Benzoic acid synthesis

Abstract

Disclosed herein are methods for the production of benzoic acid, nucleic acids for use in methods for the production of benzoic acid, methods of producing a compound utilising benzoic acid produced by a method disclosed herein, and microorganisms for the production of benzoic acid.

Description

[0001] BENZOIC ACID SYNTHESIS

[0002] This application claims priority from GB2415664.8 filed 24 October 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Technical field

[0004] The present invention relates to the fields of molecular biology and biotechnology and particularly, although not exclusively, to methods of producing phenylalanine and benzoic acid.

[0005] Background

[0006] Biological benzoic acid production can be achieved by non-native enzymatic pathways expressed within microorganisms, however available methods are not ideal. Improved methods are required to make biological benzoic acid production more efficient and economically viable.

[0007] Summary of the Invention

[0008] A method of producing benzoic acid is provided.

[0009] A bacterial cell for the production of benzoic acid is also provided.

[0010] In some embodiments, the method of producing benzoic acid comprises:

[0011] (i) the conversion of Phe to cinnamate catalysed by a phenylalanine lyase,

[0012] (ii) the conversion of cinnamate to cinnamoyl-CoA catalysed by a cinnamate-CoA ligase, (iii) and the conversion of cinnamoyl-CoA to benzoic acid by phenylpropanoid degradation pathway enzymes.

[0013] In some embodiments, the method comprises providing a bacterial cell which expresses a heterologous enzyme involved in the production of benzoic acid.

[0014] In some embodiments, the method comprises fermentation of a bacterial cell which expresses a heterologous enzyme involved in the production of benzoic acid.

[0015] In some embodiments, the cell comprises a phenylalanine lyase, a cinnamate-CoA ligase, and / or phenylpropanoid degradation pathway enzymes (e.g., phdE, phdB, and phdC).

[0016] A method of producing benzoic acid is provided, wherein the method comprises fermentation of a bacterial cell which heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, and phenylpropanoid degradation pathway enzymes,

[0017] wherein benzoic acid is produced through the following steps: (i) the conversion of phenylalanine to cinnamate catalysed by the phenylalanine lyase,

[0018] (ii) the conversion of cinnamate to cinnamoyl-CoA catalysed by the cinnamate-CoA ligase,

[0019] (iii) and the conversion of cinnamoyl-CoA to benzoic acid by the phenylpropanoid degradation pathway enzymes.

[0020] In some embodiments, the cell comprises an enoyl-CoA hydratase (e.g., phdE). In some embodiments, the cell comprises a 3-hydroxyacyl-CoA dehydrogenase (e.g., phdB). In some embodiments, the cell comprises a 3-oxoacyl-CoA ketohydrolase (e.g., phdC).

[0021] A method of producing benzoic acid is provided, wherein the method comprises fermentation of a bacterial cell which heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, and a 3-oxoacyl-CoA ketohydrolase, wherein benzoic acid is produced through the following steps:

[0022] (i) the conversion of phenylalanine to cinnamate catalysed by the phenylalanine lyase,

[0023] (ii) the conversion of cinnamate to cinnamoyl-CoA catalysed by the cinnamate-CoA ligase,

[0024] (iii) and the conversion of cinnamoyl-CoA to benzoic acid by the enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA ketohydrolase.

[0025] In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15. In some embodiments, the enzyme comprises an amino acid sequence with at least 70% sequence identity to SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15. In some embodiments, the enzyme comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15. In some embodiments, the enzyme comprises an amino acid sequence according to SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0026] In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19. In some embodiments, the enzyme comprises an amino acid sequence with at least 70% sequence identity to SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19. In some embodiments, the enzyme comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19. In some embodiments, the enzyme comprises an amino acid sequence according to SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19. In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:1. In some embodiments, the enzyme comprises an amino acid sequence with at least 70% sequence identity to SEQ ID NO:1. In some embodiments, the enzyme comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:1. In some embodiments, the enzyme comprises an amino acid sequence according to SEQ ID NO:1.

[0027] In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2. In some embodiments, the enzyme comprises an amino acid sequence with at least 70% sequence identity to SEQ ID NO:2. In some embodiments, the enzyme comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:2. In some embodiments, the enzyme comprises an amino acid sequence according to SEQ ID NO:2.

[0028] In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:3. In some embodiments, the enzyme comprises an amino acid sequence with at least 70% sequence identity to SEQ ID NO:3. In some embodiments, the enzyme comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:3. In some embodiments, the enzyme comprises an amino acid sequence according to SEQ ID NO:3.

[0029] A method of producing benzoic acid is provided, wherein the method comprises fermentation of a bacterial cell which heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, and a 3-oxoacyl-CoA ketohydrolase, wherein benzoic acid is produced through the following steps:

[0030] (i) the conversion of phenylalanine to cinnamate catalysed by the phenylalanine lyase,

[0031] (ii) the conversion of cinnamate to cinnamoyl-CoA catalysed by the cinnamate-CoA ligase,

[0032] (iii) and the conversion of cinnamoyl-CoA to benzoic acid by the enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA ketohydrolase, Wherein the the phenylalanine lyase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:4, the cinnamate-CoA ligase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:5, the enoyl-CoA hydratase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:1 , the 3-hydroxyacyl-CoA dehydrogenase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, and the 3-oxoacyl-CoA ketohydrolase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:3.

[0033] A method of producing benzoic acid is provided, wherein the method comprises fermentation of a bacterial cell which heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, and a 3-oxoacyl-CoA ketohydrolase, wherein benzoic acid is produced through the following steps during fermentation: (i) the conversion of phenylalanine to cinnamate catalysed by the phenylalanine lyase,

[0034] (ii) the conversion of cinnamate to cinnamoyl-CoA catalysed by the cinnamate-CoA ligase,

[0035] (iii) and the conversion of cinnamoyl-CoA to benzoic acid by the enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA ketohydrolase, wherein the the phenylalanine lyase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:4, the cinnamate-CoA ligase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:5, the enoyl-CoA hydratase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:1 , the 3-hydroxyacyl-CoA dehydrogenase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, and the 3-oxoacyl-CoA ketohydrolase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:3.

[0036] In some embodiments, the method of producing benzoic acid comprises fermentation of a microorganism. In some embodiments, the method of producing benzoic acid comprises fermentation of a bacterium. In some embodiments, the method of producing benzoic acid comprises bacterial fermentation.

[0037] In some embodiments, the method comprises fermentation in culture media. In some embodiments, the method comprises fermentation in high salt culture media. In some embodiments, the method comprises fermentation in culture media comprising more than 20 g / L NaCI. In some embodiments, the method comprises fermentation in culture media comprising 60 g / L NaCI. In some embodiments, the method comprises fermentation in high salt Luria broth (LB60). In some embodiments, the method comprises fermentation in modified MM63 media, comprising 60 g / L NaCI.

[0038] In some embodiments, the method comprises fermentation of a bacterial cell under non-sterile conditions.

[0039] In some embodiments, culture medium is not sterile. In some embodiments, culture medium is not sterilised. In some embodiments, culture medium is not sterilised before it is inoculated. In some embodiments, the culture medium is not autoclaved, filtered, heated, boiled, irradiated, and / or treated with a gas, before the culture medium is contacted with the microorganism.

[0040] In some embodiments, the method comprises fermentation of a bacterial cell in culture media having a pH between pH 7.5 and pH 9.5.

[0041] In some embodiments, the titre / yield of benzoic acid is greater than 4.5 g / L. In some embodiments, the titre / yield of benzoic acid is greater than 4.7 g / L. In some embodiments, the titre / yield of benzoic acid is greater than 5 g / L. In some embodiments, the titre / yield of benzoic acid is greater than 5.6 g / L. In some embodiments, the percentage yield of benzoic acid from phenylalanine is greater than 90%. In some embodiments, the percentage yield of benzoic acid from phenylalanine is greater than 95%. In some embodiments, the percentage yield of benzoic acid from phenylalanine is greater than 96%.

[0042] In some embodiments, less than 1 g / L cinnamic acid is produced. In some embodiments, less than 0.5 g / L cinnamic acid is produced. In some embodiments, less than 0.3 g / L cinnamic acid is produced. In some embodiments, less than 0.1 g / L cinnamic acid is produced.

[0043] In some embodiments, the method comprises fermentative production of benzoic acid, or biocatalytic production of benzoic acid.

[0044] In some embodiments, the method of producing benzoic acid comprises whole cell biocatalysis. In some embodiments, the method of producing benzoic acid comprises biocatalytic conversion of phenylalanine to benzoic acid by a whole cell catalyst.

[0045] In some embodiments, the method of producing benzoic acid comprises the following steps during fermentation:

[0046] (i) the conversion of Phe to cinnamate catalysed by a phenylalanine lyase,

[0047] (ii) the conversion of cinnamate to cinnamoyl-CoA catalysed by a cinnamate-CoA ligase, (iii) and the conversion of cinnamoyl-CoA to benzoic acid by phenylpropanoid degradation pathway enzymes.

[0048] In some embodiments, the method comprises two stages: (i) a growth stage, and (ii) a conversion stage.

[0049] A ‘conversion stage’ may alternatively be termed a ‘resting stage’.

[0050] In some embodiments, the microorganism is cultured in a growth medium in the growth stage.

[0051] In some embodiments, the method of producing benzoic acid comprises the following steps in a conversion stage:

[0052] (i) the conversion of phenylalanine to cinnamate catalysed by a phenylalanine lyase, (ii) the conversion of cinnamate to cinnamoyl-CoA catalysed by a cinnamate-CoA ligase, (iii) and the conversion of cinnamoyl-CoA to benzoic acid by phenylpropanoid degradation pathway enzymes.

[0053] A method of producing benzoic acid is provided, wherein the method comprises fermentation of a bacterial cell in a growth stage, wherein the bacterial cell heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, and a 3-oxoacyl-CoA ketohydrolase,

[0054] wherein benzoic acid is produced through the following steps in a conversion stage:

[0055] (i) the conversion of phenylalanine to cinnamate catalysed by the phenylalanine lyase,

[0056] (ii) the conversion of cinnamate to cinnamoyl-CoA catalysed by the cinnamate-CoA ligase,

[0057] (iii) and the conversion of cinnamoyl-CoA to benzoic acid by the enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA ketohydrolase, wherein the the phenylalanine lyase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:4, the cinnamate-CoA ligase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:5, the enoyl-CoA hydratase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:1 , the 3-hydroxyacyl-CoA dehydrogenase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2, and the 3-oxoacyl-CoA ketohydrolase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:3.

[0058] In some embodiments, the method comprises microbial fermentation. In some embodiments, the method comprises biotransformation.

[0059] In some embodiments, the growth stage comprises microbial fermentation. In some embodiments, the conversion stage comprises biotransformation. In some embodiments, the conversion stage comprises biocatalytic production of benzoic acid.

[0060] In some embodiments, the microorganism is separated from the culture media. In some embodiments, the microorganism is resuspended. In some embodiments, the microorganism is separated from the culture medium and resuspended in a buffer. In some embodiments, the microorganism is separated from the culture medium and resuspended in a buffer for the conversion stage.

[0061] In some embodiments, phenylalanine is provided exogenously to the bacterial cell.

[0062] In some embodiments, phenylalanine is provided exogenously to the bacterial cell during fermentation. In some embodiments, phenylalanine is provided exogenously to the culture media.

[0063] In some embodiments, phenylalanine is provided exogenously to the bacterial cell while the bacterial cell is in a conversion stage. In some embodiments, phenylalanine is provided exogenously to the bacterial cell while the bacterial cell is in a conversion stage following a growth stage. In some embodiments, phenylalanine is provided exogenously to a buffer. In some embodiments, phenylalanine is provided exogenously to a buffer containing a bacterial cell. In some embodiments, the bacterium is an Escherichia bacterium. In some embodiments, the bacterium is an Escherichia coli.

[0064] In some embodiments, the bacterium is a halophilic bacterium. In some embodiments, the bacterium is a Halomonas bacterium. In some embodiments, the bacterium is Halomonas bluephagenesis TD1.0, Halomonas bluephagenesis TD01 , Halomonas bluephagenesis TQ10, or Halomonas rowanensis. In some embodiments, the bacterium is Halomonas bluephagenesis. In some embodiments, the bacterium is Halomonas bluephagenesis TD1.0.

[0065] In some embodiments, the method comprises fermentative benzoic acid production.

[0066] In some embodiments, the method of producing benzoic acid comprises:

[0067] production of phenylalanine through bacterial fermentation, and

[0068] biocatalytic conversion of phenylalanine to benzoic acid by a whole cell catalyst.

[0069] In some embodiments, the microorganism heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl-CoA ketohydrolase, and an enoyl-CoA hydratase.

[0070] In some embodiments, the bacterium heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl-CoA ketohydrolase, and an enoyl-CoA hydratase.

[0071] In some embodiments, the whole cell catalyst heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl-CoA ketohydrolase, and an enoyl-CoA hydratase.

[0072] A method of producing benzoic acid is provided, wherein the method of producing benzoic acid comprises:

[0073] production of phenylalanine through bacterial fermentation, and

[0074] biocatalytic conversion of phenylalanine to benzoic acid by a whole cell catalyst, wherein the whole cell catalyst heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl-CoA ketohydrolase, and / or an enoyl-CoA hydratase.

[0075] In some embodiments, the method comprises phenylalanine (Phe) production.

[0076] In some embodiments, the method comprises fermentative Phe production.

[0077] In some embodiments, the method comprises the conversion of Phe to cinnamate. In some embodiments, the method comprises the conversion of Phe to cinnamate catalysed by a phenylalanine lyase.

[0078] In some embodiments, the method comprises the conversion of cinnamate to cinnamoyl-CoA.

[0079] In some embodiments, the method comprises the conversion of cinnamate to cinnamoyl-CoA catalysed by a cinnamate-CoA ligase.

[0080] In some embodiments, the method comprises the conversion of cinnamoyl-CoA to benzoic acid.

[0081] In some embodiments, the method comprises the conversion of cinnamoyl-CoA to benzoic acid by phenylpropanoid degradation pathway enzymes.

[0082] A method of producing benzoic acid is provided, wherein the method of producing benzoic acid comprises:

[0083] production of phenylalanine through bacterial fermentation, and

[0084] biocatalytic conversion of phenylalanine to benzoic acid by a whole cell catalyst, wherein the whole cell catalyst heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl-CoA ketohydrolase, and an enoyl-CoA hydratase, wherein the method comprises the conversion of phenylalanine to cinnamate catalysed by a phenylalanine lyase, the conversion of cinnamate to cinnamoyl-CoA catalysed by a cinnamate-CoA ligase, and the conversion of cinnamoyl-CoA to benzoic acid by phenylpropanoid degradation pathway enzymes (e.g., a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl-CoA ketohydrolase, and an enoyl-CoA hydratase).

[0085] In some embodiments, the method comprises the use of enzymes shown in the pathway of Figure 1.

[0086] In some embodiments, the method comprises the catalytic activity of enzymes shown in the pathway of Figure 1 .

[0087] In some embodiments, the method comprises the production of a whole cell biocatalyst which expresses a biocatalytic recombinant enzyme (e.g., RgPAL, ScCCL and / or phdBCE).

[0088] In some embodiments, the method comprises conversion of Phe to benzoic acid using a whole cell catalyst which expresses a biocatalytic recombinant enzyme.

[0089] In some embodiments, the method comprises biocatalytic conversion of Phe to benzoic acid. In some embodiments, the method comprises biocatalytic conversion of Phe to benzoic acid by a whole cell catalyst.

[0090] In some embodiments, the method comprises:

[0091] (i) fermentative Phe production, and

[0092] (ii) biocatalytic conversion of Phe to benzoic acid by a whole cell catalyst.

[0093] In some embodiments, fermentative Phe production comprises the fermentation of a bacterial cell. In some embodiments, fermentative Phe production comprises the fermentation of an E. coli cell.

[0094] In some embodiments, the bacterial cell has been modified to increase phenylalanine production. In some embodiments, the microorganism is an E. coli NST74 phenylalanine overproduction strain.

[0095] In some embodiments, fermentative Phe production comprises secretion of Phe into culture supernatant. In some embodiments, Phe is extracted from the culture supernatant. In some embodiments, Phe is extracted from the culture supernatant before being used as a substrate in the biocatalytic conversion of Phe to benzoic acid by a whole cell catalyst.

[0096] In some embodiments, fermentative Phe production comprises the fermentation of a first bacterial cell, and biocatalytic conversion of Phe to benzoic acid is performed by a second bacterial cell. In some embodiments, the first bacterial cell is an E. coli cell. In some embodiments, the first bacterial cell is an E. coli cell modified to increase phenylalanine production. In some embodiments, the second bacterial cell is an E. coli cell or a Halomonas cell, In some embodiments, the second bacterial cell heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, and phenylpropanoid degradation pathway enzymes.

[0097] In some embodiments, the method comprises:

[0098] 1. Fermentative Phe production,

[0099] 2. Generation of a whole cell biocatalyst which expresses a biocatalytic recombinant enzyme (e.g., RgPAL, ScCCL and / or phdBCE),

[0100] 3. Conversion of Phe to benzoic acid using the generated whole cell catalyst.

[0101] In some embodiments, the method comprises:

[0102] 1. Fermentative Phe production,

[0103] 2. Generation of a whole cell biocatalyst which expresses a biocatalytic recombinant enzyme (e.g., RgPAL, ScCCL and / or phdBCE),

[0104] 3. A growth stage comprising the growth of the whole cell biocatalyst, and

[0105] 4. A conversion stage comprising the conversion of Phe to benzoic acid by the whole cell catalyst. In some embodiments, the whole cell catalyst comprises an enzyme described herein. In some embodiments, the whole cell catalyst expresses an enzyme described herein. In some embodiments, the whole cell catalyst is a bacterial cell described herein.

[0106] In some embodiments, the whole cell biocatalyst expresses RgPAL. In some embodiments, the whole cell biocatalyst expresses ScCCL. In some embodiments, the whole cell biocatalyst expresses phdB. In some embodiments, the whole cell biocatalyst expresses phdC. In some embodiments, the whole cell biocatalyst expresses phdE.

[0107] In some embodiments, the whole cell biocatalyst is a bacterial cell. In some embodiments, the whole cell biocatalyst is E. coli. In some embodiments, the whole cell biocatalyst is a Halomonas bacterium.

[0108] In some embodiments, the whole cell biocatalyst has been modified with a plasmid. In some embodiments, the whole cell biocatalyst comprises a plasmid. In some embodiments, the whole cell biocatalyst comprises two plasmids.

[0109] In some embodiments, the plasmid is a heterologous plasmid. In some embodiments, the plasmid is a recombinant plasmid. In some embodiments, the plasmid is a pETDuet-1_RgPAL_ScCCL(A294G) plasmid. In some embodiments, the plasmid is a pETCOLADuet_phdBCE plasmid. In some embodiments, the plasmid is a pSEVA434-T7_RgPAL_ScCCL(A294G)_phdBCE plasmid.

[0110] In some embodiments, pH is monitored. In some embodiments, pH is monitored and maintained. In some embodiments, pH is monitored and maintained during fermentation.

[0111] In some embodiments, pH is maintained between pH 7.5 and pH 9.5. In some embodiments, pH is maintained at pH 8.5. In some embodiments, pH is maintained at pH 9. In some embodiments, pH is maintained through the addition of an acid (e.g., HCI) or a base (e.g., NaOH).

[0112] In some embodiments, temperature is monitored. In some embodiments, temperature is monitored and maintained. In some embodiments, temperature is monitored and maintained within an optimum range during fermentation.

[0113] In some embodiments, temperature is maintained between 20 °C and 40 °C. In some embodiments, temperature is maintained between 25 °C and 37 °C.

[0114] In some embodiments, dissolved oxygen concentration (dO2) is monitored. In some embodiments, dissolved oxygen concentration (dO2) is monitored and maintained. In some embodiments, dissolved oxygen concentration (dO2) is monitored and maintained within an optimum range during

[0115] fermentation. In some embodiments, dO2 is maintained above 20%. In some embodiments, dO2 is maintained above 25%. In some embodiments, dO2 is maintained above 30%.

[0116] In some embodiments, the method further comprises isolating the benzoic acid.

[0117] Other objects and embodiments of the invention will be apparent from the detailed description that follows.

[0118] Summary of the Figures

[0119] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0120] Figure 1: Novel pathway for the production of benzoic acid, comprising the activity of enzymes RgPAL (phenylalanine lyase), ScCCL(A294G) (cinnamate-CoA ligase), phdE (enoyl-CoA hydratase), phdB (3-hydroxyacyl-CoA dehydrogenase) and phdC (3-oxoacyl-CoA ketohydrolase).

[0121] Figure 2: Map of plasmid pETDuet-1_RgPAL_ScCCL(A294G)

[0122] Figure 3: Map of plasmid pETCOLADuet_phdBCE

[0123] Figure 4: Strategy 1 of Example 1 , for the production of benzoic acid from phenylalanine. The method comprises the fermentative production of cinnamic acid (CA) from phenylalanine (Phe) by cell line 1.1 , followed by biocatalytic production of benzoic acid (BA) from CA by cell line 1.2.

[0124] Figure 5: Strategy 2 of Example 1 , for the production of benzoic acid from Phe. The method comprises the fermentative production of L-phenylalanine (L-Phe) by cell line 2.1 , followed by biocatalytic production of cinnamic acid (CA) by cell line 2.2. and biocatalytic production of benzoic acid (BA) by cell line 2.2.

[0125] Figure 6: Strategy 3 of Example 1 , for the production of benzoic acid from Phe. The method comprises the fermentative production of L-phenylalanine (L-Phe) by cell line 2.1 , followed by biocatalytic production of benzoic acid (BA) by cell line 3.2.

[0126] Figure 7: Graphs showing the concentrations of benzoic acid (BA), cinnamic acid (CA), and phenylalanine (Phe), following small scale (10 mL) and scaled (1 L) reactions according to Strategy 3.

