CoChat
Sign Up
Cited by 3Microbial Metabolic Engineering and BioproductionMicrobial metabolism and enzyme functionEnzyme Catalysis and Immobilization

The DOI matches its Crossref record; 2 of the 2 references it cites were found in a registry or an index.

Awakening of the RuMP cycle for partial methylotrophy in the thermophile Parageobacillus thermoglucosidasius

Open access
Paredes-Barrada, Miguel, Mathissen, Annemieke, van der Molen, Roland A., Jiménez-Huesa, Pablo J., Polano, Machiel Eduardo, Donati, Stefano, Abele, Miriam, Ludwig, Christina, van Kranenburg, Richard, Claassens, Nico J.·2025·Metabolic Engineering
DOI 10.1016/j.ymben.2025.04.002

Published version of a preprint

Open this paper in CoChat — free account

Abstract

Given sustainability and scalability concerns of using sugar feedstocks for microbial bioproduction of bulk chemicals, widening the feedstock range for microbial cell factories is of high interest. Methanol is a one-carbon alcohol that stands out as an alternative feedstock for the bioproduction of chemicals, as it is electron-rich, water-miscible and can be produced from several renewable resources. Bioconversion of methanol into products under thermophilic conditions (>50 °C) could be highly advantageous for industrial biotechnology. Although progress is being made with natural, thermophilic methylotrophic microorganisms, they are not yet optimal for bioproduction and establishing alternative thermophilic methylotrophic bioproduction platforms can widen possibilities. Hence, we set out to implement methanol assimilation in the emerging thermophilic model organism Parageobacillus thermoglucosidasius. We engineered P. thermoglucosidasius to be strictly dependent for its growth on methanol assimilation via the core of the highly efficient ribulose monophosphate (RuMP) cycle, while co-assimilating ribose. Surprisingly, this did not require heterologous expression of RuMP enzymes. Instead, by laboratory evolution we awakened latent, native enzyme activities to form the core of the RuMP cycle. We obtained fast methylotrophic growth in which ∼17 % of biomass was strictly obtained from methanol. This work lays the foundation for developing a versatile thermophilic bioproduction platform based on renewable methanol.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1016/j.ymben.2025.04.002

Cites

2 of 2 references verified0 citation errors · 0 not checkable

Statistics

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Flagged first, then by citations

