Fermentation process for reforming yeast of liking for carbinol
A fermentation method and batch fermentation technology are applied in the field of fermentation of recombinant methanolophilic yeast, which can solve the problems of affecting and restricting the application of phenotype Pichia pastoris, and achieve the effect of improving the expression of recombinant gene
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Embodiment 1
[0013] The recombinant Pichia pastoris expressing angiostatin was constructed by Gan Renbao's research group at the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (see literature: Acta Biochemistry and Biophysics, 2001, 33: 291- 295), with His + Mut S Phenotype.
[0014] After thawing the 1mL glycerol frozen strain preservation tube, take 0.7mL seed solution and put it into 25mL BMGY medium (1L contains: YNB 13.4g, glycerol 10mL, biotin 0.4mg, 1mol / L pH6.0 phosphate buffer solution 100mL, Yeast Extract 10g, Polypepton 20g) in a 250mL shake flask, at 250rpm, 30°C shaker culture for 14h, to obtain first-grade seeds. Put 25mL shake flask fermentation medium into 250mL shake flask (1L contains: glycerol 5g, (NH 4 ) 2 SO 4 2g, CaSO 4 0.06g, K 2 SO 4 1.2g, MgSO 4 ·7H 2 O 1g, biotin 0.2mg, 1mol / L pH6.0 phosphate buffer solution 100mL), insert 5mL first-class seeds, culture at 250rpm, 30°C shaker for 12-14h until the glycerol in the mediu...
Embodiment 2
[0017] The primary seeds as described in Example 1 were respectively inoculated into three 500 mL shake flasks filled with 50 mL of BMGY medium, and cultured on a shaker at 250 rpm at 30° C. for 7-8 hours to obtain secondary seeds. Secondary seeds were inoculated in the BSM medium (1L containing: 85% phosphoric acid 26.7mL, CaSO 4 0.93g, K 2 SO 4 18.2g, MgSO 4 ·7H 2 O 14.9g, KOH 4.13g, glycerol 40g, PTM1 solution 4.35mL. 1L PTM1 contains: CuSO 4 6.0g, KI 0.8g, MnSO 4 3.0g, Na 2 MoO 4 0.2g, H 3 BO 3 0.2g, CoCl 2 0.5g, ZnCl 2 20.0g, FeSO 4 ·7H 2 O 65.0g, biotin 0.2g, sulfuric acid 5mL), the volume after inoculation is 2.5L (inoculum size 7%), the temperature is 30°C, and the ventilation rate is 4mL / min. Adjust the pH to 5.0 by adding 5M KOH automatically, and manually adjust the stirring speed to maintain the dissolved oxygen not lower than 20%. After glycerol was exhausted, start to feed 50% (w / w) glycerol (12mL PTM1 solution per liter) after starvation f...
Embodiment 3
[0019] The fermentation process in the thalline growth stage is as described in Example 2. After entering the induced expression stage, feed methanol (add 12mL PTM1 solution per liter), and the methanol concentration in the fermenter is maintained at 5g / L by the methanol detection and control system; At the same time, 51% (w / w) lactic acid (containing 12mL PTM1 solution per liter) was added, and the acceleration rate of lactic acid flow was gradually increased from 5.7g / h in 49.1h to 21.8g / h in 102.6h and ended with 14% (v / v) Ammonia water to adjust pH to 5.0. After 64.5 hours of induction (full fermentation time is 113 hours), the cell concentration reached 87g / L, the expression level of angiostatin reached 191mg / L, and the average specific production rate reached 0.04mg / g·h. The results of angiostatin production using different carbon sources and methanol mixed carbon sources in the induction stage are shown in Table 2. The expression level of recombinant protein was higher ...
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