Biosynthesis of plastic precursors

Abstract

The invention provides a method for preparing 5-hydroxymethylfurfural or a derivative thereof and 2, 5-furandicarboxylic acid, which comprises the following steps of: preparing 5-hydroxymethylfurfural or a derivative thereof and 2, 5-furandicarboxylic acid by using streptomyces S with the preservation number of NCIMB 43995 and an MfnB gene which is separated from the strain and is used for coding (5-formyl furan-3-yl) methyl phosphate synthase (EC 4.2. 3.153); at least one carbonaceous raw material added with glucose is biocatalytically converted into 5-hydroxymethylfurfural or a derivative thereof and 2, 5-furandicarboxylic acid through intermediates such as glyceraldehyde-3-phosphoric acid and 4-hydroxymethyl-2-furan formaldehyde phosphate.

Description

Technical Field

[0001] The present invention relates to a biosynthetic method for 5-hydroxymethylfurfural (5-HMF) and 2,5-furandicarboxylic acid (2,5-FDCA). Background Art

[0002] The production of industrial chemicals consumes more than 10% of oil and natural gas resources; this figure is expected to rise significantly as demand from the chemical industry continues to grow. The global response to the COVID-19 pandemic has accelerated the consumption of fossil-derived chemical feedstocks due to the increased production of single-use plastics.

[0003] 2,5-Furandicarboxylic acid (2,5-FDCA) is currently listed as one of 12 priority chemicals necessary for establishing a future "green" chemical industry (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). In 2017, the market value of 2,5-FDCA was $260 million and is expected to grow to $850 million by 2023. 2,5-FDCA is a potential alternative to petroleum-derived terephthalic acid (TPA), which is currently used to produce polymers such as polyesters (such as polyethylene terephthalate (PET)), polyurethanes, and polyamides. Replacing TPA with 2,5-FDCA in polymer synthesis would have a significant impact. Replacing PET with PEF (polyethylene furandicarboxylate—a copolymer of ethylene glycol and 2,5-FDCA) would significantly reduce the 60 million bottles of plastic currently landfilled and incinerated, significantly lowering the carbon footprint, including a reduction of greenhouse gas emissions by at least 50%. Furthermore, PEF's superior barrier and thermal properties make it an ideal material for a wide range of applications and a good alternative to PET. PEF also offers superior mechanical properties to PET, including a higher glass transition temperature and higher tensile modulus. Furthermore, PEF offers improved barrier properties against oxygen, carbon dioxide, and water vapor. PEF can be used in the production of water bottles, food packaging, sportswear, footwear, and more.

[0004] Currently, the production of 2,5-FDCA from biomass relies on the chemical synthesis of 5-hydroxymethylfurfural (5-HMF) from cellulose and hemicellulose under acidic conditions. This process results in furfural degradation and low 5-HMF yields (Mittal A et al., (2020) Energy Fuels 34, 3, 3284–3293). Despite efforts to optimize this process through various thermochemical catalytic strategies, the yield and selectivity of 5-HMF remain low. Subsequently, 5-HMF is primarily converted to 2,5-FDCA via chemical or electrocatalytic pathways (Yuan H et al., (2020) Appl Micro Biotech 104, 527–543). These processes share similar challenges in catalyst development, product separation, and environmental impact. Currently, the chemical production of 2,5-FDCA from 5-HMF requires the use of high temperature and pressure, metal salts (such as Co / Mn / Br / Cr), organic solvents (such as methanol / ethanol), additives (such as acetic acid), oxidants (such as KMnO4), polluting catalysts (such as Pb), and expensive catalysts (such as Pt) or nanoparticles (such as Au-CeO2 / Au-TiO2 / Au-Fe2O3). To avoid the need to isolate HMF due to its instability, a one-pot process has been developed, but its yields are not yet competitive.

[0005] 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 utilizes the HmfH enzyme (hydroxymethylfurfural oxidoreductase) originally discovered in Cupriavidus basilensis. Although a 2,5-FDCA yield of 30.6 g / L was achieved from Pseudomonas putida within 24 hours, the process still requires the addition of 5-HMF, which is currently synthesized by chemical pretreatment of biomass (Ghatta A et al., (2021) Green Chem., 23, 1716-1733). To date, the generally accepted sustainable bioproduction pathway for 2,5-FDCA is the conversion of lignocellulosic glucose into 5-HMF, which is then further converted into 2,5-FDCA.

[0006] The present invention aims to improve one or more problems in the prior art by providing a method for synthesizing 5-HMF and 2,5-FDCA. The method has one or more of the following advantages:

[0007] Sustainable and / or technically and economically viable methods;

[0008] More simplified compared to prior art methods;

[0009] Lower energy consumption compared to existing technology methods;

[0010] Allows direct production of 5-HMF and 2,5-FDCA from a single raw material (preferably readily available);

[0011] Avoid the accumulation of unstable intermediates in the reaction and their subsequent degradation;

[0012] Allowing the production of 5-HMF and 2,5-FDCA from waste raw materials;

[0013] Little or no need for harsh chemicals / catalysts;

[0014] Provides good conversion of feedstock to 5-HMF and 2,5-FDCA;

[0015] Provides good yields of 5-HMF and 2,5-FDCA; and

[0016] High selectivity.

[0017] Another object of embodiments of the present invention is to overcome or alleviate at least one problem in the prior art, whether or not explicitly disclosed herein. Summary of the Invention

[0018] According to a first aspect of the present invention, there is provided a method for preparing 5-hydroxymethylfurfural (5-HMF) or a derivative thereof, the method comprising the following steps:

[0019] (a) biocatalytically converting at least one carbonaceous feedstock into 5-hydroxymethylfurfural (5-HMF) or a derivative thereof.

[0020] According to a second aspect of the present invention, there is provided a method for preparing 2,5-furandicarboxylic acid (2,5-FDCA), the method comprising the following steps:

[0021] (a) biocatalytically converting at least one carbonaceous feedstock into 5-hydroxymethylfurfural (5-HMF) or a derivative thereof according to the method of the first aspect of the present invention; and

[0022] (b) biocatalytically converting the 5-HMF or its derivatives obtained in step (a) into 2,5-FDCA.

[0023] Step (a) of the second aspect is preferably step (a) of the first aspect of the invention.

[0024] The following statements are applicable to the first and second aspects of the present invention by analogy. A person skilled in the art can clearly determine whether a statement is applicable to the first aspect, the second aspect, or both aspects of the present invention.

[0025] Such methods allow for the direct biosynthesis of the platform chemical 5-HMF and the bioplastic monomer 2,5-FDCA from carbonaceous feedstocks, without the need to add additional raw materials to the reaction mixture during the reaction. This discovery is significant because direct bioproduction of 5-HMF and 2,5-FDCA eliminates the need for chemical pretreatment of lignocellulosic biomass, which is known to be problematic. These biosynthetic pathways for 5-HMF and 2,5-FDCA are more competitive than existing methods for synthesizing these compounds.

[0026] In a preferred embodiment, the biocatalytic conversion in step (a) and / or step (b) comprises an enzymatic conversion. In the description of the present invention, references to biocatalytic conversion hereinafter may also refer to enzymatic conversion.

[0027] In some embodiments, the at least one carbonaceous feedstock can be independently selected from the group consisting of: a lignocellulosic feedstock, an oligosaccharide or polysaccharide feedstock, a lignin feedstock, a synthetic polymer feedstock, a protein feedstock, and combinations thereof. The at least one feedstock can include a waste feedstock and / or a biomass-derived feedstock.

[0028] Lignocellulosic feedstocks may include biomass, which may include agricultural and / or urban biomass.

[0029] The agricultural biomass may include at least one starch-based feedstock.

[0030] The lignocellulosic feedstock may include lignocellulosic waste biomass, which may be independently selected from the group consisting of: straw, bagasse, miscanthus, switchgrass, reed grass, rye, rice straw, compost, wood chips, sawmill waste, paper mill waste, and combinations thereof. The lignocellulosic feedstock may preferably include lignin, hemicellulose, and cellulose. Lignin-derived lignocellulosic biomass may include, but is not limited to, at least one of: soda lignin, kraft lignin, hydrolyzed lignin, organosolv lignin, lignin sulfonate, black liquor, and combinations thereof.

[0031] At least one feedstock may include a waste biomass-derived feedstock, which may include household and / or industrial food waste, including slaughterhouse biomass (eg, feather biomass).

[0032] The oligosaccharide or polysaccharide raw material may preferably include at least one of the following: natural polysaccharides, synthetic polysaccharides, structured polysaccharides and combinations thereof. In some embodiments, the oligosaccharide and / or polysaccharide raw material includes oligosaccharides and / or polysaccharides comprising at least one glucose or glucose-derived monosaccharide unit. The oligosaccharide or polysaccharide raw material may include oligosaccharides and / or polysaccharides independently selected from the group consisting of cellulose, hemicellulose, chitin, arabinoxylan, pectin, seaweed-derived polysaccharides (including but not limited to alginate, ulva polysaccharide, carrageenan and / or fucoidan) and combinations thereof.

[0033] The synthetic polymer raw material may include at least one petroleum-based raw material and / or at least one biomass-based raw material. The synthetic polymer raw material may include at least one polymer independently selected from the group consisting of polyesters, polyethers, polyurethanes, polyamides, polystyrenes, polyolefins (polyalkenes), polyalkanes, polyhaloolefins, polyhaloalkanes (such as polyvinyl chloride), bioplastics (such as polylactic acid, aliphatic polyesters), polyepoxides, aromatic polymers, and combinations thereof. The synthetic polymer raw material may preferably include at least one polyolefin independently selected from the group consisting of polyethylene, polypropylene, and combinations thereof. The at least one polyolefin preferably includes polyethylene. The synthetic polymer raw material may include at least one polyether, the at least one polyether being a polyalkylene glycol. The synthetic polymer raw material may include at least one polyalkylene glycol independently selected from the group consisting of polyethylene glycol, polypropylene glycol, polybutylene glycol, and combinations thereof. The synthetic polymer raw material may preferably include at least one polymer independently selected from the group consisting of polyolefins, polyesters, polyalkylene glycols, and combinations thereof. The synthetic polymer raw material may include polyethylene terephthalate (PET). In some embodiments, the synthetic polymer raw material includes at least one of polyethylene terephthalate (PET), polyethylene (PE), polyether, and combinations thereof.

