WO2021058691A1

WO2021058691A1 - Method for the production of beta-alanine or salts thereof

Info

Publication number: WO2021058691A1
Application number: PCT/EP2020/076800
Authority: WO
Inventors: Kai-Uwe Baldenius; Dick B. Janssen; Hein J. WIJMA; Hugo VAN BEEK
Original assignee: Basf Se; Rijksuniversiteit Te Groningen
Priority date: 2019-09-26
Filing date: 2020-09-24
Publication date: 2021-04-01

Abstract

The invention is directed to methods to produce β-alanine or salts thereof from acrylic acid using a recombinant aspartase-like protein as catalyst.

Description

METHOD FOR THE PRODUCTION OF BETA-ALANINE OR SALTS THEREOF

Field of the Invention

The invention is directed to methods to produce b-alanine or salts thereof from acrylic acid using a recombinant aspartase-like protein as catalyst.

Description of the Invention

Aspartases (aspartate ammonia-lyases EC 4.3.1 .1 , AspB) are a class of enzymes that cata lyse the reaction from L-aspartate to fumarate + NH3. It has recently been shown, that by mutating an aspartase from Bacillus spec. YM55-1 it was possible to produce an aspartase that has a b-amino acid synthesis activity (Vogel et al., 2014, ChemCatChem 6, pages 965- 968; Li et al., 2018, Nature Chemical Biology 14, pages 664-670). For example, aspartases were produced that produced (R)^-aminobutanoic acid from crotonic acid and ammonia or for the production of (R)^-amino-pentanoic acid, (S)^-asparagine or (S)^-phenylalanine from various substrates. However, no mutant was identified producing b-alanine (3-aminopro- panoic acid, 3-aminopropionic acid). b-alanine is an intermediate in the chemical industry with primary use in the manufacturing of Calpan (Vit B5). Total market need of b-alanine is estimated to be 10,000t/a. b-alanine can be obtained by adding ammonia in aqueous solution to acrylic acid whereby significant amounts of iminodipropionic acid and nitrilotripropionic acid as by-product are pro duced. Purification of b-alanine from such reactions is cumbersome and expensive. Performing the reaction under elevated temperatures and under pressure improves the b- alanine yield but increases the cost for production and does not fully eliminate the production of the respective by-products.

Another route for synthesis of b-alanine is a multi-step synthesis which requires (i) addition of ammonia to acrylonitrile, (ii) separation by distillation, (iii) saponification, removal of am monia and (iv) acidification.

However, these chemical syntheses lack selectivity and therefore require cost and labour- intensive purification of b-alanine.

Recently a publication (Gao et al, 2017; Biocatalytic access to b-alanine by a two-enzyme cascade synthesis, Chinese Journal of Biotechnology) describes biosynthesis of b-alanine using a two-enzyme cascade wherein fumarate is used as substrate. This process lacks effi ciency, because it requires fumaric (or maleic) acid as starting material and a subsequent decarboxylation to the 3-carbon product, wasting one carbon as CO2.

A further recent publication describes a method for biosynthesis of b-alanine wherein the substrate acrylic acid is added to a fermentation broth comprising wildtype E. coli or Sarcina lutea bacteria (CN 1626665). However, the publication is silent about which enzyme or en zymes are producing b-alanine.

It is one purpose of the invention at hand to provide a method to produce b-alanine with increased selectivity and reduced costs wherein the substrate of the reaction is a 3-carbon product, preferably acrylic acid. Legend to Drawing

Figure 1 shows the reaction catalyzed by the aspartase of the invention.

Figure 2 shows the activities of recombinant aspartases calculated from the results of the screening reactions. Numbers are also listed in Table 5.

Figure 3 shows a Time course of b-alanine production by recombinant aspartases (see leg end, protein concentration in parentheses), including data in Table 6. Up to 250 mM of b- alanine was formed by variant A5. From highest to lowest activity: A5, A1 , Aint, A5 and B19.

Figure 4 shows the ¹H-N MR spectra of the starting acrylic acid mixture before the addition of recombinant aspartase (top), the composition of the reaction mixture after 24 h at 37 °C (middle) and a standard b-alanine solution (bottom).

Detailed Description of the Invention

It is one embodiment of the invention to provide a recombinant aspartase-like protein capable of catalysing the reaction from ammonia and acrylic acid to b-alanine in an aqueous medium comprising water, at least one recombinant aspartase-like protein, ammonia and acrylic acid and optionally b-alanine, wherein the concentration of b-alanine in the aqueous medium after incubation is at least 1 % (w/w), preferably at least 2%, at least 3%, at least 4% or at least 5%, more preferably the concentration is at least 6%, at least 7%, at least 8% or at least 9%. Most preferably the concentration is at least 10%. Preferably the concentration of acrylic acid in the aqueous medium after incubation is below 5% (w/w), below 4%, below 3% or below 2%. More preferably the concentration of acrylic acid is below 1 %, even more preferably below 0.5%.

A further embodiment of the invention is said recombinant aspartase-like protein, wherein said recombinant aspartase-like protein comprises at least one mutation, preferably at least two mutations, more preferably at least three mutations, more preferably all four mutations leading to an amino acid exchange in the position corresponding to the position 187, 321, 324 and/or 326 in SEQ ID NO: 2 a. T187 b. M321 c. K324 d. N326.

In a more preferred embodiment, at least the position b. M321 is mutated in the recombinant aspartase-like protein of the invention.

In a most preferred embodiment of the invention the mutation in position 321 corresponding to position 321 in SEQ ID NO: 2 is M321 I.

Preferably the mutation in any of the positions a. to d. is a mutation introducing another hy drophobic amino acid at the respective position.

The group of hydrophobic amino acids as meant herein consist of alanine, cysteine, phenyl alanine, glycine, isoleucine, leucine, methionine, proline, valine or tryptophan. Preferably the recombinant aspartase-like protein of the invention does not comprise one of the following mutations: K324P, K324F or K324L.

Preferably the recombinant aspartase-like protein of the invention comprises said mutations wherein the amino acid at the position corresponding to position 324 in SEQ ID NO: 2 is not substituted by Val, Met, lie or Cys, when the amino acid at the position corresponding to position 187 in SEQ ID NO: 2 is substituted by Val, and wherein the amino acid at the position corresponding to position 324 in SEQ ID NO: 2 is not substituted by Met or Leu, when the amino acid at the position corresponding to position 187 in SEQ ID NO: 2 is substituted by Cys.

Preferably the recombinant aspartase-like protein of the invention comprises said mutations wherein the amino acid at the position corresponding to position 324 in SEQ ID NO: 2 is not substituted by lie and the amino acid at the position corresponding to position 326 in SEQ ID NO: 2 is not substituted by Cys when the amino acid at the position corresponding to position 187 in SEQ ID NO: 2 is substituted by Val.

Most preferably the recombinant aspartase-like protein of the invention comprises at least one mutation, preferably at least two mutations, more preferably at least three mutations, more preferably all four mutations at the positions 187, 321 , 324 and 326 corresponding to the position in SEQ ID NO: 2 wherein the mutations are a. T187I or T 187V b. M321 I c. K324M or K324I d. N326C

In a more preferred embodiment of the invention the mutation in position 321 corresponding to position 321 in SEQ ID NO: 2 is M321 I.

A further embodiment of the inventions is the recombinant aspartase-like protein of the in vention comprising a sequence selected from the group consisting of a. The polypeptide molecule of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 and 54 and b. A polypeptide molecule having at least 55% identity to the polypeptide mole cule of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 or 54 or a functional fragment thereof, and c. A polypeptide molecule encoded by a nucleic acid molecule of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, and d. A polypeptide molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, and e. A polypeptide molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, wherein a protein complex consisting of four of the polypeptide molecules (tetramer) as de fined in b., d. and e. is catalysing the reaction from ammonia and acrylic acid to b-alanine in an aqueous medium, preferably wherein the mutations at positions T187, M321, K324 and N326 as defined above are conserved in the polypeptide molecules as defined in b., d. and e.

In one embodiment, at least one of the polypeptide molecules comprised in said tetramer has a nucleic acid sequence as defined in a to e and/or is comprising at least one, at least two, at least three, preferably all of the mutations as defined above. In a preferred embodiment at least two, more preferably at least three, most preferably all of the polypeptide molecules comprised in said tetramer have a nucleic acid sequence as defined in a to e and/or is com prising at least one, at least two, at least three, preferably all of the mutations as defined above.

Polypeptide molecules and nucleic acid molecules having a certain identity to any of the se quences of SEQ ID NO 1 to 54 include nucleic acid molecules and polypeptide molecules having at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to any of SEQ ID NO:1 to 54.

A further embodiment of the invention is a recombinant construct comprising at least one recombinant aspartase-like protein as defined above.

