Patent Application
20230323410
The present invention discloses bacterial cells useful for production of 2-fluoro-cis,cis-muconate and derivatives thereof. The disclosure also provides methods and nucleic acid constructs therefor.
The introduction of fluorine into polymers has been shown to significantly alter their physicochemical properties and can lead to new materials with distinct properties for diverse applications.
Chemical dehydrogenation of cis,cis-muconic acid to adipic acid and subsequent polycondensation with hexamethylenediamine can be used to form the polyamide Nylon 6-6. Nylon 6-6 is frequently used in fibers for textiles and carpets and moulded parts due to its high mechanical strength, rigidity, good stability under heat and/or chemical resistance. It has furthermore broad use in automotive applications, or in electro-insulating elements. The introduction of fluorine into Nylon 6-6 via a chemical synthesis process that uses 2-fluoro-cis,cis-muconate (2-FMA) could alter the polymer properties of Nylon 6-6 significantly, e.g., by increasing its chemical resistance and thermal stability, making it even more suitable for specific applications.
Other applications include the conversion of 2-FMA into 2-fluoro-1,6-hexanediol. The non-halogenated counterpart of this platform molecule (1,6-hexanediol) is widely used for polyester (e.g. PET) and polyurethane production, and as an intermediate in the production of acrylics, adhesives, and dyes. Cis,cis-muconate additionally can serve as one of two precursor compounds to synthesize terephthalic acid, a monomeric chemical to produce polyethylene-terephthalate (PET). Similar beneficial effects as mentioned for fluorinated Nylon 6-6 can be predicted for the incorporation of fluorine into these compounds.
Unfortunately, the fluorinated precursor molecule 2-FMA is not commercially available due to inaccessibility through organic-chemical synthesis. The organic-chemical synthesis of 2-FMA has proved to be highly difficult, if not completely impossible, due to the poisoning of the metal catalyst by fluorinated compounds.
The bioconversion of 3-fluorobenzoic acid (3-FBz) to 2-FMA by wild-type P. putida KT2440 has been shown to occur in shaken-flask experiments with a very low yield and severe growth impairment. In a wild-type strain, the consumption of the substrate 3-FBz proceeds to completion, with a significant accumulation of intermediate metabolites and compounds derived thereof, including non-fluorinated intermediates and byproducts.
Production of 2-FMA has been attempted in biological organisms, however the accumulation of the toxic growth-inhibiting intermediate 3-fluorocatechol has severely limited the yield and efficiency of these processes (Schmidt and Knackmuss, 1984). There is thus a need for reliable and efficient methods for production of fluorinated compounds such as 2-FMA. In particular, sustainable methods based on biological production of fluorinated compounds such as 2-FMA, e.g. from a microorganism such as a bacteria, are needed.
The invention is as defined in the claims.
Herein is provided a method of production of 2-fluoro-cis,cis-muconate and derivatives thereof, in a bacterial cell, such as a non-pathogenic Pseudomonas putida cell, from the precursor compound 3-fluorobenzoate. The described process is to the best of our knowledge the first one to efficiently produce 2-fluoro-cis,cis-muconate.
In one aspect, the present disclosure provides a bacterial cell capable of producing 2-fluoro-cis,cis-muconate and optionally derivatives thereof, in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
In one aspect, the present disclosure provides a bacterial cell capable of producing 2-fluoro-cis,cis-muconate and optionally derivatives thereof, in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
In one aspect, the present disclosure provides a bacterial cell capable of producing 2-fluoro-cis,cis-muconate and optionally derivatives thereof, in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
In one aspect, the present disclosure provides a method of producing 2-fluoro-cis,cis-muconate, and optionally derivatives thereof, in a bacterial cell, said method comprising the steps of providing a bacterial cell and incubating said bacterial cell in a medium in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
In one aspect, the present disclosure provides a method of producing 2-fluoro-cis,cis-muconate, and optionally derivatives thereof, in a bacterial cell, said method comprising the steps of providing a bacterial cell and incubating said bacterial cell in a medium in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
In one aspect, the present disclosure provides a method of producing 2-fluoro-cis,cis-muconate, and optionally derivatives thereof, in a bacterial cell, said method comprising the steps of providing a bacterial cell and incubating said bacterial cell in a medium in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
In one aspect, the present disclosure provides 2-fluoro-cis,cis-muconate or a derivative thereof, preferably wherein the derivative is selected from the group consisting of 2-fluoroadipic acid, [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n, 2-fluoro-1,6-hexanediol, a fluorinated polyester, a fluorinated polyurethane, a fluorinated acrylic, a fluorinated adhesive and a fluorinated dye.
In one aspect, the present disclosure provides 2-fluoro-cis,cis-muconate or a derivative thereof obtainable by the methods described herein, preferably wherein the derivative is selected from the group consisting of 2-fluoroadipic acid, [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n, 2-fluoro-1,6-hexanediol, a fluorinated polyester, a fluorinated polyurethane, a fluorinated acrylic, a fluorinated adhesive and a fluorinated dye.
In one aspect, the present disclosure provides a nucleic acid construct for modifying a bacterial cell comprising:
In one aspect, the present disclosure provides a nucleic acid construct for modifying a bacterial cell comprising:
In one aspect, the present disclosure provides a nucleic acid construct for modifying a bacterial cell comprising:
In another aspect, the present disclosure provides a kit of parts comprising:
Herein is provided a bacterial cell capable of producing 2-fluoro-cis,cis-muconate and derivatives thereof, in a bacterial cell, from the precursor compound 3-fluorobenzoate. The inventors have found that balancing the expression of enzymes encoded by the ben and cat operon in Pseudomonas putida enables efficient conversion of 3-fluorobenzoate to 2-fluoro-cis,cis-muconate with high titers in a bacterial cell, while avoiding the accumulation of the toxic intermediate 3-fluorocatechol. The cell can be engineered as described herein.
Herein are also provided methods for producing 2-fluoro-cis,cis-muconic acid and derivatives thereof. The bacterial cells and nucleic acid constructs described herein are useful for bacterial cell-based production of 2-fluoro-cis,cis-muconic acid and derivatives thereof, including 2-fluoroadipic acid, [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n (a fluorinated version of the polyamide Nylon 6-6), 2-fluoro-1,6-hexanediol, fluoro-terephthalic acid, fluoro-trimellitic acid, fluorinated polyesters, fluorinated polyurethanes, fluorinated acrylics, fluorinated adhesives and fluorinated dyes.
Herein is also provided 2-fluoro-cis,cis-muconate or a derivative thereof, preferably wherein the derivative is selected from the group consisting of 2-fluoroadipic acid, [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n, 2-fluoro-1,6-hexanediol, a fluorinated polyester, a fluorinated polyurethane, a fluorinated acrylic, a fluorinated adhesive and a fluorinated dye.
Herein is also provided 2-fluoro-cis,cis-muconate or a derivative thereof obtainable by the methods described herein, preferably wherein the derivative is selected from the group consisting of 2-fluoroadipic acid, [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n, 2-fluoro-1,6-hexanediol, a fluorinated polyester, a fluorinated polyurethane, a fluorinated acrylic, a fluorinated adhesive and a fluorinated dye.
Herein are also provided nucleic acid constructs for modifying a bacterial cell and useful to perform the present methods.
Also provided herein is a kit of parts comprising a bacterial cell, for example a bacterial cell as described herein, and/or a nucleic acid construct as described herein, and instructions for use.
2-fluoro-cis,cis-muconate is also known as 2-fluoro-cis,cis-muconic acid, 2-fluoro-2,4-hexadienedioic acid or PTYIDKBTGREHJW-TZFCGSKZSA-N. The chemical structure of 2-fluoro-cis,cis-muconate is as shown in formula I:
“2-fluoro-cis,cis-muconate derivatives” as used herein refers to compounds obtainable from 2-fluoro-cis,cis-muconate, such as 2-fluoroadipic acid, [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n or 2-fluoro-1,6-hexanediol, a fluorinated polyester, a fluorinated polyurethane, a fluorinated acrylic, a fluorinated adhesive and a fluorinated dye. The term “derivative” in this context thus refers to compounds which can be obtained by converting 2-fluoro-cis,cis-muconate into another compound, such as the previously listed compounds.
Functional variant: the term is herein applied to functional variants of enzymes, i.e. modified versions of the enzyme, or homologous enzymes originating from a different species, which retain some or all the catalytic activity of the original enzyme. Functional variants may have been modified by introducing mutations which confer e.g. increased activity, a change in intracellular localisation, prolonged half-life, among others, but retain the ability to perform the same enzymatic reaction as the enzymes they are derived from, albeit possibly to a different extent. Activities of functional variants may be determined as described elsewhere herein.
“Identity”, “similarity” and “homology” with respect to a polynucleotide (or polypeptide) is defined herein as the percentage of nucleic acids (or amino acids) in the candidate sequence that are identical with 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/similarity/homology, and considering any conservative substitutions according to the NCIUB rules (hftp://www.chem.qmul.ac.uk/iubmb/misc/naseq.html; NC-IUB, Eur J Biochem (1985) 150: 1-5) as part of the sequence identity. Neither 5′ or 3′ extensions nor insertions result in a reduction of identity, similarity or homology. Methods and computer programs for the alignments are well known in the art.
By “encoding” or “encoded”, in the context of a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid or polynucleotide encoding a protein may comprise non-translated sequences, e.g. introns, within translated regions of the nucleic acid, or may lack such intervening non-translated sequences, e.g. in cDNA. The information by which a protein is encoded is specified by the use of codons.
The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (promoter and terminator).
As used herein, “expression” in the context of nucleic acids is to be understood as the transcription and accumulation of sense mRNA or antisense RNA derived from a nucleic acid fragment. “Expression” used in the context of proteins refers to translation of mRNA into a polypeptide.
The term “overexpression” as used herein refers to a process by which a gene comprising a sequence that encodes a polypeptide is artificially expressed in a modified cell to produce a level of expression of the encoded polypeptide that exceeds the level of expression of the same polypeptide in an unmodified, or in a reference host cell. Thus, while the term is typically used in conjunction with a gene, the term “overexpression” may also be used in conjunction with a protein to refer to the increased level of a protein resulting from the overexpression of its encoding gene. In some embodiments, overexpression of a gene encoding a protein is achieved by increasing the number of copies of the gene that encodes the protein. In other embodiments, overexpression of a gene encoding a protein is achieved by increasing the binding strength of the promoter region and/or the ribosome binding site in such a way to increase the transcription and/or the translation of the gene that encodes the protein. In other embodiments, overexpression can be achieved by increasing the number of copies of a gene and increasing the binding strength of the promoter region and/or the ribosome binding site. In some embodiments, the overexpression of a gene encoding a protein results from the expression of at least one copy of the corresponding encoding polynucleotide present on a multicopy plasmid that has been introduced into a host cell. In other embodiments, the overexpression of a gene encoding a protein results from the expression of two or more copies of the corresponding encoding polynucleotide that are integrated into the genome of the host cell. For expression of heterologous proteins where no native expression level exists for comparison, “overexpression” refers to expressing the protein at a medium or strong level in the cell.
“Polycistronic” as defined herein describes a type of messenger RNA that can encode more than one polypeptide separately within the same RNA molecule. Bacterial messenger RNA (mRNA) is generally polycistronic.
“Translational coupling” as defined herein refers to the interdependence of translation efficiency of neighbouring genes encoded within an operon. The degree of coupling may be quantified by measuring how the translation rate of a gene is modulated by the translation rate of its upstream gene. The polycistronic mRNAs of bacteria enable translational coupling of two or more genes, whereby translation of a downstream gene depends on the translation of the upstream gene. A “strong translational coupler” as defined herein denotes a translational coupler whose expression strength value is within the highest 20% of synthetic translation initiation sequences developed and characterized by Mutalik et al. (2013) and, in the case of Pseudomonas species, as defined by Zobel et al. (2015). The strength of translational couplers in combination with different promoters has been tested using fluorescent proteins as described by Zobel et al. (2015). Zobel et al. characterized a promotor library using the same translational coupler (BCD2, the strongest one) as reference. Different BCD sequences were characterized in P. putida in the present disclosure (See
An “inducible promoter” in the context of the present disclosure refers to a promoter which is regulated or activated under certain conditions, such as light, chemical concentration, protein concentration, conditions in an organism, cell, or organelle, etc. An example of an inducible promoter is the Pm promoter from Pseudomonas putida mt-2 which is controlled by the activator protein XylS that responds to a range of benzoic acid derivatives by inducing expression of the gene under the control of the Pm promoter. In essence, expression under the control of an inducible promoter is “switched on” or increased in response to an applied stimulus. The nature of the stimulus varies between promoters and may include the above environmental factors. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The term “constitutive promoter” may be understood as an extensively unregulated promoter that allows continual transcription of its associated gene.
Classification of promoter strength as “weak”, “medium-strength” or “strong” refers to the degree to which the promoter alters the rate of transcription initiation from its associated gene compared to the endogenous level. This can result in either an increase or a decrease in the associated gene transcription compared to endogenous levels. The skilled person knows whether a given promoter is classified as a weak, medium-strength or strong promoter. Further examples of promoters and promoter systems and their classification as weak, medium-strength or strong are shown in
BenABC: benzoate 1,2-dioxygenase (EC: 1.14.12.10)
benzoate+NADH+H++O2<=>cyclohexadiene-cis,cis-1,2-diol-1-carboxylate+NAD+
The enzyme catalyzes conversion of a benzoate to the corresponding cyclohexadiene-cis,cis-1,2-diol-1-carboxylate in the presence of NADH, H+ and oxygen. For example it catalyzes the conversion of 3-fluorobenzoate into 3-fluorocyclohexadiene-cis,cis-1,2-diol-1-carboxylate. Benzoate 1,2-dioxygenase activity can be determined spectrophotometrically by measuring the decrease in absorbance at 340 nm of NADH or polarographically by measuring the oxygen consumption (Yamaguchi and Fujisawa al., 1980).
BenD: benzoate-1,2-dihydrodiol dehydrogenase (EC: 1.3.1.25)
cyclohexadiene-cis,cis-1,2-diol-1-carboxylate+NAD+<=>catechol+CO2+NADH+H+
The enzyme catalyzes conversion of a cyclohexadiene-cis,cis-1,2-diol-1-carboxylate to the corresponding catechol in the presence of NAD+. For example it catalyzes the conversion of 3-fluorocyclohexadiene-cis,cis-1,2-diol-1-carboxylate into 3-fluorocatechol. Benzoate-1,2-dihydrodiol dehydrogenase activity can be determined spectrophotometrically by measuring the increase in absorbance at 340 nm of NADH (Reiner, 1972).
CatA-I: catechol 1,2-dioxygenase (EC: 1.13.11.1)
catechol+O2<=>cis,cis-muconate
The enzyme catalyzes conversion of a catechol to the corresponding cis,cis-muconate in the presence of oxygen. For example it catalyzes the conversion of 3-fluorocatechol into 2-fluoro-cis,cis-muconate. Catechol 1,2-dioxygenase activity can be determined spectrophotometrically by monitoring the increase in cis,cis-muconate concentration at 260 nm (Wojcieszyńska et al., 2011).
CatA-II: catechol 1,2-dioxygenase (EC: 1.13.11.1)
catechol+O2<=>cis,cis-muconate
The enzyme catalyzes conversion of a catechol to the corresponding cis,cis-muconate in the presence of oxygen. For example it catalyzes the conversion of 3-fluorocatechol into 2-fluoro-cis,cis-muconate. Catechol 1,2-dioxygenase activity can be determined spectrophotometrically by monitoring the increase in cis,cis-muconate concentration at 260 nm (Wojcieszyńska et al., 2011).
