Striedner Gerald, Cserjan-Puschmann Monika, Pötschacher Florentina, Bayer Karl
Institute of Applied Microbiology, University of Agricultural Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria.
Biotechnol Prog. 2003 Sep-Oct;19(5):1427-32. doi: 10.1021/bp034050u.
The main goal of this work was to develop a strategy that enables tuning of recombinant gene expression relative to the metabolic capacity of the host cell synthesis machinery. In the past, strong expression systems have been developed in order to maximize recombinant gene expression. However, these systems exert an extremely high metabolic burden onto the host cell, which may even lead to cell death. Hence, the period of recombinant gene expression is significantly reduced, and therefore, maximal yield cannot be attained. To extend the production phase and to achieve optimal yields, adjustment of recombinant gene expression by modulation of the transcription rate is required. To control transcription, we designed a feed regime, which continuously supplies limiting amounts of inducer in a constant ratio to biomass. For the accurate determination of appropriate amounts of inducer, a time shifted exponential substrate and inducer feed strategy has been developed. The potential of this metabolic and engineering integrated approach was proven in fed-batch cultivation experiments using E. coli HMS174(DE3)(pET11ahSOD) as model system. Furthermore, our strategy enables the use of lactose as inducer, since its consumption can be compensated by appropriate feed profiles. The attained results fully comply with all requirements of industrial large scale cultivation and improve the applicability of strong expression systems.
这项工作的主要目标是开发一种策略，能够根据宿主细胞合成机制的代谢能力来调节重组基因的表达。过去，为了使重组基因表达最大化，人们开发了强表达系统。然而，这些系统给宿主细胞带来了极高的代谢负担，甚至可能导致细胞死亡。因此，重组基因表达的时间显著缩短，从而无法获得最大产量。为了延长生产阶段并实现最佳产量，需要通过调节转录速率来调整重组基因的表达。为了控制转录，我们设计了一种补料方式，以恒定比例向生物量持续供应限量诱导剂。为了准确确定合适的诱导剂用量，已开发出一种时间偏移指数底物和诱导剂补料策略。在以大肠杆菌HMS174(DE3)(pET11ahSOD)作为模型系统的补料分批培养实验中，证明了这种代谢与工程相结合方法的潜力。此外，我们的策略能够使用乳糖作为诱导剂，因为其消耗可以通过适当的补料曲线来补偿。所获得的结果完全符合工业大规模培养的所有要求，并提高了强表达系统的适用性。