Said recombinant construct may be integrated into the genome of an organism for producing and isolating the respective recombinant aspartase-like protein or the recombinant aspartase- like protein may be expressed from a vector such as a plasmid or viral vector that is intro duced into an organism for producing and isolating said recombinant aspartase-like protein.

The recombinant aspartase-like protein in the recombinant construct may be functionally linked to a heterologous promoter, a heterologous terminator or any other heterologous ge netic element.

A further embodiment of the invention is a recombinant vector, such as an expression vector or a viral vector comprising said recombinant construct.

A recombinant microorganism comprising said recombinant construct or said recombinant vector is also an embodiment of the invention.

In some embodiments, the recombinant microorganism is a prokaryotic cell. Suitable prokaryotic cells include Gram-positive, Gram negative and Gram-variable bacterial cells, preferably Gram-negative.

Thus, microorganisms that can be used in the present invention include, but are not limited to, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobac- ter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radio- bacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium sa- perdae, Azotobacter indicus, Brevi bacterium ammoniagenes, Brevi bacterium divaricatum, Brevi bacterium lactofermentum, Brevi bacterium flavum, Brevibacterium globosum, Brevi- bacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacte rium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immari- ophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophi- lum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoaci- dophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobac terium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucina- tus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomo nas azotoformans, Pseudomonas jluorescens, Pseudomonas ovalis, Pseudomonas stut- zeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodo- coccus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylo coccus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomy ces violaceochromogenes, Kitasatosporia parulosa, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Strepto myces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibi- oticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Esch erichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhi- murium, Salmonella schottmulleri, Xanthomonas citri, Synechocystis sp., Synechococcus elongatus, Thermosynechococcus elongatus, Microcystis aeruginosa, Nostoc sp., N. com mune, N.sphaericum, Nostoc punctiforme , Spirulina platensis, Lyngbya majuscula, L. lagerheimii, Phormidium tenue, Anabaena sp., Leptolyngbya sp and so forth.

In some embodiments, the microorganism is a eukaryotic cell. Suitable eukaryotic cells in clude yeast cells, as for example Saccharomyces spec, such as Saccharomyces cere- visiae, Hansenula spec, such as Hansenula polymorpha, Schizosaccharomyces spec, such as Schizosaccharomyces pombe, Kluyveromyces spec, such as Kluyveromyces lactis and Kluyveromyces marxianus, Yarrowia spec, such as Yarrowia lipolytica, Pichia spec, such as Pichia methanolica, Pichia stipites and Pichia pastoris, Zygosaccharomyces spec, such as Zygosaccharomyces rouxii and Zygosaccharomyces bailii, Candida spec, such as Candida boidinii, Candida utilis, Candida freyschussii, Candida glabrata and Candida sonorensis, Schwanniomyces spec, such as Schwanniomyces occidentalis, Arxula spec, such as Arxula adeninivorans, Ogataea spec such as Ogataea minuta, Klebsiella spec, such as Klebsiella pneumonia, Aspergillus spec such as Aspergillus niger, Myceliophthora thermophila.

A process for producing b-alanine or salt thereof comprising the steps of i. Providing an aqueous medium comprising water, ammonia, acrylic acid, one or more recombinant aspartase-like protein and optionally b-ala- nine, ii. Incubating the aqueous medium and iii. Optionally isolating the b-alanine or salt thereof from the reaction mix ture

Is a further embodiment of the invention.

Preferably the recombinant aspartase-like protein used in the process of the invention has more than 55% homology to an aspartase.

Preferably the one or more recombinant aspartase-like protein used in the process of the invention comprises at least one mutation, preferably at least two mutations, more preferably at least three mutations, more preferably all four mutations leading to an amino acid exchange in the position corresponding to the position 187, 321, 324 and 326 in SEQ ID NO: 2.

In a more preferred embodiment, at least the position M321 is mutated in the recombinant aspartase-like protein used in the process of the invention.

In a more preferred embodiment, the mutation in position 321 corresponding to position 321 in SEQ ID NO: 2 is M321 I.

Preferably the mutation in any of the positions corresponding to position 187, 321 , 324, and 326 of SEQ ID NO: 2 of the recombinant aspartase-like protein used in the process of the invention is a mutation introducing a hydrophobic amino acid at the respective position, wherein the group of hydrophobic amino acid consist preferably of alanine, cysteine, phenyl alanine, glycine, isoleucine, leucine, methionine, proline, valine or tryptophan.

Preferably the Lys at the position corresponding to position 324 in SEQ ID NO: 2 of the re combinant aspartase-like protein used in the process of the invention is not substituted by Pro or Phe.

The recombinant aspartase-like protein used in the process of the invention comprises at least one mutation, preferably at least two mutations, more preferably at least three muta tions, more preferably all four mutations at the position corresponding to the position 187, 321 , 324 and 326 in SEQ ID NO: 2 wherein the mutations are a. T187I or T 187V b. M321 I c. K324M or K324I or K324L d. N326C or N326A

In a most preferred embodiment, at least the position M321 I is mutated in the recombinant aspartase-like protein used in the process of the invention.

In one embodiment of the invention, the recombinant aspartase-like protein used in the pro cess of the invention is comprising a sequence selected from the group consisting of a. The polypeptide molecule of SEQ ID NO: 44, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 and 54, and b. A polypeptide molecule having at least 55% identity to the polypeptide mole cule of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 or 54 or a functional fragment thereof, and c. A polypeptide molecule encoded by a nucleic acid molecule of SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, and d. A polypeptide molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, and e. A polypeptide molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, wherein a protein complex consisting of four of the polypeptide molecules (tetramer) as de fined in b., d. and e. is catalysing the reaction from ammonia and acrylic acid to b-alanine in an aqueous medium, preferably wherein the mutations at positions T187, M321, K324 and N326 as defined above are conserved in the polypeptide molecules as defined in b., d. and e.

In one embodiment of the process of the invention the ph-value of the aqueous medium may be kept between 5 and 12, preferably between 5 and 11 , more preferably between 6 and 10, more preferably between 6 and 9, more preferably between 6 and 8.

The product of the process of the invention may be isolated by precipitation, filtration or cen trifugation after incubation.

The aqueous medium may be a solution or a suspension or a solution and a suspension, wherein any of the substances comprised in said aqueous medium may be fully or partially dissolved and / or partially or fully suspended.

In a preferred embodiment of the process for producing b-alanine, the incubation is per formed at 20°C to 70°C, preferably at 25°C to 65°C, more preferably at 30°C to 60°C, even more preferably at 35°C to 55°C, even more preferably at 37°C to 52°C, most preferably at 38°C to 50°C.

In a preferred embodiment, the incubation is performed for 30 minutes to 48 hours, preferably for 1 hour to 36 hours, more preferably for 2 hours to 24 hours, more preferably for 3 hours to 20 hours, more preferably for 5 to 15 hours, more preferably for 8 to 12 hours.

In a preferred embodiment of the process for producing b-alanine, the method is carried out using continuous process.

At the start of the process of the invention, the aqueous medium may comprise at least 0.05% acrylic acid, preferably at least 0.1 % acrylic acid, more preferably at least 0.5% acrylic acid, most preferably at least 1.0% acrylic acid (w/w). Throughout the incubation the concentration of acrylic acid may be kept at a concentration of about 0.5% to 1.5%, preferably about 1.0% acrylic acid by continuous feeding of acrylic acid.

Alternatively, the concentration of acrylic acid in the aqueous medium may be between in cluding 10 g/l to 300 g/l at the start of the incubation, preferably between including 50 g/l to 10Og/l, even more preferably between including 60 g/l to 90 g/l, most preferably between including 70 g/l to 85 g/l.

The incubation time of the aqueous medium may be at least 2h, at least 5h, at least 10h or at least 12h. Preferably the incubation time is at least 18h, for example about 24h or about 30h. More preferably the incubation time is about 36h or about 42h. Most preferably, the incubation time is about 48h. Depending on the recombinant aspartase-like protein used and the reaction rate of said recombinant aspartase-like protein, the incubation time may also exceed 48 h.

The aqueous medium may be incubated at at least 20°C, at least 25°C, at least 30°C or at least 35°C. Preferably the aqueous medium is incubated between including 30°C and 60°C. Most preferably the aqueous medium is incubated at 50°C. The aqueous medium may also be incubated at 38°C, 39°C, 40°C, 41 °C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51 °C, 52°C, 53°C, 54°C, 55°C or 56°C.

In a preferred embodiment, the method is carried out using a semi-batch process. In another preferred embodiment, the method is carried out using a continuous process.

A further aspect of the method of the invention is a method for producing a recombinant aspartase-like protein, comprising the steps of a) providing a recombinant cell expressing at least one recombinant aspartase-like protein of the invention, and b) cultivating the recombinant cell under conditions allowing for the expression of said recombinant aspartase-like protein, and c) optionally incubating the cultivated cells from step b) for at least 30 min, preferably 60 min between 50°C and 80°C, preferably 50°C, and d) optionally incubating the cultivated cells from step c) for at least 30 min, preferably at least 60 min at 60 °C.

