WO2011078667A2

WO2011078667A2 - Method of finding a biocatalyst having ammonia lyase activity

Info

Publication number: WO2011078667A2
Application number: PCT/NL2010/050876
Authority: WO
Inventors: Petronella Catharina Raemakers-Franken; Guido Meurer; Jürgen Wolfgang ECK; Klaus Jürgen LIEBETON; Axel Christoph Trefzer
Original assignee: Dsm Ip Assets B.V.
Priority date: 2009-12-22
Filing date: 2010-12-22
Publication date: 2011-06-30
Also published as: WO2011078667A3

Abstract

A method of finding a biocatalyst having ammonia lyase activity, in particular a lysine ammonia lyase, comprising - providing a library comprising a plurality of cells in one or more cell cultures, which cells are candidates for having an ammonia lyase activity, which one or more cultures comprise a culture medium containing at least one nitrogen source selected from the group of amines, including amino acids, and structural analogues thereof, as sole nitrogen source or sources for the cells; - selecting at least one candidate which grows in said culture medium; and - screening the selected candidate or candidates which grow in said culture for the ammonia lyase activity.

Description

Title: Method of finding a biocatalyst having ammonia lyase activity

The invention relates to a method of finding a biocatalyst having ammonia lyase activity (hereafter in short 'ammonia lyase activity'), in particular amino acid ammonia lyase activity. The invention further relates to a peptide having ammonia lyase activity, to a nucleic acid sequence encoding such peptide, to a host cell comprising such nucleic acid and to a method wherein a biocatalyst having ammonia lyase activity is used for the preparation of a (bio)chemical compound.

In biochemistry, an ammonia lyase is a biocatalyst (in general an enzyme) that catalyses the breaking of a carbon-nitrogen bond, whereby an amino group of a substrate is cleaved from the substrate, under formation of ammonia.

Ammonia lyases can thus be interesting catalysts for preparing (bio)chemical compounds from starting compounds containing an amino group, such as amino acids, or be useful for the synthesis of a compound comprising an amino group, e.g. an amino acid (by catalysing the reverse reaction).

The industrial use of biocatalysts for synthesis of (bio)chemical compounds is of growing interest, amongst others methodologies are provided that can serve as a sustainable alternative for processes that make use of oil or natural gas as a source for starting materials for the synthesis of bulk chemical compounds.

An example of a bulk chemical compound for which preparation it would be interesting to provide a preparation method making use of a biocatalyst is epsilon caprolactam (hereafter "caprolactam"), or a compound from which caprolactam can be prepared. Caprolactam can be used for preparing polyamides.

WO 05/68643 discloses a method for biochemically preparing 6- amino caproic acid (6-ACA), a known intermediate for the preparation of caprolactam, from 6-aminohex-2-enoic acid (6-AHEA). The 6-AHEA may e.g. be obtained by chemically converting alpha-lysine in a manner known per se. It would be desirable though to provide a method wherein lysine, which is a biomolecule and thus can be used as a carbon source for a micro-organism, is converted biochemically into 6- AHEA. This would allow the production of 6-ACA in a single fermentative process from a suitable carbon source, if desired, thereby providing a simplified process.

It is an object of the present invention to provide a method that can be used to find a biocatalyst that has ammonia lyase activity.

It is in particular an object of the present invention to provide a method that can be used to find a biocatalyst that is capable of catalysing the conversion of lysine into 6-AHEA.

It is further an object to provide a novel biocatalyst that has ammonia lyase activity, in particular such biocatalyst that has catalytic activity with respect to the conversion of lysine into 6-AHEA.

It is further an object to provide a novel method for producing a (bio)chemical compound, making use of a biocatalyst that has ammonia lyase activity.

It is further an object to provide a novel compound, that may be used as a nitrogen source in a method for finding an ammonia lyase according to the invention.

One or more further objects which may be solved in accordance with the invention will follow from the description below.

The inventors found that it is possible to provide a biocatalyst that has ammonia lyase activity from a library comprising a plurality of cells which are candidates for having ammonia lyase activity.

Accordingly, the present invention relates to a method of finding a biocatalyst having ammonia lyase activity, comprising

- providing a library comprising a plurality of cells in one or more cell cultures, which cells are candidates for having an ammonia lyase activity, which one or more cultures comprise a culture medium containing at least one nitrogen source selected from the group of amines, including amino acids, and structural analogues thereof, as sole nitrogen source or sources for the cells;

- selecting at least one candidate which grows in said culture medium; and

- screening the selected candidate or candidates which grow in said culture for the ammonia lyase activity.

In particular, said method has been found suitable for finding a biocatalyst having amino acid ammonia lyase activity, in an embodiment wherein as sole nitrogen source or sources one or more compounds are used selected from the group of amino acids and structural analogues of amino acids.

More in particular, said method has been found suitable for finding a biocatalyst having lysine ammonia lyase activity, in an embodiment wherein as sole nitrogen source or sources one or more compounds are used selected from the group of lysine and structural analogues of lysine, the lysine ammonia lyase activity preferably being L-lysine ammonia lyase activity and the sole nitrogen source or sources preferably being selected from L-lysine and structural analogues thereof. If desired, the biocatalyst having amonia lyase activity or a nucleic acid sequence encoding an ammonia lyase is isolated from the cell culture, in a method according to the invention.

It has been found that in particular a specific group of amino acids is useful as a sole nitrogen source in a method for finding an ammonia lyase, in particular a lysine ammonia lyase. Accordingly, the invention further relates to an amino acid selected from the group of 2-amino-2-methyl-6-(dimethylamino)-hexanoic acid, 3- amino-3-methyl-6-(dimethylamino)-hexanoic acid, 3-amino-3-methyl-heptanoic acid and 6-acetamido-3-amino-3-methyl-hexanoic acid. One or more of these amino acids may in particular be used in a method for finding a lysine ammonia lyase.

The present invention further relates to a biocatalyst or nucleic acid sequence encoding an ammonia lyase found by a method according to the invention.

A biocatalyst found in accordance with the invention may in particular be used as a catalyst in the preparation of a (bio)chemical compound from a suitable substrate. For example, 6-AHEA may be prepared from lysine using a biocatalyst having lysine ammonia lyase activity (found) according to the invention. Hereinafter, 'lysine ammonia lyase' is also referred to as 'LAL'.

Another application for a biocatalyst found in accordance with the invention may be in the pharmaceutical field. For instance, phenyl alanine ammonia lyases have been reported to be useful in cancer therapy, see e.g. US 2009/263369. Thus in a specific aspect, the invention is directed at a biocatalyst found in accordance with the invention for medical use.

It is noted that ammonia lyases have been mentioned in the art for a limited number of substrates (i.e. specifics amino acids). To the inventors knowledge, these are aspartate ammonia lyases, phenylalanine ammonia lyases, tyrosine ammonia lyases and histidine ammonia lyases. It is contemplated that these activities are extraordinary, since these substrate amino acids contain a group that has an enzyme-activating effect. Such activating group is not generally present in amino acids. Although a method according to the invention may be used for finding a (further) aspartate ammonia lyase, phenylalanine ammonia lyase, tyrosine ammonia lyase or histidine ammonia lyase, the peptides having ammonia lyase activity claimed herein as such are other than those known aspartate ammonia lyases, phenylalanine ammonia lyases, tyrosine ammonia lyases and histidine ammonia lyases. In particular, the peptides, biocatalysts or nucleic acids claimed herein as such have ammonia lyase activity with respect to a substrate selected from the group of alanine, valine, leucine, isoleucine, serine, threonine, methionine, cysteine, asparagine, glutamine, tryptophan, glycine, aspartic glutamic acid, lysine, arginine, and proline, or - in the case of the nucleic acids: encode ammonia lyase activity with respect to at least one of these substrates. Preferably, this substrate for an ammonia lyase activity is selected from the proteinogenic isomers of these amino acids, in particular alpha-L-lysine. Likewise, in a preferred embodiment a method of finding an ammonia lyase is directed at finding a an ammonia lyase having lyase activity for one or more amino acids selected from the group of alanine, valine, leucine, isoleucine, serine, threonine, methionine, cysteine, asparagine, glutamine, tryptophan, glycine, aspartic glutamic acid, lysine, arginine, and proline. In a particularly preferred embodiment the method is directed at finding an ammonia lyase having lyase activity for one or more proteinogenic isomers of these amino acids, in particular alpha-L-lysine.

