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WO2009153047A2 - Production biotechnologique d'acide acrylique - Google Patents

Production biotechnologique d'acide acrylique Download PDF

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Publication number
WO2009153047A2
WO2009153047A2 PCT/EP2009/004418 EP2009004418W WO2009153047A2 WO 2009153047 A2 WO2009153047 A2 WO 2009153047A2 EP 2009004418 W EP2009004418 W EP 2009004418W WO 2009153047 A2 WO2009153047 A2 WO 2009153047A2
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WIPO (PCT)
Prior art keywords
seq
decarboxylase
acrylic acid
host cell
acid
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PCT/EP2009/004418
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German (de)
English (en)
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WO2009153047A3 (fr
Inventor
Joachim W. Schmid
Klaus Mauch
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Yokogawa Insilico Biotechnology GmbH
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Insilico Biotechnology AG
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Publication of WO2009153047A2 publication Critical patent/WO2009153047A2/fr
Publication of WO2009153047A3 publication Critical patent/WO2009153047A3/fr
Anticipated expiration legal-status Critical
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    • 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
    • C12N9/88—Lyases (4.)
    • 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

Definitions

  • the invention relates to the biotechnological production of acrylic acid and related compounds as well as agents and organisms which are suitable for use therefor.
  • Acrylic acid is an important starting material in the production of polymeric compounds such as plastics.
  • Acrylic acid is known to be produced in classical chemical processes, in particular by the two-stage oxidation of propene.
  • Previously known biotechnological alternatives for the production of acrylic acid for example by means of lactate or propionate as reaction intermediates, achieve low real yields, mainly due to the unfavorable thermodynamic properties of the final product formation reaction.
  • the invention is therefore based on the technical problem of providing an improved process for the biotechnological production of acrylic acid and means for carrying out this process.
  • the technical problem is also to provide a method which can be used especially in known and used in biological / fermentative processes and established microorganisms or cell lines, which usually by means of conventional and established recombination technologies in a conventional manner and with little effort transfected and cultured.
  • the technical problem to be solved to make biotechnological processes for the synthesis of acrylic acid can be used, which can also run outside of an intact biological cell.
  • the underlying technical problem is primarily solved by a process for the production of acrylic acid, wherein, in a preferred last step of the synthesis route, fumaric acid is converted into acrylic acid.
  • the invention provides a process in which fumaric acid or fumarate is converted into acrylic acid or acrylate by cleavage of a carboxyl group, which preferably proceeds as a CO 2 radical.
  • the decarboxylation is catalyzed by one or more decarboxylase activities according to the invention. These are preferably selected from the group consisting of: 4-oxalocrotonate-carboxy-lyase (EC 4.1.1.77),
  • ACMSD Aminocarboxymuconate Semialdehyde Decarboxylase
  • EC 4.1.1.45 5-Oxopent-3-en-1
  • 2,5-tricarboxylate decarboxylase EC 4.1.1.68
  • dihydroxyphthalate-carboxy-lyase EC 4.1.1.69
  • oxaloacetate-carboxy-lyase EC 4.1.1.3
  • acetoacetate Decarboxylase EC 4.1.1.4
  • 2,3-dihydroxybenzoate decarboxylase EC 4.1.1.46
  • Uroporphyrinogen carboxy lyase (EC 4.1.1.37), methylmalonyl-CoA decarboxylase (EC 4.1.1.41), dihydroxyphthalate decarboxylase (EC 4.1.1.55), aryl methylmalonate decarboxylase (EC 4.1.1.76), uracil ⁇ - Carboxylate-carboxy-lyase (EC 4.1.1.66),
  • ACMSD ACMSD and variants derived therefrom, in particular sequence variants, with increased substrate specificity for fumaric acid / fumarate.
  • the decarboxylase activity used according to the invention is a decarboxylase activity of a modified enzyme.
  • the enzyme preferably has at least one modification of the primary structure and preferably of the tertiary structure of the enzyme protein. This modification is preferably suitable for increasing the substrate specificity with respect to the substrate fumaric acid. In a variant, this modification is preferred, preferably in addition, suitable for reducing end product inhibition with respect to acrylic acid. In a further variant, this modification is preferred, preferably additionally, suitable for increasing the chemical and structural stability of the enzyme. In a further variant, this modification is preferred, preferably in addition, suitable, the Temperature stability of the enzyme to increase. It is understood that modifications within the meaning of this invention can also be carried out on already modified or modified enzyme proteins.
