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WO2017163164A1 - Biosynthèse de 1,3-butadiène - Google Patents

Biosynthèse de 1,3-butadiène Download PDF

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Publication number
WO2017163164A1
WO2017163164A1 PCT/IB2017/051593 IB2017051593W WO2017163164A1 WO 2017163164 A1 WO2017163164 A1 WO 2017163164A1 IB 2017051593 W IB2017051593 W IB 2017051593W WO 2017163164 A1 WO2017163164 A1 WO 2017163164A1
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Prior art keywords
coa
butadiene
butanediol
adhe
enzyme
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Inventor
Sivaraman BALASUBRAMANIAM
Sneh Sanjay BADLE
Swati BADGUJAR
Vinod PUTHAN VEETIL
Vidhya Rangaswamy
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Reliance Industries Ltd
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Reliance Industries Ltd
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Priority to EP17769540.0A priority Critical patent/EP3433371A4/fr
Priority to KR1020187030042A priority patent/KR20180133426A/ko
Priority to BR112018069085A priority patent/BR112018069085A2/pt
Publication of WO2017163164A1 publication Critical patent/WO2017163164A1/fr
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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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C1/00—Preparation of hydrocarbons from one or more compounds, none of them being a hydrocarbon
    • C07C1/20—Preparation of hydrocarbons from one or more compounds, none of them being a hydrocarbon starting from organic compounds containing only oxygen atoms as heteroatoms
    • C07C1/24—Preparation of hydrocarbons from one or more compounds, none of them being a hydrocarbon starting from organic compounds containing only oxygen atoms as heteroatoms by elimination of water
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2527/00—Catalysts comprising the elements or compounds of halogens, sulfur, selenium, tellurium, phosphorus or nitrogen; Catalysts comprising carbon compounds
    • C07C2527/14—Phosphorus; Compounds thereof
    • C07C2527/16—Phosphorus; Compounds thereof containing oxygen
    • C07C2527/167—Phosphates or other compounds comprising the anion (PnO3n+1)(n+2)-
    • C07C2527/173—Phosphoric acid or other acids with the formula Hn+2PnO3n+1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2529/00—Catalysts comprising molecular sieves
    • C07C2529/04—Catalysts comprising molecular sieves having base-exchange properties, e.g. crystalline zeolites, pillared clays
    • C07C2529/06—Crystalline aluminosilicate zeolites; Isomorphous compounds thereof
    • C07C2529/08—Crystalline aluminosilicate zeolites; Isomorphous compounds thereof of the faujasite type, e.g. type X or Y
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel

