EP4619538A1

EP4619538A1 - Biosynthesis of plastics precursors

Info

Publication number: EP4619538A1
Application number: EP23813832.5A
Authority: EP
Inventors: Samantha BRYAN; Edward Spence; Pierre REITZER; Anca PORDEA; Anna CROFT; Alex CONRADIE
Original assignee: University of Nottingham
Current assignee: University of Nottingham
Priority date: 2022-11-16
Filing date: 2023-11-16
Publication date: 2025-09-24
Also published as: WO2024105408A1; GB202217116D0; CN120677246A

Abstract

The invention provides a method for the preparation of 5-hydroxymethylfurfural or a derivative thereof and 2,5-furandicarboxylic acid comprising the step of biocatalytically converting at least one carbonaceous feedstock supplemented with glucose to 5-hydroxymethylfurfural or a derivative thereof and 2,5-furandicarboxylic acid also via intermediates like glyceraldehyde-3-phosphate and 4-hydroxymethyl-2-furancarboxaldehyde-phosphate using Streptomyces sp. S deposited as NCIMB 43995 and gene MfnB encoding (5-fromylfuran-3-yl)methyl phosphate synthase (EC 4.2.3.153) isolated from said strain.

Description

Biosynthesis of Plastics Precursors

Technical Field of the Invention

The present invention relates to methods for the biosynthesis of 5-hydroxymethylfurfural (5-HMF) and 2,5-furandicarboxylic acid (2,5-FDCA).

Background to the Invention

The production of industrial chemicals consumes more than 10% of our oil and gas resources; this figure is expected to greatly increase as the demands of the chemicals industry continue to grow. The global response to the COVID-19 pandemic has accelerated our consumption of fossil-derived chemical building blocks due to an increase in the production of single-use plastics.

2,5-Furandicarboxylic acid (2,5-FDCA) is currently listed as one of the 12 priority chemicals essential for establishing the “green” chemicals industry of the future (Werpy, T., and Petersen, G. (2004) Top Value Added Chemicals from Biomass: Vol. I Results of Screening for Potential Candidates from Sugars and Synthesis Gas. US Department of Energy, DOI: 10.2172/15008859). The market value for 2,5-FDCA in 2017 was 260 million USD and is predicted to grow to 850 million USD by 2023. 2,5-FDCA is a potential alternative to petrochemically derived terephthalic acid (TP A) currently used to produce polymers such as polyesters (such as polyethylene terephthalate (PET)), polyurethanes, and polyamides. The impact of replacing TPA with 2,5-FDCA in polymer synthesis would be significant, the replacement of PET with PEF (polyethylene furanoate - a copolymer of ethylene glycol and 2,5-FDCA) would result in a significant reduction in the 60 million bottles currently sent to landfill and incineration, resulting in a significant reduction in the carbon footprint, for example in an at least 50% reduction in greenhouse gas emissions. Further, PEF has superior barrier and thermal properties, making it the ideal material for a wide range of applications, and a good replacement for PET. PEF also has improved mechanical properties compared to PET, such as higher glass transition temperature and improved tensile modulus. In addition, PEF has better gas barrier properties for oxygen, carbon dioxide, and water vapor. PEF can be used in the production of water bottles, food packaging, sports apparel, footwear, etc.

Current production of 2,5-FDCA from biomass relies on the chemical synthesis of 5- (hydroxymethyl) furfural (5-HMF) from cellulose and hemicellulose, under acidic conditions leading to furan aldehyde degradation and low 5-HMF yields (Mittal A et al., (2020) Energy Fuels 34, 3, 3284-3293). Despite intensive efforts to optimise the process using various thermochemical catalytic strategies, 5-HMF yields and selectivities remain modest. 5-HMF is then converted to 2,5-FDCA mainly via chemocatalytic or electrocatalytic routes (Yuan H et al., (2020) Appl Micro Biotechl04, 527-543). These processes suffer from similar problems relating to catalyst development, product separation and environmental impact. Currently, the chemical production of 2,5-FDCA from 5-HMF uses high temperatures and pressures, in combination with metal salts (e.g., Co/Mn/Br/Cr), organic solvents (e.g., methanol/ethanol, additives (e.g., acetic acid), oxidants (e.g., KMnCh), polluting catalysts (e.g., Pb) and expensive catalysts (e.g., Pt) or nanoparticles (e.g., Au-CeO2/Au-TiO2/Au-Fe2O3). One-pot processes have been developed to avoid the need for HMF isolation, due to its instability, however yields are not yet competitive.

Several microbial platforms have been engineered to produce 2,5-FDCA from 5-HMF, including Pseudomonas putida S12, M. radiotolerans and B. cepacia (Hsu CT et al., (2020) Microb Biotech. 13(4): 1094- 1102). This process utilises the enzyme HmfH (hydroxymethyl) furfural oxidoreductase), originally identified in Cupriavidus basilensis. Despite promising yields of 30.6 g/L of 2,5-FDCA in P. putida over a 24-hour period, the process still requires the addition of 5-HMF, currently synthesised by the chemical pretreatment of biomass (Ghatta A et al., (2021) Green Chem., 23, 1716-1733). To date, the accepted sustainable route to 2,5-FDCA bioproduction is the conversion of lignocellulosic glucose to 5-HMF and the subsequent conversion to 2,5-FDCA.

It is an aim of embodiments of the present invention to ameliorate one or more problems of the prior art by provision of a method for the synthesis of 5-HMF and 2,5-FDCA, which offers one or more of the following advantages:

• Sustainable and/or techno-economically feasible method.

• Simplified method compared to prior art methods.

• Less energy intensive compared to prior art methods.

• Allows for 5-HMF and 2,5-FDCA to be produced directly from a single feedstock, preferably a readily available feedstock.

• Avoids accumulation of unstable intermediates in the reaction and their subsequent degradation.

• Allows for production of 5-HMF and 2,5-FDCA from waste feedstocks.

• Limited to no requirement for harsh chemicals/catalysts to be used.

• Provides good conversion rates of feedstock to 5-HMF and 2,5-FDCA.

Provides good 5-HMF and 2,5-FDCA yields.

High selectivity. It is also an aim of embodiments of the present invention to overcome or mitigate at least one problem of the prior art, whether expressly disclosed herein or not.

Summary of the Invention

According to a first aspect of the invention, there is provided a method for the preparation of 5-hydroxy methylfurfural (5-HMF) or a derivative thereof comprising the step of:

(a) Biocatalytically converting at least one carbonaceous feedstock to 5- hydroxymethylfurfural (5-HMF) or a derivative thereof.

According to a second aspect of the invention, there is provided a method for the preparation of 2,5-furandicarboxylic acid (2,5-FDCA) comprising the steps of:

(a) Biocatalytically converting at least one carbonaceous feedstock to 5- hydroxymethylfurfural (5-HMF) or a derivative thereof according to the method of the first aspect of the invention; and

(b) Biocatalytically converting the 5-HMF or derivative thereof from step (a) to produce 2,5-FDCA.

Step (a) of the second aspect is preferably step (a) of the first aspect of the invention.

The following statements may be applied mutatis mutandis to the first and second aspects of the invention. It would be clear to a person skilled in the art whether a statement may be applied to the first aspect of the invention, the second aspect of the invention, or both the first and second aspects of the invention.

Such methods allow for the biosynthesis of the platform chemical 5-HMF and the bioplastics monomer 2,5-FDCA directly from a carbonaceous feedstock, without the requirement to incorporate additional feedstock into the reaction mixture during the reaction. This observation is extremely significant, as direct 5-HMF and 2,5-FDCA bioproduction removes the need for chemical pre-treatments of lignocellulosic biomass, which are known to be problematic. These biosynthetic processes offer far more competitive routes to 5-HMF and 2,5-FDCA than known methods of synthesising these compounds in the prior art.

In preferred embodiments, biocatalytic conversion in step (a) and/or (b) comprises enzymatic conversion. References to biocatalytic conversion in statements of invention below may also be references to enzymatic conversion.

In some embodiments, at least one carbonaceous feedstock may be independently selected from the group comprising: a lignocellulosic feedstock, an oligo- or polysaccharide feedstock, a lignin feedstock, a synthetic polymer feedstock, a protein feedstock, and combinations thereof. At least one feedstock may comprise a waste feedstock and/or biomass derived feedstock.

The lignocellulosic feedstock may comprise biomass, which may comprise agricultural and/or municipal biomass.

Agricultural biomass may include at least one starch-based feedstock.

The lignocellulosic feedstock may comprise lignocellulosic waste biomass that may be independently selected from the group comprising: stover, bagasse, miscanthus, switchgrass, reed canary grass, rye, straw, compost, wood chips, sawmill discards, paper mill discards, and combinations thereof. The lignocellulosic feedstock may preferably comprise lignin, hemicellulose, and cellulose. Lignin derived lignocellulosic biomass may include, but is not limited to, at least one of: soda lignin, kraft lignin, hydrolysed lignin, organosolv lignin, lignosulfonates, black liquor, and combinations thereof.

At least one feedstock may comprise a waste biomass derived feedstock, which may comprise a domestic and/or industrial food waste, including slaughter industry biomass (e.g. feather biomass).

The oligo- or polysaccharide feedstock may preferably comprise at least one of: a naturally derived polysaccharide, a synthetic polysaccharide, a structural polysaccharide, and combinations thereof. In some embodiments, the oligo- or polysaccharide feedstock comprises an oligo- and/or polysaccharide comprising at least one glucose or glucose derivative monosaccharide unit. The oligo- or polysaccharide feedstock may comprise an oligo- or polysaccharide that is independently selected from the group comprising: cellulose, hemicellulose, chitin, an arabinoxylan, a pectin, a seaweed derived polysaccharide (including, but not limited to, alginates, ulvans, carrageenans, and/or fucoidans), and combinations thereof.

