An Integrated Approach to Produce a Recombinant Yeast Pichia Pastoris For GLA Production

γ-Linolenic acid (GLA) is an important n-6 polyunsaturated fatty acid (PUFA) used in many nutritional and medicinal applications such as the treatment of cancer, inammatory disorders, and diabetes. However, GLA level of the total fatty acids in plant seeds and nuts as prominent sources of GLA is not enough to utilize on an industrial scale. The study aimed to improve the expression of delta-6 desaturase, which is one of the important enzymes in GLA production pathway. The expression vector pPICZC was selected for clone M.rouxii delta-6 desaturase. The engineered vector was rst cloned into E. coli DH5α and after plasmid extraction and conrmation of sequencing was transformed by electroporation into Pichia pastoris GS115. The results indicated that the recombinant yeast strain expressed the gene in the presence of methanol 0.5%. The lipids and essential fatty acids especially GLA were extracted to conrm the expression. The results of studies of lipid and fatty acid production by Sudan black and Nile red staining, GC, and ow cytometry revealed that recombinant strain can produce GLA levels up to 19.2% of total fatty acids. The present study may provide an opportunity for the development of an alternative host for manufacturing GLA on an industrial scale.

Introduction γ-Linolenic acid (GLA 18:3, △6, 9,12), is a prominent n-6 polyunsaturated fatty acid (PUFA) that has a structural role in lipid membranes ingredients (Needleman et al. 1986;Vrinten et al. 2007). In humans, GLA is metabolized to produce prostaglandins and eicosanoids such as leukotrienes which have many health and medicinal roles in cardiovascular disorders, atherosclerosis, cancers, in ammatory disorders, diabetes, and some other diseases by regulating the levels of expression in various genes (Fan et  GLA is synthesized from the conversion of linoleic acid (LA, 18:2, △ 9,12), an essential omega-6 series of fatty acids, using a delta-6 desaturase. Gene coding for delta-6 desaturase has been previously cloned and characterized by several prokaryotes and higher eukaryotes and is also overexpressed in many hosts, such as microalgae, yeasts, and plants. However, looking for more excellent, safe GLA-producing microorganisms is still one of the current prominent research Mucor rouxii a typical oleaginous lamentous fungus has been widely used to investigate GLA production. Previous studies have shown that the high proportion of GLA in total fatty acids of M. rouxii was up to 39·7% achieved by fed-batch cultivation (Jangbua et al. 2009). Nevertheless, the production of GLA in M. rouxii is so far not cost-competitive to the plant sources. Thus, it is necessary to explore another fungus host for GLA production on an industrial scale.
Previous studies have shown that the expression system of Pichia pastoris fungus has several advantages, including rapid growth rate along with high cell density fermentation, diverse posttranslational modi cations, high levels of productivity, elimination of endotoxin and bacteriophage contamination, and feasible genetic manipulation ). These encouraging advantages, make Pichia pastoris one of the best hosts for recombinant protein expression.
The present strategy provides an avenue for increasing the GLA level in total fatty acids by overexpression of delta-6 desaturase gene from M. rouxii that transformed to Pichia pastoris, and may provide an opportunity for the development of the GLA production on an industrial scale.

Materials And Methods
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. The M.rouxii strain was grown at 28 °C in a 250 mL shaker ask 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 ampli cation The partial cDNA fragment of M.rouxii delta-6 desaturase was ampli ed 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). Puri ed 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 CaCl 2 method (Green et al.). Colony PCR was carried out to verify that the Plasmid DNA had transformed correctly using the below primers. Construction of the expression plasmid Plasmid DNA was puri ed 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 T 4 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. Veri cation of insertion of the PCR fragment into the correct translational reading frame was con rmed 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 asks P. pastoris GS115 strain was selected to be used as a host strain. The recombinant plasmid pPICZA-delta-6 desaturase was puri ed from E. coli cells and linearized with the restriction enzyme SacI to allow integration of the vector DNA into the chromosomal DNA.
For the screening of strains and for the optimization of culture conditions in shaking asks, 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 ow cytometry on recombinant and wild-type spices as a control.

GLA puri cation and GC analysis
The modi ed 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 lter paper, mycelia were washed by distilled water three times and once by ethanol, respectively. Samples were incubated at 60 º C 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 modi ed 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 ow rate of nitrogen as the carrier gas was 1.5 mL/min.

