Biological production of polyhydroxyalkanoates and monomers therefor using engineered pseudomonas cells

Abstract

The present invention relates to cells and methods for producing polymers from acetate. The invention also relates to cells with increased tolerance to short-chain organic acids, such as acetate. In particular, the invention relates to production of polyhydroxybutyrate (PHB), fluorinated 3-hydroxybutyrate and fluorinated PHB (F-PHB) from acetate and / or F-acetate using bacterial cells with increased tolerance to acetate.

Description

[0001] BIOLOGICAL PRODUCTION OF POLYHYDROXYALKANOATES AND MONOMERS THEREFOR USING ENGINEERED PSEUDOMONAS CELLS

[0002] Technical field

[0003] The present invention relates to cells and methods for producing polymers from acetate. The invention also relates to cells with increased tolerance to shortchain organic acids, such as acetate. In particular, the invention relates to production of poly(3-hydroxybutyrate) (PHB), fluorinated 3-hydroxy butyrate and fluorinated PHB (F- PHB) from acetate and / or F-acetate using bacterial cells with increased tolerance to acetate.

[0004] Background

[0005] Bio-based polymers, and in particular fluorinated polymers, are poised to replace petrochemical-derived counterparts, offering a more environmentally friendly solution to the unsustainable practices of traditional polymer- and fluorochemistry and the global plastic challenge. In particular, polyhydroxyalkanoates (PHAs) are biobased and biodegradable polyesters. PHAs feature a wide diversity of physical and mechanical properties that can be upgraded by the introduction of functionally diverse chemical groups (e.g. F atoms) into the side chains of the polymers to fulfill the material demands of the materials market (Mezzina et al., 2021). Fluorinated polymers largely dominate the fluorochemical market presenting numerous industrial applications, e.g. as oil- and water-repellents, lubricants, easy-to-clean surfaces, coatings, and surfactants. Currently, industrial fluorochemicals production, including fluoropolymers, largely relies on unsustainable chemical processes as well as oil-derived feedstocks. Furthermore, owing to the high reactivity of F, the degree of fluorination remains difficult to control, thereby giving rise to production of harmful poly- and perfluorinated substances (PFAS) and PFAS-based materials, which are recognized as "forever chemicals" due to their persistent nature and negative health and environmental impacts.

[0006] The efficient production of bio-based polymers, including fluorinated polymers, poses various challenges, primarily due to higher production costs compared to their fossil fuel-derived counterparts. A strategy towards reducing operation costs is to implement low-cost carbon sources and readily available fluorinated precursors that can be further converted into PHAs and its derivatives. In particular, acetic acid or acetate is a feedstock produced by a variety of procedures, e.g. first- and second-generation biomass fermentation, acetaldehyde oxidation, hydrocarbon oxidation, and methanol carbonylation. From a circular economy point of view, acetate can be recovered as a by-product during the manufacture of terephthalic acid, or produced by hydrolysis of lignocellulosic biomass, microbial fermentation from renewable sources (e.g. CO2, CO, and Syngas), and also microbial electrosynthesis. Because of its chemical simplicity, growing demand, multiple ways of production, and low cost, acetate, as well as other short-chain organic acids, is considered a viable feedstock for biotechnological processes with a variety of applications. Although acetate and other short-chain organic acids offers an attractive alternative for the development of competitive bioprocesses, they have a detrimental impact on bacterial physiology and metabolism (Kirkpatrick et al. 2001).

[0007] Summary

[0008] The invention is as defined in the claims.

[0009] The invention concerns engineered bacterial cells with improved tolerance to nonnative, short-chain organic acids, such as acetate, lactate, propionate, and / or butyrate, and the application of such cells for efficient biosynthesis of acetate-derived compounds, such as short-chain length (scl-) polymers and monomers therefor and in particular fluorinated scl-polymers and monomers therefor. Surprisingly, the inventors have found that improved tolerance to non-native, short-chain organic acids by means of mutation of fabB, uvrY and / or hupB, or equivalents thereof, enables production by bacterial cells of hitherto unseen titers of the scl-polyhydroxyalkanoate (scl-PHA) polyhydroxybutyrate (PHB), the monomer 3-hydroxybutyrate (3HB) as well as fluorinated PHB (F-PHB) and fluorinated 3HB (F-3HB). For the purpose of producing F- PHB, the inventors developed engineered cells with modifications for the inactivation of medium chain length (mcl-) polymer production and for the promotion of scl-PHA production, such as F-3HB and F-PHB production, as described in further detail herein.

[0010] Thus, herein disclosed is an engineered bacterial cell, said cell comprising one or more modifications selected from the group consisting of: one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), such as a mutation resulting in substitution of residue 77 of FabB, preferably resulting in FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70); one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10); one or more mutations in HupB (SEQ ID NO: 8 or SEQ ID NO: 69), such as a mutation resulting in substitution of residue 35 of HupB, preferably resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71), and / or a mutation resulting in substitution of residue 49 of HupB (SEQ ID NO: 8 or SEQ ID NO: 69), preferably resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72), or one or more mutations in a nucleic acid encoding a functional variant thereof having at least 70% identity to any one of FabB, UvrY or HupB, preferably the engineered bacterial cell is a Pseudomonas cell, most preferably a P. putida cell.

[0011] Disclosed herein is also a method of producing a polyhydroxyalkanoate (PHA) having a short carbon chain length (scl-PHA), preferably wherein the scl-PHA is polyhydroxybutyrate (PHB), preferably fluorinated PHB (F-PHB), in a Pseudomonas cell, said method comprising the steps of: a) Providing an engineered bacterial cell, preferably an engineered Pseudomonas cell, b) Cultivating said engineered cell in a culture medium comprising a carbon source and a nitrogen source; wherein: i. the engineered cell is not capable of producing PHAs having a mediumlength carbon chain (mcl-PHAs); ii. the engineered cell is not capable of converting fatty acids into PHA monomers having a medium-length carbon chain, iii. optionally the benABCD cluster in said cell is inactivated, and iv. the engineered cell comprises a nucleic acid encoding an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting acetyl-CoA into acetoacetyl-CoA, preferably a nucleic acid encoding PhaA from Cupriavidus necator (SEQ ID NO: 1), a nucleic acid encoding an acetyl-CoA reductase (EC 1.1.1.36) capable of converting acetoacetyl-CoA into F?-(-)-3-hydroxybutyryl-CoA, preferably a nucleic acid encoding PhaB from Cupriavidus necator (SEQ ID NO: 2), and a nucleic acid encoding a short-chain-length-polyhydroxyalkanoate (scl- PHA) synthase (EC 2.3.1.304) capable of polymerizing ?-(-)-3- hydroxybutyryl-CoA into polyhydroxybutyrate (PHB), preferably a nucleic acid encoding PhaC from Cupriavidus necator (SEQ ID NO: 3), or functional variants thereof having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, preferably wherein the culture medium comprises glucose, citrate, acetate and / or F-acetate as carbon source, thereby producing a scl-PHA with a titer of at least 0.25 g / L, preferably wherein the scl-PHA comprises or consists of polyhydroxybutyrate (PHB) and / or F-PHB.

[0012] Also disclosed herein is also a method of producing a 3-hydroxybutyrate (3HB), preferably wherein the 3HB is fluorinated, in an engineered cell, preferably a bacterial cell, said method comprising the steps of: a) providing an engineered cell, preferably an engineered Pseudomonas cell, optionally a P. putida cell, b) cultivating said engineered cell in a culture medium comprising a carbon source and a nitrogen source; wherein: i. the engineered cell is not capable of producing PHAs having a medium-length carbon chain (mcl-PHAs); ii. the engineered cell is not capable of converting fatty acids into PHA monomers having a medium-length carbon chain; iii. optionally the benABCD cluster in said cell is inactivated, and iv. the engineered cell comprises a nucleic acid encoding an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting acetyl-CoA into acetoacetyl-CoA, preferably a nucleic acid encoding PhaA from Cupriavidus necator (SEQ ID NO: 1), a nucleic acid encoding an acetyl-CoA reductase (EC 1.1.1.36) capable of converting acetoacetyl-CoA into F?-(-)-3-hydroxybutyryl-CoA, preferably a nucleic acid encoding PhaB from Cupriavidus necator (SEQ ID NO: 2), and a nucleic acid encoding a thioesterase (EC 3.1.2.20) capable of hydrolysing a 3-hydroxybutyryl-CoA to CoA and a corresponding 3-hydroxybutyrate, preferably a nucleic acid encoding TesB from P. putida (PpTesB, SEQ ID NO: 38) or Escherichia coli (EcTesB, SEQ ID NO: 39), or functional variants thereof having at least 70% identity to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, preferably wherein the culture medium comprises glucose, citrate, acetate and / or F- acetate as carbon source, thereby producing 3HB, preferably fluorinated 3HB, optionally with a titer of at least 0.25 g / L, preferably wherein the fluorinated 3HB comprises or consists of 2,4-difluoro-3- hydroxy butyrate, 4-fluoro-3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate.

[0013] Description of Drawings

[0014] Figure 1. Engineering of a P. putida strain for poly(3-hydroxybutyrate) (PHB) synthesis. (A) Construction of a PHA-deaf P. putida mutant. Several deletions were performed in the parental strain P. putida EM42 to obtain strain P. putida SEMI .4, which cannot produce PHAs of any type. (B) Construction of a series of plasmids for expression of the genes encoding the PHB biosynthetic pathway. Plasmid pS6311 PHB, selected for further experiments, bears the phaCAB operon under the regulation of the orthogonal ChnR / PchnB expression system (SEQ ID NO: 5), inducible by addition of cyclohexanone to the culture medium. (C) Heterologous PHB biosynthetic pathway and metabolic routes involved in PHB synthesis from glucose and acetate in the engineered strains. Deletion of phaG and phaC1C2 prevents synthesis of mcl-PHA monomers from fatty acid synthesis (FAS) intermediates.

[0015] Figure 2. Growth curves of strain SEMI .4 transformed with pSEVA-derived plasmids bearing the synthetic phaCAB operon under the regulation of different expression systems. Culture conditions were as follows: (A) de Bont minimal medium (DBM) with 20 mM glucose, C / N ratio = 4; (B) 0.1 N DBM with 20 mM glucose, C / N ratio = 40; (C) DBM with 30 mM acetate, C / N ratio = 20; and (D) 0.1 N DBM with 30 mM acetate, C / N ratio = 20. Growth curves were performed in triplicates; mean values are shown in the graphs. OD, optical density.

[0016] Figure 3. PHB production by strain SEMI .4 transformed with pSEVA plasmids bearing the synthetic phaCAB operon under the regulation of different expression systems under balanced and N-limiting growth conditions. (A) PHB production from glucose. PHB content (% w / w), PHB titer (g / L) and final biomass density (cell dry weight, CDW, in g / L) under balanced and N-limiting growth conditions. (B) PHB production from acetate. PHB content (% w / w), PHB titer (g / L) and final biomass density (cell dry weight, CDW, in g / L) under balanced and N-limiting growth conditions. Cultures were performed in 4 replicates; mean and SD values are shown in the graphs. Statistical analysis: unpaired t-student’s test was performed in order to compare growth of the different expression systems in balanced and N-limiting growth conditions. Significant differences are indicated with *. Strain SEM1.4 / pS6311 PHB is the best-performing PHB-producing P. putida strain tested and was termed strain SEM1.4-PHB henceforth.

[0017] Figure 4. Role of 2-ketoglutarate in alleviating fluoroacetate toxicity. (A) Mechanism of fluoroacetate (F-acetate) toxicity and growth curves of strain SEM1.4-PHB in DBM with different C sources and with or without the addition of F-acetate and / or 2-ketoglutarate (2KG) when using (A) 30 mM glucose, (B) 30 mM acetate or (C) 30 mM citrate as the main C source.

[0018] Figure 5. Identification and characterization of biologically-produced fluorinated F-PHB by NMR analysis. (A)1H-NMR of the extracted F-PHB; (B) superimposed19F-1H spectra without and with decoupling; and (C) 2D19F-1H HETCOR NMR spectra.

[0019] Figure 6. Water contact angle (WCA) and surface energy measurements in the biologically-produced fluorinated polymers. (A) Static WCA analysis of the F-PHB polymers purified from fermentations F1 , F2 and F3. (B) Dynamic contact angle analysis of different solvents on the surface of the polymer produced during fermentation F3. F-PHB was found to be water repellent.

[0020] Figure 7. Growth curves of strain SEMI .4, SEM1.4evoand reverse-engineering mutants (RsmYR277H, GacAE197K, FabBL77P, HupBA35Vand GacAE197KFabBL77P) at increasing acetate concentrations. Bacterial growth was assessed at 20, 40, 60, 80, 100, 120 and 140 mM acetate. Growth curves were performed in triplicates, and mean values are shown in the graphs.

[0021] Figure 8. Growth curves of the reverse-engineering triple mutants (i.e. strain SEMI .4 carrying the FabBL77P, UvrYE197K, and HupBA35Vmutations, individually or in combinations) grown in DBM in the presence of 100 mM acetate. Growth curves were performed in triplicates, and mean values are shown in the graphs. Figure 9. Volcano plot of mass spectrometry results showing differentially expressed proteins in evolved and reverse-engineered strains. Proteome profiles for P. putida SEMI.4, as wild-type (A), the evolved SEM1.4evostrain (B), and the double mutant gacAE197KfabBL77Pstrain (C). Four biological replicates were carried out in DBM supplemented with 100 mM acetate. Cells were harvested in the mid-exponential phase to measure the genome-wide protein content. Each point in the plot represents an individual protein. The horizontal and vertical intersections were set at a 1.

[0022] Figure 10. Acetate metabolism and associated proteins in P. putida KT2440 and derivatives. (A) In vivo carbon flux distributions in the central metabolism of P. putida SEMI .4 (B), the evolved SEM1.4evostrain (C), and gacAE197KfabBL77P(D) during growth on acetate as the sole carbon source. Fluxes are expressed as a molar percentage of the average acetate uptake rate. Fluxes were expressed as a molar percentage of the average acetate uptake rate (34.1 , 25.9 and 23.8 mmol gcow'1h-1for strains SEMI.4, SEM1.4evoand the double gacAE197KfabBL77Pmutant, respectively). The ranges assigned to each flux indicate the respective 95% confidence intervals. The fluxes were represented graphically by coloring the arrows in the flux map using a gray scale, where high fluxes were depicted as dark gray and no fluxes were shown as white.

[0023] Figure 11. Growth and PHB production by strain SEM1.4evocarrying plasmid p6311 PHB in bioreactor cultures. A time-resolved chart, including bacterial growth, indicated as ODeoo, as well as the cell dry weight (CDW) density and PHB titer, is shown.

[0024] Figure 12. (A) Synthetic pathways used for the synthesis of fluorinated 3- hydroxy butyrate (3HB) from fluoroacetate (F-acetate). (B) Structure of the plasmids constructed to bear the synthetic pathway. The genes encoding the different enzyme variants tested are indicated. (C) Profiles of 3HB production by strains transformed with the different plasmids when cultivated in the presence of different acetate (H) to F- acetate (F) ratios as indicated. Detailed description

[0025] Definitions

[0026] The term short-length carbon chain or short-chain length with respect to PHAs herein refers to PHAs with monomers between two and five carbon atoms (glycolate to valerate). scl-PHAs are described in Sagong et al., 2018. The terms scl-PHA, PHA having short-length carbon chain, and PHA with or of short-length carbon chain are interchangeable and may be used so herein.

[0027] The term medium-length carbon chain or medium-chain length with respect to PHAs herein refers to PHAs with monomers longer than five carbon atoms, i.e. monomers of six (6) to sixteen (16) carbon atoms (3-hydroxyhexanoate and onwards). mcl-PHAs are described in Sagong et al., 2018. The terms mcl-PHA, PHA having medium-length carbon chain, and PHA with or of medium-length carbon chain are interchangeable and may be used so herein.

[0028] The term polyhydroxybutyrate herein refers to a polyester belonging to the PHA class of polymers. The terms polyhydroxybutyrate, poly(3-hydroxybutyrate), poly(3- hydroxybutyric acid), PHB, P3HB, poly(p-hydroxybutyrate), poly(p-hydroxybutyric acid), and P(3HB) may be used interchangeable herein.

[0029] The term 3-hydroxy butyrate herein refers to the conjugate base of 3-hydroxybutyric acid with the chemical formula CH3CH(OH)CH2CO2H. The terms 3-hydroxybutyrate, [3- hydroxybutyrate, [3-hydroxybutyric acid, BHB, 3-hydroxybutyric acid, 3HB, HB, and 3- HB may be used interchangeably herein.

[0030] The term fluorinated 3HB herein refers to fluorinated analogues of 3HB. With respect to the present disclosure, fluorinated 3HB refers to 4-fluoro-3-hydroxybutyrate, 2-fluoro-3- hydroxybutyrate, and / or 2, 4-fluoro-3-hydroxybutyrate. The terms fluorinated 3- hydroxy butyrate, fluorinated 3HB, fluorinated HB, F-HB, and FHB may be used interchangeably herein.

[0031] As described herein, the term fluorinated PHB refers to one or more polymers comprising or consisting of fluorinated 3HB monomers. One example of a fluorinated PHB may be one or more polymers comprising or consisting of the monomers 4-fluoro- 3-hydroxybutyrate, 2-fluoro-3-hydroxybutyrate, and / or 2,4-difluoro-3-hydroxybutyrate. Another example of fluorinated PHB may be one or more polymers comprising or consisting of at least the following monomers: i) one or more selected monomers selected from the group consisting of: 4- fluoro-3-hydroxybutyrate, 2-fluoro-3-hydroxybutyrate, and 2,4-difluoro-3- hydroxy butyrate; and ii) 3-hydroxybutyrate.

[0032] As will be understood to someone of skill in the art, when the fluorinated PHB is derived from more than one species of monomers, it is a co-polymer, such as a copolyester. The units derived from the monomers may be in any order within the polymer chain. For example, the units derived from 3-hydroxybutyrate, 4-fluoro-3- hydroxy butyrate, 2-fluoro-3-hydroxybutyrate, and / or 2,4-difluoro-3-hydroxybutyrate may be in any order within fluorinated PHB polymer chains. The terms fluorinated PHB, F- PHB, poly(fluoro-3-hydroxybutryate), poly(3-hydroxybutyrate-co-4-fluoro-3- hydroxybutyrate), poly(3-hydroxybutyrate-co-fluoro-3-hydroxybutyrate), P(F-HB-co- 3HB), P(FHB-co-HB), fluoro-PHB, poly(2-fluoro-3-hydroxybutyrate), poly(3- hydroxybutyrate-co-2-fluoro-3-hydroxybutyrate), poly(2,4-fluoro-3-hydroxybutyrate), poly(3-hydroxybutyrate-co-4-fluoro-3-hydroxybutyrate-co-2-fluoro-3-hydroxybutyrate), poly(3-hydroxybutyrate-co-4-fluoro-3-hydroxybutyrate-co-2-fluoro-3-hydroxybutyrate- co-2,4-fluoro-3-hydroxybutyrate), and poly(3-hydroxybutyrate-co-2,4-fluoro-3- hydroxybutyrate), all refers to F-PHB.

[0033] The term fluorinated PHB herein refers in particular to fluorinated analogues of PHB having the structure of formula (I) and / or of formula (II): wherein: x, y and z are integers between 1 and 19,999, and wherein:

[0034] - for (I): x+y<20,000

[0035] - for (II): x+y+z<20,000.

[0036] As described above, the monomeric units corresponding to x, y and / or z in formula (I) or formula (II) may be in any order within the polymer chain.

[0037] Thus, the term fluorinated PHB herein may refer to: a compound of formula (I); a compound of formula (II); or a compound of formula (I) and of a compound of formula (II).

[0038] The titer of a compound refers herein to the produced concentration of a compound. When the compound is produced by a cell, the term refers to the total concentration produced by the cell, i.e. the total amount of the compound divided by the volume of the culture medium. This means that, particularly for volatile compounds, the titer includes the portion of the compound which may have evaporated from the culture medium, and it is thus determined by collecting the produced compound from the fermentation broth and from potential off-gas from the fermenter. The titer of the compound takes into account both intracellular and extracellular amounts of the produced compound.

[0039] The conversion rate of a given compound produced from a given substrate refers to the percentage of the substrate which has been converted to the product during a given duration.

[0040] The term water contact angle (WCA) herein refers to the angle between a liquid surface and a solid surface where they meet, where the liquid is water. More specifically, it is the angle between the surface tangent on the liquid-vapor interface and the tangent on the solid-liquid interface at their intersection. It quantifies the wettability of a solid surface by a liquid. If the measured water contact angle is above 90 degrees (90°C), the solid is said to have poor wetting and is hydrophobic. If the water contact angle is below 90 degrees, the solid is said to have good wetting and is hydrophilic.

[0041] The term surface energy herein refers to the energy that exists between the surface molecules of solid materials or substances when a comparable attractive force exists. It quantifies the disruption of intermolecular bonds that occurs when a surface is created. Surface energy is commonly measured through contact angle experiments: the contact angle of the surface is measured with one or several liquids, usually water and diiodomethane. Based on the contact angle results and knowing the surface tension of the liquids, the surface energy can be calculated.

[0042] The term contact angle herein refers to the angle between a liquid surface and a solid surface where they meet. More specifically, it is the angle between the surface tangent on the liquid-vapor interface and the tangent on the solid-liquid interface at their intersection. It quantifies the wettability of a solid surface by a liquid.

[0043] The term F-acetate herein refers to acetate that is fluorinated at carbon 2. The terms F- acetate, FAc, F-Ac, 2-fluoroacetate, FA, fluoroacetate, fluoro-acetate, or fluorinated acetate are interchangeable and may be used so herein.

[0044] The term acetate tolerance herein refers to the ability of a cell to tolerate acetate, i.e. the ability of a cell to grow in the presence of acetate, to use acetate as a carbon source, and / or to metabolise acetate. The term comprises acetate fitness. Improved acetate tolerance or acetate fitness may be increased growth rate, increased final cell density (biomass), and / or reduced lag-phase compared to a reference cell not having improved acetate tolerance.

[0045] The term non-native, when used in the context of organics acids, herein refers to organic acids that are not naturally produced by a non-engineered Pseudomonas cell. Non-native organic acids do not form part of the metabolism, nor are metabolites, in a non-engineered Pseudomonas cell. The term short-chain, when used in the context of organic acids, herein refers to organic acids comprising two to five carbon atoms, such as two, three, four or five carbon atoms, and encompass acetic acid, lactic acid, propionic acid, butyric acid, acetate, lactate, propionate and butyrate. The terms short and C2-C5, may also be used interchangeably herein to indicate organic acids comprising between two and five carbon atoms. Preferably the short-acid chain organic acids as monocarboxylic acids.

[0046] The term functional variant refers herein to a functional variant of a protein or polypeptide, in particular a functional variant of an enzyme. A functional variant of a given enzyme may have a different amino acid sequence than the given enzyme, but is able to perform the same function, either partially or totally. In other words, a functional variant of a given enzyme has the same function as the given enzyme, albeit the extent of the function may be different: the functional variant may be less active than the given enzyme, or the functional variant may be more active than the given enzyme. Other features of the functional variant may differ from the given enzyme, e.g. half-life. The functional variant retains at least some of the activity exerted by the given enzyme.

[0047] The term variant in relation to a nucleic acid designates a variant of a given nucleic acid encoding a given polypeptide, i.e. a nucleic acid encoding a functional variant of the given polypeptide.

[0048] The term mutation with respect to nucleic acids, such as genes, herein refers to substitutions, insertions, deletions, and indels. A mutation in a gene refers to a mutation in the coding sequence, in the promoter of the gene, or in the terminator of the gene.

[0049] The terms innate, endogenous, and native with respect to polypeptides or nucleic acids, such as genes or gene clusters, refers herein to polypeptides or nucleic acids that has not been introduced to the engineered cell, i.e. are not heterologous. The terms innate, endogenous, native, intrinsic, and inherent may be used interchangeably herein.

[0050] The terms identity and homology, with respect to a polynucleotide (or polypeptide), are defined herein as the percentage of nucleic acids (or amino acids) in the candidate sequence that are identical or homologous, respectively, to the residues of a corresponding native nucleic acids (or amino acids), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity I similarity I homology, and considering any conservative substitutions according to the NCIIIB rules (http: / / www.chem. qmul.ac.uk / iubmb / misc / naseq.html; NC-llIB, Eur J Biochem (1985)) as part of the sequence identity. Neither 5' or 3' extensions nor insertions (for nucleic acids) or N’ or C’ extensions nor insertions (for polypeptides) result in a reduction of identity, similarity or homology. Methods and computer programs for the alignments are well known in the art. Generally, a given identity between two sequences implies that the homology between these sequences is at least equal to the identity; for example, if two sequences are 70% identical to one another, they cannot be less than 70% homologous to one another - but could be sharing 80% homology. Throughout the present disclosure, the term “at least 70%” when referring to a percentage of sequence homology or of sequence identity shall refer to a sequence homology or identity of at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0051] The term “heterologous” when referring to a polypeptide, such as a protein or an enzyme, or to a polynucleotide, shall herein be construed to refer to a polypeptide or a polynucleotide which is not naturally present in a wild type cell.

[0052] The term acetyl-CoA thiolase herein refers to an enzyme with EC 2.3.1.9, which is capable of producing an acetoacetyl-CoA from acetate, such as fluorinated acetoacetyl-CoA from fluoroacetate (F-acetate). Acetyl-CoA thiolase is capable of catalysing the reactions:

[0053] 2 acetyl-CoA = CoA + acetoacetyl-CoA acetyl-CoA + [acetyl-CoA C-acetyltransferase]-L-cysteine = [acetyl-CoA C- acetyltransferase]-S-acetyl-L-cysteine + CoA [acetyl-CoA C-acetyltransferase]-S-acetyl-L-cysteine + acetyl-CoA = acetoacetyl-CoA + [acetyl-CoA C-acetyltransferase]-L-cysteine

[0054] Herein disclosed are acetyl-CoA thiolases capable of producing fluorinated acetoacetyl-CoA (F-acetoacetyl-CoA) from F-acetate. Acetyl-CoA thiolase may also be referred to as acetyl-coenzyme A thiolase, acetyl-CoA acetyltransferase, acetoacetyl- CoA thiolase, acetyl-CoA C-acetyltransferase, beta-acetoacetyl coenzyme A thiolase, thiolase II, type II thiolase, 3-oxothiolase, or acetyl-CoA:N-acetyltransferase, and the terms may be used interchangeably herein.

[0055] The term acetyl-CoA reductase herein refers to an enzyme with EC 1.1.1.36, which is capable of producing R-(-)-3-hydroxybutyryl-CoA from acetoacetyl-CoA, such as fluorinated R-(-)-3-hydroxybutyryl-CoA from fluorinated acetoacetyl-CoA. The acetyl- CoA reductase is known to be capable of catalysing the reaction: (R)-3-hydroxyacyl-CoA + NADP+= 3-oxoacyl-CoA + NADPH + H+Acetoacetyl-CoA reductase may also be referred to as (R)-3-hydroxyacyl-CoA dehydrogenase, D-3-hydroxyacyl-CoA reductase, short chain betaketoacetyl (acetoacetyl)-CoA reductase, hydroxyacyl coenzyme-A dehydrogenase, NADP-linked acetoacetyl CoA reductase, NADPH:acetoacetyl-CoA reductase, D(-)- beta-hydroxybutyryl CoA-NADP oxidoreductase, or beta-ketoacyl-CoA reductase ( - ketoacyl-CoA reductase), and the terms may be used interchangeably herein.

[0056] The term scl-PHA synthase herein refers to an enzyme with EC 2.3.1.304, which is capable of polymerizing R-(-)-3-hydroxybutyryl-CoA into polyhydroxybutyrate (PHB), for example polymerizing fluorinated R-(-)-3-hydroxybutyryl-CoA into fluorinated PHB. The scl-PHA synthase is known to be capable of catalyzing the reactions: (3R)-3-hydroxyacyl-CoA + poly[(R)-3-hydroxyalkanoate]n= CoA + poly[(R)-3- hydroxyalkanoate]n+i (R)-3-hydroxybutanoyl-CoA + [(R)-3-hydroxybutanoate]n= [(R)-3-hydroxybutanoate]n+i + CoA 3-hydroxyacyl-CoA + [(R)-3-hydroxyacyl]n= [(R)-3-hydroxyacyl]n+i + CoA 3-hydroxybutyryl-CoA + [(R)-3-hydroxybutanoate]n= [(R)-3-hydroxybutanoate]n+i + CoA scl-PHA synthase may also be referred to as poly[(R)-3-hydroxyalkanoate] polymerase PHA polymerase, and PHA synthase, and the terms may be used interchangeably herein. The term thioesterase herein refers to an enzyme with EC 3.1.2.20, which is capable of hydrolysing a 3-hydroxybutyryl-CoA to CoA and a corresponding 3-hydroxybutyrate, such as hydrolysing a fluorinated 3-hydroxybutyryl-CoA to CoA and a corresponding fluorinated 3-hydroxybutyrate. Thioesterase is known to be capable of catalysing the reaction: an acyl-CoA + H2O = CoA + a carboxylate.

[0057] Thioesterase may also be referred to as thioester hydrolase, acyl-CoA hydrolase, acyl- CoA thioesterase, acyl-CoA thioesterase, acyl-CoA thioesterase 2, thioesterase II, thioesterase B, and TesB.

[0058] Bacterial cell

[0059] In a first aspect, provided is an engineered cell, preferably a bacterial cell, such as an engineered Pseudomonas cell, said cell comprising one or more modifications selected from the group consisting of: one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), such as a mutation resulting in substitution of residue 77 of FabB, preferably resulting in FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70); one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10); one or more mutations in HupB (SEQ ID NO: 8 or SEQ ID NO: 69), such as a mutation resulting in substitution of residue 35 of HupB, preferably resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71), and / or a mutation resulting in substitution of residue 49 of HupB (SEQ ID NO: 8 or SEQ ID NO: 69), preferably resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72), or one or more mutations in a nucleic acid encoding a functional variant thereof having at least 70% identity to any one of FabB, UvrY or HupB.

[0060] The engineered cell is useful for producing acetate-derived compounds, for example compounds as described in the section “Acetate-derived compounds” herein below, for example using a method also described herein below in the section “Methods for production of acetate-derived products”. Preferably, the engineered cell is used for producing 3HB, fluorinated 3HB, PHB, and / or fluorinated PHB. Origin of bacterial cell

[0061] In preferred embodiments, the engineered cells disclosed herein are bacterial cells. Preferably, the bacterial cells can tolerate toxic compounds, such as fluorinated compounds. In some embodiments, the engineered cell is a cell of a non-pathogenic organism. For example, the engineered cell is a cell of a GRAS (Generally Regarded As Safe) or HV1-certified organism.

[0062] Preferably, the bacterial cell is a Gram-negative bacterial cell. The skilled person will not have difficulties in identifying Gram-negative bacterial cells. In some embodiments, the cell is a bacterial cell of the Pseudomonas genus, the Cupriavidus genus, the Bacillus genus, the Streptomyces genus, the Vibrio genus or the Escherichia genus. For example, the bacterial cell is selected from Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas taiwanensis, Pseudomonas syringae, Pseudomonas stutzeri, Pseudomonas oleovorans, Pseudomonas mendocina, Cupriavidus necator, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Streptomyces albus, Streptomyces venezuelae, Streptomyces coelicolor, Vibrio natriegens, and Escherichia coli.

[0063] In preferred embodiments, the bacterial cell is a Pseudomonas putida cell.

[0064] In other preferred embodiments, the bacterial cell is a Pseudomonas putida KT2440 cell. In some embodiments, the cell is evolved and / or engineered from a P. putida KT2440 cell.

[0065] In preferred embodiments, the cell is a P. putida cell, such as a P. putida KT2440 cell. For example, the P. putida KT2440 cell may be an engineered and / or evolved P. putida KT2440 cell. In other embodiments, the cell is a genome-reduced P. putida KT2440 cell, for example the cell is a P. putida EM42 cell. P. putida EM42 is a genome-reduced derivative of P. putida 2440, harboring the following deletions: Aprophagel, Aprophage4, Aprophage3 Aprophage2, ATn7, AendA-1, endA-2, AhsdRMS, Aflagellum, and ATn4652. The skilled person is well-capable of obtaining strain P. putida EM42. P. putida EM42 is known in the field and described, see for example Martinez-Garcia et al., 2014. In preferred embodiments, the P. putida EM42 cell may be an engineered and / or evolved P. putida EM42 cell. The bacterial cell, preferably the Pseudomonas cell, is used as production organism for polymers and / or fluorinated polymers as described herein.

