CN106414753A - Compositions and methods for producing chemicals and derivatives thereof - Google Patents
Compositions and methods for producing chemicals and derivatives thereof Download PDFInfo
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- CN106414753A CN106414753A CN201580021486.XA CN201580021486A CN106414753A CN 106414753 A CN106414753 A CN 106414753A CN 201580021486 A CN201580021486 A CN 201580021486A CN 106414753 A CN106414753 A CN 106414753A
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- XGFDHKJUZCCPKQ-UHFFFAOYSA-N n-nonadecyl alcohol Natural products CCCCCCCCCCCCCCCCCCCO XGFDHKJUZCCPKQ-UHFFFAOYSA-N 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- WSGCRAOTEDLMFQ-UHFFFAOYSA-N nonan-5-one Chemical compound CCCCC(=O)CCCC WSGCRAOTEDLMFQ-UHFFFAOYSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 1
- AMWVZPDSWLOFKA-UHFFFAOYSA-N phosphanylidynemolybdenum Chemical compound [Mo]#P AMWVZPDSWLOFKA-UHFFFAOYSA-N 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920000137 polyphosphoric acid Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
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- 230000002441 reversible effect Effects 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- MSXHSNHNTORCAW-GGLLEASOSA-M sodium;(2s,3s,4s,5r,6s)-3,4,5,6-tetrahydroxyoxane-2-carboxylate Chemical compound [Na+].O[C@H]1O[C@H](C([O-])=O)[C@@H](O)[C@H](O)[C@H]1O MSXHSNHNTORCAW-GGLLEASOSA-M 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
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- 238000005891 transamination reaction Methods 0.000 description 1
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- 230000035897 transcription Effects 0.000 description 1
- 229940057402 undecyl alcohol Drugs 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
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- 239000000811 xylitol Substances 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/58—Aldonic, ketoaldonic or saccharic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
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- Bioinformatics & Cheminformatics (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention provides methods for producing a product of one or more enzymatic pathways. The pathways used in the methods of the invention involve one or more conversion steps such as, for example, an enzymatic conversion of guluronic acid into D-glucarate (Step 7); an enzymatic conversion of 5-ketogluconate (5-KGA) into L-Iduronic acid (Step 15); an enzymatic conversion of L-Iduronic acid into Idaric acid Step 7b); and an enzymatic conversion of 5-ketocluconate into 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16). In some embodiments the methods of the invention produce 2,5-furandicarboxylic acid (FDCA) as a product. The methods include both enzymatic and chemical conversions as steps. Various pathways are also provided for converting glucose into 5-dehdyro-4-deoxy-glucarate (DDG), and for converting glucose into 2,5-furandicarboxylic acid (FDCA). The methods also involve the use of engineered enzymes that perform reactions with high specificity and efficiency. Additional products that can be produce include metabolic products such as, but not limited to, guluronic acid, L-iduronic acid, idaric acid, glucaric acid. Any of the products can be produced using glucose as a substrate or using any intermediate in any of the methods or pathways of the invention.
Description
Cross-Reference to Related Applications
The application requires the US application serial No. 14/222,453 of on March 21st, 2014 submission according to U.S.C. § 119 (e)
Part continuation application, the part continuation application of US application serial No. 14/033,300 submitted to for 20th of September in 2013 preferential
Power, it requires the excellent of the U.S. Provisional Application sequence number 61/704,408 of September in 2012 submission on the 21st according to 35U.S.C. § 119 (e)
First weigh rights and interests, every application here is incorporated hereby, including all tables, figure and claim.
The merging of sequence table
Material in appended sequence table is hereby incorporated by reference in the application.Appended sequence list text presents
It is named as SGI1660_3WO_Sequence_Listing.txt, creates on March 20th, 2015, and be 191KB.Can use
Microsoft Word on the computer using Windows OS evaluates this document.
Background of invention
In recent years, put into increasing energy come to identify using renewable raw materials produce organic chemicals new and
Effective means.In numerous downstream chemical process technologies, the sugar of biomass derived is recognized to the conversion of high valuable chemicals
For being highly important.Particularly, the saccharide of six kinds of carbon containings, i.e. hexose, such as Fructose and glucose, are to be widely known by the people certainly
So the maximum amount of monosaccharide present in boundary, therefore can suitably and economically be used as chemical raw material.
Furan is produced by sugar and furan derivatives have attracted increasing concern in chemistry and catalyticing research, and according to
Letter has the potential providing a main route realizing sustainability energy supply and chemicals production.In fact, with integrating life
In the biorefining of material conversion process, the dehydration of obtainable sugar and/or oxidation can produce an extended familys product, including various
The furan of various kinds and furan derivatives.
In the furan with maximum commercial value, furan -2,5- dioctyl phthalate (also referred to as FDCA, under
It is abbreviated as FDCA in literary composition) it is to include medicine, insecticide, antibacterial agent, spice, some industries of agricultural chemicalses and extensively
The valuable intermediate serving many purposes in the manufacture application of the polymeric material (for example, biological plasticss resin) of scope.
Therefore, FDCA is considered as the green substitute of p-phthalic acid (TPA), i.e. a kind of petroleum base monomer, and it is that the whole world is annual
One of petroleum chemicals of maximum volume producing.In fact, FDCA is accredited as optimal 12 kind priority by USDOE
One of compound, these compounds are all " green " chemistry being used for establishing future by the chemicals that sugar is made for high added value,
And therefore, its be named as one of " giant being sunk into sleep " of reproducible intermediate chemicals (Werpy and Petersen,
Top Value Added Chemicals from Biomass.US Department of Energy, Biomass, volume 1,
2004).
Although have pointed out various methods for large-scale production FDCA (with regard to summary, see, e.g. Tong et al.,
Appl.Catalysis A：General, 385,1-13,2010), but the key industry of FDCA synthesis currently relies on hexose such as
The chemical dehydration of glucose or Fructose is intermediate 5 hydroxymethyl furfural (5-HMF), and then chemical oxidation is FDCA.However, according to
Currently the FDCA production process via dehydration is usually nonselective for report, unless it is newly formed, unstable intermediate product
Soon it is converted into more stable material.Therefore, the major technique barrier in the production and use of FDCA is that exploitation is effective
Dewatering process with the sugar of selective biomass derived.
Accordingly, it would be desirable to exploitation by alternative means produce this highly important compound and many other chemicals with
The method of metabolite, this means are reproducible succedaneum not only for petroleum-based feedstock, and less and are provided using the energy
This intensive technology.Particularly, the selectivity of sugar dehydration controls can be a very powerful technology, produce extensively multiple
Extra cheap construction unit.
Content of the invention
The present invention is provided to the method producing the product of one or more enzymatic route.Way used in the inventive method
Footpath includes one or more step of converting, such as guluronic acid Enzymatic transformation is D- glucosaccharic acid (step 7)；5- ketone glucose
Sour (5-KGA) Enzymatic transformation is L- iduronic acid (step 15)；L- iduronic acid enzymatic is converted into idosaccharic acid step
7b)；And 5- ketogluconate Enzymatic transformation is 4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) (step 16)；1,5- glucose
Acid lactone Enzymatic transformation is guluronic acid-lactone (step 19).In some embodiments, the method for the present invention produces 2,5-
Furandicarboxylic acid (FDCA) is as product.Methods described may include enzymatic and chemical conversion as step.Also provide various
Approach is used for for glucose or Fructose or sucrose or galactose being converted into 5- dehydrogenation -4- deoxidation-glucosaccharic acid (DDG), and will
Identical sugar is converted into FDCA.Methods described may also include the purposes of the through engineering approaches enzyme being reacted with high specific and efficiency.
In a first aspect, the present invention provides a kind of method of the product for producing enzymatic or chemistry route from starting material.
Described approach can contain any one or more following step of converting：Guluronic acid Enzymatic transformation is D- glucosaccharic acid (step
7)；5- ketogluconate (5-KGA) Enzymatic transformation is L- iduronic acid (step 15)；L- iduronic acid enzymatic is converted into Ai Du
Saccharic acid (step 7b)；And 5- ketogluconate Enzymatic transformation is 4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) (step
16)；1,5- gluconolactone enzymatic is converted into guluronic acid-lactone (step 19).
In one embodiment, the product of enzymatic route is 5- dehydrogenation -4- deoxidation-glucosaccharic acid (DDG).Different real
Apply in scheme, the substrate of method can be glucose, and product can be 5- dehydrogenation -4- deoxidation-glucosaccharic acid (DDG).Described side
Method may include following steps：D-Glucose Enzymatic transformation is 1,5- gluconolactone (step 1)；1,5- gluconolactone enzymatic turns
Turn to guluronic acid-lactone (step 19)；Guluronic acid-lactone enzymatic is converted into guluronic acid (step 1B)；Gu Luo
Alduronic acid Enzymatic transformation is D- glucosaccharic acid (step 7)；And D- glucosaccharic acid Enzymatic transformation is 5- dehydrogenation -4- deoxidation-glucose
Diacid (DDG) (step 8).
In the other method of the present invention, substrate is glucose and product is DDG, and methods described includes following step
Suddenly：D-Glucose is converted into 1,5- gluconolactone (step 1)；1,5- gluconolactone is converted into gluconic acid (step 1a)；Portugal
Saccharic acid is converted into 5- ketogluconate (5-KGA) (step 14)；5- ketogluconate (5-KGA) is converted into L- iduronic acid (step
15)；L- iduronic acid is converted into idosaccharic acid (step 7b)；And idosaccharic acid is converted into DDG (step 8a).
In the other method of the present invention, substrate is glucose and product is DDG, and methods described includes following step
Suddenly：D-Glucose is converted into 1,5- gluconolactone (step 1)；1,5- gluconolactone is converted into gluconic acid (step 1a)；Portugal
Saccharic acid is converted into 5- ketogluconate (5-KGA) (step 14)；5- ketogluconate (5-KGA) is converted into 4,6- dihydroxy 2,5- diketone
Caproic acid (2,5-DDH) (step 16)；4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) is converted into 4- deoxidation -5- threo form-hexanone
Sugared alduronic acid (DTHU) (step 4)；And 4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) is converted into DDG (step 5).
In the other method of the present invention, substrate is glucose and product is DDG, and methods described includes following step
Suddenly：D-Glucose is converted into 1,5- gluconolactone (step 1)；1,5- gluconolactone is converted into gluconic acid (step 1a)；Portugal
Saccharic acid is converted into 5- ketogluconate (5-KGA) (step 14)；5- ketogluconate (5-KGA) is converted into L- iduronic acid (step
15)；L- iduronic acid is converted into 4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) (step 7B)；And 4- deoxidation -5-
Threo form-ketohexose alduronic acid (DTHU) is converted into DDG (step 5).
Any method disclosed herein can further include the step that DDG is converted into 2,5- furan-dioctyl phthalate (FDCA).
In where method in office, DDG is converted into FDCA and may include and contacted DDG so that DDG is converted into FDCA with mineral acid.
On the other hand, the present invention provides the method that one kind is used for synthesizing the FDCA of derivatization (esterification).Methods described includes
DDG is contacted to form the FDCA of derivatization at a temperature of more than 60C with alcohol, mineral acid.In various embodiments, described
Alcohol is methanol, butanol or ethanol.
On the other hand, the present invention provides a kind of method of the derivant for synthesizing FDCA.Methods described include by DDG with
Alcohol, mineral acid and cosolvent contact to produce the derivant of DDG；The optionally derivant of purification DDG；And DDG is derivative
Thing is contacted with mineral acid to produce the derivant of FDCA.Mineral acid can be sulphuric acid and alcohol can be ethanol or butanol.Implement different
In scheme, cosolvent can for following any one：THF, acetone, acetonitrile, ether, butyl acetate, dioxane, chloroform, dichloromethane, 1,
2- dichloroethanes, hexane, toluene and dimethylbenzene.
In one embodiment, the derivant of DDG be the derivant of diethyl DDG and FDCA be diethyl FDCA, and
And in another embodiment, the derivant of DDG is the derivant of dibutyl DDG and FDCA is dibutyl FDCA.
On the other hand, the present invention provides a kind of method for synthesizing FDCA.Methods described includes DDG is existed with mineral acid
Contact in gas phase.
On the other hand, the present invention provides a kind of method for synthesizing FDCA.Methods described includes DDG is existed with mineral acid
Contact more than at a temperature of 120C.
On the other hand, the present invention provides a kind of method for synthesizing FDCA.Methods described includes DDG is existed with mineral acid
Contact under the conditions of anhydrous response.
Brief description
Fig. 1 is the thick lysate of protein 47 4,475 and 476 and the running gel of purifying enzyme.
Fig. 2A -2H be respectively route 1,2, the schematic diagram of the approach of 2A, 2C, 2D, 2E, 2F.
Fig. 3 A-3c respectively illustrates the schematic diagram of the approach of route 3,4 and 5.
Fig. 4 is gluconic acid to be dehydrated to produce the HPCL-MS analysis of DHG by pSGI-359 with gluconate dehydratase.
Fig. 5 is to measure, for measuring the semicarbazides (semicarbizide) of gluconate dehydratase activity, the curve chart drawn.
Glucuronic acid that Fig. 6 A-6B offer is carried out with three kinds of enzymes of the present invention and the oxidation of iduronic acid
Lineweaver-Burk draws.
Fig. 7 A show using the enzyme DTHU isomerase in EC 5.3.1.17 family, 5KGA and iduronic acid are carried out different
The result of the HPLC analysis of the time point of structure.Comparison：Dead enzyme is and the compareing of hot inactivator.Med B1 refers to not add
The reaction of isomerase.Time point, x-axis 1=0.5h；2=1；3=2h；4=16h.Fig. 7 b shows using EC 5.3.1.17 family
Enzyme in race carries out the HPLC analysis of the time point of isomerization to 5KGA and iduronic acid.Comparison：Dead enzyme is and heat inactivates
The comparison of enzyme.；Med B1：Refer to not add the reaction of isomerase.Time point, X-axis：1=0h；2=1h；3=2h；4=
17h.
Fig. 8 shows the product shape that with the enzyme in EC 5.3.1.nl family, 5KGA and iduronic acid are carried out with isomerization
Become.Data is obtained from enzymatic determination.
Fig. 9：The HPLC analysis of 2,5-DDH formation and 5KGA concentration reduction in time.Show total ion of 2,5-DDH
Count.
Figure 10 is to show the HPLC-MS chromatogram being produced guluronic acid lactone by 1,5- gluconolactone.Show true
The overlap of the trace of positive guluronic acid.
Figure 11 is the schematic diagram of scheme 6 reaction path.
Figure 12 A-12B shows the LC-MS chromatogram of 5-KGA and DDG product respectively.
Figure 13 shows the LC-MS chromatogram of FDCA and FDCA dibutyl ester derivatization reaction product.