In a preferred embodiment of the process for producing a recombinant aspartase-like pro tein, the cultivation in step b) is performed at 10°C to 50°C, preferably at 15°C to 40°C, more preferably at 20°C to 40°C, even more preferably at 24°C to 37°C, most preferably at 36°C to 38°C.

In a preferred embodiment, the cultivation in step b) is performed for 30 minutes to 48 hours, preferably for 1 hour to 36 hours, more preferably for 2 hours to 24 hours, more preferably for 3 hours to 20 hours, more preferably for 5 to 15 hours, more preferably for 8 to 12 hours.

The recombinant aspartase-like protein may be added to the aqueous medium by adding cells comprising said recombinant aspartase-like protein or by adding a suspension compris ing inactivated, for example disrupted cells. In another embodiment of the invention, the re combinant aspartase-like protein may be produced in recombinant organisms, preferably mi croorganisms, expressing the recombinant aspartase-like protein of the invention from a het erologous construct. The recombinant aspartase-like protein so produced may be isolated from the recombinant organism and added to the aqueous medium or the recombinant as partase-like protein may be added by inactivating, for example disrupting the cells and adding the suspension or by heat treatment of the cell suspension.

The cells or suspension comprising inactivated cells may be at least partially concentrated for example by drying before being added to the aqueous medium used in the methods of the invention or to the composition of the invention.

The recombinant aspartase-like protein may be (partly) immobilized for instance entrapped in a gel or it may be used for example as a free cell suspension. For immobilization well known standard methods can be applied like for example entrapment cross linkage such as glutaraldehyde-polyethyleneimine (GA-PEI) crosslinking, cross linking to a matrix and/or car rier binding etc., including variations and/or combinations of the aforementioned methods. Alternatively, the recombinant aspartase-like protein enzyme may be extracted and for in stance may be used directly in the process for preparing the b-alanine. When using inacti vated or partly inactivated cells, such cells may be inactivated by thermal or chemical treat ment.

A further embodiment of the invention is a method for producing b-alanine, comprising the steps of a) providing a recombinant microorganism expressing at least one recombinant aspar tase-like protein of the invention, and b) cultivating said microorganism under conditions allowing for the expression of said recombinant aspartase-like protein, and c) optionally isolating the recombinant aspartase-like protein of the invention from said microorganism.

Another embodiment of the invention is a composition comprising water, one or more recom binant aspartase-like protein of the invention, ammonia, acrylic acid and optionally b-alanine. The concentration of b-alanine in the composition of the invention is at least 50 mM, at least 100 mM or at least 500 pM, preferably at least 1 mM, preferably at least 5 mM, more preferably at least 10 mM, most preferably at least 50 mM, even more preferably at least 100 mM.

The one or more recombinant aspartase-like protein in the composition of the invention has preferably at least 55% homology to an aspartase. polypeptide molecules and nucleic acid molecules having a certain identity to an aspartase include nucleic acid molecules and polypeptide molecules having at least 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, homology to an aspartase.

The one or more recombinant aspartase-like protein in the composition of the invention com prises at least one mutation, preferably at least two mutations, more preferably at least three mutations, more preferably all four mutations leading to an amino acid exchange in the posi tion corresponding to the position 187, 321, 324 and 326 in SEQ ID NO: 2 In a most preferred embodiment, at least the position b. M321 is mutated in the recombinant aspartase-like protein of the invention.

Preferably the mutation in any of the positions 187, 321, 324 and 326 is a mutation introducing another hydrophobic amino acid at the respective position.

The group of hydrophobic amino acids as meant herein consist of alanine, cysteine, phenyl alanine, glycine, isoleucine, leucine, methionine, proline, valine or tryptophan.

Preferably the Lys in the position corresponding to position 324 in SEQ ID NO: 2 of the re combinant aspartase-like protein of the invention comprised in the composition of the inven tion is not substituted by Pro or Phe.

Most preferably the recombinant aspartase-like protein of the invention comprised in the com position of the invention comprises at least one mutation, preferably at least two mutations, more preferably at least three mutations, more preferably all four mutations at the position corresponding to the position 187, 321 , 324 and/or 326 in SEQ ID NO: 2 wherein the muta tions are a. T187I or T 187V b. M321 I c. K324M or K324I or K324L d. N326C or N326A

In a most preferred embodiment, at least the position b. M3211 is mutated in the recombinant aspartase-like protein comprised in the composition of the invention.

A further embodiment of the invention is a composition comprising water, one or more recom binant aspartase-like protein of the invention, ammonia, acrylic acid and optionally b-alanine wherein the recombinant aspartase-like protein of the invention is comprising a sequence selected from the group consisting of a. The polypeptide molecule of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 and 54, and b. A polypeptide molecule having at least 55% identity to the polypeptide mole cule of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 or 54 or a functional fragment thereof, and c. A polypeptide molecule encoded by a nucleic acid molecule of SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, and d. A polypeptide molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, and e. A polypeptide molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, wherein a protein complex consisting of four of the polypeptide molecules (tetramer) as de fined in b., d. and e. is catalysing the reaction from ammonia and acrylic acid to b-alanine in an aqueous medium, preferably wherein the mutations at positions T187, M321, K324 and N326 as defined above are conserved in the polypeptide molecules as defined in b., d. and e.

Polypeptide molecules and nucleic acid molecules having a certain identity to any of the se quences of SEQ ID NO 1 to 54 include nucleic acid molecules and polypeptide molecules having 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to any of SEQ ID NO: 1 to 54.

Preferably, the recombinant aspartase-like protein amino acid sequences having a certain identity to the aspartase of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 or 54 comprise some, preferably predominantly, more preferably only conservative amino acid substitutions. Conservative substitutions are those where one amino acid is exchanged with a similar amino acid. For determination of %-simi- larity the following applies, which is also in accordance with the BLOSUM62 matrix, which is one of the most used amino acids similarity matrix for database searching and sequence alignments:

Amino acid A is similar to amino acids S Amino acid D is similar to amino acids E; N Amino acid E is similar to amino acids D; K; Q Amino acid F is similar to amino acids W; Y Amino acid H is similar to amino acids N; Y Amino acid I is similar to amino acids L; M; V Amino acid K is similar to amino acids E; Q; R Amino acid L is similar to amino acids I; M; V Amino acid M is similar to amino acids I; L; V Amino acid N is similar to amino acids D; H; S Amino acid Q is similar to amino acids E; K; R Amino acid R is similar to amino acids K; Q Amino acid S is similar to amino acids A; N; T Amino acid T is similar to amino acids S Amino acid V is similar to amino acids I; L; M Amino acid W is similar to amino acids F; Y Amino acid Y is similar to amino acids F; H; W

Conservative amino acid substitutions may occur over the full length of the sequence of a poly peptide sequence of a functional protein such as an enzyme. In one embodiment, such muta tions are not pertaining the functional domains of an enzyme. In one embodiment, conservative mutations are not pertaining the catalytic centers of an enzyme.

A functional fragment of the polypeptide molecules selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 or 54 comprises at least 100 amino acids, preferably at least 150 amino acids, more preferably at least 200 amino acids, more preferably at least 250 amino acids, most preferably at least 300 amino acids.

DEFINITIONS

It is to be understood that this invention is not limited to the particular methodology or proto cols. It is also to be understood that the terminology used herein is for the purpose of describ ing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural reference un less the context clearly dictates otherwise. Thus, for example, reference to "a vector" is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art, and so forth. The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent, preferably 10 percent up or down (higher or lower). As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list. The words "comprise," "comprising," "in clude," "including," and "includes" when used in this specification and in the following claims are intended to specify the presence of one or more stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, or groups thereof. For clarity, certain terms used in the specifi cation are defined and used as follows:

Recombinant aspartase-like protein: The term “recombinant aspartase-like protein” or “re combinant aspartate ammonia lyase like protein” or “recombinant fumaric aminase-like pro tein” as used herein refers to an enzyme that is derived from an enzyme capable of catalyzing the reaction from L-aspartate to fumarate + NFh having the EC number EC 4.3.1.1. and that is capable to catalyze the reaction from acrylic acid plus ammonia to b-alanine wherein the enzyme capable of catalyzing the reaction from acrylic acid plus ammonia to b-alanine is produced by human intervention. Such human intervention may comprise the synthesis of the respective coding sequence, introduction of mutations by means of genome editing and the like.

