Construction of <i>Mucor circinelloides </i>cell factory for producing stearidonic acid and fermentation technology thereof

Abstract

Provided is construction of Mucor circinelloides cell factory for producing stearidonic acid (SDA) and fermentation technology. Δ15 Desaturase gene is obtained by cloning from Mortierella alpina, the gene is ligated to an integrative plasmid pMAT1552, and transformed into a Mucor circinelloides defective strain Mu402, and Δ15 Desaturase gene is integrated on Mucor circinelloides genome through homologous recombination, to obtain the recombinant strain Mc-Δ15, finally, the expression of the Δ15 Desaturase gene in Mucor circinelloides is realized. The recombinant new strain is accession number CGMCC No. 15888, and the classification name is Mucor circinelloides-D15D.

Claims

1. A recombinant cell for producing stearidonic acid, wherein the recombinant cell comprises a polynucleotide encoding Δ15 desaturase, and the polynucleotide encoding Δ15 desaturase is operably ligated to a promoter that directs the polynucleotide to be expressed in a cell, wherein the polynucleotide encoding Δ15 desaturase comprises the polynucleotide sequence set forth in SEQ ID NO: 1, and wherein the recombinant cell is obtained from a Mucor cell.

2. The recombinant cell of claim 1, wherein the recombinant cell is obtained from a Mucor circinelloides cell.

3. A method for preparing a recombinant cell for producing stearidonic acid, the method comprising: introducing into the cell a polynucleotide encoding Δ15 desaturase, wherein the polynucleotide encoding the Δ15 desaturase is operably ligated to a promoter that directs the polynucleotide to be expressed in the cell, wherein the polynucleotide encoding Δ15 desaturase comprises the polynucleotide sequence set forth in SEQ ID NO: 1, and wherein the recombinant cell is obtained from a Mucor cell.

4. A method for producing stearidonic acid in a recombinant cell, comprising: introducing into the recombinant cell a polynucleotide encoding Δ15 desaturase, wherein the polynucleotide encoding Δ15 desaturase is operably ligated to a promoter that directs the polynucleotide to be expressed in the cell; and fermenting the recombinant cell to produce stearidonic acid, wherein the polynucleotide encoding Δ15 desaturase com prises the polynucleotide sequence set forth in SEQ ID NO: 1; and wherein the recombinant cell is obtained from a Mucor cell.

5. The method of claim 4, wherein the fermenting is carried out in a fermentation culture medium, and the fermentation culture medium comprises linolenic acid, linolenic acid compounds and/or precursor compounds of linolenic acid.

6. The method of claim 5, wherein the fermentation culture medium comprises γ-linolenic acid and/or γ-linolenic acid compounds.

7. The method of claim 5, wherein the linolenic acid and/or the linolenic acid compounds are in a vegetable oil.

8. The method of claim 7, wherein the vegetable oil is a safflower seed oil and/or a sunflower seed oil.

9. The method of claim 7, wherein the vegetable oil is initially present in the fermentation culture medium, or is added to the fermentation culture medium as a supplement during fermenting.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) In order to more clearly illustrate technical solutions of embodiments of the present disclosure, the accompanying drawing which need to be used in the embodiments will be introduced below briefly.

(2) FIG. 1 is a PCR verification chart of a Mucor circinelloides recombinant strain. In the drawing, M represents a standard protein molecular weight; 0 represents a control strain Mc1552; 1-3 represent a Mucor circinelloides recombinant strain Mc-Δ15.

DETAILED DESCRIPTION OF EMBODIMENTS

(3) In order to make objects, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be described clearly and completely below. If no specific conditions are specified in the embodiments, they are carried out under normal conditions or conditions recommended by the manufacturer. If the manufacturers of reagents or instrument used are not specified, they are conventional products commercially available.

(4) Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure should have meanings that are commonly understood by those ordinarily skilled in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or test of the present disclosure.

(5) As used herein, term “identity”, “identity percentage” or “identity%” refers to a relationship of two or more nucleotide sequences (or polypeptide sequences) to one another as determined by sequence comparison. Identity may represent the degree of correlation of sequences between polynucleotides (or polypeptide sequences), and may be determined by the matching degree between character strings of such sequences. In one or more embodiments, the identity percentage of two sequences, whether nucleotide or amino acid sequences, is obtained by dividing the number of exact matches between the two compared sequences by the length of a shorter sequence, then multiplying the result by 100. For example, the polynucleotide encoding Δ15 desaturase includes a polynucleotide sequence having at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identity to the polynucleotide sequence represented by SEQ ID NO: 1.