It is also possible to use the biocatalyst to provide a nucleic acid sequence encoding an ammonia lyase, which sequence may then be used to produce a (heterologous) host cell, which thereafter may be used as a biocatalyst itself in the production of a (bio)chemical compound, or to produce an ammonia lyase.

The term "selecting" as used herein is defined as a process in which one or more biocatalysts are tested for growth using certain specific conditions (using lysine or a structural analogue as the sole nitrogen source), which growth is an indication for the presence of the desired biocatalytic activity.

The term "screening" as used herein is defined as a method in which one or more biocatalysts are tested for a desired biocatalytic conversion(s), in particular for having ammonia lyase activity, such as (alpha-L-)lysine ammonia lyase activity.

The term "or" as used herein means "and/or" unless specified otherwise.

The term "a" or "an" as used herein means "at least one" unless specified other wise.

When referring to a noun (e.g. a compound, an additive, etc.) in the singular, the plural is meant to be included. Thus, when referring to a specific moiety, e.g. "compound", this means "at least one" of that moiety, e.g. "at least one compound", unless specified otherwise.

When referred herein to carboxylic acids or carboxylates, e.g. 6-ACA, another amino acid, 5-FVA, adipic acid/adipate, succinic acid/succinate, acetic acid/acetate, these terms are meant to include the protonated carboxylic acid (free acid), the corresponding carboxylate (its conjugated base) as well as a salt thereof, unless specified otherwise. When referring herein to amino acids, e.g. 6-ACA, this term is meant to include amino acids in their zwitterionic form (in which the amino group is in the protonated and the carboxylate group is in the deprotonated form), the amino acid in which the amino group is protonated and the carboxylic group is in its neutral form, and the amino acid in which the amino group is in its neutral form and the carboxylate group is in the deprotonated form, as well as salts thereof.

When referring to a compound of which several isomers exist (e.g. a cis and a trans isomer, an R and an S enantiomer), the compound in principle includes all enantiomers, diastereomers and cis/trans isomers of that compound that may be used in the particular method of the invention.

When referring to 'lysine' in general, this term includes alpha-L-lysine, alpha-D-lysine, beta-L-lysine and beta-D-lysine.

When an enzyme is mentioned with reference to an enzyme class (EC) between brackets, the enzyme class is a class wherein the enzyme is classified or may be classified, on the basis of the Enzyme Nomenclature provided by the

Nomenclature Committee of the International Union of Biochemistry and Molecular

Biology (NC-IUBMB), which nomenclature may be found at

http://www.chem.gmul.ac.uk/iubmb/enzyme/. Other suitable enzymes that have not (yet) been classified in a specified class but may be classified as such, are meant to be included.

When referring to an enzyme or another biocatalytic moiety from a particular source, recombinant enzymes or other recombinant biocatalytic moieties, originating from a first organism, but actually produced in a (genetically modified) second organism, are specifically meant to be included as enzymes or other biocatalytic moieties, from that first organism.

As used herein, the term "functional analogue" of a nucleic acid at least includes other sequences encoding a peptide having the same amino acid sequence and other sequences encoding a homologue of such peptide.

The term "homologue" is used herein in particular for polynucleotides or polypeptides having a sequence identity of at least 30 %, preferably at least 40 %, more preferably at least 60%, more preferably at least 65%, more preferably at least 70 %, more preferably at least 75%, more preferably at least 80%, in particular at least 85 %, more in particular 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 %. The term homologue is also meant to include nucleic acid sequences (polynucleotide sequences) which differ from another nucleic acid sequence due to the degeneracy of the genetic code and encode the same polypeptide sequence.

Sequence identity or similarity is herein defined as a relationship between two or more polypeptide sequences or two or more nucleic acid sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences, but may however also be compared only for a part of the sequences aligning with each other. In the art, "identity" or "similarity" also means the degree of sequence relatedness between polypeptide sequences or nucleic acid sequences, as the case may be, as determined by the match between such sequences. Preferred methods to determine identity or similarity are designed to give the largest match between the sequences tested. In context of this invention a preferred computer program method to determine identity and similarity between two sequences includes BLASTP and BLASTN (Altschul, S. F. et al., J. Mol. Biol. 1990, 215, 403-410, publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894). Preferred parameters for polypeptide sequence comparison using BLASTP are gap open 10.0, gap extend 0.5, Blosum 62 matrix. Preferred parameters for nucleic acid sequence comparison using BLASTN are gap open 10.0, gap extend 0.5, DNA full matrix (DNA identity matrix).

The term biocatalyst is used herein in a broad sense for biological material or moieties derived from a biological source, for instance an organism or a biomolecule derived there from, having catalytic activity, in particular having ammonia lyase activity. The biocatalyst may in particular comprise one or more enzymes. A biocatalytic reaction may comprise one or more chemical conversions of which at least one is catalyzed by a biocatalyst. Thus the biocatalyst may accelerate a chemical reaction in at least one reaction step in the preparation of a (bio)chemical compound. The biocatalyst may be in any form. In an embodiment, the biocatalyst is a living organism (such as living whole cells). In particular, the biocatalyst may be an enzyme. The enzyme may perform a catalytic function inside the cell. It is also possible that the enzyme may be secreted into a medium, wherein the cells are present. In an embodiment, one or more enzymes are used isolated from the natural environment (isolated from the organism it has been produced in), for instance as a solution, an emulsion, a dispersion, (a suspension of) freeze-dried cells, a lysate, or immobilised on a support. The use of an enzyme isolated from the organism it originates from may in particular be useful in view of an increased flexibility in adjusting the reaction conditions such that the reaction equilibrium is shifted to the desired side.

Living cells may be growing cells, resting or dormant cells (e.g.

spores) or cells in a stationary phase. It is also possible to use an enzyme forming part of a permeabilised cell (i.e. made permeable to a substrate for the enzyme or a precursor for a substrate for the enzyme or enzymes).

The biocatalyst may in principle be any organism, or be obtained or derived from any organism. This organism may be a naturally occurring organism or a heterologous organism. The heterologous organism is typically a host cell which comprises at least one nucleic acid sequence encoding a heterologous enzyme, capable of catalysing at least one reaction step in a preparation method of the invention. The organism from which the heterologous nucleic acid sequence originates may be may be eukaryotic or prokaryotic. In particular said organisms may be independently selected from animals (including humans), plants, bacteria, archaea, yeasts and fungi.

The host cell may be eukaryotic or prokaryotic. In an embodiment, the host cell is selected from the group of fungi, yeasts, euglenoids, archaea and bacteria.

A heterologous biocatalyst, in particular a heterologous cell, as used herein, is a biocatalyst comprising a heterologous protein or a heterologous nucleic acid (usually as part of the cell's DNA or RNA) The term "heterologous" when used with respect to a nucleic acid sequence (DNA or RNA), or a protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. It is understood that heterologous DNA in a heterologous organism is part of the genome of that heterologous organism. Heterologous nucleic acids or proteins are not endogenous to the cell into which they are introduced, but have been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly heterologous RNA encodes for proteins not normally expressed in the cell in which the heterologous RNA is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognise as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein. The library may in particular be a metagenomic library. Such library typically comprises genomic fragments of various micro-organisms, which fragments may have been identified or which may be unidentified, and which fragments have been cloned into a suitable micro-organism for expression, such as into a organism selected from the group of Escherichia, Pseudomonas, Bacillus, Streptomyces and Saccharomyces. The fragments may in principle originate from any organism. The organism(s) may be culturable or un-culturable under the existing conditions, may have a specific habitat, requiring specific environmental factors (e.g. temperature, pH, light, oxygen, nutrients) or symbiotic partners. In particular the organisms may be endosymbionts of a multicellular organism such as a sponge, insect, mammal or plant.

As known in the art, the term "metagenome" defines the totality of all genomes of organisms of a given habitat and is furthermore defined in the art; see inter alia Handelsman et. al. (1998, Chem Biol 5, R245-9). In particular, the term

"metagenome" relates to genomic and episomal nucleic acids, preferably DNA, derived from unknown or uncultivable microorganisms, i.e. organisms that cannot be isolated by standard methods and made actively replicating in standard artificial media for indefinite periods of time. Accordingly the term "a fraction of the metagenome of a given habitat" defines in accordance with the invention nucleic acid molecules and in particular large nucleic acid molecules (>200bp) derived from the total pool of heterogenous microbial genomes present in a given habitat. This is irrespective of phylogenetic affiliation or molecular or physiological traits. Particularly the

representation of any particular microbial genome in the extracted portion of the metagenome is not influenced by or dependent on the cultivatability of this organism. Therefore nucleic acids of uncultivated and in a preferred form particularly of uncultivatable (micro)organisms are substantially represented in the extracted fraction of the metagenome.

