Ribulose Monophosphate Shunt Provides Nearly All Biomass and Energy Required for Growth of <i>E. coli</i>

He, Hai; Edlich‐Muth, Christian; Lindner, Steffen N.; Bar‐Even, Arren

doi:10.1021/acssynbio.8b00093

Cited by 52 publications

(71 citation statements)

References 42 publications

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“…This is in line with previous findings that seemingly small differences in the design of metabolic growth selection schemes (e.g. the choice of deleted enzymes) can lead to substantially dissimilar metabolic behaviors 36,41 .…”

Section: A δTktab Context Requires Additional Metabolic Adaptations Tsupporting

confidence: 92%

“…2A; for a similar approach see ref. 36,37 ). As we show below, growth via this linear shunt requires the activity of most enzymes of the GED cycle, but relies on a pentose substrate rather than regeneration of Ru5P.…”

Section: Selection For the Activity Of The Ged Shunt Within A δRpe Comentioning

confidence: 99%

“…We deleted the genes encoding for both isozymes of transketolase (ΔtktAB). This strain, in which the non-oxidative pentose phosphate pathway is effectively abolished, cannot grow when provided with a pentose as sole carbon source 36,40 . Furthermore, as E4P cannot be synthesized in this strain, small amounts of essential cellular components, the biosynthesis of which is E4P-dependent, need to be added to the media 36,40 : phenylalanine, tyrosine, tryptophan, shikimate, pyridoxine, 4aminobenzoate, 4-hydroxybenzoate, and 2,3-dihydroxybenzoate (referred to as E4P-supplements, see Methods).…”

Section: A δTktab Context Requires Additional Metabolic Adaptations Tmentioning

confidence: 99%

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Awakening a latent carbon fixation cycle inEscherichia coli

Satanowski

Dronsella

Noor

et al. 2020

Preprint

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Carbon fixation is one of the most important biochemical processes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of Escherichia coli native enzymes. We identify the GED (Gnd-Entner-Doudoroff) cycle as the simplest pathway that can operate with high thermodynamic driving force. This autocatalytic route is based on reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (Gnd), followed by reactions of the Entner-Doudoroff pathway, gluconeogenesis, and the pentose phosphate pathway. We demonstrate the in vivo feasibility of this new-to-nature pathway by constructing E. coli gene deletion strains whose growth on pentose sugars depends on the GED shunt, a linear variant of the GED cycle which does not require the regeneration of Ru5P. Several metabolic adaptations, most importantly the increased production of NADPH, assist in establishing sufficiently high flux to sustain this growth. Our study exemplifies a trajectory for the emergence of carbon fixation in a heterotrophic organism and demonstrates a synthetic pathway of biotechnological interest.

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Section: A δTktab Context Requires Additional Metabolic Adaptations Tsupporting

confidence: 92%

Section: Selection For the Activity Of The Ged Shunt Within A δRpe Comentioning

confidence: 99%

Section: A δTktab Context Requires Additional Metabolic Adaptations Tmentioning

confidence: 99%

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Awakening a latent carbon fixation cycle inEscherichia coli

Satanowski

Dronsella

Noor

et al. 2020

Preprint

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“…Considering that CGR1 soil contains higher metal concentration (Fig. 1), the possibility to use an alternative carbon source gives to CGR1 an important advantage to produce energy (ATP), a crucial metabolite used for metal efflux ATPase, also with interesting biotechnological applications 68 .…”

Section: Resultsmentioning

confidence: 99%

Genome-scale metabolic models of Microbacterium species isolated from a high altitude desert environment

