Engineering the Reductive Glycine Pathway: A Promising Synthetic Metabolism Approach for C1-Assimilation

Claassens, Nico J.; Satanowski, Ari; Bysani, Viswanada R; Dronsella, Beau; Orsi, Enrico; Rainaldi, Vittorio; Yilmaz, Suzan; Wenk, Sebastian; Lindner, Steffen N.

doi:10.1007/10_2021_181

Cited by 15 publications

(20 citation statements)

References 122 publications

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“…However, glycine and serine are central metabolites of the reductive glycine pathway, which was previously shown to enable formatotrophic growth of E. coli . The key reaction of the pathway is the formation of glycine from free ammonium, CO 2 , and 5,10-methylene-THF via the reductive activity of the glycine cleavage system ( Claassens et al, 2022 ; Kim et al, 2020 ). Transamination of glycine or serine instead of deamination can improve efficiency of the formate assimilation pathway ( Claassens et al, 2022 ).…”

Section: Resultsmentioning

confidence: 99%

On the flexibility of the cellular amination network in E coli

Schulz-Mirbach

Müller

et al. 2022

eLife

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Ammonium (NH4+) is essential to generate the nitrogenous building blocks of life. It gets assimilated via the canonical biosynthetic routes to glutamate and is further distributed throughout metabolism via a network of transaminases. To study the flexibility of this network, we constructed an Escherichia coli glutamate auxotrophic strain. This strain allowed us to systematically study which amino acids serve as amine sources and found that several amino acids complement the auxotrophy, either by producing glutamate via transamination reactions or by their conversion to glutamate. In this network, we identified aspartate transaminase AspC as a major connector between many amino acids and glutamate. Additionally, we extended the transaminase network by the amino acids β-alanine, alanine, glycine, and serine as new amine sources and identified d-amino acid dehydrogenase (DadA) as an intracellular amino acid sink removing substrates from transaminase reactions. Finally, ammonium assimilation routes producing aspartate or leucine were introduced. Our study reveals the high flexibility of the cellular amination network, both in terms of transaminase promiscuity and adaptability to new connections and ammonium entry points.

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Section: Resultsmentioning

confidence: 99%

On the flexibility of the cellular amination network in E coli

Schulz-Mirbach

Müller

et al. 2022

eLife

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“…The NAD-dependent enzymes can lead to higher biomass yields but come with disadvantageous thermodynamics. The PQQ-dependent enzymes do cause a slight reduction in biomass yield, but their higher thermodynamics could lead to higher growth rates (Claassens et al, 2022;Cotton et al, 2020;Whitaker et al, 2015). So far, none of all the published metabolic engineering efforts towards synthetic methylotrophy have demonstrated the engineered expression of a PQQ-dependent MDH.…”

Section: Discussionmentioning

confidence: 99%

“…At last, serine is converted to pyruvate through deamination and from thereon to biomass (Bar-Even et al, 2013;Claassens et al, 2022;Yishai et al, 2017Yishai et al, , 2018. This formate assimilation pathway can be further extended towards methanol by adding a module containing a methanol and a formaldehyde dehydrogenase.…”

Section: Figurementioning

confidence: 99%

Paving the way for synthetic C1- metabolism in Pseudomonas putida through the reductive glycine pathway

Bruinsma

Wenk

Claassens

et al. 2022

Preprint

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One-carbon (C1) compounds such as methanol, formate, and CO2 are alternative, sustainable microbial feedstocks for the biobased production of chemicals and fuels. In this study, we engineered the carbon metabolism of the industrially important bacterium Pseudomonas putida to assimilate these three substrates through the reductive glycine pathway. First, we demonstrated the functionality of the C1-assimilation module by coupling the growth of auxotrophic strains to formate assimilation. Next, we extended the module from formate to methanol using both NAD and PQQ – dependent methanol dehydrogenases. Finally, we demonstrated CO2-dependent growth through CO2 reduction to formate by the native formate dehydrogenase, which required short-term evolution to rebalance the cellular NADH/NAD+ ratio. This research paves the way to engineer P. putida towards growth on formate, methanol, and CO2 as sole feedstocks, thereby substantially expanding its potential as a sustainable and versatile cell factory.

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“…The spectrum of C1-derived products is broader in the presence of O 2 as generation of cellular energy and biosynthesis are uncoupled—which often comes at the price of a relatively low bioconversion efficiency (14). To tackle this issue, the linear reductive glycine (rGly) pathway has been identified as the most energy-efficient aerobic pathway supporting formate assimilation (7,15,16). In this pathway, formate is firstly condensed with tetrahydrofolate (THF) and reduced to methylene-THF.…”

Section: Introductionmentioning

confidence: 99%

Integrated rational and evolutionary engineering of genome-reducedPseudomonas putidastrains empowers synthetic formate assimilation

Turlin

Dronsella

Maria

et al. 2022

Preprint

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Formate is a promising, water-soluble C1 feedstock for biotechnology since it can be efficiently produced from CO2-but very few industrially-relevant hosts have been engineered towards formatotrophy. Here, the non-pathogenic soil bacterium Pseudomonas putida was adopted as a platform for synthetic formate assimilation. The metabolism of genome-reduced variants of P. putida was rewired to establish synthetic auxotrophies that could be functionally complemented by expressing components of the reductive glycine (rGly) pathway. The rGly pathway mediates the formate → glycine → serine transformations that yield pyruvate, ultimately assimilated into biomass. We adopted a modular engineering approach, dividing C1 assimilation in segments composed of both heterologous activities (sourced from Methylorubrum extorquens) and native reactions. Promoter engineering of chromosomally-encoded functions coupled to modular expression of rGly pathway elements enabled growth on formate as carbon source and acetate for energy supply. Adaptive laboratory evolution of two lineages of engineered P. putida formatotrophs significantly reduced doubling times to ca. 15 h. During evolution, two catabolic regimes became predominant in independently evolved clones, either via glycine hydroxymethylation (GlyA) or oxidation (ThiO). Taken together, our results expand the landscape of microbial platforms for C1-based biotechnological production towards supporting a formate bioeconomy.

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Engineering the Reductive Glycine Pathway: A Promising Synthetic Metabolism Approach for C1-Assimilation

Cited by 15 publications

References 122 publications

On the flexibility of the cellular amination network in E coli

On the flexibility of the cellular amination network in E coli

Paving the way for synthetic C1- metabolism in Pseudomonas putida through the reductive glycine pathway

Integrated rational and evolutionary engineering of genome-reducedPseudomonas putidastrains empowers synthetic formate assimilation

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