIC ENZYME

This application claims priority from U.S. Provisional Patent Application No. 60/206,789, which was filed May 24, 2000.

Field of the Invention

This invention relates to the isolation of a genomic DNA sequence encoding a condensing enzyme involved in very long chain fatty acid production in plants and its uses.

BACKGROUND Living organisms synthesize a vast array of different fatty acids which are incorporated into complex lipids. These complex lipids represent both major structural component membranes, and are a major storage product in both plants and animals. Very long chain fatty acids (NLCFAs, chain length C20 or longer) are synthesized in the epidermal cells where they are either directly incorporated into waxes, or serve as precursors for other aliphatic hydrocarbons found in waxes, including alkanes, primary and secondary alcohols, ketones aldehydes and acyl-esters. NLCFAs also accumulate in the seed oil of some plant species, where they are incorporated into triacylglycerols (TAGs), as in the Brassicaceae, or into wax esters, as in jojoba. These seed NLCFAs include the agronomically important erucic acid (C22:l), used in the production of lubricants, nylon, cosmetics, pharmaceuticals and

plasticizers. NLCFAs are synthesized by a microsomal fatty acid elongation (FAE) system which involves four enzymatic reactions: (1) condensation of malonyl-CoA with a long chain acyl- CoA, (2) reduction to -hydroxyacyl-CoA, (3) dehydration to an enoyl-CoA and (4) reduction of the enoyl-CoA, resulting in the elongated acyl-CoA by two carbons. The condensing enzyme catalyzing reaction (1) is the key activity of the FAE system. It is the rate-limiting enzyme of the NLCFA biosynthetic pathway, which controls the amount of NLCFAs produced, h addition, the condensing enzyme determines the ultimate NLCFA acyl chain length, and thus their use.

SUMMARY OF THE INVENTION

The present invention consists of a DNA sequence encoding a condensing enzyme involved in NLCFA biosynthesis. Such a DΝA fragment is desirable for use in genetic engineering projects aimed at increasing the chain length of fatty acids in seed oils. In addition, expression of this sequence in the epidermis can be used for altering the composition and accumulation of cuticular and epicuticular waxes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows DNA sequence of the Lβ CS3 genomic clone. The deduced amino acid sequence in shown below the nucleotide sequence of corresponding exons. Intron sequences are shown in bold and italics.

Figure 2 shows sequence similarity among the Brassicaceae condensing enzymes along their entire length (Figure 2). DETAILED DESCRIPTION

The present invention provides an isolated genomic DNA sequence encoding a condensing enzyme involved in very long chain fatty acid production in plants. Because condensing enzymes are pivotal enzymes in the synthesis of very long chain fatty acids (NLCFA), controlling levels of accumulation of NLCFAs and their acyl chain length (Millar and Kunst, 1997), are useful for biotechnology. For instance, the accumulation of NLCFAs in tobacco seed expressing FAEl from Arabidopsis (Millar and Kunst, 1997) indicates that VLCFAs can be produced in plant species that currently do not synthesize VLCFAs. The availability of LfKCS3 condensing enzyme may be especially useful, because it is capable of efficiently elongating hydroxy fatty acids. Thus, the expression of the LfKCS3 condensing enzyme in seeds should allow the production of crop plants capable of synthesizing hydroxylated VLCFAs in seed oil for industrial applications. The methods employed in the isolation of the nucleic acid sequence of the present invention and the uses thereof are discussed in the following non-limiting examples:

Screening of the Genomic DΝA Library:

A Lesquerella fendleri genomic DΝA library was obtained from Dr. Chris Somerville of the Carnegie Institution of Washington, Stanford, CA. The genomic library was plated on E. coli LE392 (Promega) and about 150,000 clones were screened using Arabidopsis FAEl as a probe. The probe was prepared by PCR using pGEM7-FAEl (Millar and Kunst, 1997) as a template with FAEl upstream primer, 5'-CCGAGCTCAAAGAGGATACATAC-3' and FAEl downstream primer, 5'-GATACTCGAGAACGTTGGCACTCAGATAC-3\ PCR was performed in a lOμl reaction containing 10 ng of the template, 2mM MgCl₂, 1.1 μM of each

primer, 100 μM of (dCTP + dGTP + dTTP) mix, 50 μCi of [ -32P]dATP, IX PCR buffer

and 2.5 units of Tag DNA polymerase (Life Technologies). Amplification conditions were: 2 min of initial denaturation at 94°C, 30 cycles of 94°C for 15 sec, 55°C for 30 sec, 72°C for 1 min and 40 sec, followed by a final extension at 72°C for 7 min. The amplified hot probe was purified by QIAquick PCR Purification Kit (Qiagen) and denatured by boiling before adding to the hybridization solution. Hybridization took place overnight at 65°C in a solution containing 6X SSC (lxSSC = 0.15 M NaCl, 0.015 M Na-citrate, pH 7.0), 20 mM NaH₂PO₄. 0.4% SDS, 5X Denhardt's solution [0.1 % (w/v) of Ficoll (Type 400, Pharmacia), 0.1 % (w/v) of polyvinylpyrrolidone, and 0.1 % (w/v) of bovine serum albumin (Fraction V,

Sigma)], and 50 μg/ml sonicated, denatured salmon sperm DNA (Sigma) and washing was performed three times for 20 min each in 2X SSC, 0.5% (w/v) SDS at 65°C.

