Fan, QiNeubauer, PeterGimpel, Matthias2021-12-132021-12-132021-10-18https://depositonce.tu-berlin.de/handle/11303/14037http://dx.doi.org/10.14279/depositonce-12810Background: Autoinduction systems can regulate protein production in Escherichia coli without the need to monitor cell growth or add inducer at the proper time following culture growth. Compared to classical IPTG induction, autoinduction provides a simple and fast way to obtain high protein yields. In the present study, we report on the optimization process for the enhanced heterologous production of the Ralstonia eutropha regulatory hydrogenase (RH) in E. coli using autoinduction. These autoinduction methods were combined with the EnPresso B fed-batch like growth system, which applies slow in situ enzymatic glucose release from a polymer to control cell growth and protein synthesis rate. Results: We were able to produce 125 mg L−1 RH corresponding to a productivity averaged over the whole process time of 3 mg (L h)−1 in shake flasks using classic single-shot IPTG induction. IPTG autoinduction resulted in a comparable volumetric RH yield of 112 mg L−1 and due to the shorter overall process time in a 1.6-fold higher productivity of 5 mg (L h)−1. In contrast, lactose autoinduction increased the volumetric yield more than 2.5-fold and the space time yield fourfold reaching 280 mg L−1 and 11.5 mg (L h)−1, respectively. Furthermore, repeated addition of booster increased RH production to 370 mg L−1, which to our knowledge is the highest RH concentration produced in E. coli to date. Conclusions: The findings of this study confirm the general feasibility of the developed fed-batch based autoinduction system and provide an alternative to conventional induction systems for efficient recombinant protein production. We believe that the fed-batch based autoinduction system developed herein will favor the heterologous production of larger quantities of difficult-to-express complex enzymes to enable economical production of these kinds of proteins.en540 Chemie und zugeordnete WissenschaftenEscherichia coliralstonia eutrophadifficult-to-express proteinEnPresso Bfed-batch like cultivationIPTG autoinductionlactose autoinductionregulatory hydrogenaseArticle1475-285934663324