Process for the production of 2-acetylpyrroline

Abstract

The present application provides a method of producing 2-acetylpyrroline, comprising reacting 2-carboxypyrrline with methylglyoxal. The present application also provides a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1 or a functional variant thereof, wherein the functional variant has delta(1)-pyrroline-2-carboxylate reductase activity. The present application also provides a method of producing 2-carboxypyrrline, comprising contacting the polypeptide with proline.

Description

Technical Field

[0001] This application generally relates to the fields of genetic engineering, enzyme engineering and bioinformatics; specifically, this application provides a novel method for producing 2-acetylpyrrolline. Background Technology

[0002] 2-Acetylpyrrolline (2AP) has an aroma similar to toasted bread or popcorn, and is the main aroma component of fragrant rice, being the source of its unique fragrance. Fragrant rice has a higher economic value than non-fragrant rice due to its distinctive aroma. 2-Acetylpyrrolline, with its popcorn-like aroma, can be used in the development of fermented food products.

[0003] The reported synthesis methods for 2-acetylpyrrololine are mainly chemical synthesis and natural synthesis from fragrant rice. Chemical synthesis requires complex equipment, produces numerous byproducts, and consumes a large amount of raw materials. Natural generation of 2-acetylpyrrololine from fragrant rice has a long growth cycle, resulting in significant time costs, and its varieties and applications are limited. Recent biosynthesis of 2-acetylpyrrololine has consistently used 5-carboxypyrrololine (P5C) as an intermediate product, exhibiting low conversion efficiency.

[0004] Given the unique aroma of 2AP, it possesses significant potential value and applications, and research into its synthetic route is of scientific and industrial significance. Invention Overview

[0005] In a first aspect, this application provides a method for producing 2-acetylpyrrolline, comprising reacting 2-carboxypyrrolline with acetone aldehyde.

[0006] In some embodiments of the first aspect, the method further includes contacting δ(1)-pyrrololine-2-carboxylic acid reductase with proline to generate 2-carboxypyrrololine (P2C).

[0007] In some embodiments of the first aspect, the method further includes contacting trans-3-hydroxy-L-proline dehydratase with trans-3-hydroxy-L-proline to generate 2-carboxypyrrololine.

[0008] In some embodiments of the first aspect, δ(1)-pyrrololine-2-carboxylic acid reductase comprises the amino acid sequence shown in SEQ ID NO:1 or a functional variant thereof.

[0009] In some embodiments of the first aspect, the functional variant retains the activity of δ(1)-pyrrolline-2-carboxylic acid reductase.

[0010] In a second aspect, this application provides a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or a functional variant thereof, wherein the functional variant has δ(1)-pyrrololine-2-carboxylic acid reductase activity.

[0011] In some embodiments of the second aspect, the substrate of the polypeptide is proline.

[0012] Thirdly, this application provides a nucleic acid molecule that encodes the polypeptide described in the second aspect.

[0013] Fourthly, this application provides an expression cassette containing the nucleic acid molecules described in the third aspect.

[0014] Fifthly, this application provides an expression vector comprising the nucleic acid molecule described in the third aspect or the expression cassette described in the fourth aspect.

[0015] Sixthly, this application provides a host cell comprising the nucleic acid molecule described in the third aspect, the expression cassette described in the fourth aspect, or the expression vector described in the fifth aspect.

[0016] In a seventh aspect, this application provides a method for generating 2-carboxypyrrololine, comprising contacting the polypeptide described in the second aspect with proline to generate 2-carboxypyrrololine. Attached Figure Description

[0017] Figure 1 Gene clusters in Bacillus cereus are shown.

[0018] Figure 2 The biosynthetic pathway of 2-acetylpyrrolidine was shown.

[0019] Figure 3 The SDS-PAGE analysis of the purified protein used for enzyme activity assays and biochemical characterization is shown. In this diagram, A represents BcP2CR, B represents BcT3LD, and C represents OsP2CR. Lanes 1 through 4 of each 4–20% gradient gel (Bis-Tris) contain the protein marker, 1, 2, and 4 μg of recombinant protein, respectively.

[0020] Figure 4 The results show the enzyme activity assays. Specifically, they include the A.BcP2CR activity assay, the B.OsP2CR activity assay, and the C.BcT3LD activity assay.

[0021] Figure 5 The kinetic data of BcP2CR enzyme are shown. Among them, A. the effect of pH on the BcP2CR-catalyzed L-proline oxidation activity; B. the dose-dependent enzyme activity of BcP2CR; C. the Michaelis-Menten kinetic parameters of BcP2CR on the substrate L-proline; D. the kinetic parameters of BcP2CR on the substrate NADP. + The Mie kinetic analysis is shown. The error bars represent the standard deviations of the three individual experiments.

[0022] Figure 6The kinetic data of the OsP2CR enzyme are shown. Among them, A. the effect of pH on the OsP2CR-catalyzed L-proline oxidation activity; B. the dose-dependent nature of OsP2CR; C. the Michaelis-Menten kinetic parameters of OsP2CR on the substrate L-proline; D. the effect of OsP2CR on the substrate NADP. + The Mie kinetic analysis is shown. The error bars represent the standard deviations of the three individual experiments.

[0023] Figure 7 The kinetic data for BcT3LD are shown. These include: A. the effect of pH on the activity of BcT3LD in catalyzing the oxidation of L-proline; B. the dose-dependent OsP2CR-coupled enzyme activity assay of BcT3LD; and C. the Michaelis-Menten kinetic parameters of BcT3LD with respect to the substrate t3L-HP. Error bars represent the standard deviations of the three individual experiments.

[0024] Figure 8 The LC-MS / MS enzymatic reaction analysis of P2CR is shown. A. Detection of 2-carboxypyrrololine (P2C) formation in the BcP2CR-catalyzed L-proline oxidation reaction by LC-MS / MS. In cation mode, secondary mass spectrometry (MRM mode) was used to extract P2C ion pairs from 114.1–68.1 ppm and detect P2C formation. B. Detection of 2-carboxypyrrololine (P2C) formation in the OsP2CR-catalyzed L-proline oxidation reaction by LC-MS / MS. In cation mode, secondary mass spectrometry (MRM mode) was used to extract P2C ion pairs from 114.1–68.1 ppm and detect P2C formation.