Figure 14 A is the GC-MS analysis of the crude reaction sample of diethyl FDCA synthesis of the reaction from DDG Yu ethanol.Single
Peak corresponds to diethyl-FDCA.Figure 14 B is the MS fragment of the primary product of the reaction from DDG Yu ethanol.
Figure 15 A is the GC-MS analysis of the crude reaction sample of diethyl FDCA synthesis of the reaction from DDG Yu ethanol.Single
Peak corresponds to diethyl-FDCA.Figure 15 B is the MS fragment of the primary product of the reaction from DDG Yu ethanol.
Figure 16 is the schematic diagram being synthesized FDCA and its derivant by DTHU.
Figure 17 is the schematic diagram of scheme 1.The acellular enzyme' s catalysis of the DDG by glucose.Enzyme is ST-1：Glucose
Oxidase；ST-1A：Hydrolysis-chemicals；ST-14：Gluconatephosphate dehydrogenase (pSGI-504)；ST-15：5- dehydrogenation -4- deoxidation-D-
Glucuronate isomerase (DTHU IS, pSGI-434)；ST-7B：Alditol acidohydrogenase (UroDH, pSGI-476))；ST-8A：Portugal
Saccharic acid dehydratase (GlucDH, pSGI-353)；ST-A：NAD (P) H oxidase (NADH_OX, pSGI-431)；ST-B：Peroxide
Change hydrogen enzyme.Figure 17 b shows the concentration as the reaction intermediate analyzed by HPLC in head 3h.All aobvious in two reactions
Show the formation of DDG.
Specific embodiment
The present invention is provided to the method producing the product of enzymatic route.Methods described may include Substrate Enzyme catalysed and is converted into product
Thing.By using the present invention enzymatic and chemistry route it is possible to synthesize extensive multi-products in the way of efficient and economical.Can
A kind of product being produced by the method for the present invention and approach is 2,5- furyl dioctyl phthalate (FDCA), its can according to the present invention with
Commercial scale.Methods described may include one or more enzymatics disclosed herein and/or the substrate of chemistry turns to product
Change step.In some embodiments, using enzyme using carrying out Enzymatic transformation step for the unknown activity of described enzyme.Cause
This, can be used in the present invention these novel actives using carry out step of converting and substrate to the conversion of product as enzymatic and/or
A part for chemistry route.Spawn (such as DDG, iduronic acid, the idose two of any approach disclosed herein
Acid, glucosaccharic acid, FDCA etc.) all can as disclosed herein in single bioreactor or reaction vessel on an industrial scale, that is,
At least 1 gram or at least 10 grams or at least 100 grams or at least amount of 1kg production.
The approach of the present invention comprises any one or more steps disclosed herein.It should be appreciated that the approach of the present invention
Step may include positive reaction or back reaction, that is, substrate A is converted into product B, and substrate B is converted into product in back reaction
A.In method, unless otherwise mentioned, positive reaction and back reaction are all described as step.
Methods described includes the product of production ways, and this approach can be enzymatic route.Methods described includes one or more
Enzymatic and/or chemical conversion steps, these steps convert a substrate into product.May include the step in method include for example with
Descend any one or more：Guluronic acid Enzymatic transformation is D- glucosaccharic acid (step 7)；L- iduronic acid enzymatic is converted into
4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) (17)；5- ketogluconate (5-KGA) Enzymatic transformation is L- iduronic acid
(step 15)；L- iduronic acid enzymatic is converted into idosaccharic acid step 7B)；And 5- ketogluconate Enzymatic transformation is 4,6-
Dihydroxy 2,5- diketone caproic acid (2,5-DDH) (step 16)；1,5- gluconolactone enzymatic is converted into guluronic acid-lactone
(step 19).Any one or more above-mentioned steps may comprise in the method for the present invention or approach.Enzymatic step or approach
It is to need enzyme as catalyst so that the step that carries out of step or approach in the reaction.Chemical step can not have enzyme to make in the reaction
For carrying out in the case of catalyst.In method, any one or more cited steps can be all enzymatic step.At some
In embodiment, each step of approach is enzymatic step, and in other embodiments, one or more of approach walks
Suddenly it is chemical step.
In some embodiments, any methods described may comprise to be related to be added to the substrate of reaction and turns containing execution
Step in the reactant mixture of enzyme changed.Therefore, guluronic acid is converted into the method for D- glucosaccharic acid (step 7) and may include
Guluronic acid is added in reactant mixture as starting material；L- iduronic acid enzymatic is converted into idosaccharic acid
(7B) may include and L- iduronic acid is added in reactant mixture as starting material；L- iduronic acid enzymatic is converted into
4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) (17) may include and is added to anti-L- iduronic acid as starting material
Answer in mixture.Any methods described may comprise glucose, Fructose, galactose, sucrose or mannose or another monosaccharide or
Disaccharide is added to the step in reactant mixture.Another step that may include in where method in office is purification from reactant mixture
The step of product.Therefore, purification glucosaccharic acid/D- glucosaccharic acid or L- iduronic acid/iduronic acid or idose
The step of diacid or 2,5- diketoadipic acid/DKHA may include in any method as herein described.Disclosed any method
The step that may comprise isolated or purified DDG or FDCA from reactant mixture.And any method may comprise interpolation enzyme and arrives
Step in reactant mixture, described enzyme executes any one or more steps as herein described.Reactant mixture is at least one
The mixture of kind of substrate and at least one enzyme and include at least one substrate and be converted at least one enzyme product.Any described side
Method may comprise the step being added to detached enzyme in reactant mixture, and the substrate executing approach of the present invention converts step to product
Rapid enzyme, and detached enzyme be at least 10% purification or at least 20% purification or at least 25% purification or at least 50% purification or
At least 70% purification or at least 80% purification or at least 90% purification, all is all w/w.
Because many sugar can be converted into other sugar, so any method of the present invention or approach all can relate to glucose, sugarcane
Sugar, Fructose or galactose are used as starting material.Therefore, glucose is disclosed herein any approach of starting material wherein
Or it should be understood that Fructose or sucrose or galactose or mannose or another starting material can also be that approach or anti-in reaction
The starting material answered.In some embodiments, sugar is converted into glucose, and glucose subsequently enters approach, but in other enforcements
In scheme, approach is from the beginning of Fructose or sucrose or galactose or mannose or another monosaccharide or disaccharide.
The reaction of the present invention can be in the cell lysate or acellular of the enzyme containing one or more execution Enzymatic transformation
Occur in lysate, but also can occur in the reactant mixture containing component, described component is to be added to be formed instead by user
Answer mixture, or the component of purification from cell lysate can be contained, or can be included in whole-cell biocatalyst.Reaction
Can occur in the mixture being made up of the purified components having merged, such as substrate has merged formation reaction mixing with enzyme wherein
In the mixture of thing.Reaction can occur in reacting in vitro or can occur in reconstitution cell, and therefore, one or more product
Can harvest by dissolving cell or by collecting at culture medium.Reaction can be sent out in following laboratory containers or reaction vessel
Raw, such as centrifuge tube, test tube, bottle, beaker or glass or metal or plastic containers or reactor, fermentation tank or round
Or bioreactor, algae pond, can be on a small scale or large-scale any container.Any organism as herein described is used as
Host cell is to produce the step of the present invention or the product of approach.Organism can also be used for producing one or more enzyme of the present invention
For in the method for the present invention.Various types of organisms can be used.Example includes：Antibacterial (the such as vinegar bar of acetobacter section
Pseudomonas, acidophilus Pseudomonas, Gluconobacter, the antibacterial of gluconic acid acetobacter) or pseudomonadaceae antibacterial (such as fixed nitrogen
Pseudomonas, Rhodopseudomonass) or enterobacteriaceae antibacterial (such as Escherichia (such as escherichia coli), klebsiella
Belong to).Yeast can also be used for these purposes, such as Saccharomycodeses, AshbyaCif．etFrag．, Kluyveromyceses, Lachancea, joint ferment
Female genus, Candida, the yeast of pichia genus, Arxula or Piedraia or Blastobotry.Also cyanophyceae can be used
Antibacterial, such as blue silk Pseudomonas (for example blue bar phycomycete strain ATCC 51142, PCC 7424, PCC 7425, PCC 7822, PCC 8801,
PCC 8802) or Microcystis or Synechococcus belong to (for example elongated bacterial strain PCC 7942, PCC 7002, PCC 6301, CC9311,
CC9605, CC9902, JA-2-3B ' a (2-13), JA-3-3Ab, RCC307, WH 7803, WH 8102) or synechocystis or thermophilic
Hot Synechococcus.Therefore, the present invention provides recombinant host cell, and it comprises SEQ ID NO：4-6、20-32、36-38、47-54、
56th, the recombinant nucleic acid of one or more of 62-66,69-70,72 and 79-84 or SEQ ID NO：Any one of 1-84's
Codon optimised sequence.Host cell also can the carrier containing invention as described herein." codon optimization " sequence refers to sequence
Row codon is changing into those preferentially using in specific organism so that coded protein is carrying this sequence
Organism in effectively express.Recombinant nucleic acid sequence can be included on carrier as disclosed herein.
In various embodiments, the method for the present invention is following methods：Glucose or Fructose or sucrose or galactose turn
Turn to DDG, or glucose or Fructose or sucrose or galactose are converted into FDCA, or glucose or Fructose or sucrose or gala
Sugar is converted into DTHU or DEHU, or DDG is converted into FDCA.Methods described may include and is converted into the starting material in method
Product.The chemistry of the final product that starting material is regarded as starting the chemical entities of the method and product is regarded as method is real
Body.Intermediate is to generate in (either temporarily or permanently) and the reaction path between starting material and product to exist in method
Those chemical entities.In various embodiments, the method for the present invention and approach have about four or about five intermediate or
4-5 intermediate or about 3 intermediate or 3-5 intermediate or less than 6 or less than 7 or less than 8 or less than 9 or be less than 10
Or less than 15 or less than 20 intermediate it is meant that these values are not by including starting material or final product calculation.
The present invention provides the method from glucose or Fructose or sucrose or galactose production FDCA and/or DDG, and it has height
Yield.Theoretical yield is by the amount of the product being formed if reaction completes under ideal conditions.In various embodiments,
The method of the present invention produces DDG from glucose, Fructose or galactose, and its theoretical yield is at least 50 moles % or at least 60 rubs
Your % or at least 70 moles % or at least 80 moles %, at least 90 moles % or at least 95 moles % or at least 97 moles % or
At least 98 moles % or at least 99 moles %, or theoretical yield be 100 moles of %.The method of the present invention may also provide have following
The product of carbon storage rate：At least 80% or at least 90% at least 95% or at least 97% at least 98% or at least 99% or
100% it is meant that concrete carbon atom present in initial substrate is present in the final product of method with cited percentage ratio
In.In some embodiments, methods described produces DDG via dehydration from glucose or Fructose or sucrose or galactose
And/or FDCA.
Exemplary route of synthesis
The present invention also provides the particular approach for product needed for synthesis and generation.Any described below route or approach are all
Can start from glucose or Fructose or sucrose or galactose or mannose and move to required product.In some embodiments
In, D-Glucose is starting material and is considered as downstream side towards the approach direction of the intermediate of approach or final product
To, and be considered as updrift side towards the rightabout of glucose.It should be appreciated that route or approach can be with downstream or upstreams
Direction is moved.Although glucose is used as the exemplary starting material of approach as herein described, it is also to be understood that sucrose, really
Sugar, galactose or mannose or can also be the initial bottom in any method of the present invention in any intermediate in any approach
Any intermediate in thing, and any route of DDG, DTHU, FDCA or the present invention or approach can be the inventive method
Final product.Therefore, disclosed method include disclosed in any route of the present invention or approach any one or many
Any starting material or intermediate are converted into institute using one or more of disclosed route or approach step by individual step
Any final product in disclosed route or approach or intermediate.So that it takes up a position, for example, methods described can be with lower section
Method：For glucose or Fructose or sucrose or galactose or mannose being converted into DDG or being converted into guluronic acid or turn
Turn to galactobionic acid Galactonic acid or be converted into DTHU be converted into DEHU or be converted into guluronic acid or be converted into iduronic acid,
Or be converted into idosaccharic acid or be converted into glucosaccharic acid；Or for galactobionic acid Galactonic acid is converted into DDG；Or be used for ancient sieve
Alduronic acid is converted into D- glucosaccharic acid；Or for 5-KGA is converted into L- iduronic acid；Or for turning L- iduronic acid
Turn to idosaccharic acid；Or for 5-KGA is converted into 2,5-DDH or DTHU；Or for DHG is converted into DEHU.At these
In embodiment, methods described utilizes the step disclosed in the method for the present invention and approach, final to correlation from starting material
Product.One or more of these steps also can use in method, with from approach disclosed herein " reverse " or
Updrift side moves.
Route 1 is shown in Fig. 2 a.Route 1 via a series of shown steps via enzymatic route by D-Glucose (or approach
In any intermediate) be converted into 5- dehydrogenation -4- deoxidation-glucosaccharic acid (DDG).Route 1 is via having following approach by D-
Glucose is converted into DDG：There is 1,5- gluconolactone, gluconic acid, 3- dehydrogenation-gluconic acid (DHG), 4,6- dihydroxy 2,5- bis-
As intermediate and DDG is as final product for ketone caproic acid (2,5-DDH) and 4- deoxidation-L- threo form-ketohexose alduronic acid (DTHU).
For any approach, also there may be unshowned extra intermediate.These steps are D-Glucose Enzymatic transformation is 1,5- glucose
Acid lactone (step 1)；1,5- gluconolactone enzymatic is converted into gluconic acid (step 1A)；Gluconic acid Enzymatic transformation be 3- dehydrogenation-
Gluconic acid (DHG) (step 2)；3- dehydrogenation-gluconic acid (DHG) Enzymatic transformation is 4,6- dihydroxy 2,5- diketone caproic acid (2,5-
DDH) (step 3)；4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) Enzymatic transformation is 4- deoxidation-L- threo form-ketohexose alditol
Sour (DTHU) (step 4)；And 4- deoxidation-L- threo form-ketohexose alduronic acid (DTHU) Enzymatic transformation be 5- dehydrogenation -4- deoxidation -
Glucosaccharic acid (DDG) (step 5).Route 1 also includes any centre in sub- route, the wherein glucose as substrate or approach
Body is all converted into any other downstream intermediate as final product, and every substrate to the sub- route of product is all regarded
It is open as each herein elaborates.
