Method for preparing various poly(ester amide)s

Abstract

The present invention relates to a metabolically engineered recombinant microbial enzyme and a method for preparing poly(ester amide)s using same. The recombinant microorganism and the method for preparing poly(ester amide)s using same according to the present invention can biosynthesize various poly(ester amide) polymers from various carbon sources such as glucose, amino acids, hydroxy acids, and carboxylic acids, and are thus useful for the industrial production of poly(ester amide)s.

Description

Method for producing various poly(ester amides)

[0001] The present invention relates to a recombinant microbial enzyme metabolically engineered and a method for producing poly(ester amide) using the same, and more particularly, to a recombinant microorganism into which a gene encoding a coenzyme A transferase using an organic acid containing an amino acid as a substrate and a gene encoding a PHA synthase have been introduced or amplified, and a method for producing poly(ester amide) using the same.

[0002]

[0003] Recently, due to the depletion of oil and environmental concerns, significant attention has been focused on the sustainable production of various value-added chemicals and polymers using microorganisms. Research is being conducted to produce various types of biopolymers as replacements for petrochemical-based plastics. However, to date, there have been no successful examples of the production of various poly(ester amides) using recombinant microorganisms.

[0004] Poly(ester amide) is a polymer that contains both ester and amide bonds. These properties give poly(ester amide) the advantages of both polyester and polyamide. Although there have been no successful cases of poly(ester amide) production to date, there have been many reports of studies using metabolic engineering to produce naturally occurring polyesters known as polyhydroxyalkanoates (Choi et al., Adv. Mat. 32: 1-37, 2020). Furthermore, there have been reports of the biosynthesis of poly(lactate-ran-glycolate) and poly(3-hydroxybutyrate-ran-phenyllactate), polyesters that did not exist in nature, using microorganisms through systems metabolic engineering (Choi et al., Nat. Biotech. 34: 435-440, 2016; Yang et al., 9:79, 2018).

[0005] Accordingly, the inventors of the present invention have endeavored to develop a method for efficiently producing poly(ester amide) from various carbon sources using microorganisms, and as a result, have confirmed that polyester amide can be biosynthesized using a recombinant microorganism into which genes for an enzyme that converts amino acid as a substrate into aminoacyl-CoA and an enzyme that polymerizes aminoacyl-CoA have been introduced, thereby completing the present invention.

[0006]

[0007] Summary of the invention

[0008] The purpose of the present invention is to provide a recombinant microorganism capable of producing poly(ester amide) from various carbon sources.

[0009] Another object of the present invention is to provide a method for producing poly(ester amide) using the recombinant microorganism.

[0010]

[0011] To achieve the above purpose, the present invention provides a recombinant microorganism having the ability to produce poly(ester amide) from a carbon source, in which a gene encoding coenzyme A transferase and a gene encoding polyhydroxyalkanoate synthase are introduced or amplified.

[0012] The present invention also provides a method for producing poly(ester amide), comprising the steps of: (a) culturing a recombinant microorganism of the present invention to produce poly(ester amide); and (b) recovering the produced poly(ester amide).

[0013] The present invention also comprises the steps of: (a) culturing the recombinant microorganism of the present invention in a medium supplemented with an organic acid that serves as a substrate for coenzyme A transferase to produce poly(ester amide); and

[0014] (b) Provided is a method for producing poly(ester amide), including a step of recovering the poly(ester amide) produced above.

[0015]

[0016] The present invention also provides a poly(ester amide) manufactured by the above manufacturing method.

[0017]

[0018] Figure 1 illustrates the metabolic pathways that produce various poly(ester amides) and the amino acid monomers contained in these poly(ester amides). Newly designed metabolic reactions that do not exist in nature for the biosynthesis of poly(ester amides) are indicated by red arrows. Naturally occurring metabolic pathways that synthesize polyhydroxyalkanoates are indicated by black arrows. Amino acid monomers that can be polymerized by these metabolic pathways are indicated in gray boxes. Amine and amide functional groups are indicated in pink. Hydroxyl and ester functional groups are indicated in blue. The indicated genes represent the following enzymes: act, β-alanine CoA transferase; pct540, engineered propionate CoA transferase; phaC, polyhydroxyalkanoate synthase. Abbreviations for the chemicals are as follows: 3AB, 3-aminobutyrate; 3AIB, 3-aminoisobutyrate; 3AP, 3-aminopropionate; 3AV, 3-aminovalerate; 4AB, 4-aminobutyrate; 5AV, 5-aminovalerate.

[0019] Figure 2 shows the results of analyses to confirm the biosynthesis of poly(ester amide) containing various amino acid monomers. a, Schematic diagram of culturing PEA01 strain in MR medium supplemented with (RS)-3HB and target amino acids. Multiple metabolic pathways are indicated by black dotted arrows. b, Fractions of amino acid monomers in polymers obtained by culturing PEA00 and PEA01 strains according to the schematic diagram in (a). c, Mass spectra of 3AP, (R)-3AB, 3AIB, (R)-3AV, 4AB, and 5AV monomers and their corresponding standard chemicals. The indicated genes represent the following enzymes, respectively: act, β-alanine CoA transferase; ldhA, (R)-lactate dehydrogenase; pct540, engineered propionate CoA transferase; phaC1437Ps6-19, engineered Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate synthase. Abbreviations for the chemicals are as follows: 3AB, 3-aminobutyrate; 3AIB, 3-aminoisobutyrate; 3AP, 3-aminopropionate; 3AV, 3-aminovalerate; 3HB, 3-hydroxybutyrate; 4AB, 4-aminobutyrate; 5AV, 5-aminovalerate; 6AC, 6-aminocaproate; 7AH, 7-aminoheptanoate.

[0020] Figure 3 shows the results of an analysis confirming the biosynthesis of poly(ester amide) containing multiple amino acid monomers at once. When the PEA01 strain was cultured in MR medium with (RS)-3HB and 3AP, 3AP / (RS)-3AB, or 3AP / (RS)-3AB / (RS)-3AV, the fraction of 3AP, (R)-3AB, or (R)-3AV monomers was measured. The fractions of 3AP, (R)-3AB, or (R)-3AV are indicated in pink, yellow, and orange, respectively. Abbreviations for the chemicals are as follows: 3AP, 3-aminopropionate; 3AV, 3-aminovalerate.

[0021] Figure 4 shows the results of analyzing the reactivity of purified beta-alanine coenzyme A transferase to various substrates. a, Schematic diagram showing the in vitro assay of Act for various substrates classified as amino acids, hydroxy acids, or carboxylic acids. b, Enzymatic activity of Act for amino acids, hydroxy acids, or carboxylic acids. c, Chemical structures of substrates on which Act showed activity. Abbreviations for the chemicals are as follows: 2AB, 2-aminobutyrate; 3AB, 3-aminobutyrate; 3AIB, 3-aminoisobutyrate; 3AP, 3-aminopropionate; 3AV, 3-aminovalerate; 3HB, 3-hydroxybutyrate; 4AB, 4-aminobutyrate; 5AV, 5-aminovalerate; 6AC, 6-aminocaproate; 7AH, 7-aminoheptanoate; ALA, alanine; BPA, β-phenylalanine; GLY, glycine; PAB, p-aminobenzoate.