[0025] Figure 9 The LC-MS enzymatic reaction analysis of BcT3LD-OsP2CR coupled enzyme is shown. A. Detection of L-proline formation in the BcT3LD-OsP2CR coupled-catalyzed trans-3-hydroxy-L-proline (t3L-HP) reaction by LC-MS. L-proline formation was monitored by extracted ion chromatogram (EIC) at m / z = 116 in positive ion mode (tR = 16.6 min). B. Mass spectrum of L-proline corresponding to the EIC peak in A in the total reaction set (positive ion mode).

[0026] Figure 10The LC-MS analysis of the synthesis of 2-acetylpyrrololine using t3L-HP is shown. A. LC-MS detection of P2C formation in the t3L-HP dehydration reaction catalyzed by BcT3LD. In negative ion mode, the formation of P2C was detected by the extractable ion chromatogram (EIC) at m / z = 112 (tR = 5.6 min). B. In the control group without acetone aldehyde, P2C corresponds to the EIC peak in A (negative ion mode). C. LC-MS detection of 2-pyrrololine (2AP) formation in the reaction of P2C with acetone aldehyde. In positive ion mode, the formation of 2AP was detected by the extractable ion chromatogram (EIC) at m / z = 112 (tR = 15.3 min). D. In the total reaction group, 2AP corresponds to the EIC peak in C (positive ion mode).

[0027] Figure 11 The absorption spectrum of the final product 2AP is shown.

[0028] Sequence Description

[0029] The amino acid sequence shown in SEQ ID NO:1 is as follows:

[0030] MAAPPPPANQFVYIDAAALHSVLPFPSLISHLGAGLPAFAAGIHCPHRVSFPLPTAPSASLLLMPSWSAHPSLPYLALKAVTSFPANSPRLPSVHAAVSLFDSASGVPLASLDGSALTLLRTAAVSALAASLLASPTRPPSTLALAGAGALAPYLAEAHLSALPSISR ILIWNRTKAKSAALAARLRDAHPGVAVEEADSMDEAVSAADVVSCATGSQEPIVRGELLKPGAHLDLVGSFTPAMRECDDEALRRGRVFIDFEAAMQEAGELVGALQRGVLRREDVAGTLAELAAGSVAGRRCDDEITVFKSVGTAVVDLLAAQLAYETYIATTTKKT Invention Details

[0032] Through bioinformatics analysis, the inventors of this application have discovered a novel biosynthetic pathway for 2AP, which primarily uses P2C as a precursor for 2AP. This breaks with the traditional method of using P5C as a precursor for 2AP, improving the low conversion efficiency of P5C and enabling more efficient production of 2AP. Furthermore, the inventors of this application have also discovered a novel enzyme, OsP2CR, which is δ(1)-pyrrolline-2-carboxylic acid reductase. This enzyme can catalyze the production of P2C from proline (e.g., L-proline), which then spontaneously reacts with acetone aldehyde to generate 2AP.

[0033] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0034] definition

[0035] Unless otherwise specified, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in a amino to carboxyl direction. Numerical ranges include the numbers that define the range. Amino acids may be represented herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. Similarly, nucleotides may be represented by commonly accepted single-letter codes. The terminology defined above is defined more fully in the reference specification as a whole. As used in this article, the amino acid residue abbreviations are as follows: alanine is Ala or A; arginine is Arg or R; asparagine is Asn or N; aspartic acid is Asp or D; cysteine ​​is Cys or C; glutamic acid is Glu or E; glutamine is Gln or Q; glycine is Gly or G; histidine is His or H; isoleucine is Ile or I; leucine is Leu or L; lysine is Lys or K; methionine is Met or M; phenylalanine is Phe or F; proline is Pro or P; serine is Ser or S; threonine is Thr or T; tryptophan is Trp or W; tyrosine is Tyr or Y; and valine is Val or V.

[0036] In this application, the terms “peptide” and “protein” are used interchangeably to refer to polymers of amino acid residues and their variants, as well as synthetic and naturally occurring analogs. Therefore, these terms apply to naturally occurring amino acid polymers and their naturally occurring chemical derivatives, as well as amino acid polymers in which one or more amino acid residues are synthetic, non-naturally occurring amino acids (such as chemical analogs of the corresponding naturally occurring amino acids). Such derivatives include, for example, post-translational modifications and degradation products, including phosphorylated, glycosylated, oxidized, isomerized, carboxylated, and deamination variants of peptide fragments.

[0037] As used in this article, the term "enzyme active site" refers to the part of an enzyme molecule that can directly bind to the substrate molecule and catalyze the chemical reaction of the substrate; this part is called the enzyme active site.

[0038] As used herein, the term "amino acid" refers to a compound in which a hydrogen atom on the carbon atom of a carboxylic acid is replaced by an amino group, and the amino acid molecule contains both amino and carboxyl functional groups. It includes naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimics. Naturally occurring amino acids include the 20 (L)-amino acids used in protein biosynthesis, as well as other amino acids such as 4-hydroxyproline, hydroxylysine, carboxylated lysine, desmosin, isodesmosin, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, leucine, valine, p-fluorophenylalanine, ethylthiocyanate, etc., which are known to those skilled in the art. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications may include, for example, substitution of chemical groups and moieties on the amino acid, or derivatization of the amino acid. Amino acid mimics include, for example, organic structures exhibiting functionally similar properties, such as the charge and charge-space characteristics of the amino acid. For example, an organic structure mimicking arginine (Arg or R) has a positively charged moiety located in a similar molecular space and having the same degree of mobility of the e-amino group in the side chain as the naturally occurring Arg amino acid. The mimics also include constrained structures to maintain optimal space and charge interactions of the amino acid or amino acid functional groups. Those skilled in the art can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimics.

