The invention relates to novel ethylenediamine-N, Napos; -disuccinate (EDS) synthases

Abstract

In the present invention, three novel ethylenediamine-N, N '-disuccinic acid (EDS) synthases are provided, which are derived from the group consisting of Cholatococcus asaccharoporus, Sargassum pedis, and Burkholderia sarisoli, and, in the present invention, there are provided three novel ethylenediamine-N, N'-disuccinic acid (EDDS) synthases, which are derived from the group consisting of Cholatococcus asaccharoporus, Sargassum pedis, and Burkholderia sarisoli, and, in the present invention, there are provided three novel ethylenediamine-N, N '-disuccinic acid (EDDS) synthases, which are derived from the group consisting of Cholatococcus asaccharoporus. More particularly, provided are a novel polypeptide having EDDS synthase activity, a method for producing EDDS using an EDDS synthase, a method for producing a composition comprising EDDS, and a method for preparing an EDDS synthase.

Description

Technical Field

[0001] The present invention provides novel ethylenediamine-N,N′-disuccinic acid (EDDS) synthases. More specifically, novel polypeptides having EDDS synthase activity, methods for producing EDDS using an EDDS synthase, methods for producing a composition comprising EDDS, and methods for preparing an EDDS synthase are provided. Background Art

[0002] Enzymes are protein-based biocatalysts that are widely used in a variety of different industries. They can be very specific and enantioselective catalysts and can be used under milder reaction conditions compared to traditional catalysts. For example, they can have catalytic activity at lower temperatures, which can reduce the overall energy consumption of the production process. Additionally, enzymes are biodegradable and are therefore increasingly used as a sustainable alternative to petrochemicals in various applications.

[0003] Ethylenediamine-N,N′-disuccinic acid (EDDS) is a chelating agent that can bind metal ions in a stable chelate complex. (S,S)-EDDS is readily biodegradable and has low toxicity and is therefore an attractive alternative to traditional chelating agents such as ethylenediaminetetraacetic acid (EDTA). EDTA is a synthetic chelating agent that is currently widely used in industry and has a very similar structure and properties to EDDS. However, EDTA is not easily biodegradable.

[0004] Therefore, there is a need to identify novel enzymes for the sustainable production of biodegradable EDDS. Summary of the Invention

[0005] Accordingly, the present invention relates to an isolated, synthetic or recombinant polypeptide having EDDS synthase activity, which comprises an amino acid sequence having at least 87% identity with SEQ ID NO:1, an amino acid sequence having at least 80% identity with SEQ ID NO:7, or an amino acid sequence having at least 80% identity with SEQ ID NO:9, and polynucleotides encoding these EDDS synthases.

[0006] In addition, the present invention relates to a method for producing EDDS using a polypeptide having EDDS synthase activity, the method comprising contacting fumaric acid and ethylenediamine with an EDDS synthase under conditions that permit the EDDS synthase to produce EDDS, wherein the EDDS synthase comprises an amino acid sequence having at least 87% identity with SEQ ID NO:1, an amino acid sequence having at least 80% identity with SEQ ID NO:7, or an amino acid sequence having at least 80% identity with SEQ ID NO:9.

[0007] In addition, the present invention relates to a method for preparing an EDDS synthase and a method for producing a composition (preferably a detergent composition) comprising EDDS, wherein the method comprises the steps of: a) producing EDDS using the method claimed herein, and b) formulating the EDDS produced in a) into the composition. Detailed Description

[0008] The present invention can be more readily understood by reference to the following definitions, a detailed description of the embodiments of the invention, and the examples included herein. Although the invention will be described with respect to specific embodiments, this description should not be construed in a limiting sense.

[0009] Definition

[0010] Unless otherwise indicated, the terms used herein are to be understood according to the ordinary usage of those of ordinary skill in the relevant art.

[0011] Before describing the exemplary embodiments of the present invention in detail, definitions that are important for understanding the present invention are provided. Unless otherwise stated or apparent from the nature of the definition, these definitions apply to all compounds, methods, and uses described herein.

[0012] As used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms “a / an” also include the corresponding plurals.

[0013] In the context of the present invention, the terms “about” and “approximately” indicate the range of precision that a person skilled in the art will understand still ensures the technical effect of the feature being discussed. The term typically indicates a deviation of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5% from the indicated value.

[0014] Furthermore, the terms “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable where appropriate, and the embodiments of the present invention described herein are capable of operating in other orders than those described or shown herein. In the case where the terms “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” “i,” “ii,” etc. relate to steps of a method or use or experiment, there is no temporal or time interval coherence between these steps, i.e., these steps can be carried out simultaneously or there can be a time interval of seconds, minutes, hours, days, weeks, months, or even years between such steps, unless otherwise indicated in the present application as described above or below.

[0015] Throughout the application, various publications are cited. The disclosures of all these publications and the references cited in those publications are incorporated herein by reference in their entirety to describe the state of the art to which the present invention pertains.

[0016] It should be understood that the term "comprising" is not restrictive. For the purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising at least a certain number of members, this means that a group consisting only of these members is also covered.

[0017] When describing the polypeptides of the present invention, the single-letter abbreviations of individual amino acids are used according to the well-recognized IUPAC single-letter code, or the three-letter amino acid abbreviations are used.

[0018] As used herein, "amino acid change" refers to an amino acid substitution, deletion, or insertion.

[0019] "Substitution" is described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the amino acid that replaces the original amino acid. For example, the substitution of histidine at position 120 by alanine is denoted as "His120Ala" or "H120A". Substitution can also be described by naming only the resulting amino acid without specifying the initial amino acid at that position, for example, "X120A", "120A", or "Xaa120Ala" or "120Ala".

[0020] "Deletion" is described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by an asterisk. Accordingly, the deletion of glycine at position 150 is denoted as "Gly150*" or "G150*". Alternatively, the deletion is denoted, for example, as "deletion of D183 and G184".

[0021] "Insertion" is described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the original amino acid and the additional amino acid. For example, the insertion of lysine at position 180 adjacent to glycine is named "Gly180GlyLys" or "G180GK". When more than one amino acid residue is inserted, such as the insertion of Lys and Ala after Gly180, this insertion can be denoted as: "Gly180GlyLysAla" or "G195GKA".

[0022] In cases where a substitution and an insertion occur at the same position, this can be represented as “S99SD+S99A” or, in short, “S99AD”. Sequences containing multiple changes are separated by the plus sign “+”. For example, “Arg170Tyr+Gly195Glu”, “R170Y+G195E” or “X170Y+X195E” represent that the arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamate, respectively. Alternatively, multiple changes can be separated by a space or a comma, for example, “R170Y G195E” or “R170Y,G195E”. When different alternative changes can be introduced at a single position, these different changes are separated by a comma. For example, “Arg170Tyr,Glu” and “R170T,E” represent that the arginine at position 170 is replaced by tyrosine or glutamate, respectively. Alternative substitutions at a specific position can also be represented as “X120A,G,H”, “120A,G,H”, “X120A / G / H” or “120A / G / H”. Alternatively, different changes or optional substitutions can be indicated in parentheses, for example, “Arg170[Tyr,Gly]” or “Arg170{Tyr,Gly}” or, in short, “R170[Y,G]” or “R170{Y,G}”.

[0023] The term “native” (or natural or wild-type or endogenous) cell or organism or polynucleotide or polypeptide refers to a cell or organism or polynucleotide or polypeptide as found in nature (i.e., without any human intervention).

[0024] The term “isolated” molecule, such as a polypeptide or polynucleotide, is defined herein as a molecule that has been separated from its natural environment.

[0025] The term “heterologous polypeptide” (or exogenous or foreign polypeptide) is defined herein as a polypeptide that is not naturally expressed by a host cell. The term “heterologous nucleotide” (or exogenous or foreign polynucleotide) is defined herein as a polynucleotide that is not naturally contained in a host cell.

