Dataset Information

Engineering a new-to-nature cascade for phosphate-dependent formate to formaldehyde conversion in vitro and in vivo

ABSTRACT: To realize a carbon-neutral bioeconomy, the concept of electrobiocatalysis has been developed, in which CO2 is first chemically reduced to formate and subsequently assimilated by enzymatic cascades or engineered microbes. A key step in the assimilation of formate is its reduction into formaldehyde, which is chemically challenging. Here, we developed a two-enzyme route in which formate is activated into formyl phosphate and reduced by NAD(P)H into formaldehyde. Exploiting the promiscuity of acetate kinase and N-acetyl--glutamyl phosphate reductase, we demonstrate the phosphate (Pi) route in vitro and in vivo. We further engineered a formyl phosphate reductase variant with improved formyl phosphate conversion in vivo by suppressing cross-talk with native metabolism. We further show that the Pi route can be interfaced with a recently developed formaldehyde assimilation pathway (FORCE pathway) to provide a thermodynamically and kinetically highly efficient route from formate into C2-compounds.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Escherichia Coli (ncbitaxon:562)

SUBMITTER: Tobias Erb

PROVIDER: MSV000091532 | MassIVE | Wed Mar 22 22:46:00 GMT 2023

SECONDARY ACCESSION(S): PXD041037

REPOSITORIES: MassIVE

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Engineering a new-to-nature cascade for phosphate-dependent formate to formaldehyde conversion in vitro and in vivo.

Nattermann Maren M Wenk Sebastian S Pfister Pascal P He Hai H Lee Seung Hwan SH Szymanski Witold W Guntermann Nils N Zhu Fayin F Nickel Lennart L Wallner Charlotte C Zarzycki Jan J Paczia Nicole N Gaißert Nina N Franciò Giancarlo G Leitner Walter W Gonzalez Ramon R Erb Tobias J TJ

Nature communications 20230509 1

Formate can be envisioned at the core of a carbon-neutral bioeconomy, where it is produced from CO<sub>2</sub> by (electro-)chemical means and converted into value-added products by enzymatic cascades or engineered microbes. A key step in expanding synthetic formate assimilation is its thermodynamically challenging reduction to formaldehyde. Here, we develop a two-enzyme route in which formate is activated to formyl phosphate and subsequently reduced to formaldehyde. Exploiting the promiscuity o ...[more]

PMID: 37160875

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Project description:Previously, it has been demonstrated that formate can be utilized by Saccharomyces cerevisiae as additional energy source using cells grown in a glucose-limited chemostat. Here, we investigated utilization of formaldehyde as co-substrate. Since endogenous formaldehyde dehydrogenase activities were insufficient to allow co-feeding of formaldehyde, the Hansenula polymorpha FLD1, encoding formaldehyde dehydrogenase, was introduced in S. cerevisiae. Chemostat cultivations revealed that formaldehyde was co-utilized with glucose, but the yield was lower than predicted. Moreover, formate was secreted by the cells. Upon co-expression of the H. polymorpha gene encoding formate dehydrogenase, FMD, the levels of secreted formate decreased, but the biomass yield was still lower than anticipated. Transcriptome comparisons of cells of the engineered FLD1/FMD-expressing S. cerevisiae strain grown with or without formaldehyde feed, suggested that the cells experienced biotin limitation, possibly due to inactivation of biotin by formaldehyde in the feed. When separate feeds were used for formaldehyde and biotin, the engineered S. cerevisiae strain was able to efficiently utilize formaldehyde as additional energy source. Experiment Overall Design: In industrial biotechnology, the cost of feedstocks (often sugars) are crucial for production of both biomass related products, such as proteins, or for the production of commodity chemicals. In many such processes a large fraction of the sugars is dissimilated to either provide free-energy (in the form of ATP) or to provide electrons (in the form of NADH). Co-consumption of methanol, which can be derived from fossil sources (via natural gas) or from biomass (via syngas) , via the above mentioned route can provide NADH and/or ATP, thereby creating the possibility to utilise the sugars more efficiently. It would therefore be advantageous to co-feed S. cerevisiae with methanol during fermentations since it is a cheap alternative to sugars as energy source. Experiment Overall Design: Efficient dissimilation of the intermediate formaldehyde is a crucial first step for utilisation of methanol by S. cerevisiae. Therefore, his study analyses the effect of co-expression of H. polymorpha FLD1 encoding NAD+-dependent formaldehyde and FMD encoding formate dehydrogenases, on tolerance to formaldehyde and co-consumption of formaldehyde by S. cerevisiae.

Dataset Information

Engineering a new-to-nature cascade for phosphate-dependent formate to formaldehyde conversion in vitro and in vivo

Publications

Engineering a new-to-nature cascade for phosphate-dependent formate to formaldehyde conversion in vitro and in vivo.

Similar Datasets

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