In an embodiment, the library comprises a variety of environmental samples containing candidate biocatalysts, in particular a variety of water samples (e.g. waste water samples), compost samples and/or soil samples. Such samples comprise a variety of wild-type micro-organisms.

Metagenomic libraries may be provided in a manner known perse, e.g. from a commercial supplier or by a method as described e.g. in Schmidt et al., 1991 , J Bact, 173, 4371 -4378, Zhou et al., 1996, Appl Environm Microbiol, 62„ 316- 322, Henne. et al., 1999, Appl Environm Microbiol, 65, 3901 -3907, or Ronden et al., 2000, Appl Environm Microbiol, 66, 2541 -2547. In particular, a metagenomic library may be obtained by recovering DNA for the metagenomic library from an environment rich in organisms (e.g. soil, sediments of waste water samples). Suitable methods of accomplishing this can be divided in two categories depending on whether bacteria are lysed directly in the context of the substratum (in situ, direct methods) or are first separated from the surrounding material (ex situ, indirect methods), see inter alia Co urto is et al., 2001 , Environm. Microbiol., 3_431 - 439). In either category of methods cells can be lysed by enzymatic (e.g. lysozyme, proteases) and/or detergent treatments enabling the release of DNA from the cells. Subsequent purification steps like e.g. chromatographic, electrophoretic or chemical methods are usually performed to separate the DNA from substances (e.g. humic acids) inhibitory to subsequent enzymatic treatments for the integration of the metagenomic fragments into plasmid-, fosmid-, cosmid- or bacterial artificial chromosome (BAC)-vectors. The introduction of these metagenomic DNA- containing vectors into a bacterial host allows for their infinite reproduction and provide the resource for the identification of the genes encoding the desired enzymes.

The term "structural analogue" of an amine - in particular an amino acid, more in particular lysine - is used herein to indicate that the analogue comprises a functional group that may be recognised by the biocatalyst having ammonia lyase activity and acts as a substrate for the ammonia lyase activity of the biocatalyst.

Preferred structural analogues can differ depending upon the desired activity for the ammonia lyase. In particular a structural analogue may contain one or more functional groups in a way that favour release of nitrogen from such structural analogue by an ammonia lyase type reaction over other more common biochemical reactions - for instance - aminotransferase or amino oxidase reactions - that will release nitrogen from an amine, in particular an amino acid, for instance lysine.

In case an amino acid ammonia lyase for a specific alpha amino acid is to be found, the corresponding beta amino acid may be used as a structural analogue, and vice versa.

Structural analogues for amino acids, such as lysine, are in particular defined by the general formula

Herein one of X and Y is NH₂ and the other is H.

Herein one of Z and Z' is H and the other is a protective group for avoiding keto- formation, such as an alkyl group, in particular an alkyl having 1 -6 carbons, more in particular a methyl group.

Herein R is a hydrocarbon group, in particular an alkyl group, which hydrocarbon group may comprise one or more substituents, for instance one or more substituents selected from amino groups, hydroxyl groups, thiol groups, sulphur and halogen groups. R is preferably a group comprising 1 -12 carbon atoms, in particular 2-7 carbon atoms.

The use of one or more structural analogues for the amine for which the ammonia lyase to be found should have ammonia lyase activity as the sole nitrogen source is preferred because the inventors have contemplated that the chance of finding a false positive would be higher when using said amine as sole nitrogen source.

Preferably, a structural analogue of the amine is chosen which i) elicits the desired ammonia lyase activity leading to removal of the amino group from the amino or the structural analogue (in case the amine is an alpha-amino acid, it preferably elicits the desired ammonia lyase activity leading to removal of the alpha- amino group from the amino acid or structural analogue), and

ii) has a low tendency towards eliciting side-reactions.

In an advantageous embodiment, the structural analogue or analogues used as sole nitrogen source or sources consist of one or more amino acids selected from the group of 2-amino-2-methyl-6-(dimethylamino)-hexanoic acid, 2- amino-2-methyl-hexanoic acid, 3-amino-3-methyl-(6-dimethylamino)-hexanoic acid, 3- amino-3-methyl-heptanoic acid and 6-acetamido-3-amino-3-methyl-hexanoic acid. Said structural analogue or analogues are in particular useful for finding a lysine ammonia lyase. Of these structural analogues, 2-amino-2-methyl-hexanoic acid (CAS# 6322-51 - 6 for the racemic compound, CAS# 105815-96-1 for the S-enantiomer and CAS# 105815-95-0 for the R-enantiomer) may be prepared in a manner known per se. Said other structural analogues may be prepared, e.g., as described in the examples herein below.

The exact composition of the cell culture, including culture medium, and growth conditions (such as temperature, pH etc) can be based on generally known procedures for maintaining a culture of cells of the species used. Growth selection is in particular based on the requirement of microorganism on nitrogen for growth and is known in the art; see for instance Shin & Kim, 1997, Biotech Bioeng, 55, 348 - 358. Due to the omission of any other nitrogen source in the culture-medium cells ideally depend on the presence of the desired ammonia lyase to release ammonia from the structural-analogue. In the absence of the ammonia lyase, the cells cannot grow out to colony forming units (cfu). In the presence of a ammonia lyase, cells can grow and form colony forming units.

The selection of at least one candidate which grows in the culture medium can be done in a manner known per se for verifying whether a cell culture grows, e.g. by visual inspection of plates with solid media for appearance of colonies or by measuring OD₆₀₀ (optical density at 600 nm) for liquid cultures.

The screening of a candidate for having ammonia lyase activity can be done by cultivating the selected candidate in a culture medium comprising the amine, in particular the amino acid, for which the candidate should have ammonia lyase activity (instead of the structural analogue thereof) and testing the culture medium for the presence of the corresponding product that will be formed when removing an amino group. This product usually is an enoic acid, in case of an amino acid as the amine from which an amino group is removed. For the testing, e.g. liquid chromatography (LC) with mass spectrometry (MS) detection may be used.

For instance, the screening for LAL activity can be done by cultivating the selected candidate in a culture medium comprising (alpha-)lysine and measuring whether any 6-AHEA and/or beta-homoproline (a spontaneous breakdown product of 6-AHEA) is formed, e.g. by liquid chromatography (LC) with mass spectrometry (MS) detection.

Another way to carry out the screening is to prepare cell free extracts from the selected candidate cells, to mix the extracts with the amine (for instance lysine) in a suitable buffer (e.g. pH 8), incubate the mixture for a suitable duration at a suitable temperature, e.g. 1 -12 hrs at 20-45 °C and analyse the mixture for the presence of the product that will be formed when removing the amino group from the amine (6-AHEA and/or beta-homoproline in case the amine is lysine), e.g. by LC-MS- MS. If desired, the reaction can be actively be stopped by adding acid and/or by freezing the mixture until analysis.

For instance, when screening for a LAL, cell free extracts from the selected candidate cells, the extracts can be mixed with lysine in the buffer, where after the mixture is incubated, e.g. for 1 -12 hrs at 20-45 ^<€. Thereafter the mixture can be analysed for the presence of 6-AHEA and/or beta-homoproline, e.g. by LC-MS-MS.

If desired, the biocatalyst having ammonia lyase activity can be isolated from the cell culture, as a cell. It is also possible to isolate polypeptide(s) having ammonia lyase activity produced in such cell from the cell, of which polypeptide sequence or a partial polypeptide sequence can be determined. If desired, the isolated biocatalyst may be purified further. Further, a nucleic acid may be obtained from a metagenomic fragment of the cell, of which nucleic acid the sequence can be determined. Suitable biotechnological techniques to perform any of such activities are generally known in the art, see e.g. Sambrook, J., and Russell, D.W. Molecular Cloning: A Laboratory Manual.3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001 ).