  • the decarboxylase activity used according to the invention is particularly preferably one of known enzymes
  • the invention thus provides for the biotechnological production of acrylic acid by obtaining acrylic acid by decarboxylation of fumaric acid by means of at least one decarboxylase activity.
  • the inventors surprisingly found that the process of decarboxylation, as the driving force of the acrylic acid pathway, provides high real product yield.
  • the method according to the invention makes it possible to carry out the last reaction step without co-metabolites such as NADH or ferredoxin or cofactors such as coenzyme A; It is therefore particularly suitable for extracellular biotransformation.
  • the process according to the invention also allows the use of mild environmental conditions, ie low temperature and low pressure, which has a favorable effect on the moderate chemical stability of the acrylic acid and the real yield.
  • mild environmental conditions ie low temperature and low pressure
  • the acrylic acid formed spontaneously converts into by-products, for example polymerization of acrylic acid, which may even occur at temperatures above 25 ° C.
  • the terms "fumaric acid” and "acrylic acid” regularly also fumarate and acrylate understood.
  • a preferred variant of the invention is the use of the decarboxylase activity of 4-oxalocrotonate-carboxy-lyase (EC 4.1.1.77) or an enzyme activity derived therefrom, which can catalyze the decarboxylation of the substrate fumaric acid to acrylic acid, for the production of acrylic acid.
  • This decarboxylase activity is preferably encoded by a nucleic acid molecule which contains or consists of the sequence according to SEQ ID NO: 1.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of the amino acid sequence according to SEQ ID NO: 2.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 2 or a fragment thereof which can be obtained by modification selected from deletion, addition, substitution or mutation, one or more, preferably from 1 to 10, amino acids.
  • a further preferred variant of the invention is the use of the decarboxylase activity of the aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45), ACMSD 1 or an enzyme activity derived therefrom which can catalyze the decarboxylation of the substrate fumaric acid to acrylic acid for the preparation of acrylic acid.
  • This decarboxylase activity is preferably encoded by a nucleic acid molecule which contains or consists of the sequence according to SEQ ID NO: 3.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of the amino acid sequence according to SEQ ID NO: 4.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 4 or a fragment thereof which can be obtained by modification, selected from deletion, addition, substitution or derivatization, of one or more, preferably from 1 to 10, amino acids.
  • Another preferred variant of the invention is the use of the decarboxylase activity of 5-oxopent-3-en-1, 2,5-tricarboxylate decarboxylase (EC 4.1.1.68) or an enzyme activity derived therefrom, which increases the decarboxylation of the substrate fumaric acid Acrylic acid can catalyze for the production of acrylic acid.
  • This decarboxylase activity is preferably coded by a nucleic acid molecule which contains or consists of the sequence according to SEQ ID NO: 5.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of the amino acid sequence according to SEQ ID NO: 6.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 6 or a fragment thereof which can be obtained by modification selected from deletion, addition, substitution or mutation, one or more, preferably from 1 to 10, amino acids.
  • a further preferred variant of the invention is the use of the decarboxylase activity of dihydroxyphthalate carboxy lyase (EC 4.1.1.69) or an enzyme activity derived therefrom, which can catalyze the decarboxylation of the substrate fumaric acid to acrylic acid, for the production of acrylic acid.
  • This decarboxylase activity is preferably encoded by a nucleic acid molecule which contains or is derived from the sequence according to SEQ ID NO: 7. stands.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of the amino acid sequence according to SEQ ID NO: 8.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 8 or a fragment thereof which can be obtained by modification selected from deletion, addition, substitution or mutation, one or more, preferably from 1 to 10, amino acids.
  • a further preferred variant of the invention is the use of the decarboxylase activity of the oxaloacetate carboxy lyase (EC 4.1.1.3) or an enzyme activity derived therefrom, which the
  • Decarboxylation of the substrate can catalyze fumaric acid to acrylic acid, to produce acrylic acid.
  • This decarboxylase activity is preferably coded by a nucleic acid molecule which contains or consists of the sequence according to SEQ ID NO: 9.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of the amino acid sequence according to SEQ ID NO: 10.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 10 or a fragment thereof which can be obtained by modification selected from deletion, addition, substitution or mutation, one or more, preferably from 1 to 10, amino acids.
  • a further preferred variant of the invention is the use of the decarboxylase activity of acetoacetate decarboxylase (EC 4.1.1.4) or an enzyme activity derived therefrom, which can catalyze the decarboxylation of the substrate fumaric acid to acrylic acid, for the production of acrylic acid.