[0034] The protein material may include at least one fibrous protein. The at least one fibrous protein may be independently selected from the group consisting of keratin, collagen, elastin, fibrin, casein, whey, and combinations thereof.

[0035] At least one carbonaceous feedstock may be a non-sugar feedstock.

[0036] At least one carbonaceous feedstock may include a small molecule feedstock. In some embodiments, at least one carbonaceous feedstock includes an oxycarbon compound. At least one oxycarbon feedstock may include CO2 and / or CO.

[0037] At least one carbonaceous feedstock may include a polyol. The at least one polyol may be independently selected from the group consisting of diols, triols, tetraols, and combinations thereof. In some embodiments, the at least one polyol is or includes glycerol.

[0038] In some embodiments, at least one carbonaceous feedstock (which may be biologically derived and / or synthetic) may be pretreated, preferably prior to step (a) using at least one of the following methods: physical pretreatment (e.g., heat, microwaves), chemical pretreatment (e.g., acid / base, solvents), mechanical pretreatment (e.g., cryo-grinding, ultrasonic treatment), biological pretreatment (e.g., enzymatic digestion), and combinations thereof.

[0039] In some embodiments, step (a) comprises biocatalytically converting a primary and / or secondary metabolite into 5-HMF or a derivative thereof. In some embodiments, the primary and / or secondary metabolite is a carbonaceous feedstock. In some embodiments, the primary and / or secondary metabolite is an intermediate. Step (a) may comprise biocatalytically converting a carbonaceous feedstock into a primary and / or secondary metabolite intermediate, and then biocatalytically converting the intermediate into 5-HMF or a derivative thereof. In such embodiments, at least one primary and / or secondary metabolite may be independently selected from the group consisting of ketones, ketoses, keto acids, aldoses, and combinations thereof.

[0040] In this specification, references to primary / secondary metabolites also include references to tautomers, isomers, metal complexes, salts and other derivatives thereof.

[0041] In this specification, references to ketones, ketoses, keto acids and aldoses also include references to their tautomers, isomers, metal complexes, salts and other derivatives.

[0042] In some embodiments, step (a) comprises the step of biocatalytically converting at least one substance independently selected from ketones, ketoses, keto acids, aldoses, and combinations thereof to produce 5-HMF or a derivative thereof.

[0043] The substance in step (a) may include at least one ketose, and may further include at least one ketoacid and / or at least one aldose. The substance in step (a) may include at least one ketoacid, and may further include at least one ketose and / or at least one aldose. The substance in step (a) may include at least one aldose, and may further include at least one ketose and / or at least one ketoacid.

[0044] The material in step (a) may include at least one ketose and at least one keto acid. The material in step (a) may include at least one ketose and at least one aldose. The material in step (a) may include at least one keto acid and at least one aldose.

[0045] The substance in step (a) may include at least one ketose, at least one ketoacid, and at least one aldose.

[0046] In certain embodiments, the material independently selected from ketones, ketoses, keto acids, aldoses and combinations thereof is a carbonaceous feedstock. In such embodiments, the material may preferably include ketoses and / or aldoses. At least one ketose and / or aldose in step (a) may be a saccharide. The saccharide used in step (a) may preferably include monosaccharides and / or disaccharides. The saccharide used in step (a) preferably includes monosaccharides. In certain embodiments, the saccharide includes 2 or at least 2 different saccharides, 3 or at least 3 different saccharides, 4 or at least 4 different saccharides, or 5 or at least 5 different saccharides. The saccharide may include no more than 5 different saccharides, or no more than 4 different saccharides, or no more than 3 different saccharides, or no more than 2 different saccharides. In some preferred embodiments, the saccharide may include a single saccharide, preferably a monosaccharide.

[0047] The saccharide used in step (a) may include at least one monosaccharide independently selected from the group consisting of trioses, tetroses, pentoses, hexoses, heptoses, and combinations thereof. The saccharide may preferably include at least one hexose monosaccharide. The saccharide may include at least one monosaccharide independently selected from the group consisting of glucose, dextrose, fructose, levulose, galactose, and combinations thereof. The saccharide may particularly preferably be or include glucose.

[0048] In some embodiments, the substance independently selected from the group consisting of ketones, ketoses, keto acids, aldoses, and combinations thereof in step (a) is an intermediate. In such embodiments, the substance may preferably include at least one substance independently selected from ketones, keto acids, aldoses, and combinations thereof.

[0049] The at least one aldose, preferably an intermediate aldose, may include glyceraldehyde-3-phosphate (GA3P) or a derivative thereof.

[0050] The at least one substance in step (a), preferably the intermediate product, may be a ketoacid independently selected from the group consisting of α-ketoacids, β-ketoacids, γ-ketoacids, and combinations thereof. The at least one substance may be an α-ketoacid independently selected from the group consisting of pyruvate, oxaloacetate, α-ketoglutarate, succinate, dihydroxyacetone phosphate, and phosphoenolpyruvate, and combinations thereof. The at least one substance may preferably comprise pyruvate or a derivative thereof.

[0051] At least one substance in step (a), preferably an intermediate, can be a ketone. The at least one ketone can include a carboxylic acid and / or a carboxylic acid moiety. The at least one ketone can be independently selected from the group consisting of acetate, propionate, succinate, malonate, and combinations thereof.

[0052] In some embodiments, the substance in step (a) comprises GA3P or a derivative thereof and / or pyruvate or a derivative thereof.

[0053] Step (a) may comprise biocatalytically converting the carbonaceous feedstock into at least one primary and / or secondary metabolic intermediate, and then biocatalytically converting the intermediate into 5-HMF or a derivative thereof. Step (a) may comprise biocatalytically converting the carbonaceous feedstock into at least one intermediate independently selected from ketones, ketoses, keto acids, aldoses, and combinations thereof, and then biocatalytically converting the intermediate into 5-HMF or a derivative thereof.

[0054] Step (a) may comprise biocatalytically converting the carbohydrate feedstock into at least one primary and / or secondary metabolic intermediate, and then biocatalytically converting the intermediate into 5-HMF or a derivative thereof. In some embodiments, step (a) comprises biocatalytically converting the carbohydrate feedstock into an intermediate independently selected from ketones, ketoses, keto acids, aldoses, and combinations thereof, and then biocatalytically converting the intermediate into 5-HMF or a derivative thereof.

[0055] Step (a) may comprise biocatalytically converting a non-carbohydrate feedstock into a carbohydrate, and then biocatalytically converting the carbohydrate into 5-HMF or a derivative thereof. The carbohydrate may be converted to 5-HMF or a derivative thereof directly or via another intermediate, which may include at least one primary and / or secondary metabolite. The other intermediate may be independently selected from the group consisting of ketones, ketoses, keto acids, aldoses, and combinations thereof.

[0056] In preferred embodiments, the at least one feedstock is independently selected from the group consisting of a lignocellulosic feedstock, an oligosaccharide or polysaccharide feedstock, and combinations thereof. Step (a) of the method may comprise providing a lignocellulosic or oligosaccharide / polysaccharide feedstock, and biocatalytically converting the feedstock into at least one primary and / or secondary metabolite. Step (a) of the method may comprise providing a lignocellulosic or oligosaccharide / polysaccharide feedstock, and biocatalytically converting the feedstock into at least one substance independently selected from ketones, ketoses, keto acids, aldoses, and combinations thereof. The method may comprise biocatalytically converting the feedstock into a carbohydrate, which may be a monosaccharide.

[0057] In some embodiments, the primary and / or secondary metabolites in step (a) can be directly or indirectly biocatalytically converted into 5-HMF or its derivatives. In some embodiments, the ketones, ketoses, keto acids and / or aldose substances (which may include sugars) in step (a) can be directly or indirectly biocatalytically converted into 5-HMF or its derivatives. The substance (which may be a sugar) can be directly converted into 5-HMF or its derivatives without forming an intermediate. In other embodiments, the substance (which may be a sugar) can be indirectly converted into 5-HMF or its derivatives via an intermediate. The intermediate may include primary and / or secondary metabolites. The intermediate can preferably be independently selected from the group consisting of: ketones, ketoses, keto acids, aldoses and combinations thereof.

[0058] At least one of steps (a) and step (b) of the method may be carried out in the presence of a synthetic polymer feedstock, which may preferably be as described above. In some embodiments, the carbonaceous feedstock is a saccharide (which may include monosaccharides), and at least one of steps (a) and step (b) is carried out in the presence of a synthetic polymer feedstock. In a preferred embodiment, the saccharide is or includes glucose, and at least one of steps (a) and step (b) is carried out in the presence of a synthetic polymer feedstock. In some embodiments, steps (a) and (b), preferably all steps of the method, are carried out in the presence of a synthetic polymer feedstock. In such embodiments, the synthetic polymer feedstock may be as described above, and may preferably include at least one synthetic polymer independently selected from the group consisting of PET, polyethylene (PE) and combinations thereof.

[0059] In some embodiments, step (a) and / or step (b) of the method is performed in the presence of at least one polyol. The at least one polyol can be independently selected from the group consisting of diols, triols, tetraols, and combinations thereof. In some preferred embodiments, the at least one polyol is or includes glycerol.

[0060] 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 microorganism. At least one step of the method may preferably be performed using a single microorganism. Step (a) and / or step (b) may be performed using a single microorganism. In a preferred embodiment, at least step (a) and step (b), and preferably all steps of the method, are performed using a single microorganism. Preferably, the entire method is performed using a single microorganism.

[0061] In embodiments where the method comprises generating at least one primary and / or secondary metabolite from a carbonaceous feedstock as described above, the method may preferably comprise biocatalytically converting the feedstock into the metabolite in a microorganism. In embodiments where the method comprises generating at least one substance independently selected from ketones, ketoses, keto acids, aldoses, and combinations thereof from a carbonaceous feedstock as described above, the method may preferably comprise biocatalytically converting the feedstock into the substance in a microorganism. In such embodiments, the generated substance may be biocatalytically converted to 5-HMF or a derivative thereof directly after formation from the feedstock, and this step may also preferably be performed in the microorganism.

