WO2021058691A1 - Procédé de production de bêta-alanine ou de sels de celle-ci - Google Patents

Procédé de production de bêta-alanine ou de sels de celle-ci Download PDF

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Publication number
WO2021058691A1
WO2021058691A1 PCT/EP2020/076800 EP2020076800W WO2021058691A1 WO 2021058691 A1 WO2021058691 A1 WO 2021058691A1 EP 2020076800 W EP2020076800 W EP 2020076800W WO 2021058691 A1 WO2021058691 A1 WO 2021058691A1
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seq
recombinant
protein
aspartase
nucleic acid
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PCT/EP2020/076800
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English (en)
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Kai-Uwe Baldenius
Dick B. Janssen
Hein J. WIJMA
Hugo VAN BEEK
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Basf Se
Rijksuniversiteit Te Groningen
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Publication of WO2021058691A1 publication Critical patent/WO2021058691A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01001—Aspartate ammonia-lyase (4.3.1.1), i.e. aspartase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine

Definitions

  • the invention is directed to methods to produce b-alanine or salts thereof from acrylic acid using a recombinant aspartase-like protein as catalyst.
  • Aspartases 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).
  • 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.
  • 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 proucked. 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.
  • 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).
  • the publication is silent about which enzyme or en zymes are producing b-alanine.
  • 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 1 H-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).
  • 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.
  • At least the position b. M321 is mutated in the recombinant aspartase-like protein of the invention.
  • the mutation in position 321 corresponding to position 321 in SEQ ID NO: 2 is M321 I.
  • 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.
  • the recombinant aspartase-like protein of the invention does not comprise one of the following mutations: K324P, K324F or K324L.
  • 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.
  • 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.
  • 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
  • At least the position b. M321 is mutated in the recombinant aspartase-like protein 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.
  • 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.
  • 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 Jerusalem 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.
  • the recombinant microorganism is a prokaryotic cell.
  • Suitable prokaryotic cells include Gram-positive, Gram negative and Gram-variable bacterial cells, preferably Gram-negative.
  • 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, Brevibact
  • the microorganism is a eukaryotic cell.
  • 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
  • the recombinant aspartase-like protein used in the process of the invention has more than 55% homology to an aspartase.
  • 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.
  • At least the position M321 is mutated in the recombinant aspartase-like protein used in the process of the invention.
  • the mutation in position 321 corresponding to position 321 in SEQ ID NO: 2 is M321 I.
  • 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.
  • 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
  • At least the position M321 I is mutated in the recombinant aspartase-like protein used in the process 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method is carried out using continuous process.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • a composition comprising water, one or more recom binant aspartase-like protein of the invention, ammonia, acrylic acid and optionally b-alanine.
  • 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%, 9
  • 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
  • at least the position b. M321 is mutated in the recombinant aspartase-like protein of the invention.
  • the mutation in any of the positions 187, 321, 324 and 326 is a mutation introducing another hydrophobic amino acid at the respective position.
  • hydrophobic amino acids as meant herein consist of alanine, cysteine, phenyl alanine, glycine, isoleucine, leucine, methionine, proline, valine or tryptophan.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • Recombinant aspartase-like protein 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 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).
  • 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 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.
  • 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.
  • nucleic acid sequence refers to a nucleotide sequence whose nucleic acid molecules show total complementarity to the nucleic acid molecules of the nucleic acid sequence.
  • 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.
  • 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.
  • “enhancement” or “increase” of the level of an agent 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.
  • 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.
  • a nucleotide sequence for example an endogenous gene or a heterologous 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 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.
  • nucleic acid molecule e.g., gene sequence
  • some modification e.g., a point muta tion, the presence of a selectable marker gene, etc.
  • 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.
  • 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.
  • 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 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.
  • a regulatory element e.g. a promoter
  • further regulatory elements such as e.g., a ter minator
  • operble 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.
  • 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.
  • 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.
  • 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.
  • 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 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).
  • 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 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.
  • Natural genetic environment refers to the natural genomic locus in the organism of origin, or to the presence in 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.
  • non-natural, synthetic “artificial” methods such as, for example, mutagenization.
  • 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.
  • 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.
  • heterologous DNA encodes RNA or proteins that are not normally produced by the cell into which it is expressed.
  • Hybridization 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the Tm decreases about 1°C per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids:
  • 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:
  • 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.
  • 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%).
  • progressively lowering the annealing temperature for example from 68°C to 42°C
  • progressively low ering the formamide concentration for example from 50% to 0%.
  • hybridisation typically also depends on the function of post-hybridisation washes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • sequence B is sequence B.
  • 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.
  • 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).
  • 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).
  • an identity value is determined from the align ment produced.
  • 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”.
  • Isolated 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.
  • 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.
  • 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.
  • 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.
  • non-isolated nucleic acid molecules are nucleic acid molecules such as DNA and RNA, which are found in the state they exist in nature.
  • a given DNA sequence e.g., a gene
  • 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.
  • 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.
  • 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).
  • 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 refer to naturally occurring or synthetic or artificial nucleic acid or nucleotides.
  • 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.
  • 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.
  • 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 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.
  • non-natural bases e.g., ionosin and xanthine
  • non-natural sugars e.g., 2'-methoxy ribose
  • non-natural phosphodiester link ages e.g., methylphosphonates, phosphorothioates and peptides.
  • 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.
  • a nucleic acid can be a "probe” which is a relatively short nucleic acid, usually less than 100 nucleotides in length.
  • 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 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 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 refers 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.
  • 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 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.
  • 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).
  • 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 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”).
  • 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 when referring to an organism means transformed, prefer ably stably transformed, with at least one recombinant nucleic acid molecule.
  • vector refers to a nucleic acid molecule capable of trans porting another nucleic acid molecule to which it has been linked.
  • a genomic integrated vector or "integrated vector” which can become integrated into the ge nomic DNA of the host cell.
  • 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”.
  • ex pression vectors Vectors capable of directing the expression of genes to which they are operatively linked.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • expression means the transcription of a specific gene(s) or specific genetic vector construct.
  • 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.
  • expression or “gene expression” may also include the translation of the mRNA and therewith the synthesis of the encoded protein, i.e. protein expression.
  • 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 1 H-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).
  • Example 1 Mutations in Bacillus spec YM55-1 aspartase tested for production of b-alanine
  • 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).
  • 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.
  • the enzyme was expressed by using an auto-induction medium (Li et al., 2018) containing 1 % tryptone, 0.5% yeast extract, 0.33% (NH 4 ) 2 S0 4 , 0.71 % Na 2 HP0 4 , 0.68% KH 2 P0 4 ,
  • 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).
  • 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 .
  • 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.
  • 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.

Abstract

L'invention concerne des procédés pour produire de la bêta-alanine ou des sels de celle-ci à partir d'acide acrylique à l'aide d'une protéine de type aspartase recombinante en tant que catalyseur.
PCT/EP2020/076800 2019-09-26 2020-09-24 Procédé de production de bêta-alanine ou de sels de celle-ci WO2021058691A1 (fr)

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CN113122527A (zh) * 2021-04-25 2021-07-16 江南大学 一种酶活提高及最适pH改变的天冬氨酸酶突变体
CN114934037A (zh) * 2021-08-27 2022-08-23 上海邦林生物科技有限公司 用于生产3-氨基丙腈的天冬氨酸酶突变体
CN115894267A (zh) * 2021-09-30 2023-04-04 秦皇岛华恒生物工程有限公司 β-丙氨酸母液中产物的分离方法

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