Insect-derived aspartate decarboxylases and their variants for improved beta-alanine production

Abstract

Provided are N-terminally truncated variants of insect aspartate 1-decarboxylase that exhibit improved performance for beta-alanine production.

Description

[Technical field]

[0001] This description relates to biological methods for the production of beta-alanine. More particularly, described herein are insect aspartate 1-decarboxylase (ADC) enzymes and variants thereof that are particularly advantageous for the production of beta-alanine from L-aspartic acid. [Background technology]

[0002] Beta-alanine, also known as beta-aminopropionic acid or 3-aminopropionic acid, is a naturally occurring amino acid in which the amino group is in the beta position of the carboxylate group. Beta-alanine is a versatile organic synthesis raw material, mainly used in the synthesis of pantothenic acid and calcium pantothenate, carnosine, pamidronic acid, balsalazide, etc. It is widely used in pharmaceutical, feed, food, and other fields, and has a large market demand. On an industrial scale, beta-alanine is currently produced by chemical methods that require harsh reaction conditions accompanied by safety concerns, high equipment costs, and environmental pollution. The production of beta-alanine by safer and more environmentally friendly biological methods is largely hindered by low enzyme activity, expression, and / or stability, which makes such methods commercially unviable compared to chemical synthesis approaches. Summary of the Invention

[0003] Therefore, improved enzymes useful for the biological production of beta-alanine are highly desirable.

[0004] In one aspect, described herein are recombinant truncated insect aspartate 1-decarboxylases (ADCs) that lack a sufficient number of contiguous residues within the amino-terminal region of a corresponding full-length wild-type insect ADC such that the truncated ADC exhibits increased conversion of aspartate to beta-alanine compared to the full-length wild-type insect ADC.

[0005] In a further aspect, provided herein is a recombinant protein having aspartate 1-decarboxylase activity, comprising: (a) positions 72 to 561 of the amino acid sequence of CtADC set forth in SEQ ID NO:2; (b) positions 79 to 568 of the amino acid sequence of AaADC set forth in SEQ ID NO:4; (c) positions 56 to 544 of the amino acid sequence of AtADC set forth in SEQ ID NO:3; (d) positions 52 to 540 of the amino acid sequence of TcADC set forth in SEQ ID NO:1; (e) positions 71 to 560 of the amino acid sequence of Aa2ADC set forth in SEQ ID NO:10; (f) positions 71 to 562 of the amino acid sequence of Aa3ADC set forth in SEQ ID NO:11; (g) positions 74 to 563 of the amino acid sequence of CqADC set forth in SEQ ID NO:9; (h) positions 72 to 561 of the amino acid sequence of Aa4ADC set forth in SEQ ID NO:13; (i) positions 74 to 624 of the amino acid sequence of AdADC set forth in SEQ ID NO: 14; (j) positions 83 to 572 of the amino acid sequence of AsADC set forth in SEQ ID NO: 12; (k) positions 72 to 561 of the amino acid sequence of As2ADC set forth in SEQ ID NO: 15; (l) positions 53 to 541 of the amino acid sequence of TmADC set forth in SEQ ID NO:6; (m) positions 57 to 572 of the amino acid sequence of AvADC set forth in SEQ ID NO:5; or (n) positions 1 to 491 of the amino acid sequence of SEQ ID NO: 29 The present invention relates to a recombinant protein comprising an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to

[0006] In a further aspect, the present specification describes a polynucleotide that comprises a nucleic acid sequence that encodes a recombinant truncated insect ADC described herein, or a recombinant protein described herein.

[0007] In a further aspect, described herein is an expression cassette comprising an isolated or recombinant polynucleotide described herein operably linked to a promoter that is heterologous to the insect ADC.

[0008] In further aspects, the specification describes host cells that express the recombinant truncated insect ADCs described herein, the recombinant proteins described herein, and / or that are transformed with or engineered to contain the polynucleotides described herein, or the expression cassettes described herein.

[0009] In a further aspect, described herein is a method for the production of beta-alanine, the method comprising: (a) providing an ADC enzyme source that is a truncated insect ADC described herein, a recombinant protein described herein, and / or a host cell described herein; (b) contacting the ADC enzyme source with a source of aspartic acid under conditions that allow the ADC enzyme source to catalyze the conversion of aspartic acid to beta-alanine; and (c) isolating and / or concentrating the produced beta-alanine.

[0010] In a further aspect, described herein are compositions comprising the beta-alanine produced by the methods described herein.

[0011] general definition Headings and other identifying numbers, e.g., (a), (b), (i), (ii), etc., are presented merely to facilitate the readability of the specification and claims. The use of headings or other identifying numbers in the specification or claims does not necessarily require that the steps or elements be performed in alphabetical or numerical order or in the order in which they appear.

[0012] When used in the claims and / or specification in conjunction with the term "comprising," the use of the words "a" or "an" may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0013] The term "about" is used to indicate that a value includes the standard deviation of error for the device or method being used to determine the value. Generally, the term "about" is meant to indicate a possible variation of up to 10%. Thus, the term "about" includes variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% of the value. Unless otherwise indicated, the use of the term "about" before a range refers to both ends of the range.

[0014] As used herein, the terms "comprising" (and all forms of including, such as "comprise" and "comprises"), "having" (and all forms of including, such as "have" and "has"), "including" (and all forms of including, such as "includes" and "include"), or "containing" (and all forms of including, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or methods / method steps.