The present disclosure relates to a bacterial cell capable of producing 2-fluoro-cis,cis-muconate and derivatives thereof, in a bacterial cell, from the precursor compound 3-fluorobenzoate. The inventors have found that balancing the expression of enzymes encoded by the ben and cat operon in Pseudomonas putida using suitable promoters as described herein in detail enables efficient conversion of 3-fluorobenzoate to 2-fluoro-cis,cis-muconate with high titers in a bacterial cell, while avoiding the accumulation of the toxic intermediate 3-fluorocatechol as well as the toxic effects of catechol 1,2-dioxygenase overexpression. The cell can be engineered as described herein.
Herein is provided a bacterial cell capable of producing 2-fluoro-cis,cis-muconate and optionally derivatives thereof, in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
Herein is also provided a bacterial cell capable of producing 2-fluoro-cis,cis-muconate and optionally derivatives thereof, in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
In some bacterial cells, more than one of each of the before-mentioned classes of enzymes may be expressed. Thus in some cases, the cell may contain more than one benzoate 1,2-dioxygenase, benzoate-1,2-dihydrodiol dehydrogenase and/or catechol 1,2-dioxygenase.
In some embodiments, the present disclosure provides a bacterial cell capable of producing 2-fluoro-cis,cis-muconate and optionally derivatives thereof, in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
In some embodiments, the bacterial cell is a non-pathogenic organism.
In some embodiments, the bacterial cell can tolerate toxic compounds, such as fluorinated compounds.
In some embodiments, the bacterial cell belongs to the Pseudomonas genus, the Burkholderia genus, the Escherichia genus, the Rhodococcus genus, the Bacillus genus or the Vibrio genus.
In some embodiments, the bacterial cell may be selected from the group Pseudomonas putida, Pseudomonas knackmussi, Pseudomonas fluorescens, Pseudomonas taiwanesis, Pseudomonas syringae, Pseudomonas stutzeri, Pseudomonas oleovorans, Pseudomonas mendocina, Burkholderia fungorum, Escherichia coli, Rhodococcus rhodochrous, Bacillus subtilis, Bacillus cereus, Bacillus megaterium and Vibrio natriegens. In preferred embodiments, the bacterial cell is a Pseudomonas putida cell, such as a Pseudomonas putida KT2440 cell.
The present bacterial cell requires the precursor molecule 3-fluorobenzoate or a salt thereof in order to produce 2-fluoro-cis,cis-muconate. 3-fluorobenzoate or a salt thereof may be provided directly in the medium or it may be derived from a precursor molecule through a metabolic process by the bacterial cell; said precursor molecule may be synthesized by the cell, and/or it may be provided in the medium.
Production of 2-Fluoro-Cis,Cis-Muconate
The bacterial cell of the present disclosure can produce 2-fluoro-cis,cis-muconate. This requires that the bacterial cell expresses a benzoate 1,2-dioxygenase capable of converting 3-fluorobenzoate to 3-fluorocyclohexadiene-cis,cis-1,2-diol-1-carboxylate, a benzoate-1,2-dihydrodiol dehydrogenase capable of converting 3-fluorocyclohexadiene-cis,cis-1,2-diol-1-carboxylate to 3-fluorocatechol, and a catechol 1,2-dioxygenase capable of converting 3-fluorocatechol to 2-fluoro-cis,cis-muconate.
A benzoate 1,2-dioxygenase (EC: 1.3.1.25) catalyzes the conversion of a benzoate to the corresponding cyclohexadiene-cis,cis-1,2-diol-1-carboxylate in the presence of NADH, H+ and oxygen according to the following reaction:
benzoate+NADH+H++O2<=>cyclohexadiene-cis,cis-1,2-diol-1-carboxylate+NAD+
For example, the benzoate 1,2-dioxygenase may catalyze the conversion of 3-fluorobenzoate into 3-fluorocyclohexadiene-cis,cis-1,2-diol-1-carboxylate. The benzoate 1,2-dioxygenase activity of an enzyme may be tested as per the procedure of Yamaguchi & Fujisawa (1980). A functional variant of a benzoate 1,2-dioxygenase may be identified by testing if it retains some or all of the benzoate 1,2-dioxygenase catalytic activity when tested as described herein.
A benzoate-1,2-dihydrodiol dehydrogenase (EC: 1.3.1.25) catalyzes conversion of a cyclohexadiene-cis,cis-1,2-diol-1-carboxylate to the corresponding catechol in the presence of NAD+ according to the following reaction:
cyclohexadiene-cis,cis-1,2-diol-1-carboxylate+NAD+<=>catechol+CO2+NADH+H+
For example, the benzoate-1,2-dihydrodiol dehydrogenase may catalyze the conversion of 3-fluorocyclohexadiene-cis,cis-1,2-diol-1-carboxylate into 3-fluorocatechol. The benzoate-1,2-dihydrodiol dehydrogenase activity of an enzyme may be tested as described by Reiner (1972). A functional variant of a benzoate-1,2-dihydrodiol dehydrogenase may be identified by testing if it retains some or all of the benzoate-1,2-dihydrodiol dehydrogenase catalytic activity when tested as described herein.
A catechol 1,2-dioxygenase (EC: 1.13.11.1) catalyzes the conversion of a catechol to the corresponding cis,cis-muconate in the presence of oxygen according to the following reaction:
catechol+O2<=>cis,cis-muconate
For example, the catechol 1,2-dioxygenase may catalyze the conversion of 3-fluorocatechol into 2-fluoro-cis,cis-muconate. The catechol 1,2-dioxygenase activity of an enzyme may be tested by the procedure of Wojcieszyńska et al. (2011). A functional variant of a catechol 1,2-dioxygenase may be identified by testing if it retains some or all of the catechol 1,2-dioxygenase catalytic activity when tested as described herein.
In some embodiments, the benzoate 1,2-dioxygenase is a native or heterologous benzoate 1,2-dioxygenase. In some embodiments, the benzoate 1,2-dioxygenase is BenABC or a functional variant thereof having 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% homology or identity thereto. The benzoate 1,2-dioxygenase is capable of converting 3-fluorobenzoate to 3-fluorocyclohexadiene-cis,cis-1,2-diol-1-carboxylate. In some embodiments, the benzoate 1,2-dioxygenase comprises:
In some embodiments, the benzoate 1,2-dioxygenase consists of BenA, BenB and BenC or functional variants thereof having at least 80% homology or identity thereto.
In some preferred embodiments, the benzoate 1,2-dioxygenase originates from the organism Pseudomonas putida.
In some embodiments, the benzoate 1,2-dioxygenase, for example a benzoate 1,2-dioxygenase consisting of BenA, BenB and BenC or functional variants thereof having at least 80% homology or identity thereto, is expressed under the control of a medium-strong constitutive promoter, whereby the benzoate 1,2-dioxygenase is constitutively overexpressed. In some embodiments, the medium-strong constitutive promoter is Ptac (SEQ ID NO: 15) or a variant thereof having 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% homology or identity thereto. In some embodiments, the native promoter of the benzoate 1,2-dioxygenase, such as the Pben promoter (SEQ ID NO: 13) or a variant thereof having 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% homology or identity thereto, has been replaced with a medium-strong constitutive promoter as described herein above.
In some embodiments, the medium-strong constitutive promoter is P14g (SEQ ID NO: 50) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is P14f (SEQ ID NO: 53) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is P14e (SEQ ID NO: 54) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is P14d (SEQ ID NO: 55) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is BG19 (SEQ ID NO: 56) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is BG34 (SEQ ID NO: 57) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is P14c (SEQ ID NO: 58) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is PEM7 (SEQ ID NO: 59) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is J23119 (SEQ ID NO: 60) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is J23101 (SEQ ID NO: 61) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is J23107 (SEQ ID NO: 62) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is JEc3 (SEQ ID NO: 63) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is JEa3 (SEQ ID NO: 64) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is JE1611 (SEQ ID NO: 65) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the medium-strong constitutive promoter is JEa2 (SEQ ID NO: 66) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto.
In some embodiments, the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase, for example the benzoate 1,2-dioxygenase consisting of BenA, BenB and BenC or functional variants thereof having at least 80% homology or identity thereto, is unmodified. In some embodiments, the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase subunit BenA described herein above is unmodified. In some embodiments, the translation initiation region consisting of the 34 base pairs upstream from the start codon of said gene comprises or consists of SEQ ID NO: 24 or a variant thereof having 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% homology or identity thereto.
Any suitable nucleic sequence may be used to replace the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase subunit BenA. In some embodiments, the translation initiation region consisting of the 34 base pairs upstream from the start codon of the gene encoding benzoate 1,2-dioxygenase subunit BenA comprises or consists of a translation initiation region sequence with a predicted translation rate of 100 to 5,000, predicted using the RBS Calculator available online at https://salislab.net/software/design_rbs_calculator.
In some embodiments, the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase subunit BenA comprises or consists of BCD22 (SEQ ID NO: 68) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase subunit BenA comprises or consists of BCD16 (SEQ ID NO: 69) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase subunit BenA comprises or consists of BCD23 (SEQ ID NO: 70) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase subunit BenA comprises or consists of BCD8 (SEQ ID NO: 71) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase subunit BenA comprises or consists of BCD21 (SEQ ID NO: 72) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase subunit BenA comprises or consists of MCD18 (SEQ ID NO: 73) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase subunit BenA comprises or consists of MCD8 (SEQ ID NO: 74) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase subunit BenA comprises or consists of MCD14 (SEQ ID NO: 75) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase subunit BenA comprises or consists of MCD15 (SEQ ID NO: 76) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the translation initiation region of the gene encoding the benzoate 1,2-dioxygenase subunit BenA comprises or consists of MCD22 (SEQ ID NO: 77) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto.
In some embodiments, the benzoate-1,2-dihydrodiol dehydrogenase (EC: 1.3.1.25) is a native or heterologous benzoate-1,2-dihydrodiol dehydrogenase. In some embodiments, the benzoate-1,2-dihydrodiol dehydrogenase is BenD (SEQ ID NO: 7) or a functional variant thereof having 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% homology or identity thereto. In some preferred embodiments, the benzoate-1,2-dihydrodiol dehydrogenase is native to Pseudomonas putida.
In some embodiments, the translation initiation region and/or the promoter of the gene encoding the benzoate-1,2-dihydrodiol dehydrogenase, for example BenD, is unmodified. In some embodiments, the translation initiation region and/or the promoter of the gene encoding the benzoate-1,2-dihydrodiol dehydrogenase BenD described herein above is unmodified. In some embodiments, the genomic region upstream of said gene containing said translation initiation region and/or promoter comprises or consists of SEQ ID NO: 25 or a variant thereof having 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% homology or identity thereto.
In some embodiments, the bacterial cell expresses at least a first native or heterologous catechol 1,2-dioxygenase (EC: 1.13.11.1). In some embodiments, the first catechol 1,2-dioxygenase is CatA-I (SEQ ID NO: 9) or a functional variant thereof having 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% homology or identity thereto. In some preferred embodiments, the first catechol 1,2-dioxygenase is native to the organism Pseudomonas putida.
In some embodiments, the first catechol 1,2-dioxygenase, for example CatA-I, is expressed under the control of a first expression module comprising a first promoter, said first expression module further comprising a first strong translation initiation sequence such as a first strong translational coupler.
In some embodiments, the first promoter of the first expression module is a medium-to-weak constitutive promoter, such as P14b (SEQ ID NO: 49), whereby the catechol 1,2-dioxygenase is constitutively expressed.
In some embodiments, the first catechol 1,2-dioxygenase, for example CatA-I, is expressed under the control of a first expression module comprising a medium-to-weak constitutive promoter, said first expression module further comprising a first strong translational coupler, whereby said catechol 1,2-dioxygenase is constitutively expressed. In some embodiments, the medium-to-weak constitutive promoter of said first expression module is P14b (SEQ ID NO: 49), and the first strong translational coupler is BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16), or variants thereof having 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% homology or identity thereto, preferably the first strong translational coupler is BCD10 (SEQ ID NO: 16) or a variant thereof having 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% homology or identity thereto.
In some embodiments, the first promoter of the first expression module is a first inducible promoter, optionally coupled with a sequence encoding an associated activator protein.
In some embodiments, the first catechol 1,2-dioxygenase, for example CatA-I, is expressed under the control of a first expression module comprising a first inducible promoter coupled with a sequence encoding an associated activator protein, said first expression module further comprising a first strong translational coupler, whereby said catechol 1,2-dioxygenase is expressed strongly in response to 3-fluorobenzoate. In other words, when 3-fluorobenzoate is present in the media, the strong translational coupler ensures that the expression change mediated by the inducible promoter strongly correlates with the amount of subsequently transcribed mRNA that is translated into protein. The first expression module thus ensures that the cell comprises many molecules of catechol 1,2-dioxygenase only when 3-fluorobenzoate is present. In some embodiments, the first inducible promoter of said first expression module is the Pm variant ML1-17 (SEQ ID NO: 18) or a variant thereof having 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% homology or identity thereto, the associated activator protein is XylS (SEQ ID NO: 21) or a functional variant thereof having 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% homology or identity thereto, and the first strong translational coupler is BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16), or variants thereof having 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% homology or identity thereto, preferably the first strong translational coupler is BCD10 (SEQ ID NO: 16) or a variant thereof having at least 80% homology or identity thereto.
In some embodiments the first strong translational coupler is BCD11 (SEQ ID NO: 44), BCD7 (SEQ ID NO: 45), BCD6 (SEQ ID NO: 17), BCD5 (SEQ ID NO: 19), or BCD1 (SEQ ID NO: 23) or variants thereof having 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% homology or identity thereto.
In some embodiments, the bacterial cell expresses at least a first and a second native or heterologous catechol 1,2-dioxygenases (EC: 1.13.11.1). In some embodiments, the first catechol 1,2-dioxygenase is CatA-I (SEQ ID NO: 9), and the second catechol 1,2-dioxygenase is CatA-II (SEQ ID NO: 11), or functional variants thereof having 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% homology or identity thereto. In some preferred embodiments, the first and the second catechol 1,2-dioxygenases are native to Pseudomonas putida.
In some embodiments, the expression of the first catechol 1,2-dioxygenase, for example CatA-I, described herein is under the control of a first expression module comprising a first promoter, said first expression module further comprising a first strong translation initiation sequence such as a first strong translational coupler, and the expression of said second catechol 1,2-dioxygenase is under the control of a second expression module comprising a second promoter, said second expression module further comprising a second strong translational initiation sequence such as a second strong translational coupler.
In some embodiments, the first and/or the second promoter is a medium-to-weak constitutive promoter, whereby the catechol 1,2-dioxygenase is constitutively expressed. Thus, in some embodiments, the first promoter is a first medium-to-weak constitutive promoter. In some embodiments, the second promoter is a second medium-to-weak constitutive promoter. In some embodiments, the first and the second promoters are medium-to-weak constitutive promoters.
In some embodiments, the medium-to-weak constitutive promoter is P14b (SEQ ID NO: 49) or a variant thereof having 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% homology or identity thereto.
