Enforced ATP futile cycling increases specific productivity and yield of anaerobic lactate production in Escherichia coli

Abstract

The manipulation of cofactor pools such as ATP or NAD(P)H has for long been recognized as key targets for metabolic engineering of microorganisms to improve yields and productivities of biotechnological processes. Several works in the past have shown that enforcing ATP futile cycling may enhance the synthesis of certain products under aerobic conditions. However, case studies demonstrating that ATP wasting may also have beneficial effects for anaerobic production processes are scarce. Taking lactic acid as an economically relevant product, we demonstrate that induction of ATP futile cycling in Escherichia coli leads to increased yields and specific production rates under anaerobic conditions, even in the case where lactate is already produced with high yields. Specifically, we constructed a high lactate producer strain KBM10111 (= MG1655 ΔadhE::Cam ΔackA-pta) and implemented an IPTG-inducible overexpression of ppsA encoding for PEP synthase which, together with pyruvate kinase, gives rise to an ATP consuming cycle. Under induction of ppsA, KBM10111 exhibits a 25% higher specific lactate productivity as well as an 8% higher lactate yield. Furthermore, the specific substrate uptake rate was increased by 14%. However, trade-offs between specific and volumetric productivities must be considered when ATP wasting strategies are used to shift substrate conversion from biomass to product synthesis and we discuss potential solutions to design optimal processes. In summary, enforced ATP futile cycling has great potential to optimize a variety of production processes and our study demonstrates that this holds true also for anaerobic processes.

Manipulation of the ATP pool as a tool for metabolic engineering

“…The excretion rate of fermentation products could also be increased by implementing ATP futile cycles aerobically [17,20,21]. For anaerobic conditions, only recently an elevated specific lactate production rate was observed by implementing an ATP futile cycle between pyruvate and phosphoenolpyruvate (PEP) through overexpression of the PEP synthase [22]. In concordance with other experimental results, this study also reported a decreased growth rate and significantly increased glucose uptake rate.…”

“…In such a scenario, in addition to the product yield and specific productivity, even the volumetric productivity could be increased. However, the observed increased substrate uptake rates [22] upon ATP wasting could not completely restore the original growth rate.…”

Stimulation of acetoin production in metabolically engineered Lactococcus lactis by increasing ATP demand

“…As described previously, the AAC strategies are able to increase the specific productivity and yield for chemical production (Hädicke et al 2015); however, there are no reports showing that the volumetric productivity and yield can be improved simultaneously, and this is something we have achieved in this study by fine-tuning the expression of the F 1 -ATPase. We selected acetoin production for this investigation as acetoin formation generates ATP (2 mol ATP per mol acetoin formed), and it is essential for the strategy to work that the biosynthetic pathway involved generates surplus ATP.…”

“…Patnaik et al was able to stimulate the glycolytic flux in Escherichia coli under aerobic conditions, by overexpressing phosphoenolpyruvate synthase, which generated an ATP consuming futile cycle (Patnaik et al 1992). Hädicke et al used the same futile cycle for improving lactate production in E. coli under anaerobic conditions (Hädicke et al 2015). Koebmann et al took a different approach and expressed the F 1 -ATPase in the cytoplasm of E. coli and achieved the same effect, i.e., stimulation of glycolytic flux (Koebmann et al 2002b).…”

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

“…Consequently, cells have to be forced to high glucose uptake rates even when they are not growing. As the ATP maintenance demand seems to be the main driving force for itaconic acid production in this phase, ATP‐wasting strategies (Hädicke, Bettenbrock, & Klamt, 2015; Hädicke and Klamt, 2015; Liu, Kandasamy, Wurtz, Jensen, & Solem, 2016) could be used to increase substrate turnover. Besides, manipulation of regulatory networks (Michalowski, Siemann‐Herzberg, & Takors, 2017) and certain nutrient limitations (Chubukov & Sauer, 2014) might further increase uptake rates.…”