[0015] As used herein, the term "beta-alanine" includes beta-alanine and beta-alanine salts (eg, calcium, sodium, or potassium beta-alanine salts). [Brief description of the drawings]

[0016] [Figure 1]Figure 1 shows a phylogenetic tree of ADC enzymes from various insect species grouped by 85% sequence identity. Figure 2 shows activity data for some of the ADCs tested in Example 2. [Figure 2-1] Alignment of amino acid sequences of ADCs identified from nine different mosquito species. Dashed lines indicate regions in the N-terminal portion that are less conserved among mosquito ADCs. The glycine residue at position 96, unique to CtADCs, is highlighted in black. The SEQ ID NO corresponding to each sequence is in brackets. [Figure 2-2] Continued from Figure 2. [Diagram 3] Figure 1 shows an alignment of the N-terminal amino acid sequences of the mosquito and beetle ADCs described in Examples 5 and 6. N-terminal truncations between the two residues highlighted in black resulted in truncated ADCs with increased activity compared to the corresponding full-length proteins, whereas N-terminal truncations between the two residues boxed in white resulted in enzymes with low or undetectable ADC activity. The regions marked with dashed lines indicate positions where N-terminal truncations would be expected to be less beneficial for enzyme activity.

[0017] Sequence Listing This application contains a sequence listing in computer readable format of approximately 100 kb in size, created on March 1, 2021. The computer readable format is incorporated herein by reference.

[0018] [Table 1] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Attempts at industrial-scale biological synthesis of beta-alanine by enzyme-catalyzed removal of the alpha carboxyl group of L-aspartic acid have been largely hampered by insufficient enzyme activity, expression, and / or stability, making such methods commercially unviable compared to chemical synthesis approaches. Improved enzymes catalyzing the conversion of L-aspartic acid to beta-alanine with increased activity, expression, and / or stability would greatly facilitate commercial-scale biological synthesis of beta-alanine. This description relates to the discovery that certain insect-derived enzymes with aspartate 1-decarboxylase activity are particularly advantageous for beta-alanine production, and further, that the performance of such insect-derived enzymes can be significantly improved by truncating a portion of their N-terminus.

[0020] In a first aspect, the present specification describes recombinant truncated insect aspartate 1-decarboxylase (ADC) enzymes that are particularly advantageous for beta-alanine production. As used herein, the term "aspartate 1-decarboxylase" or "ADC" refers to a polypeptide that has the ability to catalyze the enzymatic conversion of L-aspartate to beta-alanine and carbon dioxide. In some embodiments, such polypeptides may include those classified in the enzyme class EC 4.1.1.11. In some embodiments, such polypeptides may also include enzymes classified in other enzyme classes (e.g., enzymes that have activity on substrates other than L-aspartate) and / or polypeptides annotated (e.g., in public databases) as enzymes other than ADCs (e.g., glutamate decarboxylase, cysteine ​​sulfinate decarboxylase). In some embodiments, the insect ADCs and their truncated variants described herein may include enzymes that have both aspartate 1-decarboxylase activity and cysteine ​​sulfinate decarboxylase activity.

[0021] As used herein, the term "truncated" or "truncation" not only includes the removal of a segment of a protein beginning at a terminal residue (e.g., beginning at the N-terminal methionine of a recombinant protein), but may also include the deletion of consecutive residues within a terminal region or portion of a protein (e.g., a wild-type protein) such that the terminal portion of the truncated protein is shorter than the terminal portion of the untruncated protein.

[0022] In some embodiments, the truncated insect ADCs described herein lack a sufficient number of consecutive residues within the amino-terminal portion of the corresponding full-length wild-type insect ADC such that the truncated ADC exhibits increased conversion of aspartic acid to beta-alanine compared to the parent full-length wild-type protein. In some embodiments, increased conversion of aspartic acid to beta-alanine may include increased ADC catalytic activity, increased ADC stability, and / or increased expression compared to the corresponding full-length wild-type protein.

[0023] In some embodiments, the truncated ADCs described herein may be truncated variants of an organism of the class Insecta (e.g., a mosquito, fly, beetle, flea, cockroach, or termite ADC). In particular embodiments, the truncated insect ADCs described herein may be truncated variants of a mosquito, fly, or beetle ADC whose structural relationships are shown in the phylogenetic tree of Figure 1. In some embodiments, the truncated insect ADCs described herein may be truncated variants of an insect ADC of the genus Culex, Anopheles, Drosophila, Aethina, Aedes, Tribolium, Anopheles, Tenebrio, Asbolus, or Cryptotermes. In some embodiments, the truncated insect ADCs described herein may comprise truncated variants of insect ADCs from the following species: Culex tarsalis, Anopheles arabiensis, Drosophila melanogaster, Culex quinquefasciatus, Aethina tumida, Aedes albopictus, Aedes aegypti, Tribolium castaneum, Anopheles sinensis, Tenebrio molitor, Asbolus verrucosus, or Cryptotermes secundus. In some embodiments, the truncated insect ADCs described herein may be truncated variants of a wild-type ADC enzyme, where the wild-type ADC enzyme is defined by an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to the amino acid sequence of SEQ ID NO:29.

[0024] In some embodiments, the truncated ADCs described herein may be truncated variants of mosquito ADCs that comprise an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical in its entirety to any one of SEQ ID NOs: 2, 4, or 9-15. In some embodiments, the truncated ADCs described herein may be truncated variants of rhinoceros beetle ADCs that comprise an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical in its entirety to any one of SEQ ID NOs: 1, 3, or 5-6. In some embodiments, the truncated ADCs described herein may be truncated variants of the fly ADCs that comprise an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to SEQ ID NO:8.

