A key enzyme in the spermidine biosynthesis pathway

Abstract

The present invention discloses a novel biosynthetic pathway for spermidine, in which carboxyaminopropylagmatine dehydrogenase (SEQ ID NO: 2) catalyzes a reductive condensation reaction of agmatine and aspartate semialdehyde to produce a novel compound, (S)-2-amino-4-((4-guanidine)amino)butyric acid. The present invention provides a new strategy for the biosynthesis of spermidine, spermidine intermediates, and other polyamine derivatives, and has potential for research, development, and application.

Description

Technical Field

[0001] The present invention belongs to the field of biosynthesis, and in particular relates to a key enzyme in the spermidine biosynthesis pathway, carboxyaminopropylagmatine dehydrogenase (CAPADH), and its use in preparing spermidine and spermidine intermediates. Background Art

[0002] Polyamines are molecular compounds containing two or more amino groups. They are present in almost all bacteria, archaea, and eukaryotic cells and play an important role in a variety of cellular processes, including gene regulation, cell proliferation and differentiation, and adaptation to various stresses. Spermidine, as the most common triamine compound in organisms, is directly involved in the modification of the eukaryotic translation factor eIF5a to support normal protein translation and is essential for eukaryotic organisms. Spermidine also has important physiological functions in bacteria, including maintaining transcription and translation, maintaining growth, and regulating the synthesis of biofilms. In terms of human health, spermidine, as a natural polyamine, has significant cardioprotective and neuroprotective effects, as well as certain anti-inflammatory and anti-aging properties. Because polyamines are closely related to clinical and agricultural applications, polyamines and polyamine derivatives with complex and diverse structures can be used as candidate drugs, agricultural chemicals, functional foods, etc., and have received widespread attention.

[0003] Currently, there are two main known spermidine biosynthetic pathways, both of which essentially involve the aminopropylation of putrescine to form spermidine (see CN112111536A and CN113736719A). One is the aminopropylation reaction, catalyzed by the enzyme spermidine synthase, which transfers decarboxylated S-adenosylmethionine to the putrescine backbone to form spermidine. The other is the reaction of putrescine with aspartic acid semialdehyde, catalyzed by carboxyspermidine dehydrogenase, to form carboxyspermidine, which is then decarboxylated to form spermidine by carboxyspermidine decarboxylase. However, many bacteria lack spermidine synthase. Although these bacteria can synthesize spermidine, the specific biosynthetic pathway remains unknown.

[0004] Deciphering the bacterial polyamine biosynthetic pathway and its key enzymes is crucial for understanding and artificially regulating the physiological functions of polyamines in bacteria, providing new strategies and tools for the efficient synthesis of polyamines. New polyamine intermediates and their derivatives discovered in these synthetic pathways can also be incorporated into polyamine compound libraries for subsequent molecular functional studies and clinical or industrial applications. Summary of the Invention

[0005] This study successfully deciphered a novel spermidine biosynthetic pathway in cyanobacteria and functionally characterized a key enzyme in the pathway: carboxyaminopropylagmatine dehydrogenase (CAPADH), encoded by the gene CAPADH. CAPADH catalyzes the reductive condensation reaction of agmatine and aspartate semialdehyde in the presence of the coenzymes NADPH or NADH. During this decipherment, a novel intermediate product, (S)-2-amino-4-((4-guanidinobutyl)amino)butanoic acid, was discovered and named carboxyaminopropylagmatine (CAPA), and its structure was identified. The enzyme CAPADH and compound CAPA identified by our research group can be used in the artificial synthesis of spermidine, spermidine intermediates, and other polyamine derivatives. These findings lay the foundation for the present invention.

[0006] Therefore, the first aspect of the present invention provides a compound shown in Formula III, which is (S)-2-amino-4-((4-guanidine)amino)butyric acid, and is named carboxyaminopropylagmatine (CAPA):

[0007]

[0008] A second aspect of the present invention provides a method for synthesizing the above-mentioned compound, comprising the following steps: using agmatine represented by Formula I and aspartic acid semialdehyde represented by Formula II as substrate raw materials, and preparing Compound III by a reduction condensation reaction under enzyme catalysis:

[0009]

[0010] Based on this function, the enzyme was named Carboxyaminopropylagmatine Dehydrogenase (CAPADH).

[0011] Preferably, the enzyme is a polypeptide selected from the group consisting of:

[0012] (a) a polypeptide having an amino acid sequence of SEQ ID NO: 2;

[0013] (b) a polypeptide derived from (a) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 and having the functions of the polypeptide of (a);

[0014] (c) a polypeptide derived from (a) having a homology of 45% or more, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, preferably 80% or more, preferably 85% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, more preferably 99% or more with the polypeptide sequence defined in (a), and having the function of the polypeptide of (a); or

[0015] (d) A derivative polypeptide having a sequence containing the polypeptide sequence described in (a) or (b) or (c).