  • This decarboxylase activity is preferably encoded by a nucleic acid molecule which contains or is derived from the sequence according to SEQ ID NO: 11. stands.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of the amino acid sequence according to SEQ ID NO: 12.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 12 or a fragment thereof which can be obtained by modification selected from deletion, addition, substitution or mutation, one or more, preferably from 1 to 10, amino acids.
  • a further preferred variant of the invention is the use of the decarboxylase activity of 2,3-dihydroxybenzoate-decarboxylate (EC 4.1.1.46) or an enzyme activity derived therefrom, which can catalyze the decarboxylation of the substrate fumaric acid to acrylic acid, for the preparation of acrylic acid.
  • This decarboxylase activity is preferably coded by a nucleic acid molecule which contains or consists of the sequence according to SEQ ID NO: 13.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of the amino acid sequence according to SEQ ID NO: 14.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 14 or a fragment thereof which can be obtained by modification selected from deletion, addition, substitution or mutation, one or more, preferably from 1 to 10, amino acids.
  • a further preferred variant of the invention is the use of the decarboxylase activity of uroporphyrinogen carboxy lyase (EC 4.1.1.37) or an enzyme activity derived therefrom, which can catalyze the decarboxylation of the substrate fumaric acid to acrylic acid, for the production of acrylic acid.
  • This decarboxylase activity is preferably encoded by a nucleic acid molecule which contains the sequence according to SEQ ID NO: 15 or is derived therefrom. stands.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of the amino acid sequence according to SEQ ID NO: 16.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 16 or a fragment thereof which can be obtained by modification selected from deletion, addition, substitution or mutation, one or more, preferably from 1 to 10, amino acids.
  • a further preferred variant of the invention is the use of the decarboxylase activity of methylmalonyl-CoA decarboxylase (EC 4.1.1.41) or an enzyme activity derived therefrom, which can catalyze the decarboxylation of the substrate fumaric acid to acrylic acid, for the production of acrylic acid.
  • This decarboxylase activity is preferably coded by a nucleic acid molecule which contains or consists of the sequence according to SEQ ID NO: 17.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of the amino acid sequence according to SEQ ID NO: 18.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 18 or a fragment thereof which can be obtained by modification selected from deletion, addition, substitution or mutation, one or more, preferably from 1 to 10, amino acids.
  • a further preferred variant of the invention is the use of the decarboxylase activity of dihydroxyphthalate decarboxylase (EC 4.1.1.55) or an enzyme activity derived therefrom, which can catalyze the decarboxylation of the substrate fumaric acid to acrylic acid, for the production of acrylic acid.
  • This decarboxylase activity is preferably encoded by a nucleic acid molecule which contains or is derived from the sequence according to SEQ ID NO: 19. stands.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of the amino acid sequence according to SEQ ID NO: 20.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 20 or a fragment thereof which can be obtained by modification selected from deletion, addition, substitution or mutation, one or more, preferably from 1 to 10, amino acids.
  • a further preferred variant of the invention is the use of the decarboxylase activity of the aryl methylmalonate decarboxylase (EC 4.1.1.76) or an enzyme activity derived therefrom, which can catalyze the decarboxylation of the substrate fumaric acid to acrylic acid, for the production of acrylic acid.
  • This decarboxylase activity is preferably coded by a nucleic acid molecule which contains or consists of the sequence according to SEQ ID NO: 21.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of the amino acid sequence according to SEQ ID NO: 22.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 22 or a fragment thereof which can be obtained by modification selected from deletion, addition, substitution or mutation, one or more, preferably from 1 to 10, amino acids.
  • Another preferred variant of the invention is the use of the decarboxylase activity of uracil-5-carboxylate-carboxy-lyase (EC 4.1.1.66) or an enzyme activity derived therefrom, which can catalyze the decarboxylation of the substrate fumaric acid to acrylic acid, for the production of acrylic acid ,
  • This decarboxylase activity is preferably encoded by a nucleic acid molecule which contains or is derived from the sequence according to SEQ ID NO: 23. stands.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of the amino acid sequence according to SEQ ID NO: 24.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 24 or a fragment thereof which can be obtained by modification selected from deletion, addition, substitution or mutation, one or more, preferably from 1 to 10, amino acids.