[0025] In some embodiments, a truncated ADC described herein may comprise an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to an N-terminal truncated fragment of the ADC that exhibits increased activity compared to its untruncated (e.g., full-length) parent enzyme. In some embodiments, the truncated ADCs described herein are selected from the group consisting of (a) positions 72 to 561 of the amino acid sequence of CtADC set forth in SEQ ID NO: 2, (b) positions 79 to 568 of the amino acid sequence of AaADC set forth in SEQ ID NO: 4, (c) positions 56 to 544 of the amino acid sequence of AtADC set forth in SEQ ID NO: 3, (d) positions 52 to 540 of the amino acid sequence of TcADC set forth in SEQ ID NO: 1, (e) positions 71 to 560 of the amino acid sequence of Aa2ADC set forth in SEQ ID NO: 10, (f) positions 71 to 562 of the amino acid sequence of Aa3ADC set forth in SEQ ID NO: 11, (g) positions 74 to 563 of the amino acid sequence of CqADC set forth in SEQ ID NO: 9, and (h) positions 72 to 56 of the amino acid sequence of Aa4ADC set forth in SEQ ID NO: 13. (i) positions 74 to 624 of the amino acid sequence of AdADC set forth in SEQ ID NO: 14, (j) positions 83 to 572 of the amino acid sequence of AsADC set forth in SEQ ID NO: 12, (k) positions 72 to 561 of the amino acid sequence of As2ADC set forth in SEQ ID NO: 15, (l) positions 53 to 541 of the amino acid sequence of TmADC set forth in SEQ ID NO: 6, (m) positions 57 to 572 of the amino acid sequence of AvADC set forth in SEQ ID NO: 5, or (n) an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to positions 1 to 491 of the amino acid sequence of SEQ ID NO: 29. These segments correspond to fragments of wild-type full-length insect ADCs that have been shown to exhibit improved performance in beta-alanine production or that may be predicted to exhibit improved performance based on sequence conservation and multiple sequence alignments such as those in Examples 5-7 and Figures 2 and 3.

[0026] In some embodiments, the truncated ADCs described herein may lack at least X contiguous residues at the amino terminus of a corresponding full-length wild-type insect ADC, where X is any integer between 5 and 50. In some embodiments, the truncated ADCs described herein may lack at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 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, or 70 consecutive residues from the amino terminus of the corresponding full-length wild-type insect ADC.

[0027] In some embodiments, the truncated ADCs described herein may be truncated at a position immediately C-terminal (downstream) to a residue corresponding to position n of a full-length wild-type insect ADC, where n is any integer between 2 and Y, and Y is the position of the most C-terminal residue in the full-length wild-type insect ADC, and truncation at this position may result in a truncated ADC that exhibits increased conversion of aspartic acid to beta-alanine compared to the full-length wild-type ADC. As used herein in reference to amino acid residue numbering, the phrase "corresponding to a position" takes into consideration that although the numbering of amino acid residues varies between different proteins (e.g., different insect ADCs), one of skill in the art would be able to determine corresponding residue positions in two proteins that share a degree of amino acid sequence identity by performing a sequence alignment between the two proteins, optionally including additional orthologs to identify conserved residues, using widely available software (e.g., Clustal Omega), as demonstrated herein.

[0028] In some embodiments, the truncated ADCs described herein comprise: (a) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11-71 of the amino acid sequence of CtADC set forth in SEQ ID NO:2; (b) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11-78 of the amino acid sequence of AaADC set forth in SEQ ID NO:4; (c) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11-55 of the amino acid sequence of AtADC set forth in SEQ ID NO:3; (d) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11-55 of the amino acid sequence of TADC set forth in SEQ ID NO:1; (e) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 51 of the amino acid sequence of Aa2ADC described in SEQ ID NO: 10; (f) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 70 of the amino acid sequence of Aa3ADC described in SEQ ID NO: 11; (g) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 70 of the amino acid sequence of CqADC described in SEQ ID NO: 9. (h) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 73 of the amino acid sequence of Aa4ADC described in SEQ ID NO: 13; (i) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 73 of the amino acid sequence of AdADC described in SEQ ID NO: 14; (j) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 82 of the amino acid sequence of AsADC described in SEQ ID NO: 12; (k) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 82 of the amino acid sequence of As2ADC described in SEQ ID NO: 15. , 5, 6, 7, 8, 9, 10, or 11-78 of the amino acid sequence of TmADC set forth in SEQ ID NO:6, (l) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11-52 of the amino acid sequence of TmADC set forth in SEQ ID NO:6, (m) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11-56 of the amino acid sequence of AvADC set forth in SEQ ID NO:5, or (n) positions 1-491 of the amino acid consensus sequence set forth in SEQ ID NO:29. The upper limits of each of the above amino acid sequences are shown in Table 7 and FIG. 3 and refer to the residue position in the full-length wild-type insect ADC that corresponds to K71 of CtADC. N-terminal truncation of CtADC to at least K71 results in a truncated ADC (CtADC) that exhibits increased conversion of aspartic acid to beta-alanine compared to the full-length wild-type ADC. 72-561) resulted.