Research works printed but not linked: 59

  1. Abele, 2023, Unified workflow for the rapid and in-depth characterization of bacterial proteomes, Mol. Cell. Proteomics, 22, 10.1016/j.mcpro.2023.100612 · DOI
  2. Beber, 2022, eQuilibrator 3.0: a database solution for thermodynamic constant estimation, Nucleic Acids Res., 50, D603, 10.1093/nar/gkab1106 · DOI
  3. Brautaset, 2007, Bacillus methanolicus: a candidate for industrial production of amino acids from methanol at 50°C, Appl. Microbiol. Biotechnol., 74, 22, 10.1007/s00253-006-0757-z · DOI
  4. Brautaset, 2007, Bacillus methanolicus: a candidate for industrial production of amino acids from methanol at 50°C, Appl. Microbiol. Biotechnol., 74, 22, 10.1007/s00253-006-0757-z · DOI
  5. Bruinsma, 2023, Paving the way for synthetic C1 - metabolism in Pseudomonas putida through the reductive glycine pathway, Metab. Eng., 76, 215, 10.1016/j.ymben.2023.02.004 · DOI
  6. Chen, 2018, Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production, Metab. Eng., 49, 257, 10.1016/j.ymben.2018.08.010 · DOI
  7. Chen, 2020, Converting Escherichia coli to a synthetic methylotroph growing solely on methanol, Cell, 182, 933, 10.1016/j.cell.2020.07.010 · DOI
  8. Chistoserdova, 2007, Genome of methylobacillus flagellatus, molecular basis for obligate methylotrophy, and polyphyletic origin of methylotrophy, J. Bacteriol., 189, 4020, 10.1128/JB.00045-07 · DOI
  9. Cotton, 2020, Renewable methanol and formate as microbial feedstocks, vol. 62, 168
  10. Cox, 2011, Andromeda: a peptide search engine integrated into the MaxQuant environment, J. Proteome Res., 10, 1794, 10.1021/pr101065j · DOI
  11. Cox, 2014, Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ, Mol. Cell. Proteomics, 13, 2513, 10.1074/mcp.M113.031591 · DOI · Paper page
  12. Delépine, 2020, Charting the metabolic landscape of the facultative methylotroph Bacillus methanolicus, mSystems, 5, 10.1128/mSystems.00745-20 · DOI
  13. Di Lorenzo, 2022, State of the art on the microbial production of industrially relevant organic acids, vol. 12
  14. Gao, 2022, Constructing a methanol-dependent Bacillus subtilis by engineering the methanol metabolism, J. Biotechnol., 343, 128, 10.1016/j.jbiotec.2021.12.005 · DOI
  15. Jakobsen, 2006, Upregulated transcription of plasmid and chromosomal ribulose monophosphate pathway genes is critical for methanol assimilation rate and methanol tolerance in the methylotrophic bacterium Bacillus methanolicus, J. Bacteriol., 188, 3063, 10.1128/JB.188.8.3063-3072.2006 · DOI
  16. Keller, 2020, Methanol-dependent Escherichia coli strains with a complete ribulose monophosphate cycle, Nat. Commun., 11, 10.1038/s41467-020-19235-5 · DOI
  17. Keller, 2022, Generation of an Escherichia coli strain growing on methanol via the ribulose monophosphate cycle, Nat. Commun., 13, 5243, 10.1038/s41467-022-32744-9 · DOI
  18. Kim, 2020, Growth of E. coli on formate and methanol via the reductive glycine pathway, Nat. Chem. Biol., 16, 538, 10.1038/s41589-020-0473-5 · DOI
  19. Klein, 2022, Unravelling formaldehyde metabolism in bacteria: road towards synthetic methylotrophy, Microorganisms, 10, 220, 10.3390/microorganisms10020220 · DOI
  20. Klein, 2024, Identification and characterization of a novel formaldehyde dehydrogenase in Bacillus subtilis, Appl. Environ. Microbiol., 90, 10.1128/aem.02181-23 · DOI
  21. Kochetov, 2005, Binding of the Coenzyme and Formation of the Transketolase Active Center, IUBMB Life, 57, 491, 10.1080/15216540500167203 · DOI
  22. Komives, 2005, Growth of Bacillus methanolicus in seawater-based media, J. Ind. Microbiol. Biotechnol., 32, 61, 10.1007/s10295-004-0195-9 · DOI
  23. Kovina, 1998, Cooperativity and flexibility of active sites in homodimeric transketolase, FEBS (Fed. Eur. Biochem. Soc.) Lett., 440, 81, 10.1016/S0014-5793(98)01423-9 · DOI
  24. Kovina, 1996, Localization of reactive tyrosine residues of baker's yeast transketolase, FEBS (Fed. Eur. Biochem. Soc.) Lett., 392, 293, 10.1016/0014-5793(96)00836-8 · DOI
  25. Krog, 2013, Methylotrophic Bacillus methanolicus encodes two chromosomal and one plasmid born NAD+ dependent methanol dehydrogenase paralogs with different catalytic and biochemical properties, PLoS One, 8, 10.1371/journal.pone.0059188 · DOI
  26. Krüsemann, 2023, The cofactor challenge in synthetic methylotrophy: bioengineering and industrial applications, Curr. Opin. Biotechnol., 82, 10.1016/j.copbio.2023.102953 · DOI
  27. Kutuzova, 2020, SmartPeak automates targeted and quantitative metabolomics data processing, Anal. Chem., 92, 15968, 10.1021/acs.analchem.0c03421 · DOI
  28. Lee, 2020, Discovery and biochemical characterization of a methanol dehydrogenase from lysinibacillus xylanilyticus, Front. Bioeng. Biotechnol., 8, 10.3389/fbioe.2020.00067 · DOI
  29. Lessmeier, 2013, Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase, Microbiology, 159, 2651, 10.1099/mic.0.072413-0 · DOI
  30. Liao, 2016, Fuelling the future: microbial engineering for the production of sustainable biofuels, Nat. Rev. Microbiol., 14, 288, 10.1038/nrmicro.2016.32 · DOI
