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 DHA. The oleaginous yeast used in this method includes Saccaromyces cervisceae. Further, the invention relates to transformation of Delta-12 desaturase, Delta-6 desaturase, C18/C20 elongase, Delta-5 desaturase, co-3 desaturase, elongase and Delta-4 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, linoleic acid, dihomogamma linolenic acid respectively and also encode proteins having an activity of catalyzing elongation of gamalinolenic acid. Advantageously, the invention is feasible and can be used commercially.
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:
Fatty acids are the long chain hydrocarbons with carboxylic group. They always occur in the etherified form as the major components of lipids. Lipids are the source of energy and they occur as an integral part of all the membranes and play a vital role in processing biological information.
Fatty acids are of two types: The saturated fatty acids and unsaturated fatty acids. Unsaturated fatty acids have one (mono) or more double bonds (poly) in the carbon chain in the cis configuration. Certain poly unsaturated fatty acids (PUFAs) are important components of cells and these cannot be synthesized by the human body. Such fatty acids are called "essential fatty acids" and have to be obtained by diet or synthesized by the further desaturation and elongation of Linoleic acid or Alpha linolenic acid.
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. Several studies suggest that omega-3 fatty acid supplements may help reduce tenderness in joints, decrease morning stiffness, and improve mobility. Omega-3s may also help relieve inflammation.
Omega-6 fatty acid supplementation, in the form of GLA 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). The dihomo gamma-linolenic acid gives rise to the prostaglandin-1 series. The prostaglandin-! series, especially PGEl, 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 PGEl.
An important family of regulatory molecules is derived from arachidonic acid, and these molecules collectively are often called the eicosanoids. They are synthesized by most tissues and have an incredibly wide range of actions. However, many of the most important are linked to defence against damage and pathogens. We will encounter them especially in the areas of inflammation and hemostasis.
Together, omega-3 and omega-6 fatty acids play a crucial role in brain function as well as normal growth and development. They are generally necessary for stimulating skin and hair growth, maintaining bone health, regulating metabolism and maintaining reproductive capability.
It is important to maintain an appropriate balance of omega-3 and omega-6 (another essential fatty acid) in the diet, as these two substances work together to promote health. Omega-3 fatty acids help reduce inflammation and most omega-6 fatty acids tend to promote inflammation. An inappropriate balance of these essential fatty acids contributes to the development of disease while a proper balance helps maintain and even improve health. A healthy diet should consists of roughly 2 - 4 times more omega-6 fatty acids than omega-3 fatty acids.
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,
SUMMARY OF THE INVENTION:
Plants and microorganisms majorly carry out synthesis of polyunsaturated fatty acids (PUFAs). Humans have a very limited ability to synthesize these essential PUFAs. Hence humans have to obtain these essential fatty acids from plants and microorganisms through diet. Biotechnology has been considered as an efficient way to manipulate the pathway in plants and microorganisms. Since the process is highly cost-effective and renewable, tremendous industrial effort has been directed towards the production of these fatty acids through fermentation. Accordingly biotechnology has also been thought to be an attractive route to produce PUFAs in a safe and cost-efficient manner so as to obtain the maximum therapeutic value from these fatty acids.
The present invention is based on the identification and cloning of the fatty acids desaturases (SEQ ID 1 to SEQ ID 10) involved in the DHA biosynthetic pathway. The present invention features the isolated nucleic acid molecules that encode polypeptide.
In a related aspect, the invention also provides the vectors into which the nucleic acid molecules encoding the polypeptide have been cloned. And the invention also features the host system for the expression of the above said constructs.
The constructs containing the SEQ IDs 1 to 10 can be used in yeast expression systems to produce the cells capable of producing DHA, DPA, EPA, ARA, DHGLA, GLA and ALA.
3

The methodologies of the present invention include methods of producing PUFAs in cells having at least one fatty acid desaturase of the unsaturated fatty acid biosynthetic pathway manipulated such that the unsaturated fatty acids are produced. For example the invention features methods of producing a PUFA (Eg: GLA) in cells comprising of SEQID 1 or 2, and SEQID 3.Such methods can further comprise a step of recovering the LCPUFA.
The invention features methods for modulating the production of fatty acids comprising culturing cells comprising of an isolated nucleic acid molecule encoding a polypeptide having an activity of catalyzing the formation of a double bond due to which modulation in the fatty acid occurs.
Further the invention features the culturing methods for the recombinant cells to enhance the production of desired LCPUFA.
The compositions of the present invention are used as a dietary supplements that is as neutraceuticals. The compositions of the above invention can also be used in the treatment of patients suffering from diabetics, cancer, inflammatory diseases, depressions and cardiovascular disorders. Other features and benefits would be evident in the following detailed description and claims.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
FIGURE 1: Construct map for the A-12 desaturase genes in pESC-His vector.
FIGURE 2: Construct map for the A -6 desaturase in the pESC-Trp vector.
FIGURE 3: Map of the construct containing A-12 desaturase genes with A -6 desaturase gene.
FIGURE 4: Construct map for A-6 Elongase in pESC-Trp vector.
FIGURE 5: Map of the construct carrying A-6 Elongase and A-5 desaturase genes in pESC-Ura construct.
FIGURE 6: Map of the construct for the w-3 desaturase in pESC-Leu vector
FIGURE 7: Map of the construct containing A-6 elongase from X.laevis in pESC-Leu vector
FIGURE 8: Construct map for the A-5 elongase gene present in pESC-Ura vector
FIGURE 9: Construct map for the T.aureum A-4 desaturase clones in the pESC-Ura construct.
FIGURE 10: Map for the construct carrying A-5 elongase and A-4 desaturase in pESC-Ura vector.
FIGURE 11: Gas chromatographic (GC)/Mass spectroscopy (MS) analysis of the FAMEs prepared from
YPH499 cells expressing B.juncea D12 desaturase.
FIGURE 12:GC-MS analysis of FAMEs prepared from yeast cells expressing M. alpina A-12 desaturase.
FIGURE 13: GC-MS analysis of FAMEs prepared from yeast cells expressing B. juncea A-12 desaturase and
M. alpina FAD6 gene
FIGURE 14: GC-MS analysis of FAMEs prepared from yeast cells expressing M. alpina A-12 desaturase and
FAD6 gene
FIGURE 15: GC-MS analysis of FAMEs prepared from yeast cells expressing B. juncea A-12 desaturase, M.
alpina FAD6 gene and M. alpina A-6 elongase gene.