Coding region: As used herein the term "coding region" when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5'-side by the nucleotide triplet "ATG" which encodes the initiator methio nine, prokaryotes also may use the triplets “GTG” and “TTG” as start codon. On the 3'-side it is bounded by one of the three triplets which specify stop codons (i.e. , TAA, TAG, TGA). In addition, a gene may include sequences located on both the 5'- and 3'-end of the sequences which are present on the RNA transcript. These sequences are referred to as "flanking" se quences or regions (these flanking sequences are located 5' or 3' to the non-translated se quences present on the mRNA transcript). The 5'-flanking region may contain regulatory se quences such as promoters and enhancers which control or influence the transcription of the gene. The 3'-flanking region may contain sequences which direct the termination of transcrip tion, post-transcriptional cleavage and polyadenylation.

Complementary: "Complementary" or "complementarity" refers to two nucleotide sequences which comprise antiparallel nucleotide sequences capable of pairing with one another (by the base-pairing rules) upon formation of hydrogen bonds between the complementary base res idues in the antiparallel nucleotide sequences. For example, the sequence 5'-AGT-3' is com plementary to the sequence 5'-ACT-3'. Complementarity can be "partial" or "total." "Partial" complementarity is where one or more nucleic acid bases are not matched according to the base pairing rules. "Total" or "complete" complementarity between nucleic acid molecules is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid molecule strands has significant effects on the efficiency and strength of hybridization between nucleic acid molecule strands. A "complement" of a nucleic acid sequence as used herein refers to a nucleotide sequence whose nucleic acid molecules show total complementarity to the nucleic acid molecules of the nucleic acid sequence.

Endogenous: An "endogenous" nucleotide sequence refers to a nucleotide sequence, which is present in the genome of a wild type microorganism.

Enhanced expression: “enhance” or “increase” the expression of a nucleic acid molecule in a microorganism are used equivalently herein and mean that the level of expression of a nucleic acid molecule in a microorganism is higher compared to a reference microorganism, for example a wild type. The terms "enhanced” or “increased" as used herein mean herein higher, preferably significantly higher expression of the nucleic acid molecule to be ex pressed. As used herein, an “enhancement” or “increase” of the level of an agent such as a protein, imRNA or RNA means that the level is increased relative to a substantially identical microorganism grown under substantially identical conditions. As used herein, “enhance ment” or “increase” of the level of an agent, such as for example a preRNA, mRNA, rRNA, tRNA, expressed by the target gene and/or of the protein product encoded by it, means that the level is increased 50% or more, for example 100% or more, preferably 200% or more, more preferably 5 fold or more, even more preferably 10 fold or more, most preferably 20 fold or more for example 50 fold relative to a suitable reference microorganism. The enhancement or increase can be determined by methods with which the skilled worker is familiar. Thus, the enhancement or increase of the nucleic acid or protein quantity can be determined for exam ple by an immunological detection of the protein. Moreover, techniques such as protein as say, fluorescence, Northern hybridization, densitometric measurement of nucleic acid con centration in a gel, nuclease protection assay, reverse transcription (quantitative RT-PCR), ELISA (enzyme-linked immunosorbent assay), Western blotting, radioimmunoassay (RIA) or other immunoassays and fluorescence-activated cell analysis (FACS) can be employed to measure a specific protein or RNA in a microorganism. Depending on the type of the induced protein product, its activity or the effect on the phenotype of the microorganism may also be determined. Methods for determining the protein quantity are known to the skilled worker. Examples, which may be mentioned, are: the micro-Biuret method (Goa J (1953) Scand J Clin Lab Invest 5:218-222), the Folin-Ciocalteau method (Lowry OH et al. (1951 ) J Biol Chem 193:265-275) or measuring the absorption of CBB G-250 (Bradford MM (1976) Analyt Bio- chem 72:248-254).

Expression: "Expression" refers to the biosynthesis of a gene product, preferably to the tran scription and/or translation of a nucleotide sequence, for example an endogenous gene or a heterologous gene, in a cell. For example, in the case of a structural gene, expression in volves transcription of the structural gene into mRNA and - optionally - the subsequent trans lation of mRNA into one or more polypeptides. In other cases, expression may refer only to the transcription of the DNA harboring an RNA molecule.

Foreign: The term "foreign" refers to any nucleic acid molecule (e.g., gene sequence) which is introduced into a cell by experimental manipulations and may include sequences found in that cell as long as the introduced sequence contains some modification (e.g., a point muta tion, the presence of a selectable marker gene, etc.) and is therefore different relative to the naturally-occurring sequence.

Functional fragment: The term “functional fragment” refers to any nucleic acid or amino acid sequence which comprises merely a part of the full length nucleic acid or full length amino acid sequence, respectively, but still has the same or similar activity and/or function. In one embodiment, the fragment comprises at least 70%, at least 80 %, at least 85%, at least 90 %, at least 95%, at least 96%, at least 97%, at least 98 %, at least 99% of the original se quence. In one embodiment, the functional fragment comprises contiguous nucleic acids or amino acids compared to the original nucleic acid or original amino acid sequence, respec tively. Functional linkage: The term "functional linkage" or "functionally linked" is equivalent to the term “operable linkage” or “operably linked” and is to be understood as meaning, for example, the sequential arrangement of a regulatory element (e.g. a promoter) with a nucleic acid se quence to be expressed and, if appropriate, further regulatory elements (such as e.g., a ter minator) in such a way that each of the regulatory elements can fulfill its intended function to allow, modify, facilitate or otherwise influence expression of said nucleic acid sequence. As a synonym the wording “operable linkage” or “operably linked” may be used. The expression may result depending on the arrangement of the nucleic acid sequences in relation to sense or antisense RNA. To this end, direct linkage in the chemical sense is not necessarily re quired. Genetic control sequences such as, for example, enhancer sequences, can also exert their function on the target sequence from positions which are further away, or indeed from other DNA molecules. Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other. In a preferred embodiment, the nucleic acid sequence to be transcribed is located behind the promoter in such a way that the tran scription start is identical with the desired beginning of the chimeric RNA of the invention. Functional linkage, and an expression construct, can be generated by means of customary recombination and cloning techniques as described (e.g., Sambrook J, Fritsch EF and Mani- atis T (1989); Silhavy et al. (1984) Experiments with Gene Fusions, Cold Spring Harbor La boratory, Cold Spring Harbor (NY); Ausubel et al. (1987) Current Protocols in Molecular Biol ogy, Greene Publishing Assoc and Wiley Interscience; Gelvin et al. (Eds) (1990) Plant Mo lecular Biology Manual; Kluwer Academic Publisher, Dordrecht, The Netherlands). However, further sequences, which, for example, act as a linker with specific cleavage sites for re striction enzymes, or as a signal peptide, may also be positioned between the two sequences. The insertion of sequences may also lead to the expression of fusion proteins. Preferably, the expression construct, consisting of a linkage of a regulatory region for example a promoter and nucleic acid sequence to be expressed, can exist in a vector-integrated form or can be inserted into the genome, for example by transformation.

Gene: The term "gene" refers to a region operably linked to appropriate regulatory sequences capable of regulating the expression of the gene product (e.g., a polypeptide or a functional RNA) in some manner. A gene includes untranslated regulatory regions of DNA (e.g., pro moters, enhancers, repressors, etc.) preceding (up-stream) and following (downstream) the coding region (open reading frame, ORF). The term "structural gene" as used herein is in tended to mean a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide.

Genome and genomic DNA: The terms “genome” or “genomic DNA” is referring to the herit able genetic information of a host organism. Said genomic DNA comprises the DNA of the nucleoid but also the DNA of the self-replicating plasmid.

Heterologous: The term "heterologous” with respect to a nucleic acid molecule or DNA refers to a nucleic acid molecule which is operably linked to, or is manipulated to become operably linked to, a second nucleic acid molecule to which it is not operably linked in nature, or to which it is operably linked at a different location in nature. A heterologous expression con struct comprising a nucleic acid molecule and one or more regulatory nucleic acid molecule (such as a promoter or a transcription termination signal) linked thereto for example is a con structs originating by experimental manipulations in which either a) said nucleic acid mole cule, or b) said regulatory nucleic acid molecule or e) both (i.e. (a) and (b)) is not located in its natural (native) genetic environment or has been modified by experimental manipulations, an example of a modification being a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment refers to the natural genomic locus in the organism of origin, or to the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the sequence of the nucleic acid molecule is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least at one side and has a sequence of at least 50 bp, preferably at least 500 bp, especially preferably at least 1 ,000 bp, very especially preferably at least 5,000 bp, in length. A naturally occurring expression construct - for example the naturally occurring combination of a pro moter with the corresponding gene - becomes a transgenic expression construct when it is modified by non-natural, synthetic “artificial” methods such as, for example, mutagenization. Such methods have been described (US 5,565,350; WO 00/15815). For example a protein encoding nucleic acid molecule operably linked to a promoter, which is not the native pro moter of this molecule, is considered to be heterologous with respect to the promoter. Pref erably, heterologous DNA is not endogenous to or not naturally associated with the cell into which it is introduced but has been obtained from another cell or has been synthesized. Het erologous DNA also includes an endogenous DNA sequence, which contains some modifi cation, non-naturally occurring, multiple copies of an endogenous DNA sequence, or a DNA sequence which is not naturally associated with another DNA sequence physically linked thereto. Generally, although not necessarily, heterologous DNA encodes RNA or proteins that are not normally produced by the cell into which it is expressed.

