酿酒酵母pdc基因缺陷菌株的构建及其丙酮酸发酵特性

Construction of pdc-deficient Saccharomyces cerevisiae and its pyruvic acid fermentation characteristics

丙酮酸是一种重要的有机酸,在聚合物、化妆品、食品添加剂、医药等领域具有广泛应用。酿酒酵母被认为是丙酮酸生产的潜在最适微生物,但其糖酵解过程产生的丙酮酸会在胞质内的丙酮酸脱羧酶催化作用下降解为CO2和乙醛,造成碳代谢流的损失。为将更多的碳代谢流引向丙酮酸合成,该研究通过敲除丙酮酸脱羧酶的3个结构基因(pdc1、pdc5和pdc6),显著地促进了丙酮酸积累,但也造成突变菌株XY-156生长缓慢。与进化之前相比,采用适应性进化策略驯化得到的菌株XY-156A,在细胞生长、葡萄糖消耗以及丙酮酸积累方面均获得显著提高;进一步利用2 L发酵罐进行批次补料发酵试验,发酵76 h丙酮酸产量可达105 g/L,生产强度为1.38g/(L·h),得率为0.5 g/g葡萄糖。结果表明,采用失活丙酮酸脱羧酶与适应性定向进化相结合的策略改造酿酒酵母,能够实现其高效积累丙酮酸,可为生物基丙酮酸工业化生产奠定坚实的基础。

Pyruvic acid is an important organic acid with wide applications in polymer,cosmetic,food additive,pharmaceutical and other fields.Saccharomyces cerevisiae is considered as the best candidate microorganism for pyruvic acid production.However,pyruvic acid produced during glycolysis could be catalyzed by pyruvate decarboxylase in the cytoplasm to degrade into CO2 and acetaldehyde,resulting in the loss of carbon metabolic flux.In order to redirect more carbon metabolic flux to pyruvic acid synthesis,the deletions of the three structural genes of pyruvate decarboxylase(pdc1,pdc5 and pdc6)were performed,which significantly promoted pyruvic acid accumulation.Nevertheless,poor growth of the mutant strain XY-156 was also observed.Compared with the non-evolved strain XY-156,the evolved strain XY-156 A obtained by adaptive evolution showed significant improvement in cell growth,glucose consumption and pyruvate accumulation.Fed-batch fermentation was further performed in a 2 L stirred bioreactor,and up to 105 g/L pyruvic acid was obtained within 76 h,with a productivity of 1.38 g/(L·h)and a yield of0.5 g/g glucose.The results showed that the combination of inactivating pyruvate decarboxylase with adaptive evolution could achieve efficient pyruvic acid accumulation in S.cerevisiae,and could lay a solid foundation for the industrial production of biological pyruvic acid.