FIGURE 16: GC-MS analysis of FAMEs prepared from yeast cells expressing M. alpina A-6 elongase and M
alpim FADS gene while the fatty acid Gamma linolenic acid (GLA 18:3 n:6) has been added into the medium
externally.
FIGURE 17: GC-MS analysis of FAMEs prepared from yeast cells expressing M. alpina FADS gene with
exogenously added Eicosatetraenoic acid (ETA 20:4 n: 3) into the medium.
FIGURE 18: GC-MS analysis of FAMEs prepared from yeast cells expressing Thraustochytrium species
FADS gene with exogenously added Eicosatetraenoic acid (ETA 20:4 n: 3) into the medium.
FIGURE 19: GC-MS analysis of FAMEs prepared from yeast cells expressing B. jmcea A-12 desaturase, M
alpina FAD6 gene, D-6 elongase and FADS genes.
FIGURE 20: GC-MS analysis of FAMEs prepared from yeast cells expressing M. alpina A-12 desaturase,
FAD6 gene, D-6 elongase and FADS gene.
FIGURE 21: GC-MS analysis of FAMEs prepared from yeast cells expressing ci)-3 desaturase from P.
pastoris where Linoleic acid (LA 18:2) has been exogenously added into the medium.
FIGURE 22: GC-MS analysis of FAMEs prepared from yeast cells expressing co-3 desaturase from P.
pastoris where Arachidonic acid (ARA 20:4 n:6) has been exogenously added into the medium.
FIGURE 23: GC-MS analysis of FAMEs prepared from yeast cells expressing B. juncea D12 desaturase, M.
alpina FAD6, A -6 elongase,FAD5 and P. pastoris co-S desaturase genes.
FIGURE 24: GC-MS analysis of FAMEs prepared from yeast cells expressing M alpina D12 desaturase,
FAD6, A -elongase, FADS and P. pastoris co-3 desaturase genes.
FIGURE 25: GC-MS analysis of FAMEs prepared from yeast cells expressing A -S elongase gene from
Thraustochytrium aureum with the exogenously added Eicosapentaenoic acid (EPA 20:S n:3) into the medium.
FIGURE 26: GC-MS analysis of FAMEs prepared from yeast cells expressing X. laevis A -S elongase and T.
aureum FAD 4 where the fatty acid Eicosapentaenoic acid (EPA 20:5 n:3) has been added into the medium
exogenously.
DETAILED DESCRIPTION OF THE INVENTION:
The present invention is related in part to the cloning and expression of the nucleotide sequences that have the ability to catalyze the formation of double bonds in fatty acids and also relates in part to the cloning and expression of Elongases that have the ability of catalyzing the carbon chain elongation. The nucleic acid sequences that have the property of introducing a double bond in a fatty acid belong to a family of proteins called fatty acid desaturases (FADs). These novel nucleotide sequences are present in the microorganisms and plants that produce polyunsaturated fatty acids.
The controlling steps in unsaturated fatty acid biosynthetic pathway are catalyzed by fatty acid desaturases and elongases. The term "polyunsaturated fatty acid pathway" refers to a series of chemical changes that lead to a synthesis of PUFAs. Such pathways include a series of desaturation and elongation

reactions that generate unsaturated fatty acids finally leading to the production of poly unsaturated fatty acids like Alpha linolenic acid (ALA; 18:3 n:3), Gamma linolenic acid (GLA;18:3 n:6), Stearidonic acid (SDA; 18:4 n:3), Dihomogamma linolenic acid (DGLA; 20:3 n-6), Arachidonic acid(ARA; 20:4 n-6), Eicosapentaenoic acid(EPA; 20:5 n:3) and Docosahexaenoic acid (DHA; 22:6 n:3)
Examples:
M. alpina strain ATCC32221 was obtained from ATCC and grown in the laboratory. Thraustochytrivm aureum strain ATCC 34304 was obtained from ATCC and grown in the laboratory. Thraustochytrium species 26185 was obtained from ATCC and grown in the laboratory. X. laevis elongase clone in pCMV-sport6 from ATCC No 6844054
RT PCR:
RT-PCR reaction was performed for the total RNA of M alpina using Eppendorf cMaster plus kit (Eppendorf). Gene specific primers for SEQ ID 3, SEQ ID 4 and SEQ ID 5 were used in the amplification. PCR amplification consisted of 35 cycles of 94''C for 30 sec, 55''C for 30 sec and 72°C for 2 minutes followed by a final extension of 72''C for 7 minutes.
Cloning of SEQID 1 and SEQID 2:
Genomic DNA of Brassica juncea and M. alpina were used for the amplification of the delta dl2 destaurase. The gene was cloned into pGEM-T easy vector and sequenced for the confirmation. The nucleotide sequence obtained by the amplification of B. juncea genomic DNA amplification was codon optimised for the expression in yeast. Codon optimized sequence has been presented in SEQID 1.Nucleotide sequence for the gene amplified from M. alpina has been depicted in SEQID2. Both the SEQID 1 and SEQID2 were cloned in the yeast expression vector pESC-His (Stratagene) at the multiple cloning site II (MCSII) directionally. The construct depicted in FIG 1 was transformed into yeast host YPH499 by electro-poration.
Expression of SEQIDl and SEQID2:
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 A A, 2%Dextrose, 0.13% A A dropout powder minus His and 0.004%Adenine Sulphate). Cultures were incubated at 30°C for overnight. 10%(3.0ml) of inoculum was used to inoculate into 30ml of SDVSG'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 without carbon source and water.
Fatty acid analysis:

Yeast cells were transferred to glass tubes and lysed using glass beads and 2ml of hot Isopropyl alcohol. Tubes were incubated at 80°C for 10 min. Total lipid was extracted twice with 3 ml of hexane. Final extraction was done with 2ml of IPA: Hexane in the ratio 2:7. Hexane phases were combined and dried under nitrogen gas.
To the tubes containing total lipids 1ml of 5% sulfuric acid in methanol was added. Tubes were incubated at 80''C for 2 hours with intermittent vortexing. After cooling to room temperature 1ml of water was added and fatty acid methyl esters (FAMEs) were extracted twice with 3ml of hexane. Hexane was evaporated under nitrogen gas. Resulting FAMEs were subjected to GC MS analysis.
FAMEs were analyzed by GC-MS (Trace Ultra GC, DSQ II, Thermo electronic corporation, Milan, Italy) and analyzed by comparing the peaks with those of standards and also by EI detector. Nonadecanoic acid (C19:0) methyl ester (Sigma) was used as an internal standard for quantitative analysis of fatty acids. All analysis was carried out with the same polar capillary column (TR-FAME, 0.25mm ID, 60 m long). The mass spectrum of the novel peaks was compared with those of the standards for identification of the fatty acids.
Chromatogram showed a new peak in the induced samples of the clones (FIG 11 and FIG 12). Retention time and MS spectrum data confirmed the peak is of Linoleic acid (18:2) (Table 1 and Table 2). Functionality of both the SEQIDl and SEQID2 are proven with this experiment.
TABLE 1: Gas chromatographic (GC)/Mass spectroscopy (MS) analysis of the FAMEs prepared from
YPH499 cells expressing BJuncea D12 desaturase
Fatty Acid I YPH499/ pESC-His/Ay«/Jcea All
_ . __ . _ . _ I Vector Control
SD SG SD SG SD ISG SD ISG ^ ic
i4i0 0.96 1.18 1J06 L97 l88 o!99 0^99 1.31 2.04 o!98
Tsio 10.59 0.27 0!2 0i28 047 038 028 0.98 0.39 027
ieio 34.37 37.4 33.86 37.4 346 37.15 29.61 38.29 40.54 30.04
leH 20.2 29.93 29.88 29.8 27.68 28.49 34.42 29.05 27.49 32.74
nio 9.65 0.07 O03 O03 018 022 O03 0.O8 0.07 O07
18^0 5.49 T3 157 8^44 U 8^25 4!68 7.32 4.66 5^95
mil 18.1 14.06 30.14 13.62 26.09 15.37 29.47 14.34 24.08 29.6
18^2 0 HM 0 7^94 0 8^92 0 8.36 0 0

TABLE2; Gas chromatographic (GC)/Mass spectroscopy (MS) analysis of the FAMEs prepared from YPH499 cells expressing M.alplna D12 desaturase.
Fatty Add I YPH48a/pESC-Tfprt312JVIalp_9.08.07OMJ<
#1 I #2 I *3 I #4 I #S I pESC
SD I SG SD I SG " SD I SG SD | SG ~ SD | SG SD | SG
13:g 1.64 0 SIsT^ 0.62 0.97 0.66 1.97 0.72 1.06 0 0.81 0.9
15:0' 0.39 0 0.28 0 0.42 0.88 0.56 0 0.51 0 1.76 0.47
16:0' 36.01 23.48 34.08 23.85 29.03 39.03 51.07 28.13 25.41 28.97 34.21 29.75
16:1' 24.44 24.58 20.62 25.05 24.75 14.15 11.17 21.44 28.18 21.78 24.93 22.15
17:0 0.08 0.39 0.35 0.29 0.15 1.26 0.66 0.62 0.21 0 2.19 0.13
18:0 8.69 19.99 13.79 16.65 12.37 24.95 19.69 21.5 9.82 19.54 7.14 15.2
18:1' 2R81 24>43 30.33 28.3 3231 17.31 1486 22.98 34.49 244 28.72 31.3
18:2' 0 6.85 0 8.23 0 1.76 0 441 0 6.31 0 0
20:0* I 0.25 I 0.28 I 0 | 0 | 0 | 0 | 0 | 0 | 0.33 | 0 | 0.24 | 0.1
Cloning and expression of FAD6 gene from M.alplna
RT PCR reaction was performed for the total RNA isolated from M.alpina as set forth in example 1. The nucleotide sequence as in SEQID3 was obtained. The SEQID3 was cloned into pESCTrp vector in the MCSI directionally. Construct in FIG2 was transformed into the yeast host S.cerevisiae YPH499. Clones obtained were subjected to proof of function experiment as depicted in example 4 and 5. Linoleic acid (18:2 n:6) and Alphalinolenic acid (18:3 n:3)were added externally into the medium in presence of 0.1% tergitol. Retention time and MS spectrum data confirmed the results presented in Table 3 and Table 4 wherein conversion of LA to GLA and ALA to STA is seen indicating that the gene A-6 desaturase is functional in yeast.

TABLE 3; Gas chromatographic (GC)/Mass spectroscopy (MS) analysis of the FAMEs prepared from YPH499 cells expressing M.alpina D6 desaturase when Linoleic acid was externally added to the medium


Amplified products were cloned into pGEM-T easy and sequencing was done to confirm that the gene sequence was identical to the original sequences. Nucleotide sequences as set forth in SEQIDS 1 or 2 and SEQ ID 3 were cloned into pESC His vector in the MCS II and MCS I respectively (The construct in FIG3).
To confirm the functionalities of both the above genes, yeast host strain YPH499 was transformed with the construct containing both the open reading fi-araes under galactose inducible promoter. When the yeast cells were induced with the galactose, two distinct peaks appeared in the induced samples as compared to that of the uninduced and vector control samples(FIG 13 and FIG 14). A comparison of the chromatogram with the standards revealed that the retention times of the peaks obtained match with the Linoleic acid (LA) and gamma Linolenic acid (GLA). MS spectrums of the peaks were matched with the library (Table 5 and Table 6). Library search also confirmed that the two new peaks seen in the induced samples were of LA and GLA. These results confirm that both the SEQIDl and SEQID 3 are functional and bringing about the conversion of oleic acid that is indigenously present in yeast into Linoleic and Gamma Linolenic acids.