P14b has a transcriptional strength that is 25% that of the strongest promoter reported for P. putida: P14g (characterized in
The first and the second promoters may be different promoters. In some embodiments, the first and the second promoters are the same promoter. Thus, the first and the second promoters may be the same medium-to-weak constitutive promoter, such as P14b (SEQ ID NO: 49) or a variant thereof having at least 80% homology or identity thereto.
In some embodiments, the first and/or the second strong translation initiation sequence is BCD2 (SEQ ID NO: 22) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the first and/or the second strong translation initiation sequence is BCD10 (SEQ ID NO: 16) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto.
Any suitable nucleic acid sequence may be used to replace the translation initiation region of the gene encoding the first and/or the second catechol 1,2-dioxygenase. In some embodiments, the first and/or the second strong translation initiation sequence has similar translation initiation strength as BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16). In some embodiments, the first and/or the second strong translation initiation sequence has a predicted translation rate of 3,000 to 80,000, predicted using the RBS Calculator available online at https://salislab.net/software/design_rbs_calculator.
In some embodiments, the first and/or the second strong translation initiation sequence is BCD1 (SEQ ID NO: 23) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the first and/or the second strong translation initiation sequence is BCD5 (SEQ ID NO: 78) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the first and/or the second strong translation initiation sequence is BCD6 (SEQ ID NO: 17) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the first and/or the second strong translation initiation sequence is BCD7 (SEQ ID NO: 45) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the first and/or the second strong translation initiation sequence is BCD9 (SEQ ID NO: 79) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the first and/or the second strong translation initiation sequence is BCD11 (SEQ ID NO: 44) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the first and/or the second strong translation initiation sequence is BCD12 (SEQ ID NO: 80) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the first and/or the second strong translation initiation sequence is BCD13 (SEQ ID NO: 81) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the first and/or the second strong translation initiation sequence is BCD18 (SEQ ID NO: 83) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the first and/or the second strong translation initiation sequence is BCD19 (SEQ ID NO: 82) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto. In some embodiments, the first and/or the second strong translation initiation sequence is BCD20 (SEQ ID NO: 84) or a variant thereof having at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99% homology or identity thereto.
The first and the second strong translational initiation sequences may be different strong translational initiation sequences. In some embodiments, the first and the second strong translational initiation sequences are the same strong translational initiation sequence.
In some embodiments, the expression of the first catechol 1,2-dioxygenase described herein above, for example CatA-I, is under the control of a first expression module comprising a medium-low constitutive promoter, said first expression module further comprising a first strong translational coupler, whereby said first catechol 1,2-dioxygenase is constitutively expressed, and the expression of the second catechol 1,2-dioxygenase, for example CatA-II, described herein above is under the control of a second expression module comprising a second constitutive medium-low promoter, said second expression module further comprising a second strong translational coupler whereby said second catechol 1,2-dioxygenase is constitutively expressed. In some embodiments the first medium-low constitutive promoter of said first expression module is P14b (SEQ ID NO: 49) or a variant thereof having 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% homology or identity thereto, and the first strong translational coupler is BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16), or a variant thereof having 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% homology or identity thereto, preferably the first strong translational coupler is BCD10 (SEQ ID NO: 16) or a variant thereof having 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% homology or identity thereto. In some embodiments, the second medium-low constitutive promoter of said second expression module is P14b (SEQ ID NO: 49) or a variant thereof having 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% homology or identity thereto, and the second strong translational coupler is BCD10 (SEQ ID NO: 16) or a variant thereof having 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% homology or identity thereto.
In some embodiments, the first and/or the second promoter is a first and/or a second inducible promoter, optionally wherein the first and/or second promoter is coupled with a sequence encoding an associated activator protein.
In some embodiments, the first and/or the second inducible promoter is the Pm variant ML1-17 (SEQ ID NO: 18) or a variant thereof having 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% homology or identity thereto. In some embodiments, the first and/or the second inducible promoter is Pm (SEQ ID NO: 15) or a variant thereof having 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% homology or identity thereto.
In some embodiments, the associated activator protein is XylS (SEQ ID NO: 21) or a functional variant thereof having 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% homology or identity thereto.
Other useful inducible promoter systems that mediate a similar transcription level at a certain inducer compound concentration includes the LacIq/Ptrc, CprK1/PDB3, ChnR/PchnB, and RhaS-RhaR/Prha systems.
In some embodiments, the expression of the first catechol 1,2-dioxygenase described herein above is under the control of a first expression module comprising a first inducible promoter coupled with a sequence encoding an associated activator protein, said first expression module further comprising a first strong translational coupler, whereby said first catechol 1,2-dioxygenase is expressed strongly in response to 3-fluorobenzoate, and the expression of the second catechol 1,2-dioxygenase described herein above is under the control of a second expression module comprising a second inducible promoter, said second expression module further comprising a second strong translational coupler whereby said second catechol 1,2-dioxygenase is expressed strongly in response to 3-fluorobenzoate. In some embodiments the first inducible promoter of said first expression module is the Pm variant ML1-17 (SEQ ID NO: 18), the associated activator protein is XylS (SEQ ID NO: 21), and the first strong translational coupler is BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16), or variants thereof having 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% homology or identity thereto, preferably the first strong translational coupler is BCD10 (SEQ ID NO: 16) or a variant thereof having 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% homology or identity thereto. In some embodiments the first strong translational coupler is BCD11 (SEQ ID NO: 44), BCD7 (SEQ ID NO: 45), BCD6 (SEQ ID NO: 17), BCD5 (SEQ ID NO: 19), or BCD1 (SEQ ID NO: 23) or variants thereof having 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% homology or identity thereto. In some embodiments, the second inducible promoter of said second expression module is Pm (SEQ ID NO: 15), and the second strong translational coupler is BCD10 (SEQ ID NO: 16), or variants thereof having 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% homology or identity thereto.
In some embodiments, the bacterial cell expresses the benzoate 1,2-dioxygenase BenABC consisting of BenA (SEQ ID NO: 1), BenB (SEQ ID NO: 2) and BenC (SEQ ID NO: 2), the benzoate-1,2-dihydrodiol dehydrogenase BenD (SEQ ID NO: 7), and the catechol 1,2-dioxygenase CatA-I (SEQ ID NO: 9), or functional variants thereof as described herein.
In some embodiments, the bacterial cell expresses the benzoate 1,2-dioxygenase BenABC consisting of BenA (SEQ ID NO: 1), BenB (SEQ ID NO: 2) and BenC (SEQ ID NO: 2), the benzoate-1,2-dihydrodiol dehydrogenase BenD (SEQ ID NO: 7), and the catechol 1,2-dioxygenases CatA-I (SEQ ID NO: 9) and CatA-II (SEQ ID NO: 11), or functional variants thereof as described herein.
In some embodiments, the bacterial cell expresses the benzoate 1,2-dioxygenase BenABC consisting of BenA (SEQ ID NO: 1), BenB (SEQ ID NO: 2) and BenC (SEQ ID NO: 2), the benzoate-1,2-dihydrodiol dehydrogenase BenD (SEQ ID NO: 7), and the catechol 1,2-dioxygenase CatA-I (SEQ ID NO: 9), wherein BenABC is expressed under the control of the medium-strong constitutive promoter Ptac (SEQ ID NO: 15) and CatA-I is expressed under the control of the first expression module comprising or consisting of the medium-to-weak constitutive promoter P14b (SEQ ID NO: 49), and the first strong translational coupler BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16), or functional variants thereof as described herein.
In some embodiments, the bacterial cell expresses the benzoate 1,2-dioxygenase BenABC consisting of BenA (SEQ ID NO: 1), BenB (SEQ ID NO: 2) and BenC (SEQ ID NO: 2), the benzoate-1,2-dihydrodiol dehydrogenase BenD (SEQ ID NO: 7), and the catechol 1,2-dioxygenase CatA-I (SEQ ID NO: 9), wherein BenABC is expressed under the control of the medium-strong constitutive promoter Ptac (SEQ ID NO: 15) and CatA-I is expressed under the control of the first expression module comprising or consisting of the first inducible promoter Pm variant ML1-17 (SEQ ID NO: 18), the associated activator protein is XylS (SEQ ID NO: 21), and the first strong translational coupler BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16), or functional variants thereof as described herein.
In some embodiments, the bacterial cell expresses the benzoate 1,2-dioxygenase BenABC consisting of BenA (SEQ ID NO: 1), BenB (SEQ ID NO: 2) and BenC (SEQ ID NO: 2), the benzoate-1,2-dihydrodiol dehydrogenase BenD (SEQ ID NO: 7), and the catechol 1,2-dioxygenases CatA-I (SEQ ID NO: 9) and CatA-II (SEQ ID NO: 11), wherein BenABC is expressed under the control of the medium-strong constitutive promoter Ptac (SEQ ID NO: 15) and CatA-I is expressed under the control of the first expression module comprising or consisting of the medium-to-weak constitutive promoter P14b (SEQ ID NO: 49), and the first strong translational coupler BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16), and wherein CatA-II is expressed under the control of the second expression module comprising or consisting of the medium-to-weak constitutive promoter P14b (SEQ ID NO: 49) and the second strong translational coupler BCD10 (SEQ ID NO: 16), or functional variants thereof as described herein.
In some embodiments, the bacterial cell expresses the benzoate 1,2-dioxygenase BenABC consisting of BenA (SEQ ID NO: 1), BenB (SEQ ID NO: 2) and BenC (SEQ ID NO: 2), the benzoate-1,2-dihydrodiol dehydrogenase BenD (SEQ ID NO: 7), and the catechol 1,2-dioxygenases CatA-I (SEQ ID NO: 9) and CatA-II (SEQ ID NO: 11), wherein BenABC is expressed under the control of the medium-strong constitutive promoter Ptac (SEQ ID NO: 15) and CatA-I is expressed under the control of the first expression module comprising or consisting of the first inducible promoter Pm variant ML1-17 (SEQ ID NO: 18), the associated activator protein XylS (SEQ ID NO: 21), and the first strong translational coupler BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16), and wherein CatA-II is expressed under the control of the second expression module comprising or consisting of the inducible promoter Pm (SEQ ID NO: 15) and the second strong translational coupler BCD10 (SEQ ID NO: 16), or functional variants thereof as described herein.
In some embodiments, the bacterial cell is capable of producing 2-fluoro-cis,cis-muconate with a titer of at least 500 mg/L, such as at least 750 mg/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 6 g/L, such as at least 7 g/L, such as at least 8 g/L, such as at least 9 g/L, such as at least 10 g/L, such as at least 15 g/L, such as at least 20 g/L, such as at least 25 g/L, such as at least 30 g/L, such as at least 40 g/L, such as at least 50 g/L, such as at least 60 g/L, such as at least 80 g/L, such as at least 100 g/L, such as at least 150 g/L, such as at least 200 g/L, such as at least 250 g/L, such as at least 300 g/L or more.
As sugars may comprise the main carbon source for the bacterial cell, and without being bound by theory, modifications in glucose metabolism to increase growth performance on sugars and to avoid by-product formation typically associated with glucose utilization, in order to allow a faster growth rate of the cell may further increase the titers of 2-fluoro-cis,cis-muconate.
In some embodiments, the bacterial cell is as described above and further comprises modifications resulting in increased growth performance on sugars. In some embodiments, the bacterial cell further comprises deletion of the global catabolite repression control protein Crc (SEQ ID NO: 36) or a functional variant thereof having 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% homology or identity thereto.
In some embodiments, the bacterial cell further comprises deletion of the global catabolite repression control protein Crc encoded by SEQ ID NO: 37 or a variant thereof having 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% homology or identity thereto.
Without being bound by theory, two predominant by-products of glucose consumption in Pseudomonas putida are gluconate and 2-ketogluconate, due to the constitutively expressed, periplasmic glucose dehydrogenase (Gcd), and gluconate dehydrogenase (Gad). With fast-growing wildtype-cells, those two compounds are usually co-consumed together with glucose and result in no adverse effects on the growth rate. However, under growth-restricting conditions such as in the presence of growth-inhibiting agents (e.g. fluorinated compounds), or with cells showing slower growth rates due to genomic modifications, the production of gluconate and 2-ketogluconate exceeds their consumption, which can cause a decrease in pH, which may adversely affect growth rates. It may thus be beneficial to decrease the formation of gluconate and 2-ketogluconate to further increase the titers of 2-fluoro-cis,cis-muconate.
In some embodiments, the bacterial cell further comprises modifications to decrease by-product formation typically associated with glucose utilization. In some embodiments, the bacterial cell further comprises a mutation resulting in partial or total loss of activity of at least one of the proteins selected from the group consisting of the gamma subunit of Gad (SEQ ID NO: 28), the flavoprotein subunit of Gad (SEQ ID NO: 30), the cytochrome c subunit of Gad (SEQ ID NO: 32), and Gcd (SEQ ID NO: 34) or functional variants thereof having 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% homology or identity thereto.
In some embodiments, the bacterial cell further comprises a mutation resulting in partial or total loss of activity of the gamma subunit of Gad encoded by SEQ ID NO: 29 or a variant thereof having 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% homology or identity thereto. In some embodiments, the bacterial cell further comprises a mutation resulting in partial or total loss of activity of the flavoprotein subunit of Gad encoded by SEQ ID NO: 31 or a variant thereof having 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% homology or identity thereto. In some embodiments, the bacterial cell further comprises a mutation resulting in partial or total loss of activity of the cytochrome c subunit of Gad encoded by SEQ ID NO: 33 or a variant thereof having 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% homology or identity thereto. In some embodiments, the bacterial cell further comprises a mutation resulting in partial or total loss of activity of Gcd encoded by SEQ ID NO: 35 or a variant thereof having 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% homology or identity thereto.
Because the present pathways require 3-fluorobenzoate as a first substrate, and without being bound by theory, it may be advantageous to modify the bacterial cell in such a manner that the uptake of 3-fluorobenzoate into the cell is increased, so that more 3-fluorobenzoate is available for conversion into 2-fluoro-cis,cis-muconate, thereby further increasing the titers of 2-fluoro-cis,cis-muconate.
In some embodiments, the bacterial cell further comprises modifications resulting in overexpression of genes encoding transporter proteins involved in 3-fluorobenzoate uptake. In some embodiments, the bacterial cell further comprises modifications resulting in overexpression one of the proteins selected from the group consisting of BenK (SEQ ID NO: 38), BenE-II (SEQ ID NO: 40), and NicP-I (SEQ ID NO: 42) or functional variants thereof having 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% homology or identity thereto.
In some embodiments, the bacterial cell further comprises modifications resulting in overexpression of BenK encoded by SEQ ID NO: 39 or a variant thereof having 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% homology or identity thereto. In some embodiments, the bacterial cell further comprises modifications resulting in overexpression of BenE-II encoded by SEQ ID NO: 41 or a variant thereof having 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% homology or identity thereto. In some embodiments, the bacterial cell further comprises modifications resulting in overexpression of NicP-1 encoded by SEQ ID NO: 43 or a variant thereof having 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% homology or identity thereto.
As the present bioconversion pathway comprises several oxygen-consuming reactions, and without being bound by theory, increasing intracellular oxygen availability may further increase flux through the pathway, and thus further increase the titers of 2-fluoro-cis,cis-muconate.
In some embodiments, the bacterial cell further expresses a bacterial haemoglobin, whereby intracellular oxygen availability is increased. In some embodiments, the bacterial haemoglobin is a heterologous bacterial hemoglobin (EC: 1.14.12.17). In some embodiments, the heterologous bacterial hemoglobin is VHb (SEQ ID NO: 26) or a functional variant thereof having 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% homology or identity thereto, to increase oxygen availability for the oxygen-consuming reactions of the bioconversion pathway.