[0127] Figure 8: Comparative1H NMR of benzoic acid generated according to Example 2 and processed according to example 3, compared with commercially sourced benzoic acid.

[0128] Figure 9: Recovery of benzoic acid from a biocatalytic reaction.

[0129] Figure 10: Precipitation of benzylamine produced through the biocatalytic conversion of benzoic acid.

[0130] Figure 11: Schematic showing the conversion of benzoic acid (99%) to toluene, using nitrogen doped, alumina supported iron catalysts for hydrodeoxygenation reactions.

[0131] Figure 12: Map of plasmid pSEVA434-T7_RgPAL_ScCCL(A294G)_phdBCE Figure 13: Downstream processing of Halomonas culture supernatant for the recovery and purification of benzoic acid.

[0132] Figure 14: Benzoic acid production during the fermentation of H. bluephagenesis containing plasmid pSEVA434-T7-RgPAL_ScCCL (A294G)_phdBCE

[0133] Description

[0134] The inventors have developed new methods of producing phenylalanine (Phe) and benzoic acid.

[0135] In one aspect, the method of producing Phe and benzoic acid is based on an alternative 5-step cascade of enzyme reactions from Phe. An exemplary pathway is shown in Figure 1. In some embodiments, this pathway is based on the expression of enzymes RgPAL (phenylalanine lyase), ScCCL(A294G) (cinnamate-CoA ligase), phdE (enoyl-CoA hydratase), phdB (3-hydroxyacyl-CoA dehydrogenase) and phdC (3-oxoacyl-CoA ketohydrolase). Both E. coli and Halomonas can produce phenylalanine from simple carbon sources, such as glucose through genomic encoded central metabolism pathways.

[0136] In some embodiments, the method comprises fermentation. A simpler strategy for benzoic acid production by E. coli encoding the genes RgPAL, ScCCL and phdBCE would involve a fully fermentative approach from glucose. However, multiple studies investigating similar compound production showed that the production of cytotoxic compounds can lead to poor titres when using a fully fermentative approach. Therefore, different approaches using fermentation and / or biotransformations were investigated.

[0137] Inventors have surprisingly found that high yields of benzoic acid can be produced using fermentation and / or biotransformations (biocatalysis). For example, one method comprising a hybrid fermentationbiocatalysis approach (Example 2) led to a benzoic acid titre of 5.7 g / L. Additionally, a fully fermentative approach (Example 4) led to a benzoic acid titre of 4.8 g / L.

[0138] In some embodiments, the method comprises fermentative Phe production.

[0139] In some embodiments, the method comprises the production of a whole cell biocatalyst which expresses a recombinant enzyme (e.g., RgPAL, ScCCL and / or phdBCE).

[0140] In some embodiments, the method comprises conversion of Phe to benzoic acid using a whole cell catalyst which expresses a recombinant enzyme.

[0141] In some embodiments, the method comprises:

[0142] 1. Fermentative Phe production, 2. Generation of a whole cell biocatalyst which expresses a biocatalytic recombinant enzyme (e.g., RgPAL, ScCCL and / or phdBCE),

[0143] 3. Biocatalytic conversion of Phe to benzoic acid using the generated whole cell catalyst.

[0144] In some embodiments, a Phe overproduction bacterial strain is utilised in fermentative Phe production (e.g., in step 1 above). In some embodiments, an E. coli NST74 Phe overproduction strain is utilised in fermentative Phe production.

[0145] In some embodiments, the whole cell biocatalyst is a bacterial cell.

[0146] In some embodiments, the whole cell biocatalyst is E. coli.

[0147] In some embodiments, the whole cell biocatalyst is a Halomonas bacterium.

[0148] In some embodiments, the organism is capable of producing benzoic acid. In some embodiments, the organism is capable of producing benzoic acid from phenylalanine. In some embodiments, the organism is capable of producing benzoic acid from glucose.

[0149] In some embodiments, the whole cell biocatalyst expresses RgPAL. In some embodiments, the whole cell biocatalyst expresses ScCCL. In some embodiments, the whole cell biocatalyst expresses phdB. In some embodiments, the whole cell biocatalyst expresses phdC. In some embodiments, the whole cell biocatalyst expresses phdE.

[0150] In some embodiments, the whole cell biocatalyst expresses RgPAL, ScCCL and / or phdBCE.

[0151] In some embodiments, the whole cell biocatalyst has been modified with a plasmid. In some embodiments, the whole cell biocatalyst comprises a plasmid. In some embodiments, the whole cell biocatalyst comprises two plasmids.

[0152] In some embodiments, the plasmid is a heterologous plasmid. In some embodiments, the plasmid is a recombinant plasmid. In some embodiments, the plasmid is a pETDuet-1_RgPAL_ScCCL(A294G) plasmid. In some embodiments, the plasmid is a pETCOLADuet_phdBCE plasmid.

[0153] The pETDuet-1_RgPAL_ScCCL(A294G) plasmid contains RgPAL and ScCCL(A294G), and is shown in Figure 2.

[0154] The pETCOLADuet_phdBCE plasmid contains phdE, phdB and phdC, and is shown in Figure 3. In some embodiments, the method comprises a hybrid fermentation-whole cell biocatalysis approach. In some embodiments, the method comprises one or more steps according to Strategy 1 , Strategy 2, and / or Strategy 3 as outlined in Example 1. Further strategies for benzoic acid production are discussed by Luo and Lee (Metabolic Engineering 62 (2020) 298-311), the contents of which are hereby incorporated by reference in their entirety.

[0155] In some embodiments, benzoic acid undergoes downstream processing.

[0156] In some embodiments, benzoic acid is recovered through precipitation and filtration. In some embodiments, the benzoic acid is recovered through the methodology described in Example 3 herein.

[0157] In some embodiments, the benzoic acid is converted to benzylamine.

[0158] The benzoic acid is converted to benzylamine through any method known to the skilled person. The production of benzylamine from benzoic acid is a method known in the art. For example, WO2024 / 227822A1 (PCT / EP2024 / 061993), which is hereby incorporated by reference in its entirety, discloses a method of producing benzylamine, wherein the method comprises: (a) the conversion of benzoic acid into benzaldehyde catalysed by a carboxylic acid reductase (CAR), and (b) the conversion of the benzaldehyde into benzylamine catalysed by a transaminase (TA). In some embodiments, the benzoic acid is converted to benzylamine according to a method disclosed in WO2024 / 227822A1. In some embodiments, the method comprises: (a) the conversion of benzoic acid into benzaldehyde catalysed by a carboxylic acid reductase (CAR), and (b) the conversion of the benzaldehyde into benzylamine catalysed by a transaminase (TA). Inventors have shown that benzylamine can be produced from benzoic acid (Figure 10).

[0159] CAR enzymes comprise enzymatic activity which facilitates the conversion of carboxylic acids (e.g. benzoic acid) to aldehydes (e.g. benzaldehydes). Individual CAR enzymes, e.g. the Nocardia iowensis CAR, are capable of catalysing the reduction of a wide range of carboxylic acids. In some embodiments, the CAR is a wild type or mutant CAR derived from Nocardia iowensis, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium marinum, Nocardia otitidiscaviarum, or Tsukamurella paurometabola. In some embodiments, the CAR is a wild type or mutant CAR derived from Nocardia iowensis. In some embodiments, the CAR is a Nocardia iowensis CAR (UniProt Q6RKB1).

[0160] TAs, also known as aminotransferases, are a group of enzymes that mediate the transfer of an amine group between an amino acid and a keto acid. TA enzymes are capable of the enzymatic conversion of an aldehyde (e.g. benzaldehyde) to an amine (e.g. benzylamine). It has been shown that cotransaminases (co-TAs) can be efficient enzymes for the conversion of aldehyde to amine (Fuchs et al., 2012). In some embodiments, the TA is a wild type or mutant TA derived from Chromobacterium violaceum, Vibrio fluvialis, Alcaligenes denitrificans (also known as Achromobacter denitrificans), Pseudomonas aeruginosa, or Paracoccus denitrificans. In some embodiments, the TA is a wild type or mutant TA derived from Chromobacterium violaceum or Vibrio fluvialis. In some embodiments, the TA comprises an amino acid sequence with at least 40% sequence identity to the amino acid sequence of the Chromobacterium violaceum TA (CvTA; UNIPROT:Q7NWG4). In some embodiments, the TA comprises an amino acid sequence with at least 40% sequence identity to the amino acid sequence of the Vibrio fluvialis TA (VfTA; UNIPROT:F2XBU9). In some embodiments, the TA is a Chromobacterium violaceum TA (CvTA; UNIPROT:Q7NWG4). In some embodiments, the TA is a Vibrio fluvialis TA (VfTA; UNIPROT:F2XBU9).

[0161] In some embodiments, benzoic acid is converted to toluene.

[0162] Benzoic acid is commercially synthesised via the oxidation of toluene (US3816523A). The reverse reaction of toluene production from benzoic acid is not a commonly described transformation.

[0163] However, the reduction of carboxylic acids to the corresponding hydrocarbons has been studied with a variety of methods for organic synthesis.

[0164] Selective (>90%) conversion of benzoic acid to toluene using high temperatures (e.g. >400 °C) under a hydrogen gas atmosphere using metal oxide catalysts has been previously reported (Lange et al; 2002. Appl. Catal. A: Gen. 231 :17-26). A recent study has demonstrated the effectiveness of nitrogen doped, alumina supported iron catalysts for hydrodeoxygenation reactions capable of high conversion of benzoic acid (99%) and high selectivity to toluene (97.8%) at 280 °C and 4 MPa hydrogen in 8 hours (Figure 11) (Li et al; 2019. ACS Catal. 9:1564-1577).

[0165] This catalyst has been tested against a broad scope of organic acids and bioderived triglycerides with good chemoselectivity, conversion and yields. For benzoic acid the reaction is reported to proceed via hydrogenation to benzaldehyde and benzyl alcohol intermediates followed by hydrodeoxygenation to toluene. This catalyst shows significant improvements from previous methods reducing the required temperature with short reaction times.

[0166] Enzymes for benzoic acid production

[0167] Whole cell catalysts (e.g., bacterial cells) may be modified to express heterologous enzymes.

[0168] In some embodiments, a bacterial cell expresses a heterologous enzyme involved in the production of benzoic acid.

[0169] In some embodiments, the cell is capable of producing benzoic acid. In some embodiments, the cell is capable of producing benzoic acid from phenylalanine. In some embodiments, the cell is capable of producing benzoic acid from glucose.

[0170] In some embodiments, the cell comprises enzymes capable of producing benzoic acid from phenylalanine. In some embodiments, the cell comprises a phenylalanine lyase, a cinnamate-CoA ligase, and / or phenylpropanoid degradation pathway enzymes (e.g., phdE, phdB, and phdC). In some embodiments, the cell comprises the enzymes shown in the pathway of Figure 1.

[0171] In some embodiments, the cell comprises a phenylalanine lyase. In some embodiments, the cell comprises a cinnamate-CoA ligase. In some embodiments, the cell comprises phenylpropanoid degradation pathway enzymes. In some embodiments, the cell comprises an enoyl-CoA hydratase (e.g., phdE). In some embodiments, the cell comprises an 3-hydroxyacyl-CoA dehydrogenase (e.g., phdB). In some embodiments, the cell comprises an 3-oxoacyl-CoA ketohydrolase (e.g., phdC).

[0172] Phenylalanine lyases (or phenylalanine ammonia-lyases; PAL; EC 4.3.1.24) catalyze the deamination of phenylalanine to cinnamate and ammonia. In some embodiments the PAL is a Rhodotorula toruloides PAL (RgPAL). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to the amino acid sequence of an RgPAL (e.g., UNIPROT ID:

[0173] P11544).

[0174] In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of an RgPAL (e.g., UNIPROT ID: P11544). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:4. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:4.

[0175] In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of a PbPAL from Planctomyces brasiliensis. In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO: 12. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:12.

[0176] In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of a DdPAL from Dictyostelium discoideum (e.g., UniProt ID - Q556V9). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:13. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:13. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of an AvPAL from Anabaena variabilis (e.g., UniProt ID - Q3M5Z3). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:14. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:14.

[0177] In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of an SmPAL from Streptomyces maritimus (e.g., UniProt ID - Q9KHJ9). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:15. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:15.

[0178] Cinnamate-CoA ligases (CCL) catalyse the conversion of cinnamate to cinnamoyl-CoA. In some embodiments the CCL is a Streptomyces coelicolor CCL (ScCCL). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to the amino acid sequence of a ScCCL (e.g., UNIPROT ID: Q9K3W1). In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of a ScCCL (e.g., UNIPROT ID: Q9K3W1). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:5. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:5. In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:16. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:16.

[0179] In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to the amino acid sequence of a Glycine max CCL (e.g., UniProt - Q8S5C1). In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of a Glycine max CCL (e.g., UniProt - Q8S5C1). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO: 17. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:17. In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to the amino acid sequence of a Capsicum annuum CCL (e.g., UniProt - A0A2G3A9X6). In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of a Capsicum annuum CCL (e.g., UniProt - A0A2G3A9X6). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:18. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:18.

[0180] In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to the amino acid sequence of a Arabidopsis thaliana CCL (e.g., UniProt - Q9LU36). In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to the amino acid sequence of a Arabidopsis thaliana CCL (e.g., UniProt - Q9LU36). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:19. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:19.

[0181] In some embodiments, the organism capable of producing benzoic acid comprises one or more Corynebacterium glutamicum phd enzymes, variants of Corynebacterium glutamicum phd enzymes, or enzymes which are homologous to Corynebacterium glutamicum phd enzymes. In some embodiments, the organism capable of producing benzoic acid comprises one or more of Corynebacterium glutamicum phdB (3-hydroxyacyl-CoA dehydrogenase), Corynebacterium glutamicum phdC (3-oxoacyl-CoA ketohydrolase) (acetyl-CoA forming), or Corynebacterium glutamicum phdE (enoyl-CoA hydratase).

[0182] In some embodiments, the organism capable of producing benzoic acid comprises an enzyme capable of converting cinnamoyl-CoA to 3-HPP CoA. In some embodiments, the cell comprises an enzyme comprising an amino acid sequence with at least 40% sequence identity to the amino acid sequence of phdE from Corynebacterium glutamicum. In some embodiments, the cell comprises an enzyme comprising an amino acid sequence with at least 40% sequence identity to the amino acid sequence of phdE from Corynebacterium glutamicum (GENBANK ID: AGN20978.1). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:1. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:1. In some embodiments, the organism capable of producing benzoic acid comprises an enzyme capable of converting 3-HPP CoAto 3-KPP CoA. In some embodiments, the cell comprises an enzyme comprising an amino acid sequence with at least 40% sequence identity to the amino acid sequence of phdB from Corynebacterium glutamicum. In some embodiments, the cell comprises an enzyme comprising an amino acid sequence with at least 40% sequence identity to the amino acid sequence of phdB from Corynebacterium glutamicum (GENBANK ID: AGN20975). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2. In some embodiments, the enzyme comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:2.

[0183] In some embodiments, the organism capable of producing benzoic acid from phenylalanine comprises an enzyme capable of converting 3-KPP CoAto benzoic acid. In some embodiments, the organism capable of producing benzoic acid from phenylalanine comprises an enzyme which comprises an amino acid sequence with at least 40% sequence identity to the amino acid sequence of phdC from Corynebacterium glutamicum. In some embodiments, the organism capable of producing benzoic acid from phenylalanine comprises an enzyme which comprises an amino acid sequence with at least 40% sequence identity to the amino acid sequence of phdC from Corynebacterium glutamicum (GENBANK ID:AGN20976.1). In some embodiments, the enzyme comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:3. In some embodiments, the phdC comprises, or consists, of an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to one or more of the amino acid sequences of SEQ ID NO:3.

[0184] In some embodiments, the organism capable of producing a carboxylic acid is a microorganism. In some embodiments, the organism capable of producing benzoic acid from phenylalanine is a microorganism. In some embodiments, the microorganism is a bacterium, a fungus, a cyanobacterium, or an alga. In some embodiments, the microorganism is a gram-positive bacterium. In some embodiments, the microorganism is a gram-negative bacterium.

[0185] Production methods

[0186] Specific methods of producing phenylalanine and benzoic acid according to the present disclosure are disclosed within the examples herein.

[0187] The method may comprise more than one stage. For example, the method may comprise one or more of a phenylalanine production stage, a growth stage, and a conversion stage.

[0188] The method may comprise more than one method of growing or utilising a microorganism. The method may comprise more than one substrate conversion process. The method may comprise fermentation, whole cell biocatalysis, biotransformation, and / or product isolation. In some embodiments, the method of producing benzoic acid comprises whole cell biocatalysis.

[0189] Whole cell biocatalysis is reviewed in detail in Lin and Tao (Microbial Cell Factories volume 16, Article number: 106 (2017)). Whole-cell catalysis approaches can broadly be classified into biotransformation (biocatalysis) and fermentation bioprocesses. In fermentations, the products are synthesized from growth substrates via the host cells’ native metabolism and are accompanied in the fermentation broth by metabolic intermediates that make downstream processing complicated. In biotransformations, cell growth (the enzyme manufacturing phase) and production phase (conversion stage) are separated. Substrates are converted to the desired products by resting cells. The key advantages of whole-cell biocatalysis are its abilities to use cheap and abundant raw materials and to catalyse multistep reactions.

[0190] In some embodiments, the method comprises fermentative production of benzoic acid, or biocatalytic production of benzoic acid.

[0191] The fermentative production of benzoic acid comprises the fermentation of a microorganism. In some embodiments, fermentative production of benzoic acid does not comprise resuspending bacterial cells in a buffer. The method may involve culture or fermentation of a microorganism. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Secreted proteins can be collected by partitioning culture media / fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted or expressed peptide or polypeptide. Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein above).

[0192] A bioreactor may comprise a regulated bioreactor, in which one or more conditions may be controlled or monitored, for example, oxygen partial pressure. Devices for measuring and regulating these conditions are known in the art. For example, dissolved oxygen electrodes may be used for oxygen partial pressure and / or monitoring dissolved oxygen concentration (dO2). The oxygen partial pressure can be regulated via the amount and the composition of the selected gas mixture (e.g., air or a mixture of air and / or oxygen and / or nitrogen and / or carbon dioxide) and / or by adjusting the agitation rate. Suitable devices for measuring and regulating the oxygen partial pressure are described by Bailey, J E. (Bailey, J E., Biochemical Engineering Fundamentals, second edition, McGraw-Hill, Inc. ISBN 0-07-003212-2 Higher Education, (1986)) or Jackson A T. Jackson A T., Verfahrenstechnik in der Biotechnologie, Springer, ISBN 3540561900 (1993)). Alternatively, a culture may be static, i.e. where active agitation of the culture / culture media is not employed, optionally wherein mixing is performed via gas bubbling. In some examples, a fed batch method is utilised. In some examples, a fed batch fermentation is utilised. In some embodiments, a dose of a biochemical substrate (or more than one dose) is provided during microorganism growth. In some embodiments, a dose of a biochemical substrate (or more than one dose) is provided during exponential microorganism growth. In some embodiments, a dose of a biochemical substrate (or more than one dose) is provided during mid-late exponential microorganism growth.

[0193] Cultures of a microorganism, or microbial cultures, or cultures or microbiological cultures, generally comprise a culture vessel, a growth medium, and at least one microorganism cell. In some embodiments, the culture is a lab-scale culture. In some embodiments, the culture is an industrialscale culture. In some embodiments of the present disclosure, the microorganism cell is a bacterial cell.

[0194] A microbial medium, microbial growth medium or microbial culture medium is a liquid, semi-solid or solid designed to support the growth and proliferation of microbial cells. Microbial culture media are well known by scientists in the area of cell culture. Microbial cell culture media types and methods are comprehensively reviewed in A Rouf, Varsha Kanojia, HR Naik, Bazilla Naseer and Tahiya Qadri (2017) An overview of microbial cell culture, Journal of Pharmacognosy and Phytochemistry, Vol. 6, Issue 6 p 1923-1928, the contents of which are incorporated by reference.

[0195] Microbial culture media may comprise an appropriate source of energy and compounds which regulate the cell cycle. In addition to nutrients, the medium also helps maintain pH and osmolality. However, in some embodiments, there is no glucose and / or no carbon source present in the media. Microbial culture media may be liquid (i.e. aqueous), semi-solid (i.e. gelatinous), or completely solid. Semi-solid and solid media may contain agar, silicagel, acrylamide, gellan gum, or other solidification agents. Liquid media generally do not contain solidification agents.

[0196] In some embodiments, the culture media is Luria broth (LB), high salt Luria broth (LB60), minimal media, high salt minimal media, or another suitable media known by the skilled person.

[0197] LB Miller contains 5 g / L yeast extract, 10 g / L tryptone and 10 g / L NaCI pH 7.0, and is widely used and well known to the skilled person. High salt Luria broth (LB60) is identical to LB, but has a higher salt content (60 g / L NaCI). Minimal medium is culture medium for microorganisms that contains the minimal necessities for growth - containing only inorganic salts, water, and optionally a carbon source. High salinity minimal medium contains higher salt content than typical minimal medium. An exemplary high salinity minimal medium is filter sterilised sea water with or without a supplemental carbon source.

[0198] In some embodiments, the microbial culture media is taken directly from the environment, for example from sea water, brackish water, river water, lake water, pond water. In some embodiments the cells are grown directly in the water taken from the environment, in some embodiments the environmental water is processed (e.g. filtered and / or autoclaved) before use as culture media, in some embodiments further additives (e.g. carbon, mineral, and / or amino acids) are added to the water to support growth of the cells. In some embodiments, the water is polluted with excess carbon and / or excess sulfur containing compounds. The sulfur containing compound may be a sulfate, a thiosulfate, and / or a sulfite.

[0199] In some embodiments, exogenous glucose is provided. In some embodiments, exogenous glucose is provided in a conversion stage. In some embodiments, exogenous glucose is provided following a growth stage. In some embodiments, exogenous glucose is provided in a conversion stage following a growth stage.

