# Patent

## Title
Delta-9 desaturase used in the biosynthesis of polyunsaturated fatty acids

## Abstract

The present invention provides novel fatty acid desaturases genes used for synthesis of polyunsaturated fatty acids, especially delta-9 desaturases (FADS9-I). The present invention also provides nucleic acid sequence coding the above-described desaturases, expression vector of the above-described desaturases and recombinant microorganism expressing above-described desaturases.

## Background
FIELD OF INVENTION
The present invention relates to the field of microbial manufacturing of monounsaturated fatty acids (MUFAs), specifically to the fatty acid desaturases in the synthesis process of unsaturated fatty acids.
BACKGROUND OF THE INVENTION
Lipids are first synthesized as saturated fatty acids and double bonds are introduced post-synthetically by oxygen-dependent enzymes known as fatty acid desaturases, in a process that is initiated by abstraction of hydrogen from a methylene group. Fatty acid desaturases are divided into soluble and integral membrane classes, which may have been evolved independently (Shanklin J, Somerville C., “Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs”, Proc Natl Acad Sci USA 1991; 88:2510-4). The acyl-ACP desaturases are soluble enzymes found in the plastids of higher plants, whereas the more widespread class of integral membrane acyl-CoA desaturases is found in endomembrane systems in prokaryotes and eukaryotes (Shanklin J, Cahoon E B., “Desaturation and Related Modifications of Fatty Acids1”, Annu Rev Plant Physiol Plant Mol Biol 1998; 49:611-41). Fatty acid desaturases in each class are closely related homologs based on their amino acid sequences, and yet perform highly regio- and stereo-selective reactions on long-chain fatty acids composed of essentially equivalent methylene chains that lack distinguishing landmarks close to the site of desaturation. As pointed out by Nobel Laureate Dr. Konrad Bloch, this region- and stereo-specific removal of hydrogen “would seem to approach the limits of the discriminatory power of enzymes” (Bloch K., “Enzymatic synthesis of monounsaturated fatty acids”, Accounts of Chemical Research 1969; 2:193-202).
The membrane class of desaturases consists of enzymes with c5, c6, c9, c12 or ω3 regio-selectivity. Mammalian cells possess c5, c6 and c9, but lack c12 and ω3 desaturases (Berquin I M, Edwards I J, Kridel S J, Chen Y Q, “Polyunsaturated fatty acid metabolism in prostate cancer”, Cancer Metastasis Rev 2011; 30:295-309, and Chen Y Q, Edwards I J, Kridel S J, Thornburg T, Berquin I M., “Dietary fat-gene interactions in cancer”, Cancer Metastasis Rev 2007; 26:535-51). Mortierella alpina belongs to the subphylum of Mucoromycotina (Hibbett D S, Binder M, Bischoff J F, et al., “A higher-level phylogenetic classification of the Fungi”, Mycol Res 2007; 111:509-47). It can produce lipids up to 50% of its dry weight. We have recently characterized M. alpina genome (Wang L, Chen W, Feng Y, et al., “Genome Characterization of the Oleaginous Fungus Mortierella alpine”, PLoS One 2011; 6:e28319) which encodes one c5, two c6, three c9, one c12 and one ω3 desaturase. Therefore, M. alpina has all known regio-selective groups of membrane desaturases.
We have expressed M. alpina c9, c12 and ω3 desaturases (FADS9-I, FADS12 and FADS15) in the methylotrophic yeast Pichia pastoris, purified the recombinant proteins and determined their enzymatic activities.
DETAILED DESCRIPTION OF THE INVENTION
The applicant has identified a novel ω3 desaturase and a Δ9 desaturase, successfully expressed, purified and characterized their enzymatic activities. More specifically, the applicant designed primers of nucleotide aiming to three desaturases coding for Δ9-I Des, Δ12 Des and ω3 Des on the basis of whole-genome sequencing of M. alpina, the sequences of specific primers are listed in Table 1. M. alpina RNA was extracted and reverse transcribed to obtain cDNA. The amplified three sequences by PCR with three pairs of primer of FF1 and FR1, FF2 and FR2, FF3 and FR3 to PCR were inserted into pET19b (PP) and sequenced, and then subcloned into pPinkα-HC, an expression vector in Pichia pastoris. Expression vectors were linearized and transformed into PichiaPink strain 2. The recombinant strains thus obtained express the above-mentioned three desaturases. The enzyme activity analysis showed that the purified recombinant desaturases were functional.
The present invention provides genes coding for M. alpina ω3 desaturase (FADS15) and Δ9 desaturase (FADS9-I), whose nucleic acid sequences are shown as SEQ ID NO:1 and SEQ ID NO:3, respectively.
The present invention also provides expression vectors respectively containing SEQ ID NO:1 and SEQ ID NO:3 which can respectively express M. alpina ω3 desaturase (FADS15) and Δ9 desaturase (FADS9-I). Preferably, the said expression vector is Pichia pastoris expression vector.
The present invention also provides a recombinant microorganism which can respectively express ω3 desaturase (FADS15) and M. alpina Δ9 desaturase (FADS9-I). Preferably, the said recombinant microorganism is recombinant Pichia pastoris PichiaPink strain 2, which contains Pichia pastoris expression vector carrying SEQ ID NO:1 or SEQ ID NO:3.
The present invention successfully expresses and purifies the novel membrane ω3 desaturase (FADS15) and Δ9 desaturase (FADS9-I), which play a key role in the polyunsaturated fatty acid biosynthetic pathway, and whose amino acid sequences are shown as SEQ ID NO:2 and SEQ ID NO:4. Furthermore, the present invention verifies the enzyme activity of the above two kinds of membrane desaturases.
The present invention apply the above-mentioned novel membrane desaturase Δ9 desaturase (FADS9-I) in the unsaturated fatty acid synthesis, converting fatty acid C14 into C14:1Δ9, C16 into C16:1Δ9, C18 and C18:1Δ9, C20 into C20:1Δ9, which can be utilized as food supplementation to prevent human disorders, wherein said human disorders consisting of cancer, cardiovascular disease and inflammation.







