Low cost medium for recombinant endoglucanase II production by Pichia pastoris

Program in Biotechnology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand. PTT Research and Technology Institute, PTT Public Co. Ltd., Wangnoi, Ayutthaya 13170, Thailand. Department of Botany, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand. Biofuels by Biocatalysts Research Unit, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand. Aquatic Resources Research Institute, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand.


INTRODUCTION
The high cost of nutrient medium for the cultivation of recombinant Pichia pastoris in the production of recombinant proteins is a major factor that needs to be considered and optimized for large scale production (Yadav et al., 2011).Therefore, the development of a suitable culture medium using low cost raw materials is of interest.The growth and recombinant protein production level in recombinant P. pastoris depends on various conditions and on the composition of the culture medium (Batista et al., 2013).
P. pastoris is a methylotrophic yeast that is widely used as a host for the production of recombinant proteins.
Recently, the improvement of individual cellulase enzymes from Trichoderma reesei has been attempted by genetic engineering in P. pastoris in order to enhance their cellulose degradation ability (Boonvitthya et al., 2013).
Cellulases, which degrade cellulose, consist of endoglucanases (EGs), cellobiohydrolases and betaglucosidases.The EGs act on the amorphous regions of cellulose, and EGII is one of the most abundant EGs produced by T. reesei and has the highest catalytic efficiency (Qin et al., 2008;Zhang et al., 2012).
The estimated cost of enzyme production at an industrial scale depends to a large extent upon the growth medium used in the process (Hajii et al., 2008;Batista et al., 2013).Previous studies have reported on the fermentation using pure glycerol as the carbon substrate because the salts in raw glycerol exert important inhibitory effects on many microorganisms (Petitdemange et al., 1995).However, yeast strains that can grow on raw glycerol have been reported (Papanikolaou et al., 2002).In order to reduce the medium costs, the crude glycerol produced during biodiesel manufacturing, which contains macro elements, such as calcium, potassium, magnesium, sulfur and sodium (Kitcha and Cheirsilp, 2011), has been used as a carbon source in P. pastoris culture medium to increase the cell concentration in the logarithmic growth phase (Celik et al., 2008).In addition, the waste agricultural products of soybean meal and rice bran have been used as organic nitrogen sources since they provide a rich but inexpensive source of nutrients (Xiao et al., 2007;Sereewatthanawut et al., 2008).
The aim of this study was to determine the suitability of crude glycerol, obtained as the by-product of biodiesel production, and soybean meal and rice bran as a substitute for high purity commercial glycerol, peptone and yeast extract, respectively, in the formation of a low cost culture medium for the growth of, and recombinant (r)EGII production by, P. pastoris.

Microbial strain
The genetically engineered P. pastoris X-33 yeast strain, which is transfected with the pPICZαA plasmid encoding for the T. reesei EGII gene under the methanol inducible AOX1 promoter (Boonvitthya et al., 2013) was provided by the Biocatalysts Research Unit, Chulalongkorn University.The transformed line was maintained on YPD (2% (w/v) peptone, 1% (w/v) yeast extract and 2% (w/v) dextrose) agar.Expression of the rEGII as a secretory product was controlled under the methanol inducible AOX1 promoter and repressed by glycerol.

Preparation of crude glycerol, the byproduct of biodiesel production
The crude glycerol was obtained from the Energy Absolute Public Co. Ltd., biodiesel production plant at Kabinburi Industrial Zone in Prachinburi, Thailand, which uses refined bleached deodorized palm oil and methanol in the transesterification process with an alkaline catalyst.This crude glycerol preparation contained 75% (v/v) glycerol and so the volume of this crude glycerol added to the medium was set accordingly to give the desired final glycerol concentration (v/v) ignoring the impurities.The crude glycerol was adjusted to pH 7.0 prior to addition to the culture medium.

Preparation of soybean meal and rice bran hydrolysates
Soybean meal was obtained from Lactasoy Co. Ltd., Prachinburi, Thailand, while rice bran was obtained from a rice mill in Pathumthani, Thailand, and separately treated in 1 N sulfuric acid (H 2 SO 4 ) at 33% (w/v).Each mixture was autoclaved at 121°C for 40 min, cooled down and distilled water added to the required volume as well as 10 N NaOH to set the pH to 7.0.The suspension was then filtered and the filtrate stored at 4°C until use.The total nitrogen content was analyzed by the Kjeldahl method (AOAC, 1990).