[0200] In some embodiments, the method comprises two stages: (i) a growth stage, and (ii) a conversion stage.

[0201] In some embodiments, the microorganism is cultured in a growth medium in the growth stage. In some embodiments, the growth stage comprises microbial fermentation. In some embodiments, the conversion stage comprises biotransformation. In some embodiments, the conversion stage comprises biocatalytic production of benzoic acid.

[0202] The growth stage may involve culture or fermentation of cells modified to express the relevant polypeptide(s). The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. In some embodiments, the growth stage comprises culture or fermentation of a microorganism. Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein), and Doran, Chapter 1 - Bioprocess Development: An Interdisciplinary Challenge, Bioprocess Engineering Principles (Second Edition; incorporated by reference herein) 2013, Pages 3-11. Any culture vessel or media, for example those described in this disclosure, can be used in the growth stage.

[0203] In some embodiments, the growth stage occurs until the culture reaches a certain density, or the culture comprises a specific amount of biomass.

[0204] The biomass of a culture can be accurately estimated using optical density (CD) assays.

[0205] In some embodiments, microorganisms are moved from the growth stage to the conversion stage when CD at a wavelength of 600 nm (ODeoonm) is above a pre-specified threshold. In some embodiments, microorganisms are grown until the culture reaches an ODeoonm of at least 0.1. In some embodiments, microorganisms are grown until the culture reaches an ODeoonm of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or at least 1.0. In some embodiments, microorganisms are grown until the culture reaches an ODeoonm of 0.1. In some embodiments, microorganisms are grown until the culture reaches an ODeoonm of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or at least 1.0.

[0206] The method may comprise a resting stage. A resting stage may also be known as a conversion stage. In some embodiments, the method comprises a conversion stage.

[0207] In some embodiments, microorganisms are moved from a growth stage to a conversion stage. In some embodiments, microorganisms are moved from the culture media to a buffer. In some embodiments, microorganisms are moved from the culture media in the growth stage to a buffer for the conversion stage. In some embodiments, a buffer is a solution containing either a weak acid and its salt, or a weak base and its salt, which is resistant to significant changes in pH within a certain range (buffer capacity).

[0208] When moving microorganisms from a culture media, the solid cell biomass must be separated from the liquid media. The media is a liquid fraction or a liquid portion which lies above a sediment formed by the solid microorganisms. The microorganism fraction is the solid sediment fraction which forms below the liquid fraction. The microorganism fraction can be separated from the liquid fraction in a number of ways known by the skilled person, including filtration, chromatography, evaporation, sedimentation, and centrifugation. In many known methods of whole cell biocatalysis, microorganisms are freeze dried (lyophilised) after the growth stage. In some embodiments, microorganisms are lyophilised. In some embodiments, microorganisms are not lyophilised after the growth stage.

[0209] Following separation from the media (culture media, liquid fraction, liquid portion), the microorganisms are resuspended in a liquid for the conversion stage. In some embodiments, the liquid for the conversion stage is a buffer. In some embodiments, the buffer is phosphate-citrate buffer. In some embodiments, the microorganisms are washed before resuspension.

[0210] Microorganisms can be resuspended at a concentration that is higher than is typically possible through fermentation methods.

[0211] In some embodiments, the microorganism is resuspended at a cell density wherein an ODeoonm measurement is above or below a pre-specified threshold.

[0212] In some embodiments, the microorganism is resuspended at a cell density wherein an ODeoonm measurement is above 1. In some embodiments, the microorganism is resuspended at a cell density wherein an ODeoonm measurement is above 2. In some embodiments, the microorganism is resuspended at a cell density wherein an ODeoonm measurement is above 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or above 100.

[0213] In some embodiments, the microorganism is resuspended at a cell density wherein an ODeoonm measurement is below 100. In some embodiments, the microorganism is resuspended at a cell density wherein an ODeoonm measurement is below 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or below 100.

[0214] After resuspension, the microorganisms may be utilised in a method of biotransformation.

[0215] In some embodiments, phenylalanine is provided exogenously to the bacterial cell. In some embodiments, phenylalanine is provided exogenously to the bacterial cell during the conversion phase.

[0216] In some embodiments, phenylalanine is provided at a concentration of more than 1 g / L, for example at a concentration of more than 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, or more than 10 g / L. In some embodiments, phenylalanine is provided at a concentration of more than 5 g / L. In some embodiments, phenylalanine is provided at a concentration of more than 7 g / L. In some embodiments, phenylalanine is provided at a concentration of 8 g / L.

[0217] In some embodiments, phenylalanine is provided at a concentration of less than 10 g / L, for example at a concentration of less than 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, or less than 9 g / L.

[0218] In some embodiments, phenylalanine is provided at a concentration between 1 g / L and 10 g / L, for example phenylalanine is provided at a concentration between 2 g / L and 10 g / L, between 3 g / L and 10 g / L, between 4 g / L and 10 g / L, between 5 g / L and 10 g / L, between 6 g / L and 10 g / L, between 7 g / L and 10 g / L, between 8 g / L and 10 g / L, between 9 g / L and 10 g / L, between 2 g / L and 9 g / L, between 3 g / L and 9 g / L, between 4 g / L and 9 g / L, between 5 g / L and 9 g / L, between 6 g / L and 9 g / L, between 7 g / L and 9 g / L, between 8 g / L and 9 g / L, between 2 g / L and 8 g / L, between 3 g / L and 8 g / L, between 4 g / L and 8 g / L, between 5 g / L and 8 g / L, between 6 g / L and 8 g / L, or between 7 g / L and 8 g / L. In some embodiments, phenylalanine is provided at a concentration between 1 g / L and 10 gIL In some embodiments, phenylalanine is provided at a concentration between 1 g / L and 9 g / L. In some embodiments, phenylalanine is provided at a concentration between 1 g / L and 8 g / L.

[0219] In some embodiments, phenylalanine is provided at a concentration of 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, or 10 g / L. In some embodiments, phenylalanine is provided at a concentration of 5 g / L. In some embodiments, phenylalanine is provided at a concentration of 8 g / L. In some embodiments, phenylalanine is provided at a concentration of 10 g / L.

[0220] In some embodiments, supplemental phenylalanine is provided at least once in a 24 hour period. In some embodiments, supplemental phenylalanine is provided at least once in a 36 hour period. In some embodiments, supplemental phenylalanine is provided at least once in a 48 hour period.

[0221] In some embodiments, phenylalanine is provided at 1 g / L / day, 2 g / L / day, 3 g / L / day, 4 g / L / day, 5 g / L / day, 6 g / L / day, 7 g / L / day, 8 g / L / day, 9 g / L / day, or 10 g / L / day. In some embodiments, phenylalanine is provided at 5 g / L / day. In some embodiments, phenylalanine is provided at 8 g / L / day. In some embodiments, phenylalanine is provided at 10 g / L / day.

[0222] In some embodiments, phenylalanine concentration in the culture media is maintained at a concentration between 0.1 g / L and 10 g / L, for example phenylalanine is provided at a concentration between 1 g / L and 10 g / L, between 2 g / L and 10 g / L, between 3 g / L and 10 g / L, between 4 g / L and 10 g / L, between 5 g / L and 10 g / L, between 7 g / L and 10 g / L, between 8 g / L and 10 g / L, between 9 g / L and 10 g / L, between 2 g / L and 9 g / L, between 3 g / L and 9 g / L, between 4 g / L and 9 g / L, between 5 g / L and 9 g / L, between 7 g / L and 9 g / L, between 8 g / L and 9 g / L, between 2 g / L and 8 g / L, between 3 g / L and 8 g / L, between 4 g / L and 8 g / L, between 5 g / L and 8 g / L, or between 7 g / L and 8 g / L. In some embodiments, phenylalanine concentration in the culture media is maintained at a concentration between 0.1 g / L and 10 g / L. In some embodiments, phenylalanine concentration in the culture media is maintained at a concentration between 0.1 g / L and 9 g / L. In some embodiments, phenylalanine concentration in the culture media is maintained at a concentration between 0.1 g / L and 8 g / L. In some embodiments, phenylalanine concentration in the culture media is maintained at a concentration between 0.1 g / L and 7 g / L.

[0223] In some embodiments, phenylalanine concentration in the buffer is maintained at a concentration between 0.1 g / L and 10 g / L, for example phenylalanine is provided at a concentration between 1 g / L and 10 g / L, between 2 g / L and 10 g / L, between 3 g / L and 10 g / L, between 4 g / L and 10 g / L, between 5 g / L and 10 g / L, between 7 g / L and 10 g / L, between 8 g / L and 10 g / L, between 9 g / L and 10 g / L, between 2 g / L and 9 g / L, between 3 g / L and 9 g / L, between 4 g / L and 9 g / L, between 5 g / L and 9 g / L, between 7 g / L and 9 g / L, between 8 g / L and 9 g / L, between 2 g / L and 8 g / L, between 3 g / L and 8 g / L, between 4 g / L and 8 g / L, between 5 g / L and 8 g / L, or between 7 g / L and 8 g / L. In some embodiments, phenylalanine concentration in the buffer is maintained at a concentration between 0.1 g / L and 10 g / L. In some embodiments, phenylalanine concentration in the buffer is maintained at a concentration between 0.1 g / L and 9 g / L. In some embodiments, phenylalanine concentration in the buffer is maintained at a concentration between 0.1 g / L and 8 g / L. In some embodiments, phenylalanine concentration in the buffer is maintained at a concentration between 0.1 g / L and 7 g / L. Microorganisms

[0224] Microorganisms may be provided in isolated form and / or in culture. Microorganisms may be provided in vitro.

[0225] A microorganism comprising an enzyme according to the present invention may do so through expression from a nucleic acid / expression vector according to the present invention that has been introduced into the microorganism.

[0226] Microorganisms contemplated for use with the present invention include prokaryotic and eukaryotic cells. For example, the prokaryotic cell may be a bacteria or archaea, and the eukaryotic microorganism may be a fungi, protist, or microscopic animal or microscopic plant organism.

[0227] In preferred aspects, the microorganism is a bacterium. Any bacterium may be used, such as laboratory strains (such as E. coli or Bacillus subtilis), or field strains. Preferred bacteria will be those that are organotrophic, e.g. chemoheterotrophic bacteria, capable of using biomass or compounds derived therefrom as an energy source.

[0228] In particular embodiments, Escherichia bacteria such as E. coli, Saccharomyces yeast such as S. cerevisiae and cyanobacteria are contemplated for use in the present invention.

[0229] Preferred bacteria are robust bacteria, such as soil bacteria and / or extremophilic bacteria. Especially preferred are halophilic bacteria. These are capable of growing in open non-sterile conditions. As these strains are salt tolerant, they will not be outcompeted by contaminating bacteria or other microorganisms so long as there is a high enough salt content. Furthermore, the addition of a high salt buffer (e.g. at least a 3% salt solution) can be used to control competing bacteria. Halophilic bacteria include those of the genus Halomonas.

[0230] In some embodiments, the microorganism has been modified to increase phenylalanine production. In some embodiments, the microorganism is an E. coli phenylalanine overproduction strain. In some embodiments, the microorganism is an E. coli NST74 phenylalanine overproduction strain. In some embodiments, the E. coli NST74 phenylalanine overproduction strain is available as ATCC 31884 ( / .e„ aroH367, tyrR366, tna-2, lacY5, aroF394(fbr), malT384, pheA101(fbr), pheO352, aroG397(fbr)).

[0231] In some embodiments, the microorganism is an E. coli BL21 (DE3) BZE strain which has been modified to express a heterologous gene which encodes an enzyme described herein.

[0232] Nucleic acids, expression vectors, cells and compositions

[0233] The present invention also provides a nucleic acid, or a plurality of nucleic acids. In some embodiments the nucleic acid is DNA. In some embodiments the nucleic acid is RNA. The nucleic acid may be single-stranded or double-stranded. The nucleic acid may be provided in isolated / purified form, or within a host cell.

[0234] In some embodiments, the nucleic acid encodes a protein (e.g., and enzyme) described herein. In some embodiments, the nucleic acid is SEQ ID NO:6. In some embodiments, the nucleic acid is SEQ ID NO:7. In some embodiments, the nucleic acid is SEQ ID NO:8. In some embodiments, the nucleic acid is SEQ ID NO:9. In some embodiments, the nucleic acid is SEQ ID NQ:10.

[0235] In general, short polynucleotides can be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art. Longer polynucleotides will generally be produced using recombinant means, for example using PCR (polymerase chain reaction) cloning techniques. In some embodiments this will involve making a pair of primers (e.g. of about 15-30 nucleotides) to a region of the gene which it is desired to clone, bringing the primers into contact with DNA, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by separating the reaction mixture on an agarose gel) and recovering and purifying the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook etal., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Harbour Laboratory Press.

[0236] Alternatively, InFusion cloning (described e.g. in Throop and LaBaer, Curr Protoc Mol Biol. (2015) 110: 3.20.1-3.20.23) or other cloning techniques may be used, such as Gibson Assembly (Gibson et al., Nat. Methods 2009; 6, 343-345), CRISPR / Cas9-based methods (Wang etal., (2015) BioTechniques 58:161-170), Sequence and Ligation Independent Cloning (SLIC; Nucleic Acids Res.

[0237] 2012, 40: e55) and Modular Overlap-Directed Assembly with Linkers (MODAL; Nucleic Acids Res. (2014) 42.1: el -el).

[0238] The present invention further provides a vector, particularly an expression vector, comprising a nucleic acid or plurality of nucleic acids according to the present invention. The vector may be used to replicate the nucleic acid in a compatible host cell. Therefore, nucleic acids according to the present invention can be produced by introducing a polynucleotide into a replicable vector, introducing the vector into a compatible host cell and growing the host cell under conditions that bring about replication of the vector.

[0239] A “vector” as used herein is an oligonucleotide molecule (DNA or RNA) used as a vehicle to transfer foreign genetic material into a cell. The vector may be an expression vector for expression of the foreign genetic material in the cell. Such vectors may include a promoter and / or a ribosome binding site (RBS) sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, RBS, enhancers and other elements, such as for example polyadenylation signals, which may be necessary and which are positioned in the correct orientation in order to allow for protein expression.

[0240] Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a polypeptide from a vector according to the invention. In some embodiments, the vector may be a plasmid, phage, MAC, virus, etc.

[0241] In some embodiments the vector may be a prokaryotic expression vector, e.g. a bacterial expression vector. In some embodiments the vector is a pBbE2c vector or a pBbA1 k vector. In some embodiments, the vector is a pSEVA or a pHALP vector. In some embodiments, the vector is a pSEVA434, pSEVA241 , pHALP7, or pHALP102 vector.

[0242] Vectors containing hybrid promoters may be preferred. This may contain native Halomonas Pporin or other promoters that have been engineered to convert them to inducible or constitutive promoters. Hybrid vectors may contain randomised variable region sequences that confer different expression levels of recombinant proteins, e.g. constitutive promoters found in vectors pHALP7 and pHALP102.

[0243] In some embodiments, the vector is a pHALP7 or pHALP102 vector. Full details of these vectors can be found in Trisrivirat et al. (Synthetic Biology, Volume 5, Issue 1 , 2020, ysaa022), which is hereby incorporated by reference. The p7 promoter is a weak constitutive promoter, and the p102 promoter is a strong constitutive promoter.

[0244] In some embodiments, the vector is a pSEVA vector. Further details of pSEVA vectors is provided by Silva-Rocha et al. (Nucleic Acids Res. 2013 Jan; 41. Database issue: D666-D675), which is hereby incorporated by reference in its entirety. In some embodiments, the vector is a pSEVA vector comprising an inducible promoter. In some embodiments, the vector is a pSEVA vector comprising a constitutive promoter. In some embodiments, the vector is a pSEVA vector comprising a T7-like constitutive MMP1 viral promoter. In some embodiments, the vector is a pSEVA434 or pSEVA241 vector. In some embodiments, the vector is a pSEVA434 vector. In some embodiments, the pSEVA434 vector is a pSEVA434-T7 vector. In some embodiments, the vector is a pSEVA241 vector. Example 4 provided herein utilises a pSEVA434-T7 vector, with plasmid map shown in Figure 12.

[0245] In some embodiments, the vector may be a eukaryotic expression vector. In some embodiments, the vector may be a eukaryotic expression vector, e.g. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian expression vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al., 2001 , Molecular Cloning: a laboratory manual, 3rd edition, Cold Harbour Laboratory Press.

[0246] The term “operably linked” may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence (e.g. promoter and / or enhancer) are covalently linked in such a way as to place the expression of the nucleotide sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus a regulatory sequence is operably linked to the selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of the nucleotide sequence. The resulting transcript may then be translated into a desired peptide or polypeptide. The promoter may be a T7 promoter.

[0247] In some embodiments, the vector may comprise an element for facilitating translation of encoded protein from mRNA transcribed from the construct. For example, the construct may comprise a ribosomal binding site (RBS) such as a Shine-Dalgarno (SD) sequence upstream of the start codon. In some embodiments, the vector may encode one or more response elements for modulating expression of the encoded protein(s). In some embodiments, the response element is an element that causes upregulation of gene or protein expression in response to treatment with a particular agent. For example, the agent may induce transcription of DNA encoding the protein(s) from a vector including a response element for the agent. In some embodiments the agent may be isopropyl p-D-1-thiogalactopyranoside (IPTG), and the vector may comprise a lac operator. Other induction agent / response element combinations are known in the art.

[0248] In some embodiments, the vector may encode one or more response elements for constitutive expression of the encoded protein(s), such that no induction is necessary.

[0249] In some embodiments the vector may comprise a transcription terminator sequence downstream of the sequences encoding to the protein or proteins of interest. In some embodiments the terminator may be a T7 terminator sequence. In some embodiments the vector may comprise a sequence encoding a detectable marker in-frame with the sequence encoding the protein of interest to facilitate detection of expression of the protein, and / or purification or isolation of the protein (e.g. a His, (e.g.

[0250] 6XHis), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N- or C- terminus).

[0251] In some embodiments, nucleic acids (e.g., genes) disclosed herein are provided within a plasmid for the production of enzymes described herein in a host cell. In some embodiments, the plasmid is a plasmid described herein.

[0252] In some embodiments, genes are provided in a two-plasmid system. A two-plasmid system is defined herein as two plasmids which together comprise the genes necessary for the expression of an enzymatic cascade. An enzymatic cascade is defined herein as a sequence of successive enzymatic reactions.

[0253] In some embodiments, genes are provided in a single-plasmid system. A single-plasmid system is defined herein as a plasmid which comprises the genes necessary for the expression of an enzyme cascade.

[0254] The nucleic acids / expression vectors can be introduced into a cell by any suitable means, which are well known to the skilled person. In some embodiments the nucleic acids / expression vectors are introduced into a cell by transformation, transduction, conjugation, transfection or electroporation.

[0255] Cells and microorganisms contemplated for use with the present invention include prokaryotic and eukaryotic cells. For example, the prokaryotic cell may be a bacteria or archaea, and the eukaryotic microorganism may be a fungi, protist, or microscopic animal or microscopic plant organism. In some embodiments, the cells are isolated cells from a multicellular organism.

[0256] Microorganisms commonly used in commercial and industrial processes are contemplated, including microorganisms used for the commercial or industrial production of chemicals, enzymes or other biological molecules. In preferred aspects, the cells are of a bacterium. In some embodiments, the bacterium may be a Gram-positive bacterium. Gram-positive bacteria include bacteria from the genus Bacillus, bacteria from the genus Listeria, Clostridium (e.g. C. difficile), or cocci such as Staphylococcus (e.g. S. aureus), or Streptococcus. In some embodiments the bacterium may be a Gram-negative bacterium. Gram-negative bacteria may be defined as a class of bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation, making positive identification possible. Gram-negative bacteria include proteobacteria or bacteria of the family Enterobacteriaceae, such as Escherichia coli, Salmonella sp, Shigella sp, or bacteria selected from the genus Pseudomonas, Helicobacter, Neisseria, Legionella, Halomonas, Klebsiella or Yersinia bacterium.

[0257] In some embodiments, the fungi may be Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, or Neocallimastigomycota. In some embodiments, the fungi may be Dikarya (including Deuteromycota), such as fungi of the Ascomycota, including Pezizomycotina, Saccharomycotina, and Taphrinomycotina; or Basidiomycota, including Agaricomycotina, Pucciniomycotina, and Ustilaginomycotina. In some embodiments, the fungi may be fungi of the Entomophthoromycotina, Kickxellomycotina, Mucoromycotina, or Zoopagomycotina.

[0258] In some embodiments, Escherichia bacteria such as E. coli, Saccharomyces yeast such as S. cerevisiae and cyanobacteria / microalgae are contemplated for use in the present invention. In some embodiments the polypeptides may be prepared by cell-free-protein synthesis (CFPS), e.g. according to a system described in Zemella etal. Chembiochem (2015) 16(17): 2420-2431, which is hereby incorporated by reference in its entirety.

[0259] The present invention also provides compositions comprising the cells, nucleic acids, expression vectors, and enzymes / combinations of enzymes according to the present invention. The compositions find use e.g. in methods for benzoic acid production according to the present invention.

[0260] Any bacterium may be used, such as laboratory strains (such as E. coli or B. subtilis), or field strains. Preferred bacteria will be those that are organotrophic, e.g. chemoheterotrophic bacteria, capable of using biomass or compounds derived therefrom as an energy source.

[0261] Preferred bacteria are robust bacteria, such as soil bacteria and / or extremophilic bacteria.

[0262] Extremophilic bacteria include slight halophiles (able to grow in 1.7 to 4.8% NaCI), moderate halophiles (able to grow in 4.7 to 20% NaCI), extreme halophiles (able to grow in 20 to 30% NaCI), acidophiles (able to grow in conditions of low pH, such as below pH 5.0, e.g. pH 2 or below), alkaliphiles (able to grow in conditions of pH 8.5 or above), metallotolerant bacteria (able to survive in environments containing high concentrations of dissolved heavy metals), thermophiles or hyperthermophiles (with an optimal growth temperature between about 41 and 122°C, e.g. strains of Caldicellulosiruptor, Thermotoga, Thermoanaerobacterium, Pyrococcus, and Aeropyrum), or polyextremophiles (bacterial possessing two or more extremophilic characteristics).

