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

Liu, Jianming; Kandasamy, Vijayalakshmi; Würtz, Anders; Jensen, Peter Ruhdal; Solem, Christian

doi:10.1007/s00253-016-7687-1

Cited by 40 publications

(52 citation statements)

References 32 publications

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“…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.…”

Section: Resultsmentioning

confidence: 99%

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

Harder

Bettenbrock

Klamt

2017

Biotech & Bioengineering

103

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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.

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Section: Resultsmentioning

confidence: 99%

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

Harder

Bettenbrock

Klamt

2017

Biotech & Bioengineering

103

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“…It has been observed that, in cells where biomass and ATP synthesis are coupled with product synthesis, enforced ATP wasting due to futile cycles or other mechanisms dissipating ATP may (1) boost the specific substrate uptake and product synthesis rate and (2) increase the product yield . These results were obtained for OSF processes but they may have a favorable effect also for TSF processes, especially due to the potential to boost substrate consumption in the production phase.…”

Section: Resultsmentioning

confidence: 99%

“…This mechanism must be robust enough to ensure that the amount of ATP wasted is not too high as it would drive the cell into an unstable state. Regarding OSF processes, enforced ATP wasting strategies have a beneficial effect on product yield as well as on specific substrate uptake and production rates (see simulations in Figure and experimental proofs in Refs ).…”

Section: Discussionmentioning

confidence: 99%

“…However, lower overall volumetric productivities can be an undesired side effect because biomass accumulates more slowly unless the cell is able to increase the specific substrate uptake rate to a value where the loss of ATP is (almost) fully compensated by the additional substrate taken up for ATP synthesis. Liu et al showed that this might indeed be feasible. While the potential of ATP wasting strategies has already been demonstrated for OSF, applications for TSF processes have not been reported yet although our simulations suggest that the performance gain could be even much more significant.…”

Section: Discussionmentioning

confidence: 99%

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

Klamt

Mahadevan

Hädicke

2017

Biotechnology Journal

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Apart from product yield and titer, volumetric productivity is a key performance indicator for many biotechnological processes. Due to the inherent trade-off between the production of biomass as catalyst and of the actual target product, yield and volumetric productivity cannot be optimized simultaneously. Therefore, in combination with genetic techniques for dynamic regulation of metabolic fluxes, two-stage fermentations (TSFs) with separated growth and production phase have recently gained much interest because of their potential to improve the productivity of bioprocesses while still allowing high product yields. However, despite some successful case studies, so far it has not been discussed and analyzed systematically whether or under which conditions a TSF guarantees superior productivity compared to one-stage fermentation (OSF). In this study, we use mathematical models to demonstrate that the volumetric productivity of a TSF is not automatically better than of a corresponding OSF. Our analysis reveals that the sharp decrease of the specific substrate uptake rate usually observed in (non-growth) production phases severely impacts the volumetric productivity and thus raises a big challenge for designing competitive TSF processes. We discuss possible approaches such as enforced ATP wasting to improve substrate utilization rates in the production phase by which TSF processes can become superior to OSF. We also analyze additional factors influencing the relative performance of OSF and TSF and show that OSF processes can be more appropriate if a high product yield is an economic constraint. In conclusion, a careful assessment of the trade-offs between substrate uptake rates, yields, and productivity is necessary when deciding for OSF vs. TSF processes.

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“…Manipulating the adenosine triphosphate (ATP) pool has been a major target for enhancing the performance of production organisms . For pathways with ATP limitations, increasing the pool of available ATP may improve the production of desired compounds, e.g., of succinic acid or of recombinant proteins .…”

Section: Introductionmentioning

confidence: 99%

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

Boecker

Ahmed

Schramm

et al. 2019

Biotechnology Journal

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The targeted increase of cellular adenosine triphosphate (ATP) turnover (enforced ATP wasting) has recently been recognized as a promising tool for metabolic engineering when product synthesis is coupled with net ATP formation. The goal of the present study is to further examine and to further develop the concept of enforced ATP wasting and to broaden its scope for potential applications. In particular, considering the fermentation products synthesized by Escherichia coli under anaerobic conditions as a proxy for target chemical(s), i) a new genetic module for dynamic and gradual induction of the F1‐part of the ATPase is developed and it is found that ii) induction of the ATPase leads to higher metabolic activity and increased product formation in E. coli under anaerobic conditions, and that iii) ATP wasting significantly increases substrate uptake and productivity of growth‐arrested cells, which is vital for its use in two‐stage processes. To the best of the authors' knowledge, the glucose uptake rate of 6.49 mmol gCDW−1 h−1 achieved with enforced ATP wasting is the highest value reported for nongrowing E. coli cells. In summary, this study shows that enforced ATP wasting can be used to improve yield and titer (in growth‐coupled processes) as well as volumetric productivity (in two‐stage processes) depending on which of the performance measures is more crucial for the process and product of interest.

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

Cited by 40 publications

References 32 publications

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

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

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

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

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