Hybridization: The term "hybridization" as defined herein is a process wherein substantially complementary nucleotide sequences anneal to each other. The hybridisation process can occur entirely in solution, i.e. both complementary nucleic acids are in solution. The hybridi sation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation pro cess can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithog raphy to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.

The term “stringency” refers to the conditions under which a hybridisation takes place. The stringency of hybridisation is influenced by conditions such as temperature, salt concentra tion, ionic strength and hybridisation buffer composition. Generally, low stringency conditions are selected to be about 30°C lower than the thermal melting point (Tm) for the specific se quence at a defined ionic strength and pH. Medium stringency conditions are when the tem perature is 20°C below T m, and high stringency conditions are when the temperature is 10°C below Tm. High stringency hybridisation conditions are typically used for isolating hybridising sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acids may deviate in sequence and still encode a substantially identical polypeptide, due to the degeneracy of the genetic code. Therefore, medium stringency hybridisation con ditions may sometimes be needed to identify such nucleic acid molecules.

The “Tm” is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures. The maximum rate of hybridisation is obtained from about 16°C up to 32°C below Tm. The presence of monovalent cations in the hybridisa tion solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M (for higher concentrations, this effect may be ignored). Formamide reduces the melting tempera ture of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7°C for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45°C, though the rate of hybridisation will be lowered. Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes. On average and for large probes, the Tm decreases about 1°C per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids:

DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984):

Tm= 81.5°C + 16.6xlog[Na⁺]a + 0.41x%[G/Cb] - 500x[Lc]-1 - 0.61x% formamide DNA-RNA or RNA-RNA hybrids:

Tm= 79.8 + 18.5 (log10[Na⁺]a) + 0.58 (%G/Cb) + 11.8 (%G/Cb)2 - 820/Lc oligo-DNA or oligo-RNAd hybrids:

For <20 nucleotides: Tm= 2 (In)

For 20-35 nucleotides: Tm= 22 + 1.46 (In ) a or for other monovalent cation, but only accurate in the 0.01-0.4 M range b only accurate for %GC in the 30% to 75% range c L = length of duplex in base pairs. d Oligo, oligonucleotide; In, effective length of primer = 2^c(ho. of G/C)+(no. of A/T). Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heter ologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase. For non- related probes, a series of hybridizations may be performed by varying one of (i) progressively lowering the annealing temperature (for example from 68°C to 42°C) or (ii) progressively low ering the formamide concentration (for example from 50% to 0%). The skilled artisan is aware of various parameters which may be altered during hybridisation and which will either main tain or change the stringency conditions.

Besides the hybridisation conditions, specificity of hybridisation typically also depends on the function of post-hybridisation washes. To remove background resulting from non-specific hybridisation, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt con centration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridisation stringency. A positive hybridisa tion gives a signal that is at least twice of that of the background. Generally, suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. The skilled artisan is aware of various parameters which may be altered during washing and which will either maintain or change the stringency conditions.

For example, typical high stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at65°C in 1xSSC orat42°C in IxSSC and 50% forma- mide, followed by washing at 65°C in 0.3x SSC. Examples of medium stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 50°C in 4x SSC or at 40°C in 6x SSC and 50% formamide, followed by washing at 50°C in 2x SSC. The length of the hybrid is the anticipated length for the hybridising nucleic acid. When nucleic acids of known sequence are hybridised, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein. 1 xSSC is 0.15M NaCI and 15mM sodium citrate; the hybridisation solution and wash solutions may additionally in clude 5x Denhardt's reagent, 0.5-1.0% SDS, 100 pg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate. Another example of high stringency conditions is hybrid isation at 65°C in 0.1x SSC comprising 0.1 SDS and optionally 5x Denhardt's reagent, 100 pg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, followed by the washing at 65°C in 0.3x SSC.

For the purposes of defining the level of stringency, reference can be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly updates).

“Identity”: “Identity” when used in respect to the comparison of two or more nucleic acid or polypeptide molecules means that the sequences of said molecules share a certain degree of sequence similarity, the sequences being partially identical.

Enzyme variants may be defined by their sequence identity when compared to a parent en zyme. Sequence identity usually is provided as “% sequence identity” or “% identity”. To de termine the percent-identity between two amino acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e. , a pairwise global alignment). The alignment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably by using the program “NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters (gapo- pen=10.0, gapextend=0.5 and matrix=EBLOSUM62). The preferred alignment for the pur pose of this invention is that alignment, from which the highest sequence identity can be determined. The following example is meant to illustrate two nucleotide sequences, but the same calcu lations apply to protein sequences:

Seq A: AAGATACTG length: 9 bases Seq B: GATCTGA length: 7 bases

Hence, the shorter sequence is sequence B.

Producing a pairwise global alignment which is showing both sequences over their complete lengths results in

Seq A: AAGATACTG-

Seq B:

The “I” symbol in the alignment indicates identical residues (which means bases for DNA or amino acids for proteins). The number of identical residues is 6.

The symbol in the alignment indicates gaps. The number of gaps introduced by alignment within the Seq B is 1. The number of gaps introduced by alignment at borders of Seq B is 2, and at borders of Seq A is 1.

The alignment length showing the aligned sequences over their complete length is 10.

Producing a pairwise alignment which is showing the shorter sequence over its complete length according to the invention consequently results in:

Seq A:

Seq B:

Producing a pairwise alignment which is showing sequence A over its complete length ac cording to the invention consequently results in:

Seq A:

Seq B:

Producing a pairwise alignment which is showing sequence B over its complete length ac cording to the invention consequently results in:

Seq A:

Seq B:

The alignment length showing the shorter sequence over its complete length is 8 (one gap is present which is factored in the alignment length of the shorter sequence).

Accordingly, the alignment length showing Seq A over its complete length would be 9 (mean ing Seq A is the sequence of the invention). Accordingly, the alignment length showing Seq B over its complete length would be 8 (mean ing Seq B is the sequence of the invention).

After aligning two sequences, in a second step, an identity value is determined from the align ment produced. For purposes of this description, percent identity is calculated by %-identity = (identical residues / length of the alignment region which is showing the respective se quence of this invention over its complete length) *100. Thus, sequence identity in relation to comparison of two amino acid sequences according to this embodiment is calculated by di viding the number of identical residues by the length of the alignment region which is showing the respective sequence of this invention over its complete length. This value is multiplied with 100 to give “%-identity”. According to the example provided above, %-identity is: for Seq A being the sequence of the invention (6 / 9) * 100 = 66.7 %; for Seq B being the sequence of the invention (6 / 8) * 100 =75%.

Isolated: The term "isolated" as used herein means that a material has been removed by the hand of man and exists apart from its original, native environment and is therefore not a product of nature. An isolated material or molecule (such as a DNA molecule or enzyme) may exist in a purified form or may exist in a non-native environment such as, for example, in a transgenic host cell. For example, a naturally occurring nucleic acid molecule or polypeptide present in a living cell is not isolated, but the same nucleic acid molecule or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acid molecules can be part of a vector and/or such nucleic acid molecules or poly peptides could be part of a composition, and would be isolated in that such a vector or com position is not part of its original environment. Preferably, the term "isolated" when used in relation to a nucleic acid molecule, as in "an isolated nucleic acid sequence" refers to a nu cleic acid sequence that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in its natural source. Isolated nucleic acid molecule is nucleic acid molecule present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acid molecules are nucleic acid molecules such as DNA and RNA, which are found in the state they exist in nature. For ex ample, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in prox imity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs, which encode a multitude of proteins. However, an isolated nucleic acid sequence comprising for example SEQ ID NO: 1 includes, by way of example, such nucleic acid sequences in cells which ordi narily contain SEQ ID NO: 1 where the nucleic acid sequence is in a genomic or plasmid location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid sequence may be present in single- or double-stranded form. When an isolated nucleic acid sequence is to be utilized to express a protein, the nucleic acid sequence will contain at a minimum at least a portion of the sense or coding strand (i.e., the nucleic acid sequence may be single-stranded). Alterna tively, it may contain both the sense and anti-sense strands (i.e., the nucleic acid sequence may be double-stranded). Non-coding: The term "non-coding" refers to sequences of nucleic acid molecules that do not encode part or all of an expressed protein. Non-coding sequences include but are not limited enhancers, promoter regions, 3' untranslated regions, and 5' untranslated regions.

