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System-level characterization of engineered and evolved formatotrophic E. coli strains

ABSTRACT: One carbon compounds like formate provide a promising and sustainable feedstock for microbial bioproduction of fuels and chemicals, as an alternative to the petro-chemical production industry. Growth of Escherichia coli on formate was recently achieved by introducing the reductive glycine pathway (rGlyP) into its genome, which is theoretically the most efficient aerobic formate assimilation pathway. While adaptive laboratory evolution was used to enhance the growth rate and biomass yield significantly, still the best performing formatotrophic E. coli strain did not approach the theoretical optimal biomass yield of the rGlyP. In this study, we investigated the metabolism of these previously engineered formatotrophic E. coli strains in order to elucidate why the biomass yield was sub-optimal and how it may be improved. Through a combination of metabolic modelling and proteomic analysis, we identified several potential metabolic bottlenecks and targets for expression tuning. This characterization study provides insights that can inform future rational engineering efforts to improve the growth of E. coli on formate.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Escherichia Coli

TISSUE(S): Cell Culture

SUBMITTER: Timo Glatter

LAB HEAD: Timo Glatter

PROVIDER: PXD050315 | Pride | 2025-03-10

REPOSITORIES: Pride

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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.

Project description:Metabolism is fundamental to life, all life activities are sustained through metabolism. Metabolic engineering, while modifying host endogenous metabolic network or introducing heterologous pathway for biotechnological applications, often results in unfitness and suboptimal characteristics, because of the lack of full knowledge on host metabolism. Adaptive laboratory evolution (ALE), harnessing the nature of life -- evolution, has become a potent approach in phenotype optimization, environmental adaptation, and cell biology study1–3. Through ALE, alternative pathways of β-alanine biosynthesis4, pyridoxal 5’-phosphate (PLP) biosynthesis5, isoleucine biosynthesis6, as well as ATP generation7 have been uncovered; E. coli aerobic citrate utilization8,9 and various synthetic C1-trophy10–14 have been realized. In this study, we describe E. coli recruits the same workforce, Sad, in evolution through various strategies, i.e., catalytic efficiency improvement, gene dose increase, and gene expression regulations, to overcome metabolic damages (Fig. 1). Sad, i.e. NAD(P)+-dependent succinate semialdehyde dehydrogenase (SSADH), is an aldehyde dehydrogenase (ALDH) family protein. Sad oxidizes succinate semialdehyde to succinate, preventing accumulation of the toxic aldehyde intermediate in nitrogen metabolism, such as putrescine, arginine and γ-aminobutyrate (GABA) degradations15–17. Some bacteria, for example E. coli, possess also a NADP+-dependent SSADH, GabD15,18. SSADH is ubiquitous in both prokaryotes and eukaryotes. In human, it involves in catabolism of GABA, a major neurotransmitter, its malfunction leads to a disorder SSADH deficiency19. In plants, SSADH involves in leaf development and morphogenesis20 as well as defense of abiotic stress21. And in cyanobacteria, SSADH is an essential enzyme of its uncanonical tricarboxylic acid cycle. SSADH was also found to be able to oxidize glutarate semialdehyde, participating in lysine catabolism in bacteria.

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System-level characterization of engineered and evolved formatotrophic E. coli strains

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