[0039] As used in this article, the term "isoenzyme" refers to enzymes in organisms that catalyze the same reaction but have different molecular structures.

[0040] As used herein, the term "nucleic acid" refers to mRNA, RNA, cRNA, cDNA, or DNA, including single-stranded and double-stranded DNA. The term generally refers to a polymeric form of a nucleotide at least 10 bases in length, wherein the nucleotide is a modified form of a ribonucleotide, deoxynucleotide, or any type of nucleotide.

[0041] When used in the context of a specific nucleic acid, as used herein, the term "encoding" refers to the nucleic acid containing the essential information that guides the translation of that nucleotide sequence into a specific protein. Codons are used to represent the information encoding the protein. Nucleic acids encoding proteins may contain untranslated sequences (e.g., introns) located within the translational region of the nucleic acid, or may lack such intervening untranslated sequences (e.g., as in cDNA).

[0042] As used herein, the term "full-length sequence" referring to a specific polynucleotide or the protein it encodes means the entire nucleic acid sequence or the entire amino acid sequence having a natural (non-synthetic) endogenous sequence. The full-length polynucleotide encodes the full-length, catalytically active form of that specific protein.

[0043] As used herein, the term "isolated" refers to a polypeptide or nucleic acid or its biologically active portion that is substantially or essentially free of components typically associated with or reacting with the protein or nucleic acid as found in its natural environment. Therefore, when isolated polypeptides or nucleic acids are produced using recombinant techniques, they are substantially free of other cellular material or culture media; or when isolated polypeptides or nucleic acids are chemically synthesized, they are substantially free of chemical precursors or other chemicals.

[0044] As used in this article, “expression vector” is a recombinant or synthetically produced nucleic acid construct that has a series of specific nucleic acid elements that allow specific nucleic acids to be transcribed in a host cell.

[0045] As used in this article, the term "host cell" refers to a cell that receives a foreign gene during transformation and transduction (infection). Host cells can be eukaryotic cells such as yeast cells or prokaryotic cells such as Escherichia coli. Detailed Implementation Plan

[0046] In a first aspect, this application provides a method for producing 2-acetylpyrrolline, comprising reacting 2-carboxypyrrolline with acetone aldehyde.

[0047] In some embodiments of the first aspect, 2-carboxypyrrololine spontaneously reacts with acetone aldehyde to generate 2-acetylpyrrololine.

[0048] In some embodiments of the first aspect, the method further includes contacting δ(1)-pyrrololine-2-carboxylic acid reductase with proline (e.g., L-proline) to generate 2-carboxypyrrololine; optionally, the method further includes regenerating NADP+ into NADPH.

[0049] In some embodiments of the first aspect, the method further includes contacting trans-3-hydroxy-L-proline dehydratase with trans-3-hydroxy-L-proline to generate 2-carboxypyrrololine.

[0050] In some embodiments of the first aspect, δ(1)-pyrrololine-2-carboxylic acid reductase comprises the amino acid sequence shown in SEQ ID NO:1 or a functional variant thereof.

[0051] In some embodiments of the first aspect, the functional variant is a natural isoenzyme of the amino acid sequence shown in SEQ ID NO:1.

[0052] In a second aspect, this application provides a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or a functional variant thereof, wherein the functional variant has δ(1)-pyrrololine-2-carboxylic acid reductase activity.

[0053] In some embodiments of the second aspect, the functional variant is a natural isoenzyme of the amino acid sequence shown in SEQ ID NO:1.

[0054] In some embodiments of the second aspect, the substrate of the polypeptide is proline, such as L-proline.

[0055] In some embodiments, the polypeptide is δ(1)-pyrrololine-2-carboxylic acid reductase.

[0056] In some embodiments, the amino acid sequence of the polypeptide is shown in SEQ ID NO:1.

[0057] In some embodiments of the first or second aspect, the functional variant is generated by the insertion, substitution, and / or deletion of one or more amino acids based on the amino acid sequence shown in SEQ ID NO:1 or its natural isoenzyme.

[0058] In some embodiments of the first or second aspect, the insertion, substitution, and / or deletion do not occur at the active site.

[0059] In some embodiments of the first or second aspect, the number of amino acid insertions, substitutions and / or deletions is 1-30, preferably 1-20, more preferably 1-10, wherein the resulting functional variant substantially retains the unchanged δ(1)-pyrrolline-2-carboxylic acid reductase activity.

[0060] In some embodiments of the first or second aspect, the functional variant differs from the amino acid sequence shown in SEQ ID NO:1 by the insertion, substitution, and / or deletion of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0061] In some embodiments of the first or second aspect, the functional variant differs from the amino acid sequence shown in SEQ ID NO:1 by the insertion, substitution, and / or deletion of about 1, 2, 3, 4, or 5 amino acids.

[0062] In some embodiments of the second aspect, the polypeptide is a separated polypeptide.

[0063] In some embodiments of the above aspects, the δ(1)-pyrrololine-2-carboxylic acid reductase is OsP2CR. In some embodiments, the amino acid sequence of the δ(1)-pyrrololine-2-carboxylic acid reductase is shown in SEQ ID NO:1.

[0064] In some embodiments of the above aspects, the δ(1)-pyrrololine-2-carboxylic acid reductase is OsP2CR, whose amino acid sequence is shown in SEQ ID NO:1.

[0065] Thirdly, this application provides a nucleic acid molecule that encodes the polypeptide described in the second aspect.

[0066] In some embodiments of the third aspect, the nucleic acid molecule of this application comprises a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence encoding the polypeptide shown in SEQ ID NO:1, or consists of a nucleic acid sequence that specifically hybridizes with the nucleotide sequence encoding the polypeptide shown in SEQ ID NO:1 and encodes a polypeptide that is functionally equivalent to the polypeptide shown in any one of SEQ ID NO:1.