[0016] MAKVMIVGAGGVGSVVAHKCAALEDFTDILLASRTVAKCDQIAAHIGSPKVKTAALDAFQVSDTVKLLQDFGADLLINVALPYQDLVLMDACLEAGVDYL DTANYEPPDVAKFEYSWQWAYQDKFKDAGLMALLGCGFDPGVTGVFTAYALKHHFDEIHYLDIVDCNAGNHGQAFATNFNPEINIREITQKGRYHEDGVWQ EIDPLSVHRDINYPHIGDRPSYLLYHEELESLVKNIPTLKRARFWMTFSEAYINHLRVLEAVGMTRIDEVEYQGQKIVPLQFLKAVLPEPASLAENYSGQ TSIGCYIKGVKDGQAKTYYIYNNCDHAVCFAEVGSQAISYTTGVPAALGGLMMVQGKWKQAGVFNVEEMDPDPFLAKLGEMGLPWHEVVNGPFPFDD(SEQ ID NO:2).

[0017] In the reaction, the carboxyaminopropylagmatine dehydrogenase can be in the form of an enzyme or in the form of a microbial cell expressing the same.

[0018] In one embodiment, coenzyme NADPH (β-nicotinamide adenine dinucleoside phosphate, coenzyme II) or NADH (β-nicotinamide adenine dinucleotide, i.e., coenzyme I) may be added to the reaction system to provide electrons for the enzymatic reaction of CAPADH.

[0019] The third aspect of the present invention provides a spermidine biosynthetic pathway, which comprises the above-mentioned carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase (CAPADC) and aminopropylagmatine urea hydrolase (APAUH).

[0020] The above-mentioned spermidine biosynthesis pathway is that agmatine (Formula I) and aspartate semialdehyde (Formula II) are condensed to form carboxyaminopropylagmatine (CAPA) (Formula III) via carboxyaminopropylagmatine dehydrogenase (CAPADH); carboxyaminopropylagmatine (CAPA) is catalyzed to form aminopropylagmatine (APA) via carboxyaminopropylagmatine decarboxylase (CAPADC); aminopropylagmatine (APA) is catalyzed to form spermidine via aminopropylagmatine urea hydrolase (APAUH), as shown in FIG. Figure 3 As shown.

[0021] The above-mentioned spermidine biosynthetic pathway can be used for in vitro multi-stage enzyme cascade reactions or genetically engineered bacterial fermentation to produce spermidine, spermidine intermediates, and other polyamine derivatives. For example, the enzymes carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase, and aminopropylagmatine urea hydrolase can be used in combination to catalyze the synthesis of spermidine, spermidine intermediates, and other polyamine derivatives using agmatine and aspartate semialdehyde as substrates. Alternatively, genetically engineered Escherichia coli can be constructed to co-express carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase, and aminopropylagmatine urea hydrolase to fermentatively produce spermidine, spermidine intermediates, and other polyamine derivatives.

[0022] A fourth aspect of the present invention provides the use of the above-mentioned carboxyaminopropylagmatine dehydrogenase or the above-mentioned spermidine biosynthesis pathway in constructing spermidine-producing bacteria.

[0023] In a specific embodiment, the coding genes of the above-mentioned carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase and aminopropylagmatine urea hydrolase are cloned into spermidine-producing bacteria, that is, the spermidine biosynthetic pathway is constructed in spermidine-producing bacteria to form spermidine engineered bacteria.

[0024] The present invention also provides a spermidine-producing bacterium, which comprises the above-mentioned spermidine biosynthesis pathway.

[0025] Among them, the synthesis or production of carboxyaminopropylagmatine (CAPA) can be used as a landmark event of the above-mentioned spermidine engineering bacteria.

[0026] Another aspect of the present invention provides a gene encoding the above-mentioned carboxyaminopropylagmatine dehydrogenase, which is also referred to as CAPADH.

[0027] Preferably, the gene encoding the polypeptide having the amino acid sequence of SEQ ID NO: 2 may be a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1.

[0028] Accordingly, another aspect of the present invention provides a vector comprising the above polynucleotide, and a microorganism transformed with the vector, for expressing the above carboxyaminopropylagmatine dehydrogenase (CAPADH).

[0029] The above-mentioned vector can be a pET series plasmid such as pET22b, pET24a, pET28a, or other vectors such as pSH plasmid and pRSFDuet plasmid.

[0030] The microorganism can be selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, and Bacillus subtilis, preferably Escherichia coli BL21 (DE3).

[0031] This paper analyzes for the first time a new spermidine biosynthetic pathway in cyanobacteria, discovers a key enzyme in this pathway, namely carboxyaminopropylagmatine dehydrogenase (CAPADH), and identifies its function; it also identifies the hallmark compound carboxyaminopropylagmatine (CAPA) in this pathway, which greatly facilitates the design of process strategies for the biosynthesis or enzymatic preparation of spermidine and its intermediates, and is worthy of further in-depth study and evolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Figure 1 shows how polyamine concentrations rapidly fluctuate in response to external nutrient conditions. Figure A shows the changes in the concentrations of metabolites within Synechocystis during the rapid recovery of external nutrients, while Figure B shows high-resolution mass spectrometry analysis of two polyamine metabolites within Synechocystis, which rapidly increase in concentration upon nitrogen stimulation.