[0066] Short-chain organic acid tolerance

[0067] Herein is provided an engineered bacterial cell with increased tolerance to at least one non-native, short-chain organic acid, i.e. increased ability to grow on at least one nonnative, short-chain organic acid compared to a cell which has not been engineered as described below. Non-native, short-chain organic acids may be acetate, lactate, propionate, butyrate, and the like. Preferably, the non-native, short chain organic acid is a monocarboxylic acid. Preferably, the non-native organic acid is acetate. The cell may be any of the cells described herein, in particular a Pseudomonas cell such as a Pseudomonas putida cell, such as a P. putida EM42 cell.

[0068] The inventors found that the modifications described in the present section, when introduced in a bacterial cell, enhance or improve the ability of the cell to grow on non- native, short-chain organic acids and / or to use non-native, short-chain organic acids as a substrate. In particular, the non-native, short-chain organic acid may be acetate, lactate, propionate and / or butyrate. Preferably said non-native, short-chain organic acid is acetate.

[0069] Said non-native, short-chain organic acids may be monocarboxylic acids such as acetic acid, lactic acid, propionic acid, butyric acid, acetate, lactate, propionate and / or butyrate. Preferably said non-native, short-chain organic acid is acetate.

[0070] Enhanced or improved acetate tolerance may also be referred to as enhanced or improved acetate fitness. Improved or enhanced acetate tolerance or acetate fitness may be reflected in an increase in growth rate, increase in final cell density (biomass), and / or reduction in the time of the lag-phase when the cell with improved tolerance or fitness is cultivated on acetate, compared to a reference cell not having improved acetate tolerance or fitness, such as a cell which has not been engineered as described herein.

[0071] Enhanced or improved lactate tolerance may also be referred to as enhanced or improved lactate fitness. Improved or enhanced lactate tolerance or lactate fitness may be reflected in an increase in growth rate, increase in final cell density (biomass), and / or reduction in the time of the lag-phase when the cell with improved tolerance or fitness is cultivated on lactate, compared to a reference cell not having improved lactate tolerance or fitness, such as a bacterial cell of the same genus and species, which has not been engineered as described herein, but which is otherwise identical to the engineered cell.

[0072] Enhanced or improved propionate tolerance may also be referred to as enhanced or improved propionate fitness. Improved or enhanced propionate tolerance or propionate fitness may be reflected in an increase in growth rate, increase in final cell density (biomass), and / or reduction in the time of the lag-phase when the cell with improved tolerance or fitness is cultivated on propionate, compared to a reference cell not having improved propionate tolerance or fitness, such as a bacterial cell of the same genus and species, which has not been engineered as described herein, but which is otherwise identical to the engineered cell.

[0073] Enhanced or improved butyrate tolerance may also be referred to as enhanced or improved butyrate fitness. Improved or enhanced butyrate tolerance or butyrate fitness may be reflected in an increase in growth rate, increase in final cell density (biomass), and / or reduction in the time of the lag-phase when the cell with improved tolerance or fitness is cultivated on butyrate, compared to a reference cell not having improved butyrate tolerance or fitness, such as a bacterial cell of the same genus and species, which has not been engineered as described herein, but which is otherwise identical to the engineered cell.

[0074] The engineered cell thus comprises one or more of the following modifications: one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), such as a mutation resulting in substitution of residue 77 of FabB, preferably resulting in FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70); one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10); one or more mutations in HupB (SEQ ID NO: 8 or SEQ ID NO: 69), such as a mutation resulting in substitution of residue 35 of HupB, preferably resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71), and / or a mutation resulting in substitution of residue 49 of HupB (SEQ ID NO: 8 or SEQ ID NO: 69), preferably resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72), or one or more mutations in a nucleic acid encoding a functional variant thereof having at least 70% identity to any one of FabB, UvrY or HupB.

[0075] Thus in some embodiments, one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), such as a mutation resulting in substitution of residue 77 of FabB, results in FabBL77P (SEQ ID NO: 9). In some embodiments, one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), such as a mutation resulting in substitution of residue 77 of FabB, results in FabBL77P (SEQ ID NO: 70).

[0076] In some embodiments, one or more mutations in HupB (SEQ ID NO: 8), such as a mutation resulting in substitution of residue 35 of HupB, results in HupBA35V (SEQ ID NO: 11). In some embodiments, one or more mutations in HupB (SEQ ID NO: 8), such as a mutation resulting in substitution of residue 35 of HupB, results in HupBA35V (SEQ ID NO: 71). In some embodiments, one or more mutations in HupB (SEQ ID NO: 69), such as a mutation resulting in substitution of residue 35 of HupB, results in HupBA35V (SEQ ID NO: 11). In some, embodiments, one or more mutations in HupB (SEQ ID NO: 69), such as a mutation resulting in substitution of residue 35 of HupB, results in HupBA35V (SEQ ID NO: 71).

[0077] In some embodiments, one or more mutations in HupB (SEQ ID NO: 8), such as a mutation resulting in substitution of residue 49 of HupB, results in HupBT49l (SEQ ID NO: 12). In some embodiments, one or more mutations in HupB (SEQ ID NO: 8), such as a mutation resulting in substitution of residue 49 of HupB, results in HupBT49l (SEQ ID NO: 72). In some embodiments, one or more mutations in HupB (SEQ ID NO: 69), such as a mutation resulting in substitution of residue 49 of HupB, results in HupBT49l (SEQ ID NO: 12). In some, embodiments, one or more mutations in HupB (SEQ ID NO: 69), such as a mutation resulting in substitution of residue 49 of HupB, results in HupBT49l (SEQ ID NO: 72).

[0078] Thus in some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 9), and one or more mutations in HupB (SEQ ID NO: 8), which results in HupBA35V (SEQ ID NO: 11), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 9), and one or more mutations in HupB (SEQ ID NO: 8), which results in HupBA35V (SEQ ID NO: 71) , and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO:

[0079] 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 9), and one or more mutations in HupB (SEQ ID NO: 69), which results in HupBA35V (SEQ ID NO:

[0080] 11) , and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 9), and one or more mutations in HupB (SEQ ID NO: 69), which results in HupBA35V (SEQ ID NO: 71) , and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 9), and one or more mutations in HupB (SEQ ID NO: 8), which results in HupBT49l (SEQ ID NO:

[0081] 12) , and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 9), and one or more mutations in HupB (SEQ ID NO: 8), which results in HupBT49l (SEQ ID NO: 72) , and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 9), and one or more mutations in HupB (SEQ ID NO: 69), which results in HupBT49l (SEQ ID NO:

[0082] 12) , and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 9), and one or more mutations in HupB (SEQ ID NO: 69), which results in HupBT49l (SEQ ID NO: 72), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO:

[0083] 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 9), and one or more mutations in HupB (SEQ ID NO: 8), which results in HupBA35V,T49l (SEQ ID NO: 46), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 9), and one or more mutations in HupB (SEQ ID NO: 8), which results in HupBA35V,T49l (SEQ ID NO: 76), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10).

[0084] Thus in some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 70), and one or more mutations in HupB (SEQ ID NO: 8), which results in HupBA35V (SEQ ID NO:

[0085] 11), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 70), and one or more mutations in HupB (SEQ ID NO: 8), which results in HupBA35V (SEQ ID NO: 71), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO:

[0086] 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 70), and one or more mutations in HupB (SEQ ID NO: 69), which results in HupBA35V (SEQ ID NO:

[0087] 11), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 70), and one or more mutations in HupB (SEQ ID NO: 69), which results in HupBA35V (SEQ ID NO: 71), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 70), and one or more mutations in HupB (SEQ ID NO: 8), which results in HupBT49l (SEQ ID NO: 12), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 70), and one or more mutations in HupB (SEQ ID NO: 8), which results in HupBT49l (SEQ ID NO: 72), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 70), and one or more mutations in HupB (SEQ ID NO: 69), which results in HupBT49l (SEQ ID NO: 12), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 70), and one or more mutations in HupB (SEQ ID NO: 69), which results in HupBT49l (SEQ ID NO: 72), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 70), and one or more mutations in HupB (SEQ ID NO: 8), which results in HupBA35V,T49l (SEQ ID NO: 76), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10). In some embodiments, the cell comprises one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), which results in FabBL77P (SEQ ID NO: 70), and one or more mutations in HupB (SEQ ID NO: 8), which results in HupBA35V,T49l (SEQ ID NO: 76), and optionally one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10)

[0088] The equivalent sequences provided in the present disclosure arise from discrepancies between the annotated wild-type sequences and the sequences determined from sequencing of the experimental strains used in the examples of the present disclosure. The experimental strain used in the examples has single nucleotide polymorphisms (SNPs) relative to the annotated sequence, which result in single nucleotide substitutions in polynucleotide sequences, and single amino acid substitutions in the corresponding polypeptide sequences. The equivalence of the annotated sequences and the experimentally determined sequences is obvious to a person skilled in the art.

[0089] The sequences set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 46 and SEQ ID NO: 47 are, or are derived from the annotated wild type sequences.

[0090] The sequences set forth in SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 77 are, or are derived from sequencing of the experimental cells used in the examples of the present disclosure.

[0091] The sequences set forth in SEQ ID NO: 8 and SEQ ID NO: 69 are considered to be equivalent herein throughout; both are functional HupB. The sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 70 are considered to be equivalent herein throughout; both are FabBL77P. The sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 71 are considered to be equivalent herein throughout; both are HupBA35V. The sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 72 are considered to be equivalent herein throughout; both are HupBT49l. The sequences set forth in SEQ ID NO: 33 and SEQ ID NO: 73 are considered to be equivalent herein throughout; both encode functional HupB. The sequences set forth in SEQ ID NO: 36 and SEQ ID NO: 74 are considered to be equivalent herein throughout; both encode HupBA35V. The sequences set forth in SEQ ID NO: 37 and SEQ ID NO: 75 are considered to be equivalent herein throughout; both encode HupBT49l. The sequences set forth in SEQ ID NO: 46 and SEQ ID NO: 76 are considered to be equivalent herein throughout; both are HupBA35V,T49l. The sequences set forth in SEQ ID NO: 47 and SEQ ID NO: 77 are considered to be equivalent herein throughout; both encode HupBA35V,T49l.

[0092] GacA / UvrY

[0093] The DNA-binding response regulator UvrY (also termed GacA herein and elsewhere) is a member of the two-component system GacS / GacA, where GacS is a sensor kinase. This complex regulatory system, found in a wide range of Gram-negative bacteria, supports adaptations to various environments and colonization of environmental niches in response to external signals. A phospho-transfer event is thought to activate GacA, when the GacS / GacA system receives the proper extracellular inputs via the GacS sensor kinase. Phosphorylated GacA then acts as a transcriptional activator of several non-operonic sRNA, exerting control on both transcriptional regulation and protein activities.

[0094] The engineered cell may thus comprise one or more mutations in one or more genes encoding proteins involved in the regulation of innate PHA synthesis, such as uvrY (SEQ ID NO: 32).

[0095] The inventors have identified that mutating uvrY in the engineered cell, in particular introducing a mutation resulting in a change of residue 197 of UvrY, such as a substitution, preferably a glutamic acid to lysine substitution, enhances the cell’s tolerance to non-native, short-chain organic acids, such as acetate. The engineered cell may thus comprise one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10), or the cell may comprise one or more mutations in a nucleic acid encoding a functional variant of UvrY (SEQ ID NO: 7), wherein the functional variant has at least 70% identity to UvrY (SEQ ID NO: 7).

[0096] The engineered cell may comprise one or more mutations in an orthologue, analogues, homologue or paralogue of uvrY (SEQ ID NO: 32) encoding UvrY (SEQ ID NO: 7). The mutation in a gene encoding a protein involved in the regulation of innate PHA synthesis, such as uvrY (SEQ ID NO: 32), may be a loss-of-function mutation or a gain- of-function mutation. In some embodiments, the engineered cell comprises a deletion of uvrY (SEQ ID NO: 32), or a variant thereof having at least 70% identity to uvrY (SEQ ID NO: 32), or an orthologue, analogues, homologue or paralogue of uvrY (SEQ ID NO: 32) encoding UvrY (SEQ ID NO: 7).

[0097] In some embodiments, the cell is a Pseudomonas putida cell and the nucleic acid encoding UvrY (SEQ ID NO: 7) is uvrY (SEQ ID NO: 32), or a variant thereof having at least 70% identity to SEQ ID NO: 7. In some embodiments, the cell is a Pseudomonas putida cell, the nucleic acid is mut_uvrY (SEQ ID NO: 35) encoding UvrYE197K (SEQ ID NO: 10), or a variant thereof having at least 70% identity to SEQ ID NO: 35.

[0098] HupB

[0099] HupB encodes a DNA-binding protein belonging to bacterial histone-like protein HU family. The HU protein is found to bind DNA, not in a sequence-specific manner, increasing the flexibility of the bound DNA and thus facilitating the formation of bent structures. DNA bending is widely used as a signal transmission technique to integrate various environmental cues determining an organism's ecological fitness under challenging environmental conditions.

[0100] The engineered cell may thus comprise one or more mutations in one or more genes encoding proteins which are involved in DNA bending, such as genes encoding histone-like proteins, in particular of the HU family, such as hupB (SEQ ID NO: 33 or SEQ ID NO: 73), orthologue, analogues, homologue or paralogue of hupB encoding HupB (SEQ ID NO: 8 or SEQ ID NO: 69).

[0101] The inventors have identified that mutating hupB (SEQ ID NO: 33 or SEQ ID NO: 73) in the engineered cell, in particular introducing a mutation of residue 35 of HupB (SEQ ID NO: 8 or SEQ ID NO: 69), such as a substitution, preferably an alanine to valine substitution, enhances the cell’s tolerance to non-native, short-chain organic acids, such as acetate. The engineered cell may thus comprise one or more mutations in a nucleic acid encoding HupB (SEQ ID NO: 8 or SEQ ID NO: 69), such as a mutation resulting in substitution of residue 35 of HupB, preferably resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71), or the cell may comprise one or more mutations in a nucleic acid encoding a functional variant of HupB (SEQ ID NO: 8 or SEQ ID NO: 69), wherein the functional variant has at least 70% identity to HupB (SEQ ID NO: 8 or SEQ ID NO: 69).

[0102] Likewise, the inventors have identified that introducing a mutation of residue 49 of HupB, such as a substitution, preferably a threonine to isoleucine mutation, enhances the cell’s tolerance to non-native, short-chain organic acids, such as acetate. The engineered cell may comprise one or more mutations in a nucleic acid encoding HupB (SEQ ID NO: 8 or SEQ ID NO: 69), such as a mutation resulting in substitution of residue 49 of HupB, preferably resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72), or the cell may comprise one or more mutations in a nucleic acid encoding a functional variant of HupB (SEQ ID NO: 8 or SEQ ID NO: 69), wherein the functional variant has at least 70% identity to HupB (SEQ ID NO: 8 or SEQ ID NO: 69).

[0103] The engineered cell may comprise one or more mutations in an orthologue, analogues, homologue or paralogue of hupB (SEQ ID NO: 33 or SEQ ID NO: 73) encoding HupB (SEQ ID NO: 8 or SEQ ID NO: 69).

[0104] The mutation in a gene encoding a protein involved in DNA bending, such as genes encoding histone-like proteins, in particular of the HU family, such as hupB (SEQ ID NO: 33 or SEQ ID NO: 73), may be a loss-of-function mutation or a gain-of-function mutation. In some embodiments, the engineered cell comprises a deletion of hupB (SEQ ID NO: 33 or SEQ ID NO: 73), or a variant thereof having at least 70% identity to hupB (SEQ ID NO: 33 or SEQ ID NO: 73), or an orthologue, analogues, homologue or paralogue of hupB (SEQ ID NO: 33 or SEQ ID NO: 73) encoding HupB (SEQ ID NO: 8 or SEQ ID NO: 69).

[0105] In some embodiments, the cell is a Pseudomonas putida cell and the nucleic acid encoding HupB (SEQ ID NO: 8 or SEQ ID NO: 69) is HupB (SEQ ID NO: 33 or SEQ ID NO: 73), or a variant thereof having at least 70% identity to SEQ ID NO: 33 or SEQ ID NO: 73. In some embodiments, the cell is a Pseudomonas putida cell, the nucleic acid is mut1_hupB (SEQ ID NO: 36 or SEQ ID NO: 74) encoding HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71), or a variant thereof having at least 70% identity to SEQ ID NO: 36 or SEQ ID NO: 74. In some embodiments, the cell is a Pseudomonas putida cell, the nucleic acid is mut2_hupB (SEQ ID NO: 37 or SEQ ID NO: 75) encoding HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72), or a variant thereof having at least 70% identity to SEQ ID NO: 37 or SEQ ID NO: 75. In some embodiments, the cell is a Pseudomonas putida cell, the nucleic acid is mut3_hupB (SEQ ID NO: 47 or SEQ ID NO: 77) encoding HupBA35V,T49l (SEQ ID NO: 46 or SEQ ID NO: 76), or a variant thereof having at least 70% identity to SEQ ID NO: 47 or SEQ ID NO: 77.

[0106] FabB fabB (SEQ ID NO: 34) encodes the 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase I (EC 2.3.1.41) FabB (SEQ ID NO: 6) in P. putida. FabB belongs to a well-studied class of enzymes that catalyze the elongation reaction of fatty acid synthesis. FabB performs a decarboxylative condensation between a malonyl-ACP and an acyl-ACP with n carbon atoms, initiating the cycle of elongation of unsaturated fatty acids (UFAs). With the simultaneous release of ACP and CO2, this reaction produces an n+2 carbon oxoacyl-ACP. This process is repeated until the acyl chain reaches C14 to C18, when ACP is cleaved off, and the fatty acid is released.

[0107] The 3-oxoacyl-ACP synthase II FabF (EC 2.3.1.179) performs the same reaction as FabB. While in E. coli fabB is essential for stable growth, in P. putida it is suggested that the function of FabB can be back-upped by one of the four FabF isozymes encoded in its genome.

[0108] The engineered cell may thus comprise one or more mutations in one or more genes encoding 3-oxoacyl-[acyl-carrier-protein (ACP)] synthases, in particular 3-oxoacyl-[acyl- carri er- protein (ACP)] synthase I or 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase II, such as fabB (SEQ ID NO: 34) encoding FabB (SEQ ID NO: 6) or fabF (SEQ ID NO: 66) encoding FabF (SEQ ID NO: 65), respectively.

[0109] The inventors have identified that mutating fabB in the engineered cell, in particular introducing a mutation resulting in a change of residue 77 of FabB, such as a substitution, preferably a lysine to proline substitution, enhances the cell’s tolerance to non-native, short-chain organic acids, such as acetate. The engineered cell may thus comprise one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), such as a mutation resulting in substitution of residue 77 of FabB, preferably resulting in FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70), or the cell may comprise one or more mutations in a nucleic acid encoding a functional variant of FabB (SEQ ID NO: 6), wherein the functional variant has at least 70% identity to FabB (SEQ ID NO: 6).

[0110] The engineered cell may comprise one or more mutations in an orthologue, analogue, homologue or paralogue of fabB encoding FabB (SEQ ID NO: 6). fabF may be considered an homologue of fabB in P. putida.

[0111] The mutation in a gene encoding a encoding 3-oxoacyl-[acyl-carrier-protein (AGP)] synthases, in particular 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase I or 3-oxoacyl- [acyl-carrier-protein (AGP)] synthase II, such as fabB (SEQ ID NO: 34) encoding FabB (SEQ ID NO: 6) or fabF (SEQ ID NO: 66) encoding FabF (SEQ ID NO: 65), respectively, may be a loss-of-function mutation or a gain-of-function mutation. In some embodiments, the engineered cell comprises a deletion of fabB (SEQ ID NO: 34) or fabF (SEQ ID NO: 66), or a variant thereof having at least 70% identity to fabB (SEQ ID NO: 34) or fabF (SEQ ID NO: 66), respectively, or an orthologue, analogue, homologue, or paralogue of fabB (SEQ ID NO: 34) encoding FabB (SEQ ID NO: 6) or fabF (SEQ ID NO: 66) encoding FabF (SEQ ID NO: 65).

[0112] In some embodiments, the cell is a Pseudomonas putida cell and the nucleic acid encoding FabB (SEQ ID NO: 6) is fabB (SEQ ID NO: 31), or a variant thereof having at least 70% identity to SEQ ID NO: 31. In some embodiments, the cell is a Pseudomonas putida cell, the nucleic acid is mut_FabB (SEQ ID NO: 34) encoding FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70), or a variant thereof having at least 70% identity to SEQ ID NO: 34.

[0113] Other useful modifications for increasing tolerance to short-chain organic acids The engineered cell may carry only one of the modifications described herein above. Additional modifications which the skilled person might find useful to include for other purposes may also be present. In the present section, when it is stated that the engineered cell comprises mutations only in a given gene, this means that it does not also comprise the mutations described herein above, but may comprise mutations in other genes, for example as described in the section “Further modifications” herein below. In some embodiments, the engineered cell comprises mutations only in a gene encoding a protein involved in the regulation of innate PHA synthesis, such as uvrY (SEQ ID NO: 32). In some embodiments, the engineered cell comprises a mutation of residue 197 of UvrY (SEQ ID NO: 7), such as a substitution, preferably a glutamic acid to lysine substitution such as a mutation resulting in UvrYE197K (SEQ ID NO: 10).

[0114] In other embodiments, the engineered cell comprises mutations only in a gene encoding a protein involved in DNA bending, such as a gene encoding a histone-like protein, in particular of the HU family, such as hupB (SEQ ID NO: 8 or SEQ ID NO: 69). In some embodiments, the engineered cell comprises a mutation of residue 35 of HupB, such as a substitution, preferably an alanine to valine substitution such as a mutation resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71), or a threonine to isoleucine substitution such as a mutation resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72).

[0115] In other embodiments, the engineered cell comprises mutations only in a gene encoding a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthases, in particular a 3-oxoacyl- [acyl-carrier-protein (AGP)] synthase I or a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase II, such as fabB encoding FabB (SEQ ID NO: 6). In some embodiments, the engineered cell comprises a mutation of residue 77 of FabB, such as a substitution, preferably a lysine to proline substitution such as a mutation resulting in FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70).

[0116] In some embodiments, the engineered cell may comprise two of the above-described modifications. The cell may thus comprise: one or more mutations in a gene encoding a protein involved in the regulation of innate PHA synthesis, such as uvrY (SEQ ID NO: 7), as described herein above; and one or more mutations in a gene encoding a protein involved in DNA bending, such as a gene encoding a histone-like protein, in particular of the HU family, such as hupB (SEQ ID NO: 8 or SEQ ID NO: 69), as described herein above, such as a mutation resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71) or such as a mutation resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72). In other embodiments, the engineered cell comprises: one or more mutations in a gene encoding a protein involved in the regulation of innate PHA synthesis, such as uvrY (SEQ ID NO: 7), as described herein above; and one or more mutations in a gene encoding a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthases, in particular a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase I or a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase II, such as fabB encoding FabB (SEQ ID NO: 6), as described herein above.

[0117] In other embodiments, the engineered cell comprises: one or more mutations in a gene encoding a protein involved in DNA bending, such as a gene encoding a histone-like protein, in particular of the HU family, such as hupB (SEQ ID NO: 8 or SEQ ID NO: 69), as described herein above, such as a mutation resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71) or such as a mutation resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72); and one or more mutations in a gene encoding a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthases, in particular a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase I or a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase II, such as fabB encoding FabB (SEQ ID NO: 6), as described herein above.

[0118] In some embodiments, the engineered cell comprises all three modifications, i.e. the cell comprises: one or more mutations in a gene encoding a protein involved in the regulation of innate PHA synthesis, such as uvrY (SEQ ID NO: 7), as described herein above; one or more mutations in a gene encoding a protein involved in DNA bending, such as a gene encoding a histone-like protein, in particular of the HU family, such as hupB (SEQ ID NO: 8 or SEQ ID NO: 69), as described herein above, such as a mutation resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71) or such as a mutation resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72); and one or more mutations in a gene encoding a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthases, in particular a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase I or a 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase II, such as fabB encoding FabB (SEQ ID NO: 6), as described herein above.

[0119] In some embodiments, the engineered cell comprises two mutations in hupB (SEQ ID NO: 8 or SEQ ID NO: 69), preferably a mutation resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71) and a mutation resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72), resulting in HupBA35V,T49l (SEQ ID NO: 46 or SEQ ID NO: 76).

[0120] Thus, herein is also disclosed an engineered cell comprising one or more mutations in either one of uvrY, hupB and fabB, or in homologues, paralogues, analogues, or orthologues thereof encoding a functional variant of UvrY, HupB and FabB, respectively. The cell may in particular comprise a mutation resulting in UvrYE197K (SEQ ID NO: 10); or the cell may in particular comprise a mutation resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71); or the cell may in particular comprise a mutation resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72); or the cell may comprise a mutation resulting in FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70).

[0121] In other embodiments, the engineered cell comprises three of the above-described modification. In particular, the engineered cell may comprise: one or more mutations in a gene encoding a protein involved in the regulation of innate PHA synthesis, such as uvrY (SEQ ID NO: 7), as described herein above; and one or more mutations in a gene encoding a protein involved in DNA bending, such as a gene encoding a histone-like protein, in particular of the HU family, such as hupB (SEQ ID NO: 8 or SEQ ID NO: 69), as described herein above, such as a mutation resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71) or such as a mutation resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72); and one or more mutations in a gene encoding a 3-oxoacyl-[acyl-carrier-protein (ACP)] synthases, in particular a 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase I or a 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase II, such as fabB encoding FabB (SEQ ID NO: 6), as described herein above.

[0122] The engineered cell may in some embodiments comprise: one or more mutations in a gene encoding a protein involved in the regulation of innate PHA synthesis, such as uvrY (SEQ ID NO: 7), as described herein above; and two mutations in a gene encoding a protein involved in DNA bending, such as a gene encoding a histone-like protein, in particular of the HU family, such as two mutations in hupB (SEQ ID NO: 8 or SEQ ID NO: 69), as described herein above, such as a mutation resulting in HupBA35V,T49l (SEQ ID NO: 46 or SEQ ID NO: 76).

[0123] In some embodiments the engineered cell comprises: two mutations in a gene encoding a protein involved in DNA bending, such as a gene encoding a histone-like protein, in particular of the HU family, such as two mutations in hupB (SEQ ID NO: 8 or SEQ ID NO: 69), as described herein above, such as a mutation resulting in HupBA35V,T49l (SEQ ID NO: 46 or SEQ ID NO: 76); and one or more mutations in a gene encoding a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthases, in particular a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase I or a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase II, such as fabB encoding FabB (SEQ ID NO: 6), as described herein above.

[0124] In some embodiments, the engineered cell comprises all of the above-described modifications, i.e. the engineered cell comprises: one or more mutations in a gene encoding a protein involved in the regulation of innate PHA synthesis, such as uvrY (SEQ ID NO: 7), as described herein above; and two mutations in a gene encoding a protein involved in DNA bending, such as a gene encoding a histone-like protein, in particular of the HU family, such as two mutations in hupB (SEQ ID NO: 8 or SEQ ID NO: 69), as described herein above, such as a mutation resulting in HupBA35V,T49l (SEQ ID NO: 46 or SEQ ID NO: 76); and one or more mutations in a gene encoding a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthases, in particular a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase I or a 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase II, such as fabB encoding FabB (SEQ ID NO: 6), as described herein above. mcl-PHA-deaf

[0125] It is preferred that the endogenous synthesis of mcl-PHAs by the bacterial cell is either partially or totally inactivated. In other words, it is preferable that the engineered cell is not capable of producing mcl-PHAs as they may function as a “metabolic sink” or so- called bypass pathway, wherein consumed carbon is distributed instead of being used for the production of the product of interest, in this disclosure acetate-derived compounds, such as PHB and / or F-PHB.

[0126] Hence, in preferred embodiments, the engineered cell is not capable of producing polyhydroxyalkanoates (PHAs) having a medium-length carbon chain, i.e. mcl-PHAs.

[0127] Thus, in some embodiments of the present disclosure, the endogenous pha cluster in said engineered cell is partially or totally inactivated, wherein the endogenous pha cluster consists of: i) a first operon comprising nucleic acids encoding PhaC1 (SEQ ID NO: 16), PhaC2 (SEQ ID NO: 17), PhaZ (SEQ ID NO: 18), and PhaD (SEQ ID NO: 19); and ii) a second operon comprising nucleic acids encoding PhaF (SEQ ID NO: 20), and Phal (SEQ ID NO: 21), or a functional variant thereof having at least 70% identity to PhaC1, PhaC2, PhaZ, PhaD, PhaF, or Phal, respectively.

[0128] The inactivation of the endogenous pha cluster may comprise or consist of a mutation or a deletion of all or part of the endogenous pha cluster. Preferably the inactivation consists of a mutation or deletion of at least one of the first and second operons, such as a mutation or deletion of the first operon, a mutation or deletion of the second operon, or a mutation or deletion of both the first and the second operons.

[0129] In further embodiments, the inactivation comprises or consists of a mutation in or a deletion of at least one or more of a nucleic acid encoding PhaC1 (SEQ ID NO: 16), PhaC2 (SEQ ID NO: 17), PhaZ (SEQ ID NO: 18), PhaD (SEQ ID NO: 19); PhaF (SEQ ID NO: 20), and Phal (SEQ ID NO: 21), or a functional variant thereof having at least 70% identity to PhaC1 , PhaC2, PhaZ, PhaD, PhaF, or Phal, respectively. In preferred embodiments, the engineered cell is a P. putida cell, for example a P. putida EM42 or derivative thereof.

[0130] In some embodiments, the engineered cell comprises one or more of the modifications described herein above in the section “Short-chain organic acid tolerance”.

[0131] In some embodiments, the cell is a Pseudomonas putida cell and the nucleic acid encoding PhaC1 (SEQ ID NO: 16) is phaC1 (SEQ ID NO: 22), or a variant thereof having at least 70% identity to SEQ ID NO: 22.

[0132] In some embodiments, the cell is a Pseudomonas putida cell and the nucleic acid encoding PhaC2 (SEQ ID NO: 17) is phaC2 (SEQ ID NO: 23), or a variant thereof having at least 70% identity to SEQ ID NO: 23.

[0133] In some embodiments, the cell is a Pseudomonas putida cell and the nucleic acid encoding PhaZ (SEQ ID NO: 18) is phaZ (SEQ ID NO: 24), or a variant thereof having at least 70% identity to SEQ ID NO: 24.

[0134] In some embodiments, the cell is a Pseudomonas putida cell and the nucleic acid encoding PhaD (SEQ ID NO: 19) is phaD (SEQ ID NO: 25), or a variant thereof having at least 70% identity to SEQ ID NO: 25.

[0135] In some embodiments, the cell is a Pseudomonas putida cell and the nucleic acid encoding PhaF (SEQ ID NO: 20) is phaF (SEQ ID NO: 26), or a variant thereof having at least 70% identity to SEQ ID NO: 26.

[0136] In some embodiments, the cell is a Pseudomonas putida cell and the nucleic acid encoding Phal (SEQ ID NO: 21) is pha\ (SEQ ID NO: 27), or a variant thereof having at least 70% identity to SEQ ID NO: 27.

[0137] Inactivation of phaG

[0138] In other preferred embodiments, the engineered cell is not capable of converting fatty acids into PHA monomers having a medium-length carbon chain.

[0139] This may be achieved by partially or totally inactivating the conversion of fatty acids into said PHA monomers. For example, in some embodiments of the present disclosure, an (F?)-3-hydroalkanoyl-ACP:CoA transferase, preferably an (F?)-3- hydroxydecanoyl-ACP:CoA transacylase, is inactivated in said engineered cell, such as by mutation or deletion of all or part of the nucleic acid encoding said transferase or transacylase. In other embodiments, PhaG (SEQ ID NO: 28), or a functional variant thereof having at least 70% identity to PhaG, is inactivated, such as by mutation or deletion of all or part of the nucleic acid encoding said PhaG or functional variant thereof, for example phaG (SEQ ID NO: 29), or a variant thereof having at least 70% identity to phaG (SEQ ID NO: 29). In preferred embodiments, the engineered cell is a P. putida cell, for example a P. putida EM42 or derivative thereof.

[0140] The engineered cell may, in addition to inactivation of an (F?)-3-hydroalkanoyl-ACP:CoA transferase, for example an (F?)-3-hydroxydecanoyl-ACP:CoA transacylase such as PhaG (SEQ ID NO: 28), comprise any of the modifications described in the section “Short-chain organic acid tolerance” or Further modifications”, and / or comprises inactivation of the pha cluster as described above.

