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

Boecker, Simon; Ahmed, Zahoor; Schramm, Thorben; Link, Hannes; Klamt, Steffen

doi:10.1002/biot.201800438

Cited by 42 publications

(45 citation statements)

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“…), the two strains expressing the three subunit genes of the E. coli F 1 -ATPase exhibit a clear increase in ATPase activity (Fig. 1 ) by 78% and 103%, respectively, which is in the range observed when overexpressing these genes directly in E. coli [ 17 ]. In contrast, the ATPase activity even decreased by 21% in the strain expressing the T. reesei F 1 -ATPase β subunit (5D- atp2 ), providing first evidence that this strain might not behave as intended.…”

Section: Resultsmentioning

confidence: 99%

“…The reduced volumetric productivity, observed despite improved product yield and partially improved specific productivities, is a well-documented effect of enforced ATP wasting as more product is made at the expense of biomass [ 11 , 17 , 18 , 37 ]. Similar as in [ 17 ], we hypothesized that this trade-off can be overcome if growth and production are separated. This was tested in a growth-decoupled (nitrogen-starved) approach in which the strains were inoculated with a higher starting OD 600 of ca.…”

Section: Resultsmentioning

confidence: 99%

“…In prokaryotic hosts, expression of the cytosolic F 1 -ATPase has recently been shown to improve the production of metabolites that are coupled to ATP generation. For example, overexpressing genes of the native F 1 -ATPase in E. coli improved titer, yield and specific productivities of the fermentation products under growth-coupled conditions, as well as volumetric productivity during two-stage fermentation [ 17 ]. Similarly, expression of the F 1 -ATPase led to improved acetoin production in an engineered Lactococcus lactis strain [ 18 ].…”

Section: Introductionmentioning

confidence: 99%

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ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production

Ahmed

Messerschmidt

Boecker

et al. 2020

Biotechnol Biofuels

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Background Enforced ATP wasting has been recognized as a promising metabolic engineering strategy to enhance the microbial production of metabolites that are coupled to ATP generation. It also appears to be a suitable approach to improve production of ethanol by Saccharomyces cerevisiae. In the present study, we constructed different S. cerevisiae strains with heterologous expression of genes of the ATP-hydrolyzing F1-part of the ATPase enzyme to induce enforced ATP wasting and quantify the resulting effect on biomass and ethanol formation. Results In contrast to genomic integration, we found that episomal expression of the αβγ subunits of the F1-ATPase genes of Escherichia coli in S. cerevisiae resulted in significantly increased ATPase activity, while neither genomic integration nor episomal expression of the β subunit from Trichoderma reesei could enhance ATPase activity. When grown in minimal medium under anaerobic growth-coupled conditions, the strains expressing E. coli’s F1-ATPase genes showed significantly improved ethanol yield (increase of 10% compared to the control strain). However, elevated product formation reduces biomass formation and, therefore, volumetric productivity. We demonstrate that this negative effect can be overcome under growth-decoupled (nitrogen-starved) operation with high and constant biomass concentration. Under these conditions, which mimic the second (production) phase of a two-stage fermentation process, the ATPase-expressing strains showed significant improvement in volumetric productivity (up to 111%) compared to the control strain. Conclusions Our study shows that expression of genes of the F1-portion of E. coli’s ATPase induces ATPase activity in S. cerevisiae and can be a promising way to improve ethanol production. This ATP-wasting strategy can be easily applied to other metabolites of interest, whose formation is coupled to ATP generation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production

Ahmed

Messerschmidt

Boecker

et al. 2020

Biotechnol Biofuels

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“…Consequently, the HEX1 glucose transport system has previously been studied for its importance in creating platform strains for microbial chemical production 42 . Furthermore, extra usage of ATP has been explored as a means to increase substrate uptake rates, potentially increasing product flux under low growth conditions 43 , suggesting the importance of studying alternatives to the native phosphotransferase system. The reaction THD2pp (NADP transhydrogenase) is also required to be switched on for many products.…”

