An improved high cell density cultivation—iHCDC—strategy for leucine auxotrophic Escherichia coli K12 ER2507

Abstract

An improved high cell density cultivation-iHCDC-strategy for leucine auxotrophic Escherichia coli K12 ER2507Fed-batch processes are commonly used tools in high cell density cultivation of Escherichia coli. However, the setup of a fed-batch process can be challenging, especially when working with genetically modified strains. This work deals with the design of a specific strategy for the leucine auxotrophic E. coli strain K12 ER2507 that is of interest for arabinose-inducible gene expression systems due to its ara-14 mutation. To set up this process the optimum yield coefficient of biomass from leucine was determined by use of a design of experiments tool. Unfortunately, the yield coefficient is negatively influenced by both leucine and glucose concentrations. Furthermore, an inhibitory effect of leucine reduced the specific growth rate even at low concentration. Under consideration of these problems, an improved high cell density cultivation (iHCDC) for E. coli K12 ER2507 was established. The start medium was set up without any C-source to omit a batch phase with high glucose concentration and substrate feeding was initiated directly upon inoculation. Due to the poor solubility of leucine in feed medium, the amino acid was dissolved in the base. With the improved high cell density cultivation process a final cell density of more than 90 g/L was obtained reproducibly.

High-density Cultivation in the Production of Microbial Products

“…Use of defined media is, however, not without its own pitfalls, and requires medium optimization and tweaking of feeding strategies in high-density cultivations. As an example, Beckmann et al 38 reported Leucine was inhibitory to leucine-auxotrophic Escherichia coli K12 ER2507 even at a concentration of 0.3 g L -1 . This effect could be overcome by optimizing the feeding strategy and medium composition.…”

“…While early approaches were trial and error processes, statistical techniques such as Response Surface Methodology (RSM) or factorial design can provide a more organized avenue for the Design of Experiments (DOE). Beckmann et al 38 applied full factorial design of experiments to optimize the concentration of glucose and leucine in the feed medium and achieved a cell density of 97 g L -1 for the leucine auxotrophic E. coli strain K12 ER2507. Subramaniam et al [48][49][50] developed a mi nimal medium by replacing costly yeast extract with vitamins such as biotin, calcium pantothenol and inositol.…”

High Cell Density Cultivation (HCDC) of E. coli with Intensive Multiple Sequential Batches Together with Fed-Batch at Cell Level (FBC) Superior to the Fed-Batch Technique

“…Most HCDC were designed only as the fed-batch technique and researched to the optimization of substrate feeding strategies to eliminate the problems of using too high concentration of substrate especially for glucose. Glucose or sucrose at a very high concentration enhances the biosynthesis of growth inhibitory by-products, i.e., acetate in bacteria and ethanol in yeasts due to the Crabtree effect [3,6]. The HCDC technique development often studies the optimum conditions of two main factors, i.e., media and cultivation technique, such as substrate feeding strategies.…”

“…It is a representative model microorganism of bacteria due to its holds several advantages, e.g., having more data both in genetics and culture conditions from long time investigations, well-explored characteristics, fast growth (doubling time of 15-20 min) on chemically defined media, cost effectiveness and scalability [6,13].…”