[0033] Figure 2 is the nuclear magnetic resonance spectrum of carboxyaminopropylagmatine. Wherein, A is the nuclear magnetic resonance hydrogen spectrum ( 1 H-NMR, 400 MHz, in D2O), B is the carbon nuclear magnetic resonance spectrum ( 13 C-NMR, 100 MHz, in D2O), C is the two-dimensional hydrogen nuclear magnetic resonance spectrum ( 1 H- 1 H), D is the NMR HSQC spectrum, and E is the NMR HMBC spectrum.

[0034] Figure 3The spermidine synthesis pathway mediated by CAPADH discovered in the present invention is shown.

[0035] Figure 4 Stable isotope tracing was used to detect the labeling of intermediate metabolites in the CAPA pathway.

[0036] Figure 4 A shows the carbon and nitrogen fully labeled arginine ([U- 13 C,U- 15 Marking of intermediate metabolites during tracing with N]arginine

[0037] Figure 4 B shows the fully carbon-labeled asparagine ([U- 13 C]asparagine) tracing intermediate metabolite markers

[0038] Figure 5 The concentrations of polyamine metabolites in CAPA pathway mutants and complementing strains are shown. A represents the concentration of polyamine metabolites in CAPA pathway mutants, and B represents the concentration of polyamine metabolites in CAPA pathway mutants and complementing strains analyzed by high-resolution mass spectrometry.

[0039] Figure 6 The in vitro enzymatic reaction of CAPADH is shown. A shows a photograph of purified CAPADH detected by SDS-PAGE, B shows HPLC analysis of the products of the in vitro enzymatic reaction of CAPADH, C shows high-resolution mass spectrometry analysis of the products of the in vitro enzymatic reaction of CAPADH, and D shows a high-resolution mass spectrometry analysis comparing the products of the in vitro enzymatic reaction of intracellular CAPA with those of CAPADH.

[0040] Figure 7 This figure shows the construction of the CAPA pathway in E. coli to synthesize spermidine and other polyamine derivatives. Figure A shows the strategy for constructing the CAPA pathway in E. coli, and Figure B shows high-resolution mass spectrometry analysis of the fermentation products of the engineered E. coli strain. DETAILED DESCRIPTION

[0041] Spermidine is a polyamine that maintains important physiological functions of bacteria. However, many bacteria lack the traditional pathway for synthesizing spermidine from S-adenosylmethionine via spermidine synthase, and how they synthesize spermidine remains to be elucidated. 13 C and 15Using nitrogen tracing experiments combined with metabolomics, genetic manipulation, and biochemical characterization, a spermidine biosynthesis pathway mediated by the carboxyaminopropylagmatine dehydrogenase gene (CAPADH) was discovered in the model cyanobacterium Synechocystis sp. PCC 6803. This pathway uses agmatine and aspartate semialdehyde as substrates. CAPADH, encoded by the enzyme carboxyaminopropylagmatine dehydrogenase (CAPADH), catalyzes the production of a novel intermediate, carboxyaminopropylagmatine (CAPA, (S)-2-amino-4-((4-guanidine)amino)butyric acid), under the action of NADPH or NADH. CAPA then generates aminopropylagmatine (APA) via carboxyaminopropylagmatine decarboxylase (CAPADC). Finally, APA is catalyzed by aminopropylagmatine ureahydrolase (APAUH) to produce spermidine. At the same time, when the cyanobacterium Synechocystis sp. PCC 6803 was switched from nutrient-limited to nutrient-rich culture conditions, the inventors also detected a new intermediate compound, carboxyaminopropyl agmatine (CAPA), which accumulated in large quantities in the bacteria.

[0042] For simplicity of description, the term "carboxyaminopropylagmatine dehydrogenase" (CAPADH) is sometimes used interchangeably with the name of the gene encoding it. Those skilled in the art will understand that these refer to different substances in different contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing the function or class of carboxyaminopropylagmatine dehydrogenase, CAPADH refers to the protein; when describing a gene, it refers to the gene encoding the enzyme.

[0043] Based on the above findings, the present invention discloses a novel bacterial enzyme involved in the spermidine biosynthesis pathway: carboxyaminopropylagmatine dehydrogenase, which catalyzes the condensation of agmatine with aspartate semialdehyde to produce carboxyaminopropylagmatine. Preferably, the carboxyaminopropylagmatine dehydrogenase has the amino acid sequence set forth in SEQ ID NO: 2.