[0029] In some embodiments, the truncated ADCs described herein are selected from the group consisting of (a) positions 72 to 80 of the amino acid sequence of CtADC set forth in SEQ ID NO: 2, (b) positions 79 to 87 of the amino acid sequence of AaADC set forth in SEQ ID NO: 4, (c) positions 56 to 64 of the amino acid sequence of AtADC set forth in SEQ ID NO: 3, (d) positions 52 to 60 of the amino acid sequence of TcADC set forth in SEQ ID NO: 1, (e) positions 71 to 79 of the amino acid sequence of Aa2ADC set forth in SEQ ID NO: 10, (f) positions 71 to 79 of the amino acid sequence of Aa3ADC set forth in SEQ ID NO: 11, (g) positions 74 to 82 of the amino acid sequence of CqADC set forth in SEQ ID NO: 9, (h) positions 75 to 79 of the amino acid sequence of CqADC set forth in SEQ ID NO: 9, (i) positions 76 to 79 of the amino acid sequence of CtADC set forth in SEQ ID NO: 2, (ii) positions 79 to 87 of the amino acid sequence of AaADC set forth in SEQ ID NO: 4, (iii) positions 56 to 64 of the amino acid sequence of AtADC set forth in SEQ ID NO: 3, (iv) positions 52 to 60 of the amino acid sequence of TcADC set forth in SEQ ID NO: 1, (v) positions 71 to 79 of the amino acid sequence of Aa2ADC set forth in SEQ ID NO: 10, (vi) positions 71 to 79 of the amino acid sequence of Aa3ADC set forth in SEQ ID NO: 11, (vii) positions 74 to 82 of the amino acid sequence of CqADC set forth in SEQ ID NO: 9, (v) positions The amino acid sequence may be truncated at the N-terminus (upstream) of any one of the following residues: (i) positions 72 to 82 of the amino acid sequence of Aa4ADC described in column number 13, (i) positions 74 to 82 of the amino acid sequence of AdADC described in SEQ ID NO: 14, (j) positions 83 to 91 of the amino acid sequence of AsADC described in SEQ ID NO: 12, (k) positions 72 to 87 of the amino acid sequence of As2ADC described in SEQ ID NO: 15, (l) positions 53 to 61 of the amino acid sequence of TmADC described in SEQ ID NO: 6, (m) positions 57 to 65 of the amino acid sequence of AvADC described in SEQ ID NO: 5, or (n) positions 1 to 491 of the amino acid sequence of SEQ ID NO: 29. The positions of these residues correspond to the positions indicated by dashed lines in Table 7 and FIG. 3.

[0030] In some embodiments, the truncated ADCs described herein are those having the amino acid sequence: (a) position 75 of the amino acid sequence of CtADC set forth in SEQ ID NO:2; (b) position 82 of the amino acid sequence of AaADC set forth in SEQ ID NO:4; (c) position 59 of the amino acid sequence of AtADC set forth in SEQ ID NO:3; (d) position 55 of the amino acid sequence of TcADC set forth in SEQ ID NO:1; (e) position 74 of the amino acid sequence of Aa2ADC set forth in SEQ ID NO:10; (f) position 74 of the amino acid sequence of Aa3ADC set forth in SEQ ID NO:11; (g) position 77 of the amino acid sequence of CqADC set forth in SEQ ID NO:9; (h) position 86 of the amino acid sequence of CqADC set forth in SEQ ID NO:10; The truncation may be N-terminal (upstream) to a residue corresponding to any one of the following: (i) position 77 of the amino acid sequence of Aa4ADC described in SEQ ID NO: 13; (i) position 77 of the amino acid sequence of AdADC described in SEQ ID NO: 14; (j) position 86 of the amino acid sequence of AsADC described in SEQ ID NO: 12; (k) position 82 of the amino acid sequence of As2ADC described in SEQ ID NO: 15; (l) position 56 of the amino acid sequence of TmADC described in SEQ ID NO: 6; (m) position 60 of the amino acid sequence of AvADC described in SEQ ID NO: 5; or (n) positions 1 to 491 of the amino acid sequence described in SEQ ID NO: 29. These residue positions correspond to S75 of CtADC present within the tripeptide sequence "SLP" that is conserved across all insect sequences aligned in FIG. 3.

[0031] In a further aspect, the present specification provides a recombinant protein having aspartate 1-decarboxylase activity, comprising: (a) positions 72 to 561 of the amino acid sequence of CtADC as set forth in SEQ ID NO: 2; (b) positions 79 to 568 of the amino acid sequence of AaADC as set forth in SEQ ID NO: 4; (c) positions 56 to 544 of the amino acid sequence of AtADC as set forth in SEQ ID NO: 3; (d) positions 52 to 540 of the amino acid sequence of TcADC as set forth in SEQ ID NO: 1; (e) positions 71 to 560 of the amino acid sequence of Aa2ADC as set forth in SEQ ID NO: 10; (f) positions 71 to 562 of the amino acid sequence of Aa3ADC as set forth in SEQ ID NO: 11; (g) positions 74 to 563 of the amino acid sequence of CqADC as set forth in SEQ ID NO: 9; and (h) positions 75 to 563 of the amino acid sequence of Aa4ADC as set forth in SEQ ID NO: 13. (i) positions 74 to 624 of the amino acid sequence of AdADC set forth in SEQ ID NO:14; (j) positions 83 to 572 of the amino acid sequence of AsADC set forth in SEQ ID NO:12; (k) positions 72 to 561 of the amino acid sequence of As2ADC set forth in SEQ ID NO:15; (l) positions 53 to 541 of the amino acid sequence of TmADC set forth in SEQ ID NO:6; (m) positions 57 to 572 of the amino acid sequence of AvADC set forth in SEQ ID NO:5; or (n) positions 1 to 491 of the amino acid sequence set forth in SEQ ID NO:29, which are at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to positions 72 to 561 of the amino acid sequence of AdADC set forth in SEQ ID NO:14; (i) positions 74 to 624 of the amino acid sequence of AdADC set forth in SEQ ID NO:14; (j) positions 83 to 572 of the amino acid sequence of AsADC set forth in SEQ ID NO:12; (k) positions 72 to 561 of the amino acid sequence of As2ADC set forth in SEQ ID NO:15; These regions are herein not only found to be highly conserved at least among mosquito and beetle ADCs, but also to represent increased conversion of aspartate to beta-alanine compared to the full-length wild-type CtADC. 72-561 It also supports truncated variants of

