Scale-up bioprocess development for production of the antibiotic valinomycin in Escherichia coli based on consistent fed-batch cultivations.
Scale-up bioprocess development for production of the antibiotic valinomycin in Escherichia coli based on consistent fed-batch cultivations.
复制标题
DOI：
10.1186/s12934-015-0272-y
复制
发表时间：
2015-06-12
影响因子：
6.4
通讯作者：
Neubauer P
中科院分区：
文献类型：
--
作者：
Li J;Jaitzig J;Lu P;Süssmuth RD;Neubauer P
在大肠杆菌中异源生产天然产物已经成为获得感兴趣分子的有吸引力的策略。尽管技术上可行，但其中大多数仍限于实验室规模生产。因此，有必要针对工业规模条件下目标天然产物的过量生产，制定合理的生物过程放大策略。为此，我们使用在E.大肠杆菌作为一个模型系统，可扩展的生物工艺开发的基础上一致的补料分批培养。在这项工作中，葡萄糖限制补料分批策略的基础上纯无机盐培养基用于缬氨霉素生产的所有规模。最佳葡萄糖进料速率最初通过使用生物催化控制的葡萄糖释放（EnBase®技术）在24孔板中平行培养并连续监测pH和溶解氧来检测。这些结果在摇瓶中得到了证实，其中当比生长速率降低到0.1 h-1以下时，缬氨霉素的积累最高。在实验室规模生物反应器中的高细胞密度补料分批培养中也观察到这种相关性。生物反应器发酵产生的缬氨霉素滴度超过2 mg L−1，基于浓缩葡萄糖溶液的进料。缬氨霉素生产不受按比例缩小的两室反应器中的振荡条件（即葡萄糖和氧气）的影响，这可以模拟工业生物反应器中的类似情况，表明该工艺非常稳健，并且将该工艺放大到更大的工业规模似乎是现实的情况。缬氨霉素生产从mL体积扩大到10 L，并始终使用补料分批技术。这项工作为可扩展的生物过程开发提供了一种稳健且可靠的方法，并代表了异源表达天然产品工艺向工业规模一致开发的示例。
Heterologous production of natural products in Escherichia coli has emerged as an attractive strategy to obtain molecules of interest. Although technically feasible most of them are still constrained to laboratory scale production. Therefore, it is necessary to develop reasonable scale-up strategies for bioprocesses aiming at the overproduction of targeted natural products under industrial scale conditions. To this end, we used the production of the antibiotic valinomycin in E. coli as a model system for scalable bioprocess development based on consistent fed-batch cultivations. In this work, the glucose limited fed-batch strategy based on pure mineral salt medium was used throughout all scales for valinomycin production. The optimal glucose feed rate was initially detected by the use of a biocatalytically controlled glucose release (EnBase® technology) in parallel cultivations in 24-well plates with continuous monitoring of pH and dissolved oxygen. These results were confirmed in shake flasks, where the accumulation of valinomycin was highest when the specific growth rate decreased below 0.1 h−1. This correlation was also observed for high cell density fed-batch cultivations in a lab-scale bioreactor. The bioreactor fermentation produced valinomycin with titers of more than 2 mg L−1 based on the feeding of a concentrated glucose solution. Valinomycin production was not affected by oscillating conditions (i.e. glucose and oxygen) in a scale-down two-compartment reactor, which could mimic similar situations in industrial bioreactors, suggesting that the process is very robust and a scaling of the process to a larger industrial scale appears a realistic scenario. Valinomycin production was scaled up from mL volumes to 10 L with consistent use of the fed-batch technology. This work presents a robust and reliable approach for scalable bioprocess development and represents an example for the consistent development of a process for a heterologously expressed natural product towards the industrial scale.
登录
查看更多内容
影响因子：
6.4
作者：
Soini J;Falschlehner C;Liedert C;Bernhardt J;Vuoristo J;Neubauer P
通讯作者：
Neubauer P
影响因子：
4.4
作者：
Gruenewald, S;Mootz, HD;Stachelhaus, T
通讯作者：
Stachelhaus, T
影响因子：
2.9
作者：
Zhang, Haoran;Skalina, Karin;Pfeifer, Blaine A.
通讯作者：
Pfeifer, Blaine A.
影响因子：
4.7
作者：
Jaitzig, Jennifer;Li, Jian;Neubauer, Peter
通讯作者：
Neubauer, Peter
影响因子：
4.1
作者：
Enfors, SO;Jahic, M;Manelius, Å
通讯作者：
Manelius, Å