TABLE 6 Gas chromatographic (GC)/Mass spectroscopy (MS) analysis of the FAMEs prepared from YPH499 cells co-expressing M.alplna D12 desaturase and M.alpina D6 desaturase


Cloning and expression of A-6 Elongase:
Total RNA of M.alpina was subjected to reverse transcription as set forth in example 1. 957 bp amplicon obtained as set forth in SEQID 4 was directionally cloned into yeast expression (pESC-Trp) vector. Construct as set forth in FIG 4 was transformed into yeast host S.cerevisiae YPH499. Transformants were subjected to proof of function experiment with the addition of Gammalinolenic acid into yeast. GC-MS data as presented in Table 7 reveals conversion of GLA to DGLA in the induced samples indicating that the gene Elongase is functional in yeast..
TABLE 7 Gas chromatographic (GC)/Mass spectroscopy (MS) analysis of the FAMEs prepared from YPH499 cells expressing M.alpina D6 Elongase when Gammalinolenic acid was externally added to the medium.


Co-expression of SEQIDl, SEQroS and SEQID 4:
S. cerevisiae host cells YPH 499 was transformed with the constructs as given in FIG 3 and FIG 4. Transformants were selected on SDHisTrp" medium. Clones were confirmed for the presence of all three genes and subjected to proof of function experiment. Protocol followed was as set forth before. Retention times for the new peaks obtained (Refer FIG 15) in the induced samples corresponded to GLA (18:3 n:3) and DHGLA(20:3 n:3). The results were also confirmed with mass spectroscopy data as provided in Table 8 wherein the conversion of OA to DGLA via GLA is seen in the induced samples indicating that all the three genes are functional and produce DGLA in yeast.
Table 8 Gas chromatographic(GC)/Mass spectroscopy(MS) analysis of the FAMEs prepared from YPH499 cells expressing BJuncea D12 desaturase, Af.a/pina D6 desaturase and M.alpina D6 Elongase

Cloning of FAD5 gene into D-6 elongase construct (FIG 4):
Total RNA isolated from M.alpina was amplified with the gene specific primers for SEQID 5. 1341 bp nucleotide fragment obtained was cloned directionally into the multiple cloning site of construct carrying D-6 elongase (FIG 4). Final construct obtained (FIG 5) containing both the open reading frames under galactose inducible promoter was transformed into yeast host cell YPH499.To examine the activities of both the transgenic enzymes in yeast, the 18:3(n-6) substrate should be supplied exogenously into the medium. In this experiment induced, uninduced and vector control samples were supplied with gamma Linolenic acid in presence of 0.1% tergitol. Exogenously added fatty acids were taken up by all the samples. But the conversion of GLA to Dihomogamma Linolenic acid (DHGLA 20:3 (n-6)) and Arachidonic acid (20:4 (n-6)) was seen only in case of galactose-induced transformants (FIG 16). Retention times and MS spectrum confirmed the presence of DHGLA and ARA in the induced samples (Table 9).

Table 9: Gas chromatographic(GC)/Mass spectroscopy(MS) analysis of the FAMEs prepared from YPH499 cells expressing M.alplna D6 Elonagase and M.alpina D5 desaturase when Gammalinolenic acid was externally added to the medium.

Cloning of FADS gene from Thraustochyrium species:
An open reading frame of 1320bp(SEQ ID 6) was amplified from the genomic DNA of Thraustochytrium species and cloned into yeast expression vector. The construct was introduced into yeast and clones were subjected to proof of function experiment with the addition of DHGLA (Dihomogamma linolenic acid) and ETA (Eicosatetraenoic acid) into the medium
Fatty acid profiling of the FAMEs showed a distinct peak in the induced samples of all the clones. The RT of the peak correlates with that of the standard Arachidonic acid in the set into which DHGLA was added

and the Mass spectrum also shows that the peak corresponds to that of Arachidonic acid (Table 11 and Table 12).
Table 11; Gas chromatography (GC)/Mass spectroscopy(MS) analysis of the FAMEs prepared from YPH 499 cells expressing T.species D5 desaturase when Dihomogamma linolenic acid was externally added to the medium

jiff':->.% 1 0.7 '•ml m tftm. 4 m ':4mi 'dm }^mi ^^^.. '■."■J

2 0.9 1.5 1.2 1.1 1 1.4 1.1 1.6 1.5 1.4 1.2
'■k ' - V: 0.2 1.9 0.7 1 1.2 0.5 1 0.7 3.2 1.1 1.1 1 0.8
20 45.6 26.1 35.2 32 27 30.6 29 29.9 40.1 34 35.2 26.1
30.5 24.7 22.7 29.3 25 34.2 23.1 30.6 21.2 26.1 20.7 31.3 24
f|;j.'.<.:.-;!;,.j:i| 1 0.9 - 0.5 0.5 - - - 0.4 - 0.5 0.4 0.3
B«MM 5.8 4.4 6 4 4.8 4.4 5.3 3.7 5.2 5.1 6.6 4.2 6.9
26.5 16.7 22 19.4 17.6 24.7 20.1 24.2 15.7 16.9 16.9 19.2 19.7

0.7 0.4 2 0.8 1.6 0.8 2.4 1 1.7 1 2.6 0.6 2.4
20:3 15.6 3.4 19.6 8.3 9.1 7.3 10.3 9.4 8.2 8.1 9.3 6.7 11.1
20:4 - • 7 6.1 6 6.8 7.5
- - - - 1.3 - -

- - 6.1 - -
The set of experiments into which ETA (Eicosatetraenoic acid) was added into the medium showed the conversion of ETA to EPA (Eicosapentaenoic acid)(FIG 18).
Table 12 Gas chromatography(GC)/Mass spectroscopy(MS) analysis of the FAMEs prepared from YPH 499 cells expressing T.species D5 desaturase when Eicosatetraenoic acid was externally added to the medium