[0044] The CAPADH active polypeptides described herein, whose amino acid sequence is shown in SEQ ID NO:2, can be recombinant, natural, or synthetic polypeptides. The polypeptides described herein can be purified from natural sources, chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, or higher plants). Depending on the host used in the recombinant production protocol, the polypeptides described herein can be glycosylated or non-glycosylated. The polypeptides described herein may or may not include an initial methionine residue.

[0045] It should be understood that one of the objects of the present invention is to provide an enzyme having the function of catalyzing the reductive condensation of agmatine and aspartic acid semialdehyde to form carboxyaminopropylagmatine, which includes but is not limited to mutants of the polypeptide having an amino acid sequence as shown in SEQ ID NO: 2. As long as it has a high homology with the amino acid sequence of SEQ ID NO: 2, for example, more than 45% homology, and has the function of catalyzing the reduction of the substrates agmatine and aspartic acid semialdehyde to carboxyaminopropylagmatine, it preferably has higher catalytic activity.

[0046] The terms "(catalytic activity) higher", "increase" or "enhance" can mean an increase of at least 10% compared to a reference level (such as wild-type CAPADH), for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase compared to a reference level, or any increase between 10% and 100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold compared to a reference level.

[0047] Amino acid mutations include substitutions, deletions, or additions. Amino acid substitutions include conservative and non-conservative substitutions. "Conservative substitutions" refer to the interchangeability of residues with similar side chains and therefore generally include replacing an amino acid in a polypeptide with an amino acid from the same or similar amino acid-defined class. For example, but not limited to, an amino acid with an aliphatic side chain can be substituted with another aliphatic amino acid, such as alanine, valine, leucine, and isoleucine; an amino acid with a hydroxyl side chain can be substituted with another amino acid with a hydroxyl side chain, such as serine and threonine; an amino acid with an aromatic side chain can be substituted with another amino acid with an aromatic side chain, such as phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain can be substituted with another amino acid with a basic side chain, such as lysine and arginine; an amino acid with an acidic side chain can be substituted with another amino acid with an acidic side chain, such as aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid can be substituted with another hydrophobic or hydrophilic amino acid, respectively. "Non-conservative substitutions" refer to replacing an amino acid in a polypeptide with an amino acid with significantly different side chain properties. Non-conservative substitutions can utilize amino acids between defined groups rather than within them and affect: (a) the structure of the peptide backbone in the region of the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain bulk. For example, but not limited to, exemplary non-conservative substitutions can be substitutions of acidic amino acids with basic or aliphatic amino acids; substitutions of aromatic amino acids with small amino acids; and substitutions of hydrophilic amino acids with hydrophobic amino acids.

[0048] These mutations include, but are not limited to, deletions, insertions, and / or substitutions of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and additions or deletions of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For another example, substitutions with amino acids having similar or similar properties generally do not alter the function of the protein in the art. The present invention also provides analogs of the polypeptides. These analogs may differ from the native polypeptide in terms of amino acid sequence, modifications that do not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biology techniques. Analogs also include analogs with residues other than natural L-amino acids (e.g., D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0049] The amino or carboxyl terminus of the CAPADH polypeptide with the amino acid sequence shown in SEQ ID NO:2 may also contain one or more polypeptide fragments as protein tags. These tags can be used to purify the protein. To ensure secretory expression of the translated protein (e.g., extracellular secretion), a signal peptide sequence may be added to the amino terminus of the CAPADH polypeptide. The signal peptide may be cleaved during the process of secretion of the polypeptide from the cell.

[0050] The polynucleotide encoding the CAPADH polypeptide can be in the form of DNA or RNA. The polynucleotide encoding the mature CAPADH polypeptide includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0051] The full-length nucleotide sequence encoding CAPADH or fragments thereof can generally be obtained using PCR amplification, recombinant methods, or synthetic methods. Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the propagated host cells using conventional methods. In addition, synthetic methods can also be used to synthesize the relevant sequence, especially for shorter fragments. Mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.

[0052] The present invention also relates to a vector comprising a CAPADH polynucleotide, a host cell produced by genetic engineering using the vector of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0053] The CAPADH polynucleotide sequence can be inserted into a recombinant expression vector. Methods well known to those skilled in the art can be used to construct an expression vector containing the CAPADH encoding DNA sequence and appropriate transcription / translation control signals.

[0054] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0055] In order to enable the carboxyaminopropylagmatine dehydrogenase CAPADH to be widely used in the enzymatic preparation of carboxyaminopropylagmatine or other compounds, expressing the polypeptide through microorganisms is the best method for preparing the enzyme.

[0056] Since the amino acid sequence of CAPADH of the present invention is clearly defined as SEQ ID NO: 2, those skilled in the art can easily obtain its encoding genes, expression cassettes and plasmids containing these genes, and transformants containing these plasmids. These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering methods well known to those skilled in the art.

[0057] The transformant host can be any microorganism suitable for expressing SEQ ID NO: 2, including bacteria and fungi. Preferably, the microorganism is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, and Bacillus subtilis. More preferably, it is Escherichia coli BL21 (DE3).

