López Javiera, Cataldo Vicente F, Peña Manuel, Saa Pedro A, Saitua Francisco, Ibaceta Maximiliano, Agosin Eduardo
Centro de Aromas and Sabores, DICTUC S.A., Santiago, Chile.
Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Santiago, Chile.
Front Bioeng Biotechnol. 2019 Jul 18;7:171. doi: 10.3389/fbioe.2019.00171. eCollection 2019.
Robust fermentation performance of microbial cell factories is critical for successful scaling of a biotechnological process. From shake flask cultivations to industrial-scale bioreactors, consistent strain behavior is fundamental to achieve the production targets. To assert the importance of this feature, we evaluated the impact of the yeast strain design and construction method on process scalability -from shake flasks to bench-scale fed-batch fermentations- using two recombinant strains capable of producing β-carotene; SM14 and βcar1.2 strains. SM14 strain, obtained previously from adaptive evolution experiments, was capable to accumulate up to 21 mg/g of β-carotene in 72 h shake flask cultures; while the βcar1.2, constructed by overexpression of carotenogenic genes, only accumulated 5.8 mg/g of carotene. Surprisingly, fed-batch cultivation of these strains in 1L bioreactors resulted in opposite performances. βcar1.2 strain reached much higher biomass and β-carotene productivities (1.57 g/L/h and 10.9 mg/L/h, respectively) than SM14 strain (0.48 g/L/h and 3.1 mg/L/h, respectively). Final β-carotene titers were 210 and 750 mg/L after 80 h cultivation for SM14 and βcar1.2 strains, respectively. Our results indicate that these substantial differences in fermentation parameters are mainly a consequence of the exacerbated Crabtree effect of the SM14 strain. We also found that the strategy used to integrate the carotenogenic genes into the chromosomes affected the genetic stability of strains, although the impact was significantly minor. Overall, our results indicate that shake flasks fermentation parameters are poor predictors of the fermentation performance under industrial-like conditions, and that appropriate construction designs and performance tests must be conducted to properly assess the scalability of the strain and the bioprocess.
微生物细胞工厂强大的发酵性能对于生物技术工艺的成功放大至关重要。从摇瓶培养到工业规模的生物反应器，一致的菌株行为是实现生产目标的基础。为了强调这一特性的重要性，我们使用两种能够产生β-胡萝卜素的重组菌株（SM14和βcar1.2菌株）评估了酵母菌株设计和构建方法对工艺可扩展性的影响——从摇瓶到实验室规模的补料分批发酵。SM14菌株先前通过适应性进化实验获得，在72小时的摇瓶培养中能够积累高达21mg/g的β-胡萝卜素；而通过类胡萝卜素生成基因过表达构建的βcar1.2菌株仅积累了5.8mg/g的胡萝卜素。令人惊讶的是，这些菌株在1L生物反应器中的补料分批培养产生了相反的结果。βcar1.2菌株的生物量和β-胡萝卜素生产率（分别为1.57g/L/h和10.9mg/L/h）比SM14菌株（分别为0.48g/L/h和3.1mg/L/h）高得多。SM14和βcar1.2菌株在培养80小时后的最终β-胡萝卜素滴度分别为210mg/L和750mg/L。我们的结果表明，发酵参数的这些显著差异主要是SM14菌株加剧的Crabtree效应的结果。我们还发现，将类胡萝卜素生成基因整合到染色体上的策略影响了菌株的遗传稳定性，尽管影响非常小。总体而言，我们的结果表明，摇瓶发酵参数不能很好地预测类似工业条件下的发酵性能，必须进行适当的构建设计和性能测试，以正确评估菌株和生物工艺的可扩展性。