The synthetic polymer feedstock may comprise at least one petroleum-based feedstock and/or at least one biomass-based feedstock. The synthetic polymer feedstock may comprise at least one polymer that is independently selected from the group comprising: a polyester, a polyether, a polyurethane, a polyamide, a polystyrene, a polyolefin (polyalkene), a polyalkane, a polyhaloalkene, a polyhaloalkane (e.g. polyvinyl chloride), a bioplastic (e.g. polylactic acid, aliphatic polyester), a polyepoxide, an aromatic polymer, and combinations thereof. The synthetic polymer feedstock may preferably comprise at least one polyolefin that may be independently selected from the group comprising: polyethylene, polypropylene, and combinations thereof. At least one polyolefin preferably comprises polyethylene. The synthetic polymer feedstock may comprise at least one polyether that is a polyalkylene glycol. The synthetic polymer feedstock may comprise at least one polyalkylene glycol that is independently selected from the group comprising: polyethylene glycol, polypropylene glycol, polybutylene glycol, and combinations thereof. The synthetic polymer feedstock may preferably comprise at least one polymer that is independently selected from the group comprising: a polyolefin, a polyester, a polyalkylene glycol, and combinations thereof. The synthetic polymer feedstock may comprise polyethylene terephthalate (PET). In some embodiments, the synthetic polymer feedstock comprises at least one of: polyethylene terephthalate (PET), polyethylene (PE), polyether, and combinations thereof.

The protein feedstock may comprise at least one fibrous protein. At least one fibrous protein may be independently selected from the group comprising: a keratin, a collagen, an elastin, a fibrin, a casein, a whey, and combinations thereof.

At least one carbonaceous feedstock may be a non-saccharide feedstock.

At least one carbonaceous feedstock may comprise a small molecule feedstock. In some embodiments, at least one carbonaceous feedstock comprises an oxocarbon. At least one oxocarbon feedstock may comprise CO2 and/or CO.

At least one carbonaceous feedstock may comprise a polyol. At least one polyol may be independently selected from the group consisting of: a diol, a triol, a tetrol, and combinations thereof. In some embodiments, at least one polyol is or comprises glycerol.

In some embodiments, at least one carbonaceous feedstock, which may be a bio-derived and/or synthetic feedstock may be pre-treated, preferably prior to step (a), using at least one of: a physical pre-treatment (e.g., thermal, microwave), a chemical pre-treatment (e.g. acid/alkaline, solvent), a mechanical pre-treatment (e.g. cryomilling, sonication), a biological pre-treatment (e.g. enzymatic digestion), and combinations thereof.

In some embodiments, step (a) comprises the step of biocatalytically converting a primary and/or secondary metabolite species to 5-HMF or a derivative thereof. In some embodiments, the primary and/or secondary metabolite species is a carbonaceous feedstock. In some embodiments, the primary and/or secondary metabolite species is an intermediate. Step (a) may comprise biocatalytically converting the carbonaceous feedstock to a primary and/or secondary metabolite intermediate species; and then biocatalytically converting the intermediate species to 5-HMF or a derivative thereof. In such embodiments, at least one primary and/or secondary metabolite species may be independently selected from the group comprising: a ketide, a ketose, a keto acid, an aldose, and combinations thereof.

Throughout the specification, reference made to primary/secondary metabolites also includes reference to tautomers, isomers, metal complexes, salts, and other derivatives thereof.

Throughout the specification, reference made to ketides, ketoses, keto acids, and aldoses also includes reference to tautomers, isomers, metal complexes, salts, and other derivatives thereof.

In some embodiments, step (a) comprises the step of biocatalytically converting at least one species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof, to produce 5-HMF or a derivative thereof.

The species in step (a) may comprise at least one ketose, and may further comprise at least one keto acid and/or at least one aldose. The species in step (a) may comprise at least one keto acid, and may further comprise at least one ketose and/or at least one aldose. The species in step (a) may comprise at least one aldose, and may further comprise at least one ketose and/or at least one keto acid.

The species in step (a) may comprise at least one ketose and at least one keto acid. The species in step (a) may comprise at least one ketose and at least one aldose. The species in step (a) may comprise at least one keto acid and at least one aldose.

The species in step (a) may comprise at least one ketose, at least one keto acid, and at least one aldose.

In some embodiments, the species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof is a carbonaceous feedstock. In such embodiments, the species may preferably comprise a ketose and/or an aldose. At least one ketose and/or aldose in step (a) may be a saccharide. The saccharide used in step (a) may preferably comprise a monosaccharide and/or disaccharide. The saccharide used in step (a) preferably comprises a monosaccharide. In some embodiments, the saccharide comprises 2 or at least 2 different saccharides, 3 or at least 3, 4 or at least 4, or 5 or at least 5 different saccharides. The saccharide may comprise no greater than 5 different saccharides, or no greater than 4, 3, or no greater than 2 different saccharides. In some preferred embodiments, the saccharide may comprise a single saccharide, preferably a monosaccharide.

The saccharide used in step (a) may comprise at least one monosaccharide that is independently selected from the group comprising: a triose, a tetrose, a pentose, a hexose, a heptose, and combinations thereof. The saccharide may preferably comprise at least one hexose monosaccharide. The saccharide may comprise at least one monosaccharide that is independently selected from the group comprising: glucose, dextrose, fructose, levulose, galactose, and combinations thereof. The saccharide may particularly preferably be or comprise glucose.

In some embodiments, the species in step (a) that is independently selected from the group comprising: a ketide, a ketose, a keto acid, an aldose, and combinations thereof is an intermediate. In such embodiments, the species may preferably comprise at least one species independently selected from: a ketide, a keto acid, an aldose, and combinations thereof.

At least one aldose, preferably an intermediate aldose may comprise glyceraldehyde-3- phosphate (GA3P) or a derivative thereof.

At least one species in step (a), preferably an intermediate species may be a keto acid that is independently selected from the group comprising: an alpha-keto acid, a beta-keto acid, a gamma-keto acid, and combinations thereof. At least one species may be an alphaketo acid, which may be independently selected from: pyruvate, oxaloacetate, alphaketoglutarate, succinic acid, dihydroxy acetone phosphate and phosphoenolpyruvate, and combinations thereof. At least one species may preferably comprise pyruvate or a derivative thereof.

At least one species in step (a), preferably an intermediate species may be a ketide. At least one ketide may comprise a carboxylic acid and/or carboxylate moiety. At least one ketide may be independently selected from the group comprising: acetate, propionate, succinate, malonate, and combinations thereof.

In some embodiments, the species in step (a) comprises GA3P or a derivative thereof and/or pyruvate or a derivative thereof. Step (a) may comprise biocatalytically converting the carbonaceous feedstock to at least one primary and/or secondary metabolite intermediate species; and then biocatalytically converting the intermediate species to 5-HMF or a derivative thereof. Step (a) may comprise biocatalytically converting the carbonaceous feedstock to at least one intermediate species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof; and then biocatalytically converting the intermediate species to 5-HMF or a derivative thereof.

Step (a) may comprise biocatalytically converting a saccharide feedstock to at least one primary and/or secondary metabolite intermediate species; and then biocatalytically converting the intermediate species to 5-HMF or a derivative thereof. In some embodiments, step (a) comprises biocatalytically converting a saccharide feedstock to an intermediate species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof; and then biocatalytically converting the intermediate species to 5-HMF or a derivative thereof.

Step (a) may comprise biocatalytically converting a non-saccharide feedstock to a saccharide; and then biocatalytically converting the saccharide to 5-HMF or a derivative thereof. The saccharide may be converted to 5-HMF or a derivative thereof directly or via a further intermediate, which may comprise at least one primary and/or secondary metabolite species. The further intermediate may be independently selected from the group comprising: a ketide, a ketose, a keto acid, an aldose, and combinations thereof.

In preferred embodiments, at least one feedstock is independently selected from the group comprising: a lignocellulosic feedstock, an oligo- or polysaccharide feedstock, and combinations thereof. Step (a) of the method may comprise providing a lignocellulosic or oligo-/polysaccharide feedstock, and biocatalytically converting the feedstock to at least one primary and/or secondary metabolite species. Step (a) of the method may comprise providing a lignocellulosic or oligo-/polysaccharide feedstock, and biocatalytically converting the feedstock to at least one species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof. The method may comprise biocatalytically converting the feedstock to a saccharide, which may be a monosaccharide.

In some embodiments, the primary and/or secondary metabolite species in step (a) may be biocatalytically converted to 5-HMF or a derivative thereof directly or indirectly. In some embodiments, the ketide, ketose, keto acid, and/or aldose species in step (a), which may comprise a saccharide may be biocatalytically converted to 5-HMF or a derivative thereof directly or indirectly. The species, which may be a saccharide may be converted directly to 5-HMF or a derivative thereof without formation of an intermediate species. In other embodiments, the species, which may be a saccharide may be converted indirectly to 5-HMF or a derivative thereof via an intermediate species. The intermediate species may comprise a primary and/or secondary metabolite species. The intermediate species may preferably be independently selected from the group comprising: a ketide, a ketose, a keto acid, an aldose, and combinations thereof.

At least one of steps (a) and (b) of the method may be performed in the presence of a synthetic polymer feedstock, which may preferably be as described in statements of invention above. In some embodiments, the carbonaceous feedstock is a saccharide, which may comprise a monosaccharide and at least one of steps (a) and (b) is performed in the presence of a synthetic polymer feedstock. In preferred embodiments, the saccharide is or comprises glucose and at least one of steps (a) and (b) is performed in the presence of a synthetic polymer feedstock. In some embodiments, steps (a) and (b), and preferably all steps of the method, are performed in the presence of a synthetic polymer feedstock. In such embodiments, the synthetic polymer feedstock may be as described in statements of invention above and may preferably comprise at least one synthetic polymer that is independently selected from the group comprising: PET, polyethylene (PE), and combinations thereof.

In some embodiments, at step (a) and/or (b) of the method is performed in the presence of at least one polyol. At least one polyol may be independently selected from the group consisting of: a diol, a triol, a tetrol, and combinations thereof. In some preferred embodiments, at least one polyol is or comprises glycerol.