Fatty acid D5 (T.Sp).ETA- EPA
#3 #6 URA Control
SD SG SD SG SD SG
16:00 30.86 34.95 30.62 31.26 32.97 32.94
16:01 28.35 13.68 29.35 14.57 26.76 18.05
18:00 8.45 14.3 8.06 10.95 9.06 12.85
18:01 26.98 24.68 27.06 22.73 27.81 22.28
20:00 0 0 0 0 0 0
20:4 (ETA 5.35 5.92 4.91 9.56 3.41 13.88
26:$ ilPA 0 6.48 0 10.93 0 0

14

Co-expression of SEQIDl, SEQID3, SEQID4 and SEQID 5 in yeast
The constructs that have been presented in FIG 3 (Consisting of SEQID lor 2 and SEQID3) and FIG 5 (SEQID 4 and SEID 5 or SEQID 6) were co-transformed into S.cerevisiae strain YPH499 by electro-poration. Transformants were selected on SD HisTrp" plates. Transformants obtained were cultured in an induction medium containing galactose. Fatty acid methyl esters isolated from these samples were analyzed. Chromatograms of all the induced transformants containing all four genes showed the presence of 4 new peaks (FIG 19 and FIG 20), which were absent in uninduced and vector control samples. Retention times of the peaks obtained corresponded to that of LA(18:2), GLA(18:3 n:6), DHGLA(20:3 n:6) and ARA(20:4 n:6) (Table 13 and Table 14).
Table 13 Gas chromatographic(GC)/Mass spectroscopy(MS) analysis of the FAMEs prepared from YPH499 cells expressing BJuncea D12 desaturase, Af.a/pina D6 desaturase, M.alpina D6 Elongase and M.alpina DS desaturase.

i*Sw Saii-" H IMI '1 :»«|lalfii:»JS«eii:
Si*iC/;,','-v'iSil 1.57 0.86 1.1 1 2 0.93 0.78 1 13 0.86
U n A r U. IV U. lO U.OO U.OO r\ nA

34.44 19.78 31.08 53.89 25.33 22.61 26.18 24.19

35.3 35.31 36.7 18.92 43.67 34.86 42.84 34.25

0.14 5.58 0 4.07 0.12 5.7 0.2 6.81

3.93 5.07 4.52 4.6 3.71 4.95 3.69 5.03
18:1 OA 22.91 27.84 25.62 12.82 26.12 25.22 25.75 22.74
18:2 GLA 0 4.86 0 2.99 0 4.82 0 5.92
18:3 DGLA 0 0.19 0 0.16 0 0.31 0 0
18:4 ARA 0 0.36 0 0.71 0 0.19 0 0
15

Table 14 Gas chromatographic(GC)/Mass spectroscopy(MS) analysis of the FAMEs prepared from YPH499 cells expressing M.alpina D12 desaturase, M.alpina D6 desaturase, M.alpina D6 Elongase and M.alpina D5 desaturase

0.99
0.69
26.84
26.19
4.74
8.74
20.32
19.16
4.7
4.9
1.17
0.72
'ISlii^lf^^S3i'iS^'S*fe'® rC'-^ :■■''::
'^P^^SSS W'
0.98
1.29
0.83
14:00
6.77
0.48
0.88
0.34
15:00
7.51
30.79
29.33
31.56
7.94
29.3
18.73
16:00
27.71
1.95
16:01
8.6
2.72
4.52
9.22
17:00
5.79
5.9
12.8
18:00
14.41
32.36
9.61
30.76
0A(18;1)
6.84
10.31
LA(18:2)
6.73
10.82
GLA(18;3 n-6)
13.27
2.1
DGLA(20:3 n-6)
13.74
1.91
ARA(20;4 n-6)

0.97
0.36
31.44
28.13
1.03
5.71
32.36

i'i-:!-...m '9'dis-^*r^4>M^M ■
1.57
0.82
0.37
0.49
31.22
28.95
29.5
25.05
4.57
0.24
8.72
5.8
32.05
4.67
4.87
1.26
0.81

m-^
0.73
0.32
20.57
37.08
0.21
5.03
3ijr

Result: All the clones showed the conversion of OA-LA-GLA-DHGLA-ARA, proving all the four genes are functional and bringing about the desired conversions.
Cloning and expression of 0)-3 desaturase from P.pastoris:
Open reading frame of the gene was amplified from genomic DNA of P.pastoris GS115 and cloned directionally into yeast expression vector pESCLeu between BamHI and Xho I sites directionally. Final construct obtained (FIG 6) containing the open reading frames under galactose inducible promoter was transformed into yeast host cell YPH499.To examine the activities of the transgenic enzyme in yeast, the Linoleic acid (18:2) and Arachiodonic acid (20:4 n:6) should be supplied exogenously into the medium. In this experiment induced, uninduced and vector control samples were supplied with Linoleic acid and Arachidonic acid in presence of 0.1% tergitol. Exogenously added fatty acids were taken up by all the samples. But the conversion of LA (18:2) to ALA Alpha Linolenic acid (18:3 n:3) and Arachidonic acid (20:4 (n-6)) to Eicosapentaenoic acid(20:5 n:3) were seen only in case of galactose-induced transformants (Table 15 and Table 16). Retention times and MS spectrum confirmed the presence of ALA and EPA in the induced samples (Fig 21 and FIG 22).
16

Table 15 Gas chromatography(GC)/Mass spectroscopy(MS) analysis of the FAMEs prepared from YPH 499 cells expressing P.pastoris w3 desaturase when Linoleic add was externally added to the medium