  31. Liu, J., Han, X., Tao, F., & Xu, P. (n.d.). Reprogramming a high robust Geobacillus thermoglucosidasius 1 for efficient synthesis of polymer-grade lactic acid under 2 extremely high temperature (60° C). https://doi.org/10.1101/2023.04.14.536835. · DOI
  32. Long, 2019, High-resolution 13C metabolic flux analysis, Nat. Protoc., 14, 2856, 10.1038/s41596-019-0204-0 · DOI
  33. Meyer, 2018, Methanol-essential growth of Escherichia coli, Nat. Commun., 9, 10.1038/s41467-018-03937-y · DOI
  34. Millgaard, 2023, An improved integrative GFP-based vector for genetic engineering of Parageobacillus thermoglucosidasius facilitates the identification of a key sporulation regulator, AMB Express, 13, 44, 10.1186/s13568-023-01544-9 · DOI
  35. Mougiakos, 2017, Characterizing a thermostable Cas9 for bacterial genome editing and silencing, Nat. Commun., 8, 10.1038/s41467-017-01591-4 · DOI
  36. Müller, 2015, Methylotrophy in the thermophilic Bacillus methanolicus, basic insights and application for commodity production from methanol, vol. 99, 535
  37. Müller, 2015, Core pathways operating during methylotrophy of Bacillus methanolicusMGA3 and induction of a bacillithiol-dependent detoxification pathway upon formaldehyde stress, Mol. Microbiol., 98, 1089, 10.1111/mmi.13200 · DOI
  38. Müller, 2015, Engineering Escherichia coli for methanol conversion, Metab. Eng., 28, 190, 10.1016/j.ymben.2014.12.008 · DOI
  39. Nash, 1953, The colorimetric estimation of formaldehyde by means of the Hantzsch reaction, Biochem. J., 55, 416, 10.1042/bj0550416 · DOI
  40. Nieh, 2024, Evolutionary engineering of methylotrophic E. coli enables fast growth on methanol, Nat. Commun., 15, 8840, 10.1038/s41467-024-53206-4 · DOI
  41. Paredes-Barrada, 2024, Parageobacillus thermoglucosidasius as an emerging thermophilic cell factory, Metab. Eng., 83, 39, 10.1016/j.ymben.2024.03.001 · DOI
  42. Perez-Riverol, 2022, The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences, Nucleic Acids Res., 50, D543, 10.1093/nar/gkab1038 · DOI · Paper page
  43. Raita, 2016, Production of ethanol by thermophilic oligosaccharide utilising Geobacillus thermoglucosidasius TM242 using palm kernel cake as a renewable feedstock, Biomass Bioenergy, 95, 45, 10.1016/j.biombioe.2016.08.015 · DOI
  44. Rappsilber, 2007, Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips, Nat. Protoc., 2, 1896, 10.1038/nprot.2007.261 · DOI · Paper page
  45. Reeve, 2016, The Geobacillus plasmid set: a modular toolkit for thermophile engineering, ACS Synth. Biol., 5, 1342, 10.1021/acssynbio.5b00298 · DOI
  46. Reiter, 2024, A synthetic methylotrophic Escherichia coli as a chassis for bioproduction from methanol, Nat. Catal., 7, 560, 10.1038/s41929-024-01137-0 · DOI
  47. Schann, 2024, Design, construction and optimization of formaldehyde growth biosensors with broad application in biotechnology, Microb. Biotechnol., 17, 10.1111/1751-7915.14527 · DOI
  48. Tyanova, 2016, The MaxQuant computational platform for mass spectrometry-based shotgun proteomics, Nat. Protoc., 11, 2301, 10.1038/nprot.2016.136 · DOI
  49. Wahl, 2004, New tools for mass isotopomer data evaluation in 13 C flux analysis: mass isotope correction, data consistency checking, and precursor relationships, Biotechnol. Bioeng., 85, 259, 10.1002/bit.10909 · DOI
  50. Whitaker, 2015, Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization, Curr. Opin. Biotechnol., 33, 165, 10.1016/j.copbio.2015.01.007 · DOI
  51. Witthoff, 2013, C 1 metabolism in Corynebacterium glutamicum: an endogenous pathway for oxidation of methanol to carbon dioxide, Appl. Environ. Microbiol., 79, 6974, 10.1128/AEM.02705-13 · DOI
  52. Wu, 2016, Characterization and evolution of an activator-independent methanol dehydrogenase from Cupriavidus necator N-1, Appl. Microbiol. Biotechnol., 100, 4969, 10.1007/s00253-016-7320-3 · DOI
  53. Yang, 2023, A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering, Nat. Commun., 14, 6193, 10.1038/s41467-023-41973-5 · DOI
  54. Yang, 2024, Bacillus methanolicus: an emerging chassis for low-carbon biomanufacturing, Trends Biotechnol.
  55. Yasueda, 1999, Bacillus subtilis yckG and yckF encode two key enzymes of the ribulose monophosphate pathway used by methylotrophs, and yckH is required for their expression, J. Bacteriol., 181, 7154, 10.1128/JB.181.23.7154-7160.1999 · DOI
  56. Yurimoto, 2005, HxlR, a member of the DUF24 protein family, is a DNA‐binding protein that acts as a positive regulator of the formaldehyde‐inducible hxlAB operon in Bacillus subtilis, Mol. Microbiol., 57, 511, 10.1111/j.1365-2958.2005.04702.x · DOI
  57. Zhan, 2023, Reprogramming methanol utilization pathways to convert Saccharomyces cerevisiae to a synthetic methylotroph, Nat. Catal., 6, 435, 10.1038/s41929-023-00957-w · DOI
  58. Zhou, 2016, Evolutionary engineering of Geobacillus thermoglucosidasius for improved ethanol production; Evolutionary engineering of Geobacillus thermoglucosidasius for improved ethanol production, Biotechnol. Bioeng., 113, 2156, 10.1002/bit.25983 · DOI
  59. Zhu, 2021, Genetically encoded formaldehyde sensors inspired by a protein intra-helical crosslinking reaction, Nat. Commun., 12, 581, 10.1038/s41467-020-20754-4 · DOI · Paper page

