WO2009153047A2 - Biotechnological production of acrylic acid - Google Patents

Abstract

The invention relates to the biotechnological production of acrylic acid and related compounds and also means and organisms which are suitable therefor.

Description

 BIOTECHNOLOGICAL MANUFACTURE OF ACRYLIC ACID

description

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.

State of the art

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.

There is a need for an improved route to the production of acrylic acid, especially a process for recovering acrylic acid from readily available substrates, and high real yield biotechnological processes. task

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. Next, 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),

Aminocarboxymuconate Semialdehyde Decarboxylase (ACMSD) (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 δ- Carboxylate-carboxy-lyase (EC 4.1.1.66),

4-hydroxyphenylacetate-carboxy-lyase (EC 4.1.1.83),

4-hydroxybenzoate-carboxy-lyase (EC 4.1.1.61) and activities derived therefrom modified enzymes.

Particular preference is given to the ACMSD and variants derived therefrom, in particular sequence variants, with increased substrate specificity for fumaric acid / fumarate.

In a preferred embodiment, 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

(see above) realized modified enzyme with altered substrate specificity, which improves the conversion of fumaric acid to acrylic acid.

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. Thus it can be avoided that the acrylic acid formed spontaneously converts into by-products, for example polymerization of acrylic acid, which may even occur at temperatures above 25 ° C. In the context of this invention, the terms "fumaric acid" and "acrylic acid" regularly also fumarate and acrylate understood. On the basis of the pK _s values (fumaric acid: pK _s -ι: 4.5, pK _S2 : 3.0, acrylic acid: pK _s : 4.25) and the respective prevailing and pH values and concentrations, the person skilled in the art can readily recognize whether and to what extent the de-protonated forms exist.

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.

Finally, another preferred variant of the invention for

Preparation of Acrylic Acid The use of the decarboxylase activity of 4-hydroxybenzoate carboxy lyase (EC 4.1.1.61) or an enzyme activity derived therefrom which inhibits decarboxylation. tion of the substrate fumaric acid to acrylic acid can catalyze. 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.

In a preferred embodiment of the invention, the enzymatically catalyzed conversion of fumaric acid to acrylic acid takes place following a biotechnological, preferably fermentative synthesis of fumaric acid. Variants of 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). Such 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.

Alternatively, known microorganisms belonging to the fumaric acid

Synthesis are suitable used. Rhizopus species are preferred here, preferably selected from R. nigricans, R. arrhizzu, R. oryzae and R. formosa. In a preferred embodiment, 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

Variant of this aspect of the invention, therefore, the fumaric acid overproducer additionally expresses at least one protein which mediates or supports the transmembrane transport of intracellularly synthesized fumaric acid.

Alternatively or additionally, the host cell becomes at least short-term

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.

By the preferably provided subdivision of the biotechnological synthesis of acrylic acid in a substep (a) of the biotechnological production of fumaric acid within a host cell and the second substep (b) of the decarboxylation of the fumaric acid to acrylic acid, which preferably takes place as extracellular biotransformation (see below ), the toxic effect of the final acrylic acid product on the fumaric acid-producing host cell can be avoided. In another preferred embodiment of the invention, 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.

In a preferred embodiment of the invention, 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. In this preferred variant of the invention, 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. Finally, 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:

a) 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

b) derived or modified amino acid molecules or fragments which have at least 80%, more preferably at least 90% or more, sequence identity with the amino acid molecules described under a) and encode the decarboxylase activity.

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.

This also means so-called protein analogues. In the context of this invention, these 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.

For technical applications, 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. In the context of the invention, 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. According to the invention, 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.

The person skilled in 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. Preferably, 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.

Preferably, 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.

Low swelling behavior, good chemical stability towards acids and bases, good microbiological stability and good compressive strength are further prerequisites which have to be met by the wearer. be submitted to. The surface-accessible functional groups also play an important role.

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 expresses at least one of the above-characterized Decar- boxylase activities.

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. In a preferred variant of this aspect of the invention, 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. Mediated by an additional dedicated transport system to exclude the acrylic acid from the cell, preferably in combination with a process for removing acrylic acid from the culture medium, the acrylic acid tolerance of the host cell and the yield can be improved. Without wishing to be bound by theory, 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. Of the

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.

Finally, 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

Preparation of acrylic acid containing a carrier and at least one transgenic host cell immobilized thereon that expresses at least one decarboxylase activity.

The invention provides for a method, wherein this transgenic host cell and / or the biocatalyst with immobilized transgenic

Host cell with fumaric acid, especially with fumaric acid-containing medium, is brought into contact, so that an intracellular conversion of fumaric acid to acrylic acid takes place in the cells. 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.

In an optional further step, the product is isolated from the culture medium and / or the cell and optionally purified. Usually, 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. According to the invention, the nucleic acid molecule is selected from the group consisting of:

a) 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,

23, 25, 27, 29, 31, 33 and 35 and their complementary sequences contain or consist of;

b) 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

c) 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

Substrate reacted and acrylic acid is formed.

In an alternative variant of this aspect of the invention, 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. In an alternative variant, 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. Alternative examples are 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

Fumaric acid to acrylic acid to allow.

