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 Vhb在胞内的成功表达，使重组毕赤酵母高甜度monellin的分泌表达量提高了20 % ，达0 . 3g / l 。
2.
Expression of monellin gene and vitreoscilla hemoglobin gene in the methylotrophic yeast pichia pastoris according to the monellin amino acid sequence and pichia pastoris preference codon usage , the monellin gene was designed and synthesized 同时探讨vgb基因在毕赤酵母中的功能，并应用于高甜度monellin重组酵母，解决毕赤酵母高密度发酵中存在的供氧不足的问题，进一步提高高甜度monellin的产量。
3.
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 进一步通过电击转化将vgb基因整合到重组酵母gs115 / ppic9m的基因组中，获得了由高甜度monellin和vgb两个基因同时整合的重组酵母gs115 / ppic9m / ppic3 . 5kv 。
4.
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 同时，采用毕赤酵母偏爱密码子，人工合成了透明颤菌血红蛋白基因( vgb ) ；并构建了vgb基因的酵母胞内表达载体ppic3 . 5kv ，通过电击转化获得了能在胞内表达有生物活性vhb的重组毕赤酵母gs115 / ppic3 . 5kv 。