[0141] Inactivation of the benABCD cluster

[0142] In addition to the above-described modifications, it may be advantageous to also delete or inactivate the benABCD cluster. In P. putida, the benABCD cluster consists of the genes benA (SEQ ID NO: 42), benB (SEQ ID NO: 43), benC (SEQ ID NO: 44) and benD (SEQ ID NO: 45). Inactivation of the benABCD cluster is within reach of the skilled person, and can be achieved for example by deleting part or all of one or more of benA (SEQ ID NO: 42), benB (SEQ ID NO: 43), benC (SEQ ID NO: 44) and benD (SEQ ID NO: 45). In some embodiments, benA (SEQ ID NO: 42), benB (SEQ ID NO: 43), benC (SEQ ID NO: 44) and benD (SEQ ID NO: 45) are all deleted in the engineered cell, or otherwise inactivated by mutating the genes to result in loss-of- function. Thus, in some embodiments, the benABCD cluster is partially or completely inactivated, such as by mutation or deletion of all or part of benABCD cluster.

[0143] This modification prevents synthesis of metabolic intermediates resulting from benzoate degradation, which intermediates yields a brownish color interfering with optical density measurements during cultivation. Further modifications

[0144] Also disclosed herein is an engineered bacterial cell, preferably a Pseudomonas cell such as a P. putida cell, or an Escherichia cell, such as an E. coli cell, having one or more of the following modifications, which also improve tolerance to non-native, shortchain organic acids, such as acetate, lactate, propionate, or butyrate, preferably acetate: increased methionine metabolism, in particular increased expression of proteins involved in methionine metabolism, such as increased expression of MetN (SEQ ID NO: 49) and / or Metl (SEQ ID NO: 67), or of functional variants thereof having at least 70% homology or identity to SEQ ID NO: 49 and / or SEQ ID NO: 67; decreased methionine metabolism, in particular decreased expression of proteins involved in methione utilisation, such as decreased expression of an O-acetylhomoserine sulfhydrylase such as MetY (SEQ ID NO: 51) or of a functional variant thereof having at least 70% homology or identity thereto; increased sulfate metabolism, in particular increased expression of proteins involved in sulfate transport, such as increased expression of CysZ (SEQ ID NO: 53) or of a functional variant thereof having at least 70% homology or identity thereto; decreased sulfate metabolism, in particular decreased expression of proteins related to sulfate utilisation, such as decreased expression of an alkanesulfonate monooxygenase such as SsuD (SEQ ID NO: 55), decreased expression of an organosulfonate utilisation protein such as SsuE (SEQ ID NO: 57), and / or decreased expression of a thiosulfate sulfurtransferase such as GIpE (SEQ ID NO: 59), or functional variants thereof having at least 70% homology or identity thereto; and / or increased expression of activators or catalysts of the citric acid cycle, such as increased expression of a succinyl-CoA ligase, in particular a succinyl- CoA ligase [ADP-forming] subunit beta, such as SucC (SEQ ID NO: 61), or in particular a succinyl-CoA ligase [ADP-forming] subunit alpha, such as SucD (SEQ ID NO: 63)3or functional variants thereof having at least 70% homology or identity thereto.

[0145] The skilled person knows how to alter the expression of a given protein. This can be done for instance by introducing a strong, constitutive promoter upstream of the nucleic acid encoding said protein, which will increase the expression of said protein. In cases where decreased expression is desired, this can be achieved by numerous techniques, including the introduction of a repressible promoter upstream of the nucleic acid encoding said protein.

[0146] MetN (SEQ ID NO: 49) in P. putida is encoded by MetN (SEQ ID NO: 50). Metl (SEQ ID NO: 67) in P. putida is encoded by Metl (SEQ ID NO: 68). MetY (SEQ ID NO: 51) in P. putida is encoded by MetY (SEQ ID NO: 52). CysZ (SEQ ID NO: 53) in P. putida is encoded by CysZ (SEQ ID NO: 54). SsuD in P. putida is encoded by SsuD (SEQ ID NO: 55). SsuE (SEQ ID NO: 57) in P. putida is encoded by SsuE (SEQ ID NO: 58). GIpE (SEQ ID NO: 59) in P. putida is encoded by GIpE (SEQ ID NO: 60). SucC (SEQ ID NO: 61) in P. putida is encoded by SucC (SEQ ID NO: 62). SucD (SEQ ID NO: 63) in P. putida is encoded by SucD (SEQ ID NO: 64).

[0147] Any of these modifications can be done in addition to any of the other modifications described herein, or can be done as an alternative to any of the other modifications described herein, in particular the modifications described under “Acetate tolerance” herein above.

[0148] Improved short-chain organic acid tolerance

[0149] The above described engineered cells have improved tolerance to non-native, shortchain organic acids, such as acetate, lactate, propionate or butyrate, preferable acetate; in particular engineered cells comprising one or more mutations in one or more genes encoding proteins involved in the regulation of innate PHA synthesis, such as uvrY (SEQ ID NO: 32); and / or one or more mutations one or more genes encoding proteins which are involved in DNA bending, such as genes encoding histone-like proteins, in particular of the HU family, such as hupB (SEQ ID NO: 33 or SEQ ID NO: 73); and / or one or more mutations in one or more genes encoding 3-oxoacyl-[acyl- carri er- protein (AGP)] synthases, in particular 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase I or 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase II, such as fabB (SEQ ID NO: 34) encoding FabB (SEQ ID NO: 6) or fabF (SEQ ID NO: 66) encoding FabF (SEQ ID NO: 65), respectively. The engineered cells may further comprise additional modifications which help further improve tolerance to non-native short-chain organic acids, such as acetate, lactate, propionate, butyrate, preferably acetate, as described in the section “Other useful modifications for increasing tolerance". In , the engineered cells described above have i acetate tolerance, improved lactate tolerance, i tolerance and / or i tolerance. Preferably, the above described engineered cells have improved acetate tolerance.

[0150] Such cells can tolerate at least 80 mM acetate, such as at least 100 mM acetate, such as at least 110 mM acetate, such as at least 120 mM acetate, such as at least 130 mM acetate, such as at least 140 mM acetate, such as at least 150 mM acetate, such as at least 160 mM acetate, such as at least 170 mM acetate, such as at least 180 mM acetate, such as at least 190 mM acetate, such as at least 200 mM acetate, such as at least 210 mM acetate, such as at least 220 mM acetate, such as at least 230 mM acetate, such as at least 240 mM acetate, such as at least 250 mM acetate, such as at least, 260 mM acetate, such as at least 275 mM acetate, such as at least 280 mM acetate, such as at least 290 mM acetate, such as at least 295 mM acetate, such as at least 300 mM acetate, or more.

[0151] In some embodiments, the acetate is F-acetate. In some embodiments, the engineered cells can tolerate at least 80 mM F-acetate, such as at least 100 mM F-acetate, such as at least 110 mM F-acetate, such as at least 120 mM F-acetate, such as at least 130 mM F-acetate, such as at least 140 mM F-acetate, such as at least 150 mM F-acetate, such as at least 160 mM F-acetate, such as at least 170 mM F-acetate, such as at least 180 mM F-acetate, such as at least 190 mM F-acetate, such as at least 200 mM F-acetate, such as at least 210 mM F-acetate, such as at least 220 mM F-acetate, such as at least 230 mM F-acetate, such as at least 240 mM F-acetate, such as at least 250 mM F-acetate, such as at least, 260 mM F-acetate, such as at least 275 mM F-acetate, such as at least 280 mM F-acetate, such as at least 290 mM F-acetate, such as at least 295 mM F-acetate, such as at least 300 mM F-acetate, such as at least 325 mM F-acetate, such as at least 350 mM F-acetate, such as at least 380 mM F-acetate, such as at least 400 mM F-acetate, such as at least 425 mM F-acetate, or more, for example 450 mM F-acetate.

[0152] Such cells may tolerate at least 60 mM lactate, such as at least 70 mM, such as at least 80 mM, such as at least 90 mM ,such as at least 100 mM, such as at least 110 mM, such as at least 120 mM, such as at least 130 mM, such as at least 140 mM, such as at least 150 mM, such as at least 160 mM, such as at least 170 mM, such as at least 180 mM, such as at least 190 mM, such as at least 200 mM, such as at least 210 mM, such as at least 220 mM, such as at least 230 mM, such as at least 240 mM, such as at least 250 mM, such as at least, 260 mM, such as at least 275 mM, such as at least 280 mM, such as at least 290 mM, such as at least 295 mM, such as at least 300 mM lactate, or more.

[0153] Such cells may tolerate at least 60 mM propionate, such as at least 70 mM, such as at least 80 mM, such as at least 90 mM ,such as at least 100 mM, such as at least 110 mM, such as at least 120 mM, such as at least 130 mM, such as at least 140 mM, such as at least 150 mM, such as at least 160 mM, such as at least 170 mM, such as at least 180 mM, such as at least 190 mM, such as at least 200 mM, such as at least 210 mM, such as at least 220 mM, such as at least 230 mM, such as at least 240 mM, such as at least 250 mM, such as at least, 260 mM, such as at least 275 mM, such as at least 280 mM, such as at least 290 mM, such as at least 295 mM, such as at least 300 mM propionate, or more.

[0154] Such cells may tolerate at least 40 mM butyrate, such as at least 50 mM, such as at least 60 mM, such as at least 70 mM, such as at least 80 mM, such as at least 90 mM ,such as at least 100 mM, such as at least 110 mM, such as at least 120 mM, such as at least 130 mM, such as at least 140 mM, such as at least 150 mM, such as at least 160 mM, such as at least 170 mM, such as at least 180 mM, such as at least 190 mM, such as at least 200 mM, such as at least 210 mM, such as at least 220 mM, such as at least 230 mM, such as at least 240 mM, such as at least 250 mM, such as at least, 260 mM, such as at least 275 mM, such as at least 280 mM, such as at least 290 mM, such as at least 295 mM, such as at least 300 mM butyrate, or more.

[0155] 3HB and / or PHB production

[0156] Also provided herein are engineered cells which can, when grown in a culture medium comprising a carbon source and a nitrogen source, produce a PHA having a short carbon chain length (scl-PHA), preferably wherein the scl-PHA is polyhydroxybutyrate (PHB), or monomers therefor, such as 3-hydroxybutyrate (3HB). In some embodiments, the produced PHA, such as PHB, or monomer therefor, such as 3HB, is fluorinated. Herein are thus provided engineered, bacterial cells which can, when grown in a culture medium comprising a carbon source and a nitrogen source, produce 3HB, which may be partially fluorinated, for example 2,4-difluoro-3-hydroxybutyrate, 4-fluoro- 3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate, most preferably 4-fluoro-3- hydroxybutyrate. Preferably the culture medium comprises glucose, citrate, acetate, and / or F-acetate as carbon source.

[0157] In order to achieve this, the bacterial cell is engineered by introduction of nucleic acids, such as nucleic acids encoding heterologous proteins, wherein said nucleic acids encode: an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting acetyl-CoA into acetoacetyl-CoA; an acetyl-CoA reductase (EC 1.1.1 .36) capable of converting acetoacetyl- CoA into F?-(-)-3-hydroxybutyryl-CoA; and a scl-PHA synthase (EC 2.3.1.304) capable of polymerizing ?-(-)-3- hydroxybutyryl-CoA into polyhydroxybutyrate (PHB); or a thioesterase (EC 3.1.2.20) capable of hydrolysing a 3-hydroxybutyryl-CoA to CoA and a corresponding 3-hydroxybutyrate.

[0158] The above nucleic acids encoding an acetyl-CoA thiolase, an acetyl-CoA reductase, and an scl-PHA synthase may be provided as a synthetic operon and / or be expressed using one or more inducible promoters, expression systems and inducers. The skilled person will know how to design such a synthetic operon and / or inducible expression systems. The operon may be introduced in the cell by integration in the genome, or by introduction of a vector, such as a plasmid, comprising said synthetic operon in the cell, by methods known in the art.

[0159] Acetyl-CoA thiolase

[0160] The engineered, bacterial cell may comprises a nucleic acid encoding an acetyl-CoA thiolase, which converts acetyl-CoA into acetoacetyl-CoA and / or fluorinated acetyl-CoA (F-acetyl-CoA) into fluorinated acetoacetyl-CoA (F-acetoacetyl-CoA).

[0161] The acetyl-CoA thiolase may be an acetyl-CoA thiolase native to Cupriavidus, such as C. necator, for example PhaA as set forth in SEQ ID NO: 1 , or an acetyl-CoA thiolase native to Azotobacter, Streptomyces, or Paracoccus. In preferred embodiments, the nucleic acid encoding the acetyl-CoA thiolase is a nucleic acid encoding PhaA from Cupriavidus necator, as set forth in SEQ ID NO: 1 , or encoding a functional variant thereof having at least 70% identity to SEQ ID NO: 1.

[0162] Acetyl-CoA reductase

[0163] The engineered, bacterial cell further comprises a nucleic acid encoding an acetyl-CoA reductase, which converts acetoacetyl-CoA into R-(-)-3-hydroxybutyryl-CoA and / or fluorinated acetoacetyl-CoA (F- acetoacetyl-CoA) into fluorinated R-(-)-3- hydroxybutyryl-CoA (F-3-hydroxybutyryl-CoA).

[0164] The acetyl-CoA reductase may be an acetyl-CoA reductase native to Cupriavidus, such as C. necator, for example PhaB as set forth in SEQ ID NO: 2, or an acetyl-CoA reductase native to Azotobacter, Streptomyces, or Paracoccus.

[0165] In preferred embodiments, the nucleic acid encoding the acetyl-CoA reductase is a nucleic acid encoding PhaB from Cupriavidus necator (SEQ ID NO: 2), or encoding a functional variant thereof having at least 70% identity to SEQ ID NO: 2. scl-PHA synthase or thioesterase

[0166] The cell further comprises a nucleic acid encoding an additional enzyme selected from a scl-PHA synthase and a thioesterase. The scl-PHA synthase polymerizes R-(-)-3- hydroxybutyryl-CoA into polyhydroxybutyrate (PHB) and / or fluorinated R-(-)-3- hydroxybutyryl-CoA (F-3-hydroxybutyryl-CoA) into fluorinated polyhydroxybutyrate (F- PHB). The thioesterase hydrolyses 3-hydroxybutyryl-CoA to CoA and a corresponding 3-hydroxybutyrate; the term “corresponding” in this context simply means that if the 3- hydroxybutyryl-CoA is further substituted in other positions, the resulting 3- hydroxy butyrate is also further substituted in the same position. Thus, the thioesterase may hydrolyse a fluorinated 3-hydroxybutyryl-CoA to CoA and a corresponding fluorinated 3-hydroxybutyrate.

[0167] The scl-PHA synthase is an scl-PHA synthase native to Cupriavidus, such as C. necator, for example PhaC as set forth in SEQ ID NO: 3, Azotobacter, Streptomyces, or Paracoccus. In preferred embodiments, where the engineered cell comprises a nucleic acid encoding an scl-PHA synthase, the nucleic acid preferably encodes PhaC from Cupriavidus necator (SEQ ID NO: 3), or encodes a functional variant thereof having at least 70% identity to SEQ ID NO: 3.

[0168] Thus, in some embodiments, the engineered cell comprises: a nucleic acid encoding a scl-PHA synthase, preferably PhaC (SEQ ID NO: 3), or encoding a functional variant of PhaC having at least 70% identity to SEQ ID NO: 3.

[0169] In embodiments where the cell comprises a nucleic acid encoding a thioesterase, the nucleic acid preferably encodes TesB from Pseudomonas putida (herein PpTesB, SEQ ID NO: 38) and / or TesB from Escherichia coli (herein EcTesB, SEQ ID NO: 39), or encodes a functional variant thereof having at least 70% identity to SEQ ID NO: 38 or SEQ ID NO: 39.

[0170] In embodiments where the cell is a Pseudomonas putida cell or an E. coli cell, the cell may already comprise a nucleic acid encoding TesB. In this case, the native enzyme can be used; however, the cell may be further modified so that TesB is overexpressed, and / or so that the cell also expresses a scl-PHA synthase.

[0171] Cells engineered as described in the present section (“3HB and / or PHB production”) can be further engineered so that their tolerance to non-native short-chain organic acids, such as acetate, lactate, propionate, butyrate, preferably acetate, is improved, as described in the sections “Short-chain organic acid tolerance” and “Other useful modifications for increasing tolerance". This may greatly improve the ability of the cell to grow on acetate and / or its ability to metabolise non-native short-chain organic acids, such as acetate, lactate, propionate, butyrate, preferably acetate, particularly when the culture medium comprises non-native short-chain organic acids, such as acetate, lactate, propionate, butyrate, preferably acetate as a carbon source. The non-native short-chain organic acids, such as acetate, lactate, propionate, butyrate, preferably acetate, may be fluorinated acetate (F-acetate).

[0172] The engineered cell may thus further comprise the above-described mutations, in particular: one or more mutations in the nucleic acids encoding UvrY, HupB and / or FabB; inactivation of an innate pha cluster or endogenous pathways or key metabolic proteins for mcl-PHA biosynthesis; inactivation of phaG; and / or inactivation of the ben A BCD cluster.

[0173] The engineered cell may be as described herein above, i.e. a Pseudomonas cell, such as a P. putida cell, such as a P. putida KT440 cell, such as a P. putida EM42 cell.

[0174] Promoters and other modifications

[0175] It may be advantageous to further modify the engineered cell, such as overexpress endogenous or exogenous transporters, or delete by-pass pathways.

[0176] In some embodiments, the synthetic operon described above, which encodes the polypeptides necessary for production of 3HB and / or PHB, which may be fluorinated as described herein, further comprises a promoter, such as an inducible regulatorpromoter and / or expression system, preferably a cyclohexanone-inducible expression system such as ChnR / Pc / ine(SEQ ID NO: 5), a 3-methylbenzoate-inducible expression system such as XylS / Pm (SEQ ID NO: 30), Lacl / Ptac, Lacl / Ptrc, a rhamnose-inducible promoter, or an arabinose-inducible promoter. Other inducible promoters suitable for this purpose are known to the skilled person.

[0177] In some embodiments, the synthetic operon comprises one or more ribosome binding sites (RBSs), such as an RBS of SEQ ID NO: 4, upstream of the nucleic acids encoding an acetyl-CoA thiolase, an acetyl-CoA reductase and an scl-PHA synthase.

[0178] Any of the genetic modifications detailed herein may require codon optimisation for the specific cell to be engineered, as is known in the art; the cell may be as described herein above, in particular a Pseudomonas cell, such as a Pseudomonas putida cell, or an Escherichia cell, such as an Escherichia coli cell. In particular, the nucleic acids encoding an acetyl-CoA thiolase, an acetyl-CoA reductase, an scl-PHA synthase and / or a thioesterase, may be codon-optimised for proper expression in P. putida and / or in E. coli. Fluorinated 3HB (F-HB) and / or fluorinated PHB (F-PHB) production

[0179] The inventors have found that engineered cells which can produce 3HB and / or PHB can accommodate fluorinated substrates, whereby fluorinated 3HB (F-HB) and / or fluorinated PHB (F-PHB) are produced.

[0180] The cells described herein above which can produce 3HB and / or PHB can, through the same modifications, produce F-HB and / or F-PHB, preferably 2,4-difluoro-3- hydroxy butyrate, 4-fluoro-3-hydroxybutyrate, 2-fluoro-3-hydroxybutyrate, and / or poly(fluoro-3-hydroxybutryate) (F-PHB), most preferably 4-fluoro-3-hydroxybutyrate and / or poly(fluoro-3-hydroxybutryate), when the cells are grown in a culture medium comprising a carbon source and a nitrogen source. Preferably the culture medium comprises F-acetate, and optionally acetate, as carbon source; alternatively, F-acetate or F-acetyl-CoA may be synthesised by the cells themselves, for example advantage can be taken of engineered cells as described in WO 2023 / 170058.

[0181] The acetyl-CoA thiolase described herein above can convert F-acetyl-CoA to [2,4]- fluoro-acetoacetyl-CoA. The acetyl-CoA reductase described herein above can convert 2,4-difluoro-acetoacetyl-CoA to 2,4-difluoro-F?-(-)-3-hydroxybutyryl-CoA, which in turn can be converted to 2,4-difluoro-3-hydroxybutyrate by an scl-PHA synthase and / or a thioesterase. Useful examples of acetyl-CoA thiolases include PhaA from C. necator (SEQ ID NO: 1). Useful examples of acetyl-CoA reductases include PhaB from C. necator (SEQ ID NO: 2). Useful examples of (scl)-PHA synthases include PhaC from C. necator (SEQ ID NO: 3). The thioesterase described herein above, is also capable of hydrolysing a fluorinated 3-hydroxybutyryl-CoA (F-3-hydroxybutyryl-CoA) to CoA and a corresponding fluorinated 3-hydroxy butyrate, for example; hydrolysing 4-fluoro-3-hydroxybutyryl-CoA to CoA and 4-fluoro-3- hydroxybutyrate; hydrolysing 2-fluoro-3-hydroxybutyryl-CoA to CoA and 2-fluoro-3- hydroxy butyrate; and / or hydrolysing 2,4-difluoro-3-hydroxybutyryl-CoA to CoA and 2,4-difluoro-3- hydroxybutyrate.

[0182] Useful examples of thioesterases include TesB as set forth in SEQ ID NO: 38 or as set forth in SEQ ID NO: 39. Expression systems

[0183] Also disclosed herein are expression systems useful for introducing the herein described modifications in the cell to be engineered.

[0184] In particular, herein are provided expression systems, for example vectors, such as plasmids, comprising the nucleic acids encoding the polypeptides that should be expressed by the engineered cell.

[0185] In some embodiments, the expression system comprises:

[0186] - A nucleic acid encoding an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting acetyl-CoA into acetoacetyl-CoA;

[0187] - A nucleic acid encoding an acetyl-CoA reductase (EC 1.1.1.36) capable of converting acetoacetyl-CoA into R-(-)-3-hydroxybutyryl-CoA; and

[0188] - A nucleic acid encoding an scl-PHA synthase (EC 2.3.1.304) capable of polymerizing R-(-)-3-hydroxybutyryl-CoA into polyhydroxybutyrate (PHB); or a thioesterase (EC 3.1.2.20) capable of hydrolysing a 3-hydroxybutyryl-CoA to CoA and a corresponding 3-hydroxybutyrate.

[0189] The nucleic acids may comprise an inducible promoter and / or expression system, such as described herein in the section “Promoters and other modifications”.

[0190] In some embodiments, the expression system comprises a nucleic acid encoding an acetyl-CoA thiolase from Cupriavidus, such as the acetyl-CoA thiolase PhaA from C. necator, as set forth in SEQ ID NO: 1 , or a nucleic acid encoding a functional variant thereof having at least 70% identity to SEQ ID NO: 1. In some embodiments, the nucleic acid is phaA from C. necator (SEQ ID NO: 13), or a homologue thereof having at least 70% homology or identity thereto, and preferably encoding a functional variant of PhaA.

[0191] In some embodiments, the expression system comprises a nucleic acid encoding an acetyl-CoA reductase native to Cupriavidus, such as the acetyl-CoA reductase PhaB from C. necator, as set forth in SEQ ID NO: 2, or a nucleic acid encoding a functional variant thereof having at least 70% identity to SEQ ID NO: 2. In some embodiments, the nucleic acid is phaB from C. necator (SEQ ID NO: 14), or a homologue thereof having at least 70% homology or identity thereto, and preferably encoding a functional variant of PhaB.

[0192] In some embodiments, the expression system comprises a nucleic acid encoding an scl-PHA synthase native to Cupriavidus, such as the scl-PHA synthase PhaC from C. necator, as set forth in SEQ ID NO: 3, or a nucleic acid encoding encoding a functional variant thereof having at least 70% identity to SEQ ID NO: 3. In some embodiments, the nucleic acid is phaC from C. necator (SEQ ID NO: 15), or a homologue thereof having at least 70% homology or identity thereto, and preferably encoding a functional variant of PhaC.

[0193] In some embodiments, the expression system comprises a nucleic acid encoding a thioesterase native to P. putida, such as thioesterase PpTesB (SEQ ID NO: 38) from P. putida, as set forth in SEQ ID NO: 38, or a nucleic acid encoding a functional variant thereof having at least 70% identity to SEQ ID NO: 38. In some embodiments, the nucleic acid is Pp_tesB from C. necator (SEQ ID NO: 40), or a homologue thereof having at least 70% homology or identity thereto, and preferably encoding a functional variant of PpTesB.

[0194] In some embodiments, the expression system comprises a nucleic acid encoding a thioesterase native to E. coli, such as thioesterase EcTesB (SEQ ID NO: 39) from E. coli, as set forth in SEQ ID NO: 39, or a nucleic acid encoding a functional variant thereof having at least 70% identity to SEQ ID NO: 39. In some embodiments, the nucleic acid is Ec_tesB from E. coli (SEQ ID NO: 41), or a homologue thereof having at least 70% homology or identity thereto, and preferably encoding a functional variant of EcTesB.

[0195] In some embodiments, the expression system comprises the following:

[0196] - phaA from C. necator (SEQ ID NO: 13);

[0197] - phaB from C. necator (SEQ ID NO: 14); and

[0198] - phaC from C. necator (SEQ ID NO: 15), or variants thereof having at least 70% homology or identity thereto, and preferably encoding functional variants.

[0199] In some embodiments, the expression system comprises the following: - phaA from C. necator (SEQ ID NO: 13);

[0200] - phaB from C. necator (SEQ ID NO: 14); and

[0201] Pp_tesB from Pseudomonas putida (SEQ ID NO: 40), or variants thereof having at least 70% homology or identity thereto, and preferably encoding functional variants.

[0202] In some embodiments, the expression system comprises the following:

[0203] - phaA from C. necator (SEQ ID NO: 13);

[0204] - phaB from C. necator (SEQ ID NO: 14); and

[0205] - Ec_tesB from E. coli (SEQ I D NO: 41 ), or variants thereof having at least 70% homology or identity thereto, and preferably encoding functional variants.

[0206] In some embodiments, the expression system comprises the following:

[0207] - phaA (SEQ ID NO: 13);

[0208] - phaB (SEQ ID NO: 14); and

[0209] / . phaC (SEQ ID NO: 15) and Pp_tesB (SEQ ID NO: 40); or

[0210] / / . phaC (SEQ ID NO: 15) and Ec_tesB (SEQ ID NO: 41); or

[0211] Hi. Pp_tesB (SEQ ID NO: 40) and Ec_tesB (SEQ ID NO: 41); or iv. phaC (SEQ ID NO: 15), Ec_tesB (SEQ ID NO: 41) and Pp_tesB

[0212] (SEQ ID NO: 40), or variants thereof having at least 70% homology or identity thereto, and preferably encoding functional variants.

[0213] Compounds and compositions

[0214] Disclosed herein is an acetate-derived compounds obtained by the engineered cells and / or the methods disclosed herein, and / or compositions comprising an acetatederived compound obtained by the engineered cells and / or methods disclosed herein, preferably wherein the acetate-derived compound is a fluorinated acetate-derived compound. In particular, disclosed is fluorinated 3HBs and fluorinated scl-PHAs, such as F-PHB, obtained using the engineered cells and / or methods disclosed herein. Acetate-derived compounds

[0215] In the present context, the term acetate-derived compound refers to compounds that are derived from acetate, acetic acid or acetyl-CoA. In other words, acetyl-CoA derived compounds are herein also acetate-derived compounds.

[0216] The engineered cells disclosed herein, and in particular the engineered cells with increased tolerance towards acetate as described herein, for example in the section “Short-chain organic acid tolerance” or “Further modifications”, may be useful for producing a variety of compounds derived from acetate, acetic acid and acetyl-CoA, such as polyketides, lipids, biopolymers, alcohols and organic acids, for instance using the pathways described in Chen et al. (2013) and Zhang et al. (2019). Useful methods for producing acetate-derived compounds are disclosed in the section “Methods for production of acetate-derived products” herein.

[0217] Acetate-derived compounds include compounds derived from acetyl-CoA, i.e. acetyl- CoA-derived compounds. PHAs are acetate-derived compounds, including 3HB and PHB, as well as fluorinated 3HB and F-PHB.

[0218] 3HB and fluorinated 3HB

[0219] The enzymes disclosed herein, in particular the polypeptides acetyl-CoA thiolase, acetyl-CoA reductase, scl-PHA synthase, and / or thioesterase can act on fluorinated compounds.

[0220] Thus, herein disclosed are engineered cells, preferably bacterial cells, and methods for producing fluorinated 3-hydroxybutyrate (3HB), wherein said fluorinated 3HB may be 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3-hydroxybutyrate, and / or 2-fluoro-3- hydroxybutyrate. Thus, the methods disclosed herein may be methods for producing 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3-hydroxybutyrate, and / or 2-fluoro-3- hydroxybutyrate.

[0221] In other words, the fluorinated 3HB produced by a bacterial cell and / or a method disclosed herein, may be a mixture of 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3- hydroxy butyrate, and / or 2-fluoro-3-hydroxybutyrate. Thus, disclosed herein is fluorinated 3-hydroxybutyrate (3HB), wherein said fluorinated 3HB may be 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3-hydroxybutyrate, and / or 2- fluoro-3-hydroxybutyrate. Thus, the methods disclosed herein may be methods for producing 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3-hydroxybutyrate, and / or 2-fluoro-3- hydroxybutyrate.

[0222] PHB and fluorinated PHB

[0223] The enzymes disclosed herein, in particular the polypeptides acetyl-CoA thiolase, acetyl-CoA reductase, scl-PHA synthase, and / or thioesterase can act on fluorinated compounds.

[0224] In one embodiment, the molecular weight (MW) of the PHB produced by the engineered cells and / or methods disclosed herein is between 0.1 mega Dalton (MDa) and 1.5 MDa.

[0225] In some embodiments said F-PHB is poly(fluoro-3-hydroxybutyrate). In other embodiments, said F-PHB has the structure of formula (I): wherein: x and y independently are integers between 1 and 19,999; and x+y < 20,000. A co-polymer of formula (I) may be termed poly(4-fluoro-3- hydroxybutyrate-co-3-hydroxybutyrate).

[0226] In other embodiments, said F-PHB has the structure of formula (II): wherein: x, y and z independently are integers between 1 and 19998, and x+y+z<20000. A copolymer of formula (I) may be termed poly(4-fluoro-3-hydroxybutyrate-co-2-fluoro-3- hydroxybutyrate-co-3-hydroxybutyrate).

[0227] Without being bound by theory, the fluorinated PHB (F-PHB) produced by the engineered cells disclosed herein may be: a compound of formula (I) a compound of formula (II) a mixture of PHB, a compound of formula (I) and of a compound of formula (II).

[0228] In other words, when the present methods involve fluorinated substrates, preferably F- acetate, to obtain fluorinated compounds, the compounds may comprise a mixture of PHB and F-PHB, in particular of F-PHB of formula (I), of formula (II), or of a mixture of both.

[0229] Thus, herein disclosed is engineered cells, preferably bacterial cells, and methods for producing fluorinated PHB (F-PHB), wherein said F-PHB is poly(fluoro-3- hydroxy butyrate). In other words, the F-PHB produced by an engineered cell and / or a method disclosed herein, may be a mixture of PHB and of F-PHB, in particular of F- PHB of formula (I), of formula (II), or of a mixture of both.

[0230] In other words, herein disclosed is engineered cells, preferably bacterial cells, and methods for producing F-PHB, wherein said F-PHB comprises one or more monomers of 3-hydroxybutyrate, 4-fluoro-3-hydroxybutyrate, 2-fluoro-3-hydroxybutyrate, and / or 2,4-fluoro-3-hydroxybutyrate. Without being bound by theory, the produced F-PHB is not considered a poly- or perfluorinated (PFAS) polymer or material, although F-PHB shares physicochemical properties therewith.

[0231] In some embodiments, where the engineered cell produces a scl-PHA, said scl-PHA comprises or consists of F-PHB and optionally PHB and has a fluorine (F) content of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (mol / mol).

[0232] In some embodiments, where the engineered cell produces a scl-PHA, said scl-PHA comprises or consists of F-PHB and optionally PHB and has a fluorine (F) content of at least 0.1%, such as at least 0.2%, such as at least 0.5%, such as at least 0.7%, such as at least 1.0%.

[0233] In some embodiments, where the engineered cell produces a scl-PHA, said scl-PHA comprises or consists of F-PHB and optionally PHB and has a fluorine (F) content between 0.1% and 1.5%, for example between 0.2% and 1.25%, such as between 0.3% and 1.15%, for example between 0.4% and 1.1%, such as between 0.5% and 1%.

[0234] In some embodiments, where the engineered cell produces a scl-PHA, said scl-PHA comprises or consists of F-PHB and optionally PHB and has a static water contact angle of at least 75°, such as at least 80°, such as at least 85°, such as at least 90°, such as at least 95°, such as at least 100°, such as at least 105°, such as at least 110°, such as at least 115°, such as at least 120°, such as 121°, such as 122°, such as 123°, such as 124°, or more.