[0026] For the purposes of the present invention, "recombinant" (or non-natural or unnatural) with respect to a cell or organism means that the cell or organism contains a polynucleotide introduced using genetic techniques. Recombinant with respect to a polynucleotide or polypeptide means that the polynucleotide or polypeptide has been newly combined or rearranged with respect to its genetic environment by using recombinant DNA techniques. Thus, a recombinant polynucleotide or polypeptide includes a polypeptide or polynucleotide that is native to the host cell, the expression of which has been quantitatively altered or the expression of which is from a genomic location different from that of the native host cell due to manipulation of the DNA of the host cell by recombinant DNA techniques (such as a stronger promoter). A recombinant polynucleotide or polypeptide can also be heterologous, which means that they can be foreign sequences, but they can also be derived from the same organism into which they are introduced.

[0027] With respect to the relationship between two or more polynucleotides or between two or more polypeptides, the term "recombinant" is used to characterize that two or more polynucleotides or two or more polypeptides do not naturally occur in a particular combination with each other.

[0028] A "modified recombinant" polynucleotide or polypeptide means a recombinant polynucleotide or recombinant polypeptide that has been modified by introducing alterations (such as deletions, substitutions and / or insertions) by using recombinant DNA techniques to alter a native polypeptide or native polynucleotide.

[0029] A "synthetic" compound is obtained by in vitro chemical and / or enzymatic synthesis.

[0030] Sequence identity is usually provided in the form of "sequence identity %" or "identity %". To calculate sequence identity, a sequence alignment is generated in a first step. According to the present invention, a pairwise global alignment is generated, which means that the two sequences are aligned over their entire length. The alignment is usually generated by using a mathematical method called an alignment algorithm.

[0031] According to the present invention, alignments are generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. [Journal of Molecular Biology] (1979) 48, pages 443 - 453). Preferably, the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the present invention, using the program default parameters (for polynucleotides: gap open = 10.0, gap extend = 0.5, and matrix = EDNAFULL; for polypeptides: gap open = 10.0, gap extend = 0.5, matrix = EBLOSUM62). After aligning two sequences, in a second step, the identity value is determined based on the resulting alignment. For this purpose, the % identity is calculated as follows: the number of identical residues is divided by the length of the alignment region (which shows the full length of the corresponding sequence of the present invention) and multiplied by 100: % identity = (identical residues / length of the alignment region of the corresponding sequence of the present invention showing the full length) * 100.

[0032] For calculating the percent identity of two polynucleotides, the same applies for calculating the percent identity with certain specifications as outlined above. For polynucleotides encoding proteins, pairwise alignments should be carried out over the full length of the coding region of the sequences of the present invention (from the start codon to the stop codon, excluding introns). Introns present in other sequences compared to the sequences of the present invention should also be removed for pairwise alignments. After aligning two polynucleotide sequences, in a second step, the identity value is determined based on the resulting alignment. Then the percent identity is calculated as follows: % identity = (identical residues / length of the alignment region, which shows the full length of the sequence of the present invention from the start codon to the stop codon and excluding introns) * 100.

[0033] In the present context, an exchange of one amino acid with a similar amino acid can be referred to as a "conservative substitution". Similar amino acids according to the present invention are defined as follows:

[0034] Amino acid A is similar to amino acid S

[0035] Amino acid D is similar to amino acids E, N

[0036] Amino acid E is similar to amino acids D, K, Q

[0037] Amino acid F is similar to amino acids W, Y

[0038] Amino acid H is similar to amino acids N, Y

[0039] Amino acid I is similar to amino acids L, M, V

[0040] Amino acid K is similar to amino acids E, Q, R

[0041] Amino acid L is similar to amino acids I, M, V

[0042] Amino acid M is similar to amino acids I, L, and V.

[0043] Amino acid N is similar to amino acids D, H, and S.

[0044] Amino acid Q is similar to amino acids E, K, and R.

[0045] Amino acid R is similar to amino acids K and Q.

[0046] Amino acid S is similar to amino acids A, N, and T.

[0047] Amino acid T is similar to amino acid S.

[0048] Amino acid V is similar to amino acids I, L, and M.

[0049] Amino acid W is similar to amino acids F and Y.

[0050] Amino acid Y is similar to amino acids F, H, and W.

[0051] As used herein, "fragment" or "subsequence" is a part of a polynucleotide or amino acid sequence. The term "functional fragment" refers to any nucleic acid or amino acid sequence that contains only a part of the full-length amino acid sequence but still has the same or similar activity and / or function. Preferably, the functional fragment has at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% identity, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99% or at least 99.5% identity to the original full-length amino acid sequence. The functional fragment contains consecutive nucleic acids or amino acids of the original nucleic acid or original amino acid sequence.

[0052] As used herein, "genetic construct" or "expression cassette" is a nucleic acid molecule consisting of at least one target sequence to be expressed, the at least one target sequence being operably linked to one or more control sequences as described herein (linked at least to a promoter).

[0053] As used herein, the term "vector" encompasses any kind of construct suitable for carrying a polynucleotide sequence for transfer into a cell or for stable or transient expression within a given cell. This encompasses any kind of cloning vehicle, such as but not limited to plasmids, phagemids, viral vectors (e.g., phage), bacteriophages, baculoviruses, cosmids, fosmids, artificial chromosomes, and any other vector specific for a particular intended host. The foreign polynucleotide sequence typically contains a coding sequence, which may be referred to herein as the "gene of interest". The gene of interest may contain introns and exons, depending on the kind of source or destination host cell.

[0054] The term "introduction of a polynucleotide" or "transformation of a polynucleotide" as referred to herein encompasses the transfer of a polynucleotide outside of a host cell into the host cell, regardless of the method used for transfer. That is, as used herein, the term "transformation of a polynucleotide" is independent of the vector, shuttle system, or host cell, and it encompasses not only transformation by polynucleotide transfer methods known in the art (see, e.g., Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), it also encompasses any other kind of polynucleotide transfer method, such as but not limited to transduction or transfection.

[0055] A polynucleotide encoding a polypeptide may be "expressed". The term "expression" or "gene expression" means the transcription of one or more genes or genetic constructs into structural RNA (e.g., rRNA, tRNA) or mRNA, followed by the translation or not of the structural RNA into protein. The process includes DNA transcription and the processing of the resulting mRNA product.

[0056] When compared to the corresponding wild-type cells, cells expressing a recombinant polynucleotide or polypeptide can exhibit "increased" or "decreased" expression. As used herein, the terms "increased expression", "enhanced expression", or "overexpression" mean any form of expression in addition to the original wild-type expression level (which may be lack of expression or non-measurable expression). As referred to herein, "increased expression", "enhanced expression", or "overexpression" is considered to mean an increase in gene expression relative to a control organism and / or, in the case of a polypeptide, an increased polypeptide level and / or increased polypeptide activity. The increase in expression can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or 100% or even more compared to the control organism.

[0057] The term "purification" or "purifying" refers to a process in which at least one component (e.g., the protein of interest) is separated from at least another component (e.g., particulate matter in a fermentation broth) and transferred to a different compartment or phase, where the different compartment or phase does not necessarily need to be separated by a physical barrier. Examples of such different compartments are two compartments separated by a filter membrane or cloth, i.e., the filtrate and the retentate; examples of such different phases are the precipitate and the supernatant or the filter cake and the filtrate. The resulting solution after purifying the enzyme of interest from the fermentation broth is referred to herein as the "purified enzyme solution".

[0058] A "composition" is a mixture of multiple components (also referred to as ingredients) prepared according to a specific formulation. Compositions include, but are not limited to, detergent compositions, fertilizer compositions, pharmaceutical compositions, household care compositions or personal care compositions, nutritional supplement compositions or industrial compositions or formulations, such as compositions used in the pulp and paper industry.

[0059] "Enzyme properties" include, but are not limited to, catalytic activity, substrate / cofactor specificity, product stability, stability over time, thermal stability, pH stability, and chemical stability.