Construction of Plasmids (refer to Table 1): From tertiary screening, nine positive clones were purified from the Lesquerella fendleri genomic library. The phage DNA from those nine clones was extracted and purified using QIAGEN Lambda Mini Kit (Qiagen) according to the manufacturer's protocol. One of them was digested with EcoRI and a 4.3 kb fragment was subcloned into the pGΕM-7Zf(+) vector (Promega) cut with EcoRI, resulting in the vector pMHS15. The whole insert was sequenced with ABI automatic 373 DNA sequencer using fluorescent dye terminators. The upstream region of the genomic DNA was amplified using the high fidelity Pfu polymerase (Stratagene) with a forward primer 5'-CGCAAGCTTGAATTCGGAAATGGGCCAAG-3' and a reverse primer 5'-CGCGTCGACTGTTTTGAGTTTGTGTCGGG-3\ The amplified 573 bp promoter was inserted upstream of the GUS gene in pBHOl (Clontech) cut with HindxTI and Sail, resulting in the vector pLfKCS3-GUS. The fragment containing the promoter and the coding sequence was removed from pMHS15 by digestion with EcoRI and Hpal and the insert fragment was ligated to pRD400 cut with EcoRI and Smal, resulting in the vector pLfKCS3. As a comparison, another construct was made with LFAH12 promoter (Broun et al., 1998) and the coding sequence, which was named pLFAH12-LfKCS3.

Table 1 Plasmids

All the plasmids shown in Table 1, pLfKCS3-GUS, pLfKCS3, and pLFAH12-

LfKCS3, were introduced into Agrobacterium tumefaciens strain GV3101 (pMP90; Koncz

and Schell, 1986) by heat-shock and selected for resistance to kanamycin (50 μg/mL). They were then used to transform Arabidopsis (Columbia wild type) and/or fad2/fael double mutant by floral dip method (Clough and Bent, 1998). The fad2/fael double mutant is characterized by a very high level (>80%) of oleic acid (18:1) in its seed oil due to deficiency

in the activities of both cytoplasmic oleate Δ12 desaturase and the condensing enzyme, FAEl.

Screening for transformed seed was done on 50 μg/mL kanamycin as described previously (Katavic et al., 1994). Analysis of Fatty Acid Composition:

To determine the fatty acid composition of the tissues, fatty acid methyl esters were prepared by refluxing the samples in 2 ml of IN methanolic-HCl for 90 min at 80°C. After

cooling 2 ml of 0.9% NaCl solution and 200 μl of hexane were added and the mixture was

vortexed vigorously. The fatty acid methyl esters in the hexane layer were analyzed by gas chromatography.

GUS Assay: GUS assay was performed by immersing tissues in GUS histochemical staining solution (Jefferson, 1989) for 4 to 7 hours at 37°C. The assay solution was composed of 50 mM sodium phosphate, pH 7.0, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM EDTA, 0.05%(w/v) triton X-100, and 0.35 mg/ml 5-bromo-4-chloro-3-

indolyl-β-D-glucuronide (X-Gluc). Following staining the blue-stained samples were fixed in

70% ethanol.

Isolation and characteristics of the Lβ CS3 gene:

A genomic clone of a putative condensing enzyme was isolated using the Arabidopsis FAEl (James et al., 1995) to probe filters of a genomic library of Lesquerella fendleri. The EcoRI fragment subcloned into the plasmid pMHS15 was fully sequenced and a 4313 bp consensus sequence was assembled from individual sequence fragments using GCG program (Εdelman et al., 1994). The sequence included 573 bp of 5' flaking region, a 2062 bp coding region, and an 1678 bp 3' flanking sequence (Figure 1). A sequence comparison between the 4313 bp genomic DNA and the Arabidopsis cDNA made using the BCM Search Launcher: Multiple Sequence Alignments (Smith et al., 1996) revealed two introns in the E. fendleri

genomic DNA. Then the deduced amino acid sequence was obtained after removing the two

introns by aid of a translation tool, "Translate" (http://www.expasv.ch/tools/dna.html'). The amino acid sequence indicates that the gene, designated L/KCS3, encodes a polypeptide of 496

amino acids, and an estimated molecular mass of the LfKCS3 protein is 55.3 kD. Amino acid

sequence comparisons, using the BCM Search Launcher: Pairwise Sequence Alignments

(Smith et al., 1996), show that LfKCS3 shares high sequence identity, ranging from 49.9% to

80.2%, with VLCFA condensing enzymes, including Arabidopsis FAΕ1 (James et al, 1995),

Brassica napus KCS (Roscoe et al., 1996: GenBank accession number U50771), CUT1

(Miller et ah, 1999), and jojoba KCS (Lassner et al., 1996). Multiple sequence alignments reveal remarkable similarity among the Brassicaceae condensing enzymes along their entire

length (Figure 2).