(6) In one or more embodiments, the recombinant cell may be a cell of an organism suitable for fermentation, for example, a unicellular microorganism, which may be a prokaryote or a eukaryote such as yeast, or a plant cell. In one or more embodiments, the cell is a microbial cell. In one or more embodiments, the cell is a fungal cell.

(7) In one or more embodiments, the recombinant cell of the present disclosure can be obtained upon transformation of a nucleic acid molecule into a cell accomplished by any method that involves inserting the nucleic acid molecule into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. The recombinant cell may remain unicellular or may grow into tissue, organ or multicellular organism. The transformed nucleic acid molecule may remain extrachromosomal or the transformed nucleic acid molecule may be integrated into one or more sites within the chromosome of the transformed cell (i.e., recombinant cell) in a manner that retains its ability to be expressed.

(8) In one or more embodiments, transformation of a nucleic acid molecule into a cell may be mediated by a recombinant vector. One type of recombinant vector includes a nucleic acid molecule of the present disclosure operably ligated to an expression vector. As indicated above, the words “operably ligate” refers to inserting a nucleic acid molecule into an expression vector in such a manner that the nucleic acid molecule can be expressed. The expression vector (which may be a DNA or RNA vector) is then capable of transforming a host cell and achieving expression of a specific nucleic acid molecule. In one or more embodiments, the expression vector is also capable of replication in a host cell. The expression vector may be a prokaryotic vector or a eukaryotic vector, and is typically virus or plasmid. The expression vector in the present disclosure includes any vector that is capable of functioning (i.e., directing gene expression) within a recombinant cell of the present disclosure.

(9) As used herein, the term “biologically active fragment” herein refers to a portion of a given polypeptide/enzyme that still retains desaturase activity. These biologically active fragments can be readily determined by making a series of deletions to the full-length protein and testing activity of the resulting fragments.

(10) When compared to a naturally occurring molecule, polynucleotides of the present disclosure may have one or more mutations that are deletions, insertions, or substitutions of nucleotide residues. The mutant may be naturally occurring (that is to say, isolated from a natural source) or synthesized (for example, synthesized by site-directed mutagenesis of a nucleic acid).

Example 1: Cloning of Mortierella alpina Δ15 Des

(11) A Mortierella alpina strain was inoculated into a 500 mL Erlenmeyer flask equipped with a baffle and containing 100 mL of Kendrick culture medium (30 g/L glucose, 1.5 g/L MgSO.sub.4.7H.sub.2O, 3.3 g/L ammonium tartrate, 7.0 g/L KH.sub.2PO.sub.4, 2.0 g/L Na.sub.2HPO.sub.4, 1.5 g/L yeast extract, 0.076 g/L CaCl.sub.2, 8 mg/L FeCl.sub.3.6H.sub.2O, 1 mg/L ZnSO.sub.4.7H.sub.2O, 0.1 mg/L CuSO.sub.4.5H.sub.2O, 0.1 mg/L Co(NO.sub.3).sub.2.6H.sub.2O, 0.1 mg/L MnSO.sub.4.5H.sub.2O), cultured at 28° C. and 150 rpm for 48 h, and thalli were collected by suction filtration. RNA was extracted, and reversely transcribed into cDNA, which was carried out with reference to instructions of a reverse transcription kit. According to the genome information of Mortierella alpina in NCBI database, Δ15 Des (KF 433065.1, 1212 bp) was found.

(12) A nucleotide sequence (SEQ ID NO: 1) of Δ15 Des gene is as follows:

(13) TABLE-US-00001 atggcacccc 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 ag

(14) Specific primers Δ15-F and Δ15-R were designed according to the gene sequence, and PCR was carried out taking Mortierella alpina cDNA as a template.