In some embodiments, at least one step of the method and preferably the entire method is performed in vivo. In some embodiments, at least one step of the method is performed using a microbial species. At least one step of the method may preferably be performed using a single microbial species. Step (a) and/or step (b) may be performed using a single microbial species. In preferred embodiments, at least steps (a) and (b), and preferably all steps of the method, are performed using a single microbial species. Preferably, the entire method is performed using a single microbial species.

In embodiments wherein the method comprises generating at least one primary and/or secondary metabolite species from the carbonaceous feedstock as described above, the method may preferably comprise biocatalytically converting the feedstock to the species in the microbial species. In embodiments wherein the method comprises generating at least one species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof from the carbonaceous feedstock as described above, the method may preferably comprise biocatalytically converting the feedstock to the species in the microbial species. In such embodiments, the generated species may be biocatalytically converted to 5-HMF or a derivative thereof directly after formation from the feedstock, and this step may preferably also be performed in the microbial species.

The microbial species may be independently selected from the group comprising: a bacterium, fungus, algal chassis, and combinations thereof. The microbial species may preferably comprise a bacterium, which may be a gram-positive, gram-negative, and/or filamentous bacterium.

In some embodiments, the microbial species may comprise a gram- negative bacterium. The microbial species may comprise a cyanobacterium. The microbial species may comprise a bacterium of the Synechocystis genus. The microbial species may be Synechocystis sp. PCC 6803.

The microbial species may comprise a proteobacterium. The microbial species may comprise a bacterium of the Pseudomonas genus. The microbial species may be Pseudomonas putida.

The bacterium may preferably comprise a gram-positive and/or filamentous bacterium. The microbial species may comprise an Actinomycete bacterium. The microbial species may preferably comprise a bacterium of the Streptomyces genus.

The microbial species may preferably be Streptomyces sp. S. Streptomyces sp. S. Has been deposited at the National Collection of Industrial Food and Marine Bacteria in Aberdeen, Scotland on 14 June 2022 under Deposit Reference NCIMB 43995. The microorganism Streptomyces sp. S is a bacteria species that is a member of the genus

Streptomyces. The Streptomycetes are part of the Strep tomycetaceae family which is a member of the Actinomycetales order found within the Phylum Actinomycetota. The bacteria Streptomyces sp. S is a Gram positive, aerobic multicellular filamentous bacteria. Streptomyces sp. S typically develop branched vegative hyphae and forms spores. When grown on solid medium colonies typically take between 24 and 48 hours to appear. The initial colony morphology of Streptomyces sp. S, when grown on soya flour mannitol (SFM) medium, are smooth beige-coloured circular colonies. Following sporulation the colony becomes white and hard. A yellow pigment may develop within the colony. Streptomyces sp. S can metabolise a wide range of carbon sources, including sugars, amino acids, lipids and both natural and synthetic polymers.

In some embodiments, the microbial species may be independently selected from the group comprising: E. coli, Sacchromyces, Aspergillus, Pseudomonas, Corynbactrium, Bacillus, and combinations thereof.

In some embodiments, step (a) and/or step (b) may be performed by heterologous expression of a pathway using a microbial chassis, preferably an industrial microbial chassis. The microbial chassis may be independently selected from the group comprising: E. coli, Sacchromyces, Aspergillus, Pseudomonas, Corynbactrium, Bacillus, and combinations thereof.

In some embodiments, the step (a) comprises incubating, growing and/or culturing the microbial species with the carbonaceous feedstock. The microbial species is preferably as described in statements of invention above. The microbial species may preferably comprise a bacterium of the Streptomyces genus, and may preferably be Streptomyces sp.

S. In some embodiments, step (a) comprises incubating, growing and/or culturing the microbial species with the carbonaceous feedstock, preferably as described above, to produce at least one primary and/or secondary metabolite species. In some embodiments, step (a) comprises incubating, growing and/or culturing the microbial species with the carbonaceous feedstock, preferably as described above, to produce at least one species independently selected from: aketide, a ketose, a keto acid, an aldose, and combinations thereof. The species may comprise a ketose and/or aldose saccharide. The saccharide preferably comprises a monosaccharide as described in statements of invention above. Particularly preferably, the saccharide may be or comprise glucose. The microbial species is preferably as described in statements of invention above. The microbial species may preferably comprise a bacterium of the Streptomyces genus, and may preferably be Streptomyces sp. S.

In some embodiments, the method comprises incubating, growing and/or culturing the microbial species with the carbonaceous feedstock under aerobic conditions. In some embodiments, the method comprises incubating, growing and/or culturing the microbial species with the carbonaceous feedstock using a bioreactor, which may be under aerobic conditions. The method may comprise incubating, growing and/or culturing the microbial species with the feedstock under anaerobic or aerobic conditions either as a batch, fed- batch or a continuous process.

The method may comprise incubating, growing and/or culturing the microbial species with the carbonaceous feedstock in a medium. The medium may be a liquid. The medium may comprise at least one salt. At least one salt of the medium may be independently selected from the group comprising: a phosphate, a monohydrogen phosphate, a dihydrogen phosphate, a sulfate, a halide (which may be independently selected from: fluoride, chloride, bromide, iodide, and combinations thereof), a citrate, a carbonate, a molybdate, a nitrate, a nitrite, and combinations thereof. At least one salt of the medium may have a cation that is independently selected from the group comprising: an alkali metal cation, an alkaline earth metal cation, an ammonium cation, and combinations thereof. In some embodiments, the medium is or comprises at least one medium independently selected from the group comprising: M9 medium, supplemented liquid minimal medium (SMM), basic minimal medium, minimal liquid medium (NMMP), minimal medium (MM), and combinations thereof.

The method may comprise the step of incubating, growing and/or culturing the microbial species with the carbonaceous feedstock in a medium at a total feedstock concentration of at least 0.5 g/L, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 g/L. The method may comprise incubating, growing and/or culturing the microbial species with the carbonaceous feedstock in a medium at a total feedstock concentration of no greater than 100 g/L, or no greater than 90, 80, 70, 60, 50, 45, 40, 35, 30, or no greater than 25 g/L. The method may comprise incubating, growing and/or culturing the microbial species with the carbonaceous feedstock in a medium at a total feedstock concentration of between 1-100 g/L, or between 1-50 g/L, or between 2-40, 5-30, 5-25, or between 7-22 g/L. The method may comprise incubating, growing and/or culturing the microbial species with the feedstock in a medium at a total feedstock concentration of between 50- 100 g/L.

The method may comprise incubating, growing and/or culturing the microbial species with at least one carbonaceous feedstock at a temperature of at least 10 °C, or at least 15, 20, or at least 25 °C. The method may comprise incubating, growing and/or culturing the microbial species with at least one carbonaceous feedstock at a temperature of no greater than 60 °C, or no greater than 50, 40, or no greater than 35 °C. The method may comprise incubating, growing and/or culturing the microbial species with at least one carbonaceous feedstock at a temperature of between 10-50 °C, or between 15-45, or preferably between 20-40, or between 25-35 °C. In some embodiments, the entire method may be performed at a temperature as described above.

The method may comprise incubating, growing and/or culturing the microbial species with at least one carbonaceous feedstock at a pH of at least 3, or at least 4, 5, or at least 6. The method may comprise incubating, growing and/or culturing the microbial species with the carbonaceous feedstock at a pH of no greater than 10, or no greater than 9, or no greater than 8. The method may comprise incubating, growing and/or culturing the microbial species with the carbonaceous feedstock at a pH of between 4-10, or between 5-9, or between 6-8, or at around pH 7. In some embodiments, the entire method may be performed at a pH as described above.

In some embodiments, the method comprises incubating, growing and/or culturing the microbial species with the carbonaceous feedstock, preferably in a medium for a total time of at least 5 hours, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or at least 60, 70, 80, 90, 100, 110, 120, 130, 140, or at least 160, 170, 180, 190, or at least 200 hours, or at least 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or at least 750 hours. The method may comprise incubating, growing and/or culturing the microbial species with the carbonaceous feedstock, preferably in a medium for a total time of no greater than 1000 hours, or no greater than 950, 900, 850, 800, 750, or no greater than 700 hours, 650, 600, 550, 500, 450, 400, or no greater than 350 hours. The method may comprise incubating, growing and/or culturing the microbial species with the carbonaceous feedstock, preferably in a medium for a total time of between 10-800 hours, or between 15-750, 25- 525 hours, or between 50-500, 100-450, 150-400, or between 200-350 hours.

The method may comprise incubating, growing and/or culturing the microbial species with the carbonaceous feedstock in a medium to form a reaction mixture. The method may comprise the step of agitating the reaction mixture. The method may comprise agitating the mixture during at least part of step (a), preferably during the whole of step (a). The method may comprise agitating the mixture during both steps (a) and (b). The method may comprise agitating the mixture throughout the entire method. The method may comprise agitating the reaction mixture at an agitation speed of at least 70 rpm, or at least 80, 90, 100, 110, 120, 130, or at least 140, 150, 200, 250, 300, 350, or at least 400 rpm. The method may comprise agitating the reaction mixture at a speed of no greater than 1500 rpm, or no greater than 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400 rpm, or no greater than 350, 300, 250, 200, or no greater than 190, 180, 170, or no greater than 160 rpm. The method may comprise agitating the reaction mixture at a speed of between 70-230, 80-220, 90-210, 100-200, 110-190, 120-180, 130-170, or between 140-160 rpm. The method may comprise agitating the reaction mixture at a speed of between 100-1500 rpm, or between 250-1200, or between 400-1000 rpm.

At least step (a) and preferably the entire method may be performed at an aeration rate of at least 0.1 vvm, or at least 0.2, 0.3, or at least 0.4 vvm, or at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, or at least 4.5. At least step (a) and preferably the entire method may be performed at an aeration rate of no greater than 5 vvm, or no greater than 4, 3, 2, 1, 0.9, 0.8, 0.7, or no greater than 0.6 vvm. At least step (a) and preferably the entire method may be performed at an aeration rate of between 0.1-5 vvm, 0.1-4, 0.1-3, 0.1-2, 0.1-1, 0.1-0.9, or between 0.2-0.8, 0.3-0.7, or between 0.4-0.6 vvm. At least step (a) and preferably the entire method may be performed at an aeration rate of between 0.5-5 vvm, 1-2.5, 2-4, or between 4-5 vvm.

