毕赤酵母Gpn12异戊烯基转移酶NovQ催化合成MK-3

Synthesis of vitamin K_2 by isopentenyl transferase NovA in Pichia pastoris Gpn12

对异戊烯基转移酶NovQ在毕赤酵母Gpn12异源表达过程中诱导剂甲醇添加量进行了探究,并以毕赤酵母Gpn12全细胞为酶源,以甲萘醌、异戊烯醇为前体,催化合成维生素K2(MK-3)。每24 h添加2%甲醇时,NovQ表达量提高约36%。考察摇瓶中初始pH、温度、甲醇添加量、前体(甲萘醌、异戊烯醇)添加量、催化时间、十六烷基三甲基溴化铵(CTAB)添加量等7个因素对Gpn12全细胞催化合成MK-3的影响,发现催化温度、甲萘醌添加量、催化时间影响显著,对3个显著因素进行响应面优化得出催化条件为:催化温度31.56℃,甲萘醌添加量295.54 mg/L,催化时间15.87 h,优化后的摇瓶MK-3产量达到98.47 mg/L,与响应面预测结果一致,较优化前对照组提高了35%。在30 L发酵罐进行生物催化实验,催化时间24 h,细胞催化剂浓度220 g(干重)/L,MK-3产量达到189.67 mg/L。该方法为Gpn12规模化生产MK-3奠定了一定的基础。

The effect of methanol addition on the heterologous expression of isoprenyl transferase NovQ was studied in Pichia pastoris Gpn12, with menadione and isopentenol as precursors to catalyze vitamin K2(MK-3) synthesis. The expression of NovQ increased by 36% when 2% methanol was added every 24 h. The influence of initial p H, temperature, methanol addition, precursors(menadione, isopentenol) addition, catalytic time and cetyltrimethyl-ammonium bromide(CTAB) addition were explored in the P. pastoris whole-cell catalytic synthesis process of MK-3 in shaking flask. Three significant factors were then studied by response surface method. The optimal catalytic conditions obtained were as follows: catalytic temperature 31.56 ℃, menadione 295.54 mg/L, catalytic time 15.87 h. Consistent with the response surface prediction results, the optimized yield of MK-3 reached 98.47 mg/L in shaking flask, 35% higher than that of the control group. On this basis, the production in a 30-L fermenter reached 189.67 mg/L when the cell catalyst of 220 g/L(dry weight) was used to catalyze the synthesis for 24 h. This method laid the foundation for the large-scale production of MK-3 by P. pastoris Gpn12.