(485bo) Adaptive Glucose Feeding for An E. Coli Recombinant Production Process

A recombinant E. coli fermentation process was developed to reach greater than 80 gdw/L and a peak respiration of 5 mMol O2 / L-min in order to ensure that an adequate quantity of therapeutic protein was generated. The production host was a W3110-based strain containing a pBR322-based plasmid encoding the recombinant protein of interest behind the phoA promoter.

A common element of high cell density microbial fermentation process design is the requirement of adding sufficient nutrients to the fermentor without suffering the ill effects of overflow metabolism. Under conditions of excess glucose or sometimes in glucose-limited conditions, E. coli produces acetate via the Crabtree effect which can accumulate and inhibit growth and recombinant product formation. In order to reach commercially desirable cell densities of ~250 OD550, or greater than 80 gdw/L, it may be necessary to feed up to 450 g/L of glucose by the end of a fermentation and it is necessary to do so in a way that minimizes acetate accumulation.

A typical approach to glucose feeding is to batch glucose into the fermentor and then feed glucose on a time-based schedule until a target dissolved oxygen tension is reached. Previous work has demonstrated that glucose pulses can be used to measure glucose uptake rates. The present work deals with the solution to the problem posed by a host/plasmid/product combination that proved particularly prone to acetate formation when exposed to frequent glucose feed pulse tests. Here, we describe an approach that was inspired by securities analyses and is based on moving average comparisons (the MACD technical chart) which are used to detect growth stalls when using a feed schedule that was optimized to minimize acetate accumulation. When a growth stall is detected using this method the remaining feed schedule is increased by 5% in order to maintain a targeted rate of respiration increase.

Implementation of this adaptive glucose feed schedule allowed the project to meet its goals for product yield by improving process robustness and eliminating runaway overfeeding. The previously attempted adaptive algorithm was not optimal due to this production system's propensity to generate acetate when exposed to feed pulses. The fermentation reaches a final OD550 of 150 and cell density of approximately 80 g/L dcw in 36 hours.