[0060] According to the present invention, the term "enzyme stability" relates to the retention of enzymatic activity as a function of time during storage or operation. The retention of enzymatic activity as a function of time during storage is referred to as "storage stability" and is preferred in the context of the present invention.

[0061] To determine and quantify the change in catalytic activity of an enzyme stored or used under certain conditions over time, the "starting enzymatic activity" is measured at time zero (100%) and at a later time point (x%) under defined conditions. By comparing these values, the potential loss of enzymatic activity can be determined. The degree of loss of enzymatic activity determines the stability or instability of the enzyme.

[0062] "pH stability" refers to the ability of an enzyme to exhibit enzymatic activity after exposure to a particular pH value.

[0063] "Detergent composition" or "detergent" means a composition designated for cleaning soiled materials. The detergent compositions according to the present invention include detergent compositions for different applications such as laundry washing and hard surface cleaning. The term "detergent component" is defined herein to mean the types of chemicals that can be used in a detergent composition. A typical detergent component is a surfactant. A "surfactant" (used synonymously herein with "surface active agent") means an organic chemical that, when added to a liquid, changes the properties of that liquid at the interface. Surfactants are called nonionic, anionic, cationic, or amphoteric according to their ionic charge. The term "effective amount of a detergent component" includes the amount of certain components that provide effective stain removal and / or effective cleaning conditions (such as pH, temperature, water hardness), the amount of certain components that effectively provide optical benefits (such as optical brightening, dye transfer inhibition, color care), and the amount of certain components that effectively assist in processing (maintaining physical properties during processing, storage, and use; such as rheology modifiers, hydrotropes, desiccants).

[0064] The term "hard surface cleaning" relates to both household and industrial hard surface cleaning and means the process of treating a hard surface with a solution containing the detergent composition according to the present invention. Hard surfaces can include any hard surface in the home or industry, such as floors, decorations, walls, sanitary ceramics, glass, metal surfaces, including medical devices, cutlery, and tableware. A specific form of hard surface cleaning is dishwashing, including manual dishwashing (MDW) or automatic dishwashing (ADW).

[0065] The term "dishwashing" refers to all forms of dishwashing, such as manual or automatic dishwashing. Washing dishes includes, but is not limited to, cleaning all forms of pottery (such as plates, cups, glasses, bowls), all forms of cutlery (such as spoons, knives, forks, and serving utensils), and ceramics, plastics (such as melamine), metals, porcelain, glass, and acrylics.

[0066] The term "medical device cleaning" refers to the cleaning step in the reprocessing of reusable medical devices. Medical device cleaning methods can be divided into two categories: manual and mechanical / automated cleaning methods. Manual cleaning is used only when mechanical devices are not available or the medical device to be cleaned is too fragile or difficult to clean with a mechanical device. Mechanical / automated cleaning methods remove dirt and microorganisms through an automated cleaning and rinsing process, which includes ultrasonic cleaning and washing.

[0067] A composition “substantially free of” a compound shall mean herein that the corresponding compound is not intentionally added to the composition, meaning that there is at most a non-effective amount, and most preferably the composition contains 0% of the compound.

[0068] Detailed description

[0069] In the present invention, novel EDDS synthases are provided. More particularly, novel polypeptides having EDDS synthase activity, methods of producing EDDS using an EDDS synthase, methods of producing a composition comprising EDDS, and methods of preparing an EDDS synthase are provided.

[0070] EDDS synthase

[0071] The present invention relates to an EDDS synthase. The EDDS synthase according to the present invention has “EDDS synthase activity”. “EDDS synthase activity” describes the ability to catalyze the formation of EDDS using one molecule of ethylenediamine and two molecules of fumaric acid as substrates. The EDDS synthase activity can be determined as described in the literature (e.g., by Poddar et al., Biochemistry 2018, 57, 26, 3752–3763; by Wang et al., Process Biochemistry 2022, 116, 38-48; or by Takahashi et al., Biosci. Biotechnol. Biochem 1999, 63(7), 1269-1273). For example, the EDDS synthase activity can be determined by using ethylenediamine and fumaric acid as substrates and measuring the synthesized EDDS by HPLC analysis.

[0072] As a side note, the EDDS synthase has also been shown, for example by Poddar et al., Biochemistry 2018, 57, 26, 3752–3763, to accept a variety of different monoamine and diamine substrates in place of ethylenediamine to produce different aminocarboxylic acid products, including EDDS derivatives, together with fumarate.

[0073] The EDDS synthase of the present invention is an EDDS synthase having an amino acid sequence with at least 87% identity to SEQ ID NO:1, an amino acid sequence with at least 80% identity to SEQ ID NO:7, or an amino acid sequence with at least 80% identity to SEQ ID NO:9.

[0074] In one embodiment, the EDDS synthase comprises an amino acid sequence that is increasingly preferably at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:1.

[0075] In another embodiment, the EDDS synthase comprises an amino acid sequence that is increasingly preferably at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:7.

[0076] In another embodiment, the EDDS synthase comprises an amino acid sequence that is increasingly preferably at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:9.

[0077] Preferably, the EDDS synthase comprises an amino acid sequence that is increasingly preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:1, 7 or 9.

[0078] More preferably, the EDDS synthase comprises an amino acid sequence that is increasingly preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:1 or 7.

[0079] Most preferably, the EDDS synthase comprises an amino acid sequence that is increasingly preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:1.

[0080] In one embodiment, the EDDS synthase consists of an amino acid sequence that is 100% identical to SEQ ID NO:1, 7, or 9, preferably SEQ ID NO:1.

[0081] In one embodiment, the EDDS synthase comprises or consists of an amino acid sequence that is 100% identical to SEQ ID NO:1, 7, or 9, preferably SEQ ID NO:1, but contains 1 - 20, preferably 1 - 15, more preferably 1 - 10, and even more preferably 1 - 5 conservative amino acid substitutions.

[0082] Methods for introducing amino acid changes (such as substitutions, preferably conservative substitutions) into a protein sequence are well known in the art. Substitutions can be introduced by using any mutagenesis procedure known in the art (such as site - directed mutagenesis, synthetic gene construction, semi - synthetic gene construction, random mutagenesis, and shuffling), followed by the relevant screening procedures. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949 - 4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349 - 4966, US Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773 - 776; Kren et al., 1998, Nat. Med. 4:285 - 290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15 - 16, Tian et al., 2004, Nature 432:1050 - 1054; Reidhaar - Olson and Sauer, 1988, Science 241:53 - 57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152 - 2156; WO 95 / 17413; or WO 95 / 22625, Lowman et al., 1991, Biochemistry 30:10832 - 10837; US Patent No. 5,223,409; WO 92 / 06204, Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127.

[0083] In a preferred embodiment, the EDDS synthase of the present invention has a specific activity (kU / L * signal factor) higher than 1.5, preferably higher than 2.0, more preferably higher than 2.5, and most preferably higher than 3.0, where the signal factor is the SDS page signal intensity relative to the intensity of SEQ ID NO:1.

[0084] In a preferred embodiment, the EDDS synthase of the present invention exhibits the highest EDDS synthase activity within a temperature range between 20 °C and 40 °C, preferably between 30 °C and 40 °C, and more preferably between 35 °C and 40 °C.

[0085] In a preferred embodiment, the EDDS synthase of the present invention exhibits EDDS synthase activity within the range of pH 6 - 10, preferably 7 - 9, and more preferably 8 - 9.

[0086] In one embodiment, the EDDS synthase of the present invention is a purified, isolated, synthetic, and / or recombinant EDDS synthase. Preferably, the EDDS synthase of the present invention is a recombinant EDDS synthase.

[0087] Nucleic acid construct

[0088] The present invention also relates to a polynucleotide encoding the EDDS synthase of the present invention or its complementary sequence. Preferably, the polynucleotide is a codon-optimized polynucleotide for improving expression in a specific host cell (preferably an Escherichia coli cell).