  • a further preferred variant of the invention is the use of the decarboxylase activity of 4-hydroxyphenylacetate carboxy lyase (EC 4.1.1.83) or an enzyme activity derived therefrom which can catalyze the decarboxylation of the substrate fumaric acid to acrylic acid for the production of acrylic acid ,
  • This decarboxylase activity is preferably encoded by one or more of the nucleic acid molecules which contain or consist of the sequence according to SEQ ID NO: 25, SEQ ID NO: 27 and / or SEQ ID NO: 29.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of one or more amino acid sequences according to SEQ ID NO: 26, SEQ ID NO: 28 and / or SEQ ID NO: 30.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 26, SEQ ID NO: 28 and / or SEQ ID NO: 30 or a fragment thereof which, by modification, selected from deletion, addition, substitution or mutation, one or more , preferably from 1 to 10, amino acids can be obtained.
  • This decarboxylase activity is preferably encoded by one or more of the nucleic acid molecules which contain or consist of the sequence according to SEQ ID NO: 31, SEQ ID NO: 33 and / or SEQ ID NO: 35.
  • This decarboxylase activity is preferably an enzyme protein which contains or consists of one or more amino acid sequences according to SEQ ID NO: 32, SEQ ID NO: 34 and / or SEQ ID NO: 36.
  • the enzyme protein particularly preferably contains a sequence derived from SEQ ID NO: 32, SEQ ID NO: 34 and / or SEQ ID NO: 36 or a fragment thereof which, by modification, selected from deletion, addition, substitution or mutation, one or more , preferably from 1 to 10, amino acids can be obtained.
  • the enzymatically catalyzed conversion of fumaric acid to acrylic acid takes place following a biotechnological, preferably fermentative synthesis of fumaric acid.
  • biotechnological fumaric acid synthesis are both anaerobic and aerobic. Preference is given to the fermentative synthesis of fumarate using a transgenic host cell which has an increased fumaric acid metabolism (fumaric acid overproducer).
  • fumaric acid overproducers can be prepared in a manner known per se and can readily be provided for carrying out the process according to the invention.
  • Rhizopus species are preferred here, preferably selected from R. nigricans, R. arrhizzu, R. oryzae and R. formosa.
  • the fumaric acid producing cell releases the fumaric acid into the surrounding (culture) medium.
  • Preferably realized ways are the proton symport, passive diffusion, passive carriers, primary-active transport, and / or other secondary-active transports or symposiums. In a preferred
  • the fumaric acid overproducer additionally expresses at least one protein which mediates or supports the transmembrane transport of intracellularly synthesized fumaric acid.
  • the host cell becomes at least short-term
  • Transmembrantransport Exposed to conditions that the integrity of the cell membrane, short-term and reversible or permanently repealed so far that a Transmembrantransport is simplified, supported or enabled.
  • Such methods include but are not limited to electroporation and chemical and thermal disintegration.
  • the fumaric acid can be isolated in a conventional manner from the culture medium and optionally purified.
  • the fumaric acid is conventionally chemically synthesized and the decarboxylation according to the invention to acrylic acid takes place following a chemical synthesis of fumaric acid.
  • fumaric acid is reacted outside a biological cell in a so-called extracellular decarboxylation, wherein the decarboxylase activity is immobilized on a carrier.
  • the at least one decarboxylase activity-mediating enzyme is preferably present outside a biological cell, either dissolved or preferably immobilized in a manner known per se, on a substrate.
  • the fumaric acid is preferably dissolved in a carrier liquid, brought directly into contact with the decarboxylase activity, so that there takes place the inventive decarboxylation to acrylic acid.
  • the thus synthesized acrylic acid is isolated in a conventional manner from the carrier medium.
  • the invention relates to a process for the production of acrylic acid, which consists at least or exclusively of the steps: providing a host cell which is suitable for synthesizing fumaric acid, culturing the host cell in culture medium under conditions under which fumaric acid is formed, optionally isolating the fumaric acid from the host cell, and contacting the fumaric acid formed with at least one decarboxylase activity, preferably the above-characterized decarboxylases and modifying variants thereof, under conditions which allow or promote conversion of the fumaric acid to acrylic acid, the reaction being preferred outside a biological see cell preferably on a biocatalyst having said decarboxylase activity takes place.
  • the invention also provides a biocatalyst for the production of acrylic acid, consisting of or at least comprising: at least one carrier and immobilized isolated enzyme protein having at least one decarboxylase activity.