TABLE 1







Primers′ sequence table and its restriction enzyme cutting site:









Name
Sequence
Targeted vector





FF1(SEQ ID: 5)
atatCATATGATGGCCCCCCCTCACGTTGTCGACGAGCA(Nde I)
pET19b-FADS15


 


FF2(SEQ ID: 6)
atatATTAATATGGCACCTCCCAACACTATTGATGCCGG(Ase I)
pET19b-FADS12


 


FF3(SEQ ID: 7)
atatCATATGATGGCAACTCCTCTTCCCCCCTCCTTTGT(Nde I)
pET19b-FADS9-I


 


FR1(SEQ ID: 8)
atatGGATCCTAATGCTTGTAGAACACTACGTC(BamH I)
pET19b-FADS15


 


FR2(SEQ ID: 9)
atatGGATCCTTACTTCTTGAAAAAGACCACGTC(BamH I)
pET19b-FADS12


 


FR3(SEQ ID: 10)
atatAGATCTTTATTCGGCCTTGACGTGGTCAGT(Bgl II)
pET19b-FADS9-I


 


SF1(SEQ ID: 11)
atatatTGCGCACATCATCATCATCATCATCAT(Fsp I)
pPink-FADS


 


SR1(SEQ ID: 12)
atatGAATTCATATTTAAATTAATGCTTGTAGAACACTACGTC(Eco
pPink-FADS15



RI, Swa I)



 


SR2(SEQ ID: 13)
atatatGGTACCTTACTTCTTGAAAAAGACCACGTC(Kpn I)
pPink-FADS12


 


SR3(SEQ ID: 14)
atatatGGTACCTTATTCGGCCTTGACGTGGTCAGT(Kpn I)
pPink-FADS9-I












DESCRIPTION OF THE ATTACHED DRAWINGS
FIG. 1: Diagram of the cloning strategy for desaturase expression vectors. FADScoding sequences were PCR amplified using primers listed in Table 1. PCR fragment were digested with indicated restriction enzymes, column purified and inserted into the pET-19b(PP) vector linearized with corresponding restriction enzymes. The FADScoding sequence plus His tag and Precision protease recognition sequence were PCR amplified and inserted into the pPinkalpha-HC vector. TRP2: TRP2 gene, AmpR: ampicillin resistance gene, pUC ori: oriental promoter of pUC, PAOX1: 5′AOX1 promoter region, α-factor: α-mating factor secretion signal, CYC1 TT: CCY1 transcription termination region, PADE2 HC: high-copy ADE2 promoter region, ADE2: ADE2 open reading frame.
FIG. 2A-F: the sequencing results of the clone sequence of ω3 desaturase (FADS15), in which FADS15 nucleotide sequence from M. alpina ATCC#32222 shows 93.1% identity with AB182163 from M. alpina 1s-4.
FIG. 3A-E: the sequencing results of the clone sequence of Δ12 desaturase (FADS12), in which FADS12 nucleotide sequence from M. alpina ATCC#32222 shows 99.9% identity with AF110509 from M. alpina 1s-4.
FIG. 4A-F: the sequencing results of the clone sequence of Δ9 desaturase (FADS9-I), in which FADS9-I nucleotide sequence from M. alpina ATCC#32222 shows 98.4% identity with AF085500 from M. alpina 1s-4.
FIG. 5A: Growth curve of the recombinant P. pastoris measured by cell density, wet weight and total protein concentration.
FIG. 5B: Kinetics of recombinant protein induction. Desaturase expression was determined by Western blotting using anti-His tag antibody. The normalized level of highest expression was set at one arbitrary unit. Three independent experiments were performed and bars represent standard deviations.
FIG. 5C: Quantification of the recombinant desaturase proteins by Coomassie blue staining after SDS-PAGE. Known concentrations of BSA were used as quantification standard.
FIG. 5D: InVisionTM His-tag In-Gel Stain of recombinant FADSproteins. The arrow head indicates the addition of methanol for induction of recombinant protein expression. The triangles indicate the expressed recombinant proteins. M: protein marker, Cont: negative control which was PichiaPinkTM harboring pPinkα-HC, 15: FADS15, 12: FADS12, 9-I: FADS9-I.
FIG. 6A: Fractionation of recombinant desaturases. InVisionTM His-tag In-Gel Staining after SDS-PAGE analysis of FADS9-I membrane (top panel) and supernatant fraction (bottom panel), using different speeds of centrifugation. The triangles indicate the recombinant FADS9-I.
FIG. 6B: Coomassie blue Staining and InVisionTM His-tag In-Gel Staining of recombinant desaturases after fractionation. T: total protein after grinded by glass beads, D: debris after centrifugation at 500 g for 10 min, M: membrane fraction after centrifugation at 10,000 g for 10 min, S: supernatant after the centrifugation. The triangles indicate the recombinant desaturase proteins.
FIG. 7A: Solubilization of recombinant desaturases. Membrane fractions were suspended in 1% concentrations of various detergents and incubated at 4° C. for 2 hr. Proteins were visualized by InVisionTM His-tag In-Gel Staining (upper panel) and Western blot (lower panel). T20: Tween-20, T80: Tween-80, N40: NP-40, DDM: n-Dodecyl-β-D-maltoside, F12: Fos-Choline 12, F16: Fos-Choline 16, S: supernatant, P: pellet.
FIG. 7B: Membrane fractions of recombinant FADS9-I were suspended in 1% Fos-Choline 16 and incubated at 4° C. for various time (0, 0.5, 1.5, 3, 12 hr). Aliquots were analyzed by InVisionTM His-tag In-Gel Staining. S: supernatant, P: pellet.
FIG. 7C: One-step purification using His Mag Sepharose Ni beads under the high yield (upper panel) and high stringency conditions (lower panel). Proteins were analyzed by SDS-PAGE and Coomassie blue staining. M: protein marker, S: supernatant, F: flow through, E: eluate.