[0062] The microorganisms may be independently selected from the group consisting of bacteria, fungi, algae substrates, and combinations thereof. The microorganisms may preferably include bacteria, which may be gram-positive bacteria, gram-negative bacteria, and / or filamentous bacteria.

[0063] In some embodiments, the microorganism may include a Gram-negative bacterium. The microorganism may include a cyanobacterium. The microorganism may include a bacterium of the genus Synechocystis. The microorganism may be Synechocystis sp. PCC 6803.

[0064] The microorganism may include Proteus. The microorganism may include bacteria of the genus Pseudomonas. The microorganism may be Pseudomonas putida.

[0065] The bacteria may preferably include Gram-positive and / or filamentous bacteria. The microorganism may include actinomycetes. The microorganism may preferably include bacteria of the genus Streptomyces.

[0066] The microorganism may preferably be Streptomyces sp. S. The Streptomyces sp. S was deposited in the National Collection of Industrial Food and Marine Bacteria in Aberdeen, Scotland on June 14, 2022, with the deposit number NCIMB 43995. Streptomyces S is a bacterium in the genus Streptomyces. Streptomyces belongs to the family Actinomycetales, order Actinomycetales, and family Streptomycetaceae. Streptomyces S is a Gram-positive, aerobic, multicellular filamentous bacterium. Streptomyces S typically forms branched vegetative hyphae and produces spores. When grown on solid culture medium, colonies usually take 24 to 48 hours to appear. When grown on soy flour mannitol (SFM) medium, the initial colony morphology of Streptomyces S is a smooth beige round colony. After spore formation, the colony becomes white and hard. Yellow pigments may be produced in the colony. Streptomyces S can metabolize a variety of carbon sources, including sugars, amino acids, lipids, and natural and synthetic polymers.

[0067] In some embodiments, the microorganism can be independently selected from the group consisting of Escherichia coli, Saccharomyces, Aspergillus, Pseudomonas, Corynebactrium, Bacillus, and combinations thereof.

[0068] In some embodiments, step (a) and / or step (b) can be performed by utilizing a microbial chassis, preferably an industrial microbial chassis, for heterologous expression. The microbial chassis can be independently selected from the group consisting of Escherichia coli, yeast, Aspergillus, Pseudomonas, Corynebacterium, Bacillus, and combinations thereof.

[0069] In some embodiments, step (a) comprises incubating, growing and / or culturing a microorganism with a carbonaceous feedstock. The microorganism is preferably as described above. The microorganism may preferably include a Streptomyces bacterium, and may preferably be Streptomyces S.

[0070] In some embodiments, step (a) comprises incubating, growing and / or culturing the microorganism with the carbonaceous feedstock, preferably as described above, to produce at least one primary and / or secondary metabolite. In some embodiments, step (a) comprises incubating, growing and / or culturing the microorganism with the carbonaceous feedstock, preferably as described above, to produce at least one substance independently selected from ketones, ketoses, keto acids, aldoses and combinations thereof. The substance may include ketose and / or aldose sugars. The sugar preferably includes a monosaccharide as described above. Particularly preferably, the sugar may be or include glucose. The microorganism is preferably as described above. The microorganism may preferably include a Streptomyces bacterium, and may preferably be Streptomyces S.

[0071] In some embodiments, the method comprises incubating, growing, and / or culturing the microorganisms with the carbonaceous feedstock under aerobic conditions. In some embodiments, the method comprises incubating, growing, and / or culturing the microorganisms with the carbonaceous feedstock using a bioreactor, which may be performed under aerobic conditions. The method may comprise incubating, growing, and / or culturing the microorganisms with the feedstock under anaerobic or aerobic conditions in a batch, fed-batch, or continuous process.

[0072] The method may include incubating, growing and / or culturing the microorganism with the carbonaceous feedstock in a culture medium. The culture medium may be liquid. The culture medium may include at least one salt. The at least one salt of the culture medium may be independently selected from the group consisting of: phosphate, monohydrogen phosphate, dihydrogen phosphate, sulfate, halide (independently selected from: fluoride, chloride, bromide, iodide and combinations thereof), citrate, carbonate, molybdate, nitrate, nitrite and combinations thereof. The at least one salt of the culture medium may contain a cation independently selected from the group consisting of: alkali metal cations, alkaline earth metal cations, ammonium cations and combinations thereof. In some embodiments, the culture medium is or includes at least one culture medium independently selected from the group consisting of: M9 medium, supplemented liquid minimal medium (SMM), basic minimal medium (BMS), minimal liquid medium (NMMP), minimal medium (MM) and combinations thereof.

[0073] The method can include incubating, growing and / or culturing the microorganisms with the carbonaceous feedstock in a culture medium at a total feedstock concentration of at least 0.5 g / L, or at least 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 at least 10 g / L. The method can include incubating, growing and / or culturing the microorganisms with the carbonaceous feedstock in a culture medium at a total feedstock concentration of no more than 100 g / L, or no more than 90 g / L, 80 g / L, 70 g / L, 60 g / L, 50 g / L, 45 g / L, 40 g / L, 35 g / L, 30 g / L, or no more than 25 g / L. The method can include incubating, growing and / or culturing the microorganism with the carbonaceous feedstock in a culture medium at a total feedstock concentration of between 1-100 g / L, or between 1-50 g / L, or between 2-40 g / L, 5-30 g / L, 5-25 g / L, or between 7-22 g / L. The method can include incubating, growing and / or culturing the microorganism with the feedstock in a culture medium at a total feedstock concentration of between 50-100 g / L.

[0074] The method may include incubating, growing and / or culturing the microorganism with at least one carbonaceous feedstock at a temperature of at least 10°C, or at least 15°C, 20°C, or at least 25°C. The method may include incubating, growing and / or culturing the microorganism with at least one carbonaceous feedstock at a temperature of no more than 60°C, or no more than 50°C, 40°C, or no more than 35°C. The method may include incubating, growing and / or culturing the microorganism with at least one carbonaceous feedstock at a temperature between 10-50°C, or between 15-45°C, or preferably between 20-40°C, or between 25-35°C. In some embodiments, the entire method may be carried out at the above-mentioned temperatures.

[0075] The method may include incubating, growing and / or culturing the microorganism 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 include incubating, growing and / or culturing the microorganism with the carbonaceous feedstock at a pH of no more than 10, or no more than 9, or no more than 8. The method may include incubating, growing and / or culturing the microorganism with the carbonaceous feedstock at a pH of between 4-10, or between 5-9, or between 6-8, or about 7. In some embodiments, the entire method may be carried out at the above-mentioned pH.

[0076] In some embodiments, the method comprises incubating, growing and / or culturing the microorganism with the carbonaceous feedstock, preferably in a culture medium, for a total of at least 5 hours, or at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or at least 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 140 hours, or at least 160 hours, 170 hours, 180 hours, 190 hours, or at least 200 hours, or at least 250 hours, 300 hours, 350 hours, 400, 450 hours, 500 hours, 550 hours, 600 hours, 650 hours, 700 hours, or at least 750 hours. The method can include hatching, growing and / or cultivating microorganism and carbonaceous feedstock (preferably in substratum) and be no more than 1000 hours, or be no more than 950 hours, 900 hours, 850 hours, 800 hours, 750 hours, or be no more than 700 hours, 650 hours, 600 hours, 550 hours, 500 hours, 450 hours, 400 hours, or be no more than the total duration of 350 hours. The method can include hatching, growing and / or cultivating microorganism and carbonaceous feedstock (preferably in substratum) and be no more than 10-800 hours, or between 15-750 hours, 25-525 hours, or between 50-500 hours, 100-450 hours, 150-400 hours, or the total duration between 200-350 hours.

[0077] The method may include incubating, growing and / or cultivating the microorganism and carbonaceous feedstock in a culture medium to form a reaction mixture. The method may include the step of stirring the reaction mixture. The method may include stirring the mixture during at least part of step (a), preferably during the entire step (a). The method may include stirring the mixture during both step (a) and step (b). The method may include stirring the mixture during the entire method. The method may include stirring the reaction mixture at a stirring speed of at least 70rpm, or at least 80rpm, 90rpm, 100rpm, 110rpm, 120rpm, 130rpm, or at least 140rpm, 150rpm, 200rpm, 250rpm, 300rpm, 350rpm, or at least 400rpm. The method can include stirring the reaction mixture at a speed of no more than 1500rpm, or no more than 1400rpm, 1300rpm, 1200rpm, 1100rpm, 1000rpm, 900rpm, 800rpm, 700rpm, 600rpm, 500rpm, 400rpm, or no more than 350rpm, 300rpm, 250rpm, 200rpm, or no more than 190rpm, 180rpm, 170rpm, or no more than 160rpm. The method can include stirring the reaction mixture at a speed of no more than 160rpm, or no more than 70-230rpm, 80-220rpm, 90-210rpm, 100-200rpm, 110-190rpm, 120-180rpm, 130-170rpm, or 140-160rpm. The method may comprise stirring the reaction mixture at a speed between 100-1500 rpm, or between 250-1200, or between 400-1000 rpm.

[0078] At least step (a), preferably the entire method can be carried out at a ventilation rate of at least 0.1 vvm, or at least 0.2 vvm, 0.3 vvm, or at least 0.4 vvm, or at least 0.5 vvm, 1 vvm, 1.5 vvm, 2 vvm, 2.5 vvm, 3 vvm, 3.5 vvm, 4 vvm, or at least 4.5 vvm. At least step (a), preferably the entire method can be carried out at a ventilation rate of no more than 5 vvm, or no more than 4 vvm, 3 vvm, 2 vvm, 1 vvm, 0.9 vvm, 0.8 vvm, 0.7 vvm, or no more than 0.6 vvm.

[0079] At least step (a), preferably the entire method can be carried out at an aeration rate of between 0.1-5 vvm, 0.1-4 vvm, 0.1-3 vvm, 0.1-2 vvm, 0.1-1 vvm, 0.1-0.9 vvm, or between 0.2-0.8 vvm, 0.3-0.7 vvm, or between 0.4-0.6 vvm. At least step (a), preferably the entire method can be carried out at an aeration rate of between 0.5-5 vvm, 1-2.5 vvm, 2-4 vvm, or between 4-5 vvm.