Definitions

  • the present disclosure relates to the biosynthesis of 1,3-butadiene.
  • DEFINITIONS As used in the present disclosure, the following terms are generally intended to have the meaning as set forth below, except to the extent that the context in which they are used indicates otherwise.
  • Microbial based conversion refers to any conversion/reaction taking place in the presence of an enzyme.
  • Decarboxylative claisen condensation reaction The term “decarboxylative claisen condensation reaction” refers to the carbon-carbon bond forming reaction that takes place in the presence of an enzyme.
  • 1,3-Butadiene is a conjugated diene with the formula C43 ⁇ 4. It is an important industrial chemical used as a monomer in the production of synthetic rubber, including styrene- butadiene -rubber (SBR), polybutadiene (PB), styrene-butadiene latex (SBL), acrylonitrile- butadiene-styrene resins (ABS), nitrile rubber, and adiponitrile.
  • SBR styrene- butadiene -rubber
  • PB polybutadiene
  • SBL styrene-butadiene latex
  • ABS acrylonitrile- butadiene-styrene resins
  • nitrile rubber and adiponitrile.
  • 1,3-Butadiene is a co-product obtained from the steam cracking process of petrochemical-based feedstocks and purified via extractive distillation. Due to the increasing demand for 1,3-but
  • An object of the present disclosure to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
  • Another object of the present disclosure to provide a method for producing 1,3 -butadiene.
  • Still another object of the present disclosure is to provide a method for producing 1,3- butadiene using biological catalysts.
  • Yet another object of the present disclosure is to provide a method for producing 1,3- butadiene using chemical reagents.
  • Yet another object of the present disclosure is to provide a method for producing 1,3- butadiene which is environmentally-friendly and sustainable.
  • the present disclosure envisages a method for producing 1,3-butadiene involving the key intermediate 3-hydroxybutyryl-CoA.
  • the method comprises microbial based decarboxylative claisen condensation reaction of malonyl-CoA and acetaldehyde in the presence of at least one acyltransferase enzyme and acyl carrier protein, to obtain 3-hydroxybutyryl-CoA, microbial based conversion of the 3-hydroxybutyryl-CoA to 1,3-butanediol in the presence of a dehydrogenase enzyme and NADH, and further dehydrating the 1,3-butanediol in the presence of at least one chemical reagent to obtain 1,3-butadiene.
  • the acyltransferase enzyme is beta-ketoacyl-ACP synthase III.
  • the acetaldehyde can be obtained by the reduction of acetyl-CoA using acetaldehyde dehydrogenase.
  • the intermediate, 3-hydroxybutyryl-CoA can be converted to 1, 3-butanediol in the presence of a dehydrogenase enzyme and nicotinamide adenine dinucleotide (NADH), and further dehydrating 1, 3-butanediol in the presence of chemical reagents to obtain 1,3-butadiene.
  • NADH nicotinamide adenine dinucleotide
  • the intermediate, 3-hydroxybutyryl-CoA can be hydrolysed in the presence of thiolester hydrolase enzyme to obtain 3-hydroxybutanoic acid.
  • the 3-hydroxybutanoic acid is reduced in the presence of carboxylate reductase to obtain 3-hydroxybutanal, which is further reduced in the presence of an aldehyde reductase to obtain 1,3-butanediol.
  • 1,3- butanediol is dehydrated in the presence of a catalyst to obtain 1,3-butadiene.
  • malonyl-CoA can be obtained by converting syngas to acetyl-CoA, and further conversion of acetyl-CoA to malonyl-CoA.
  • the conversion of syngas to acetyl-CoA is carried out in the presence of a microorganism comprising a tetrahydrofolate metabolism pathway.
  • Figure 1 represents the PCR amplification of fabH gene
  • Figure 2 represents the screening for fabH positive clones
  • Figure 3 represents the PCR amplification of acp gene
  • Figure 4 represents the screening for acp positive clones
  • Figure 5 represents the PCR amplification of adhE (Cbei_3832) gene
  • Figure 6 represents the screening for adhE (Cbei_3832) positive clones
  • Figure 7 represents the PCR amplification of adhE (Cbei-0223), adhE (Cbei-0528), adhE (Cbei-1722), adhE (Cbei-0869), adhE (Cbei-2243), adhE (Cbei-2421 ), adhE (Cbei-2421) and adhE (Cbei_4053) genes from C. beijerinckii;
  • Figure 8 represents the screening for adhE (Cbei-0223), adhE (Cbei-0528), adhE (Cbei- 1722), adhE (Cbei-0869), adhE (Cbei-2243), adhE (Cbei-2421), and adhE (Cbei-2421) positive clones;
  • Figure 9 represents the SDS-PAGE image of the purified overexpressed ACP, AdhE and FabH proteins
  • Figure 10 depicts an HPLC spectrum of 3-hydroxybutyryl-CoA obtained from FabH enzyme assay and overlaid with standard
  • Figure 11 depicts an HPLC spectrum of 1,3-butanediol obtained from cascade reaction (FabH and AdhE) and overlaid with control (without FabH and AdhE);
  • Figure 12 depicts an HPLC spectrum of 1,3-butanediol obtained from cascade reaction (FabH and AdhE) and overlaid with standard 1,3-butanediol;
  • Figure 13 depicts a GC-MS spectrum of 1,3-butanediol (standard), spiked, from cascade reaction (FabH and AdhE) and control (without FabH and AdhE);
