发酵过程优化提高毕赤酵母合成血红素

Enhancing heme biosynthesis in Pichia pastoris by fermentation process optimization

血红素(heme)是一种卟啉衍生物,是血红蛋白的重要组成部分,在氧气运输以及电子传递链中有着重要作用。该研究以一株具有血红素高生产能力的毕赤酵母(Pichia pastoris)为出发菌株,在5 L发酵罐上,从pH、亚铁离子添加量、温度和溶氧水平等方面进行发酵条件优化,提高其积累血红素的水平。最后,对发酵条件最优的一组结果进行了生长动力学和生产动力学的模拟。结果表明,当控制pH值为4时最有利于血红素的生产积累,每日添加2 mL FeSO_(4)·7H_(2)O溶液(100 mg/L)时即可满足毕赤酵母生产血红素对亚铁离子的需求,血红素产量达到160.1 mg/L。应用毕赤酵母甲醇补料发酵过程常用的降低温度提高蛋白表达水平的策略在甘油补料发酵生产血红素过程并未发挥预期作用,血红素产量反而降低。另外,对比不同溶氧水平发现,提高溶氧对提高血红素的积累效果不明显,但是能有效改善菌体的生长状况。最后,通过动力学模型拟合发现,发酵过程中血红素的积累与细胞生长是耦合的。研究成果为促进发酵法生产血红素的工业化提供了借鉴意义。

As an important component of hemoglobin,heme is a porphyrin derivative which involved in the transport and combination of oxygen and other metabolic pathways.In this study,a Pichia pastoris strain with high heme productivity was used as the starting strain.The fermentation conditions were optimized to enhance the heme production in a 5 L fermenter,from the factors of pH,ferrous ion concentration,temperature,and dissolved oxygen level.Then the growth kinetics and production kinetics of the optimal fermentation condition were simulated.It was determined that controlling pH at 4 was the most favorable for heme production.The daily addition of 2 mL of FeSO_(4)·7H_(2)O solution(100 mg/L)adequately could satisfy the ferrous ion requirements for heme production,resulting in a heme production of 160.1 mg/L.The strategy of reducing temperature to enhance expression levels employed in the methanol supplementation fermentation process did not obtain the anticipated outcome in the glycerol supplementation fermentation process.Conversely,it resulted in a decline in heme production.In addition,by comparing different dissolved oxygen levels,it was found that increasing dissolved oxygen had no obvious effect on the accumulation of heme,but could effectively improve the cell growth.Finally,result of kinetic model fitting presented that the accumulation of heme was coupled with the cell growth.The obtained results could serve as references for the industrial fermentation production of heme.