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 present invention pertains to methods of expressing enzymes comprising omega-6 fatty acid biosynthetic pathway in an oleaginous yeast host for the production of DGLA. The oleaginous yeast used in this method includes Saccaromyces cervisceae. Further, the invention relates to transformation of Delta-12 desaturase, Delta-6 desaturase and elongase 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, linoleic acid and elongating gamma linolenic acid respectively. Advantageously, the invention is feasible and 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

Dihomo gamma-linolenic acid (DGLA) is an Omega 6 PUFA. DGLA has more of a functional rather than a structural role in the brain. Together with EPA and AA, DGLA is made into a complex group of compounds known as eicosanoids. Eicosanoids are involved in numerous regulatory functions in the brain and body.

Arachidonic acid and Dihomo-gamma-linolenic acid (DGLA) are some of the important Omega- 6 polyunsaturated fatty acids (PUFA) in the human body.

Dietary DGLA raises the DGLA level in the tissues of rats and guinea pigs much more
effectively than dietary gamma-linolenic acid. Since only a little linoleic acid seems to be
converted to DGLA and arachidonic acid in the human body, dietary arachidonic acid and DGLA are necessary nutrients for humans, at feast for people in which the conversion of linoleic acid to arachidonic acid is decreased.

The dihomogamma-linolenic acid gives rise to the prostaglandin-1 series, The prostaglandin-1 series, especially PGE1, is anti-inflammatory in nature, promotes T-cell function, has both anticoagulant and hypotensive actions, as well as decreases cholesterol production. In contrast, members of the PG2 series, with the exception of thromoboxane (PGI2), are inflammatory, opposing the action of PGE1.

DGLA is considered a healthy omega-6 fatty acid because it promotes the body to produce a series of prostaglandins that control platelets and blood pressure.

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.

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

D-6 elongase from M. alpina is capable of introducing a double bond into GLA to convert it to DHGLA.

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

Fig 7: Amplification of Elongase from Total RNA of M. alpina through RT PCR

Fig 8: Map of the construct pESC-Trp/M.alpElongase

Fig 9: GC-MS profiling for the clones subjected to proof of function experiment.

Description of the Sequence Listings:

SEQ ID NO 1: Codon optimized sequence of deltal2 desaturase from Brassica juncea BPR559 with nucleotide substitutions.

SEQ ID NO 2: Nucleotide Sequence of delta-6 desaturase ORF isolated from M.alpina 32222 SEQ ID NO 3: Nucleotide Sequence of Cig/C2oelongase from M.alpina 32222

DETAILED DESCRIPTION

DHGLA is considered to be a healthy n-6 fatty acid as it produces a series of prostaglandins and also plays important role in maintaining the blood pressure. DHGLA is produced in the human body by the conversion of GLA obtained through dietary source. But the conversion of GLA to DHGLA is limited. Biotechnology provides convenient and efficient methods to produce these essential fatty acids in the microbial systems. These microbial systems offer cost-effective and renewable 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 and an elongase required for the conversion of OA which is indigenously present in the yeast to DHGLA an essential fatty acid. Present invention also provides the methods for producing DHGLA 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 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.

l00ng 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 A-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 A-12 desaturase for expression in yeast. The optimized sequence of A-12 desaturase is given below: The A-12 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.

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

Amplification and cloning of Elongase:

Cloning of Elongase from M. alpina has already been reported (Parker-Barnes, J. M. et al., 2000). 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. (Fig 7). The PCR product (957bp) was cloned into pGEM-T easy vector. (SEQ ID 3) M.alpina Elongase present in pGEM-T easy vector has been digested with EcoRI and Clal enzymes and cloned directionally into pESC-Trp vector digested with the same enzymes. Map of the construct is given in FIG 8.

Yeast host YPH 499 was transformed with the construct. Five of the positive colonies were subjected to proof of function experiments as detailed earlier. The pellets were used for the extraction of total lipids. The fatty acids extracted were converted into FAMES and analysed by GC-MS. SD and SG correspond to Uninduced and Induced samples. GCMS analysis gave the following result.

Result: Conversion of GLA to DGLA is seen in the induced samples indicating that the gene Elongase is functional in yeast.

Proof of Function of gene combinations (D12+D6)+(Elo)

Host yeast YPH 499 cells was co-transformed with pESC-His/(B.jD12+M.alpD6)(FIG 6) and pESC-Trp/(M.alp Elongase)(FIG 8). Clones were induced with galactose and were grown without the addition of fatty acid into the medium. FAMEs were analysed by GC-MS. SD and SG correspond to Uninduced and Induced samples. GCMS analysis gave the following result.(Chromatograms FIG 9).

We claim:

1. A method for the production of Dihomo 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

(iii) an isolated nucleotide molecule encoding a cl8/c20 elongase polypeptide
sequence as set forth in SEQ ID NO: 3;

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

(c) optionally recovering the Dihomo 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:l.

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. An isolated nucleotide molecule encoding a C18/C20 elongase enzyme, as described in Claim 1, having sequence as set forth in SEQ ID NO:3.

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

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

7. A chimeric gene as claimed in 6 comprising the isolated nucleic acid molecule of claim 4 operably linked to suitable regulatory sequences.

8. An isolated transformed host cell comprising the isolated nucleic acid molecule of claim 7.

9. The transformed host cell of claim 8 selected from the group consisting of an oleaginous yeast.

10. The transformed host cell of claim 9 wherein the yeast is Saccharomyces cerevisiae.