Leveraging engineered pseudomonas putida minicells for bioconversion of organic acids into short-chain methyl ketones

Kozaeva, Ekaterina; Nieto Domínguez, Manuel; Tang, Kent Kang Yong; Stammnitz, Maximilian R.; Nikel, Pablo Iván

Leveraging engineered pseudomonas putida minicells for bioconversion of organic acids into short-chain methyl ketones

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dc.contributor.author Kozaeva, Ekaterina
dc.contributor.author Nieto Domínguez, Manuel
dc.contributor.author Tang, Kent Kang Yong
dc.contributor.author Stammnitz, Maximilian R.
dc.contributor.author Nikel, Pablo Iván
dc.date.accessioned 2025-02-24T07:19:56Z
dc.date.available 2025-02-24T07:19:56Z
dc.date.issued 2025
dc.description.abstract Methyl ketones, key building blocks widely used in diverse industrial applications, largely depend on oil-derived chemical methods for their production. Here, we investigated biobased production alternatives for short-chain ketones, adapting the solvent-tolerant soil bacterium Pseudomonas putida as a host for ketone biosynthesis either by whole-cell biocatalysis or using engineered minicells, chromosome-free bacterial vesicles. Organic acids (acetate, propanoate and butanoate) were selected as the main carbon substrate to drive the biosynthesis of acetone, butanone and 2-pentanone. Pathway optimization identified efficient enzyme variants from Clostridium acetobutylicum and Escherichia coli, tested with both constitutive and inducible expression of the cognate genes. By implementing these optimized pathways in P. putida minicells, which can be prepared through a simple three-step purification protocol, the feedstock was converted into the target short-chain methyl ketones. These results highlight the value of combining morphology and pathway engineering of noncanonical bacterial hosts to establish alternative bioprocesses for toxic chemicals that are difficult to produce by conventional approaches.
dc.description.sponsorship We are indebted to the Microscopy Team, especially the characterization specialist Marie Karen Tracy Hong Lin at DTU NanoLab (National Centre for Nano Fabrication and Characterization, Technical University of Denmark), for their help with the processing of the samples and visualization by cryo-SEM. E.K. was the recipient of a fellowship from the Novo Nordisk Foundation as part of the Copenhagen Bioscience Ph.D. Programme (Cohort 2017), supported through grant NNF17CC0026768. M.N.D. acknowledges the support received from the European Union’s Horizon2020 Research and innovation program under the Marie Sklodowska-Curie grant agreement No. 713683 (COFUNDfellowsDTU) and from the VELUX Foundation under the Villum Experiment program (grant No. 40979). The financial support from the Novo Nordisk Foundation (grants NNF18OC0034818, NNF20CC0035580, and NNF21OC0067996) to P.I.N. is also gratefully acknowledged.
dc.format.mimetype application/pdf
dc.identifier.citation Kozaeva E, Nieto-Domínguez M, Tang KKY, Stammnitz M, Nikel PI. Leveraging engineered pseudomonas putida minicells for bioconversion of organic acids into short-chain methyl ketones. ACS Synth Biol. 2025 Jan 17;14(1):257-72. DOI: 10.1021/acssynbio.4c00700
dc.identifier.doi http://dx.doi.org/10.1021/acssynbio.4c00700
dc.identifier.issn 2161-5063
dc.identifier.uri http://hdl.handle.net/10230/69684
dc.language.iso eng
dc.publisher American Chemical Society (ACS)
dc.relation.ispartof ACS Synth Biol. 2025 Jan 17;14(1):257-72
dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/713683
dc.rights.accessRights info:eu-repo/semantics/openAccess
dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.keyword 2-pentanone
dc.subject.keyword Pseudomonas putida
dc.subject.keyword Acetone
dc.subject.keyword Butanone
dc.subject.keyword Ketones
dc.subject.keyword Metabolic engineering
dc.subject.keyword Minicells
dc.subject.keyword Synthetic biology
dc.title Leveraging engineered pseudomonas putida minicells for bioconversion of organic acids into short-chain methyl ketones
dc.type info:eu-repo/semantics/article
dc.type.version info:eu-repo/semantics/publishedVersion

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