Expression Of Monellin Gene And Vitreoscilla Hemoglobin Gene In The Methylotrophic Yeast Pichia Pastoris

Expression of Monellin Gene and Vitreoscilla Hemoglobin Genein the Methylotrophic Yeast Pichia pastorisAccording to the monellin amino acid sequence and Pichia pastoris preference codon usage, the monellin gene was designed and synthesized. Then it was cloned into vector pPIC9(with a -factor signal), and the recombinant expression vector pPIC9M was obtained. Pichia pastoris stain GS115/pPIC9M was constructed by transfering the expression vector pPIC9M into Pichia pastoris GS115 competent cells via electroporation method. Under the induction by methanol, the expressed monellin was secreted into medium at the level of 0.25g/L.The gene(vgb) coding for the Vitreoscilla hemoglobin (VHb) molecule was cloned and funtionally expressed in Pichia pastoris. Shake flask fermentation experiments showed that the presence of the intracellular VHb in Pichia pastoris efficiently enhanced cell growth under oxygen-poor conditions.The recombinant expression vector pPIC3.5KV with vgb gene was used to transform the Pichia pastoris GS115/pPIC9M via electroporation, yeast strain Pichia pastoris GS115/pPIC9M/pPIC3.5K containing the integrated monellin gene and vgb gene was obtained by G418 (0.5mg/ml) plate. Shake flask fermentation experiments showed that the expression of the intracellular VHb in the yeast strain Pichia pastoris GS115/pPIC9M/pPIC3.5K improved monellin production. Secreted monellin is about 0.3g/L.In order to enhance the expression level of monellin in Pichia pastoris, we optimized the transformant growth conditions in shake flasks including carbon sources in growth medium, inoculation ratio, biotin concentration, methanol concentration , pH and inducing time. Monellin productivity of 2.0g/L was obtained in the 30-liter fermentor .