  • Figure 14 depicts the fragmentation pattern of GC-MS spectrum of 1,3-butanediol from cascade reaction (FabH and AdhE);
  • Figure 15 depicts a standard plot for 1,3-butanediol by GC;
  • Figure 16 depicts a standard plot for 1,3-butanediol by HPLC
  • Figure 17 depicts the GC-MS spectrum of 1,3-butadiene obtained by reacting 1,3-butanediol with H 3 PO4 and Zeolite NaY;
  • Figure 18 depicts the standard plot for propene by GC; and Figure 19 depicts the ⁇ -NMR spectrum of 1,3-butadiene obtained by reacting 1,3- butanediol with H 3 PO4 and Zeolite NaY, recorded in CDCI 3 .
  • 1,3-Butadiene is an important industrial chemical used as a monomer in the production of synthetic rubber having various applications across the industry. Moreover, the strong increase in demand for the elastomeric products, drawn by the expansion of fields such as the automotive industry, has consequently led to an ever- increasing demand for 1,3 - butadiene, used as raw material for the production of a large range of synthetic rubbers. 1,3- butadiene is largely produced by methods like naphtha cracking, direct dehydrogenation of n- butene, and oxidative dehydrogenation of «-butene. Among them, the naphtha cracking process is energy intensive and requires high infrastructure costs.
  • the naphtha cracking process is problematic because of production of several side products, so that the investment and operation for a naphtha cracker cannot be optimally matched with the production and demand of 1,3-butadiene, and other basic fractions besides 1,3- butadiene are excessively produced.
  • the naphtha cracking process being dependent on the petrochemical feedstocks, along with the increasing environmental concerns and the limited resources for producing 1,3-butadiene using chemical processes, the present disclosure envisages an alternative, environmentally-friendly and sustainable processes for the production of 1,3-butadiene.
  • a method for producing 1,3- butadiene by biocatalysis is envisaged.
  • the present disclosure discloses a method for synthesizing 1,3-butadiene from syngas by biocatalysis.
  • a method for producing 1,3- butadiene involves condensing malonyl-CoA and acetaldehyde to obtain 3- hydroxybutyryl-CoA, and converting 3-hydroxybutyryl-CoA to 1,3-butadiene in the presence of at least one of an enzyme and/or at least one chemical reagent.
  • the condensation reaction between malonyl-CoA and acetaldehyde is a microbial based decarboxylative claisen condensation reaction, which is catalysed by acyl transferase to form 3-hydroxylbutyryl-CoA.
  • the acyltransferase enzyme is beta-ketoacyl- ACP synthase III.
  • the acetaldehyde is obtained by reducing acetyl-CoA using a dehydrogenase, such as, acetaldehyde dehydrogenase.
  • 3-hydroxybutyryl-CoA is converted to 1,3-butanediol using a microbial based conversion in the presence of a dehydrogenase enzyme, such as, aldehyde dehydrogenase, and NADH.
  • a dehydrogenase enzyme such as, aldehyde dehydrogenase, and NADH.
  • 1,3-Butanediol is then dehydrated in the presence of at least one chemical reagent to obtain 1,3-butadiene.
  • the chemical reagent is at least one selected from orthophosphoric acid, aqueous hydrogen iodide, trifluoroacetic acid, sulphuric acid, zeolites, ionic liquids, and combinations thereof.
  • the chemical reagent is at least one selected from the group consisting of orthophosphoric acid, zeolite NaY, faujasite, and a combination thereof.
  • 3-hydroxybutyryl-CoA is sequentially converted to 1,3-butadiene, in a series of steps. Initially, 3-hydroxybutyryl-CoA is hydrolysed in the presence of a thiolester hydrolase enzyme to obtain 3-hydroxybutanoic acid. In one embodiment, the thiolester hydrolase enzyme is thioesterase. 3-hydroxybutanoic acid is reduced in the presence of carboxylate reductase to obtain 3- hydroxybutanal.
  • 3-hydroxybutanal is further reduced in the presence of an aldehyde reductase to obtain 1,3-butanediol.
  • 1,3-butanediol, as described previously is dehydrated in the presence of at least one catalyst to obtain 1,3-butadiene.
  • malonyl-CoA is obtained by converting syngas to acetyl-CoA in the presence of at least one microorganism; and further converting the acetyl-CoA to malonyl- CoA using a carbonyl donor in the presence of a carboxylase enzyme.
  • Malonyl-CoA along with acetaldehyde is condensed to obtain 3-hydroxybutyryl-CoA, followed by conversion of 3-hydroxybutyryl-CoA to 1,3-butanediol.
  • 1,3-Butadiene is then obtained by dehydrating 1,3- butanediol either by using a biocatalyst like a dehydratase enzyme or by using a dehydrating chemical reagent.
  • Scheme-I represents the method for synthesizing 1,3-butadiene in an embodiment of the present disclosure.
  • Synthesis gas (hereinafter referred to as syngas) is a mixture of hydrogen (3 ⁇ 4) and carbon monoxide (CO).
  • Syngas is a platform intermediate in the chemical and bio refining industries and has a vast number of uses.
  • Syngas can be converted into alkanes, olefins, oxygenates, and alcohols. These chemicals can be blended into, or used directly as, diesel fuel, gasoline, and other liquid fuels.