(15) TABLE-US-00002 Δ15-F: (SEQ ID NO: 2) 5′-ACTTTTATATACAAAATAACTAAATCTCGAGATGGCACCCCCTCACG TTG-3′ Δ15-R: (SEQ ID NO: 3) 5′-ACTAGTCGCAATTGCCGCGGCTCGAGCTAATGCTTGTAGAACACTAC G-3′

(16) A 50 μL PCR reaction system: 10 μL of 5 ×PS buffer solution, 5 μL of dNTPs mixture (2 mM of each type), 1 μL of upstream primer, 1 μL of downstream primer, 100˜200 ng of total cDNA, 1 μL of PrimeSTAR HS DNA polymerase, and ddH.sub.2O for complementation to 50 μL. Reaction conditions were as follows: denaturation at 95° C. for 3 min, then starting cycling, then denaturation at 95° C. for 30 sec, annealing at 55° C. for 30 sec, extension at 72° C. for 1.5 min, after 30 cycles in total, extension again at 72° C. for 10 min, and reducing the temperature to 4° C. and keeping the temperature for 5 min. A 957 bp PCR fragment was obtained from amplification, the recovered fragment was ligated with a pMAT1552 vector, a ligated product transformed Escherichia coli Top10 competent cell, a transformed product was spread on an LB flat plate (10 g/L peptone, 5 g/L yeast cream, 10 g/L NaCl, and 1.5% agar) containing 100 mg/L ampicillin. The LB flat plate was cultured at 37° C. overnight, colonies were selected, and inoculated into an LB liquid culture medium (10 g/L peptone, 5 g/L yeast cream, and 10 g/L NaCl), after 8-10 h, plasmids were extracted for sequence determination, and the plasmids with correct sequences were named as pMAT1552-Δ15.

(17) Preparation of Mucor circinelloids protoplasts: spores of Mucor circinelloides Mu402 strain were inoculated into a flat plate of YPG culture medium (3 g/L yeast extract, 10 g/L peptone, 20 g/L glucose, 20 μg/mL leucine, 200 μg/mL uracil, pH 4.5) and cultured at 28° C. for 1 day. The monoclonal hyphae were taken and dot-planted on a flat plate of YPG culture medium, and cultured at 28° C. for 3˜4 days, then the spores could grow well. To each flat plate, on which the spores grew well, 5˜6 mL of YPG culture medium was added, the spores were scraped with a sterilized spreading rod, a spore suspension was collected in a sterilized 50 mL centrifuge tube, and the concentration was calculated with a hemocytometer and the spore concentration was adjusted to 1×10.sup.7/mL with YPG of pH 4.5. 12.5 mL of the above spore suspension was placed in a sterilized 250 mL Erlenmeyer flask, and placed in a refrigerator at 4° C. overnight to allow the spores to fully adsorb water and swell. The Erlenmeyer flask was placed on a shaker at 30° C. and 250 rpm to culture until the spores germinated. After centrifugation at 1100 rpm, the spores were washed twice with 5 mL of PS buffer of pH 6.5 [18.22 g of sorbitol and 20 mL of PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 2 mM KH.sub.2PO.sub.4)], to wash away the culture medium. The spores were resuspended in 5 mL of PS buffer, and lyase at a final concentration of 4 mg/mL and 0.06 U/mL chitosanase were added, followed by incubation on a shaker at 30° C. and 60 rpm for 90 min so as to remove cell walls. After centrifugation at 100×g, the spores were washed twice with 0.5 M sorbitol pre-cooled at 4° C., and 800 μL of 0.5 M sorbitol was added, followed by gentle pipetting for resuspension and precipitation to obtain protoplasts, which were dispensed for later use, with 100 μL in each tube.

