Glauche Florian, Glazyrina Julia, Cruz Bournazou Mariano Nicolas, Kiesewetter Gregor, Cuda Fabian, Goelling Detlef, Raab Andreas, Lang Christine, Neubauer Peter
Chair of Bioprocess Engineering Technische Universität Berlin Berlin Germany.
Organobalance GmbH Berlin Germany.
Eng Life Sci. 2017 Aug 29;17(11):1215-1220. doi: 10.1002/elsc.201600029. eCollection 2017 Nov.
is a popular expression system for recombinant proteins. In most cases, production processes are performed as carbon-limited fed-batch cultures to avoid aerobic ethanol formation. Especially for constitutive expression systems, the specific product formation rate depends on the specific growth rate. The development of optimal feeding strategies strongly depends on laboratory-scale cultivations, which are time and resource consuming, especially when continuous experiments are carried out. It is therefore beneficial for accelerated process development to look at alternatives. In this study, AH22 secreting a heterologous endo-polygalacturonase (EPG) was characterized in microwell plates with an enzyme-based fed-batch medium. Through variation of the glucose release rate, different growth profiles were established and the impact on EPG secretion was analyzed. Product formation rates of 200-400 U (g h) were determined. As a reference, bioreactor experiments using the change-stat cultivation technique were performed. The growth-dependent product formation was analyzed over dilution rates of = 0.01-0.35 with smooth change of at a rate of 0.003 h. EPG production was found to be comparable with a of 400 U (g h) at = 0.27 h. The presented results indicate that parallel miniaturized fed-batch cultures can be applied to determine product formation profiles of putative production strains. With further automation and parallelization of the concept, strain characterization can be performed in shorter time.
是一种用于重组蛋白的常用表达系统。在大多数情况下，生产过程以碳限制补料分批培养的方式进行，以避免有氧乙醇的形成。特别是对于组成型表达系统，特定产物形成速率取决于特定生长速率。最佳补料策略的开发强烈依赖于实验室规模的培养，这既耗时又耗资源，尤其是在进行连续实验时。因此，寻找替代方法有利于加速工艺开发。在本研究中，使用基于酶的补料分批培养基在微孔板中对分泌异源内切多聚半乳糖醛酸酶（EPG）的AH22进行了表征。通过改变葡萄糖释放速率，建立了不同的生长曲线，并分析了其对EPG分泌的影响。测定了200 - 400 U/（g·h）的产物形成速率。作为参考，使用变化统计培养技术进行了生物反应器实验。在稀释率D = 0.01 - 0.35且D以0.003 h⁻¹的速率平稳变化的条件下，分析了生长依赖性产物形成。发现在D = 0.27 h⁻¹时，EPG产量与400 U/（g·h）相当。所呈现的结果表明，平行小型化补料分批培养可用于确定假定生产菌株的产物形成曲线。随着该概念的进一步自动化和平行化，菌株表征可在更短时间内完成。
