Expression Of Monellin In Pichia Pastoris And Optimization Of Fermentation Medium By Response Surface Methodology

The recombinant strain of high efficiency in expression of sweet protein Monellin were constructed by use of Pichia pastoris, and then base salt medium was optimized with Response surface methodology (RSM). The yield of Monellin in optimized medium and the preoptimized were compared in 5 liter fermenter.According to the Monellin amino acid sequence and Pichia pastoris preference codon useage, the Monellin gene was designed and synthesized. The synthesized gene was cloned in pUC18. Using the plasmid of pUC18-Mon as templates, monellin were amplified by PCR with addition of BspT104 I and Kpn I in the 5'primer and 3'primer, respectively. The amplified gene was cloned into vector pPICZaA by BspT104 I / Kpn I, the recombinant vector pPICZA-Mon was constructed. The recombinant plasmid was linearized with SacI and transformed into GS115 strain by electroporation.The positive recombinant was confirmed by PCR. Under induction by methanol, the product was analysed by Trincine SDS-PAGE, the result demonstrated that Monellin was successfully expressed of which molecular weight was about 11 kD.Response surface methodology (RSM) was adopted to optimize the basal salt medium (BSM) components for Pichia pastoris. With software Design Expert 7.0, the influence of initial medium constituents were studied by Plackett-Burma. The results showed that yeast extract, Calcium sulfate and potassium phosphate buffer were the main factors. The path of steepest ascent was used to approach the optimal response area. Box-Behnken design and RSM analysis showed that the optimal conditions were as follows: yeast extract 6 g/L, Calcium sulfate 0.3 g/L, magnesium sulfate 4.5 g/L, glycerol 40 g/L, ammonium sulfate 12.5 g/L, potassium phosphate buffer 7.7%. The optimized condition led to a 0.5 fold increase in yeast biomass.With the optimized medium, the feed-batch fermentation was investigated in 5 liter Bamn fermentor. Firstly, glycerol was added until the biomass optical density up to 200 at 600nm; then, methanol and glycerol was added, after about 4-5 hours; lastly, methanol was used to induced protein expression. It is important to maintain the dissolved oxygen (DO) concentration at a certain level (>20%) to ensure growth of Pichia pastoris during fermention. After induced 72 hours, the yeast biomass achieved final optical density to 512 at 600nm, and the Monellin yield reached 1.2 g/L.