[0022] Figure 5 shows a schematic diagram of a strain producing poly(ester amide) using glucose as the sole carbon source, the results of analysis, and the production of poly(ester amide) through fed-batch fermentation. a, Schematic diagram of culturing the PEA06 strain in MR medium. b, The fraction of 3AP monomer in the polymer obtained by culturing the PEA06 strain according to the schematic diagram in (a). c, Schematic diagram of culturing the PEA07 strain in MR medium. d, The fraction of 4AB monomer in the polymer obtained by culturing the PEA07 strain according to the schematic diagram in (c). Multiple metabolic pathways are indicated by black dotted arrows. Inactivated reactions are indicated by red “X” marks. e, Fed-batch fermentation profile of the PEA06 strain using glucose as the sole carbon source. Cell concentration, polymer content, and glucose concentration are indicated by gray, orange, white, and black circles, respectively. Abbreviations for the chemicals are: 3-aminopropionate; 3HB, 3-hydroxybutyrate; 4AB, 4-aminobutyrate; AKG, α-ketoglutarate; PEP, phosphoenolpyruvate; LA, lactate.

[0023] Figure 6 shows the results showing that poly(3-hydroxybutyrate-ran-3-aminopropionate), a representative poly(ester amide), can be biosynthesized using various polyhydroxyalkanoate synthases. The indicated genes represent the following enzymes: wild-type class I PhaCs (PhaCCne, PhaCAca, PhaCAhy, PhaCAsa, respectively) derived from C. necator, Aeromonas caviae, Aeromonas hydrophila, and Aeromonas salmonicida, and Pseudomonas sp. MBEL 6-19, Pseudomonas sp. Engineered (containing E130D, S325T, S477G, Q481K mutations) class II PhaCs (PhaC1437Ps6-19, PhaC1437Ps61-3, PhaC1437Ppu, PhaC1437Pae, PhaC1437Pch, PhaC1437Pre, respectively) from strains 61-3, Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1, Pseudomonas chlororaphis, and Pseudomonas resinovorans.

[0024]

[0025] Detailed description of the invention and preferred embodiments

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0027] In one embodiment of the present invention, it was confirmed that poly(ester amide) can be synthesized from various carbon sources (e.g., amino acids, sugars, organic acids, etc.) using a recombinant microorganism into which a gene encoding coenzyme A transferase using various amino acids as substrates and a gene encoding polyhydroxyalkanoate synthase were introduced.

[0028] In addition, in another embodiment of the present invention, it was confirmed that a recombinant microorganism additionally introduced with a gene capable of synthesizing hydroxy acids and amino acids from glucose can synthesize poly(ester amide) using glucose as a single carbon source.

[0029]

[0030] Accordingly, the present invention relates, from one aspect, to a recombinant microorganism having the ability to synthesize poly(ester amide) into which a gene encoding coenzyme A transferase and a gene encoding polyhydroxyalkanoate synthase are introduced or amplified.

[0031] In the present invention, the recombinant microorganism may be characterized in that a gene encoding one or more types of coenzyme A transferase and a gene encoding one or more types of PHA synthase (polyhydroxyalkanoate synthase) are introduced.

[0032] In one embodiment of the present invention, the recombinant microorganism can synthesize a poly(ester amide) containing an organic acid containing an amino acid as a precursor and a monomer thereof.

[0033] In another embodiment of the present invention, the recombinant microorganism can synthesize a poly(ester amide) containing amino acids and hydroxy acids as precursors.

[0034] The recombinant microorganism of the present invention may be characterized in that i) Coenzyme A transferase introduced or amplified catalyzes the synthesis of acyl-CoA using an organic acid containing an amino acid as a precursor, and ii) PHA synthase catalyzes the synthesis of poly(ester amide) through polymerization of the synthesized acyl-CoA.

[0035]

[0036] The term “Coenzyme A transferase” of the present invention refers to a transferase that catalyzes the transfer reaction of a coenzyme A group from an acyl-CoA donor to a carboxylic acid.

[0037] In the present invention, the coenzyme A transferase may be characterized by catalyzing the synthesis of aminoacyl-CoA using an amino acid as a substrate. In the present invention, the amino acid is used to mean both protein-forming amino acids and non-protein-forming amino acids that constitute proteins.

[0038] In the present invention, the protein constituent amino acids may be, for example, 21 kinds of natural amino acids found in eukaryotes or isomers thereof, but are not limited thereto.

[0039] In the present invention, more preferably, the amino acid may be a non-protein amino acid. In the present invention, the non-protein amino acid means all organic acids containing an amino group (-NH2) and a carboxyl group (-COOH) in addition to the protein-forming amino acids. In the present invention, the non-protein amino acid may be selected from the group consisting of, for example, 2-aminobutyrate, 3-aminopropionate, isoserine, 3-aminobutyrate, 3-aminovalerate, beta-phenylalanine (BPA), 4-aminobutyrate, 4-amino-3-methylbutyrate, 5-aminovalerate, 6-aminocaproate, 7-aminoheptanoate, 3-aminoisobutyrate, para-aminobenzoate (PAB) and isomers thereof. It is not limited to this.

[0040] In the present invention, the coenzyme A transferase may be characterized by synthesizing acyl-CoA using other carboxylic acids as substrates in addition to amino acids.

[0041] In the present invention, the carboxylic acid may preferably be a hydroxy acid containing a hydroxyl group. In the present invention, the hydroxy acid includes all organic acids containing a hydroxyl group (-OH) and a carboxyl group (-COOH) in addition to the protein constituent amino acids. In the present invention, the hydroxy acid may be selected from the group consisting of, for example, glycolate, lactate, 2-hydroxybutyrate, 3-hydroxypropionate, 3-hydroxybutyrate, carboxylic acids containing a carbon chain longer than that, and isomers thereof, but is not limited thereto.

[0042] In the present invention, the carboxylic acid may be selected from the group consisting of, but is not limited to, amino acids and hydroxy acids, carboxylic acids and isomers thereof, including, for example, propionate, butyrate, isobutyrate, valerate, isovalerate, 2-methylbutyrate, caproate, malonate, succinate, and carboxylic acids having a carbon chain longer than or equal to that of the amino acid and the hydroxy acid.

[0043] In the present invention, the isomer may be a structural isomer or a stereoisomer. In the present invention, when the carboxylic acid is an amino acid or a hydroxy acid, the isomer includes, but is not limited to, a (R)-form or (S)-form stereoisomer.

[0044] In particular, in the present invention, the isomer may preferably be an (R)-type stereoisomer.

[0045] In the present invention, the coenzyme A transferase includes a wild type, a naturally occurring mutant, or an artificially engineered mutant. Preferably, it may be characterized by being at least one selected from the group consisting of beta-alanine coenzyme A transferase, propionate coenzyme A transferase, and isocaprenyl coenzyme A:2-hydroxyisocaproyl coenzyme A transferase.

[0046] In the present invention, the gene for the coenzyme A transferase may be derived from various organisms. More preferably, it may be characterized by at least one selected from the group consisting of the act gene derived from Clostridium propionicum, the engineered pct gene (pct540) derived from Clostridium propionicum, and the hadA gene derived from Clostridium difficile, but is not limited thereto.

[0047] In the present invention, for example, the coenzyme A transferase may be characterized by including a sequence selected from SEQ ID NOs: 1 to 3 or a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, but is not limited thereto.