Mandaković

Cintolesi

Maldonado

et al. 2020

Sci Rep

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The Atacama Desert is the most arid desert on Earth, focus of important research activities related to microbial biodiversity studies. In this context, metabolic characterization of arid soil bacteria is crucial to understand their survival strategies under extreme environmental stress. We investigated whether strain-specific features of two Microbacterium species were involved in the metabolic ability to tolerate/ adapt to local variations within an extreme desert environment. Using an integrative systems biology approach we have carried out construction and comparison of genome-scale metabolic models (GEMs) of two Microbacterium sp., CGR1 and CGR2, previously isolated from physicochemically contrasting soil sites in the Atacama Desert. Despite CGR1 and CGR2 belong to different phylogenetic clades, metabolic pathways and attributes are highly conserved in both strains. However, comparison of the GEMs showed significant differences in the connectivity of specific metabolites related to pH tolerance and co 2 production. The latter is most likely required to handle acidic stress through decarboxylation reactions. We observed greater GEM connectivity within Microbacterium sp. CGR1 compared to CGR2, which is correlated with the capacity of CGR1 to tolerate a wider pH tolerance range. Both metabolic models predict the synthesis of pigment metabolites (β-carotene), observation validated by HPLC experiments. Our study provides a valuable resource to further investigate global metabolic adaptations of bacterial species to grow in soils with different abiotic factors within an extreme environment. In recent years, there has been increased interest in the study of Actinobacteria from desert areas. As noted by previous researchers, this class possess bacteria with a variety of attractive metabolic qualities that make them good candidates to survive in desert areas 1 , including their extensive metabolic capability to degrade and utilize compounds from nutrient poor environments and their ability to synthesize secondary metabolites and natural antibiotics 2. In fact, Actinobacteria is the dominant class present in desert locations 3 , and their ability to adapt to this kind of environment is the focus of intense research. For example, Lynch and collaborators found that over 98% of the lineages present in the vicinity of a volcano in the Atacama Desert corresponded to Actinobacteria, and that their metabolisms were adapted to utilize atmospheric gases present in this ecosystem 4. One genus that

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“…Similar results have also been reported in other model organisms such as Corynebacterium glutamicum, Pseudomonas putida and Saccharomyces cerevisiae (as reviewed recently by (Heux S. et al, 2018)). Improvements in methanol assimilation have been achieved using different strategies such as (i) optimizing the cultivation medium (Gonzalez et al, 2018), (ii) lowering the thermodynamic and kinetic constraints associated with NAD-dependent methanol oxidation (Roth et al, 2019; Wu et al, 2016), (iii) improving formaldehyde assimilation (Price et al, 2016; Woolston et al, 2018), (iv) increasing carbon fluxes through the autocatalytic cycle (Bennett et al, 2018), and (v) coupling the activity of the RuMP cycle to the growth of the host microorganism and then using adaptive laboratory evolution (Chen et al, 2018; He et al, 2018; Meyer et al, 2018). However, none of these synthetic strains are able to grow on methanol alone.…”

Section: Introductionmentioning

confidence: 99%

Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway inE. coli

Simone

Vicente

Peiro

et al. 2020

Preprint

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25One-carbon (C1) compounds, such as methanol, have recently gained attention as 26 alternative low-cost and non-food feedstocks for microbial bioprocesses. Considerable 27 research efforts are thus currently focused on the generation of synthetic 28 methylotrophs by transferring methanol assimilation pathways into established 29 bacterial production hosts. In this study, we used an iterative combination of dry and 30 wet approaches to design, implement and optimize this metabolic trait in the most 31 common chassis, E. coli. Through in silico modeling, we designed a new route that 32 "mixed and matched" two methylotrophic enzymes: a bacterial methanol 33 dehydrogenase (Mdh) and a dihydroxyacetone synthase (Das) from yeast. To identify 34 the best combination of enzymes to introduce into E. coli, we built a library of 266 35 pathway variants containing different combinations of Mdh and Das homologues and 36 screened it using high-throughput 13 C-labeling experiments. The highest level of 37 incorporation, 22% of labeled methanol carbon into the multi-carbon compound PEP, 38 was obtained using a variant composed of a Mdh from A. gerneri and a codon-39 optimized version of P. angusta Das. Finally, the activity of this new synthetic pathway 40 was further improved by engineering strategic metabolic targets identified using omics 41 and modelling approaches. The final synthetic strain had 1.5 to 5.9 times higher 42 methanol assimilation in intracellular metabolites and proteinogenic amino acids than 43 the starting strain did. Broadening the repertoire of methanol assimilation pathways is 44 one step further toward synthetic methylotrophy in E. coli. 45

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Ribulose Monophosphate Shunt Provides Nearly All Biomass and Energy Required for Growth of E. coli

Cited by 52 publications

References 42 publications

Awakening a latent carbon fixation cycle inEscherichia coli

Awakening a latent carbon fixation cycle inEscherichia coli

Genome-scale metabolic models of Microbacterium species isolated from a high altitude desert environment

Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway inE. coli

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