Systematic design and in vitro validation of novel one-carbon assimilation pathways

Yang, Xue; Yuan, Qianqian; Luo, Haiyun; Li, Feiran; Mao, Yufeng; Zhao, Xin; Du, Jiawei; Li, Peishun; Ju, Xiaozhi; Zheng, Yangyang; Yang, Chong‐Ren; Liu, Yuwan; Jiang, Huifeng; Yao, Yonghong; Ma, Hongwu; Ma, Yanhe

doi:10.1016/j.ymben.2019.09.001

Cited by 63 publications

(96 citation statements)

References 73 publications

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“…The identification of the most active FDH and MDH variants in this bacterium, as well as the determination of optimal concentrations of formate and methanol, could assist in supporting carbon fixation, whether via the Calvin Cycle (Antonovsky et al, 2016), the 3‐hydroxypropionate bicycle (Mattozzi et al, 2013), or any other route. Similarly, recent efforts to establish E. coli growth on formate or methanol (Chou, Clomburg, Qian, & Gonzalez, 2019; He, Edlich‐Muth, Lindner, & Bar‐Even, 2018; Kim et al, 2020; Lu et al, 2019; Meyer et al, 2018; Siegel et al, 2015; Wang et al, 2017; Yang et al, 2019; Yishai, Bouzon, Doring, & Bar‐Even, 2018) would greatly benefit from the information gathered in this study, showing how these feedstocks—besides assimilated by the synthetic pathways—can efficiently provide reducing power and energy for cell growth.…”

Section: Discussionmentioning

confidence: 94%

An “energy‐auxotroph” Escherichia coli provides an in vivo platform for assessing NADH regeneration systems

Wenk

Schann

et al. 2020

Biotech & Bioengineering

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An efficient in vivo regeneration of the primary cellular resources NADH and ATP is vital for optimizing the production of value-added chemicals and enabling the activity of synthetic pathways. Currently, such regeneration routes are tested and characterized mainly in vitro before being introduced into the cell. However, in vitro measurements could be misleading as they do not reflect enzyme activity under physiological conditions. Here, we construct an in vivo platform to test and compare NADH regeneration systems. By deleting dihydrolipoyl dehydrogenase in Escherichia coli, we abolish the activity of pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. When cultivated on acetate, the resulting strain is auxotrophic to NADH and ATP: acetate can be assimilated via the glyoxylate shunt but cannot be oxidized to provide the cell with reducing power and energy. This strain can, therefore, serve to select for and test different NADH regeneration routes. We exemplify this by comparing several NAD-dependent formate dehydrogenases and methanol dehydrogenases. We identify the most efficient enzyme variants under in vivo conditions and pinpoint optimal feedstock concentrations that maximize NADH biosynthesis while avoiding cellular toxicity. Our strain thus provides a useful platform for comparing and optimizing enzymatic systems for cofactor regeneration under physiological conditions.

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Section: Discussionmentioning

confidence: 94%

An “energy‐auxotroph” Escherichia coli provides an in vivo platform for assessing NADH regeneration systems

Wenk

Schann

et al. 2020

Biotech & Bioengineering

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“…Upon entrance into bacterial cells, almost all C1 compounds such as methanol, methane, formate and dichloromethane are first oxidized or reduced to formaldehyde and subsequently assimilated into the central part of the metabolism of methylotrophic bacteria [10]. Formaldehyde can be further metabolized either via the serine pathway, the ribulose monophosphate (RuMP) pathway or the Calvin-Benson-Bassham (CBB) cycle in native methylotrophs [11]. Although methylotrophic bacteria can assimilate C1 compounds, their low specific growth rate is less suitable for large industrial production, and the improvement of natural C1 metabolic pathways in native hosts is often difficult due to lack of efficient genetic tools [12].…”

Section: Introductionmentioning

confidence: 99%

Formaldehyde formation in the glycine cleavage system and its use for an aldolase-based biosynthesis of 1,3-propanediol