[0048]

[0049]

[0050]

[0051] In the present invention, the sequence of the gene encoding the coenzyme A transferase can be easily obtained from a database widely used in the art or from the amino acid sequence of the coenzyme A transferase described above. In the present invention, the sequence of the gene encoding the coenzyme A transferase can be codon optimized to be suitable for the host microorganism through a method well known in the art.

[0052] In an embodiment of the present invention, it was confirmed that coenzyme A transferase comprising sequence number 1 can use various organic acids as substrates. For example, the substrate is a carboxylic acid, preferably an amino acid or a hydroxy acid, most preferably, glycine, alanine (preferably (S)-alanine), 2-aminobutyrate, 3-aminobutyrate, 3-aminoisobutyrate, 3-aminovalerate, beta-phenylalanine (BPA), 4-aminobutyrate, para-aminobenzoate (PAB), 5-aminovalerate, 6-aminocaproate, 7-aminoheptanoate, glycolate, lactate, 2-hydroxybutyrate, It may be characterized by being selected from the group consisting of 3-hydroxypropionate, 3-hydroxybutyrate, propionate, butyrate, isobutyrate, valerate, isovalerate, 2-methylbutyrate, caproate, malonate, succinate and isomers thereof, but is not limited thereto.

[0053] In the present invention, the isomer may be a structural isomer or a stereoisomer. In the present invention, when the carboxylic acid is an amino acid or a hydroxy acid, the isomer may be, for example, an (R)-form or (S)-form stereoisomer, more preferably an (R)-form isomer.

[0054]

[0055] The term "polyhydroxyalkanoate synthase" of the present invention refers to an enzyme that synthesizes polyhydroxyalkanoate from hydroxyacyl-CoA. In the present invention, it was confirmed that the PHA synthase can synthesize poly(ester amide) using aminoacyl-CoA as a substrate.

[0056] In the present invention, the PHA synthase includes a wild type, a naturally occurring mutant, or an artificially engineered mutant. In the present invention, the PHA synthase may be characterized as being Class I PhaC or Class II PhaC, but is not limited thereto. In the present invention, the gene sequence of the engineered PHA synthase is described, for example, in Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010, but is not limited thereto.

[0057] In the present invention, the gene encoding the PHA synthase may be characterized as being a phaC gene derived from various organisms or a mutant thereof.

[0058] In the present invention, the gene encoding the PHA synthase may be derived from various organisms. In the present invention, the gene encoding the PHA synthase may be derived from any microorganism having PHA synthesis activity without limitation. For example, the gene encoding the PHA synthase may be selected from the group consisting of Cupriavidus necator, Aeromonas caviae, Aeromonas hydrophila, Aeromonas salmonicida, Pseudomonassp. MBEL 6-19, Pseudomonassp. 61-3, Pseudomonas putida KT2440, Pseudomonas aeruginosaPAO1, Pseudomonas chlororaphis, and Pseudomonas resinovorens. It may be characterized by a phaC gene derived from a strain selected from the group consisting of, but not limited to, resinovorans.

[0059] In the present invention, for example, the protein sequence of the PHA synthase may be characterized by including a sequence selected from SEQ ID NOs: 4 to 13 or a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, but is not limited thereto.

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] In the present invention, the sequence of the gene encoding the PHA synthase can be easily obtained from a database widely used in the art or from the amino acid sequence of the PHA synthase described above. In the present invention, the sequence of the gene encoding the PHA synthase can be codon optimized to suit the host microorganism using a method well known in the art.

[0071]

[0072] The term “poly(ester amide)” in the present invention is used to mean, without limitation, a polymer having both an ester bond and an amide bond. The structures and utilities of various poly(ester amides), including amino acid-based poly(ester amides), are well known in the art (e.g., Polym. Chem., 2016, 7, 7039-7046).

[0073] In the present invention, the poly(ester amide) may be characterized in that it contains an organic acid that serves as a substrate for the coenzyme A transferase as a monomer.

[0074] In the present invention, the poly(ester amide) may be characterized in that it contains an amino acid that serves as a substrate for the coenzyme A transferase as a monomer.

[0075] In the present invention, the poly(ester amide) may be characterized by including an amino acid and a hydroxy acid as a substrate of the coenzyme A transferase as monomers.

[0076] In the present invention, for non-limiting examples, the poly(ester amide) may be characterized by including a structure represented by the following chemical formula, but is not limited thereto:

[0077]

[0078] Here, R1 to R4 can each independently be any chemical moiety without limitation, preferably each independently hydrogen, alkyl, alcohol or amine,

[0079] x, y, n, and m are each independently integers greater than or equal to 0 or 1.

[0080] (0 means non-existent)

[0081] In the present invention, the poly(ester amide) may be characterized as being a polymer containing one or more monomers.

[0082] In the present invention, it will be apparent to those skilled in the art that the monomers and their composition ratios included in the poly(ester amide) synthesized by the recombinant microorganism can be controlled depending on the level of organic acids (e.g., amino acids and hydroxy acids) that become monomers (i.e., precursors) of the poly(ester amide).

[0083] In one embodiment of the present invention, for example, the monomers and their composition ratios included in the poly(ester amide) synthesized by the recombinant microorganism can be controlled by the concentration and ratio of the supplied carbon source (sugar or organic acid).

[0084] In another embodiment of the present invention, for example, the monomers and their composition ratios included in the poly(ester amide) synthesized by the recombinant microorganism can be controlled according to the precursor biosynthesis level of the recombinant microorganism, and the precursor biosynthesis level can be controlled through, but is not limited to, the creation, strengthening, weakening, or blocking of the precursor biosynthesis pathway through genetic engineering.

[0085] In the present invention, the host microorganism into which the gene encoding the coenzyme A transferase and the gene encoding the polyhydroxyalkanoate synthase are introduced or amplified is Escherichia coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter, Acinetobacter, It may be characterized by being selected from the group consisting of Ralstonia, Agrobacterium, Aspergillus, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, Cyanobacterium, Saccharomyceses and Cyclobacterium, but is not limited thereto.

[0086] In the present invention, for example, the host microorganism may be preferably selected from the group consisting of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus reuteri, Lactococcus lactis, Aspergillus niger, Saccharomyceses cerevisiae, and Saccharomyces pombe, but is not limited thereto.

[0087] In the present invention, the host microorganism may most preferably be E. coli, but is not limited thereto.

[0088]

[0089] In one embodiment of the present invention, it was confirmed that when various carbon sources (hydroxy acids, sugars, amino acids) are supplied to the recombinant microorganism, poly(ester amide) can be synthesized using these as substrates, and when the recombinant microorganism is engineered to have the ability to synthesize precursors (e.g., hydroxy acids and amino acids) through additional genetic modification, it was confirmed that poly(ester amide) can be synthesized from one carbon source (e.g., glucose).

[0090] In one embodiment of the present invention, the recombinant microorganism may be characterized in that it does not have the ability to produce a substrate for coenzyme A transferase or pha. The substrate for coenzyme A transferase or pha may be supplied from outside to synthesize poly(ester amide).