In some embodiments, the bacterial cell further expresses the heterologous bacterial haemoglobin VHb encoded by SEQ ID NO: 27 or a variant thereof having 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% homology or identity thereto.
The bacterial cell may be as described herein.
In some embodiments, one or more of the genes encoding the benzoate 1,2-dioxygenase, the medium-strong constitutive promoter, the benzoate-1,2-dihydrodiol dehydrogenase, the first catechol 1,2-dioxygenase, the second catechol 1,2-dioxygenase, the first expression module and/or the second expression module are codon-optimized for the bacterial cell.
In some embodiments, one or more of the genes encoding the benzoate 1,2-dioxygenase, the medium-strong constitutive promoter, the benzoate-1,2-dihydrodiol dehydrogenase, the first catechol 1,2-dioxygenase, the second catechol 1,2-dioxygenase, the first expression module and/or the second expression module are present in multiple copy number.
In some embodiments, one or more of the genes encoding the benzoate 1,2-dioxygenase, the medium-strong constitutive promoter, the benzoate-1,2-dihydrodiol dehydrogenase, the first catechol 1,2-dioxygenase, the second catechol 1,2-dioxygenase, the first expression module and/or the second expression module are integrated in the genome of the bacterial cell.
In some embodiments, one or more of the genes encoding the benzoate 1,2-dioxygenase, the medium-strong constitutive promoter, the benzoate-1,2-dihydrodiol dehydrogenase, the first catechol 1,2-dioxygenase, the second catechol 1,2-dioxygenase, the first expression module and/or the second expression module are expressed from a vector such as a plasmid.
The present disclosure relates to methods for producing 2-fluoro-cis,cis-muconic acid and derivatives thereof. The bacterial cells and nucleic acid constructs described herein are useful for bacterial cell-based production of 2-fluoro-cis,cis-muconic acid and derivatives thereof, including 2-fluoroadipic acid, [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—], (a fluorinated version of the polyamide Nylon 6-6), 2-fluoro-1,6-hexanediol, fluoro-terephthalic acid, fluoro-trimellitic acid, fluorinated polyesters, fluorinated polyurethanes, fluorinated acrylics, fluorinated adhesives and fluorinated dyes.
Herein is provided a method of producing 2-fluoro-cis,cis-muconate, and optionally derivatives thereof, in a bacterial cell, said method comprising the steps of providing a bacterial cell and incubating said bacterial cell in a medium in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
Herein is provided a method of producing 2-fluoro-cis,cis-muconate, and optionally derivatives thereof, in a bacterial cell, said method comprising the steps of providing a bacterial cell and incubating said bacterial cell in a medium in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
In some embodiments, the present disclosure provides a method of producing 2-fluoro-cis,cis-muconate, and optionally derivatives thereof, in a bacterial cell, said method comprising the steps of providing a bacterial cell and incubating said bacterial cell in a medium in the presence of 3-fluorobenzoate or a salt thereof, said bacterial cell expressing:
In some embodiments, the benzoate 1,2-dioxygenase (EC: 1.14.12.10) expressed by the bacterial cell is a native or heterologous benzoate 1,2-dioxygenase, as described herein above in the section “Bacterial cell”, and may further comprise any of the modifications described in “Other modifications”. In particular, the bacterial cell may express BenABC or a functional variant thereof having at least 80% homology or identity thereto, as detailed above. In particular, the gene(s) encoding the benzoate 1,2-dioxygenase may be under the control of a specific promoter, such as Ptac or a variant thereof having at least 80% homology or identity thereto, as described herein above in the section “Bacterial cell”.
In some embodiments, the benzoate-1,2-dihydrodiol dehydrogenase (EC: 1.3.1.25) expressed by the bacterial cell is a native or heterologous benzoate-1,2-dihydrodiol dehydrogenase, as described herein above in the section “Bacterial cell”, and may further comprise any of the modifications described in “Other modifications”. In particular, the bacterial cell may express BenD or a functional variant thereof having at least 80% homology or identity thereto, as detailed above.
In some embodiments, the first and/or second catechol 1,2-dioxygenase (EC: 1.13.11.1) expressed by the bacterial cell is a native or heterologous catechol 1,2-dioxygenase, as described herein above in the section “Bacterial cell”, and may further comprise any of the modifications described in “Other modifications”. In particular, the bacterial cell may express CatA-I and/or CatA-II as detailed above. In particular, the expression of the gene(s) encoding the benzoate 1,2-dioxygenase(s) may be under the control of specific first and/or second expression module(s) as described herein above in the section “Bacterial cell”. In particular, as described herein above, the first expression module may comprise the P14b promoter or a variant thereof having at least 80% homology or identity thereto, and the strong translational coupler BCD2 or BCD10 or a variant thereof having at least 80% homology or identity thereto. In particular, as described herein above, the second expression module may comprise the P14b promoter or a variant thereof having at least 80% homology or identity thereto and the strong translational coupler BCD10 or a variant thereof having at least 80% homology or identity thereto. In particular, as described herein above, the first expression module may comprise the Pm promoter variant ML1-17 or a variant thereof having at least 80% homology or identity thereto, the associated activator protein XylS or a functional variant thereof having at least 80% homology or identity thereto, and the strong translational coupler BCD2 or BCD10 or a variant thereof having at least 80% homology or identity thereto. In particular, as described herein above, the second expression module may comprise the Pm promoter or a variant thereof having at least 80% homology or identity thereto and the strong translational coupler BCD10 or a variant thereof having at least 80% homology or identity thereto.
The present method requires the precursor molecule 3-fluorobenzoate or a salt thereof. 3-fluorobenzoate or a salt thereof may be provided directly in the medium or it may be derived from a provided precursor molecule through a metabolic process by the bacterial cell.
In some embodiments, the concentration of 3-fluorobenzoate or a salt thereof in said medium is at least 0.05 mM, such as at least 0.1 mM, such as at least 0.2 mM, such as at least 0.25 mM, such as at least 0.3 mM, such as at least 0.4 mM, such as at least 0.5 mM, such as at least 0.6 mM, such as at least 0.7 mM, such as at least 0.75 mM, such as at least 0.8 mM, such as at least 0.9 mM, such as 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 6 mM, such as at least 7 mM, such as at least 8 mM, such as at least 9 mM, such as at least 10 mM, such as at least 11 mM, such as at least 12 mM, such as at least 13 mM, such as at least 14 mM, such as at least 15 mM, such as at least 20 mM such as at least 25 mM, such as 30 mM or more. In some embodiments, the concentration of 3-fluorobenzoate or a salt thereof in said medium is at least 35 mM, such as at least 40 mM, such as at least 45 mM, such as 50 mM or more. In some embodiments, the concentration of 3-fluorobenzoate or a salt thereof in said medium is between 5 mM and 15 mM. In some specific embodiments, the concentration of 3-fluorobenzoate or a salt thereof in said medium is 10 mM. In some embodiments, the concentration of 3-fluorobenzoate or a salt thereof in said medium is between 10 mM and 50 mM. In some specific embodiments, the concentration of 3-fluorobenzoate or a salt thereof in said medium is 20 mM. In some specific embodiments, the concentration of 3-fluorobenzoate or a salt thereof in said medium is 30 mM. In some specific embodiments, the concentration of 3-fluorobenzoate or a salt thereof in said medium is 40 mM. In some specific embodiments, the concentration of 3-fluorobenzoate or a salt thereof in said medium is 50 mM.
In some embodiments, the method is for production of 2-fluoroadipic acid, and further comprises a step of converting the 2-fluoro-cis,cis-muconate to 2-fluoroadipic acid. In some embodiments, the method is for production of [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n, and further comprises a step of converting the 2-fluoro-cis,cis-muconate to [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n. In some embodiments, the method is for production of 2-fluoro-1,6-hexanediol, and further comprises a step of converting the 2-fluoro-cis,cis-muconate to 2-fluoro-1,6-hexanediol. In some embodiments, the method is for production of fluoro-terephthalic acid, and further comprises a step of converting the 2-fluoro-cis,cis-muconate to fluoro-terephthalic acid. In some embodiments, the method is for production of fluoro-trimellitic acid, and further comprises a step of converting the 2-fluoro-cis,cis-muconate to fluoro-trimellitic acid. In some embodiments, the method is for production of a fluorinated polyester, and further comprises a step of converting the 2-fluoro-cis,cis-muconate to a fluorinated polyester. In some embodiments, the method is for production of a fluorinated polyurethane, and further comprises a step of converting the 2-fluoro-cis,cis-muconate to a fluorinated polyurethane. In some embodiments, the method is for production of a fluorinated acrylic, and further comprises a step of converting the 2-fluoro-cis,cis-muconate to a fluorinated acrylic. In some embodiments, the method is for production of a fluorinated adhesive, and further comprises a step of converting the 2-fluoro-cis,cis-muconate to a fluorinated adhesive. In some embodiments, the method is for production of a fluorinated dye, and further comprises a step of converting the 2-fluoro-cis,cis-muconate to a fluorinated dye.
The present methods may comprise a further step of recovering the 2-fluoro-cis,cis-muconate obtained by the methods disclosed herein. Methods for recovering the products obtained by the present disclosure are known in the art, for example organic solvent extraction followed by lyophilisation and purification by preparative HPLC or similar column purification techniques. Another example includes pH/temperature shift crystallization (acidification and cooling), as described by Vardon, Rorrer et al. (2016).
The bacterial cell is preferably as defined herein.
The present methods are useful for producing 2-fluoro-cis,cis-muconate with high titers.
In some embodiments, 2-fluoro-cis,cis-muconate is produced with a titer of at least 500 mg/L, such as at least 750 mg/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 6 g/L, such as at least 7 g/L, such as at least 8 g/L, such as at least 9 g/L, such as at least 10 g/L, such as at least 15 g/L, such as at least 20 g/L, such as at least 25 g/L, such as at least 30 g/L, such as at least 40 g/L, such as at least 50 g/L, such as at least 60 g/L, such as at least 80 g/L, such as at least 100 g/L, such as at least 150 g/L, such as at least 200 g/L, such as at least 250 g/L, such as at least 300 g/L or more.
The accumulation of 2-fluoro-cis,cis-muconate in the growth medium may be toxic to the bacterial cell, and may thus impair cell growth. In some embodiments, the tolerance of the bacterial cell to 2-fluoro-cis,cis-muconate is further improved via adaptive laboratory evolution. In preferred embodiments, the bacterial cell is a Pseudomonas putida cell.
Herein is provided 2-fluoro-cis,cis-muconic acid and derivatives thereof.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconate.
In some embodiments, the present disclosure provides 2-fluoroadipic acid. 2-fluoroadipic acid may be obtained through chemical conversion of 2-fluoro-cis,cis-muconate to 2-fluoroadipic acid.
In some embodiments, the present disclosure provides [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n (a fluorinated version of the polyamide Nylon 6-6). [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n may be obtained through chemical conversion of 2-fluoro-cis,cis-muconate to [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n, such as by condensing the 2-fluoro-cis,cis-muconate derivative 2-fluoroadipic acid with hexamethylenediamine to form [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n.
In some embodiments, the present disclosure provides 2-fluoro-1,6-hexanediol. 2-fluoro-1,6-hexanediol may be obtained through chemical conversion of 2-fluoro-cis,cis-muconate to 2-fluoro-1,6-hexanediol. 2-fluoro-1,6-hexanediol may be used for fluorinated polyester and polyurethane production, and as an intermediate in the production of fluorinated acrylics, adhesives and dyes.
In some embodiments, the present disclosure provides fluoro-terephthalic acid. Fluoro-terephthalic acid may be obtained through chemical conversion of 2-fluoro-cis,cis-muconate to fluoro-terephthalic acid.
In some embodiments, the present disclosure provides fluoro-trimellitic acid. Fluoro-trimellitic acid may be obtained through chemical conversion of 2-fluoro-cis,cis-muconate to fluoro-trimellitic acid.
In some embodiments, the present disclosure provides a fluorinated polyester.
Fluorinated polyesters may be obtained through chemical conversion of 2-fluoro-cis,cis-muconate to a fluorinated polyester.
In some embodiments, the present disclosure provides a fluorinated polyurethane.
Fluorinated polyurethanes may be obtained through chemical conversion of 2-fluoro-cis,cis-muconate to a fluorinated polyurethane.
In some embodiments, the present disclosure provides a fluorinated acrylic.
Fluorinated acrylics may be obtained through chemical conversion of 2-fluoro-cis,cis-muconate to a fluorinated acrylic.
In some embodiments, the present disclosure provides a fluorinated adhesive.
Fluorinated adhesives may be obtained through chemical conversion of 2-fluoro-cis,cis-muconate to a fluorinated adhesive.
In some embodiments, the present disclosure provides a fluorinated dye. Fluorinated dyes may be obtained through chemical conversion of 2-fluoro-cis,cis-muconate to a fluorinated dye.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconic acid and derivatives thereof and derivatives thereof obtainable by a method as disclosed herein.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconate. The thus obtained 2-fluoro-cis,cis-muconate can be further converted into other compounds or derivatives, as detailed herein. The methods disclosed herein may thus further comprise a step of converting the 2-fluoro-cis,cis-muconate to another compound or derivative.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconate which is further converted to 2-fluoroadipic acid. 2-fluoroadipic acid may be obtained through conversion, for example chemical conversion of 2-fluoro-cis,cis-muconate to 2-fluoroadipic acid. Accordingly, in some embodiments the method further comprises a step of conversion of the 2-fluoro-cis,cis-muconate which is further converted to 2-fluoroadipic acid.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconate which is further converted to [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n (a fluorinated version of the polyamide Nylon 6-6). [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n may be obtained through conversion, for example chemical conversion of 2-fluoro-cis,cis-muconate to [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n, such as by condensing the 2-fluoro-cis,cis-muconate derivative 2-fluoroadipic acid with hexamethylenediamine to form [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n. Accordingly, in some embodiments the method further comprises a step of conversion of the 2-fluoro-cis,cis-muconate which is further converted to [—OC—(F)—(CH2)4—CO—NH—(CH2)6—NH—]n.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconate which is further converted to 2-fluoro-1,6-hexanediol. 2-fluoro-1,6-hexanediol may be obtained through conversion, for example chemical conversion of 2-fluoro-cis,cis-muconate to 2-fluoro-1,6-hexanediol. 2-fluoro-1,6-hexanediol may be used for fluorinated polyester and polyurethane production, and as an intermediate in the production of fluorinated acrylics, adhesives and dyes. Accordingly, in some embodiments the method further comprises a step of conversion of the 2-fluoro-cis,cis-muconate which is further converted to 2-fluoro-1,6-hexanediol.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconate which is further converted to fluoro-terephthalic acid. Fluoro-terephthalic acid may be obtained through conversion, for example chemical conversion of 2-fluoro-cis,cis-muconate to fluoro-terephthalic acid. Accordingly, in some embodiments the method further comprises a step of conversion of the 2-fluoro-cis,cis-muconate which is further converted to fluoro-terephthalic acid.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconate which is further converted to fluoro-trimellitic acid. Fluoro-trimellitic acid may be obtained through conversion, for example chemical conversion of 2-fluoro-cis,cis-muconate to fluoro-trimellitic acid. Accordingly, in some embodiments the method further comprises a step of conversion of the 2-fluoro-cis,cis-muconate which is further converted to fluoro-trimellitic acid.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconate which is further converted to a fluorinated polyester. Fluorinated polyesters may be obtained through conversion, for example chemical conversion of 2-fluoro-cis,cis-muconate to a fluorinated polyester. Accordingly, in some embodiments the method further comprises a step of conversion of the 2-fluoro-cis,cis-muconate which is further converted to a fluorinated polyester.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconate which is further converted to a fluorinated polyurethane. Fluorinated polyurethanes may be obtained through conversion, for example chemical conversion of 2-fluoro-cis,cis-muconate to a fluorinated polyurethane. Accordingly, in some embodiments the method further comprises a step of conversion of the 2-fluoro-cis,cis-muconate which is further converted to a fluorinated polyurethane.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconate which is further converted to a fluorinated acrylic. Fluorinated acrylics may be obtained through conversion, for example chemical conversion of 2-fluoro-cis,cis-muconate to a fluorinated acrylic. Accordingly, in some embodiments the method further comprises a step of conversion of the 2-fluoro-cis,cis-muconate which is further converted to a fluorinated acrylic.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconate which is further converted to a fluorinated adhesive. Fluorinated adhesives may be obtained through conversion, for example chemical conversion of 2-fluoro-cis,cis-muconate to a fluorinated adhesive. Accordingly, in some embodiments the method further comprises a step of conversion of the 2-fluoro-cis,cis-muconate which is further converted to a fluorinated adhesive.