  • a preferred subject matter of the invention is also a biocatalyst which has at least one enzymatically active polyamino acid molecule (enzyme protein) which is selected from the group consisting of:
  • amino acid molecules which contain at least one of the sequences selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36, or consist of, and
  • Preferred amino acid molecules of the invention contain at least one of the modifications characterized in more detail above and thus form modified enzyme proteins.
  • protein analogues are protein-related molecules which contain at least one unnatural or derivatized amino acid. and, in general, all macromolecules are polymers which are based on amino acids and are suitable for the formation of protein structures and can exert protein functions, in particular enzyme functions.
  • the use of cell-free soluble enzymes is usually unfavorable, since they can not or only with difficulty be recovered. Therefore, it is advantageous to bring them into a reusable form by immobilization.
  • the enzymes immobilized in this way offer all the advantages of classical heterogeneous catalysis.
  • such fixed enzymes are also referred to as biocatalysts. They are easily recoverable, for example, by filtration or centrifugation. They can be used in continuous processes as a bed in solid-state reactors and in fluidized bed reactors or stirred reactors. Thus, a continuous reaction is possible, which usually combines simple technical versions with extensive automation.
  • the enzymes can preferably be immobilized by being enclosed as dissolved molecules in a defined space, for example in pores of a carrier material, or by being converted into an insoluble state.
  • enzyme immobilization methods are known. Preference is given to processes for attachment to the surface of a support, preferably by adsorption, ionic or covalent bonding, and cross-linking with the support, carrier-free cross-linking and inclusion immobilization, preferably membrane entrapment and gel entrapment.
  • the person skilled in the art also knows the relationships between immobilization and enzyme activity. To avoid or compensate for a reduction in enzyme activity, he uses known measures.
  • modified enzyme proteins are used which undergo no or only an insignificant shift or reduction of the catalytic activity by the immobilization chosen.
  • the modification of the enzyme which is preferably provided according to the invention is preferably selected such that the desired improvement in the substrate specificity and / or stability of the enzyme is achieved in connection with the immobilization.
  • the support is selected from matrices, membranes, gels, and porous structures, especially tissues, nonwovens, polymer membranes, and gels which are suitable for immobilizing enzyme proteins having at least one decarboxylase activity thereon.
  • Preferred natural organic carriers are polysaccharides such as cellulose, starch, dextran, agarose or chitin. Proteins like
  • Collagen, gelatin or albumin are also preferred.
  • Preferred synthetic organic polymers are polyacrylates, polymethacrylates, polyacrylamides, vinyl and allyl polymers, polycarbonates and other polyesters or polyamides.
  • Preferred inorganic carriers are porous materials, preferably based on silicon or aluminum oxides or mixtures thereof. Of particular importance is the porosity of the carrier. Porous carriers have a large surface area for enzyme immobilization, resulting in high activities. The pore radii should be large enough to ensure access for the enzyme protein.
  • a preferred alternative embodiment of the invention provides for a direct synthesis of acrylic acid within a recombinant microorganism or transgenic host cell, the so-called intracellular decarboxylation.
  • the host cell is capable of decarboxylating fumaric acid, in particular due to the expression of homologous or heterologous genes for enzyme activities of fumaric acid synthesis, and contains according to the invention, preferably additionally, at least one, preferably heterologous, nucleic acid molecule in expressible form, preferably in the form of one or more Expression cassettes, in the genome and / or on a plasmid or vector which encodes at least one decarboxylase activity, especially at least one modified decarboxylase, wherein the host cell expresses or optionally overexpress decarboxylase activity, for example, if a homologous decarboxylase activity in the host cell is available.
  • the invention relates to a transgenic host cell which is suitable for the production of acrylic acid, which is characterized in that the host cell express
  • Preferred host cells are selected from: E. coli strains, Pseudomonas strains, Rhizopus strains, preferably the aforementioned fumaric acid producers and Clostridium strains. Preference is given to strains with high tolerance to acrylic acid, especially with at least one mechanism of their own for transporting the acrylic acid formed out of the cell (transmembrane transport) in order to minimize the intracellular action.
  • the host cell capable of producing acrylic acid from fumarate additionally expresses a primary-active, secondary-active or passive transporter or symporter system for transmembrane transport of the intracellularly synthesized acrylic acid.
  • the acrylic acid tolerance of the host cell and the yield can be improved.
  • the achievable yield is limited by the synthetic route for producing acrylic acid provided according to the invention, above all by its toxic effect on the host cells.
  • Objects of the invention are also an expression cassette and its use for transformation of a host cell.