SPECIFIC EMBODIMENTS
Example 1
Mortierella alpina Culture
Mortierella alpina (#32222, American Type Culture Collection, Manassas, Va., USA) was inoculated on Potato Dextrose Agar (PDA) plates (BD DifcoTM Potato Dextrose Agar cat#213400) and incubated for 20-30 days at 25° C. 5 mL broth (20 g/L Glucose, 5 g/L Bacto yeast extract BD Biosciences cat#212750, 1 g/L KH2PO4, 0.25 g/L MgSO4, 10 g/L KNO3) were added to three plates. Spores were gently scraped off the surface with a sterile loop, and then filtrated through a 40 micron cell strainer. Spores were concentrated by centrifuging at 12,000×g for 15 min, suspended in a small volume of broth, enumerated using a hemocytometer, and kept at −80° C. in 30% glycerol at a density of approximately 107 spores/mL. Alternatively, 3 mL of unconcentrated spore suspension were directly added into 45 mL broth without KNO3 in a 250-mL flask covered with 8 layers of cheese cloth, and shaken at 200 rpm, 25° C. for 5 days. Cultures were blended using a Braun hand blender for 5 sec/pulse, 8 pulses, then 0.3 g wet mycelia were inoculated into 45 mL broth without KNO3 in a 250-ml flask and shaken at 200 rpm, 25° C. for 24 h. The above step was repeated once, by which time the whole fungal culture was in proliferative phase and ready for experiments. Mycelia were collected by filtration and weighed. Samples were snap-frozen in liquid nitrogen, pulverized and kept at −80° C. for RNA extraction.
Example 2
Expression Vector Construction
M. alpina RNA extraction was performed using Trizol Reagent (Invitrogen, CA) according to the manufacturer's instructions. Total RNA was reverse transcribed with SuperScript® III First-Strand Synthesis SuperMix (Invitrogen) following the manufacturer's instructions. Using both C- and N-terminal sequences as primers (Table 1), desaturase coding sequences were PCR amplified as follows: denaturation at 95° C. for 30 sec, annealing at 55° C. for 45 sec and extension at 72° C. for 1 min for 25 cycles. The amplified products were cloned into a modified pET19 vector (Novagen) derivative containing a PreScission protease cleavage site (GE Healthcare) between the multiple cloning site and N-terminal His tag (Jonsson T J, Johnson L C, Lowther W T (2009) Protein engineering of the quaternary sulfiredoxin.peroxiredoxin enzyme.substrate complex reveals the molecular basis for cysteine sulfinic acid phosphorylation. J Biol Chem 284: 33305-33310.) to construct pET19b-FADS15, pET19b-FADS12 and pET19b-FADS94). The desaturase genes, including the His-Tag and PreScission protease cleavage site, were then PCR amplified using primers SF1 and SR1-SR3 (Table 1). The PCR conditions used were the same as the first step for cDNAs. The PCR fragments were then purified and inserted into pPinkα-HC to generate the expression vectors pPinkα-HC-FADS15, pPinkα-HC-FADS12 and pPinkα-HC-FADS9-I. The presence of the inserts in the plasmids was confirmed by restriction digestion analysis and sequencing. The strategy used for constructing desaturase expression vectors is shown in FIG. 1. Sequencing results from the amplified fragment of these desaturases are in FIG. 2-4. The FADS12 and FADS9-I genes from M. alpina ATCC#32222 are 99.9% and 98.4% identical, respectively, to the corresponding genes from M. alpina 1s-4. The FADS12 and FADS9-I proteins from M. alpina ATCC#32222 are 100% and 99.6% identical, respectively, to these proteins from M. alpina 1s-4. The high similarity of FADS12 and FADS9-I genes between two strains indicates that these genes are highly conserved in M. alpina. Interestingly, the FADS15 gene is much less conserved at both DNA (93.1% identity) and protein (97.9%) levels.
Example 3
Protein Expression
Desaturase expression vectors and pPinkα-HC (negative control vector) were linearized with restriction enzyme Spe I and transformed into P. pastoris strains (PichiaPink strain 1, 2, 3 and 4) using the MicroPulser Electroporator (Bio-Rad Laboratories, Hercules, Calif.) according to the User Manual of PichiaPink Expression System (Invitrogen). P. pastoris were incubated with YPDS media (YPD with 1 M sorbitol) in the Gene Pulser Cuvettes at 28° C. for 2 hr without shaking, spread onto PAD (Pichia Adenine Dropout) agar selection plates, and then incubated at 28° C. for 4 days until distinct colonies were formed. Eight white colonies for each transformation were picked and plasmid integration in the yeast genome was confirmed by PCR.