[0263] Especially preferred are halophilic bacteria. These are capable of growing in open non-sterile conditions. As these strains are salt tolerant, they will not be outcompeted so long as there is a high enough salt content. Furthermore, the addition of a high salt buffer (e.g. at least a 3% salt solution) can be used to control competing bacteria. Halophilic bacteria include those of the genus Halomonas. Exemplary species of Halomonas have been described, including H. alimentaria, H. alkaliantarctica, H. alkaliphila, H. almeriensis, H. andesensis, H. anticariensis, H. aquamarina, H. arcis, H. axialensis, H. beimenensis, H. bluephagenesis, H. boliviensis, H. campaniensis, H. campisalis, H. caseinilytica, H. cerina, H. cibimaris, H. cupida, H. daqiaonensis, H. daqingensis, H. denitrificans, H. desiderata, H. elongata, H. eurihalina, H. flava, H. fontilapidosi, H. garicola, H. gomseomensis, H. gudaonensis, H. halmophila, H. halocynthiae, H. halodenitrificans, halophila, H. hamiltonii, H. heilong / jiangensis, H. huangheensis, H. hydrothermalis, H. ilicicola, H. janggokensis, H. jeotgali, H. johnsoniae, H. kenyensis, H. koreensis, H. korlensis, H. kribbensis, H. lutea, H. lutescence, H. magadiensis, H. maura, H. meridian, H. mongoliensis, H. muralis, H. nanhaiensis, H. neptunia, H. nitroreducens, H. olivaria, H. organivorans, H. pacifica, H. pantelleriensis, H. qiaohouensis, H. qijiaojingensis, H. ramblicola, H. rifensis, H. rowanensis, H. sabkhae, H. saccharevitans, H. salicampi, H. salifodinae, H. salina, H. sediminicola, H. shengliensis, H. sinaiensis, H. smyrnensis, H. songnenensis, H. stenophila, H. stevensii, H. subglaciescola, H. subterranean, H. sulfidaeris, H. taeanensis, H. titanicae, H. urumqiensis, H. variabilis, H. ventosae, H. venusta, H. vilamensis, H. xianhensis, H. xinjiangensis, H. zhang / jiangensis, and H. zincidurans. Preferred Halomonas strains include H. bluephagenesis st. TQ10, H. bluephagenesis st. TD1.0 and H. bluephagenesis st. TD01. Strains TD1.0 and TQ10 are genetically modified versions of the native TD01 strain where the gene encoding MmP1 has been chromosomally integrated into the bacterium. The gene MmP1 is a T7-like promoter that enables the IPTG-inducible expression of recombinant proteins in Halomonas (Zhao H et al 2017 Novel T7-like expression systems used for Halomonas. Metabolic Engineering 39: p. 128-140 which is herein incorporated by reference in its entirety).

[0264] Preferably, the Halomonas strain comprises the MmP1 gene, either chromosomally integrated or on a vector or plasmid. Strain TQ10 additionally has been genetically modified to knock out gene(s) involved in polyhydroxyalkanoate (PHA) biosynthesis.

[0265] Preferred Halomonas strains also include H. rowanensis, and modified versions of H. rowanensis (i.e., H. rowanensis derived strains). H. rowanensis is described in PCT / EP2024 / 061994.

[0266] Yields and percentage conversion

[0267] The yield of an enzymatic process, (also referred to as titre, or reaction yield) is the amount of product obtained in a reaction or enzymatic process. The absolute yield can be given as the weight in grams or in moles (molar yield). The fractional yield or relative yield, which serve to measure the effectiveness of a synthetic procedure, is calculated by dividing the amount of the obtained product in moles by the theoretical yield in moles. To obtain a percentage yield, the fractional yield is simply multiplied by 100.

[0268] The present disclosure provides different methods which have been has been shown to produce benzoic acid titres / yields of higher than 4.5 g / L. For example, one method comprising a hybrid fermentation-whole cell biocatalysis approach (Example 2) led to a benzoic acid titre of 5.7 g / L.

[0269] Additionally, a fully fermentative approach (Example 4) led to a benzoic acid titre of 4.8 g / L.

[0270] In some embodiments, the titre of benzoic acid is greater than 2.0 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L, 3.0 g / L, 3.1 g / L, 3.2 g / L, 3.3 g / L, 3.4 g / L, 3.5 g / L, 3.6 g / L, 3.7 g / L, 3.8 g / L, 3.9 g / L, 4.0 g / L, 4.1 g / L, 4.2 g / L, 4.3 g / L, 4.4 g / L, 4.5 g / L, 4.6 g / L, 4.7 g / L, 4.8 g / L, 4.9 g / L, 5.0 g / L, 5.1 g / L, 5.2 g / L, 5.3 g / L, 5.4 g / L, 5.5 g / L, 5.6 g / L, or greater than 5.7 g / L.

[0271] In some embodiments, the titre of benzoic acid is greater than 2.4 g / L. In some embodiments, the titre of benzoic acid is greater than 3.0 g / L. In some embodiments, the titre of benzoic acid is greater than 3.5 g / L. In some embodiments, the titre of benzoic acid is greater than 4.0 g / L. In some embodiments, the titre of benzoic acid is greater than 4.5 g / L. In some embodiments, the titre of benzoic acid is greater than 5.0 g / L. In some embodiments, the titre of benzoic acid is greater than 5.5 g / L.

[0272] In some embodiments, the percentage yield of benzoic acid from phenylalanine is greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or greater than 99%.

[0273] In some embodiments, the percentage yield of benzoic acid from phenylalanine is greater than 80%. In some embodiments, the percentage yield of benzoic acid from phenylalanine is greater than 85%. In some embodiments, the percentage yield of benzoic acid from phenylalanine is greater than 90%. In some embodiments, the percentage yield of benzoic acid from phenylalanine is greater than 95%. In some embodiments, the percentage yield of benzoic acid from phenylalanine is greater than 96%. In chemistry and biochemistry, and within this document, conversion refers to the conversion of molecule A to molecule B, or molecule A to molecule C (via molecule B). This can be calculated in a similar way to percentage yield.

[0274] Cinnamic acid is a precursor in disclosed methods of benzoic acid production, and may be a contaminant in some cases. In some embodiments, the amount of cinnamic acid produced in less than 1 g / L, for example less than 0.9 g / L, 0.8 g / L, 0.7 g / L, 0.6 g / L, 0.5 g / L, 0.4 g / L, 0.3 g / L, 0.2 g / L, 0.1 g / L, 0.05 g / L, or less than 0.02 g / L cinnamic acid.

[0275] In some embodiments, the percentage yield of cinnamic acid from phenylalanine is less than 3%, for example, less than 2%, or less than 1%.

[0276] Whole cell biocatalysis

[0277] Specific methods of producing phenylalanine and benzoic acid according to the present disclosure are disclosed within the examples herein.

[0278] The method may comprise more than one stage. For example, the method may comprise one or more of a phenylalanine production stage, a growth stage, and a conversion stage.

[0279] The method may comprise more than one method of growing or utilising a microorganism. The method may comprise more than one substrate conversion process. The method may comprise fermentation, biotransformation, and / or product isolation.

[0280] In some embodiments, the method of producing benzoic acid comprises whole cell biocatalysis.

[0281] Whole cell biocatalysis is reviewed in detail in Lin and Tao (Microbial Cell Factories volume 16, Article number: 106 (2017)). Whole-cell catalysis approaches can broadly be classified into biotransformation (biocatalysis) and fermentation bioprocesses. In fermentations, the products are synthesized from growth substrates via the host cells’ native metabolism and are accompanied in the fermentation broth by metabolic intermediates that make downstream processing complicated. In biotransformations, cell growth (the enzyme manufacturing phase) and production phase (conversion stage) are separated. Substrates are converted to the desired products by resting cells. The key advantages of whole-cell biocatalysis are its abilities to use cheap and abundant raw materials and to catalyse multistep reactions.

[0282] The method may involve culture or fermentation of a microorganism. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Secreted proteins can be collected by partitioning culture media / fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted or expressed peptide or polypeptide. Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein above).

[0283] In some examples, a fed batch method is utilised. In some examples, a fed batch fermentation is utilised. In some embodiments, a dose of a biochemical substrate (or more than one dose) is provided during microorganism growth. In some embodiments, a dose of a biochemical substrate (or more than one dose) is provided during exponential microorganism growth. In some embodiments, a dose of a biochemical substrate (or more than one dose) is provided during mid-late exponential microorganism growth.

[0284] Cultures of a microorganism, or microbial cultures, or cultures or microbiological cultures, generally comprise a culture vessel, a growth medium, and at least one microorganism cell. In some embodiments, the culture is a lab-scale culture. In some embodiments, the culture is an industrialscale culture. In some embodiments of the present disclosure, the microorganism cell is a bacterial cell.

[0285] A microbial medium, microbial growth medium or microbial culture medium is a liquid, semi-solid or solid designed to support the growth and proliferation of microbial cells. Microbial culture media are well known by scientists in the area of cell culture. Microbial cell culture media types and methods are comprehensively reviewed in A Rouf, Varsha Kanojia, HR Naik, Bazilla Naseer and Tahiya Qadri (2017) An overview of microbial cell culture, Journal of Pharmacognosy and Phytochemistry, Vol. 6, Issue 6 p 1923-1928, the contents of which are incorporated by reference.

[0286] Microbial culture media may comprise an appropriate source of energy and compounds which regulate the cell cycle. In addition to nutrients, the medium also helps maintain pH and osmolality. However, in some embodiments, there is no glucose and / or no carbon source present in the media. Microbial culture media may be liquid (i.e. aqueous), semi-solid (i.e. gelatinous), or completely solid. Semi-solid and solid media may contain agar, silicagel, acrylamide, gellan gum, or other solidification agents. Liquid media generally do not contain solidification agents. In some embodiments, the culture media is Luria broth (LB), high salt Luria broth (LB60), minimal media, high salt minimal media, or another suitable media known by the skilled person.

[0287] LB Miller contains 5 g / L yeast extract, 10 g / L tryptone and 10 g / L NaCI pH 7.0, and is widely used and well known to the skilled person. High salt Luria broth (LB60) is identical to LB, but has a higher salt content (60 g / L NaCI). Minimal medium is culture medium for microorganisms that contains the minimal necessities for growth - containing only inorganic salts, water, and optionally a carbon source. High salinity minimal medium contains higher salt content than typical minimal medium. An exemplary high salinity minimal medium is filter sterilised sea water with or without a supplemental carbon source.

[0288] In some embodiments, the microbial culture media is taken directly from the environment, for example from sea water, brackish water, river water, lake water, pond water. In some embodiments the cells are grown directly in the water taken from the environment, in some embodiments the environmental water is processed (e.g. filtered and / or autoclaved) before use as culture media, in some embodiments further additives (e.g. carbon, mineral, and / or amino acids) are added to the water to support growth of the cells. In some embodiments, the water is polluted with excess carbon and / or excess sulfur containing compounds. The sulfur containing compound may be a sulfate, a thiosulfate, and / or a sulfite.

[0289] In some embodiments, exogenous glucose is provided. In some embodiments, exogenous glucose is provided in a conversion stage. In some embodiments, exogenous glucose is provided following a growth stage. In some embodiments, exogenous glucose is provided in a conversion stage following a growth stage.

[0290] In some embodiments, the microorganism is halophilic. Halophilic microorganisms are known to the skilled person and are described both herein and in the prior art. In some embodiments, the microorganism is a recombinant halophilic microorganism. A recombinant halophilic microorganism is a halophilic microorganism comprising heterologous genetic material. In some embodiments, the recombinant halophilic microorganism comprises a recombinant plasmid. In some embodiments, the recombinant halophilic microorganism expresses a heterologous enzyme.

[0291] In some embodiments, the recombinant microorganism comprises a plasmid described herein. In some embodiments, the recombinant microorganism expresses an enzyme described herein.

[0292] The method of producing benzoic acid may comprise fermentation, for example, fermentation of a recombinant microorganism which is described herein.

[0293] Microbial fermentation is a metabolic process in which microorganisms utilise a carbon source to obtain energy for growth and the production of metabolites. Fermentation enables the use of microorganisms to produce biochemicals of commercial importance. Consequently, bacterial fermentation is used in industrial settings for the manufacture of biopharmaceuticals, foods, chemicals, and biofuels. In one aspect, the current disclosure describes the use offermentation in the production of benzoic acid.

[0294] In some embodiments, the method may involve culture or fermentation of a microorganism. The culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein), and Doran, Chapter 1 -Bioprocess Development: An Interdisciplinary Challenge, Bioprocess Engineering Principles (Second Edition; incorporated by reference herein) 2013, Pages 3-11.

[0295] In some embodiments, the fermentation conditions are monitored during the method. In some embodiments, temperature, pH, agitation, airflow, optical density, carbon-source concentration, and / or benzoic acid concentration are monitored during the method.

[0296] In some embodiments, pH is monitored. In some embodiments, pH is monitored and maintained. In some embodiments, pH is monitored and maintained within an optimum range during the method. In some embodiments, pH is monitored and maintained within an optimum range during certain steps of the method. In some embodiments, pH is monitored and maintained within an optimum range during fermentation. In some embodiments, pH is monitored and maintained within an optimum range during biocatalytic conversion of phenylalanine to benzoic acid.

[0297] In some embodiments, pH is maintained between pH 7.5 and pH 9.5. In some embodiments, pH is maintained between pH 7 and pH 9.5. In some embodiments, pH is maintained between pH 8 and pH 9.5. In some embodiments, pH is maintained between pH 8.5 and pH 9.5. In some embodiments, pH is maintained between pH 8.5 and pH 9. In some embodiments, pH is maintained at pH 8.5. In some embodiments, pH is maintained at pH 9. In some embodiments, pH is maintained through the addition of an acid (e.g., HCI) or a base (e.g., NaOH).

[0298] In some embodiments, temperature is monitored. In some embodiments, temperature is monitored and maintained. In some embodiments, temperature is monitored and maintained within an optimum range during the method. In some embodiments, temperature is monitored and maintained within an optimum range during certain steps of the method. In some embodiments, temperature is monitored and maintained within an optimum range during fermentation. In some embodiments, temperature is monitored and maintained within an optimum range during biocatalytic conversion of phenylalanine to benzoic acid. In some embodiments, temperature is maintained between 20 °C and 40 °C. In some embodiments, temperature is maintained between 25 °C and 37 °C. In some embodiments, temperature is maintained between 25 °C and 30 °C.

[0299] In some embodiments, temperature is adjusted to 37 °C for biomass production. In some embodiments, temperature is adjusted to a temperature between 35 °C and 40 °C. In some embodiments, temperature is adjusted to a temperature between 36 °C and 38 °C.

[0300] In some embodiments, temperature is adjusted to 25 °C for improved constitutive expression of heterologous enzymes. In some embodiments, temperature is adjusted to a temperature between 20 °C and 30 °C. In some embodiments, temperature is adjusted to a temperature between 22 °C and 28 °C.

[0301] In some embodiments, temperature is adjusted to 30 °C for initiation of benzoic acid production. In some embodiments, temperature is adjusted to a temperature between 25 °C and 35 °C. In some embodiments, temperature is adjusted to a temperature between 28 °C and 32 °C.

[0302] In some embodiments, initial bacterial growth is performed at a temperature between 32 °C and 40 °C, followed by a reduction in temperature for improved constitutive expression of heterologous enzymes.

[0303] In some embodiments, initial bacterial growth is performed at a temperature between 32 °C and 40 °C, followed by a reduction in temperature for improved constitutive expression of heterologous enzymes, followed by an increase in temperature for increased benzoic acid production.

[0304] In some embodiments, initial bacterial growth is performed at a temperature between 32 °C and 40 °C, followed by a reduction in temperature to a temperature between 20 °C and 28 °C for improved constitutive expression of heterologous enzymes, followed by an increase in temperature to a temperature between 28 °C and 32 °C for increased benzoic acid production.

[0305] In some embodiments, initial bacterial growth is performed at a temperature between 32 °C and 40 °C, followed by a reduction in temperature to a temperature between 20 °C and 28 °C, followed by an increase in temperature to a temperature between 28 °C and 32 °C.

[0306] In some embodiments, initial bacterial growth is performed at a temperature of 37 °C, followed by a reduction in temperature to 25 °C for improved constitutive expression of heterologous enzymes, followed by an increase in temperature to 30 °C for increased benzoic acid production.

[0307] In some embodiments, dissolved oxygen concentration (dO2) is monitored. In some embodiments, dissolved oxygen concentration (dO2) is monitored and maintained. In some embodiments, dissolved oxygen concentration (dO2) is monitored and maintained within an optimum range during the method. In some embodiments, dissolved oxygen concentration (dO2) is monitored and maintained within an optimum range during certain steps of the method. In some embodiments, dissolved oxygen concentration (dO2) is monitored and maintained within an optimum range during fermentation. In some embodiments, dissolved oxygen concentration (dO2) is monitored and maintained within an optimum range during biocatalytic conversion of phenylalanine to benzoic acid.

[0308] In some embodiments, dO2 is maintained above 20%. In some embodiments, dO2 is maintained above 25%. In some embodiments, dO2 is maintained above 30%.

[0309] In some embodiments, dO2 is maintained at a concentration between 20% and 50%. In some embodiments, dO2 is maintained at a concentration between 20% and 40%. In some embodiments, dO2 is maintained at a concentration between 25% and 40%. In some embodiments, dO2 is maintained at a concentration between 25% and 35%. In some embodiments, dO2 is maintained at a concentration between 28% and 32%. In some embodiments, dO2 is maintained at a concentration between 30% and 32%. In some embodiments, dO2 is maintained at a concentration between 30% and 35%. In some embodiments, dO2 is maintained at a concentration between 30% and 40%.

[0310] In some embodiments, the fermentation is open fermentation. Open fermentation is the name given to fermentations that take place in vessels that are “open” to the environment in which they are situated.

[0311] In some embodiments, NaCI concentration is monitored. In some embodiments, NaCI concentration is maintained within a predefined range. In some embodiments, NaCI concentration is monitored and maintained within a predefined range.

[0312] In some embodiments, the concentration of NaCI provided in the culture media at the start of fermentation is greater than 20 g / L. In some embodiments, the concentration of NaCI at the start of fermentation is greater than 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 g / L.

[0313] In some embodiments, the concentration of NaCI provided in the culture media at the start of fermentation is less than 70 g / L. In some embodiments, the concentration of NaCI at the start of fermentation is less than 80, 90, 100, 110, or 120 g / L.

[0314] In some embodiments, the concentration of NaCI provided in the culture media at the start of fermentation is between 20 g / L and 120 g / L. In some embodiments, the concentration of NaCI provided in the culture media at the start of fermentation is between 30 and 120, 40 and 120, 50 and 120, 60 and 120, 70 and 120, 80 and 120, 90 and 120, 100 and 120, or 110 and 120 g / L.

[0315] In some embodiments, the concentration of NaCI provided in the culture media at the start of fermentation is greater than 0.4 M. In some embodiments, the concentration of NaCI provided in the culture media at the start offermentation is greater than 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M, or 2.5 M.

[0316] In some embodiments, the concentration of NaCI provided in the culture media at the start of fermentation is lower than 1.0 M. In some embodiments, the concentration of NaCI provided in the culture media at the start offermentation is lower than 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M, or 2.5 M.

[0317] In some embodiments, the concentration of NaCI provided in the culture media at the start of fermentation is between 0.4 M and 2.0 M. In some embodiments, the concentration of NaCI provided in the culture media at the start offermentation is between 0.5 M and 2.0 M, 0.6 M and 2.0 M, 0.7 M and 2.0 M, 0.8 M and 2.0 M, 0.9 M and 2.0 M, 1.0 M and 2.0 M, 1.1 M and 2.0 M, 1.2 M and 2.0 M, 1.3 M and 2.0 M, 1.4 M and 2.0 M, 1.5 M and 2.0 M, 1.6 M and 2.0 M, 1.7 M and 2.0 M, 1.8 M and 2.0 M, or 1.9 M and 2.0 M.

[0318] In some embodiments, the NaCI concentration within the culture media is maintained at a concentration of at least 20 g / L during fermentation. In some embodiments, NaCI concentration within the culture media is maintained at a concentration of at least 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 g / L during fermentation.

[0319] In some embodiments, the NaCI concentration within the culture media is maintained at a concentration below 70 g / L during fermentation. In some embodiments, NaCI concentration within the culture media is maintained at a concentration of below 80, 90, 100, 110, or 120 g / L during fermentation.

[0320] In some embodiments, the NaCI concentration within the culture media is maintained at a concentration between 20 g / L and 120 g / L. In some embodiments, the NaCI concentration within the culture media is maintained at a concentration between 30 and 120, 40 and 120, 50 and 120, 60 and 120, 70 and 120, 80 and 120, 90 and 120, 100 and 120, or 110 and 120 g / L.

[0321] In some embodiments, the NaCI concentration within the culture media is maintained at a concentration of at least 0.4 M during fermentation. In some embodiments, NaCI concentration within the culture media is maintained at a concentration of at least 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M, or 2.5 M during fermentation.

[0322] In some embodiments, the NaCI concentration within the culture media is maintained at a concentration below 1.0 M during fermentation. In some embodiments, NaCI concentration within the culture media is maintained at a concentration of below 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M, or 2.5 M during fermentation. In some embodiments, the NaCI concentration within the culture media is maintained at a concentration between 0.4 M and 2.0 M. In some embodiments, the NaCI concentration within the culture media is maintained at a concentration between 0.5 M and 2.0 M, 0.6 M and 2.0 M, 0.7 M and 2.0 M, 0.8 M and 2.0 M, 0.9 M and 2.0 M, 1.0 M and 2.0 M, 1.1 M and 2.0 M, 1.2 M and 2.0 M, 1.3 M and 2.0 M, 1.4 M and 2.0 M, 1.5 M and 2.0 M, 1.6 M and 2.0 M, 1.7 M and 2.0 M, 1.8 M and 2.0 M, or 1.9 M and 2.0 M.

