Goers Lisa, Ainsworth Catherine, Goey Cher Hui, Kontoravdi Cleo, Freemont Paul S, Polizzi Karen M
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.
Centre for Synthetic Biology and Innovation, Imperial College London, London, UK.
Biotechnol Bioeng. 2017 Jun;114(6):1290-1300. doi: 10.1002/bit.26254. Epub 2017 Feb 23.
Many high-value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole-cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole-cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole-cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole-cell biosensors. Biotechnol. Bioeng. 2017;114: 1290-1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
许多高附加值的重组蛋白，如治疗性糖蛋白，是通过哺乳动物细胞培养生产的。为了优化这些培养物的生产力，监测细胞代谢很重要，例如营养物质的利用和代谢废物的积累。一种值得关注的代谢废物是乳酸，其过量积累会降低细胞生长和蛋白质产量。目前检测乳酸的方法在成本、灵敏度和稳健性方面存在局限性。因此，我们基于lldPRD操纵子开发了一种全细胞大肠杆菌乳酸生物传感器，并成功用于监测哺乳动物细胞培养物中的乳酸浓度。通过实际样品和分析验证，我们证明我们的生物传感器可用于对复杂样品中的代谢物进行高精度、高灵敏度和高稳健性的绝对定量。重要的是，我们的全细胞生物传感器能够检测到比行业标准方法低两个数量级以上浓度的乳酸，这使其可用于监测早期培养物中的乳酸浓度。鉴于乳酸在各种工业和临床环境中的重要性，我们预计我们的全细胞生物传感器可用于解决一系列有趣的生物学问题。它还为如何利用自然界中可用于代谢物传感的丰富基因操纵子来开发其他全细胞生物传感器提供了蓝图。《生物技术与生物工程》2017年；114: 1290 - 1300。© 2017作者。《生物技术与生物工程》由威利期刊公司出版。