In some embodiments, the present disclosure provides 2-fluoro-cis,cis-muconate which is further converted to a fluorinated dye. Fluorinated dyes may be obtained through conversion, for example chemical conversion of 2-fluoro-cis,cis-muconate to a fluorinated dye. Accordingly, in some embodiments the method further comprises a step of conversion of the 2-fluoro-cis,cis-muconate which is further converted to a fluorinated dye.
In some embodiments, the enantiomeric purity of the 2-fluoro-cis,cis-muconate may be at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, 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%, such as 100%.
Also provided herein are nucleic acid constructs useful for engineering a bacterial cell capable of producing 2-fluoro-cis,cis-muconate or derivatives thereof as described above. The present nucleic acid constructs may be provided as one or more nucleic acid molecules or polynucleotides, for example they may be comprised in one or more vectors. Such nucleic acids may be introduced in the bacterial cell by methods known in the art.
It will be understood that throughout the present disclosure, the term ‘nucleic acid encoding an activity’ shall refer to a nucleic acid molecule capable of encoding a peptide, a protein or a fragment thereof having said activity. Such nucleic acid molecules may be open reading frames or genes, or fragments thereof.
Herein is provided a nucleic acid construct for modifying a cell comprising:
Herein is provided a nucleic acid construct for modifying a cell comprising:
Herein is also provided a nucleic acid construct for modifying a cell comprising:
The polynucleotide encoding the benzoate 1,2-dioxygenase may herein be referred to as the “first polynucleotide”. The polynucleotide encoding the benzoate-1,2-dihydrodiol dehydrogenase may be referred to as the “second polynucleotide”. The polynucleotide encoding the first catechol 1,2-dioxygenase may be referred to as the “third polynucleotide”. The polynucleotide encoding the second catechol 1,2-dioxygenase may be referred to as the “fourth polynucleotide”. This does not imply that the construct comprises four polynucleotides in total; in some embodiments the cell comprises only the first, second and third polynucleotides.
In some embodiments, the benzoate 1,2-dioxygenase (EC: 1.14.12.10) is a native or heterologous benzoate 1,2-dioxygenase, as described herein above in the section “Bacterial cell”, and may further comprise any of the modifications described in “Other modifications”. In particular, the benzoate 1,2-dioxygenase may be BenABC or a functional variant thereof having at least 80% homology or identity thereto, the expression of which may be regulated as detailed above. In particular, the gene(s) encoding the benzoate 1,2-dioxygenase may be under the control of a specific promoter, such as Ptac or a variant thereof having at least 80% homology or identity thereto, as described herein above in the section “Bacterial cell”.
In some embodiments, the benzoate-1,2-dihydrodiol dehydrogenase (EC: 1.3.1.25) is a native or heterologous benzoate-1,2-dihydrodiol dehydrogenase, as described herein above in the section “Bacterial cell”, and may further comprise any of the modifications described in “Other modifications”. In particular, the benzoate-1,2-dihydrodiol dehydrogenase may be BenD or a functional variant thereof having at least 80% homology or identity thereto, the expression of which may be regulated as detailed above.
In some embodiments, the first and/or second catechol 1,2-dioxygenase (EC: 1.13.11.1) is a native or heterologous catechol 1,2-dioxygenase, as described herein above in the section “Bacterial cell”, and may further comprise any of the modifications described in “Other modifications”. In particular, the first catechol 1,2-dioxygenase may be CatA-I and/or the second catechol 1,2-dioxygenase may be CatA-II as detailed above. In particular, the expression of the gene(s) encoding the benzoate 1,2-dioxygenase(s) may be under the control of specific first and/or second expression module(s) as described herein above in the section “Bacterial cell”. In particular, as described herein above, the first expression module may comprise the P14b promoter or a variant thereof having at least 80% homology or identity thereto, and the strong translational coupler BCD2 or BCD10 or a variant thereof having at least 80% homology or identity thereto. In particular, as described herein above, the second expression module may comprise the P14b promoter or a variant thereof having at least 80% homology or identity thereto and the strong translational coupler BCD10 or a variant thereof having at least 80% homology or identity thereto. In particular, as described herein above, the first expression module may comprise the Pm promoter variant ML1-17 or a variant thereof having at least 80% homology or identity thereto, the associated activator protein XylS or a functional variant thereof having at least 80% homology or identity thereto, and the strong translational coupler BCD2 or BCD10 or a variant thereof having at least 80% homology or identity thereto. In particular, as described herein above, the second expression module may comprise the Pm promoter or a variant thereof having at least 80% homology or identity thereto and the strong translational coupler BCD10 or a variant thereof having at least 80% homology or identity thereto.
In some embodiments, the first polynucleotide encoding a benzoate 1,2-dioxygenase, such as the benzoate 1,2-dioxygenase BenABC, comprises or consists of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or variants thereof having 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% homology or identity thereto, said first polynucleotide further comprising a medium-strong constitutive promoter, such as the medium-strong constitutive promoter Ptac, comprising or consisting of SEQ ID NO: 14 or a variant thereof having 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% homology or identity thereto.
In some embodiments, the second polynucleotide encoding a benzoate-1,2-dihydrodiol dehydrogenase, such as the benzoate-1,2-dihydrodiol dehydrogenase BenD, comprises or consists of SEQ ID NO: 8 or a variant thereof having 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% homology or identity thereto.
In some embodiments, the third polynucleotide encoding a first catechol 1,2-dioxygenase, such as the catechol 1,2-dioxygenase CatA-I, comprises or consists of SEQ ID NO: 10 or a variant thereof having 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% homology or identity thereto, said third polynucleotide further comprising a first expression module comprising a first promoter, such as the medium-to-weak constitutive promoter P14b comprising or consisting of SEQ ID NO: 49 or a variant thereof having 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% homology or identity thereto, and a first strong translational coupler, such as the translational coupler BCD10 comprising or consisting of SEQ ID NO: 16, or the translational coupler BCD2 comprising or consisting of SEQ ID NO: 22, or variants thereof having 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% homology or identity thereto.
In some embodiments, the third polynucleotide encoding a first catechol 1,2-dioxygenase, such as the catechol 1,2-dioxygenase CatA-I, comprises or consists of SEQ ID NO: 10 or a variant thereof having 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% homology or identity thereto, said third polynucleotide further comprising a first expression module comprising a first inducible promoter, such as the inducible promoter Pm variant ML1-17 comprising or consisting of SEQ ID NO: 18 or a variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology or identity thereto, an associated activator protein, such as the associated activator protein XylS, comprising or consisting of SEQ ID NO: 20 or a functional variant thereof having 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% homology or identity thereto, and a first strong translational coupler, such as the strong translational coupler BCD10 comprising or consisting of SEQ ID NO: 16 or a variant thereof having 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% homology or identity thereto, or the strong translational coupler BCD2 comprising or consisting of SEQ ID NO: 22 or a variant thereof having 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% homology or identity thereto.
In some embodiments, the fourth polynucleotide encoding a second catechol 1,2-dioxygenase, such as the catechol 1,2-dioxygenase CatA-II, comprises or consists of SEQ ID NO: 12 or a variant thereof having 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% homology or identity thereto, said fourth polynucleotide further comprising a second expression module comprising a second promoter, such as the medium-to-weak constitutive promoter P14b comprising or consisting of SEQ ID NO: 49 or a variant thereof having 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% homology or identity thereto, and a second strong translational coupler, such as the strong translational coupler BCD10, comprising or consisting of SEQ ID NO: 16 or a variant thereof having 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% homology or identity thereto.
In some embodiments, the fourth polynucleotide encoding a second catechol 1,2-dioxygenase, such as the catechol 1,2-dioxygenase CatA-II, comprises or consists of SEQ ID NO: 12 or a variant thereof having 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% homology or identity thereto, said fourth polynucleotide further comprising a second expression module comprising a second inducible promoter, such as the inducible Pm promoter, comprising or consisting of SEQ ID NO: 15 or a variant thereof having 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% homology or identity thereto, and a second strong translational coupler, such as the strong translational coupler BCD10, comprising or consisting of SEQ ID NO: 16 or a variant thereof having 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% homology or identity thereto.
In some embodiments, the nucleic acid construct encodes the benzoate 1,2-dioxygenase BenABC consisting of BenA (SEQ ID NO: 1), BenB (SEQ ID NO: 2) and BenC (SEQ ID NO: 2), the benzoate-1,2-dihydrodiol dehydrogenase BenD (SEQ ID NO: 7), and the catechol 1,2-dioxygenase CatA-I (SEQ ID NO: 9).
In some embodiments, the nucleic acid construct encodes the benzoate 1,2-dioxygenase BenABC consisting of BenA (SEQ ID NO: 1), BenB (SEQ ID NO: 2) and BenC (SEQ ID NO: 2), the benzoate-1,2-dihydrodiol dehydrogenase BenD (SEQ ID NO: 7), and the catechol 1,2-dioxygenases CatA-I (SEQ ID NO: 9) and CatA-II (SEQ ID NO: 11).
In some embodiments, the nucleic acid construct encodes the benzoate 1,2-dioxygenase BenABC consisting of BenA (SEQ ID NO: 1), BenB (SEQ ID NO: 2) and BenC (SEQ ID NO: 2), the benzoate-1,2-dihydrodiol dehydrogenase BenD (SEQ ID NO: 7), and the catechol 1,2-dioxygenase CatA-I (SEQ ID NO: 9), wherein BenABC is expressed under the control of the medium-strong constitutive promoter Ptac (SEQ ID NO: 15) and CatA-I is expressed under the control of the first expression module comprising or consisting of the medium-to-weak constitutive promoter P14b (SEQ ID NO: 49), and the first strong translational coupler BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16).
In some embodiments, the nucleic acid construct encodes the benzoate 1,2-dioxygenase BenABC consisting of BenA (SEQ ID NO: 1), BenB (SEQ ID NO: 2) and BenC (SEQ ID NO: 2), the benzoate-1,2-dihydrodiol dehydrogenase BenD (SEQ ID NO: 7), and the catechol 1,2-dioxygenase CatA-I (SEQ ID NO: 9), wherein BenABC is expressed under the control of the medium-strong constitutive promoter Ptac (SEQ ID NO: 15) and CatA-I is expressed under the control of the first expression module comprising or consisting of the first inducible promoter Pm variant ML1-17 (SEQ ID NO: 18), the associated activator protein is XylS (SEQ ID NO: 21), and the first strong translational coupler BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16).
In some embodiments, the nucleic acid construct encodes the benzoate 1,2-dioxygenase BenABC consisting of BenA (SEQ ID NO: 1), BenB (SEQ ID NO: 2) and BenC (SEQ ID NO: 2), the benzoate-1,2-dihydrodiol dehydrogenase BenD (SEQ ID NO: 7), and the catechol 1,2-dioxygenases CatA-I (SEQ ID NO: 9) and CatA-II (SEQ ID NO: 11), wherein BenABC is expressed under the control of the medium-strong constitutive promoter Ptac (SEQ ID NO: 15) and CatA-I is expressed under the control of the first expression module comprising or consisting of the medium-to-weak constitutive promoter P14b (SEQ ID NO: 49), and the first strong translational coupler BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16), and wherein CatA-II is expressed under the control of the second expression module comprising or consisting of the medium-to-weak constitutive promoter P14b (SEQ ID NO: 49) and the second strong translational coupler BCD10 (SEQ ID NO: 16).
In some embodiments, the nucleic acid construct encodes the benzoate 1,2-dioxygenase BenABC consisting of BenA (SEQ ID NO: 1), BenB (SEQ ID NO: 2) and BenC (SEQ ID NO: 2), the benzoate-1,2-dihydrodiol dehydrogenase BenD (SEQ ID NO: 7), and the catechol 1,2-dioxygenases CatA-I (SEQ ID NO: 9) and CatA-II (SEQ ID NO: 11), wherein BenABC is expressed under the control of the medium-strong constitutive promoter Ptac (SEQ ID NO: 15) and CatA-I is expressed under the control of the first expression module comprising or consisting of the first inducible promoter Pm variant ML1-17 (SEQ ID NO: 18), the associated activator protein XylS (SEQ ID NO: 21), and the first strong translational coupler BCD2 (SEQ ID NO: 22) or BCD10 (SEQ ID NO: 16), and wherein CatA-II is expressed under the control of the second expression module comprising or consisting of the inducible promoter Pm (SEQ ID NO: 15) and the second strong translational coupler BCD10 (SEQ ID NO: 16).
In some embodiments, one or more of the nucleic acids encoding each of the present activities, i.e. a benzoate 1,2-dioxygenase, a benzoate-1,2-dihydrodiol dehydrogenase, and catechol 1,2-dioxygenase, may be codon-optimized for said bacterial cell.
In some embodiments, each of the nucleic acids encoding each of the present activities, i.e. a benzoate 1,2-dioxygenase, a benzoate-1,2-dihydrodiol dehydrogenase, and catechol 1,2-dioxygenase, may be designed to be integrated within the genome of the cell or they may be within one or more vectors comprised within the cell.
In some embodiments, one or more of the nucleic acids encoding each of the present activities may be integrated in the genome of said cell. Methods for integrating a nucleic acid are well known in the art. Thus in some embodiments the activity of interest is encoded by introduction of a heterologous nucleic acid in the bacterial cell.
The heterologous nucleic acid encoding said activity may be codon-optimized, or may comprise features that can help improve the activity. Such modifications include, but are not limited to, the introduction of localization signals, gain-of-function or loss-of-function mutations, fusion of the protein to a marker or a tag such as fluorescent tag, insertion of an inducible promoter, introduction of modifications conferring increased stability and/or half-life.
The introduction of the heterologous nucleic acid encoding the activity of interest can be performed by methods known in the art. The skilled person will recognize that such methods include, but are not limited to: cloning and homologous recombination-based methods. Cloning methods may involve the design and construction of a plasmid e.g. in an organism such as Escherichia coli. The plasmid may be an integrative or a non-integrative vector. Cloning-free methods comprise homologous recombination-based methods such as adaptamer-mediated PCR or gap repair. Such methods often result in integration of the heterologous nucleic acid in the genome of the bacterial cell.