Isolated clones were individually inoculated into 10 mL of BMGY medium (Buffered Glycerol-complex Medium, 1% yeast extract; 2% peptone; 100 mM potassium phosphate, pH 6.0; 1.34% YNB-Yeast Nitrogen Base; 0.0004% biotin; 1% glycerol) in 50 mL conical tubes. The cells were grown for 48 hr at 28° C. with vigorous shaking at 250 rpm. Then, the cultures were centrifuged at 1,500 g for 5 mM at room temperature, the cell pellets were resuspended in 2 mL of BMMY medium (Buffered Methanol-complex Medium, 1% yeast extract; 2% peptone; 100 mM potassium phosphate, pH 6.0; 1.34% YNB; 0.0004% biotin; 0.5% methanol) and cultured at 28° C. with shaking at 250 rpm to induce the expression. After continuous cultivation for 72 hr with daily addition of 0.5% methanol, cells were harvested by centrifuging for 10 mM at 1500 g. Supernatant was transferred to a separate tube and both the supernatant and cell pellet were stored at −80° C. until ready for assay. Supernatants and cell pellets were analyzed for protein expression by SDS-PAGE Coomassie blue staining and Western blot. Our data showed that PichiaPink strain 2(ade2, pep4) supported the highest level of expression for FADS15, 12 and 9-I.
Example 4
Expression Condition Optimization and Protein Analysis
Individual colonies of P. pastoris-FADS15, FADS12 and FADS9-I were inoculated into 10 mL of BMGY medium in 50 mL conical tubes and cultured for 48 hr at 28° C. at shaking speed of 250 rpm. Then, 2.5 mL of culture were inoculated into 50 mL of BMGY medium in 250-mL volume shaker flasks and grown at 28° C. for 24 hr at 250 rpm. The cells were collected by centrifugation at 1500 g for 10 mM, and resuspended in 10 mL induction medium (BMMY medium with 0.5% methanol) in a 100-mL shaker flask. The induction of protein expression was performed for 96 hr at 28° C. with 250 rpm agitation and daily addition of 0.5% methanol. Samples were collected at 0, 6, 24, 48, 72 and 96 hr for measuring cell density at OD600, wet cell weight and total protein concentration, and for Western blot analysis of desaturase expression levels.
The cell pellets and supernatants were collected by centrifuging 100 μL cell culture at 1500 g for 10 mM Cell pellets were resuspended in 100 μL lysis buffer (20 mM Tris.Cl pH7.9, 1 mM EDTA, 5% Glycerol) with an equal volume of 0.5 mm Glass Beads (Biospec products, Inc.), and vortexed for 10 mM at 4° C. Cell lysates were mixed with 4×SDS sample buffer and heated for 5 mM at 95° C. About 5 μl sample was loaded onto Mini-Protein Precast Gels (4-15%, Bio-Rad Laboratories, Cat #456-1086), and ran for 40 mM at 150 V. Then, the SDS-PAGE gels were used for Coomassie blue stain, Invision His-Tag in-gel stain (Invitrogen) or Western blot.
For Western blot analysis, protein gels were transferred onto a nitrocellulose transfer membrane (Schleicher & Schuell GmbH, Germany) by electroblotting (100 V, 2 hr) using Mini Trans-Blot electrophoretic transfer cell (Bio-Rad Laboratories). The membrane was blocked with 3% BSA in TBST (150 mM NaCl, 10 mM Tris-Cl pH 7.5, 0.05% Tween20), and probed with mouse Penta.His antibody (Invitrogen) followed by HRP-conjugated goat anti-mouse IgG (GE Healthcare). Blots were then incubated with enhanced chemiluminescence reagent (ECL, GE healthcare) and analyzed using Fluorchem E (Cell Biosciences, Inc.).
The total protein concentration was determined with Pierce BCA protein assay kit (Thermo Scientific). The quantification of target protein on Coomassie blue stained gel was performed using known concentrations of BSA as standard, and analyzed with the AlphaView SA software (Cell Biosciences, Inc.).
To determine potential toxicity of recombinant proteins, we first examined cell growth density, weight and total protein synthesis of the PichiaPink pPinkα-HC-FADSclones. The recombinant PichiaPink pPinkα-HC-FADScells had growth characteristics similar to the control (FIG. 5A). A time course experiment showed that desaturase expression was detectable after 24 hr induction with 0.5% methanol and remained high for at least 72 hr post-induction (FIG. 5B). There were no significant differences in protein expression when cells were induced at different temperatures (16° C., 22° C., 28° C.) or with a different concentration of methanol (0.5%, 1%). Therefore, we used an optimized procedure as described in the Materials and Methods for the expression of recombinant desaturase. Under this condition, expression levels of recombinant desaturase proteins reached approximately 130 mg/L of culture for FADS15, 110 mg/L for FADS12 and 350 mg/L for FADS9-I (FIG. 5C).
Example 5
Protein Purification