In some embodiments, the method comprises incubating, growing and/or culturing the microbial species with the carbonaceous feedstock in a bioreactor. In some embodiments, the entire method is performed in a bioreactor.

In some embodiments, the method comprises incubating, growing and/or culturing the microbial species with the carbonaceous feedstock in a batch, batch-fed or continuous bioreactor.

In some embodiments, the method comprises adding a medium, preferably as described above to the bioreactor before addition of the carbonaceous feedstock, and microbial species. In some embodiments, the method comprises adding the carbonaceous feedstock to the bioreactor before the microbial species.

In some embodiments, the method may comprise sterilising the bioreactor before addition of the carbonaceous feedstock. The bioreactor may be sterilised by autoclaving. The bioreactor may be sterilised at a temperature of between 80-160 °C, or between 90- 150, 100-140, or between 110-130 °C. Sterilisation may also be achieved using steam, chemical treatments, heat, or radiation. The bioreactor may be sterilised for between 5-60 minutes, or between 10-40, or between 15-30 minutes. The bioreactor may be sterilised after addition of a medium thereto.

In some embodiments, the carbonaceous feedstock may be sterilised before addition to the bioreactor. The carbonaceous feedstock may be sterilised by autoclaving, streaming, heating, radiation, or chemical treatments. Statements relating to the sterilisation of the bioreactor may also be applied to sterilisation of the carbonaceous feedstock.

In some embodiments, the carbonaceous feedstock may be added as at least one culture independently selected from: a batch culture, a fed-batch culture, a continuous culture, and a combination thereof. In some embodiments, a single carbonaceous feedstock may be added. In other embodiments, more than one carbonaceous feedstock may be added. The or each carbonaceous feedstock may be added individually. In some embodiments, greater than one carbonaceous feedstock may be added together.

In some embodiments, the microbial species is added to the bioreactor as part of an inoculum (sometimes known as a starter culture or inoculation culture). The inoculum may be a liquid inoculum. The inoculum may comprise at least one monosaccharide, which may be as described in the statements of invention above. At least one monosaccharide may be glucose. The inoculum may contain the monosaccharide in a total concentration of between 0.5-10 g/L, or between 1-8, or between 10-100 g/L. In some embodiments, the inoculum may comprise at least one carbonaceous feedstock. In some embodiments, the inoculum comprises at least one yeast extract, which may be present in a total concentration as described for the monosaccharide above. In some embodiments, the inoculum comprises at least one malt extract, which may be present at a total concentration of between 1-20 g/L, or between 5-15 g/L. In some embodiments, spores of the microbial species may be added to the bioreactor, preferably as part of the inoculum. The inoculum may comprise one or more of the following: spores, germinating cells, dormant cells, live cells, and combinations thereof. In embodiments wherein the method comprises generating at least one primary and/or secondary metabolite species from the carbonaceous feedstock (which may be from microbial degradation of the carbonaceous feedstock), the biocatalytic conversion of the species to 5-HMF or a derivative thereof may occur directly and/or spontaneously after production of the species.

In embodiments wherein the method comprises generating a species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof from the carbonaceous feedstock, (which may be from microbial degradation of the carbonaceous feedstock), the biocatalytic conversion of the species to 5-HMF or a derivative thereof may occur directly and/or spontaneously after production of the species.

In some embodiments, step (b) occurs directly and/or spontaneously after generation of the 5-HMF or derivative thereof in step (a). In some embodiments, steps (a) and (b) are performed under identical conditions. In preferred embodiments, the entire method comprises a single biocatalytic process. At least steps (a) and (b), and preferably the whole method, may be performed as a one-pot process.

In some embodiments, the method comprises forming 5-HMF (or a derivative thereof) and/or 2,5-FDCA by direct conversion from the carbonaceous feedstock. The method may comprise forming 5-HMF (or a derivative thereof) and/or 2,5-FDCA by direct conversion from the microbial degradation of the carbonaceous feedstock. The carbonaceous feedstock may comprise a primary and/or secondary metabolite. The carbonaceous feedstock may comprise a species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof. The method may comprise forming 5-HMF (or a derivative thereof) and/or 2,5-FDCA by direct conversion from a ketose and/or aldose, which may be a saccharide, preferably as described in statements of invention above.

In some embodiments, the method comprises forming 5-HMF (or a derivative thereof) and/or 2,5-FDCA by indirect conversion from the carbonaceous feedstock. The method may comprise forming 5-HMF (or a derivative thereof) and/or 2,5-FDCA by indirect conversion from the microbial degradation of the carbonaceous feedstock.

In some embodiments, step (a) comprises indirectly biocatalytically converting the carbonaceous feedstock to 5-HMF or a derivative thereof via a primary and/or secondary metabolite intermediate species. In some embodiments, step (a) comprises indirectly biocatalytically converting the carbonaceous feedstock to 5-HMF or a derivative thereof via an intermediate species that is independently selected from the group comprising: a ketide, a ketose, a keto acid, an aldose, and combinations thereof. In some embodiments, step (a) comprises indirectly converting the carbonaceous feedstock to 5-HMF or a derivative thereof via a phosphate intermediate.

The method may comprise forming 5-HMF (or a derivative thereof) and/or 2,5-FDCA by microbial secondary metabolism. The method may comprise forming 5-HMF (or a derivative thereof) and/or 2,5-FDCA from microbial secondary metabolism of the carbonaceous feedstock. The method may comprise forming 5-HMF (or a derivative thereof) and/or 2,5-FDCA from direct and/or spontaneous microbial secondary metabolism of the carbonaceous feedstock, which may result from the microbial degradation of the carbonaceous feedstock. The method may comprise incubating, growing and/or culturing the microbial species with the carbonaceous feedstock to induce the microbial secondary metabolism. In some embodiments, the feedstock may comprise at least one species independently selected from the group comprising: a ketide, a ketose, a keto acid, and an aldose. The carbonaceous feedstock may be a ketose and/or an aldose, which may be a saccharide as described in statements of invention above.

In such embodiments wherein the method comprises forming 5-HMF and/or 2,5-FDCA from microbial secondary metabolism of a carbonaceous feedstock (which may result from the microbial degradation of the carbonaceous feedstock) comprising at least one species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof, said species may be formed from a precursor carbonaceous feedstock.

In such embodiments wherein the method comprises forming 5-HMF and/or 2,5-FDCA from microbial secondary metabolism of a carbonaceous feedstock, the method may occur via a polyketide synthase (PKS) enzyme. The method may comprise converting the carbonaceous feedstock to 5-HMF and/or 2,5-FDCA via at least one ketide species. The ketide species may be independently selected from the group comprising: acetate, propionate, succinate, malonate, and combinations thereof. The method may comprise converting the carbonaceous feedstock to 2,5-FDCA via 2-oxohexanedioic acid. The method may comprise the step of converting at least one ketide species to 2- oxohexanedioic acid using a PKS enzyme. The method may further comprise the step of converting 2-oxohexanedioic acid to 2,5-FDCA using a hydratase and/or isomerase enzyme, which may be an enoyl-CoA hydratase and/or isomerase enzyme. In such embodiments wherein the method comprises forming 5-HMF and/or 2,5-FDCA from microbial secondary metabolism of a carbonaceous feedstock, the method may occur via a non-ribosomal peptide synthase enzyme (NRPS)

At least one species independently selected from the group comprising: a ketide, a ketose, a keto acid, an aldose, and combinations thereof may be a primary metabolic intermediate. The primary metabolic intermediate may be formed from degradation of the carbonaceous feedstock, preferably from biological degradation of the carbonaceous feedstock, which may be a biological or synthetic carbonaceous feedstock. In some embodiments, step (a) comprises biocatalytically converting the carbonaceous feedstock to the primary metabolic intermediate and then biocatalytically converting said intermediate to form 5-HMF or a derivative thereof. The carbonaceous feedstock may be a pre-treated carbonaceous feedstock (which may be a biological or synthetic feedstock). The carbonaceous feedstock may be as described in statements of invention above.

In some embodiments, the 5-HMF (or derivative thereof) and/or 2,5-FDCA may be produced by secondary metabolism that may be derived from primary metabolism. The primary metabolism may be derived from the biological degradation and/or metabolism of the carbonaceous feedstock (which may be a biological or synthetic feedstock). The carbonaceous feedstock may be a pre-treated carbonaceous feedstock (which may be a biological or synthetic feedstock). The primary metabolism may result in the production of at least one species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof.

In some embodiments, the 5-HMF (or derivative thereof) and/or 2,5-FDCA may be produced by secondary metabolism that may be derived from the activity of at least one enzyme. At least one enzyme may be a synthase enzyme. At least one enzyme may be independently selected from the group comprising: apolyketide synthase enzyme (PKS), a non-ribosomal peptide synthase enzyme (NRPS), a PKS-NRPS hybrid type enzyme, an NRPS-PKS hybrid type enzyme, and combinations thereof.

In some embodiments, activity of at least one enzyme, preferably at least one PKS and/or NRPS enzyme, may result from condensation of at least one starter unit. At least one starter unit may be independently selected from the group comprising: a ketide (which may be independently selected from the group comprising: acetate, propionate, succinate, malonate, and combinations thereof), an aromatic unit, a peptide unit, a carboxylic ester (which may be a cyclic ester), a hydrocarbon (which may be independently selected from an alkane, alkene, alkyne, and combinations thereof), an alcohol, a carboxylic acid, an aldehyde, and combinations thereof.

