Engineering and Fermenter Optimization of Fungi GLA Productions in Pichia Pastoris GS115 Using Oil Waste


 γ-Linolenic acid (GLA) is an important n-6 polyunsaturated fatty acid (PUFA) that has received considerable attention in both levels in human and animal feed. GLA is used in many nutritional and medicinal applications such as the treatment of cancer, inflammatory disorders, and diabetes. Currently, plant seed is the main dietary source of GLA that is not enough to utilize on an industrial scale. To generate a sustainable novel source of GLA, the gene of delta-6 desaturase, which is one of the important enzymes in the GLA production pathway was isolated from Mucor rouxii DSM1194 and expressed in Pichia pastoris GS115 by pPICZC vector. The recombinant yeast expressed the GLA up to 19.2% (72 mg/g) of total fatty acids. GLA production of recombinant yeast was studied in fermenter by oil waste along 5 days and results detected 6.3 g / l lipid and 103 mg/g GLA was produced in 72 hours. 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 [1,2]. 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 expression levels of various genes [3,4].
GLA is rarely found in some nuts and oil plants seeds such as evening primrose (Oenothera biennis L.), borage (Borago o cinalis L.) and, blackcurrant (Ribes nigrum L.) [5][6][7]. Although up to 22% of the total fatty acid content can be GLA in plant seeds, the quantities and qualities of this PUFA production are easily impacted by climate, region, and seasons. Studies have shown that the high proportion of GLA in total fatty acids may be up to 70% in Genetically-engineered sa ower seeds [8,9]. Nevertheless, there is continued interest in developing alternative sources from oleaginous microorganisms with high GLA contents including Cunninghamella echinulata, Mucor rouxii, Mortierella alpina, Mucor circinelloides, and Mortierella isabellina due to the acceptance uncertainties of genetically modi ed (GM) materials [5,6,[10][11][12] 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. The gene for delta-6 desaturase has been previously cloned and characterized for several prokaryotes and higher eukaryotes. It was also overexpressed in many hosts, such as microalgae, yeasts, and plants. However, looking for more developed GLA-producing microorganisms is still one of the current prominent researches, which considerable interest has focused on oleaginous microorganisms, such as Thamnidium elegans, Cunninghamella echinulata, Mortierella alpina, Mocur rouxii, Pythium irregulare, Rhizopus arrhizus, and R. nigricans. [8, 13-17].
Mucor rouxii a typical oleaginous lamentous fungus has been widely used to investigate GLA production. Previous studies have shown that a high proportion of GLA in total fatty acids of M. rouxii was up to 39.7% achieved by fed-batch cultivation [18]. Nevertheless, the production of GLA in M. rouxii is not cost-competitive compared with plants up to now. Thus, it is necessary to explore another fungus host for GLA production on an industrial scale.
Previous studies have shown the gene expression in 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 [19]. 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.

Material 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. All chemicals were obtained from Merck with analytical grade and the restaurant oil wastes sample were collected from Naz plant oil factory.
Cultures were grown in Luria Bertani Agar (LB Agar) [20]. 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 [22], 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 48 h 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 10 µL reaction containing 4 µL 5× PCR buffer, 1 µL Reverse Transcriptase, 2 µL of 10 mM dNTPs, 1 µL of primers, and 1 µg of the RNA sample was used. After mixing and centrifuge for 5 sec, 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 10 min. 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 [20]. 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 50ml YPD with methanol 0.5% and linoleic acid 2% 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 experiment, samples were obtained from production media after 72 h and stained.
We also evaluated the amount of GLA production using ow cytometry on recombinant and wild-type species as a control. First, the FL2 channel of orescent recombinant P. pastoris lipid production was compared with wild type. Subsequently, SSC dot blots of the recombinant P. pastoris along with FL2 studies (FL2=1, SSC=10) against orescent were studied.

GLA puri cation and GC analysis
The modi ed Bligh and Dyer method was performed to extract lipid from P. pastoris cells [23]. 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 4 M), 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 [24].
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. Optimization in fermenter by using of oil waste Under optimal conditions, recombinant yeast was cultured in a 3-liter fermenter (with 1.5 liters of culture uid) and the results of lipid production and dry weight, and GLA production were obtained during 5 days of growth. It should be noted that oil waste was used as a carbon source and the experiment was done three times.

Results And Discussion
In 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 [5,13]. 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. 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.
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 50 mL 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 and Nile red staining (Fig. 1) 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.  2). 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. 3). 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 species 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. 4).

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. 5). Recombinant species express 19.2% GLA compared to 0% in wild-type spices.
The GC assay of lipid combination was also evaluated on both species that cultured in restaurant oil waste culture and revealed increasing of GLA manufacturers (Table 1).

Optimization in fermenter by using of oil waste
The results of growth of recombinant yeast in fermenter ( Figure 6) showed that the amount of dry weight on the 4th day of fermentation reached its highest level of 12.5 g / l and remained in the same range until the end of incubation time. Lipid production showed an increasing trend until the end of incubation and reached 6.3 g / l (50.4%) on day 5 of fermentation. The amount of GLA showed a signi cant difference and increased to 103 mg / g on the third day. Compared to the cultivation of the recombinant strain in ask, this strain showed upper production in the fermenter. The fermenter conditions were also optimized for aeration and the amount of dissolved oxygen in the medium was changed, but there was no signi cant difference in the production of this fatty acid.
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 [19]. Because of the lack of delta-6 desaturases gene, wild-type Pichia pastoris species could not produce GLA from their LA and LAL products. Wei  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 wild-type samples. Florescent analysis including, FL2 channel, SSC dot blots, and FL2 differentiation against FSC were 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 species cultured in the 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.    FL2 channel orescent histogram of Nile red stained A) Auto-orescent cells B) wild type P. pastoris C) recombinant P. pastoris. All experiments described were performed at least three times and statistical signi cance p value was below 5%.

Figure 3
Evaluation of SSC dot blots against orescent of A) Auto-orescent cells B) wild type P. pastoris C) recombinant P. pastoris. All experiments described were performed at least three times and statistical signi cance p value was below 5%.

Figure 4
Identifying FL2 differentiation against FSC of A) Auto-orescent cells B) wild type P. pastoris C) recombinant P. pastoris. All experiments described were performed at least three times and statistical signi cance p value was below 5%. GC lipid combination chromatograph of A) wild type P. pastoris B) recombinant P. pastoris. All experiments described were performed at least three times and statistical signi cance p value was below 5%.