Section: Resultsmentioning

confidence: 99%

Novel two-stage processes for optimal chemical production in microbes

Raj

Venayak

Mahadevan

2019

Preprint

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1Microbial metabolism can be harnessed to produce a broad range of industrially important chemicals. 2 Often, three key process variables: Titer, Rate and Yield (TRY) are the target of metabolic engineering 3 efforts to improve microbial hosts toward industrial production. Previous research into improving the 4 TRY metrics have examined the efficacy of having distinct growth and production stages to achieve 5 enhanced productivity. However, these studies assumed a switch from a maximum growth to a maximum 6 production phenotype. Hence, the choice of operating points for the growth and production stages of 7 two-stage processes is yet to be explored. The impact of reduced growth rates on substrate uptake adds 8 to the need for intelligent choice of operating points while designing two-stage processes. In this work, we 9 present a computational framework that scans the phenotypic space of microbial metabolism to identify 10 ideal growth and production phenotypic targets, to achieve optimal TRY values. Using this framework, 11 with Escherichia coli as a model organism, we compare two-stage processes that use dynamic pathway 12 regulation, with one-stage processes that use static intervention strategies. Our results indicate that 13 two-stage processes with intermediate growth during the production stage always result in the highest 14 productivity. By analyzing the flux distributions for the production enhancing strategies, we identify 15 key reactions and reaction subsystems that need to be downregulated for a wide range of metabolites 16 in E. coli. We also elucidate the importance of flux perturbations that increase phosphoenolpyruvate 17 and NADPH availability among strategies to design production platforms. Furthermore, reactions in 18 the pentose phosphate pathway emerge as key control nodes that function together to increase the 19 availability of precursors to most products in E. coli. Due to the presence of these common patterns 20 in the flux perturbations, we propose the possibility of a universal production strain that enhances the 21 production of a large number of metabolites. 22 23 Keywords: dynamic pathway engineering, two-stage processes, industrial bioprocesses, phenotypic choices, 24 production platforms, substrate uptake effects 25 1 26The use of microbes for the production of chemicals through metabolic engineering has garnered significant 27 interest in the past few decades. The naturally modular arrangement of metabolic networks makes micro-28 bial strains amenable to be used as chemical production platforms 1 . Metabolic networks have a bow-tie 29 architecture which allows a large number of metabolites to be produced from a few universal precursors 2 . 30This has allowed us to successfully engineer microbes to be biocatalysts for the production of a wide range of 31 commodity chemicals 3,4 , pharmaceuticals 5,6 , biofuels, 7,8 and other natural and non-natural compounds 9 . 32While few such processes have been successful at an industrial scale 10,11 , large strain development costs 3...

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“…Interestingly, iJO1366* predicts increasing lactate production rates (and reduced rates for all other fermentation products) for very high glucose uptake rates, however, the net gain in growth rate for this shift is only marginal and thus probably not relevant in vivo. However, in [33] it was shown that under conditions with large fluxes in the central metabolism, lactate synthesis might become the preferred fermentation pathway, possibly due to its reduced protein costs compared to the combined action of the ethanol, acetate and formate fermentation pathways.…”

Section: Prediction Of Exchange Fluxes and Of Flux Rangesmentioning

confidence: 99%

Automatic construction of metabolic models with enzyme constraints

2020

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Background: In order to improve the accuracy of constraint-based metabolic models, several approaches have been developed which intend to integrate additional biological information. Two of these methods, MOMENT and GECKO, incorporate enzymatic (kcat) parameters and enzyme mass constraints to further constrain the space of feasible metabolic flux distributions. While both methods have been proven to deliver useful extensions of metabolic models, they may considerably increase size and complexity of the models and there is currently no tool available to fully automate generation and calibration of such enzyme-constrained models from given stoichiometric models. Results: In this work we present three major developments. We first conceived short MOMENT (sMOMENT), a simplified version of the MOMENT approach, which yields the same predictions as MOMENT but requires significantly fewer variables and enables direct inclusion of the relevant enzyme constraints in the standard representation of a constraint-based model. When measurements of enzyme concentrations are available, these can be included as well leading in the extreme case, where all enzyme concentrations are known, to a model representation that is analogous to the GECKO approach. Second, we developed the AutoPACMEN toolbox which allows an almost fully automated creation of sMOMENT-enhanced stoichiometric metabolic models. In particular, this includes the automatic read-out and processing of relevant enzymatic data from different databases and the reconfiguration of the stoichiometric model with embedded enzymatic constraints. Additionally, tools have been developed to adjust (kcat and enzyme pool) parameters of sMOMENT models based on given flux data. We finally applied the new sMOMENT approach and the AutoPACMEN toolbox to generate an enzyme-constrained version of the E. coli genome-scale model iJO1366 and analyze its key properties and differences with the standard model. In particular, we show that the enzyme constraints improve flux predictions (e.g., explaining overflow metabolism and other metabolic switches) and demonstrate, for the first time, that these constraints can markedly change the spectrum of metabolic engineering strategies for different target products. Conclusions: The methodological and tool developments presented herein pave the way for a simplified and routine construction and analysis of enzyme-constrained metabolic models.

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

Cited by 42 publications

References 45 publications

ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production

ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production

Novel two-stage processes for optimal chemical production in microbes

Automatic construction of metabolic models with enzyme constraints

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