A biocatalyst having ammonia lyase activity may in principle be used for any purpose for which such activity may be useful. In particular such biocatalyst may be used for preparing a (bio)chemical compound. The biocatalyst used for a specific activity may in principle be in any form that allows it to perform its intended function. It is for instance not necessary in general to isolate and purify a specific enzyme having ammonia lyase activity from a cell in which it has been expressed in order to be able to prepare a (bio)chemical compound. Thus, the invention further relates to a composition comprising a biocatalyst having ammonia lyase activity. As an alternative to an isolated and purified enzyme having ammonia lyase activity, a biocatalyst used for catalysing a specific reaction may in particular be a cell having ammonia lyase activity, a cell lysate having ammonia lyase activity or an enzyme mixture having ammonia lyase activity.

Accordingly, the invention further relates to a method of producing a (bio)chemical compound, comprising:

a) providing a biocatalyst having ammonia lyase activity found by a method according to the invention or a peptide according to the invention;

b) providing a production strain having ammonia lyase activity from said biocatalyst;

c) providing a reaction mixture comprising said production strain and a substrate for the ammonia lyase and if desired one or more cofactors for the chemical conversion reaction catalysed by said biocatalyst, and

d) allowing said substrate to be converted by said biocatalyst, thereby providing the (bio)chemical compound as the reaction product.

The substrate for the ammonia lyase may be obtained in a manner known per se, e.g. lysine may be obtained in a manner as described in Appl. Microbiol. Biotech (2005) 69, 1 -8.

In a preferred method of producing a (bio)chemical compound, the ammonia lyase is alpha-lysine ammonia lyase, the substrate is alpha-lysine and the reaction product is 6-AHEA, which may thereafter be converted into 6-ACA, if desired.

To the best of the inventors knowledge, the use of lysine ammonia lyase in a method for producing 6-ACA has not been disclosed in the art before.

Accordingly, the inventors are thought to be the first to disclose such a method. Hence, the invention further relates to a method for preparing 6-amino caproic acid, wherein 6- aminohex-2-enoic acid is prepared from alpha-lysine in a reaction catalysed by a biocatalyst having alpha-lysine ammonia lyase activity, which may be a biocatalyst found in a method according to the invention, and converting the 6-aminohex-2-enoic acid into 6-amino caproic acid.

The conversion of 6-AHEA into 6-ACA may be carried out in a manner known per se. In particular the conversion of 6-aminohex-2-enoic acid into 6- amino caproic acid is catalysed by a biocatalyst having alpha, beta-enoate reductase activity towards 6-AHEA. A suitable biocatalyst having alpha, beta-enoate reductase activity and suitable reaction conditions for this step may e.g. be found in WO 05/68643 of which the contents with respect to these biocatalysts and suitable reaction conditions are incorporated by reference, in particular claims 2-19, page 28, line 17 to 31 line 39.

If desired, a host cell can be used which - in addition to exhibiting LAL activity - exhibits said alpha, beta-enoate reductase activity.

If a host cell comprising an ammonia lyase is used for preparing a (bio)chemical compound of interest, the host cell may advantageously be a host cell that is capable of producing the amino group containing substrate for the ammonia lyase that is to be used in the preparation of the (bio)chemical compound from said substrate. Thus, the host cell may be cultured in a medium comprising a precursor for said substrate, which precursor may be more readily available on a large scale than said substrate. The precursor usually comprises a carbon source and a nitrogen source.

In particular, if in a method for preparing a (bio)chemical compound an amino acid is converted in a reaction catalysed by an amino acid ammonia lyase, the host cell may advantageously be a host cell that is capable of producing said amino acid.

For instance, if the ammonia lyase is a LAL used for preparing a (bio)chemical compound from lysine, the host cell advantageously is capable of synthesising lysine. In particular, in case an organism (usually a cell culture) is used that is capable of lysine synthesis, one or more precursors for lysine may be used that allow the organism to synthesise lysine. In general, a nitrogen source and a carbon source is used for the synthesis of lysine.

The carbon source for synthesis of lysine or for synthesis of another substrate for an ammonia lyase may in particular contain at least one compound selected from the group of monohydric alcohols, polyhydric alcohols, carboxylic acids, carbon dioxide, fatty acids, glycerides, tri- and di-acyl-glycerides including mixtures comprising any of said compounds. Suitable monohydric alcohols include methanol and ethanol, Suitable polyols include glycerol and carbohydrates. Suitable fatty acids or glycerides may in particular be provided in the form of an edible oil, preferably of plant origin.

In particular a carbohydrate may be used as a carbon source, because usually carbohydrates can be obtained in large amounts from a biologically renewable source, such as an agricultural product, preferably an agricultural waste- material. Preferably a carbohydrate is used selected from the group of glucose, fructose, sucrose, lactose, saccharose, starch, cellulose and hemi-cellulose.

Particularly preferred are glucose, oligosaccharides comprising glucose and polysaccharides comprising glucose.

As a nitrogen source for synthesis of lysine or for synthesis of another substrate for an ammonia lyase in principle an amino acid different from lysine respectively said other substrate may be suitable, which may then also serve as a nitrogen source. In particular an anorganic nitrogen source may be used, such as a nitrate salt or an ammonium salt.

Reaction conditions in a method of the invention making use of a biocatalyst may be chosen depending upon known conditions for the biocatalyst, in particular the enzyme, the information disclosed herein and optionally some routine experimentation.

In principle, the pH of the reaction medium used may be chosen within wide limits, as long as the biocatalyst is active under the pH conditions. Alkaline, neutral or acidic conditions may be used, depending on the biocatalyst and other factors. In case the method includes the use of a micro-organism, e.g. for expressing an enzyme catalysing a method of the invention, the pH is selected such that the micro-organism is capable of performing its intended function or functions. The pH may in particular be chosen within the range of four pH units below neutral pH and two pH units above neutral pH, i.e. between pH 3 and pH 9 in case of an essentially aqueous system at 25 °C. A system is considered aqueous if water is the only solvent or the predominant solvent (> 50 wt. %, in particular > 90 wt. %, based on total liquids), wherein e.g. a minor amount (< 50 wt. %, in particular < 10 wt. %, based on total liquids) of alcohol or another solvent may be dissolved (e.g. as a carbon source) in such a concentration that micro-organisms which may be present remain active. In particular in case a yeast and/or a fungus is used, acidic conditions may be preferred, in particular the pH may be in the range of pH 3 to pH 8, based on an essentially aqueous system at 25 °C. If desired, the pH may be adjusted using an acid and/or a base or buffered with a suitable combination of an acid and a base.

In principle, the incubation conditions can be chosen within wide limits as long as the biocatalyst shows sufficient activity and/ or growth. This includes aerobic, micro-aerobic, oxygen limited and anaerobic conditions.

Anaerobic conditions are herein defined as conditions without any oxygen or in which substantially no oxygen is consumed by the biocatalyst, in particular a micro-organism, and usually corresponds to an oxygen consumption of less than 5 mmol/l.h, in particular to an oxygen consumption of less than 2.5 mmol/l.h, or less than 1 mmol/l.h.

Aerobic conditions are conditions in which a sufficient level of oxygen for unrestricted growth is dissolved in the medium, able to support a rate of oxygen consumption of at least 10 mmol/l.h, more preferably more than 20 mmol/l.h, even more preferably more than 50 mmol/l.h, and most preferably more than 100 mmol/l.h.

Oxygen-limited conditions are defined as conditions in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The lower limit for oxygen-limited conditions is determined by the upper limit for anaerobic conditions, i.e. usually at least 1 mmol/l.h, and in particular at least 2.5 mmol/l.h, or at least 5 mmol/l.h. The upper limit for oxygen-limited conditions is determined by the lower limit for aerobic conditions, i.e. less than 100 mmol/l.h, less than 50 mmol/l.h, less than 20 mmol/l.h, or less than to 10 mmol/l.h.

Whether conditions are aerobic, anaerobic or oxygen limited is dependent on the conditions under which the method is carried out, in particular by the amount and composition of ingoing gas flow, the actual mixing/mass transfer properties of the equipment used, the type of micro-organism used and the micro-organism density. In a preferred method of the invention, at least the reaction step wherein the ammonia lyase is used is carried out under fermentative conditions. The terms 'fermentation', 'fermentative' and the like, are used herein in the broad sense to describe an industrial process wherein a (bio)chemical compound is converted into another (bio)chemical compound making use of a micro-organism.