(18) Construction of recombinant strain Mc-Δ15: 100 μL of the protoplasts prepared above were uniformly mixed with 1 μg of plasmids pMAT1552-Δ15 or pMAT1552 to be subjected to electric shock transformation, after the electric shock was ended, 1 mL of pre-cooled YPGS (0.5 mol/L sorbitol, 3 g/L yeast extract, 10 g/L peptone, and 20 g/L glucose) was immediately added, for incubation at 26° C. and 100 rpm for 1 h, followed by centrifugation at 100×g to remove YPGS, after being resuspended in YNBS [91.1 g/L sorbitol, 1.5 g/L glutamic acid, 1.5 g/L (NH.sub.4).sub.2SO.sub.4, 0.5 g/L yeast basic nitrogen source, 10 g/L glucose, pH adjusted to 4.5, and after sterilization, thiamine and nicotinic acid were added to a final concentration of 1 μg/mL], the mixture was uniformly spread on an MMC selective culture medium [10 g/L casein amino acid, 0.5 g/L yeast basic nitrogen source, 20 g/L glucose, and 15 g/L agar, pH adjusted to 3.2, and after sterilization, thiamine and nicotinic acid were added to a final concentration of 1 μg/mL], followed by culturing in a light-tight condition at 28° C. for 3-4 days. Single colony hyphae growing on 8 selective flat plates were randomly selected to a new MMC flat plate, and cultured at 28° C. for 2-3 days to collect spores. About 200-300 spores were respectively inoculated to the MMC flat plate and the MMC flat plate containing uracil, and cultured at 28° C. for 2-3 days to count. The above screening steps were repeated until the number of the spores growing in the two flat plates was substantially the same, which indicated that the transformants with stable inheritance were obtained. The transformant hyphae with stable inheritance were cultured on a YPG culture medium flat plate at 30° C. for 5-7 days, then spores were collected, the concentration of the spores was adjusted to be 1×10.sup.7/mL, and the spores were stored in a 30% glycerin tube at −80° C. Finally, the Mucor circinelloides recombinant strain Mc-Δ15 and the control strain Mc1552 were obtained. The residual thalli, cultured in the shake flask after the spreading, were isolated by vacuum filtration with a Buchner funnel, and the Mucor circinelloides genomic DNA was extracted (which was carried out by referring to the instructions of a plant rapid DNA extraction kit), which was taken as a template for PCR verification, with taking 1552-F and 1552-R as primers (this pair of primers were positions of 300bp upstream and downstream of a site of target gene inserted into the plasmid).

(19) TABLE-US-00003 1552-F: (SEQ ID NO: 4) 5′-CCTCGGCGTCATGATGTTTTTGTGTACCT-3′ 1552-R: (SEQ ID NO: 5) 5′-GGGATGTCTGCTGCTACCATGTCTCAT-3′

(20) The reaction system and amplification conditions were as follows: pre-denaturation at 95° C. for 3 min, denaturation at 95° C. for 30 sec, annealing at 60° C. for 30 sec, extension at 72° C. for 2 min, with 30 cycles, and compensation extension at 72° C. for 10 min. The PCR verification results are shown as in FIG. 1. A fragment obtained for the Mucor circinelloides recombinant strain Mc-Δ15 has 1812 bp, while a fragment at the corresponding position of the control strain Mc1552 has 600 bp, which indicated that the plasmids had been successfully transformed into the Mucor circinelloides.

(21) Fermentation Production of SDA Cell Factory:

(22) (1) Fatty Acid Composition and Content Determination of Mucor circinelloides Recombinant Strain Mc-Δ15

(23) Preparation of sample to be tested: the Mucor circinelloides recombinant strain Mc-Δ15 was cultured in a 2 L fermentation tank using Kendrick culture medium. The fermentation conditions were as follows: 28° C., 700 rpm, an air inflow of 1 v/v min.sup.−1, and pH being maintained at 6.0. According to the oil production rule of Mucor circinelloides, a whole fermentation broth sample was collected and subjected to vacuum filtration with a Buchner funnel, then the fermentation broth and thalli were separated, the fermentation broth was collected and stored at −20° C. for later use, and the thalli were washed with distilled water for 3 times, and then freeze-dried for later use.

(24) Using the cell disruption method of acid treatment combined with multigelation, lipids were extracted from thalli of the recombinant bacteria Mc-Δ15 with an organic solvent, referring to the method (Folch J, Lees M, Sloane-Stanley G, et al. A simple method for the isolation and purification of total lipids from animal tissues. Biol Chem, 1957, 226, 497-509) with appropriate modifications. The specific method was as follows: (1) after grinding the freeze-dried thalli, weighing 20 mg dry weight of thalli in a 5 mL glass bottle, and adding 2 mL of 4 M hydrochloric acid thereto; (2) subjecting the glass bottle to a 80° C. water bath for 1 h, and at −80° C. for 15 min, and repeating the process once; (3) after the glass bottle returns to room temperature, adding 1 mL of methanol and 1 mL of chloroform, and adding 100 μL of internal standard C15:0 with a concentration of 2.02 μg/μL by a microsyringe; (4) subjecting the glass bottle to rotational extraction for 0.5 h with a mixer, followed by centrifugation at 3000 rpm for 3 min, and collecting a chloroform layer in a new 5 mL glass bottle; (5) adding 1 mL of chloroform to the original glass bottle again, repeating the process of (4) and combining the chloroform layers; (6) blowing dry with nitrogen; (7) adding 1 mL of 10% methanolic hydrochloric acid solution, subjecting the original glass bottle to a 60° C. water bath for 3 h, during which the bottle was shaken for 30 sec every half hour; (8) after the bottle was cooled to room temperature, adding 2 mL of n-hexane and 1 mL of saturated NaCl solution, mixing well by vortex oscillation, centrifuging at 4000 rpm for 3 min, then taking 1 mL of n-hexane layer, and transferring to a gas-phase bottle to obtain a fatty acid methyl ester solution.

