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 omega-6 fatty acid biosynthetic pathway in an oleaginous yeast host for the production of GLA. The oleaginous yeast used in this method includes Saccaromyces cerevisceae. Further, the invention relates to transformation of Delta-12 desaturase and Delta-6 desaturase into yeast host. 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. 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

Linoleic acid is converted to GLA by the enzyme delta-6-desaturase.

Omega-6 fatty acid supplementation, in the form of GLA from evening primrose oil (EPO) or other sources, may assist nerve function and help prevent nerve disease experienced by those with diabetes (called peripheral neuropathy and felt as numbness, tingling, pain, burning, or lack of sensation in the feet or legs). People found they had increased mobility and a reduction of stiffness in their joints. Other research has found that GLA reduced joint swelling and tenderness by more than 40% more than the placebo takers.

GLA may be beneficial in dry-eye conditions such as Sjogren's syndrome (a condition with symptoms of dry eyes, dry mouth, and, often, arthritis).

A deficiency in essential fatty acids, including GLA and eicosapentaenoic acid (EPA, an omega-3 fatty acid) can lead to severe bone loss and osteoporosis. Clinical studies have reported that supplements of GLA and EPA help maintain or increase bone mass. Essential fatty acids may also enhance calcium absorption, increase calcium deposits in bones, diminish calcium loss in urine, improve bone strength, and enhance bone growth, all of which may contribute to improved bone mass and, therefore, strength. GLA may also allow for reduction in the amount of pain medication used by those with rheumatoid arthritis.

People who are prone to allergies may require more essential fatty acids (EFAs) and often have difficulty converting LA to GLA. In fact, women and infants who are prone to allergies appear to have lower levels of GLA in breast milk and blood.

Animal studies suggest that GLA either alone or in combination with two important omega-3 fatty acids, EPA and DHA, both found in fish and fish oil may lower the blood pressure in laboratory animals. Together with EPA and DHA, the GLA helped to prevent the development of heart disease in these animals as well.

In one clinical study that evaluated people with peripheral artery disease (blockage in the blood vessels in the legs from atherosclerosis or hardening of the arteries causing cramping pain when walking), men and women with did experience improvement in their blood pressure from the combination of EPA and GLA.

Dietary sources of GLA include borage oil. Since the oil is highly heterogeneous, extraction of GLA from the oil becomes very expensive process. These fatty acids essential to human body have tobe produced by alternate methods to meet the demand.

SUMMARY OF INVENTION

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

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

D-6 desaturase from M. alpina is capable of introducing a double bond into Linoleic acid to convert it to gamma linolenic acid.

Accordingly, the present invention features methods of producing DHGLA 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, linoleic acid and gamma linolenic 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 D-12 desaturase from B. juncea.

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

Fig 3: D12 desaturase cloned into the MCSII site under the GAL1 promoter of pESC-His.

Fig 4: Amplification of D-6 desaturase from total RNA of M.alpina

Fig 5: Map of the construct pESC-Trp/M.alp-D6 desaturase

Fig 6: Construct map for pESCHis-BjD12+M.alpD6

Fig7: GC-MS profiles for the clones containing both the D12 and D6 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 delta-6 desaturase ORF isolated from M.alpina 32222

DETAILED DESCRIPTION

GLA is an essential fatty acid. GLA is produced in the human body by the conversion of LA obtained through the dietary source. But the conversion of LA to GLA is very limited. 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. S. 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 GLA an essential fatty acid. Present invention also provides the methods for producing GLA in the yeast host.

Amplification and cloning of D-12 desaturase from Brassicajuncea

The D-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 Brassicajuncea are given below:

DNA was isolated from three popular varieties of Brassicajuncea - 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 D-12 desaturase. (Fig 1)

Comparison of the nucleotide and amino acid sequences of D-12 desaturase of B. juncea
varieties RL-99-27, BPR-559 and B. rapa SKM-9816 with that of B. napus (Ace. No. AAS92240) 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 D-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 D12 desaturase (B.j) in yeast.

Amplification and cloning of D-6 desaturase from M.alpina

Cloning of D6 desaturase has been reported (Sakuradani, E, 1999). Gene specific primers with the restriction sites were designed to amplify the fragment.

RT PCR reaction was performed using cMaster RT Plus PCR system (Eppendorf) in order to amplify the gene from total RNA.(Fig: 4)

The 1.3 KB PCR fragment was cloned into pGEM-T easy vector. D-6 desaturase of M.alpina present in pGEM-T easy vector was cloned between EcoRI and Spel sites of the pESC-Trp vector(Fig 5). The clones were confirmed by RE digestion and DNA sequencing.(SEQ ID 2).

YPH499was transformed with this construct (Fig 5) and POF performed with the addition of LA and ALA into the medium. SD and SG correspond to Uninduced and Induced samples. GCMS analysis gave the following result.

Result: Conversion of LA to GLA and ALA to STA is seen indicating that the gene A-6 desaturase is functional in yeast.

Cloning of B.juncea D12 desaturase and M. alpina D6 desaturase into single construct
D6-Desaturase of M.alpina was cloned into EcoRI and Clal sites of the pESC-His construct having codon optimized D12 desaturase present in. construct map (Fig :6).

Proof of Function:

YPH 499 was transformed with the plasmid containing the B.j D12 desaturase and M.alp D-6 desaturase.

Clones were induced with galactose and were grown without the addition of fatty acid into the medium. SD and SG correspond to Uninduced and Induced samples. GCMS analysis gave the following result(Chromatograms in FIG 7)

Result: Conversion of OA to GLA via LA is seen in the induced samples indicating that both the genes are functional.

We claim:

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

(i) an isolated nucleotide molecule encoding a DELTA 12 desaturase polypeptide
sequence as set forth in SEQ ID NO: 1; (ii) an isolated nucleotide molecule encoding a DELTA-6 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 gamma linolenic acid; and (c) optionally recovering the gamma 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 DELTA 6 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.