[0058] It is well known in the art that the expression results of the same nucleotide sequence in different microbial hosts often vary greatly. In order to optimally express carboxyaminopropylagmatine dehydrogenase CAPADH or its mutants in Escherichia coli, which is most commonly used in genetic engineering, the expression genes of these enzymes can be codon-optimized.

[0059] Codon optimization is a kind of technology that can be used for making protein expression in organism maximized by increasing the translation efficiency of gene of interest.Different organisms usually illustrate the special preference of one of some codons for encoding identical amino acids due to mutation tendency and natural selection.For example, in fast-growing microorganisms such as Escherichia coli, the composition of its respective genome tRNA library is reflected by optimizing codon.Therefore, in fast-growing microorganisms, the low frequency codon of amino acid can be used for identical amino acid but the codon replacement of high frequency.Therefore, the expression of optimized dna sequence dna is improved in fast-growing microorganisms.

[0060] The above-mentioned CAPADH or its derivative polypeptides can be applied to the synthesis reaction of carboxyaminopropyl agmatine discovered in the present invention, using agmatine (Formula I) and aspartic acid semialdehyde (Formula II) as substrates to obtain the product carboxyaminopropyl agmatine (III). The uses of the above-mentioned CAPADH or its derivative polypeptides are not limited to the synthesis of carboxyaminopropyl agmatine (III), but also include the production of spermidine or its intermediates by in vitro multi-stage enzyme cascade reactions or fermentation with genetically engineered Escherichia coli. The spermidine intermediates described include at least carboxyaminopropyl agmatine and aminopropyl agmatine.

[0061] The present inventors conducted in vitro enzyme activity experiments by recombinantly expressing CAPADH and demonstrated the catalytic activity of CAPADH.

[0062] When used as a biocatalyst to catalyze the synthesis of spermidine intermediates such as carboxyaminopropylagmatine, the CAPADH of the present invention can be in the form of an enzyme or a bacterial cell. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, and enzymes immobilized on carriers; and the bacterial cells include living and dead cells.

[0063] In specific applications, particularly in in vitro enzyme-linked catalysis, the polypeptide CAPADH of the present invention or its derivative polypeptide can also be immobilized on other solid-phase carriers to obtain immobilized enzymes for use in in vitro reactions with substrates. Examples of such solid-phase carriers include microspheres and tubular bodies made of inorganic materials. There are two main methods for preparing immobilized enzymes: physical and chemical. Physical methods include physical adsorption and embedding. Chemical methods include binding and cross-linking. Binding methods are further divided into ionic binding and covalent binding. All of the above-mentioned methods for immobilizing enzymes can be applied in the present invention.

[0064] In addition, the present invention also relates to a new compound having a structure of formula (III), which is the product of the condensation of agmatine and aspartic acid semialdehyde catalyzed by CAPADH, is an important intermediate in the cyanobacterial spermidine biosynthesis pathway, can be used in the artificial synthesis of spermidine or spermidine intermediates, and has industrial application value.

[0065] The present invention will be further described in detail below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0066] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned therein, unless otherwise specified, refer to the percentage by mass.

[0067] In the examples herein, if no specific description is given for the reaction temperature or the operating temperature, the temperature generally refers to room temperature (15-30° C.).

[0068] Example

[0069] Materials and methods

[0070] The primer synthesis and sequencing in the examples were commissioned to Nanjing GenScript Biotechnology Co., Ltd.

[0071] The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., were performed primarily with reference to Molecular Cloning: A Laboratory Manual (3rd edition), edited by J. Sambrook and D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. Specific experimental conditions can be determined by simple experiments when necessary.

[0072] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. These conditions can be adjusted through simple experiments if necessary.

[0073] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (Add 20 g / L agar powder to LB solid medium.)

[0074] In the following examples, when a culture medium containing kanamycin (kan) was used, the final concentration of the antibiotic in the culture medium was 50 μg / ml.

[0075] The PCR primers used in the examples are shown in Table 1.

[0076] Table 1. Primers used in the examples

[0077]

[0078]

[0079] Example 1: Discovery of the Spermidine Synthesis Pathway in Cyanobacteria

[0080] After extensive preliminary research, the inventors discovered that when the model cyanobacterium Synechocystis sp. PCC 6803 was cultured under nutrient-rich conditions instead of nitrogen-, phosphorus-, or sulfur-restricted conditions, the concentrations of two polyamine compounds showed significant accumulation in the cells. One of the polyamine compounds was a new compound, such as Figure 1 The NMR data of the compound are shown in Table 2, and the NMR hydrogen spectrum, carbon spectrum and other two-dimensional spectra are shown in Table 2. Figure 2The structure of the compound was determined by secondary mass spectrometry and nuclear magnetic resonance spectroscopy to be (S)-2-amino-4-((4-guanidinobutyl)amino)butanoic acid. Herein, it is named carboxyaminopropylagmatine, or CAPA for short.