[0235] In some embodiments, where the engineered cell produces a scl-PHA, said scl-PHA comprises or consists of F-PHB and optionally PHB and has a water contact angle (WCA) in diiodomethane of at least 35°, such as at least 40°, such as at least 45°, such as at least 46°, such as at least 47°, such as at least 48°, such as at least 49°, or more.

[0236] In some embodiments, where the engineered cell produces a scl-PHA, said scl-PHA comprises or consists of F-PHB and optionally PHB and has a surface energy of at most 59 mJ nr2, such as at most 55 mJ rrr2, such as at most 50 mJ rrr2. Thus, disclosed herein are fluorinated scl-PHAs, preferably wherein said fluorinated scl-PHA is F-PHB, and optionally wherein said F-PHB comprises one or more monomers of 3-hydroxy butyrate, 4-fluoro-3-hydroxybutyrate, 2-fluoro-3- hydroxybutyrate, and / or 2,4-fluoro-3-hydroxybutyrate.

[0237] In some embodiments, said F-PHB may have the structure of formula (I). In other embodiments, said F-PHB may have the structure of formula (II).

[0238] In some embodiments, said F-PHB may comprise a mixture, wherein said mixture comprises or consists of F-PHB with formula (I) and F-PHB with formula (II).

[0239] In some embodiments, said F-PHB has a static water contact angle of at least 75°, such as at least 80°, such as at least 85°, such as at least 90°, such as at least 95°, such as at least 100°, such as at least 105°, such as at least 110°, such as at least 115°, such as at least 120°, such as 121 °, such as 122°, such as 123°, such as 124°, or more.

[0240] In some embodiments, said F-PHB has a water contact angle (WCA) in diiodomethane of at least 35°, such as at least 40°, such as at least 45°, such as at least 46°, such as at least 47°, such as at least 48°, such as at least 49°, or more.

[0241] In some embodiments, said F-PHB has a surface energy of at most 59 mJ rrr2, such as at most 55 mJ nr2, such as at most 50 mJ rrr2.

[0242] Compositions

[0243] Compositions comprising 3HB, a fluorinated 3HB, PHB, and / or F-PHB obtainable by the methods described herein.

[0244] Thus, provided is a composition comprising 3HB obtainable by the engineered cells and / or methods disclosed herein. Provided is also a composition comprising a fluorinated 3HB obtainable by the engineered cells and / or methods disclosed herein, wherein the fluorinated 3HB is 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3- hydroxy butyrate, and / or 2-fluoro-3-hydroxybutyrate.

[0245] Thus, provided is a composition comprising PHB obtainable by the engineered cells and / or methods disclosed herein. Provided is also a composition comprising F-PHB obtainable by the engineered cells and / or methods disclosed herein, preferably wherein the F-PHB is as defined in the section “PHB and fluorinated PHB” herein above.

[0246] Methods for production of acetate-derived products

[0247] The present disclosure relates to methods for production of acetate-derived compounds using engineered cells, preferably bacterial cells. In particular, disclosed herein are methods for production of 3HB, fluorinated 3HB, PHB, and / or fluorinated PHB.

[0248] Thus, disclosed herein is a method of producing an acetate-derived compound in an engineered cell, preferably a bacterial cell, such as a Pseudomonas cell, said method comprising the steps of: a) Providing an engineered cell, optionally an engineered Pseudomonas cell, preferably an engineered P. putida cell, b) Cultivating said engineered cell in a culture medium comprising acetate; wherein said engineered cell comprising one or more modifications selected from the group consisting of: one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), such as a mutation resulting in substitution of residue 77 of FabB, preferably resulting in FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70); one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10); one or more mutations in HupB (SEQ ID NO: 8 or SEQ ID NO: 69), such as a mutation resulting in substitution of residue 35 of HupB, preferably resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71), and / or a mutation resulting in substitution of residue 49 of HupB (SEQ ID NO: 8 or SEQ ID NO: 69), preferably resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72), or one or more mutations in a nucleic acid encoding a functional variant thereof having at least 70% identity to any one of FabB, UvrY or HupB, thereby producing the acetate-derived compound. Useful bacterial cells for the methods described in this section are described elsewhere herein, in particular in the section “Bacterial cell”. Preferably the engineered cell is a Pseudomonas cell, such as a P. putida cell, for example a P. putida KT440 cell, such as a P. putida EM42 cell.

[0249] The acetate-derived product is preferably a compound described in the section “Acetate-derived compounds” herein above. In preferred embodiments, the acetatederived compound comprises or consists of 3HB or fluorinated 3HB, or a scl-PHA, such as PHB and / or fluorinated PHB (F-PHB).

[0250] Thus, herein described are engineered cells, preferably bacterial cells, and / or methods useful for producing acetate-derived compounds, such as 3HB, fluorinated 3HB, PHB, and / or F-PHB with a high titer. The titer(s) obtained with any of the engineered cells and / or methods of the present disclosure may be as described in the section “Titers” herein below.

[0251] Culture medium and cultivation

[0252] Throughout the present disclosure, it will be understood that the engineered cells disclosed herein can produce the compound(s) of interest when incubated in a culture medium, also known as a cultivation medium, under conditions that enable the engineered cells to grow and produce the desired compound(s). From the description of the engineered cells, also known as production organisms, production host cells, production hosts, or host cells, provided herein, and knowing the type of engineered cell used, the skilled person will not have difficulties in identifying a suitable culture medium and / or conditions to achieve production of the compound(s) of interest. Thus, the type of culture medium used for propagating the engineered cell will depend on the type of cell used for the method.

[0253] In particular, the cultivation may be performed at temperatures and at pH suitable for supporting growth of the engineered cells. The culture medium should include the required nutrients, and may be supplemented with precursors, inducer(s) and / or substrate(s) as applicable, i.e. at any stage prior to or during the cultivation of the engineered cell in the culture medium. Thus, in some embodiments, the culture medium comprises glucose. In other embodiments, the culture medium comprises acetate.

[0254] The time of cultivation will vary depending on which cell is used and what compound may be produced by said cell, but can easily be adapted by the skilled person. The engineered cell may for example be cultivated for at least 24 hours, such as at least 32 hours, such as at least 48 hours, such as at least 75 hours, or more.

[0255] Any of the engineered cells described herein can be used to produce acetate-derived products. In particular, the engineered cell preferably has increased tolerance to acetate compared to the tolerance to acetate of a reference cell from the same organism, which has not been engineered.

[0256] C / N ratio

[0257] It may be advantageous to provide a culture medium wherein the C / N ratio is high. Without being bound by theory, a high C / N ratio induces polymer synthesis.

[0258] In some embodiments, the C / N ratio of the culture medium is of at least 4, such as at least 5, such as at least 10, such as at least 20, such as at least 30, such as at least 40, or more.

[0259] The culture medium may be supplied with additional substrates and / or inducer compounds. The choice of substrate or inducer will typically be dictated by the intention of the method, i.e. the compound of interest to be produced and strain design.

[0260] In some embodiments, the culture medium comprises acetate and one or more of glucose, or citrate, and PHB is produced.

[0261] In some embodiment, the culture medium comprises inducer compounds, such as 3- mBz or cyclohexanone. This is particularly relevant for embodiments where the cell comprises an inducible promoter and / or expression system as described herein above in the section “Promoters and other modifications”. In some embodiments, the culture medium comprises F-acetate and one or more of glucose, citrate, a TCA cycle intermediate such as 2-ketoglutarace (2KG), or acetate, and 3HB, fluorinated 3HB, PHB and / or F-PHB are produced.

[0262] The substrates may be provided in the form of salts. Preferred salts are soluble salts.

[0263] Acetate

[0264] In embodiments wherein the culture medium comprises acetate, the acetate is preferably added to the culture medium as a salt, preferably as sodium acetate or potassium acetate.

[0265] Thus, in some embodiments, the culture medium comprises at least 10 mM acetate, such as at least 20 mM acetate, such as at least 30 mM acetate, such as at least 40 mM acetate, such as at least 50 mM acetate, such as at least 60 mM acetate, such as at least 70 mM acetate, such as at least 80 mM acetate, such as at least 90 mM acetate, such as at least 100 mM acetate, such as at least 110 mM acetate, such as at least 120 mM acetate, such as at least 130 mM acetate, such as at least 140 mM acetate, such as at least 150 mM acetate, such as at least 160 mM acetate, such as at least 170 mM acetate, such as at least 180 mM acetate, such as at least 190 mM acetate, such as at least 200 mM acetate, such as at least 210 mM acetate, such as at least 220 mM acetate, such as at least 230 mM acetate, such as at least 240 mM acetate, such as at least 250 mM acetate, such as at least, 260 mM acetate, such as at least 275 mM acetate, such as at least 280 mM acetate, such as at least 290 mM acetate, such as at least 295 mM acetate, such as at least 300 mM acetate, or more.

[0266] In embodiments wherein the culture medium comprises F-acetate, the F-acetate is preferably added to the culture medium as a salt, such a potassium salt and / or a sodium salt, preferably as potassium F-acetate and / or sodium F-acetate.

[0267] In other embodiments, the culture medium comprises at least 10 mM F-acetate, such as at least 20 mM F-acetate, such as at least 30 mM F-acetate, such as at least 40 mM F-acetate, such as at least 50 mM F-acetate, such as at least 60 mM F-acetate, such as at least 70 mM F-acetate, such as at least 80 mM F-acetate, such as at least 90 mM F-acetate, such as at least 100 mM F-acetate, such as at least 110 mM F-acetate, such as at least 120 mM F-acetate, such as at least 130 mM F-acetate, such as at least 140 mM F-acetate, such as at least 150 mM F-acetate, such as at least 160 mM F-acetate, such as at least 170 mM F-acetate, such as at least 180 mM F-acetate, such as at least 190 mM F-acetate, such as at least 200 mM F-acetate, such as at least 210 mM F-acetate, such as at least 220 mM F-acetate, such as at least 230 mM F-acetate, such as at least 240 mM F-acetate, such as at least 250 mM F-acetate, such as at least, 260 mM F-acetate, such as at least 275 mM F-acetate, such as at least 280 mM F-acetate, such as at least 290 mM F-acetate, such as at least 295 mM F-acetate, such as at least 300 mM F-acetate, such as at least 325 mM F-acetate, such as at least 350 mM F-acetate, such as at least 380 mM F-acetate, such as at least 400 mM F-acetate, such as at least 425 mM F-acetate, or more, for example 450 mM F-acetate.

[0268] TCA cycle intermediates for alleviating F-acetate toxicity

[0269] F-acetate is well known for its toxicity towards bacterial cells. To alleviate this toxicity, the inventors have observed that intermediates of the tricarboxylic acid cycle (TCA cycle) may be provided to the engineered cell, for example supplied to the culture medium, when F-acetate is also supplied or will be supplied. In other words, the engineered cell may be cultivated in the presence of one or more native TCA cycle intermediates prior to or together with cultivation with F-acetate.

[0270] Thus, in some embodiments, the culture medium further comprises 2-ketoglutarate (2KG), isocitrate, or succinate and glutamate, preferably at a concentration of at least 1 mM, such as at least 2 mM, such as at least 3 mM, such as at least 4 mM, such as at least 5 mM, such as at least 10 mM, such as at least 15 mM, or more. Preferably, the culture medium comprises 2KG at a concentration of at least 1 mM, such as at least 15 mM, or more.

[0271] Methods for production of 3HB and fluorinated 3HB

[0272] As detailed herein above, the engineered cells disclosed elsewhere herein may be useful for producing 3HB and fluorinated 3HB. These may be used as monomers for the synthesis of different polymers, for example as described herein below in the section “Methods for manufacturing a fluorinated polymer”.

[0273] The fluorinated 3HB may be selected from 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3- hydroxy butyrate, and / or 2-fluoro-3-hydroxybutyrate. Thus, the method may be a method for producing 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate.

[0274] Disclosed are methods of producing a 3HB, preferably wherein the 3HB is fluorinated, in an engineered cell, preferably a bacterial cell, said methods comprising the steps of: a) providing an engineered cell, preferably an engineered Escherichia cell, optionally a E. coli cell, b) cultivating said engineered cell in a culture medium comprising a carbon source and a nitrogen source; wherein the engineered cell comprises a nucleic acid encoding an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting acetyl-CoA into acetoacetyl-CoA, preferably a nucleic acid encoding PhaA from Cupriavidus necator (SEQ ID NO: 1), a nucleic acid encoding an acetyl-CoA reductase (EC 1.1.1.36) capable of converting acetoacetyl-CoA into F?-(-)-3-hydroxybutyryl-CoA, preferably a nucleic acid encoding PhaB from Cupriavidus necator (SEQ ID NO: 2), and a nucleic acid encoding a thioesterase (EC 3.1.2.20) capable of hydrolysing a 3-hydroxybutyryl-CoA to CoA and a corresponding 3- hydroxybutyrate, preferably a nucleic acid encoding TesB from P. putida (PpTesB, SEQ ID NO: 38) or Escherichia coli (EcTesB, SEQ ID NO: 39), or functional variants thereof having at least 70% identity to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, preferably wherein the culture medium comprises glucose, citrate, acetate and / or F- acetate as carbon source, thereby producing 3HB, preferably fluorinated 3HB, optionally with a titer of at least 0.25 g / L, preferably wherein the fluorinated 3HB comprises or consists of 2,4-difluoro-3- hydroxy butyrate, 4-fluoro-3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate.

[0275] Disclosed herein is methods of producing a 3HB, preferably wherein the 3HB is fluorinated, in an engineered cell, preferably a bacterial cell, said methods comprising the steps of: a) providing an engineered cell, preferably an engineered Pseudomonas cell, optionally a P. putida cell, b) cultivating said engineered cell in a culture medium comprising a carbon source and a nitrogen source; wherein: i. the engineered cell is not capable of producing PHAs having a medium-length carbon chain (mcl-PHAs); ii. the engineered cell is not capable of converting fatty acids into PHA monomers having a medium-length carbon chain; iii. optionally the benABCD cluster in said cell is inactivated, and iv. the engineered cell comprises a nucleic acid encoding an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting acetyl-CoA into acetoacetyl-CoA, preferably a nucleic acid encoding PhaA from Cupriavidus necator (SEQ ID NO: 1), a nucleic acid encoding an acetyl-CoA reductase (EC 1.1.1.36) capable of converting acetoacetyl-CoA into F?-(-)-3-hydroxybutyryl-CoA, preferably a nucleic acid encoding PhaB from Cupriavidus necator (SEQ ID NO: 2), and a nucleic acid encoding a thioesterase (EC 3.1.2.20) capable of hydrolysing a 3-hydroxybutyryl-CoA to CoA and a corresponding 3-hydroxybutyrate, preferably a nucleic acid encoding PpTesB from P. putida (SEQ ID NO: 38) or EcTesB from Escherichia coli (SEQ ID NO: 39), or functional variants thereof having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, preferably wherein the culture medium comprises glucose, citrate, acetate and / or F- acetate as carbon source, thereby producing 3HB, preferably fluorinated 3HB, optionally with a titer of at least 0.25 g / L, preferably wherein the fluorinated 3HB comprises or consists of 2,4-difluoro-3- hydroxy butyrate, 4-fluoro-3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate.

[0276] Engineered cells useful for the production of 3HB and / or fluorinated 3HB comprise nucleic acids encoding: an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting acetyl-CoA into acetoacetyl-CoA; an acetyl-CoA reductase (EC 1.1.1.36) capable of converting acetoacetyl- CoA into F?-(-)-3-hydroxybutyryl-CoA; and a thioesterase (EC 3.1.2.20) capable of hydrolysing a 3-hydroxybutyryl-CoA to CoA and a corresponding 3-hydroxybutyrate.

[0277] The nucleic acid encoding the acetyl-CoA thiolase and / or the acetyl-CoA reductase may be a nucleic acid native to a bacterial cell, such as native to Cupriavidus, such as Cupriavidus necator, Azotobacter, Streptomyces, or Paracoccus. The nucleic acid encoding the the thioesterase may be a nucleic acid native to a bacterial cell, such as native to Pseudomonas, for example P. putida, or native to Escherichia, such as E. coli.

[0278] In some embodiments, one or more of the above nucleic acids encode heterologous polypeptides, such as enzymes and / or proteins.

[0279] In other embodiments the engineered cell comprises nucleic acids encoding the following polypeptides, which are expressed in the cell:

[0280] PhaA (SEQ ID NO: 1), or a functional variant thereof having at least 70% identity to SEQ ID NO: 1 ;

[0281] PhaB (SEQ ID NO: 2), or a functional variant thereof having at least 70% identity to SEQ ID NO: 2; and

[0282] PpTesB (SEQ ID NO: 38), or a functional variant thereof having at least 70% identity to SEQ ID NO: 38.

[0283] In other embodiments the engineered cell comprises nucleic acids encoding the following polypeptides, which are expressed in the cell:

[0284] PhaA (SEQ ID NO: 1), or a functional variant thereof having at least 70% identity to SEQ ID NO: 1 ;

[0285] PhaB (SEQ ID NO: 2), or a functional variant thereof having at least 70% identity to SEQ ID NO: 2; and

[0286] EcTesB (SEQ ID NO: 39), or a functional variant thereof having at least 70% identity to SEQ ID NO: 39.

[0287] These nucleic acids may be provided in the form of a synthetic operon as described herein above, and may comprise an inducible promoter and / or as described herein above, in particular a cyclohexanone-inducible expression system such as ChnR / Pc / »iB (SEQ ID NO: 5), a 3-methylbenzoate-inducible expression system such as XylS / Pm (SEQ ID NO: 30), Lacl / Ptac, Lacl / Ptrc, a rhamnose-inducible promoter, or an arabinose inducible promoter.

[0288] The engineered cell may further comprise any of the modifications described in the section “Short-chain organic acid tolerance”, “Other useful modifications for increasing tolerance" and “Further modifications”, in particular:

[0289] One or more mutations in one or more genes encoding proteins involved in the regulation of innate PHA synthesis, such as uvrY (SEQ ID NO: 32), preferably resulting in UvrYE197K (SEQ ID NO: 10);

[0290] One or more mutations in one or more genes encoding proteins which are involved in DNA bending, such as genes encoding histone-like proteins, in particular of the HU family, such as hupB (SEQ ID NO: 33 or SEQ ID NO: 73), preferably resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71), HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72); or resulting in HupBA35V,T49l (SEQ ID NO: 46 or SEQ ID NO: 76); and

[0291] One or more mutations in one or more genes encoding 3-oxoacyl-[acyl- carri er- protein (AGP)] synthases, in particular 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase I or 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase II, such as fabB (SEQ ID NO: 34), preferably resulting in FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70).

[0292] In addition, the engineered cell may be further modified to partially or totally inactivate mcl-PHA synthesis as described in the section “mcl-PHA-deaf”. The engineered cell may also, alternatively or additionally, be hampered in converting fatty acids into PHA monomers having a medium-length carbon chain as described in the section “Inactivation ofphaG". The benABCD cluster may also, alternatively or additionally, be partially or totally inactivated as described in the section “Inactivation of the benABCD cluster”.

[0293] Methods for production of PHB and fluorinated PHB

[0294] The engineered cells disclosed elsewhere herein may be useful for producing PHB and fluorinated PHB, such as F-PHB. Fluorinated PHB may be used as or in water repellent compositions, such as materials. In particular, disclosed herein is a method of producing a PHA having a short carbon chain length (scl-PHA), preferably wherein the PHA is PHB, preferably fluorinated PHB (F-PHB), in a engineered cell, such as a Pseudomonas cell, said method comprising the steps of: a) Providing an engineered cell, such as an Pseudomonas cell, b) Cultivating said engineered cell in a culture medium comprising a carbon source and a nitrogen source; wherein: the engineered cell is not capable of producing PHAs having a medium-length carbon chain; i. the engineered cell is not capable of converting fatty acids into PHA monomers having a medium-length carbon chain; ii. optionally the benABCD cluster in said cell is inactivated, and iii. the engineered cell comprises a nucleic acid encoding an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting two acetyl-CoAs into acetoacetyl-CoA, preferably a nucleic acid encoding PhaA from Cupriavidus necator (SEQ ID NO: 1), a nucleic acid encoding an acetyl-CoA reductase (EC 1.1.1.36) capable of converting acetoacetyl-CoA into F?-(-)-3-hydroxybutyryl-CoA, preferably a nucleic acid encoding PhaB from Cupriavidus necator (SEQ ID NO: 2), and a nucleic acid encoding a short-chain-length-polyhydroxyalkanoate scl-PHA synthase (EC 2.3.1.304) capable of polymerizing F?-(-)-3-hydroxybutyryl- CoA into polyhydroxybutyrate (PHB), preferably a nucleic acid encoding PhaC from Cupriavidus necator (SEQ ID NO: 3), or functional variants thereof having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, preferably wherein the culture medium comprises glucose, citrate, acetate and / or F- acetate as carbon source, thereby producing a short carbon chain length (scl-PHA) with a titer of at least 0.25 g / L, preferably wherein the PHA comprises or consists of polyhydroxybutyrate (PHB), preferably fluorinated PHB (F-PHB).

[0295] Engineered cells useful for the production of PHB and / or fluorinated PHB comprise nucleic acids encoding: an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting acetyl-CoA into acetoacetyl-CoA; an acetyl-CoA reductase (EC 1.1.1 .36) capable of converting acetoacetyl- CoA into P-(-)-3-hydroxybutyryl-CoA; and a scl-PHA synthase (EC 2.3.1.304) capable of polymerizing P-(-)-3- hydroxybutyryl-CoA into polyhydroxybutyrate (PHB).

[0296] The nucleic acid encoding the acetyl-CoA thiolase, the acetyl-CoA reductase, and / or the scl-PHA synthase may be a nucleic acid native to a bacterial cell, such as native to Cupriavidus, such as Cupriavidus necator, Azotobacter, Streptomyces, or Paracoccus.

[0297] In some embodiments, one or more of the above nucleic acids encode heterologous polypeptides, such as enzymes and / or proteins.

[0298] In some embodiments the engineered cell comprises nucleic acids encoding the following polypeptides, which are expressed in the cell:

[0299] PhaA (SEQ ID NO: 1), or a functional variant thereof having at least 70% identity to SEQ ID NO: 1 ;

[0300] PhaB (SEQ ID NO: 2), or a functional variant thereof having at least 70% identity to SEQ ID NO: 2; and

[0301] PhaC (SEQ ID NO: 3), or a functional variant thereof having at least 70% identity to SEQ ID NO: 3.

[0302] These nucleic acids may be provided in the form of a synthetic operon as described herein above, and may comprise an inducible promoter and / or expression system as described herein above, in particular a cyclohexanone-inducible expression system such as ChnR / PchnB (SEQ ID NO: 5), a 3-methylbenzoate-inducible expression system such as XylS / Pm (SEQ ID NO: 30), Lacl / Ptac, Lacl / Ptrc, a rhamnose-inducible promoter, or an arabinose inducible promoter.

[0303] The engineered cell may further comprise any of the modifications described in the section “Short-chain organic acid tolerance”, in particular:

[0304] One or more mutations in one or more genes encoding proteins involved in the regulation of innate PHA synthesis, such as uvrY (SEQ ID NO: 32), preferably resulting in UvrYE197K (SEQ ID NO: 10); One or more mutations in one or more genes encoding proteins which are involved in DNA bending, such as genes encoding histone-like proteins, in particular of the HU family, such as hupB (SEQ ID NO: 33 or SEQ ID NO: 73), preferably resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71), HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72); or resulting in HupBA35V,T49l (SEQ ID NO: 46 or SEQ ID NO: 76); and

[0305] One or more mutations in one or more genes encoding 3-oxoacyl-[acyl- carri er- protein (AGP)] synthases, in particular 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase I or 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase II, such as fabB (SEQ ID NO: 34), preferably resulting in FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70).

[0306] In addition, the engineered cell may be further modified to partially or totally inactivate mcl-PHA synthesis as described in the section “mcl-PHA-deaf”. The engineered cell may also, alternatively or additionally, be hampered in converting fatty acids into PHA monomers having a medium-length carbon chain as described in the section “Inactivation ofphaG". The benABCD cluster may also, alternatively or additionally, be partially or totally inactivated as described in the section “Inactivation of the benABCD cluster”. The cell may further be modified as described in “Further modifications”.

[0307] Yields and conversion rates

[0308] The methods described herein are effective and both results in high yields and conversion rates of acetate into an acetate-derived compound, such as a 3HB and / or a scl-PHA, preferably PHB and / or fluorinated PHB.

[0309] Thus, provided herein is methods, wherein the yield of PHB and / or F-PHB is at least 5% (w / w) on biomass, such as at least 7.5% (w / w) on biomass, or more.

[0310] In some embodiments, wherein the method is a method for production of PHB and / or F-PHB, the conversion rate of acetate and / or F-acetate to PHB and / or F-PHB is at least 0.1% (w / w) per day, such as 0.2% (w / w) per day, or more. The methods disclosed herein may further comprise a step of recovering the produced compound(s) of interest, such as the acetate-derived compounds, for example 3HB, fluorinated 3HB, PHB and / or F-PHB.

[0311] In other words, the methods disclosed herein may further comprise a step of isolating the compound(s) of interest, such as the 3HB, F-HB, PHB, and / or the F-PHB.

[0312] The step of recovering the compound(s) of interest, such as the 3HB, F-HB, PHB, and / or F-PHB may comprise solvent extraction, such as Sohxlet extraction. In some embodiments, the 3HB, F-HB, PHB, and / or F-PHB is recovered by subjecting dry cell biomass to Sohxlet extraction, followed by precipitation of the 3HB, F-HB, PHB and / or F-PHB, and solvent evaporation.

[0313] In some embodiments, the methods further comprises a step of converting the produced compounds into a downstream compound. For example, the produced PHB and / or F-PHB may be further modified, such as modified by enzymatic and / or chemical methods. For example, PHB and / or F-PHB may be further modified by the addition of one or more substituents, such as described in El-Malek and Steinbuchel (2022).

[0314] Titers

[0315] In some embodiments, wherein the engineered cell is capable of producing 3HB, fluorinated 3HB, PHB, and / or F-PHB, said 3HB, fluorinated 3HB, PHB, and / or F-PHB is produced with a titer of at least 0.25 g / L, or more. With respect to the methods disclosed herein, wherein 3HB, fluorinated 3HB, PHB, and / or F-PHB is produced, said 3HB, fluorinated 3HB, PHB, and / or F-PHB is produced with a titer of at least 0.25 g / L, or more.

[0316] In other words, in some embodiments, wherein the engineered cell is capable of producing a scl-PHA and / or wherein scl-PHA is produced with a method disclosed herein, said scl-PHA is produced with a titer of at least 0.25 g / L, preferably wherein the sIc-PHA comprises or consists of PHB and / or F-PHB. Titers of 3HB and fluorinated 3HB

[0317] The cells disclosed herein can produce 3HB and / or fluorinated 3HB with high titers.

[0318] The titers of a given product, i.e. 3HB or fluorinated 3HB, as understood herein take into account the product within the cell (intracellular product), as well as the product excreted in the medium (extracellular product). The titers herein below thus refer to the titers of 3HB and / or fluorinated 3HB both within the cell and within the culture medium or supernatant, i.e. outside the cell.

[0319] In some embodiments, wherein the engineered cell is capable of producing 3HB, said 3HB is produced with a titer of at least 0.25 g / L, such as at least 0.3 g / L, such as at least 0.35 g / L, such as at least 0.5 g / L, such as at least 0.75 g / L, such as at least 1 g / L, such as at least 1.1 g / L, such as at least 1.5 g / L, or more.

[0320] Accordingly, in other embodiments, wherein 3HB is produced in a method disclosed herein, said 3HB is produced with a titer of at least 0.25 g / L, such as at least 0.3 g / L, such as at least 0.35 g / L, such as at least 0.5 g / L, such as at least 0.75 g / L, such as at least 1 g / L, such as at least 1.1 g / L, such as at least 1.2 g / L, such as at least 1.5 g / L, or more.

[0321] In further embodiments, wherein the engineered cell is capable of producing fluorinated 3HB, said fluorinated 3HB is produced with a titer of at least 0.25 g / L, such as at least 0.3 g / L, such as at least 0.35 g / L, such as at least 0.5 g / L, such as at least 0.75 g / L, such as at least 1 g / L, such as at least 1.1 g / L, such as at least 1.5 g / L, or more.

[0322] Accordingly, in further other embodiments, wherein fluorinated 3HB is produced in a method disclosed herein, said fluorinated 3HB is produced with a titer of at least 0.25 g / L, such as at least 0.3 g / L, such as at least 0.35 g / L, such as at least 0.5 g / L, such as at least 0.75 g / L, such as at least 1 g / L, such as at least 1.1 g / L, such as at least 1.5 g / L, or more.

[0323] The produced fluorinated 3HB may be 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3- hydroxy butyrate, and / or 2-fluoro-3-hydroxybutyrate. Titers of PHB and fluorinated PHB

[0324] In further embodiments, wherein the engineered cell is capable of producing PHB, PHB is produced with a titer of at least 30 g / L, such as at least 31 g / L, such as at least 32 g / L, such as at least 33 g / L, for example at least 33.5 g / L, such as at least 35 g / L, such as at least 37.5 g / L, or more, preferably wherein the titer is determined after at least 48 hours, or more.

[0325] Accordingly, in other embodiments, wherein PHB is produced in a method disclosed herein, PHB is produced with a titer of at least 30 g / L, such as at least 31 g / L, such as at least 32 g / L, such as at least 33 g / L, for example at least 33.5 g / L, such as at least 35 g / L, such as at least 37.5 g / L, or more, preferably wherein the titer is determined after at least 48 hours, or more.

[0326] In some embodiments, wherein the engineered cell is capable of producing F-PHB, F- PHB is produced with a titer of at least 0.25 g / L, such as at least 0.5 g / L, such as at least 0.75 g / L, such as at least 0.9 g / L, such as at least 0.95 g / L, such as at least 1 g / L, such as at least 1.1 g / L, such as at least 1.25 g / L, such as at least 1.5 g / L, such as at least 1.75 g / L, such as at least 2 g / L, such as at least 2.5 g / L, such as at least 5 g / L, or more.

[0327] Accordingly, in other embodiments, wherein F-PHB is produced in a method disclosed herein, F-PHB is produced with a titer of at least 0.25 g / L, such as at least 0.5 g / L, such as at least 0.75 g / L, such as at least 0.9 g / L, such as at least 0.95 g / L, such as at least 1 g / L, such as at least 1.1 g / L, such as at least 1.25 g / L, such as at least 1.5 g / L, such as at least 1.75 g / L, such as at least 2 g / L, such as at least 2.5 g / L, such as at least 5 g / L, or more.

[0328] Methods for manufacturing a fluorinated polymer

[0329] Also provided herein is a method for manufacturing an acetate-derived compound, said method comprising the steps of: i) providing an acetate-derived compound or compounds by the methods described herein; and ii) optionally converting said acetate-derived compound to a another compound of interest. The acetate-derived compound obtained by the methods described herein can be used directly, i.e. without further modification.

[0330] The acetate-derived compound may be a compound described in the section “Acetatederived compounds” herein above. Preferably, the acetate-derived compound is a PHA, for example PHB and / or fluorinated PHB, or a monomer therefor, for example 3HB and / or fluorinated 3HB. Said fluorinated 3HB may be 2,4-difluoro-3- hydroxy butyrate, 4-fluoro-3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate.

[0331] In other embodiments, however, the acetate-derived compound is further converted into another compound of interest. Indeed, the acetate-derived compound obtained by the present methods may be used as a drug precursor, a building block, and / or a precursor of polymers.

[0332] The methods of manufacturing an acetate-derived compound of interest may thus comprise a step of converting the acetate-derived compound obtained by the present methods into another compound of interest, such as a drug or a therapeutic compound.

[0333] Thus, provided herein is a method for manufacturing a fluorinated polymer, preferably F-PHB, said method comprising the steps of: i) providing a fluorinated 3HB by the methods described herein; and ii) converting said fluorinated 3HB to a fluorinated polymer, such as fluorinated PHB, preferably wherein the fluorinated 3HB is 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3- hydroxy butyrate, and / or 2-fluoro-3-hydroxybutyrate.

[0334] In another embodiment, provided is a method for manufacturing a fluorinated polymer, preferably F-PHB, said method comprising the steps of: i) providing a F-PHB by the methods described herein; and ii) converting said fluorinated 3HB to a fluorinated polymer, such as fluorinated PHB.

[0335] In another embodiment, provided is a method for manufacturing a fluorinated polymer, preferably F-PHB, said method comprising the steps of: i) providing a F-PHB by the methods described herein; and ii) converting said fluorinated PHB to a fluorinated polymer, such as fluorinated PHB.