In some embodiments, the biocatalytic conversion of the carbonaceous feedstock to generate 5-HMF or a derivative thereof in step (a) may involve the use of an aldolase and/or synthase enzyme. Step (a) may comprise biocatalytically converting the feedstock to at least one species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof; and then biocatalytically converting the species to 5- HMF or a derivative thereof using an aldolase and/or synthase enzyme. The species may comprise an aldose and/or keto acid. The carbonaceous feedstock may comprise a saccharide and the aldose and/or keto acid may be formed by glycolysis of the saccharide. In other embodiments, the feedstock may comprise a feedstock that is independently selected from the group comprising: a lignocellulosic feedstock, an oligo- or polysaccharide feedstock, a lignin feedstock, a synthetic polymer feedstock, a protein feedstock, an agricultural feedstock and combinations thereof. The feedstock may 1 preferably be as described in statements of invention above. In some embodiments, the feedstock may comprise cellulose and/or starch. In some embodiments, the species may comprise glyceraldehyde- 3 -phosphate and/or pyruvate, or derivatives thereof. In some embodiments, the glyceraldehyde-3 -phosphate and/or pyruvate, or derivatives thereof, may be converted to 5-HMF or a derivative thereof using an aldolase and/or synthase enzyme. For statements of invention above, the synthase enzyme may preferably be as described in statements of invention below.

In some embodiments, step (a) comprises the condensation of 2 aldose molecules to form 5-HMF or a derivative thereof. Step (a) may comprise the condensation of a GA3P molecule with another species to form 5-HMF or a derivative thereof. Step (a) may comprise the condensation of a GA3P molecule with another species that is a primary metabolite or a secondary metabolite species. Step (a) may comprise the condensation of a GA3P molecule with another species that is independently selected from: a ketide, a ketose, a keto acid, and an aldose. Step (a) may comprise the condensation of 2 GA3P molecules to form 5-HMF or a derivative thereof. The condensation reaction may preferably be catalysed by a synthase enzyme. The condensation reaction may preferably be catalysed by a synthase enzyme that is independently selected from the group comprising: a lyase, an aldolase, a cyclase, and combinations thereof. In a particular embodiment, the 5-HMF derivative is 4-(hydroxymethyl)-2-furancarboxaldehyde- phosphate (4-HFC-P). In some embodiments, the synthase enzyme may comprise 4- HFC-P synthase (MfnB), or a homologue thereof. Such synthase enzymes are believed to have a more open active site, which assists in the selective formation of 5-HMF as opposed to other isomers. In some embodiments, the synthase enzyme may comprise 4- HFC-P synthase (MfnB) or homologues of 4-HFC-P synthase (MfnB) with an E value of 0.0 or lower, using canonical 4-HFC-P synthase (MfnB) amino acid or nucleotide sequences. In some embodiments, the synthase enzyme may comprise 4-HFC-P synthase (MfnB) or homologues of 4-HFC-P synthase (MfnB) with an E value of le-150 or greater using canonical 4-HFC-P synthase (MfnB) amino acid or nucleotide sequences. In some embodiments, the synthase enzyme may comprise 4-HFC-P synthase (MfnB) or homologues of 4-HFC-P synthase (MfnB) with an E value of le-80 or greater using canonical 4-HFC-P synthase (MfnB) amino acid or nucleotide sequences. In some embodiments, the synthase enzyme may comprise 4-HFC-P synthase (MfnB) or homologues of 4-HFC-P synthase (MfnB), with an E value of le-50 or greater using canonical 4-HFC-P synthase (MfnB) amino acid or nucleotide sequences. In some embodiments, the synthase enzyme may comprise 4-HFC-P synthase (MfnB) or homologues of 4-HFC-P synthase (MfnB), with an E value of le-20 or greater using canonical 4-HFC-P synthase (MfnB) amino acid or nucleotide sequences. In some embodiments, the synthase enzyme may comprise 4-HFC-P synthase (MfnB) or homologues of 4-HFC-P synthase (MfnB) that may include DUF556 proteins and/or UPF0264 proteins. In some embodiments, the synthase enzyme may comprise 4-HFC-P synthase (MfnB) or homologues of 4-HFC-P synthase (MfnB) identified using standard homology searched using canonical 4-HFC-P synthase (MfnB) amino acid or nucleotide sequences. In some embodiments, the synthase enzyme comprises a 4-HFC-P (MfnB) homologue having an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 1, or at least 85, 90, 95, 96, 97, 98, or at least 99% identity with the sequence shown in SEQ ID NO: 1. In some embodiments, the synthase enzyme comprises a 4-HFC-P (MfnB) homologue having an amino acid sequence having

100% identity with the sequence shown in SEQ ID NO: 1 (the amino acid sequence of MfnB of Steptomyces sp.S). Step (a) may further comprise the step of biocatalytically converting a derivative of 5-HMF, preferably 4-HFC-P to 5-HMF.

In some embodiments, step (a) comprises forming 5-HMF or a derivative thereof from at least one keto acid and at least one aldose. Step (a) may comprise forming 5-HMF or a derivative thereof using GA3P and at least one keto acid, preferably pyruvate or a derivative thereof. At least one keto acid may be a glycolysis intermediate. Step (a) may preferably comprise forming 5-HMF or a derivative thereof using a synthase enzyme, preferably MfnB or an MfnB homologue.

In some embodiments, step (a) comprises biocatalytically dehydrating the carbonaceous feedstock, which may be a saccharide, to generate the 5-HMF or derivative thereof. The saccharide may be as described in statements of invention above.

Step (a) may comprise biocatalytically converting the carbonaceous feedstock, which may comprise a saccharide, preferably glucose, to generate the 5-HMF or derivative thereof via an intermediate comprising fructose or a derivative thereof. Step (a) may comprise the step of converting the carbonaceous feedstock, preferably a saccharide, preferably glucose, to fructose or a derivative thereof, preferably via an isomerisation reaction. Step (a) may comprise converting the carbonaceous feedstock, preferably a saccharide, preferably glucose, to fructose or a derivative thereof using an isomerase enzyme. Step (a) may comprise biocatalytically converting the carbonaceous feedstock, preferably a saccharide, preferably glucose, to fructose or a derivative thereof; and then biocatalytically converting the fructose or derivative thereof to 5-HMF or a derivative thereof. In some embodiments, the biocatalytic conversion of the carbonaceous feedstock, which may be a saccharide to generate the 5-HMF or derivative thereof in step (a) may involve the use of a dehydratase enzyme. The dehydratase enzyme may comprise a monosaccharide dehydratase. Step (a) may comprise isomerising the feedstock, preferably a saccharide, preferably glucose, to fructose or a derivative thereof, preferably using an isomerase enzyme; and then converting the fructose or derivative thereof to 5- HMF or a derivative thereof using a dehydratase enzyme.

Step (a) may comprise isomerising a feedstock, preferably a saccharide feedstock, preferably glucose, to fructose or a derivative thereof, preferably using an isomerase enzyme; and then converting the fructose or derivative thereof to glyceraldehyde 3- phosphate (GA3P) or a derivative thereof using an aldolase enzyme. In such embodiments, the fructose derivative may be a fructose phosphate derivative, which may comprise fructose 1,6-bisphosphate and/or 4-(hydroxymethyl)-2-furancarboxaldehy dephosphate (4-HFC-P). The fructose phosphate derivative may be formed by treatment of the feedstock, which is preferably a saccharide, preferably glucose, with a kinase enzyme before isomerisation to the fructose phosphate derivative. The fructose phosphate derivative may alternatively be formed by treatment of fructose with a kinase enzyme. Step (a) may further comprise the step of converting GA3P or a derivative thereof to 5- HMF or a derivative thereof using a synthase enzyme. The step of converting GA3P or a derivative thereof to 5-HMF or a derivative thereof may involve the condensation of 2 molecules of GA3P. Step (a) may comprise isomerising the feedstock, which is preferably a saccharide, preferably glucose, to fructose or a derivative thereof, preferably using an isomerase enzyme; converting the fructose or derivative thereof to glyceraldehyde 3-phosphate (GA3P) or a derivative thereof using an aldolase enzyme; and then converting GA3P or a derivative thereof to 5-HMF or a derivative thereof using a synthase enzyme.

In some embodiments, the biocatalytic conversion of the feedstock to generate the 5- HMF or derivative thereof in step (a) may proceed via a protocatechuate intermediate or a derivative thereof. The protocatechuate or derivative thereof may be formed from the feedstock, preferably a saccharide feedstock via the shikimate pathway, preferably via a 4-hydroxybenzoic acid intermediate or derivative thereof. In such embodiments, step (a) may generate a 5-HMF derivative, preferably a carboxylic acid derivative, more preferably 5-hydroxymethyl-2-furoic acid. In some embodiments, step (a) comprises forming the carboxylic acid 5-HMF derivative from protocatechuate or a derivative thereof using at least one dioxygenase, hydroxylase, isomerase, and decarboxylase enzyme, and optionally at least one dehydratase enzyme. In such embodiments, step (b) of the method may comprise biocatalytically converting the carboxylic acid 5-HMF derivative to produce 2,5-FDCA using at least one dehydrogenase enzyme.

In some embodiments, step (a) provides a 5-HMF derivative. The 5-HMF derivative may comprise an alkylated 5-HMF derivative, which may be a methylated 5-HMF derivative, such as methoxy methylfurfural. In some embodiments, the 5-HMF derivative may comprise a carboxylic acid derivative, which may be independently selected from: 5- hydroxymethyl-2-furoic acid and 5-formyl-2-furoic acid. In some embodiments, step (a) produces at least one 5-HMF derivative that is independently selected from the group comprising: a methylated 5-HMF derivative, a carboxylic acid 5-HMF derivative, and combinations thereof. In some embodiments, step (b) comprises biocatalytically oxidising the 5-HMF or derivative thereof from step (a) to produce 2,5-FDCA.

In some embodiments, step (b) comprises biocatalytically converting the 5-HMF or derivative thereof from step (a) to produce 2,5-FDCA using an oxidoreductase enzyme, preferably an HMF oxidoreductase enzyme.

Step (b) may comprise biocatalytically converting the 5-HMF or derivative thereof to produce 2,5-FDCA using an oxidase or dehydrogenase enzyme.

In some embodiments, step (b) comprises biocatalytically converting the 5-HMF or derivative thereof to produce 2,5-FDCA using an HMF oxidase enzyme (HmfH). In such embodiments, the 5-HMF or derivative thereof may be converted to 2,5-FDCA using an oxidase enzyme and molecular oxygen.