[0067] Those skilled in the art can routinely select stringent conditions for DNA hybridization. Generally, longer probes require higher temperatures to allow for proper annealing, while shorter probes require lower temperatures. Hybridization typically depends on the re-annealing capability of denatured DNA when the complementary strand is exposed to an environment below its melting temperature. The higher the homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Thus, higher relative temperatures tend to result in more stringent reaction conditions, while lower temperatures result in less stringent conditions. For a detailed description of stringent conditions for hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

[0068] In some implementations of the third aspect, the stringent conditions used for DNA hybridization include: 1) washing with low ionic strength and high temperature, such as 0.015M sodium chloride / 0.0015M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; 2) using denaturing agents such as formamide during hybridization, such as 50% (v / v) formamide plus 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polydienepyrrolidone / 50mM sodium phosphate buffer at pH 6.5 at 42°C, and 750mM sodium chloride and 75mM sodium citrate; or (3) overnight hybridization at 42°C, with the hybridization solution containing 50% formamide, 5×SSC (0.75M sodium chloride, 0.075M sodium citrate), and 50mM sodium phosphate (pH 6.5). 6.8) 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 mg / mL), 0.1% SDS, and 10% dextran sulfate were added, followed by washing in 0.2× SSC (sodium chloride / sodium citrate) at 42°C for 10 min, and then high-toughness washing with 0.1× SSC containing EDTA at 55°C. Moderately tough conditions can be determined as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989. Moderately tough conditions include using wash solutions and hybridization conditions (such as temperature, ionic strength, and SDS percentage) with less toughness than described above. For example, moderately tough conditions include hybridization at 42°C with at least about 16% v / v to at least about 30% v / v formamide and at least about 0.5 M to at least about 0.9 M salt, and washing at 55°C with at least about 0.1 M to at least about 0.2 M salt. Moderately stringent conditions may also include hybridization at 65°C with 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), and 7% SDS, followed by washing with (i) 2×SSC and 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), and 5% SDS at 60–65°C. Experts will adjust the temperature, ionic strength, etc., according to factors such as probe length. The stringency of nucleic acid hybridization depends on the length and complementarity of the nucleic acid molecules, as well as other variables well known in the art. The greater the similarity or homology between two nucleotide sequences, the higher the Tm of the nucleic acid hybrid containing those sequences. The relative stability of nucleic acid hybridization (corresponding to a higher Tm) decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. Preferably, the minimum length of the hybridizable nucleic acid is at least about 12 nucleotides, more preferably at least about 16, more preferably at least about 24, and most preferably at least about 36 nucleotides.

[0069] The nucleic acid molecules of this application can be combined with other DNA sequences, such as promoters, polyadenylation signals, other restriction enzyme sites, multiple cloning sites, other coding segments, etc., such that their total length can vary significantly. Therefore, it is considered that almost any length of polynucleotide fragment can be used; the total length is preferably limited by the ease of preparation and use in the intended recombinant DNA scheme.

[0070] Polynucleotides and their fusions can be prepared, manipulated, and / or expressed using any of a variety of well-established techniques known and available in the art. For example, nucleic acid molecules encoding the polypeptides of this application or functional variants thereof can be used in recombinant DNA molecules to direct polypeptide expression in appropriate host cells. Due to the inherent degeneracy of the genetic codon, other DNA sequences encoding substantially the same or functionally equivalent amino acid sequences can also be used in this application, and these sequences can be used to clone and express a given polypeptide.

[0071] Furthermore, the nucleic acid molecules of this application can be modified using methods known in the art, including but not limited to the cloning, processing, expression, and / or alteration of the activity of gene products.

[0072] In some embodiments of the third aspect, the nucleic acid molecule is produced artificially, such as through direct chemical synthesis or enzyme synthesis.

[0073] In some implementations of the third aspect, the nucleic acid molecules are generated through recombination technology.

[0074] In some embodiments of the third aspect, the nucleic acid molecule is an isolated nucleic acid molecule.

[0075] Fourthly, this application provides an expression cassette containing the nucleic acid molecules described in the third aspect.

[0076] In some specific implementations of the fourth aspect, the expression box may additionally include a 5' leader sequence, which can enhance translation.

[0077] In preparing expression cassettes, various DNA fragments can be manipulated to provide DNA sequences with the appropriate orientation and reading frame. To achieve this, adapters or linkers can be used to ligate the DNA fragments, or other manipulations can be involved to provide convenient restriction sites, remove excess DNA, and so on. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, and substitution techniques such as transition and transversion can be employed.

[0078] Fifthly, this application provides an expression vector comprising the nucleic acid molecule described in the third aspect or the expression cassette described in the fourth aspect.

[0079] In some embodiments of the fifth aspect, any suitable expression vector may be used in this application; for example, the expression vector may be a vector suitable for the E. coli system of this application. In some embodiments, the expression vector may be any one of HT, pACYC, etc.

[0080] In some embodiments of the fifth aspect, a nucleic acid molecule encoding the polypeptide shown in SEQ ID NO:1 is cloned into a vector to form a recombinant vector containing the nucleic acid molecule described in this application.

[0081] In some embodiments of the fifth aspect, the expression vector used for cloning polynucleotides is a plasmid vector.

[0082] In some embodiments of the fifth aspect, the expression vector further comprises a regulatory sequence for regulating the expression of a nucleic acid molecule, wherein the nucleic acid molecule is operatively linked to the regulatory sequence.

[0083] As used herein, the term "regulatory sequence" refers to the polynucleotide sequence required to achieve the expression of the coding sequence linked to it. The nature of these regulatory sequences varies depending on the host organism. In prokaryotes, these regulatory sequences generally include promoters, ribosome binding sites, and terminators; in eukaryotes, they generally include promoters, terminators, and, in some cases, enhancers. Therefore, the term "regulatory sequence" includes all sequences whose presence is the minimum necessary for the expression of the target gene, and may also include other sequences, such as leader sequences, whose presence is beneficial for the expression of the target gene.

