Engineering Escherichia coli for methanol conversion

Müller, Jean Moritz; Meyer, Fabian; Litsanov, Boris; Kiefer, Patrick; Potthoff, Eva; Heux, Stéphanie; Quax, Wim J.; Wendisch, Volker F.; Brautaset, Trygve; Portais, Jean‐Charles; Vorholt, Julia A.

doi:10.1016/j.ymben.2014.12.008

Cited by 168 publications

(246 citation statements)

References 59 publications

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“…Error bars represent the SD of at least three replicate experiments. than the 0.12 mM/h reported by Müller et al in a similar resting cell assay (15). It should be noted that the overall enhancement in methanol consumption was only 2.3-fold after 24 h incubation, likely due to limitations in R5P in resting cell cultures.…”

Section: Lactate Dehydrogenase As An "Nadh Sink" Further Enhances Metmentioning

confidence: 52%

“…Engineering E. coli as a synthetic methylotroph has been a challenging problem to date, as very little methanol utilization and no growth on methanol have been demonstrated (15). One of the key obstacles is the unfavorable kinetic properties of NAD-dependent Mdhs, such as Mdh3 (9), making the reversible reduction of formaldehyde far more favorable than methanol oxidation.…”

Section: Discussionmentioning

confidence: 99%

“…Methanol utilization and 13 C-methanol-derived labeling of intracellular metabolites were first demonstrated in Corynebacterium glutamicum by expressing the mdh gene from B. methanolicus and the hps and phi genes from Bacillus subtilis (16,17). A similar approach was also demonstrated in E. coli, where Mdh2 from B. methanolicus PB1 was coexpressed with Hps and Phi from B. methanolicus MGA3 to increase the production of 13 C-labeled intracellular metabolites compared with the control strain expressing Mdh3 alone (15). Although these reports provide evidence that the RuMP pathway can be transferred to nonnative methylotrophs, the rate of methanol consumption is significantly lower than those reported for native methyltrophs.…”

mentioning

confidence: 98%

“…Recent work has demonstrated the ability to transfer methylotrophic genes to other organisms to enable nonnative methanol oxidation (15)(16)(17). Methanol utilization and 13 C-methanol-derived labeling of intracellular metabolites were first demonstrated in Corynebacterium glutamicum by expressing the mdh gene from B. methanolicus and the hps and phi genes from Bacillus subtilis (16,17).…”

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confidence: 99%

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Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Price

Chen

Whitaker

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3-ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an "NADH Sink" was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol. methane | methylotophs | supramolcular | scaffold | substrate channeling

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Section: Lactate Dehydrogenase As An "Nadh Sink" Further Enhances Metmentioning

confidence: 52%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 98%

mentioning

confidence: 99%

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Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Price

Chen

Whitaker

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…One approach is to engineer biological systems to convert one-carbon compounds into multicarbon molecules such as fuels and other high value chemicals. Many synthetic pathways to produce value-added chemicals from common feedstocks, such as glucose, have been constructed in organisms that lack one-carbon anabolic pathways, such as Escherichia coli or Saccharomyces cerevisiae (1-3); however, despite considerable effort, it has been difficult to introduce heterologous one-carbon fixing pathways into these organisms (4). Likely problems include the inherent complexity, environmental sensitivity, inefficiency, or unfavorable chemical driving force of naturally occurring one-carbon metabolic pathways (5).…”

mentioning

confidence: 99%

Computational protein design enables a novel one-carbon assimilation pathway

Siegel¹,

Smith²,

Poust

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.computational protein design | pathway engineering | carbon fixation N ovel strategies are needed to address current challenges in energy storage and carbon sequestration. One approach is to engineer biological systems to convert one-carbon compounds into multicarbon molecules such as fuels and other high value chemicals. Many synthetic pathways to produce value-added chemicals from common feedstocks, such as glucose, have been constructed in organisms that lack one-carbon anabolic pathways, such as Escherichia coli or Saccharomyces cerevisiae (1-3); however, despite considerable effort, it has been difficult to introduce heterologous one-carbon fixing pathways into these organisms (4). Likely problems include the inherent complexity, environmental sensitivity, inefficiency, or unfavorable chemical driving force of naturally occurring one-carbon metabolic pathways (5).An optimal pathway for one-carbon utilization in common synthetic biology platforms would be (i) composed of a minimal number of enzymes, (ii) linear and disconnected from other metabolic pathways, (iii) thermodynamically favorable with a significant driving force at most or all steps, and (iv) capable of functioning in a robust manner under both aerobic and anaerobic conditions (5). A pathway with these properties could enable the assimilation of one-carbon molecules as the sole carbon source for the production of fuels and chemicals. Although no such pathway is known in nature, the established electrochemical reduction of carbon dioxide to formate under ambient temperatures and pressures in neutral aqueous solutions provides an attractive starting point for a onecarbon fixation pathway (5-8).We describe the computational design of an enzyme that catalyzes the carboligation of three one-carbon molecules into a single three-carbon molecule. This enzyme enables the construction of a new pathway, the formolase pathway, in which formate is converted into the central metabolite dihydroxyacetone phosphate (DHAP; Fig. 1). The use of computational protein design to reengineer catalytic activities opens up the pathway design space beyond that available based o...

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Industrial Biotechnology:Escherichia colias a Host

Theisen

Liao

2016

Industrial Biotechnology

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Engineering Escherichia coli for methanol conversion

Cited by 168 publications

References 59 publications

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Computational protein design enables a novel one-carbon assimilation pathway

Industrial Biotechnology:Escherichia colias a Host

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