[0091] In another embodiment of the present invention, the recombinant microorganism may be characterized by having a biosynthetic pathway from a carbon source to an organic acid (preferably an amino acid) that serves as a substrate for coenzyme A transferase. In the present invention, the organic acid may be characterized by being, for example, an amino acid.

[0092] In the present invention, the carbon source may be, for example, a sugar or an organic acid, but is not limited thereto.

[0093] In the present invention, the sugar as the carbon source may be selected from the group consisting of monosaccharides, disaccharides, and polysaccharides, including, for example, glucose, sucrose, galactose, maltose, xylose, glycerol, fructose, and sugar cane, but is not limited thereto.

[0094] In the present invention, the organic acid may be an organic acid (e.g., pyruvate, glutamate, etc.) produced in the metabolic pathway of the carbon source, but is not limited thereto.

[0095] The term 'biosynthetic pathway' of the present invention refers to a biological pathway that can produce a target substance through an enzymatic reaction from a precursor of the organic acid.

[0096] In the present invention, the recombinant microorganism may be characterized by having an innate biosynthetic pathway for an organic acid that serves as a substrate for coenzyme A transferase.

[0097] In the present invention, the recombinant microorganism may be engineered to have a biosynthetic pathway for an organic acid that serves as a substrate for coenzyme A transferase. For example, the recombinant microorganism may be characterized in that the biosynthetic pathway for an organic acid that serves as a substrate for coenzyme A transferase is newly established, strengthened, weakened, or blocked through the introduction, amplification, attenuation, or deletion of a gene related to the biosynthetic pathway.

[0098] For example, as shown in Table 2 of the examples of the present invention, the recombinant microorganism has enhanced production of hydroxyacyl-CoA and amino acids through additional engineering of one or more of deletion or attenuation of the ldhA gene; introduction or amplification of the phaAB gene; introduction or amplification of the davAB gene; introduction or amplification of the panD gene or gadB gene, but is not limited thereto.

[0099]

[0100] The term “host microorganism” of the present invention means any microorganism capable of expressing functional genes and / or gene products derived from other cells or organs, and more specifically, means a microorganism before a gene encoding a coenzyme A transferase of the present invention and a gene encoding a PHA synthase are introduced or amplified.

[0101] The term "recombinant" in the present invention means including a polynucleotide or protein that does not occur naturally within the host cell.

[0102] In the present invention, "introduction" means that a gene is made replicable either as an extrachromosomal element or by completion of chromosomal integration. The transformed gene may be either integrated into the chromosome of the microorganism or located extrachromosomally, as long as it can be expressed within the microorganism. Furthermore, the gene may be a polynucleotide capable of encoding a protein, including DNA and RNA.

[0103] In this disclosure, the term "gene" should be considered in its broadest sense and may encode a structural or regulatory protein. Regulatory proteins include transcription factors, heat shock proteins, or proteins involved in DNA / RNA replication, transcription, and / or translation.

[0104] In the present invention, “amplification” or “strengthening” is a concept encompassing increasing the activity of a corresponding enzyme or consequently strengthening the progression of a corresponding pathway by mutating, substituting, or deleting some bases of the corresponding gene, introducing some bases, or introducing a gene derived from another microorganism encoding the same enzyme.

[0105] In the present invention, “weakening” is a concept encompassing reducing the activity of an enzyme expressed by a corresponding gene by mutating, substituting, or deleting some bases of the corresponding gene or introducing some bases, and includes everything that blocks part or a significant part of a biosynthetic pathway in which the enzyme of the corresponding gene is involved.

[0106] In the present invention, 'deletion' is a concept that encompasses mutating, substituting, or deleting part or all of the bases of the corresponding gene, or introducing some bases to prevent the gene from being expressed, or to prevent the gene from exhibiting enzymatic activity even if expressed, and includes everything that blocks the biosynthetic pathway in which the enzyme of the corresponding gene is involved.

[0107] As used herein, a "vector" refers to a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, can integrate into the genome itself. Since plasmids are currently the most commonly used form of vector, the terms "plasmid" and "vector" are sometimes used interchangeably herein. For the purposes of the present invention, the use of a plasmid vector is preferred. A typical plasmid vector that can be used for this purpose has a structure that includes (a) an origin of replication to allow efficient replication, allowing for several to several hundred copies of the plasmid vector per host cell, (b) an antibiotic resistance gene to allow selection of host cells transformed with the plasmid vector, and (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if a suitable restriction enzyme cleavage site does not exist, the vector and the foreign DNA can be easily ligated using conventional synthetic oligonucleotide adapters or linkers. After ligation, the vector must be transformed into an appropriate host cell. Transformation can be readily accomplished using the calcium chloride method or electroporation (Neumann, et al. EMBO J., 1:841, 1982).

[0108] In the present invention, a base sequence is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. This may be a gene and regulatory sequence(s) that are linked in such a way that gene expression is enabled when an appropriate molecule (e.g., a transcriptional activating protein) binds to the regulatory sequence(s). For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and are in reading frame. However, an enhancer need not be in contact. These sequences are joined by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used according to conventional methods.

[0109] In the present invention, all genes can be introduced into a host microorganism together with an appropriate promoter. For example, in the present invention, the genes may be introduced together with a promoter selected from the group consisting of lac, lacUV5, trc, tac, trp, araBAD, T3, T5, T7, L10, I16, H30, H36, sod, tuf, eftu, Pm, and Ptet, but are not limited thereto.

[0110]

[0111] From another aspect, the present invention relates to a method for producing poly(ester amide), comprising the steps of (a) culturing a recombinant microorganism of the present invention to produce poly(ester amide); and (b) recovering the produced poly(ester amide).

[0112] In the present invention, step (a) can produce poly(ester amide) by culturing the culture medium by adding a carbon source from outside. In the present invention, the carbon source may be characterized by being at least one selected from the group consisting of sugars, amino acids, and hydroxy acids, but is not limited thereto.

[0113] In the present invention, as described above with respect to recombinant microorganisms, if the host microorganism has a biosynthetic pathway for an organic acid (e.g., hydroxy acid or amino acid) that is a substrate for the coenzyme A transferase of the present invention, either innately or through additional genetic engineering, poly(ester amide) can be produced through culturing the recombinant microorganism of the present invention without adding a single carbon source or adding a separate carbon source from the outside.

[0114] In another aspect, the present invention relates to a method for producing poly(ester amide), comprising the following steps:

[0115] (a) a step of producing poly(ester amide) by culturing the recombinant microorganism of the present invention in a medium containing an organic acid that serves as a substrate for coenzyme A transferase; and

[0116] (b) A step of recovering the poly(ester amide) produced above.

[0117] In the present invention, the culturing process of the recombinant microorganism can be performed using a commonly known culturing method, and in addition to the specific medium and specific culturing method used in the examples of the present invention, a saccharified liquid such as whey or CSL (corn steep liquor) and other media can be used, and various methods such as fed-batch culture and continuous culture can be used (Lee et al., Bioprocess Biosyst. Eng., 26: 63, 2003; Lee et al., Appl. Microbiol. Biotechnol., 58: 663, 2002; Lee et al., Biotechnol. Lett., 25: 111, 2003; Lee et al., Appl. Microbiol. Biotechnol., 54: 23, 2000; Lee et al., Biotechnol. Bioeng., 72: 41, 2001).