Route 2 is shown in Fig. 2 b and D-Glucose is converted into DDG.Step in route 2 approach is D-Glucose enzyme
Promote to be converted into 1,5- gluconolactone (step 1)；1,5- gluconolactone enzymatic is converted into gluconic acid (step 1A)；Gluconic acid enzyme
Promote to be converted into guluronic acid (step 6)；Guluronic acid Enzymatic transformation is D- glucosaccharic acid (step 7)；D- glucosaccharic acid enzyme
Promote to be converted into DDG (step 8).Route 2 also includes any intermediate in sub- route, the wherein glucose as substrate or approach
All it is converted into any other downstream intermediate as final product, and every strip route is considered as each is at this
Elaborate typically open in literary composition.For example, in some embodiments, methods described is included using described in route 2
One or more steps will be converted into guluronic acid or D- glucose two as product as the glucose of substrate or gluconic acid
The step of acid.
Route 2A is shown in Fig. 2 c.Step in approach 2A is D-Glucose Enzymatic transformation is 1,5- gluconolactone (step
Rapid 1)；1,5- gluconolactone enzymatic is converted into guluronic acid lactone (step 19)；Guluronic acid lactone enzymatic is converted into
Guluronic acid (step 1B)；Guluronic acid Enzymatic transformation is D- glucosaccharic acid (step 7)；D- glucosaccharic acid Enzymatic transformation is
5- dehydrogenation -4- deoxidation-glucosaccharic acid (DDG) (step 8).Route 2A also includes sub- route, wherein as the Fructus Vitis viniferae of starting material
Any intermediate in sugar or approach is all converted into any other downstream intermediate as final product, and every strip route
It is considered as open as each herein elaborates.For example, in some embodiments, methods described
To be converted into as the glucose of substrate or guluronic acid lactone including the one or more steps described in route 2A is used
Glucosaccharic acid as product or the step of DDG.
Route 2B is shown in Fig. 2 d.Step in route 2B is D-Glucose Enzymatic transformation is gluconic acid (step 1 and 1A)；
Gluconic acid Enzymatic transformation is 5- ketogluconate (5-KGA) (step 14)；5-KGA Enzymatic transformation is L- iduronic acid (step
15)；L- iduronic acid enzymatic is converted into idosaccharic acid (step 7B)；Idosaccharic acid Enzymatic transformation is DDG (step 8A).
Any intermediate that route 2B is also included in sub- route, the wherein glucose as starting material or approach is all converted into any
Other downstream intermediate are as final product, and every strip route is considered as herein elaborating one such as each
As open.For example, in some embodiments, methods described is included using the one or more steps described in route 2B
The step of iduronic acid as product or idosaccharic acid will be converted into as the glucose of substrate or 5-KGA.
Route 2C is shown in Fig. 2 e.Step in route 2C is D-Glucose Enzymatic transformation is gluconic acid (step 1 and 1A)；
Gluconic acid Enzymatic transformation is 5- ketogluconate (5-KGA) (step 14)；5-KGA Enzymatic transformation is 4,6- dihydroxy 2, and 5- diketone is own
Sour (2,5-DDH) (step 16)；4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) Enzymatic transformation be 4- deoxidation -5- threo form-oneself
Ketose alduronic acid (DTHU) (step 4)；DTHU Enzymatic transformation is DDG (step 5).Route 2C also includes sub- route, wherein conduct
Any intermediate in the glucose of starting material or approach is all converted into any other downstream intermediate as final product,
And every strip route is considered as open as each herein elaborates.For example, implement at some
In scheme, methods described includes being used the one or more steps described in route 2C using as the glucose of substrate or gluconic acid
The step being converted into 2,5-DDH or DTHU.
Route 2D is shown in Fig. 2 f.Step in route 2D is D-Glucose Enzymatic transformation is gluconic acid (step 1 and 1A)；
Gluconic acid Enzymatic transformation is 5- ketogluconate (5-KGA) (step 14)；5-KGA Enzymatic transformation is iduronic acid (step 15)；
L- iduronic acid enzymatic is converted into DTHU (step 17)；DTHU Enzymatic transformation is DDG (step 5).Route 2D also includes sub- road
Any intermediate in line, the wherein glucose as starting material or approach is all converted into any other downstream intermediate and makees
For final product, and every strip route be considered as open as each herein elaborates.For example,
In some embodiments, methods described includes being used the one or more steps described in route 2D using the Fructus Vitis viniferae as substrate
The step that sugared or 5-KGA is converted into L- iduronic acid or DTHU.
Route 2E is shown in Fig. 2 g.Step in route 2D is D-Glucose Enzymatic transformation is 1,5- gluconolactone (step
Rapid 1)；1,5- gluconolactone enzymatic is converted into guluronic acid lactone (step 19)；Guluronic acid lactone enzymatic is converted into
Guluronic acid (step 1B)；Guluronic acid Enzymatic transformation is 4- deoxidation-erythro-ketohexose alduronic acid (DEHU) (step
17A)；DEHU Enzymatic transformation is 3- deoxidation-D- erythro-methyl-n-butyl ketone saccharic acid (DDH) (step 7A).Route 2E also includes sub- road
Any intermediate in line, the wherein glucose as starting material or approach is all converted into any other downstream intermediate and makees
For final product, and every strip route be considered as open as each herein elaborates.For example,
In some embodiments, methods described includes being used the one or more steps described in route 2E using the Fructus Vitis viniferae as substrate
The step that sugar is converted into guluronic acid or DEHU.
Route 2F is shown in Fig. 2 h.Step in route 2F is D-Glucose Enzymatic transformation is gluconic acid (step 1 and 1A)；
Gluconic acid Enzymatic transformation is guluronic acid (step 6)；Guluronic acid Enzymatic transformation is 4- deoxidation-erythro-ketohexose alditol
Sour (DEHU) (step 17A)；DEHU Enzymatic transformation is 3- deoxidation-D- erythro-methyl-n-butyl ketone saccharic acid (DDH) (step 7A).Route
2F also includes sub- route, be wherein used the one or more steps of route 2F using as the glucose of starting material or gluconic acid or
Any intermediate in approach is converted into guluronic acid or DDH or any other downstream intermediate as final product, and
Every strip route is considered as open as each herein elaborates.
Route 3 is shown in Fig. 3 a.Step in route 3 is D-Glucose Enzymatic transformation is gluconic acid (step 1 and 1A)；Portugal
Saccharic acid Enzymatic transformation is 3- dehydrogenation-gluconic acid (DHG) (step 2)；DHG Enzymatic transformation is 4- deoxidation-erythro-ketohexose alduronic acid
(DEHU) (step 6A)；DEHU Enzymatic transformation is DDG (step 7A).Route 3 also includes sub- route, wherein using the one of route 3
Any intermediate as in the glucose or Fructose or sucrose or galactose or approach of starting material is turned by individual or multiple steps
Any other downstream intermediate turning to gluconic acid or DDH or route 3 is as final product, and every strip route is considered as
It is open as each herein elaborates.
Route 4 is shown in Fig. 3 b.Step in route 4 is D-Glucose Enzymatic transformation is a-D- glucose-hexandial sugar -1,
5- pyranose (step 9)；A-D- glucose-hexandial sugar -1,5- pyranose Enzymatic transformation is a-D- Portugal pyrans alduronic acid (step
10)；A-D- Portugal pyrans alduronic acid Enzymatic transformation is D- glucosaccharic acid 1,5- lactone (step 11)；D- glucosaccharic acid 1,5- lactonase
Promote to be converted into D- glucosaccharic acid (step 1C)；D- glucosaccharic acid Enzymatic transformation is DDG (step 8).Route 4 also includes sub- route,
Any intermediate as in the glucose or approach of starting material is converted into by the one or more steps that route 4 is wherein used
Glucosaccharic acid or DDG or any other downstream intermediate are as final product, and every strip route is considered as such as each
All herein elaborate typically open.
Route 5 is shown in Fig. 3 c.Step in route 5 is that D- galactose enzymatic is converted into D- galactose-hexandial sugar (step
Rapid 9A)；D- galactose-hexandial carbohydrase promotees to be converted into galacturonic acid (step 10A)；Galacturonic acid Enzymatic transformation is gala
Saccharic acid (step 11A)；Galactobionic acid Galactonic acid Enzymatic transformation is DDG (step 13).Route 5 also includes sub- route, wherein as initial bottom
Any intermediate in the galactose of thing or approach is all converted into any other downstream intermediate as final product, and often
Strip route is considered as open as each herein elaborates.For example, in some embodiments,
Methods described includes, using the step described in route 5, galactose or another substrate are converted into galacturonic acid hydrochlorate or galactose
The step of acid.
In various other embodiments, the present invention provides and a kind of produces enzymatic and/or chemistry route from starting material
The method of product, methods described includes execution step 1, followed by step 19, followed by step 1B, to produce guluronic acid product
Thing.Optionally, described approach can proceed with step 7 to produce glucosaccharic acid.In another embodiment, methods described includes
Execution step 1 and 1A, followed by step 14, followed by step 15, to produce iduronic acid.Optionally, methods described can continue
Continue and carry out step 7B to produce idosaccharic acid product or to carry out step 17 to produce DTHU.In another embodiment, described
Method includes execution step 1 and 1A, followed by step 14, and followed by step 16 is to produce 2,5-DDH product.In another embodiment party
In case, methods described includes execution step 1, followed by step 19, to produce guluronic acid lactone.
Enzymatic step
The enzyme (and nucleic acid of codase) of extensively multiple executable method described herein steps has been disclosed.In the present invention
Enzymatic step in using enzyme can be protein or polypeptide.Except for executing the enzyme man disclosed herein of step of the present invention
Outside race and classification, with any enzyme disclosed herein or nucleic acid or with any one of SEQ ID NO 1-84 have sequence with
The congener of one property is also applied for the present invention.It is SEQ ID NO：The enzyme of the congener of 1-84 and nucleic acid and SEQ ID NO：1-84
Any nucleic acid enzyme or with enzyme disclosed herein other member have at least 40% at least 50% or at least 60% or
At least 70% or at least 80% or at least 90% or at least 95% or at least 97% or at least 98% or at least 99% sequence is same
One property.It is defined herein as aminoacid with respect to the sequence iden of aminoacid or nucleotide sequence or percent homology
Or nucleotide residue with known peptide identical candidate sequence in percentage ratio, if necessary, the maximum homogeneity of aligned sequences
Percentage ratio simultaneously introduces breach, to realize maximum homogeneity or percent homology.Under nucleotide or amino acid sequence level
Homology or homogeneity can be measured using method as known in the art, including but not limited to use by program blastp,
Blastn, blastx, tblastn and tblastx (Altschul (1997), Nucleic Acids Res.25,3389-3402,
And Karlin (1990), Proc.Natl.Acad.Sci.USA 87,2264-2268) BLAST (the basic office of algorithm that adopted
Portion compares gopher) analysis, these programs are to aim at sequence similarity search.Or, it is possible to use any enzyme or coding institute
State nucleic acid or any enzyme of SEQ ID NO 1-84 disclosed herein or the function fragment of nucleic acid of enzyme.Term " function fragment "
Refer to there is amino terminal and/or carboxyl-terminal deletion and/or inside lacks the many of (it can be substituted to form chimeric protein)
Relevant position in peptide, wherein remaining amino acid sequence and canonical sequence has at least 40%, 45%, 50%, 55%, 60%,
65%th, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%th, 97%, 98%, 99% or 100% sequence iden, and/or keep about 75%, 80%, 85%, 90%, 91%,
92%th, the activity of 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% full-length polypeptide.EC numbering provides international raw
Thing chemistry and NK of molecular biology community (Nomenclature Committee of the
International Union of Biochemistry and Molecular Biology) enzyme name.In other enforcements
In scheme, function fragment retains to by SEQ ID NO：The requirement that the cofactor needed for protein active of 1-84 coding exists.
Also disclose and there is following sequence of expression vector：SEQ ID NO：4-6、20-32、36-38、47-54、56、62-
66th, 69-70,72 and 79-84.Described carrier can be antibacterial, yeast or Sargassum carrier.The carrier designing for gene expression is also
May include promoter that is active in the organism of carrying carrier and being operably connected with sequence of the present invention.Carrier can contain
The promoter being operably connected with following sequence or expression control sequenc：SEQ ID NO：4-6、20-32、36-38、47-54、
56th, 62-66,69-70,72 and 79-84 or its codon optimised sequence of any one." promoter " is to refer to combine RNA
Polymerase is so that the nucleotide sequence that promotor gene is transcribed on 5 ' to 3 ' (" downstream ") direction.When RNA polymerase and promoter
In conjunction be described genetic transcription immediate cause when, sequence " being operably connected " is to promoter.
Step 1- glucose conversion (oxidation or dehydrogenation) is 1,5- gluconolactone.This step can be carried out with various enzymes, such as
Oxygen dependence glucoseoxidase family (EC 1.1.3.4) or NAD (P)-dependent glucose dehydrogenase family (EC
1.1.1.118、EC 1.1.1.119).Show that, when growing in fermentation tank, gluconobacter oxydans are by glucose oxygen effectively
Turn to gluconic acid and 5- ketogluconate (5-KGA).Can using see solubility in Gluconobacter and other oxidizing bacterias and
The enzyme (EC 1.1.99.35 and EC 1.1.5.2) of film combination PQQ- dependent enzyme family.Quinoprotein glucose applies to carry out
Another kind of enzyme of this step.The certain enzyme selected will depend upon required reaction condition and will be present in react in necessary auxiliary because
Son, it is shown in Table 1.
Step 1A-1,5- gluconolactone conversion (for example hydrolyzing) is gluconic acid.This step can be to change in water-bearing media
Mode is carried out and hydrolysis rate depends on pH (Shimahara, K, Takahashi, T., Biochim.Biophys.Acta
(1970), 201,410).Hydrolysis is very fast and slower at acidic under alkaline pH (such as pH 7.5).Many microorganisms also contain
There is specific 1,5- gluconolactone hydrolytic enzyme, and have some to be cloned in them and characterize (EC 3.1.1.17；
Shinagawa, E Biosci.Biotechnol.Biochem.2009,73,241-244).
Step 1B- guluronic acid lactone is converted into guluronic acid.The chemical hydrolysis of guluronic acid lactone can pass through
Spontaneous reaction in aqueous completes.The enzyme that this hydrolysis can be catalyzed is identified (EC among numerous lactonases
3.1.1.XX and more specifically, 3.1.1.17,3.1.1.25).
Step 2- gluconic acid is converted into 3- dehydrogenation gluconic acid (DHG)：Several enzymes, such as gluconate dehydratase, can be used for glucose
Acid is dehydrated in the reaction of dehydrogenation gluconic acid (DHG).Example includes the those (EC belonging to gluconate dehydratase family
4.2.1.39).One particular instance of described dehydratase shown gluconic acid be dehydrated (Kim, S.Lee,
S.B.Biotechnol.Bioprocess Eng. (2008), 13,436).Carry out the instantiation of the enzyme of family and its clone since then
It is shown in embodiment 1.
