Leveraging Engineered Pseudomonas putida Minicells for Bioconversion of Organic Acids into Short-Chain Methyl Ketones

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.

Keywords: 2-pentanone; Pseudomonas putida; acetone; butanone; ketones; metabolic engineering; minicells; synthetic biology.

Figure 1

Exploring the tolerance of P. putida and E. coli to 2-pentanone and short-chain organic acids. (A) Physiological response of P. putida SEM1.3 to increasing concentrations of 2-pentanone, added to either de Bont minimal (DBM) medium or rich LB medium in microtiter plate cultures. Cell densities, estimated as the optical density at 600 nm (OD₆₀₀), were measured after 24 h of cultivation. (B) Growth profile of P. putida SEM1.3 cultivated on DBM medium containing either glucose or 2-pentanone as the sole carbon source. (C) Growth of E. coli MG1655 and P. putida SEM1.3 cultivated in DBM medium with different carbon sources (i.e., glucose, acetate, butanoate, or a combination of the two short-chain organic acids). Cell densities in these shaken-flask cultures, estimated as the OD₆₀₀, were measured after 24 h of cultivation; specific growth rates (μ) were calculated during exponential growth. In all cases, mean values ± standard deviations were derived from independent biological triplicates. Significance levels of the cell density values when compared to control conditions [0 g L^–1 2-pentanone for panel (A) and 2 g L^–1 glucose for panel (B)] are indicated as follows: *P-value <0.05, **P-value <0.01 and ***P-value <0.001.

Figure 2

Pathway engineering and optimization for 2-pentanone biosynthesis from short-chain organic acids. (A) Biosynthetic pathway for 2-pentanone production from butanoate. Methyl ketone biosynthesis relies on the enzymes of the canonical acetone pathway from C. acetobutylicum, comprising Thl, thiolase (acetyl-CoA acetyltransferase, EC 2.3.1.9), CtfAB (acetoacetyl-CoA:acetate/butanoate CoA transferase, α and β subunits, EC 2.8.3.9), and Adc (acetoacetate decarboxylase, EC 4.1.1.4). Enzyme variants are likewise indicated whenever relevant. Abbreviations: CoA, coenzyme A; PPi, inorganic pyrophosphate. (B) Synthetic operons constructed for 2-pentanone biosynthesis. The elements in this diagram are not drawn to scale. (C) Testing 2-pentanone biosynthesis from short-chain organic acids in engineered P. putida. Bacteria were individually transformed with plasmids pS2313·MK[c,s1-s3], carrying the synthetic operons shown in panel (B) under control of the constitutive P_EM7 promoter (Table 1), and incubated for 24 h in DBM medium with the different carbon source combinations indicated; 2-pentanone titers were quantified in culture supernatants by GC-FID. (D) Plasmids for inducible expression of the synthetic operons for 2-pentanone biosynthesis. The synthetic XylS/Pm and RhaRS/P_rhaBAD expression systems are induced by addition of 3-methylbenzoate and rhamnose, respectively; both plasmids carry a streptomycin-resistance determinant (Str^R). (E) Exploring strain performance by growing P. putida SEM1.3 with the selected plasmids in DBM medium containing 3 g L^–1 butanoate for 24 h. 2-Pentanone titers in the culture supernatant were quantified by GC-FID; methyl ketone titers in strains carrying inducible expression systems are compared to the constitutive expression mediated by the P_EM7 promoter. Results shown in panel (C) and (E) correspond to mean values ± standard deviations from independent biological triplicates. Significance levels of 2-pentanone titers when compared to control conditions [pathway variant 1, spanning the canonical set of enzymes, for panel (C), and constitutive gene expression mediated by the P_EM7 promoter for panel (E)] are indicated as follows: *P-value <0.05, **P-value <0.01 and ***P-value <0.001.

