Strains, plasmids, and culture conditions
Mucor rouxii DSM1194 was used as the source of delta-6 desaturase gene. The Escherichia coli DH5α (Stratagene. The USA) and pTZ57R/T (Invitrogen) were used as the host– vector system. Pichia pastoris host strain GS115 (Invitrogen. The UK) and pPICZC (Invitrogen) were used for protein expression.
Cultures were grown in Luria Bertani Agar (LB Agar) (Merck) (Green et al.). Media were supplemented with zeocine (25 μg/ml) when required. The pH was adjusted to 7.5 and 7 for mycelia and colonial growth, respectively.
In vitro assays of delta-6 desaturase gene existence
To verify that the enzyme gene existed in strains of M.rouxii and to ensure that mutations were not formed, DNA alignment was carried out. To this end, DNA extraction, PCR with special primers, and DNA sequencing were done using the Yamada et al (Yamada et al. 2002), White et al, and Sanger methods, respectively. The primers used for the PCR are described below.
F primer: 5`-CAAGAATTCAAAATGGCTCCCCCAAATACTGCGGC3'
R primer: 5'-CAACTCGAGTTCTTTATCATTAGCCCAAATCTC'3
Total RNA isolation
The M.rouxii strain was grown at 28 °C in a 250 mL shaker flask containing 50 mL liquid medium in a shaking incubator (250 rpm). After a 48h growth period, the strains were harvested and the total RNA from M.rouxii was isolated as described below.
Cells of the digestive gland were disrupted with a sterile homogenizer with liquid nitrogen. Total RNA was extracted using the RNAX-PLUS™ Total RNA Extraction Kit (CinnaGen) according to the manufacturer's instructions.
cDNA synthesis and PCR amplification
The partial cDNA fragment of M.rouxii delta-6 desaturase was amplified by RT-PCR. The primers used were Random Hexamer Primers, which were designed according to all kinds of cellular RNAs.
The reactions were performed using RevertAid M-MuLV Reverse Transcriptase. A 10µL reaction contains 4µL 5× PCR buffer, 1µL Reverse Transcriptase, 2 µL of 10mM dNTPs, 1µL of primers, and 1µg of the RNA sample. After mixing and centrifuge for 5sec, samples were incubated at 65·C for 5min and 42·C for 1h. Finally, for deactivation of Reverse Transcriptase, samples were incubated at 70·C for 10min. RT-PCR was performed on the cDNA product using GAPDH primers as a control.
Cloning of PCR product and transformation
The PCR products were characterized by agarose gel electrophoresis and extracted using a DNA extraction kit (Fermentase). Purified products were ligated into pTZ57R/T vector according to the manufacturer's instructions and transformed using the heat shock method into E. coli DH5α competent cells prepared by chemical CaCl2 method (Green et al.). Colony PCR was carried out to verify that the Plasmid DNA had transformed correctly using the below primers.
F primer: 5`-CAAGAATTCAAAATGGCTCCCCCAAATACTGCGGC3'
R primer: 5'-CAACTCGAGTTCTTTATCATTAGCCCAAATCTC'3
Construction of the expression plasmid
Plasmid DNA was purified using the plasmid extraction kit (#K0502; Fermentase) and digested using XhoI and EcoRI (Fermentase). Furthermore, pPICZC vector was also digested using XhoI and EcoRI, respectively and after incubation with Alkaline phosphatase (Fermentase), the digested plasmid was cleaned up. Digested pPICZC vector and delta-6 desaturase gene were ligated using DNA T4 ligase (#EL0014; Fermentase).
The ligation product was transformed into E. coli DH5α competent cells. The E. coli transformants were selected on plates containing the antibiotic, Zeocin™ at a concentration of 25 µg/ml. Verification of insertion of the PCR fragment into the correct translational reading frame was confirmed by colony PCR, double digestion, and DNA sequencing before the introduction of the chimeric plasmid into P. pastoris host cells. Sequencing primers (5′AOX1 and 3′AOX1) were obtained from Invitrogen.
Transformation of P. pastoris and expression in shaken flasks
P. pastoris GS115 strain was selected to be used as a host strain. The recombinant plasmid pPICZA-delta-6 desaturase was purified from E. coli cells and linearized with the restriction enzyme SacI to allow integration of the vector DNA into the chromosomal DNA.
Pichia transformation was performed using the electroporation (Eppendorf), and the high-level expression transformants were screened in the YPD (1% yeast extract, 2% peptone, 2% dextrose, 1.8% agar) plates supplemented with Zeocin™ (100 µg·mL−1).
For the screening of strains and for the optimization of culture conditions in shaking flasks, methanol 0.5% was used for induction, according to the Invitrogen instructions. GLA‐producing P. pastoris cells were grown in 200mL YPD with methanol 0.5% at 30 °C for 4 days.
In vitro assays of GLA production
To verify that the delta-6 desaturase had been expressed and to ensure its activity, Sudan black and Nile red staining were carried out. For that experience, samples were obtained from production media after 72 h and stained according to the Zhou method ([CSL STYLE ERROR: reference with no printed form.]).
We also evaluated the amount of GLA production using flow cytometry on recombinant and wild-type spices as a control.
GLA purification and GC analysis
The modified Bligh and Dyer method was performed to extract lipid from P. pastoris cells (Pan et al.). After passing P. pastoris cells through Whatman No.1 filter paper, mycelia were washed by distilled water three times and once by ethanol, respectively. Samples were incubated at 60ºC for 2 h with HCl solution (10 mL of 4M), and. The hydrolyzed solution was shaken with 20 mL of chloroform/methanol (1:1) at room temperature for 3 hours and centrifuged at 2000 ×g for 5 min. After the supernatant phase was separated, the lower phase containing lipids was evaporated under reduced pressure for 10 min. The extracted fatty acids were modified to fatty acid methyl esters (FAMEs) according to the Christie method (Christie 1993).
The Gas Chromatography (GC) was performed using Agilent 19091J-413 Series with an FID and the capillary column DB-23 (USA). The injector and detector temperatures were maintained at 260 and 300 °C, and the oven program was 100 °C: 2 min; 160°C: 3 min; 215 °C: 2 min; 217 °C: 2 min; 218 °C: 2 min, and 260 °C for 2 min. The flow rate of nitrogen as the carrier gas was 1.5 mL/min.