[0032] In some embodiments, the truncated ADCs and / or recombinant proteins described herein may contain a glycine residue at a position corresponding to position 96 of the amino acid sequence of CtADC as set forth in SEQ ID NO:2. CtADC was significantly superior to other insect-derived ADCs, including a greater than 39% increase in activity over the corresponding mosquito enzyme CqADC, with which it shares approximately 97% amino acid sequence identity, as demonstrated by enzyme activity studies performed in Example 2. Comparison of amino acid differences between CtADC and CqADC in the catalytic portion of the enzyme, performed in Example 8, revealed a single glycine residue at position 96 of CtADC that is unique among all other insect sequences analyzed (see FIG. 3), suggesting that this residue may play a role in the increased beta-alanine production associated with CtADC.

[0033] In a further aspect, the present specification describes a polynucleotide comprising a nucleic acid sequence that encodes a recombinant truncated insect ADC or a recombinant protein described herein.In some embodiments, the polynucleotide is DNA.In some embodiments, the polynucleotide is RNA.

[0034] In a further aspect, the present specification describes polynucleotide molecules that hybridize under stringent conditions to the complete complement of any one of the nucleic acid sequences of SEQ ID NOs: 16-27, nucleic acid sequences encoding recombinant truncated insect ADCs described herein, nucleic acid sequences encoding truncated recombinant proteins described herein, or any combination thereof.

[0035] In a further aspect, described herein is an expression cassette comprising an isolated or recombinant polynucleotide described herein operably linked to a promoter (eg, heterologous with respect to the insect ADC).

[0036] In a further aspect, the present specification describes a host cell that expresses a recombinant truncated insect ADC or recombinant protein described herein and / or is transformed with or engineered to contain a polynucleotide or expression cassette described herein. In some embodiments, the host cell may be a microbial cell. In some embodiments, the host cell may be a bacterial, insect, mammalian, yeast, or fungal cell.

[0037] In a further aspect, the recombinant truncated insect ADC, recombinant protein, or host cell described herein may be for use in the industrial production of beta-alanine from aspartic acid. In a further aspect, the present invention describes a method for the production of beta-alanine, comprising: (a) providing an ADC enzyme source that is a truncated insect ADC described herein, a recombinant protein described herein, and / or a host cell described herein; (b) contacting the ADC enzyme source with an aspartic acid source under conditions that allow the ADC enzyme source to catalyze the conversion of aspartic acid to beta-alanine; and (c) isolating and / or concentrating the produced beta-alanine. In some embodiments, the host cell expressing the recombinant truncated insect ADC or recombinant protein described herein may be utilized as an intact cell, which advantageously prevents cell debris from lysed cells from contaminating the produced beta-alanine.

[0038] In a further aspect, described herein are compositions comprising the beta-alanine produced by the methods described herein. EXAMPLES

[0039] Example 1: General Materials and Methods Cloning and expression of L-aspartate-α-decarboxylase (ADC) enzyme The codon-optimized cDNA sequences of ADCs cloned and expressed in bacteria are shown in SEQ ID NOs: 16-27. The cDNA sequences of ADCs were cloned into separate expression vectors and transformed into Escherichia coli to enhance expression of ADCs by the addition of inducers. For N-terminal truncations, the desired number of amino acids downstream of the initiator methionine were deleted.

[0040] ADC activity measurement ADC activity was measured by first growing BL21(DE3) E. coli cells expressing the ADC of interest in 500 μL of LB broth containing kanamycin and 0.2% isopropyl β-d-1-thiogalactopyranoside (IPTG) for 24 hours at 30°C. The cells were then pelleted, the supernatant removed, and resuspended and sonicated. The plates were then centrifuged to remove debris and the supernatant containing the cell lysate was collected. The activity of the cell lysate containing the ADC was then tested by incubating 50 μL of the supernatant in 50 mL of a solution containing L-aspartic acid at a final concentration of 60 g / L and pyridoxal phosphate (PLP) at a final concentration of 0.2 g / L at pH 6.5, at 37°C, and with agitation at 200 rpm. 1 M sulfuric acid was then titrated into the reaction solution to maintain the pH. After 1 hour, the amount of sulfuric acid used in the reaction was determined to directly measure the ADC activity. Experiments were performed at least three times and the average activity value was calculated.

[0041] Example 2: Activity of insect-derived ADCs A large-scale screen was conducted to compare the expression and activity of several different prokaryotic and eukaryotic ADC enzymes when recombinantly expressed in bacterial host cells. This screen revealed that bacterial cell lysates transformed with insect ADCs consistently exhibit higher beta-alanine production than ADCs from other organisms. Table 2 shows the relative ADC activity of bacterial lysates transformed with codon-optimized cDNAs of mosquito, fly, and beetle ADCs, measured as described in Example 1. Interestingly, bacterial lysates transformed with ADC from mosquito Culex tarsalis (CtADC; SEQ ID NO: 2) performed significantly better than all other enzymes tested.