  • Carbon is fixed in anaerobic microorganisms from gaseous substrates like carbon monoxide or carbon dioxide via the Wood-Ljungdahl pathway.
  • Carbon monoxide from syngas is also converted to acetyl-CoA through the Wood-Ljungdahl pathway utilizing tetrahydrofolate metabolism.
  • Malonyl-CoA and acetaldehyde are derived from acetyl-CoA, which are the starting materials for producing 1,3-butadiene.
  • the microorganism comprises tetrahydrofolate metabolism pathway, and is selected from the group consisting of genera Acetitomaculum, Acetobacterium, Blautia, Clostridium, Eubacterium, Methanothermobacter, Moorella, Sporomusa, Syntrophococcus and Butyribacterium.
  • the carbonyl donor can be selected from the group consisting of hydrogen carbonate and carboxylated biotin adducts.
  • the carboxylase enzyme is acetyl-CoA carboxylase.
  • live microorganisms which produce the enzyme(s) of interest or enzymes extracted from the microorganism can be used.
  • the live microorganisms used are genetically modified microorganisms.
  • the present disclosure envisages the use of syngas as a source for producing the starting materials used in the method for synthesizing 1,3-butadiene.
  • Syngas which is generally a byproduct of many industrial processes, can be used here to produce 1,3-butadiene, thus making the method of the present disclosure economical and produce a product having value addition.
  • Escherichia coli DH5a was used for cloning procedures, and was cultivated at 37 °C in Luria-Bertani (LB) medium containing 50 ⁇ g/mL kanamycin for selection with shaking at 220 rpm.
  • the two clostridial strains, Clostridium acetobutylicum ATCC55383 and Clostridium beijerinckii ATCC 10132 were procured from American type culture collection and anaerobically grown in RCB medium (Reinforced clostridial broth). Genomic DNA from Escherichia coli K12 (Novagen, USA), C. acetobutylicum ATCC55383 and C.
  • glycerol- stock was inoculated into a tube containing 10 mL of LB medium with respective antibiotic if required for overnight growth.
  • the preculture was inoculated into a 500 mL of shake-flask containing 250 mL of fresh LB medium at an initial OD600 of 0.05, grown at 37 °C for 1-2 hours till OD of 0.5 is achieved and induced with 0.5 mM IPTG. Induced cultures were either incubated further at 37 °C for 4 hours or overnight at 20 °C.
  • 3-Hydroxybutyryl-CoA and 1,3-butanediol were analyzed on HPLC.
  • the conditions for the analysis of 3-hydroxybutyryl-CoA included using CI 8 column and isocratic mobile phase consisting of 100 mM ammonium acetate with 9% methanol and 0.1% formic acid at a flow rate of 0.8 mL min 1 .
  • Column temperature was maintained at 25 °C.
  • the retention time for standard 3-hydroxybutyryl-CoA was 10.3 minutes.
  • 1,3-butanediol The conditions for the analysis of 1,3-butanediol included the use of an anion exchange column and the mobile phase used was 5 mM sulfuric acid solution at a flow rate of 0.6 mL min "1 . Column temperature was maintained at 50 °C. The retention time for standard 1,3- butanediol was 18.0 minutes.
  • Sample preparation for ⁇ -NMR The head space aliquot was sampled and bubbled into the CDC1 3 solution in NMR tube which was kept at -10 °C. The NMR tube was sealed and recorded instantly using Bruker NMR instrument.
  • oligonucleotides were purchased from Eurofins.
  • One Taq DNA polymerase, restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (USA).
  • DNA purification, plasmid isolation and PCR purification kits were purchased from Qiagen.
  • Ni- NTA resin was purchased from Qiagen. All other reagents were of analytical grade.
  • the pET- 28a (+) plasmid and E. coli BL21 (DE3)/DH5a strains (Novagen, USA) were used as expression vector and host strains.
  • the primers used in the present disclosure are summarized in Table- 1.
  • the 3-oxoacyl-[acyl-carrier-protein] synthase (fabH), and acyl carrier protein (acp) genes having a size of about 955 and 237 bp respectively were amplified from genomic DNA of E. coli K12.
  • acyl carrier protein (adhE) were amplified using C. acetobutylicum (Ca_adhE) and C. beijerinckii (Cbei_adhE) genomic DNA using One Taq polymerase.
  • the genes were then cloned into pET30a (+) vector with respective restriction enzymes mentioned in above table.
  • the recombinant plasmid carrying the fabH, and acp genes were validated by digesting with same restriction enzymes pairs i.e. BamHI/Hindlll and Kpnl/Xhol, respectively.
  • the different adhE genes carrying pET30a (+) recombinant vector were also confirmed by restriction digestion using same pair of enzymes as mentioned in table 1.
  • Lane 1 to 3 represents PCR amplified fabH product
  • Lane 4 and 5 represents pET30 plasmid
  • Lane 6 and 7 represents Xhol and Kpnl digested pET30 (+) fragment
  • Lane-8 represents the gene ruler (1 kb).
  • Lanes 2-10 represent colonies screened for fabH
  • Lanes 1 and 11 represent the gene ruler (1 kb) ladder.
  • Lane-3 Lanes 1 to 3 represent PCR amplified acp product
  • Lane-4 represents the 100 bp ladder.
  • Lane 4 Lane 1 to 10 represent colonies screened for acp
  • Lane-11 represents the 100 bp ladder.
  • Lane 1-2 represent PCR amplified adhE Cbei-3832 product, and Lane-4 represents 1 kb ladder.