[0089] Therefore, the present invention also relates to a nucleic acid, preferably an isolated, synthetic, and / or recombinant nucleic acid, which comprises:

[0090] (a) A polynucleotide that is increasingly preferably at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2, SEQ ID NO:8, or SEQ ID NO:10, where the nucleic acid encodes a polypeptide having EDDS synthase activity;

[0091] (b) A polynucleotide encoding a polypeptide, which polynucleotide is increasingly preferably at least 87%, at least 90%, at least 92%, at least 95%, at least 97% or 100% identical to SEQ ID NO:1, or at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 97% or 100% identical to SEQ ID NO:7, or at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 97% or 100% identical to SEQ ID NO:9, wherein the polypeptide has EDDS synthase activity;

[0092] (c) A polynucleotide which hybridizes under high stringency conditions, preferably under very high stringency conditions, to the complementary sequence of:

[0093] (i) the coding sequence of SEQ ID NO:1, SEQ ID NO:7 or SEQ ID NO:9; or

[0094] (ii) the polynucleotide shown in SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:10;

[0095] (d) A fragment of (a), (b) or (c), wherein the fragment encodes a polypeptide having EDDS synthase activity; or

[0096] (e) A polynucleotide that is fully complementary to any one of (a) to (d).

[0097] (f) A polynucleotide that differs from any one of the polynucleotides described in (a) to (e) only by the degeneracy of the genetic code.

[0098] In a further embodiment, the present invention also relates to a polypeptide having EDDS synthase activity, which is encoded by a polynucleotide as described herein, preferably an isolated, synthetic or recombinant polypeptide having EDDS synthase activity, which is encoded by a polynucleotide as described herein. Preferably, the polypeptide having EDDS synthase activity is encoded by a polynucleotide that is increasingly preferably at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:10.

[0099] In another embodiment, a polypeptide having EDDS synthase activity is encoded by a polynucleotide that hybridizes under high stringency conditions, preferably under very high stringency conditions, to the full-length complementary sequence of a polynucleotide that is increasingly preferably at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:10.

[0100] The invention also relates to nucleic acid constructs, preferably expression cassettes, comprising a polynucleotide as described herein.

[0101] Typically, an expression cassette comprises three elements: a promoter sequence, an open reading frame, and a 3′ untranslated region that usually contains a polyadenylation site in eukaryotes. Additional regulatory elements may include transcriptional as well as translational enhancers. Intron sequences can also be added to the 5′ untranslated region (UTR) or the coding sequence to increase the amount of mature messenger RNA accumulating in the cytosol. The expression cassette can be part of a vector or can be integrated into the genome of a host cell and replicated with the genome of its host cell. The expression cassette is generally capable of increasing or decreasing expression.

[0102] The invention also relates to expression vectors comprising a polynucleotide or nucleic acid construct as described herein. The expression vector can be a low-copy number vector or a high-copy number vector.

[0103] As used herein, a vector can provide segments for transcription and translation of a polynucleotide when transformed into a host cell or host organelle. Such additional segments can include regulatory nucleotide sequences, one or more origins of replication required for maintenance and / or replication in a particular cell type, one or more selectable markers, polyadenylation signals, suitable sites for insertion of foreign coding sequences (such as multiple cloning sites, etc.). An example is when the vector is to be maintained as an episomal genetic element (such as a plasmid or cosmid molecule) in a bacterial cell. Non-limiting examples of suitable origins of replication include f1-ori and colE1.

[0104] The vector can replicate without integrating into the genome of the host cell, such as a plasmid in a bacterial host cell, or it can integrate a part or all of its DNA into the genome of the host cell and thus result in replication and expression of its DNA.

[0105] The polynucleotide encoding the EDDS synthase of the present invention can be introduced into a vector by standard recombinant DNA techniques. Once introduced into the vector, the polynucleotide containing the coding sequence can be suitably introduced (transformed, transduced, transfected, etc.) into a host cell or host organelle. A cloning vector suitable for expressing the polynucleotide sequence in the host cell or host organelle can be selected.

[0106] Host cell

[0107] The present invention also relates to a host cell comprising a polynucleotide encoding an EDDS synthase as described herein, a nucleic acid construct as described herein, or an expression vector as described herein. In one embodiment of the invention, the vector is used for transformation of the host cell.

[0108] The polynucleotide encoding the EDDS synthase as described herein can be introduced into the host cell transiently or stably and can be maintained as non-integrated, for example as a plasmid. Generally, stable transformation is due to the integration of the nucleic acid containing the recombinant coding sequence into the chromosome or as an episome (a separate nuclear DNA fragment). Generally, transient transformation is due to the non-integration of the nucleic acid containing the recombinant nucleic acid sequence into the chromosome or not as an episome.

[0109] The introduction of the nucleic acid into the host cell can be, for example but not limited to, by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168:111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81:823-829 or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751) or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169:5271-5278). Specific transformation protocols are known in the art for various types of host cells (see, e.g., for E. coli protoplast transformation see Hanahan, 1983, J. Mol. Biol. 166:557-580).

[0110] A variety of host cells can be used to express the nucleic acid constructs described herein. Host cells containing the genetic constructs described herein can be obtained by one of the methods described herein for introducing polynucleotides into such host cells. The host cells of the present invention do not naturally express EDDS synthase. Thus, the host cells are recombinant host cells, and the nucleic acid constructs described herein are heterologous to the host cells.

[0111] In one embodiment, the host cell is a prokaryote or a eukaryote. Preferably, the host cell is a bacterial cell, an archaeal cell, a fungal cell, a yeast cell, an insect cell, a mammalian cell, or any other eukaryotic cell. In another embodiment, the host cell is a non-human host cell.

[0112] In one embodiment, the host cell is a bacterial cell. The bacterial host cell can be any Gram-positive bacterium or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, the genera Bacillus, Brevibacterium, Corynebacterium, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Paenibacillus, Geobacillus, and Oceanobacillus. Gram-negative bacteria include, but are not limited to, the genera Escherichia, Pseudomonas, Salmonella, Campylobacter, Basfia, Ensifer, Sinorhizobium, Helicobacter, Acetobacter, Flavobacterium, Fusobacterium, and Gluconobacter. In one embodiment, the host cell is a bacterial cell. In a particular embodiment, the host cell belongs to the genus Escherichia or Bacillus. In a particular embodiment, the bacterial host cell is an Escherichia cell. In a preferred embodiment, the bacterial host cell is an Escherichia coli cell.

[0113] In the method of the present invention, the bacterial host cell can be a Bacillus cell. Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus methylotrophicus, Bacillus cereus, Bacillus paralicheniformis, Bacillus subtilis, and Bacillus thuringiensis cells. In one embodiment, the bacterial host cell is a Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell. In a preferred embodiment, the bacterial host cell is a Bacillus licheniformis cell, Bacillus pumilus, or Bacillus subtilis cell. Preferably, the bacterial host cell is a Bacillus licheniformis cell.

[0114] In the method of the present invention, the bacterial host cell can be Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus bulgaricus, Lactobacillus reuteri, Staphylococcus aureus, Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium callunae, Corynebacterium ammoniagenes, Corynebacterium thermoaminogenes, Corynebacterium melassecola, Corynebacterium effiziens, Corynebacterium efficiens, Corynebacterium deserti, Brevibacterium fulvum, Brevibacterium lactofermentum, Brevibacterium divarecatum, Pseudomonas putida, Pseudomonas syringae, Streptomyces coelicolor, Streptomyces lividans, Streptomyces albus, Streptomyces avermitilis, Gluconobacter oxydans, Gluconobacter morbifer, Gluconobacter thailandicus, Acetobacter sp., Clostridium acetobutylicum, Clostridium saccharobutylicum, Clostridium beijerinckii, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, Streptococcus equi subsp. zooepidemicus or Basfiasucciniciproducens.

[0115] In another embodiment, the bacterial host cell can additionally contain modifications of other genes that may be harmful to the production, recovery or application of the polypeptide of interest, such as deletions or disruptions.