[0118] The term "cultivation" in the present invention refers to culturing microorganisms to derive a desired effect. In the present invention, the desired effect refers to the production of a hydrophobic substance. The cultivation can generally be performed in an incubator equipped with an appropriate culture medium optimized for the microorganism being used, containing at least one simple energy source and, if necessary, a co-substrate.

[0119] In the present invention, the term “cultivation” may be used interchangeably with “fermentation” in the sense of production of a hydrophobic substance through microbial culture.

[0120] In the present invention, the step of culturing the recombinant microorganism to produce a hydrophobic substance may be characterized in that it is performed through fed-batch fermentation.

[0121] The term “fed-batch culture” or “fed-batch fermentation” of the present invention means culturing / fermenting by intermittently supplying additional medium to control the concentration of the culture solution.

[0122] A “suitable culture medium” may include a carbon source or carbon substrate; a nitrogen source, such as peptone, glucose, glycerol, yeast extract, meat extract, malt extract, urea, ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium phosphate; a phosphorus source, such as monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), such as magnesium salts, cobalt salts, and / or manganese salts; as well as nutrients essential or beneficial to the maintenance and / or growth of cells, such as growth factors, amino acids, and vitamins; yeast extracts, antibiotics, and the like. In the present invention, the culture may be characterized in that it is cultured at a temperature in an appropriate range to produce a hydrophobic substance, and may be characterized in that it is cultured at, for example, 20°C to 40°C, preferably 25°C to 35°C, but is not limited thereto, and may be cultured under different conditions depending on the species of the host microorganism, the substance to be expressed, etc.

[0123]

[0124] As used herein, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, integrate into the genome itself. Since plasmids are currently the most commonly used form of vector, the terms "plasmid" and "vector" are sometimes used interchangeably herein. However, the present invention encompasses other forms of vectors known or becoming known in the art that have equivalent functions. Typical expression vectors for mammalian cell culture expression are, for example, based on pRK5 (EP 307,247), pSV16B (WO 91 / 08291), and pVL1392 (Pharmingen).

[0125] The term "expression control sequence" refers to a DNA sequence essential for the expression of an operably linked coding sequence in a particular host organism. Such control sequences include a promoter for initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences that control the termination of transcription and translation. For example, a control sequence suitable for prokaryotes includes a promoter, optionally an operator sequence, and a ribosome binding site. For eukaryotes, this includes a promoter, a polyadenylation signal, and an enhancer. The factor most influencing the level of gene expression in a plasmid is the promoter. Promoters suitable for high expression include the SRα promoter and cytomegalovirus-derived promoters.

[0126] Any of a wide variety of expression control sequences may be used in the vector to express the DNA sequence of the present invention. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter region of phage lambda, the regulatory region of the fd-encoded protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of the phosphatases, e.g., Pho5, the promoter of the yeast alpha-mating system, and any other sequence known to regulate the expression of genes in prokaryotes or eukaryotes or their viruses, and various combinations thereof. The T7 RNA polymerase promoter Φ10 can be usefully used to express the protein NSP in E. coli.

[0127] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. This can be a gene and regulatory sequence(s) that are linked in such a way that an appropriate molecule (e.g., a transcriptional activating protein) enables gene expression when bound to the regulatory sequence(s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and are in reading frame. However, an enhancer need not be in contact. These sequences are joined by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used according to conventional methods.

[0128] The term "expression vector" as used herein typically refers to a recombinant carrier into which a fragment of heterologous DNA has been inserted, typically a fragment of double-stranded DNA. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in the host cell. Once within a host cell, the expression vector can replicate independently of the host chromosomal DNA, producing multiple copies of the vector and its inserted (heterologous) DNA.

[0129] As is well known in the art, to increase the level of expression of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function within the selected expression host. Preferably, the expression control sequences and the gene are contained within a single expression vector that also includes a bacterial selection marker and an origin of replication. If the expression host is a eukaryotic cell, the expression vector must further include an expression marker useful in the eukaryotic expression host.

[0130] A host cell transformed or transfected with the expression vector described above constitutes another aspect of the present invention. As used herein, the term "transformation" refers to the introduction of DNA into a host cell, such that the DNA becomes replicable either as an extrachromosomal element or through chromosomal integration. As used herein, the term "transfection" refers to the uptake of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed.

[0131] The host cell of the invention may be a prokaryotic or eukaryotic cell. Furthermore, a host cell with high DNA introduction efficiency and high expression efficiency of the introduced DNA is typically used. Examples of host cells that can be used include well-known eukaryotic and prokaryotic hosts such as E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, and yeast; insect cells such as Spodoptera frugiperda (SF9); animal cells such as CHO and mouse cells; African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10; and tissue-cultured human cells. When cloning the cDNA encoding the NSP protein of the present invention, it is preferable to use an animal cell as the host. When using COS cells, since the SV40 large T antigen is expressed in COS cells, the plasmid containing the SV40 replication origin will exist as multiple copies of episomes in the cells, and higher expression than usual can be expected. The introduced DNA sequence can be obtained from the same species as the host cell, can be from a different species from the host cell, or can be a hybrid DNA sequence containing any heterologous or homologous DNA.

[0132] It should be understood that not all vectors and expression control sequences are equally effective in expressing the DNA sequences of the present invention. Similarly, not all hosts will function equally well in the same expression system. However, those skilled in the art can appropriately select from among various vectors, expression control sequences, and hosts without undue experimental burden and without departing from the scope of the present invention. For example, when selecting a vector, consideration should be given to the host, as the vector must replicate within it. The vector's copy number, its ability to control copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered. When selecting an expression control sequence, various factors should be considered, such as the relative strength of the sequence, its controllability, and its compatibility with the DNA sequences of the present invention, particularly with respect to potential secondary structures. The unicellular host should be selected by considering factors such as the selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, secretion characteristics, the ability to accurately fold the protein, culture and fermentation requirements, and the ease of purifying the product encoded by the DNA sequence of the present invention from the host. Within the scope of these variables, those skilled in the art can select various vector / expression control sequence / host combinations that can express the DNA sequence of the present invention in fermentation or large-scale animal culture. When attempting to clone the cDNA of the NSP protein by expression cloning, the binding method, the panning method, the film emulsion method, etc. can be applied as screening methods.

[0133] In the context of the present invention, “substantially pure” means that the polypeptide according to the present invention and the DNA sequence encoding the polypeptide are substantially free of other proteins derived from bacteria.

[0134] Prokaryotic cells such as Escherichia coli and Bacillus subtilis have been widely used as host cells for expressing recombinant proteins because they can be cultured at high concentrations in a short period of time, are easy to genetically manipulate, and have well-defined genetic and physiological characteristics. However, in order to solve problems in post-translational modification of proteins, the secretion process, the three-dimensional structure of the active form, and the activity state of proteins, higher organisms such as unicellular eukaryotic yeasts (Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, etc.), filamentous fungi, insect cells, plant cells, and mammalian cells have recently been utilized as host cells for recombinant protein production. Therefore, those skilled in the art can easily apply the use of other host cells in addition to the E. coli exemplified in the examples.

[0135]

[0136] In another aspect, the present invention relates to a poly(ester amide) produced by the above method.