  • Lane 1- 9 represent colonies screened for adhE (Cbei-3832)
  • Lane-10 represents 1 kb ladder.
  • Lane-1 represents gene ruler (1 kb)
  • Lane-2 represents PCR amplified adhE (Cbei- 0223 ) product
  • Lane-3 represents PCR amplified adhE ( Cbei-0528 )
  • Lane-4 represents PCR amplified adhE (Cbei-1722) product
  • Lane-5 represents PCR amplified adhE (Cbei-0869) product
  • Lane-6 represents PCR amplified adhE (Cbei-2243) product
  • Lane-7 represents PCR amplified adhE ( Cbei-2421 ) product
  • Lane-8 represents PCR amplified adhE ( Cbei- 4053) product.
  • Figure-8 represents colonies screened for adhE (Cbei-0223), adhE (Cbei- 0528), adhE (Cbei-1722), adhE (Cbei-0869), adhE (Cbei-2243), adhE (Cbei-2421), and adhE ( Cbei-2421 ) on individual gel respectively.
  • the arrows in Figures 1 to 8 point to the amplification bands observed at the correct size for the respective genes.
  • the cells harvested from 500 mL of culture grown and induced under optimum conditions were suspended in 10 mL of Lysis buffer (50 mM NaH 2 P0 4 , pH 8.0, 300 mM NaCl, and 5 mM imidazole) containing lysozyme (1 mg.mL "1 ) and cells were disrupted by sonication. Soluble and insoluble cell fractions were separated by centrifugation at 15000 rpm for 10 min in cold. Supernatants carrying the soluble fractions were mixed with Ni-NTA resin to purify fusion proteins according to manufacturer's manual.
  • Lysis buffer 50 mM NaH 2 P0 4 , pH 8.0, 300 mM NaCl, and 5 mM imidazole
  • Soluble and insoluble cell fractions were separated by centrifugation at 15000 rpm for 10 min in cold.
  • Supernatants carrying the soluble fractions were mixed with Ni-NTA resin to purify fusion proteins according to manufacturer's manual
  • Bound His-tagged proteins were eluted in 1 mL of elution buffer containing 50 mmol NaH 2 P0 4 , 300 mmol NaCl, and 250 mmol imidazole, at pH 8.0. Equal volume of purified proteins were mixed with 2 X SDS loading buffer and boiled for 10 min to prepare samples for SDS-PAGE. The target proteins were detected by comparison with protein standard markers, the proteins were purified to >90 purity.
  • the SDS-PAGE of purified overexpressed proteins is shown in Figure-9.
  • the purified proteins bands of 14 kDa, 52 kDa and 35 kDa size corresponding to the enzymes ACP (A; 16 % Tricin gel), AdhE (B; 12 % SDS-PAGE), and FabH (C; 12 % SDS-PAGE), respectively are shown in Figures-9A, 9B and 9C.
  • the reaction mixture constituted of malonyl-CoA 200 ⁇ , acetaldehyde 250 ⁇ , purified ACP protein 100 ⁇ g which was mixed thoroughly followed by the addition of cerulenin 250 ⁇ (solution made in ethanol) and purified FabH 100 ⁇ g was added later.
  • the enzyme mixture was incubated 37 °C for 2.5 hours. The mixture was then centrifuged at room temperature for 5 minutes at 8000 rpm. The supernatant was separated and tested for production of 3- hydroxybutyryl-CoA using HPLC and the result obtained is depicted in Figure-10.
  • peak A represents the overlay of the standard
  • peak B represents the reaction peak of 3-hydroxybutyryl-CoA obtained from FabH enzyme.
  • the reaction mixture obtained from FabH enzyme assay was used as substrate for AdhE enzyme activity.
  • the total protein content as estimated by bicinchoninic acid assay (BCA) method for AdhE was 15 mg.
  • L "1 . 25 ⁇ of 5 mM NADH solution and 100 ⁇ g of purified AdhE enzyme were added to 100 ⁇ of FabH reaction mixture (containing 3-hydroxybutyryl- CoA).
  • the reaction mixture was mixed thoroughly and incubated for 2.5 hours at 37 °C.
  • the enzymatic mixture was centrifuged for 5 min at 8000 rpm.
  • 1 represents the control reaction (without FabH and AdhE)
  • 2 represents the standard 1, 3-butanediol (1 g/1)
  • 3 represents the reaction spiked with 1,3- butanediol
  • 4 represents the reaction mixture obtained from the cascade reaction: FabH followed by AdhE.
  • Figure-14 depicts a GC-MS spectrum of 1, 3-butanediol obtained from cascade reaction. The data obtained from the analysis using HPLC and GC-MS proved the formation of 1, 3-butanediol without any ambiguity.
  • the headspace was analyzed using GC-MS and the product 1,3-butadiene was confirmed along with the presence of propene and trace amounts of E or Z-butenes based on the mass spectra ( Figure 17).
  • the peak at retention time 5.5 minutes in GC spectrum corresponds to propene.
  • loadings of NaY was varied from 100 mg to 400 mg for 1 g of 1,3-butanediol with 0.5 mL of orthophosphoric acid to optimize 1,3-butadiene production.
  • Table 11 Details of GC-MS spectra of 1,3-butadiene obtained by reacting 1,3- butanediol with H 3 PO4 and zeolite NaY Retention Compound Molecular
  • the yield of 1,3-butadiene was 66 % and that of propene was 4 % as inferred from the standard graphs ( Figure-16 and Figure-18).
  • the residual 1,3-butanediol which was analyzed using HPLC and was found to be 20 %, which is 2g.
  • TECHNICAL ADVANCEMENTS The present disclosure described herein above has several technical advantages including, but not limited to, the realization of a method for producing 1,3-butadiene via biocatalysis which is environmental friendly and economical.