  • the expression cassette contains at least one nucleic acid molecule characterized in more detail above, which encodes one of the decarboxylase activities characterized in more detail above.
  • the cassette has at least one promoter and optionally a termination sequence.
  • the promoter is preferably constitutive.
  • Promoter is preferably modified so that it can mediate overexpression of the encoded decarboxylase activity. Modified promoters and methods for modifying promoters are known.
  • Objects of the invention are also a corresponding vector suitable for mediating the expression of decarboxylase activity in a host cell and its use for transformation a host cell containing the above-characterized expression cassette.
  • the invention also relates to the use of the above-characterized transgenic host cell, for the biotechnological production of acrylic acid and a biocatalyst for
  • the invention provides for a method, wherein this transgenic host cell and / or the biocatalyst with immobilized transgenic
  • the invention therefore also provides a process for the preparation of acrylic acid, which, at least or exclusively, from the
  • Steps providing a transgenic host cell expressing at least one of the above-characterized decarboxylase activities, and culturing the host cell in culture medium containing the substrate fumaric acid under conditions in which the substrate is reacted and acrylic acid is formed.
  • the product is isolated from the culture medium and / or the cell and optionally purified.
  • the intracellularly formed product is released from the cell into the extracellular medium.
  • the cultivation preferably takes place in preferably liquid culture medium. It is preferably provided to cultivate the cell under anaerobic conditions. It depends on the host organism in an alternative variant also provided to cultivate the cell under aerobic conditions. On the basis of the above description of the invention, the skilled person can choose the respectively favorable enzyme equipment.
  • the invention thus relates to a transgenic host cell which comprises at least one element selected from: a nucleic acid molecule, preferably in expressible form, at least one expression cassette containing the at least one nucleic acid molecule optionally together with at least one promoter and optionally a terminator sequence, and at least one vector which contains at least one such expression cassette.
  • the nucleic acid molecule is selected from the group consisting of:
  • nucleic acid molecules which comprise at least one of the sequences selected from: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
  • nucleic acid molecules encoding amino acid molecules having at least one sequence selected from: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 , 34 and 36 contain or consist of code; and
  • nucleic acid molecules in particular fragments and derived molecules, which have at least 50%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% or more homology or sequence identity with the nucleic acid molecules described under a) and b), which has at least one decarboxylase activity, preferably one of the above Characterized decarboxylase activities, particularly preferably encode a decarboxylase activity modified according to the invention.
  • the invention also provides a process for preparing acrylic acid which comprises at least or exclusively the following steps: providing a transgenic host cell expressing at least one of the above-characterized decarboxylase activities, preferably a host cell characterized above; and cultivating the host cell in culture medium containing the substrate fumaric acid under conditions in which the
  • the transgenic host cell according to the invention is itself a fumaric acid overproducer and both the synthesis of fumaric acid and its conversion into acrylic acid according to the invention take place in the same host cell.
  • Preferred examples here are host cells of the genera Escherichia and Pseudomonas.
  • the host cell is selected from fumaric acid-forming cells, especially the genus Rhizopus.
  • the invention also provides a process for preparing acrylic acid which comprises at least or exclusively the following steps: providing a host cell which is suitable for synthesizing fumaric acid and expressing at least one of the decarboxylase activities characterized in claims 2 to 4 ; and culturing the host cell in culture medium under conditions under which acrylic acid is formed.
  • Preferred examples of such host cells are organisms of the genus Rhizopus, which contain a homologous enzyme for the synthesis of fumaric acid, preferably from C3-C6 substrates, especially glycerol and glucose, and thus are capable of producing fumaric acid.