[0084] As used herein, the term "operably linked" refers to a situation where the sequences involved are in a relationship that allows them to function in a desired manner. Thus, for example, "operably linked" to a regulatory sequence of a coding sequence enables the expression of that coding sequence under conditions compatible with said regulatory sequence.

[0085] In some embodiments of the fifth aspect, an expression vector comprising a nucleotide sequence encoding the polypeptide shown in SEQ ID NO:1 and suitable transcription / translation regulatory elements is constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory, New York, 1989). The nucleotide sequence is operatively linked to a suitable promoter in the expression vector to direct mRNA synthesis. Representative examples of such promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of an enhancer sequence into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors of DNA expression, typically consisting of approximately 10 to 300 base pairs, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0086] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells, such as those encoding resistance to kanamycin sulfate, ampicillin, etc.

[0087] Sixthly, this application provides a host cell comprising the nucleic acid molecule described in the third aspect, the expression cassette described in the fourth aspect, or the expression vector described in the fifth aspect.

[0088] In some embodiments of the sixth aspect, the available host cell is a cell containing the above-described expression vector, which can be a eukaryotic cell, such as a yeast cell culture system used for the expression of the polypeptide of this application. The host cell can also be a prokaryotic cell containing the above-described expression vector, for example, selected from Escherichia coli (e.g., Escherichia coli), Klebsiella, Streptococcus, Lactobacillus, Bifidobacterium, Bacteroides, and Firmicutes.

[0089] In some specific implementations of the sixth aspect, the host cell is a yeast cell or Escherichia coli.

[0090] In some specific embodiments of the sixth aspect, nucleic acid molecules encoding one or more enzymes of this application may exist in host cells as free vectors or may be integrated into the genome of host cells.

[0091] In some embodiments of any of the foregoing aspects, the isolated nucleic acid is operatively linked to a regulatory sequence that can be recognized by host cells transformed with the expression vector.

[0092] Expression vectors can be introduced into host cells using any technique known in the art, including transformation, transduction, transfection, viral infection, gene gun, or Ti-mediated gene transfer. Specific methods include calcium phosphate transfection, DEAE-glucan-mediated transfection, lipid transfection, or electroporation (Davis, L., Dibner, M., Batty, I., Basic Methods in Molecular Biology, (1986)). As an example, when the host is a prokaryote such as *Escherichia coli*, competent cells can be harvested after the exponential growth phase and transformed using the CaCl2 method well known in the art.

[0093] In a seventh aspect, this application provides a method for generating 2-carboxypyrrololine, comprising contacting the polypeptide described in the second aspect with proline to generate 2-carboxypyrrololine.

[0094] In some implementations of the seven aspects, proline is L-proline.

[0095] The following embodiments are merely illustrative and are not intended to limit the scope of the embodiments of this application or the scope of the appended claims.

[0096] Example

[0097] Identification, characterization and testing of 2-acetylpyrrolidine (2AP)-related pathways

[0098] Materials and Methods

[0099] Experimental materials

[0100] Tryptone and yeast extract used to prepare LB medium were purchased from Oxoid Limited (Hampshire, UK). Ultrapure deionized water from Millipore Direct-Q was used. TALON resin was purchased from Clontech Laboratories Inc (California, USA). All protein purification chromatographic experiments were performed in [location missing]. Performed on a pure FPLC system (GE Healthcare, USA).

[0101] Gene synthesis

[0102] The BcP2CR (Uniprot accession number: Q81HB0), BcT3LD (Uniprot accession number: Q81HB1), and E. coli codon-optimized OsP2CR (Uniprot accession number: Q8LN35) gene fragments were synthesized by General Biotechnology (Anhui) Co., Ltd. The BcP2CR, BcT3LD, and OsP2CR gene fragments were inserted into the SspI site of the HT plasmid (optimized pET28 vector) to express a protein with an N-terminal His6 tag. The BcP2CR and OsP2CR genes encode δ(1)-pyrrolline-2-carboxylic acid reductase. The BcT3LD gene encodes trans-3-hydroxy-L-proline dehydratase.

[0103] Expression and purification of BcP2CR, BcT3LD and OsP2CR

[0104] Recombinant plasmids HT-BcP2CR, HT-BcT3LD, and HT-OsP2CR were transformed into E. coli BL21(DE3) competent cells for heterologous protein expression and purification. BcP2CR, BcT3LD, and OsP2CR were screened using LB agar plates containing 50 μg / mL kanamycin. A single colony was picked and inoculated into 20 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C and 220 rpm. 5 mL of the activated bacterial culture was then inoculated into 1 L of LB liquid medium and cultured on a shaker at 37°C and 220 rpm until the OD value was reached. 600nm When the pH reached approximately 0.8, isopropyl β-D-thiogalactoside (IPTG) was added to a final concentration of 0.3 mM to induce the expression of the target protein. After induction, the cells were cultured at 18°C ​​and 220 rpm for 16 h. After culture, the cells were centrifuged at 4°C and 5000 rpm for 15 min to collect the *E. coli* cells. The collected cells were resuspended in 35 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, 200 mM KCl, 1 mM benzyl sulfonyl fluoride (PMSF), 0.03% Triton X-100, 0.2 mg / mL lysozyme, 0.02 mg / mL DNase I) to prepare a cell suspension, which was then frozen at -80°C and lysed using a freeze-thaw method.

[0105] Thaw the frozen cells and incubate at room temperature (RT, 25℃) for 20 minutes, during which cell lysis will occur. Add 1% streptomycin sulfate to precipitate the DNA in the bacterial cells, centrifuge at 10,000 rpm for 15 minutes at 4℃, remove the cell pellet, filter the supernatant through a 0.22 μm nylon filter membrane, and load the supernatant into 5 mL of TALON Co2 pre-equilibrated with buffer A (20 mM Tris / HCl, pH 7.5, 200 mL M KCl) at 4℃. + The sample was loaded onto a column (Takara Bio USA, Inc.) and washed with 10 column volumes of buffer A to remove contaminating proteins, followed by 5 column volumes of buffer B (20 mM Tris / HCl, pH 7.5, 200 mM KCl, 150 mM imidazole, 5 mM MME) to elute the target protein. The eluted target protein (~12 mL) was placed in a dialysis bag, immersed in 2 L of dialysis buffer, and dialyzed at 4°C for 3 h to remove imidazole. The target protein was collected, mixed thoroughly with 10% glycerol, aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C.