In principle, the temperature used is not critical, as long as the biocatalyst, in particular the enzyme, shows substantial activity. Generally, the temperature may be at least 0 °C, in particular at least 15 °C, more in particular at least 20 °C. A desired maximum temperature depends upon the biocatalyst. In general such maximum temperature is known in the art, e.g. indicated in a product data sheet in case of a commercially available biocatalyst, or can be determined routinely based on common general knowledge and the information disclosed herein. The temperature is usually 90 °C or less, preferably 70 °C or less, in particular 50 °C or less, more in particular or 40 °C or less.

In particular if a biocatalytic reaction is performed outside a host organism, a reaction medium comprising an organic solvent may be used in a high concentration (e.g. more than 50 %, or more than 90 wt. %), in case an enzyme is used that retains sufficient activity in such a medium.

As indicated above, the present invention further relates to a biocatalyst or nucleic acid sequence encoding an ammonia lyase, in particular a lysine ammonia lyase, found by a method according to the invention. Methods for identification of a gene encoding a biocatalyst are generally known in the art and include gain of function methods (e.g. described in Ά novel method for efficient expression cloning of fungal enzyme genes'. Dalboge H, Heldt-Hansen HP.Mol Gen Genet. 1994 May 10;243(3):253-60.) or loss of function methods (e.g. transposon mediated as described in In vivo mutagenesis using EZ-Tn5. Kirby JR. Methods Enzymol. 2007;421 :17-21 .). The peptide encoded by a gene can unequivocally be derived from the gene sequence.

The invention further in particular relates to a nucleic acid encoding a peptide having lysine ammonia lyase activity (optionally in combination with one or more cofactors as may be needed), the nucleic acid comprising the open reading frame encoding said peptide of a nucleic acid sequence represented by any of the SEQ ID NO: 1 -14 or a functional analogue of said nucleic acid sequences.

In particular, the invention further relates to peptide having lysine ammonia lyase activity encoded by a nucleic acid according to SEQ ID NO: 1 -14, a homologue or a functional analogue of said nucleic acid sequences.

The invention further relates to an organism other than a human, in particular a host cell or a wild-type micro-organism, comprising an ammonia lyase, in particular a lysine ammonia lyase or another amino acid ammonia lyase, which may be used as a biocatalyst in the synthesis of a (bio)chemical compound, or for the production of the ammonia lyase, which may thereafter be isolated from the organism.

The micro-organism may be a wild-type organism that was hitherto unknown or a host cell which is heterologous in that it comprises a heterologous nucleic acid sequence encoding an ammonia lyase or a host cell which is heterologous in that it comprises at least one heterologous nucleic acid sequence encoding at least one other peptide, e.g. an enzyme useful in the synthesis of the substrate for the ammonia lyase, in particular lysine, or an enzyme useful to convert a (bio)chemical product obtained in a ammonia lyase-catalysed reaction into a further (bio)chemical product.

In particular, an organism according to the invention may comprise a nucleic acid encoding a peptide having lysine ammonia lyase activity (optionally in combination with one or more cofactors as may be needed), wherein the nucleic acid comprises the open reading frame encoding said peptide of a nucleic acid sequence represented by any of SEQ ID NO: 1 -14 or a functional analogue of any of said nucleic acid sequencesl

The host cell may be eukaryotic or prokaryotic. In an embodiment, the host cell is selected from the group of fungi, yeasts, euglenoids, archaea and bacteria. The host cell may in particular be selected from the group of genera consisting of Aspergillus, Penicillium, Ustilago, Cephalosporium, Trichophytum, Paecilomyces, Pichia, Hansenula, Saccharomyces, Candida, Kluyveromyces, Yarrowia, Bacillus, Corynebacterium, Escherichia, Azotobacter, Frankia, Rhizobium, Brady rhizobium, Anabaena, Synechocystis, Microcystis, Klebsiella, Rhodobacter, Pseudomonas, Thermus, Deinococcus Gluconobacter, Methanococcus, Methanobacterium,

Methanocaldococcus and Methanosarcina. In particular, the host strain and, thus, host cell may be selected from the group of Escherichia coli, Azotobacter vinelandii, Klebsiella pneumoniae, Anabaena sp., Synechocystis sp., Microcystis aeruginosa, Deinococcus radiourans, Deinococcus geothermalis, Thermus thermophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus methanolicus, Corynebacterium glutamicum, Aspergillus niger, Penicillium chrysogenum, Penicillium notatum,

Paecilomyces carneus, Cephalosporium acremonium, Ustilago maydis, Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis, Candida maltosa, Yarrowia lipolytica, Hansenula polymorpha, Sulfolobus solfataricus, Methanobacterium thermoautothrophicum, Methanococcus ma paludis, Methanocaldococcus jannashii, Methanosphaera stadtmanae, Methanococcus voltae, Methanosarcina acetivorans, Methanosarcina barkeri and Methanosarcina mazei host cells.

Advantageously, the host cell is an organism capable of lysine biosynthesis. When using the host cell for synthesis of a bio(chemical) compound of interest this allows, e.g., the use of a carbon source for the host cell that is less scarce than lysine.

A host cell may be selected that is naturally capable of synthesising lysine or a host cell may have been genetically modified such that it is capable of lysine synthesis.

Several lysine synthesis pathways are known to exist. For example, the diaminopimelic pathway (DAP pathway) is characteristic of bacteria, algae, Oomycetes, Myxomycetes, Hypnochytrids and higher plants.

Another lysine synthesis pathway is the amino adipate pathway for lysine biosynthesis (also termed AAA pathway). In particular, the biocatalyst forming part of the AAA pathway for lysine biosynthesis may be found in an organism selected from the group of yeasts, fungi, archaea and bacteria, more in particular from the group of Penicillium, Cephalosporium, Paecilomyces, Trichophytum, Aspergillus,

Phanerochaete, Emericella, Ustilago, Schizosaccharomyces, Saccharomyces, Candida, Kluyveromyces, Yarrowia, Pichia, Hansenula, Thermus, Deinococcus, Pyrococcus, Sulfolobus, Thermococcus, Methanococcus, Methanosarcina,

Methanocaldococcus, Methanosphaera, Methanopyrus, Methanobrevibacter, and Methanothermobacter.

In a preferred embodiment, the host cell has lysine ammonia lyase activity and is an organism with a high flux through the lysine biosynthesis pathway, such as Penicillium chrysogenum, Ustilago maydis or an organism adapted, preferably optimised, for lysine production. Organisms adapted for lysine production may in particular be selected from Corynebacterium, Bacillus methanolicus, Escherichia coli and Saccharomyces.

A high flux is defined as at least 20%, more preferred at least 100%, even more preferred at least 500%, most preferred at least 1000% of the rate required to supply lysine for biosynthesis of cellular protein in the respective organism under the chosen production conditions. As indicated above, in accordance with the invention a host cell may be used for the production of 6-ACA. Accordingly, a host cell according to the invention encoding a LAL, may further encode (the peptide of) an enzyme having catalytic activity with respect to the formation of 6-ACA from 6-AHEA. Such enzyme may in particular be selected from alpha, beta-enoate reductases having alpha, beta-enoate reductase activity towards 6-AHEA. Examples of such enzymes are found in

WO 05/68643, as mentioned above. A nucleic acid sequence encoding said reductase may be obtained from an organism described therein and used for genetic modification of a different organism (having LAL activity), a nucleic acid sequence encoding a LAL according to the invention may be incorporated into an organism mentioned in

WO 05/68643 or said nucleic acid sequences may be used to modify a suitable organism to express both LAL and said reductase activities.

A heterologous cell comprising one or more enzymes for catalysing a reaction step in a method of the invention can be constructed using molecular biological techniques, which are known in the art per se. For instance, such techniques can be used to provide a vector which comprises one or more genes encoding one or more of said biocatalysts. A vector comprising one or more of such genes can comprise one or more regulatory elements, e.g. one or more promoters, which may be operably linked to a gene encoding an biocatalyst.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements (or coding sequences or nucleic acid sequence) in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain.

The promoter that could be used to achieve the expression of the nucleotide sequences coding for an enzyme for use in a method of the invention, such as described herein, may be native to the nucleotide sequence coding for the enzyme to be expressed, or may be heterologous to the nucleotide sequence (coding sequence) to which it is operably linked. Preferably, the promoter is homologous, i.e. endogenous to the host cell.

If a heterologous promoter (to the nucleotide sequence encoding for the enzyme of interest) is used, the heterologous promoter is preferably capable of producing a higher steady state level of the transcript comprising the coding sequence (or is capable of producing more transcript molecules, i.e. mRNA molecules, per unit of time) than is the promoter that is native to the coding sequence. Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art.