[0081] Table 2. Nuclear magnetic resonance data of carboxyaminopropyl agmatine

[0082]

[0083]

[0084] In order to explore the synthetic source of CAPA, the inventors 13 C and 15 N tracing experiments, combined with metabolomics, genetic manipulation and biochemical identification methods, discovered a new pathway for spermidine synthesis in the model cyanobacterium Synechocystis sp. PCC 6803, named the carboxyaminopropylagmatine pathway, or CAPA pathway, such as Figure 3 As shown. Adding carbon and nitrogen fully labeled arginine ([U- 13 C,U- 15 N]arginine), or fully carbon-labeled asparagine ([U- 13 C]asparagine), and after 4 hours of incubation, the labeling status of the intermediate metabolites in Synechocystis was detected. Figure 4 As shown, fully carbon- and nitrogen-labeled arginine is converted into fully carbon- and nitrogen-labeled agmatine by arginine decarboxylase in a one-step reaction inside the cell, and then the labeling of agmatine enters carboxyaminopropylagmatine (CAPA), aminopropylagmatine (APA) and spermidine, indicating that agmatine is directly involved in the synthesis of spermidine; similarly, fully carbon-labeled asparagine is hydrolyzed by asparaginase inside the cell and converted into fully carbon-labeled aspartic acid, and then the labeling enters carboxyaminopropylagmatine (CAPA), aminopropylagmatine (APA) and spermidine, indicating that aspartic acid, after forming aspartate semialdehyde, serves as a substrate for the synthesis of carboxyaminopropylagmatine (CAPA) and spermidine.

[0085] The inventors knocked out multiple genes that may be related to spermidine synthesis in the model cyanobacterium Synechocystis sp. PCC 6803. Figure 5As shown in the figure, by comparing the metabolome differences between the wild-type strain and the knockout strain, it was found that the concentration of agmatine in the bacteria increased significantly after the knockout of CAPADH, and carboxyaminopropyl agmatine (CAPA) and its downstream products could not be detected; after the knockout of CAPADC, carboxyaminopropyl agmatine (CAPA) accumulated in large quantities, and aminopropyl agmatine (APA) and spermidine could not be detected; after the knockout of APAUH, aminopropyl agmatine (APA) accumulated in large quantities and spermidine could not be detected, as shown in the figure. Figure 6 shown. 13 C and 15 This was also confirmed by N tracer experiments (see Figure 4 After knocking out CAPADH and then replenishing it, the intracellular concentrations of agmatine and spermidine returned to those of the wild-type strain. This suggests that CAPADH catalyzes the condensation reaction of agmatine and aspartate semialdehyde to produce carboxyaminopropylagmatine (CAPA); CAPADC catalyzes the decarboxylation of carboxyaminopropylagmatine (CAPA) to produce aminopropylagmatine (APA); and APAUH catalyzes the hydrolysis of aminopropylagmatine (APA), removing a molecule of urea to produce spermidine.

[0086] The nucleotide sequence of the CAPADH gene is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded polypeptide is shown in SEQ ID NO: 2.

[0087] Example 2: Construction and purification of CAPADH expression vector

[0088] 2.1 Vector construction

[0089] Using the pET28a vector (Novagen), the restriction endonucleases NcoI and XhoI were used to generate sticky ends. Using primers P1 and P2, the genome of Synechocystis sp. PCC 6803 was used as a template and a high-fidelity DNA polymerase (PhantaMax, Novagen) was used to amplify the DNA fragment. The recovered DNA fragment was mixed with the digested vector and a cloning kit and incubated at 50°C for 30 minutes to allow ligation. The target protein was constructed with a C-terminal 6×Histidine tag for subsequent affinity purification.

[0090] 2.2 E. coli transformation

[0091] Remove E. coli DH5α competent cells (Novagen) from the -80°C freezer and slowly thaw on ice. Once the competent cells have thawed, slowly add the ligation product from step 2.1 to the competent cells along the wall. Gently flick with your fingertips to mix thoroughly. After mixing, place on ice for 30 minutes. Heat shock the cells in a 42°C water bath for 90 seconds and place on ice again for 2 minutes. Add 1 mL of LB medium and allow the cells to recover in a shaker at 37°C, 170 rpm, for 1 hour. Centrifuge at 8000 × g for 1 minute to concentrate the cells. Discard the excess supernatant in a clean hood and pipette the cells with the remaining approximately 100 μL of medium. Use a sterile spreading rod to evenly spread the mixed bacterial solution onto a solid LB plate containing 50 μg / mL kanamycin and incubate at 37°C for 10 hours. The next day, verify the correct bands by PCR and electrophoresis for single clones. Once sequencing is complete, extract the plasmid.