All purification procedures were performed at 4° C. Cells harvested from 800 μL of culture were suspended in 800 μL of lysis buffer. After addition of 0.5 mm glass beads to the cell suspension, P. pastoris cells were disrupted by vortexing at 4° C. for 10 min. Cell lysis efficiency was usually more than 95% evaluated using a light microscope. Intact cells and cell debris were removed from the membrane suspension by low speed centrifugation (500 g, 10 min at 4° C.). Then various centrifugation speeds and time (1,000 g for 10 min; 10,000 g for 10 min; 10,000 g for 20 min; 20,000 g for 10 min; 20,000 g for 20 min) were used to determine the best centrifugation conditions for collecting the membrane fraction.
Fractions containing recombinant desaturases were solubilized in buffer, containing 20 mM Tris.Cl, pH 7.9, 500 mM NaCl, 10% glycerol, 0.1 mM EDTA, and different concentrations (0.5%, 1%, 2%) of various detergents (Tween 20, Tween 80, Nonidet P-40, DDM, Fos-Choline 12, Fos-Choline 16) at 4° C. for different times (0.5, 1, 1.5, 2 hr and overnight). The insoluble materials were removed by centrifugation at 25,000 g for 30 min at 4° C.
Optimized culture and protein solubilization conditions were used for the subsequent purification process. His Mag SepharoseTM Ni affinity beads (GE Healthcare) were washed with binding buffer (20 mM Tris.Cl, pH 7.9, 500 mM NaCl, 10% glycerol, 0.1 mM EDTA, 0.5% Fos-Choline 16, 5 or 20 mM imidazole) and added to the solubilized fractions after detergent incubation. The bead-protein sample mixtures were incubated for 45 min at 4° C. with end-over-end mixing. After washing three times with binding buffer containing 5 mM or 20 mM imidazole, desaturase enzymes were eluted with elution buffer (20 mM Tris.Cl, pH 7.9, 500 mM NaCl, 10% glycerol, 0.1 mM EDTA, 0.5% Fos-Choline 16, 500 mM imidazole). The purified FADS15, FADS12 and FADS9-I proteins were stored at −80° C. in aliquots. The quantity and quality of these purified enzymes were analyzed by SDS-PAGE and desaturase activity assay.
In order to solubilize and purify the recombinant desaturases from cell membrane for in vitro enzymatic activity, we first tested conditions to enrich the cell membrane containing recombinant FADS15, FADS12 and FADS9-I. Different centrifugation speeds and times were examined for the separation of the membrane fractions containing target proteins. Efficient recovery of each recombinant desaturase produced in P. pastoris was achieved by centrifuging the cell homogenates at 500 g for 10 min to remove cell debris, then at 10,000 g for 10 min to collect membrane fractions (FIG. 6).
Solubilization of membrane proteins requires the presence of detergents. Therefore, we tested the conditions for solubilization of the recombinant FADS15, FADS12 and FADS9-I from enriched cell membrane fractions using a panel of detergents: Tween-20, Tween-80, NP-40, n-Dodecyl-β-D-maltoside (DDM), Fos-Choline 12 or Fos-Choline 16. After treatment with 1% (w/v) of Fos-Choline 12 or Fos-Choline 16, FADS9-I and FADS12 were totally solubilized, and approximately 50% and 80% of FADS15 was solubilized with Fos-Choline 12 and Fos-Choline 16, respectively (FIG. 7A). Tween-20, Tween-80, NP-40 and DDM had little effect on extracting these desaturase enzymes from the membrane. In addition, we noticed that FADS9-I protein degradation occurred during protein solubilization. This phenomenon was visible for proteins solubilized by both Fos-Choline 12 and 16. Thus, we investigated detergent incubation time during solubilization to optimize for the least protein degradation. Our results showed that the solubilization of FADS9-I protein reached its maximum level after incubation with detergent for 1.5 hr. Degradation of desaturase protein increased after more than 3 hr of incubation (FIG. 7B). To maximize the ratio of intact vs. degraded proteins, we used 1.5 hr as our standard detergent incubation time for protein solubilization. We also compared the effect of detergent concentrations on protein solubilization efficiency and found that 0.5%, 1% or 2% of Fos-Choline 16 had similar effects. Taken together, our results indicate that all three recombinant desaturase enzymes can be solubilized efficiently from the cell membrane with 0.5% Fos-Choline 16 for 1.5 hr at 4° C.
Solubilized FADS15, FADS12 and FADS9-I were affinity-purified on His Mag Sepharose Ni beads (GE healthcare) with aims of high purity or high yield. High purity (>95%) was achieved after one step purification using the His Mag Sepharose Ni beads with high stringency wash before elution (FIG. 7C). High yield (2-fold higher than that in the high purity process) was achieved with low stringency wash. Yield and quantity of each desaturase enzyme are summarized in Table 2. Our estimated yields of desaturases with purity >95% are approximately 22.5 mg/L for FADS15, 12 mg/L for FADS12 and 188 mg/L for FADS9-I.