Step 3：3- dehydrogenation-gluconic acid (DHG) is converted into 4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH).Have described that
For carrying out the enzyme of this conversion, 2- dehydrogenation -3- deoxidation-D- Gluconate 5-dehydrogenase (or DHG dehydrogenase) (EC 1.1.1.127).
Step 4：4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) is converted into 4- deoxidation-L- threo form-ketohexose alduronic acid
(DTHU).The enzyme of EC 5.3.1.12 family can be used for this step, and step 15 shows that five kinds of described enzymes are cloned and represent tool
There is the activity for 5-KGA dehydration.These enzymes also show that the activity for 2,5-DDH and DTHU.
Step 5：DTHU is converted into 5- dehydrogenation -4- deoxidation-glucosaccharic acid (DDG).DDG can be by for example being urged with gentle chemistry
The chemistry of the DTHU that agent is carried out or enzymatic oxidation produce, and described chemical catalyst can aoxidize aldehyde in the presence of alcohol.Oxidation of aldehydes
Enzyme can be used for being catalyzed this oxidation reaction.Oxidizing bacteria such as Acetobacter sp. and Gluconobacter (Hollmann et al. Green
Chern.2011,13,226) screening will be applied to.The enzyme of following family can carry out this reaction：Aldehyde oxidase EC1.2.3.1, aldehyde
Ferrodoxinss oxidoreductase (EC1.2.7.5) and in all families of EC 1.2.1.-XX.Alduronic acid dehydrogenation
The enzyme (for example, with reference to step 7) of enzyme (EC 1.1.1.203) family also will have this activity.Can be lived using having alcohols and aldehydes oxidation
Property other enzymes, including aldehyde alcohol aoxidize enzyme family in enzyme (referring to step 19 and 6).Other extensive substrate oxidation enzymes include
The PQQ- dependency alcohol/aldehyde oxidase of solubility and film combination.More specifically, I type (EC 1.1.9.1) and II type (EC are belonged to
1.1.2.8) the solubility pericentral siphon PQQ oxidase of family and its congener and the film combination belonging to EC 1.1.5.X family
PQQ oxidase is applicable.In other embodiments, can be using the aldehyde dehydrogenase/oxidase acting on DTHU.
Step 5 also can be using from acetic acid bacteria such as Gluconobacter and acetobacter and gluconic acid acetobacter
Deng dehydrogenase carry out.Microorganism is screened by the oxidation with regard to DTHU and identifies full cytoactive.Identified activity simultaneously clones one kind
Or multiple enzyme.Also screen there is the enzyme of alditol dehydrogenase activity and find that there is this activity described in step 7 and 7B.
Also clone the library of PQQ dependent enzyme of solubility pericentral siphon and film combination and find that some enzymes have this activity.Find wherein
Some active enzymes are NAD (P)-or PQQ- dependent dehydrogenase, and other are FAD dependency aldehyde dehydrogenases.SEQ ID
NO：71-72 is the example of NADP dependent dehydrogenase, and any one or combination therein can be used in carrying out step 5.SEQ
ID NO：73-84 is the example of suitable PQQ dependent dehydrogenase, and any one or combination therein can be used in being walked
Rapid 5.
Step 6 and 6A：Gluconic acid is converted into guluronic acid (6) and 3- dehydrogenation-gluconic acid (DHG) be converted into 4- deoxidation-
5- erythro-ketohexose alduronic acid (DEHU) (6A).Described enzyme is applied to these conversions in steps of 5.Other useful enzyme bags
Include NAD (the P)-dependent dehydrogenase in EC 1.1.1.XX family and more specifically glucuronic acid dehydrogenase (EC
1.1.1.19), glucuronolactone reductase (EC 1.1.1.20).In addition, having the O of the wide substrate spectrum including sugar in a large number2
Dependency alcohol oxidase will be useful (EC 1.1.3.XX), including sorbitol, mannitol oxidase (EC 1.1.3.40), oneself
Carbohydrate oxidase (EC 1.1.3.5), alcohol oxidase (EC 1.1.3.13) and Rhizoma et radix valerianae aldehyde oxidase (EC 1.1.3.38).PQQ according to
Bad property enzyme and enzyme present in oxidizing bacteria can also be used for these conversions.
Step 7 and 7B：Guluronic acid is converted into D- glucosaccharic acid (7) and L- iduronic acid is converted into idosaccharic acid
(7B).These steps can be completed with the enzyme (EC 1.1.1.203) of alduronic acid dehydrogenase family or oxidase as described herein.
The example of alditol acidohydrogenase includes SEQ ID NO：1-6, and any one or any combinations therein can be used in being walked
Rapid 7 and 7B.
Step 7A：4- deoxidation -5- erythro-ketohexose alduronic acid (DEHU) is converted into 3- deoxidation-D- erythro-methyl-n-butyl ketone sugar two
Sour (DDH).Identical enzyme described in step 5 will be applied to and carry out this conversion.Similar to step 5, for step 7 and 7B,
Identification has the enzyme of described activity, and it is NAD (P)-or PQQ- dependent dehydrogenase, and other are FAD dependency aldehyde dehydrogenases.
The example of NADP dependent glucose acid -5- dehydrogenase includes SEQ NO：The example of 71-72 and PQQ dependent dehydrogenase includes SEQ
ID NO：73-84, and any one or any combinations therein can be used in carrying out step 7 and 7B.
Step 8 and 8A：D- glucosaccharic acid is converted into 5- dehydrogenation -4- deoxidation-glucosaccharic acid (DDG) (step 8) and idose
Diacid is converted into DDG (step 8A).The enzyme (EC 4.2.1.40) of glucarate dehydratase family can be used for carrying out these steps.
The enzyme of this family has been cloned and has shown that glucosaccharic acid is effectively converted into DDG.Glucosaccharic acid dehydration as following clone
Two kinds of D- glucarate dehydratases (EC 4.2.1.40) are cloned shown in the table of enzyme.Two kinds of enzymes are dehydrated as DDG for glucosaccharic acid
Show high activity, measured using semicarbazides, as described in step 2.
The glucarate dehydratase of clone
Step 9 and 9A：D-Glucose is converted into α-D- glucose-hexandial sugar -1,5- pyranose (9) and the conversion of D- galactose
For D- galactose-hexandial sugar (9A).Oxidase, as galactose oxidase enzyme family (EC 1.1.3.9), can be used for this
Step.Mutation beta-Galactose oxidase is also engineered with active to glucose and have been described (Arnold, F.H. et al.
ChemBioChem, 2002,3 (2), 781).The enzyme of step 9A available categories EC 1.1.3.9 is carried out.
Step 10：α-D- glucose-hexandial sugar -1,5- pyranose is converted into α-D- Portugal's pyrans alduronic acid (step 10) and D-
Galactose-hexandial sugar is converted into galacturonic acid (10A) this step can be carried out using the enzyme of aldehyde dehydrogenase family.Equally, by
The enzyme that those of step 5 are identified will be applied to this two conversions.
Step 11 and 11A：α-D- Portugal pyrans alduronic acid is converted into glucuronic acid 1,5- lactone.Aldehyde as described in step 5
Dehydrogenase and oxidase will be applied to and carry out this step.Alditol acidohydrogenase described in step 7 and 7B be equally applicable to into
This step of row.Step 11A is that galacturonic acid is converted into galactobionic acid Galactonic acid.Alditol acidohydrogenase (EC 1.1.1.203), for example, exist
Those described in step 7 and 7B, will be applied to and carry out this step.
Step 12：Fructose converting for glucose.Glucose and fucose isomerase (EC 5.3.1.5) will be applied to and carry out this
Step.
Step 13：Galactobionic acid Galactonic acid is converted into 5- dehydrogenation -4- deoxidation-D- glucosaccharic acid (DDG).Galactobionic acid Galactonic acid dehydrogenase family
Enzyme (EC 4.2.1.42) can be used for carrying out this step, and extra enzyme can be engineered for carrying out this step.
Step 14：Gluconic acid is converted into 5- ketogluconate (5-KGA).NAD (P)-dependent dehydrogenase family (EC
1.1.1.69 many enzymes) have been cloned and the reduction of the oxidation to gluconic acid or 5KGA is active.For example, synthesize to come
From the sour 5- dehydrogenase of the NADPH dependent glucose of Gluconobacter (Expasy P50199) for as shown here in large intestine bar
Optimum expression in bacterium and being cloned in pET24 (pSGI-383).Express described enzyme and show that it has required activity.
Include those in the PQQ dependent enzyme family being present in Gluconobacter be applied to the extra enzyme carrying out this step
(Peters, B. et al. Appl.Microbiol Biotechnol., (2013), 97,6397) and described in step 6
Enzyme.Can also be used for synthesizing 5KGA from gluconic acid from the enzyme of these families.
Step 15：5-KGA is converted into L- iduronic acid.This step can use the various enzymes from different isomerization enzyme family to enter
OK, as further described in example 4.Example includes SEQ ID NO：The isomerase of 7-19 or with SEQ ID NO：7-19's
Isomerase has the congener of at least 70% sequence iden；Or by SEQ ID NO：The isomerase of the nucleic acid coding of 20-32 or
The congener of any of which.
Step 16：5-KGA is converted into (4S) -4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH).The available glucose of this dehydration
Enzyme (EC 4.2.3.39) in acid dehydration enzyme family, as described in embodiment 5 or step 17 carry out.Can be used for walking
The example of rapid 16 gluconate dehydratase includes SEQ ID NO 33-35 (by SEQ ID NO：36-38 encodes, and therein
One or any combinations can be used in carrying out step 16 or its congener.
Step 17 and 17A：L- iduronic acid is converted into 4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) and ancient sieve
Alduronic acid is converted into 4- deoxidation-erythro -5- ketohexose alduronic acid (DEHU).
Identification can be used for carrying out the enzyme of the dehydration enzyme family of this step.From gluconic acid or glucarate dehydratase family
Enzyme carries out the required activity of these steps by having.Additionally, many dehydrations enzyme family (EC 4.2.1.X) are applied to carries out these
Step.Particularly, 1,2- dihydroxy acid being dehydrated optionally to produce the enzyme of 2- keto acid to be useful, as following family
Enzyme：EC 4.2.1.6 (galactonate dehydratase), EC 4.2.1.8 (mannonate dehydratase), EC 4.2.1.25 (arabinose
Sour dehydratase), EC 4.2.1.39 (gluconate dehydratase), EC 4.2.1.40 (glucarate dehydratase), EC 4.2.1.67
(fucose acid dehydratase), EC 4.2.1.82 (xylonate dehydratase), EC 4.2.1.90 (Fructus rhamni (Rhamnus davurica Pall.) saccharic acid dehydratase) and two
Hydroxy acid dehydratase (4.2.1.9).Because known enzyme selectivity is the generation of 2-ketoacid, so the enzyme of identification is produced respectively
DEHU and DTHU is as product.Step 19：1,5- gluconolactone is converted into guluronic acid lactone.This step can be passed through
Aldehyde alcohol aoxidizes the enzyme (EC 1.1.3.41) of enzyme family or enzyme described in step 6 is carried out.Can be used for the aldehyde alcohol oxidation of step 19
The example of enzyme includes SEQ ID NO39-54 or its congener of any one, or by SEQ ID NO：The nucleic acid coding of 47-54
Aldehyde alcohol oxidase or its congener of any one；And any one or any combinations therein can be used in carrying out.Step
19.DDG the method being converted into FDCA and manufacturing DDG and FDCA of esterification.The present invention also provides DDG to be converted into FDCA and FDCA
The novel method of ester.The ester of FDCA includes diethylester, dibutyl ester and other esters.Methods described is included by DDG and alcohol, mineral acid
And DDG is converted into DDG ester to produce the derivant of DDG by optional cosolvent contact.Alcohol can be methanol, ethanol, propanol, different
Propanol, butanol, isobutanol, amylalcohol, hexanol, enanthol, capryl alcohol, nonyl alcohol, decanol, undecyl alcohol, lauryl alcohol, tridecanol, tetradecyl alchohol, ten
Pentol, hexadecanol, Heptadecyl alcohol, octadecanol, nonadecanol, eicosanol, dimethyl sulfoxide, dimethylformamide, Polyethylene Glycol, methyl are different
Butyl ketone or any C1-C20 alcohol.Mineral acid can be sulphuric acid, phosphoric acid, perchloric acid, nitric acid, hydrochloric acid, Fluohydric acid., hydrobromic acid and hydrogen
Iodic acid.Cosolvent can be following any one or any mixture：THF, acetone, acetonitrile, ether, butyl acetate, dioxane, chloroform,
Dichloromethane, 1,2- dichloroethanes, hexane, toluene and dimethylbenzene.Any combinations of alcohol, mineral acid and cosolvent can be used in instead
Ying Zhong.The DDG of esterification can be subsequently converted to the FDCA being esterified, for example, realized by contacting it with acid catalyst.
DDG purification
For be dehydrated or be esterified DDG purification be by be acidified DDG, for example pass through by means of add dense HCl by react
PH is down to pH～2.5 to carry out.At this ph, protein is filtered to remove and any remaining glucosaccharic acid precipitates and right
Mixture carries out lyophilizing to obtain the white powder being made up of DDG and reacting salt.After enzyme is filtered to remove, can be in neutrality
Lyophilizing mixture under pH.In the case of not being further purified, DDG can subsequently dehydrated obtaining 2,5-FDCA, or de-
Esterified one-tenth dibutyl DDG (or diethyl DDG) before water.The one or more steps of purification or esterification DDG can be added to this
In any method and approach of generation DDG disclosed in literary composition.Also the additive method of purification DDG from aqueous mixture can be used.
These methods include carrying out separating using the film of capture salt or DDG etc. or ion exchange resin.
Therefore, the present invention provides a kind of method of purification DDG, and it includes being acidified in the solution DDG, molten via membrane filtration
Liquid, and from solution, (for example by lyophilization or spray drying) removes water.Solution containing DDG can be acidified to 2.5-
The pH of pH or 5.0-6.0 of pH or 4.5-5.5 of pH or 4.0-5.0 of pH or 3.5-4.5 of 3.5 pH or 3.0-4.0 or
The pH of pH or 7.5-8.5 of pH or 7.0-8.0 of pH or 6.5-7.5 of pH or 6.0-7.0 of 5.5-6.5 or about 8 pH.Remove
The water yield can more than 80% or more than 85% more than 87% water or more than 90% water more than 95% water or be more than
97% or more than 98% or more than 99% self-contained DDG solvent water.Can obtain more than 25 moles of % or 30 mole of % or
The yield of 35 moles of % or 40 mole of % or 45 mole of %.In one embodiment, methods described does not include ion exchange color
Spectrum step.