[0092] 2.3 Prokaryotic expression and protein purification

[0093] Transform the plasmid constructed in step 2.2 into competent E. coli BL21 (DE3) cells. Pick a single clone and inoculate it into a 4 mL LB liquid culture medium test tube containing 50 μg / mL Kanamycin, and culture it at 37°C, 190 rpm for 10 h. Inoculate the cultured bacterial liquid into a 50 mL LB liquid culture medium triangular flask containing 50 μg / mL Kanamycin at a 1% v / v inoculum volume, and culture it at 37°C, 190 rpm for about 1.5 h. When the OD 600 When the pH value is within the range of 0.4-0.6, add 10 μL of 1 M IPTG solution and induce the culture at 16°C and 110 rpm for 16 hours. After induction, cool the bacterial solution on ice and collect the precipitated bacteria by low-temperature centrifugation at 8000×g and 4°C for 5 minutes. Ultrasonicate the cells and collect the total protein after the disruption. Centrifuge it at 12000×g and 4°C for 60 minutes to obtain the supernatant. The target protein with 6×Histidine tag is affinity purified by Ni-NTA resin, the protein concentration is measured by Bradford colorimetry, and the protein molecular weight and purity are detected by SDS-PAGE electrophoresis. Figure 6 The protein was stored at -80℃.

[0094] Example 3: In vitro enzyme activity assay of CAPADH

[0095] 3.1 Enzyme activity assay

[0096] The reaction system consisted of 200 μL of 50 mM phosphate buffer solution (pH 7.4), containing 5 mM agmatine, 1.25 mM aspartate semialdehyde, 0.25 mM NADPH, and 1 mM dithiothreitol (DTT). The reaction was initiated by adding 0.5 μg of purified CAPADH and the temperature was maintained at 30°C. The reaction was terminated after 2 h and the product was detected by liquid chromatography HPLC or high-resolution mass spectrometry. The enzyme reaction kinetics were detected by spectrophotometry. 340 The rate over time reflects the consumption of NADPH.

[0097] The inventors used HPLC and high-resolution mass spectrometry in a reaction system with agmatine, aspartic acid semialdehyde and NADPH as substrates to detect that after the addition of CAPADH protein, the substrate was consumed and a new product was generated ( Figure 6 ), and the product's chromatographic retention time, precise molecular weight, and MS / MS analysis were consistent with intracellular carboxyaminopropylagmatine (CAPA) from Synechocystis sp. PCC 6803. No CAPA production was detected in the reaction system without CAPADH or NADPH.

[0098] 3.2 Preparation and purification of CAPADH products using HPLC

[0099] The enzymatic reaction was performed using purified CAPADH. The reaction system contained 50 mM phosphate buffer (pH 7.4), 5 mM agmatine, 2 mM L-aspartate semialdehyde, 2 mM NADPH, 1 mM dithiothreitol, and 500 μg of purified CAPADH. After incubation overnight at 30°C, the product was purified using preparative HPLC (1290 Infinity II-6125, Agilent). The product was separated by chromatography on an Xbridge Amide column (150 mm × 4.6 mm, 3.5 μm, Waters). Mobile phases A and B consisted of 0.5% formic acid in water and 0.5% formic acid in acetonitrile. The column temperature was maintained at 40°C, the solvent flow rate was 1 mL / min, and the elution gradient was: 0 min, 95% B; 4 min, 70% B; 10 min, 60% B; 11 min, 20% B; 15 min, 20% B; 15.1 min, 95% B; 24 min, 95% B. Fractions were collected every 0.5 min and analyzed for purity using high-resolution mass spectrometry. Nuclear magnetic resonance analysis confirmed that the product structure of the in vitro CAPADH enzymatic reaction was consistent with carboxyaminopropylagmatine (CAPA). In summary, the following reactions in the spermidine biosynthesis pathway involved in CAPADH can be determined:

[0100] Agmatine+L-Aspartate semialdehyde+NAD(P)H+H + →

[0101] Carboxyaminopropylagmatine+NAD(P) +

[0102] Example 4: Heterologous construction of the CAPA pathway to synthesize spermidine and other polyamine derivatives

[0103] 4.1 Strain construction

[0104] By overexpressing the key enzyme CAPADH in the CAPA pathway and other related proteins, it can be used to synthesize spermidine intermediates CAPA, APA, spermidine and other polyamine derivatives. Figure 7 The pET28a vector was used and digested with restriction enzymes NcoI and XhoI to generate sticky ends.

[0105] Using the genome of Synechocystis sp. PCC 6803 as a template, primers P1 and P2 were used to amplify a DNA fragment containing the CAPADH gene by PCR. The recovered DNA fragment was mixed with the digested vector and a cloning kit and incubated at 50°C for 30 minutes to allow ligation. The plasmid was transformed into the BL21(DE3) strain, resulting in strain 1, which can express CAPADH upon IPTG induction.