TABLE 2







Purification of M. alpina desaturases











FADS15 protein
FADS12 protein
FADS9-I protein


















Vol
Con.
Total
Yield
Con.
Total
Yield
Con.
Total
Yield


Process
(μL)
(mg/L)
(μg)
(%)
(mg/L)
(μg)
(%)
(mg/L)
(μg)
(%)




















Cell lysates
200
130.0
26.0
100.0
110.0
22.0
100.0
350.0
70.0
100.0


Centrifugal
200
112.0
22.4
86.2
82.0
16.0
72.7
254.0
50.8
72.6


collections


(500-10 kg)


Detergent
200
76.0
15.2
58.5
74.8
15.0
68.2
223.0
44.6
63.7


treatment


extracts


Ni-NTA
40
22.5
0.9
3.5
12.0
0.5
2.3
188.0
7.5
10.7


(20 mM ID)


Ni-NTA
100
10.5
1.1
4.2
7.5
0.8
3.6
185.0
18.5
26.4


(5 mM ID)





ID: imidazole






Example 6
Desaturase Activity Assay
Approximately 20 mg of P. pastoris cell pellets were collected and used for each lipid extraction with the method of Bligh and Dyer (Bligh E G, Dyer W J (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911-917). under acidified conditions with pentadecanoic acid and heneicosanoic acid added as internal standards. The solvent from the extract was removed under a stream of nitrogen. Lipids were saponified in 1 mL of freshly prepared 5% ethanolic potassium hydroxide at 60° C. for 1 hr under an argon atmosphere. After cooling, 1 mL of water was added to the samples and non-saponifiable lipids were extracted into 3 mL of hexane. The aqueous layer was acidified with 220 μL of 6 M hydrochloric acid and the fatty acids extracted into 3 mL of hexane. After removing the hexane in a stream of nitrogen, fatty acids were converted to methyl esters by first treating with 1 mL of 0.5 M methanolic sodium hydroxide at 100° C. for 5 min under argon followed by 1 mL of 14% methanolic boron trifluoride at 100° C. for 5 min under argon (Metcalfe L D, Schmitz A A, Pelka J R (1966) Rapid preparation of fatty acids esters from lipids for gas chromatographic analysis. Analytical Chemistry 38: 514-515). After cooling, the sample was mixed with 2 mL of hexane followed by 4 mL of saturated aqueous sodium chloride. After separating the phases, aliquots of the hexane layers were diluted 24-fold with hexane and then analyzed by GC/MS. One μL was injected in the splitless mode onto a 30 m×250 μm DB-WAXETR column (Agilent Technologies, Santa Clara, Calif.) with 0.25 μm film thickness. The temperature program was as follows: 100° C. for 2 mM, ramp to 200° C. at 16° C. per mM, hold for one mM, ramp to 220° C. at 4° C. per mM, hold one mM, ramp to 260° C. at 10° C. per mM, and hold for 11 mM Helium was the carrier gas at a constant flow of 1.5 mL/min. The mass spectrometer was operated in positive-ion electron impact mode with interface temperature 260° C., source temperature 200° C., and filament emission 250 μA. Spectra were acquired from m/z 50 to 450 with a scan time of 0.433 s. Lower-boiling fatty acid methyl esters were quantified using the pentadecanoic acid internal standard, whereas higher-boiling methyl esters were quantified using the heneicosanoic acid internal standard.
In Vivo Desaturase Activity Analysis:
Individual colonies of P. pastoris-FADS15, FADS12 and FADS9-I were cultured as described in the Recombinant protein expression section. Protein expression was induced for 72 hr with 0.5% methanol. Cell pellets were collected by centrifugation and stored at −80° C. for fatty acid analysis.
In Vitro Desaturase Activity Analysis:
20 μL of the purified protein was added to 200 μL of yeast EGY49 cell homogenate, prepared by breaking cells with 0.5 mm glass beads in lysis buffer (20 mM Tris-HCl pH7.9, 1 mM EDTA, 5% Glycerol). The enzyme reactions were performed at 28° C. for 3 h with shaking (250 rpm), and the assay mixture (220 μL) were stored at −80° C. for fatty acid analysis.
To determine the functional activity of the recombinant M. alpina desaturase in vivo, PichiaPink cells were cultured and induced to express desaturases. Fatty acid methyl esters (FAME) analysis of cell pellets showed that expression of recombinant desaturases in PichiaPink cells altered their fatty acid contents compared to the control. Table 3 shows the percentage increase of C16:1Δ9, C18:1Δ9, C18:2Δ9,12 and C18:2Δ9,12,15 compared to the negative control. The C16:1Δ9 and C18:1Δ9 were increased 40% and 20%, respectively, in PichiaPink cells expressing FADS9-I, suggesting that FAD9-I can insert the first double bond into both C16:0 and C18:0 with a preference for C16:0 as substrate. The C18:2Δ9,12 content was 27% higher in cells expressing FADS12, suggesting that FADS12 can desaturate C18:1Δ9 at the c12-position to produce C18:2Δ9,12. There was a 5% increase in C18:3Δ9,12,15 in cells expressing FADS15, suggesting that FADS15 can desaturate C18:2Δ9,12 at the c15-position to produce C18:3Δ9,12,15. These results suggest that the recombinant desaturases, FADS9-I, FADS12 and FADS15, were active in P. pastoris. 
We used yeast EGY49 cell homogenate for our in vitro assay of recombinant desaturase activity. Our results showed that purified recombinant FADS12 converted C18:1Δ9 to C18:21Δ9,12 in vitro, and C18:2Δ9,12 level was increased 116% compared to the control (Table 3). Activities of purified FADS9-I and FADS15 were relatively low in vitro.







TABLE 3







M. alpina desaturases in-vivo and in-vitro activities








In vivo
In vitro












FADS9-I
FADS12
FADS15
FADS9-I
FADS12
FADS15














C16:1Δ9
C18:1Δ9
C18:2Δ9,12
C18:3Δ9,12,15
C16:1Δ9
C18:1Δ9
C18:2Δ9,12
C18:3Δ9,12,15


(%a)
(%)
(%)
(%)
(%)
(%)
(%)
(%)





40 ± 6
20 ± 7
27 ± 4
5 ± 3
6 ± 8
7 ± 7
116 ± 40
8 ± 4










60 ± 7b

13 ± 8





aPercent increase over control;


bSum of two products
















SEQUENCE LISTING















SEQ ID NO: 1


atggcccccc ctcacgttgt cgacgagcaa gtacgacgca gaatcgtcgt cgaggacgag


 


atccagtcca agaagcagtt tgagcgcaac tatgtgccta tggactttac aatcaaggag


 


attcgagatg cgatcccagc ccacctcttc atccgtgata ccacaaagtc gatcctgcat


 


gtcgtcaagg atctggtcac tatcgccatc gttttttact gtgcaacctt catcgagact


 


ctgccctcgc tcgctctgag agttcctgcc tggatcacct actggatcat ccaaggaact


 


gtcatggtcg gcccctggat tctggcccac gagtgcggcc atggagcgtt ctcggacagc


 


aagacgatca acaccatctt tggatgggtc cttcactctg ctcttttggt gccctaccag


 


gcttgggcca tgtcgcattc caagcaccac aagggcactg gatccatgag caaggatgtc


 


gttttcatcc ctgccactcg atcctacaag ggccttcccc cactggagaa gcctgccgcg


 


gaagaggagg ttttggagca ggagcatcac caccatgaag agtccatctt tgctgagact


 


cccatctaca ctctcggagc gctttttttc gtcctgacct tgggatggcc cttgtacttg


 


atcatgaact tttctggaca cgaagcccct cactgggtca accacttcca gacggtcgcc


 


cctctgtatg agcctcacca gcgcaagaac attttctact ccaactgcgg cattgtcgct


 


atgggctcga tcctcactta cctctcgatg gtcttctcgc ccttgactgt gttcatgtac


 


tatggcatcc cctacctcgg agtcaatgct tggatcgtct gcatcaccta tctccagcac


 


accgatccca aggtgcctca tttccgtgat aacgagtgga acttccagcg cggtgctgcc


 


tgcactatcg accgatcctt cggtaccatt gtcaaccact tgcaccacca cattggtgac


 


tctcatcaat gtcatcatat gttctcgcag atgcccttct acaacgccgt tgaggctaca


 


aagcatctca aagccaagct tggcaagtac tacatatttg acgacactcc cattgccaag


 


gccctctacc gcaattggag agagtgcaaa ttcgtggagg acgagggaga cgtagtgttc


 


tacaagcatt aa


 