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  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

La présente invention décrit un procédé de production de 1,3-butadiène par une approche biochimique. Le matériau de départ utilisé pour la biosynthèse du 1,3-butadiène, c'est-à-dire, le malonyl-CoA, peut être obtenu en convertissant du gaz de synthèse en acétyl-CoA et en outre par carboxylation en malonyl-CoA. L'étape suivante implique la condensation du malonyl-CoA et d'acétaldéhyde au moyen d'une réaction de condensation de Claisen décarboxylatrice, pour obtenir du 3-hydroxybutyryl-CoA. Le gaz de synthèse, un sous-produit de nombreux procédés industriels, est utilisé ici pour produire du 1,3-butadiène, qui rend le procédé de la présente invention économique, et produit un produit à valeur ajoutée.
PCT/IB2017/051593 2016-03-21 2017-03-20 Biosynthèse de 1,3-butadiène Ceased WO2017163164A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP17769540.0A EP3433371A4 (fr) 2016-03-21 2017-03-20 Biosynthèse de 1,3-butadiène
KR1020187030042A KR20180133426A (ko) 2016-03-21 2017-03-20 1,3-부타디엔의 생합성
BR112018069085A BR112018069085A2 (pt) 2016-03-21 2017-03-20 biossíntese de 1,3-butadieno