FA
14:00 6.15 0.89 0.58 0.96 0.8 1.22 1.43 1.05 0.79
15:00 6.61 0.78 0.43 1.15 0.38 1.02 1.73 1.08 0.81
16:00 7.55 30.96 28.27 43.5 27.91 34.1 30.73 33.23 29.11
16:01 7.94 18.23 14.45 12.45 14.62 14.76 14.36 13.79 15.77
17:00 8.83 0.58 0.3 1.36 0.61 1.43 2.48 0.93 0.39
18:00 9.22 6.04 5.96 5.77 5.34 5.37 5.51 5.63 5.06
OA 9.61 13.15 11.92 10.31 11.43 12.34 9.37 12.2 12.97
LA 10.31 29.36 30.06 24.5 29.68 29.75 27.86 32.08 35.11
ALA 11.21 0 8.02 0 7.67 0 6.52 0 0
Table 16 Gas chromatography(GC)/Mass spectroscopy(MS) analysis of the FAMEs prepared from YPH 499 cells expressing P.pastoris w3 desaturase when Arachidonic acid was externally added to the medium

HliiiUBC fj;ft*>'\j ■■'i,'J: ': fi:'vi'K^-[ :i" 'i\. ■,»„>V^ ■" ,r,:,,^:/ ?-ti|jii»SRl'£SS*3'ir'.<'3^y'i;^-s-.
FA Apex RT #1SD #1SG #2SD #2SG #3SD #3SG SD
Leu SG
Leu
14:00 6.16 1.12 0.89 0.81 0.98 1.28 0.66 1.79 0.73
15:00 6.61 0.64 1.18 0.54 0.63 0.45 0.54 1.05 0.76
16:00 7.56 23.65 28.68 25.17 24.69 37.45 20.53 26.58 34.02
16:01 7.94 41.19 26.37 38.19 30.14 28.96 34.13 30.32 21.94
17:00 8.83 0.42 0.45 0.42 0.73 0.35 1.06 0.55 1.71
18:00 9.22 4.38 5.09 4.69 4.6 4.36 5.05 4.63 5.9
OA 9.61 17.68 20.06 20.06 18.61 17.92 18.19 21.36 18.75
ARA 13.78 10.92 15.64 10.12 16.9 9.22 17.17 13.73 16.18
EPA 15.1 0 1.66 0 2.73 0 2.67 0 0
Co-expression of SEQ ID 1, SEQ ID 3, SEQ ID 4, SEQ ID 5 and SEQ ID 7 in a single yeast host
To confirm the functionalities of all the first five nucleotide sequences of the DHA biosynthetic pathway, yeast host strain YPH499 was transformed with the constructs as depicted in FIG 3(SEQID1 or 2 and SEQID3), FIG 5 (SEQID 4 and SEQID 5) AND FIG 6 (SEQID 7) containing all five open reading fi-ames under galactose inducible promoter. When the yeast cells were induced with the galactose, distinct peaks appeared in the induced samples as compared to that of the uninduced and vector control samples. A comparison of the chromatogram (FIG 23 and FIG 24) with the standards revealed that the retention times of the peaks obtained match with the Linoleic acid (LA 18:2) and (o-6 fatty acids gamma Linolenic acid (GLA, 18:3 n:6), Dihomogammalinolenic acid (DHGLA 20:3 n:6), Arachidonic acid (ARA, 20:4 n:6). Along with these fatty acids peaks were also obtained for co-3 fatty acids Alpha linolenic acid (ALA 18: 3 n: 3), Stearidonic acid (18:4 n:3), Eicosatetraenoic acid (ETA, 20:4 n:3) and Eicosapentaenoic acid (EPA 20:5 n:3). MS
17

spectrums of the peaks were matched with the library. Library search also confirmed the presence of both (o-6 and co-3 fatty acids (Table 17 and Table 18). These results confirm that all the nucleotide sequences introduced into the yeast in this experiment are functional and bringing about the conversion of oleic acid that is indigenously present in yeast.
Table 17 Gas chromatography(GC)/Mass spectroscopy (MS) analysis of FAMEs prepared from YPH499 cells expressing B.juncea D12 desaturase, M.alpina D6 desaturase, M.alpina D6 Elongase, M.alpina D5 desaturase and P.pastoris w3


YPH499-pESC-His(Bj-D12+M.alp-D6)+pESC-Trp(M.alp-Elo+D5)+pESC-Leu-ppw3 OA-LA-GLA-ALA-SDA-DGLA-ARA-
ETA-EPA
■n-
1.06
104
0 68
I 07
I 12
0 76
0.19
0.14
0.21
0,14
0.34
0.32
28.36
25.49
28.68
25.81
24.66
20.99
40.69
21.05
10,53
20.65
32.5
10.37
3.1
2.89
16,1
7.29
11,39
18,19
6.45
11,5
28,36
36.54
24.19
36.25
33,86
24.36
0.81
2.57
1,45
9.14
1.72
6.37
2.82
2.93
10.94

. i-t^flfJ.jii g.'>•«■!■
1.09
0 89
0.26
0.12
25.94
29.37
10.62
21.86
2.83
9,94
17,13
25.28
35.94
7,44
2.8
1.69

M.37

0.04

0.08

0.02

^W"
TTEK:
riM

11.91
13.51
13.94
14.75
15.26

0.29
3.68
1.92
0.52
0.36

0.37
3.6
1.96
0.62
0.54

0.34
3.99
2.08
0.68
0.59

18

Table 18 Gas cbroniatography(GC)/Mass spectroscopy (MS) analysis of FAMEs prepared from YPH499 cells expressing M.alpina D12 desaturase, M.alpina D6 desaturase, M.alpina D6 Elongase, M.alpina D5 desaturase and P.pastoris w3

YPH499/pESC-Hi8-M.alp-(D12+6)/pESC-Trp-M.alp-(Elo+D5)/pESC-Leu-Pp-w3
OA-LA-GLA-ALA-SDA-DGLA-ARA-ETA-EPA
05 ■fcEH §E^/i iiiiii 0.73 0.88 0.5S 0.97
JBiL'f 7 0.13 0.22 0.14 0.34 0.29 0.34 0.46 0.5