[0080] In some embodiments, the method comprises incubating, growing and / or culturing the microorganism with the carbonaceous feedstock in a bioreactor. In some embodiments, the entire method is performed in a bioreactor.

[0081] In some embodiments, the method comprises incubating, growing, and / or culturing a microorganism with a carbonaceous feedstock in a batch, fed-batch, or continuous bioreactor.

[0082] In some embodiments, the method comprises adding a culture medium, preferably as described above, to the bioreactor prior to adding the carbonaceous feedstock and the microorganisms. In some embodiments, the method comprises adding the carbonaceous feedstock to the bioreactor prior to adding the microorganisms.

[0083] In some embodiments, the method may include sterilizing the bioreactor before adding the carbonaceous feedstock. The bioreactor may be sterilized by autoclaving. The bioreactor may be sterilized at a temperature between 80-160°C, or between 90-150°C, 100-140°C, or between 110-130°C. Sterilization may also be achieved by steam, chemical treatment, heating or radiation. The sterilization time of the bioreactor may be between 5-60 minutes, or between 10-40 minutes, or between 15-30 minutes. The bioreactor may be sterilized after adding the culture medium.

[0084] In some embodiments, the carbonaceous feedstock may be sterilized before being added to the bioreactor. The carbonaceous feedstock may be sterilized by autoclaving, steam, heating, radiation, or chemical treatment. Statements regarding bioreactor sterilization also apply to the sterilization of the carbonaceous feedstock.

[0085] In some embodiments, the carbonaceous feedstock may be added as at least one culture independently selected from the group consisting of a batch culture, a fed-batch culture, a continuous culture, and combinations thereof. In some embodiments, a single carbonaceous feedstock may be added. In other embodiments, more than one carbonaceous feedstock may be added. The single or each carbonaceous feedstock may be added separately. In some embodiments, more than one carbonaceous feedstock may be added simultaneously.

[0086] In some embodiments, the microorganism is added to the bioreactor as part of an inoculum (sometimes referred to as a starter culture or inoculum culture). The inoculum may be a liquid inoculum. The inoculum may include at least one monosaccharide, which may be as described above. The at least one monosaccharide may be glucose. The total concentration of monosaccharides in the inoculum may be between 0.5-10 g / L, or between 1-8 g / L, or between 10-100 g / L. In some embodiments, the inoculum may include at least one carbonaceous feedstock. In some embodiments, the inoculum includes at least one yeast extract, and the total concentration of the at least one yeast extract may be as described above. In some embodiments, the inoculum includes at least one malt extract, and the total concentration of the at least one malt extract may be between 1-20 g / L, or between 5-15 g / L. In some embodiments, spores of the microorganism may be added to the bioreactor, preferably as part of the inoculum. The inoculum may include one or more of the following: spores, germinating cells, dormant cells, living cells, and combinations thereof.

[0087] In embodiments where the method comprises generating at least one primary and / or secondary metabolite from a carbonaceous feedstock (which may be microbial degradation of a carbonaceous feedstock), the biocatalytic conversion of the metabolite to 5-HMF or a derivative thereof may occur directly and / or spontaneously following the generation of the metabolite.

[0088] In embodiments where the method comprises generating a substance independently selected from ketones, ketoses, keto acids, aldoses, and combinations thereof from a carbonaceous feedstock (which may be microbial degradation of a carbonaceous feedstock), biocatalytic conversion of the substance to 5-HMF or a derivative thereof may occur directly and / or spontaneously after the substance is generated.

[0089] In some embodiments, step (b) is performed directly and / or spontaneously after the production of 5-HMF or a derivative thereof in step (a). In some embodiments, steps (a) and (b) are performed under the same conditions. In a preferred embodiment, the entire method comprises a single biocatalytic process. At least steps (a) and (b), and preferably the entire method, can be performed as a one-pot process.

[0090] In some embodiments, the method comprises forming 5-HMF (or its derivatives) and / or 2,5-FDCA by direct conversion from a carbonaceous feedstock. The method may comprise forming 5-HMF (or its derivatives) and / or 2,5-FDCA by direct conversion from microbial degradation of a carbonaceous feedstock. The carbonaceous feedstock may comprise primary and / or secondary metabolites. The carbonaceous feedstock may comprise substances independently selected from ketones, ketoses, keto acids, aldoses, and combinations thereof. The method may comprise forming 5-HMF (or its derivatives) and / or 2,5-FDCA by direct conversion from ketoses and / or aldoses (which may be sugars, preferably as described above).

[0091] In some embodiments, the method comprises forming 5-HMF (or a derivative thereof) and / or 2,5-FDCA by indirect conversion from a carbonaceous feedstock. The method may comprise forming 5-HMF (or a derivative thereof) and / or 2,5-FDCA by indirect conversion from microbial degradation of a carbonaceous feedstock.

[0092] In some embodiments, step (a) comprises indirectly biocatalytically converting the carbonaceous feedstock to 5-HMF or a derivative thereof via primary and / or secondary metabolic intermediates. In some embodiments, step (a) comprises indirectly biocatalytically converting the carbonaceous feedstock to 5-HMF or a derivative thereof via an intermediate independently selected from the group consisting of ketones, ketoses, keto acids, aldoses, and combinations thereof. In some embodiments, step (a) comprises indirectly converting the carbonaceous feedstock to 5-HMF or a derivative thereof via a phosphoric acid intermediate.

[0093] The method may include forming 5-HMF (or its derivatives) and / or 2,5-FDCA by microbial secondary metabolism. The method may include forming 5-HMF (or its derivatives) and / or 2,5-FDCA from the microbial secondary metabolism of the carbonaceous feedstock. The method may include forming 5-HMF (or its derivatives) and / or 2,5-FDCA by direct and / or spontaneous microbial secondary metabolism of the carbonaceous feedstock (which may be produced by microbial degradation of the carbonaceous feedstock). The method may include incubating, growing and / or culturing the microorganism with the carbonaceous feedstock to induce microbial secondary metabolism. In some embodiments, the feedstock may include at least one substance independently selected from the group consisting of ketones, ketoses, keto acids and aldoses. The carbonaceous feedstock may be ketoses and / or aldoses, and the ketoses and / or aldoses may be sugars as described above.

[0094] In embodiments where the method comprises forming 5-HMF and / or 2,5-FDCA from microbial secondary metabolism of a carbonaceous feedstock (which may result from microbial degradation of the carbonaceous feedstock), the carbonaceous feedstock comprises at least one substance independently selected from ketones, ketoses, keto acids, aldoses, and combinations thereof, which may be formed from a carbonaceous feedstock precursor.

[0095] In embodiments where the method comprises forming 5-HMF and / or 2,5-FDCA from microbial secondary metabolism of a carbonaceous feedstock, the method may be performed by a polyketide synthase (PKS). The method may comprise converting the carbonaceous feedstock into 5-HMF and / or 2,5-FDCA via at least one ketone. The ketone may be independently selected from the group consisting of acetate, propionate, succinate, malonate, and combinations thereof. The method may comprise converting the carbonaceous feedstock into 2,5-FDCA via 2-oxoadipate. The method may comprise the step of converting the at least one ketone into 2-oxoadipate using a PKS enzyme. The method may further comprise the step of converting 2-oxoadipate into 2,5-FDCA using a hydratase and / or an isomerase, which may be an enoyl-CoA hydratase and / or an enoyl-CoA isomerase.

[0096] In embodiments where the method comprises forming 5-HMF and / or 2,5-FDCA from microbial secondary metabolism of a carbonaceous feedstock, the method can be performed by a non-ribosomal peptide synthase (NRPS).

[0097] At least one substance independently selected from the group consisting of the following can be a primary metabolic intermediate: ketones, ketose, keto acid, aldose and a combination thereof. This primary metabolic intermediate can be formed from the degradation of carbonaceous feedstock, preferably from the biodegradation of carbonaceous feedstock, and this carbonaceous feedstock can be a biological carbonaceous feedstock or a synthetic carbonaceous feedstock. In certain embodiments, step (a) includes biocatalytically converting the carbonaceous feedstock into a primary metabolic intermediate, and then biocatalytically converting this intermediate into 5-HMF or a derivative thereof. This carbonaceous feedstock can be a pretreated carbonaceous feedstock (can be a biological feedstock or a synthetic feedstock). This carbonaceous feedstock can be as described above.

[0098] In some embodiments, 5-HMF (or its derivatives) and / or 2,5-FDCA can be produced by secondary metabolism derived from primary metabolism. The primary metabolism can be derived from the biodegradation and / or metabolism of a carbonaceous feedstock (which can be a biological feedstock or a synthetic feedstock). The carbonaceous feedstock can be a pretreated carbonaceous feedstock (which can be a biological feedstock or a synthetic feedstock). The primary metabolism can result in the production of at least one substance independently selected from ketones, ketoses, keto acids, aldoses, and combinations thereof.

[0099] In some embodiments, 5-HMF (or a derivative thereof) and / or 2,5-FDCA can be produced by secondary metabolism derived from the activity of at least one enzyme. The at least one enzyme can be a synthase. The at least one enzyme can be independently selected from the group consisting of a polyketide synthase (PKS), a non-ribosomal peptide synthase (NRPS), a PKS-NRPS hybrid enzyme, an NRPS-PKS hybrid enzyme, and combinations thereof.

[0100] In some embodiments, the activity of at least one enzyme, preferably at least one PKS enzyme and / or NRPS enzyme, can be generated by the condensation of at least one starting unit. The at least one starting unit can be independently selected from the group consisting of ketones (which can be independently selected from the group consisting of acetate, propionate, succinate, malonate, and combinations thereof), aromatic units, peptide units, carboxylates (which can be cyclic esters), hydrocarbons (which can be independently selected from alkanes, alkenes, alkynes, and combinations thereof), alcohols, carboxylic acids, aldehydes, and combinations thereof.