SEQ ID NO: 2


Met Ala Pro Pro His Val Val Asp Glu Gln Val Arg Arg Arg Ile Val


 


Val Glu Asp Glu Ile Gln Ser Lys Lys Gln Phe Glu Arg Asn Tyr Val


 


Pro Met Asp Phe Thr Ile Lys Glu Ile Arg Asp Ala Ile Pro Ala His


 


Leu Phe Ile Arg Asp Thr Thr Lys Ser Ile Leu His Val Val Lys Asp


 


Leu Val Thr Ile Ala Ile Val Phe Tyr Cys Ala Thr Phe Ile Glu Thr


 


Leu Pro Ser Leu Ala Leu Arg Val Pro Ala Trp Ile Thr Tyr Trp Ile


 


Ile Gln Gly Thr Val Met Val Gly Pro Trp Ile Leu Ala His Glu Cys


 


Gly His Gly Ala Phe Ser Asp Ser Lys Thr Ile Asn Thr Ile Phe Gly


 


Trp Val Leu His Ser Ala Leu Leu Val Pro Tyr Gln Ala Trp Ala Met


 


Ser His Ser Lys His His Lys Gly Thr Gly Ser Met Ser Lys Asp Val


 


Val Phe Ile Pro Ala Thr Arg Ser Tyr Lys Gly Leu Pro Pro Leu Glu


 


Lys Pro Ala Ala Glu Glu Glu Val Leu Glu Gln Glu His His His His


 


Glu Glu Ser Ile Phe Ala Glu Thr Pro Ile Tyr Thr Leu Gly Ala Leu


 


Phe Phe Val Leu Thr Leu Gly Trp Pro Leu Tyr Leu Ile Met Asn Phe


 


Ser Gly His Glu Ala Pro His Trp Val Asn His Phe Gln Thr Val Ala


 


Pro Leu Tyr Glu Pro His Gln Arg Lys Asn Ile Phe Tyr Ser Asn Cys


 


Gly Ile Val Ala Met Gly Ser Ile Leu Thr Tyr Leu Ser Met Val Phe


 


Ser Pro Leu Thr Val Phe Met Tyr Tyr Gly Ile Pro Tyr Leu Gly Val


 


Asn Ala Trp Ile Val Cys Ile Thr Tyr Leu Gln His Thr Asp Pro Lys


 


Val Pro His Phe Arg Asp Asn Glu Trp Asn Phe Gln Arg Gly Ala Ala


 


Cys Thr Ile Asp Arg Ser Phe Gly Thr Ile Val Asn His Leu His His


 


His Ile Gly Asp Ser His Gln Cys His His Met Phe Ser Gln Met Pro


 


Phe Tyr Asn Ala Val Glu Ala Thr Lys His Leu Lys Ala Lys Leu Gly


 


Lys Tyr Tyr Ile Phe Asp Asp Thr Pro Ile Ala Lys Ala Leu Tyr Arg


 


Asn Trp Arg Glu Cys Lys Phe Val Glu Asp Glu Gly Asp Val Val Phe


 


Tyr Lys His


 


SEQ ID NO: 3


atggcaactc ctcttccccc ctcctttgtc gtccctgcga cacagacgga aacccgcaga


 


gatcctctcc agcacgagga actgccccct ctcttccccg agaaaatcac catcttcaac


 


atctggagat atcttgacta caagcatgtt ctcggtctgg ggctgacgcc tttgatcgct


 


ctctatggtc tcttgacgac cgagatccag acgaagacac tgatctggtc catcatctac


 


tattatgcta cgggacttgg catcacagca ggttaccatc gactctgggc ccatcgtgct


 


tacaacgcag gaccagccat gagcttcgtg ctcgcactgc tcggcgccgg tgcagttgaa


 


ggatctatca agtggtggtc ccgcggccac cgtgctcacc accgctggac tgacaccgag


 


aaggacccct acagcgctca ccgaggactc ttcttctcgc acattggctg gatgttgatc


 


aagcgccctg gatggaagat tggccatgcc gatgttgacg acctcaacaa gagcaaactc


 


gttcagtggc agcacaagaa ctaccttcct cttgttctta tcatgggtgt cgtcttcccc


 


acggttgttg ctggactcgg ctggggcgat tggcgtggag gctacttcta tgctgctatc


 


ctccgtcttg tctttgttca ccacgccacc ttctgtgtca actccctggc ccattggctc


 


ggtgatggac cctttgatga ccgccactct ccccgcgacc acttcatcac tgcctttgtc


 


actttgggag agggctacca caactttcat caccaattcc cccaggacta ccgcaacgct


 


atccgttttt accagtacga tcctaccaag tgggtcatcg ccctctgtgc tttctttggc


 


ctcgctacgc acctcaagac cttccctgag aatgaagtcc gcaagggtca gctccagatg


 


attgagaagc gtgtcctgga gaagaagacc aagctccagt ggggcactcc cattgccgat


 


ctgcccattc tgagctttga ggacttccag catgcttgca aaaacgacaa caagaagtgg


 


atcctcttgg agggtgtcgt ttacgacgtt gccgacttta tgaccgagca ccctggtggt


 


gagaagtacc tcaagatggg cgtcggcaag gacatgaccg cagctttcaa cggcggtatg


 


tacgatcaca gcaatgccgc ccgcaacctg ctgagcttga tgcgcgtcgc cgtcgttgag


 


tatggtggtg aggtggaggc acagaagaag aacccttcga tgcccatcta cggcactgac


 


cacgtcaagg ccgaataa


 