[0323] In some embodiments, the microbial culture media is taken directly from the environment or comprises water taken from the environment, for example from sea water, brackish water, river water, lake water, pond water. In some embodiments the cells are grown directly in the water taken from the environment. In some embodiments, the environmental water is processed (e.g. filtered and / or autoclaved) before use as culture media. In some embodiments, further additives (e.g. carbon, mineral, additional NaCI, and / or amino acids) are added to the water to support growth of the cells. In some embodiments, the water is polluted with excess carbon and / or excess sulfur containing compounds. The sulfur containing compound may be a sulfate, a thiosulfate, and / or a sulfite.

[0324] In some embodiments, the culture media comprises tap water. Tap water is water that is obtained directly from a faucet or tap, that has not been further purified, distilled, or otherwise treated after delivery from the faucet or tap.

[0325] In some embodiments, the culture media comprises grey water. Greywater (or grey water, sullage) refers to domestic wastewater generated in households or office buildings from streams without faecal contamination, i.e., all streams except for the wastewater from toilets. Sources of greywater include sinks, showers, baths, washing machines or dishwashers.

[0326] Typically, culture media, culture vessels, and other equipment used in the culture of microorganisms is sterilized before it is used in methods of culturing microorganisms (e.g., fermentation). Sterilisation may comprise autoclaving, filtering, heating, boiling, irradiating, and / or treating with a gas.

[0327] Additionally, other measures can be put in place to prevent subsequent contamination of cultures and equipment. Some methods employ the use of antibiotics, flow cabinets, flow chambers. This is to prevent contamination of subsequent cultures. Contamination could lead to reduced efficiency of a method involving the culture of microorganisms, and could lead to contaminating microorganisms outcompeting recombinant microorganisms which are important to said method. Such methods of culture are known as sterile culture, or culture under sterile conditions.

[0328] The methods described herein may comprise the use of a culture medium. In some embodiments, culture medium is non-sterile. In some embodiments, culture medium is not sterile. In some embodiments, culture medium is not sterilised. In some embodiments, culture medium is not sterilised before it is inoculated. In some embodiments, culture medium is not sterilised before it is contacted with a halophilic microorganism. In some embodiments, the culture medium is not autoclaved. In some embodiments, the culture medium is not filtered. In some embodiments, the culture medium is not autoclaved, filtered, heated, boiled, irradiated, and / or treated with a gas, before the culture medium is contacted with the halophilic microorganism.

[0329] The culture medium is prepared using a solvent. In some embodiments, the solvent is water. In some embodiments, the water is tap water. In some embodiments, the water is non-sterile. In some embodiments, a solvent is not sterilised before culture medium preparation. In some embodiments, the culture medium comprises a non-sterile solvent. In some embodiments, the culture medium is prepared using a non-sterile solvent. In some embodiments, the culture medium is prepared using a non-sterile solvent, and the culture medium is not subsequently sterilized.

[0330] In some embodiments, methods described herein do not comprise the use of aseptic technique. Aseptic technique is designed to provide a barrier between the microorganisms in the environment and the cell culture (and microorganisms intentionally included in the cell culture). Aseptic technique depends upon a set of procedures to reduce the probability of contamination from environmental sources. The elements of aseptic technique are a sterile work area, good personal hygiene, sterile reagents and media, and sterile handling.

[0331] In some embodiments, the culture (e.g., fermentation) of a microorganism requires nitrogen, salts, minerals, and vitamins. Seawater is a cost-effective natural mineral and salt broth (3.5%), while clarified wastewater streams provide an abundant alternative for inland sites. Additional salt can be added in the form of crude sea salt. Provision of vitamins, where necessary, can be achieved from autolysed spent brewery yeast, an abundant waste product.

[0332] In some embodiments, the medium comprises a nitrogen source. In some embodiments, the nitrogen source is yeast extract, urea, ammonium sulphate, ammonium chloride, ammonia, and / or potassium nitrate.

[0333] In some embodiments of this invention, microorganisms (e.g. Halomonas) utilise atmospheric carbon dioxide as a carbon source. In some embodiments a carbon supply is provided in addition to atmospheric carbon dioxide. Suitable additional carbon supplies may be provided by the addition of sugar, NaHCOs, KHCO3, Na2CO3, waste glycerol, pre-treated food waste, pre-treated plant waste, pre-treated seaweed, or supplemental gaseous CO2 to the culture. In some embodiments the microorganism utilises carbon dioxide as a carbon source and also uses another carbon source (e.g. sugar, NaHCOs, waste glycerol, pre-treated food waste, pre-treated plant waste, or pre-treated seaweed).

[0334] The additional carbon may be supplied as a gas, liquid (including a solution in water) or a solid. The additional carbon supply may be added continuously or at selected times during the culture or growth cycle of the chemoautotrophic microorganism.

[0335] In some embodiments, gaseous carbon dioxide (CO2) is provided to the culture. In some embodiments, atmospheric CO2 is provided to the culture. In some embodiments, further carbon dioxide is provided in addition to atmospheric carbon dioxide.

[0336] Carbon dioxide (CO2) is produced as a by-product of many industrial processes such as oil and gas production, cement production, iron and steel production, and electricity generation, as well as many others. This carbon dioxide is typically released into the environment through the combustion of fuels. However, it can be captured, separated, isolated and stored. This disclosure provides methods of utilising this waste carbon dioxide in a process of chemoautotrophic carbon fixation producing products of value and industrial application. In some embodiments, the culture is grown in a CO2 incubator.

[0337] In some embodiments, additional carbon dioxide is provided to the atmosphere surrounding the culture. In some embodiments, carbon dioxide is provided directly to the culture media.

[0338] Carbon dioxide may be provided at a concentration of about 420 parts per million (ppm) which is a typical average concentration of atmospheric carbon dioxide.

[0339] In some embodiments, gaseous CO2 is provided at one of about 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200 ppm. In some embodiments, gaseous CO2 is provided at a concentration of at least one of 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or 1200 ppm.

[0340] In some embodiments, gaseous CO2 is provided at a concentration between 300 ppm and 2000 ppm, 350 ppm and 2000 ppm, 400 ppm and 2000 ppm, 450 ppm and 2000 ppm, 500 ppm and 2000 ppm, 550 ppm and 2000 ppm, 600 ppm and 2000 ppm, 650 ppm and 2000 ppm, 700 ppm and 2000 ppm, 750 ppm and 2000 ppm, 800 ppm and 2000 ppm, 850 ppm and 2000 ppm, 900 ppm and 2000 ppm, 950 ppm and 2000 ppm, or 1000 ppm and 2000 ppm.

[0341] In some embodiments, gaseous CO2 is provided at a concentration between 300 ppm and 1000 ppm, 350 ppm and 1000 ppm, 400 ppm and 1000 ppm, 450 ppm and 1000 ppm, 500 ppm and 1000 ppm, 550 ppm and 1000 ppm, 600 ppm and 1000 ppm, 650 ppm and 1000 ppm, 700 ppm and 1000 ppm, 750 ppm and 1000 ppm, 800 ppm and 1000 ppm, 850 ppm and 1000 ppm, 900 ppm and 1000 ppm, or 950 ppm and 1000 ppm.

[0342] In some embodiments, gaseous CO2 is provided to the culture at a volume concentration of about one of 0.1%, 0.5.%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% volume / volume (v / v). In some embodiments, gaseous CO2 is provided to the culture at a volume concentration of at least one of 0.1%, 0.5.%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% volume / volume (v / v).

[0343] In some embodiments, gaseous CO2 is provided to the culture at a volume concentration of at most one of 0.1%, 0.5.%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% volume / volume (v / v).

[0344] In some embodiments, gaseous CO2 is provided to the culture at a volume concentration of between 0.1% and 100%, 0.1% and 90%, 0.1% and 80%, 0.1% and 70%, 0.1% and 60%, 0.1% and 50%, 0.1% and 40%, 0.1% and 30%, 0.1% and 20%, 0.1% and 10%, 0.1% and 5%, 0.1% and 2.5%, or 0.1% and 1% volume / volume (v / v). In some embodiments, gaseous CO2 is provided to the culture at a volume concentration of between 0.5% and 100%, 0.5% and 90%, 0.5% and 80%, 0.5% and 70%, 0.5% and 60%, 0.5% and 50%, 0.5% and 40%, 0.1% and 30%, 0.5% and 20%, 0.5% and 10%, 0.5% and 5%, 0.5% and 2.5%, or 0.5% and 1% volume / volume (v / v).

[0345] In some embodiments, NaHCOsis provided to the culture. In some embodiments, NaHCOsis provided to the culture to supplement CO2.

[0346] In some embodiments, NaHCOs, or an equimolar mixture of NaHCOs with KHCO3, is provided to the culture at a concentration of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mM. In some embodiments, NaHCOs, or an equimolar mixture of NaHCOs with KHCO3, is provided to the culture at a concentration of at most 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mM.

[0347] In some embodiments, NaHCOs, or an equimolar mixture of NaHCOs with KHCO3, is provided to the culture at a concentration between 1 mM and 10 mM, 1 mM and 20 mM, 1 mM and 30 mM, 1 mM and 40 mM, 1 mM and 50 mM, 1 mM and 60 mM, 1 mM and 70 mM, 1 mM and 80 mM, 1 mM and 90 mM, 1 mM and 100 mM, 1 mM and 110 mM, 1 mM and 120 mM, 1 mM and 130 mM, 1 mM and 140 mM, 1 mM and 150 mM, 1 mM and 160 mM, 1 mM and 170 mM, 1 mM and 180 mM, 1 mM and 190 mM, or 1 mM and 200 mM.

[0348] In some embodiments, NaHCOs, or an equimolar mixture of NaHCOs with KHCO3, is provided to the culture at a concentration between 5 mM and 10 mM, 5 mM and 20 mM, 5 mM and 30 mM, 5 mM and 40 mM, 5 mM and 50 mM, 5 mM and 60 mM, 5 mM and 70 mM, 5 mM and 80 mM, 5 mM and 90 mM, 5 mM and 100 mM, 5 mM and 110 mM, 5 mM and 120 mM, 5 mM and 130 mM, 5 mM and 140 mM, 5 mM and 150 mM, 5 mM and 160 mM, 5 mM and 170 mM, 5 mM and 180 mM, 5 mM and 190 mM, or 5 mM and 200 mM.

[0349] Antibiotics are often used in the culture or fermentation of microorganisms. Antibiotics are used to prevent contamination or select for cells containing genetic modifications ( / .e., recombinant microorganisms). If antibiotics are not used to maintain the majority of microorganisms expressing recombinant plasmids carrying antibiotic resistance, the stability of the plasmid is greatly reduced. However, the use of antibiotics to maintain plasmids carrying antibiotic resistance genes escalates the costs offermentation and heightens the risk of environmental pollution. In some embodiments, the method comprises the use of antibiotics. In some embodiments, the method does not comprise the use of antibiotics. In some embodiments, the method of microbial culture or fermentation of a microorganism does not comprise the use of an antibiotic. In some embodiments, the fermentation does not comprise the use of an antibiotic. In some embodiments, the fermentation comprises the use of an antibiotic.

[0350] Any suitable container or culture vessel may be used to propagate a microorganism according to the methods and compositions described here.

[0351] The skilled person is aware of many different types of culture vessels. Exemplary culture vessels include: plates, dishes, flasks, bottles, bioreactors, or fermenters. Also known as culture plates, culture dishes, culture flasks, culture bottles, culture bioreactors, or culture fermenters. Culture vessels can be constructed from a number of materials such as metal, glass, plastics and polymers such as polystyrene and polyester.

[0352] Culture plates are low flat-bottomed laboratory containers for growing cells on a thin layer of nutrient medium. Plates and dishes can be used with liquid and solid media. Culture dishes are similar to culture plates. The most common types of culture plates and dishes are the Petri dish and the multiwell plate. Multiwell plates are available in many sizes, for example as a 2-well plate, 4-well plate, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384 well plate. Petri dishes and multiwell plates are available from many manufacturers (e.g. Thermo Fisher Scientific) with different specifications. Plates are available in different colours (e.g. clear, black and white), and are usually made from polystyrene or glass.

[0353] Culture flasks are generally used with liquid cultures and typically resemble bottles. Flasks are generally constructed from glass, polycarbonate or polystyrene. It is possible to reuse and sterilise glass culture flasks, whereas disposable polycarbonate or polystyrene flasks are widely available (e.g. Thermo Fisher Scientific Nunc EasYFIasks). Culture flasks are available in many sizes, such as T25, T75, T175, T225, T300, T1000 and T2000. Different size flasks have different volumes and different surface areas. In some embodiments, the culture vessel has a volume of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0, 125.0, 150.0, 175.0, 200.0, 300.0, 400.0, 500.0, 600.0, 700.0, 800.0, 900.0, or 1000.0 mL. In some embodiments, the culture vessel has a volume of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0, 125.0, 150.0, 175.0, 200.0, 225.0, or 250.0 litres (L).

[0354] A bioreactor may comprise a regulated bioreactor, in which one or more conditions may be controlled or monitored, for example, oxygen partial pressure. Devices for measuring and regulating these conditions are known in the art. For example, dissolved oxygen electrodes may be used for oxygen partial pressure and / or monitoring dissolved oxygen concentration (dO2). The oxygen partial pressure can be regulated via the amount and the composition of the selected gas mixture (e.g., air or a mixture of air and / or oxygen and / or nitrogen and / or carbon dioxide) and / or by adjusting the agitation rate. Suitable devices for measuring and regulating the oxygen partial pressure are described by Bailey, J E. (Bailey, J E., Biochemical Engineering Fundamentals, second edition, McGraw-Hill, Inc. ISBN 0-07-003212-2 Higher Education, (1986)) or Jackson A T. Jackson A T., Verfahrenstechnik in der Biotechnologie, Springer, ISBN 3540561900 (1993)). Alternatively, a culture may be static, i.e. where active agitation of the culture / culture media is not employed, optionally wherein mixing is performed via gas bubbling.

[0355] Isolating benzoic acid

[0356] Benzoic acid may be present in the media or buffer and are isolated from the liquid fraction (e.g. media or buffer). In other embodiments, benzoic acid is isolated from a microorganism fraction. In some embodiments, benzoic acid is isolated from the liquid fraction and / or from microorganisms.

[0357] The first step for isolation methods is usually to separate the microorganism fraction from the liquid fraction. The liquid fraction is the liquid portion (e.g. culture media or buffer) which lies above a sediment formed by the solid microorganisms. The microorganism fraction is the solid sediment fraction which forms below the liquid fraction.

[0358] The microorganism fraction can be separated from the liquid fraction in a number of ways, including filtration, chromatography, evaporation, sedimentation, and centrifugation. Following this crude separation, the compounds of interest can be separated from their fraction.

[0359] Compounds that are secreted into the media from microorganisms and present in the liquid fraction can be separated in a number of ways, including physical separation, distillation and pervaporation.

[0360] Exemplary methods of benzoic acid isolation are provided in Example 3 and Example 4 (4.4.2). In some embodiments, the method of isolating benzoic acid comprises one or more steps highlighted in Example 3 herein. In some embodiments, the method of isolating benzoic acid comprises one or more steps highlighted in Example 4 herein.

[0361] In some embodiments, the method comprises solubilising benzoic acid. In some embodiments, the method comprises basification of the liquid portion to solubilise benzoic acid.

[0362] In some embodiments, the method comprises removal of biomass (i.e., the solid fraction). In some embodiments, the method comprises filtration or centrifugation to separate the solid fraction and liquid fraction.

[0363] In some embodiments, the method comprises the addition of an organic solvent. In some embodiments, the method comprises the addition of an organic solvent to the culture media. In some embodiments, the method comprises the addition of an organic solvent to the buffer. In some embodiments, the method comprises the addition of an organic solvent to the liquid fraction. In some embodiments, the organic solvent is ethyl acetate. In some embodiments, an organic phase and an aqueous phase is formed. In some embodiments, the organic layer comprises benzoic acid. In some embodiments, the organic layer is recovered. In some embodiments, the benzoic acid is re-extracted into water from the organic phase.

[0364] In some embodiments, the method comprises precipitation of benzoic acid. In some embodiments, the method comprises precipitation of benzoic acid through cooling and / or acidification.

[0365] In some embodiments, the method comprises harvesting the precipitate comprising benzoic acid. In some embodiments, the method comprises harvesting the precipitate comprising benzoic acid, by filtration. In some embodiments, the method comprises harvesting the precipitate comprising benzoic acid, by filtration in vacuo.

[0366] The method of isolating benzoic acid may comprise one or more of the following steps:

[0367] (i) solubilising benzoic acid (e.g., in culture media or buffer),

[0368] (ii) separation of solids from a liquid fraction (e.g., by filtration or centrifugation),

[0369] (iii) addition of an organic solvent to the liquid fraction,

[0370] (iv) re-extraction into water from an organic phase,

[0371] (iii) precipitation of benzoic acid (e.g., through cooling and / or acidification), and / or

[0372] (iv) harvesting the benzoic acid.

[0373] General definitions

[0374] As used herein, a “fragment”, “variant” or “homologue” of a protein may optionally be characterised as having at least 50%, preferably one of 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein. Fragments, variants, isoforms and homologues of a reference protein may be characterised by the ability to perform a function performed by the reference protein.

[0375] Pairwise and multiple sequence alignment for the purpose of determining percent identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame etal. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

[0376] A “fragment” generally refers to a fraction of the reference protein. A “variant” generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of the reference protein, but retaining a considerable degree of sequence identity (e.g. at least 60%) to the amino acid sequence of the reference protein. An “isoform” generally refers to a variant of the reference protein expressed by the same species as the species of the reference protein. A “homologue” generally refers to a variant of the reference protein produced by a different species as compared to the species of the reference protein.

[0377] A “fragment” of a reference protein may be of any length (by number of amino acids), although may optionally be at least 25% of the length of the reference protein (that is, the protein from which the fragment is derived) and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein.

[0378] A fragment of a polypeptide may have a minimum length of one of 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids, and may have a maximum length of one of 15, 20, 25, 30, 40, 50, 100, 110, 120 or 130 amino acids.

[0379] Sequence identity

[0380] Pairwise and multiple sequence alignment for the purpose of determining percent identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame etal. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6(298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used. Sequences

[0381] SEQ DESCRIPTION SEQUENCE ID NO:

[0382] 1 phdE from MTTSTTPNTIISFEDAPTLTGQDLGFSQWRTVTQEMVNTFADATDDQ Corynebacterium QWIHTDPERAKDGPFGGAIAHGFLTLSMIIPFWGELLDVTGVTTKVN glutamicum YGLDKVRFTSPVKVGSRIRMGAVVREISEVKGNGLHLVADGTIEIEG (GENBANK: QERPAVVATFLTRFYA

[0383] AGN20978.1)- amino

[0384] acid sequence

[0385] 2 phdB from MSLNGKVAIVTGSGAGLGRSFAQELARQGASVIVNDVNQAAADETV Corynebacterium AAITEAGGKAAAVIAPVGPSESAALLVREAVDKFGSLDILVTNAGILRD glutamicum RSLLKMTDDDFDAVINVHLKGTFTCVREAFGYFKENGIAGRIVTIGSP (GENBANK: TGQRGNFGQSNYAAAKAGIVGMVRTWALEMKRAGVTINAIIPEAATD AGN20975.1)- amino MTKTVPYFQKAVEADERGEAMPAFFRETLGFGTPQDVAGLVAFLSS acid sequence DEAANISGQAIGAGGDRMQVWKHPEPAVTEFNPGGWTYEALQERG KNIIEGNLQSVGVVFPELPAELQPQIPVKA

[0386] 3 phdC from MSNNVVKYECAVDADNIVAVDMHVHLEVDSCGHKSMPADIMAASSK Corynebacterium YFKTAERTPSADAIADIYREHKMAAVVFTIDARTQMGHLPNSIDDLVA glutamicum SCARNNDVLIPFGSVDPRTGEDALVEARRQVEELGVRGFKFHPSVQ (GENBANK: GFDPSAPEFYPLWELLESFGLPCVFHTGQNGMGAGLPGGRGIKLRF AGN20976.1)- amino SNPMLLDDVAADFPNLTIIMAHPSVPWQDEANSIATHKANVFIDLSG acid sequence WSPKYFPESLVRQSNNVLSKKVLFGTDFPLITPEKWLAAFANLPLKD EVRPGILKDNAVKVLGLAASTERGSQAEKVVQHA

[0387] 4 Phenylalanine / tyrosine MAPSLDSISHSFANGVASAKQAVNGASTNLAVAGSHLPTTQVTQVDI ammonia-lyase from VEKMLAAPTDSTLELDGYSLNLGDVVSAARKGRPVRVKDSDEIRSKI Rhodotorula toruloides DKSVEFLRSQLSMSVYGVTTGFGGSADTRTEDAISLQKALLEHQLCG (also known as VLPSSFDSFRLGRGLENSLPLEVVRGAMTIRVNSLTRGHSAVRLVVL Rhodosporidium EALTNFLNHGITPIVPLRGTISASGDLSPLSYIAAAISGHPDSKVHVVH toruloides). UNIPROT: EGKEKILYAREAMALFNLEPVVLGPKEGLGLVNGTAVSASMATLALH P11544 - amino acid DAHMLSLLSQSLTAMTVEAMVGHAGSFHPFLHDVTRPHPTQIEVAG sequence NIRKLLEGSRFAVHHEEEVKVKDDEGILRQDRYPLRTSPQWLGPLVS DLIHAHAVLTIEAGQSTTDNPLIDVENKTSHHGGNFQAAAVANTMEK TRLGLAQIGKLNFTQLTEMLNAGMNRGLPSCLAAEDPSLSYHCKGLD IAAAAYTSELGHLANPVTTHVQPAEMANQAVNSLALISARRTTESND