Method for synthesizing FDCA and FDCA derivant
The present invention also provides the various methods of synthesis FDCA.A kind of method for synthesizing FDCA is included DDG and alcohol, no
Machine acid contacts at high temperature to form FDCA.Alcohol can be any alcohol (for example, as mentioned above in those of any one), and real
Example includes but is not limited to methanol, ethanol, propanol and butanol.Also glycol can be used.High temperature may be greater than 70 DEG C or is more than 80 DEG C
Or more than 90 DEG C or more than 100 DEG C or more than 110 DEG C or more than 120 DEG C or more than 130 DEG C or more than 140 DEG C or it is more than 150 DEG C
Temperature to form FDCA.Can achieve the reaction yield more than 20% or more than 30% or more than 35% or more than 40%.
The present invention also provides the method for synthesizing FDCA derivant.Methods described include by the derivant of DDG with inorganic
Acid contact is to produce the derivant of FDCA.Mineral acid can be such as sulphuric acid or any mineral acid, as described above those.Optionally
Ground, can before it is contacted with the second mineral acid purification DDG derivant.The non-limiting examples of the derivant of DDG or FDCA
Including but not limited to：Methyl DDG, ethyl DDG, propyl group DDG, butyl DDG, isobutyl group DDG, dimethyl DDG, diethyl DDG, two
Propyl group DDG, dibutyl DDG.The derivant of produced FDCA can for but be not limited to methyl FDCA, ethyl FDCA, propyl group FDCA,
Butyl FDCA, dimethyl FDCA, diethyl FDCA, dipropyl FDCA, dibutyl FDCA and isobutyl group FDCA.Produced
The derivant of FDCA corresponds to the derivant of DDG used in method.The derivant of FDCA can subsequently by deesterify to produce
FDCA.Methods described also can for example be carried out using parameter as described below in the gas phase.
Another kind of method for synthesizing FDCA or FDCA derivant includes DDG or DDG derivant is (any described herein
) contact in the gas phase with mineral acid, this can complete under following short residence time, e.g., less than 10 seconds or be less than 8 seconds or little
Or in 6 seconds or or less than 4 seconds or less than 2 seconds or it was less than 1 second less than 3 seconds less than 5 seconds.The time of staying refers to that sample is present in high temperature
Flow through the time in the reaction zone of type reactor.Methods described also can be carried out at high temperature, such as more than 150 DEG C, more than 200
DEG C, more than 250 DEG C, more than 300 DEG C or more than at a temperature of 350 DEG C.Can obtain more than 25 moles of % or more than 30 moles of % or
Yield more than 40 moles of % or more than 45 moles of % or more than 50 moles of %.Another kind of method for synthesizing FDCA include by
DDG is being contacted more than 80 DEG C or 90 DEG C or at a temperature of 100 DEG C or 110 DEG C or 120 DEG C with mineral acid.For synthesizing the another of FDCA
A kind of method includes contacting DDG under the conditions of anhydrous response with mineral acid.In various embodiments, anhydrous condition can pass through
In any method of synthesis FDCA disclosed herein, lyophilizing DDG is so that the water that DDG contains following content to be set up：It is less than
10 weight % or less than 9 weight % or less than 8 weight % or less than 7 weight % or less than 6 weight % or be less than 5 weight % or little
Water in 4 weight % or less than 3 weight % water or less than 2 weight %.
The inventive method for synthesizing FDCA and its derivant as described herein provide than more available obvious more
High yield.The molar yield of following FDCA (to DDG) in different embodiments, can be obtained：More than 10% or be more than
15% or more than 20% or more than 25% or more than 30% or more than 35% or more than 40% or more than 45% or more than 50% or
More than 60% or more than 65% or about 40% to about 70% or about 45% to about 65% or about 50% to about 60%.
Embodiment
Embodiment 1- step 2, gluconic acid is converted into 3- dehydrogenation-gluconic acid (DHG)
The enzyme with the natural activity being dehydrated for gluconic acid is be applied to the present invention (EC 4.2.1.39).From this
Three kinds of enzymes of family are cloned as shown in table 1.Enzyme pSGI-365 is cloned and is proven with the dehydration of wide substrate spectrum
Enzyme, its for gluconic acid dehydration have strongly active (Kim, S.Lee, S.B.Biotechnol.Bioprocess Eng.2008,
13,436).
Table 1：Enzyme and homogeneity homology used in here experiment.All expression all in pseudomonas fluorescens
| 359_ achromobacter | 365_E3HJU7 | |
| PSGI-360_ acinetobacter (SGI) | 78 | 79 |
| PSGI-359_ achromobacter (SGI) | 95 | |
| PSGI-365_ Aeromonass |
Albumen 359,360 and 365 (respectively SEQ ID NO 33-35) for gluconic acid dehydration synthesize (gel does not show
Go out) show the thick enzymatic lysises thing activity that every mg is 2-5 μm of ol/min.PSGI-359 is passed through with ammonium sulfate precipitation and again to dissolve
To separate in buffer and to be measured by semicarbazides and to be analyzed.Calculated for gluconic acid dehydration by semicarbazides mensure figure
46.2U/mL or 5.3U/mg (1 unit=μm ol/min) activity.Containing Kpi 10mMpH 8.0 and will have 2mM
The reaction buffer (93mL) of MgCl2 and 3.5gr (0.016 mole) the gluconic acid sodium salt gluconate dehydratase solution previous with 7mL
Mixing.Reactant is cultivated at 45 DEG C 16h, afterwards an aliquot (Fig. 4) is analyzed by HPLC-MS.As shown in figure 4,
Produce a kind of new primary product of the molecular weight with DHG.This product also shows the activity with DHG dehydratase.
All proteins are all in pRANGERTMClone on (Lucigen, Middleton, WI) expression vector and false in fluorescence
Express in aeromonas strain.pRANGERTMThe commercially available plasmid vector of extensive host, its contain pBBR1 replicon,
Kalamycin resistance and the pBAD promoter of the inducible expression for gene.For enzymatic determination, will be used for quantifying the ammonia of α keto acid
The revision that base urea measures is used for calculating activity (Kim, the S. of every kind of enzyme；Lee, S.B.Biochem J.2005,387,
271).SEQ ID NO：30-32 and 33-35 shows the aminoacid being respectively gluconate dehydratase #0385, #0336 and E3HJU7
And nucleotide sequence.
Embodiment 2- step 3-3- dehydrogenation-gluconic acid (DHG) is converted into (4S) -4,6- dihydroxy 2,5- diketone caproic acid (2,
5-DDH).
Enzyme family (EC 1.1.1.127) can be used for carrying out this step.Two examples are 2- dehydrogenation -3- deoxidation-D- gluconic acids
5- dehydrogenase and DHG dehydrogenase.Five kinds of enzymes coming family since then are cloned as shown in Table 2 below.pRANGERTMCarrier is used for
Each situation.
Table 2：The clone of DHG oxidoreductase (or 2- dehydrogenation -3- deoxidation-D- Gluconate 5-dehydrogenase)
It is used as the substrate of the lysate of analytical table 2 by the product of the dehydration preparation of the gluconic acid in step 2.As table 3 below
Shown in, identify DHG aoxidize to the active enzyme of display (absorbance carries under 340nm in the mensure that measurement NADH is formed
High).
Table 3：It is oxidized to the activity calculating of 2,5-DDH using the DHG of DHG oxidoreductase.
The NADH of unit=μm ol/min
It is shown in step 16 by the checking further that these enzymes form 2,5-DDH, wherein show and use at acidic
PSGI-395 reductase 12,5-DDH (is obtained by the dehydration of 5KGA).
Embodiment 3- step 7 and 7B- guluronic acid is converted into D- glucosaccharic acid (7) and L- iduronic acid is converted into Chinese mugwort
Du's saccharic acid (7B).
In order to prove step 7 and 7B, carry out following research.Alditol acidohydrogenase (EC 1.1.1.203) is Gluconobacter oxydans aldehyde
Acid and the enzyme of galacturonic acid.Bacterial strain clone shown in table 4 and 5 has sequence similarity with known alduronic acid dehydrogenase
Three kinds of enzyme (Expasy：Q7CRQ0；Prather, K.J et al., J.Bacteriol.2009,191,1565).
The alditol acidohydrogenase of table 4- clone
| Organism | pSGI(pET28) | Gene I/D | Expression |
| Edaphic bacilluss | #474 | #8807 | BL21DE3 |
| Root nodule bacteria | #475 | #8958 | BL21DE3 |
| Pseudomonass | #476 | #1770 | BL21DE3 |
Table 5- sequence iden
| #475 | #476 | Q7CRQ0 | |
| 474_ edaphic bacilluss | 73 | 49 | 90 |
| 475_ root nodule bacteria | 51 | 74 | |
| 476_ pseudomonass | 50 |
With carry out purification from the every kind of albumen of His tag expression of pET28 and before its screening.Thick lysate and pure
The protein gel changing enzyme is shown in the gel of Fig. 1.After purification, with regard to the activity for glucuronate and for Gu
The activity of sieve glycuronate and iduronate is tested to all enzymes.Carry out kinetics survey under different concentration of substrate
Measure and be shown in Table 6 for the activity and Km value of every kind of enzyme calculating.All enzymes all show for glucuronate and are directed to L-
Iduronic acid and the excellent activity of guluronic acid.
Table 6：The activity of alditol acidohydrogenase of purification and Km value.
Each plasmid shown in table 4 converts in BL21DE3 Bacillus coli cells.By the lysate of clarification and grade body
The level pad of long-pending (25mL) mixes and is incorporated in purification on Ni NTA post.By by the various dilutions of every kind of for 0.050mL purifying enzyme
Liquid and 0.95mL reaction buffer (100mM TrisHCl, pH 8.0,50mM NaCl, 0.75mM NAD+) mix every to measure
Plant the activity of purifying enzyme.Measure reaction process by monitoring the formation of NADH under 340nm.Fig. 6 a and 6b provides glucuronic acid
The Lineweaver-Burk of the oxidation of salt and iduronate draws, and wherein all three enzyme is shown in Fig. 6.Be given
Obvious positive slope is all obtained in the case of all enzymes of activity shown in upper table.The protein sequence of alditol acidohydrogenase shows
For SEQ ID NO：1-3 and gene is shown as SEQ ID NO：4-6.
Pyrroloquinoline (PQQ) dependency aldehyde dehydrogenase displays that the oxygen for guluronic acid salt and iduronate
The excellent activity changed.These are solubility pericentral siphon enzymes, after their pericentral siphon target sequence removes in escherichia coli kytoplasm liquid
Expression.The activity of the thick lysate in terms of the unit (μm ol/min) of every milligram of total lysate protein is shown in table 6 below A.As
If fruit purification, the substantial activity of every kind of enzyme is at least 2-5 times high (expression referring in Fig. 3).
Table 6A：The activity (unit=μm ol/min) of PQQ dependent dehydrogenase under iduronic acid and guluronic acid
| Enzyme | Iduronic acid U/mg | Guluronic acid U/mg |
| P75804(SEQ ID NO：73) | 8.7 | 3.2 |
| 9522(SEQ ID NO：74) | 7.3 | 6.1 |
| 6926(SEQ ID NO：75) | 9.2 | 4.1 |
| 7510(SEQ ID NO：76) | 7.3 | 3.7 |
| 7215(SEQ ID NO：77) | 14.2 | 8.3 |
| 8386(SEQ ID NO：78) | 4.3 | 1.5 |
According to below scheme, using the activity shown in artificial electron receptor DCPIP (2,6- dichloroindophenol) measurement table 6A：
Contain the 20mM of 0.2mM DCPIP, 0.2mM PMS (phnazine ethosulffate) and substrate (10-40mM) in 0.95mL
In triethanolamine (pH 8.0), add 0.050mL enzyme (as thick lysate or with buffer 10-100x dilute) and react into
Exhibition is the change according to DCPIP absorbance under 600nm.Because these enzymes will in film electron transport chain in its relaxed state
Electron transfer gives other protein or cofactor, so being subject to bulk measurement external activity using artificial electron, wherein DCPIP is the most normal
See.
It is active, including butyraldehyde, butyraldehyde and glycerol (and non-glucose) that enzyme in table 6A is directed to other aldehyde many.Therefore,
These enzymes will aoxidize the aldehyde radical of iduronate and guluronic acid salt to respectively obtain iduronic acid and glucosaccharic acid.For
Confirm this selectivity, two in these enzymes, #403 and #412, by pericentral siphon with #403 (native E. coli enzyme) by it
Target sequence merges and expresses in colibacillary pericentral siphon.Two kinds of protein are expressed all in pericentral siphon, but water compared with cytosol
Flat relatively low.Reconstitution cell before with good yield by oxidation of Benzaldehyde as benzoic acid and with compared with low-yield by gulose aldehyde
Hydrochlorate and iduronate produce glucosaccharic acid and idosaccharic acid.
Embodiment 4- step -15：5- ketogluconate (5-KGA) is converted into L- iduronic acid (15) or guluronic acid
(15A).
This example illustrates and 5-KGA can be isomerized to iduronic acid (step 15) or guluronic acid (step
The identification of enzyme 15A).13 kinds of enzymes from three different isomerization enzyme families are cloned as shown in table 7, and its sequence is same
Property % is shown in Table 8.
Table 7：The isomerase cloned
Table 8：Homogeneity % of isomerase
As shown in table 8, the enzyme in each family with medium homology (underlining) is selected to be cloned.Data proves
Enzyme from all families all shows activity for the isomerization (obtaining L- iduronate as primary product) of 5-KGA.
Two kinds of enzymes (433 and 434) from 5.3.1.17 family also use in an embodiment, and display is formed from 5- ketogluconate (5KGA)
DDG.
Use the activity for 5KGA and the isomerization of iduronic acid for the enzyme of table 7, the method using enzymatic method measurement
The formation of the Activity determination product of two kinds of different enzymes is directed to by it.For example, the isomerization of 5KGA is by using alditol
Acidohydrogenase (pSGI-476) measures the activity of product iduronate detecting.The isomerization of iduronic acid is by surveying
5KGA reductase (pSGI-383, the EC 1.1.1.69) activity of volume production thing 5KGA is detecting.Detect product also by GC-MS
Exist.