In some embodiments, step (b) comprises biocatalytically converting the 5-HMF or derivative thereof to produce 2,5-FDCA using an aldehyde dehydrogenase enzyme (AldH), preferably an HMF dehydrogenase enzyme. In such embodiments, the 5-HMF or derivative thereof may be converted to 2,5-FDCA via a 5-formyl-2-furoic acid (FFA) intermediate or derivative thereof.

In some embodiments, step (a) comprises biocatalytically converting the carbonaceous feedstock, preferably a saccharide, preferably comprising glucose, to generate 5-HMF via an intermediate comprising fructose or a derivative thereof; and step (b) comprises biocatalytically converting the 5-HMF or derivative thereof to produce 2,5-FDCA using an oxidase or dehydrogenase enzyme.

In some embodiments, step (a) comprises biocatalytically isomerising the carbonaceous feedstock, preferably a saccharide, preferably glucose, to fructose or a derivative thereof, preferably using an isomerase enzyme; and then converting the fructose or derivative thereof to 5-HMF or a derivative thereof using a dehydratase enzyme; and step (b) comprises biocatalytically converting the 5-HMF or derivative thereof to produce 2,5- FDCA using an oxidase or dehydrogenase enzyme.

In some embodiments, step (a) comprises biocatalytically isomerising the carbonaceous feedstock, preferably a saccharide, preferably glucose, to fructose or a derivative thereof, preferably using an isomerase enzyme; converting the fructose or derivative thereof to glyceraldehyde 3-phosphate (GA3P) or a derivative thereof using an aldolase enzyme; and then converting GA3P or a derivative thereof to 5-HMF or a derivative thereof using a synthase enzyme; and step (b) comprises biocatalytically converting the 5-HMF or derivative thereof to produce 2,5-FDCA using an oxidase or dehydrogenase enzyme.

The enzymes referenced in statements of invention above are preferably derived from the microbial species described above. The enzymes may be isolated enzymes. The microbial species may preferably comprise an actinobacterium species, which may comprise a bacterium species of the Streptomyces genus. The microbial species may preferably be Streptomyces sp. S. Streptomyces sp. S. has been deposited at the National Collection of Industrial Food and Marine Bacteria in Aberdeen, Scotland on 14 June 2022 under Deposit Reference NCIMB 43995.

According to a third aspect of the invention, there is provided the biosynthesis of 5- hydroxymethylfurfural (5-HMF) from a carbonaceous feedstock, wherein the biosynthesis is performed in a bacterium of the Streptomyces genus. The biosynthesis of the third aspect of the invention may be performed by the method of the first aspect of the invention. Statements of invention for the first aspect of the invention above may also be applied mutatis mutandis to the third aspect of the invention.

According to a fourth aspect of the invention, there is provided the biosynthesis of 2,5- furandicarboxylic acid (2,5-FDCA) from a carbonaceous feedstock, wherein the biosynthesis is performed in a bacterium of the Streptomyces genus.

The biosynthesis of the fourth aspect of the invention may be performed by the method of the second aspect of the invention. Statements of invention for the first and second aspects of the invention above may also be applied mutatis mutandis to the fourth aspect of the invention.

The following statements may be applied mutatis mutandis to the third and fourth aspects of the invention.

The carbonaceous feedstock may be independently selected from the group comprising: a lignocellulosic feedstock, a monosaccharide, an oligo- or polysaccharide feedstock, a lignin feedstock, a synthetic polymer feedstock, a protein feedstock, an agricultural feedstock and combinations thereof. The carbonaceous feedstock may also be subjected to at least one pre-treatment, which may be as described for the first and second aspects of the invention above.

The carbonaceous feedstock may preferably be the carbonaceous feedstock of the first aspect of the invention. The biosynthesis may comprise the biocatalytic conversion of the carbonaceous feedstock to 5-HMF and/or 2,5-FDCA via an intermediate species. The intermediate species may comprise a primary and/or secondary metabolite species. The intermediate species may be independently selected from the group comprising: aketide, a ketose, a keto acid, an aldose, and combinations thereof. The intermediate species may preferably be as described for the first and second aspects of the invention.

The carbonaceous feedstock may comprise a primary and/or secondary metabolite species. The carbonaceous feedstock may comprise a species that is independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof. The carbonaceous feedstock may comprise a saccharide, preferably as described for the first and second aspects of the invention above.

The carbonaceous feedstock may preferably be the carbonaceous feedstock of the first aspect of the invention. The biosynthesis may comprise the biocatalytic conversion of the carbonaceous feedstock directly to 5-HMF (or a derivative thereof) and/or 2,5-FDCA. The production of 5-HMF (or a derivative thereof) and 2,5-FDCA may proceed via microbial secondary metabolism of the carbonaceous feedstock.

In some embodiments, the carbonaceous feedstock comprises pre-treated carbonaceous feedstock, preferably as described for the first and second aspects of the invention above.

The bacterium of the Streptomyces genus is preferably a bacterium of Streptomyces sp. S. Streptomyces sp. S. has been deposited at the National Collection of Industrial Food and Marine Bacteria in Aberdeen, Scotland on 14 June 2022 under Deposit Reference NCIMB 43995.

According to a fifth aspect of the invention, there is provided the biosynthesis of 5- hydroxymethylfurfural (5-HMF) or a derivative thereof from a saccharide feedstock, wherein the biosynthesis is performed in isolated Streptomyces sp. S. The biosynthesis of the fifth aspect of the invention may be performed by the method of the first aspect of the invention. Statements of invention for previous aspects of the invention above may also be applied mutatis mutandis to the fifth aspect of the invention.

The biosynthesis of the fifth aspect of the invention may be the biosynthesis of the third aspect of the invention.

Statements of invention above relating to any previous aspect of the invention may also be applied mutatis mutandis to the fifth aspect of the invention.

According to a sixth aspect of the invention, there is provided the biosynthesis of 2,5- furandicarboxylic acid (2,5-FDCA) from a saccharide feedstock, wherein the biosynthesis is performed in isolated Streptomyces sp. S.

The biosynthesis of the sixth aspect of the invention may be performed by the method of the second aspect of the invention. Statements of invention for previous aspects of the invention above may also be applied mutatis mutandis to the sixth aspect of the invention.

The biosynthesis of the sixth aspect of the invention may be the biosynthesis of the fourth aspect of the invention.

Statements of invention above relating to any previous aspect of the invention may also be applied mutatis mutandis to the sixth aspect of the invention.

The following statements apply to the fifth and sixth aspects of the invention.

The saccharide is preferably as described for the first and second aspects of the invention above. 31

The biosynthesis may comprise the biocatalytic conversion of the saccharide to 5-HMF or 2,5-FDCA via microbial secondary metabolism. In some embodiments, the biosynthesis may comprise the biocatalytic conversion of the saccharide to 5-HMF or 2,5-FDCA via at least one intermediate species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof. The intermediate species may be generated by glycolysis and may comprise a keto acid and/or aldose.

According to a seventh aspect of the invention, there is provided a method for the preparation of 2,5-furandicarboxylic acid (2,5-FDCA) comprising the steps of:

(a) Biocatalytically converting glucose to fructose with an isomerase enzyme;

(b) Biocatalytically converting the fructose to 5 -hydroxy methylfurfural (5-HMF) with a dehydratase enzyme; and

(c) Biocatalytically converting the 5-HMF to 2,5-FDCA with an HMF oxidoreductase.

According to an eighth aspect of the invention, there is provided a method for the preparation of 2,5-furandicarboxylic acid comprising the steps of:

(a) Biocatalytically converting a carbonaceous feedstock to at least one intermediate species that is independently selected from: a keto acid, an aldose, and combinations thereof;

(b) Biocatalytically converting the at least one intermediate species to 5- hydroxymethylfurfural or a derivative thereof with a MfnB or MfnB homologue synthase enzyme; (c) Biocatalytically converting the 5 -hydroxy methylfurfural or derivative thereof to 2,5-furandicarboxylic acid with an HMF oxidoreductase or aldehyde dehydrogenase enzyme.

According to a ninth aspect of the invention, there is provided a method for the preparation of 2,5-furandicarboxylic acid comprising the steps of:

(a) Biocatalytically converting a carbonaceous feedstock to at least one ketide species, preferably independently selected from the group comprising: acetate, propionate, succinate, malonate, and combinations thereof;

(b) Biocatalytically converting the at least one ketide species via secondary metabolism to 5 -hydroxy methylfurfural or a derivative thereof with a poly ketide synthase enzyme or a non-ribosomal peptide synthase enzyme;

(c) Biocatalytically converting the 5 -hydroxy methylfurfural or derivative thereof to 2,5-furandicarboxylic acid with an HMF oxidoreductase or aldehyde dehydrogenase enzyme.

The method of the seventh, eighth and ninth aspect of the invention is preferably the method of the second aspect of the invention. Statements of invention above relating to the second aspect of the invention may also be applied mutatis mutandis to the seventh, eighth and ninth aspects of the invention.

Other statements of invention above relating to any previous aspect of the invention may also be applied mutatis mutandis to the seventh, eighth and ninth aspects of the invention.

Sequence Listings

SEQ ID NO: 1 (MfnB of Streptomyces sp.S) MRWKESTEEEEISPDGVEEAEECAKAAEHEDIVDVKKPDEGSEGANFPWVIREIR

GAVPADKPVSATVGDVPFKPGTVAQAALGAAVSGATYIKVGLYGCTTPDQAIEV MRGVVRAVKDYRPDAFVVASGYADAHRIGCVNPLALPDIARRSGSDAAMLDTA IKDGTRLFDHVPPEACGEFVRLAHEAGLLAALAGSVKSADLATLTRIGTDIVGVR GAVCEGGDRNKGRIQPRLVADFRAEMDRHAREHAATLAAS

Detailed Description of the Invention

In order that the invention may be more clearly understood, embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

Figure 1 displays growth of Streptomyces sp. S on glucose based on colony forming units (CFU). Samples are taken from three biological replicates.