Applications Claiming Priority (2)

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IN201621009837 2016-03-21
IN201621009837 2016-03-21

Publications (1)

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WO2017163164A1 true WO2017163164A1 (fr) 2017-09-28

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EP (1) EP3433371A4 (fr)
KR (1) KR20180133426A (fr)
BR (1) BR112018069085A2 (fr)
WO (1) WO2017163164A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120276606A1 (en) * 2009-10-30 2012-11-01 Daicel Corporation Recombinant microorganisms with 1,3-butanediol-producing function and uses thereof
US20160040172A1 (en) * 2013-03-15 2016-02-11 Genomatica, Inc. Microorganisms and methods for producing butadiene and related compounds by formate assimilation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120276606A1 (en) * 2009-10-30 2012-11-01 Daicel Corporation Recombinant microorganisms with 1,3-butanediol-producing function and uses thereof
US20160040172A1 (en) * 2013-03-15 2016-02-11 Genomatica, Inc. Microorganisms and methods for producing butadiene and related compounds by formate assimilation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3433371A4 *

Also Published As

Publication number Publication date
EP3433371A1 (fr) 2019-01-30
BR112018069085A2 (pt) 2019-01-29
EP3433371A4 (fr) 2019-11-20
KR20180133426A (ko) 2018-12-14

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