  • transgenic host cells which are capable of producing fumaric acid by expression of heterologous genes. Such host cells are expediently used as starting organism for the transfection with at least one above-characterized nucleic acid, expression cassettes or vector, for the intracellular decarboxylation of the formed
  • 4-oxalocrotonate-carboxy-lyase (EC 4.1.1.77) from Pseudomonas sp. CF600, preferably encoded by a nucleic acid molecule having the sequence according to SEQ ID NO: 1 and / or preferably with an amino acid sequence according to SEQ ID NO: 2;
  • ACMSD (EC 4.1.1.45) from Pseudomonas fluorescens strain KU-7 (NCBI: sequence BAC65312.1, Gl 28971629), preferably coded by a nucleic acid molecule having the sequence according to SEQ ID NO: 3 and / or preferably having an amino acid sequence according to SEQ ID NO: 4;
  • OPET decarboxylase (EC 4.1.1.68), a monomeric enzyme from Escherichia coli C pJJ801, preferably encoded by a nucleic acid molecule having the sequence according to SEQ ID NO: 5 and / or preferably having an amino acid sequence according to SEQ ID NO: 6;
  • Dihydroxyphthalate carboxy lyase (EC 4.1.1.69) from Arthrobacter keyiseri 12B, preferably encoded by a nucleic acid molecule with the sequence according to SEQ ID NO: 7 and / or preferably with an amino acid sequence according to SEQ ID NO: 8;
  • Oxaloacetate carboxy lyase (EC 4.1.1.3) from Clostridium glutamicum or Lactococcus lactis subsp. lactis bv. diacetylactis (strain CRL 264), preferably encoded by a nucleic acid molecule having the sequence according to SEQ ID NO: 9 and / or preferably having an amino acid sequence according to SEQ ID NO: 10;
  • Acetoacetate decarboxylase (EC 4.1.1.4) from Clostridium acetobutyl cum ATCC 824, preferably encoded by a nucleic acid molecule having the sequence according to SEQ ID NO: 11 and / or preferably having an amino acid sequence according to SEQ ID NO: 12;
  • 2,3-dihydroxybenzoate decarboxylase (EC 4.1.1.46) from Aspergillus niger CBS 513.88, preferably encoded by a nucleic acid molecule having the sequence according to SEQ ID NO: 13 and / or preferably having an amino acid sequence according to SEQ ID NO: 14;
  • Uroporphyrinogen carboxylyase (EC 4.1.1.37) from Escherichia coli K-12 MG1655, preferably encoded by a nucleic acid molecule having the sequence according to SEQ ID NO: 15 and / or preferably having an amino acid sequence according to SEQ ID NO: 16;
  • Methylmalonyl-CoA-Decarboxylase (EC 4.1.1.41) Escherichia coli K-
  • Dihydroxyphthalate decarboxylase (EC 4.1.1.55) from Pseudomonas putida (PHT plasmid), Pseudomonas fluorescens or Pseudomonas testosteroni, preferably encoded by a nucleic acid molecule having the sequence according to SEQ ID NO: 19 and / or preferably having an amino acid sequence according to SEQ ID NO: 20 ;
  • Aryl methylmalonate decarboxylase (EC 4.1.1.76) from Alcaligenes bronchisepticus (Bordetella bronchiseptica KU 1201), preferably encoded by a nucleic acid molecule having the sequence according to SEQ ID NO: 21 and / or preferably having an amino acid sequence according to SEQ ID NO: 22;
  • Uracil-5-carboxylate-carboxylyase (EC 4.1.1.66) from Neurospora crassa, preferably encoded by a nucleic acid molecule with the sequence according to SEQ ID NO: 23 and / or preferably with an amino acid sequence according to SEQ ID NO: 24;
  • 4-hydroxybenzoate-carboxy-lyase (EC 4.1.1.61) from Clostridium hydroxybenzoicum or Sedimentibacter hydroxybenzoicus (strain JW / Z-1), an existing current knowledge of three subunits enzyme, preferably encoded by nucleic acid molecules having the sequences shown in SEQ ID NO: 31, 33 and 35 and / or preferably with the amino acid sequences according to SEQ ID NO: 32, 34 and 36.
  • Example 1 Synthesis of acrylic acid by intracellular decarboxylation of fumaric acid in a recombinant E. coli strain
  • the starting point for the transformation was a transgenic E. coli strain K12, which is capable of overproduction of fumaric acid from C3 to C6 substrates (glucose, glycerol).
  • the synthesis also provides for (re) fixation of CO 2 in the reaction of PEP carboxylase.
  • expression cassettes for decarboxylase activity of the enzyme ACMSD (EC 4.1.1.45) containing SEQ ID NO: 3 were cloned / ligated into expression vectors pUC or pPCU18 or the like.
  • the ligation of the plasmid DNA was carried out in a manner known per se.
  • the vector is opened by hydrolysis with restriction endonuclease and the corresponding DNA sequences are inserted.
  • the ligation was carried out, for example, by cassette mutagenesis.
  • the transformation of transformation-competent E. coli cells and the production of transformation-competent E. coli cells was preferably carried out according to the protocol of Hanahan, 1985 (Hananan, D. in Glover, DM (ed.)) DNA Cloning: "A Practical Approach "IRL Press Oxford 109-135).