Figure 3

Production and characterization of P. putida minicells. (A) Key elements involved in cell division in Gram-negative bacteria. In normally shaped cells, the MinC–MinD–MinE system and the cell wall structural, actin-like MreB protein establish a dynamic equilibrium with FtsZ, the division protein, to ensure proper septation and segregation of daughter cells. Mutations in the Min components, e.g., the Z-ring positioning MinD protein, lead to minicell segregation. (B) Overview of the protocol for P. putida minicells production, purification, storage, and downstream applications. The minicell suspension can be either used immediately or stored at −20 °C or −70 °C upon buffer exchange. Whenever needed, the minicell preparation is incubated in microtiter plates under specific conditions to support product formation, and the metabolites of interest are detected by dedicated analytical methods. (C) Representative scanning electron cryomicroscopy (CryoSEM) images of strains P. putida SEM1.3 and its minicell-producing derivative. CryoSEM microscopy was performed on freeze-fractured samples from these cultures, imaged at 15 kV in an X-Max^N 150 silicon drift detector (sensor-active area = 150 mm²; Oxford Instruments NanoAnalysis) after a Pt-coating treatment. Parental cells and minicells are identified with green and blue arrowheads, respectively; the asymmetrical segregation of daughter cells leads to the formation of chromosome-free minicells that retain plasmid(s) from the parental bacterium. (D) Representative phase-contrast (bright field) and fluorescence merged images of GFP-fluorescence and DAPI staining of a ΔminD derivative of P. putida KT2440 carrying plasmid pS4413·msfGFP. The arrow marks the position of a bacterial minicell. (E) Representative thin-sectioned negative-stain micrographs (transmission electron microscopy, TEM) of the same strains (wild-type, WT, and a ΔminD mutant) shown in panel (D).

Figure 4

Exploring (methyl) ketone biosynthesis by P. putida. (A–C) Synthetic pathways tested for the bioconversion of different organic acids into short-chain (methyl) ketones by P. putida minicells. The selected pathways mediate the production of acetone from acetate (C₂) (A), butanone from propanoate (C₃) (B), and 2-pentanone from butanoate (C₄) (C). The theoretical yield for all (methyl) ketones shown in the diagram is Y_P/S = 50% mol mol^–1. Abbreviations: CoA, coenzyme A; PPi, inorganic pyrophosphate. (D) Whole-cell biocatalysis experiments with P. putida SEM 1.3 and KT2440 were performed in the presence of acetate, propanoate or butanoate as the substrates for short-chain (methyl) ketone (MK) production. In all cases, P. putida strains were incubated with 50 mM of selected organic acid and 15% (v/v) glycerol. Both organic acid (OA) consumption and product (acetone, butanone and 2-pentanone) titers were determined after 24 h. Results correspond to mean values ± standard deviations from three independent biological triplicates.

Figure 5

Short-chain (methyl) ketone production byP. putidaminicells. (A) Bioconversion experiments performed using acetate, propanoate or butanoate as the substrate for short-chain (methyl) ketone biosynthesis by minicells. P. putida minicells were incubated in the presence of the selected organic acid (50 mM) and 15% (v/v) glycerol. Both product (acetone, butanone and 2-pentanone) titers (A) and substrate (acetate, propanoate and butanoate) consumption (B) were determined after 24 h. (C) The effect of glycerol on bioconversion of organic acids into (methyl) ketones was assessed after 18 h of incubation with 15% (v/v) glycerol and/or 50 mM of the corresponding organic acid substrate (acetate, propanoate or butanoate). Ctrl., control. Substrate consumption by purified P. putida minicells under the same experimental conditions is indicated in panel (D). Acetate, propanoate or butanoate could not be detected in samples that were not supplemented with the corresponding substrates; therefore, these experiments are not included in the figure. (E) Acetone titers were used as a proxy for evaluating production phenotypes in P. putida minicells after storage at −70 °C in the presence of 15% (v/v) glycerol. Engineered minicells, prepared as indicated in Figure 3B, were tested for their ability to mediate the bioconversion of acetate (50 mM) into acetone. Acetone titers were compared to a control (Ctrl.) experiment conducted with freshly prepared P. putida minicells. Results shown in panels (A–E) represent mean values ± standard deviations from independent biological triplicates. (F) Plasmid DNA content in P. putida SEM1.3 and its minicell-producing derivative. Lyophilized samples (corresponding to 6.5 mg cell dry weight) were used to isolate plasmid DNA with a commercial kit; plasmid DNA content is expressed in ng DNA mg cell dry weight^–1. Results correspond to mean values ± standard deviations from three independent biological triplicates; significance levels are indicated with **P-value <0.01.