[0388]

[0389] VLSLLLATHLYCVLQAIDLRAIEFEFKKQFGPAIVSLIDQHFGSAMTGS NLRDELVEKVNKTLAKRLEQTNSYDLVPRWHDAFSFAAGTVVEVLS STSLSLAAVNAWKVAAAESAISLTRQVRETFWSAASTSSPALSYLSP RTQILYAFVREELGVKARRGDVFLGKQEVTIGSNVSKIYEAIKSGRIN NVLLKMLA

[0390] 4-coumarate:CoA MFRSEYADVPPVDLPIHDAVLGGAAAFGSTPALIDGTDGTTLTYEQV ligase from DRFHRRVAAALAETGVRKGDVLALHSPNTVAFPLAFYAATRAGASVT Streptomyces TVHPLATAEEFAKQLKDSAARWIVTVSPLLSTARRAAELAGGVQEILV coelicolor A3(2) with CDSAPGHRSLVDMLASTAPEPSVAIDPAEDVAALPYSSGTTGTPKGV amino acid mutation MLTHRQIATNLAQLEPSMPSAPGDRVLAVLPFFHIYGLTALMNAPLRL A294G (DNA base GATVVVLPRFDLEQFLAAIQNHRITSLYVAPPIVLALAKHPLVADYDLS change Q9K3W1). SLRYIVSGAAPLDARLAAACSQRLGLPPVGQAYGMTELSPGTHVVPL UNIPROT: Q9K3W1 - DAMADAPPGTVGRLIAGTEMRIVSLTDPGTDLPAGESGEILIRGPQIM amino acid sequence KGYLGRPDATAAMIDEEGWLHTGDVGHVDADGWLFVVDRVKELIKY KGFQVAPAELEAHLLTHPGVADAAVVGAYDDDGNEVPHAFVVRQPA APGLAESEIMMYVAERVAPYKRVRRVTFVDAVPRAASGKILRRQLRE PR

[0391] phdE from ATGACTACTTCCACCACCCCAAACACCATCATTTCTTTCGAAGAC Corynebacterium GCACCAACCCTCACCGGCCAGGACCTGGGC I l l i CGCAGTGGCG glutamicum AACTGTCACCCAGGAGATGGTCAACACCTTCGCAGACGCAACCG (GENBANK: ATGATCAGCAGTGGATCCACACCGATCCTGAGCGCGCCAAGGAC AGN20978.1)- DNA GGTCC I l l i GGCGGCGCGATTGCCCACGGTTTCCTCACCTTGTC sequence CATGATCATTCCGTTCTGGGGCGAGCTGCTCGATGTCACCGGCG TGACCACCAAGGTGAACTATGGCCTGGATAAGGTGCGTTTCACCT CTCCCGTCAAGGTCGGTTCCCGCATCCGCATGGGCGCTGTGGTC CGCGAGATCTCTGAGGTGAAGGGCAATGGCCTGCACCTGGTCGC CGATGGCACCATTGAGATCGAAGGGCAGGAGCGCCCGGCCGTC GTAGCCACCTTCCTCACCCGCTTCTACGCTTAA

[0392] phdB from ATGAGCCTGAATGGTAAAGTTGCAATTGTTACCGGTAGCGGTGCA Corynebacterium GGTCTGGGTCGTAGCTTTGCACAAGAACTGGCACGTCAGGGTGC glutamicum AAGCGTTATTGTTAATGATGTTAATCAGGCAGCAGCCGATGAAAC (GENBANK: CGTTGCAGCAATTACCGAAGCCGGTGGTAAAGCAGCAGCAGTTA AGN20975.1)- DNA TTGCACCGGTTGGTCCGAGCGAAAGCGCAGCACTGCTGGTTCGT sequence GAAGCAGTTGATAAATTTGGTAGCCTGGATATTCTGGTTACCAAT GCAGGTATTCTGCGTGATCGTAGCCTGCTGAAAATGACCGATGAT GA I I I I GATGCCGTGATTAACGTTCATCTGAAAGGCACCTTTACCT GTGTGCGTGAAGCATTTGGTTATTTCAAAGAAAATGGTATTGCCG GTCGCATTGTTACCATTGGTAGCCCGACCGGTCAGCGTGGTAATT TTGGTCAGAGCAATTATGCAGCAGCAAAAGCAGGTATTGTTGGTA TGGTTCGTACCTGGGCATTAGAAATGAAACGTGCCGGTGTGACC ATTAATGCAATTATTCCGGAAGCAGCAACCGATATGACCAAAACC GTTCCGTA I l l i CAGAAAGCCGTTGAAGCGGATGAACGTGGTGAA GCAATGCCTGCA I I I I I DUG I GAAACC I I AGG I l l i GGTACACCG CAGGATGTTGCCGGTCTGGTTGCATTTCTGAGCAGTGATGAAGCA

[0393]

[0394] GCCAATATTAGCGGTCAGGCAATTGGTGCCGGTGGTGATCGTAT GCAGGTTTGGAAACATCCGGAACCGGCAGTTACCGAATTTAATCC TGGTGGTTGGACCTATGAAGCACTGCAAGAACGTGGTAAAAACAT TATTGAAGGTAATCTGCAGAGCGTTGGTGTTGTTTTTCCGGAACT GCCTGCAGAACTGCAGCCGCAGATTCCGGTTAAAGCATAA

[0395] phdC from ATGAGCAACAACGTGGTGAAATATGAATGTGCAGTTGATGCCGAT Corynebacterium AATATTGTTGCCGTTGATATGCATGTTCACCTGGAAGTTGATAGCT glutamicum GTGGTCATAAAAGCATGCCTGCAGATATTATGGCAGCAAGCAGCA (GENBANK: AATATTTCAAAACCGCAGAACGTACCCCGAGCGCAGATGCAATTG AGN20976.1)- DNA CCGATATTTATCGTGAACATAAAATGGCAGCCGTGGTGTTTACCA sequence TTGATGCACGTACCCAGATGGGTCATCTGCCGAATAGCATTGATG ATCTGGTTGCAAGCTGTGCCCGTAATAATGATGTTCTGATTCCGT TTGGTAGCGTTGATCCGCGTACCGGTGAAGATGCACTGGTTGAA GCACGTCGTCAGGTTGAAGAACTGGGTGTTCGTGGTTTCAAATTT CATCCGAGCGTTCAGGGTTTTGATCCGAGCGCACCGGAA I I I I AT CCGCTGTGGGAACTGCTGGAAAGCTTTGGTCTGCCGTGTGTTTTT CATACCGGTCAGAATGGTATGGGTGCAGGTCTGCCTGGTGGTCG TGGTATTAAACTGCG I l l i AGCAATCCGATGCTGCTGGATGATGT TGCAGCAGA I l l i CCGAATCTGACCATTATTATGGCCCATCCGAG TGTTCCGTGGCAGGATGAAGCAAATAGTATTGCAACCCATAAAGC CAACGTGTTTATTGATCTGAGCGGTTGGAGCCCGAAATACTTTCC GGAATCACTGGTTCGTCAGAGCAATAATGTTCTGAGCAAAAAGGT TCTGTTCGGCACCGA I l l i CCGCTGATTACACCGGAAAAATGGCT GGCAGCATTTGCCAATCTGCCGCTGAAAGATGAAGTTCGTCCGG GTATTCTGAAAGATAATGCCGTTAAAGTTCTGGGTTTAGCAGCCA GCACCGAACGTGGTAGCCAGGCAGAAAAAGTTGTTCAGCATGCA TAA

[0396] Phenylalanine / tyrosine ATGGCACCGAGCCTGGATAGCATTAGCCATAGCTTTGCGAACGG ammonia-lyase from CGTGGCGAGCGCGAAACAGGCGGTGAATGGCGCGAGCACCAAT Rhodotorula toruloides CTGGCCGTGGCGGGTAGCCATCTGCCGACCACCCAGGTGACCC (also known as AGGTTGATATTGTGGAAAAAATGCTGGCAGCGCCGACCGATAGC Rhodosporidium ACCCTGGAACTGGATGGCTATAGCCTGAACCTGGGTGATGTGGT toruloides). UNIPROT: GAGCGCAGCGCGTAAAGGTCGTCCGGTGCGTGTGAAAGATAGC P11544 - DNA GATGAAATCCGCAGCAAAATCGATAAAAGCGTGGAATTTCTGCGT sequence AGCCAGCTGTCTATGAGCGTGTATGGCGTGACCACCGGCTTTGG CGGTAGCGCGGATACCCGTACCGAAGATGCGATTAGCCTGCAGA AAGCGCTGCTGGAACATCAGCTGTGCGGCGTGCTGCCGAGCAG CTTTGATAGCTTTCGTCTGGGCCGTGGCCTGGAAAATAGCCTGCC GCTGGAAGTGGTGCGTGGCGCCATGACCATTCGTGTGAACAGCC TGACCCGTGGCCATAGCGCGGTGCGTCTGGTGGTTCTGGAAGCG CTGACCAACTTTCTGAATCATGGCATTACCCCGATTGTGCCGCTG CGTGGCACCATTAGCGCGAGCGGCGATCTGAGCCCGCTGTCTTA TATTGCAGCGGCGATTAGCGGCCATCCGGATAGCAAAGTGCATG TGGTGCATGAAGGCAAAGAAAAAATTCTGTATGCGCGTGAAGCGA

[0397]

[0398] TGGCGCTGTTTAACCTGGAACCGGTGGTGCTGGGCCCGAAAGAA GGCCTGGGCCTGGTTAATGGCACGGCGGTTAGCGCGAGCATGG CGACCCTGGCCCTGCACGATGCACACATGCTGAGTCTGCTGTCT CAGAGCCTGACCGCCATGACCGTGGAAGCGATGGTGGGCCATG CGGGCAGCTTTCATCCGTTTCTGCATGATGTGACCCGTCCGCATC CGACCCAGATTGAAGTGGCGGGCAACATTCGTAAACTGCTGGAA GGCAGCCG I l l i GCGGTGCATCATGAAGAAGAAGTGAAAGTTAAA GACGATGAAGGCATTCTGCGTCAGGATCGTTATCCGCTGCGTAC CAGCCCGCAGTGGCTGGGTCCGCTGGTGAGCGATCTGATTCATG CGCATGCGGTGCTGACCATTGAAGCGGGCCAGAGCACCACCGAT AATCCGCTGATTGATGTGGAAAACAAAACCAGCCATCATGGCGGC AACTTTCAGGCGGCAGCGGTGGCGAACACGATGGAAAAAACCCG TCTGGGCCTGGCGCAGATTGGCAAACTGAACTTTACCCAGCTGA CCGAAATGCTGAACGCGGGCATGAACCGTGGTCTGCCGAGCTGT CTGGCCGCGGAAGATCCGAGCCTGAGCTATCATTGCAAAGGCCT GGATATCGCGGCAGCGGCGTATACCAGCGAACTGGGCCATCTGG CCAATCCGGTGACCACCCATGTGCAGCCGGCGGAAATGGCGAAC CAGGCGGTTAACTCTCTGGCCCTGATTAGCGCGCGTCGTACCAC CGAAAGCAACGATGTTCTGAGCCTGCTGCTGGCCACCCATCTGT ATTGCGTGCTGCAGGCGATTGATCTGCGTGCGATCGAGTTCGAA TTCAAAAAACAGTTTGGTCCGGCGATTGTGAGCCTGATTGATCAG CA I I I I GGCAGCGCCATGACCGGCAGCAACCTGCGTGACGAACT GGTTGAAAAAGTGAACAAAACCCTGGCCAAACGTCTGGAACAGA CCAACAGCTATGATCTGGTGCCGCGTTGGCATGATGCGTTTAGCT TTGCAGCGGGCACCGTGGTGGAAGTTCTGAGCAGCACCAGCCTG AGCCTGGCCGCGGTGAACGCGTGGAAAGTGGCGGCAGCGGAAA GCGCCATTAGCCTGACCCGCCAGGTGCGTGAAACC I l l i GGAGC GCGGCGAGCACCAGCAGCCCGGCACTGAGCTACCTGAGCCCGC GTACCCAGATCCTGTATGCGTTTGTGCGTGAAGAACTGGGCGTG AAAGCGCGTCGTGGTGATGTGTTTCTGGGCAAACAGGAAGTGAC CATTGGCAGCAACGTGAGCAAAATTTATGAAGCGATTAAAAGCGG CCGTATTAACAACGTGCTGCTGAAAATGCTGGCCTAA

[0399] 4-coumarate:CoA ATGTTCCGTTCAGAATACGCGGATGTTCCTCCAGTCGACTTACCC ligase from ATCCATGATGCGGTATTAGGGGGCGCCGCGGCATTCGGATCCAC Streptomyces GCCAGCGCTGATTGACGGCACCGACGGGACCACGTTGACATATG coelicolor A3(2) with AGCAGGTCGACCGCTTTCACCGTCGTGTCGCCGCGGCTCTGGCC amino acid mutation GAAACCGGAGTGCGTAAGGGAGACG I l l i GGCATTGCACTCGCC A294G (DNA base GAACACAGTAGCTTTCCCGCTTGCG I l l i ACGCAGCAACCCGTGC change Q9K3W1). TGGAGCCTCCGTTACGACAGTACATCCCTTGGCCACTGCCGAGG UNIPROT: Q9K3W1 - AATTTGCGAAGCAACTGAAGGATTCCGCAGCCCGCTGGATTGTTA DNA sequence CGGTATCGCCCTTACTTTCGACAGCACGTCGCGCCGCCGAACTG GCGGGAGGAGTGCAAGAGATCCTGGTCTGCGATAGTGCCCCGG GGCACCGCTCTCTGGTCGACATGTTGGCTTCGACCGCACCTGAG CCTTCTGTTGCTATCGACCCCGCTGAGGATGTCGCTGCTTTGCCA

[0400]

[0401] TATTCGTCCGGAACTACAGGCACTCCGAAGGGTGTGATGTTGAC CCACCGCCAGATTGCCACTAATTTGGCACAACTGGAACCTAGTAT GCCATCCGCCCCCGGGGATCGCGTACTTGCTGTGCTGCCG I l l i TTCATATCTATGGATTAACTGCATTGATGAACGCGCCATTACGCTT AGGGGCTACCGTCGTAGTCCTTCCGCGTTTCGATCTGGAACAGTT TTTGGCAGCCATTCAGAACCACCGTATTACCAGCCTGTACGTAGC ACCTCCGATCGTCCTTGCCCTGGCAAAACACCCATTGGTTGCAGA TTATGACCTGTCGAGTTTACGCTATATCGTATCCGGCGCTGCCCC CTTAGACGCCCGCTTGGCCGCCGCATGCTCCCAACGTCTTGGTT TGCCACCAGTGGGCCAAGCATACGGAATGACCGAACTTTCCCCA GGCACCCATGTTGTCCCTTTGGATGCGATGGCTGACGCTCCCCC TGGCACTGTCGGGCGCTTAATTGCGGGGACTGAAATGCGCATCG TGTCATTAACTGATCCTGGAACGGACCTGCCCGCTGGTGAGTCC GGCGAGATCCTGATTCGCGGACCACAAATTATGAAGGGCTATTTG GGCCGCCCAGATGCGACAGCCGCGATGATTGACGAGGAGGGTT GGTTGCACACTGGCGACGTAGGGCATGTAGACGCCGACGGTTG GTTGTTTGTCGTGGATCGTGTCAAAGAGTTGATTAAGTACAAGGG ATTCCAAGTAGCCCCCGCTGAATTAGAGGCACACTTACTTACACA CCCAGGCGTGGCGGATGCTGCTGTTGTCGGTGCCTATGATGACG ATGGGAACGAGGTTCCGCATGCATTTGTTGTGCGCCAGCCCGCA GCCCCTGGGTTAGCGGAAAGCGAGATCATGATGTACGTGGCGGA GCGCGTCGCGCCTTACAAGCGTGTTCGCCGTGTCACCTTCGTCG ACGCAGTACCGCGTGCCGCTAGTGGGAAGATCCTGCGCCGTCAG TTGCGCGAGCCTCGTTAA

[0402] pSEVA434- TTAATTAAAGCGGATAACAATTTCACACAGGAGGCCGCCTAGGCA T7_RgPAL_ScCCL(A2 AGGATGCCTCCACACCGCTCGTCACATCCTGCCCATGAGTTAATT 94G)_phdBCE DNA ATATTTGTGGCATTATAGGGAATTGTGAGCGCTCACAATTAGCTG sequence TCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAA CAGCCTCTACAAATAA I l l i GTTTAAGATTAACTTTATAAGGAGGA AAAACATATGGCACCGAGCCTGGATAGCATTAGCCATAGCTTTGC GAACGGCGTGGCGAGCGCGAAACAGGCGGTGAATGGCGCGAGC ACCAATCTGGCCGTGGCGGGTAGCCATCTGCCGACCACCCAGGT GACCCAGGTTGATATTGTGGAAAAAATGCTGGCAGCGCCGACCG ATAGCACCCTGGAACTGGATGGCTATAGCCTGAACCTGGGTGAT GTGGTGAGCGCAGCGCGTAAAGGTCGTCCGGTGCGTGTGAAAG ATAGCGATGAAATCCGCAGCAAAATCGATAAAAGCGTGGAATTTC TGCGTAGCCAGCTGTCTATGAGCGTGTATGGCGTGACCACCGGC TTTGGCGGTAGCGCGGATACCCGTACCGAAGATGCGATTAGCCT GCAGAAAGCGCTGCTGGAACATCAGCTGTGCGGCGTGCTGCCGA GCAGCTTTGATAGCTTTCGTCTGGGCCGTGGCCTGGAAAATAGC CTGCCGCTGGAAGTGGTGCGTGGCGCCATGACCATTCGTGTGAA CAGCCTGACCCGTGGCCATAGCGCGGTGCGTCTGGTGGTTCTGG AAGCGCTGACCAACTTTCTGAATCATGGCATTACCCCGATTGTGC CGCTGCGTGGCACCATTAGCGCGAGCGGCGATCTGAGCCCGCT

[0403]

[0404] GTCTTATATTGCAGCGGCGATTAGCGGCCATCCGGATAGCAAAGT GCATGTGGTGCATGAAGGCAAAGAAAAAATTCTGTATGCGCGTGA AGCGATGGCGCTGTTTAACCTGGAACCGGTGGTGCTGGGCCCGA AAGAAGGCCTGGGCCTGGTTAATGGCACGGCGGTTAGCGCGAG CATGGCGACCCTGGCCCTGCACGATGCACACATGCTGAGTCTGC TGTCTCAGAGCCTGACCGCCATGACCGTGGAAGCGATGGTGGGC CATGCGGGCAGCTTTCATCCGTTTCTGCATGATGTGACCCGTCCG CATCCGACCCAGATTGAAGTGGCGGGCAACATTCGTAAACTGCT GGAAGGCAGCCG I l l i GCGGTGCATCATGAAGAAGAAGTGAAAG TTAAAGACGATGAAGGCATTCTGCGTCAGGATCGTTATCCGCTGC GTACCAGCCCGCAGTGGCTGGGTCCGCTGGTGAGCGATCTGATT CATGCGCATGCGGTGCTGACCATTGAAGCGGGCCAGAGCACCAC CGATAATCCGCTGATTGATGTGGAAAACAAAACCAGCCATCATGG CGGCAACTTTCAGGCGGCAGCGGTGGCGAACACGATGGAAAAAA CCCGTCTGGGCCTGGCGCAGATTGGCAAACTGAACTTTACCCAG CTGACCGAAATGCTGAACGCGGGCATGAACCGTGGTCTGCCGAG CTGTCTGGCCGCGGAAGATCCGAGCCTGAGCTATCATTGCAAAG GCCTGGATATCGCGGCAGCGGCGTATACCAGCGAACTGGGCCAT CTGGCCAATCCGGTGACCACCCATGTGCAGCCGGCGGAAATGGC GAACCAGGCGGTTAACTCTCTGGCCCTGATTAGCGCGCGTCGTA CCACCGAAAGCAACGATGTTCTGAGCCTGCTGCTGGCCACCCAT CTGTATTGCGTGCTGCAGGCGATTGATCTGCGTGCGATCGAGTT CGAATTCAAAAAACAGTTTGGTCCGGCGATTGTGAGCCTGATTGA TCAGCA I l l i GGCAGCGCCATGACCGGCAGCAACCTGCGTGACG AACTGGTTGAAAAAGTGAACAAAACCCTGGCCAAACGTCTGGAAC AGACCAACAGCTATGATCTGGTGCCGCGTTGGCATGATGCGTTTA GCTTTGCAGCGGGCACCGTGGTGGAAGTTCTGAGCAGCACCAGC CTGAGCCTGGCCGCGGTGAACGCGTGGAAAGTGGCGGCAGCGG AAAGCGCCATTAGCCTGACCCGCCAGGTGCGTGAAACC 1 1 1 1 GG AGCGCGGCGAGCACCAGCAGCCCGGCACTGAGCTACCTGAGCC CGCGTACCCAGATCCTGTATGCGTTTGTGCGTGAAGAACTGGGC GTGAAAGCGCGTCGTGGTGATGTGTTTCTGGGCAAACAGGAAGT GACCATTGGCAGCAACGTGAGCAAAATTTATGAAGCGATTAAAAG CGGCCGTATTAACAACGTGCTGCTGAAAATGCTGGCCTAATGCTT AAGTCGAACAGAAAGTAATCGTATTGTACACGGCCGCATAATCGA AATAAGGAGATATACATATGTTCCGTTCAGAATACGCGGATGTTC CTCCAGTCGACTTACCCATCCATGATGCGGTATTAGGGGGCGCC GCGGCATTCGGATCCACGCCAGCGCTGATTGACGGCACCGACG GGACCACGTTGACATATGAGCAGGTCGACCGCTTTCACCGTCGT GTCGCCGCGGCTCTGGCCGAAACCGGAGTGCGTAAGGGAGACG