In some embodiments, the nucleic acids encoding the activities of interest may be present in high copy number.
In some embodiments, the nucleic acid construct further comprises or consists of one or more vectors, such as an integrative vector or a replicative vector. In some embodiments, the vector is a high copy replicative vector.
The bacterial cell may be as described herein.
Also provided herein is a kit of parts comprising a bacterial cell, for example a bacterial cell as described herein, and/or a nucleic acid construct as described herein, and instructions for use.
In some embodiments, the kit comprises a bacterial cell that can be used in the methods for producing 2-fluoro-cis,cis-muconate described herein. In other embodiments, the kit comprises a nucleic acid construct that can be used to engineer a bacterial cell useful for the methods for producing 2-fluoro-cis,cis-muconate described herein. In some embodiments, the kit comprises a bacterial cell and a nucleic acid construct as described herein.
In some embodiments, the kit comprises a bacterial cell capable of producing 2-fluoro-cis,cis-muconate, wherein the bacterial cell expresses a benzoate 1,2-dioxygenase, a benzoate-1,2-dihydrodiol dehydrogenase and at least one catechol 1,2-dioxygenase. The yeast cell may be further modified as detailed in the section “Other modifications”.
In some embodiments, the kit comprises a bacterial cell capable of producing 2-fluoro-cis,cis-muconate, wherein the bacterial cell expresses a benzoate 1,2-dioxygenase, a benzoate-1,2-dihydrodiol dehydrogenase and at least two catechol 1,2-dioxygenases. The yeast cell may be further modified as detailed in the section “Other modifications”.
In some embodiments, the kit comprises a nucleic construct comprising a first polynucleotide encoding a benzoate 1,2-dioxygenase and further comprising a medium-strong constitutive promoter. In some embodiments, the kit comprises a nucleic construct comprising a second polynucleotide encoding a benzoate-1,2-dihydrodiol dehydrogenase. In some embodiments, the kit comprises a nucleic construct comprising a third polynucleotide encoding a first catechol 1,2-dioxygenase, a first expression module comprising a first promoter, said first expression module further comprising a first strong translational coupler. In some embodiments, the kit comprises a nucleic construct comprising a fourth polynucleotide encoding a second catechol 1,2-dioxygenase, a second expression module comprising a second promoter, said second expression module further comprising a second strong translational coupler. In some embodiments, the kit comprises a nucleic construct comprising the first, second and third polynucleotides described herein above. In some embodiments, the kit comprises a nucleic construct comprising the first, second, third and fourth polynucleotides described herein above.
In some embodiments, the kit comprises a nucleic construct comprising a first polynucleotide encoding a benzoate 1,2-dioxygenase and further comprising a medium-strong constitutive promoter. In some embodiments, the kit comprises a nucleic construct comprising a second polynucleotide encoding a benzoate-1,2-dihydrodiol dehydrogenase. In some embodiments, the kit comprises a nucleic construct comprising a third polynucleotide encoding a first catechol 1,2-dioxygenase, a first expression module comprising a first inducible promoter and a sequence encoding an associated activator protein, said first expression module further comprising a first strong translational coupler. In some embodiments, the kit comprises a nucleic construct comprising a fourth polynucleotide encoding a second catechol 1,2-dioxygenase, a second expression module comprising a second inducible promoter, said second expression module further comprising a second strong translational coupler. In some embodiments, the kit comprises a nucleic construct comprising the first, second and third polynucleotides described herein above. In some embodiments, the kit comprises a nucleic construct comprising the first, second, third and fourth polynucleotides described herein above.
In some embodiments, the kit comprises the nucleic acid construct as described herein and the bacterial cell to be modified. In some embodiments, the bacterial cell to be modified is a Pseudomonas putida cell, preferably a Pseudomonas putida KT2440 cell.
The bacterial strains employed in the present study are listed in Table 1, below. E. coli and P. putida were incubated at 37° C. and 30° C., respectively. For cell propagation and storage, 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 NaCl). Liquid cultures were performed using either 50-ml centrifuge tubes with a medium volume of 5-10 mL, or in 500-mL Erlenmeyer flasks covered with cellulose plugs (Carl Roth, Karlsruhe, Germany) and with a medium volume of 50 mL. All liquid cultures were agitated at 250 rpm (MaxQ™8000 incubator; ThermoFisher Scientific, Waltham, MA, USA). Solid culture media contained an additional 15 g/L agar. Selection of plasmid-harboring cells was achieved by adding kanamycin (Km), gentamicin (Gm), or streptomycin (Sm) when required at 50 μg mL−1, 10 μg mL−1, and 50 μg mL−1, respectively. For phenotypic characterizations in microtiter plates as well as fermentations in shaken flasks, P. putida was pre-grown in LB medium, and the experiments were performed in de Bont synthetic minimal medium (Hartmans, Smits et al. 1989) additionally buffered with 10 g/L 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.0 and supplemented with different carbon compounds as explained in the text.
For shaken flask experiments, LB precultures were harvested by centrifugation at 4000×g for 10 min, washed with de Bont medium without the addition of any carbon compound, and resuspended in the final media of the experiment at the desired start-OD600 nm. Cell growth was monitored by measuring the absorbance at 630 nm (for plate reader experiments) or 600 nm (for shaken flask experiments). The optical density at 630 nm (OD630 nm) was estimated from plate reader absorbance values by multiplying the values by 1.751, a value which had been previously determined with pure water using the method described by Lampinen, Raitio et al. (2012). Alternatively, cell growth was monitored by measuring the absorbance at 630 nm (for plate reader experiments with ELx808, BioTek Instruments; Winooski, VT, USA) or 600 nm (for shaken flask experiments) and estimating the optical density at 600 nm (OD600 nm) from plate reader absorbance values by multiplying the values by correlation factors, which had been previously determined for the employed microtiter plate-readers and spectrophotometers. Biomass concentrations (cell dry weight, gCDW L−1) in shaken flask experiments were derived from OD600 nm measurements with a correlation factor 0.35, previously determined for the spectrophotometer employed with exponentially growing P. putida KT2440.
Comparative phenotypical characterizations and quantifications of green fluorescence for bioreporters were performed in 96-well plates in a Synergy HI plate reader (BioTek Instruments; Winooski, VT, USA). Therefore, the LB preculture was diluted 1:100 in the respective screening medium (de Bont medium supplemented with various organic compounds). Fluorescence was measured at an excitation wavelength of 488 nm and an emission wavelength of 588 nm. The transcription and translation initiation strengths of various expression systems tested in bioreporter experiments are provided by the relative normalized fluorescence of GFP at an OD630 nm value of 1.0 for each culture, which represents a point within the mid-exponential growth phase of P. putida. For this purpose, the absolute GFP emission values were normalized by the OD630 nm (˜1.0) and divided by the normalized fluorescence value of the Ptac reporter strain.
Escherichia coli
Pseudomonas
P. putida KT2440
putida mt-2 (Worsey and Williams
P. knackmussii
P. putida Ptac→benABC *1
P. putida KT2440 with the
P. putida Ptac→benD *1
P. putida KT2440 with the
P. putida PEM7→catA *1
P. putida KT2440 with the the
P. putida ΔcatBC *1
P. putida KT2440 with both
P. putida ΔcatBC::catA *1
P. putida KT2440 with the genes
P. putida ΔcatBC::catA-
P. putida KT2440 with the genes
P. putida ΔcatBC
P. putida ΔcatBC
P. putida ΔcatBC::catA
P. putida ΔcatBC::catA-II
P. putida
P. putida KT2440 with the
P. putida
P. putida KT2440 with the
P. putida [Pmuc3] *2
P. putida [Pmuc3x] *2
P. putida [Pmuc4] *3
P. putida [Pmuc4vhb] *3
stercoraria vhb gene inserted into
P. putida [Pmuc4tat-
P. putida [Pmuc4c] *3
P. putida [Pmuc4g] *3
P. putida [Pmuc5] *4
P. putida [Pmuc6] *4
P. putida [Pmuc7] *4
P. putida [Pmuc8] *4
P. putida [Pmuc9] *4
P. putida [Pmuc10] *5
P. putida [Pmuc11] *5
P. putida [Pmuc12] *5
P. putida [Pmuc13] *5
P. putida [Pmuc14] *5
All plasmids and oligonucleotides used in this work are listed in Table 2, below. Uracil-excision (USER) cloning (Cavaleiro, Kim et al. 2015) was used for the construction of all plasmids. The AMUSER tool was employed for the design of oligonucleotides (Genee, Bonde et al. 2015). All genetic manipulations followed the protocol published previously (Wirth, Kozaeva et al. 2019). DNA fragments employed in assembly reactions were amplified using the Phusion™ U high-fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's specifications. The identity and correctness of all plasmids and DNA constructs were confirmed by sequencing. For genotyping experiments after cloning procedures and genome manipulations, colony PCRs were performed using the commercial OneTaq™ master mix (New England BioLabs; Ipswich, MA, USA) according to the manufacturer's instructions. E. coli DH5α λ pir was employed for all cloning purposes. Chemically competent E. coli cells were prepared and transformed with plasmids according to well-established protocols (Elder 1983). P. putida was rendered electro-competent following the protocol of Choi, Kumar et al. (2006), i.e., by washing the biomass from saturated (16 h) LB medium cultures with 0.3 M sucrose, followed by transformation with plasmids by electroporation with a voltage of 2.5 kV, 25 μF capacitance and 200) resistance in a Gene Pulser Xcell™ Electroporation System (Bio-Rad Laboratories, Hercules, CA, USA).
putida KT2440
putida KT2440
P. putida KT2440
P. putida KT2440 and replace it with the
P. putida KT2440 and replace it with the
putida KT2440.
aAntibiotic markers: Km, kanamycin; Nal, nalidixic acid; Gm, gentamicin; Km,
For the detection of extracellular metabolites, supernatants were obtained via centrifugation of bacterial culture broths for 2 min at 13,000×g. 2-Fluoromuconic acid (2-FMA), cis,cis-muconic acid (ccMA), catechol, 3-fluorocatechol (3-FC), 4-fluorocatechol (4-FC), 2-fluorobenzoic acid (2-FBz), and 3-fluorobenzoic acid (3-FBz) were quantified using a Dionex 3000 HPLC system equipped with a Zorbax Eclipse Plus C18 column (Agilent Technologies, Santa Clara, CA, USA) that was heated to 30° C., and a guard column from Phenomenex. Separation was achieved with a mobile phase consisting of 0.05% (w/v) acetic acid and varying amounts of acetonitrile. The total runtime per sample was 8.3 min (with a separation time of 8.0 min), during which the fraction of acetonitrile was increased from 1% to 3% within the first 3 min, followed by a steady increase to 20% within 12 s, and a further steady increase to 75% within 4 min. From 7.2 to 7.5 min, the acetonitrile concentration was held at 75% and subsequently reduced to 1% within 18 s. The column was then equilibrated again at 1% acetonitrile for further 30 s before injecting the next sample. The flow rate was set to 1 ml min−1, and the injection volume was 0.75 μl. Elution of the compounds was detected by UV light at wavelengths of 210 nm, 240 nm, 280 nm, and 300 nm. HPLC data were processed using the Chromeleon 7.1.3 software (Thermo Fisher Scientific), and compound concentrations were calculated using calibration curves.
Data manipulations and calculations were performed in Microsoft Excel 2016 and OriginPro 2019 (OriginLab Corporation). Figures and Illustrations were created in OriginPro 2019 and Adobe Illustrator 2020. Geneious Prime 2020.1.2 (Biomatters Ltd.) served as a database for any kind DNA sequences, to design plasmids and constructs, and to analyze Sanger sequencing results. Maximum exponential growth rates μmax in Example 1 were calculated using the tool GrowthRates published by Hall, Acar et al. (2014). Allternatively, maximum exponential growth rates μmax were determined by Gaussian process regression using the Python-based tool deODorizer published by Swain et al. (2016). The prediction of translation initiation strengths for 5′ untranslated region (5′ UTR) mRNA sequences was performed using the online RBS Calculator v2.1 which can be found at https://www.denovodna.com/software/predict_rbs_calculator. The results are given in arbitrary units (au) on the RBS Calculator v2.0 scale, representing the relative strength of translation.
Biomass-specific 3-FBz consumption rates qs and 2-FMA production rates qp were determined over the timeframes of fermentations in which there was 3-FBz detectable in the culture media with the following equations:
The qs and qp rates displayed in Table 5 are averages of the differential values determined for the individual time intervals.
To examine the suitability of the ortho-cleavage pathway enzymes in P. putida for the bioconversion of 2- or 3-FBz to 2-FMA, both P. knackmussii and P. putida KT2440 were cultured in microtiter plates with minimal medium supplemented with 30 mM glucose (or 30 mM potassium benzoate) and varying concentrations of metabolites involved in the bioconversion process (Figures A-C). Maximum exponential growth rates (μmax) observed, determined in the early exponential phase for each growth curve individually, are listed in Table 3, below.
P. putida KT2440
P. knackmussii
Within the course of 50 h, no growth was detectable for P. knackmussii on benzoate as the sole source of carbon, while P. putida grew with a μmax of 0.37 h−1. With glucose as the primary source of carbon and energy, P. putida grew with a maximum rate μmax of 0.6 h−1, which is about 1.6 times higher than that of P. knackmussii (μmax, 0.38 h−1). In the presence of 2-FBz, both species demonstrated some degree of sensitivity to the compound, which became more prominent with increasing concentrations. However, while P. putida still grew with 80% of its maximum rate on glucose and without any noticeable effect on the duration of the lag phase in the presence of 20 mM 2-FBz, P. knackmussii was unable to grow at all within the course of the experiment (50 h) and showed significantly increased lag phases at 10 mM and 15 mM of 2-FBz. At 15 mM 2-FBz, P. knackmussii grew with 58% of the μmax observed on only glucose. The detrimental impact of the second bioconversion substrate 3-FBz on the growth of both species was more severe. P. putida was able to grow in the presence of maximum 10 mM 3-FBz with a substantially reduced μmax value. Even at 5 mM, the growth of P. knackmussii was almost completely impaired and no growth was observable at concentrations of 10 mM.
Depending on the position of the fluoro-substitution on the benzoate derivative, the three 1,2-dihydroxybenzenes pyrocatechol (here referred to as catechol), 3-FC, and 4-FC are produced as intermediary metabolites. No toxic effect was observed in the presence of up to 2 mM catechol on P. putida. In fact, with increasing catechol concentrations, μmax increased by up to 30%. For P. knackmussii, catechol demonstrated a slightly toxic effect, evident in a decrease in μmax with increasing catechol concentrations. The physiological response of the two species to the two fluorinated 1,2-dihydroxybenzene derivatives, 3-FC and 4-FC, was more pronounced, but was reflected in different growth characteristics for each strain. While 3-FC affected predominantly the cells' growth rate, the presence of 4-FC had a much more prominent effect on the duration of lag phases. However, in both cases, P. putida demonstrated a higher resistance to the toxic effects of both 3-FC and 4-FC than P. knackmussii.