SEQ ID NO: 4


Met Ala Thr Pro Leu Pro Pro Ser Phe Val Val Pro Ala Thr Gln Thr


 


Glu Thr Arg Arg Asp Pro Leu Gln His Glu Glu Leu Pro Pro Leu Phe


 


Pro Glu Lys Ile Thr Val Tyr Asn Ile Trp Arg Tyr Leu Asp Tyr Lys


 


His Val Phe Gly Leu Gly Leu Thr Pro Leu Ile Ala Leu Tyr Gly Leu


 


Leu Thr Thr Glu Ile Gln Thr Lys Thr Leu Ile Trp Ser Ile Ile Tyr


 


Tyr Tyr Ala Thr Gly Leu Gly Ile Thr Ala Gly Tyr His Arg Leu Trp


 


Ala His Arg Ala Tyr Asn Ala Gly Pro Ala Met Ser Phe Val Leu Ala


 


Leu Leu Gly Ala Gly Ala Val Glu Gly Ser Ile Lys Trp Trp Ser Arg


 


Gly His Arg Ala His His Arg Trp Thr Asp Thr Glu Lys Asp Pro Tyr


 


Ser Ala His Arg Gly Leu Phe Phe Ser His Ile Gly Trp Met Leu Ile


 


Lys Arg Pro Gly Trp Lys Ile Gly His Ala Asp Val Asp Asp Leu Asn


 


Lys Ser Lys Leu Val Gln Trp Gln His Lys Asn Tyr Leu Pro Leu Val


 


Leu Ile Met Gly Val Val Phe Pro Thr Val Val Ala Gly Leu Gly Trp


 


Gly Asp Trp Arg Gly Gly Tyr Phe Phe Ala Ala Ile Leu Arg Leu Val


 


Phe Val His His Ala Thr Phe Cys Val Asn Ser Leu Ala His Trp Leu


 


Gly Asp Gly Pro Phe Asp Asp Arg His Ser Pro Arg Asp His Phe Ile


 


Thr Ala Phe Val Thr Leu Gly Glu Gly Tyr His Asn Phe His His Gln


 


Phe Pro Gln Asp Tyr Arg Asn Ala Ile Arg Phe Tyr Gln Tyr Asp Pro


 


Thr Lys Trp Val Ile Ala Leu Cys Ala Phe Phe Gly Leu Ala Thr His


 


Leu Lys Thr Phe Pro Glu Asn Glu Val Arg Lys Gly Gln Leu Gln Met


 


Ile Glu Lys Arg Val Leu Glu Lys Lys Thr Lys Leu Gln Trp Gly Thr


 


Pro Ile Ala Asp Leu Pro Ile Leu Ser Phe Glu Asp Phe Gln His Ala


 


Cys Lys Asn Asp Asn Lys Lys Trp Ile Leu Leu Glu Gly Val Val Tyr


 


Asp Val Ala Asp Phe Met Thr Glu His Pro Gly Gly Glu Lys Tyr Ile


 


Lys Met Gly Val Gly Lys Asp Met Thr Ala Ala Phe Asn Gly Gly Met


 


Tyr Asp His Ser Asn Ala Ala Arg Asn Leu Leu Ser Leu Met Arg Val


 


Ala Val Val Glu Phe Gly Gly Glu Val Glu Ala Gln Lys Lys Asn Pro


 


Ser Met Pro Ile Tyr Gly Thr Asp His Val Lys Ala Glu


 


SEQ ID NO: 5


atatcatatg atggcccccc ctcacgttgt cgacgagca


 


SEQ ID NO: 6


atatattaat atggcacctc ccaacactat tgatgccgg


 


SEQ ID NO: 7


atatcatatg atggcaactc ctcttccccc ctcctttgt


 


SEQ ID NO: 8


atatggatcc taatgcttgt agaacactac gtc


 


SEQ ID NO: 9


atatggatcc ttacttcttg aaaaagacca cgtc


 


SEQ ID NO: 10


atatagatct ttattcggcc ttgacgtggt cagt


 


SEQ ID NO: 11


atatattgcg cacatcatca tcatcatcat cat


 


SEQ ID NO: 12


atatgaattc atatttaaat taatgcttgt agaacactac gtc


 


SEQ ID NO: 13


atatatggta ccttacttct tgaaaaagac cacgtc


 


SEQ ID NO: 14


atatatggta ccttattcgg ccttgacgtg gtcagt

## Claims

1. An isolated recombinant DNA which consists of a nucleotide sequence, wherein said nucleotide sequence is shown as SEQ ID NO:3 that encodes a delta-9 desaturase that is shown as SEQ ID NO:4.

2. An expression vector comprising the recombinant DNA molecule of claim 1, wherein said expression vector can be hosted in a fungi.

3. The vector of claim 2, wherein said fungi is Mortierella alpina.

4. The vector of claim 2, wherein said expression vector is pPinkα-HC.

5. A process for producing a purified delta-9 desaturase, comprising:
(a) obtaining the vector of claim 2;
(b) transforming a suitable host cell with said vector of Step (a), wherein said host cell is a yeast cell;
(c) culturing said host cell under conditions which allow the expression of said delta-9 desaturase by said host cell; and
(d) isolating said delta-9 desaturase expressed by said host cell of Step (c) from the culture media;
wherein said purified delta-9 desaturase is produced.

6. The process of claim 5, wherein said yeast cell is Pichia pastoris.

7. The process of claim 6, wherein said Pichia pastoris is selected from a group of hosts consisting of PichiaPink strain 1, PichiaPink strain 2, PichiaPink strain 3, and PichiaPink strain 4.

8. The process of claim 7, wherein said delta-9 desaturase is shown as SEQ ID NO:4.