Meng

Ren

et al. 2020

J Biol Eng

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Glycine cleavage system (GCS) occupies a key position in one-carbon (C1) metabolic pathway and receives great attention for the use of C1 carbons like formate and CO 2 via synthetic biology. In this work, we demonstrate that formaldehyde exists as a substantial byproduct of the GCS reaction cycle. Three causes are identified for its formation. First, the principal one is the decomposition of N 5 ,N 10-methylene-tetrahydrofolate (5,10-CH 2-THF) to form formaldehyde and THF. Increasing the rate of glycine cleavage promotes the formation of 5,10-CH 2-THF, thereby increasing the formaldehyde release rate. Next, formaldehyde can be produced in the GCS even in the absence of THF. The reason is that T-protein of the GCS can degrade methylamine-loaded H-protein (H int) to formaldehyde and ammonia, accompanied with the formation of dihydrolipoyl H-protein (H red), but the reaction rate is less than 0.16% of that in the presence of THF. Increasing T-protein concentration can speed up the release rate of formaldehyde by H int. Finally, a certain amount of formaldehyde can be formed in the GCS due to oxidative degradation of THF. Based on a formaldehyde-dependent aldolase, we elaborated a glycine-based one carbon metabolic pathway for the biosynthesis of 1,3-propanediol (1,3-PDO) in vitro. This work provides quantitative data and mechanistic understanding of formaldehyde formation in the GCS and a new biosynthetic pathway of 1,3-PDO.

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“…The other artificial pathway from the literature that was included in this study, the Reductive Glycine Pathway is the only artificial C1-fixing pathway that has been implemented in vivo to support growth on formate as the sole carbon and energy source and is also the potentially most efficient pathway to support formatotrophic growth. Exotic pathways that rely on high concentrations of formaldehyde (Chou et al, 2019;He et al, 2020;Siegel et al, 2015) and pathways that are variations of existing pathways (Yang et al, 2019; were also not considered.…”

Section: Overview Of the Natural And Artificial Pathways Used In Thismentioning

confidence: 99%

In-depth computational analysis of natural and artificial carbon fixation pathways

Löwe

Kremling

2021

Preprint

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In the recent years, engineering new-to-nature CO2 and C1 fixing metabolic pathways made a leap forward. These new, artificial pathways promise higher yields and activity than natural ones like the Calvin-Benson-Bassham cycle. The question remains how to best predict their in vivo performance and what actually makes one pathway “better” than another.In this context, we explore aerobic carbon fixation pathways by a computational approach and compare them based on their ATP-efficiency and specific activity considering the kinetics and thermodynamics of the reactions. Beside natural pathways, this included the artificial Reductive Glycine Pathway, the CETCH cycle and two completely new cycles with superior stoichiometry: The Reductive Citramalyl-CoA cycle and the 2-Hydroxyglutarate-Reverse Tricarboxylic Acid cycle. A comprehensive kinetic data set was collected for all enzymes of all pathways and missing kinetic data was sampled with the Parameter Balancing algorithm. Kinetic and thermodynamic data were fed to the Enzyme Cost Minimization algorithm to check for respective inconsistencies and calculate pathway specific activities.We found that the Reductive Glycine Pathway, the CETCH cycle and the new Reductive Citramalyl-CoA cycle were predicted to have higher ATP-efficiencies and specific activities than the natural cycles. The Calvin Cycle performed better than previously thought, however. It can be concluded that the weaker overall characteristics in the design of the Calvin Cycle might be compensated by other benefits like robustness, low nutrient demand and a good compatibility with the host’s physiological requirements. Nevertheless, the artificial carbon fixation cycles hold great potential for future applications in Industrial Biotechnology and Synthetic Biology.

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Systematic design and in vitro validation of novel one-carbon assimilation pathways

Cited by 63 publications

References 73 publications

An “energy‐auxotroph” Escherichia coli provides an in vivo platform for assessing NADH regeneration systems

An “energy‐auxotroph” Escherichia coli provides an in vivo platform for assessing NADH regeneration systems

Formaldehyde formation in the glycine cleavage system and its use for an aldolase-based biosynthesis of 1,3-propanediol

In-depth computational analysis of natural and artificial carbon fixation pathways

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