[0106] Using the genome of Synechocystis sp. PCC 6803 as a template, primers P1 and P3, and primers P4 and P5, were used to amplify DNA fragments containing the CAPADH and CAPADC genes, respectively. The recovered DNA fragments were mixed with the digested vector and a cloning kit and incubated at 50°C for 30 minutes to allow ligation. The plasmids were transformed into the BL21(DE3) strain to generate strain 2, which can express CAPADH and CAPADC upon induction with IPTG. The plasmids from strain 2 were extracted and digested with the restriction endonuclease XhoI to generate sticky ends.

[0107] Using the genome of Synechocystis sp. PCC 6803 as a template, primers P6 and P7 were used to amplify a DNA fragment containing the APAUH gene by PCR. The recovered DNA fragment was mixed with the digested vector and a cloning kit and incubated at 50°C for 30 minutes to allow ligation. The plasmid was transformed into the BL21(DE3) strain, resulting in strain 3, which can express CAPADH, CAPADC, and APAUH upon IPTG induction.

[0108] In addition, the pET28a vector was directly transformed into the BL21(DE3) strain as a negative control.

[0109] 4.2 Strain fermentation

[0110] Streak each of the above strains on LB plates, pick a single colony and inoculate it into a 4 mL tube of LB liquid medium containing Kanamycin and incubate at 37°C, 190 rpm for 10 hours. Inoculate the culture into a 50 mL Erlenmeyer flask of LB liquid medium containing Kanamycin at a 1% v / v inoculum and incubate at 37°C, 220 rpm for approximately 3 hours. Add 10 μL of 1 M IPTG (5000×) and ferment at 16°C, 150 rpm for 15 hours.

[0111] 4.3 Fermentation product analysis

[0112] After fermentation, 1 mL of cell culture medium was taken and centrifuged at 4°C and 14000 rpm for 3 min. The supernatant was collected and the polyamines produced by the fermentation and the supernatant were detected by high-resolution mass spectrometry. Figure 7 As shown, in the three constructed Escherichia coli strains containing the CAPA pathway, a variety of polyamines and polyamine derivatives were detected in their fermentation products.

[0113] In Strain 1, overexpression of CAPADH allows cells to synthesize carboxyaminopropylagmatine (CAPA) and its acetylated product, acetylcarboxyaminopropylagmatine (Acetyl-CAPA);

[0114] In Strain 2, overexpression of CAPADH and CAPADC allows cells to synthesize aminopropylagmatine (APA) and its acetylated product, acetylaminopropylagmatine (Acetyl-APA);

[0115] In Strain 3, overexpression of CAPADH, CAPADC, and APAUH allowed cells to synthesize spermidine and its acetylated products, N 1 -Acetylspermidine, N 8-N8-Acetylspermidine and Diacetylspermidine.

[0116] Therefore, the biosynthesis of carboxyaminopropylagmatine, aminopropylagmatine, spermidine and their derivatives can be achieved by heterologously constructing the CAPA pathway in the E. coli chassis.

[0117] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. It will be apparent to those skilled in the art that various improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The compound shown in formula III is (S)-2-amino-4-((4-guanidine)amino)butyric acid, which is named carboxyaminopropylagmatine:

2. A method for synthesizing the compound according to claim 1, characterized in that: The method comprises the following steps: using agmatine represented by formula I and aspartic acid semialdehyde represented by formula II as substrate raw materials, and preparing compound III through a reduction condensation reaction under the catalysis of an enzyme: The amino acid sequence of the enzyme is SEQ ID NO: 2, and the enzyme is named carboxyaminopropylagmatine dehydrogenase.

3. The method according to claim 2, wherein The carboxyaminopropylagmatine dehydrogenase is in the form of an enzyme or in the form of a microbial cell expressing the same.

4. The method according to claim 2, wherein Coenzyme NADPH or NADH is added to the reaction system.

5. An enzyme composition for synthesizing spermidine, characterized in that The invention comprises the carboxyaminopropylagmatine dehydrogenase as claimed in claim 2, as well as carboxyaminopropylagmatine decarboxylase and aminopropylagmatine urea hydrolase.

6. The enzyme composition according to claim 5, wherein Starting from agmatine and aspartic acid semialdehyde, carboxyaminopropyl agmatine is condensed and catalyzed by carboxyaminopropyl agmatine dehydrogenase to generate carboxyaminopropyl agmatine; carboxyaminopropyl agmatine is catalyzed by carboxyaminopropyl agmatine decarboxylase to generate aminopropyl agmatine; and aminopropyl agmatine is catalyzed by aminopropyl agmatine urea hydrolase to generate spermidine.

7. Use of the enzyme composition as claimed in claim 5 in constructing spermidine-producing bacteria.

8. The use according to claim 7, characterized in that The coding genes of carboxyaminopropylagmatine dehydrogenase, carboxyaminopropylagmatine decarboxylase and aminopropylagmatine urea hydrolase are cloned into spermidine producing bacteria to form spermidine engineering bacteria.

9. A spermidine-producing bacterium, characterized in that Comprising the enzyme composition as claimed in claim 5.

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