Figure 2 displays a C18 reverse phase HPLC analysis of: (A) an authentic 2,5- FDCA standard; (B)-(G) Streptomyces sp. S supernatant taken during the biosynthesis of 2,5-FDCA from glucose at 0 (B), 72 (C), 120 (D), 168 (E), 240 (F), and 336 (G) hours. Results show that a peak corresponding to the 2,5-FDCA authentic standard was observed in the reaction mixture after 120 hours.

Figure 3 displays 2,5-FDCA (A and B) and 5-HMF (C and D) productivity in whole cell pellets (WC) (A and C) and supernatant extracts (Sup) (B and D) from Streptomyces sp. S at various time points during the biosynthesis of 2,5-FDCA from glucose.

Figure 4 shows ID NMR of (A) 2,5-FDCA reference sample, (B) sample extract spiked with 2,5-FDCA, (C) bioreactor sample taken during the biosynthesis of 2,5-FDCA from glucose. The ID NMR shows a clear increase in intensity and identification of the 2,5-FDCA signal.

Figure 5 shows 2,5-FDCA production in Streptomyces sp. S on: glucose (10. g/L); polyethylene terephthalate (PET) supplemented with glucose (10 g/L); and polyethylene (PE) supplemented with glucose (10 g/L). Supernatant was extracted and subjected to LC-MS/MS analysis.

Figure 6 shows the enzyme reaction performed by the Methanocaldococcus jannaschii MfnB enzyme.

Figure 7 shows the DNA plasmid used to express the Streptomyces sp. S MfnB protein in Pseudomonas putida for 5-HMF production.

Figure 8 displays a 12% SDS-PAGE gel showing the expression of the MfnB protein in Pseudomonas putida. The arrow indicates the MfnB band. The predicted molecular weight of MfnB is 27 kDa. The SDS-PAGE gel confirms the expression of the Streptomyces sp. S MfnB enzyme.

Figure 9 displays a C18 reverse phase HPLC analysis of: (A) an authentic 5-HMF standard; (B) wild type Pseudomonas putida supernatant; (C) Pseudomonas putida MfnB expression strain supernatant taken during growth on glycerol (96 hours); (D) 5-HMF spike of sample (C). Results show that a peak corresponding to the 5-HMF authentic standard was observed in the MfnB expression strain after 96 hours.

Figure 10 displays HPLC analysis of the conversion of CO2 to 5-HMF in Synechocystis sp. PCC 6803 (PCC6803). (a) shows 5-HMF standard; (b) PCC6803 wild type; (c) PCC6803 expressing the pathway for conversion of CO2 to 5-HMF (PCC6803 mutant); (d) PCC6803 mutant spiked with 5-HMF.

Isolation of Streptomyces sp. S

The Streptomyces sp. designated as Streptomyces sp. S (Streptomyces sp. S. has been deposited at the National Collection of Industrial Food and Marine Bacteria in Aberdeen, Scotland on 14 June 2022 under Deposit Reference NCIMB 43995) was isolated from soil in Warwickshire using Inorganic Salt Starch Agar (ISP4) plates. The soil samples were dried at 60 °C for 1 hour. The samples were then diluted using sterile Ringer’s solution and spread onto solid actinomycetes isolation plates, ISP4. Individual Streptomyces colonies were selected and repeatedly re-streaked onto new ISP4 plates to single colonies, colonies were wrinkled, raised and white. Amplification of the 16s rRNA gene sequence and subsequent phylogenetic analysis confirmed that Streptomyces sp. S was closely related to Streptomyces fulvissimus . S fulvissimus is a mesophilic bacterium, with a linear chromosome of 7.9 Mbp, which contains 32 gene clusters, involved in the biosynthesis of secondary metabolites. Two of these biosynthetic clusters have very high similarity to the cyclic peptide valinomycin and macrotetrolide antibiotic nonactin.

Biosynthesis of 2,5-FDCA directly from glucose

Streptomyces sp. S was routinely maintained using liquid and solid GYM liquid medium (4 g/L glucose, 4 g/L yeast extract, 10 g/L malt extract). Spore stocks were made using the solid sporulation medium SFM (soya flour 20 g/L, mannitol 20 g/L, agar 20 g/L).

Batch fermentations were conducted in a 2 L Applikon bioreactor (Gd'teborg, Sweden) at 30 °C under controlled pH and temperature. M9 minimal media (Sigma) was used as the cultivation medium with an initial glucose concentration of 20 g/L and trace elements 1 ml/L (ferric ammonium citrate 0.006 g/L, EDTA (disodium salt) 0.001 g/L, Na2C030.02 g/L, H3BO3 2.86 g/L, MnCl₂-4H₂O 1.81 g/L, ZnSO₄-7H₂O 0.222 g/L, NaMoO₄-2H₂O 0.39 g/L, CUSO₄-5H₂O 0.079 g/L, Co (NO₃)₂-6H₂O 49.4 mg/L). For the batch fermentation, the bioreactor containing 700 ml of M9 with trace elements was sterilised by autoclaving at 121 °C for 20 minutes. A 200 ml glucose solution containing 20 g of glucose was sterilised separately by autoclaving at 121 °C for 15 min and added aseptically to the bioreactor to give a concentration of 20 g/L. The bioreactor was then inoculated aseptically, with 100 ml of a preprepared Streptomyces sp. S inoculum at 1 % (v/v). The fermentation temperature, agitation speed and aeration rate were set to 30 °C, 150 rpm and 0.5 vvm respectively. The dissolved oxygen concentration was maintained at above 30 % of air saturation by regulating the air supply. The pH was set to 7.0 and maintained with 10 M NaOH or 10 M HC1. The fermentations were allowed to run for 2 weeks. Growth of Streptomyces sp. S was monitored using colony forming units (CFU), reaching a maximum cell density of 4.12x10⁹ CFU per ml (Figure 1).

Samples (10 ml each) were collected aseptically and prepared for LC-MS analysis as described. Samples were taken at Tl= Ohrs, T2=72hrs, T3=120hrs, T4=168hrs, T5=240hrs and T6=336hrs. Technical replicates were taken for each time point (n=6). Both the cell pellet and the supernatant were subject to methanol extraction and the extracts were analysed using both HPLC and LC-MS/MS.

The extracted supernatant from the fermentation was subject to HPLC analysis using C 18 reverse phase HPLC. A 50 mM 2,5-FDCA standard was prepared in 50% methanol to confirm that the presence of 2,5-FDCA. Both the standard and the samples from Tl= Ohrs, T2=72hrs, T3=120hrs, T4=168hrs, T5=240hrs and T6=336hrs were all analysed.

2,5-FDCA was detectable as a peak at the same retention time as the standard (Figure 2A and Figures 2B-G). 2,5-FDCA was initially detected as a small peak at around T3=120hrs, the peak area subsequently increased, reaching a maximum at T5=240hrs and then decreasing. Maximum productivity was determined to be 48 mg/L. The supernatant from T6=336hrs was subsequently spiked with 50 mM 2,5-FDCA, resulting in an increase in the peak area compared to the un-spiked supernatant. The extracted cell pellets and supernatants were subjected to targeted LC-MS/MS, specifically targeting 2,5-FDCA and 5-HMF. Standards were prepared for both 2,5-FDCA and 5-HMF to confirm the presence of both in the whole cell pellets and the supernatant. The concentration of both 2,5-FDCA and 5-HMF increased over time in both the supernatant and the whole cell pellets (Figures 3A-D), with peak productivity seen after 120hrs. Interestingly, increases in the peak area of 5-HMF also correlated with increased 2,5- FDCA productivity, indicating that 2,5-FDCA is being produced from 5-HMF. MS/MS in negative ionisation mode confirmed the presence of both 2,5-FDCA and 5-HMF, the spectrums of both the 2,5-FDCA and 5-HMF standards being identical to the corresponding spectrum for the T6=336 hrs sample.

Confirmation of 2,5-FDCA production by NMR analysis

Extracted supernatant samples from T6=336hrs were sent for NMR analysis. Despite its limitations in sensitivity compared to LC-MS, NMR has several advantages, its versatility, and rigour in elucidating structures, analysis of positional isotopomer distributions and isotope filtered selection of molecules. Both the samples and the standard were analysed using ID and 2D proton NMR. Samples were also analysed using ID and 2D proton NMR to confirm the presence of 2,5-FDCA. A 2,5-FDCA standard was used as a control and the sample supernatant from T6=336 was also spiked with 2,5-

FDCA. The 2D NMR showed the 13C and 1H positions and avoided any ambiguity around the identity of the signal. The standard, un-spiked and spiked samples all overlapped. It was also very clear that there were no overlapping similar signals which could be confused with the 2,5-FDCA signal. The ID analysis was quantitative and clearly showed an increase in the signal intensity in the spiked sample compared to the un-spiked sample, the standard and both samples also had the same chemical shift (ppm). The ID 1H NMR analysis is shown in Figure 4.

Biosynthesis of 2,5-FDCA on different substrates

Biosynthesis of 2,5-FDCA was performed as described above, but on different substrates. Figure 5 shows 2,5-FDCA production on: glucose (10. g/L); polyethylene terephthalate (PET) supplemented with glucose (10 g/L); and polyethylene glycol (PEG) supplemented with glucose (10 g/L). The graph demonstrates that 2,5-FDCA production increases over the first 8 days of the fermentation. The data showed that by day 5, the glucose has been completely consumed. This suggests that 2,5-FDCA production coincides with cell growth. It is likely that 2,5-FDCA biosynthesis originates from glucose in the PET sample. However, 2,5-FDCA biosynthesis also appears to correspond with PEG degradation. This again suggests that 2,5-FDCA biosynthesis is likely to be the result of enzyme catalysis and not a spontaneous reaction.