Nucleic acids and nucleotides: The terms "nucleic acids" and "Nucleotides" refer to naturally occurring or synthetic or artificial nucleic acid or nucleotides. The terms “nucleic acids” and "nucleotides” comprise deoxyribonucleotides or ribonucleotides or any nucleotide analogue and polymers or hybrids thereof in either single- or double-stranded, sense or antisense form. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and comple mentary sequences, as well as the sequence explicitly indicated. The term "nucleic acid" is used inter-changeably herein with "gene", "cDNA, "mRNA", "oligonucleotide," and "nucleic acid molecule". Nucleotide analogues include nucleotides having modifications in the chem ical structure of the base, sugar and/or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitution of 5-bromo-uracil, and the like; and 2'-position sugar modifications, in cluding but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN. Short hairpin RNAs (shRNAs) also can comprise non-natural elements such as non-natural bases, e.g., ionosin and xanthine, non-natural sugars, e.g., 2'-methoxy ribose, or non-natural phosphodiester link ages, e.g., methylphosphonates, phosphorothioates and peptides.

Nucleic acid sequence: The phrase "nucleic acid sequence" refers to a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5'- to the 3'- end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role. "Nucleic acid sequence" also refers to a consecutive list of abbreviations, letters, characters or words, which represent nucleotides. In one embodiment, a nucleic acid can be a "probe" which is a relatively short nucleic acid, usually less than 100 nucleotides in length. Often a nucleic acid probe is from about 50 nucleotides in length to about 10 nucleotides in length. A "target region" of a nucleic acid is a portion of a nucleic acid that is identified to be of interest. A "coding region" of a nucleic acid is the portion of the nucleic acid, which is transcribed and translated in a se quence-specific manner to produce into a particular polypeptide or protein when placed under the control of appropriate regulatory sequences. The coding region is said to encode such a polypeptide or protein.

Oligonucleotide: The term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and in creased stability in the presence of nucleases. An oligonucleotide preferably includes two or more nucleomonomers covalently coupled to each other by linkages (e.g., phosphodiesters) or substitute linkages.

Overhang: An "overhang" is a relatively short single-stranded nucleotide sequence on the 5'- or 3'-hydroxyl end of a double-stranded oligonucleotide molecule (also referred to as an "ex tension," "protruding end," or "sticky end").

Polypeptide: The terms "polypeptide", "peptide", "oligopeptide", "polypeptide", "gene prod uct", "expression product" and "protein" are used interchangeably herein to refer to a polymer or oligomer of consecutive amino acid residues.

Promoter: The terms "promoter", or "promoter sequence" are equivalents and as used herein, refer to a DNA sequence which when operably linked to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into RNA. A promoter is located 5' (i.e., upstream), proximal to the transcriptional start site of a nucleotide sequence of interest whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription. The promoter does not comprise coding regions or 5^' untranslated regions. The promoter may for example be heterologous or homologous to the respective cell. A nucleic acid molecule se quence is "heterologous to" an organism or a second nucleic acid molecule sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety). Suitable promoters can be derived from genes of the host cells where expression should occur or from pathogens for this host.

Purified: As used herein, the term "purified" refers to molecules, either nucleic or amino acid sequences that are removed from their natural environment, isolated or separated. "Substan tially purified" molecules are at least 60% free, preferably at least 75% free, and more pref erably at least 90% free from other components with which they are naturally associated. A purified nucleic acid sequence may be an isolated nucleic acid sequence.

Significant increase: An increase for example in enzymatic activity, gene expression, produc tivity or yield of a certain product, that is larger than the margin of error inherent in the meas urement technique, preferably an increase by about 10% or 25% preferably by 50% or 75%, more preferably 2-fold or-5 fold or greater of the activity, expression, productivity or yield of the control enzyme or expression in the control cell, productivity or yield of the control cell, even more preferably an increase by about 10-fold or greater.

Significant decrease: A decrease for example in enzymatic activity, gene expression, produc tivity or yield of a certain product, that is larger than the margin of error inherent in the meas urement technique, preferably a decrease by at least about 5% or 10%, preferably by at least about 20% or 25%, more preferably by at least about 50% or 75%, even more preferably by at least about 80% or 85%, most preferably by at least about 90%, 95%, 97%, 98% or 99%.

Substantially complementary: In its broadest sense, the term "substantially complementary", when used herein with respect to a nucleotide sequence in relation to a reference or target nucleotide sequence, means a nucleotide sequence having a percentage of identity between the substantially complementary nucleotide sequence and the exact complementary se quence of said reference or target nucleotide sequence of at least 60%, more desirably at least 70%, more desirably at least 80% or 85%, preferably at least 90%, more preferably at least 93%, still more preferably at least 95% or 96%, yet still more preferably at least 97% or 98%, yet still more preferably at least 99% or most preferably 100% (the later being equivalent to the term “identical” in this context). Preferably identity is assessed over a length of at least 19 nucleotides, preferably at least 50 nucleotides, more preferably the entire length of the nucleic acid sequence to said reference sequence (if not specified otherwise below). Se quence comparisons are carried out using default GAP analysis with the University of Wis consin GCG, SEQWEB application of GAP, based on the algorithm of Needleman and Wun- sch (Needleman and Wunsch (1970) J Mol. Biol. 48: 443-453; as defined above). A nucleo tide sequence "substantially complementary " to a reference nucleotide sequence hybridizes to the reference nucleotide sequence under low stringency conditions, preferably medium stringency conditions, most preferably high stringency conditions (as defined above).

Transgene: The term "transgene" as used herein refers to any nucleic acid sequence, which is introduced into the genome of a cell by experimental manipulations. A transgene may be an "endogenous DNA sequence," or a "heterologous DNA sequence" (i.e. , "foreign DNA"). The term "endogenous DNA sequence" refers to a nucleotide sequence, which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the natu rally-occurring sequence.

Transgenic: The term transgenic when referring to an organism means transformed, prefer ably stably transformed, with at least one recombinant nucleic acid molecule.

Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of trans porting another nucleic acid molecule to which it has been linked. One type of vector is a genomic integrated vector, or "integrated vector", which can become integrated into the ge nomic DNA of the host cell. Another type of vector is an episomal vector, i.e., a plasmid or a nucleic acid molecule capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "ex pression vectors". In the present specification, "plasmid" and "vector" are used interchange ably unless otherwise clear from the context.

Wild type: The term "wild type", "natural" or "natural origin" means with respect to an organism that said organism is not changed, mutated, or otherwise manipulated by man. With respect to a polypeptide or nucleic acid sequence, that the polypeptide or nucleic acid sequence is naturally occurring or available in at least one naturally occurring organism which is not changed, mutated, or otherwise manipulated by man.

A wild type of a microorganism refers to a microorganism whose genome is present in a state as before the introduction of a genetic modification of a certain gene. The genetic modification may be e.g. a deletion of a gene or a part thereof or a point mutation or the introduction of a gene.

The terms "production" or "productivity" are art- recognized and include the concentration of the fermentation product (for example, dsRNA) formed within a given time and a given fer mentation volume (e.g., kg product per hour per liter). The term "efficiency of production" includes the time required for a particular level of production to be achieved (for example, how long it takes for the cell to attain a particular rate of output of a fine chemical).

The term "yield" or "product/carbon yield" is art-recognized and includes the efficiency of the conversion of the carbon source into the product (i.e., fine chemical). This is generally written as, for example, kg product per kg carbon source. By increasing the yield or production of the compound, the quantity of recovered molecules or of useful recovered molecules of that com pound in a given amount of culture over a given amount of time is increased.

The term “recombinant microorganism” includes microorganisms which have been genet ically modified such that they exhibit an altered or different genotype and/or phenotype (e. g., when the genetic modification affects coding nucleic acid sequences of the microorganism) as compared to the wild type microorganism from which it was derived. A recombinant micro organism comprises at least one recombinant nucleic acid molecule.

The term "recombinant" with respect to nucleic acid molecules refers to nucleic acid mole cules produced by man using recombinant nucleic acid techniques. The term comprises nu cleic acid molecules which as such do not exist in nature or do not exist in the organism from which the nucleic acid molecule is derived, but are modified, changed, mutated or otherwise manipulated by man. Preferably, a "recombinant nucleic acid molecule" is a non-naturally occurring nucleic acid molecule that differs in sequence from a naturally occurring nucleic acid molecule by at least one nucleic acid. A “recombinant nucleic acid molecules” may also comprise a “recombinant construct” which comprises, preferably operably linked, a sequence of nucleic acid molecules not naturally occurring in that order. Preferred methods for produc ing said recombinant nucleic acid molecules may comprise cloning techniques, directed or non-directed mutagenesis, gene synthesis or recombination techniques.