[0116] In one embodiment, the bacterial host cell is a host cell having reduced fumarase activity, preferably a fumarase-deficient cell. The reduction or absence of fumarase activity can occur naturally in the host cell or can be achieved by genetic techniques. In a more specific embodiment, the bacterial host cell is a fumarase-deficient Escherichia coli cell. In another embodiment, the bacterial host cell (e.g., an Escherichia coli cell) comprises disruption and / or deletion of the fumarase genes fumA, B, and / or C. The FumA, B, and / or C genes are described, for example, in Derbikov, D.D. et al., 2017, Appl Biochem Microbiol 53, 859–866 or Zhang et al., Appl Microbiol Biotechnol. June 2015; 99(12):5033-44. Without wishing to be bound by theory, the reduction or absence of fumarase activity in the host cell (preferably Escherichia coli) results in a lower concentration of by-products such as maleic acid compared to a host cell without reduced or absent fumarase activity.

[0117] In an alternative embodiment, the host cell does not comprise reduced or absent fumarase activity.

[0118] In another embodiment, the host cell comprises a genetic construct encoding one or more molecular chaperones capable of assisting the correct folding of the EDDS synthase. In a preferred embodiment, the host cell is a bacterial host cell, preferably an Escherichia coli cell, which comprises a genetic construct encoding the GroEL / GroES protein complex. The genetic construct encoding the GroEL / GroES complex can be located on the same vector or a different vector as the construct encoding the EDDS synthase as described herein.

[0119] In another embodiment, the bacterial host cell is a standard Escherichia coli host cell used for cloning or protein expression, including but not limited to DH5α (Invitrogen), DH10B (Invitrogen), Omnimax (Invitrogen), INV110 (Invitrogen), TOP10 (Invitrogen), HB101 (Promega), SURE (Stratagene), XL1-Blue (Stratagene), TG1 (Lucigen), BL21 and its variants (Invitrogen), and JM109 (NEB). In another embodiment, variants and derivatives of the listed E. coli cells are used, such as the E. coli strain TG10 (see the experimental description details of the strain). In a particularly preferred embodiment, the bacterial host cell is an E. coli TG10 cell. In another embodiment, the bacterial host cell is a standard Bacillus subtilis cloning host cell, including but not limited to Bacillus subtilis carrying a defective hsd(RI)R-M-locus (such as Bacillus subtilis IG-20 (BGSC 1A436)) or Bacillus subtilis carrying a defective hsdRM1 mutation (such as Bacillus subtilis 1012WT (Mobitec)).

[0120] Alternative additional host cells include but are not limited to: Aspergillus niger, Aspergillus oryzae, Hansenula polymorpha, Thermomyces lanuginosus, Fusarium oxysporum, Fusarium heterosporum, Pichia pastoris (also known as Komagataella phaffii), Myceliophthora thermophila (C1), Thothelomyces thermophila, Schizosaccharomyces pombe, Trichoderma (preferably Trichoderma reesei), and Saccharomyces (preferably Saccharomyces cerevisiae) or Rhizomucor.

[0121] Method for preparing EDDS synthase

[0122] The EDDS synthase of the present invention can be produced by any method known in the art for protein synthesis. Preferably, the EDDS synthase of the present invention is produced on an industrial scale. Industrial production of enzymes is typically accomplished by culturing host cells that express the enzyme (also known as fermentation). Suitable host cells are described herein. The polynucleotide encoding the EDDS synthase described herein can be transformed into a host cell and subsequently cultured under conditions suitable for the host cell to produce the EDDS synthase of the present invention.

[0123] Thus, in yet another embodiment, the present invention relates to a method for producing the EDDS synthase described herein, the method comprising the steps of

[0124] (a) providing a host cell comprising a recombinant nucleic acid construct by introducing into a host cell a nucleic acid construct comprising a polynucleotide encoding the EDDS synthase described herein, the recombinant nucleic acid construct comprising a polynucleotide encoding the EDDS synthase described herein;

[0125] (b) culturing the recombinant host cell of step (a) under conditions conducive to the expression of the polynucleotide; and

[0126] (c) optionally, recovering the EDDS synthase encoded by the polynucleotide.

[0127] Preferably, the host cell for preparing the EDDS synthase is an Escherichia coli cell, preferably an Escherichia coli cell comprising a genetic construct encoding one or more molecular chaperones, preferably the GroEL / GroES protein complex. In one embodiment, the Escherichia coli cell has a reduced or absent fumarase activity.

[0128] The culturing of the host cell is generally carried out in a suitable nutrient medium, allowing the recombinant cells to grow and express the desired protein. At the end of fermentation, the fermentation broth is collected and can be further processed, wherein the fermentation broth comprises a liquid fraction and a solid fraction. The target enzyme can be further purified from the fermentation broth.

[0129] The EDDS synthase described herein can be secreted (into the liquid fraction of the fermentation broth) or can remain non-secreted from the microbial cell (and thus be contained in the cells of the fermentation broth). Depending on this, the EDDS synthase can be recovered from the liquid fraction of the fermentation broth or from the host cells, or the recovered cells can be used as whole cell catalysts using methods known in the art. Recovery of the EDDS synthase can be achieved by methods known to those skilled in the art. Suitable methods for recovering proteins from fermentation broths include, but are not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. The isolated polypeptide can then be further purified by a variety of procedures known in the art, which include, but are not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, eds., VCH Publishers, New York, 1989). The purified polypeptide can then be concentrated by procedures known in the art, which include, but are not limited to, ultrafiltration and evaporation, particularly thin film evaporation. If the product of interest precipitates or crystallizes in the fermentation broth or is at least partially bound to particulate matter in the fermentation broth, additional processing steps may be required to release the protein of interest from the biomass or to dissolve the protein crystals and precipitates. If the desired protein is contained in the cells of the fermentation broth and its release is assumed, release of the protein of interest from the cells may be required. Release from the cells can be achieved, for example but not limited to, by cell lysis using techniques well known to those skilled in the art (e.g., treatment with lysozyme, sonication, French press, or combinations thereof).

[0130] Formulation or enzyme preparation comprising EDDS synthase

[0131] The invention also relates to a formulation comprising a polypeptide having EDDS synthase activity and at least one additional component, preferably the additional compound is selected from the group consisting of solvents, enzyme stabilizing compounds, and preservatives, wherein the polypeptide having EDDS synthase activity comprises an amino acid sequence having at least 87% identity to SEQ ID NO:1, at least 80% identity to SEQ ID NO:7, or at least 80% identity to SEQ ID NO:9.

[0132] Method for producing EDDS

[0133] The present invention also relates to a method for producing EDDS using a polypeptide having EDDS synthase activity, the method comprising contacting fumaric acid and ethylenediamine with an EDDS synthase under conditions that permit the EDDS synthase to produce EDDS, wherein the EDDS synthase comprises an amino acid sequence having at least 87% identity to SEQ ID NO:1, an amino acid sequence having at least 80% identity to SEQ ID NO:7, or an amino acid sequence having at least 80% identity to SEQ ID NO:9.

[0134] EDDS produced by the methods described herein can be analyzed using HPLC measurements.

[0135] For the method for producing EDDS as described herein, the EDDS synthase can be purified and used as a catalyst in pure form, or whole cells expressing the EDDS synthase can be used as whole cell catalysts.

[0136] In a preferred embodiment, EDDS is produced using a whole cell catalyst expressing the EDDS synthase described herein. Preferably, the whole cell catalyst is a host cell as described herein.

[0137] Thus, in one embodiment, the present invention relates to a method for producing EDDS, the method comprising the steps

[0138] a) providing a host cell comprising a recombinant nucleic acid construct by introducing a nucleic acid construct comprising a polynucleotide encoding the EDDS synthase described herein into a host cell, the recombinant nucleic acid construct comprising a polynucleotide encoding the EDDS synthase described herein;

[0139] b) culturing the host cell of step (a) under conditions conducive to the expression of the polynucleotide encoding the EDDS synthase;

[0140] c) optionally harvesting the host cell of step (b) and

[0141] d) contacting the host cell of step (b) and / or (c) with fumaric acid and ethylenediamine under conditions that permit the fumaric acid and ethylenediamine to contact the EDDS synthase to produce EDDS;

[0142] wherein the EDDS synthase comprises an amino acid sequence having at least 87% identity to SEQ ID NO:1, an amino acid sequence having at least 80% identity to SEQ ID NO:7, or an amino acid sequence having at least 80% identity to SEQ ID NO:9.