Show that from the enzyme of all families the change activity of the isomerization for 5KGA and iduronate will be derived from
Two kinds of enzymes of EC 5.3.1.12 are used in acellular reaction so that isomerization 5KGA and finally generation are as be shown in the examples
DDG.Show single band by enzyme purification and by gel electrophoresiss.The isomerase of purification is used for using containing 5KGA or Chinese mugwort
In the reaction of the lysate of Du's alduronic acid and buffer.Prove that product is formed using HPLC and previously described enzymatic method.Make
It is shown in Fig. 7 a with the result of the culture 17h of HPLC and enzymatic determination.All enzymes all show to the isomerization of 5KGA and iduronic acid
Show excellent activity.Worked as by the yield of the iduronic acid isomerization of pSGI433, pSGI 434, pSGI 435 and p SGI 436
56%, 48%, 42% (436 is unmeasured) respectively during enzymatic measurement and be 78.8% respectively when measurement is measured by HPLC,
78.5%th, 73.3% and 76.6%.By the yield after the 16h of the 5KGA isomerization of same enzyme when by enzymatic mensure measurement
When be 18%, 17% and 19% (436 is unmeasured) respectively, and be 16.6% respectively when measurement is measured by HPLC, 17.8%,
16.3% and 16.9%.
EC 5.3.1.12 enzyme
From EC 5.3.1.12 family enzyme (glucuronate isomerase) also by gel purified, separate, and pass through
It is mixed with reaction with the buffer (50mM HEPES, 1mM ZnCl2, pH 8.0) of the 5KGA containing 5mM or iduronic acid
Thing.Reactant is cultivated at 30 DEG C and is formed using HPLC and enzymatic method assay products.Result is shown in Fig. 7 b.
5.3.1.17 enzyme
Enzyme pSGI-478 and pSGI-479 (5- dehydrogenation -4- deoxidation-D- glucuronate isomerase) show for 5KGA and
The isomerization activity of both iduronic acids.The same enzymatic as above of this activity measures to confirm.By pSGI-478 and-
The isomerization yield of 479 iduronic acid is 50% and 37% respectively when enzymatic measures, and ought measure the time-division by HPLC
It is not 20% and 18%.The yield of 5KGA isomerization is 23% and 26% respectively when enzymatic measures, and ought be measured by HPLC
When be 24% and 16% respectively.Result is shown in Fig. 7 a.
5.3.1.n1 enzyme
By the enzyme in this family gel purified.Measure measurement product using enzymatic as above to be formed and result
Shown in Figure 8.In this family, all enzymes of clone all show that the isomerization for 5KGA and iduronic acid is active.
In every case, plasmid is converted and protein purification on Ni NTA post in BL21DE3.
Embodiment 5：Step 16-5- ketogluconate (5KGA) is converted into (4S) -4,6- dihydroxy 2,5- diketone caproic acid (2,5-
DDH).
Three kinds of gluconate dehydratases described in step 2 (embodiment 1) are expressed as described in example 1 above, together with being derived from
The glucarate dehydratase of the purification of step 8 is together.Carry out for active enzymatic reaction and HPLC-MS analysis display 2,5-
The formation (Fig. 9) of DDH, it to confirm also by following facts：The formation of new product is accompanied by and is only containing gluconate dehydratase
Sample in 5-KGA reduction, and also by being measured with the enzymatic of DHG dehydratase (pSGI-395).Good under 340nm
Good slope represents and obtains larger enzymatic activity when mixing the aliquot of NADH, pSGI-395 lysate and previous reactant
(data is not shown).5KGA is dehydrated as 2,5-DDH the gluconate dehydratase that this result joint HPLC analytical proof is checked.
Embodiment 6：Step 19-1,5- gluconolactone is converted into guluronic acid delta-lactone.
1,5- gluconolactone oxidation is the secondary activity of the enzyme from aldehyde alcohol oxidase (EC 1.1.3.41) family.These
The various aldehyde alcohol of oxydasises, such as Sorbitol, xylitol, glycerol etc..Identification is active to the oxidation of 1,5- gluconolactone
Enzyme, as shown in Table 6 below.
The table 6. aldehyde alcohol oxidase active to 1,5- gluconolactone.
* thick lysate
Lysate using all purifying enzyme shown in table 6 prepares reactant.In 50mM, there is 0.5mg/mL peroxidating
Prepare reactant in the K- phosphate buffer pH 7.0 of hydrogen enzyme and cultivate at 30 DEG C.Newly produced by HPLC-MS analysis and observation
Thing, is shown and guluronic acid identical retention time (Figure 10) after being compared with trusted standard.This is to be demonstrate,proved by GC-MS
Real, wherein product also has and guluronic acid identical MS fingerprint.It is therefore apparent that the aldehyde alcohol oxidase oxygen described in table
The 6-OH changing 1,5- gluconolactone is to produce guluronic acid lactone.All of aldehyde is cloned in the pET28a have HisTag
Alcohol oxidase and in BL21DE3 expression and on Ni NTA post purification.
Embodiment 7-FDCA and the synthesis of other intermediate
The DDG monopotassium salt of purification is used for being dehydrated into 2,5-FDCA.Add sulphuric acid to stir in DDG and at 60 DEG C instead
Should.The original position yield that (by HPLC-MS) calculates is～24% and～27%.
Merge reaction solution, then to be diluted by pouring (with the heat of neutralization) in ice into.Add the THF of roughly equal volume,
And solution is transferred to separatory funnel.Add sodium chloride salt until realizing separating.Agitation solution between adding is so that optimal
Possible dissolving.Remove water layer, and wash THF layer 3x with NaCL saturated solution.Add sodium sulfate by solution left standstill overnight.
Form two-layer overnight again.Abandon water layer, then add silica gel in solution.Subsequently it is condensed into solid via rotary evaporator
Body.By solids laden in the quick post of silicon dioxide, then separate via chromatography.It is concentrated and dried fraction.Separating yield is
173.9mg.Correcting yield：24.9%.1H and13C NMR and HPLC-MS analysis confirms product.
DDG dibutyl -2,5-FDCA is in BuOH/H2SO4In dehydration
Complete the dehydration of the lyophilizing DDG of the non-derivative containing the dehydration salt in BuOH using Dean-Stark device.
Under these conditions, add DDG to BuOH in, then add H2SO4 and at 140 DEG C reacting by heating.After stirring 4h,
HPLC-MS analysis display DDG disappears and forms dibutyl 2,5-FDCA.The original position yield that (by HPLC-MS) calculates is
36.5%..
Use water extraction by mixture water, 1%NaOH and again.Subsequently concentration of organic layers to 37.21g final mass.
Remove a part (3.4423g) and dibutyl -2 using HPLC purification 0.34g of this quality, 5-FDCA.By separation product
Yield be extrapolated to by the compound of Reaction Separation total amount (37.21g) and in view of the salt amount in original DDG of being present in
(～60 weight % are pure), reaction yield is calculated as 42%.1H and13C NMR and HPLC-MS analysis confirms product.
The synthesis of dibutyl DDG
On the other hand, the present invention provides a kind of method for synthesizing DDG derivant.Methods described include by DDG and alcohol,
Mineral acid and optional cosolvent contact to produce the derivant of DDG.Optionally, can purification DDG derivant.Reaction can have
The yield of following DDG derivant：At least 10 moles % yields or at least 15 moles % yields or at least 20 moles % yields or extremely
Few 25 moles of % or at least 30 moles % or at least 35 moles % yields or at least 40 moles % yields.Mineral acid can for sulphuric acid and
Alcohol can be methanol, ethanol, propanol, butanol, isobutanol or any C1-C20 alcohol.In various embodiments, cosolvent can be with
Descend any one：THF, acetone, acetonitrile, ether, butyl acetate, dioxane, chloroform, dichloromethane, 1,2- dichloroethanes, hexane, first
Benzene and dimethylbenzene.When alcohol is for ethanol, DDG derivant will be DDG mono- ethyl ester and/or DDG diethylester.When alcohol is for butanol, DDG
Derivant will be DDG mono- butyl ester and/or DDG dibutyl ester.
DDG monopotassium salt is used for the derivatization carrying out according to below scheme.In the 1L equipped with mechanical agitator and heating mantle
60: 40DDG: KCl (31.2mmol), BuOH and heptane is loaded in the recessed reaction vessel of Morton type.In single bottle, add
Plus sulphuric acid is in water, and allow to cool down after dissolution.Subsequently add solution in flask.Solution is maintained at 30 DEG C.
Precipitation is filtered, concentrates.Remaining gel is dissolved in EtOAc, then uses solution point sample TLC plate and use phosphorus molybdenum
Acid blend is sprayed to plate, is then heated at least 150 DEG C on hot plate to identify DDG-DBE fraction.Separate yield：4.62g
(15.2mmol, 47% yield), ＞ 98% purity.1H and13C NMR and HPLC-MS analysis confirms product.
Different solvents can be used in the synthesis of DDG ester, and the mixture of such as BuOH (5%-95%v/v) and following cosolvent is such as
THF, acetone, acetonitrile, ether (butyl oxide, diethyl ether etc.), ester for example butyl acetate, 1,6- dioxane, chloroform, dichloromethane, 1,2-
Dichloroethanes, hexane, toluene and dimethylbenzene.Catalysts such as acid (sulphuric acid, hydrochloric acid, polyphosphoric acid or immobilization acid such as DOWEX)
Or substrate (pyridine, ethamine, diethylamine, boron trifluoride) or other catalyst are generally used for the esterification of carboxylic acidss.
In n-BuOH/H
2
SO
4
Middle dibutyl DDG is dehydrated as dibutyl FDCA
Prepare the stock solution of DDG-DBE (dibutyl ester) and transfer to cleaning, dry 100mL round bottom in butanol
In flask, this flask is equipped with stirring rod.Add 25mL concentrated sulphuric acid in flask.Sealed flask, then at 60 DEG C, stirring 2 is little
When.Original position yield is calculated as～56%.Concentrated reaction solution and residue is dissolved in MTBE and transfers to separatory funnel,
Then wash with water.The organic layer of concentration and recovery, then separates via HPLC, separates yield：250.7mg (～90% purity) and
Separate yield：35% (correcting for purity).1C and13C NMR and HPLC-MS analysis confirms product.
Embodiment 8- is from 5-KGA acellular synthesis DDG and FDCA and derivant (route 2A)
This example illustrates and use purifying enzyme according to scheme 6 (subscheme of 2B) is DDG by 5KGA Enzymatic transformation, and
Also illustrate and using chemical step, produced DDG is dehydrated as FDCA.Described scheme comprises the following steps：The isomerization of 5KGA
(step 15) and then it is oxidized to idosaccharic acid (step 7B).DDG is also dehydrated as FDCA under different electrochemical conditions.Using next
Carry out final step (step 8A) from colibacillary glucarate dehydratase.
Scheme 6 is shown in Figure 11.Described scheme is carried out using the acellular enzyme' s catalysis of the DDG starting from 5-KGA.Described
Scheme includes execution step 15,7B and 8A (referring to Fig. 2 d).Complete response path using two kinds of extra protein, the first
For nadh oxidase (step A), it is in the presence of oxygen by NAD+ cofactor recirculation；With catalase (step B), its point
The peroxide that solution is produced by the effect of nadh oxidase.Enzyme is shown in table 7 below.All enzymes all contain HisTag and use
Ni-NTA post carrys out purification.
The yield of DDG synthesis is calculated as at least 88-97%.
Table 7：
Every kind of isomery enzyme purification 500mL liquid culture for reaction.In addition to the enzyme shown in table 7, each reaction
Also contain 50mM TrisHCl (pH 8.0), 50mM NaCl, 1mM ZnCl2With 2mM MgCl2、1mM MnCl2With 1mM NAD+.
By HPLC analytical reactions and Figure 12 assumes chromatogram after culture 16h.
It is FDCA for dehydration, the reactant mixture of two samples is merged and is lyophilized into white powder, be divided into
Two samples simultaneously each are dissolved in 0.25M H2SO4AcOH in or there is 0.25M H2SO44.5mL BuOH in.?
At 120 DEG C, in sealed vial, two reaction 2-4h product of heating are shown in Figure 13.
Sample 1 and 2 represents trusted standard respectively and is derived from AcOH/H2SO4In reaction 3h time point.With sample 1 to sample
Product 2 carry out admixture and obtain unimodal, further demonstrate that FDCA product.Sample 1 and 3 (Figure 13) represents trusted standard respectively and is derived from
BuOH/H2SO4In reaction 4h time point.FDCA is formed by enzymatic reaction and further demonstrate that DDG depositing in these samples
?.
Embodiment 9- synthesizes DDG by glucose and gluconic acid
This embodiment display glucose and gluconic acid Enzymatic transformation are DDG.Reacted with purifying enzyme, and thick lysate is made
For catalyst.Merge enzyme and substrate in bioreactor as shown in following table：
1. it is derived from the lysate of the 500mL liquid culture of recombination bacillus coli with plasmid
2. it is derived from the lysate of the 2L liquid culture of BL21DE3/pSGI-434
3. purifying enzyme, the activity (or GlucD of 3mg purification) of～30 units
4. it is derived from the lysate of 250mL culture
Cultivate reactant and dissolved oxygen and pH are kept at 20% and 8 at 35 DEG C.By HPLC-MS analysis time
Put and result is shown in the chromatogram checking DDG mass (not shown) extract in Figure 17 b and corresponding MS fragment.Result is clearly
It is shown in the generation of DDG during being cultivated with the enzyme that glucose or gluconic acid are carried out.
Embodiment 10- is used for the structure of the expression cassette of glucarate dehydratase of recombinating
The weight of the following example coded sequence containing D- glucarate dehydratase activity (GDH, EC 4.2.1.40) for the description
The formation of group nucleic acid construct is used for heterogenous expression in Bacillus coli cells.
Coding is derived from colibacillary D- glucarate dehydratase (Expasy：P0AES2；), acinetobacter ADP1
(Expasy：) and the gene of proprietary pseudomonad strain (#8114) enters performing PCR amplification by genomic DNA P0AES2.
Subsequently by each PCR amplification gene be cloned into bacterial transformation vectors pET24a (+) in, wherein each GDH gene
Expression is all located under the control of T7 promoter.Confirm to confirm the nucleotide sequence that each PCR expands insert also by sequencing.
The coli strain of embodiment 11- expression restructuring glucarate dehydratase
Each expression vector building as described in example 9 above is introduced NovaBlue by the conversion that heat shock mediates
(DE3) in escherichia coli.The transformant of presumption is selected on the LB agar being supplemented with kanamycin (50 μ g/ml).Will be suitable
PCR primer is used in colony-PCR mensure to determine the positive colony containing each expression vector.
For each expression vector, picking colony allow it being supplemented with kanamycin at 30 DEG C from reformer plate
Grow two days in the LB liquid medium of (50 μ g/ml).Subsequently culture is transferred in the bottle containing 15% glycerol and make
At pure culture for freezing is saved in -80 DEG C.
Embodiment 12- synthesizes DDG's in vitro by using the cell lysate expressing the recombinant Bacillus coli cells of GDH enzyme
Demonstration
This embodiment describes and how to use the restructuring glucarate dehydratase (GDH) producing in Bacillus coli cells real
The external synthesis of existing DDG intermediate.