[0137]

[0138] [Example]

[0139] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0140]

[0141] Example 1: Production of vectors for genetic engineering

[0142] Example 1-1: pTac15k_davAB vector

[0143] The davAB gene fragment encoding 5-aminovaleramide aminohydrolase and L-lysine monooxygenase was constructed by PCR using pKE112_davAB vector (Park et al., Metab. Eng. 16:42-47 2013) as a DNA template and primers of sequence numbers 14 and 15.

[0144] [SEQ ID NO: 14] P1:

[0145] 5'-AGACAGGAATTCATGCGCATCGCTCTGTACC-3'

[0146] [Sequence number 15] P2:

[0147] 5'-AGACAGGGATCCTCAATCCGCCAG-3'

[0148] Next, the pTac15k (US patent 20110269183) plasmid, which promotes strong gene expression of the davAB fragment and the tac promoter, was treated with restriction enzymes (EcoRI and BamHI), and then treated with T4 DNA ligase to conjugate the davAB fragment cut with the restriction enzyme and the pTac15k plasmid, thereby constructing a recombinant plasmid, pTac15k_davAB.

[0149]

[0150] Example 1-2: pTac15k_act_davAB vector

[0151] The davAB gene fragment encoding 5-aminovaleramide aminohydrolase and L-lysine monooxygenase was constructed by PCR using pKE112_davAB vector (Park et al., Metab. Eng. 16:42-47 2013) as a DNA template and primers of sequence numbers 16 and 17.

[0152] [Sequence number 16] P3:

[0153] 5'-AGACAGGAGCTCTTTCACACAGGAAACAATGCGCATCGCTCTTGTACC-3'

[0154] [Sequence number 17] P4:

[0155] 5'-AGACAGCTGCAGTGCAATCCGCCAGGGCG-3'

[0156] Next, the pTac15k_act vector (Chae et al., Metab. Eng. 41: 82-91, 2017), which promotes strong gene expression of the davAB fragment and the tac promoter, was treated with restriction enzymes (SacI and PstI), and then treated with T4 DNA ligase to conjugate the davAB fragment cleaved with the restriction enzyme and the pTac15k_act vector, thereby constructing a recombinant plasmid, pTac15k_act_davAB.

[0157]

[0158] Example 1-3: pTac15k_act_panD vector

[0159] A panD gene fragment encoding aspartate decarboxylase was produced by PCR using the chromosomal DNA of a Corynebacterium glutamicum strain as a template and primers of sequence numbers 18 and 19.

[0160] [Sequence number 18] P5:

[0161] 5'-AGACAGGAGCTCTTTCACACAGGAAACAATGCTGCGCACCATCC-3'

[0162] [Sequence number 19] P6:

[0163] 5'-AGACAGGGTACCCTAAATGCTTCTCGACGTCAAAAG-3'

[0164] Next, the panD fragment and the pTac15k_act plasmid (Chae et al., Metab. Eng. 41: 82-91, 2017) were treated with restriction enzymes (SacI and KpnI), and then treated with T4 DNA ligase to ligate the panD fragment cut with the restriction enzyme and the pTac15k_act plasmid, thereby constructing a recombinant plasmid, pTac15k_act_panD.

[0165]

[0166] Example 1-4: pPs619C1437PhaAB vector

[0167] Using the chromosomal DNA of the Cupriavidus necator strain as a template, PCR was performed with primers of sequence numbers 20 and 21 to produce a phaAB gene fragment encoding acetyl-CoA acetyltransferase and acetoacetyl-CoA reductase.

[0168] [SEQ ID NO: 20] P7:

[0169] 5'-AGACAGTTCGAATAGTGACGGCAGAGAGACAATCAAATC-3'

[0170] [SEQ ID NO: 21] P8:

[0171] 5'-AGACAGCCTGCAGGCCTGCCG-3'

[0172] Next, the phaAB fragment and the pPs619C1437Pct540 plasmid (Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010) were treated with restriction enzymes (BstBI and SbfI), and then treated with T4 DNA ligase to conjugate the phaAB fragment cut with the restriction enzyme and the pPs619C1437Pct540 plasmid, thereby constructing a recombinant plasmid, pPs619C1437PhaAB.

[0173]

[0174] Example 1-5: pCneCPct540 vector

[0175] A phaCCne gene fragment encoding polyhydroxyalkanoate synthase was produced by performing PCR using the chromosomal DNA of the Cupriavidus necator strain as a template and primers of sequence numbers 22 and 23.

[0176]

[0177]

[0178] Next, the phaCCne fragment and the pPs619C1437Pct540 plasmid (Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010) were treated with restriction enzymes (BstBI and SbfI), and then treated with T4 DNA ligase to conjugate the phaCCne fragment cut with the restriction enzyme and the pPs619C1437Pct540 plasmid, thereby constructing a recombinant plasmid, pCneCPct540.

[0179]

[0180] Example 1-6: pAcaCPct540, pAhyCPct540, pAsaCPct540 vectors

[0181] Using the chromosomal DNA of Aeromonas caviae, Aeromonas hydrophila, and Aeromonas salmonicida strains as templates, PCR was performed with primers of SEQ ID NO: 24 and 25 to produce phaCAca, phaCAhy, and phaCAsa gene fragments encoding polyhydroxyalkanoate synthase.

[0182]

[0183]

[0184] Next, the phaCAca, phaCAhy, and phaCAsa gene fragments and the pPs619C1437Pct540 plasmid (Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010) were treated with restriction enzymes (BstBI and SbfI), and then treated with T4 DNA ligase to conjugate the phaCAca, phaCAhy, and phaCAsa gene fragments cut with the restriction enzymes and the pPs619C1437Pct540 plasmid, thereby constructing recombinant plasmids pAcaCPct540, pAhyCPct540, and pAsaCPct540.

[0185]

[0186] Information on the vectors produced in Examples 1-1 to 1-6 is as shown in Table 1 below.

[0187]

[0188]

[0189] Example 2: Production of recombinant microorganisms

[0190] The recombinant microorganisms shown in Table 2 below were produced using the vectors prepared in Example 1.

[0191]

[0192]

[0193]

[0194] The pTac15k_act plasmid, which was constructed to express the act gene encoding beta-alanine coenzyme A transferase in microorganisms (Chae et al., Metab. Eng. 41: 82-91, 2017), and the pPs619C1437Pct540 plasmid, which was constructed to express the phaC1437Ps6-19 gene encoding engineered polyhydroxyalkanoate synthase and the pct540 gene encoding engineered propionate coenzyme A transferase (Jung et al., Biotechnol. Bioeng., 105: 161- 171, 2010), were introduced into Escherichia coli XL1-Blue (Stratagene) to produce a recombinant microorganism (PEA01), and E. coli (PEA00) into which the blank pTac15k was introduced was used as a control strain.

[0195] In addition, in order to confirm the possibility of production from various carbon sources, the pTac15k_act_davAB vector constructed in Example 1-2 was introduced into E. coli XL1-Blue / pPs619C1437Pct540 so that the act gene encoding beta-alanine coenzyme A transferase, the davA gene encoding 5-aminovaleramide aminohydrolase, and the davB gene encoding L-lysine monooxygenase were expressed in the microorganism, thereby producing a recombinant microorganism (PEA03), and E. coli (PEA02) into which pTac15k_davAB constructed in Example 1-1 was introduced was used as a control strain.