  • the resulting recombinant cells were prepared after inoculation in a 50 L fermenter with culture medium and cultured at 25 to 40 0 C.
  • Example 2 Extracellular decarboxylation of fumaric acid in a transformed biocatalvator
  • the starting point for fumarate synthesis was a transgenic E. coli strain K12, which is capable of overproduction of fumaric acid from C3 to C6 substrates (glucose, glycerol).
  • the synthesis also provides for (re) fixation of CO 2 in the reaction of PEP carboxylase.
  • the cells were prepared after inoculation in a 50 L fermentor with culture medium and cultured at 25 to 40 0 C.
  • Fumarate was released by the cells directly into the culture medium; the transmembrane transport of the intracellularly formed fumarate was mediated for example via a proton symport.
  • a modified enzyme protein which was derived from SEQ ID NO: 4 (aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45)) and adapted for immobilization to a carrier was prepared by covalent binding to a carrier membrane (polycarbonate ).
  • the decarboxylation of the fumarate was carried out by contacting the culture medium after fermentative fumarate formation extracellularly with the bound enzyme protein.
  • the extracellular decarboxylation of fumarate on the immobilized enzyme protein is also possible in a high yield.

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Abstract

L'invention concerne la production biotechnologique d'acide acrylique et de composés apparentés ainsi que des agents et des organismes se prêtant à cette production.
PCT/EP2009/004418 2008-06-19 2009-06-19 Production biotechnologique d'acide acrylique Ceased WO2009153047A2 (fr)

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DE102008029302.4 2008-06-19
DE102008029302.4A DE102008029302B4 (de) 2008-06-19 2008-06-19 Biotechnologische Herstellung von Acrylsäure

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011131667A1 (fr) 2010-04-21 2011-10-27 Dsm Ip Assets B.V. Cellule adaptée pour la fermentation d'une composition de sucre mélangé
WO2012049173A1 (fr) 2010-10-13 2012-04-19 Dsm Ip Assets B.V. Polypeptides avec activité de perméase
WO2012143513A2 (fr) 2011-04-22 2012-10-26 Dsm Ip Assets B.V. Cellule de levure pouvant convertir des sucres comprenant l'arabinose et le xylose
WO2014060377A1 (fr) 2012-10-16 2014-04-24 Dsm Ip Assets B.V. Cellules à conversion de pentose améliorée
WO2014072232A1 (fr) 2012-11-07 2014-05-15 Dsm Ip Assets B.V. Propagation de levure à ph contrôlé
WO2016012429A1 (fr) 2014-07-24 2016-01-28 Dsm Ip Assets B.V. Cellule de levure présentant un transport amélioré du pentose

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB933102A (en) * 1959-11-03 1963-08-08 Exxon Research Engineering Co Decarboxylation of unsaturated dicarboxylic acids and their derivatives
PT1183385E (pt) * 1999-05-21 2006-11-30 Cargill Dow Llc Métodos e materiais para a síntese de produtos orgânicos

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011131667A1 (fr) 2010-04-21 2011-10-27 Dsm Ip Assets B.V. Cellule adaptée pour la fermentation d'une composition de sucre mélangé
WO2011131674A1 (fr) 2010-04-21 2011-10-27 Dsm Ip Assets B.V. Procédé de production de cellules qui sont capables de convertir l'arabinose
WO2012049173A1 (fr) 2010-10-13 2012-04-19 Dsm Ip Assets B.V. Polypeptides avec activité de perméase
WO2012049179A2 (fr) 2010-10-13 2012-04-19 Dsm Ip Assets B.V. Polypeptides à activité kinase
WO2012143513A2 (fr) 2011-04-22 2012-10-26 Dsm Ip Assets B.V. Cellule de levure pouvant convertir des sucres comprenant l'arabinose et le xylose
WO2014060377A1 (fr) 2012-10-16 2014-04-24 Dsm Ip Assets B.V. Cellules à conversion de pentose améliorée
EP3492579A1 (fr) 2012-10-16 2019-06-05 DSM IP Assets B.V. Cellules présentant une meilleure conversion de pentose
WO2014072232A1 (fr) 2012-11-07 2014-05-15 Dsm Ip Assets B.V. Propagation de levure à ph contrôlé
WO2016012429A1 (fr) 2014-07-24 2016-01-28 Dsm Ip Assets B.V. Cellule de levure présentant un transport amélioré du pentose

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DE102008029302B4 (de) 2016-08-11
WO2009153047A3 (fr) 2010-02-18

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