A "strong constitutive promoter" is one which causes mRNAs to be initiated at high frequency compared to a native host cell. Examples of such strong constitutive promoters in Gram-positive micro-organisms include SP01 -26, SP01 -15, veg, pyc (pyruvate carboxylase promoter), and amyE.

Examples of inducible promoters in Gram-positive micro-organisms include, the IPTG inducible Pspac promoter, the xylose inducible PxylA promoter.

Examples of constitutive and inducible promoters in Gram-negative microorganisms include, but are not limited to, tac, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, T5, 73, gal, trc, ara (P_BAD), SP6, A-P_R> and A-P_L.

Promoters for (filamentous) fungal cells are known in the art and can be, for example, the glucose-6-phosphate dehydrogenase gpdk promoters, protease promoters such as pepk, pep , pepC, the glucoamylase glak promoters, amylase amyk, amyB promoters, the catalase catR or catA promoters, glucose oxidase goxC promoter, beta-galactosidase lack promoter, alpha-glucosidase aglk promoter, translation elongation factor tefk promoter, xylanase promoters such as xlnk, xlnB, xlnC, xlnD, cellulase promoters such as eglk, eglB, cbhk, promoters of transcriptional regulators such as arek, crek, xlnR, pacC, prfT, etc or any other, and can be found among others at the NCBI website (http://www.ncbi.nlm.nih.gov/entrez/

The invention further relates to a method for preparing caprolactam, wherein 6-amino caproic acid prepared in a method according to the invention is converted into caprolactam. This conversion can be accomplished in a manner known per se, e.g. as described in US 6,194,572, of which the contents with respect to reaction conditions are enclosed herein by reference.

In accordance with the invention, 6-amino caproic acid or caprolactam prepared in a method according the invention may be used to prepare a polyamide or other polymer, optionally in the presence of at least one other monomer. Suitable conditions for such preparation are commonly known in the art.

The invention will now be illustrated by the following example.

Example 1 Identification of LAL activity encoded in a metagenomic library Design of screening substrates

Several structural analogues of alpha- and beta-lysine which would favour a LAL type reaction and block other biochemical reactions resulting in release of nitrogen were designed and prepared by chemical synthesis.

3-amino-3-methyl-heptanoic acid

6-acetamido-3-amino-3-methyl-hexanoic acid

The synthesis of these compounds is described next

Synthesis of 2-amino-2-methyl-6-(dimethylamino)- hexanoic acid

6-(Dimethylamino ) -hexan-2-one

To a solution of 1 -chloro-5-hexanone (259.7 g, 1 .93 mol) in acetonitrile (1 .4 L) was added dimethylamine hydrochloride (143.2 g, 1 .76 mol, 0.91 eq) and potassium carbonate (533.3 g, 3.86 mol). The reaction mixture was refluxed for 10.5 h and partioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane (3x 300 mL). The combined organic layers were dried over Na₂S0₄ and after filtration concentrated in vacuo yielding crude 6- (dimethylamino)-hexan-2-one (214 g). Vacuum distillation furnished nearly pure 6- (dimethylamino)-hexan-2-one (139 g, 50 %).

2-Amino-6- ( dimethylamino ) -2-methylhexanenitrile To a solution of NaCN (53.23 g, 1 .09 mol, 1 .12 eq) in water (105 ml) was added drop-wise a solution of ammonium chloride (63.82 g, 1.19 mol, 1 .23 eq) in a mixture of ammonia-solution (88 mL, 1 .24 mol, 1 .28 eq, 25 %) and water (130 mL). To the reaction mixture was added drop-wise a solution of 6-(dimethylamino)-hexan-2-one (138.9, 970 mmol, 1 eq) in methanol (210 mL) and kept at 20-25 ^eC in a water bath. After stirring for 2 h at room temperature the reaction mixture was heated to 60 ^eC for 1 h. After cooling to room temperature the reaction mixture was extracted with dichloromethane (3x) and the combined organic layers were dried with Na₂S0₄. After filtration the solvent was removed in vacuo furnishing crude 2-amino-6- (dimethylamino)-2-methylhexanenitrile (165.3 g, 100%) as a orange oil. The product was used in the next step without further purification.

2-Amino-2-methyl-6-(dimethylamino)- hexanoic acid dihydrochloride.

2-Amino-6-(dimethylamino)-2-methylhexanenitrile (179.3 g, 1.06 mol, 1 eq) was dissolved in an aqueous 6 M HCl-solution (1 .7 L). After refluxing the reaction mixture for 40 h the solvent was evaporated and the residue was triturated with ethanol. Subsequent filtration yielded crude 2-amino-6-(dimethylamino)-2- methylhexanoic acid dihydrochloride (1 16.1 g, 49%).

2-Amino-6- ( dimethylamino ) -2-methylhexanoic acid.

Dowex 50WX8, 200-400 mesh, 1 .1 kg) was suspended in a 5% NH₃- solution. The resin was washed until neutral and 237 g of crude 2-amino-6-

(dimethylamino)-2-methylhexanoic acid dihydrochloride was subjected to the column After washing with plenty of water the product was eluted with 9-10 eq 5%-NH₃- solution. Subsequent freeze-drying yielded 2-amino-6-(dimethylamino)-2- methylhexanoic acid. (65.4 g, 33 %).

Synthesis of 3-amino-3-methyl-6-(dimethylamino)hexanoic acid

Ethyl 3-acetamido-6-hydroxy-3-methylhexanoate

Ethyl 3-acetamido-3-methylhept-6-enoate (22.1 g, 97.4 mmol) was dissolved in dichloromethane: methanol 1 :1 (900 mL) and cooled to -65 °C. Ozone was bubbled through the solution until a blue colour appeared. Finally, the excess of ozone was removed by bubbling nitrogen gas through the solution until the blue colour disappeared. NaBH₄ (12.9 g, 341 mmol, 3.5 eq.) was added in portions to the solution. The mixture was allowed to warm-up slowly to room temperature in the cooling bath and left stirring overnight. Saturated aqueous NH₄CI (800 mL) was added and the mixture was extracted with dichloromethane (4 x 400 mL). The combined organic layers were dried on Na₂S0₄, filtered and evaporated in vacuo affording ethyl 3- acetamido-6-hydroxy-3-methylhexanoate (20.9 g, 93 %).

Ethyl 3-acetamido-3-methyl-6-(methylsulfonyloxy)hexanoate Ethyl 3-acetamido-6-hydroxy-3-methylhexanoate (20.9 g, 90.5 mmol) was dissolved in dichloromethane (400 mL). To the reaction mixture was added triethylamine (12.5 mL, 181 mmol, 2 eq.) followed by the drop-wise addition of methanesulfonyl chloride (8.4 mL, 108.6 mmol, 1 .2 eq.), keeping the temperature below 20 °C with an ice-bath. The mixture was stirred overnight at room temperature. The mixture was washed with a 2 N HCl solution (2 x 200 mL) and the organic layer was dried on Na₂S0₄. After filtration and evaporation of the solvents in vacuo, ethyl 3- acetamido-3-methyl-6-(methylsulfonyloxy)hexanoate (21.7 g, 78 %) was obtained.

Ethyl 3-acetamido-6-(dimethylamino)-3-methylhexanoate

Ethyl 3-acetamido-3-methyl-6-(methylsulfonyloxy)hexanoate (4.6 g, 15 mmol) was dissolved in 2 M dimethylamine in tetrahydrofurane (35 mL, 70 mmol, 4.6 eq.) in a pressure tube. The mixture was left overnight at room temperature, whereafter ¹ H NMR showed no more starting material present. The solvents were evaporated in vacuo and the residue was dissolved in 1 N HCl solution (100 mL). The mixture was washed with toluene (2 x 10 mL) and the water layer was basified with aqueous Na₂C0₃ (sat). The product was removed from the water layer with dichloromethane (10 x 50 ml) concentration of the solvents yielded ethyl 3-acetamido- 6-(dimethylamino)-3-methylhexanoate (2.8 g, 74%). The product was used in the next step without any further purification.

3-Amino-6- ( dimethylamino ) -3-methylhexanoic acid A mixture of ethyl 3-acetamido-6-(dimethylamino)-3- methylhexanoate(1 .8 g, 6.9 mmol) in 2M HCl solution (40 ml) was heated to reflux for two days. The reaction mixture was concentrated and subsequent purification using a Dowex column (gradient of HCl solution from 1 M to 2M) first afforded some side- products followed by the compound aimed for. Concentration furnished compound 3- amino-6-(dimethylamino)-3-methylhexanoic acid (0.9 g, 45%).