Medium optimization by a sequential univariate approach
The effect of the nutrient composition, in terms of the concentration of crude glycerol, methanol, soybean meal and rice bran hydrolysates and ammonium sulfate, as well as the medium pH and culture temperature and time, was investigated upon the growth of, and the production of rEGII by, P. pastoris in that order as follows.
For the initial growth of P. pastoris to form the starter inoculum, the crude glycerol concentration was varied in BMGY medium (1% (w/v) yeast extract, 2% (w/v) peptone, 100 mM potassium phosphate buffer pH 6.0, 0.4% (v/v) Pichia trace minerals (PTM) and 1% (v/v) glycerol) by substitution of the pure glycerol with the crude glycerol preparation at a final glycerol concentration of 0, 0.5, 1, 1.5, 2 and 2.5% (v/v).P. pastoris was grown in each of these media (50 mL) in 250-mL baffled flasks at 30°C with shaking at 250 rpm for 21 h.The growth of the yeast was then monitored in terms of the yeast dry cell weight (DCW) and optical density, as detailed below.
For the induction of rEGII production by the recombinant P. pastoris, the yeast was first grown in the optimum crude glycerol concentration supplemented BMGY medium (BMcGY), harvested by centrifugation (10,000 xg, 3 min), washed in sterile water and then resuspended and transferred into 100 mL BMMY medium (as per BMGY except the glycerol was replaced with methanol) except that the methanol concentration was varied at 0, 0.5, 1, 2, 3 and 4% (v/v) for induction of the AOX-1 promoter and as a carbon source for the yeast.The cell suspension was then grown in 250-mL baffled flasks at 30°C with shaking at 250 rpm for 144 h.For each selected methanol concentration, the same amount (final concentration) of fresh methanol was added to the culture each day (0, 24, 48, 72, 96 and 120 h).At 12, 24 h and then every 24 h thereafter a 5-mL aliquot of the cell suspension was removed to ascertain the yeast DCW and extracellular rEGII activity.
The effect of substitution of peptone with soybean meal hydrolysate (at 0, 0.025, 0.05, 0.1, 0.125, 0.15, 0.175 and 0.2 (v/v)) upon P. pastoris growth in the BMcGY medium (optimal crude glycerol level determined as above), and upon the subsequent growth and rEGII production level in BMMY medium was then evaluated as above, except using the optimal methanol concentration in the BMMY medium, respectively.The replacement of both the peptone and yeast extract in the BMGY and BMMY media with a combined organic nitrogen source of soybean meal and rice bran hydrolysates was then evaluated as above, except using the optimal soybean hydrolysate level and varying the rice bran hydrolysate at 0, 0.1, 0.25, 0.5, 1.0, 1.5 and 2.0% (v/v) in place of the yeast extract.
Table 1.Effect of the crude glycerol concentration as a replacement for pure glycerol in BMGY medium on the growth of recombinant P. pastoris.

Enzyme assay
The evaluation of the extracellular rEGII activity in the culture supernatant assay was performed by monitoring the hydrolysis of carboxymethyl cellulose (CMC) as the total reducing sugar, evaluated using the dinitrosalicylic acid DNS method (Miller, 1959).Each reaction contained 0.5 mL of 2% (w/v) CMC solution in 50 mM acetate buffer (pH 4.8) and 0.5 mL of the diluted test enzyme solution.The reaction was incubated at 50°C for 30 min and then 3 mL of DNS reagent was added, incubated in a boiling water bath for 5 min, and then cooled.Thereafter, the absorbance was measured at 540 nm to estimate the quantity of reducing sugars produced in the assay (Miller, 1959).One unit (U) of enzyme activity was defined as the amount of enzyme that released 1 µmol of reducing sugar/mL/min under the above assay conditions.Glucose (0-2 µg/mL) was used to form the standard curve.

Measurement of protein concentration
The total protein content was determined following the method of Bradford (1976) using bovine serum albumin (0-25 µg/mL) as a standard and measuring the absorbance at 595 nm with a microplate reader (ANTHOS Zenyth 200, USA).

Determination of yeast cell concentration
The yeast DCW was determined by centrifugation (5000 xg for 5 min) of 5 mL of the culture broth, washing the cells with distilled water, and then drying at 105°C for 24 h.From the obtained mass and the starting culture volume analyzed the DCW was then calculated (g/L).In addition, the yeast cell concentration was estimated in terms of the optical density (OD) of the suspension at 600 nm (OD 600 ) AS compared to the medium alone (Charoenrat et al., 2013).

Data analysis
Data WEre presented as the mean ± one standard deviation (SD).The statistical significance of the difference between means was tested by analysis of variance (ANOVA) and Duncan's multiple range tests (DMRT), with significance accepted at the P < 0.05 level.