(25) Fatty acid methyl ester was analyzed by gas chromatography with taking commercial fatty acid methyl ester standards (37 fatty acid methyl ester mixed standards) as standard samples. The gas chromatography was performed by GC-6890N of Agilent, USA, and the measurement conditions were as follows: gas chromatography conditions: splitless sampling, using DM-FFAP (30 m×0.32 mm, 0.22 μm) as chromatographic column, a hydrogen ion flame detector, and nitrogen as the carrier gas, the temperature of a gasification chamber and the temperature of the detector both being 250° C., and a sample size of 1 μL. A temperature rising process: an initial temperature was 80° C., the temperature was first increased to 200° C. at a heating rate of 8° C./min, then increased to 205° C. at a heating rate of 1° C./min, and finally increased to 240° C. at a heating rate of 4° C./min, and the temperature was kept for 5 min. Taking pentadecanoic acid (C15:0) as a reference, the size of the peak area of each fatty acid composition was recorded, and the content of total fatty acid was calculated. The results are as shown in Table 1, in which the intracellular lipid content of the over-expressed strain Mc-Δ15 is increased.

(26) TABLE-US-00004 TABLE 1 Lipid Contents of Control Strain and Δ15 Over-expressed Strain by Fermentation Culture Fermentation Time (h) 12 24 36 48 60 72 84 96 Strains Mc-Δ15  3.75  6.78 12.11 14.92 15.53 15.34 15.32 15.29 Mc1552 5.8 9.2 11.7  12.3  12.4  12.6  13.2  13.1 

(27) The fatty acid composition of intracellular lipid of the over-expressed strain Mc-Δ15 changed greatly, and SDA was contained in the lipid of the over-expressed strain Mc-Δ15, of which the content reached 5.2% of the total fatty acid content. The results are as shown in Table 2.

(28) TABLE-US-00005 TABLE 2 Fatty Acid Composition of Lipids of Control Strain and Δ15 Des Over-expressed Strain by Fermentation Culture Fatty Acid C (18:1) C (18:2) C (18:3) C (18:4) composition (%) C (16:0) C (18:0) OA LA GLA SDA Strains Mc-Δ15 16.4 3.7 30.2 16.2 21.2 5.2 Mc1552 18.4 4.2 31.5 16.8 29.8 0  

(29) (2) Fatty Acid Composition and Content Determination of Mucor circinelloides Recombinant Strain Mc-Δ15 Under Optimized Fermentation Conditions

(30) Preparation of sample to be tested: the Mucor circinelloides recombinant strain Mc-Δ15 was cultured in a 2 L fermentation tank using improved Kendrick culture medium (adding safflower oil rich in linoleic acid to the culture medium). The fermentation conditions were as follows: 28° C., 700 rpm, an air inflow of 1 v/v min.sup.−1, and pH being maintained at 6.0. According to the oil production rule of Mucor circinelloides, a whole fermentation broth sample was collected and subjected to vacuum filtration with a Buchner funnel, to separate the fermentation broth and thalli, the fermentation broth was collected and stored at −20° C. for later use, and the thalli were washed with distilled water for 3 times, and then freeze-dried for later use.