[0101] In some embodiments, the biocatalytic conversion of the carbonaceous feedstock to 5-HMF or its derivatives in step (a) may involve the use of an aldolase and / or synthase. Step (a) may include biocatalytically converting the feedstock into at least one substance independently selected from ketones, ketoses, keto acids, aldoses, and combinations thereof, and then biocatalytically converting the substance into 5-HMF or its derivatives using an aldolase and / or synthase. The substance may include an aldose and / or a keto acid. The carbonaceous feedstock may include a sugar, and the aldose and / or keto acids may be formed by fermentation of the sugar. In other embodiments, the feedstock may include a feedstock independently selected from the group consisting of a lignocellulosic feedstock, an oligosaccharide or polysaccharide feedstock, a lignin feedstock, a synthetic polymer feedstock, a protein feedstock, an agricultural feedstock, and combinations thereof. The feedstock may preferably be as described above. In some embodiments, the feedstock may include cellulose and / or starch. In some embodiments, the substance may include glyceraldehyde-3-phosphate and / or pyruvate, or a derivative thereof. In some embodiments, glyceraldehyde-3-phosphate and / or pyruvate, or a derivative thereof, may be converted into 5-HMF or its derivatives using an aldolase and / or synthase. The above-mentioned synthase may preferably be as described below in the present invention.

[0102] In some embodiments, step (a) comprises condensing two aldose molecules to form 5-HMF or a derivative thereof. Step (a) may comprise condensing a GA3P molecule with another substance to form 5-HMF or a derivative thereof. Step (a) may comprise condensing a GA3P molecule with another substance, the other substance being a primary metabolite or a secondary metabolite. Step (a) may comprise condensing a GA3P molecule with another substance independently selected from the group consisting of ketones, ketoses, keto acids, and aldoses. Step (a) may comprise condensing two GA3P molecules to form 5-HMF or a derivative thereof. The condensation reaction may preferably be catalyzed by a synthase. The condensation reaction may preferably be catalyzed by a synthase independently selected from the group consisting of a lyase, an aldolase, a cyclase, and combinations thereof. In specific embodiments, the 5-HMF derivative is 4-(hydroxymethyl)-2-furfural phosphate (4-HFC-P). In some embodiments, the synthase may comprise 4-HFC-P synthase (MfnB) or a homolog thereof. Such synthases are believed to have a more open active site that facilitates the selective formation of 5-HMF over other isomers. In some embodiments, the synthase may comprise 4-HFC-P synthase (MfnB) or a homolog thereof, and have an E-value of 0.0 or less using a standard 4-HFC-P synthase (MfnB) amino acid or nucleotide sequence. In some embodiments, the synthase may comprise 4-HFC-P synthase (MfnB) or a homolog thereof, and have an E-value of 1e-150 or greater using a standard 4-HFC-P synthase (MfnB) amino acid or nucleotide sequence. In some embodiments, the synthase may comprise 4-HFC-P synthase (MfnB) or a homolog thereof, and have an E-value of 1e-80 or greater using a standard 4-HFC-P synthase (MfnB) amino acid or nucleotide sequence. In some embodiments, the synthase may comprise 4-HFC-P synthase (MfnB) or a homolog thereof, and having an E-value of 1e-50 or greater using a standard 4-HFC-P synthase (MfnB) amino acid or nucleotide sequence. In some embodiments, the synthase may comprise 4-HFC-P synthase (MfnB) or a homolog thereof, and having an E-value of 1e-20 or greater using a standard 4-HFC-P synthase (MfnB) amino acid or nucleotide sequence. In some embodiments, the synthase may comprise 4-HFC-P synthase (MfnB) or a homolog thereof, and the homolog may comprise DUF556 protein and / or UPF0264 protein. In some embodiments, the synthase may comprise 4-HFC-P synthase (MfnB) or a homolog thereof identified by a standard homology search using a standard 4-HFC-P synthase (MfnB) amino acid or nucleotide sequence.In some embodiments, the synthase comprises a 4-HFC-P (MfnB) homolog having an amino acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 1, or at least 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 1. In some embodiments, the synthase comprises a 4-HFC-P (MfnB) homolog having an amino acid sequence that is 100% identical to the sequence set forth in SEQ ID NO: 1 (amino acid sequence of MfnB from Streptomyces sp.). Step (a) may further comprise the step of biocatalytically converting a 5-HMF derivative, preferably 4-HFC-P, into 5-HMF.

[0103] In some embodiments, step (a) comprises forming 5-HMF or a derivative thereof from at least one ketoacid and at least one aldose. Step (a) may comprise forming 5-HMF or a derivative thereof using GA3P and at least one ketoacid, preferably pyruvate, or a derivative thereof. The at least one ketoacid may be a glycolytic intermediate. Step (a) may preferably comprise forming 5-HMF or a derivative thereof using a synthase, preferably MfnB or a homolog thereof.

[0104] In some embodiments, step (a) comprises biocatalytically dehydrating a carbonaceous feedstock to produce 5-HMF or a derivative thereof, wherein the carbonaceous feedstock may be a sugar, and the sugar may be as described above.

[0105] Step (a) may comprise biocatalytically converting a carbonaceous feedstock (which may comprise a saccharide, preferably glucose) to produce 5-HMF or a derivative thereof through an intermediate, wherein the intermediate comprises fructose or a derivative thereof. Step (a) may comprise the step of converting the carbonaceous feedstock (preferably a saccharide, preferably glucose) into fructose or a derivative thereof, preferably through an isomerization reaction. Step (a) may comprise converting the carbonaceous feedstock (preferably a saccharide, preferably glucose) into fructose or a derivative thereof using an isomerase. Step (a) may comprise biocatalytically converting the carbonaceous feedstock (preferably a saccharide, preferably glucose) into fructose or a derivative thereof, and then biocatalytically converting the fructose or a derivative thereof into 5-HMF or a derivative thereof.

[0106] In some embodiments, the biocatalytic conversion of the carbonaceous feedstock (which may be a saccharide) to 5-HMF or its derivatives in step (a) may involve the use of a dehydratase. The dehydratase may include a monosaccharide dehydratase. Step (a) may include isomerizing the feedstock (preferably a saccharide, preferably glucose) to fructose or its derivatives, preferably using an isomerase, and then converting the fructose or its derivatives to 5-HMF or its derivatives using a dehydratase.

[0107] Step (a) may include isomerizing a feedstock (preferably a carbohydrate feedstock, preferably glucose) to fructose or a derivative thereof, preferably using an isomerase, and then converting the fructose or a derivative thereof to glyceraldehyde-3-phosphate (GA3P) or a derivative thereof using an aldolase. In such embodiments, the fructose derivative may be a fructose phosphate derivative, which may include fructose-1,6-diphosphate and / or 4-(hydroxymethyl)-2-furaldehyde phosphate (4-HFC-P). The fructose phosphate derivative may be formed by treating the feedstock (preferably a carbohydrate, preferably glucose) with a kinase prior to isomerization to the fructose phosphate derivative. Alternatively, the fructose phosphate derivative may be formed by treating fructose with a kinase. Step (a) may further include converting GA3P or a derivative thereof to 5-HMF or a derivative thereof using a synthase. The step of converting GA3P or a derivative thereof to 5-HMF or a derivative thereof may involve the condensation of two GA3P molecules. Step (a) may comprise isomerizing a feedstock (preferably a saccharide, preferably glucose) to fructose or a derivative thereof, preferably using an isomerase, followed by converting the fructose or a derivative thereof to glyceraldehyde-3-phosphate (GA3P) or a derivative thereof using an aldolase, and then converting the GA3P or a derivative thereof to 5-HMF or a derivative thereof using a synthase.

[0108] In some embodiments, the biocatalytic conversion of the feedstock into 5-HMF or its derivatives in step (a) can be performed via a protocatechuic acid intermediate or its derivatives. The protocatechuic acid or its derivatives can be formed from a feedstock (preferably a carbohydrate feedstock) via a shikimate pathway, preferably via a 4-hydroxybenzoic acid intermediate or its derivatives. In such embodiments, step (a) can produce a 5-HMF derivative, preferably a carboxylic acid derivative, more preferably 5-hydroxymethyl-2-furoic acid. In some embodiments, step (a) comprises forming a carboxylic acid-type 5-HMF derivative from protocatechuic acid or its derivatives using at least one dioxygenase, hydroxylase, isomerase, and decarboxylase, and optionally at least one dehydratase. In such embodiments, step (b) of the method can comprise biocatalytically converting the carboxylic acid-type 5-HMF derivative to 2,5-FDCA using at least one dehydrogenase.

[0109] In some embodiments, step (a) provides a 5-HMF derivative. The 5-HMF derivative may include an alkylated 5-HMF derivative, which may be a methylated 5-HMF derivative, such as methoxymethylfurfural. In some embodiments, the 5-HMF derivative may include 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, which is independently selected from the group consisting of a methylated 5-HMF derivative, a carboxylic acid-type 5-HMF derivative, and combinations thereof.

[0110] In some embodiments, step (b) comprises biocatalytically oxidizing 5-HMF or a derivative thereof obtained from step (a) to produce 2,5-FDCA.

[0111] In some embodiments, step (b) comprises biocatalytically converting 5-HMF or a derivative thereof obtained in step (a) to 2,5-FDCA using an oxidoreductase, preferably an HMF oxidoreductase.

[0112] Step (b) may comprise biocatalytically converting 5-HMF or a derivative thereof to 2,5-FDCA using an oxidase or a dehydrogenase.

[0113] In some embodiments, step (b) comprises biocatalytically converting 5-HMF or a derivative thereof to 2,5-FDCA using HMF oxidase (HmfH). In such embodiments, the 5-HMF or a derivative thereof can be converted to 2,5-FDCA using an oxidase and molecular oxygen.

[0114] In some embodiments, step (b) comprises biocatalytically converting 5-HMF or a derivative thereof to 2,5-FDCA using an aldehyde dehydrogenase (AldH), preferably an HMF dehydrogenase. In such embodiments, the 5-HMF or a derivative thereof can be converted to 2,5-FDCA via a 5-formyl-2-furoic acid (FFA) intermediate or a derivative thereof.

[0115] In some embodiments, step (a) comprises biocatalytically converting a carbonaceous feedstock (preferably a sugar, preferably glucose) to produce 5-HMF through an intermediate, wherein the intermediate comprises fructose or a derivative thereof; and step (b) comprises biocatalytically converting the 5-HMF or a derivative thereof to produce 2,5-FDCA using an oxidase or a dehydrogenase.