The preparation of cell lysate：At 30 DEG C, being preset as on the gyrate shaker of 250rpm in rotating speed will be as previously existed
The recombinant bacterial strain building described in embodiment 2 is individually supplemented with raw in the LB liquid medium of kanamycin (50 μ g/ml) in 3mL
Long 1 day.This pre-culture is used for inoculating the 100mL TB culture medium that contains kanamycin (50ug/ml), then at 30 DEG C
It is preset as on the gyrate shaker of 250rpm cultivating 2-3 hour until early stage logarithmic (log) phase (OD600～0.5-0.6), add different afterwards
Propyl group D-1 thiogalactoside (IPTG；0.25mM ultimate density) with inducible protein expression.Cell is allowed to regenerate at 30 DEG C
Long 18 hours, pass through afterwards to be harvested by centrifugation, be resuspended in 15mL dissolve buffer (10mM phosphate buffer pH 7.8,
2mM MgCl2) in and dissolved by supersound process.Quantified using the prefabricated PAGE gel system (BioRad) of standard
Generation in Bacillus coli cells for the recombinase, and according to Gulick et al. (Biochemistry 39,4590-4602,
2000) the operation measurement specific activity described in.Subsequently test either in crude cell lysates or purifying enzyme (using HisTag) are by certain gram
The glucosaccharic acid of number is converted into the ability of DDG, described in greater detail below.
The enzymatic dehydration of glucosaccharic acid
Preparation is using the extensive oxidation of the glucosaccharic acid of glucarate dehydratase.By 350mL water, 25g glucosaccharic acid sodium
Salt (0.1 mole) and 4.5 grams of KOH (0.8 mole) mix in conical flask.Add 5M KOH solution (～3mL) by slowing down
Dissolve the solid glucosaccharic acid of residual and adjust pH to 7.4.Add the glucarate dehydratase of 100mg purification in this solution
With 2mM MgCl2, and at 30 DEG C, mixture is placed in 20h in orbit shaker.Next day, precipitation is filtered to remove.Instead
The pH answering substantially does not change.Response analysises represent only DDG in the solution to be existed, and indicates ＞ 95% yield.
Purification from the DDG product of enzymatic reaction：
The DDG producing via enzymatic dehydration by using any one purification in following two technology.With 6M HCl by enzyme
Promote dehydration and be acidified to pH～3.0, filter to remove precipitation, and subsequent lyophilizing is to produce the white powder being made up of DDG and salt
End.If reaction via 10KDa membrane filtration to remove protein and then lyophilizing, identical DDG purity (but relatively low amount can be obtained
Salt).In the case of less than further purification, first the first two freeze-dried powder can be dehydrated into FDCA (or its ester) or esterifiable
For dibutyl DDG, as shown in the other embodiment of the application.
The result of HPLC-MS analysis indicates that DDG product is constituted in the sample being obtained by any one of two purification techniques and receives
At least the 95% of thing.
Embodiment 13- is synthesized the demonstration of FDCA in a step chemical reaction in vitro by DDG
Applicants have found that the synthesis of FDCA (i.e. free acid form) can be by H2SO4In the presence of DDG be chemically converted to
FDCA and realize.Reaction is carried out as follows.By about 20mg DDG acid (be previously purified as described in example 3 above containing salt
Thick freeze-dried powder) and 0.25M H2SO4 be added in the airtight seal pipe containing 1mL water and 1mL DMSO.Find that DDG is complete
It is dissolved in this solution.18 hours hours of stirring reaction at 105 DEG C.The HPLC-MS analysis result that crude reaction sample is carried out is referred to
Show formation FDCA free acid (FDCA：FDCA) as primary product and micro some other unidentified pair
Product.As comparison in HPLC-MS analysis, analyze the FDCA of commercialization under the same conditions.
The demonstration of the external synthesis of embodiment 14-FDCA ester (dimethyl esters, diethyl ester, dibutyl ester and isopropyl esters)
Diethyl -2,5FDCA are synthesized by the DDG of purification：
In airtight seal pipe, add 18mL EtOH, 0.2 gram of (1 mM) DDG acid (previously as institute in embodiment 11
State purification) and 0.25M H2SO4.DDG acid is simultaneously incompletely dissolved in this solution.It is gently stirred for reactant 18 at 105 DEG C
Hour.The GC-MS analysis result display of crude reaction sample forms diethyl-FDCA as primary product.As comparison, will be real
FDCA synthesize in chemistry, be esterified as diethyl-FDCA and analyze at identical conditions.
Embodiment 15- synthesizes dibutyl 2,5FDCA by the DDG of purification
In airtight seal pipe, add 18mL n-BuOH, 0.2 gram of (1 mM) DDG acid (previously as in embodiment 11
Described purification) and 0.25M H2SO4.DDG acid is simultaneously incompletely dissolved in this solution.It is gently stirred for reactant at 105 DEG C
18 hours.As shown in figure 15, the GC-MS analysis result display of response sample forms diethyl FDCA (FDCA：2,5- furan diformazans
Acid) as primary product.As comparison, true FDCA is synthesized in chemistry, be esterified for diethyl-FDCA and in identical bar
Analyze under part.
Embodiment 16- synthesizes dibutyl 2,5FDCA (unpurified) by thick DDG：
Thick to 0.2 gram (1 mM) DDG acid is added in the gas-tight seal pipe containing 18mL n-BuOH, then adds
0.25M H2SO4, thick DDG acid is the unpurified jelly of the enzymatic dehydration acquisition of the directly glucosaccharic acid described in embodiment 11
Dry powder.Thick DDG acid is simultaneously incompletely dissolved in this solution.It is gently stirred for reactant 18 hours at 105 DEG C.Crude reaction sample
GC-MS analysis result display formed diethyl FDCA (FDCA：FDCA) as primary product.GC-MS result
Presence in thick/unpurified freeze-dried powder for the display pollutant salt is not significantly affected by reaction result.As comparison, will be true
Synthetic ester turns to diethyl-FDCA and analyzes at identical conditions real FDCA in chemistry.
Embodiment 17- uses the FDCA of immobilization acid and/or the external generation of ester
In industrial practice, immobilization acid provides many advantages for the carrying out of dehydration, and this is because it is generally in several species
Operation in the solvent (aqueous, organic or mixing etc.) of type.In addition, it can easy recirculation reusing.Follow some enforcements
Example, using immobilized15 (Rohm and Haas, Philadelphia, PA) and
50WX8 (Dow Chemical Co, Midland, MI) synthesizes the ester of FDCA.
By using
50 WX8 synthesize dibutyl FDCA by thick DDG
In a gas-tight seal pipe, by 2mL n-butyl alcohol, 20mg thick DDG acid (the unpurified freeze-dried powder containing salt)
And 200mg50 WX8 merge.DDG is simultaneously incompletely dissolved in this solution.It is gently stirred for reacting at 105 DEG C
Thing 18 hours.The GC-MS analysis result display of crude reaction sample forms diethyl-FDCA (FDCA：FDCA) make
For primary product.This GC-MS result shows pollutant salt (phosphate and NaCl) depositing in thick/unpurified freeze-dried powder
It is being not significantly affected by reaction result.As comparison, by true FDCA, synthetic ester turns to diethyl-FDCA and in phase in chemistry
Analyze under conditions of same.
By using
15 synthesize dibutyl FDCA by thick DDG
In a gas-tight seal pipe, by 2mL n-butyl alcohol, 20mg thick DDG acid (the thick freeze-dried powder containing salt) and 200mg15 (Rohm and Haas, Philadelphia, PA) merges.DDG is simultaneously incompletely dissolved in this solution.
It is gently stirred for reactant 18 hours at 105 DEG C.The GC-MS analysis result display of crude reaction sample forms diethyl-FDCA
(FDCA：FDCA) as primary product.This GC-MS result display pollutant salt (phosphate and NaCl) is thick
/ unpurified freeze-dried powder in presence be not significantly affected by reaction result.As comparison, true FDCA is closed in chemistry
Esterification is become to analyze for diethyl-FDCA and at identical conditions.
By using
15 synthesize ethyl FDCA by thick DDG
In a gas-tight seal pipe, by 2mL ethanol, 20mg thick DDG acid (the unpurified freeze-dried powder containing salt) and
200mg15 (Rohm and Haas, Philadelphia, PA) merges.DDG is simultaneously incompletely dissolved in this
In solution.It is gently stirred for reactant 18 hours at 105 DEG C.The GC-MS analysis result display of crude reaction sample forms diethyl
Base-FDCA (FDCA：FDCA) as primary product.This GC-MS result display pollutant salt (phosphate and
NaCl) presence in thick/unpurified freeze-dried powder is not significantly affected by reaction result.As comparison, will be commercial
FDCA is esterified in chemistry as diethyl-FDCA and analyzes at identical conditions.
By using
50 WX8 synthesize diethyl FDCA by thick DDG
In a gas-tight seal pipe, by 2mL ethanol, 20mg thick DDG acid (the unpurified freeze-dried powder containing salt) and
200mg50 WX8 merge.DDG is simultaneously incompletely dissolved in this solution.It is gently stirred for reactant at 105 DEG C
18 hours.The GC-MS analysis result display of crude reaction sample forms diethyl-FDCA (FDCA：FDCA) conduct
Primary product.This GC-MS result shows presence in thick/unpurified freeze-dried powder for the pollutant salt (phosphate and NaCl)
It is not significantly affected by reaction result.As comparison, commercial FDCA is esterified in chemistry for diethyl-FDCA and in identical
Under the conditions of analyze.
The generation of embodiment 18-FDCA derivant
The synthesis of many high level FDCA derivants is described in Figure 16, and wherein DTHU dehydration produces furfural -5- formic acid, that is,
FCA, it subsequently by chemistry or enzymatic oxygen chemical conversion FDCA, is reduced into FCH, or turns ammonia (using electronation amination or transamination
Enzyme) become aminoacid-AFC.
Generation in gas phase reaction for the embodiment 19- dibutyl FDCA
In this embodiment, the entrance of GC is used as high-temperature reactor to be dehydrated dibutyl DDG for dibutyl FDCA.
The product of generation is carried out chromatographic isolation, is detected by mass spectrography.By dibutyl DDG (10mM) and sulphuric acid (100mM) in fourth
Solution in alcohol is placed in GC bottle.Bottle is injected in GC and observes FDCA dibutyl ester.Reaction occurs in 300 DEG C of entrances
(time of staying=4 second).The average yield of 6 injections is 54%.
GC is arranged：Directly liquid injection/MS detector
Entrance：300 DEG C, total flow 29.51ml/min, split ratio 10: 1, shunt volume 24.1ml/min, septum purge flow
Amount 3mL/min
GC bushing pipe：4mm, glass cotton (P/N 5183-4647)
Column flow：2.41ml/min He Isobarically Control
Baking oven program：Keep 2min at 40 DEG C, be then warming up to 275 DEG C with 25 DEG C/min, then with 40 DEG C/min liter
To 325 DEG C, keep 2min
Post：HP-5MS, Agilent Technologies, 30mx0.25mmx0.25um
Total run time：14.65 minutes
MSD transfer line：290℃
MS source：250℃
MS Quad：150℃
Retention time：
2,3-FDCA dibutyl esters：9.3min
2,5-FDCA dibutyl esters：9.7min
The all announcements mentioned in this specification and patent application are incorporated herein by reference, and its degree is just as each
Individually publication or patent application specifically and is individually indicated that introducing is for reference.
Do not recognize that any list of references constitutes prior art.The judgement of its author, Shen have been said in the discussion of reference material
Ask someone to retain and query the accuracy of reference document and the right of appropriateness.Although it is to be clearly understood that herein with reference to many existing
There is technology open source literature, but this reference is not constituted to any one formation general knowledge known in the art in these documents
Ingredient license.
It should also be understood that providing above example to illustrate, and the unrestricted present invention.
Claims (47)
1. a kind of method of the product for producing enzymatic or chemistry route from starting material, described approach comprises one or more
Step of converting selected from group consisting of：
Guluronic acid Enzymatic transformation is D- glucosaccharic acid (step 7)；
5- ketogluconate (5-KGA) Enzymatic transformation is L- iduronic acid (step 15)；
L- iduronic acid enzymatic is converted into idosaccharic acid step 7b)；And
5- ketogluconate Enzymatic transformation is 4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) (step 16)；
1,5- gluconolactone enzymatic is converted into guluronic acid-lactone (step 19).
2. the method for claim 1, wherein said one or more step of converting are that guluronic acid Enzymatic transformation is
D- glucosaccharic acid (step 7).
3. the method for claim 1, wherein said one or more step of converting are 5- ketogluconate (5-KGA) enzymatics
It is converted into L- iduronic acid (step 15).
4. the method for claim 1, wherein said one or more step of converting are the conversions of L- iduronic acid enzymatic
For idosaccharic acid step 7b).
5. the method for claim 1, wherein said one or more step of converting are that 5- ketogluconate Enzymatic transformation is
4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) (step 16).
6. the method for claim 1, wherein said one or more step of converting are that 1,5- gluconolactone enzymatic turns
Turn to guluronic acid-lactone (step 19).
7. the method for claim 1, the product of wherein said enzymatic route is 5- dehydrogenation -4- deoxidation-glucosaccharic acid
(DDG).
8. the method for claim 1, wherein said substrate is glucose and described product is 5- dehydrogenation -4- deoxidation-Portugal
Saccharic acid (DDG), the method comprising the steps of：
D-Glucose Enzymatic transformation is 1,5- gluconolactone (step 1)；
1,5- gluconolactone enzymatic is converted into guluronic acid-lactone (step 19)；
Guluronic acid-lactone enzymatic is converted into guluronic acid (step 1B)；
Guluronic acid Enzymatic transformation is D- glucosaccharic acid (step 7)；
D- glucosaccharic acid Enzymatic transformation is 5- dehydrogenation -4- deoxidation-glucosaccharic acid (DDG) (step 8).
9. the method for claim 1, wherein said substrate is glucose and described product is DDG, and methods described includes
Following steps：
D-Glucose is converted into 1,5- gluconolactone (step 1)；
1,5- gluconolactone is converted into gluconic acid (step 1a)；
Gluconic acid is converted into 5- ketogluconate (5-KGA) (step 14)；
5- ketogluconate (5-KGA) is converted into L- iduronic acid (step 15)；
L- iduronic acid is converted into idosaccharic acid (step 7b)；And
Idosaccharic acid is converted into DDG (step 8a).
10. the method for claim 1, wherein said substrate is glucose and described product is DDG, and methods described includes
Following steps：
D-Glucose is converted into 1,5- gluconolactone (step 1)；
1,5- gluconolactone is converted into gluconic acid (step 1a)；
Gluconic acid is converted into 5- ketogluconate (5-KGA) (step 14)；
5- ketogluconate (5-KGA) is converted into 4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) (step 16)；
4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) is converted into 4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) (step
Rapid 4)；And
4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) is converted into DDG (step 5).