After establishing the physiological response of both P. knackmussii and P. putida to the chemical compounds involved in the bioconversion of FBz to 2-FMA, the two species were subjected to a fermentation experiment in shaken flasks containing synthetic de Bont medium with 10 g/L MOPS at pH 7.0, and supplemented with 30 mM glucose as well as 10 mM of 2-FBz or 3-FBz (
Exposed to 3-FBz, P. putida consumed the substrate up until a time between 7 and 20 h with a qs of 0.162 mmol gCDW−1 after which the 3-FBz concentration in the culture supernatant remained constant. Within the first 7 h, all 3-FBz consumed was converted into 3-FC until it reached a concentration of 1 mM. In the course of the next 13 h, 40% of 3-FC in the supernatant was converted into 2-FMA, combined with a fraction of additionally consumed 3-FBz at a combined product formation rate qp of 0.06 mmol gCDW−1 h−1. At the end of the fermentation, 3.48 mM 3-FBz had been converted into 1.38 mM 2-FMA, corresponding to a product yield YSP of 0.41 mol mol-1, as well as 0.52 mM 3-FC. The combined concentrations of metabolites constituting the 1,2-dioxygenation route (2-FMA and 3-FC) add up to 1.90 mM, representing 55% of the consumed 3-FBz. Considering its previously established toxicity, the accumulation of 3-FC likely represents a major bottleneck for an efficient and prolonged bioconversion process in P. putida. Also with P. knackmussii, 3-FBz consumption occurred only within the first 7-20 h of the fermentation, with a qs of 0.090 mmol gCDW−1 h−1. After an initial increase of 3-FC up to 0.94 mM in the culture supernatant, the catechol derivative was completely re-consumed until 20 h. From a total concentration of 2.93 mM 3-FBz consumed, P. knackmussii produced 1.64 mM 2-FMA [corresponding to 56% of the substrate (YSP: 0.41 mol mol-1)] with a rate qp of 0.05 mM gCDW−1 h−1. In none of the conditions tested significant amounts of 4-FC could be detected in the culture supernatants. The enzyme machinery performing the di-oxygenation on fluorinated catechol derivatives that both species are equipped with thus seems to demonstrate a higher efficiency towards 4-FC compared to 3-FC.
With the asymmetrical use of the non-productive 1,2-dioxygenation route when using 2-FBz as substrate for the bioconversion, almost 90% of fluorine gets lost, in contrast to about 50% with 3-FBz as substrate. The 3-F-substituted benzoate consequently represents the most suitable precursor compound to produce 2-FMA. A faster growth rate, higher biomass yield on glucose, higher qs and qp values, and a higher resistance to toxic effects of pathway intermediates make P. putida stand out as the bacterial platform better suited to perform this bioconversion.
As apparent from metabolite analyses of the supernatant during bioconversion experiments with P. putida and 3-FBz, the last biochemical step of the upper ortho-cleavage pathway, conversion of 3-FC into 2-FMA, represents a major bottleneck for a balanced conversion without the accumulation of toxic 3-FC. Following an approach commonly employed in the field of microbial strain engineering, a first set of genome manipulations was performed on P. putida KT2440 with the aim of increasing the enzyme availability of different steps of the bioconversion pathway, especially that of the two catechol 1,2-dioxygenases, by means of constitutive promoters (the strains are listed in Table 1, above).
These modifications comprised the replacement of the native Pben promoter upstream of benA with the constitutive Ptac promoter sequence [predicted translation strength with the native 5′ untranslated region (UTR): 220], the insertion of Ptac upstream of the 28 bp 5′ UTR (predicted translation strength: 818) of benD, and the insertion of the PEM7 promoter sequence upstream of the 29 bp 5′ UTR of catA (predicted translation strength: 3211).
Approaches to insert a regulatory element comprising Ptac and a translational coupling sequence [bi-cistronic designs—BCD2 or BCD10, published by Mutalik, Guimaraes et al. (2013)] upstream of catA [translation strengths predicted using the RBS Calculator v2.1; BCD2: 28177 (SD1) and 18908 (SD2); BCD10: 28177 (SD1) and 331 (SD2)] or catA-II [predicted translation strengths BCD2: 28177 (SD1) and 7687 (SD2); BCD10: 28177 (SD1) and 134 (SD2)] failed due to the consistent emergence of mutations within the −35 region of Ptac in the cloning host E. coli harboring the respective genome editing plasmid, suggesting a toxic effect of catechol 1,2-dioxygenase overexpression under some conditions.
As an alternative strategy to increase catechol 1,2-dioxygenase activity, the three catA homologues from P. knackmussii were cloned into the replicable plasmid pSEVA634 under the control of the IPTG-inducible LacIq/Ptrc system and inserted into P. putida KT2440. Following the strategy employed by Kohlstedt, Starck et al. (2018), further P. putida KT2400 strains were engineered with in-frame deletions of the two genes catB and catC to avoid (3-F-)cis,cis-muconate degradation, as well as variants where the catBC gene sequences were replaced by additional copies of catA or catA-II, putting them under the control of the native CatR/Pcat regulatory system (with predicted translation rates of 1934 for catA, and 503 for catA-II). These modifications were also combined with the aforementioned Ptac→benD design, which had been found to enhance productivities within the two upstream reactions of the ortho-cleavage pathway in preliminary experiments (not shown).
All ‘first generation’ strains (Table 1, above) were subjected to a bioconversion experiment in 500 ml Erlenmeyer flasks filled with 50 ml de Bont medium supplemented with 10 g/L MOPS (pH 7.0), 30 mM glucose and 10 mM 3-FBz. Within 24 h, all strain cultures reached a terminal state in which no further 3-FBz was consumed and all extracellular metabolites measured, with the exception of minor concentrations of 4-FC that continued to be consumed, remained at constant concentrations in the culture supernatants. These concentrations of the three most relevant metabolites of the bioconversion pathway 3-FBz, 3-FC, and 2-FMA are compared between each member of the first generation strains, as well as wild-type P. putida and P. knackmussii, in
This apparent bottleneck was addressed by means of a catA overexpression by the constitutive PEM7 promoter with putative high transcription initiation strength. The constitutive overexpression of catA, however, had detrimental effects on the catechol 1,2-dioxygenase activity, leading to an almost complete accumulation of all 3-FBz entering the 1,2-dioxygenation route in the form of 3-FC. The deletion of catBC also lead to a higher accumulation of 3-FC in all strains that had been subject to this genomic manipulation. Additional expression of benABC or benD by Ptac brought about comparable effects in a ΔcatBC background as with catBC intact. Also the implementation of additional copies of catA or catA-II under Pcat control in place of catBC did not show any positive effect on the conversion of 3-FC into 2-FMA.
In addition to >2 mM 3-FC and <0.93 mM 2-FMA, each of the seven ΔcatBC strains accumulated a new by-product eluting at 4.0 min (HPLC) with an absorption maximum at 277.5 nm and with a molecular weight of 299.06 g mol−1 (as determined by LC-MS). The molecular weight and fragmentation pattern corresponded to either of the two C12H13NO3 compounds 3-[Bis(carboxymethyl)amino]methyl-2,4-dihydroxybenzoic acid or 3-[Bis(carboxymethyl)amino]methyl-4,5-dihydroxybenzoic acid (data not shown). The greatest detriment on the bioconversion performance was caused by the expression of the three P. knackmussii catA orthologues from replicable plasmids. While 3-FBz uptake was greatly enhanced with most of the substrate having been consumed within 24 h, almost 50% of the initial 3-FBz was converted into 3-FC, with final concentrations of about 5 mM.
Of the manipulations tested, only Ptac→benABC had a balancing effect on the enzyme activities within the pathway, and efforts to increase the enzyme abundance of catechol 1,2-dioxygenase appeared to further reduce the rate at which its reaction is performed.
To get a better understanding of the effects that were caused by the defined genomic manipulation within first generation strains, all regulatory sequences involved in the strain engineering process were subjected to a comparative analysis. To this end, a set of standardized reporter plasmids was constructed utilizing pSEVA62X as backbone (Martinez-Garcia, Aparicio et al. 2014). Besides the low-copy number origin of vegative replication oriV(RK2) and a gentamicin resistance determinant, each plasmid harbors the msfGFP reporter gene under the control of i) the bi-cistronic translational coupling sequence BCD10 (Mutalik, Guimaraes et al. 2013) preceded by varying sequences of promoters relevant in this study, or ii) the XylS/Pm expression system followed by varying bi-cistronic translational coupling sequences to compare their relative translation initiation strength in P. putida.
A complete list of reporter constructs tested is summarized in Table 2, above. P. putida KT2440 harboring each of the reporter plasmid was cultured in de Bont medium supplemented with 30 mM glucose and, if applicable, varying concentrations of inducer compounds. A relative comparison of the effects of different bi-cistronic translational coupling sequences (BCD) is shown in
The five chosen BCD variants showed the same relative effect on translation initiation as published in E. coli by Mutalik, Guimaraes et al. (2013), with BCD1 and BCD2 providing the highest GFP expression, followed by BCD7, BCD10, and BCD20. With the set of translational coupling sequences tested, expression of target genes can be tuned within an about three-fold range. With the BCD sequences behaving comparable as originally published for E. coli, it is expected that any of the other published variants can be chosen to provide translation initiation at even lower rates.
With both 3-mBz and 3-FBz as inducers, GFP expression under Pm(ML1-17) control was only 70% of the expression provided by Pm. The XylS/Pm system was induced to 94% by 1 mM of 3-mBz as compared to 5 mM of the inducer (data not shown). With Bz and 3-FBz on the other hand, there was an almost linear dependency between inducer concentration and GFP expression within the range between 1.0 and 5.0 mM. Even with 5.0 mM inducer concentration, the GFP expression from Pm amounted to only 20% (for Bz) and 7% (for 3-FBz) of the one brought about by 1.0 mM 3-mBz. With 1.0 mM of 3-FBz, the expression strength was close to the basal, uninduced expression. The CatR/Pcat system was most strongly and fully induced already in the presence of 0.5 mM 3-FBz. The supplementation of 1.0 mM 3-mBz caused no response at all and with 5 mM Bz, only 35% of GFP fluorescence was observed as compared to the induction by 3-FBz. The system furthermore responded 6.7-fold stronger to 1.0 mM 2-FMA as compared to 1.0 mM ccMA. The fact that CatR/Pcat responds much more strongly to 3-FBz supplied in the medium than to 2-FMA suggests a more efficient uptake of 3-FBz which gets converted intracellularly to 2-FMA. The stronger response to 3-FBz and 2-FMA compared to Bz and ccMA could be caused by either a stronger interaction of CatR with the fluoro-substituted muconic acid or the stronger intracellular accumulation of 2-FMA, since ccMA is further consumed by the CatR/Pcat-regulated CatB enzyme.
The BenR/Pben system displayed a high basal expression without the addition of any inducer. No further increase in GFP expression could be observed with 1.0 mM 3-mBz, 2.0 mM Bz, and 1.0 mM 3-FBz. Only with 5 mM of Bz or 3-FBz, induction increased by 70% compared to the uninduced system. It should be noted that the expression of BenR in wild-type P. putida KT2440 is negatively controlled by the translational repressor Crc which might have caused a decreased response of the BenR/Pben system in the presence of glucose as the main source of carbon and energy.
Next, the inducible promoter systems BenR/Pben, CatR/Pcat, and XylS (and its single substitution variants XylSThr45 and XylSVal288)/Pm were compared under bioconversion conditions (with 5 mM 3-FBz) to the constitutive promoters Ptac, PEM7, and P14g (
The expression strengths of the promoter systems employed in the first round of strain engineering on P. putida KT2440 in the presence of 5 mM 3-FBz provide explanations for the phenotypic responses observed in the engineered strains. The expression of benABC by means of Ptac represents an about 2-fold overexpression compared to the native control by BenR/Pben. A more complicated picture is drawn by the manipulations of catA(-II) expression by various expression modules. Any effort to increase the expression of catA or catA-II resulted in the increased accumulation of catechol 1,2-dioxygenase substrate 3-FC, suggesting a decrease in the respective biochemical activity.
This effect was particularly pronounced with the plasmid-based expression of P. knackmussii catA orthologues. With a reported copy number in P. putida of 30±7, the expression levels are expected to exceed those of any of the expression systems employed for the chromosomal manipulations. In the cloning host E. coli, the catA transcript levels provided by the chosen constitutive promoters are expected to be even higher or equal to those in P. putida under full induction of CatR/Pcat exposed to 3-FBz, as the plasmids are maintained at copy numbers of ˜10-15. In addition to that, CatA levels are further increased by the effect of the translational coupling sequences which are predicted to increase the translation initiation rate by a factor of 15 (BCD2) or 10 (BCD10) compared to the native catA translation initiation sequence (see Table 1, above).
In E. coli, the very high expression levels of catA appear to be toxic, thus providing a selection pressure to mutate the promoter sequences, while in P. putida, high CatA(-II) enzyme levels caused a disruption of catechol 1,2-dioxygenase activity. The given data on expression system performances suggests that a reduction in catA expression might lead to an increased bioconversion efficiency in P. putida.
Guided by the observations made on the effects of genetic manipulations within first generation strains and the expression system comparison, a set of new strains was designed, in which catA and/or catA-II was put under the control of the XylS/Pm system that responds weakly to the bioconversion substrate 3-FBz and showed no induction in the absence of any inducer compound. The latter was thought to facilitate the cloning process in E. coli. Because 3-FBz-induced XylS/Pm provides transcript levels at a rate of only 7% compared to CatR/Pcat (see
All strains created within the second round of strain engineering are listed in Table 1, above. Shaken flask bioconversion fermentations were performed in minimal medium with 30 mM glucose and 10 mM 3-FBz and the extracellular metabolites were analyzed via HPLC after 24 h of cultivation (see
With 2-FMA as the only metabolite detected in the supernatant of Pmuc3 pSEVA228, the culture broth was subjected to the purification protocol published for ccMA by Vardon, Rorrer et al. (2016). This enabled the purification of about 30 mg 2-FMA from 40 ml of culture broth, corresponding to about 94% of 2-FMA initially measured in the culture supernatant. The chromatogram of an HPLC analysis with a UV-absorbance at 280 nm of the purified product is shown in
With Pmuc3 harboring pSEVA228, a suitable set of genetic manipulation had been established that enables a well-balanced conversion of 3-FBz into 2-FMA without the accumulation of by-products. Using Pmuc3 as a genetic background, a further set of engineered strains was designed with the aim of further increasing the overall productivity of the bioconversion process.
As a first target the gene encoding the global catabolite repression control regulator Crc was deleted, which had been shown to exhibit post-transcriptional control over the BenR activator as well as to bind to certain mRNA targets of genes involved in the degradation of aromatic compounds. The rationale behind a crc deletion in Pmuc3 (resulting in strain Pmuc4) was to further increase the expression of benD, whose transcriptional control is unclear, as well as to enhance 3-FBz uptake since Crc was shown to have a repressing effect on the expression of a diverse range of porins and transporters. Because of the repressing effect that Crc reportedly has on benABC, its deletion was also combined with Pm-BCD10→catA and Pm-BCD10→catA-II without changing the regulation of benABC.
The three genes located within the ben gene cluster encoding membrane proteins that are thought to be involved in benzoate uptake, benK (transporter), benE-II (transporter), and nicP-I (also named benF, a porin), were also chosen as targets for expression control by Pm(ML1-17)-BCD10.