[0405] I l l i GGCATTGCACTCGCCGAACACAGTAGCTTTCCCGCTTGCGT TTTACGCAGCAACCCGTGCTGGAGCCTCCGTTACGACAGTACATC CCTTGGCCACTGCCGAGGAATTTGCGAAGCAACTGAAGGATTCC GCAGCCCGCTGGATTGTTACGGTATCGCCCTTACTTTCGACAGCA

[0406]

[0407] CGTCGCGCCGCCGAACTGGCGGGAGGAGTGCAAGAGATCCTGG TCTGCGATAGTGCCCCGGGGCACCGCTCTCTGGTCGACATGTTG GCTTCGACCGCACCTGAGCCTTCTGTTGCTATCGACCCCGCTGA GGATGTCGCTGCTTTGCCATATTCGTCCGGAACTACAGGCACTCC GAAGGGTGTGATGTTGACCCACCGCCAGATTGCCACTAATTTGG CACAACTGGAACCTAGTATGCCATCCGCCCCCGGGGATCGCGTA CTTGCTGTGCTGCCG 1 1 1 1 1 1 CATATCTATGGATTAACTGCATTGA TGAACGCGCCATTACGCTTAGGGGCTACCGTCGTAGTCCTTCCG CGTTTCGATCTGGAACAGTTTTTGGCAGCCATTCAGAACCACCGT ATTACCAGCCTGTACGTAGCACCTCCGATCGTCCTTGCCCTGGCA AAACACCCATTGGTTGCAGATTATGACCTGTCGAGTTTACGCTAT ATCGTATCCGGCGCTGCCCCCTTAGACGCCCGCTTGGCCGCCGC ATGCTCCCAACGTCTTGGTTTGCCACCAGTGGGCCAAGCATACG GAATGACCGAACTTTCCCCAGGCACCCATGTTGTCCCTTTGGATG CGATGGCTGACGCTCCCCCTGGCACTGTCGGGCGCTTAATTGCG GGGACTGAAATGCGCATCGTGTCATTAACTGATCCTGGAACGGA CCTGCCCGCTGGTGAGTCCGGCGAGATCCTGATTCGCGGACCAC AAATTATGAAGGGCTATTTGGGCCGCCCAGATGCGACAGCCGCG ATGATTGACGAGGAGGGTTGGTTGCACACTGGCGACGTAGGGCA TGTAGACGCCGACGGTTGGTTGTTTGTCGTGGATCGTGTCAAAGA GTTGATTAAGTACAAGGGATTCCAAGTAGCCCCCGCTGAATTAGA GGCACACTTACTTACACACCCAGGCGTGGCGGATGCTGCTGTTG TCGGTGCCTATGATGACGATGGGAACGAGGTTCCGCATGCATTT GTTGTGCGCCAGCCCGCAGCCCCTGGGTTAGCGGAAAGCGAGA TCATGATGTACGTGGCGGAGCGCGTCGCGCCTTACAAGCGTGTT CGCCGTGTCACCTTCGTCGACGCAGTACCGCGTGCCGCTAGTGG GAAGATCCTGCGCCGTCAGTTGCGCGAGCCTCGTTAACCTGTAG AAATAA I l l i GTTTAACTTTAATAAGGAGATATACCATGAGCCTGA ATGGTAAAGTTGCAATTGTTACCGGTAGCGGTGCAGGTCTGGGT CGTAGCTTTGCACAAGAACTGGCACGTCAGGGTGCAAGCGTTATT GTTAATGATGTTAATCAGGCAGCAGCCGATGAAACCGTTGCAGCA ATTACCGAAGCCGGTGGTAAAGCAGCAGCAGTTATTGCACCGGT TGGTCCGAGCGAAAGCGCAGCACTGCTGGTTCGTGAAGCAGTTG ATAAATTTGGTAGCCTGGATATTCTGGTTACCAATGCAGGTATTCT GCGTGATCGTAGCCTGCTGAAAATGACCGATGATGA I l l i GATGC CGTGATTAACGTTCATCTGAAAGGCACCTTTACCTGTGTGCGTGA AGCATTTGGTTATTTCAAAGAAAATGGTATTGCCGGTCGCATTGTT ACCATTGGTAGCCCGACCGGTCAGCGTGGTAA I l l i GGTCAGAG CAATTATGCAGCAGCAAAAGCAGGTATTGTTGGTATGGTTCGTAC CTGGGCATTAGAAATGAAACGTGCCGGTGTGACCATTAATGCAAT TATTCCGGAAGCAGCAACCGATATGACCAAAACCGTTCCGTATTT TCAGAAAGCCGTTGAAGCGGATGAACGTGGTGAAGCAATGCCTG CA 1 1 1 1 1 CCG 1 GAAACC 1 1 AGG I l l i GGTACACCGCAGGATGTTG CCGGTCTGGTTGCATTTCTGAGCAGTGATGAAGCAGCCAATATTA

[0408]

[0409] GCGGTCAGGCAATTGGTGCCGGTGGTGATCGTATGCAGGTTTGG AAACATCCGGAACCGGCAGTTACCGAATTTAATCCTGGTGGTTGG ACCTATGAAGCACTGCAAGAACGTGGTAAAAACATTATTGAAGGT AATCTGCAGAGCGTTGGTGTTGTTTTTCCGGAACTGCCTGCAGAA CTGCAGCCGCAGATTCCGGTTAAAGCATAATGCTTAAGTCGAACA GAAAGTAATCGTATTGTACACGGCCGCATAATCGAAATAAGGAGA TATACATATGAGCAACAACGTGGTGAAATATGAATGTGCAGTTGA TGCCGATAATATTGTTGCCGTTGATATGCATGTTCACCTGGAAGTT GATAGCTGTGGTCATAAAAGCATGCCTGCAGATATTATGGCAGCA AGCAGCAAATATTTCAAAACCGCAGAACGTACCCCGAGCGCAGAT GCAATTGCCGATATTTATCGTGAACATAAAATGGCAGCCGTGGTG TTTACCATTGATGCACGTACCCAGATGGGTCATCTGCCGAATAGC ATTGATGATCTGGTTGCAAGCTGTGCCCGTAATAATGATGTTCTG ATTCCGTTTGGTAGCGTTGATCCGCGTACCGGTGAAGATGCACTG GTTGAAGCACGTCGTCAGGTTGAAGAACTGGGTGTTCGTGGTTTC AAATTTCATCCGAGCGTTCAGGG I l l i GATCCGAGCGCACCGGAA I l l i ATCCGCTGTGGGAACTGCTGGAAAGCTTTGGTCTGCCGTGT GTTTTTCATACCGGTCAGAATGGTATGGGTGCAGGTCTGCCTGGT GGTCGTGGTATTAAACTGCG I l l i AGCAATCCGATGCTGCTGGAT GATGTTGCAGCAGA I l l i CCGAATCTGACCATTATTATGGCCCAT CCGAGTGTTCCGTGGCAGGATGAAGCAAATAGTATTGCAACCCAT AAAGCCAACGTGTTTATTGATCTGAGCGGTTGGAGCCCGAAATAC TTTCCGGAATCACTGGTTCGTCAGAGCAATAATGTTCTGAGCAAA AAGGTTCTGTTCGGCACCGA I l l i CCGCTGATTACACCGGAAAAA TGGCTGGCAGCATTTGCCAATCTGCCGCTGAAAGATGAAGTTCGT CCGGGTATTCTGAAAGATAATGCCGTTAAAGTTCTGGGTTTAGCA GCCAGCACCGAACGTGGTAGCCAGGCAGAAAAAGTTGTTCAGCA TGCATAACACAC I l l i AAGGAGAACATCATGACCACCAGTACCAC ACCGAATACCATTGTTAGCTTTGAAGATGCTCCGACACTGACAGG TCAGGATCTGGG I l l i AGCCAGTGGCGTACCGTTACACAAGAAAT GGTTAATACCTTTGCAGATGCCACCGATGATCAGCAGTGGATTCA TACCGATCCGGAACGTGCAAAAGATGGTCCGTTTGGCGGTGCCA TTGCACATGG I l l i CTGACCCTGAGCATGATTATTCCGTTTTGGG GTGAACTGTTAGATGTTACCGGTGTTACCACCAAAGTTAATTATG GTCTGGATAAAGTGCGCTTTACCAGTCCGGTTAAAGTTGGTAGCC GTATTCGCATGGGTGCCGTTGTTCGTGAAATTAGCGAAGTTAAAG GTAATGGTCTGCATCTGGTGGCAGATGGCACCATTGAAATTGAAG GTCAAGAACGTCCGGCAGTTGTTGCAACCTTTCTGACACGTTTTT ATGCCTAAGCGGCCGCGTCGTGACTGGGAAAACCCTGGCGACTA GTCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCC ATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCG 1 1 1 1 1 1 A TTGGTGAGAATCCAGGGGTCCCCAATAATTACGATTTACGTATTTA AATGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCG

[0410] G 1 1 1 1 CA 1 GGC 1 1 G 1 1 A 1 GAU 1 G 1 1 1 1 1 1 1 GGGGTACAGTCTATGC

[0411]

[0412] CTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGT TTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGGGCAGTCG CCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGTGATCGCCGA AGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCA TCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG TGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTA CGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATC AACGACC I l l i GGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATT CTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATT CCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATG GCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGAT CGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAG CGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGG TTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGC TATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTA GTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAA ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCC TGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCT TATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCA GTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGT AGTCGGCAAATAAGACAATTGTCC I l l i CCGCTGCATAACCCTGC TTCGGGGTCATTATAGCGA 1 1 1 1 1 1 UGG 1 A 1 A 1 CCA 1 CC 1 1 1 1 1 CG CACGATATACAGGA I l l i GCCAAAGGGTTCGTGTAGACTTTCCTT GGTGTATCCAACGGCGTCAGCCGGGCAGGATAGGTGAAGTAGG CCCACCCGCGAGCGGGTGTTCCTTCTTCACTGTCCCTTATTCGCA CCTGGCGGTGCTCAACGGGAATCCTGCTCTGCGAGGCTGGCCGT AGGCCGGCCCTACCGGCGCGGCAGCGTTACCCGTGTCGGCGGC TCCAACGGCTCGCCATCGTCCAGAAAACACGGCTCATCGGGCAT CGGCAGGCGCTGCTGCCCGCGCCGTTCCCATTCCTCCGTTTCGG TCAAGGCTGGCAGGTCTGGTTCCATGCCCGGAATGCCGGGCTGG CTGGGCGGCTCCTCGCCGGGGCCGGTCGGTAGTTGCTGCTCGC CCGGATACAGGGTCGGGATGCGGCGCAGGTCGCCATGCCCCAA CAGCGATTCGTCCTGGTCGTCGTGATCAACCACCACGGCGGCAC TGAACACCGACAGGCGCAACTGGTCGCGGGGCTGGCCCCACGC CACGCGGTCATTGACCACGTAGGCCGACACGGTGCCGGGGCCG TTGAGCTTCACGACGGAGATCCAGCGCTCGGCCACCAAGTCCTT GACTGCGTATTGGACCGTCCGCAAAGAACGTCCGATGAGCTTGG AAAGTGTCTTCTGGCTGACCACCACGGCGTTCTGGTGGCCCATC TGCGCCACGAGGTGATGCAGCAGCATTGCCGCCGTGGGTTTCCT CGCAATAAGCCCGGCCCACGCCTCATGCGCTTTGCGTTCCGTTT GCACCCAGTGACCGGGCTTGTTCTTGGCTTGAATGCCGATTTCTC TGGACTGCGTGGCCATGCTTATCTCCATGCGGTAGGGGTGCCGC ACGGTTGCGGCACCATGCGCAATCAGCTGCAAC I l l i CGGCAGC GCGACAACAATTATGCGTTGCGTAAAAGTGGCAGTCAATTACAGA

[0413]

[0414] I I I I C I TTAACCTACGCAATGAGCTATTGCGGGGGGTGCCGCAAT GAGCTGTTGCGTACCCCCC I I I I I I AAG I I G I I GA I I I I I AAGTCT TTCGCATTTCGCCCTATATCTAGTTCTTTGGTGCCCAAAGAAGGG CACCCCTGCGGGGTTCCCCCACGCCTTCGGCGCGGCTCCCCCT CCGGCAAAAAGTGGCCCCTCCGGGGCTTGTTGATCGACTGCGCG GCCTTCGGCCTTGCCCAAGGTGGCGCTGCCCCCTTGGAACCCCC GCACTCGCCGCCGTGAGGCTCGGGGGGCAGGCGGGCGGGCTT CGCCCTTCGACTGCCCCCACTCGCATAGGCTTGGGTCGTTCCAG GCGCGTCAAGGCCAAGCCGCTGCGCGGTCGCTGCGCGAGCCTT GACCCGCCTTCCACTTGGTGTCCAACCGGCAAGCGAAGCGCGCA GGCCGCAGGCCGGAGGCTTTTCCCCAGAGAAAATTAAAAAAATT GATGGGGCAAGGCCGCAGGCCGCGCAGTTGGAGCCGGTGGGTA TGTGGTCGAAGGCTGGGTAGCCGGTGGGCAATCCCTGTGGTCAA GCTCGTGGGCAGGCGCAGCCTGTCCATCAGCTTGTCCAGCAGG GTTGTCCACGGGCCGAGCGAAGCGAGCCAGCCGGTGGCCGCTC GCGGCCATCGTCCACATATCCACGGGCTGGCAAGGGAGCGCAG CGACCGCGCAGGGCGAAGCCCGGAGAGCAAGCCCGTAGGGGG GGCGCGCCCAGCTGTCTAGGGCGGCGGATTTGTCCTACTCAGGA GAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTT TCGACTGAGCCTTTCG I l l i ATTTGATGCCT

[0415] PdPAL from MLASSPSGHTNPVLSGAPLSINVVADIGRQRLIPSLTDDEQVLNRVHA Planctomyces CRDVVQKAVRNNERIYGITTGFGGMSDIPIPPQHVAQTQDNLLAFLS brasiliensis TSTGASLDPRHVRAAMALRANVLLQGRSGVRLELIERLVEFLRQDAI - Amino acid sequence PVVCDLGSIGASGDLVPLGVIARSIIGHPSTTQVKYQGEQADSHDVL QQLNYSALQLEAKEGLALVNGTSFSSAIAANCVFESQRLLSLSLVLQS IMVRALGGHPEAFHPFVDENKPHPGQGWSAQMMRDLLSYSPNDSK RNGDLAQDRYSLRCLAQYFAPIVEGIAQISQSISTEMNAVSDNPLIDV DTGRFHQSGNFLGQYVAMSMDQLRRHLGLLAKHLDVQIAQLVAPAF NNGLPASLRGNSSRPFNMGLKGLQITGNSIMPLLTYLGNPLTEHFPT HAEEFNQNINGLSWGSANLAWRSVQLFQHYLSVASIFAVQAIDLRAG LEADHCDGRELLGETATELYETVYDLLERNCGQESPFLFNDDEQSLE VDLQMLNGDLAGAGRMHEAVSSVTDSFLAEFCE

[0416] DdPAL from MIETNHKDNFLIDGENKNLEINDIISISKGEKNIIFTNELLEFLQKGRDQL Dictyostelium ENKLKENVAIYGINTGFGGNGDLIIPFDKLDYHQSNLLDFLTCGTGDF discoideum fUniProt FNDQYVRGIQFIIIIALSRGWSGVRPMVIQTLAKHLNKGIIPQVPMHGS ID - Q556V9) VGASGDLVPLSYIANVLCGKGMVKYNEKLMNASDALKITSIEPLVLKS - Amino acid sequence KEGLALVNGTRVMSSVSCISINKFETIFKAAIGSIALAVEGLLASKDHY DMRIHNLKNHPGQILIAQILNKYFNTSDNNTKSSNITFNQSENVQKLD KSVQEVYSLRCAPQILGIISENISNAKIVIKREILSVNDNPLIDPYYGDVL SGGNFMGNHIARIMDGIKLDISLVANHLHSLVALMMHSEFSKGLPNSL SPNPGIYQGYKGMQISQTSLVVWLRQEAAPACIHSLTTEQFNQDIVS LGLHSANGAASMLIKLCDIVSMTLIIAFQAISLRMKSIENFKLPNKVQKL YSSIIKIIPILENDRRTDIDVREITNAILQDKLDFINLNL

[0417]

[0418] AvPAL from Anabaena MKTLSQAQSKTSSQQFSFTGNSSANVIIGNQKLTINDVARVARNGTL variabilis (UniProt ID - VSLTNNTDILQGIQASCDYINNAVESGEPIYGVTSGFGGMANVAISRE Q3M5Z3) - Amino acid QASELQTNLVWFLKTGAGNKLPLADVRAAMLLRANSHMRGASGIRL sequence ELIKRMEIFLNAGVTPYVYEFGSIGASGDLVPLSYITGSLIGLDPSFKV DFNGKEMDAPTALRQLNLSPLTLLPKEGLAMMNGTSVMTGIAANCV YDTQILTAIAMGVHALDIQALNGTNQSFHPFIHNSKPHPGQLWAADQ MISLLANSQLVRDELDGKHDYRDHELIQDRYSLRCLPQYLGPIVDGIS QIAKQIEIEINSVTDNPLIDVDNQASYHGGNFLGQYVGMGMDHLRYYI GLLAKHLDVQIALLASPEFSNGLPPSLLGNRERKVNMGLKGLQICGN SIMPLLTFYGNSIADRFPTHAEQFNQNINSQGYTSATLARRSVDIFQN YVAIALMFGVQAVDLRTYKKTGHYDARACLSPATERLYSAVRHVVG QKPTSDRPYIWNDNEQGLDEHIARISADIAAGGVIVQAVQDILPCLH

[0419] SmPAL from MTFVIELDMNVTLDQLEDAARQRTPVELSAPVRSRVRASRDVLVKFV Streptomyces QDERVIYGVNTSMGGFVDHLVPVSQARQLQENLINAVATNVGAYLD maritimus (UniProt ID - DTTARTIMLSRIVSLARGNSAITPANLDKLVAVLNAGIVPCIPEKGSLG Q9KHJ9) - Amino acid TSGDLGPLAAIALVCAGQWKARYNGQIMPGRQALSEAGVEPMELSY sequence KDGLALINGTSGMVGLGTMVLQAARRLVDRYLQVSALSVEGLAGMT KPFDPRVHGVKPHRGQRQVASRLWEGLADSHLAVNELDTEQTLAG EMGTVAKAGSLAIEDAYSIRCTPQILGPVVDVLDRIGATLQDELNSSN DNPIVLPEEAEVFHNGHFHGQYVAMAMDHLNMALATVTNLANRRVD RFLDKSNSNGLPAFLCREDPGLRLGLMGGQFMTASITAETRTLTIPM SVQSLTSTADFQDIVSFGFVAARRAREVLTNAAYVVAFELLCACQAV DIRGADKLSSFTRPLYERTRKIVPFFDRDETITDYVEKLAADLIAGEPV DAAVAAH

[0420] 4-coumarate:CoA MFRSEYADVPPVDLPIHDAVLGGAAAFGSTPALIDGTDGTTLTYEQV ligase from DRFHRRVAAALAETGVRKGDVLALHSPNTVAFPLAFYAATRAGASVT Streptomyces TVHPLATAEEFAKQLKDSAARWIVTVSPLLSTARRAAELAGGVQEILV coelicolor A3(2) - CDSAPGHRSLVDMLASTAPEPSVAIDPAEDVAALPYSSGTTGTPKGV wild-type amino acid MLTHRQIATNLAQLEPSMPSAPGDRVLAVLPFFHIYGLTALMNAPLRL sequence GATVVVLPRFDLEQFLAAIQNHRITSLYVAPPIVLALAKHPLVADYDLS SLRYIVSAAAPLDARLAAACSQRLGLPPVGQAYGMTELSPGTHVVPL DAMADAPPGTVGRLIAGTEMRIVSLTDPGTDLPAGESGEILIRGPQIM KGYLGRPDATAAMIDEEGWLHTGDVGHVDADGWLFVVDRVKELIKY KGFQVAPAELEAHLLTHPGVADAAVVGAYDDDGNEVPHAFVVRQPA APGLAESEIMMYVAERVAPYKRVRRVTFVDAVPRAASGKILRRQLRE PR

[0421] 4-coumarate: MADDGSRRELIFRSKLPDIYIPKHMPLHSYCFENLRECGSRPCLINAP coenzyme A ligase TGDVYSYHEVDSTARKVARGLKKEGVEQGQVIMILLPNCPEFVFSFL from Glycine max GASHRGAMATAANPFFTPAEIAKQAHASNAKLLITQASYYDKVKDLR (UniProt - Q8S5C1) - DIKLVFVDSCPPHTEEKQHLHFSHLCEDNGDADVDVDVDIKPDDVVA Amino acid sequence LPYSSGTTGLPKGVMLSHKGLVTSIAQQVDGDNPNLYYHCHDTILCV LPLFHIYSLNSVLLCGLRAKATILLMPKFDINSLLALIHKHKVTIAPVVPP IVLAISKSPDLHKYDLSSIRVLKSGGAPLGKELEDTLRAKFPNAKLGQ GYGMTEAGPVLTMSLAFAKEPIDVKPGACGTVVRNAEMKIVDPETG

[0422]

[0423] HSLPRNQSGEICIRGDQIMKGYLNDGEATERTIDKDGWLHTGDIGYID DDDELFIVDRLKELIKYKGFQVAPAELEALLLTHPKISDAAVVPMKDEA AGEVPVAFVVISNGYTDTTEDEIKQFISKQVVFYKRINRVFFIDAIPKSP SGKILRKDLRAKIAASVPK