Construction of pPICZC -delta-6 desaturase expression plasmid
The delta-6 desaturase gene was ampli ed from the pTZ57R/T -delta-6 desaturase plasmid and inserted into the expression vector pPICZC. After transformation into E. coli DH5α, positive clones were selected with direct colony PCR screening ( Figure. 1A). Finally, pPICZC -delta-6 desaturase plasmid was additionally con rmed by restriction enzyme digestion. The expression plasmid was fragmented, as a result of two EcoRI and XhoI restriction enzyme activities ( Figure. 1B).
Lipid production evaluation in recombinant P. pastoris To obtain the recombinant P. pastoris, pPICZC -delta-6 desaturase plasmid was linearized by SacI and electro-transformed into P. pastoris. After 20h incubation of transformants in YPD, colonies were added to 50ml lipid production culture with methanol 0.5% and linoleic acid 2%.
To evaluate the activity of delta-6 desaturase in recombinant P. pastoris, the production of the lipid was studied by Sudan black (Fig. 2) and Nile red staining (Fig. 3) and visualized under the light microscope. It was found that both methods, showed a signi cant difference in lipid production, compared to the control samples.

Fluorescent assay
To identify the effects of the plasmid on the lipid production of the P. pastoris, the FL2 channel of orescent was obtained and the histogram of recombinant P. pastoris was compared with wild type (Fig. 4). The results showed that the orescent extension of wild-type P. pastoris was lower than that of recombinant species, which suggests that most of the recombinant cells had lipid production capability.
Moreover, SSC dot blots against orescent were studied to evaluate the granules of the recombinant P. pastoris along with FL2 studies, which distributed based on FL2=1 and SSC=10 (Fig. 5). It was found that the QA1 group (FI>1 SSC<10), revealed a signi cant difference between species that suggests more lipid manufacturing in recombinant spices compared to wild type. The results of FL2 differentiation against FSC were also in agreement with the previous study, which showed an increase in not only lipid production but also cell size (Fig. 6).

Gas Chromatography (GC) analysis of GLA production
The lipid combination of the recombinant P. pastoris was studied by measuring the ester fatty acids using the GC chromatography method. The fungal cells were disrupted, and the lipids were extracted to assay the types and quantities of lipids. The lipid-containing fractions were separated and modi ed to obtain fatty acid methyl esters (FAMEs). The amount had been compared with the wild-type P. pastoris GS115, which showed signi cant differences in lipid components (Fig. 7). Recombinant spices express 19.2% GLA compared to 0% in wild-type spices.
The GC assay of lipid combination was also evaluated on both spices that cultured in restaurant oil waste culture and revealed increasing of GLA manufacturers (Table 1). Generally, the biosynthesis of GLA derives from saturated stearic acid (18:0), which is converted by three desaturases, the delta-9, delta-12, and delta-6, respectively ( As mentioned above, the expression system of Pichia pastoris fungus has several advantages such as diverse posttranslational modi cations, high levels of expression with a limited amount of methanol, feasible genetic manipulation, and low-cost medium culture ). Because of the lack of delta-6 desaturases gene, wild-type Pichia pastoris spices could not produce GLA from their LA and LAL products. Wei et al. identi ed the existence of delta-12 and delta-15 desaturases in Pichia pastoris and transformed these genes to Saccharomyces cerevisiae (Wei et al. 2006 (Wan et al.).

Discussion
In this study, after gene cloning and veri cation tests, a new species of fungus, designated for express delta-6 desaturase, was obtained by insertion of an M. rouxii delta-6 desaturase gene into P. pastoris GS115 strain using electroporation and the analysis of changes in the level of lipid production was studied. Results from Sudan black and Nile red staining showed a signi cant difference in the lipid production in recombinant P. pastoris, compared to the control wildtype samples. Florescent analysis including, FL2 channel, SSC dot blots, and FL2 differentiation against FSC was also in agreement with previous studies, which showed an increase in lipid production and cell size. GC analysis of the ester fatty acids of recombinant P. pastoris revealed 19.2% GLA production compared to 0% in wild-type spices. The GC assay of lipid combination was also evaluated on both spices cultured in restaurant oil waste culture and revealed 46% lipid production with 72.3 mg GLA in recombinant spices, compared to 30% lipid production without any GLA manufacturing in wild type spices. Thus, this host may provide an opportunity for the development of the method for industrial-scale GLA manufacturing.  Tables   Table 1. Comparison of lipid, biomass, yields (%) lipid/ biomass w/w), omega 6 and omega 3 production (mg. L-1) by two strains in medium contains oil waste.
Spices Biomass   Identifying lipid production by Nile red staining under 1000x light microscope in A) recombinant P. pastoris B) wild type P. pastoris FL2 channel orescent histogram of Nile red stained A) Auto-orescent cells B) wild type P. pastoris C) recombinant P. pastoris Figure 5 Evaluation of SSC dot blots against orescent of A) Auto-orescent cells B) wild type P. pastoris C) recombinant P. pastoris Identifying FL2 differentiation against FSC of A) Auto-orescent cells B) wild type P. pastoris C) recombinant P. pastoris GC lipid combination chromatograph of A) wild type P. pastoris B) recombinant P. pastoris