[0196] In addition, in order to confirm the possibility of production using glucose as the sole carbon source and high-efficiency production through fed-batch fermentation, the pPs619C1437PhaAB plasmid constructed in Example 1 was introduced into E. coli XL1-Blue ΔldhA so that the phaC1437Ps6-19 gene encoding engineered polyhydroxyalkanoate synthase, the phaA gene encoding acetyl-CoA acetyltransferase, and the phaB gene encoding acetoacetyl-CoA reductase were expressed, and a recombinant microorganism was produced (XL1-Blue ΔldhA / pPs619C1437PhaAB). In the strain, the pTac15k_act_panD vector constructed in Example 1 or beta-alanine coenzyme A transferase was introduced so that the act gene encoding beta-alanine coenzyme A transferase and the panD gene encoding aspartate decarboxylase were expressed. Escherichia coli strains (PEA06 and PEA07) containing pTac15k_act_gadB_mut (Chae et al., Metab. Eng. 41: 82-91, 2017), which was engineered to express the act gene encoding the panD gene and the gadB_mut gene encoding engineered glutamate decarboxylase, were used. As a control, E. coli strain (PEA05) containing pTac15k_act (Chae et al., Metab. Eng. 41: 82-91, 2017), which does not express the panD or gadB_mut genes, was used as a control.

[0197] In addition, to confirm the possibility of utilizing polyhydroxyalkanoate synthases from various microorganisms, Cupriavidus necator, Aeromonas caviae, Aeromonas hydrophila, Aeromonas salmonicida, Pseudomonas sp. MBEL 6-19, Pseudomonas sp. 61-3, Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1, Pseudomonas chlororaphis, and Pseudomonas resinovorens were used. The vectors pCneCPct540, pAcaCPct540, pAhyCPct540, pAsaCPct540 constructed in Example 1 to express the phaC gene encoding polyhydroxyalkanoate synthase derived from (Pseudomonas resinovorans) and the vectors pPs619C1437Pct540, pPs613C1437Pct540, pPpuC1437Pct540, pPaeC1437Pct540, pPchC1437Pct540, pPreC1437Pct540 previously reported (Yang et al., Appl. Microbiol. Biotechnol. 90: 603-614, 2011) were introduced into E. coli XL1-Blue ΔldhA / pTac15k_act to produce recombinant microorganisms. (PEA08, PEA09, PEA10, PEA11, PEA04, PEA12, PEA13, PEA14, PEA15, PEA16)

[0198]

[0199] Example 3: Confirmation of production of poly(ester amide) containing 3-aminopropionate and 4-aminobutyrate as monomers using recombinant microorganisms.

[0200] The recombinant microorganism (PEA00, PEA01) manufactured in Example 2 was inoculated into 10 mL of LB medium and pre-cultured at 37°C for 8 hours. 1.0 mL of the pre-cultured culture was inoculated into 100 mL of MR medium in a 350 mL flask and cultured.

[0201] The composition of MR medium (pH 7.0) is as follows: 20 g glucose, 6.67 g KH2PO4, 4.0 g (NH4)2HPO4, 0.8 g citric acid, 0.8 g MgSO4ㆍ7H2O, 0.01 g CaCl2ㆍ2H2O, 5 mL trace metal solution (10 g FeSO4ㆍ7H2O, 2.2 g ZnSO4ㆍ4H2O, 0.58 g MnSO4ㆍ4H2O, 1 g CuSO4ㆍ5H2O, 0.1 g (NH4)6Mo7O per liter of distilled water 24 ㆍ4H2O, 0.02g Na2B4O7ㆍ10H2O) is a medium composed of the following components. In the above composition, amino acids known as substrates of existing beta-alanine coenzyme A transferase, 3-aminopropionate, 4-aminobutyrate, 5-aminovalerate, 6-aminocaproate, and 7-aminoheptanoate, were additionally supplied as carbon sources at a concentration of 2g / L. In addition, 2g / L of sodium (RS)-3-hydroxybutyrate was also additionally supplied. The culture was performed in a shaking incubator (jSR, Korea) operating at 30℃ and 200rpm for 96 hours. After the culture was completed, the culture solution was centrifuged at 13,200rpm for 10 minutes, and the cells were collected and analyzed to confirm polyester amide production.

[0202] As a result, as disclosed in FIG. 2, it was confirmed that the recombinant microorganism according to the present invention produces a poly(ester amide) having 3-aminopropionate and 4-aminobutyrate as monomers.

[0203] From these results, it was confirmed that the recombinant microorganism according to the present invention produces a poly(ester amide) having 3-aminopropionate and 4-aminobutyrate as monomers.

[0204]

[0205] Example 4: Confirmation of production of poly(ester amide) containing 5-aminovalerate as a monomer using recombinant microorganisms.

[0206] The recombinant microorganism (PEA02, PEA03) manufactured in Example 2 was inoculated into 10 mL of LB medium and pre-cultured at 37°C for 8 hours. 1.0 mL of the pre-cultured culture was inoculated into 100 mL of MR medium in a 350 mL flask and cultured.

[0207] The composition of MR medium (pH 7.0) is as follows: 20 g glucose, 6.67 g KH2PO4, 4.0 g (NH4)2HPO4, 0.8 g citric acid, 0.8 g MgSO4ㆍ7H2O, 0.01 g CaCl2ㆍ2H2O, 5 mL trace metal solution (10 g FeSO4ㆍ7H2O, 2.2 g ZnSO4ㆍ4H2O, 0.58 g MnSO4ㆍ4H2O, 1 g CuSO4ㆍ5H2O, 0.1 g (NH4)6Mo7O per liter of distilled water 24ㆍ4H2O, 0.02g Na2B4O7ㆍ10H2O) is a medium composed of components. In the above composition, L-lysine, which can be converted to 5-aminovalerate due to the davAB gene, was supplied as an additional carbon source at a concentration of 2g / L. In addition, 2g / L of sodium (RS)-3-hydroxybutyrate was also additionally supplied. Cultivation was performed in a shaking incubator (jSR, Korea) operating at 30℃ and 200rpm for 96 hours. After cultivation, the culture medium was centrifuged at 13,200rpm for 10 minutes, and cells were collected and analyzed to confirm polyester amide production.

[0208] As a result, as disclosed in FIG. 2, it was confirmed that the recombinant microorganism according to the present invention produces a poly(ester amide) having 5-aminovalerate as a monomer using L-lysine as a carbon source.

[0209] From these results, it was confirmed that the recombinant microorganism according to the present invention successfully produces a poly(ester amide) having 5-aminovalerate as a monomer.

[0210]

[0211] Example 5: In vitro assay for identification of additional substrates for beta-alanine coenzyme A transferase.

[0212] To expand the amino acid monomer types of poly(ester amide), in vitro assays were performed to find substrates on which beta-alanine coenzyme A transferase has activity, in addition to the existing known substrates. For purification of beta-alanine coenzyme A transferase, the pETa_his_act vector (Chae et al., Metab. Eng. 41: 82-91, 2017) was introduced into Escherichia coli BL21(DE3) (F-ompT hsdSB(rB-mB-) gal dcm (DE3) a prophage carrying the T7 RNA polymerase gene) (New England Biolabs, USA).

