Correlation of fermentation redox potential and induction of recombinant E. coli expression system

Abstract

Currently, Escherichia coli (E. coli) is widely adopted as a host for recombinant genes to overexpress. It is common to regard optical density (OD) reading as an indicator to guide IPTG induction of recombinant E. coli and harvest them at an empirical moment. However, it is found that OD reading cannot truly reflect the actively growing cells in the culture. With labor involvement, the frequent sampling for monitoring fermentation progress is prone to result in contamination. Besides, with an improperly designed sampling schedule, the optimal induction moment may be missed. Due to these reasons, a new measurement using redox potential (ORP) to replace OD as a more appropriate method to monitor the fermentation of recombinant E. coli was proposed, implemented and validated. ORP is known as an instantaneous reflection of organisms, either oxidized or reduced, which is related to the fermentation dissolved oxygen (DO) level and the intracellular metabolic reactions. During the culture of recombinant E. coli at 16 ºC, OD ORP and DO profiles were collected and compared. ORP profile was found as a portrait of a real-time growth stage of recombinant cells. And this relationship has been verified to be reproducible and reliable. With the cells rapid build up at the start of exponential phase, ORP declines promptly. Then ORP stayed at this minimal level for over sixty hours until E. coli grew to the end of stationary phase. When the culture was ready for harvest, ORP raised noticeably in a short period of time due to a living cell reduction. In addition, two special features of ORP profile were related to the crucial moments of E. coli growth. Specifically, the fluctuation during the decreasing phase of ORP was recognized as a certain timing for induction. Besides, harvesting cells when ORP increased rapidly was known as an optimal period for both enlarging the protein expression and improving the enzyme activity. DO supply was then noticed insufficient during the fermentation which had a negative effect on cells’ growth. Two methods increasing agitation rate and aeration rate were implemented in order to improve and control DO level. The results demonstrated that DO level was able to be controlled at a sufficient amount by combining these two methods. Comparing the fermentation technology developed in this research with the typical recombinant cell cultivation technique when OD is chosen to guide the IPTG addition, the expression level of prolinase of both techniques was compatible. Our fermentation technology (compared to the conventional cultivation technology) has various advantages, including less prone to culture contamination, less labor involvement, and low operating cost. This proposed technology can be further improved to become an automatic fermentation process towards the production of recombinant products.