11. the method for claim 1, wherein said substrate is glucose and described product is DDG, and methods described includes
Following steps：
D-Glucose is converted into 1,5- gluconolactone (step 1)；
1,5- gluconolactone is converted into gluconic acid (step 1a)；
Gluconic acid is converted into 5- ketogluconate (5-KGA) (step 14)；
5- ketogluconate (5-KGA) is converted into L- iduronic acid (step 15)；
L- iduronic acid is converted into 4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) (step 7b)；And
4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) is converted into DDG (step 5).
12. methods as claimed in claim 8, also include the step that DDG is converted into 2,5- furan-dioctyl phthalate (FDCA).
13. methods as claimed in claim 9, also include the step that DDG is converted into 2,5- furan-dioctyl phthalate (FDCA).
14. methods as claimed in claim 10, also include the step that DDG is converted into 2,5- furan-dioctyl phthalate (FDCA).
15. methods as claimed in claim 11, also include the step that DDG is converted into 2,5- furan-dioctyl phthalate (FDCA).
DDG is wherein converted into FDCA and includes being contacted to convert DDG DDG with acid by 16. methods as claimed in claim 12
For FDCA.
DDG is wherein converted into FDCA and includes being contacted to convert DDG DDG with acid by 17. methods as claimed in claim 13
For FDCA.
DDG is wherein converted into FDCA and includes being contacted to convert DDG DDG with acid by 18. methods as claimed in claim 14
For FDCA.
DDG is wherein converted into FDCA and includes being contacted to convert DDG DDG with acid by 19. methods as claimed in claim 15
For FDCA.
A kind of 20. methods of the derivant for synthesizing FDCA, it includes：
DDG is contacted to form FDCA at a temperature of more than 60C with alcohol, mineral acid.
21. methods as claimed in claim 20, wherein said alcohol is butanol or ethanol.
22. methods as claimed in claim 20, it has the yield of at least 25 moles of %.
A kind of 23. methods of the derivant of synthesis DDG, it includes：
DDG is contacted with alcohol, mineral acid and optional cosolvent to produce the derivant of DDG.
24. methods as claimed in claim 23, wherein：
A) described alcohol is ethanol or butanol；
B) described mineral acid is sulphuric acid；And
C) described cosolvent is selected from group consisting of：THF, acetone, acetonitrile, ether, ethyl acetate, butyl acetate, dioxane,
Chloroform, dichloromethane, 1,2- dichloroethanes, hexane, heptane, toluene, carbon tetrachloride, petroleum ether and dimethylbenzene.
A kind of 25. methods of the derivant for synthesizing FDCA, it includes：
The derivant of DDG is contacted with mineral acid to produce the derivant of FDCA.
26. methods as claimed in claim 25, it has the yield more than 25 moles of %.
27. methods as claimed in claim 26, the derivant of wherein said DDG is selected from group consisting of：Methyl D DG,
Ethyl-DDG, butyl-DDG, dimethyl-DDG, diethyl-DDG and dibutyl-DDG.
28. methods as claimed in claim 25, the derivant also including deesterify FDCA is to produce FDCA.
29. methods as claimed in claim 25, also include the step being polymerized the derivant of FDCA.
A kind of 30. methods for synthesizing FDCA, it includes：
DDG is contacted in the gas phase with mineral acid.
A kind of 31. methods for synthesizing FDCA, it includes：
DDG is contacted at a temperature of more than 120C with mineral acid.
A kind of 32. methods for synthesizing FDCA, it includes：
DDG is contacted under the conditions of anhydrous response with mineral acid.
A kind of 33. methods of the product for producing enzymatic or chemistry route from starting material, described approach comprises one or many
The individual step of converting selected from group consisting of：
DTHU is converted into DDG (step 5)；
Gluconic acid is converted into guluronic acid (step 6)；
DEHU is converted into DDH (step 7A)；
Guluronic acid is converted into DEHU (step 17A)；
34. methods as claimed in claim 33, wherein said substrate is glucose and described product is DDG, methods described bag
Include following steps：
D-Glucose is converted into 1,5- gluconolactone (step 1)；
1,5- gluconolactone is converted into gluconic acid (step 1a)；
Gluconic acid is converted into 3- dehydrogenation-gluconic acid (DHG) (step 2)
3- dehydrogenation-gluconic acid (DHG) is converted into 4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) (step 3)
2,5-DDH are converted into 4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) (step 4)
DTHU is converted into DDG (step 5).
35. methods as claimed in claim 33, wherein said substrate is glucose and described product is DDG, methods described bag
Include following steps：
D-Glucose is converted into 1,5- gluconolactone (step 1)；
1,5- gluconolactone is converted into gluconic acid (step 1a)；
Gluconic acid is converted into guluronic acid (step 6)
Guluronic acid is converted into glucosaccharic acid (step 7)
Glucosaccharic acid is converted into DDG (step 8)
36. methods as claimed in claim 33, wherein said substrate is glucose and described product is DDG, methods described bag
Include following steps：
D-Glucose is converted into 1,5- gluconolactone (step 1)；
1,5- gluconolactone is converted into gluconic acid (step 1a)；
Gluconic acid is converted into 5- ketogluconate (5-KGA) (step 14)；
5- ketogluconate (5-KGA) is converted into 4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) (step 16)；
4,6- dihydroxy 2,5- diketone caproic acid (2,5-DDH) is converted into 4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) (step
Rapid 4)；And
4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) is converted into DDG (step 5).
37. methods as claimed in claim 33, wherein said substrate is glucose and described product is DDG, methods described bag
Include following steps：
D-Glucose is converted into 1,5- gluconolactone (step 1)；
1,5- gluconolactone is converted into gluconic acid (step 1a)；
Gluconic acid is converted into 5- ketogluconate (5-KGA) (step 14)；
5- ketogluconate (5-KGA) is converted into L- iduronic acid (step 15)；
L- iduronic acid is converted into 4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) (step 7B)；
4- deoxidation -5- threo form-ketohexose alduronic acid (DTHU) is converted into DDG (step 5).
38. methods as claimed in claim 33, wherein said substrate is glucose and described product is DDH, methods described bag
Include following steps：
D-Glucose is converted into 1,5- gluconolactone (step 1)；
1,5- gluconolactone is converted into guluronic acid lactone (step 19)；
Guluronic acid lactone is converted into guluronic acid (step 1B)；
Guluronic acid is converted into DEHU (step 17A)；
DEHU is converted into DDH (step 7A).
39. methods as claimed in claim 33, wherein said substrate is glucose and described product is DDH, methods described bag
Include following steps：
D-Glucose is converted into 1,5- gluconolactone (step 1)；
1,5- gluconolactone is converted into gluconic acid (step 1a)；
Gluconic acid is converted into guluronic acid (step 6)；
Guluronic acid is converted into DEHU (step 17A)；
DEHU is converted into DDH (step 7A).
40. methods as claimed in claim 33, wherein said one or more step of converting are that DTHU is converted into DDG (step
5).
41. methods as claimed in claim 33, wherein said one or more step of converting are that gluconic acid is converted into gulose
Aldehydic acid (step 6).
42. methods as claimed in claim 33, wherein said one or more step of converting are that DEHU is converted into DDH (step
7A).
43. methods as claimed in claim 33, wherein said one or more step of converting are that guluronic acid is converted into
DEHU (step 17A).
44. methods as claimed in claim 2, it is by SEQ ID that wherein said guluronic acid is converted into D- glucosaccharic acid
NO：The alditol acidohydrogenase of 1-3 or with SEQ ID NO：1-3 has the congener of at least 70% sequence iden；Or by by
SEQ ID NO：The alditol acidohydrogenase of the nucleic acid coding of 4-6 or with SEQ ID NO：The nucleic acid of 4-6 has at least 70% sequence
The congener of homogeneity is carried out.Method as claimed in claim 4, wherein said L- iduronic acid is converted into idosaccharic acid
It is by SEQ ID NO：The alditol acidohydrogenase of 1-3 or with SEQ ID NO：1-3 has the same of at least 70% sequence iden
Source thing；Or by by SEQ ID NO：The alditol acidohydrogenase of the nucleic acid coding of 4-6 or with SEQ ID NO：The nucleic acid of 4-6 has
The congener of at least 70% sequence iden is carried out.
45. methods as claimed in claim 3, it is by SEQ ID NO that wherein said 5-KGA is converted into L- iduronic acid：
The isomerase of 7-19 or with SEQ ID NO：The isomerase of 7-19 has the congener of at least 70% sequence iden；Or by by
SEQ ID NO：The isomerase of the nucleic acid coding of 20-32 or with SEQ ID NO：It is same that the nucleic acid of 20-32 has at least 70% sequence
The congener of one property is carried out.
46. methods as claimed in claim 5, it is by SEQ ID NO that wherein said 5-KGA is converted into 2,5-DDH：33-35
Gluconate dehydratase or with SEQ ID NO：The gluconate dehydratase of 33-35 has the homology of at least 70% sequence iden
Thing；Or by by SEQ ID NO：The gluconate dehydratase of the nucleic acid coding of 36-38 or with SEQ ID NO：The nucleic acid tool of 36-38
The congener having at least 70% sequence iden is carried out.
47. methods as claimed in claim 6, it is logical that wherein said 1,5- gluconolactone is converted into guluronic acid-lactone
Cross SEQ ID NO：The aldehyde alcohol oxidase of 39-46 or with SEQ ID NO：The aldehyde alcohol oxidase of 39-46 has at least 70% sequence
The congener of homogeneity；Or by by SEQ ID NO：The aldehyde alcohol oxidase of the nucleic acid coding of 47-54 or with SEQ ID NO：47-
The congener that 54 nucleic acid has at least 70% sequence iden is carried out.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US14/222,453 US9528133B2 (en) | 2012-09-21 | 2014-03-21 | Compositions and methods for producing chemicals and derivatives thereof |
| US14/222,453 | 2014-03-21 | ||
| PCT/US2015/021848 WO2015143381A2 (en) | 2014-03-21 | 2015-03-20 | Compositions and methods for producing chemicals and derivatives thereof |
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| CN106414753A true CN106414753A (en) | 2017-02-15 |
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| EP (1) | EP3119899A4 (en) |
| CN (1) | CN106414753A (en) |
| AU (1) | AU2015231000B2 (en) |
| BR (1) | BR112016021298A2 (en) |
| CA (1) | CA2943348A1 (en) |
| SG (1) | SG11201607745WA (en) |
| WO (1) | WO2015143381A2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107954959A (en) * | 2017-11-22 | 2018-04-24 | 南京工业大学 | Method for preparing 2, 5-furandicarboxylic acid and precursor substance thereof |
| CN113527374A (en) * | 2020-04-22 | 2021-10-22 | 中国科学院上海有机化学研究所 | Preparation method of mannuronic acid oligosaccharide and intermediate thereof |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017175694A1 (en) | 2016-04-04 | 2017-10-12 | 国立大学法人三重大学 | Alginate lyase and method using enzyme for producing unsaturated uronic acid monosaccharide |
| CN109824630B (en) * | 2019-03-13 | 2020-08-04 | 浙江大学 | Method for preparing furfuryl alcohol by one-pot xylose method under hydrogen supply of formic acid |
| EP3963067A1 (en) * | 2019-07-11 | 2022-03-09 | BP Corporation North America Inc. | Gluconate dehydratase enzymes and recombinant cells |
| JP2023138462A (en) | 2022-03-16 | 2023-10-02 | 国立大学法人三重大学 | Novel use of 4-deoxy-l-erythro-5-hexoseulose uronic acid |
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| DK2281034T3 (en) * | 2008-04-30 | 2016-01-25 | Dupont Nutrition Biosci Aps | A method using alcohol dehydrogenase Pseudoglucanobacter saccharoketogenes |
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| US10975396B2 (en) * | 2012-06-04 | 2021-04-13 | Ensuiko Sugar Refining Co., Ltd. | D-glucaric acid producing bacterium, and method for manufacturing D-glucaric acid |
| SG10201702263QA (en) * | 2012-09-21 | 2017-05-30 | Synthetic Genomics Inc | Compositions and methods for producing chemicals and derivatives thereof |
-
2015
- 2015-03-20 CA CA2943348A patent/CA2943348A1/en not_active Abandoned
- 2015-03-20 AU AU2015231000A patent/AU2015231000B2/en not_active Ceased
- 2015-03-20 SG SG11201607745WA patent/SG11201607745WA/en unknown
- 2015-03-20 WO PCT/US2015/021848 patent/WO2015143381A2/en active Application Filing
- 2015-03-20 EP EP15764217.4A patent/EP3119899A4/en not_active Withdrawn
- 2015-03-20 BR BR112016021298A patent/BR112016021298A2/en not_active Application Discontinuation
- 2015-03-20 CN CN201580021486.XA patent/CN106414753A/en active Pending
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| US5298411A (en) * | 1990-09-25 | 1994-03-29 | Toyo Boseki Kabushiki Kaisha | Glucose dehydrogenase from pseudomonas |
| CN101679936A (en) * | 2007-05-08 | 2010-03-24 | 盐水港精糖株式会社 | Method for producing glucuronic acid by glucuronic acid fermentation |
| CN102066552A (en) * | 2008-04-04 | 2011-05-18 | 麻省理工学院 | Cellular production of glucaric acid |
| WO2010072902A1 (en) * | 2008-12-23 | 2010-07-01 | Valtion Teknillinen Tutkimuskeskus | Conversion of hexuronic acid to hexaric acid |
| WO2013151428A1 (en) * | 2012-04-04 | 2013-10-10 | Stichting Dienst Landbouwkundig Onderzoek | Catalytic oxidation of uronic acids to aldaric acids |
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| CN107954959A (en) * | 2017-11-22 | 2018-04-24 | 南京工业大学 | Method for preparing 2, 5-furandicarboxylic acid and precursor substance thereof |
| CN113527374A (en) * | 2020-04-22 | 2021-10-22 | 中国科学院上海有机化学研究所 | Preparation method of mannuronic acid oligosaccharide and intermediate thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2943348A1 (en) | 2015-09-24 |
| BR112016021298A2 (en) | 2017-10-03 |
| AU2015231000B2 (en) | 2017-10-26 |
| WO2015143381A2 (en) | 2015-09-24 |
| SG11201607745WA (en) | 2016-11-29 |
| WO2015143381A3 (en) | 2015-11-12 |
| EP3119899A2 (en) | 2017-01-25 |
| AU2015231000A1 (en) | 2016-10-13 |
| EP3119899A4 (en) | 2017-11-29 |
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