[0042] [Table 2] Example 3: Sequence analysis of insect-derived ADCs The amino acid sequence of the CtADC was used as the basis for Protein BLAST™ to identify other ADCs from various species. Over 5000 hit sequences were searched, and then 188 sequences with the highest BLAST scores were selected and combined with the sequences of insect-derived ADCs in Table 2, grouped by 85% sequence identity, and finally assembled into a broad insect phylogenetic tree (Figure 1). The phylogenetic tree shown in Figure 1 indicates that the ADCs of mosquitoes and flies are structurally related, and the ADCs of beetles, fleas, cockroaches, and termites are structurally related.

[0043] Example 4: Sequence analysis of mosquito-derived ADCs An alignment of the amino acid sequences of the ADCs identified from nine different mosquito species was performed using Clustal Omega (1.2.4) and is shown in Figure 2. The alignment revealed a relatively high degree of sequence conservation across the various mosquito species, as shown in the percent identity matrix in Table 3 below.

[0044] [Table 3] Example 5: N-terminal truncation of mosquito ADC resulted in high beta-alanine production Interestingly, the alignment in FIG. 2 revealed a region of low sequence conservation toward the amino terminus of the mosquito ADC, shown by the dashed line in FIG. 2, immediately following a 15 amino acid segment (SGSDSAGVSEDEDVQ; SEQ ID NO: 28) that was 100% conserved across all mosquito ADCs analyzed. To study the role of the N-terminus of CtADC in its activity, progressive N-terminal truncations were generated and expressed in bacteria, and their ADC activity was characterized as described in Example 1. As shown in Table 4, N-terminal truncations ranging from 11 to 71 amino acids significantly increased beta-alanine production by 24% to 100%. However, truncations of 81 amino acids or more from the N-terminus of CtADC resulted in no detectable ADC enzymatic activity.

[0045] [Table 4] N-terminal truncations were also made and characterized in another mosquito enzyme, AaADC, as shown in Table 5. A 70% increase in beta-alanine production was observed by truncating the N-terminal 63 amino acids of AaADC. However, truncations of AaADC beyond 137 amino acids resulted in no detectable enzymatic activity of ADC.

[0046] [Table 5-1] Example 6: N-terminal truncation of the beetle ADC resulted in high beta-alanine production The two beetle ADCs were expressed in bacteria with progressive N-terminal truncations and their ADC activity was characterized as described in Example 1. The results are shown in Table 5 for AtADC and Table 6 for TcADC. For AtADC, truncation of the N-terminal 45 amino acids resulted in a significant 256% increase in beta-alanine production. However, truncation of AtADC beyond 114 amino acids resulted in no detectable ADC enzymatic activity (Table 5). For TcADC, N-terminal truncations ranging from 10 to 50 amino acids increased beta-alanine production by 10% to 330%. However, truncation of TcADC 60 amino acids from the N-terminus resulted in no detectable ADC enzymatic activity (TcADCN6, Table 6).

[0047] [Table 5-2]

[0048] [Table 6] Example 7: Analysis of the location of N-terminal truncations leading to high beta-alanine production The alignment of the N-terminal sequences of the mosquito and beetle ADCs described in Examples 5 and 6 is shown in Figure 3. The alignment in Figure 3 helps visualize and understand the results of the N-terminal truncations in Examples 5 and 6, where N-terminal truncations between the two residues highlighted in black resulted in truncated ADCs with increased activity compared to the corresponding full-length proteins. Conversely, N-terminal truncations between the two residues boxed in white resulted in truncated ADCs with no detectable ADC activity. Thus, in the truncation experiments for CtADCs and TcADCs that provide the highest resolution, the region marked with dashed lines indicates the position where N-terminal truncation is expected to be no longer beneficial for beta-alanine production. The respective positions of the residues in the mosquito and beetle ADCs are shown in Table 7. The region marked with dashed lines in Figure 3 also coincides with the beginning of greater sequence conservation between the mosquito and beetle ADCs, with the tripeptide sequence "SLP" being 100% conserved among all aligned sequences. Without wishing to be bound by any particular theory, truncation N-terminal to (or upstream of) the serine within the conserved "SLP" tripeptide may be beneficial in increasing beta-alanine production, while truncation downstream of the conserved serine may be detrimental (Table 7).

[0049] [Table 7] The N-terminally truncated CtADC sequence beginning at S75 was used as the basis for further Protein BLAST™ searches to identify other ADCs in various species. Multiple sequence alignment analysis was performed for ADC orthologous sequences that share at least 70% amino acid identity with the truncated CtADC sequence, and the consensus ADC sequence is shown in SEQ ID NO:29.

[0050] Example 8: Comparison of CtADC with ADCs of other mosquito species CtADC significantly outperformed other insect-derived ADCs from the enzymatic activity tests performed in Example 2. Based on the activities shown in Table 2, CtADC exhibited a 25% increase in beta-alanine production over the next best insect-derived ADCs from mosquito (AaADC) and fly (DmADC). Interestingly, although CtADC shares approximately 97% overall amino acid sequence identity with CqADC (also derived from mosquito), the results in Table 2 reveal that CtADC exhibits 39% higher beta-alanine production than CqADC. The results shown in Table 4 reveal that at least the N-terminal 71 residues of CtADC can be cleaved without inhibiting ADC activity (CtADCN7). Thus, examination of the amino acid differences between CtADC and CqADC within residues 72-561 of CtADC revealed only seven amino acid substitutions. Six of the seven amino acid substitutions correspond to residues found in various mosquito ADC orthologs. Interestingly, the only residue unique to CtADC was the glycine at position 96 (see residues highlighted in black in Figure 2). Indeed, the glycine corresponding to position 96 in full-length CtADC (SEQ ID NO:2) was not found in any other mosquito or beetle sequences analyzed (see Figure 3), suggesting that this residue may play a role in the enhanced beta-alanine production associated with CtADC.