Synthesis of 3-amino-3-methylheptanoic acid

Ethyl 3-hydroxy-3-methylhept-6-enoate

Diisopropylamine (140 ml_, 0.78 mol) was dissolved in tetrahydrofurane (1 L) and the mixture was cooled to 0 °C. n-BuLi (314 ml_, 0.78 mol) was added drop-wise to the mixture at 0 °C and left stirring for 15 minutes. Ethyl acetate (61 ml_, 0.69 mol) in tetrahydrofurane (100 ml_) was added drop-wise to the freshly made LDA solution at -78 °C. The mixture was stirred for 30 minutes with the temperature below -78 °C. 5-Hexene-2-one (32.6 g, 0.34 mol) in tetrahydrofurane (450 ml_) was added slowly, keeping the temperature below -78 °C. The mixture was allowed to warm slowly in the cooling bath for 2 hours, with an end temperature of -35 °C. The reaction was quenched with aqueous NH₄CI (sat. 250 ml_) and acidified to pH = 2 with 2 N HCl solution. The product was extracted with a ethyl acetate : heptane 4:1 mixture (3 x 500 ml_). The combined organic layers were dried over brine and Na₂S0₄. Filtration and evaporation of the solvents furnished ethyl 3-hydroxy-3-methylhept-6- enoate (61 .3 g, 97 %). The reaction mixture was used as such in the next step.

Ethyl 3-acetamido-3-methylhept-6-enoate

Ethyl 3-hydroxy-3-methylhept-6-enoate (61 .3 g, 0.33 mol) was dissolved in acetonitrile (1200 ml_) and cooled to 0 °C. Chlorosulfonic acid (43.9 ml_, 0.659 mol, 2 eq.) was added drop-wise, keeping the temperature below 0 °C. The reaction was left at 0 °C for 20 minutes and subsequently poured on ice (400 ml_). The product was extracted with ethyl acetate : heptane 9:1 (2 x 500 ml_). The combined organic layers were dried over brine and Na₂S0₄ and after filtration, concentrated in vacuo. Furthermore the water layer was extracted two more times with ethyl acetate, which was combined with the earlier obtained product. Column chromatography on silicagel (ethyl acetate : heptane 2:3, after removal of impurities ethyl acetate : heptane 1 :1 ) gave ethyl 3-acetamido-3-methylhept-6-enoate (29.3 g, 0.129 mol, 39 %).

Ethyl 3-acetamido-3-methylheptanoate

Ethyl 3-acetamido-3-methylhept-6-enoate (7.5 g, 33 mmol) was dissolved in EtOH (100 ml_). Palladium on carbon (5 wt%, 0.3 g) was added and the mixture was put under a 1 bar hydrogen atmosphere. The reaction mixture was filtered over Celite and concentration in vacuo and afforded ethyl 3-acetamido-3- methylheptanoate (7.5 g, 100 %).

3-Amino-3-methyl-heptanoate

A mixture of ethyl 3-acetamido-3-methylheptanoate (7.5 g 33 mmol) and a 6M HCl solution (100ml) was refluxed overnight The mixture was extracted with n-pentane (100 ml) and the water layer was concentrated in vacuo. The white solid obtained was stirred with diethyl ether (20 ml), filtered and dried on air furnishing 3- amino-3-methyl-heptanoate (5.7 g, 100%).

Synthesis of 6-acetamido-3-amino-3-methylhexanoic acid

Ethyl 3-acetamido-6-azidc-3-methylhexanoate

Ethyl 3-acetamido-3-methyl-6-(methylsulfonyloxy)hexanoate (19.3 g, 84.5 mmol) was dissolved in dimethylformamide (150 ml_). Sodium azide (8.1 g, 125 mmol, 1 .5 eq.) was added and the mixture was stirred overnight at room temperature. The mixture was stirred at 50 °C for 1 .5 h. and subsequently water (700 ml_) was added. The reaction mixture was extracted with tert-butyl methyl ether ( 2 x 300 ml_). The organic layer was dried over brine and Na₂S0₄. Filtration and evaporation of the solvents in vacuo, yielded ethyl 3-acetamido-6-azido-3-methylhexanoate (1 1 .4 g, 71 %).

Ethyl 3-acetamido-6-amino-3-methylhexanoate

Ethyl 3-acetamido-6-azido-3-methylhexanoate (1 1.4 g, 44.5 mmol) was dissolved in methanol (300 ml_). Palladium on carbon (5 wt%, 2 g, 50 % water) was added and the mixture was put under a 1 bar hydrogen atmosphere over the weekend. The mixture was filtered over Celite and evaporation of the solvents yielded crude ethyl 3-acetamido-6-amino-3-methylhexanoate (10.0 g, 98 %) which was used as such in the next step.

Benzyl-6-acetamido-3-amino-3-methylhexanoate

A mixture of crude ethyl 3-acetamido-6-amino-3-methylhexanoate (18 g, 80 mmol) in a 6M HCl solution (200 ml) was extracted with a mixture of heptanes/ethyl acetate 10:1 (50 ml). The water layer was separated and refluxed over the weekend. Additional 2M HCl solution (200 ml) was addded and the mixture was refluxed overnight and concentrated in vacuo. The obtained crude 6-amino-3-amino-3- methylhexanoic acid was dissolved in water (100 ml). Potassium carbonate (22.6 g, 160 mmol) followed by p-nitrophenylacetate (14.5 g, 80 mmol) were added. After stirring overnight ethyl acetate (25 ml) was added and the water layer was separated and extracted with ethyl acetate (25 ml). To the water layer was added 6M HCl solution to reach pH 1 -2 and the reaction mixture was extracted with toluene (3 x 100 ml). Finally, the water layer was concentrated in vacuo. To the remaining crude 6- acetamido-3-amino-3-methyl-hexanoic acid was added benzyl alcohol (50 ml, 0.46 mol) and trimethylchlorosilane (20 ml, 17 g, 156 mmol). The reaction mixture was stirred overnight and concentrated in vacuo. Dichloromethane (100 ml) was added and the reaction mixture was made basic with sodium carbonate. After separation of the organic layer the water layer was extracted with dichloromethane and the combined organic layers were dried (Na₂S0₄) and concentrated in vacuo. Column

chromatography (Silicagel; dichloromethane - 1 % methanol) yielded benzyl-6- acetamido-3-amino-3-methylhexanoate (1.7 g, 7 %). 6-Acetamido-3-amino-3-methyl-hexanoic acid

Benzyl 6-acetamido-3-amino-3-methylhexanoate (1.7 g, 5.8 mmol) was dissolved in water (30 ml_). Palladium on carbon (10wt%, 170 mg, 50 % water) was added and the mixture was put under a 1 bar hydrogen atmosphere and stirred overnight at room temperature. The mixture was filtered over Celite. After concentration in vacuo the residue obtained was washed with ethyl acetate (5 ml). After filtration and drying 6-acetamido-3-amino-3-methyl-hexanoic acid (1 .1 g, 93 %). was obtained as a white solid.

Library construction

Obtaining Waste Water and Soil Samples

The waste water and soil samples were obtained from various locations in Germany and the Netherlands. Soil samples were initially processed mechanically while dry (screening through 4 mm and 1 mm sieves).