[0116] In some embodiments, step (a) comprises biocatalytically isomerizing a carbonaceous feedstock (preferably a sugar, preferably glucose) to fructose or a derivative thereof, preferably using an isomerase, and then converting the fructose or a derivative thereof to 5-HMF or a derivative thereof using a dehydratase; and step (b) comprises biocatalytically converting the 5-HMF or a derivative thereof to 2,5-FDCA using an oxidase or a dehydrogenase.

[0117] In some embodiments, step (a) comprises isomerizing a carbonaceous feedstock (preferably a sugar, more preferably glucose) to fructose or a derivative thereof by biocatalysis (preferably using an isomerase), converting the fructose or a derivative thereof to glyceraldehyde-3-phosphate (GA3P) or a derivative thereof using an aldolase, and then converting the GA3P or a derivative thereof to 5-HMF or a derivative thereof using a synthase; and step (b) comprises biocatalytically converting 5-HMF or a derivative thereof to 2,5-FDCA using an oxidase or a dehydrogenase.

[0118] The enzymes are preferably derived from the aforementioned microorganisms. These enzymes may be isolated enzymes. The microorganism may preferably include an actinomycete species, which may include a Streptomyces species. The microorganism may preferably be Streptomyces S. Streptomyces S was deposited with the National Collection of Industrial Food and Marine Bacteria in Aberdeen, Scotland on June 14, 2022, with the accession number NCIMB43995.

[0119] According to a third aspect of the present invention, there is provided a method for biosynthesizing 5-hydroxymethylfurfural (5-HMF) from a carbonaceous feedstock, wherein the biosynthesis is carried out in a bacterium of the genus Streptomyces.

[0120] The biosynthesis according to the third aspect of the present invention can be carried out by the method according to the first aspect of the present invention. The statements made in relation to the first aspect of the present invention also apply mutatis mutandis to the third aspect of the present invention.

[0121] According to a fourth aspect of the present invention, there is provided a method for biosynthesizing 2,5-furandicarboxylic acid (2,5-FDCA) from a carbonaceous feedstock, wherein the biosynthesis is carried out in a bacterium of the genus Streptomyces.

[0122] The biosynthesis according to the fourth aspect of the invention may be carried out by the method according to the second aspect of the invention. The statements made in relation to the first and second aspects of the invention also apply mutatis mutandis to the fourth aspect of the invention.

[0123] The following statements apply mutatis mutandis to the third and fourth aspects of the present invention.

[0124] The carbonaceous feedstock can be independently selected from the group consisting of: lignocellulosic feedstock, monosaccharide, oligosaccharide or polysaccharide feedstock, lignin feedstock, synthetic polymer feedstock, protein feedstock, agricultural feedstock and combinations thereof. The carbonaceous feedstock can also be subjected to at least one pretreatment, which can be as described in the first and second aspects of the present invention.

[0125] The carbonaceous feedstock may preferably be the carbonaceous feedstock of the first aspect of the present invention. The biosynthesis may include biocatalytic conversion of the carbonaceous feedstock into 5-HMF and / or 2,5-FDCA via an intermediate. The intermediate may include primary and / or secondary metabolites. The intermediate may be independently selected from the group consisting of ketones, ketoses, keto acids, aldoses, and combinations thereof. The intermediate may preferably be as described in the first and second aspects of the present invention.

[0126] The carbonaceous feedstock may include primary and / or secondary metabolites. The carbonaceous feedstock may include substances independently selected from ketones, ketoses, keto acids, aldoses and combinations thereof. The carbonaceous feedstock may include sugars, preferably as described in the first and second aspects of the present invention.

[0127] The carbonaceous feedstock may preferably be the carbonaceous feedstock of the first aspect of the present invention. Biosynthesis may include direct biocatalytic conversion of the carbonaceous feedstock into 5-HMF (or its derivatives) and / or 2,5-FDCA. The production of 5-HMF (or its derivatives) and 2,5-FDCA may be carried out by microbial secondary metabolism of the carbonaceous feedstock.

[0128] In some embodiments, the carbonaceous feedstock comprises a pretreated carbonaceous feedstock, preferably as described in the first and second aspects of the present invention.

[0129] The Streptomyces bacterium is preferably Streptomyces S. The Streptomyces S was deposited in the National Collection of Industrial Food and Marine Bacteria in Aberdeen, Scotland on June 14, 2022, with the deposit number being NCIMB 43995.

[0130] According to a fifth aspect of the present invention, there is provided a method for biosynthesizing 5-hydroxymethylfurfural (5-HMF) or a derivative thereof from a carbohydrate raw material, wherein the biosynthesis is carried out in an isolated Streptomyces sp.

[0131] The biosynthesis according to the fifth aspect of the present invention can be carried out by the method according to the first aspect of the present invention. The above statements on the aforementioned aspects of the present invention also apply mutatis mutandis to the fifth aspect of the present invention.

[0132] The biosynthesis according to the fifth aspect of the invention may be the biosynthesis according to the third aspect of the invention.

[0133] The above statements in relation to any of the preceding aspects of the invention also apply mutatis mutandis to the fifth aspect of the invention.

[0134] According to a sixth aspect of the present invention, there is provided a method for biosynthesizing 2,5-furandicarboxylic acid (2,5-FDCA) from a carbohydrate feedstock, wherein the biosynthesis is carried out in an isolated Streptomyces sp.

[0135] The biosynthesis according to the sixth aspect of the present invention can be carried out by the method according to the second aspect of the present invention. The above statements on the aforementioned aspects of the present invention also apply mutatis mutandis to the sixth aspect of the present invention.

[0136] The biosynthesis according to the sixth aspect of the invention may be the biosynthesis according to the fourth aspect of the invention.

[0137] The above statements in relation to any of the preceding aspects of the invention also apply mutatis mutandis to the sixth aspect of the invention.

[0138] The following statements apply to the fifth and sixth aspects of the invention.

[0139] The carbohydrate is preferably as described above in relation to the first and second aspects of the invention.

[0140] Biosynthesis can include biocatalytic conversion of carbohydrates to 5-HMF or 2,5-FDCA via microbial secondary metabolism. In some embodiments, biosynthesis can include biocatalytic conversion of carbohydrates to 5-HMF or 2,5-FDCA via at least one intermediate independently selected from ketones, ketoses, keto acids, aldoses, and combinations thereof. The intermediate can be generated by glycolysis and can include keto acids and / or aldoses.

[0141] According to a seventh aspect of the present invention, there is provided a method for preparing 2,5-furandicarboxylic acid (2,5-FDCA), the method comprising the following steps:

[0142] (a) Biocatalytic conversion of glucose into fructose using isomerase;

[0143] (b) biocatalytically converting the fructose into 5-hydroxymethylfurfural (5-HMF) using a dehydratase;

[0144] (c) The 5-HMF was biocatalytically converted into 2,5-FDCA using HMF oxidoreductase.

[0145] According to an eighth aspect of the present invention, there is provided a method for preparing 2,5-furandicarboxylic acid, the method comprising the following steps:

[0146] (a) biocatalytically converting a carbonaceous feedstock into at least one intermediate product independently selected from the group consisting of: keto acids, aldoses, and combinations thereof;

[0147] (b) biocatalytically converting the at least one intermediate into 5-hydroxymethylfurfural or a derivative thereof using MfnB or an MfnB homologous synthase;

[0148] (c) biocatalytically converting the 5-hydroxymethylfurfural or its derivatives into 2,5-furandicarboxylic acid using HMF oxidoreductase or aldehyde dehydrogenase.

[0149] According to a ninth aspect of the present invention, there is provided a method for preparing 2,5-furandicarboxylic acid, the method comprising the following steps:

[0150] (a) biocatalytically converting a carbonaceous feedstock into at least one ketone species, the at least one ketone species preferably being independently selected from the group consisting of acetate, propionate, succinate, malonate, and combinations thereof;

[0151] (b) biocatalytically converting the at least one ketone substance into 5-hydroxymethylfurfural or a derivative thereof by secondary metabolism using a polyketide synthase or a non-ribosomal peptide synthase;

[0152] (c) biocatalytically converting the 5-hydroxymethylfurfural or its derivatives into 2,5-furandicarboxylic acid using HMF oxidoreductase or aldehyde dehydrogenase.

[0153] The methods according to the seventh, eighth and ninth aspects of the present invention are preferably methods according to the second aspect of the present invention. The above statements regarding the second aspect of the present invention also apply mutatis mutandis to the seventh, eighth and ninth aspects of the present invention.

[0154] The other statements above in relation to any of the aforementioned aspects of the invention also apply mutatis mutandis to the seventh, eighth and ninth aspects of the invention.

[0155] Sequence Listing

[0156] SEQ ID NO: 1 (MfnB of Streptomyces S)

[0157] MRWKESTLLLLISPDGVEEALECAKAAEHLDIVDVKKPDEGSLGANFPWVIREIRGAVPADKPVSATVGDVPFKPGTVAQAALGAAVSGATYIKVGLYGCTTPDQAIEVMRGVVRAVKDYRPDAFVVA SGYADAHRIGCVNPLALPDIARRSGSDAAMLDTAIKDGTRLFDHVPPEACGEFVRLAHEAGLLAALAGSVKSADLATLTRIGTDIVGVRGAVCEGGDRNKGRIQPRLVADFRAEMDRHAREHAATLAAS DETAILED DESCRIPTION

[0158] To make the present invention easier to understand, embodiments of the present invention are now described by way of examples with reference to the accompanying drawings, in which:

[0159] Figure 1Figure 2 shows the growth of Streptomyces sp. on glucose substrate, based on colony forming units (CFU) determination. Data are from three biological replicates.

[0160] Figure 2 Shown are the results of C18 reverse-phase HPLC analysis of the following samples: (A): High-purity 2,5-FDCA standard; (B)-(G): Streptomyces S supernatants collected at 0 hours (B), 72 hours (C), 120 hours (D), 168 hours (E), 240 hours (F), and 336 hours (G) during the biosynthesis of 2,5-FDCA from glucose. The results show that a peak corresponding to the high-purity 2,5-FDCA standard appeared in the reaction mixture after 120 hours.