Claims

1. A recombinant truncated insect aspartate 1-decarboxylase (ADC), wherein the truncated insect ADC lacks a sufficient number of contiguous residues within the amino-terminal region of a corresponding full-length wild-type insect ADC such that the truncated ADC exhibits increased conversion of aspartate to beta-alanine compared to the corresponding full-length wild-type insect ADC.

2. 2. The recombinant truncated insect ADC of claim 1, which is a truncated variant of a mosquito, fly, beetle, flea, cockroach, or termite ADC or is a truncated variant of a wild-type ADC enzyme, wherein the wild-type ADC enzyme is defined by an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to the amino acid sequence of SEQ ID NO:

29.

3. 3. The recombinant truncated insect ADC of claim 1 or 2, which is a truncated variant of an insect ADC of the genus Culex, Anopheles, Drosophila, Aethina, Aedes, Tribolium, Anopheles, Tenebrio, Asbolus, or Cryptotermes, A recombinant truncated insect ADC which may be a truncated variant of an insect ADC from the species Culex tarsalis, Anopheles arabiensis, Drosophila melanogaster, Culex quinquefasciatus, Aethina tumida, Aedes albopictus, Aedes aegypti, Tribolium castaneum, Anopheles sinensis, Tenebrio molitor, Asbolus verrucosus, or Cryptotermes secundus.

4. The corresponding full-length wild-type insect ADC is (a) a mosquito ADC comprising an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to any one of SEQ ID NOs: 2, 4, or 9-15; (b) a beetle ADC comprising an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to any one of SEQ ID NOs: 1, 3, or 5-6; (c) a fly ADC comprising an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to SEQ ID NO:8; or (d) the recombinant truncated insect ADC of any one of claims 1 to 3, which is an ADC comprising an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to SEQ ID NO:

29.

5. The truncated ADC is (a) positions 72 to 561 of the amino acid sequence of CtADC set forth in SEQ ID NO:2; (b) positions 79 to 568 of the amino acid sequence of AaADC set forth in SEQ ID NO:4; (c) positions 56 to 544 of the amino acid sequence of AtADC set forth in SEQ ID NO:3; (d) positions 52 to 540 of the amino acid sequence of TcADC set forth in SEQ ID NO:1; (e) positions 71 to 560 of the amino acid sequence of Aa2ADC set forth in SEQ ID NO:10; (f) positions 71 to 562 of the amino acid sequence of Aa3ADC set forth in SEQ ID NO:11; (g) positions 74 to 563 of the amino acid sequence of CqADC set forth in SEQ ID NO:9; (h) positions 72 to 561 of the amino acid sequence of Aa4ADC set forth in SEQ ID NO:13; (i) positions 74 to 624 of the amino acid sequence of AdADC set forth in SEQ ID NO:14; (j) positions 83 to 572 of the amino acid sequence of AsADC set forth in SEQ ID NO:12; (k) positions 72 to 561 of the amino acid sequence of As2ADC set forth in SEQ ID NO:15; (l) positions 53 to 541 of the amino acid sequence of TmADC set forth in SEQ ID NO:6; (m) positions 57 to 572 of the amino acid sequence of AvADC set forth in SEQ ID NO:5; or (n) positions 1 to 491 of the amino acid sequence set forth in SEQ ID NO:29; 5. The recombinant truncated insect ADC of any one of claims 1-4, comprising an amino acid sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical overall to

6. 6. The recombinant truncated insect ADC of any one of claims 1 to 5, wherein the truncated ADC comprises a glycine residue at a position corresponding to position 96 of the amino acid sequence of CtADC set forth in SEQ ID NO:

2.

7. 7. The recombinant truncated insect ADC of any one of claims 1 to 6, wherein the truncated ADC lacks at least X contiguous residues at the amino terminus of a corresponding full-length wild-type insect ADC, where X is any integer between 5 and 50.

8. 8. The recombinant truncated insect ADC of any one of claims 1-7, wherein the truncation occurs at a position immediately C-terminal (downstream) to a residue corresponding to position n of the full-length wild-type insect ADC, where n is any integer between 2 and Y, and Y is the position of the most C-terminal residue within the full-length wild-type insect ADC, and where truncation from the N-terminus at this position can result in a truncated ADC that exhibits increased conversion of aspartic acid to beta-alanine compared to the full-length wild-type ADC.