[0143] Preferably, the whole cell catalyst is an Escherichia coli cell expressing the EDDS synthase described herein. Preferably, the host cell (preferably an Escherichia coli host cell) comprises a genetic construct encoding one or more molecular chaperones (preferably the GroEL / GroES protein complex) as described herein. Preferably, the host cell (preferably an Escherichia coli host cell) has reduced or absent fumarase activity as described herein. In an alternative embodiment, the host cell does not have reduced or absent fumarase activity, and the method for preparing EDDS using the EDDS synthase of the present invention comprises inactivating the fumarase side activity by heat treatment or alkali treatment.

[0144] Accordingly, in another embodiment, the present invention relates to a method for producing EDDS, the method comprising the steps

[0145] a) providing a host cell comprising a recombinant nucleic acid construct by introducing a nucleic acid construct comprising a polynucleotide encoding the EDDS synthase described herein into the host cell, the recombinant nucleic acid construct comprising a polynucleotide encoding the EDDS synthase described herein;

[0146] b) culturing the recombinant host cell of step (a) under conditions conducive to the expression of the polynucleotide;

[0147] c) optionally harvesting the host cell of step (b);

[0148] d) exposing the host cell obtained in step (b) and / or (c) to conditions suitable for inactivating the fumarase side activity of the host cell, such as by heat treatment or alkali treatment, and

[0149] e) contacting the host cell of step (d) with fumaric acid and ethylenediamine under conditions allowing the fumaric acid and ethylenediamine to contact the EDDS synthase to produce EDDS,

[0150] wherein the EDDS synthase comprises an amino acid sequence having at least 87% identity with SEQ ID NO:1, an amino acid sequence having at least 80% identity with SEQ ID NO:7, or an amino acid sequence having at least 80% identity with SEQ ID NO:9.

[0151] Chelating agents such as EDDS and their respective alkali metal salts are useful multidentate chelating agents for metal ions (such as metal ions like Mg2+, Ca2+, Ba2+, Cu2+, Fe2+, Zn2+, Fe3+). Thus, in one embodiment, a method for producing EDDS using a polypeptide having EDDS synthase activity comprises contacting fumaric acid and ethylenediamine and additionally a metal ion, preferably a metal salt, more preferably magnesium, most preferably magnesium hydroxide with the EDDS synthase under conditions that allow the EDDS synthase to produce EDDS, wherein the EDDS synthase comprises an amino acid sequence having at least 87% identity to SEQ ID NO:1, an amino acid sequence having at least 80% identity to SEQ ID NO:7, or an amino acid sequence having at least 80% identity to SEQ ID NO:9. In a preferred embodiment, the EDDS synthesized using the EDDS synthase of the present invention is in the form of a metal-EDDS complex, preferably in the form of a Mg-EDDS complex.

[0152] EDDS has two chiral centers and generally can produce three stereoisomeric forms, namely (R,R)-EDDS, (S,S)-EDDS, and the achiral meso-(R,S)-EDDS.

[0153] In a preferred embodiment, the EDDS synthase of the present invention predominantly produces (S,S)-EDDS. In a preferred embodiment, the EDDS synthase of the present invention produces (S,S)-EDDS with an optical purity of more than at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% enantiomeric excess (ee) or 100% (S,S)-EDDS.

[0154] In a preferred embodiment, the (S,S)-EDDS synthesized by the EDDS synthase of the present invention is biodegradable.

[0155] Thus, in a preferred embodiment, a method for producing EDDS using a polypeptide having EDDS synthase activity comprises contacting fumaric acid and ethylenediamine with an EDDS synthase under conditions that permit the EDDS synthase to produce (S,S)-EDDS, wherein the EDDS synthase comprises an amino acid sequence having at least 87% identity to SEQ ID NO:1, at least 80% identity to SEQ ID NO:7, or at least 80% identity to SEQ ID NO:9, and wherein the optical purity of the produced (S,S)-EDDS is increasingly preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% enantiomeric excess (ee) or is 100% (S,S)-EDDS.

[0156] In a preferred embodiment, the method steps for producing EDDS using a polypeptide having EDDS synthase activity as described herein are carried out in a temperature range between 20 °C and 40 °C, preferably between 30 °C and 40 °C, more preferably between 35 °C and 40 °C.

[0157] In a further preferred embodiment, the method steps for producing EDDS using a polypeptide having EDDS synthase activity as described herein are carried out in a pH range between 6 and 10, preferably between 7 and 9, more preferably between 8 and 9.

[0158] In an alternative embodiment, the invention also relates to a method for producing an EDDS derivative using a polypeptide having EDDS synthase activity, the method comprising contacting fumaric acid and an ethylenediamine derivative (which is a monoamine or diamine substrate different from EDDS) with an EDDS synthase under conditions that permit the EDDS synthase to produce the EDDS derivative, wherein the EDDS synthase comprises an amino acid sequence having at least 87% identity to SEQ ID NO:1, at least 80% identity to SEQ ID NO:7, or at least 80% identity to SEQ ID NO:9.

[0159] Method for using EDDS

[0160] EDDS obtained by any of the methods described herein can be used in a composition. The composition can in principle be any composition. Preferably, the composition is a composition in which the chelating agent provides a beneficial effect. Preferably, EDDS produced by any of the methods described herein is used as a builder in the composition.

[0161] Compositions comprising EDDS obtained by any of the methods described herein include, but are not limited to, detergent compositions, fertilizer compositions, pharmaceutical compositions, household care compositions or personal care compositions, nutritional supplement compositions, compositions suitable for the production of pulp or for the paper industry, for photographic equipment, for soil remediation, and for wastewater treatment.

[0162] Most preferably, EDDS obtained by any of the methods described herein can be used in detergent compositions. The detergent compositions are preferably laundry or hard surface cleaning detergent compositions suitable for household care and / or industrial and institutional (I&I) cleaning. Both the laundry and hard surface cleaning compositions can be in the form of hand-wash or automatic wash compositions. The hard surface cleaning detergent composition can preferably be a dishwashing composition or a medical instrument cleaning composition. Preferably, the dishwashing composition is an automatic dishwashing (ADW) composition.

[0163] Method for producing a composition comprising EDDS

[0164] In one embodiment, the present invention relates to a method for producing a composition comprising EDDS, wherein the method comprises the following steps:

[0165] a) producing EDDS using a method for producing EDDS using a polypeptide having EDDS synthase activity, the method comprising contacting fumaric acid and ethylenediamine with EDDS synthase under conditions allowing the EDDS synthase to produce EDDS, wherein the EDDS synthase comprises an amino acid sequence having at least 87% identity with SEQ ID NO:1, at least 80% identity with SEQ ID NO:7, or at least 80% identity with SEQ ID NO:9 as described herein, and

[0166] b) formulating the EDDS produced in a) into a composition.

[0167] In a preferred embodiment, the present invention relates to a method for producing a detergent composition comprising EDDS, wherein the method comprises the following steps:

[0168] a) producing EDDS using a method for producing EDDS using a polypeptide having EDDS synthase activity, the method comprising contacting fumaric acid and ethylenediamine with EDDS synthase under conditions allowing the EDDS synthase to produce EDDS, wherein the EDDS synthase comprises an amino acid sequence having at least 87% identity with SEQ ID NO:1, at least 80% identity with SEQ ID NO:7, or at least 80% identity with SEQ ID NO:9 as described herein, and

[0169] b) Formulate the EDDS produced in a) into a detergent composition.