[0106] The purified proteins were detected by SDS-PAGE using commercial gels (SurePAGE, Bis-Tris, 4-20%). The absorbance of the proteins at 280 nm was measured using an ultra-micro UV-Vis spectrophotometer (Hangzhou Mio Instruments Co., Ltd.), and their concentrations were calculated. [BcP2CR(ε 280 =14900M -1 cm -1 ), BcT3LD(ε 280 =30370M -1 cm -1 ) and OsP2CR(ε 280 =18450M -1 cm -1 )).

[0107] Spectrophotometric determination of the activities of δ(1)-pyrrolline-2-carboxylic acid reductase BcP2CR and OsP2CR

[0108] 200 μL of a solution containing 100 mM CAPS, pH 10.8, 100 mM KCl, 50 mM L-proline, 20 μM MB cP2CR, and 3 mM NADP was added. + The reaction mixture was monitored for absorbance at 340 nm every 10 seconds using a microplate reader. A negative control was prepared without the addition of L-proline or BcP2CR to verify the catalytic activity of BcP2CR for L-proline.

[0109] 200 μL of a solution containing 100 mM CAPS, pH 10.0, 100 mM KCl, 50 mM L-proline, 5 μM OsP2CR, and 3 mM NADP was added. + The reaction mixture was monitored for absorbance at 340 nm every 10 seconds using a microplate reader. A negative control was prepared without the addition of L-proline or OsP2CR to verify the catalytic activity of OsP2CR for L-proline.

[0110] OsP2CR and BcT3LD coupled spectrophotometric determination of trans-3-hydroxy-L-proline dehydratase BcT3LD activity

[0111] 200 μL of a reaction mixture containing 50 mM Tris / HCl, pH 8.0, 100 mM KCl, 20 mM trans-3-hydroxy-L-proline (t3L-HP), 0.5 μM BcT3LD, 5 μM OsP2CR, and 0.4 mM NADPH was used, and the absorbance at 340 nm was monitored every 10 seconds using a microplate reader. A negative control was prepared without the addition of t3L-HP, BcT3LD, or OsP2CR to verify the catalytic activity of BcT3LD against t3L-HP.

[0112] Michaelis-Menten kinetic parameters of BcP2CR, OsP2CR and BcT3LD were determined.

[0113] 200 μL of a solution containing 100 mM CAPS, pH 10.0, 100 mM KCl, L-proline at different substrate concentrations, 10 μM MB cP2CR, and 3 mM NADP was prepared. + The reaction mixture was monitored every 10 seconds in an ELISA reader to measure the increase in absorbance at 340 nm, and the kinetic parameters of BcP2CR were determined.

[0114] 200 μL of a solution containing 100 mM CAPS, pH 10.5, 100 mM KCl, L-proline at different substrate concentrations, 5 μM sP2CR, and 3 mM NADP was prepared. + The reaction mixture was monitored every 10 seconds in an ELISA reader to measure the increase in absorbance at 340 nm, and the kinetic parameters of OsP2CR were determined.

[0115] 200 μL of a reaction mixture containing 50 mM Tris / HCl, pH 8.0, 100 mM KCl, different substrate concentrations of t3L-HP, 0.1 μM BcT3LD, 5 μM OsP2CR, and 0.4 mM NADPH was used. The decrease in absorbance at 340 nm was monitored every 10 seconds in an ELISA reader to determine the kinetic parameters of BcT3LD.

[0116] LC-MS / MS analysis of BcP2CR-catalyzed P2C production

[0117] Detection of products from the BcP2CR-catalyzed L-proline reaction: 200 μL reaction system, 100 mM CAPS (pH 10.0), 100 mM KCl buffer solution, 100 mM L-proline, 10 μM BcP2CR, 5 mM NADP. + The increase in NADPH absorbance at 340 nm was detected using a microplate reader. Once the absorbance stopped increasing, a 10 kDa concentrator was used to centrifuge at 10,000 rpm for 2 min to remove proteins from the reaction system, terminating the reaction. The filtrate was transferred to a liquid chromatography tube and analyzed by LC-MS / MS in MRM mode. LC-MS / MS was performed on an Agilent 6420 triple quadrupole LC-MS instrument. The dry gas temperature was maintained at 300 °C, the flow rate was 9 L / min, the nebulizer pressure was 15 psi, and the separation column was an amino column (5 mm). 150×4.6mm; Merck). HPLC detection conditions: Initially, mobile phase B was 90%, gradually decreasing to 65% at 10 min, and further decreasing to 50% at 30 min. Mobile phase B was acetonitrile, and mobile phase A was 90% 20mM ammonium acetate and 10% acetonitrile. The flow rate was 0.5 mL / min. Mass spectrometry conditions were MRM cation mode. The m / z of P2C was 113.1, and its ion fragment m / z was 67.1, meaning that in P2C cation mode, the parent ion m / z was 114.1, and the daughter ion m / z was 68.1. The sample loading volume was 20 μL.

[0118] LC-MS / MS analysis of OsP2CR-catalyzed P2C production

[0119] Detection of products from the OsP2CR-catalyzed L-proline reaction: 200 μL reaction system, 100 mM CAPS (pH 10.5), 100 mM KCl buffer solution, 200 mM L-proline, 5 μM OsP2CR, 5 mM NADP. + The increase in NADPH absorbance at 340 nm was detected using a microplate reader. Once the absorbance stopped increasing, 200 μL of acetonitrile solution was added to terminate the reaction. The mixture was centrifuged at 10,000 rpm for 2 min, filtered through a 0.22 mm nylon filter to remove proteins, and then transferred to a liquid chromatography tube for LC-MS / MS detection. The detection method was the same as that used for the LC-MS / MS detection of P2C catalyzed by BcP2CR.