9. The disconnection (a) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 71 of the amino acid sequence of CtADC set forth in SEQ ID NO:2; (b) positions 2, 3, 4, 5, 6, 7, 8, 9 10, or 11 to 78 of the amino acid sequence of AaADC set forth in SEQ ID NO:4; (c) positions 2, 3, 4, 5, 6, 7, 8, 9 10, or 11 to 55 of the amino acid sequence of AtADC set forth in SEQ ID NO:3; (d) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 51 of the amino acid sequence of the TcADC set forth in SEQ ID NO:1; (e) positions 2, 3, 4, 5, 6, 7, 8, 9 10, or 11 to 70 of the amino acid sequence of Aa2ADC set forth in SEQ ID NO:10; (f) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 70 of the amino acid sequence of Aa3ADC set forth in SEQ ID NO:11; (g) positions 2, 3, 4, 5, 6, 7, 8, 9 10, or 11 to 73 of the amino acid sequence of CqADC set forth in SEQ ID NO:9; (h) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 73 of the amino acid sequence of Aa4ADC set forth in SEQ ID NO:13; (i) positions 2, 3, 4, 5, 6, 7, 8, 9 10, or 11 to 73 of the amino acid sequence of the AdADC set forth in SEQ ID NO:14; (j) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 82 of the amino acid sequence of AsADC set forth in SEQ ID NO:12; (k) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 78 of the amino acid sequence of As2ADC set forth in SEQ ID NO:15; (l) positions 2, 3, 4, 5, 6, 7, 8, 9 10, or 11 to 52 of the amino acid sequence of the TmADC set forth in SEQ ID NO:6; (m) positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 56 of the amino acid sequence of AvADC set forth in SEQ ID NO:5; or (n) positions 1 to 491 of the amino acid sequence set forth in SEQ ID NO:29; 9. The recombinant truncated insect ADC of any one of claims 1 to 8, wherein the ADC occurs at a position C-terminal (downstream) to a residue corresponding to any one of:

10. The disconnection (a) positions 72 to 80 of the amino acid sequence of CtADC set forth in SEQ ID NO:2; (b) positions 79 to 87 of the amino acid sequence of AaADC set forth in SEQ ID NO:4; (c) positions 56 to 64 of the amino acid sequence of AtADC set forth in SEQ ID NO:3; (d) positions 52 to 60 of the amino acid sequence of TcADC set forth in SEQ ID NO:1; (e) positions 71 to 79 of the amino acid sequence of Aa2ADC set forth in SEQ ID NO:10; (f) positions 71 to 79 of the amino acid sequence of Aa3ADC set forth in SEQ ID NO:11; (g) positions 74 to 82 of the amino acid sequence of CqADC set forth in SEQ ID NO:9; (h) positions 72 to 82 of the amino acid sequence of Aa4ADC set forth in SEQ ID NO:13; (i) positions 74 to 82 of the amino acid sequence of AdADC set forth in SEQ ID NO:14; (j) positions 83 to 91 of the amino acid sequence of AsADC set forth in SEQ ID NO:12; (k) positions 72 to 87 of the amino acid sequence of As2ADC set forth in SEQ ID NO:15; (l) positions 53 to 61 of the amino acid sequence of TmADC set forth in SEQ ID NO:6; (m) positions 57 to 65 of the amino acid sequence of AvADC set forth in SEQ ID NO:5; or (n) positions 1 to 491 of the amino acid sequence set forth in SEQ ID NO:29; 10. The recombinant truncated insect ADC of any one of claims 1 to 9, wherein the ADC occurs at a position N-terminal (upstream) of a residue corresponding to any one of:

11. The disconnection (a) position 75 of the amino acid sequence of CtADC set forth in SEQ ID NO:2; (b) position 82 of the amino acid sequence of AaADC set forth in SEQ ID NO:4; (c) position 59 of the amino acid sequence of AtADC set forth in SEQ ID NO:3; (d) position 55 of the amino acid sequence of TcADC set forth in SEQ ID NO:1; (e) position 74 of the amino acid sequence of Aa2ADC set forth in SEQ ID NO:10; (f) position 74 of the amino acid sequence of Aa3ADC set forth in SEQ ID NO:11; (g) position 77 of the amino acid sequence of CqADC set forth in SEQ ID NO:9; (h) position 77 of the amino acid sequence of Aa4ADC set forth in SEQ ID NO:13; (i) position 77 of the amino acid sequence of AdADC set forth in SEQ ID NO:14; (j) position 86 of the amino acid sequence of AsADC set forth in SEQ ID NO:12; (k) position 82 of the amino acid sequence of As2ADC set forth in SEQ ID NO:15; (l) position 56 of the amino acid sequence of TmADC set forth in SEQ ID NO:6; (m) position 60 of the amino acid sequence of AvADC set forth in SEQ ID NO:5; or (n) positions 1 to 491 of the amino acid sequence set forth in SEQ ID NO:29; 11. The recombinant truncated insect ADC of any one of claims 1 to 10, wherein the ADC occurs at a position N-terminal (upstream) of a residue corresponding to any one of:

12. A polynucleotide comprising a nucleic acid sequence encoding a recombinant truncated insect ADC as defined in any one of claims 1 to 11.

13. 13. An expression cassette comprising the polynucleotide of claim 12 operably linked to a promoter that is heterologous with respect to the insect ADC.

14. A host cell expressing a recombinant truncated insect ADC as defined in any one of claims 1 to 11 and / or transformed with or engineered to contain a polynucleotide as defined in claim 12 or an expression cassette as defined in claim 13, The host cell may be, for example, a bacterial, insect, mammalian, yeast, or fungal cell.

15. 1. A method for producing beta-alanine, comprising: (a) providing a source of ADC enzyme, which is a truncated insect ADC as defined in any one of claims 1 to 11, and / or a host cell as defined in claim 14; (b) contacting an ADC enzyme source with a source of aspartic acid under conditions capable of catalyzing the conversion of aspartic acid to beta-alanine; and (c) isolating and / or concentrating the beta-alanine produced.

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