[0336] Uses

[0337] Provided herein is the use of an acetate-derived compound obtainable by the present methods or of a composition comprising such compound as a medicament or a building block, or a precursor of polymers. Preferably, said acetate-derived compound is 3HB, fluorinated 3HB, PHB, and / or F-PHB.

[0338] Thus, provided herein is the use of a fluorinated 3HB obtainable by the present methods or of a composition comprising such fluorinated 3HB as a building block, or a precursor of polymers, preferably wherein the fluorinated 3HB is 2,4-difluoro-3- hydroxy butyrate, 4-fluoro-3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate.

[0339] Also, provided herein is the use of a fluorinated scl-PHA, preferably F-PHB, obtainable by the present methods or of a composition comprising such fluorinated scl-PHA, preferably F-PHB, as a building block, or a precursor of polymers, or a polymer.

[0340] Also disclosed is the use of F-PHB of formula (I) as water repellent, where F-PHB is of general structure (I): wherein: x and y independently are integers between 1 and 19,999; and x+y < 20,000.

[0341] Also disclosed herein is the use of F-PHB of formula (II) as water repellent, where F- PHB is of general structure (II): wherein: x, y and z independently are integers between 1 and 19,998, and x+y+z<20,000.

[0342] Further disclosed herein is the use of F-PHB as disclosed herein as a water repellent.

[0343] Also disclosed herein is the use of bacterial cells, nucleic acids, and / or expression systems and / or bacterial cells disclosed anywhere herein such as in the section “Bacterial cell” and “Expression systems” herein above, for production of PHB and / or F- PHB, preferably in a method also described herein, such as in the section “Methods for production of acetate-derived products” herein above.

[0344] Examples

[0345] Example 1: Construction of a PHB-producing P. putida strain (strain SEM1.4-PHB)

[0346] Results

[0347] P. putida EM42 (Martinez-Garcia et al., 2014), a genome-reduced variant of P. putida KT2440, was used as a platform for the construction of a medium-chain-length PHA (mcl-PHA)-deaf host (Figure 1). To this end, the complete endogenous pha cluster (phaC1ZC2DFI) was deleted by l-Scel— mediated recombination methods as described by Wirth et al., 2020. The genome loci deleted comprise two operons: one that encodes two mcl-PHA synthases (phaC1 (or PP_5003) and phaC2 (or PP_5005)), a depolymerase (phaZ or PP_5004), and a transcriptional activator (phaD or PP_5006), and an adjacent one, facing the opposite direction, that encodes phasins (phaF (or PP_5007) and phal (or PP_5008)). In the endogenous P. putida metabolism, PHA non-related substrates such as glycolytic (e.g. sugars) and gluconeogenic (e.g. aromatic compounds, alcohols, and organic acids) carbon I sources are directed through central catabolic pathways to the key intermediate acetyl-coenzyme A (CoA) and channeled towards PHA formation via de novo fatty acid synthesis (FAS). The enzyme that acts as a link between these two metabolic routes is PhaG, which mediates the conversion of FAS intermediates into mcl-PHA monomers (Mezzina et al., 2021). To completely decouple PHA synthesis from FAS, and thus, to avoid synthesis of monomers of different carbon lengths that could act as an acetyl-CoA sink or may end up in the polymer affecting monomer composition, phaG, was deleted by the Sce-I method. Lastly, the benABCD cluster (PP_3161 to PP_3164, SEQ ID NOs: 42 to 45) was also deleted to avoid synthesis of metabolic intermediates from benzoates degradation that yield a brownish color in the cultures, interfering with optical density (OD) measurements (Figure 1A). The resulting engineered strain, which cannot produce mcl-PHAs, was termed P. putida SEMI.4.

[0348] Growth of P. putida SEMI .4 was compared to the parental EM42 strain in de Bont minimal (DBM) medium with glucose or acetate as carbon sources. No significant differences were observed in the overall physiology and growth parameters of these strains. Loss of mcl-PHA production of strain SEMI.4 was firstly verified in a qualitative manner in plates of DBM containing glucose and Nile Red, a stain that binds to hydrophobic PHA granules. Strain SEMI .4 did not show any staining as compared to the parental strain EM42, indicating a PHA-negative phenotype. PHA accumulation was also quantitatively assessed by biomass hydrolysis and methanolysis of intracellular PHA followed by detection of methyl esters by gas chromatography coupled to mass spectrometry (GC-MS). Again, strains SEMI .4 did not produce any detectable PHA in medium with glucose or acetate, confirming the PHA-negative phenotype.

[0349] Subsequently, a PHB biosynthetic pathway from Cupriavidus necator, was inserted in strain SEMI .4 towards supporting PHB accumulation. This pathway comprises the phaCAB genes that encode (i) PhaA (SEQ ID NO: 1), a thiolase that condenses two acetyl-CoA molecules into acetoacetyl-CoA, (ii) PhaB (SEQ ID NO: 2), a reductase that converts acetoacetyl-CoA into R-(-)-3-hydroxybutyryl-CoA (the PHB monomer), and (iii) PhaC (SEQ ID NO: 3), a short-chain-length (scl)-PHA synthase that polymerizes R- (-)-3-hydroxybutyryl-CoA monomers to yield PHB. These three genes were cloned in a plasmid system in different configurations (Figure 1B). To this end, synthetic operons comprising optimized ribosome binding sites (RBSs, 5’-AGG AGG AAA AAC AT-3’ (SEQ ID NO: 4)) upstream of each of the three genes and different promoters were assembled in the pSEVA631 vector (Silva-Rocha et al., 2013) by the USER cloning method. Three different promoters were adopted in order to control the expression of these synthetic operons. Two inducible promoters, especially developed for controlled gene expression in P. putida, were used to this end: (i) XylS / Pm (SEQ ID NO: 30), inducible by addition of 3-methylbenzoate (3-mBz) to the culture medium (Gawin et al., 2017; Herrero et al., 1993), resulting in plasmid pS638 PHB, and (ii) ChnR / Pc / ine(SEQ ID NO: 5), induced by addition of cyclohexanone to the culture medium (Benedetti et al., 2016), resulting in plasmid pS6311 PHB. In addition, the native promoter (SEQ ID NO: 48) from the phaCAB cluster in C. necatorxNas used, resulting in plasmid pS630 PHB. This promoter allows for gene expression in P. putida without the addition of an inducer (i.e. it is a constitutive promoter in P. putida and other Gram-negative bacteria). In all these experiments, the empty pSEVA631 vector was used as control. All plasmids were individually transformed in strain SEMI .4, and the physiology of the resulting P. putida strains was compared in different culture conditions, either glycolytic or gluconeogenic. Metabolic pathways for the synthesis of PHB from sugars (e.g. glucose) and gluconeogenic substrates (e.g. acetate) expected to be active in the engineered SEMI.4 strain are depicted in Figure 1C.

[0350] A phenotypic characterization of all constructed strains was performed (Figure 2). Firstly, growth in glucose or acetate under two different carbon-to-nitrogen (C / N) ratios was assessed with the engineered strains. These experiments were designed to determine if there is any influence of the C / N ratio on PHB accumulation through the synthetic pathway. As the addition of high acetate concentrations is toxic to the cells (Yang et al., 2019), cultivations were performed with 30 mM acetate (corresponding to 60 mM C atoms) in order to prevent any growth impairment. On the other hand, due to the partial conversion of glucose to gluconate when P. putida is fed with this sugar substrate, cultivations were performed with 20 mM glucose (corresponding to 120 mM C atoms) to avoid any limitations in PHB production due to low or limiting substrate availability. With these objectives in mind, 24-h growth experiments were performed in microtiter plates with the different engineered strains in two different setups: (i) balanced growth conditions using DBM, resulting in a C / N = 4 or 2, for glucose or acetate, respectively; and (ii) N-limiting conditions using 0.1N DBM (10-fold reduction in the N content in DBM), resulting in a C / N = 40 or 20, for glucose or acetate, respectively.

[0351] When feeding glucose, P. putida SEM1.4 / pS630 PHB, the strain where the phaCAB genes are expressed from its own native promoter from C. necator, behaved in a similar manner to the control strain (SEM1.4 / pSEVA631 , i.e. no phaCAB genes). In contrast, when N was reduced in the medium, this strain reaches a higher OD (cell density) at 24 hours compared to the control strain, even though there were no significant changes in growth rate and duration of lag phase. This difference in the OD values could be due to the induction of PHB genes under the regulation of the native promoter at high C / N ratios. The strains bearing plasmids with the inducible expression systems (i.e. P. putida SEM1.4 / pS638 PHB and SEM1.4 / pS6311 PHB) showed lower growth rates than both the control strain and P. putida SEM1.4 / pS630 PHB under the two C / N ratios tested. In addition, strain SEM1.4 / pS638 PHB not only showed the lowest growth rate among the strains analyzed, but it also displayed a very long lag phase under both conditions tested. Differences in growth rates could be due to overexpression of the heterologous phaCAB genes and channeling of resources (e.g. key metabolic intermediates) to PHB synthesis instead of biomass (Figure 2A and 2B).

[0352] When feeding acetate, strains bearing inducible expression systems also showed lower growth rates under the two C / N ratios tested as compared to the control strain and the P. putida strain, where the phaCAB genes were kept under their native transcriptional regulation, but the difference was less pronounced when using this C source than when using glucose. In this case, SEMI .4 / pS6311 PHB was the strain with the lowest growth rate. The overall duration of lag phases was not affected under these culture conditions, and exponential growth was detected for all cultures essentially upon inoculation. This observation could be linked to the fact that cells were previously adapted to grow on acetate, as precultures were performed with this C source. In addition, the same effect as when feeding with glucose was observed for final cell densities under balanced conditions, as all strains reached a similar OD. Lastly, contrary to what was observed when feeding with glucose, the final cell density (OD) under N-limiting conditions was higher in strains engineered with inducible promoters driving the expression of the phaCAB genes. This increment in cell density correlated to a higher PHB production in these strains (Figure 2C and 2D).

[0353] Subsequently, PHB accumulation was analyzed in the engineered strains (Figure 3). When feeding glucose, the PHB content of strain SEM1.4 / pS630 PHB was significantly higher under N-limiting conditions, indicating that high C / N ratios induce polymer synthesis. Interestingly, both engineered strains carrying inducible expression systems to express the phaCAB genes also had a significantly higher PHB content when N was limited (Figure 3A). Furthermore, whereas all strains reached a similar PHB content under N-limiting conditions, under N-balanced conditions, the strains bearing inducible promoters accumulated PHB to a higher content than SEM1.4 / pS630 PHB, the strain bearing the plasmid with the native phaCAB promoter from C. necator. Taken together, these results indicate that when nitrogen (N) is not limited, expression of the phaCAB genes by the XylS / Pm (SEQ ID NO: 30) and ChnR / Pc / ine(SEQ ID NO: 5) systems could be induced in the presence of 3-mBz or cyclohexanone, respectively.

[0354] On the other hand, even though PHB content (normalized to the cell dry weight, CDW) was higher under N-limiting conditions for strains engineered with inducible promoters, the total amount of PHB produced after 24 hours showed no significant differences between balanced and limiting N conditions. This observation is partially due to the fact that the cultures could reach higher biomass values when N was not limited in the medium. In contrast, P. putida SEM1.4 / pS630 PHB produced both higher PHB content and higher total amount of PHB at 24 hours when N was limited. Taken together, these results show that strains SEM1.4 / pS638 PHB and SEM1.4 / pS6311 PHB are the best PHB producers from glucose, and that high PHB contents can be obtained when growing these strains in N-limiting conditions. N-balanced conditions allowed for the formation of more biomass than in limiting conditions, resulting in similar PHB titers for both strains under both balanced and limiting N conditions. With strain

[0355] SEM1.4 / pS638 PHB having a longer lag phase when grown with glucose, strain SEM1.4 / pS611 PHB seems to be a candidate for PHB production from this C source.

[0356] When feeding with acetate, the strains bearing inducible expression systems produced higher PHB contents than the one bearing the native phaCAB promoter from C. necator both under balanced and limiting N conditions, with strain SEM1.4 / pS611 PHB, engineered with the ChnR / Pc / inesystem (SEQ ID NO: 5), reaching the highest PHB content (Figure 3B). As it was the case in glucose cultures, PHB contents were higher for all the analyzed strains when N was limited, as compared to balanced N conditions, indicating that other targets for C / N regulation are also impacted when feeding acetate. In line with this observation, when PHB titers were studied, the inducible expression systems led to higher titers of PHB than those observed in strain SEM1.4 / pS630 PHB. Strain SEM1.4 / pS6311 PHB produced the highest amount of total PHB. In contrast to what has been observed for glucose cultures, the total PHB amounts produced after 24 hours in acetate cultures were higher under N-limiting conditions. This result is in line with the similar levels of biomass formation observed when comparing both N conditions. According to these results, the best PHB producer from acetate is strain SEMI .4 / pS631 T PHB, as it accumulated a higher PHB content (g PHB / g CDW) and titer under both N conditions (Figure 3).

[0357] In conclusion, P. putida SEM1.4 / pS6311 PHB is the best performing strain for PHB production when cultivated in shaken-flask cultures (Figure 3). In glucose cultures, this engineered P. putida strain produced 15 ± 3% (w / w) and 0.22 ± 0.04 g / L PHB at a C / N ratio = 4 (balanced N / growth conditions), whereas it produced 31 ± 7% (w / w) and 0.23 ± 0.07 g / L when cultivated at a C / N ratio = 40 (N-limiting condition). When acetate was used as the C source, the strain produced 37 ± 1% (w / w) and 0.18 ± 0.01 g / L PHB at a C / N ratio = 2 (balanced N / growth condition), and 44 ± 6% (w / w) and 0.25 ± 0.04 g / L at a C / N ratio = 20 (N-limiting condition). Based on these results, P. putida SEM1.4 / pS6311 PHB was selected as the best performing strain for further experiments and named SEM1.4-PHB henceforth.

[0358] Methods

[0359] Procedures for general cloning, plasmid construction and genomic manipulations. E. coli DH5a (used for cloning procedures) and P. putida cultures were incubated at 37°C and 30°C, respectively. For routine cloning procedures and during genome engineering manipulations, cells were grown in lysogeny broth (LB) medium (10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCI). All liquid cultures were agitated at 180 rpm in a rotary shaking incubator. Solid culture media also contained 15 g / L agar. Kanamycin (Km), or gentamicin (Gm), were added whenever needed at 50 pg / mL or 10 pg / mL, respectively. Uracil-excision (USER) cloning was used for the construction of all plasmids. The AMUSER tool was employed for the design of oligonucleotides. Phusion™ U and Phusion™ Hot Start II high-fidelity DNA polymerases (ThermoFisher Scientific) were used according to the manufacturer’s specifications for amplifications intended for USER cloning or site-directed PCR mutagenesis, respectively. For colony PCR, the commercial OneTaq™ master mix (New England BioLabs; Ipswich, MA, USA) was used according to the manufacturer’s instructions. E. coli DH5a was employed for general cloning purposes, while E. coli DH5a p / rwas employed when cloning and maintaining replicons with the conditional, fl-dependent origin of replication RK6. Chemically-competent E. coli cells were prepared and transformed with plasmids according to well established protocols. P. putida was rendered electrocom petent by washing the biomass from saturated (24 hours) LB medium cultures with 0.3 M sucrose, and cells were routinely transformed with plasmids by electroporation. The identity of all plasmids and DNA constructs was confirmed by sequencing. Deletions and insertions in the chromosome were performed by the l-Scel— mediated recombination method described in Wirth et al. (2020), with the following modification: pQURE6-H plasmid was used for expressing l-Scel as described in Volke et al. (2020). Growth curves experiments were performed in 96-well plates in a Synergy HI plate reader (BioTek Instruments; Winooski, VT, USA).

[0360] Culture conditions for PHB production. Engineered strains were grown in DBM for balanced growth / N conditions or 0.1 N DBM for N-limiting conditions [identical to DBM but containing 0.2 g / L of (NH^SCL], supplemented with 20 mM glucose or 30 mM acetate, 1 mM cyclohexanone or 3-mBz and 10 pg / mL gentamicin. PHB production experiments were performed in 500-mL Erlenmeyer flasks containing 50 mL of the corresponding culture medium.

[0361] Procedures for PHB quantification. The intracellular PHB content was determined after 24 hours of growth by GC-MS of the methanolysed polyester as previously described (Lageveen et al., 1988). Briefly, 5-8 mg of lyophilized culture pellets were suspended in 2 mL of methanol containing 15% (v / v) sulfuric acid (Sigma-Aldrich, Germany) and 2 mL of chloroform containing 0.5 mg / mL 3-mBz as internal standard (Sigma-Aldrich). The mixture was then incubated in screw-capped tubes at 100°C for 4 hours. After cooling, 1 mL of distilled water was added to the mixture to extract cell debris and any remaining sulfuric acid. Two-phase extraction was performed twice on the organic phase, and the water phase was completely removed in order to prevent fouling of the GO column. Finally, a small amount of anhydrous Na2SO4 was added to remove any water from the chloroform phase. Then, the organic phase containing the resulting methyl esters of monomers was analyzed by GC-MS. An Agilent (Waldbronn, Germany) series 7890A coupled with a 5975C MS detector (El, 70 eV) and a splitsplitless injector were used for analysis. An aliquot (1 pL) of organic phase was injected into the gas chromatograph at a split ratio of 1:50. A DB-5HTDB-5HT column (400°C, 30 m x 0.25 mm x 0.1 pm film thickness) was employed. Helium was used as carrier gas at a flow rate of 0.9 mL / min. The injector and transfer line temperature were set at 275°C and 300°C respectively. The oven temperature program was initial temperature 80°C for 2 min, then from 80°C up to 175°C at a rate of 5°C / min for efficient separation of peaks. El mass spectra were recorded in full scan mode (m / z = 40-550).

[0362] Commercial PHB (Sigma-Aldrich) was also subjected to methanolysis and used as standard.

[0363] Example 2: A method for the production of PHB from glucose or acetate by P. putida SEM1.4-PHB in bioreactors

[0364] Results

[0365] Strain SEM1.4-PHB, the engineered P. putida strain that harbors the genes encoding the PHB biosynthesis pathway under the regulation of the ChnR / Pc / ineexpression system (SEQ ID NO: 5), was selected for bioreactor fermentations based on the higher polymer (PHB) titers obtained in shaken-flask cultures. To study if this strain could support consistent PHB biosynthesis from PHA non-related substrates during process upscaling, P. putida SEM1.4-PHB was tested in 1-L bioreactor fermentations, using DBM supplemented either with glucose or acetate as sole C source under N balanced conditions. These conditions were selected to avoid growth limitations and to reach a high biomass density in the bioreactor.

[0366] For the glucose fermentations, a fed batch pulse feeding strategy was performed for PHB production. An initial concentration of 20 g / L (111 mM) glucose was supplied to the bioreactor, and then two glucose pulses were added at two different times. The first pulse of 20 g / L glucose was added after 10 hours of cultivation and a second and final pulse of 40 g / L (222 mM) glucose was added after 24 hours. The fermentation was carried on for an extra 48 hours (i.e. 72 hours in total). The first glucose pulse was consumed at a high rate, and C resources were evenly shared between biomass formation and PHB production in this first stage, allowing for growth to a cell density of 4.7 g / L CDWwith a 6% (w / w) PHB content. After the second pulse, the sharpest increase in PHB production was observed and polymer content increased up to a 19% (w / w), indicating that even though biomass formation also increased during this stage, C was preferentially channeled towards polymer synthesis. The third pulse (2-fold concentrated) was consumed at a slower rate than the previous ones and allowed for a continuous increment in biomass as well as in PHB production during this 48-h stage. The described pulse feeding strategy allowed for growth to high biomass amounts, with a final CDW concentration of 29 g / L with a PHB content of 39% (w / w), which corresponds to a PHB titer of 11 g / L after 72 hours of fermentation. For acetate fed fermentations, a fed-batch pulse feeding approach was also tried at first. An initial concentration of 30 mM acetate was added to the bioreactor and 4 further pulses of 30 mM acetate were spiked along the fermentation. The pulses were based on dissolved oxygen (DO), and 30-mM pulses of acetate were added at time points when a stirring decrease / DO increase was observed, as this is an indication that the main C substrate has been completely utilized. This strategy allowed to achieve a PHB content of 12.8% (w / w) at 6 hours, corresponding to a PHB titer of 0.017 g / L. After that time point, a reduction in the PHB content was observed, whereas the biomass density continued to increase until 2.5 g / L of CDWwas reached at 29 hours. The results obtained in this fermentation suggest that PHB accumulation is dependent on acetate concentration and when acetate is limited, it is mainly used for biomass formation. Next, as the concentration of available acetate seemed to be critical to PHB accumulation by the engineered strain, an exponential fed-batch fermentation was performed in order to increase PHB titers, securing that acetate is never limiting in the bioreactor, but that it also does not build up to a level that will become toxic to the cells. An initial concentration of 30 mM acetate was supplied to the reactor, and, after a batch phase of 8 hours, 20% (v / v) acetic acid was fed at different rates ranging from 0.05 h-1to 0.38 h-1(which was the maximum specific growth rate achieved by this strain, based on batch, shaken-flask cultures and batch bioreactor fermentations). The best results were obtained with an exponential feed rate of 0.075 h’1, as the highest PHB content of 31% (w / w) (corresponding to 1 g / L of PHB) was obtained after 20 hours of fermentation. When the fermentation was further continued, the CDW concentration increased further, whereas PHB content decreased until it reached a value of 20% (w / w) after 32 hours, resulting in a final PHB titer of 2 g L’1. After 34 hours, the fermentation was stopped as acetate began to build up in the reactor to toxic levels, impairing cell growth. Higher exponential feed rates resulted in acetate accumulation in the reactor causing growth and PHB accumulation to stop after around 20 hours.

[0367] Taken together, these results evidence that the development of strategies for acetate feeding in bioreactor fermentations that prevent the accumulation of this toxic feedstock extend the growth period and enable high PHB titers. Methods

[0368] Procedures for PHB production in bioreactor fermentations. A single colony of strain SEM1.4-PHB, previously grown on LB medium plates added with gentamicin, was inoculated into 4 mL of 2x DBM containing 0.1 % (w / v) yeast extract, 30 mM acetate and 10 pg / mL Gm. The cultures were grown at 30°C with constant shaking at 200 rpm for 8 hours. These cultures were then used to inoculate 400 mL of DBM containing 30 mM acetate and 10 pg / mL Gm in a 2-L Erlenmeyer flask. The culture was grown at 30°C with shaking at 200 rpm for 16 hours. Bioreactor experiments were performed in a reactor with a 1-L starting operating volume. DBM, supplemented with 10 pg mL-1Gm, was adopted as the growth medium, and 10% (v / v) inoculum was used in these experiments. Also, 1 mM cyclohexanone was added when the reactor was inoculated to induce expression of the pha genes. The pH was controlled at 7.0 ± 0.1 by automatic addition of 20% (v / v) NH4OH or 15% (v / v) H2SO4. NH4OH also acted as a N source. Foaming was controlled by addition of antifoam solution (polypropylene glycol P2000). Agitation was set initially at 500 rpm, air was supplied at an initial rate of 1 liter / min. Dissolved oxygen (DO) was controlled at 20% of the maximum by increasing the agitation speed. For acetate fermentations, an initial concentration of 30 mM acetate was supplied to the reactor. After a batch phase of 8 hours, 20% (v / v) acetic acid was fed at a constant rate of 0.075 IT1. After 34 hours, acetic acid began to build up in the reactor and the fermentation was stopped. For glucose fermentations, an initial concentration of 20 g / L glucose was supplied to the reactor. After 10 hours, a pulse of 20 g / L glucose was added and, after 24 hours, a further pulse of 40 g / L glucose was incorporated into the bioreactor. After 72 hours, the fermentation was stopped and the relevant parameters (e.g. PHB content) were analyzed as explained in Example 1 .

[0369] Example 3: A method for the biological production of a fluorinated scl-PHA, poly(fluoro- 3-hydroxybutryate) (F-PHB), by strain SEM1.4-PHB

[0370] Results

[0371] We tested the ability of the engineered P. putida SEMI .4 to incorporate fluorinated precursors into the PHB metabolism for the production of fluorinated polyesters, in particular fluorinated PHB, P(F-HB-co-HB) or F-PHB. To this end, F-acetate (FA) was added to the culture media as a fluorinated precursor. FA is converted to F-acetyl-CoA when entering the cells by acetyl-CoA synthases (Acs). F-acetyl-CoA results in cell toxicity as it can enter the tricarboxylic acid (TCA) cycle by condensation with oxaloacetate by means of citrate synthase (GltA). The resulting 2-fluorocitrate is further converted to 4-hydroxy-transaconitate (4-HTA) by the action of the aconitase. 4-HTA inhibits the aconitase irreversibly due to its extremely slow off-rate, blocking the TCA cycle and thus impairing growth (Lauble et al., 1996).

[0372] A cultivation strategy to reduce FA toxicity in bacterial cells was developed and consists of the addition of 2-ketoglutarate (2KG), a TCA cycle intermediate that bypasses the reaction catalyzed by the aconitase. This strategy is expected to replenish essential TCA cycle intermediates, thus reducing the toxic effects of F-acetyl- CoA misrouting (Figure 4). Hence, the culture media was supplemented with 15 mM of 2KG and growth of P. putida strains was evaluated when exposed to / fed with FA under these conditions. Growth experiments of strain SEM1.4-PHB were performed in 24-well plates in a plate reader using DBM added with glucose, acetate or citrate as main C source, with and without 15 mM 2KG and with and without FA. The addition of 2KG significantly increased growth rates and final biomass after 48 hours growth in FA- added culture medium with either glucose, acetate or citrate (Figure 4B, 4C and 4D, respectively), indicating that the addition of this TCA cycle intermediate to the medium significantly diminished FA toxicity. Hence, these results showed that 2KG addition is an efficient strategy for preventing growth impairment and cell toxicity due to FA (or any other toxic molecule that acts at the level of the TCA cycle).

[0373] In light of these results, F-PHB production was analyzed in cultures either with glucose, acetate or citrate as main C source and supplemented with FA and 2KG as indicated in Example 1. As a first screening experiment, cell growth, polymer production and F incorporation by strain SEM1.4-PHB were analyzed in glucose cultures supplemented with 15 mM 2KG. At mid exponential phase (6 h), 50 mM FA was added to these cultures to promote F incorporation in the polymer. Acetate (i.e. no F) was added in control experiments. Cells were harvested after 24 hours, and 78.73 mg of biomass (as CDW) were recovered at the end of the experiment. Polymer was extracted from the cells, yielding 3.79 mg of pure polymer, which corresponds to a polymer content of 4.8% (w / w). NMR analysis of the purified polymer showed no F incorporated in its structure, indicating that glucose is not a suitable C source for the synthesis of F-PHB. Polymer production and F incorporation was also analyzed in strain SEM1.4-PHB grown under the same conditions but using acetate or citrate as the main C source and supplemented with 15 mM 2KG. Again, at mid exponential phase (6 h), 50 mM FA or acetate was added to these cultures as precursor for fluorinated polymer synthesis or as a control, respectively. In these experiments, the bacterial biomass was harvested after 48 hours of incubation due to the slower growth of the cells in the presence of FA. Bacterial pellets were freeze-dried (lyophilized), and an aliquot of the freeze-dried cells was used for total biomass and polymer quantification as explained above. The remaining biomass was used for polymer extraction. The polymer sample was studied by 1 D and 2D NMR. Two types of19F-NMR spectroscopy were performed to analyze the F content: relative (1H-NMR A’) and absolute concentration (19F-NMR A’) determinations (Table 1). Figure 5A shows the1H-NMR of the extracted polymer, whereas Figure 5B and 5C indicate the superimposed19F-1H spectra without and with decoupling, and the 2D19F-1H HETCOR NMR spectra, respectively.

[0374] Table 1. Total biomass, PHB content and PHB titers in acetate- or citrate-fed cultures supplemented with acetate or fluoroacetate and F content in the produced polymers. N.D., not detected.

[0375] To elucidate the position of F in the polymer, the19F-1H spectra were superimposed, with and without decoupling. A signal with a defined multiplicity was consistently obtained at -231 ppm (Figure 5B), compatible with a F atom sitting at the terminal position (C4 in the fluorinated monomers), in the lateral chain of the polymer (Figure 5A). Taken together, these experiments show that the engineered SEM1.4-PHB strain, equipped with the synthetic PHB biosynthetic pathway, can use FA as a precursor to incorporate F into scl-PHAs in vivo. Moreover, addition of 2KG circumvents FA toxicity and enables its incorporation into the PHA material. Under these conditions, a copolymer containing fluorinated and non-fluorinated C4 monomers was produced.

[0376] Hence, F incorporation into the structure of PHB gives rise to a new-to-Nature biopolymer, termed F-PHB.

[0377] Methods

[0378] Procedures for determination of the F content in the F-PHB polymers. The solvent used in NMR experiments was CDCh (deuterated chloroform, chloroform-d, 99.8%) containing 0.03% (v / v) tetramethylsilane (TMS) (ref. 225789, Sigma-Aldrich). The internal standard (calibrant) used in the fluoride (19F) nuclear magnetic resonance (NMR) spectroscopy determinations was fluoroacetic acid (FA) 95% (ref. 796875, Sigma-Aldrich). The 1 D (1H,19F) and 2D COSY (correlated spectroscopy) and 2D (19F-1H) HETCOR (heteronuclear correlation) NMR spectra of the extracted polymers were recorded on a Bruker AV 500MHz spectrometer (Bruker, Rheinstetten, Germany) using a SEF 500MHz S2 5-mm probe with Z-gradient. DOSY experiments were recorded on Bruker AVANCE DMX 600MHz spectrometer using a BBO probe in CDCI3. Two types of quantitative NMR were performed to analyze the F content in PHB. Relative concentration determination, by comparing integrals of fluorine-adjacent protons, allowed for the accurate measurement of ratios of the different species, but not their actual concentrations. In these analyses, NMR samples were prepared by dissolving the sample in CDCI3. The weight of the sample was not relevant. Absolute concentration determination, by comparing the integrals of the fluorine polymers with known concentration compound as calibrant (FA), enabled quantitative determinations of the F content. In this case, samples were weighed and dissolved in a given volume of CDCI3 to determine the concentration of the polymer in the tube. Subsequently, a known concentration of the calibrant (0.35 mM) is added to the tube. Proton NMR spectra (1H-NMR) were recorded using a 90°-pulse experiment under the following acquisition parameters: 128 scans with a fixed receiver gain value of 228.1 , spectral width of 14.0019 ppm, 32768 points in the time domain, and acquisition time of 5 s. Pulse sequence zgfhigqn was used for the acquisition of19F (19F-NMR) with and without 1 H decoupling spectra. Data were collected in qsim mode with a fixed receiver gain value of 26008, 128 scans, an acquisition time of 5 s and a spectral width of 50.6057 ppm and 50.1283 ppm, respectively. COSY spectra were recorded using the standard Bruker sequence cosygpqf. Spectra were recorded under the following acquisition parameters: fixed receiver gain value of 912.3, 64 scans and a spectral width of 9.9974 x 9.9974 ppm. The standard Bruker sequence hfcoqfqn was used for acquiring the 2D 19F-1 H HETCOR NMR spectra, which were recorded with a fixed receiver gain value of 36780.8, 128 scans, and a spectral width of 37.9544 x 8.0000 ppm. DOSY spectra were recorded using the standard Bruker sequence Iedbpgp2s. All the DOSY samples were prepared in 3-mm tubes to reduce the convection and run with spinning option. The number of linear steps was set to 16. Data were collected with a receiver gain value of 812, 16 scans, a pulse duration P30 of 3 ms and a delay duration D20 of 0.3 s. The gradient recovery delay time was 0.2 ms and the eddy- current delay was 5 ms. The resulting NMR spectra were processed with the Mestrelab Mnova software (version 14.2.3-29241), and DOSY maps were generated using Mnova Bayesian transformation. Phasing and baseline correction were manually completed.

[0379] Example 4: A method for the biological production of F-PHB in bioreactors Results

[0380] The production of PHB by the engineered strain was also tested using different C sources to increase F-PHB production. Firstly, fermentations for PHB production from citrate and acetate by SEM1.4-PHB were carried out, which we called F1 experiments. In these bioreactor cultures, cells grew in an initial batch phase in a medium containing 111 mM citrate. At 7 hours, 30 mM acetate was added to the bioreactor. At 8 hours, PHB accumulation was induced by addition of 1 mM cyclohexanone and a total of 30 mM of a mixture of sodium acetate (7.06 mM), potassium acetate (17.65 mM) and 2KG (5.29 mM) was added to the bioreactor. A further 30 mM in total of this mixture (sodium acetate, potassium acetate and 2KG) was added at 9 hours, 10 hours and 11 hours. Also, 15 mM in total of a mixture containing sodium acetate (3.53 mM), potassium acetate (8.825 mM) and 2KG (2.645 mM) was added at 8.5 hours, 9.5 hours and 10.5 hours. A total of 38.83 mM sodium acetate, 127.075 mM potassium acetate and 29.095 mM 2KG was fed to the bioreactor. The fermentation was stopped after 26 hours and the biomass was harvested for analysis as explained in the examples above. A final biomass concentration of 7.1 g / L CDW, containing 25% (w / w) PHB, was obtained in these experiments. The PHB was extracted from the lyophilized biomass, and 1.5 g of PHB was recovered. This polymer did not contain F, since no fluorinated precursor was added to the cultures.