[0120] LC-MS analysis of P2C production catalyzed by BcT3LD

[0121] To detect the product of the BcT3LD-catalyzed t3L-HP reaction, a 200 μL reaction system was prepared, with the total reaction mixture consisting of 50 mM PB (pH 7.0), 100 mM KCl, 20 mM t3L-HP, and 10 μM BcT3LD. Two negative control reactions were set up: one without BcT3LD enzyme and the other without substrate t3L-HP. The reaction was carried out at 30℃ in the dark for 30 min. After the reaction was completed, an equal volume of methanol was added to terminate the reaction. After centrifugation at 10000 rpm for 2 min, the sample was filtered through a 0.22 mm nylon filter to remove protein precipitate. The sample was then filtered into a liquid chromatography tube for LC-MS detection. The chromatogram was performed using a C18 column (5 mm). 150×4.6 mm; Merck) to separate the products generated by BcT3LD catalysis of t3L-HP. Mobile phase A consisted of water and 0.1% formic acid, and mobile phase B consisted of methanol and 0.1% formic acid. During HPLC separation, the injection volume was 20 μL, the mobile phase flow rate was 0.5 mL / min, and the maximum column pressure was 400 bar. Mobile phase B gradually increased from 5% initially, reaching 55% at 10 min, continuing to increase to 95% at 15 min, and rapidly decreasing to 5% at 17 min, followed by isometric elution with 5% mobile phase B until 18 min. After separation of each sample, the column was washed with 95% mobile phase B and 5% mobile phase B for 2 min.

[0122] MS conditions were as follows: initial molecular weight 50, final molecular weight 500, scan time 120 seconds, fragmenter values ​​of 50V and 135V. Both anionic and cationic modes were used. The drying gas temperature was 300℃, flow rate 9L / min, and nebulizer pressure 15psi.

[0123] LC-MS analysis of OsP2CR-coupled BcT3LD-catalyzed t3L-HP products

[0124] The reaction product of BcT3LD catalyzing t3L-HP and the reaction product of OsP2CR coupled enzyme were detected. A 200 μL reaction system was prepared, containing 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 20 mM t3L-HP, 10 μM BcT3LD, 5 μM OsP2CR, and 0.4 mM NADPH as reactants. Three negative controls were set up, with BcT3LD, OsP2CR, and the substrate t3L-HP omitted, while other reactants and concentrations remained unchanged. The reaction termination time was determined by the change in NADPH absorbance at 340 nm using a microplate reader. After the absorbance stopped decreasing, an equal volume of acetonitrile was added to terminate the reaction. After centrifugation at 10000 rpm for 2 min, the protein in the system was removed using a sterile syringe through a 0.22 mm nylon filter membrane. The filtered solution was transferred to a liquid chromatography tube for LC-MS detection. The chromatography column was 5 mm thick. A 150×4.6mm (Merck) column was used to separate the intermediate and final products generated by BcT3LD-OsP2CR coupling enzyme catalyzed by t3L-HP. Mobile phase A consisted of 90% 20mM ammonium acetate solution and 10% acetonitrile, while mobile phase B consisted of 100% acetonitrile. During HPLC separation, the injection volume was 20 μL, the mobile phase flow rate was 0.5 mL / min, and the column pressure was up to 400 bar. The concentration of mobile phase B was gradually reduced from 90% initially, to 70% at 10 min, and to 50% at 30 min. After each sample analysis, the column was flushed with 50% of both mobile phase A and mobile phase B for 5 min. LC-MS was performed on an Agilent 6420 triple quadrupole LC / MS instrument (Agilent Technologies). The dry gas temperature was maintained at 300 °C, the flow rate was 9 L / min, and the nebulizer pressure was 15 psi. A ZIC-HILIC column was used. Mass spectrometry was performed in ESI cation mode. Meanwhile, using 0.25 mM L-proline as a standard for the final product, the formation of the t3L-HP product catalyzed by OsP2CR coupled with BcT3LD was verified by mass spectrometry.

[0125] LC-MS analysis of the reaction products of P2C and acetone aldehyde

[0126] The t3L-HP to 2AP synthesis pathway was investigated. BcT3LD catalyzes the t3L-HP reaction, and its product spontaneously reacts with acetone aldehyde to generate the final product. The 200 μL reaction system consisted of 50 mM PB (pH 7.0), 100 mM KCl, 20 mM t3L-HP, 10 μM BcT3LD, and 25 mM acetone aldehyde. Three negative control reactions were set up: no BcT3LD enzyme, no t3L-HP substrate, and no acetone aldehyde. Because excess acetone aldehyde would inactivate the protein, the reaction was performed in steps. In the first step, all reactants except acetone aldehyde were added, and the reaction was carried out at 30°C in the dark for 30 min. Then, 25 mM acetone aldehyde was added, and the reaction was allowed to proceed overnight. After the reaction was complete, an equal volume of methanol was added to terminate the reaction. After centrifugation at 10,000 rpm for 2 min, the sample was filtered through a 0.22 mm nylon filter to remove protein precipitate. The filtered sample was then transferred to a liquid chromatography tube for LC-MS analysis.

[0127] The final product of the full reaction group was pale yellow, while the other three negative control reaction systems did not produce any pale yellow substance. Before the reaction was terminated, the ultraviolet spectra of the four reactants were measured to determine the ultraviolet absorbance of the products.

[0128] Chromatographic column (5 mm) was used. (150×4.6 mm; Merck) The intermediate product generated by BcT3LD catalysis of t3L-HP and its final product from the reaction with acetone aldehyde were separated. Mobile phase A consisted of water and 0.1% formic acid, and mobile phase B consisted of methanol and 0.1% formic acid. During HPLC separation, the injection volume was 20 μL, the mobile phase flow rate was 0.5 mL / min, and the maximum column pressure was 400 bar. Mobile phase B gradually increased from 5% initially, reaching 55% at 10 min, continuing to increase to 95% at 15 min, and rapidly decreasing to 5% at 17 min, followed by isostatic elution with 5% mobile phase B until 18 min. After separation of each sample, the column was washed with 95% mobile phase B and 5% mobile phase B for 2 min.