[0170] In one embodiment, step b) of formulating the EDDS produced in a) into a composition comprises the steps of:

[0171] b1) EDDS produced by the method as described herein; and

[0172] b2) One or more components different from EDDS, preferably formulated into a composition with detergent components as described herein.

[0173] The one or more detergent components may be selected from the group consisting of: additional chelating agents and / or builders different from EDDS, enzymes, enzyme stabilization systems, surfactants, antifoaming agents, polymers, bleaching systems (bleaching agents), rheology modifiers, hydrotropes, softeners, desiccants, optical brighteners, buffers, preservatives, anti-corrosion additives, dyes, and fragrances. Preferably, one or more of the detergent components are biodegradable and / or bio-based.

[0174] Preferred embodiment

[0175] In particular, what is preferred herein is:

[0176] 1. A method for producing EDDS using a polypeptide having ethylenediamine-N,N'-disuccinic acid (EDDS) synthase activity, the method comprising contacting fumaric acid and ethylenediamine with the EDDS synthase under conditions that permit the EDDS synthase to produce EDDS, wherein the EDDS synthase comprises an amino acid sequence having at least 87% identity with SEQ ID NO:1, at least 80% identity with SEQ ID NO:7, or at least 80% identity with SEQ ID NO:9.

[0177] 2. The method for producing EDDS according to embodiment 1, wherein the EDDS synthase increasingly preferably comprises at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:1.

[0178] 3. The method for producing EDDS according to embodiment 1, wherein the EDDS synthase increasingly preferably comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:7.

[0179] 4. The method for producing EDDS according to embodiment 1, wherein the EDDS synthase increasingly preferably comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:9.

[0180] 5. The method for producing EDDS according to embodiment 1, wherein the EDDS synthase increasingly preferably comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:1, SEQ ID NO:7 or SEQ ID NO:9, more preferably to SEQ ID NO:1 or to SEQ ID NO:7, and most preferably to SEQ ID NO:1.

[0181] 6. The method for producing EDDS according to any one of the preceding embodiments, wherein the step of contacting fumaric acid and ethylenediamine with the EDDS synthase under conditions allowing the EDDS synthase to produce EDDS is carried out in the presence of magnesium hydroxide, preferably wherein the produced EDDS chelates magnesium to form Mg-EDDS.

[0182] 7. The method for producing EDDS according to any one of the preceding embodiments, wherein (S,S)-EDDS is produced, and wherein the optical purity of the produced (S,S)-EDDS increasingly preferably has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% enantiomeric excess (ee) or is 100% (S,S)-EDDS.

[0183] 8. A method for producing EDDS according to any one of the foregoing embodiments, wherein the step of producing EDDS using a polypeptide having EDDS synthase activity is carried out in a temperature range between 20 °C and 40 °C, preferably between 30 °C and 40 °C, more preferably between 35 °C and 40 °C.

[0184] 9. A method for producing EDDS according to any one of the foregoing embodiments, wherein the step of producing EDDS using a polypeptide having EDDS synthase activity is carried out in a pH range between 6 and 10, preferably between 7 and 9, more preferably between 8 and 9.

[0185] 10. An isolated, synthetic or recombinant polypeptide having EDDS synthase activity, wherein the recombinant polypeptide having EDDS synthase activity increasingly preferably comprises at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 1.

[0186] 11. An isolated, synthetic or recombinant polypeptide having EDDS synthase activity, wherein the recombinant polypeptide having EDDS synthase activity increasingly preferably comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 7.

[0187] 12. An isolated, synthetic or recombinant polypeptide having EDDS synthase activity, wherein the recombinant polypeptide having EDDS synthase activity increasingly preferably comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 9.

[0188] 13. An isolated, synthetic or recombinant polypeptide having EDDS synthase activity, wherein the recombinant polypeptide having EDDS synthase activity increasingly preferably comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:1, SEQ ID NO:7 or SEQ ID NO:9, more preferably to SEQ ID NO:1 or to SEQ ID NO:7, most preferably to SEQ ID NO:1.

[0189] 14. An isolated, synthetic or recombinant polypeptide according to any one of preferred embodiments 8 to 11, wherein the polypeptide comprises SEQ ID NO:1, SEQ ID NO:7 or SEQ ID NO:9, more preferably SEQ ID NO:1 or SEQ ID NO:7, most preferably SEQ ID NO:1 or consists of the same, or wherein the amino acid sequence of the polypeptide having EDDS synthase activity comprises SEQ ID NO:1, SEQ ID NO:7 or SEQ ID NO:9, more preferably SEQ ID NO:1 or SEQ ID NO:7, most preferably SEQ ID NO:1 or consists of the same with 1-20 amino acid substitutions, preferably 1-10 or more preferably 1-5 amino acid substitutions, preferably wherein the substitutions are conservative substitutions.

[0190] 15. A method for preparing an isolated, synthetic or recombinant polypeptide according to any one of preferred embodiments 10 to 14, the method comprising the steps of: a) providing a polynucleotide encoding a polypeptide having EDDS synthase activity, b) transforming the polynucleotide into a host cell, c) culturing the host cell to produce a polypeptide having EDDS synthase activity, and optionally d) purifying the polypeptide having EDDS synthase activity.

[0191] 16. An isolated, synthetic or recombinant polynucleotide encoding an isolated, synthetic or recombinant polypeptide according to any one of preferred embodiments 10 to 14, wherein the isolated, synthetic or recombinant polynucleotide preferably has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:10, more preferably, wherein when a polypeptide having EDDS synthase activity has at least 90% sequence identity with SEQ ID NO:1, the polynucleotide encoding the polypeptide having EDDS synthase activity has at least 70% sequence identity with SEQ ID NO:2, or wherein, when a polypeptide having EDDS synthase activity has at least 90% sequence identity with SEQ ID NO:7, the polynucleotide encoding the polypeptide having EDDS synthase activity has at least 70% sequence identity with SEQ ID NO:8, or wherein, when a polypeptide having EDDS synthase activity has at least 90% sequence identity with SEQ ID NO:9, the polynucleotide encoding the polypeptide having EDDS synthase activity has at least 70% sequence identity with SEQ ID NO:10.

[0192] 17. A nucleic acid construct comprising the polynucleotide according to embodiment 16.

[0193] 18. An expression vector comprising the polynucleotide according to preferred embodiment 16 or the nucleic acid construct according to preferred embodiment 17.

[0194] 19. A host cell comprising the polynucleotide according to preferred embodiment 16, the nucleic acid construct according to preferred embodiment 17 or the expression vector according to preferred embodiment 18.

[0195] 20. The host cell according to preferred embodiment 19, wherein the host cell is an Escherichia coli (E. coli) cell.

[0196] 21. The host cell according to any one of preferred embodiments 19 or 20, wherein the host cell has reduced or absent fumarase activity.

[0197] 22. A method for producing EDDS, wherein the method comprises the following steps

[0198] a) Providing a host cell according to any one of preferred embodiments 19 to 21;

[0199] b) Culturing the host cell of step (a) under conditions conducive to the expression of the polynucleotide encoding the EDDS synthase;

[0200] c) Optionally harvesting the host cell of step (b); and

[0201] d) Contacting the host cell of step (b) and / or (c) with fumaric acid and ethylenediamine under conditions that permit the fumaric acid and ethylenediamine to contact the EDDS synthase to produce EDDS, preferably under the conditions described in any one of preferred embodiments 1 to 9.

[0202] 23. A method for producing a composition comprising EDDS, preferably a detergent composition, the method comprising the following steps:

[0203] a) Producing EDDS using the method described in any one of preferred embodiments 1 to 9, and

[0204] b) Formulating the EDDS produced in a) into a composition.

[0205] 24. The method for producing a composition according to embodiment 23, wherein the composition comprising EDDS is a detergent composition, and wherein the EDDS is in the form of the magnesium salt of EDDS.

[0206] 25. The method for producing a composition according to embodiment 23 or 24, wherein the optical purity of the EDDS is increasingly preferably (S,S)-EDDS having an enantiomeric excess (ee) of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, or is 100% (S,S)-EDDS.