Each of these strains, Pmuc3, Pmuc4, P. putida Pm-BCD10-→catA/catA-II Δcrc, Pmuc3 Pm(ML1-17)-BCD10→benK (Pmuc3K), Pmuc3 Pm(ML1-17)-BCD10→benE-II (Pmuc3E), and Pmuc3 Pm(ML1-17)-BCD10→nicP-I (Pmuc3F) were transformed with pSEVA228 to provide enough XylS activator for the various genes under Pm control and tested for their performance in the conversion of 10 mM 3-FBz in shaken flasks with 30 mM glucose as the source of carbon and energy. The concentration changes in biomass (estimated cell dry weight—CDW), 3-FBz, 3-FC, 4-FC, and 2-FMA for each strain in the course of 80 h of culture are displayed in
All strains except P. putida Pm-BCD10→catA/catA-11 Δcrc completely consumed 10 mM 3-FBz within the course of the experiment and after an initial accumulation of ˜1 mM 3-FC within the first 5 h, likely due to a lack of catA(0) induction before the start of the fermentation, the only 3-FC detectable at the end of the fermentations was at a concentration of 2.6 mM 3-FC for P. putida Pm-BCD10→catA/catA-II Δcrc and 0.5 mM 3-FC for Pmuc3 Pm(ML-17)-BCD10→nicP-1 (Pmuc3F). All other Pmuc3 derivatives completely converted about 50% of the 3-FBz provided into 2-FMA. However, there were striking differences in the rates at which 3-FBz was consumed and 2-FMA was produced. The respective biomass-specific 3-FBz uptake rates qs and 2-FMA formation rates qp for each strain are listed in Table 4 and 5, below.
P. knackmussii
P. putida KT2440
a no complete consumption of 3-FBz
b qP represents about 50% of qS
P. knackmussii
P. putida KT2440
0.344 b, c
a no complete consumption of 3-FBz
b no detectable fluorocatechol at the end of the fermentation
c Pmuc4 showed a very long lag phase of about 30 h with low biochemical activity, followed by a fast conversion of 3-FBz and consumption of glucose
A direct comparison of qs and qp allows to draw conclusions about the balance of biochemical activities within the ortho-cleavage pathway acting on 3-FBz. With approximately 50% of the substrate channeled into the 2-FMA producing 1,2-dioxygenation route, the qp value should be half of the qs value if no significant accumulation of intermediates or by-product occurs. The deletion of crc in Pmuc4 resulted in an increase in productivity by about 1.5-fold compared to Pmuc3, suggesting an enhanced substrate uptake or increased BenD activity. The deletion of crc in Pmuc4 also resulted in a long lag phase of about 30 h after exposure to 3-FBz, in which the biomass-specific 3-FBz consumption rate, the 2-FMA production rate, and especially the glucose consumption rate was severely decreased. After this extended time of adaptation, all specific rates increased four to seven-fold so that about half of the supplied 3-FBz and glucose were consumed within the last three hours of fermentation. The deletion of the global carbon catabolite regulator Crc appears to create inherent instabilities in the metabolic state of Δcrc cells, which can result in either an immediate fast growth and 3-FBz utilization followed by metabolic inactivity as observed in strain Pm-BCD10→catA1/catA-II Δcrc pSEVA228, or extended phases of “dormancy” followed by very high metabolic activity as displayed by Pmuc4. It will require further investigation on the underlying mechanisms responsible for this drastic shift in physiology and suitable cultivation conditions that allow for a reliable utilization of the maximum productivities observed for these two strains. The individual expression of nicP-I, benE-II or benK resulted in a decrease in the 3-FBz uptake rate and thus 2-FMA formation, providing evidence for their contributing role in 3-FBz transport which was reduced due to the weak induction of the genes by Pm. A crc deletion without the replacement of the native Pben promoter driving benABC expression caused a qs increase by about 3.5-fold compared to strain Pmuc3 Δcrc harboring the Ptac→benABC modification. However, this strong increase in 3-FBz uptake was not counteracted by a sufficient catechol 1,2-dioxygenase activity, consequently leading to a high accumulation of 3-FC. This result demonstrates the importance of a static control of benABC decoupled from the host's regulatory network.
A further set of strains was created to further disclose the importance of each catA and catA-II via their individual expression with Pm and the stronger translational coupler BCD2, as well as to reveal the presence of a potential oxygen limitation (two reactions involved in the bioconversion consume molecular oxygen) via the introduction of the Vitreoscilla stercoraria vhb gene, encoding a bacterial hemoglobin. For the latter, vhb was inserted into the chromosomal crc locus under Pm-BCD10 control with (tat-vhb) and without (vhb) an N-terminal signal peptide for the P. putida type-II secretion system. Two last strains, the gene encoding xylS was integrated into the chromosome of Pmuc3 Δcrc under the control of BCD10 as well as the very strong P14g promoter or P14 with a reported strength of about 30% compared to P14g, in an attempt to create an efficient plasmid-free bioconversion strain. The courses of fermentations with these six strains are shown in
The expressional control of only catA resulted in a significant decrease in 1,2-dioxygenase, apparent in the strong accumulation of 3-FC with a concentration of 3.6 mM. The expression control of catA-II by Pm-BCD2, however, provided sufficient 1,2-dioxygenase activity to enable a complete conversion of 3-FBz into 2-FMA at a maximum yield (50%), despite an initial 3-FC accumulation to 1.4 mM. Yet, the product formation rate qp was only less than 50% of the qp for Pmuc3, which has both catA and catA-II controlled by Pm-BCD10. Although no complete consumption of 3-FBz occurred with P. putida Ptac-BCD10→benABC Pm-BCD10→catA Pm-BCD10→catA-II, no accumulation of intermediates was observed and both qp and qs were significantly enhanced compared to strain Pmuc3 with benABC under its native translational control. The cytoplasmic expression of V. stercoraria vhb had no significant effect on the performance of Pmuc4, while tat-vhb expression impaired growth, even with glucose as the sole carbon compound (data not shown). The two Pmuc4 strains with xylS integrated into the genome upstream of catA under control of either P14g (see
Based on the previous observations, a fourth set of strains was designed with all of the modifications that had a beneficial effect on 3-FBz uptake or bioconversion rates combined. These genomic modifications comprised Ptac-BCD10→benABC and the deletion of crc, as well as different types of regulatory sequences for catA and catA-II.
In Pmuc5, catA and catA-II were put under the control of Pm-BCD10 and Pm-BCD2, respectively, and XylS was provided from pSEVA228. In the plasmid-free strains Pmuc6, Pmuc7, Pmuc8, and Pmuc9, catA and catA-II were translationally controlled by BCD10 as well as the medium-weak, constitutive promoters J23115, J23108, J23114, and P14b, respectively. The fermentation profiles of these five strains are shown in
A fifth set of strains was created to characterize the specific effects of Ptac-BCD10→benABC and Δcrc, to enable an efficient bioconversion without the use of plasmids, and to establish the role of the transcription strength for catA-II. In the resulting strains Pmuc10, Pmuc11, Pmuc12, and Pmuc13, either only catA-II or both catA genes were put under the control of the constitutive promoter P14b and BCD10. The combination of Ptac→benABC, P14b-BCD10→catA, and P14b-BCD10→catA-II in Pmuc10 enabled a complete conversion of all supplied 3-FBz at the maximum theoretical yield of 50% at the highest specific productivities observed (Table 4), without the necessity to provide the activator XylS on a plasmid. Furthermore, the constitutive expression of catA and catA-II decreased the initial accumulation of 3-FC as compared to Pmuc3, in which the catechol 1,2-dioxygenases were induced only at the start of the fermentation with the addition of 3-FBz. With both catA and catA-II constitutively expressed by P14b, the additional deletion of crc (Pmuc11) or the introduction of BCD10 for benABC (Pmuc12) resulted in an incomplete consumption of 3-FBz at low specific rates. Thus, both genetic modifications brought about effects that are detrimental to the bioconversion. In strain Pmuc13, only catA-II was constitutively expressed, while catA was under the regulation of Pm. To achieve different induction strengths for catA, XylS was provided either with its wild-type sequence (on pSEVA228) or as variant XylSThr45 which had been shown to cause a higher induction strength in response to 3-FBz compared to the wild-type variant by a factor of 2 [Ramos et al. (1990) and
It can be expected that the regulatory sequences successfully employed to control the expression of benABC, catA, and catA-II can be replaced with promoters and translation initiation sequences of comparable strength without disrupting the functionality of the pathway. Alternative sequences for Ptac include other medium-to-high strength promoters (
Pmuc10 was tested in microtiter plate cultivations regarding its ability to utilize benzoate as the sole carbon source, as well as the maximum 3-FBz concentrations tolerated by the strain in the presence of 30 mM glucose as the source of carbon and energy (
With the 2-FMA purified from fermentation broths, the toxicity of the product on the chosen bacterial platform P. putida was investigated in microtiter plate cultivations in minimal medium with 30 mM glucose and varying concentrations of the bioconversion product (
To examine the suitability of the ortho-cleavage pathway enzymes in P. putida for the bioconversion of 2- or 3-FBz to 2-FMA, both P. knackmussii and P. putida KT2440 were cultured in microtiter plates with minimal medium supplemented with 30 mM glucose (or 30 mM potassium benzoate) and varying concentrations of metabolites involved in the bioconversion process (
P. putida KT2440
P. knackmussii
Within the course of 50 h, no growth was detectable for P. knackmussii on benzoate as the sole source of carbon, while P. putida grew with a μmax of 0.62 h−1. With glucose as the primary source of carbon and energy, P. putida grew with a maximum rate μmax of 0.86 h−1, which is 2.5 times higher than that of P. knackmussii (μmax 0.35 h−1). In the presence of 2-FBz, both species demonstrated some degree of sensitivity to the compound, which became more prominent with increasing concentrations. However, while P. putida still grew with 53% of its maximum rate on glucose and without any noticeable effect on the duration of the lag phase in the presence of 20 mM 2-FBz, P. knackmussii was unable to grow at all within the course of the experiment (50 h) and showed significantly increased lag phases at 10 mM and 15 mM of 2-FBz. At 15 mM 2-FBz, P. knackmussii grew with 54% of the μmax observed on only glucose. The detrimental impact of the second bioconversion substrate 3-FBz on the growth of both species was more severe. P. putida was able to grow in the presence of maximum 10 mM 3-FBz with a substantially reduced μmax value. Even at 5 mM, the growth of P. knackmussii was almost completely impaired and no growth was observable at concentrations of 10 mM.
Depending on the position of the fluoro-substitution on the benzoate derivative, the three 1,2-dihydroxybenzenes pyrocatechol (here referred to as catechol), 3-FC, and 4-FC are produced as intermediary metabolites. Only a little toxic effect was observed in the presence of up to 2 mM catechol on both P. putida and P. knackmussii. The physiological response of the two species to the two fluorinated 1,2-dihydroxybenzene derivatives, 3-FC and 4-FC, was more pronounced. While both 3-FC and 3-FC affected the cells' growth rate in a similar manner, the presence of 4-FC had a more prominent effect on the duration of lag phases. However, in both cases, P. putida demonstrated a higher resistance to the toxic effects of both 3-FC and 4-FC than P. knackmussii.
After establishing the physiological response of both P. knackmussii and P. putida to the chemical compounds involved in the bioconversion of FBz to 2-FMA, the two species were subjected to a fermentation experiment in shaken flasks containing synthetic de Bont medium with 10 g L−1 MOPS at pH 7.0, and supplemented with 30 mM glucose as well as 10 mM of 2-FBz or 3-FBz (
Exposed to 3-FBz, P. putida consumed the substrate up until a time between 7 and 20 h with a qs of 0.98 mmol gCDW−1 after which the 3-FBz concentration in the culture supernatant remained constant. Within the first 7 h, all 3-FBz consumed was converted into 3-FC until it reached a concentration of 1 mM. In the course of the next 13 h, 40% of 3-FC in the supernatant was converted into 2-FMA, combined with a fraction of additionally consumed 3-FBz at a combined product formation rate qp of 0.06 mmol gCDW−1 h−1. At the end of the fermentation, 3.48 mM 3-FBz had been converted into 1.38 mM 2-FMA, corresponding to a product yield YSP of 0.41 mol mol-1, as well as 0.52 mM 3-FC. The combined concentrations of metabolites constituting the 1,2-dioxygenation route (2-FMA and 3-FC) add up to 1.90 mM, representing 55% of the consumed 3-FBz. Considering its previously established toxicity, the accumulation of 3-FC was identified as a major bottleneck for an efficient and prolonged bioconversion process in P. putida. Also with P. knackmussii, 3-FBz consumption occurred only within the first 7-20 h of the fermentation, with a qs of 1.01 mmol gCDW−1 h−1. After an initial increase of 3-FC up to 0.94 mM in the culture supernatant, the catechol derivative was completely re-consumed until 20 h. From a total concentration of 2.93 mM 3-FBz consumed, P. knackmussii produced 1.64 mM 2-FMA [corresponding to 56% of the substrate (YSP: 0.41 mol mol-1)] with a rate qp of 0.09 mM gCDW−1 h−1. In none of the conditions tested significant amounts of 4-FC could be detected in the culture supernatants. The enzyme machinery performing the di-oxygenation on fluorinated catechol derivatives that both species are equipped with thus seems to demonstrate a higher efficiency towards 4-FC compared to 3-FC.
With the asymmetrical use of the non-productive 1,2-dioxygenation route when using 2-FBz as substrate for the bioconversion, almost 90% of fluorine gets lost, in contrast to about 50% with 3-FBz as substrate. The 3-F-substituted benzoate consequently represents the most suitable precursor compound to produce 2-FMA. A faster growth rate, higher biomass yield on glucose, and a higher resistance to toxic effects of pathway intermediates make P. putida stand out as the bacterial platform better suited to perform this bioconversion.
P. putida KT2440 (UniProt accession number Q88I40)
P. putida KT2440 (UniProt accession number Q88I39)
P. putida KT2440 (UniProt accession number Q88I38)
P. putida KT2440 (RefSeq NP_745305.1)
P. putida KT2440 (RefSeq NP_745306.1)
P. putida KT2440 (RefSeq NP_745307.1)
P. putida KT2440 (UniProt accession number Q88I37)
P. putida KT2440 (RefSeq NP_745308.1)
P. putida KT2440 (UniProt accession number Q88GK8)
P. putida KT2440 (RefSeq NP_745846.1)
P. putida KT2440 (UniProt accession number Q88I35)
P. putida KT2440 (RefSeq NP_745310.1)
P. putida KT2440
P. putida mt-2
P. putida mt-2 (RefSeq NP_542858.1)
P. putida mt-2 (UniProt accession number P07859)
P. putida KT2440
P. putida KT2440
Vitreoscilla stercoraria (UniProt accession number:
P. putida KT2440 (UniProt accession number Q88HH4)
P. putida KT2440 (Gene Symbol PP_3383)
P. putida KT2440 (UniProt accession number Q88HH5)
P. putida KT2440 (Gene Symbol PP_3383)
P. putida KT2440 (UniProt accession number Q88HH4)
P. putida KT2440 (Gene Symbol PP_3382)
P. putida KT2440 (UniProt accession number Q88MX4)
P. putida KT2440 (Gene Symbol PP_1444)
P. putida KT2440 (UniProt accession number Q88C91)
P. putida KT2440 (Gene Symbol PP_5292)
P. putida KT2440 (UniProt accession number Q88I36)
P. putida KT2440 (Gene Symbol PP_3165)
P. putida KT2440 (UniProt accession number Q88I34)
P. putida KT2440 (Gene Symbol PP_3167)
P. putida KT2440 (UniProt accession number Q88I33)
P. putida KT2440 (Gene Symbol PP_3168)
| Number | Date | Country | Kind |
|---|---|---|---|
| 20183692.1 | Jul 2020 | EP | regional |
| 21170337.6 | Apr 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/068282 | 7/2/2021 | WO |
