The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed:

FIELD OF INVENTION

The instant invention pertains to methods of expressing enzymes comprising polyunsaturated fatty acid biosynthetic pathway in an oleaginous yeast host for the production of Alpha linolenic acid (ALA). The oleaginous yeast used in this method includes Saccaromyces cervisceae. Further, the invention relates to transformation of Delta-12 desaturase and co-3 desaturase into yeast host. Such methods include, transforming a yeast cell with nucleic acid molecules, which encode proteins having an activity of catalyzing the formation of double bonds in the oleic acid and linoleic acid respectively. Advantageously, the invention is feasible and also commercially viable.
The nucleic acid sequences depicted herein are incorporated in a vector and are operably linked to a promoter or other regulatory elements for the expression of the genes in host cell.

BACKGROUND OF THE INVENTION

a-Linolenic acid, an n-3 fatty acid, is a member of the group of essential fatty acids, so called because they cannot be produced within the body and must be acquired through diet. Most seeds and seed oils are much richer in an n-6 fatty acid, linoleic acid. Linoleic acid is also an essential fat, but it, and the other n-6 fats, compete with n-3s for positions in cell membranes and have very different effects on human health.

Omega-3 fatty acids have been shown to reduce inflammation and may help prevent chronic diseases such as heart disease and arthritis. In the body, these essential fatty acids are highly concentrated in the brain and may be particularly important for cognitive and behavioral health as well as normal growth and development.

Studies suggest that alpha-linolenic acid and other omega-3 fatty acids may help treat a variety of conditions.

One of the best ways to help prevent and treat heart disease is to eat a diet that is low in saturated and trans fats, and rich in monounsaturated and polyunsaturated fats (particularly omega-3 fatty acids). Evidence suggests that people who eat an alpha-linolenic acid-rich diet are less likely to suffer a fatal heart attack. Alpha linolenic acid has been shown to lower cholesterol and triglycerides in people with high cholesterol. Diets or supplements rich in omega-3 fatty acids (including alpha-linolenic acid) lower blood pressure significantly in people with hypertension.

Omega-3 fatty acid supplements may help reduce tenderness in joints, decrease morning stiffness, and improve mobility. Omega-3s may also help relieve inflammation.
Preliminary research suggests that omega-3 fatty acid supplements (particularly alpha-linolenic acid) may decrease inflammation and improve lung function in adults with asthma.

Women who regularly eat foods rich in omega-3 fatty acids over many years may be less likely to develop breast cancer and to die from the disease than women who do not follow such a diet.

People who do not get enough omega-3 fatty acids in their diet may be at an increased risk for depression. The omega-3 fatty acids are important components of nerve cell membranes. They help nerve cells communicate with each other, which is essential in maintaining good mental health.

Alpha-linolenic acid cannot be synthesized by the human body, hence it is of utmost importance to obtain this essential fatty acid through dietary source. Alpha-linolenic acid can be obtained from flaxseed oil and, to a lesser extent, in canola, soy, Perilla, and walnut oils. Since the oil is highly heterogeneous in composition, extraction of ALA from the oil becomes very expensive process.

OBJECTIVES OF THE PRESENT INVENTION

The main objective of the present invention is to produce Alpha-linolenic acid in greater
quantities.

Another main objective of the present invention is to produce Alpha-linolenic acid through a novel recombinant method.

Still another objective of the present invention is bioconversion of oleuic acid to ALA through a series of conversions and to obtain the final expression of the ALA in the host cell, Saccaromyces cerevisiae.

SUMMARY OF INVENTION

The present invention is directed to the transformation of Delta-12 desaturase, Delta-6 desaturase and Delta-6 elongase into yeast host.

The D-12 desaturase from B. juncea is a desaturase that can introduce double bond into oleic acid to convert it to Linoleic acid.

A-3 desaturase from M. alpina is capable of introducing a double bond into Linoleic acid to convert it to Alpha linolenic acid.

Accordingly, the present invention features methods of producing Alpha linolenic acid in yeast.

Such methods include, transforming a yeast cell with a nucleic acid molecules, which encode proteins having an activity of catalyzing the formation of double bonds in the oleic acid and linoleic acid respectively.

The nucleic acid sequences depicted herein are incorporated in a vector and are operably linked to a promoter or other regulatory elements for the expression of the genes in host cell.

DESCRIPTION OF THE ACCOMPANYING DRWAINGS

Fig 1: Amplification of A-12 desaturase from B. juncea.

Fig 2: Fatty acid desaturase domain in the 1.16kb sequence of A-12 desaturase of BPR 559

Fig 3: A 12 desaturase cloned into the MCSII site under the GAL1 promoter of pESC-His.

Fig 4: Map of the construct pESC-Leu/P.pastoris A-3 desaturase.

Fig5: GC-MS profiles for the clones carrying both the D12 and A-3 desaturases subjected to proof of function experiment.

Description of the Sequence Listings:

SEQ ID NO 1: Codon optimized sequence of delta 12 desaturase from Brassica juncea BPR559 with nucleotide substitutions.

SEQ ID NO 2: Nucleotide Sequence of A -3 desaturases isolated from P.pastoris.

DETAILED DESCRIPTION OF THE INVENTION

Alpha linolenic acid is an essential fatty acid. ALA cannot be produced in the human body as humans lack w-3 destaurase. ALA is essential fatty acids and is further converted to A-3 polyunsaturated fatty acids like EPA and DHA that play very important biological roles. Hence ALA has to be obtained through external dietary sources. Biotechnology provides convenient and efficient methods to produce these essential fatty acids in the microbial systems. These microbial systems offer cost-effective methods for the large-scale production of essential fatty acids. 5. cerevisiae has been established as a platform for the production of these essential fatty acids. Present invention is based in part, on the cloning of the two fatty acid desaturases required for the conversion of OA which is indigenously present in the yeast to ALA an essential fatty acid. Present invention also provides the methods for producing ALA in the yeast host.