[0129] MS conditions were as follows: initial molecular weight 50, final molecular weight 500, scan time 120 seconds, fragmenter values ​​of 50V and 135V. Both anionic and cationic modes were used. The drying gas temperature was 300℃, flow rate 9L / min, and nebulizer pressure 15psi.

[0130] Results, Analysis and Discussion

[0131] Bioinformatics analysis of P2CR sequences in the UniProt database revealed a dehydratase gene cluster adjacent to the P2CR gene cluster in Bacillus cereus. This dehydratase (T3LD) catalyzes the production of 2-carboxypyrrolidine from trans-3-hydroxy-L-proline. Figure 1 Meanwhile, we noticed an ornithine cyclization deaminase superfamily enzyme (OsP2CR) in the rice genome with unknown function, which is homologous to P2CR in Bacillus cereus. Based on molecular docking results, we speculated that its substrate is L-proline and proposed a reaction pathway. In this pathway, L-proline is oxidized by P2CR to generate 2-carboxypyrrololine. This product further reacts with acetone aldehyde to produce 2-acetylpyrrololine (…). Figure 2 ).

[0132] To prove our hypothesis, we selected the P2CR (BcP2CR) and T3LD (BcT3LD) genes from Bacillus cereus and the P2CR (OsP2CR) gene from rice (Oryza sativa), and then expressed them heterologously in Escherichia coli BL21 (DE3) cells, and characterized the activity of these proteins.

[0133] In the spectrophotometric determination of BcP2CR or OsP2CR activity, a significant increase in A340nm was observed only in the total reaction group, indicating that the substrate L-proline underwent an oxidation reaction catalyzed by BcP2CR or OsP2CR. Figure 4 A, 4B), combined with the LC-MS / MS results, a new peak with m / z = 114.1 -> 68.1 can be seen in the whole reaction group (A, 4B). Figure 8 A, 8B), demonstrating that L-proline undergoes oxidation catalyzed by BcP2CR or OsP2CR to produce 2-carboxypyrrolidine. The Michaelis-Menten kinetic parameters of BcP2CR and OsP2CR were also determined. Figure 5 The kinetic parameters for BcP2CR enzyme are kcat = 0.86 ± 0.01 min⁻¹ and Km = 7.72 ± 0.43 mM. Figure 6 The kinetic parameters for OsP2CR enzyme are kcat = 4.60 ± 0.05 min⁻¹ and Km = 24.66 ± 1.08 mM.

[0134] In the OsP2CR-coupled enzyme assay for BcT3LD activity, a significant decrease in A340 nm was observed only in the whole reaction group, indicating that the substrate t3L-HP underwent a dehydration reaction catalyzed by BcT3LD, resulting in the cleavage of the CO bond and the production of 2-carboxypyrrolidine. Combined with LC-MS results, a new peak with m / z(+) = 116 was observed in the whole reaction group at tR = 16.6 min (…). Figure 9 A, 9B), demonstrating that t3L-HP reacts via coupling catalysis of BcT3LD and OsP2CR to produce L-proline. The Michaelis-Menten kinetic parameters of BcT3LD were also determined (…). Figure 7 The kinetic parameters for BcT3LD enzyme are kcat = 8.60 ± 0.51 s⁻¹ and Km = 9.73 ± 1.60 mM.

[0135] The 2-carboxypyrrolidine produced by BcT3LD catalysis from t3L-HP was reacted with acetone aldehyde, and the reaction was analyzed by LC-MS. A new peak with m / z(-) = 112.0 was observed at tR = 5.6 min in both the full reaction group and the control group without acetone aldehyde. Figure 10 A, 10B). In the total reaction group, there is a new peak with m / z(+) = 112.0 at tR = 15.3 min ( Figure 10 (C, 10D). This demonstrates that t3L-HP generates P2C via BcT3LD catalysis, and P2C spontaneously reacts with acetone aldehyde to generate 2-acetylpyrrolline.

Claims

1. A method for producing 2-acetylpyrrolline, comprising reacting 2-carboxypyrrolline with acetone aldehyde.

2. The method of claim 1, further comprising contacting δ(1)-pyrrololine-2-carboxylic acid reductase with proline to generate 2-carboxypyrrololine; preferably, the proline is L-proline; Optionally, the method further includes NADP + It is regenerated into NADPH.

3. The method of claim 1, further comprising contacting trans-3-hydroxy-L-proline dehydratase with trans-3-hydroxy-L-proline to generate 2-carboxypyrrololine.

4. The method of claim 2, wherein δ(1)-pyrrololine-2-carboxylic acid reductase comprises the amino acid sequence shown in SEQ ID NO:1 or a functional variant thereof.

5. A polypeptide comprising the amino acid sequence shown in SEQ ID NO:1 or a functional variant thereof, wherein the functional variant has δ(1)-pyrrolline-2-carboxylic acid reductase activity.

6. The polypeptide of claim 5, wherein the substrate is proline; preferably, the proline is L-proline.

7. A nucleic acid molecule encoding the polypeptide as described in claim 5 or 6.

8. An expression cassette comprising the nucleic acid molecule of claim 7.

9. An expression vector comprising the nucleic acid molecule of claim 7 or the expression cassette of claim 8.

10. A host cell comprising the nucleic acid molecule of claim 7, the expression cassette of claim 8, or the expression vector of claim 9; preferably, the host cell is a eukaryotic cell or a prokaryotic cell.

11. A method for producing 2-carboxypyrrololine, comprising contacting the polypeptide of claim 5 or 6 with proline to produce 2-carboxypyrrololine; preferably, the proline is L-proline.

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