[0207] Examples

[0208] Example 1 Cloning of EDDS Synthase

[0209] Standard codon usage in Escherichia coli was used to generate DNA sequences encoding a potential EDDS synthase and EDDS06 as a control. The synthetic DNA sequences (BioCat GmbH) were cloned into the plasmid pDHE19.2 (Ress-Loeschke, M. et al., DE 19848129, 1998, (BASF AG)). The genes of interest were under the control of the rhamnose-inducible promoter (rhaBAD). The resulting plasmids were used to transform competent cells of the Escherichia coli strain TG10 (Kesseler, M. et al., WO 2004050877 A1, 2004, (BASF AG)) (Chung, C.T. et al., Proc Natl Acad Sci U S A, 1989, 86, 2172). The Escherichia coli strain TG10 is an rhaA derivative of Escherichia coli TG1 (DSMZ 6056) transformed with pHSG575 (Takeshita, S. et al., Gene, 1987, 61, 63) and pAgro4 (pBB541 in Tomoyasu, T. et al., Mol. Microbiol., 2001, 40, 397). - - derivative.

[0210] Table 1: EDDS synthase sequences

[0211] Name SEQ ID NO polypeptide SEQ ID NO polynucleotide EDDS01 1 2 EDDS02 3 4 EDDS03 5 6 EDDS04 7 8 EDDS05 9 10 EDDS06 (control) 11 12

[0212] Example 2 Recombinant production of EDDS synthase

[0213] Escherichia coli TG10 and the control carrying the recombinant plasmid encoding the potential EDDS synthase were inoculated into 20 ml of LB medium (Bertani, G., J Bacteriol, 1951, 62, 293) in a 100 ml baffled conical flask, which was supplemented with 100 μg / ml ampicillin, 50 μg / ml spectinomycin, 20 μg / ml chloramphenicol, 12.5 μg / ml tetracycline, 0.2 mM isopropyl-β-D-thiogalactoside and 0.5 g / l rhamnose. The cultures were incubated at 37 °C under shaking conditions (200 rpm) for 18 h.

[0214] The whole cell samples were normalized to OD15 (600 nm) and analyzed using SDS PAGE. The band intensities of the EDDS synthase were compared and the signal factor was calculated as a measure of the expression level, see Table 2. Thus, the EDDS01 synthase signal was set to 1 and the relative factors (signal factors) were calculated compared to EDDS01.

[0215] Table 2: Determination of the expression level of EDDS synthase (signal factor) using SDS PAGE

[0216]

[0217] Example 3 Activity assay of EDDS synthase

[0218] The enzymatic activity was measured by incubating 20 μl of thawed cells expressing EDDS synthase (which had previously been frozen in medium at -20 °C) at 37 °C, where the thawed cells were normalized to OD 15 and the incubation was carried out at pH 8.5 in 100 μl of substrate buffer containing 71.6 g / l fumaric acid, 26.95 g / l magnesium hydroxide and 18.5 g / l ethylenediamine in 1 M Tris buffer. A 10 μl sample was taken 30 min after the start of the reaction. The reaction was terminated by adding 290 μl of termination / elution solution (500 ml of 25% methanol, 1500 g of water, 1 g / L copper(II) acetate monohydrate, 2 g / L tetrabutylammonium hydroxide, pH 2.8 (adjusted with H3PO4)). The sample was filtered through a 0.22 μm filter before analysis by HPLC. For HPLC measurements, the following settings were used: column: Aqua C18 (Phenomenex), temperature: 40 °C, UV detection at 254 nm, isocratic run with the elution solution.

[0219] The volumetric enzymatic activity was calculated using the following formula:

[0220]

[0221] where ΔC is the concentration of EDDS produced over the time span Δt (here 0 to 30 min), and k is the dilution factor, which reflects the sample dilution.

[0222] To calculate the specific activity, the volumetric activity was divided by the respective signal factor (Table 3).

[0223] Table 3: Activity of EDDS synthase

[0224]

[0225] As shown in Table 3, EDDS01 (SEQ ID NO:1), EDDS04 (SEQ ID NO:7) and EDDS05 (SEQ ID NO:9) showed EDDS synthase activity. Surprisingly, EDDS02 (SEQ ID NO:3) and EDDS03 (SEQ ID NO:5) did not show any EDDS synthase activity. EDDS01 (SEQ ID NO:1) and EDDS04 (SEQ ID NO:7) showed particularly high EDDS synthase activity.

Claims

1. A method for producing ethylenediamine-N,N'-disuccinic acid (EDDS) using a polypeptide having EDDS synthase activity, the method comprising contacting fumaric acid and ethylenediamine with the EDDS synthase under conditions that permit the EDDS synthase to produce EDDS, wherein the EDDS synthase comprises an amino acid sequence having at least 87% identity to SEQ ID NO:1, an amino acid sequence having at least 80% identity to SEQ ID NO:7, or an amino acid sequence having at least 80% identity to SEQ ID NO:

9.

2. An isolated, synthetic or recombinant polypeptide having EDDS synthase activity, the polypeptide comprising an amino acid sequence having at least 87% identity to SEQ ID NO:1, an amino acid sequence having at least 80% identity to SEQ ID NO:7, or an amino acid sequence having at least 80% identity to SEQ ID NO:

9.

3. The isolated, synthetic or recombinant polypeptide according to claim 2, wherein the polypeptide comprises an amino acid sequence having at least 87%, at least 90%, at least 92%, at least 95%, at least 97% or 100% identity to SEQ ID NO:

1.

4. The isolated, synthetic or recombinant polypeptide according to claim 2, wherein the polypeptide comprises an amino acid sequence having at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 97% or 100% identity to SEQ ID NO:

7.

5. The isolated, synthetic or recombinant polypeptide according to claim 2, wherein the polypeptide comprises an amino acid sequence having at least 80%, at least 82%, at least 85%, at least 87%, at least 90%, at least 92%, at least 95%, at least 97% or 100% identity to SEQ ID NO:

9.

6. The isolated, synthetic or recombinant polypeptide according to claims 2-5, wherein the polypeptide comprises or consists of SEQ ID NO:1, SEQ ID NO:7 or SEQ ID NO:9, or wherein the amino acid sequence of the polypeptide having EDDS synthase activity comprises or consists of SEQ ID NO:1, SEQ ID NO:7 or SEQ ID NO:9 with 1-20 amino acid substitutions, preferably 1-10 amino acid substitutions, or more preferably 1-5 amino acid substitutions, preferably wherein these substitutions are conservative substitutions.

7. An isolated, synthetic or recombinant polynucleotide encoding the isolated, synthetic or recombinant polypeptide according to any one of claims 2 to 6, wherein the isolated, synthetic or recombinant polynucleotide preferably has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:2, SEQ ID NO:8 or SEQ ID NO:

10.

8. A nucleic acid construct, the nucleic acid construct comprising the polynucleotide according to claim 7.

9. An expression vector, the expression vector comprising the polynucleotide according to claim 7 or the nucleic acid construct according to claim 8.

10. A host cell, the host cell comprising the polynucleotide according to claim 7, the nucleic acid construct according to claim 8 or the expression vector according to claim 9.

11. The host cell according to claim 10, wherein the host cell is an Escherichia coli cell.

12. The host cell according to claim 10 or 11, wherein the host cell has reduced or absent fumarase activity.

13. A method for preparing an isolated, synthetic or recombinant polypeptide according to any one of claims 2-6, the method comprising the following steps: a) providing a host cell according to any one of claims 10 - 12, b) culturing the host cell to produce a polypeptide having EDDS synthase activity, and optionally c) purifying the polypeptide having EDDS synthase activity.

14. A method for producing a composition comprising EDDS, preferably a detergent composition, wherein the method comprises the following steps: a) producing EDDS using the method according to claim 1, and b) formulating the EDDS produced in a) into a composition.

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