[0161] Figure 3 Shown are the 2,5-FDCA production (A and B) and 5-HMF production (C and D) at different time points in the whole cell pellet (WC) (A and C) and supernatant extract (Sup) (B and D) of Streptomyces S during the biosynthesis of 2,5-FDCA from glucose.

[0162] Figure 4 Shown are one-dimensional NMR spectra of the following samples: (A) a 2,5-FDCA reference sample; (B) a sample extract spiked with 2,5-FDCA; and (C) a bioreactor sample collected during the biosynthesis of 2,5-FDCA from glucose. The one-dimensional NMR spectra show a significant increase in the signal intensity of 2,5-FDCA, and its signature is clearly identified.

[0163] Figure 5 The 2,5-FDCA production by Streptomyces sp. S on glucose (10 g / L), polyethylene terephthalate (PET) substrate supplemented with glucose (10 g / L), and polyethylene (PE) substrate supplemented with glucose (10 g / L) is shown. The supernatant was extracted and subjected to LC-MS / MS analysis.

[0164] Figure 6 The enzymatic reaction catalyzed by the Methanocaldococcus jannaschii MfnB enzyme is shown.

[0165] Figure 7 Shown is the DNA plasmid used to express the Streptomyces S MfnB protein in Pseudomonas putida to produce 5-HMF.

[0166] Figure 8The results of 12% SDS-PAGE gel electrophoresis are shown, revealing the expression of MfnB protein in Pseudomonas putida. The arrow indicates the MfnB band with a predicted molecular weight of 27 kDa. The SDS-PAGE gel electrophoresis results confirm the expression of Streptomyces SMfnB enzyme.

[0167] Figure 9 Shown are the results of C18 reversed-phase HPLC analysis of the following samples: (A) a high-purity 5-HMF standard; (B) supernatant from wild-type Pseudomonas putida; (C) supernatant from a Pseudomonas putida strain expressing MfnB grown on a glycerol substrate (96 hours); and (D) a sample spiked with 5-HMF (C). The results show that a peak corresponding to the high-purity 5-HMF standard appeared in the MfnB-expressing strain after 96 hours.

[0168] Figure 10 Shown are the results of HPLC analysis of the conversion of CO2 to 5-HMF in Synechocystis sp. PCC 6803 (PCC6803): (a) 5-HMF standard; (b) PCC6803 wild type; (c) PCC6803 expressing the pathway for the conversion of CO2 to 5-HMF (PCC6803 mutant); (d) PCC6803 mutant spiked with 5-HMF added.

[0169] Isolation of Streptomyces S

[0170] The Streptomyces sp., named Streptomyces S (deposited at the National Collection of Industrial Food and Marine Bacteria in Aberdeen, Scotland on June 14, 2022, with the deposit number NCIMB 43995), was isolated from a soil sample in Warwickshire, England, using an inorganic salt starch agar (ISP4) culture medium plate. The soil sample was dried at 60°C for 1 hour. The sample was then diluted with sterile Ringer's solution and spread on a solid actinomycete isolation culture medium plate ISP4. A single Streptomyces colony was selected and repeatedly streaked onto a new ISP4 culture medium plate to obtain a single colony, which was wrinkled, raised and white. 16s rRNA gene sequence amplification and subsequent phylogenetic analysis confirmed that Streptomyces S is closely related to Streptomyces fulvissimus. Streptomyces fulvissimus is a mesophilic bacterium with a 7.9Mbp linear chromosome containing 32 gene clusters involved in the biosynthesis of secondary metabolites. Two of the biosynthetic clusters share high similarity with the cyclic peptide valinomycin and the macrotetrolide antibiotic nonactin.

[0171] Biosynthesis of 2,5-FDCA directly from glucose

[0172] Streptomyces S is routinely maintained using liquid and solid GYM medium (4 g / L glucose, 4 g / L yeast extract, 10 g / L malt extract). Spore stocks are prepared using solid sporulation medium SFM (20 g / L soy flour, 20 g / L mannitol, 20 g / L agar).

[0173] Batch fermentations were performed in a 2-L Applikon bioreactor (Gothenburg, Sweden) at 30°C with controlled pH and temperature. M9 minimal medium (Sigma) was used as the culture medium with an initial glucose concentration of 20 g / L and a trace element concentration of 1 mL / L (containing 0.006 g / L ammonium ferric citrate, 0.001 g / L EDTA (disodium salt), 0.02 g / L Na2CO3, 2.86 g / L H3BO3, 1.81 g / L MnCl2·4H2O, 0.222 g / L ZnSO4·7H2O, 0.39 g / L NaMoO4·2H2O, 0.079 g / L CuSO4·5H2O, and 49.4 mg / L Co(NO3)2·6H2O). For batch fermentations, the bioreactor containing 700 mL of M9 medium supplemented with trace elements was autoclaved at 121°C for 20 min. 200 mL of glucose solution containing 20 g of glucose was sterilized separately at 121°C for 15 minutes and aseptically added to the bioreactor to a final concentration of 20 g / L. The bioreactor was then aseptically inoculated with 100 mL of pre-made Streptomyces S inoculum at a volume of 1% (v / v). The fermentation temperature, stirring speed, and aeration rate were set to 30°C, 150 rpm, and 0.5 vvm, respectively. The dissolved oxygen concentration was maintained above 30% of air saturation by adjusting the air supply. The pH was set to 7.0 and maintained using 10 M NaOH or 10 M HCl. The fermentation was run continuously for 2 weeks. The growth of Streptomyces S was monitored based on colony forming units (CFU), and the highest cell density reached 4.12 x 10 9 CFU / mL( Figure 1 ).

[0174] Samples (10 mL each) were collected aseptically and prepared for LC-MS analysis as described. Samples were taken at T1 = 0 hours, T2 = 72 hours, T3 = 120 hours, T4 = 168 hours, T5 = 240 hours, and T6 = 336 hours. Technical replicates (n = 6) were performed at each time point. Both the cell pellet and the supernatant were extracted with methanol, and the extracts were analyzed using HPLC and LC-MS / MS.

[0175] The fermentation supernatant extract was analyzed using C18 reverse phase HPLC. A 50 mM 2,5-FDCA standard was prepared and dissolved in 50% methanol to confirm the presence of 2,5-FDCA. The standard and samples with T1 = 0 hours, T2 = 72 hours, T3 = 120 hours, T4 = 168 hours, T5 = 240 hours, and T6 = 336 hours were analyzed. The 2,5-FDCA peak was detected at the same retention time as the standard ( Figure 2 A and Figure 2 B to Figure 2 G). A small 2,5-FDCA peak was initially detected around T3 = 120 hours, and the peak area subsequently increased, reaching a maximum at T5 = 240 hours and then decreasing. The maximum yield was determined to be 48 mg / L. 50 mM 2,5-FDCA was then added to the supernatant at T6 = 336 hours, and the peak area increased compared to the unsupplemented supernatant. Targeted LC-MS / MS analysis was performed on the extracted cell pellet and supernatant, specifically targeting 2,5-FDCA and 5-HMF. 2,5-FDCA and 5-HMF standards were prepared to confirm their presence in the whole cell pellet and supernatant. In both the supernatant and the whole cell pellet, the concentrations of 2,5-FDCA and 5-HMF increased over time ( Figure 3 A to Figure 3 D), reaching peak yield after 120 hours. Interestingly, the increase in 5-HMF peak area also correlates with the increase in 2,5-FDCA yield, indicating that 2,5-FDCA is generated from 5-HMF. MS / MS in negative ion mode confirmed the presence of 2,5-FDCA and 5-HMF. The spectra of 2,5-FDCA and 5-HMF standards were identical to those of the sample obtained at T6 = 336 hours.

[0176] Confirmation of 2,5-FDCA production by NMR analysis

[0177] A supernatant sample extracted at T6 = 336 hours was submitted for NMR analysis. Although NMR has limitations in sensitivity compared to LC-MS, it offers several advantages, including versatility and stringency in elucidating structures, analyzing positional isotopic patterns, and selecting molecules using isotopic filtering. Samples and standards were analyzed using both one- and two-dimensional proton NMR. Samples were also analyzed by both one- and two-dimensional proton NMR to confirm the presence of 2,5-FDCA. A 2,5-FDCA standard was used as a control, and the supernatant sample from T6 = 336 hours was also spiked with 2,5-FDCA. Two-dimensional NMR reveals the positions of 13C and 1H, eliminating any ambiguity in signal identification. The results for the standard, unspiked sample, and spiked sample were completely overlapping. The absence of any similar, overlapping signals that could be confused with the 2,5-FDCA signal was also clear. The one-dimensional analysis was quantitative and clearly demonstrated an increase in signal intensity for the spiked sample compared to the unspiked sample. The standard and both samples had identical chemical shifts (ppm). One-dimensional 1H NMR analysis results are as follows Figure 4 shown.

[0178] Biosynthesis of 2,5-FDCA on different substrates

[0179] The biosynthesis of 2,5-FDCA was performed as described above, but using different substrates. Figure 5 The graph shows 2,5-FDCA production on a glucose (10 g / L) substrate, a polyethylene terephthalate (PET) substrate supplemented with glucose (10 g / L), and a polyethylene (PEG) substrate supplemented with glucose (10 g / L). The graph indicates that 2,5-FDCA production continues to increase over the first 8 days of fermentation. The data show that glucose was completely consumed by day 5, indicating that 2,5-FDCA production is synchronized with cell growth. In the PET sample, 2,5-FDCA biosynthesis is likely derived from glucose. However, 2,5-FDCA biosynthesis also appears to correspond to PEG degradation. This further suggests that 2,5-FDCA biosynthesis is likely the result of enzymatic catalysis rather than a spontaneous reaction.

[0180] Pathway identification – biosynthesis of 5-HMF

[0181] The MfnB enzyme was previously identified in Methanococcus jannaschii (Biochemistry 2015, 54, 19, 2997–3008) and was Figure 6 The reaction shown catalyzes the conversion of two molecules of glyceraldehyde-3-phosphate. Streptomyces S encodes a putative MfnB homolog, the amino acid sequence of which is shown in SEQ ID NO:1.