[0381] Next, fermentations for F-PHB production from citrate, acetate and FA by SEM1.4-PHB were performed in bioreactors based on the information obtained in previous experiments. Two regimes (F1 and F3) were tested towards maximizing F-PHB production. In F3 fermentations, the initial fermentation scheme (F1) was repeated replacing the sodium acetate in the pulses at 8, 8.5, 9 and 9.5 hours with an equivalent amount of sodium FA. These fermentations were stopped at 30 hours, and the biomass was harvested and processed as explained above. A total biomass of 4.4 g / L CDW containing 25% (w / w) F-PHB was obtained. The F-PHB was extracted from the lyophilized biomass, and 1 g of F-PHB was obtained. The F content in the polymer obtained in these experiments was 0.36% (mol / mol). Again, the F atom was positioned in C4 in the monomer (i.e. lateral chain of the polymer).

[0382] Methods

[0383] Fermentations were carried out in a 2-L F0 baby bioreactor (Bionet, Murcia, Spain) with a starting volume of 1 L. The culture medium contained 3.88 g / L K2HPO4, 1.63 g / L NaH2PO4, 2 g / L (NH4)2SO4, 0.1 g / L MgCI2-6H2O, 10 mg / L EDTA, 2 mg / L ZnSO4-7H2O, 1 mg / L CaCI2-2H2O, 5 mg / L FeSO4-7H2O, 0.2 mg / L Na2MoO4-2H2O, 0.2 mg / L CUSO45H2O, 0.4 mg / L CoCI2-6H2O and 1 mg / L MnCI2-2H2O. The medium was supplemented with 10 pg / mL Gm to maintain the plasmid. 2KG was added as needed; 1 mM cyclohexanone was also added to induce expression of the pha genes. The initial agitation was set at 500 rpm with air sparging at 1 liter / min. The pH was controlled at 7.0 ± 0.1 by addition of 25% (v / v) NH4OH or 15% (v / v) H2SO4. Foaming was controlled by manual addition of antifoam solution (polypropylene glycol P2000) as needed. The dissolved oxygen (DO) was controlled at 20% of maximum by increasing agitation to a maximum of 1 ,500 rpm. Once agitation had reached 1 ,500 rpm, DO was further controlled by increasing air supply to a maximum of 10 liter / min.

[0384] Example 5: Enhanced surface properties of biobased F-PHB Results

[0385] The surface properties of the fluorinated polymers obtained in the fermentations were studied using static and dynamic contact angle measurements (Figure 6). F atoms in polymers induce an increase in hydrophobicity and a decrease in surface energy (Ebnesajjad, 2013). Therefore, we measured the water contact angles (WCA) on the polymer films with different contents of fluorinated units. For WCA and surface energy measurements, polymer films were cast over a glass plate from a chloroform solution and allowed to stand for a week at room temperature. Figure 6A shows the water droplet placed over the glass surface coated with a PHB sample without any fluorination (i.e. the polymer purified from cells of fermentation F1) and with fluorination (i.e. the polymers purified from cells of fermentation F3), which allowed us to measure and quantify the static contact angle values. Non-fluorinated PHB showed a

[0386] WCA = 70°, whereas F-PHB purified from F3 had a WCA = 124°. Hence, a significant increase in the water contact angle value was observed in the F-PHB samples despite the low fluorination degree in the material. Table 2 and Figure 6B show the dynamic measurements and the surface energy values of the fluorinated and non-fluorinated PHB samples. The surface energy decreased with the introduction of F atoms in the chain, which correlates with the increase in the hydrophobicity of the samples.

[0387] Table 2. Surface energy values of fluorinated polymers purified biomass samples obtained in fermentations F1 and F3.

[0388] Methods

[0389] Contact angle (CA) measurements. This analysis was carried out to calculate the surface energy of the polymer samples using three different solvents: water, glycerol and diiodomethane. Dynamic CA (0) measurements were taken at five representative regions of each sample using a high-speed camera. A 60-Hz sampling rate was used to capture advancing and receding water Cas. Flow rates of 5 nL / s were used to dispense liquids through a 35-pm gauge needle (135-pm OD) with droplet volumes ranging from 40 to 80 nL.

[0390] Example 6: Construction and characterization of an acetate-tolerant P. putida strain (evoSEM1.4) for the production of scl-PH As from acetate and haloacetate

[0391] Results

[0392] Sustainable production of biodegradable polymers continues to be challenging, especially because the production costs are relatively high in comparison to polymers derived from fossil fuels. Biodegradable polymers can replace petrochemicals offering a greener solution to the global plastic problem. One strategy towards reducing operation costs is to implement low-cost carbon sources, such as acetate, which can be further converted into several biopolymers widely used in different industries (e.g. PHB, among others). Although acetate itself offers an economic alternative for the development of a competitive bioprocess, it generates a detrimental effect in bacterial metabolism. High titers as well as volumetric productivity can be boosted by improving microbial tolerance to this organic acid. The development of these strategies will also enable the optimized use of fluoroacetate as F-precursor in the engineered bacterial strains.

[0393] In order to obtain a P. putida mutant highly tolerant to acetate, strains of the non- pathogenic bacterium P. putida were subjected to an adaptive laboratory evolution (ALE) process. The evolved P. putida strain was termed evoSEM1.4 and / or SEM1.4evo. The genome of the evolved strain was analyzed by whole genome sequencing (WGS) methods, yielding a set of genes affected by point mutations that were responsible for the improved acetate tolerance. The list of point mutations (and cognate amino acid substitutions) and genes identified are: fabBL77P(SEQ ID NO: 70), uvrY7K(SEQ ID NO: 10), hupB749' (SEQ ID NO: 72) and / ?upBA35V(SEQ ID NO: 71). The single mutations, as well as the double mutation with amino acid changes in the genes fabB and uvrY, were introduced in the parental host and the reverse-engineered strains were tested against different acetate concentrations ranging from 20 up to 140 mM. The double P. putida mutant (FabBL77P+ UvrYE197K) was able to grow in the presence of 140 mM acetate, while the parental strain had no growth under the same conditions (Figure 7). The combination and synergistic effect of both amino acid changes (FabBL77Pand UvrYE197K) contribute to enhanced acetate resistance.

[0394] In the genus Pseudomonas, the UvrY (i.e. GacA) / GacS two-component system regulates the synthesis of extracellular proteases and hydrogen cyanide biosynthesis and has a role in the regulation of innate PHA synthesis (Mezzina et al., 2021). fabB encodes the 3-oxoacyl-[acyl-carrier-protein (AGP)] synthase I (i.e. FabB), which belongs to a well-studied class of enzymes that catalyze the elongation reaction of fatty acid synthesis (Winsor et al., 2016).

[0395] Finally, HupB encodes a DNA-binding protein belonging to bacterial histone-like protein HU family. The HU protein is found to bind DNA, not in a sequence-specific manner, increasing the flexibility of the bound DNA and thus facilitating the formation of bent structures (Perez-Martin and de Lorenzo, 1997). We generated the triple mutant FabBL77Pand UvrYE197KHupBA35Vthat was subjected to a physiological analysis in DBM containing 100 mM potassium acetate. The results are shown in Figure 8. The mutant retained acetate tolerance at the same levels as the evolved (SEM1.4evo) strain, indicating that these three point mutations are largely responsible for the acetate tolerance phenotype obtained during evolution.

[0396] Proteome analysis under high acetate concentrations in evolved and double mutant strains

[0397] Note: From now on, UvrY will be noted as GacA, and these two terms will be used interchangeably.

[0398] P. putida SEMI .4, evoSEM1.4 and GacAE197KFabBL77Pwere cultivated in the presence of 100 mM of potassium acetate and harvested in mid-exponential phase (ODeoo~1.0). As a first step, protein changes, Iog2(fold-changes) were analyzed by LC-MS / MS. For data visualization, three volcano plots were created to help identify proteins that were expressed differently by plotting on the X-axis the Iog2(fold-change) of proteins against their statistical significance on the y-axis [represented by the negative log (p-value)] (Figure 9A, 9B and 9C).

[0399] There were 23 up-regulated and 259 down-regulated proteins when the SEM1.4evowas compared to the wild-type. Similarly, when comparing the double mutant to the wild type, 29 proteins were up-regulated and 198 were down-regulated. Lastly, only 9 proteins were significantly up regulated and 3 were down regulated when comparing evoSEM1.4 to the double mutant under high acetate concentration growth conditions. In general, the evoSEM1.4 strain and double mutant exhibited comparable volcano plots, suggesting that the mutations that were previously incorporated significantly rewired the cellular proteome. Subsequently, based on the proteomics analysis, we investigated which protein groups or pathways were impacted by the presence of elevated acetate concentrations. We observed substantial alterations in methionine metabolism of both the evoSEM1.4 and double mutant strains. Methionine transport system ATP-binding protein (MetN and Metl) and sulfate permeate (CysZ) were significantly upregulated by a Iog2(fold-change) of 8.5 and 7.3, respectively. Also, related to sulfur metabolism, alkanesulfonate monooxygenase (SsuD), the organosulfonate utilization protein (SsuF) together with thiosulfate sulfurtransferase (GIpE) were significantly downregulated with a Iog2(fold-change) of -9. In addition, the enzyme that convert O-acetylhomoserine and sulfide into L-homocysteine, O- acetylhomoserine sulfhydrylase (MetY) was downregulated. We also noticed an increase in the relative amount of SucC (succinyl-CoA ligase [ADP-forming] subunit beta) and SucD (succinyl-CoA ligase [ADP-forming] subunit alpha). The findings indicate that P. putida might utilize this system in the TCA cycle as an additional mechanism to detoxify the acetate within the cell.

[0400] Acetate toxicity exposes rewiring of metabolic fluxes in the EDEMP cycle to counteract oxidative stress

[0401] High levels of acetate induce oxidative stress in several microorganisms. In order to capture the metabolic effects of acetate, toxicity in P. putida strain used in this study, we carried out a13C fluxome analysis where the cells were exposed to high acetate concentration in DBM. We implemented a parallel labelling strategy (1 -13C1 ]-, [2-13C1]- and [50% U-13C6 ]-acetate) to resolve the relative contribution of TCA cycle, EDEMP, PP, and ED pathway. Samples were collected at mid-exponential phase to assess proteinogenic amino acids and sugar monomers (glucose and glucosamine). The calculated relative fluxes for the three strains cultured on labeled acetate are shown in Figure 10.

[0402] Methods

[0403] Bacterial strains and culture conditions. E. coli and P. putida KT2440 strains were employed in this study. The strains were incubated at 37°C and 30°C, respectively. To perform cell propagation and storage as well as routine cloning and genome engineering manipulations, cells were grown in lysogeny broth (LB) medium (10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCI). Liquid pre-cultures were done in 50-mL Falcon® centrifuge tubes with a medium volume of 10 mL or; for cultures, 250-mL Erlenmeyer flask containing 50 mL of medium. All liquid pre-cultures were agitated at 250 rpm (MaxQ™8000 incubator; ThermoFisher Scientific, Waltham, MA, USA) while cultures were incubated at 200 rpm (New Brunswick™ Innova® 42 / 42R Shaker, sticky pad). Solid culture media contained an additional 15 g / L agar. Selection of plasmidharboring cells was achieved by adding kanamycin (Km) or gentamicin (Gm), when required at 50 pg / mL and 10 pg / mL, respectively. For adaptive laboratory evolution (ALE) experiments, clone screening, phenotypic characterizations as well as targeted metabolomics and proteomics analyses in shaken flasks, the experiments were performed in de Bont minimal medium (DBM) additionally buffered with 5 g / L 3-( / V-morpholino)propane sulfonic acid (MOPS) at pH 7.0 and supplemented with different concentrations of potassium acetate. To inoculate the cultures, the aliquot taken from the pre-cultures were harvested by centrifugation at 8,000 g for 5 min, washed with DBM without the addition of any carbon source, and resuspended in the final media used in the experiment at the desired starting optical density at 600 nm (ODeoo).

[0404] Adaptive Laboratory Evolution (ALE) in acetate. Pseudomonas putida SEMI .4 was evolved in 250-mL shaken baffled flask containing increasing concentrations of potassium acetate pH 7.0. The strain was streaked out onto a LB plate from the glycerol stock and it was incubated overnight. Several colonies were selected, pooled and inoculated in a 250-mL Erlenmeyer baffled flask containing 50 mL of minimal medium with an initial potassium acetate concentration of 20 mM. Cultures were kept well mixed and aerated at 200 rpm at 30°C in. When the culture reached stationary phase, 500 pL were passed into the next 250-mL Erlenmeyer baffled flask with fresh media. Three consecutive passages were performed within the same concentration prior the inoculation of the following Erlenmeyer with higher concentration. Concentrations in the flask were gradually increased a lag phase of 12 hours was observed.

[0405] Clone screening from populations and candidate evaluation. For done screening, the cultures from the last Erlenmeyer was streaked out onto selective condition in minimal medium de Bont containing 180 mM of potassium acetate. Ten clones with different colony sizes were selected with a sterile toothpick and, were individually inoculated in the same selected condition as mentioned above. The clones were stored at -80°C in 2-mL cryotubes with cryopreservation solution until further characterization. The cryopreservation solution consists of 50% v / v glycerol (of the highest purity available), 0.1 M MgSCL, and 50 mM Tris HCI (pH = 8.0). The selected clones were evaluated in Growth Profiler 960 (System Duetz, Enzyscreen, Heemstede, The Netherlands) using 96-squared well microtiter plates sealed with a gas permeable sandwich cover (Enzyscreen, Heemstede, The Netherlands). Briefly, 300 pL of de Bont minimal medium with several concentrations of potassium acetate (20, 60, 100 and 140 mM) were inoculated with an initial ODeoo of 0.1 . Bacterial growth was followed every 15 minutes by optimal image scanning through the Growth Profiler 960. G-values (integrated green values) taken from each scan and well were transformed into equivalent ODeoo values to determine microbial growth. This was determined by means of calibration values fitted to a Monod function: ODgnn

[0406] G — value - (Equation 1) 0D6004with a and b parameters determined by non-linear regression, where the optical density of a culture at different dilutions was measured a priori in a UV-1600PC spectrophotometer (VWR, Radnor, USA) and the corresponding images of the culture were taken in the Growth Profiler 960.

[0407] Whole genome sequencing and mutational analysis. Genomic DNA (gDNA) was extracted from the cultures using PureLink® Genomic DNA Mini Kit. Sequencing libraries were prepared using Plexwell, and the sequencing was performed on Nextseq500 using Illumina Nextseq mid output kit, 300 cycles. The sequencing files were analyzed using an in-house script based on bowties2, using the reference genome with GenBank accession number AE015451. The average coverage for the clonal and the population samples was approximately 60x. For population samples, mutations with frequencies less than 0.50 were excluded from the analysis in order to filter out artifacts in the mutation callings and focus on causal mutations.

[0408] Reverse engineering of relevant mutations into Pseudomonas putida SEMI.4, USER cloning and plasmid construction. In order to re-introduce the mutations obtained from ALE experiments, plasmids were constructed for homologous recombination mediate gene replacement. USER was used for the construction of all plasmids. In order to build the plasmids for gene substitution by homology recombination, USER primers were designed with AMUSER, containing the targeted single-nucleotide mutations. Phusion™ U high-fidelity DNA polymerase (ThermoFisher Scientific) was used for PCR amplification, mutagenesis, and USER adaptors construction, in a one-step reaction. PCR products were then run on 1% (w / v) agarose gel for confirmation and purified with NucleoSpin™ gel purification kit (Macherey- Nagel, Germany). The purified amplicons were ligated into pSNW2 vector with USER enzyme (New England BioLabs, Ipswich, MA, USA), following manufacturer’s specifications, and transformed into Escherichia coli by heat shock for cryostock storage. To perform gene substitution by homology recombination, l-Sce / -mediated recombination engineering strategy (Martinez-Garcia and de Lorenzo, 2011 ; Wirth et al., 2020) was followed, with the addition of inducible self-curing vectors (Volke et al., 2020). Briefly, the protocol of Wirth et al. (2020) was implemented for genomic integration and confirmed by green fluorescence (i.e. GFP) of individual colonies and colony PCR of selected amplicons. Positive colonies were grown in LB medium. Cultures were then washed with 0.3 M sucrose and transformed by electroporation with the self-curing plasmid (i.e. pQURE6 H). Cells were recovered overnight in LB medium containing 5 mM 3-mBz, and then streaked onto LB medium agar with 10 pg / mL Gm and incubated overnight. Correct gene substitutions were confirmed by red fluorescence (i.e. mRFP) of individual colonies, colony PCR of selected amplicons and sequencing. Single colonies were then inoculated and grown in LB, for plasmid curing. Eventually, the cultures were streaked onto solid media and non-fluorescent colonies were selected: the loss of the plasmid was confirmed by colony PCR, parallel plating in LB and Gm, and DNA sequencing. To store the positive mutants, single colonies were grown in LB and preserved as cryostocks.

[0409] Phenotypic characterization of reverse-engineered strains. The evolved, double mutant P. putida SEMI .4 uvrYE'i97KfabB]~77Pand reduced genome SEMI .4 strains were phenotypically characterized in 250-mL shaking baffled flask with 50 mL of DBM containing 100 mM of potassium acetate supplemented and 5 g / L MOPS. Optical density at 600 (ODeoo) and potassium acetate consumption (mmol gcow'1h’1) were determined. The shaking flasks were inoculated at an initial ODeoo of 0.1 and incubated at 30°C with an agitation of 200 rpm. When appropriated, samples were collected along the cultures; the supernatants were collected by centrifugation (10,000 g for 5 minutes at 4°C) and storage at -20°C for HPLC analysis. Cell dry weight (CDW) was determined by growing the cells in the same conditions as is mentioned before. At different cultivation times, six samples (two per replicate) of 10 mL each were collected per strain in pre-weighted 15-mL Falcon® tubes. Optical density at 600nm was measured and the collected samples were centrifuged at 8,000 g for 10 minutes at 4°C, washed twice with de Bont minimal medium without carbon sources. The tubes containing the biomass were frozen at -20°C and lyophilized under vacuum until constant weight.

[0410] Metabolite analyses via HPLC

[0411] Potassium acetate consumption was analyzed using a Dionex Ultimate 3000 HPLC with an Aminex® HPX-87X Ion Exclusion (300 x 7.8 mm) column (BioRad, Hercules, CA) as well as RI-150 refractive index and UV (260, 277, 304 and 210 nm) detectors. For analysis, the column was maintained at 45°C and a 5 mM H2SO4 solution was used as mobile phase at a flowrate of 0.6 mL / min. HPLC data were processed using the Chromeleon 7.1.3 software (Thermo Fisher Scientific), and compound concentrations were calculated from peak areas using calibration curves with six different standard concentrations covering the range from 0 to 250 mM.

[0412] Semi-quantitative proteomics by mass spectrometry (MS)

[0413] P. putida strains were pre-cultured overnight in DBM supplemented with 20 mM potassium acetate. Experimental cultures were done in flasks containing de Bont minimal medium with 100 mM potassium acetate inoculated at an initial ODeoo of 0.1. Samples were taken in the mid-log phase, and the cells were harvested by centrifugation at 10,000 g for 5 minutes at 4°C. Then, the supernatant was removed and the cell pellet was frozen at -80°C until the processing of samples. Samples were desalted and isolated using a variation of a previously-described chloroform / methanol extraction protocol (Flugge and Wessel, 1984). Cell pellets were first thawed at 4°C and resuspended in 100 pL HPLC water. Then, 400 pL of HPLC grade methanol, 100 pL of HPLC grade chloroform, 300 pL of HPLC grade water were added to each sample in sequential order with thorough vortexing after each addition. Later, samples were centrifuged for 60 seconds at 21 ,000 g to promote phase separation. The top layer (water and methanol) was discarded by centrifugation leaving on the protein pellet the chloroform layer remaining. Additional 300 pL of HPLC grade methanol was added, the samples were vortexed and centrifuged for 2 minutes at ~21 ,000 g. The remaining liquid was discarded and the cell pellets were dried in a fume hood for 5 minutes. Then, protein pellets were resuspended in freshly-prepared 100 mM ammonium bicarbonate buffer in HPLC water containing 20% (v / v) methanol HPLC grade. Protein concentrations in the resuspended samples were quantified using a DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA). In total, 100 pg of protein was transferred to a PCR strip and tris(2-carboxyethyl)phosphine (TCEP) was added until a final concentration of 5 mM. Samples were incubated at 22°C for 30 minutes and; subsequently, iodoacetamide (IAA) was added until a final concentration of 10 mM. Again, samples were incubated at 22°C in the dark for 30 minutes. Finally, trypsin was added to a final ratio of 1:25 (w / w) trypsimsample, and samples were incubated at 37°C for 5-8 hours in order to digest the proteins prior being placed into a conical LC vials for LC-MS analysis. For shotgun proteomic experiments, peptides were analyzed by using an Agilent 6550 iFunnel Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA) coupled to an Agilent 1290 LIHPLC system as described previously (Gonzalez Fernandez-Nino et al., 2015). 20 pg of peptides were separated on a Sigma-Aldrich Ascentis Peptides ES-C18 column (2.1 mm * 100 mm, 2.7 pm particle size, operated at 60°C) at a 0.4 mL / min flow rate and eluted using following gradient: 98% solvent A (0.1% (v / v) formic acid) and 2% solvent B [99.9% (v / v) acetonitrile, 0.1% (v / v) formic acid]. Solvent B was increased to 35% over 30 minutes, and then increased to 80% over 2 minutes, then was constant for 6 minutes, followed by a ramp back down to 2% Solvent B over 1 minute where it was kept constant for 4 minutes in order to re-equilibrate the column to original conditions. Peptides were introduced to the mass spectrometer from the LC by using a Jet Stream source (Agilent Technologies) operating in positive-ion mode (3500 V). Source parameters employed gas temp (250 C), drying gas (14 liter / min), nebulizer (35 psig), sheath gas temp (250°C), sheath gas flow (11 liter / min), Vcap (3500 V), fragmentor (180 V), OCT 1 RF Vpp (750 V). Agilent MassHunter Workstation Software was used to acquire the data, LC / MS Data Acquisition B.06.01 operating in Auto MS / MS mode whereby the 20 most intense ions (charge states, 2-5) within 300-1400 m / z mass range above a threshold of 1500 counts were selected for MS / MS analysis. The quadrupole was set to “Medium” resolution in order to collect MS / MS spectra (100-1700 m / z) and were acquired until 45,000 total counts were collected or for a maximum accumulation time of 333 ms. Former parent ions were excluded for 0.1 minute following MS / MS acquisition. The acquired data were exported as .mgf files and searched against the latest Pseudomonas putida KT2440 protein database with Mascot search engine version 2.3.02 (Matrix Science). The resulting search results were filtered and analyzed by Scaffold v 4.3.0 (Proteome Software Inc.).

[0414] Statistical analysis of proteomics data

[0415] The data from proteomics was examined using a customized R script in Rstudio (version 1.3.1093), which was developed as part of the thesis. Only proteins that were detected in all samples were considered. The abundance values were normalized using quantile normalization through the R-package qsmooth. A Student’s t-test was then conducted using the built-in dt() function. The data was Iog2-transformed for foldchange comparison between groups, and volcano plots were generated using VolcaNoseR online tool. Proteins that showed a Iog2-fold change of 1.0 in abundance and a -Iog10(ad / . p-value) more than or equal to 1 .0 were considered to be differentially expressed.

[0416] 13C metabolic flux analysis using labeled acetate

[0417] Pre-cultures were initially inoculated with a loop of freshly plated evolved, double mutant P. putida SEMI .4 with the uvrYE'i97KfabB]~77Pmutations and reduced genome SEMI .4 strains in de Bont minimal medium supplemented with 20 mM non-labeled potassium acetate and 5 g / L MOPS. Prior inoculation into the experimental culture, cells were centrifuged at 10,000 g for 5 minutes and washed twice using de Bont minimal medium without carbon source. Then, cells were separately inoculated with an initial ODeoo of 0.02 in 100 mL shaken flask containing 20 mL of de Bont medium with 100 mM of the following labeled carbon sources: 99% [1—13C] sodium acetate or [2-13C] sodium acetate or a molar 1 :1 mixture of [U-13C2] sodium acetate. The inoculum level of the cultures with13C-labeled tracer was kept below 1% (initial OD of <0.02) of the final sampled cell concentration. This was done to exclude potential interference of non-labeled inoculum with subsequent calculations of flux. All experiments were performed in two biological replicates and two technical replicates, and samples were harvested once the cultures reached ODeoo~2.0 for the analysis of either proteinogenic amino acids or cellular sugars. The samples were centrifuged at 10,000 g for 5 minutes, the supernatant was removed and the pellet was frozen at -80°C until further processing.

[0418] GC-MS labeling analysis of proteinogenic amino acids

[0419] Pellets were thawed on ice at 4°C and re-suspended in 200 pL of 6 M HCI and incubated at 105°C for 16-24 hours in order to hydrolyze the biomass. Hydrolyzed samples were then filtered on a 96-filter plate (MultiScreenHTS, HV Filter Plate 0.45 pm, hydrophilic, clear, non-sterile, Millipore, Catalogue number MSHVN45) by centrifugation at 1 ,500 g for 2 minutes and were freeze dried and stored at -80°C. Then, dried samples were derivatized in a two-step reaction: 1) by re-suspending the hydrolysate with 50 pL dimethylformamide (DMF) until it turns brownish and 2) by adding the previous solution into a glass vial with 50 pL of / V-tertbutyldimethylsilyl-A / - methyltrifluoroacetamide with 1% (wt / wt) tertbutyldimethyl-chlorosilane (TBDMSTFA) and incubating it at 85°C for 1 hour. The derivatized samples were aliquoted in glass vials with inlets for GC-MS analysis within 12 hours from derivatization. The samples were injected on a Agilent 7890A GC-MS system with an Agilent HP-5ms capillary column (30m, inner diameter of 0.25 mm, film thickness of 0.25 pm). Samples were measured in full-scan mode with the following gradient: start at 120°C, hold for 1 minute (Initial step); ramp to 160°C at 4°C / min, hold for 5 min (Ramp 1); ramp to 270°C at 4°C / min, hold for 3 min (Ramp 2); ramp to 310°C at 20°C / min (Ramp 3), hold for 1 minute and; ramp to 120°C at 60°C / min (Ramp 4).

[0420] GC-MS labeling analysis of sugar monomers

[0421] For cellular monomers analysis (glucose and glucosamine) pellets were hydrolyzed in 250 pL of 2 M HCI for 2 hours at 100°C. Next, cell debris was removed by filtration using a 96-filter plate (MultiScreenHTS, HV Filter Plate 0.45 pm, hydrophilic, clear, non-sterile, Millipore, Catalogue number MSHVN45). Subsequently, the hydrolysate were freeze dried and stored at -80°C. Analytes contained in the dried residue were incubated in 100 pL methoxylamine 2% (m / v) in pyridine at 80 °C for 1 hour. The obtained O-methyl oxime forms of the analytes were silylated at 80 °C for 30 minutes into trimethylsilyl (TMS) derivatives in a second step using N,O-bis-trimethylsilyl- trifluoroacetamide (BSTFA, Macherey-Nagel). The derivatized analytes were quantified by GC-MS using an Agilent 7890A GC-MS system with an Agilent HP-5ms capillary column (30m, inner diameter of 0.25 mm, film thickness of 0.25 pm). Samples were measured in full-scan mode with the following gradient: start at 120°C, hold for 2 minutes (Initial step); ramp to 280°C at 10°C / min, hold for 12 min (Ramp 1) and; ramp to 300°C at 10°C / min, hold for 0 minute (Ramp 2).

[0422] GC-MS chromatographic data analysis

[0423] Raw chromatographic data was integrated using SmartPeak (Kutuzova S. et al., 2020). Processed data was further corrected for the natural abundance of isotopes in the derivatization agents used for GC-MS analysis using INCA software (Young, 2014). Altogether, 14 amino acids yielded ion clusters with clean mass isotopomer distributions (MIDs), which were considered as inputs for flux estimation: alanine (m / z 232, m / z 260), aspartate (m / z 302, m / z 390, m / z 418), glutamate (m / z 330, m / z 432), glycine (m / z 218, m / z 246), histidine (m / z 338, m / z 440), isoleucine (m / z 274), leucine (m / z 274), lysine (m / z 329, m / z 431), methionine (m / z 218, m / z 320), phenylalanine (m / z 302, m / z 308, m / z 336), proline (m / z 258), serine (m / z 362, m / z 390), threonine (m / z 376, m / z 404), tyrosine (m / z 302) and valine (m / z 260, m / z 288). These fragments have been previously demonstrated to be suitable for13C metabolic flux analysis in Pseudomonas spp. (Kohlstedt and Wittmann, 2019). Glutamate and aspartate also reflected the pools of glutamine and asparagine, which underwent deamination during protein hydrolysis. The proteinogenic amino acids cysteine and tryptophan were not detected due to their degradation in the hydrolysis process. For sugar analysis, the fragments considered for13C-MFA were: glucose (m / z 319, m / z 554) and glucosamine (m / z 319, m / z 553) as previously reported (Kohlstedt and Wittmann, 2019).

[0424] Reaction network and computational design for flux estimation

[0425] The metabolic networks of P. putida SEMI.4 strains were built based on the most recent genome-scale metabolic model (Nogales et al., 2020). In total, 72 reactions were included as part of the central carbon metabolism. The INCA software package was utilized for13C-metabolic flux analysis (13C-MFA) (Young, 2014). Specific growth rates, and uptake or secretion rates for acetate were used to constrain the MFA model. The biomass equation was taken from (Czajka et al., 2022). To estimate the relative intracellular fluxes, a weighted sum-of-squared-residuals (SSR) was minimized through iterative analysis of simulated and experimental labeling data for various molecules, including proteinogenic amino acids, extracellular sugars and sugar acids, glycogen, and glucosamine. To ensure the most accurate results, the flux estimation was repeated at least 20 times with random initial values until convergence. To assess the goodness-of-fit, a x2test was applied, and precise 95% confidence intervals were calculated by determining the sensitivity of the sum of squared residuals to flux parameter variations. The resulting flux results, including best fits, standard deviations, and upper and lower bounds of the 95% confidence intervals for all fluxes were computed, and used to visualize the flux distributions of different strains, computed flux values were mapped onto custom metabolic maps using the R package fluctuator.

[0426] Alphafold prediction and structure characterization

[0427] The 3D structure prediction of GacA were created with AlphaFold 2.0 with default settings (Jumper et al., 2021). The tertiary structure fold of the 5 highest ranking predictions were analysed and validated with Local Distance Difference Test (IDDT), SPServer and ProSa-Web. Visualization, superimposition analysis and mutagenesis were performed with PyMOL v.2.3.4 (PyMOL Molecular Graphics System, Version

[0428] 2.3.4 Schrodinger, LLC.). Original sequences were obtained from Pseudomonas Database .

[0429] Example 7: Engineering a PHB biosynthetic pathway in the acetate-tolerant strain, and biological production of scl-PH As and halogenated scl-PHA by strain evoSEM1.4-PHB

[0430] Results

[0431] The evolved bacterial host cells were also engineered to produce scl-PHAs from acetate or acetate derivatives by the implementation of the inducible system borne by plasmid pS6311 PHB, comprising the PHB biosynthetic pathway previously used in strain SEMI.4 for scl-PHA production. PHB was produced with this engineered strain in a 1-L bioreactor to demonstrate the potential of the acetate-to-scl-PHAs P. putida platform. The cells were grown in 4.72 g / L acetate (i.e. 80 mM) at a C / N ratio = 50. Cultures were developed for 18 hours in batch mode, at which point the 45% DO trigger initiated an exponential feeding. Afterwards, P. putida evoSEM1.4-PHB growth in exponential fed-batch mode was monitored for 48 hours. PHB titers reached its maximum of 33.5 g / L at 48h with a productivity of 0.698 g PHB L'1h-1(Figure 11). These results set a record as the highest PHB titer ever obtained for the genus Pseudomonas, for an engineered strain, and from acetate as the carbon substrate.