(31) Using the cell disruption method of acid treatment combined with multigelation, lipids were extracted from thalli of the recombinant bacteria Mc-Δ15 with an organic solvent, referring to the method (Folch J, Lees M, Sloane-Stanley G, et al. A simple method for the isolation and purification of total lipids from animal tissues. Biol Chem, 1957, 226, 497-509) with appropriate modifications. The specific method was as follows: (1) after grinding the freeze-dried thalli, weighing 20 mg dry weight of thalli in a 5 mL glass bottle, and adding 2 mL of 4 M hydrochloric acid thereto; (2) subjecting the glass bottle to a 80° C. water bath for 1 h, and at −80° C. for 15 min, and repeating the process once; (3) after the glass bottle returns to room temperature, adding 1 mL of methanol and 1 mL of chloroform, and adding 100 μL of internal standard C15:0 with a concentration of 2.02 μg/μL by a microsyringe; (4) subjecting the glass bottle to rotational extraction with a mixer for 0.5 h, followed by centrifugation at 3000 rpm for 3 min, and collecting a chloroform layer in a new 5 mL glass bottle; (5) adding 1 mL of chloroform to the original glass bottle again, repeating the process of (4) and combining the chloroform layers; (6) blowing dry with nitrogen; (7) adding 1 mL of 10% methanolic hydrochloric acid solution, subjecting the original glass bottle to a 60° C. water bath for 3 h, during which the bottle was shaken for 30 sec every half hour; (8) after the bottle was cooled to room temperature, adding 2 mL of n-hexane and 1 mL of saturated NaCl solution, mixing well by vortex oscillation, centrifuging at 4000 rpm for 3 min, taking 1 mL of n-hexane layer, and transferring to a gas-phase bottle to obtain a fatty acid methyl ester solution.

(32) Fatty acid methyl ester was analyzed by gas chromatography with taking commercial fatty acid methyl ester standards (37 fatty acid methyl ester mixed standards) as standard samples. The gas chromatography was performed by GC-6890N of Agilent, USA, and the measurement conditions were as follows: gas chromatography conditions: splitless sampling, using DM-FFAP (30 m×0.32 mm, 0.22 μm) as chromatographic column, a hydrogen ion flame detector, and nitrogen as the carrier gas, the temperature of a gasification chamber and the temperature of the detector both being 250° C., and a sample size of 1 μL. A temperature rising process: an initial temperature was 80° C., the temperature was first increased to 200° C. at a heating rate of 8° C./min, then increased to 205° C. at a heating rate of 1° C./min, and finally increased to 240° C. at a heating rate of 4° C./min, and the temperature was kept for 5 min. Taking pentadecanoic acid (C15:0) as a reference, the size of the peak area of each fatty acid composition was recorded, and the content of total fatty acid was calculated. The results are as shown in Table 3, in which the intracellular lipid content of the over-expressed strain Mc-Δ15 is increased.

(33) TABLE-US-00006 TABLE 3 Lipid Contents of Control Strain and Δ15 Over-expressed Strain by Fermentation Culture Fermentation Time (h) 12 24 36 48 60 72 84 96 Strains Mc-Δ15 4.2 6.5 11.9 14.8 15.7 15.6 15.4 15.3 Mc1552 5.8 9.4 11.5 12.2 12.7 12.6 13.3 13.2

(34) The fatty acid composition of intracellular lipid of the over-expressed strain Mc-Δ15 changed greatly, and SDA appeared in the lipid of the over-expressed strain Mc-Δ15, of which the content reached 8.3% of the total fatty acid content. The results are as shown in Table 4.

(35) TABLE-US-00007 TABLE 4 Fatty Acid Composition of Lipids of Control Strain and Δ15 Over-expressed Strain by Fermentation Culture Fatty Acid C (18:1) C (18:2) C (18:3) C (18:4) composition (%) C (16:0) C (18:0) OA LA GLA SDA Strains Mc-Δ15 16.7 3.6 3.07 15.7 21.3 8.3 Mc1552 18.5 4.3 31.3 16.7 29.6 0  

(36) The above-mentioned are merely for preferred embodiments of the present disclosure, and not intended to limit the present disclosure. For one skilled in the art, various modifications and variations may be made to the present disclosure. Any amendments, equivalent replacements, improvements and so on, made within the spirit and principle of the present disclosure, should be covered within the scope of protection of the present disclosure.

INDUSTRIAL APPLICABILITY

(37) The present disclosure provides construction of Mucor circinelloides cell factory for producing SDA and fermentation technology thereof; the SDA content of the recombinant strain Mc-Δ15 can reach 8.3% of the total fatty acid. Therefore, the recombinant strain Mc-Δ15 can be used to produce stearidonic acid.