[0182] The Streptomyces S MfnB homolog was amplified by PCR using Streptomyces S genomic DNA as a template. The MfnB gene was cloned into a broad host range plasmid under the control of the Pseudomonas putida constitutive promoter ( Figure 7 ). The expression plasmid was used to transform Pseudomonas putida by electroporation.

[0183] Figure 8 The SDS-PAGE analysis shown confirms the expression of the MfnB enzyme in P. putida.

[0184] Using Pseudomonas putida as the substrate and 1-10% glycerol as the raw material (aimed at maximizing the intracellular glyceraldehyde-3-phosphate content), 5-HMF production was achieved by expressing the Streptomyces S MfnB gene.

[0185] like Figure 9 As shown, HPLC analysis confirmed the production of a compound with the same retention time as 5-HMF.

[0186] Production of 5-HMF from CO2

[0187] 5-HMF was produced from CO2 in another strain, Synechocystis sp. PCC 6803 (PCC6803). HPLC analysis results are shown in Figure 2. Figure 10 As stated.

[0188] Figure 10 c shows the production of 5-HMF from CO2 in PCC6803, which produces peaks similar to Figure 10 The peak of 5-HMF standard in a matches that of PCC6803 wild type ( Figure 10 b) does not exist.

[0189] However, expression of this biosynthetic pathway in this novel strain negatively affected PCC6803 cell viability and expression was abolished when 5-HMF was added (see Figure 10 d).

[0190] It is believed that CO2 is converted in situ into sugars, including glucose, which are subsequently converted to 5-HMF through central metabolic processes.

[0191] discuss

[0192] The present invention allows for the biosynthesis of the platform chemical 5-HMF and the bioplastic monomer 2,5-furandicarboxylic acid (2,5-FDCA) directly from carbonaceous feedstocks, without the need to add additional feedstocks to the reaction mixture during the reaction – the first time this phenomenon has been documented in nature. Several microbial platforms have previously been engineered for the production of 2,5-FDCA from 5-HMF, including Pseudomonas putida S12, Deinococcus radiodurans, and Burkholderia cepacia. However, these processes require the separate addition of 5-HMF, which, prior to the present invention, required the synthesis of 5-HMF through chemical pretreatment of biomass. Therefore, known processes still cannot avoid the defects associated with the chemical synthesis of 2,5-FDCA. The complete bioconversion of the feedstocks used in the present invention (such as sugars such as glucose) to the production of 5-HMF and 2,5-FDCA provides a truly sustainable and transformative approach.

[0193] By combining powerful analytical methods such as HPLC, LC-MS / MS, and NMR, the inventors confirmed that Streptomyces S can produce 2,5-FDCA directly from glucose. LC-MS / MS and HPLC analysis showed that 2,5-FDCA production typically begins around T3 = 120 hours, then gradually increases, reaching a peak at T6 = 336 hours, which coincides with the onset of secondary metabolism ( Figure 3 A to Figure 3 B) Increased 2,5-FDCA production correlated with both glucose consumption and 5-HMF formation. 5-HMF concentrations began to increase at T2 = 72 hours, correlating with the increase in 2,5-FDCA, clearly indicating that the 2,5-FDCA originated from 5-HMF or its derivatives. One- and two-dimensional NMR analysis confirmed the molecular structure to be 2,5-FDCA.

[0194] As demonstrated in this invention, a method for bioproducing 5-HMF and 2,5-FDCA directly from carbonaceous feedstocks eliminates the need for chemical pretreatment of lignocellulose to produce 5-HMF and then convert it into 2,5-FDCA. This provides a more competitive route for the biosynthesis of 2,5-FDCA than existing technologies. 2,5-FDCA is an important renewable bioplastic monomer with the potential to replace various petrochemicals, such as terephthalic acid and adipic acid.

[0195] The above embodiments are merely illustrative and various modifications may be made without departing from the scope of the invention as defined in the appended claims.

Claims

1. A method for preparing 5-hydroxymethylfurfural or its derivatives, characterized in that: The method comprises the following steps: a. biocatalytically converting at least one carbonaceous feedstock into the 5-hydroxymethylfurfural or its derivatives.

2. The method according to claim 1, characterized in that The biocatalytic conversion in step (a) comprises enzymatic conversion.

3. A method according to any preceding claim, characterised in that The at least one carbonaceous feedstock is independently selected from the group consisting of a lignocellulosic feedstock, an oligosaccharide or polysaccharide feedstock, a lignin feedstock, a synthetic polymer feedstock, a protein feedstock, an agricultural feedstock, and combinations thereof.

4. A method according to any preceding claim, characterised in that The at least one carbonaceous feedstock comprises at least one ketose and / or aldose.

5. The method according to claim 4, characterized in that The at least one ketose and / or aldose carbonaceous feedstock comprises sugars.

6. The method according to claim 5, characterized in that The sugars include monosaccharides.

7. A method according to any preceding claim, characterised in that The step (a) comprises directly biocatalytically converting the at least one carbonaceous feedstock into the 5-hydroxymethylfurfural or a derivative thereof.

8. The method according to any one of claims 1 to 6, characterized in that The step (a) comprises indirectly biocatalytically converting the at least one carbonaceous feedstock into the 5-hydroxymethylfurfural or a derivative thereof.

9. The method according to claim 8, characterized in that The step (a) comprises converting the at least one carbonaceous feedstock into at least one intermediate independently selected from ketones, ketoses, keto acids, aldoses and combinations thereof, and then biocatalytically converting the intermediate into the 5-hydroxymethylfurfural or its derivatives.

10. The method according to claim 9, characterized in that The at least one intermediate product comprises GA3P or a derivative thereof and / or pyruvate or a derivative thereof.

11. A method for preparing 2,5-furandicarboxylic acid, characterized in that: The method comprises the following steps: a. A method according to any preceding claim, wherein at least one carbonaceous feedstock is biocatalytically converted into 5-hydroxymethylfurfural or a derivative thereof; and b. biocatalytically converting the 5-hydroxymethylfurfural or its derivatives obtained in step (a) into 2,5-furandicarboxylic acid.

12. The method according to claim 11, characterized in that The biocatalytic conversion in step (a) and / or step (b) comprises enzymatic conversion.

13. The method according to claim 11 or 12, characterized in that The step (b) comprises biocatalytically oxidizing the 5-hydroxymethylfurfural or its derivative obtained in the step (a) to produce the 2,5-furandicarboxylic acid.

14. The method according to any one of claims 11 to 13, wherein the step (b) comprises biocatalytically converting the 5-hydroxymethylfurfural or its derivative obtained in the step (a) into the 2,5-furandicarboxylic acid using an oxidoreductase, preferably an HMF oxidoreductase.

15. The method according to claim 14, characterized in that The step (b) comprises biocatalytically converting the 5-hydroxymethylfurfural or its derivatives into the 2,5-furandicarboxylic acid using an oxidase or a dehydrogenase.

16. A method according to any preceding claim, characterised in that At least step (a), and preferably the entire method, is performed in vivo.

17. The method according to claim 16, characterized in that At least step (a), and preferably the entire process, is performed using a single microorganism.

18. The method according to claim 17, characterized in that The microorganism is actinomycetes.

19. The method according to claim 18, characterized in that The microorganism is a Streptomyces bacterium.

20. The method according to claim 19, wherein The microorganism is Streptomyces S.

21. Biosynthesis of 5-hydroxymethylfurfural from carbonaceous feedstock, characterized in that The biosynthesis is carried out in bacteria of the genus Streptomyces.

22. Biosynthesis of 2,5-furandicarboxylic acid from carbonaceous feedstock, characterized in that The biosynthesis is carried out in bacteria of the genus Streptomyces.

23. The biosynthesis according to claim 21 or 22, characterized in that The Streptomyces bacterium is Streptomyces S.

24. The biosynthesis according to any one of claims 21 to 23, characterized in that The carbonaceous feedstock is independently selected from the group consisting of a lignocellulosic feedstock, a monosaccharide, oligosaccharide or polysaccharide feedstock, a lignin feedstock, a synthetic polymer feedstock, a protein feedstock, an agricultural feedstock, and combinations thereof.

25. Biosynthesis of 5-hydroxymethylfurfural from carbohydrate raw materials, characterized in that The biosynthesis was carried out in an isolated Streptomyces sp.

26. Biosynthesis of 2,5-furandicarboxylic acid from carbohydrate raw materials, characterized in that: The biosynthesis was carried out in an isolated Streptomyces sp.

27. A method for preparing 2,5-furandicarboxylic acid, characterized in that: The method comprises the following steps: a. Biocatalytic conversion of glucose into fructose using isomerase; b. biocatalytically converting the fructose into 5-hydroxymethylfurfural using a dehydratase; and c. biocatalytically converting the 5-hydroxymethylfurfural into 2,5-furandicarboxylic acid using HMF oxidoreductase.

28. A method for preparing 2,5-furandicarboxylic acid, characterized in that: The method comprises the following steps: a. biocatalytically converting a carbonaceous feedstock into at least one intermediate product, wherein the at least one intermediate product is independently selected from: keto acids, aldoses, and combinations thereof; b. biocatalytically converting the at least one intermediate into 5-hydroxymethylfurfural or a derivative thereof using MfnB or a homologous MfnB synthase; c. biocatalytically converting the 5-hydroxymethylfurfural or its derivatives into the 2,5-furandicarboxylic acid using HMF oxidoreductase or aldehyde dehydrogenase.

29. A method for preparing 2,5-furandicarboxylic acid, characterized in that: The method comprises the following steps: a. biocatalytically converting a carbonaceous feedstock into at least one ketone species, the at least one ketone species being preferably independently selected from the group consisting of: acetate, propionate, succinate, malonate and combinations thereof; b. biocatalytically converting the at least one ketone substance into 5-hydroxymethylfurfural or a derivative thereof by secondary metabolism using a polyketide synthase or a non-ribosomal peptide synthase; c. biocatalytically converting the 5-hydroxymethylfurfural or its derivatives into the 2,5-furandicarboxylic acid using HMF oxidoreductase or aldehyde dehydrogenase.

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