[0432] In addition, when evoSEM1.4-PHB was fed with 50 mM fluoroacetate, it produced 3 g / L F-PHB with a F content in the range of 0.3-1.3% (mol / mol). The developed evolved strain can be used for the optimized production of fluorinated scl-PHAs, in particular F- PHB, using FA as fluorinated precursor.

[0433] Methods

[0434] Procedures for PHB production in evoSEM1.4-PHB in bioreactor

[0435] The bacterial cells were precultured directly from cryostock into 50 mL DBM with 20 mM acetate in 500 mL baffled flasks and incubated at 30 °C overnight. The reactor was autoclaved and initially filled with 400 mL DBM containing 80 mM acetate, while 10 pg / mL Gm, 1 mM cyclohexanone were added afterwards to avoid heat degradation. The set up was inoculated with P. putida SEM 1.4 at a final GD600 of 0.1 , and run in batch fermentation mode, after which an exponential feeding strategy was applied. The latter was initiated by a dissolved oxygen (DO) trigger: when DO decreased below 45 % during batch mode, the feed medium was started to be added to the fermentation vessel by a peristaltic pump using an exponential feeding policy, with pmax = 0.065 h’1, extrapolated from preliminary studies. The feed solution was prepared following the RSM optimization, concentrated at 3.4 M potassium acetate with C / N ratio of 50. The STBR working volume was 0.8 L. All the pumps and probes were controlled by Lucullus® (Securecell, Switzerland) through a control tower (Biostat Q plus, Sartorius). The temperature was kept constant at 30 °C by a water-heated jacket and monitored by a temperature probe. The pH of the culture medium was controlled by a pH probe (EasyFerm plus PHIK8160, Hamilton) and it was maintained at 7.0 ± 0.05 through the controlled addition of 7 M sodium hydroxide (NaOH) and 85% (w / v) phosphoric acid (H3PO4). The DO was controlled by a multiple controlled cascade. Stirring was provided by two, six-blade Rushton turbines at speeds ranging from 500 to 1000 rpm. Sterile air was supplied through a ring-sparger at 1 vvm and the DO concentration was monitored by a DO probe (VisiFerm DO160, Hamilton) and it was programmed to be maintained above 40%. Samples for CDW, acetate, and PHB determinations were taken at time points from the dedicated outlet. CDW was quantified with a halogen moisture analyzer (HC103, Mettler Toledo).

[0436] For HPLC quantification of acetate concentration, samples were thawed at room temperature and 300 pL of supernatant was filtered in a 0.20 pm 96-well plate. The samples were then injected in a Dionex Ultimate 3000 system (Thermo Scientific, USA) with a HPx87H ion exclusion column (125-0140, Aminex, Ireland), equipped with a guard column (125-0129, Bio-Rad, Hercules, USA) and guard column holder (125- 0131 , Bio-Rad, USA). The samples were eluted with 5 mM H2SO4 at an isocratic flow of 0.6 ml / min at 30°C for 45 minutes. Acetate was analyzed by Rl detection using a Smartline Rl detector 2300 (KNAUER Wissenschaftliche Gerate, Germany). PHB was quantified as described in Example 1.

[0437] Example 8: Engineering fluoro-3-hydroxybutyrate biosynthesis from F-acetate

[0438] In vivo biosynthesis of fluorinated analogues of 3-hydroxybutyrate (3HB), which can be used as a monomer for the synthesis of fluorinated analogues of PHB and other copolyesters, was explored in engineered bacteria using a synthetic pathway (Figure 12A). Two different modules encoding the enzymes needed for the first metabolic step (condensation of F-acetyl-CoA to F-acetoacetyl-CoA) were tested: either (i) a single condensation reaction, catalyzed by PhaA, or (ii) the two-step route that proceeds via malonyl-CoA, catalyzed by the native Acc (acetyl-CoA carboxylase) complex and the heterologous NphT7 enzyme. The subsequent NADPH-dependent reduction of F- acetoacetyl-CoA to F-3-hydroxybutyryl-CoA is catalyzed by PhaB from C. necator (SEQ ID NO: 2), followed by CoA release by the TesB thioesterase. The biosynthetic pathway used in this study encoded two different tesB variants, from either E. coli (Ec_tesB, SEQ ID NO: 41) or P. putida (Pp_tesB, SEQ ID NO: 40). By combining phaB with the two thioesterase variants (Ec_tesB termed Ec_B, Pp_tesB termed Pp_B) and genes for either of the two metabolic routes for acetoacetyl-CoA formation (phaA = AB, nphT7 = TB) we generated four plasmids using the pSEVA6311 vector as backbone for pathway expression under the regulated and cyclohexanone-inducible ChnR / Pc / »iB expression system (SEQ ID NO: 5) (Figure 12B). The resulting plasmids, termed pSEVA6311::Ec_BAB, Ec_BTB, Pp_BAB, and Pp_BTB, were individually transformed into an E. coli strain previously constructed and characterized for growth in the presence of up to 150 mM F-acetate. Furthermore, an empty plasmid pSEVA6311 was transformed into the same background, and the resulting strain was used as a negative control.

[0439] Production of 3HB and its fluorinated analogues was tested by cultivating the engineered strains in M9 minimal medium supplemented with 10 mM glucose and 4 mM 2KG to reduce F-acetate toxicity. In particular, 2KG was added to circumvent the inhibition of fluorometabolites on aconitase (Acn), thereby maintaining the TCA cycle active. Additionally, acetate and F-acetate were added at three different ratios to the culture for the production of 3HB and F-3HB. Also, 10 mM of acetate was fed together with 0, 10 and 20 mM of F-acetate for three different ratios of substrates (H / F = 10:0; 10:10; and 10:20) towards producing fluorinated 3HB. Supernatants of exponentially growing cultures were collected for analysis with High Performance Liquid Chromatography (HPLC) and LC-MS / MS.

[0440] HPLC chromatograms revealed production of several organic acids in all conditions, among which we could identify lactate and 3HB using commercially available standards-except for the negative control which did not show any peak corresponding to 3HB. These results indicate that the pathway assembled for 3HB synthesis is active under the conditions tested. In cultures with acetate supplementation, the engineered strains harboring the BAB module showed the highest 3HB accumulation in the supernatant, reaching titers of 4.4 and 3.0 mM for the E. coli (Ec_BAB) and P. putida (Pp_BAB) variants, respectively (Figure 12C). Similar peaks were identified for the F- acetate condition (where we expect formation of fluorinated 3HB), and we resorted to LC-MS / MS for identification of the fluorinated acid by matching the mass-to-charge (m / z) values of the peak corresponding to 3HB. We could confirm the presence of a compound with a chemical formula matching F-3HB. Furthermore, fragmentation data in MS2 suggest that the F atom to be in position 4, consistent with the formula 4-fluoro- 3-hydroxybutyrate. We note that the pathway could also give rise to 2-fluoro-3- hydroxy butyrate or 2,4-difluoro-3-hydroxybutyrate.

[0441] Example 9: Characterisation of point mutations in KT2440

[0442] Results

[0443] To confirm the phenotypic effect of the point mutations identified in Example 6, FabBL77P(SEQ ID NO:70), GaCE197K(SEQ ID NO: 10), and HupBA35V(SEQ ID NO: 71), the mutations were reverse-engineered into the wild-type strain P. putida strain KT2440 (Belda et al., 2016), from which SEMI.4 is derived. Strains were grown in minimal medium either in a control condition (33 mM citrate) or in the presence of a high acetate concentration (100 mM) for 72 h (Table 3).

[0444] Table 3. Comparison of performance of evolved and reverse-engineered strains.

[0445] These concentrations were chosen as to provide the same amount of total carbon (200 mM) in all experimental conditions. Citrate is the preferred substrate for P. putida, and is a native organic acid to which P. putida has innate tolerance, hence its selection as a control condition.

[0446] The results indicate that the individual mutations have a positive effect, reducing the lag phase and supporting better growth (as indicated by increased specific growth rate and final cell density) when the cells are grown on acetate, with a cumulative effect. These results show that the positive effect of the mutations, FabBL77P, GaCE197K, and HupBA35V, are independent of the genetic background.

[0447] The final density achieved by cultures of these mutants was also tested against other short-chain organic acids as shown in Table 4. All culture conditions were kept as above, with carbon source as indicated in the table, and the cell density was measured at 24 h of culture. As for acetate, the individual mutations have a positive effect on the strains’ tolerance to lactate, propionate and butyrate with a cumulative effect, again showing the positive effect of the mutations, independent of the genetic background.

[0448] Table 4. Max ODeoo at 24 hours of evolved and reverse-engineered strains in alternative short-chain monocarboxylic acids.

[0449] Methods

[0450] To generate single, double and triple mutant strains the point mutations (FabBL77P, GaCE197K, and HupBA35V) were sequentially accumulated in the wild-type background by allelic replacement (Volke et al., 2020). The engineering process was as described in “Reverse engineering of relevant mutations into Pseudomonas putida SEMI .4, USER cloning and plasmid construction” in the methods of example 6.

[0451] Phenotypic characterisation of the KT2440 FabBL77P, KT2440 FabBL77PGacAE197Kand KT2440 FabBL77PGacAE197KHupBA35Vstrains was performed. Strains KT2440, SEMI .4 and SEM1.4evo were included as controls. The P. putida strains were incubated in de Bont minimal medium added with 5 g / L MOPS and citrate (33 mM) or acetate (100 mM) as indicated. Cultures, inoculated at an initial optical density at 600 nm (ODeoo) of 0.01 , were incubated in microtiter plates (150 .L of medium in 200 pL wells) for 72 h. All growth and kinetic parameters were calculated with the QurvE software (Wirth et al., 2023). Results represent averages ± standard deviation from three independent experiments.

[0452] Sequence overview

[0453] References

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[0455] Benedetti et al., 2016. Genetic programming of catalytic Pseudomonas putida biofilms for boosting biodegradation of haloalkanes. Metab. Eng. 33, 109-118.

[0456] Chen et al., 2013. Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism. Metab Eng. 15:48-54. Czajka et al., 2022. Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production. Metab. Eng. Commun. 15, e00206.

[0457] Ebnesajjad, S., 2013. Introduction to fluoropolymers: Materials, technology, and applications. Elsevier, Boston, USA.

[0458] El-malek and Steinbuchel, 2022. Post-Synthetic Enzymatic and Chemical Modifications for Novel Sustainable Polyesters. Front. Bioeng. Biotechnol. 9:817023.

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[0466] Kirkpatrick et al., 2001. Acetate and formate stress: Opposite responses in the proteome of Escherichia coli. J. Bacteriol. 183, 6466-6477.

[0467] Kohlstedt and Wittmann, 2019. GC-MS-based 13C metabolic flux analysis resolves the parallel and cyclic glucose metabolism of Pseudomonas putida KT2440 and Pseudomonas aeruginosa PAO1. Metab. Eng. 54, 35-53.

[0468] Lageveen et al., 1988. Formation of polyesters by Pseudomonas oleovorans: effect of substrates on formation and composition of poly-l-3-hydroxyalkanoates and pol(R)-3- hydroxyalkenoates. Appl. Environ. Microbiol. 54, 2924-2932.

[0469] Lauble et al., 1996. The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex. Proc. Natl. Acad. Sci. USA. 93, 13699-13703.

[0470] Martinez-Garcia and de Lorenzo, 2011. Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ. Microbiol. 13, 2702-2716. Martinez-Garcia et al., 2014. Pseudomonas 2.0: genetic upgrading of P. putida

[0471] KT2440 as an enhanced host for heterologous gene expression. Microb. Cell Fact. 13: 159.

[0472] Mezzina et al., 2021. Engineering native and synthetic pathways in Pseudomonas putida for the production of tailored polyhydroxyalkanoates. Biotechnol. J. 16, 2000165. Nikel et al., 2016. From dirt to industrial applications: Pseudomonas putida as a Synthetic Biology chassis for hosting harsh biochemical reactions. Curr. Opin. Chem. Biol. 34, 20-29.

[0473] Nogales et al., 2020. High-quality genome-scale metabolic modelling of Pseudomonas putida highlights its broad metabolic capabilities. Environ. Microbiol. 22, 255-269. Perez-Martin and de Lorenzo, 1997. Coactivation in vitro of the s54-dependent promoter Pu of the TOL plasmid of Pseudomonas putida by HU and the mammalian HMG-1 protein. J. Bacteriol. 179, 2757-2760.

[0474] Sagong et al., 2018. Structural Insights into Polyhydroxyalkanoates Biosynthesis.

[0475] Trends Biochem, Sci. 43; 10, 790-805

[0476] Silva-Rocha et al., 2013. The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res. 41, D666-D675.

[0477] Volke et al., 2020. Synthetic control of plasmid replication enables target- and selfcuring of vectors and expedites genome engineering of Pseudomonas putida. Metab. Eng. Commun. 10, e00126.

[0478] Winsor et al., 2016. Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Nucleic Acids Res. 44, D646-653.

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[0481] Yang et al., 2019. Production of medium chain length polyhydroxyalkanoate from acetate by engineered Pseudomonas putida KT2440. J. Ind. Microbiol. Biotechnol. 46, 793-800.

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[0483] Items

[0484] 1. An engineered Pseudomonas cell, said cell comprising one or more modifications selected from the group consisting of: one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), such as a mutation resulting in substitution of residue 77 of FabB, preferably resulting in FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70); one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10); one or more mutations in HupB (SEQ ID NO: 8 or SEQ ID NO: 69), such as a mutation resulting in substitution of residue 35 of HupB, preferably resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71), and / or a mutation resulting in substitution of residue 49 of HupB (SEQ ID NO: 8 or SEQ ID NO: 69), preferably resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72), or one or more mutations in a nucleic acid encoding a functional variant thereof having at least 70% identity to any one of FabB, UvrY or HupB.

[0485] 2. The engineered cell according to any one of the preceding items, wherein the engineered cell is a Pseudomonas putida cell, preferably the engineered cell is a P. putida KT2440 cell, preferably a P. putida EM42 cell.

[0486] 3. The engineered cell according to any one of the preceding items, wherein the endogenous pha cluster in said engineered cell is partially or totally inactivated, wherein the endogenous pha cluster consists of: i) a first operon comprising nucleic acids encoding PhaC1 (SEQ ID NO: 16), PhaC2 (SEQ ID NO: 17), PhaZ (SEQ ID NO: 18), and PhaD (SEQ ID NO: 19); and ii) a second operon comprising nucleic acids encoding PhaF (SEQ ID NO: 20), and Phal (SEQ ID NO: 21), or one or more mutations in a nucleic acid encoding a functional variant thereof having at least 70% identity to PhaC1 , PhaC2, PhaZ, PhaD, PhaF, or Phal, respectively.

[0487] 4. The engineered cell according to any one of the preceding items, wherein the inactivation of the endogenous pha cluster comprises or consists of a mutation or a deletion of all or part of the endogenous pha cluster, preferably wherein the inactivation consists of a mutation or deletion of at least one of the first and second operons, such as a mutation or deletion of the first operon, a mutation or deletion of the second operon, or a mutation or deletion of both the first and the second operons.

[0488] 5. The engineered cell according to any one of the preceding items, wherein the inactivation comprises or consists of a mutation in or a deletion of at least one or more of a nucleic acid encoding PhaC1 (SEQ ID NO: 16), PhaC2 (SEQ ID NO: 17), PhaZ (SEQ ID NO: 18), PhaD (SEQ ID NO: 19); PhaF (SEQ ID NO: 20), and Phal (SEQ ID NO: 21), or a functional variant thereof having at least 70% identity to PhaC1 , PhaC2, PhaZ, PhaD, PhaF, or Phal, respectively.

[0489] 6. The engineered cell according to any one of the preceding items, wherein (F?)- 3-hydroalkanoyl-ACP:CoA transferase, preferably an (F?)-3-hydroxydecanoyl- ACP:CoA transacylase, is inactivated, such as by mutation or deletion of all or part of the nucleic acid encoding said transferase or transacylase.

[0490] 7. The engineered cell according to any one of the preceding items, wherein PhaG (SEQ ID NO: 28), or a functional variant thereof having at least 70% identity to PhaG, is inactivated, such as by mutation or deletion of all or part of the nucleic acid encoding said PhaG or functional variant thereof, for example phaG (SEQ ID NO: 29), or a variant thereof having at least 70% identity to phaG.

[0491] 8. The engineered cell according to any one of the preceding items, wherein the benABCD cluster is partially or completely inactivated, wherein the benABCD cluster consists of benA (SEQ ID NO: 42), benB (SEQ ID NO: 43), benC (SEQ ID NO: 44) and benD (SEQ ID NO: 45), such as by mutation or deletion of all or part of benABCD cluster. The engineered cell according to any one of the preceding items, wherein the engineered cell is capable of producing a PHA having a short carbon chain length (scl-PHA), preferably wherein the scl-PHA is polyhydroxybutyrate (PHB), preferably fluorinated PHB (F-PHB), when grown in a culture medium comprising a carbon source and a nitrogen source, preferably wherein the culture medium comprises glucose, citrate, acetate and / or F-acetate as carbon source. The engineered cell according to any one of the preceding items, wherein the engineered cell is capable of producing 3-hydroxybutyrate, optionally wherein the 3-hydroxybutyrate is fluorinated, such as 2,4-difluoro-3-hydroxybutyrate, 4- fluoro-3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate, most preferably 4- fluoro-3-hydroxybutyrate, when grown in a culture medium comprising a carbon source and a nitrogen source, preferably wherein the culture medium comprises glucose, citrate, acetate and / or F-acetate as carbon source. The engineered cell according to any one of the preceding items, wherein the engineered cell comprises: a nucleic acid encoding an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting acetyl-CoA into acetoacetyl-CoA, preferably a nucleic acid encoding PhaA from Cupriavidus necator (SEQ ID NO: 1), a nucleic acid encoding an acetyl-CoA reductase (EC 1.1.1.36) capable of converting acetoacetyl-CoA into F?-(-)-3-hydroxybutyryl-CoA, preferably a nucleic acid encoding PhaB from Cupriavidus necator (SEQ ID NO: 2), and a nucleic acid encoding a short-chain-length-polyhydroxyalkanoate (scl- PHA) synthase (EC 2.3.1.304) capable of polymerizing ?-(-)-3- hydroxybutyryl-CoA into polyhydroxybutyrate (PHB), preferably a nucleic acid encoding PhaC from Cupriavidus necator (SEQ ID NO: 3), or a nucleic acid encoding a thioesterase (TesB, EC 3.1.2.20) capable of hydrolysing a 3-hydroxybutyryl-CoA to CoA and a corresponding 3-hydroxybutyrate, preferably a nucleic acid encoding PpTesB from P. putida (SEQ ID NO: 38) or EcTesB from Escherichia coli (SEQ ID NO: 39), or functional variants thereof having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, further wherein: i. the engineered cell is not capable of producing polyhydroxyalkanoates (PHAs) having a medium-length carbon chain; ii. the engineered cell is not capable of converting fatty acids into PHA monomers having a medium-length carbon chain; and iii. optionally the benABCD cluster in said engineered cell is inactivated. . The engineered cell according to item 10, wherein the acetyl-CoA thiolase is an acetyl-CoA thiolase native to Cupriavidus, such as C. necator, for example PhaA as set forth in SEQ ID NO: 1 , Azotobacter, Streptomyces, or Paracoccus. . The engineered cell according to item 10, wherein the scl-PHA synthase is an scl-PHA synthase native to Cupriavidus, such as C. necator, for example PhaC as set forth in SEQ ID NO: 3, or an acetyl-CoA reductase native to Azotobacter, Streptomyces, or Paracoccus. . The engineered cell according to item 10, wherein the acetyl-CoA reductase is an acetyl-CoA reductase native to Cupriavidus, such as C. necator, for example PhaB as set forth in SEQ ID NO: 2, or an acetyl-CoA reductase native to Azotobacter, Streptomyces, or Paracoccus. . The engineered cell according to item 10, wherein the thioesterase is native to a bacteria, such as Pseudomonas, for example P. putida, such as PpTesB as set forth in SEQ ID NO: 38, or a thioesterase and / or acyl-CoA thioesterase 2 native to Escherichia, for example E. coli, such as EcTesB as set forth in SEQ ID NO: 39. . The engineered cell according to any one of the preceding items, wherein the nucleic acids encoding PhaA (SEQ ID NO: 1), PhaB (SEQ ID NO: 2), and PhaC (SEQ ID NO: 3) or PpTesB (SEQ ID NO: 38) and / or EcTesB (SEQ ID NO: 39), or functional variants thereof having at least 70% identity to PhaA, PhaB, PhaC, PpTesB, or EcTesB, respectively, are independently integrated in the genome of the engineered cell or are present on a vector within the engineered cell, such as a plasmid. 17. The engineered cell according to any one of the preceding items, wherein the nucleic acid encoding PhaA (SEQ ID NO: 1), PhaB (SEQ ID NO: 2), PhaC (SEQ ID NO: 3), PpTesB (SEQ ID NO: 38), and / or EcTesB (SEQ ID NO: 39), or functional variants thereof having at least 70% identity to PhaA, PhaB, PhaC, PpTesB, or EcTesB, respectively, are codon-optimised for P. putida.

[0492] 18. The engineered cell according to any one of the preceding items, wherein the nucleic acids encoding PhaA (SEQ ID NO: 1), PhaB (SEQ ID NO: 2), PhaC (SEQ ID NO: 3), PpTesB (SEQ ID NO: 38), and / or EcTesB (SEQ ID NO: 39), or functional variants thereof having at least 70% identity to PhaA, PhaB, PhaC, PpTesB (SEQ ID NO: 38), or EcTesB, respectively, are provided as a synthetic operon, optionally said synthetic operon comprising synthetic ribosome binding sites (RBSs) upstream of each of PhaA, PhaB, PhaC, PpTesB and / or EcTesB, or functional variants thereof having at least 70% identity to PhaA, PhaB and PhaC, respectively, such as RBSs having the sequence as set forth in SEQ ID NO: 4.

[0493] 19. The engineered cell according to any one of the preceding items, wherein the synthetic operon further comprises a promoter, such as an inducible promoter and / or expression system, preferably a cyclohexanone-inducible expression system such as ChnR / Pc / ine(SEQ ID NO: 5), a 3-methylbenzoate-inducible expression system such as XylS / Pm (SEQ ID NO: 30), Lacl / Ptac, Lacl / Ptrc, a rhamnose-inducible promoter, or an arabinose inducible promoter.

[0494] 20. The engineered cell according to any one of the preceding items, wherein: the nucleic acid encoding FabB (SEQ ID NO: 6), or a functional variant thereof having at least 70% identity thereto, is fabB (SEQ ID NO: 31), or a variant thereof having at least 70% identity thereto; the nucleic acid encoding UvrY (SEQ ID NO: 7), or a functional variant thereof having at least 70% identity thereto, is uvrY (SEQ ID NO: 32), or a variant thereof having at least 70% identity thereto; and / or the nucleic acid encoding HupB (SEQ ID NO: 8 or SEQ ID NO: 69), or a functional variant thereof having at least 70% identity thereto, is hupB (SEQ ID NO: 33 or 73), or a variant thereof having at least 70% identity thereto.

[0495] 21. The engineered cell according to any one of the preceding items, wherein: the nucleic acid encoding FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70), or a functional variant thereof having at least 70% identity thereto, is mut_fabB (SEQ ID NO: 34), or a variant thereof having at least 70% identity thereto; the nucleic acid encoding UvrYE197K (SEQ ID NO: 10), or a functional variant thereof having at least 70% identity thereto, is mut_uvrY (SEQ ID NO: 35), or a variant thereof having at least 70% identity thereto; the nucleic acid encoding HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71), or a functional variant thereof having at least 70% identity thereto, is mut1_hupB (SEQ ID NO: 36 or SEQ ID NO: 74), or a variant thereof having at least 70% identity thereto; the nucleic acid encoding HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72), or a functional variant thereof having at least 70% identity thereto, is mut2_hupB (SEQ ID NO: 37 or SEQ ID NO: 75), or a variant thereof having at least 70% identity thereto: and / or the nucleic acid encoding HupBA35V,T49l (SEQ ID NO: 46 or SEQ ID NO: 76), or a functional variant thereof having at least 70% identity thereto, is mut3_hupB (SEQ ID NO: 47 or SEQ ID NO: 77), or a variant thereof having at least 70% identity thereto.

[0496] 22. The engineered cell according to any one of the preceding items, wherein: the nucleic acid encoding PhaA (SEQ ID NO: 1), or a functional variant thereof having at least 70% identity thereto, is phaA (SEQ ID NO: 13), or a variant thereof having at least 70% identity thereto; the nucleic acid encoding PhaB (SEQ ID NO: 2), or a functional variant thereof having at least 70% identity thereto, is phaB (SEQ ID NO: 14), or a variant thereof having at least 70% identity thereto; the nucleic acid encoding PpTesB (SEQ ID NO: 38) or EcTesB (SEQ ID NO: 39), or a functional variant thereof having at least 70% identity to PpTesB or EcTesB, respectively, is Pp_tesB (SEQ ID NO: 40) or Ec_tesB (SEQ ID NO: 41), or a variant thereof having at least 70% identity to PptesB or EctesB, respectively; and / or the nucleic acid encoding PhaC (SEQ ID NO: 3), or a functional variant thereof having at least 70% identity thereto, is phaC (SEQ ID NO: 15), or a variant thereof having at least 70% identity thereto. The engineered cell according to any one of the preceding items, wherein: the nucleic acid encoding PhaC1 (SEQ ID NO: 16), or a functional variant thereof having at least 70% identity thereto, is phaC'l (SEQ ID NO: 22), or a variant thereof having at least 70% identity thereto; the nucleic acid encoding PhaC2 (SEQ ID NO: 17), or a functional variant thereof having at least 70% identity thereto, is phaC2 (SEQ ID NO: 23), or a variant thereof having at least 70% identity thereto; the nucleic acid encoding PhaZ (SEQ ID NO: 18), or a functional variant thereof having at least 70% identity thereto, is phaZ (SEQ ID NO: 24), or a variant thereof having at least 70% identity thereto; the nucleic acid encoding PhaD (SEQ ID NO: 19), or a functional variant thereof having at least 70% identity thereto, is phaD (SEQ ID NO: 25), or a variant thereof having at least 70% identity thereto; the nucleic acid encoding PhaF (SEQ ID NO: 20), or a functional variant thereof having at least 70% identity thereto, is phaF (SEQ ID NO: 26), or a variant thereof having at least 70% identity thereto; and / or the nucleic acid encoding Phal (SEQ ID NO: 21), or a functional variant thereof having at least 70% identity thereto, is pha\ (SEQ ID NO: 27), or a variant thereof having at least 70% identity thereto. The engineered cell according to any one of the preceding items, wherein the engineered cell has increased tolerance to a non-native, short-chain organic acid, such as acetate, lactate, propionate and / or butyrate, compared to the tolerance to the same non-native, short-chain organic acid of a reference cell from the same organism, which has not been engineered as defined in any one of the preceding items, and when cultivated under the same conditions. The engineered cell according to any one of the preceding items, wherein the engineered cell has increased tolerance to acetate compared to the tolerance to acetate of a reference cell from the same organism, which has not been engineered as defined in any one of the preceding items, and when cultivated under the same conditions.

[0497] 26. The engineered cell according to any one of the preceding items, wherein the engineered cell can tolerate at least 80 mM acetate, such as at least 100 mM acetate, such as at least 110 mM acetate, such as at least 120 mM acetate, such as at least 130 mM acetate, such as at least 140 mM acetate, such as at least 150 mM acetate, such as at least 160 mM acetate, such as at least 170 mM acetate, such as at least 180 mM acetate, such as at least 190 mM acetate, such as at least 200 mM acetate, such as at least 210 mM acetate, such as at least 220 mM acetate, such as at least 230 mM acetate, such as at least 240 mM acetate, such as at least 250 mM acetate, such as at least, 260 mM acetate, such as at least 275 mM acetate, such as at least 280 mM acetate, such as at least 290 mM acetate, such as at least 295 mM acetate, such as at least 300 mM acetate, or more.

[0498] 27. The engineered cell according to any one of the preceding items, wherein the engineered cell can tolerate lactate, such as at least 60 mM, such as at least 70 mM, such as at least 80 mM, such as at least 90 mM ,such as at least 100 mM, such as at least 110 mM, such as at least 120 mM, such as at least 130 mM, such as at least 140 mM, such as at least 150 mM, such as at least 160 mM, such as at least 170 mM, such as at least 180 mM, such as at least 190 mM, such as at least 200 mM, such as at least 210 mM, such as at least 220 mM, such as at least 230 mM, such as at least 240 mM, such as at least 250 mM, such as at least, 260 mM, such as at least 275 mM, such as at least 280 mM, such as at least 290 mM, such as at least 295 mM, such as at least 300 mM lactate, or more.

[0499] 28. The engineered cell according to any one of the preceding items, wherein the engineered cell can tolerate propionate, such as at least 60 mM, such as at least 70 mM, such as at least 80 mM, such as at least 90 mM ,such as at least

[0500] 100 mM, such as at least 110 mM, such as at least 120 mM, such as at least

[0501] 130 mM, such as at least 140 mM, such as at least 150 mM, such as at least

[0502] 160 mM, such as at least 170 mM, such as at least 180 mM, such as at least 190 mM, such as at least 200 mM, such as at least 210 mM, such as at least 220 mM, such as at least 230 mM, such as at least 240 mM, such as at least 250 mM, such as at least, 260 mM, such as at least 275 mM, such as at least 280 mM, such as at least 290 mM, such as at least 295 mM, such as at least 300 mM propionate, or more. The engineered cell according to any one of the preceding items, wherein the engineered cell can tolerate butyrate, such as at least 40 mM, such as at least 50 mM, such as at least 60 mM, such as at least 70 mM, such as at least 80 mM, such as at least 90 mM ,such as at least 100 mM, such as at least 110 mM, such as at least 120 mM, such as at least 130 mM, such as at least 140 mM, such as at least 150 mM, such as at least 160 mM, such as at least 170 mM, such as at least 180 mM, such as at least 190 mM, such as at least 200 mM, such as at least 210 mM, such as at least 220 mM, such as at least 230 mM, such as at least 240 mM, such as at least 250 mM, such as at least, 260 mM, such as at least 275 mM, such as at least 280 mM, such as at least 290 mM, such as at least 295 mM, such as at least 300 mM butyrate, or more. The engineered cell according to item 26, wherein the acetate is F-acetate. The engineered cell according to any one of the preceding items, wherein the engineered cell is capable of producing 2,4-difluoro-3-hydroxybutyrate, 4-fluoro- 3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate, most preferably 4-fluoro- 3-hydroxybutyrate, at a titer of at least 0.25 g / L. The engineered cell according to any one of the preceding items, wherein the engineered cell is capable of producing scl-PHA with a titer of at least 0.25 g / L, preferably wherein the scl-PHA comprises or consists of PHB and / or F-PHB. A method of producing a 3-hydroxy butyrate (3HB), preferably a fluorinated 3HB, or a polyhydroxyalkanoate (PHA) having a short carbon chain length (scl-PHA), preferably wherein the scl-PHA is polyhydroxybutyrate (PHB), preferably fluorinated PHB (F-PHB), in a Pseudomonas cell, said method comprising the steps of: a) Providing an engineered Pseudomonas cell, b) Cultivating said engineered cell in a culture medium comprising a carbon source and a nitrogen source; wherein: i. the engineered cell is not capable of producing PHAs having a medium-length carbon chain; ii. the engineered cell is not capable of converting fatty acids into PHA monomers having a medium-length carbon chain; iii. optionally the benABCD cluster in said cell is inactivated, and iv. the engineered cell comprises a nucleic acid encoding an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting two acetyl-CoAs into acetoacetyl-CoA, preferably a nucleic acid encoding PhaA from Cupriavidus necator (SEQ ID NO: 1), a nucleic acid encoding an acetyl-CoA reductase (EC 1.1.1.36) capable of converting acetoacetyl-CoA into F?-(-)-3-hydroxybutyryl-CoA, preferably a nucleic acid encoding PhaB from Cupriavidus necator (SEQ ID NO: 2), and

[0503] I. a nucleic acid encoding a scl-PHA synthase (EC 2.3.1.304) capable of polymerizing F?-(-)-3-hydroxybutyryl-CoA into polyhydroxybutyrate (PHB), preferably a nucleic acid encoding PhaC from Cupriavidus necator (SEQ ID NO: 3), or

[0504] II. a nucleic acid encoding a thioesterase (EC 3.1.2.20) capable of hydrolysing a 3-hydroxybutyryl-CoA to CoA and a corresponding 3- hydroxybutyrate, preferably a nucleic acid encoding PpTesB from P. putida (SEQ ID NO: 38) or EcTesB from Escherichia coli (SEQ ID NO: 39), or functional variants thereof having at least 70% identity to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, preferably wherein the culture medium comprises glucose, citrate, acetate and / or F-acetate as carbon source, thereby producing a 3HB or a scl-PHA with a titer of at least 0.25 g / L, preferably wherein the 3HB is a fluorinated 3HB or wherein the scl-PHA comprises or consists of polyhydroxybutyrate (PHB), preferably fluorinated PHB (F-PHB). 34. The method according to item 33, wherein the fluorinated 3-hydroxybutyrate is 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3-hydroxybutyrate, and / or 2-fluoro-3- hydroxybutyrate.