7.78 25.09 17.46 23.01 30.28 25.34 26.61 27.19 25.71
WMiV :-'"^-: 8.13 34.54 43.74 31.01 16.61 18.53 21.99 20.93 19.91
8.79 0.03 0.21 0 1.88 2.38 3.79 4.71 4.38

9.69 5.78 3.71 6.59 14.05 11.08 10.62 11.45 10.56
if■liii! 1 ■ ■ ';,v ^ 10 33.94 34.15 38.67 24.56 27.4 20.78 19.79 20.68
Ifllk- * 10.66 0 0.06 0.23 3 3.22 3.33 3.36 4.15
11.13 0 0 0 3.23 3.48 3.9 4.44 4.05
11.52 0 0 0 0.17 0.46 0.56 0.4 0.73
W|ft • 12.06 0 0 0 0.25 0.64 0.32 0.34 0.55

13.74 0 0 0 1.7 1.96 2.21 2.24 2.18
14.13 0 0 0 2.71 3.57 4.16 3.76 4.6
■RafiB 14.95 0 0 0 0.24 0.38 0.18 0.07 0.4
SIK- 15.49 0 0 0 0.14 0.53 0.36 0.28 0.63
Example 14:cloning and expression of A-S elongase:
cDNA clone of XJaevis elongase gene was obtained from ATCC and the nucleotide sequence was amplified using the gene-specific primers (SEQID 8). Amplified fragment was cloned into yeast expression vector. The construct as set forth in FIG 7 was transformed into yeast host YPH 499. Clones obtained were subjected to proof of function experiment with the addition of EPA into the medium. FAMEs extracted were analyzed by GC-MS. The chromatograms of the induced samples clearly showed the presence of a new peak that was not seen in the control samples. The library search revealed that the peak corresponded to Docosapentaenoic acid (DPA 22:5), the elongation product of Eicosapentaenoic acid (EPA 20:5). These results confirm that the nucleotide sequence cloned has an elongase activity (Table 19).
Table 19 Gas chromatography(GC)/Mass spectroscopy (MS) analysis of FAMEs prepared from YPH499 cells expressing Xdaevis D5 Elongase when Eicosapentaenoic acid was externally added to the medium.

«i*'!iSii«ii«^»^^^^^Miaiji:uat.?i #3 11^ wm^i^^^^M ^^^^■1^^^^
Fatty acid #1 #2 ~1

4 1 #5 Control Leu
SD SG SD SG SD SG SD SG SD SG SD SG
EPA 100 84.38 100 84.67 100 84.76 100 84.7 1 100 86.41 100 100
DPA 0 15.62 0 16.43 0 18.26 0 16.3 j 0 14.64 0 0
Result: Convers on from EPA to DPA seen in all clones
19

Example 15:cloning and expression of A-5 elongase from T.aureum
Genomic DNA of T.aureum was amplified with the gene-specific primers of T.aureum elongase. 825 bp amplicon (as set forth in SEQID 9) was directionally cloned in the MCSII of pESC-Ura vector. Construct that has been shown in FIG 8 was transformed into the YPH499 electro-competent cells. Transformants were checked for the enzyme activity of the newly introduced nucleotide sequence by proof of function. Eicosapentaenoic acid was added into the medium. FAMEs extracted from the cultures subjected to proof of function experiment. Analysis of FAMEs in the GC-MS (FIG 25) clearly showed the presence of a fatty acid peak corresponding to the retention time of that of the DPA (docosapentaenoic acid). Library search also confirmed the presence of DPA in all the induced samples containing the gene (Table 20).
Table 20 Gas chrom8tography(GC)/Mass spectroscopy (MS) analysis of FAMEs prepared from YPH499 cells expressing T.aureum D5 Elongase when Eicosapentaenoic acid was externally added to the medium

Fatty Acid YPH499/pESC-Ura/Elo(Ta) EPA-DPA 29.05.01 3
#1 #2 #3 PESC
SD SG SD SG SD SG SD SG
14:0' 3.11 2.53 2.13 2.29 2.55 2.48 2.07 2.1
15:0' 0.4 1.03 0.4 0.62 0.5 0.64 0.44 0.61
16:0' 28.27 33.13 31.43 32.95 32.65 30.58 35.02 29.71
16:1' 30.49 28.38 32.64 27.47 31 24.65 32.88 19.62
17:0' 0.11 0.22 0.07 0.17 0.11 0.11 0.14 0.19
18:0' 3.72 4.93 4.41 4.97 3.83 4.12 3.9 4.55
18:1' 19.57 16.5 23.92 17.62 17.79 14.9 20.95 11.32
19:0' 10.23 0.27 0.08 0.14 6.71 8.98 0.25 20.42
20:0' 0.31 0.59 0.22 0.37 0.46 0.34 0.26 0.3
20:5 (EPA) 3.78 11.14 4.71 11.51 4.4 11.75 4.09 11.2
22:5 (DPA) 0 1.3 0 1.89 0 1.44 0 0
Cloning of FAD4 from Thraustochytrium aureum
Genomic DNA of T.aureum (ATCC 34304) was used for the amplification of a nucleotide fragment with gene-specific primers. The resultant nucleotide sequence of 1548bp as given in the SEQID 10 was cloned into pGEM-T easy vector and sequenced for the complete sequence confirmation. The nucleotide sequence was released from the pGEM-T easy vector and the fragment was cloned into pESC-Ura vector. The construct as shown in FIG 9 was co-transformed with the construct in FIG 7. Transformants were selected on SD Ura-Leu-media. Clones obtained were subjected to proof of function experiment. Both the nucleotide sequences were cloned under the control of galactose inducible promoter. Cells were induced with galactose in presence of EPA in the medium. 0.1% tergitol was used in the experiment to facilitate the entry of fatty acid EPA into the medium. Total lipids and FAMEs were extracted from the cells subjected to proof of function experiment and analysed by GC-MS.
20