Expression studies of L/KCS3 in plants: In order to determine the LfKCS3 expression pattern a 573 bp upstream fragment

from the L/KCS3 genomic clone was translationally fused to the uidA reporter gene encoding

β-glucuronidase (GUS), and introduced into Arabidopsis by floral dip method (Clough and

Bent, 1998). LJKCS3 expression pattern was determined for more than 30 independent

primary transgenic plants using GUS histochemical assays on leaves, stems, inflorescences,

roots, and siliques at different stages of development. GUS staining was observed

exclusively in the embryos. No GUS expression was detected in other tissues. Thus, the Arabidopsis LfKCS3 promoter is regulated in a tissue specific manner. REFERENCES

Clough,S.J. and Bent,A.F. (1998) Floral dip: a simplified method for Agrobacterium- mediated transformation of Arabdiopsis thaliana. Plant J. 16, 735-743.

Broun, P., Boddupalli, S., and Somerville, C. (1998) A bifunctional oleate 12- hydroxylase:desaturase from Lesq erellafendleri. Plant J. 13, 201-210

Doyle, J.J., and Doyle, J.L. (1990) Isolation of plant DNA from fresh tissue. Focus 12, 13- 15

Edelman, I., Olson, D., and Devereux, J. (1994) Wisconsin Sequence Analysis Package, version 8.0, Genetic Computer Group, Inc., University Research Park, 575 Science Drive, Madison, Wisconsin, 53711, USA

James, D.W., Lim, E., Keller, J., Plooy, I., Ralston, E., and Dooner, H.K. (1995) Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAEl) gene with the maize transposon activator. Plant Cell 7, 309-319

Jefferson, R.A. (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol. Bol. Rep. 5, 387-405 Katavic, V., Haughn, G.W., Reed, D., Martin, M., and Kunst, L. (1994) In planta transformation of Arabidopsis thaliana. Mol.Gen.Genet 245, 363-370.

Koetsier, P.A., Schorr, J., and Doerfler, W. (1993) A rapid optimized protocol for downward alkaline Southern blotting of DNA. BioTechniques 15, 260-262

Koncz, C. and Schell, J. (1986) The promoter of T_L-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204, 383-396.

Lassner, M. W., Lardizabal, K., and Metz, J.G. (1996) A jojoba β-ketoacyl-CoA synthase

cDNA complements the canola fatty acid elongation mutation in transgenic plants. Plant Cell 8, 281-292

Millar, A.A., and Kunst , L. (1997) Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme. Plant J. 12, 121-131

Millar, A.A., Clemens, S., Zachgo, S., Giblin, E.M., Taylor, D.C., and Kunst, L. (1999) CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant Cell 11, 825-838

Smith, R.F., Wiese, B.A., Wojzynski, M.K., Davison, D.B., Worley, K.C. (1996) Search- Launcher - An integrated interface to molecular biology database search and analysis services available on the world wide web. Genome Res. 6, 454-462 van de Loo, F.J., Broun, P., Turner, S., and SomerviUe, C. (1995) An oleate 12- hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog. Proc. Natl. Acad. Sci. USA 92, 6743-6747

Claims (9)

What we claim is:

1. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a condensing enzyme involved in very long chain fatty acid production in plants.

2. An isolated nucleic acid fragment according to claim 1 , wherein said sequence is defined by the nucleic acid sequence of SEQ. ID. NO. 1.

3. An isolated nucleic acid fragment according to claim 1 , wherein said sequence has a sequence identity of 95% or greater to the nucleic acid sequence of SEQ. ID. NO. 1.

4. An isolated nucleic acid fragment according to claim 1, wherein said sequence has a sequence identity of 85% or greater to the nucleic acid sequence of SEQ. ID. NO. 1.

5. An isolated nucleic acid fragment according to claim 1 , wherein said sequence has a sequence identity of 65% or greater to the nucleic acid sequence of SEQ. ID. NO. 1.

6. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding an enzyme for enhancing expression of endogenous and foreign genes in seeds for the enhanced production of seed oils.

7. An isolated nucleic acid fragment according to claim 1, wherein said sequence promotes expression of genes, which enhance seed production of long chain fatty acids in plants

8. An isolated nucleic acid fragment according to claim 3, wherein said genes enhance the nutritive value of seed production in plants.

9. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a condensing enzyme, which promotes production of very long chain fatty acids production in plants, which naturally do not synthesize very long chain fatty acids.