RESULTS AND DISCUSSION
In this study, different cultivation media compositions and cultivation conditions were evaluated in order to attempt to reduce the cost of efficient rEGII production by P. pastoris.The culture medium composition is known to be a major influence on the growth and enzyme production, especially the carbon and nitrogen sources (Broach, 2012;Charoenrat et al., 2013).

Effectiveness of crude glycerol to replace commercially available glycerol in high cell density cultivation of P. pastoris in BMGY medium
Glycerol is regularly used as the principal initial carbon source in P. pastoris cultivation in order to increase the cell concentration.When utilizing the cheaper crude glycerol, a surplus byproduct of the expanding biodiesel production industry, in the BMGY medium, the highest level of yeast biomass production (8.17 g/L, DCW basis) was obtained with 1% (v/v) of biodiesel glycerol (Table 1).Crude glycerol concentrations above or below 1% (v/v) resulted in a lower yeast yield, in accord with the previously reported repression of yeast growth at high crude glycerol levels (Tang et al., 2009).Accordingly, P. pastoris was initially grown in BMGY medium with 1% (v/v) crude glycerol in place of the commercial glycerol (hereafter called BMcGY).Although this was a similar glycerol concentration to the commercial glycerol containing BMcGY medium, a likely economic saving would still be gained since, as compared to the pure glycerol (US$1.11 per kg), the crude glycerol is some tenfold cheaper (US$0.11per kg).Indeed, as a cheaper carbon source for biotechnological applications that may make them economically feasible, crude glycerol has been used as carbon source in many industrial fermentations (Chatzifragkou et al., 2011).However, the crude glycerol derived from biodiesel production typically possesses a low glycerol concentration of low value because of the impurities.Further refining of the crude glycerol will depend on the economy of production scale and/or the availability of a glycerol purification facility (Naresh and Brian, 2006).

Effects of the methanol concentration on BMMY medium
Methanol is typically the widely accepted industrial choice  of carbon source for P. pastoris fermentations, and also serves as inducer for recombinant protein expression under the control of the AOX1 promoter of P. pastoris.Following the initial growth of P. pastoris in BMcGY medium and transfer to BMMY medium without methanol no rEGII production was detected, as expected with the AOX1 promoter.Increasing the methanol concentration up to 3% (v/v) correspondingly increased the rEGII level produced, especially at 2 and 3% (v/v) methanol, with the highest rEGII level obtained (68.5 U/mL) at 3% (v/v) methanol after 120 h (Figure 1).Increasing the methanol concentration further to 4% (v/v), however, resulted in almost no rEGII production, consistent with that previously reported in the production of human serum albumen in this yeast (Bushell et al., 2003).Accordingly, a methanol concentration of 3% (v/v) was used in the BMMY medium (hereafter called BMM 3 Y).

Effect of organic nitrogen sources
As determined by Kjeldahl's method, the total nitrogen content of the soybean meal and rice bran hydrolysates were 0.52 and 0.31% (w/v), respectively, as compared to ~12% and ~9.5% (w/v) (Technical Data from HiMedia Laboratories Pvt.Ltd.) for the peptone and yeast extract, respectively.

Conclusions
The culturing of recombinant P. pastoris for the production of rEGII was attained using BMcGYRS and BMM 3 YRS media, which are the use of a 1% (v/v) crude glycerol (75% (v/v) glycerol content from a biodiesel production plant) plus 0.175% (v/v) soybean meal and 0.15% (v/v) rice bran hydrolysates as a replacement for the 2% (v/v) glycerol, 2% (w/v) peptone and 1% (w/v) yeast extract in standard BMGY and BMMY media, respectively.Although the optimal rEGII production required supplementation of this modified medium with 0.5% (w/v) ammonium sulphate, overall these media will provide a likely economic advantage for the industrial scale production of recombinant enzymes by P. pastoris.

a
Data are shown as the mean ± 1 SD and are derived from triplicate experiments.Means followed by a different lowercase letter are significantly different (P < 0.05; ANOVA, DMRT).

Figure 1 .
Figure 1.Effect of the methanol concentration on the growth of, and the extracellular recombinant endoglucanase II production (U/mL) level by, recombinant P. pastoris in BMMY medium.Data are shown as the mean ± 1 SD and are derived from triplicate experiments.

Table 2 .
The growth of recombinant P. pastoris in BMcGY a medium with different soybean meal hydrolysate concentrations as a replacement for the peptone.