The spectrum obtained for the induced samples (FIG 26) of the clones showed two distinct peaks that were not present in controls of the clones and also vector control samples. Retention times for these peaks were same as that of Docosapentaenoic acid (DPA) and Docosahexaenoic acid (DHA). Library search for the mass spectrum of these two peaks showed a high percentage of similarity to the above said fatty acids (Table 21). This confirms the activities of both the nucleotide sequences introduced into the yeast host.
Table 21 Gas chromatography(GC)/Mass spectroscopy (MS) analysis of FAMEs prepared from YPH499 cells expressing XJaevls D5 Elongase and T.aureum D4 when Eicosapentaenoic acid was externally added to the medium

Fatty Acid YPH499/pESC-Leu/Elo(XI)+pESC-Ura/D4(Ta)cotransformation 22.10.07 (EPA-DPA-DHA)
#3 #4 #5 pESG
SD SG SD SG SD SG SD SG
14:0' 0.75 0.96 0.68 0.84 0.71 0.61 0.7 0.71
15:0' 0.3 0.25 0.19 0.82 0.28 0.28 0.29 0.28
16:0' 33.43 35.75 36.66 45.54 33.88 35.76 34.3 36.22
16:1' 29.88 32.95 29.82 19.8 28.11 30.71 28.93 27.65
17:0' 0.07 0.03 0.03 0.52 0.07 0.02 0.05 0.03
18:0" 4.87 5.06 4.99 4.54 5.25 5.33 5.09 6.34
18:1' 27.46 21.2 26.32 21 28.27 23.54 27.15 25.19
20:0' 0.12 0.25 0.11 0.23 0.05 0.21 0.04 0.06
20:5 (EPA) 3.12 3.49 1.2 6.55 3.37 3.47 3.46 3.5
22:5 (DPA) 0 0.04 0 0.07 0 0.04 0 0
22:6 (DHA) 0 0.04 0 0.08 0 0.04 0 0
Cloning of SEQ ID 8 or SEQ ID 9 with SEQ ID 10 in the same construct:
SEQ ID 8 or SEQ ID 9 was cloned into pESCUra vector into the multiple cloning site I directionally between EcoRI and Clal sites. Into the same construct SEQIDIO was cloned in the multiple cloning site II between BamHI and KpnI. The construct as depicted in the FIG 10 was used for the transformation of yeast host containing first five genes of the DHA biosynthetic pathway.
21


We Claim:
1 .A method for the production of Docosahexaenoic acid comprising: (a) providing a host cell comprising: (i) an isolated nucleotide molecule encoding a DELTA-12 desaturase polypeptide sequence as set
forth in SEQ ID NO: 1 or SEQ ID No:2; (ii) an isolated nucleotide molecule encoding a DELTA-6 desaturase polypeptide sequence as set
forth in SEQ ID NO: 3; and (iii) an isolated nucleotide molecule encoding a cl8/c20 elongase polypeptide sequence as set forth in
SEQ ID NO: 4; (iv) an isolated nucleotide molecule encoding a DELTA - 5 desaturase polypeptide sequence as set
forth in SEQ ID N0:5 or SEQ ID NO: 6 ; (v) an isolated nucleotide molecule encoding a a-3 desaturase polypeptide sequence as set forth in
SEQ ID NO: 7; (vi) an isolated nucleotide molecule encoding a elongase polypeptide sequence as set forth in SEQ ID
NO: 8 or 9
I
(vii) an isolated nucleotide molecule encoding a Delta-4 desaturase polypeptide sequence as set forth i
in SEQ ID NO: 10
(b) growing the host cell of step (a) under conditions wherein the nucleic acid molecule encoding the DELTA
12 and DELTA 6 desaturase, Delta -6 elongase Delta 5 desaturase, co-3 desaturase , Delta-5 elongase and i
Delta -4 Desaturase polypeptides are expressed and the Oleic acid is converted to Docosahexaenoic acid; and j
(c) optionally recovering the Docosahexaenoic acid of step (b). I
I
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 and SEQID No:2.
3. An isolated nucleotide molecule encoding a DELTA 6 desaturase enzyme, as described in Claim 1, having j sequence as set forth in SEQ ID NO: 3. j
4. An isolated nucleotide molecule encoding a C18/C20 elongase enzyme, as described in Claim 1, having I sequence as set forth in SEQ ID NO: 4. I
5. An isolated nucleotide molecule encoding a DELTA 5 desaturase enzyme, as described in Claim 1, having j sequence as set forth in SEQ ID N0:5 and SEQID No:6.
6. An isolated nucleotide molecule encoding a (o-3 desaturase enzyme, as described in Claim 1, having I sequence as set forth in SEQ ID NO: 7. I
7. An isolated nucleotide molecule encoding a Delta-5 elongase enzyme, as described in Claim 1, having I sequence as set forth in SEQ ID NO: 8 and SEQID No:9
8. An isolated nucleotide molecule encoding a Delta 4 desaturase enzyme, as described in Claim 1, having i sequence as set forth in SEQ ID NO: 10. |

9. A chimeric gene comprising the isolated nucleic acid molecule of claim 2, operably linked to suitable
regulatory sequences.
10. A chimeric gene as claimed in claim 9 comprising the isolated nucleic acid molecule of claim 3 operably
linked to suitable regulatory sequences.
11. A chimeric gene as claimed in 10 comprising the isolated nucleic acid molecule of claim 4 operably linked to suitable regulatory sequences.
12. A chimeric gene as claimed in 11 comprising the isolated nucleic acid molecule of claim 5 operably linked to suitable regulatory sequences.
13. A chimeric gene as claimed in 12 comprising the isolated nucleic acid molecule of claim 6 operably linked to suitable regulatory sequences

14. A chimeric gene as claimed in 13 comprising the isolated nucleic acid molecule of claim 7 operably linked to suitable regulatory sequences
15. A chimeric gene as claimed in 14 comprising the isolated nucleic acid molecule of claim 8 operably linked to suitable regulatory sequences
16. An isolated transformed host cell comprising the isolated nucleic acid molecules of claim 15 is an
oleaginous yeast, such as but not limited to Saccharomyces cervisiae.