Temperature‐dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli

Abstract

Based on the recently constructed Escherichia coli itaconic acid production strain ita23, we aimed to improve the productivity by applying a two‐stage process strategy with decoupled production of biomass and itaconic acid. We constructed a strain ita32 (MG1655 ΔaceA Δpta ΔpykF ΔpykA pCadCs), which, in contrast to ita23, has an active tricarboxylic acid (TCA) cycle and a fast growth rate of 0.52 hr−1 at 37°C, thus representing an ideal phenotype for the first stage, the growth phase. Subsequently we implemented a synthetic genetic control allowing the downregulation of the TCA cycle and thus the switch from growth to itaconic acid production in the second stage. The promoter of the isocitrate dehydrogenase was replaced by the Lambda promoter (p R) and its expression was controlled by the temperature‐sensitive repressor CI857 which is active at lower temperatures (30°C). With glucose as substrate, the respective strain ita36A grew with a fast growth rate at 37°C and switched to production of itaconic acid at 28°C. To study the impact of the process strategy on productivity, we performed one‐stage and two‐stage bioreactor cultivations. The two‐stage process enabled fast formation of biomass resulting in improved peak productivity of 0.86 g/L/hr (+48%) and volumetric productivity of 0.39 g/L/hr (+22%) in comparison to the one‐stage process. With our dynamic production strain, we also resolved the glutamate auxotrophy of ita23 and increased the itaconic acid titer to 47 g/L. The temperature‐dependent activation of gene expression by the Lambda promoters (p R/p L) has been frequently used to improve protein or, in a few cases, metabolite production in two‐stage processes. Here we demonstrate that the system can be as well used in the opposite direction to selectively knock‐down an essential gene (icd) in E. coli to design a two‐stage process for improved volumetric productivity. The control by temperature avoids expensive inducers and has the potential to be generally used to improve cell factory performance.

Broadening the Scope of Enforced ATP Wasting as a Tool for Metabolic Engineering in Escherichia coli

“…As was shown earlier, the metabolism usually shuts down in nongrowing cells just to cover cellular maintenance, leading to low substrate uptake rates and productivities and thus severely limiting TSF processes. [23,24] This can also be observed in the present study: the specific glucose uptake rate of the control strain slows down under nitrogen starvation conditions until it comes almost to a complete rest even though the substrate was not completely consumed. In recent years, there have been several attempts to maintain high metabolic rates in the stationary phase, e.g.…”

“…We therefore analyzed the effect of overexpressed ATPase under growth‐arrested conditions (mimicking the production phase of a TSF) caused by nitrogen starvation, where the biomass of the ATPase strain and the control strain remain constant. As was shown earlier, the metabolism usually shuts down in nongrowing cells just to cover cellular maintenance, leading to low substrate uptake rates and productivities and thus severely limiting TSF processes . This can also be observed in the present study: the specific glucose uptake rate of the control strain slows down under nitrogen starvation conditions until it comes almost to a complete rest even though the substrate was not completely consumed.…”

“…One approach to overcome such inherent trade‐offs between high product yield and high volumetric productivity is to use two‐stage fermentation (TSF) with decoupled growth and production phase in contrast to one‐stage fermentation (OSF) with growth‐coupled product synthesis as used in the aforementioned studies. In a recent theoretical study where the productivities of OSF and TSF processes were systematically compared, we found that enforced ATP wasting in the second (production) phase can significantly increase the productivity and thus the competitiveness of TSFs as it keeps a high driving force for substrate uptake also in the production phase where cell growth (usually consuming a large fraction of the substrate taken up) is missing . Accordingly, we here want to give experimental evidence that ATP wasting can serve as a tool to elevate substrate uptake and product synthesis rates in growth‐arrested cells.…”

“…In a recent theoretical study where the productivities of OSF and TSF processes were systematically compared, [22] we found that enforced ATP wasting in the second (production) phase can significantly increase the productivity and thus the competitiveness of TSFs as it keeps a high driving force for substrate uptake also in the production phase where cell growth (usually consuming a large fraction of the substrate taken up) is missing. [23,24] Accordingly, we here want to give experimental evidence that ATP wasting can serve as a tool to elevate substrate uptake and product synthesis rates in growth-arrested cells. A second important goal of this study is to test the use of an inducible ATPase as an ATP wasting mechanism under anaerobic conditions, the preferred operation mode for industrial applications.…”

When Do Two‐Stage Processes Outperform One‐Stage Processes?

“…For example, typical values for Escherichia coli have been reported to be in the range of 0.5–4.3 mmol glucose/gDW/h in the production phase compared to 10–15 mmol/gDW/h usually observed in the growth phase. For example, in a recent study implementing a TSF process for production of itaconate with E. coli , a drastically reduced glucose uptake rate (below 1 mmol/gDW/h) was observed when switching from growth to production . Stoichiometric calculations indicate that substrate uptake in non‐growing cells mainly covers the demand of ATP required for non‐growth associated maintenance processes .…”