[0424] 18 4-coumarate: MGSHFVEENMEGLIRLCPANHIPLTPLSFMERAAFVYRKNIALIQENIS coenzyme A ligase YSWEELHERCVKLASALSQLGGVTHGHVVAAVSPNVPALYELHFSV from Capsicum PMAGAVLSALNTKLDAATLAQILQQLEAKVIFVHTGFIELVLQALVTLQ annuum (UniProt - EKDKPYLLVLIHDSSNVKELPVSSVPPKILDYQTLLDMGQPNFKIVYP A0A2G3A9X6) - KNEFDPISINFTSGSTGMPKGVVYSHRAVYLNAIADIFRYEMSKSPVF Amino acid sequence LWTVDMFRCNGWCLPWTVAALGGTNICLGEIDGKEILESIYLHNVTH FCGAPMMLPKIADAIGTNQPLLPNNVNVTVAGVLPPPDIMTKLEKLGF SISHAYGMSEALGPMISMPRKSQDDFSNSNEDANVKIREGIHNIMME QVDVKDPESMQSVPADGKTIGEIMFRGNAVMLGYLKDPSRTEEAFE GEWYRTKDLGIKYPNGYIKLKDRVVDVIKSGGEIISTLEIEGVLIRHPM VLEAAVVARPDDVLGETPCAFVKLKEGCFVTDEEIIKFCEDKLQDYM VPKAVIFGDLPLNSSGKIQKFILRAKTKALKLCLPL

[0425] 19 4-coumarate: MVLQQQTHFLTKKIDQEDEEEEPSHDFIFRSKLPDIFIPNHLPLTDYVF coenzyme A ligase QRFSGDGDGDSSTTCIIDGATGRILTYADVQTNMRRIAAGIHRLGIRH from Arabidopsis GDVVMLLLPNSPEFALSFLAVAYLGAVSTTANPFYTQPEIAKQAKASA thaliana (UniProt - AKMIITKKCLVDKLTNLKNDGVLIVCLDDDGDNGVVSSSDDGCVSFT Q9LU36)- Amino acid ELTQADETELLKPKISPEDTVAMPYSSGTTGLPKGVMITHKGLVTSIA sequence QKVDGENPNLNFTANDVILCFLPMFHIYALDALMLSAMRTGAALLIVP RFELNLVMELIQRYKVTVVPVAPPVVLAFIKSPETERYDLSSVRIMLS GAATLKKELEDAVRLKFPNAIFGQGYGMTESGTVAKSLAFAKNPFKT KSGACGTVIRNAEMKVVDTETGISLPRNKSGEICVRGHQLMKGYLND PEATARTIDKDGWLHTGDIGFVDDDDEIFIVDRLKELIKFKGYQVAPA ELEALLISHPSIDDAAVVAMKDEVADEVPVAFVARSQGSQLTEDDVK SYVNKQVVHYKRIKMVFFIEVIPKAVSGKILRKDLRAKLETMCSK

[0426]

[0427] ***

[0428] The present disclosure includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0429] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0430] Aspects and embodiments of the present disclosure will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0431] Throughout this specification, unless the context requires otherwise, the word ‘comprise,’ and variations such as ‘comprises’ and ‘comprising,’ will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0432] As used herein, an amino acid sequence or a region of a polypeptide which ‘corresponds’ to a specified reference amino acid sequence or region of a polypeptide has at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the amino acid sequence / polypeptide / region. An amino acid sequence / region / position of a polypeptide / amino acid sequence which ‘corresponds’ to a specified reference amino acid sequence / region / position of a polypeptide / amino acid sequence can be identified by sequence alignment of the subject sequence to the reference sequence, e.g. using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21 , 951-960).

[0433] It must be noted that, as used in the specification and the appended claims, the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment.

[0434] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0435] Methods described herein may preferably be performed in vitro. The term ‘in vitro’ is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with / on intact multi-cellular organisms.

[0436] Examples

[0437] Example 1: Evaluation of novel hybrid fermentation-whole cell biocatalysis approaches for benzoic acid production

[0438] Multiple strategies for the production of phenylalanine and benzoic acid were evaluated by the inventors, for example see Strategies 1 to 3 below.

[0439] Strategy 1

[0440] In this strategy, an E. coli Phe overproduction strain was further engineered to incorporate a plasmid containing RgPAL (Figure 4). This extra gene converts Phe to cinnamic acid, which would be excreted from the cell. The cinnamic acid-containing supernatant is recovered and is used as the substrate for the second biocatalytic step. In this step the cinnamic acid would be converted to benzoic acid during a biocatalysis using a different E. coli strain containing a single plasmid with the ScCCL and phdBCE genes.

[0441] Strategy 2

[0442] In the second strategy, an E. coli Phe overproduction strain generates and secretes Phe into the culture supernatant (Figure 5). This compound is retrieved from the culture supernatant and used as the substrate for the biocatalytic step that converts Phe to benzoic acid using two E. coli cell lines. The first strain contains a single plasmid with the gene RgPAL. The second strain contains a single plasmid with the genes ScCCL and phdBCE. Both strains are co-cultivated together with Phe from the fed-batch fermentation. Strain 1 converts Phe into cinnamic acid, which is excreted into the culture supernatant. The second co-culture strain uptakes the cinnamic acid and converts it into benzoic acid using the multiple enzymes in the plasmid.

[0443] Strategy 3

[0444] In the third strategy, an E. coli Phe overproduction strain generates and secretes Phe into the culture supernatant, similar to Strategy 2 (Figure 6). This compound is used as the substrate for biocatalytic step that converts Phe into benzoic acid using a second E. coli cell line. However, the genes RgPAL, ScCCL are co-located on one plasmid (pETDuet-1_RgPAL_ScCCL(A294G)) and the phdBCE genes are found in a second plasmid (pETCOLADuet_phdBCE). Both plasmids are co-expressed within the same E. coli strain, which is generated by fermentation.

[0445] This third strategy proved the most successful in generating the highest benzoic acid titres, so was taken forward for optimisation and scaling studies.

[0446] Example 2: Scaling of benzoic acid production

[0447] Small scale biocatalytic benzoic acid production trials in shake flasks showed near complete conversion of phenylalanine after 136 hours (Figure 7) using Strategy 3. However, the majority of the product produced was cinnamic acid, which is generated by the activity of PAL on Phe. This shows an incomplete conversion of Phe under these reaction conditions.

[0448] Scaled reactions (1 L) were performed under more controlled conditions within a fermenter, which provided pH control to mitigate the drop in pH by the production of benzoic acid. In this case, the reaction achieved a complete conversion of Phe (8 g / L) to benzoic acid (5.7 g / L) over 6 days, which is close to the theoretical maximum product achievable (96.6% yield). Only 0.6% of the product was cinnamic acid. Therefore, the use of a more controlled environment within the bioreactor led to improvements in both titre and purity of the final product. Although E. co / / is normally handled under aseptic conditions, these biotransformations were performed under non-sterile conditions in the absence of antibiotics. The cell biomass had previously generated the recombinant enzymes during the earlier fermentation, so plasmid maintenance was not necessary here. The large quantity of live cells present in the absence of a carbon source meant there was a low chance of microbial contamination in the short term. These factors simplified the biocatalytic reactions.

[0449] Example 3: Benzoic acid downstream processing

[0450] The solubility of benzoic acid is only 3.44 g / L at 25 °C. Therefore, high titre production of benzoic acid would lead to precipitation of the product. To improve the benzoic acid solubility in water, the culture supernatant was basified to a high pH prior to the removal of the culture biomass by centrifugation. This was followed by cooling to 4 °C and acidification to pH 1 , resulting in benzoic acid precipitation. The precipitate was recovered by filtration in vacuo as an off white solid (Figure 9). This process resulted in the recovery of 68% of benzoic acid from a heavily contaminated culture slurry.

[0451] The final product was recovered as a dried product without further purification stages and its identity was confirmed by comparative1H NMR against commercially sourced benzoic acid (Figure 8). The final yield of benzoic acid was 3.9 g from a possible 5.7 g (by HPLC), with a 99.4% purity (Figure 10). The identified impurity was 0.6% cinnamic acid, which is produced in the first stage of the biocatalytic reaction from Phe.

[0452] Example 4: Benzoic acid production in Halomonas

[0453] 4.1 Overview

[0454] A series of plasmid-based systems were designed and generated to produce benzoic acid from phenylalanine. This was based on the cultivation of microbial strains to generate a cascading series of enzymes that can generate benzoic acid starting from glucose (bacterial carbon source) or feeding in a phenylalanine precursor. These whole cell biocatalysts generate benzoic acid during fermentation or by employing a biocatalytic approach with phenylalanine as the feedstock. This system was demonstrated and validated using Escherichia coli as the microbial chassis.

[0455] Further development and exemplification has been completed in a Halomonas microbial chassis. This development has enabled benzoic acid production to be performed using a low-cost non-sterile fermentation and biocatalytic approaches.

[0456] A more simplified methodology for benzoic acid production was also developed. Benzoic acid production within Halomonas was shown to proceed efficiently and at comparable titres to the E. coli system, even when switching from a biocatalytic to a fermentative process. Additionally, benzoic acid production occurred at the same time as Halomonas growth during fermentation. In contrast, high benzoic acid titres were obtained with the E. coli system when the cells were cultivated, harvested and used in a secondary biocatalytic reaction. The direct fermentation approach is a simpler and lower cost option and benefits from the ability of Halomonas to be cultivated under non-sterile conditions in low-cost bioreactors. This new fermentative approach, using Halomonas, generated up to 4.8 g / L of benzoic acid after nine days, with negligible accumulation of cinnamic acid (0.015 g / L). High purity benzoic acid (>99%) was obtained using a series of selective extractions with a final precipitation of purified product.

[0457] 4.2 Microbial chassis

[0458] H. bluephagenesis TD1.0 was chosen as an exemplary microbial chassis, although other strains of H. bluephagenesis are also compatible with benzoic acid production (e.g. strains TD01 and TQ10). The standard growth medium used for H. bluephagenesis TD1.0 is shown below:

[0459] Modified MM63 media recipe

[0460] • 60 g / L NaCI

[0461] • 13.6 g / L KH2PO4

[0462] • 5 g / L KOH

[0463] • 4 g / L Urea

[0464] • 1 mL 10,000 x MM63 Trace Elements Solution

[0465] • 1 mL 1 M MgSO4.7H2O

[0466] • 1 mL 5 mM Fe(NH4)2(SO4)2.6H2O solution

[0467] • 100 mL 50 mM CaCI2.6H2O

[0468] • 5-20 g / L carbon source, such as glucose, sucrose and glycerol.

[0469] pH adjusted to 9 by the addition of NaOH

[0470] 10,000 x MM63 Trace Elements Solution

[0471] • 0.494 g / L Co(NO3)2.6H2O

[0472] • 18.1 g / L MnCI2.4H2O

[0473] • 0.79 g / L CuSO4.5H2O

[0474] • 28.6 g / L H3BO3

[0475] • 3.9 g / L Na2MoO4.2H2O

[0476] • 2.2 g / L ZnSO4.7H2O

[0477] Cultivation of H. bluephagenesis strains was performed aerobically at 30-37 °C under non-sterile (non-aseptic) conditions using a variety of simple carbon sources. Cultivation can be performed under batch, fed batch and continuous fermentation conditions in a variety of vessel types (glass, steel and plastic bioreactors).

[0478] 4.3 Novel plasmid construct The Halomonas benzoic acid production system was designed as a multi-gene single plasmid system under the control of a single T7-like constitutive MMP1 viral promoter. The T7-like MMP1 inducible promoter is controlled by the MMP1 viral polymerase that is integrated into the genome of H. bluephagenesis strains TD1.0 and TQ10. A constitutive version of this promoter was generated by removing the lacl gene from the pSEVA434 plasmid (Figure 12). Therefore, the genes for benzoic acid production are expressed constitutively throughout the growth of Halomonas. Higher expression levels are obtained by lowering the growth temperature during high Halomonas growth. Expression of the recombinant genes within the H. bluephagenesis TD01 strain requires the switching of the promoter to a porin-based constitutive or inducible promoter (Trisrivirat, Hughes and Hoeven, Synth Biol (Oxf) 5.1 (2020): ysaa022), as it does not contain the genomic copy of the MMP1 viral polymerase. Here we describe benzoic acid production using the TD1.0 strain that does contain the MMP1 viral polymerase.

[0479] This DNA construct (pSEVA434-T7_RgPAL_ScCCL(A294G)_phdBCE) utilised a modified plasmid backbone (pSEVA434) that is compatible for propagation and expression of recombinant genes within H. bluephagenesis strains. To facilitate the assembly of the new Halomonas benzoic acid plasmid, the genes RgPAL, ScCCL(A294G) and phdBCE were commercially synthesised and ligated into the linearised modified pSEVA vector (pSEVA434-T7) by In-Fusion cloning. This vector was previously modified in-house to incorporate a single T7-like promoter. The gene and Shine Dalgarno sequences were not changed between the plasmids, and the new gene arrangements are shown in Figure 12. The new plasmid was transformed into the E. coli SM10 strain, followed by conjugation into H. bluephagenesis TD1.0. All sequences were confirmed by full plasmid sequencing.

[0480] 4.4 Exemplary benzoic acid production by Halomonas

[0481] 4.4.1 Halomonas fermentation for benzoic acid production

[0482] A glycerol stock of H. bluephagenesis TD1.0 containing plasmid pSEVA434-T7-RgPAL_ScCCL (A294G)_phdBCE was cultivated overnight at 37 °C on an LBA60 agar plate pH 9.0 (10 g / L tryptone, 5 g / L yeast extract, 15 g / L agar and 60 g / L NaCI) containing 100 pg / mL spectinomycin. A single colony was used to inoculate a 10 mL LB60 growth medium pH 9.0 (LBA60 minus the agar) containing 100 pg / mL spectinomycin. This starter culture was incubated for 18 hours at 37 °C with 180 rpm agitation. This starter culture was used to inoculate 400 mL LB60 pH 9.0 (seed culture) containing 100 pg / mL spectinomycin in 2 L baffled flasks. The seed culture was incubated for 24 hours at 37 °C with 180 rpm agitation.

[0483] A 200 mL seed culture was used as the inoculum for a fermentation using 1.8 L MM63 growth medium containing 100 pg / mL spectinomycin in an I KA 5 L Habitat vessel. Cultivation was performed using the following process conditions:

[0484] • Thermoregulation - 37 °C

[0485] • pH - 9.0 with pH probe and active pH correction using 5 M HCI and 5 M NaOH. • Dissolved oxygen - with a dO2 probe and active dO2 maintenance at 40% using cascade control (agitation: 200 - 600 rpm and airflow: 1000 - 9000 ccm).

[0486] After a 4-hour cultivation the thermoregulation was reset to 25 °C for 24 hours to favour an improved constitutive expression of the recombinant enzymes. Biocatalytic benzoic acid production was initiated by the addition of 8 g / L phenylalanine, and a temperature increase to 30 °C. The culture was sampled every 24 hours to monitor H. bluephagenesis growth (OD 600 nm). In addition, the samples underwent HPLC analysis to determine the yield of benzoic acid, residual phenylalanine concentration and intermediate / side product formation (e.g. precursor molecule cinnamic acid). After the phenylalanine concentration was determined, additional solid powder was added daily to bring the concentration back up to 8 g / L. This process was repeated over 6 days (9 days total fermentation). The culture was harvested using a by tangential flow filtration (SuPRO AMI ultrafiltration 1812 membrane) to generate a clarified supernatant.

[0487] 4.4.2 Downstream processing for benzoic acid purification

[0488] The culture broth (5 vol) was acidified with HCL to pH 3.5 (Figure 13). This protonates the benzoic acid, making it less water soluble and extractable into organic solvent. Some benzoic acid precipitation occurs during this step and is recovered by filtration. Ethyl acetate (1 vol) was added to extract the protonated benzoic acid, and the organic layer was recovered using a separation funnel. The benzoic acid was re-extracted into water by the addition of a 2.5 M NaOH solution (0.4 vol) and shaking in the separating funnel. This generates a water-soluble benzoate that transfers into the aqueous phase. The aqueous phase is recovered and acidified to pH 3.5, which leads to the precipitation of benzoic acid. The solid is recovered by filtration using a sintered filter and vacuum pump.

[0489] 4.4.3 Analytical methods

[0490] Fermentation samples were analysed by HPLC to determine the concentration of benzoic acid, cinnamic acid and residual phenylalanine. Culture samples were diluted 10-fold in water and centrifuged at 13,000 g for 10 minutes to pellet the Halomonas biomass. Aliquots (5 mL) were analysed using an Agilent 1260 Infinity II HPLC with a 1260 Infinity II vial autosampler, 1260 Infinity II multicolumn thermostat and a 1260 Infinity II refractive index detector (RID). Compounds were separated using a Thermo Scientific™ Hypersil GOLD™ C18 Selectivity HPLC column (100 mm x 4 mm x 3 mm).

[0491] Analysis was performed using the following solvents and gradient (Table 1):

[0492] • Solvent A: H2O + 0.1% trifluoroacetic acid (TFA)

[0493] • Solvent B: Acetonitrile + 0.1% TFA

[0494] Table 1. HPLC running conditions for the quantitation of benzoic acid, cinnamic acid and

[0495] phenylalanine. Time (min) Solvent A (%) Solvent B (%)

[0496] 0 95 5

[0497] 6 5 95

[0498] 6.5 5 95

[0499] 6.6 95 5

[0500] 10 95 5

[0501]

[0502] The column temperature was set to 40 °C with a solvent flow rate of 1 mL / min and total running time of 10 minutes. Peak detection was performed using a UV / Vis detector set to dual wavelengths of 220 and 254 nm. Quantitation of benzoic acid, phenylalanine and cinnamic acid were performed by comparing peak areas to standard curves generated under the same running conditions. The retention times were phenylalanine = 2.7 min; benzoic acid = 4.2 min and cinnamic acid = 4.7 min.

[0503] 4.4.4 Results and Discussion

[0504] Halomonas can use the amino acid phenylalanine as a carbon source for growth. Therefore, phenylalanine consumption (8 g / L / day) was expected to be due to a combination of Halomonas respiration (and / or growth) and benzoic acid production. During the 9 days offermentation, the benzoic acid titre linearly increased to a maximum of 4.8 g / L (Figure 14). No other carbon and nitrogen source was added once phenylalanine dosing occurred. As the Halomonas biomass continued to increase, this showed phenylalanine was being used partially as a carbon and nitrogen source.

[0505] Virtually no cinnamic acid accumulation was detected. This compound is the direct precursor for benzoic acid production, suggesting the last catalytic step is not significantly rate limiting under these conditions. This is important as the downstream processes for benzoic acid purification co-purify both acid products. The purification method of benzoic acid described here achieves >99% pure benzoic acid as cinnamic acid did not accumulate during the fermentation.

Claims

Claims:

1. A method of producing benzoic acid, wherein the method comprises:(i) the conversion of phenylalanine to cinnamate catalysed by a phenylalanine lyase, (ii) the conversion of cinnamate to cinnamoyl-CoA catalysed by a cinnamate-CoA ligase, and(iii) the conversion of cinnamoyl-CoA to benzoic acid by phenylpropanoid degradation pathway enzymes.

2. The method according to claim 1 , wherein the method comprises fermentation of a bacterial cell.

3. The method according to claim 2, wherein the bacterial cell heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, and phenylpropanoid degradation pathway enzymes.

4. The method according to any previous claim, wherein benzoic acid is produced at a titre of greater than 4.5 g / L.

5. The method according to any previous claim, wherein the method comprises fermentation of a bacterial cell in culture media comprising more than 20 g / L NaCI.

6. The method according to any previous claim, wherein the method comprises fermentation of a bacterial cell in culture media having a pH between pH 7.5 and pH 9.5.

7. The method according to any previous claim, wherein the method comprises fermentation of a bacterial cell under non-sterile conditions.

8. The method according to any of claims 2 to 7, wherein phenylalanine is provided exogenously to the bacterial cell.

9. The method according to claim 8, wherein the phenylalanine is provided exogenously to the bacterial cell during fermentation.

10. The method according to claim 8, wherein the phenylalanine is provided exogenously to the bacterial cell while the bacterial cell is in a conversion stage.

11. The method according to any previous claim, wherein the method comprises:(a) fermentative phenylalanine production, and(b) biocatalytic conversion of phenylalanine to benzoic acid by a bacterial cell.

12. The method according to claim 11 , wherein the bacterial cell heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, and phenylpropanoid degradation pathway enzymes.

13. The method according to any previous claim, wherein the method further comprises isolating the benzoic acid.

14. A bacterial cell, wherein the bacterial cell heterologously expresses a phenylalanine lyase, a cinnamate-CoA ligase, and phenylpropanoid degradation pathway enzymes.

15. The method according to any of claims 2 to 13, or the bacterial cell according to claim 14, wherein the bacterial cell is an Escherichia coli cell or a Halomonas cell.

16. The method according to claim 15, or bacterial cell according to claim 15, wherein the Halomonas cell is a Halomonas bluephagenesis cell.

17. The method according to any of claims 1 to 13, or the bacterial cell according to any of claims 14 to 16, wherein the phenylalanine lyase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

18. The method according to any of claims 1 to 13, or the bacterial cell according to any of claims 14 to 17, wherein the cinnamate-CoA ligase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19.

19. The method according to any of claims 1 to 13, or the bacterial cell according to any of claims 14 to 18, wherein the phenylpropanoid degradation pathway enzymes comprise an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, and / or a 3-oxoacyl-CoA ketohydrolase.

20. The method according to claim 19, or the bacterial cell according to claim 19, wherein the enoyl-CoA hydratase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:1.

21. The method according to claim 19 or claim 20, or the bacterial cell according to claim 19 or claim 20, wherein the 3-hydroxyacyl-CoA dehydrogenase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:2.

22. The method according to any one of claims 19 to 21 , or the bacterial cell according to any one of claims 19 to 21 , wherein the 3-oxoacyl-CoA ketohydrolase comprises an amino acid sequence with at least 40% sequence identity to SEQ ID NO:3.

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