[0505] 35. The method according to any one of items 33 to 34, wherein the engineered cell is as defined in any one of items 1 to 32, 48 to 56 or 61 to 66.

[0506] 36. The method according to any one of items 33 to 35, wherein the culture medium comprises glucose.

[0507] 37. The method according to any one of items 33 to 36, wherein the culture medium comprises acetate.

[0508] 38. The method according to any one of items 33 to 37, wherein the culture medium has a C / N ratio of at least 4, such as at least 5, such as at least 10, such as at least 20, such as at least 30, such as at least 40, or more.

[0509] 39. The method according to any one of items 33 to 38, wherein the conversion rate of acetate and / or F-acetate to PHB and / or F-PHB is at least 0.1% (w / w) per day, such as 0.2% (w / w) per day, or more.

[0510] 40. The method according to any one of items 33 to 39, wherein the yield of PHB and / or F-PHB is at least 5% (w / w) on biomass, such as at least 7.5% (w / w) on biomass, or more.

[0511] 41. The method according to any one of items 33 to 40, wherein the engineered cell has increased tolerance to a non-native, short-chain organic acid, such as acetate, lactate, propionate and / or butyrate, compared to the tolerance to a non-native, short-chain organic acid, such as acetate, lactate, propionate and / or butyrate, respectively, of a reference cell from the same organism, which has not been engineered as defined in any one of the preceding items.

[0512] 42. The method according to any one of items 33 to 40, wherein the engineered cell has increased tolerance to acetate compared to the tolerance to acetate of a reference cell from the same organism, which has not been engineered as defined in any one of the preceding items. The method according to any one of items 33 to 42, further comprising a step of recovering the 3HB, fluorinated 3HB, PHB, and / or the F-PHB. The method according to item 43, wherein the step of recovering PHB and / or F- PHB comprises solvent extraction, for example Sohxlet extraction. The method according to any one of items 33 to 43, wherein the culture medium comprises F-acetate and one or more of glucose, citrate, or acetate, and wherein PHB and / or F-PHB are produced. The method according to any one of items 33 to 45, wherein the culture medium comprises at least 10 mM acetate, such as at least 20 mM acetate, such as at least 30 mM acetate, such as at least 40 mM acetate, such as at least 50 mM acetate, such as at least 60 mM acetate, such as at least 70 mM acetate, such as at least 80 mM acetate, such as at least 90 mM acetate, such as at least 100 mM acetate, such as at least 110 mM acetate, such as at least 120 mM acetate, such as at least 130 mM acetate, such as at least 140 mM acetate, such as at least 150 mM acetate, such as at least 160 mM acetate, such as at least 170 mM acetate, such as at least 180 mM acetate, such as at least 190 mM acetate, such as at least 200 mM acetate, such as at least 210 mM acetate, such as at least 220 mM acetate, such as at least 230 mM acetate, such as at least 240 mM acetate, such as at least 250 mM acetate, such as at least, 260 mM acetate, such as at least 275 mM acetate, such as at least 280 mM acetate, such as at least 290 mM acetate, such as at least 295 mM acetate, such as at least 300 mM acetate, or more. The method according to any one of the items 33 to 46, wherein the culture medium comprises at least 10 mM F-acetate, such as at least 20 mM F-acetate, such as at least 30 mM F-acetate, such as at least 40 mM F-acetate, such as at least 50 mM F-acetate, such as at least 60 mM F-acetate, such as at least 70 mM F-acetate, such as at least 80 mM F-acetate, such as at least 90 mM F- acetate, such as at least 100 mM F-acetate, such as at least 110 mM F-acetate, such as at least 120 mM F-acetate, such as at least 130 mM F-acetate, such as at least 140 mM F-acetate, such as at least 150 mM F-acetate, such as at least 160 mM F-acetate, such as at least 170 mM F-acetate, such as at least 180 mM F-acetate, such as at least 190 mM F-acetate, such as at least 200 mM F- acetate, such as at least 210 mM F-acetate, such as at least 220 mM F-acetate, such as at least 230 mM F-acetate, such as at least 240 mM F-acetate, such as at least 250 mM F-acetate, such as at least, 260 mM F-acetate, such as at least 275 mM F-acetate, such as at least 280 mM F-acetate, such as at least 290 mM F-acetate, such as at least 295 mM F-acetate, such as at least 300 mM F- acetate, such as at least 325 mM F-acetate, such as at least 350 mM F-acetate, such as at least 380 mM F-acetate, such as at least 400 mM F-acetate, such as at least 425 mM F-acetate, or more. The engineered cell and / or method according to any one of the preceding items, wherein said F-PHB is poly(fluoro-3-hydroxybutyrate). The engineered cell and / or method according to any one of the preceding items, wherein said F-PHB comprises one or more monomers of 3- hydroxy butyrate, 4-fluoro-3-hydroxybutyrate, 2-fluoro-3-hydroxybutyrate, and / or 2,4-fluoro-3-hyd roxy b uty rate . The engineered cell and / or method according to any one of the preceding items, wherein said F-PHB has the general structure of: o formula (I) wherein: x and y independently are integers between 1 and 19,999; and x+y < 20,000; or o formula (II): wherein: x, y and z independently are integers between 1 and 19998, and x+y+z<20,000.

[0513] 51. The engineered cell and / or method according to any one of the preceding items, wherein the produced scl-PHA comprises or consists of F-PHB and optionally PHB and has a fluorine (F) content of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (mol / mol).

[0514] 52. The engineered cell and / or method according to any one of the preceding items, wherein the produced scl-PHA comprises or consists of F-PHB and optionally PHB and has a fluorine (F) content of at least 0.1%, such as at least 0.2%, such as at least 0.5%, such as at least 0.7%, such as at least 1.0% (mol / mol).

[0515] 53. The engineered cell and / or method according to any one of the preceding items, wherein the produced scl-PHA comprises or consists of F-PHB and optionally PHB and has a fluorine (F) content between 0.1% and 1.5%, for example between 0.2% and 1.25%, such as between 0.3% and 1.15%, for example between 0.4% and 1.1%, such as between 0.5% and 1% (mol / mol).

[0516] 54. The engineered cell and / or method according to any one of the preceding items, wherein the produced scl-PHA comprises or consists of F-PHB and has a static water contact angle of at least 75°, such as at least 80°, such as at least 85°, such as at least 90°, such as at least 95°, such as at least 100°, such as at least 105°, such as at least 110°, such as at least 115°, such as at least 120°, such as 121°, such as 122°, such as 123°, such as 124°, or more.

[0517] 55. The engineered cell and / or method according to any one of the preceding items, wherein the produced scl-PHA comprises or consists of PHB and / or F- PHB and has a water contact angle in diiodomethane of at least 35°, such as at least 40°, such as at least 45°, such as at least 46°, such as at least 47°, such as at least 48°, such as at least 49°, or more.

[0518] 56. The engineered cell and / or method according to any one of the preceding items, wherein the produced scl-PHA comprises or consists of PHB and / or F- PHB and has a surface energy of at most 59 mJ rrr2, such as at most 55 mJ nr 2, such as at most 50 mJ rrr2.

[0519] 57. The method according to any one of items 33 to 56, wherein the culture medium further comprises 2-ketoglutarate (2KG), isocitrate, or succinate and glutamate, preferably at a concentration of at least 1 mM, such as at least 2 mM, such as at least 3 mM, such as at least 4 mM, such as at least 5 mM, such as at least 10 mM, such as at least 15 mM.

[0520] 58. The engineered cell and / or method according to any one of the preceding items, wherein the engineered cell is capable of producing PHB at a titer of at least 30 g / L, such as at least 31 g / L, such as at least 32 g / L, such as at least 33 g / L, for example at least 33.5 g / L, such as at least 35 g / L, such as at least 37.5 g / L, or more, preferably wherein the titer is determined after at least 48 hours, or more.

[0521] 59. The engineered cell and / or method according to any one of the preceding items, wherein F-PHB is produced with a titer of at least 0.5 g / L, such as at least 0.75 g / L, such as at least 0.9 g / L, such as at least 0.95 g / L, such as at least 1 g / L, such as at least 1.1 g / L, such as at least 1.25 g / L, such as at least 1.5 g / L, such as at least 1.75 g / L, such as at least 2 g / L, such as at least 2.5 g / L, such as at least 5 g / L, or more.

[0522] 60. Use of F-PHB as defined in any one of the preceding items, such as F-PHB as defined in any one of items 48 to 56 or 68 to 73, as a water repellent,.

[0523] 61. An engineered Pseudomonas cell, said cell comprising: a nucleic acid encoding an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting acetyl-CoA into acetoacetyl-CoA; a nucleic acid encoding an acetyl-CoA reductase (EC 1.1.1.36) capable of converting acetoacetyl-CoA into R-(-)-3-hydroxybutyryl-CoA; a nucleic acid encoding a short-chain-length-polyhydroxyalkanoate (scl- PHA) synthase (EC 2.3.1.304) capable of polymerizing R-(-)-3- hydroxybutyryl-CoA into polyhydroxybutyrate (PHB), or a nucleic acid encoding a thioesterase (TesB, EC 3.1.2.20) capable of hydrolysing a 3-hydroxybutyryl-CoA to CoA and a corresponding 3- hydroxybutyrate; and optionally one or more modifications selected from the group consisting of: partial or total inactivation of the endogenous pha cluster, such as by one or more mutations in a nucleic acid encoding a pha cluster gene or a functional variant thereof having at least 70% identity thereto, wherein said endogenous pha cluster consists of: i. a first operon comprising nucleic acids encoding PhaC1 (SEQ ID NO: 16), PhaC2 (SEQ ID NO: 17), PhaZ (SEQ ID NO: 18), and PhaD (SEQ ID NO: 19), and ii. a second operon comprising nucleic acids encoding PhaF (SEQ ID NO: 20), and Phal (SEQ ID NO: 21); inactivation of PhaG (SEQ ID NO: 28), such as by mutation or deletion of all or part of the nucleic acid encoding said PhaG or functional variant thereof, for example phaG (SEQ ID NO: 29), or a functional variant thereof having at least 70% identity to phaG. The engineered Pseudomonas cell according to item 61 , wherein: the acetyl-CoA thiolase (EC 2.3.1.9) is PhaA from Cupriavidus necator (SEQ ID NO: 1) or a functional variant thereof having at least 70% identity to SEQ ID NO: 1; the acetyl-CoA reductase (EC 1.1.1.36) is PhaB from Cupriavidus necator (SEQ ID NO: 2) or a functional variant thereof having at least 70% identity to SEQ ID NO: 2; the short-chain-length-polyhydroxyalkanoate (scl-PHA) synthase (EC 2.3.1.304) is PhaC from Cupriavidus necator (SEQ ID NO: 3) or a functional variant thereof having at least 70% identity to SEQ ID NO: 3; and / or the thioesterase (TesB, EC 3.1.2.20) is PpTesB from P. putida (SEQ ID NO: 38) or EcTesB from Escherichia coli (SEQ ID NO: 39) or a functional variant thereof having at least 70% identity to SEQ ID NO: 38 and SEQ ID NO: 39, respectively. The engineered cell according to any one of items 61 to 62, wherein the engineered cell is capable of producing 3-hydroxybutyrate, optionally wherein the 3-hydroxybutyrate is fluorinated, such as 2,4-difluoro-3-hydroxybutyrate, 4- fluoro-3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate, most preferably 4- fluoro-3-hydroxybutyrate, when grown in a culture medium comprising a carbon source and a nitrogen source, preferably wherein the culture medium comprises glucose, citrate, acetate and / or F-acetate as carbon source. The engineered cell according to any one of items 61 to 63, wherein the engineered cell is capable of producing 2,4-difluoro-3-hydroxybutyrate, 4-fluoro- 3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate, most preferably 4-fluoro- 3-hydroxybutyrate, at a titer of at least 0.25 g / L, such as at least 0.5 g / L, such as at least 0.75 g / L, such as at least 1 g / L, such as at least 1.5 g / L, such as at least 2 g / L, such as at least 2.5 g / L, such as at least 3 g / L, such as at least 3.5 g / L, such as at least 4 g / L, such as at least 4.5 g / L, such as at least 5 g / L, such as at least 5.5 g / L, such as at least 6 g / L, such as at least, 6.5 g / L, such as at least, 7 g / L, such as at least, 7.5 g / L, such as at least 8 g / L, such as at least 8.5 g / L, such as at least 9 g / L, such as at least 9.5 g / L, such as at least 10 g / L, or more. The engineered cell according to any one of items 61 to 64, wherein the engineered cell is capable of producing scl-PHA with a titer of at least 0.25 g / L, preferably wherein the scl-PHA comprises or consists of PHB and / or F-PHB. The engineered cell according to any one of items 61 to 65 wherein the engineered cell comprises the modifications as defined in any one of items 1 , or 3 to 8, or 11 to 30. A method for manufacturing a fluorinated polymer, preferably F-PHB, said method comprising the steps of: i. obtaining a fluorinated 3HB by the method according to any one of items 33 to 59; and ii. converting said fluorinated 3HB to a fluorinated polymer, such as fluorinated PHB, preferably wherein the fluorinated 3HB is 2,4-difluoro-3-hydroxybutyrate, 4- fluoro-3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate, optionally wherein the F-PHB is as defined in any one of items 48 to 56, or wherein the fluorinated 3HB is as defined in any one of items 68 to 73.

[0524] 68. Fluorinated 3-hydroxybutyrate (F-3HB); poly(3-hydroxybutyrate) (PHB); or fluorinated poly(3-hydroxybutyrate) (F-PHB).

[0525] 69. The fluorinated 3-hydroxybutyrate (F-3HB) according to item 68, wherein said fluorinated 3HB is 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3-hydroxybutyrate, or 2-fluoro-3-hydroxybutyrate.

[0526] 70. The poly(3-hydroxybutyrate) (PHB) according to item 68, wherein said PHB has a molecular weight between 0.1 MDa and 1.5 MDa.

[0527] 71. The fluorinated poly(3-hydroxybutyrate) (F-PHB) according to item 68, wherein said F-PHB has the structure of formula (I): wherein: x and y independently are integers between 1 and 19,999; and x+y < 20,000.

[0528] 72. The fluorinated poly(3-hydroxybutyrate) (F-PHB) according to item 68, wherein said F-PHB has the structure of formula (II): wherein: x, y and z independently are integers between 1 and 19998; and x+y+z<20000.

[0529] 73. The fluorinated poly(3-hydroxybutyrate) (F-PHB) according to item 68, wherein said F-PHB is a mixture of the F-PHB of formula (I) and of the F-PHB of formula (II).

Claims

Claims1 . An engineered Pseudomonas cell, said cell comprising one or more modifications selected from the group consisting of: one or more mutations in a nucleic acid encoding FabB (SEQ ID NO: 6), such as a mutation resulting in substitution of residue 77 of FabB, preferably resulting in FabBL77P (SEQ ID NO: 9 or SEQ ID NO: 70); one or more mutations in a nucleic acid encoding UvrY (SEQ ID NO: 7), such as a mutation resulting in substitution of residue 197 of UvrY, preferably resulting in UvrYE197K (SEQ ID NO: 10); one or more mutations in HupB (SEQ ID NO: 8 or SEQ ID NO: 69), such as a mutation resulting in substitution of residue 35 of HupB, preferably resulting in HupBA35V (SEQ ID NO: 11 or SEQ ID NO: 71), and / or a mutation resulting in substitution of residue 49 of HupB (SEQ ID NO: 8 or SEQ ID NO: 69), preferably resulting in HupBT49l (SEQ ID NO: 12 or SEQ ID NO: 72), or one or more mutations in a nucleic acid encoding a functional variant thereof having at least 70% identity to any one of FabB, UvrY or HupB, preferably wherein the engineered cell is a Pseudomonas putida cell, preferably the engineered cell is a P. putida KT2440 cell, preferably a P. putida EM42 cell.

2. The engineered cell according to claim 1 , wherein the endogenous pha cluster in said engineered cell is partially or totally inactivated, wherein the endogenous pha cluster consists of: i) a first operon comprising nucleic acids encoding PhaC1 (SEQ ID NO: 16), PhaC2 (SEQ ID NO: 17), PhaZ (SEQ ID NO: 18), and PhaD (SEQ ID NO: 19); and ii) a second operon comprising nucleic acids encoding PhaF (SEQ ID NO: 20), and Phal (SEQ ID NO: 21), or one or more mutations in a nucleic acid encoding a functional variant thereof having at least 70% identity to PhaC1 , PhaC2, PhaZ, PhaD, PhaF, or Phal, respectively, preferably wherein the inactivation comprises or consists of a mutation in or a deletion of at least one or more of a nucleic acid encoding PhaC1 (SEQ ID NO:16), PhaC2 (SEQ ID NO: 17), PhaZ (SEQ ID NO: 18), PhaD (SEQ ID NO: 19); PhaF (SEQ ID NO: 20), and Phal (SEQ ID NO: 21), or a functional variant thereof having at least 70% identity to PhaC1 , PhaC2, PhaZ, PhaD, PhaF, or Phal, respectively.

3. The engineered cell according to any one of the preceding claims, wherein PhaG (SEQ ID NO: 28), or a functional variant thereof having at least 70% identity to PhaG, is inactivated, such as by mutation or deletion of all or part of the nucleic acid encoding said PhaG or functional variant thereof, for example phaG (SEQ ID NO: 29), or a variant thereof having at least 70% identity to phaG.

4. The engineered cell according to any one of the preceding claims, wherein the engineered cell is capable of producing a PHA having a short carbon chain length (scl-PHA), preferably wherein the scl-PHA is polyhydroxybutyrate (PHB), preferably fluorinated PHB (F-PHB), when grown in a culture medium comprising a carbon source and a nitrogen source, preferably wherein the culture medium comprises glucose, citrate, acetate and / or F-acetate as carbon source, preferably wherein the scl-PHA comprises or consists of 3-hydroxybutyrate, optionally wherein the 3-hydroxybutyrate is fluorinated, such as 2,4-difluoro-3- hydroxy butyrate, 4-fluoro-3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate, most preferably 4-fluoro-3-hydroxybutyrate.

5. The engineered cell according to any one of the preceding claims, wherein the engineered cell comprises: a nucleic acid encoding an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting acetyl-CoA into acetoacetyl-CoA, preferably a nucleic acid encoding PhaA from Cupriavidus necator (SEQ ID NO: 1), a nucleic acid encoding an acetyl-CoA reductase (EC 1.1.1.36) capable of converting acetoacetyl-CoA into F?-(-)-3-hydroxybutyryl-CoA, preferably a nucleic acid encoding PhaB from Cupriavidus necator (SEQ ID NO: 2), and a nucleic acid encoding a short-chain-length-polyhydroxyalkanoate (scl- PHA) synthase (EC 2.3.1.304) capable of polymerizing ?-(-)-3- hydroxybutyryl-CoA into polyhydroxybutyrate (PHB), preferably a nucleicacid encoding PhaC from Cupriavidus necator (SEQ ID NO: 3), or a nucleic acid encoding a thioesterase (TesB, EC 3.1.2.20) capable of hydrolysing a 3-hydroxybutyryl-CoA to CoA and a corresponding 3-hydroxybutyrate, preferably a nucleic acid encoding PpTesB from P. putida (SEQ ID NO: 38) or EcTesB from Escherichia coli (SEQ ID NO: 39), or functional variants thereof having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, further wherein: i. the engineered cell is not capable of producing polyhydroxyalkanoates (PHAs) having a medium-length carbon chain; ii. the engineered cell is not capable of converting fatty acids into PHA monomers having a medium-length carbon chain; and iii. optionally, the benABCD cluster in said engineered cell is inactivated.

6. The engineered cell according to any one of the preceding claims, wherein the engineered cell has increased tolerance to a non-native, short-chain organic acid, such as acetate, lactate, propionate and / or butyrate, compared to the tolerance to the same non-native, short-chain organic acid of a reference cell from the same organism, which has not been engineered as defined in any one of the preceding items, and when cultivated under the same conditions.

7. The engineered cell according to any one of the preceding claims, wherein the engineered cell has increased tolerance to acetate compared to the tolerance to acetate of a reference cell from the same organism, which has not been engineered as defined in any one of the preceding claims, and when cultivated under the same conditions, preferably wherein the engineered cell can tolerate at least 80 mM acetate, such as at least 100 mM acetate, such as at least 110 mM acetate, such as at least 120 mM acetate, such as at least 130 mM acetate, such as at least 140 mM acetate, such as at least 150 mM acetate, such as at least 160 mM acetate, such as at least 170 mM acetate, such as at least 180 mM acetate, such as at least 190 mM acetate, such as at least 200 mM acetate, such as at least 210 mM acetate, such as at least 220 mM acetate, such as at least 230 mM acetate, such as at least 240 mM acetate, such as at least 250 mM acetate, such as at least, 260 mM acetate, such as atleast 275 mM acetate, such as at least 280 mM acetate, such as at least 290 mM acetate, such as at least 295 mM acetate, such as at least 300 mM acetate, or more, optionally wherein the acetate is F-acetate.

8. The engineered cell according to any one of the preceding claims, wherein the engineered cell can tolerate lactate, such as at least 60 mM, such as at least 70 mM, such as at least 80 mM, such as at least 90 mM ,such as at least 100 mM, such as at least 110 mM, such as at least 120 mM, such as at least 130 mM, such as at least 140 mM, such as at least 150 mM, such as at least 160 mM, such as at least 170 mM, such as at least 180 mM, such as at least 190 mM, such as at least 200 mM, such as at least 210 mM, such as at least 220 mM, such as at least 230 mM, such as at least 240 mM, such as at least 250 mM, such as at least, 260 mM, such as at least 275 mM, such as at least 280 mM, such as at least 290 mM, such as at least 295 mM, such as at least 300 mM lactate, or more.

9. The engineered cell according to any one of the preceding claims, wherein the engineered cell can tolerate propionate, such as at least 60 mM, such as at least 70 mM, such as at least 80 mM, such as at least 90 mM ,such as at least 100 mM, such as at least 110 mM, such as at least 120 mM, such as at least130 mM, such as at least 140 mM, such as at least 150 mM, such as at least160 mM, such as at least 170 mM, such as at least 180 mM, such as at least190 mM, such as at least 200 mM, such as at least 210 mM, such as at least220 mM, such as at least 230 mM, such as at least 240 mM, such as at least250 mM, such as at least, 260 mM, such as at least 275 mM, such as at least 280 mM, such as at least 290 mM, such as at least 295 mM, such as at least 300 mM propionate, or more.

10. The engineered cell according to any one of the preceding claims, wherein the engineered cell can tolerate butyrate, such as at least 40 mM, such as at least 50 mM, such as at least 60 mM, such as at least 70 mM, such as at least 80 mM, such as at least 90 mM ,such as at least 100 mM, such as at least 110 mM, such as at least 120 mM, such as at least 130 mM, such as at least 140 mM, such as at least 150 mM, such as at least 160 mM, such as at least 170 mM, such as at least 180 mM, such as at least 190 mM, such as at least 200mM, such as at least 210 mM, such as at least 220 mM, such as at least 230 mM, such as at least 240 mM, such as at least 250 mM, such as at least, 260 mM, such as at least 275 mM, such as at least 280 mM, such as at least 290 mM, such as at least 295 mM, such as at least 300 mM butyrate, or more.

11. The engineered cell according to any one of the preceding claims, wherein the engineered cell is capable of producing 2,4-difluoro-3-hydroxybutyrate, 4-fluoro- 3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate, most preferably 4-fluoro- 3-hydroxybutyrate, at a titer of at least 0.25 g / L, and / or wherein the engineered cell is capable of producing scl-PHA with a titer of at least 0.25 g / L, preferably wherein the scl-PHA comprises or consists of PHB and / or F-PHB.

12. A method of producing a 3-hydroxy butyrate (3HB), preferably a fluorinated 3HB, or a polyhydroxyalkanoate (PHA) having a short carbon chain length (scl-PHA), preferably wherein the scl-PHA is polyhydroxybutyrate (PHB), preferably fluorinated PHB (F-PHB), in a Pseudomonas cell, said method comprising the steps of: a) Providing an engineered Pseudomonas cell, preferably wherein the cell is as defined in any one of claims 1 to 11 ; b) Cultivating said engineered cell in a culture medium comprising a carbon source and a nitrogen source; wherein: i. the engineered cell is not capable of producing PHAs having a medium-length carbon chain; ii. the engineered cell is not capable of converting fatty acids into PHA monomers having a medium-length carbon chain; iii. optionally the benABCD cluster in said cell is inactivated, and iv. the engineered cell comprises a nucleic acid encoding an acetyl-CoA thiolase (EC 2.3.1.9) capable of converting two acetyl-CoAs into acetoacetyl-CoA, preferably a nucleic acid encoding PhaA from Cupriavidus necator (SEQ ID NO: 1), a nucleic acid encoding an acetyl-CoA reductase (EC 1.1.1.36) capable ofconverting acetoacetyl-CoA into F?-(-)-3-hydroxybutyryl-CoA, preferably a nucleic acid encoding PhaB from Cupriavidus necator (SEQ ID NO: 2), andI. a nucleic acid encoding a scl-PHA synthase (EC 2.3.1.304) capable of polymerizing F?-(-)-3-hydroxybutyryl-CoA into polyhydroxybutyrate (PHB), preferably a nucleic acid encoding PhaC from Cupriavidus necator (SEQ ID NO: 3), orII. a nucleic acid encoding a thioesterase (EC 3.1.2.20) capable of hydrolysing a 3-hydroxybutyryl-CoA to CoA and a corresponding 3- hydroxybutyrate, preferably a nucleic acid encoding PpTesB from P. putida (SEQ ID NO: 38) or EcTesB from Escherichia coli (SEQ ID NO: 39), or functional variants thereof having at least 70% identity to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 38, and SEQ ID NO: 39, respectively, preferably wherein the culture medium comprises glucose, citrate, acetate and / or F-acetate as carbon source, thereby producing a 3HB or a scl-PHA with a titer of at least 0.25 g / L, preferably wherein the 3HB is a fluorinated 3HB, such as 2,4-difluoro-3-hydroxybutyrate, 4-fluoro-3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate; or wherein the scl-PHA comprises or consists of polyhydroxybutyrate (PHB), preferably fluorinated PHB (F-PHB).

13. The method according to claim 12, wherein: the culture medium comprises glucose and / or acetate, and optionally further comprises 2-ketoglutarate (2KG), isocitrate, or succinate and glutamate, preferably at a concentration of at least 1 mM, such as at least 2 mM, such as at least 3 mM, such as at least 4 mM, such as at least 5 mM, such as at least 10 mM, such as at least 15 mM; the culture medium has a C / N ratio of at least 4, such as at least 5, such as at least 10, such as at least 20, such as at least 30, such as at least 40, or more; the conversion rate of acetate and / or F-acetate to PHB and / or F-PHB is at least 0.1% (w / w) per day, such as 0.2% (w / w) per day, or more; the yield of PHB and / or F-PHB is at least 5% (w / w) on biomass, such as at least 7.5% (w / w) on biomass, or more.

14. The method according to any one of claims 12 to 13, wherein the engineered cell has increased tolerance to acetate compared to the tolerance to acetate of a reference cell from the same organism, which has not been engineered as defined in any one of the preceding claims.

15. The method according to any one of claims 12 to 14, wherein: the culture medium comprises at least 10 mM acetate, such as at least 20 mM acetate, such as at least 30 mM acetate, such as at least 40 mM acetate, such as at least 50 mM acetate, such as at least 60 mM acetate, such as at least 70 mM acetate, such as at least 80 mM acetate, such as at least 90 mM acetate, such as at least 100 mM acetate, such as at least 110 mM acetate, such as at least 120 mM acetate, such as at least 130 mM acetate, such as at least 140 mM acetate, such as at least 150 mM acetate, such as at least 160 mM acetate, such as at least 170 mM acetate, such as at least 180 mM acetate, such as at least 190 mM acetate, such as at least 200 mM acetate, such as at least 210 mM acetate, such as at least 220 mM acetate, such as at least 230 mM acetate, such as at least 240 mM acetate, such as at least 250 mM acetate, such as at least, 260 mM acetate, such as at least 275 mM acetate, such as at least 280 mM acetate, such as at least 290 mM acetate, such as at least 295 mM acetate, such as at least 300 mM acetate; and / or- wherein the culture medium comprises at least 10 mM F-acetate, such as at least 20 mM F-acetate, such as at least 30 mM F-acetate, such as at least 40 mM F-acetate, such as at least 50 mM F-acetate, such as at least 60 mM F-acetate, such as at least 70 mM F-acetate, such as at least 80 mM F- acetate, such as at least 90 mM F-acetate, such as at least 100 mM F- acetate, such as at least 110 mM F-acetate, such as at least 120 mM F- acetate, such as at least 130 mM F-acetate, such as at least 140 mM F- acetate, such as at least 150 mM F-acetate, such as at least 160 mM F- acetate, such as at least 170 mM F-acetate, such as at least 180 mM F- acetate, such as at least 190 mM F-acetate, such as at least 200 mM F- acetate, such as at least 210 mM F-acetate, such as at least 220 mM F- acetate, such as at least 230 mM F-acetate, such as at least 240 mM F- acetate, such as at least 250 mM F-acetate, such as at least, 260 mM F-acetate, such as at least 275 mM F-acetate, such as at least 280 mM F- acetate, such as at least 290 mM F-acetate, such as at least 295 mM F- acetate, such as at least 300 mM F-acetate, such as at least 325 mM F- acetate, such as at least 350 mM F-acetate, such as at least 380 mM F- acetate, such as at least 400 mM F-acetate, such as at least 425 mM F- acetate, or more.

16. The method according to any one of claims 12 to 15, wherein said F-PHB is poly(fluoro-3-hydroxybutyrate), preferably wherein said F-PHB comprises one or more monomers of 3-hydroxybutyrate, 4-fluoro-3-hydroxybutyrate, 2-fluoro-3- hydroxybutyrate, and / or 2,4-fluoro-3-hydroxybutyrate, optionally wherein said F-PHB has the general structure of: o formula (I)wherein: x and y independently are integers between 1 and 19,999; and x+y < 20,000; or o formula (II):wherein: x, y and z independently are integers between 1 and 19998, and x+y+z<20,000.

17. The method according to any one of claims 12 to 16, wherein the produced scl-PHA comprises or consists of F-PHB and optionally PHB and:- has a fluorine (F) content of 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% (mol / mol); has a fluorine (F) content of at least 0.1%, such as at least 0.2%, such as at least 0.5%, such as at least 0.7%, such as at least 1.0% (mol / mol); has a water contact angle in diiodomethane of at least 35°, such as at least 40°, such as at least 45°, such as at least 46°, such as at least 47°, such as at least 48°, such as at least 49°, or more; has a surface energy of at most 59 mJ rrr2, such as at most 55 mJ rrr2, such as at most 50 mJ rrr2.

18. The method according to any one of claims 12 to 17, wherein the engineered cell is capable of producing:PH B at a titer of at least 30 g / L, such as at least 31 g / L, such as at least 32 g / L, such as at least 33 g / L, for example at least 33.5 g / L, such as at least 35 g / L, such as at least 37.5 g / L, or more, preferably wherein the titer is determined after at least 48 hours, or more; and / orF-PHB a titer of at least 0.5 g / L, such as at least 0.75 g / L, such as at least 0.9 g / L, such as at least 0.95 g / L, such as at least 1 g / L, such as at least 1.1 g / L, such as at least 1.25 g / L, such as at least 1.5 g / L, such as at least 1.75 g / L, such as at least 2 g / L, such as at least 2.5 g / L, such as at least 5 g / L, or more.

19. A method for manufacturing a fluorinated polymer, preferably F-PHB, said method comprising the steps of: i. obtaining a fluorinated 3HB by the methods according to any one of claims 12 to 18; and ii. converting said fluorinated 3HB to a fluorinated polymer, such as fluorinated PHB, preferably wherein the fluorinated 3HB is 2,4-difluoro-3-hydroxybutyrate, 4- fluoro-3-hydroxybutyrate, and / or 2-fluoro-3-hydroxybutyrate, optionally wherein the F-PHB is as defined in any one of claims 16 to 17.

20. Use of F-PHB as defined in any one of the preceding claims as a water repellent, such as F-PHB as defined in any one of claims 16 to 17.

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