Pathway identification - Biosynthesis of 5-HMF

Previously the MfnB enzyme has been identified in Methanocaldococcus jannaschii (Biochemistry 2015, 54, 19, 2997-3008) catalysing the conversion of two molecules of glyceraldehyde 3-phosphate by the reaction shown in Figure 6. Streptomyces sp.S encodes a putative MfnB homologue, with an amino acid sequence as shown in SEQ ID

NO: 1. The Streptomyces sp. S MfnB homologue was PCR amplified using Steptomyces sp. S genomic DNA as a template. The MfnB gene was cloned in a broad-host range plasmid under control of a constitutive Pseudomonas putida promoter (Figure 7). This expression plasmid was used to transform Pseudomonas putida using electroporation.

Expression of the MfnB enzyme using Pseudomonas putida was confirmed with SDS- PAGE shown in Figure 8.

The production of 5-HMF using Pseudomonas putida as a chassis via expression of the Streptomyces sp. S MfnB gene was achieved using glycerol 1-10% as a feedstock in order to maximise intracellular glyceraldehyde- 3 -phosphate content.

HPLC analysis confirmed the production of a compound with the same retention time as 5-HMF, as shown in Figure 9.

Production of 5-HMF from CO2

The production of 5-HMF from CO2 in another species, Synechocystis sp. PCC 6803 (PCC6803). HPLC results are displayed in Figure 10.

Figure 10c shows that 5-HMF is produced from CO2 in PCC6803, as a peak is produced which matches the peak of the 5-HMF standard in Figure 10a, and which peak is not present in the PCC6803 wild type (Figure 10b).

However, expression of this biosynthetic pathway in the new species has a negative impact on cell viability of PCC6803, and expression is ceased when the species is spiked with 5-HMF (see Figure lOd).

It is believed that CO2 is converted in situ to saccharides, including glucose, which is then converted to 5-HMF via a central metabolic process. Discussion

The present invention allows for the biosynthesis of the platform chemical 5-HMF and the bioplastics monomer 2,5-furandicarboxylic acid (2,5-FDCA) directly from a carbonaceous feedstock, without the requirement to incorporate additional feedstock into the reaction mixture during the reaction - the first time this phenomenon has been documented in nature. Several microbial platforms have been previously engineered to produce 2,5-FDCA from 5-HMF, including Pseudomonas putida S 12, radiotolerans and B. cepacian. However, these processes required the separate addition of 5-HMF, which prior to the present invention has been synthesised by the chemical pre-treatment of biomass. Therefore, known processes are still subject to the same pitfalls associated with the chemical synthesis of 2,5-FDCA. The complete biotransformation of feedstocks e.g. saccharides, such as glucose employed in the present invention offers a truly sustainable and game changing route to 5-HMF and 2,5-FDCA production.

Using a combination of powerful analytical methods, HPLC, LC-MS/MS and NMR the Inventors have been able to demonstrate that Streptomyces sp. S produces 2,5-FDCA directly from glucose. LC-MS/MS and HPLC demonstrated that 2,5-FDCA productivity typically starts around T3=120hrs and subsequently increases, with peak activity at T6=336hrs, which corresponds with the onset of secondary metabolism (Figure 3A-B). Increases in 2,5-FDCA productivity are linked to both glucose consumption and 5-HMF formation. The concentration of 5-HMF increased from T2=72hrs correlating with an increase in 2,5-FDCA and strongly suggesting that the 2,5-FDCA is derived from 5-HMF or a derivative thereof. ID and 2D NMR confirmed that the structure of the molecule was 2,5-FDCA. Direct 5-HMF and 2,5-FDCA bioproduction from carbonaceous feedstocks as demonstrated in the present invention removes the need for lignocellulose chemical pretreatments to produce 5-HMF for conversion to 2,5-FDCA. This offers a far more competitive route to 2,5-FDCA biosynthesis than methods of the prior art. 2,5-FDCA is an important renewable bioplastics monomer, which has the potential to substitute a variety of petrochemicals, such as terephthalic acid and adipic acid.

The above embodiments are described by way of example only. Many variations are possible without departing from the scope of the invention as defined in the appended claims.

Claims

1. A method for the preparation of 5 -hydroxy methylfurfural or a derivative thereof comprising the step of: a. Biocatalytically converting at least one carbonaceous feedstock to 5- hydroxy methylfurfural or a derivative thereof.

2. A method as claimed in claim 1, wherein biocatalytic conversion in step (a) comprises enzymatic conversion.

3. A method as claimed in any preceding claim, wherein at least one carbonaceous feedstock is independently selected from the group comprising: a lignocellulosic feedstock, an oligo- or polysaccharide feedstock, a lignin feedstock, a synthetic polymer feedstock, a protein feedstock, an agricultural feedstock, and combinations thereof.

4. A method as claimed in any preceding claim, wherein the at least one carbonaceous feedstock comprises at least one ketose and/or aldose.

5. A method as claimed in claim 4, wherein the at least one ketose and/or aldose carbonaceous feedstock comprises a saccharide.

6. A method as claimed in claim 5, wherein the saccharide comprises a monosaccharide.

7. A method as claimed in any preceding claim, wherein step (a) comprises directly biocatalytically converting the at least one carbonaceous feedstock to

5 -hydroxy methylfurfural or a derivative thereof. A method as claimed in any one of claims 1 to 6, wherein step (a) comprises indirectly biocatalytically converting the at least one carbonaceous feedstock to 5 -hydroxy methylfurfural or a derivative thereof. A method as claimed in claim 8, wherein step (a) comprises converting the at least one carbonaceous feedstock to at least one intermediate species independently selected from: a ketide, a ketose, a keto acid, an aldose, and combinations thereof; and then biocatalytically converting the intermediate species to 5 -hydroxy methylfurfural or a derivative thereof. A method as claimed in claim 9, wherein the at least one intermediate species comprises GA3P or a derivative thereof and/or pyruvate or a derivative thereof. A method for the preparation of 2,5-furandicarboxylic acid comprising the steps of: a. Biocatalytically converting at least one carbonaceous feedstock to 5- hydroxymethylfurfural or a derivative thereof according to the method of any preceding claim; and b. Biocatalytically converting the 5 -hydroxy methylfurfural or derivative thereof from step (a) to produce 2,5-furandicarboxylic acid. A method as claimed in claim 11, wherein biocatalytic conversion in step (a) and/or (b) comprises enzymatic conversion. A method as claimed in claim 11 or 12, wherein step (b) comprises biocatalytically oxidising the 5 -hydroxy methylfurfural or derivative thereof from step (a) to produce 2,5-furandicarboxylic acid. A method as claimed in any one of claims 11 to 13, wherein step (b) comprises biocatalytically converting the 5-hydroxymethylfurfural or derivative thereof from step (a) to produce 2,5-furandicarboxylic acid using an oxidoreductase enzyme, preferably an HMF oxidoreductase enzyme. A method as claimed in claim 14, wherein step (b) comprises biocatalytically converting the 5-hydroxymethylfurfural or derivative thereof to produce 2,5- furandicarboxylic acid using an oxidase or dehydrogenase enzyme. A method as claimed in any preceding claim, wherein at least step (a) and preferably the entire method is performed in vivo. A method as claimed claim 16, wherein at least step (a) and preferably the entire method, is performed using a single microbial species. A method as claimed in claim 17, wherein the microbial species is an Actinomycete bacterium. A method as claimed in claim 18, wherein the microbial species is a bacterium of the Streptomyces genus. A method as claimed in claim 19, wherein the microbial species is

Streptomyces sp. S. Biosynthesis of 5 -hydroxy methylfurfural from a carbonaceous feedstock, wherein the biosynthesis is performed in a bacterium of the Streptomyces genus. Biosynthesis of 2,5-furandicarboxylic acid from a carbonaceous feedstock, wherein the biosynthesis is performed in a bacterium of the Streptomyces genus. Biosynthesis as claimed in claim 21 or 22, wherein the bacterium of the Streptomyces genus is Streptomyces sp. S. Biosynthesis as claimed in any one of claims 21 to 23, wherein the carbonaceous feedstock is independently selected from the group comprising: a lignocellulosic feedstock, a monosaccharide, an oligo- or polysaccharide feedstock, a lignin feedstock, a synthetic polymer feedstock, a protein feedstock, an agricultural feedstock, and combinations thereof. Biosynthesis of 5 -hydroxy methylfurfural from a saccharide feedstock, wherein the biosynthesis is performed in isolated Streptomyces sp. S. Biosynthesis of 2,5-furandicarboxylic acid from a saccharide feedstock, wherein the biosynthesis is performed in isolated Streptomyces sp. S. A method for the preparation of 2,5-furandicarboxylic acid comprising the steps of: a. Biocatalytically converting glucose to fructose with an isomerase enzyme; b. Biocatalytically converting the fructose to 5 -hydroxy methylfurfural with a dehydratase enzyme; and c. Biocatalytically converting the 5 -hydroxy methylfurfural to 2,5- furandicarboxylic acid with an HMF oxidoreductase. A method for the preparation of 2,5-furandicarboxylic acid comprising the steps of: a. Biocatalytically converting a carbonaceous feedstock to at least one intermediate species that is independently selected from: a keto acid, an aldose, and combinations thereof; b. Biocatalytically converting the at least one intermediate species to 5- hydroxy methylfurfural or a derivative thereof with a MfnB or MfnB homologue synthase enzyme; c. Biocatalytically converting the 5 -hydroxy methylfurfural or derivative thereof to 2,5-furandicarboxylic acid with an HMF oxidoreductase or aldehyde dehydrogenase enzyme. A method for the preparation of 2,5-furandicarboxylic acid comprising the steps of: a. Biocatalytically converting a carbonaceous feedstock to at least one ketide species, preferably independently selected from the group comprising: acetate, propionate, succinate, malonate, and combinations thereof; b. Biocatalytically converting the at least one ketide species via secondary metabolism to 5 -hydroxy methylfurfural or a derivative thereof with a polyketide synthase enzyme or a non-ribosomal peptide synthase enzyme; c. Biocatalytically converting the 5 -hydroxy methylfurfural or derivative thereof to 2,5-furandicarboxylic acid with an HMF oxidoreductase or aldehyde dehydrogenase enzyme.