Amplification and cloning of D-12 desaturase from Bras ska juncea

The A-12 desaturase has been cloned from genomic DNA of Brassica juncea and has been found to have a single exon, with an ORF of 1155bp (Katavic and Taylor, 2000). Primers designed to amplify the gene from Brassica juncea are given below:

DNA was isolated from three popular varieties of Brassica juncea - RL-99-27, BPR-559 and Brassica rapa Skm-9816.

100ng of genomic DNA of RL-99-27, Skm-9816 and BPR-559 varieties of B. juncea were used to amplify the ORF of A-12 desaturase. (Fig 1)

Comparison of the nucleotide and amino acid sequences of A -12 desaturase of B. juncea
varieties RL-99-27, BPR-559 and B. rapa SKM-9816 with that of B. napus (Ace. No. AAS 92240) clearly showed that the sequence of BPR-559 was the closest to that of B. napus.

The cDNA sequence of all varieties translates into a protein of 384aa. Search for motifs confirmed that the sequence isolated has the fatty acid desaturase domain required for desaturase activity. (Fig 2)

BPR-559 D12 desaturase sequence differs from that of B. napus by three amino acids. To ensure that the gene isolated works as well as that of B. napus whose function has been proven (Katavic and Taylor, 2000) the three amino acids (marked in red in the Fig. 18) were replaced with those of the B. napus A-12 desaturase.

The codon bias of Brassica is considerably different from that of yeast; hence, the codons used in the D-12 desaturase of Brassica had to be optimized for expression in yeast - the target organism for production of PUFAs. A total of 23 changes had to be made in the sequence for optimizing D-12 desaturase for expression in yeast. The optimized sequence of D-12 desaturase is given below: The D12 desaturase of BPR559 was subjected to nucleotide changes using the Quickchange Multi site-directed mutagenesis Kit from Stratagene. Codon optimized sequence for D12 desaturase is given in SEQID 1.

The YPH 499 cells were transformed with the construct (FIG 3) and the transformants were selected on SDHis- medium. The transformants were subjected to PoF (Proof Of Function) experiment by the protocol outlined below.

Twenty four hour old culture of yeast cells carrying plasmid of interest was inoculated into 7ml of SD-AA selection medium (0.67%YNB without AA, 2%Dextrose, 0.13% AA dropout powder minus His). Cultures were Incubated at 30°C for overnight .10%(3.0ml) of inoculum was used to inoculate into 30ml of SD-/ SG-AA selection medium.(0.67%YNB without AA, 2% Galactose, 0.13% AA drop out powder minus His) Cultures were incubated for 24 hours at 25°C. Cells were pelleted, washed with media w/o carbon source and water. Fatty acid extraction was done using hexane and profiling was done using GC-MS.

GCMS analysis of FAMEs from yeast strain YPH 499 transformed with pESC-His/B. j A12 by simple induction. OA-LA

* SD and SG correspond to Uninduced and Induced samples.

Result

It can be noted that conversion from OA to LA, was observed in all the induced clones. These results confirm the activity of A-12 desaturase {B. j) in yeast.

Isolation and cloning of w-3 desaturase gene from Pichia pastoris w-3 desaturase gene was amplified from Pichia pastoris (SEQ ID 2) and cloned into pESCLeu vector between BamHI and Xhol in the MCS H(Fig 4).Proof of function for the Yeast clones carrying Pichia pastoris w3 desaturase was performed by simple induction.

Transformation of D12 desaturase and ca-3 desaturase for the production of EPA
Constructs in Fig 3 and Fig 4 were co-transformed into S. cerevisiae strain YPH499.

Transformants were selected on SD medium lacking Tryptophan and Leucine. Clones were subjected to proof of function experiment by simple induction.

Result: Conversion of OA-LA-ALA in the clones carrying both the D12 and w-3 desaturases. (Refer FIG 5)

We claim:

1. A process for the production of Alpha linolenic acid comprising of the following steps: (a) a host cell comprising:

(i) an isolated nucleotide molecule encoding a DELTA12 desaturase polypeptide sequence as set forth in SEQID NO: 1;

(ii) an isolated nucleotide molecule encoding a omega-3 desaturase polypeptide sequence as set forth in SEQ ID NO: 2; and

(b) growing the host cell of step (a) under conditions wherein the nucleic acid molecule encoding the DELTA 12 and DELTA 6 desaturase polypeptide is expressed and the oleic acid is converted to Alpha linolenic acid; and

(c) optionally recovering the Alpha linolenic acid of step (b).

2. An isolated nucleotide molecule encoding a DELTA 12 desaturase enzyme, as described in Claim 1, having sequence as set forth in SEQ ID NO:1.

3. An isolated nucleotide molecule encoding a omega-3 desaturase enzyme, as described in Claim 1, having sequence as set forth in SEQ ID NO:2.

4. A chimeric gene comprising the isolated nucleic acid molecule of claim 2, operably linked to suitable regulatory sequences.

5. A chimeric gene as claimed in claim 4 comprising the isolated nucleic acid molecule of claim 3 operably linked to suitable regulatory sequences.

6. An isolated transformed host cell comprising the isolated nucleic acid molecule of claim 5.

7. The transformed host cell of claim 6 selected from the group consisting of an oleaginous yeast.

8. The transformed host cell of claim 7 wherein the yeast is Saccharomyces cerevisiae.