Metadata

Authors
Paredes-Barrada, M., Mathissen, A., van der Molen, R. A., Jiménez-Huesa, P. J., Polano, M. E., Donati, S., Abele, M., Ludwig, C., van Kranenburg, R., & Claassens, N. J.
Affiliations
  • Wageningen University & Research (NL)
  • Novo Nordisk Foundation (DK)
  • Technical University of Munich (DE)
Corresponding author
Claassens, Nico J.
ORCID
Venue
Metabolic Engineering
Publisher
Elsevier BV
Published
2025-09
Volume
91
Pages
145-157
ISSN
1096-7176 (ISSN-L)
DOI
10.1016/j.ymben.2025.04.002
OpenAlex
W4409464402
Cited by
3
References
2
Citations by year
2025: 3
FWCI
0.90
Type
journal article
Language
en
Open access
Licence
Locations
  • Metabolic Engineering (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • Socio-Environmental Systems Modeling (submittedVersion, cc-by)
Funders
  • German Research Foundation (INST 95/1435-1 FUGG)
  • Dutch Research Council (VI.Veni.192.156)
  • Novo Nordisk Foundation (NNF20CC0035580)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial metabolism and enzyme function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
Keywords
Parageobacillus thermoglucosidasius, ribulose monophosphate cycle, methanol assimilation, methanol dehydrogenase, experimental evolution, genome editing, one-carbon feedstocks, microbial cell factories
MeSH terms
Methanol, Bacillaceae, Ribulosephosphates, Metabolic Engineering

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review