The invention and the means for its implementation are described in more detail below, without the invention being limited to the specific embodiments mentioned. Due to the structure of their natural substrates and products, as well as the experimental detection of sequence and recombinant expression, the following decarboxylases are preferred for the invention. These are also the preferred "wild types" for modification preferred according to the invention for improving the enzyme function, in particular the substrate specificity, and are listed in the attached Sequence Listing;

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-

12 MG1655, preferably encoded by a nucleic acid molecule having the sequence according to SEQ ID NO: 17 and / or preferably having an amino acid sequence according to SEQ ID NO: 18;

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-hydroxyphenylacetate carboxylysis (EC 4.1.1.83) from Clostridium difficile (strain DSMZ 1296T), an enzyme consisting of three subunits according to the current state of knowledge, preferably encoded by nucleic acid molecules having the sequences according to SEQ ID NO: 25, 27 and 29 and / or preferably with the amino acid sequences according to SEQ ID NO: 26, 28 and 30; and

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.

EXEMPLARY EMBODIMENTS

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.

To realize the decarboxylase activity, 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.

For example, 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. The addition of a C3 - C ^β -Substrates, preferably glucose, and in an alternative approach, glycerol, was carried out directly in the culture medium.

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. The addition of a C3-Cβ substrate, preferably glucose, and in an alternative glycerol approach, was carried out directly into the culture medium.

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.

Results: From the substrates glucose and glycerol can be obtained in high yield via the intermediate fumarate acrylate.

The extracellular decarboxylation of fumarate on the immobilized enzyme protein is also possible in a high yield.

Claims

claims 1 . Process for the biotechnological production of acrylic acid, comprising the step: Decarboxylation of fumaric acid to acrylic acid, wherein the conversion of fumaric acid to acrylic acid is catalyzed by decarboxylase activity. 2. The method of claim 1, wherein the decarboxylase activity is selected from the group consisting of: 4-oxalocrotonate-carboxy-lyase (EC 4.1.1.77), 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-δ-carboxylate-carboxy-lyase (EC 4.1.1.66), 4-hydroxyphenylacetate-carboxy-lyase (EC 4.1.1.83), 4-hydroxybenzoate-carboxy-lyase (EC 4.1.1.61) and Activities of derived modified enzymes. 3. The method of claim 1 or 2, wherein the decarboxylase activity is that of a modified enzyme and wherein the modification of the enzyme protein is suitable: to increase the substrate specificity with respect to the substrate fumaric acid, to reduce the end product inhibition with respect to acrylic acid, to increase the chemical and structural stability of the enzyme and / or to increase the temperature stability of the enzyme. 4. The method of claim 3, wherein the modification is one or more changes in the primary structure of the enzyme protein and which is selected from: deletion, insertion and addition of one or more amino acids and replacement of at least one amino acid by at least one other amino acid. 5. The method according to any one of claims 1 to 4, wherein the decarboxylase activity is immobilized on a support. 6. A method according to any one of claims 1 to 4, wherein the decarboxylation takes place within a host cell and the host cell expresses decarboxylase activity. 7. Process for the preparation of acrylic acid, comprising the steps: Providing a transgenic host cell expressing at least one of the decarboxylase activities characterized in claims 2 to 4, 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. 8. A process for the preparation of acrylic acid, comprising the steps: Providing a host cell capable of 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. 9. Process for the preparation of acrylic acid, comprising the steps: Providing a host cell capable of synthesizing fumaric acid, Culturing the host cell in culture medium under conditions in which fumaric acid is formed, and Contacting the formed fumaric acid with at least one of the characterized in the claims 2 to 4 decarboxylase activity under conditions that allow conversion of the fumaric acid to acrylic acid. 10. An expression cassette for transforming a host cell, comprising one or more nucleic acid molecules, which codes for one or more characterized in the claims 2 to 4 decarboxylase activities, wherein the nucleic acid molecule is selected from: a) Nucleic acid molecules with sequences selected from: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33 SEQ ID NO: 35 and complementary sequences thereof; b) Nucleic acid molecules encoding amino acid molecules having sequences selected from: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO : 30, SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 36; and c) Fragments and deduced molecules thereof which have at least 70% homology and encode decarboxylase activity. 11. A vector containing the expression cassette according to claim 10 which is suitable for mediating the expression of the at least one decarboxylase activity in a host cell. 12. A transgenic host cell suitable for the production of acrylic acid, characterized in that the host cell expresses at least one of characterized in the claims 2 to 4 decarboxylase activities. 13. Transgenic host cell according to claim 12, containing at least one of: A nucleic acid molecule characterized in claim 10 in expressible form, at least one expression cassette characterized in claim 10, and at least one characterized in claim 11 vector. 14. Use of the host cell according to claim 12 or 13 for the biotechnological production of acrylic acid. 15. A biocatalyst for the production of acrylic acid, comprising: a carrier and at least one biocatalytic immobilized thereon Element selected from: Transgenic host cell according to claim 12 or 13, Cell fragment of the host cell having at least one of characterized in the claims 2 to 4 decarboxylase activities, and - Isolated enzyme protein or protein analogue having at least one of characterized in the claims 2 to 4 decarboxylase activity.