An example of such a recombinant nucleic acid molecule is a plasmid into which a heterolo gous DNA-sequence has been inserted or a gene or promoter which has been mutated com pared to the gene or promoter from which the recombinant nucleic acid molecule derived. The mutation may be introduced by means of directed mutagenesis technologies known in the art or by random mutagenesis technologies such as chemical, UV light or x-ray mutagen esis or directed evolution technologies. The term “directed evolution” is used synonymously with the term “metabolic evolution” herein and involves applying a selection pressure that favors the growth of mutants with the traits of interest. The selection pressure can be based on different culture conditions, ATP and growth coupled selection and redox related selection. The selection pressure can be carried out with batch fermentation with serial transferring inoculation or continuous culture with the same pressure.

The term “expression” or “gene expression” means the transcription of a specific gene(s) or specific genetic vector construct. The term “expression” or “gene expression” in particular means the transcription of gene(s) or genetic vector construct into mRNA. The process in cludes transcription of DNA and may include processing of the resulting RNA-product. The term “expression” or “gene expression” may also include the translation of the mRNA and therewith the synthesis of the encoded protein, i.e. protein expression.

Figures

Figure 1 shows the reaction catalyzed by the recombinant aspartase-like protein of the inven tion.

Figure 2 shows the enzymatic activities calculated from the results of the screening reactions. Numbers are also listed in Table 5.

Figure 3 shows a Time course of b-alanine production by selected enzymes (see legend, protein concentration in parentheses), including data in Table 6. Up to 250 mM of b-alanine was formed by variant A5. From highest to lowest activity: A5, A1 , Aint, A5 and B19.

Figure 4 shows the ¹H-NMR spectra of the starting acrylic acid mixture before the addition of enzyme (top), the enzymatic reaction after 24 h at 37 °C (middle) and a b-alanine solution (bottom).

EXAMPLES

Example 1 : Mutations in Bacillus spec YM55-1 aspartase tested for production of b-alanine

Recombinant variants of aspartase from Bacillus spec YM55-1 of SEQ ID NO 2 are shown in Table 3. In total 15 variants, referred to as A1 to A15, were generated. One variant published as being capable to catalyze the reaction from crotonic acid to b-aminobutanoic acid named B7 was also tested. It was noticed that all variants feature an isoleucine at position 187.

Recombinant aspar- mutations tase-like protein

A1 T 187I_M321 l_K324M_N326C

A2 T 187I_M321 M_K324F_N326C

A3 T 187I_M321 l_K324M_N326A

A4 T 187I_M321 l_K324V_N326C A5 T187LM321 LK324LN326C

A6 T187LM321 l_K324C_N326C

A7 T 187I_M 321 l_K324 L_N 326C

A8 T187LM321 M_K324F_N326A

A9 T187LM321 M_K324P_N 326C

A10 T187LM321 l_K324L_N326A

A11 T187LM321 l_K324l_N326A

A12 T187LM321 l_K324V_N326A

A13 T187LM321 l_K324C_N326A

A14 T187LM321 l_K324F_N326C

B7 T 187V_M 321 l_K324V_N 326C

A15 T 187I M 321 l K324A N 326C Table 3

In order to obtain more variation at position 187 further variants were produced for testing. The variants are shown in Table 4. Six variants were generated. Of these, four were identical to variants known in the art (B1 , B2, B5, and B10) and two were new (A16-A17).

Recombinant aspartase- mutations like protein

B10 T 187V_M321 l_K324M_N326C

B2 T 187V_M321 l_K324C_N326C

B5 T 187V_M321 l_K324M_N326A

B1 T 187V_M321 l_K324l_N326C

A16 T 187V_M321 l_K324C_N326A

A17 T 187V M321I K324I N326A Table 4

Summary of recombinant aspartase-like proteins tested

Table 5

All variants were tested for their capability of b-alanine production. Example 2: Mutant construction and verification

For construction of the mutants, the plasmid pET21 ::AspB SEQ ID NO: 55 containing genes for the AspB mutants B19, N5, F29 and P1 was used. A mutagenesis strategy was employed based on these templates: three mutants, A1 , A10 and variant Aint, were constructed as intermediates using QuikChange mutagenesis. Based on these three intermediates and the available templates, all other mutants were constructed. The complete coding sequence of every AspB variant was confirmed by Sanger sequencing.

Example 3: Protein production

The enzyme was expressed by using an auto-induction medium (Li et al., 2018) containing 1 % tryptone, 0.5% yeast extract, 0.33% (NH₄)₂S0₄ , 0.71 % Na₂HP0₄, 0.68% KH₂P0₄,

0.024% MgS0₄, 0.2% glycerol (v/v), 0.05% glucose, 0.2% lactose and 50 mg/L ampicillin. Sufficient enzyme could be obtained from 1 or 2 mL culture, but to be able to perform addi tional experiments a 50 mL culture was set up for each mutant enzyme. Cultures were started with 1 % preculture, grown for 24 h at 30 °C (200 rpm, 1 inch throw). Cells were harvested by centrifugation for 15 min at 3220 ^c g. Cell pellets were resuspended in 2 mM 50 mM T ris/HCI, pH 7.5, with 2 mM MgCh, sonicated on ice (2:30, 70% amplitude, 3 s on, 6 s off) and heated in a water bath (60 °C, 1 h). The resulting mixture was centrifuged (1 h, 18,500 ^c g, 4 °C) resulting in approximately 2 ml_ of cell free extract devoid of all heat labile E. coli proteins (Li et al., 2018). The total protein content of the enzyme solutions was determined using the Bradford method, using 20-fold diluted samples. 5 pL of sample was mixed with 195 pL Brad ford reagent (Bio-Rad). Protein concentrations were calculated using a BSA standard curve.

Example 4: Screening assays

Screening assays were set up as follows: 200 pL 500 mM NH3, 25 mM Na2HPC>4, 250 mM acrylic acid, set to pH 8.0 with HCI, 25% v/v enzyme solution. 16 h at 37 °C in a flat-bottom 96-well plate (1050 rpm, 3 mm throw).

For HPLC, samples were diluted 40-fold to get an ammonia + b-alanine concentration of less than 50 mM. Sample workup was done similar to the procedure described (Li et al., 2018), only on a 2.5-fold increased scale. 100 pL 40-fold diluted sample was mixed with 40 pL 1 M NaHCOs and 160 pL DNFB (36.7 mM in acetone). After heating for 30 min at 60 °C (in a PCR machine with heated lid) 80 pL 1 M HCI was added and precipitates were removed by cen trifugation.

HPLC conditions were as follows. Column: Nucleosil C18 5 p (250 ^c 4.6 mm); temperature: 25 °C; eluent A: 0.1% formic acid in water, eluent B: acetonitrile; flow rate: 1 ml-min^-1. DNFB- b-alanine detection with UV at 395 nm. Gradient: 15% B 25% B in 6.5 min, 25% B isocratic until 24.5 min, 25% B 50% B from 24.5 min to 39 min, 15% B from 39 min to 43.2 min. Retention time of DNFB^-alanine: 23.3 min. Conversions were calculated using a standard curve based on samples with a known concentration of b-alanine.

Protein concentrations obtained after cell lysis and heat treatment did not vary over a large range, so reactions were started and the specific activity was corrected for the exact protein concentration in the reaction mixture.

Table 6. Results from the enzymatic reactions. The b-alanine concentrations were meas ured by HPLC, enzyme concentrations using Bradford, and the activities in mU (nmol prod uct per min) were calculated over the full 16 h reaction time. b-Ala enzyme (mg/ml_) mU / mg (mM)

B19 14 4.0 4

N5 1 5.6 0

P1 15 5.5 3

V9 14 4.3 3

Aint 43 4.5 10

A1 57 4.5 13

A2 2 5.9 0

A3 38 2.8 14

A4 14 4.8 3

A5 64 4.4 15 A6 11 4.4 3

A7 22 5.4 4

A9 1 4.4 0

A10 17 4.3 4

A11 8 4.3 2

A12 2 1.3 2

A13 9 4.8 2

A14 8 3.6 2

A15 4 3.8 1

A16 7 4.0 2

A17 20 5.7 4

B1 34 4.6 8

B10 31 3.5 9

B2 4 3.3 1

B5 8 5.7 1

B7 4 2.3 2

Recombinant aspartase-like proteins A2, A8 (not tested) and A9 all have a methionine at position 321, which seems to completely abolish activity with acrylic acid. Example 7: Larger Scale testing of selected proteins.

For several confirmed recombinant aspartase-like proteins, i.e. variants A1 , A3, A5 and Aint and B19 the reaction was repeated on a larger scale (1 mL). Conditions were identical to the screening reactions (250 mM acrylic acid, 500 mM NH3), only performed at 1 mL in 1.5 mL vials instead of in microtiter plates b-alanine concentrations measured by HPLC, enzyme concentration using Bradford. The reactions were followed over time (Fig. 3), and initial ac tivities were calculated (Table 7) over the time period from 0 to 8 h.