For DNA preparation, 5 g of soil were slurried by addition of 13.5 ml of extraction buffer (100 mM Tris-HCl, pH 8.0; 100 mM EDTA, pH 8.0; 100 mM sodium phosphate, pH 8.0; 1 .5 M NaCl ; 1 % hexadecyltrimethylammonium bromide). Extraction buffer was added to the suspension of waste water samples. The suspension was shock-frozen 3 times with addition of liquid nitrogen (N2), ground in a mortar and boiled in a microwave. After being transferred into a 50 ml screw-cap vessel and adding 1 .5 ml of lysozyme solution (50 mg/ml), the suspension was incubated at 37° C. for 30 min and inverted at intervals of 5 min. Addition of 200 μΙ of a proteinase K solution (10 mg/ml) was followed by incubation at 37° C. for 30 minutes with inversion as before. This was followed by treatment with SDS (by addition of 3 ml of a 10% solution) at 65° C. for two hours (inversion of the vessel every 20 min). After centrifugation (6000xg, 10 min) and slurrying with 4.5 ml of extraction buffer plus 1 ml of 10% SDS, the sediment was again extracted and incubated at 65° C. for 10 min and centrifuged as before. The combined supernatants from the two centrifugations were mixed with 1 volume of phenol/chloroform (1 :1 ) and centrifuged as before. The DNA was precipitated from the upper aqueous phase by addition of 0.6 volume of isopropanol and incubation at room temperature for one hour and centrifugation at 16 OOOxg for 20 minutes. The DNA pellet was washed with 70% ethanol and dried in air and then dissolved in 200 μΙ of TE buffer (100 mM Tris-HCl pH 8.0; 1 mM EDTA pH 8.0). DNA isolation from soil

The high molecular weight genomic DNA was finally separated from dissolved inhibitory humic acids by preparative gel electrophoresis (0.7% agarose) and subsequent extraction (QIAex II Gel Extraction Kit; from Qiagen, Hilden). Selection conditions

Expression cloning in the heterologous host organism Escherichia co// was chosen for the cloning and isolation of complete lysine ammonia lyase genes from metagenome. The ligation of metagenomic fragments to vector-DNA was performed by techniques known in the art. Since heterologous recognition of the promoters of unknown donor organisms cannot be assumed in the host organism E. coli, use was made of E. coli promoters which are inducible in a known manner as e.g the β-galactosidase-promoter by IPTG (isopropyl thiogalactoside).

To examine the quality of the gene library, the number of primary transformants generated in total, and the number of insert-harboring clones was determined by blue/white selection in a test plating. For this purpose, 1 and 10 μΙ portions of the ligation mixture were plated out on LB medium with appropriate antibiotic, IPTG, X-Gal (as described above) and incubated at 37° C. overnight. To confirm the actually cloned insert sizes, the plasmids were isolated from at least 10 white colonies of the test plating, and a suitable restriction digestion was carried out with subsequent size analysis by gel electrophoresis.

In the screening of the gene libraries, the ability of individual recombinant E. coli DH1 1 B (available from Brain AG, Zwingenberg, Germany) clones in metagenomic libraries to grow under nitrogen limiting conditions with the screening substrates as sole nitrogen source was determined. For this purpose, M9 agar without nitrogen source but supplemented with 0,5 mM of the respective screening substrate and 60 Mg/ml of leucine was used. About 1 x 10⁵ - 2.5 x 10⁵colony forming units were spread per plate and plates were incubated at 37 °C. for up to 3days and subsequently at RT for up to 20days. Plates were monitored for growth at regular intervals and growing colonies were picked for further analysis. To exclude false positives, clones may be grown again in the presence and absence of the screening substrate and only clones requiring the screening substrate for growth were then use for further analysis.

Detection of 6-AHEA and β-homo-proline by LC-MS-MS

6-AHEA and its spontaneous breakdown product β-homo-proline produced in biochemical reactions from a-lysine were measured by LC-MS-MS as follows. Analysis was performed on the Agilent 6410 QQQ LC-MS. For 6-AHEA, β- homoproline and a-homoproline the LC-MS-MS method 808595-MRM in MRM mode was used.

Bio-chemical characterization of hit clones

Hits from metagenomic screening were grown either in 5 ml or 50ml LB medium containing the appropriate antibiotic at 37 °C. After over night incubation on a rotary shaker (180rpm) cells were harvested by centrifugation, washed twice with preferentially ammonium carbonate buffer (10 mM pH 7.5) or a Tris/HCl-buffer (50mM, pH8) and crude cell extracts were prepared by sonication. To determine enzymatic activities a volume of crude cell extract corresponding to the number of cells contained in 1 ml of a culture with an optical density (O.D.₅₈₀) of 10 were combined with 500μΜ lysine in preferentially ammonium carbonate buffer (10 mM pH 7.5) or a 50mM Tris/HCl buffer (pH8) in a total volume of 600 μΙ_ and incubated at 37 °C for 4h. Assays were stopped by adding 30μΙ of 10% formic acid, centrifuged at 16200 x g for 5 min at RT in a microcentrifuge, filtered (Millex-LG 0,2 μΜ Millipore) and stored at -80C prior to analysis by LC-MS.

Genetic characterization of hit clones

Plasmid DNA from metagenomic hits were isolated according to standard procedures. For further characterization they were analyzed by restriction digestion to determine insert size. After retransformation single colony transformants were selected and plasmid DNA was re-isolated from these. In several cases it was found that the original hit clone contained more than one plasmid.

Sequences

Claims

1 . A method of finding a biocatalyst having ammonia lyase activity, comprising

- selecting at least one candidate which grows in said culture medium; and

2. A method according to claim 1 , wherein the ammonia lyase activity is an amino acid ammonia lyase activity and as sole nitrogen source or sources at least one compound is used selected from the group of amino acids and structural analogues of amino acids.

3. A method according to claim 2, wherein the amino acid ammonia lyase activity is a lysine ammonia lyase activity and as sole nitrogen source or sources at least one compound is used selected from the group of lysine and structural analogues of lysine, the lysine ammonia lyase activity preferably being L-lysine ammonia lyase activity and the sole nitrogen source or sources preferably being selected from L-lysine and structural analogues thereof.

4. A method according to claim 1 ,2 or 3, wherein the culture medium contains at least one structural analogue selected from the group of 2-amino-2-methyl-6- (dimethylamino)-hexanoic acid, 2-amino-2-methyl-hexanoic acid, 3-amino-3-methyl-(6- dimethylamino)-hexanoic acid, 3-amino-3-methyl-heptanoic acid and 6-acetamido-3- amino-3-methyl-hexanoic acid.

5. A method according to any of the preceding claims, wherein the library is a metagenomic library.

6. A method according to any of the preceding claims, wherein the biocatalyst having amonia lyase activity or a nucleic acid sequence encoding an ammonia lyase is isolated from the cell culture.

7. Biocatalyst or nucleic acid sequence encoding an ammonia lyase found by a method according to the preceding claims.

8. Nucleic acid encoding a peptide having ammonia lyase activity (optionally in combination with one or more cofactors as may be needed), wherein the nucleic acid comprises the open reading from encoding said peptide of a nucleic acid sequence represented by any of SEQ ID NO: 1 -14 or a functional analogue of any of said nucleic acid sequences.

9. Peptide having ammonia lyase activity encoded by a nucleic acid according to claim 8.

10. Host cell of which the genome comprises a nucleic acid according to claim 8.

11 Composition comprising a biocatalyst according to claim 7, a peptide according to claim 9 or a host cell according to claim 10.

12. A method of producing a (bio)chemical compound, comprising:

a) providing a biocatalyst found by a method according to any of the claims 1 -6, a peptide according to claim 9, a host cell according to claim 10 or a composition according to claim 1 1 ;

b) providing a production strain having ammonia lyase activity from said biocatalyst; c) providing a reaction mixture comprising said production strain and a substrate and if desired one or more cof actors for the chemical conversion reaction catalysed by said biocatalyst, and

13. A method according to claim 12, wherein the biocatalyst has lysine ammonia activity, the substrate is alpha-lysine and the reaction product is 6-aminohex-2-enoic acid.

14. A method for preparing 6-amino caproic acid, wherein 6-aminohex-2-enoic acid is prepared from alpha-lysine in a reaction catalysed by a biocatalyst having lysine ammonia lyase activity, such as a biocatalyst according to claim 7 or a biocatalyst comprising a peptide according to claim 9, and converting the 6-aminohex-2-enoic acid into 6-amino caproic acid.

15. A method according to claim 14, wherein the conversion of 6-aminohex-2- enoic acid into 6-amino caproic acid is catalysed by a biocatalyst having alpha, beta- enoate reductase activity towards 6-aminohex-2-enoic acid.

16. A method for preparing caprolactam, wherein 6-amino caproic acid prepared in a method according to claim 14 or 15 is converted into caprolactam.

17. A method for preparing a polyamide or other polymer, comprising polymerising 6-amino caproic acid prepared in a method according to claim 14 or 15 or caprolactam prepared in a method according to claim 16, optionally in the presence of at least one other monomer.

18. Amino acid selected from the group of 2-amino-2-methyl-6-(dimethylamino)- hexanoic acid, 3-amino-3-methyl-(6-dimethylamino)-hexanoic acid, 3-amino-3-methyl- heptanoic acid and 6-acetamido-3-amino-3-methyl-hexanoic acid.