Table 7. Larger-scale enzymatic b-alanine synthesis reactions. Activities were calculated over the initial 8 h of the reaction. 1 U of activity corresponds to a an amount of enzyme activity that catalyzes 1 pmol of product formation per min.

Variant Sp. activity (mU/mg)

A1 49

A3 45

A5 49

Aint 35

B19 12

The results of this reactions deviate from the results in the screening reactions because the specific activity (mU/mg) is calculated over a shorter initial period here. Fig. 3 also shows that under these conditions a conversion approaching completeness can be obtained.

Example 8: NMR To confirm the formation of b-alanine, a reaction with variant A5 was carried out on a 400 mI_ scale using the same conditions as above. After 24 h, 100 mI_ D20 was added and an 1H- NMR spectrum was recorded. The spectra (Fig. 4) show that 2/3 of acrylic acid is converted in 24 h. Some contaminating compounds are visible in the spectrum of the substrate; the dimer of acrylic acid present in small amounts in the starting material shows up as two triplets.

Claims

What is claimed is:

1. A recombinant aspartase-like protein capable of catalysing the reaction from ammonia and acrylic acid to b-alanine in an aqueous medium comprising water, at least one recombinant aspartase-like protein, ammonia and acrylic acid, wherein the concentra tion of b-alanine in the aqueous medium after incubation is at least 2% (w/w).

2. The recombinant aspartase-like protein of claim 1 , wherein the recombinant aspartase- like protein comprises a M321 I mutation in a position corresponding to position 321 in SEQ ID NO: 2 and at least one further mutation leading to an amino acid exchange in the position corresponding to the position 187, 324 and/or 326 in SEQ ID NO: 2.

3. The recombinant aspartase-like protein of claim 1 or 2 wherein the mutation in any of the positions 187, 324 and 326 is a mutation introducing a hydrophobic amino acid at the respective position.

4. The recombinant aspartase-like protein of claim 3, wherein the hydrophobic amino acid is alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine or tryptophan.

5. The recombinant aspartase-like protein of claim 2 to 4, wherein the amino acid at the position corresponding to position 324 in SEQ ID NO: 2 is not substituted by Val, Met or Cys, when the amino acid at the position corresponding to position 187 in SEQ ID NO: 2 is substituted by Val, and wherein the amino acid at the position corresponding to position 324 in SEQ ID NO: 2 is not substituted by Met or Leu, when the amino acid at the position corresponding to position 187 in SEQ ID NO: 2 is substituted by Cys.

6. The recombinant aspartase-like protein of any of claim 1 to 5, wherein the recombinant aspartase-like protein comprises a M321 I mutation in a position corresponding to po sition 321 in SEQ ID NO: 2 and at least one mutation at the position corresponding to the position 187, 324 and/or 326 in SEQ ID NO: 2 a. T 1871 b. K324M or K324I c. N326C

7. The recombinant aspartase-like protein of claim 1 or 2 comprising a sequence selected from the group consisting of a. The polypeptide molecule of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 and 54, and b. a polypeptide molecule having at least 55% identity to the polypeptide molecule of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 or 54 or a functional fragment thereof, and c. A polypeptide molecule encoded by a nucleic acid molecule of SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, and d. A polypeptide molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, and e. A polypeptide molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, wherein the polypeptide molecule as defined in b., d. and e. is catalysing the reac tion from ammonia and acrylic acid to b-alanine in an aqueous medium.

8. A recombinant expression construct comprising a sequence encoding a recombinant aspartase-like protein as defined in any of claims 1 to 7.

9. A recombinant vector comprising the expression construct of claim 8.

10. A recombinant cell comprising a recombinant aspartase-like protein as defined in claim 1 to 7, a recombinant construct of claim 8 or a recombinant vector of claim 9.

11. The recombinant cell of claim 10 wherein the cell is a microorganism.

12. The recombinant microorganism of claim 11 wherein the microorganism is Rhodococ- cus rhodocrous, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris.

13. A process for producing b-alanine or salt thereof comprising the steps of i. Providing an aqueous medium comprising ammonia, acrylic acid, one or more recombinant aspartase-like protein and optionally b-alanine, ii. Incubating the aqueous medium and iii. Optionally isolating the b-alanine or salt thereof from the reaction mix ture.

14. The process of claim 13 wherein the one or more recombinant aspartase-like protein has more than 55% identity to an aspartase.

15. The process of claim 13 or 14 wherein the one or more recombinant aspartase-like protein comprises a M321I mutation in a position corresponding to position 321 in SEQ ID NO: 2 and at least one mutation leading to an amino acid exchange in the position corresponding to the position 187, 324 and/or 326 in SEQ ID NO: 2.

16. The process of claim 13 to 15, wherein the mutation in any of the positions 187, 324 and 326 is a mutation introducing a hydrophobic amino acid at the respective position.

17. The process of claim 13 to 16, wherein the hydrophobic amino acid is alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine or tryptophan.

18. The process of claim 13 to 17, wherein the Lys in the position corresponding to position 324 in SEQ ID NO: 2 is not substituted by Pro or Phe.

19. The process of claim 13 to 18, wherein the recombinant aspartase-like protein com prises a M321 I mutation in a position corresponding to position 321 in SEQ ID NO: 2 and at least one of the mutations at the position corresponding to the position 187, 324 and/or 326 in SEQ ID NO: 2 a. T187I or T 187V b. K324M or K324I or K324L c. N326C or N326 A.

20. The process of claim 13 to 19 wherein the recombinant aspartase-like protein is com prising a sequence selected from the group consisting of a. The polypeptide molecule of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 and 54, and b. A polypeptide molecule having at least 55% identity to the polypeptide mole cule of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 or 54 or a functional fragment thereof, and c. A polypeptide molecule encoded by a nucleic acid molecule of SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, and d. A polypeptide molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, and e. A polypeptide molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, wherein the polypeptide molecule as defined in b., d. and e. is catalysing the reac tion from ammonia and acrylic acid to b-alanine in an aqueous medium.

21. The process of claim 14 to 21 wherein the pH is kept between 5 and 12.

22. The process of claim 14 to 22 wherein the product is isolated by precipitation, filtration or centrifugation after incubation.

23. The process of claim 14 to 23 wherein the aqueous medium is incubated for at least 8 h.

24. The process of claims 14 to 24 wherein the aqueous medium is incubated between 20 and 65°C.

25. The process of claims 14 to 25 wherein the recombinant aspartase-like protein is added as isolated protein.

26. The process of claim 26 wherein the recombinant aspartase-like protein is produced by fermentation.

27. The process of claims 14 to 25 wherein the recombinant aspartase-like protein is added comprised in a cell or in a cell extract.

28. A method for producing a recombinant aspartase-like protein, comprising the steps of a) providing a recombinant cell according to claim 10 to 12, and b) cultivating the recombinant cell under conditions allowing for the expression of said recombinant aspartase-like protein.

29. The method of claim 28 further comprising the step of c) incubating the cultivated cells for at least 30 min between 50°C and 80°C.

30. A composition comprising one or more recombinant aspartase-like protein as defined in claims 1 to 7, ammonia, acrylic acid and optionally b-alanine.

31. The composition of claim 30 wherein the one or more recombinant aspartase-like pro tein has more than 55% homology to an aspartase.

32. The composition of claims 30 and 31 wherein the one or more recombinant aspartase- like protein comprises a M321 I mutation in a position corresponding position 321 in SEQ ID NO: 2 and at least one mutation leading to an amino acid exchange in the position corresponding to the position 187, 324 and/or 326 in SEQ ID NO: 2.

33. The composition of claim 30 to 32, wherein the mutation in any of the positions 187, 324 and 326 is a mutation introducing a hydrophobic amino acid at the respective po sition.

34. The composition of claim 30 to 33, wherein the hydrophobic amino acid is alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine or tryp tophan.

35. The composition of claim 30 to 34, wherein the Lys in the position corresponding to position 324 in SEQ ID NO: 2 is not substituted by Pro or Phe.

36. The composition of claim 30 to 35, wherein the recombinant aspartase-like protein comprises a M321I mutation in a position corresponding to position 321 in SEQ ID NO: 2 and at least one of the mutations at the position corresponding to the position 187, 324 and/or 326 in SEQ ID NO: 2 a. T187I or T 187V b. K324M or K324I or K324L c. N326C or N326A.

37. The composition of claim 30 to 36 wherein the recombinant aspartase-like protein is comprising a sequence selected from the group consisting of a. The polypeptide molecule of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 and 54, and b. A polypeptide molecule having at least 55% identity to the polypeptide mole cule of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 or 54 or a functional fragment thereof, and c. A polypeptide molecule encoded by a nucleic acid molecule of SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, and d. A polypeptide molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, and e. A polypeptide molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53 or a functional fragment thereof, wherein the polypeptide molecule as defined in b., d. and e. is catalysing the reac tion from ammonia and acrylic acid to b-alanine in an aqueous medium.