[0213] Enzyme assays were performed in 50 mM potassium phosphate buffer (pH 7.5). The substrates and enzymes required for the enzyme assay were added in the amounts shown below. 10 mM target substrate, 1 mM acetyl-CoA, and 2.5 μg of purified beta-alanine coenzyme A transferase were added, and the reaction was performed at 30°C for 2 hours.

[0214] As a result, as disclosed in FIG. 4, in addition to the previously known 3-aminopropionate, 4-aminobutyrate, 5-aminovalerate, 6-aminocaproate, and 7-aminoheptanoenite, it was confirmed that it also had activity toward amino acid substrates such as (RS)-3-aminobutyrate, 3-aminoisobutyrate, and (RS)-3-aminovalerate. In addition, it was confirmed that it also had activity toward five hydroxy acids (glycolate, (RS)-lactate, (RS)-2-hydroxybutyrate, 3-hydroxypropionate, and (RS)-3-hydroxybutyrate) and seven carboxylic acids (propionate, butyrate, isobutyrate, valerate, isovalerate, 2-methylbutyrate, and caproate) that were not amino acids.

[0215] From these results, it was confirmed that beta-alanine coenzyme A transferase according to the present invention successfully converts various amino acids, hydroxy acids, and carboxylic acids, in addition to existing known substrates, into coenzyme A derivatives.

[0216]

[0217] Example 6: Confirmation of production of poly(ester amide) containing (R)-3-aminobutyrate, 3-aminoisobutyrate, and (R)-3-aminovalerate as monomers using recombinant microorganisms.

[0218] The recombinant microorganism (PEA00, PEA01) manufactured in Example 2 was inoculated into 10 mL of LB medium and pre-cultured at 37°C for 8 hours. 1.0 mL of the pre-cultured culture was inoculated into 100 mL of MR medium in a 350 mL flask and cultured.

[0219] The composition of MR medium (pH 7.0) is as follows: 20 g glucose, 6.67 g KH2PO4, 4.0 g (NH4)2HPO4, 0.8 g citric acid, 0.8 g MgSO4ㆍ7H2O, 0.01 g CaCl2ㆍ2H2O, 5 mL trace metal solution (10 g FeSO4ㆍ7H2O, 2.2 g ZnSO4ㆍ4H2O, 0.58 g MnSO4ㆍ4H2O, 1 g CuSO4ㆍ5H2O, 0.1 g (NH4)6Mo7O per liter of distilled water 24ㆍ4H2O, 0.02g Na2B4O7ㆍ10H2O) is a medium composed of the following components. In the above composition, (RS)-3-aminobutyrate, 3-aminoisobutyrate, and (RS)-3-aminovalerate, which are amino acids newly known as substrates of beta-alanine coenzyme A transferase in Example 5, were supplied as an additional carbon source at a concentration of 2g / L. In addition, 2g / L of sodium (RS)-3-hydroxybutyrate was also additionally supplied. The culture was performed in a shaking incubator (jSR, Korea) operating at 30℃ and 200rpm for 96 hours. After the culture was completed, the culture solution was centrifuged at 13,200rpm for 10 minutes, and the cells were collected and analyzed to confirm polyester amide production.

[0220] As a result, as disclosed in FIG. 2, it was confirmed that the recombinant microorganism according to the present invention produces a poly(ester amide) having (R)-3-aminobutyrate, 3-aminoisobutyrate, and (R)-3-aminovalerate as monomers.

[0221] From these results, it was confirmed that the recombinant microorganism according to the present invention produces a poly(ester amide) having (R)-3-aminobutyrate, 3-aminoisobutyrate, and (R)-3-aminovalerate as monomers.

[0222]

[0223] Example 7: Confirmation of production of poly(ester amide) containing various amino acids as monomers using recombinant microorganisms.

[0224] The recombinant microorganism (PEA00, PEA01) manufactured in Example 2 was inoculated into 10 mL of LB medium and pre-cultured at 37°C for 8 hours. 1.0 mL of the pre-cultured culture was inoculated into 100 mL of MR medium in a 350 mL flask and cultured.

[0225] The composition of MR medium (pH 7.0) is as follows: 20 g glucose, 6.67 g KH2PO4, 4.0 g (NH4)2HPO4, 0.8 g citric acid, 0.8 g MgSO4ㆍ7H2O, 0.01 g CaCl2ㆍ2H2O, 5 mL trace metal solution (10 g FeSO4ㆍ7H2O, 2.2 g ZnSO4ㆍ4H2O, 0.58 g MnSO4ㆍ4H2O, 1 g CuSO4ㆍ5H2O, 0.1 g (NH4)6Mo7O per liter of distilled water 24 ㆍ4H2O, 0.02g Na2B4O7ㆍ10H2O) is a medium composed of components. In the above composition, two (3-aminopropionate / (RS)-3-aminobutyrate) or three (3-aminopropionate / (RS)-3-aminobutyrate / (RS)-3-aminoisobutyrate), amino acids known as substrates of existing beta-alanine coenzyme A transferase, were combined and supplied as an additional carbon source. In addition, 2 g / L of sodium (RS)-3-hydroxybutyrate was also additionally supplied. The culture was performed in a shaking incubator (jSR, Korea) operating at 30℃ and 200 rpm for 96 hours. After the culture was completed, the culture solution was centrifuged at 13,200 rpm for 10 minutes, and the cells were collected and analyzed to confirm polyester amide production.

[0226] As a result, as disclosed in FIG. 3, it was confirmed that the recombinant microorganism according to the present invention produces poly(ester amide) having two (3-aminopropionate and (R)-3-aminobutyrate) and three (3-aminopropionate, (R)-3-aminobutyrate, and (R)-3-aminoisobutyrate) monomers simultaneously.

[0227] From these results, it was confirmed that the recombinant microorganism according to the present invention produces a poly(ester amide) having various amino acids as monomers.

[0228]

[0229] Example 8: Confirmation of poly(ester amide) production using glucose as the sole carbon source using recombinant microorganisms.

[0230] The recombinant microorganism (PEA05, PEA06, or PEA07) manufactured in Example 2 was inoculated into 10 mL of LB medium and pre-cultured at 37°C for 8 hours, and 1.0 mL of the pre-cultured culture was inoculated into 100 mL of MR medium in a 350 mL flask and cultured. The PEA06 and PEA07 strains are strains in which the panD and gadB_mut genes are overexpressed to increase the metabolic flux for biosynthesizing 3-aminopropionate and 4-aminobutyrate, respectively. In addition, the phaAB genes are introduced into both strains to enable biosynthesis of 3-hydroxybutyryl coenzyme A from glucose, thereby providing hydroxy acid monomers without the introduction of external hydroxy acids. Additionally, to prevent lactate from polymerizing into hydroxy acid monomers other than the targeted 3-hydroxybutyrate, a strain lacking the ldhA gene required for lactate biosynthesis was used.

[0231] The composition of MR medium (pH 7.0) is as follows: 20 g glucose, 6.67 g KH2PO4, 4.0 g (NH4)2HPO4, 0.8 g citric acid, 0.8 g MgSO4ㆍ7H2O, 0.01 g CaCl2ㆍ2H2O, 5 mL trace metal solution (10 g FeSO4ㆍ7H2O, 2.2 g ZnSO4ㆍ4H2O, 0.58 g MnSO4ㆍ4H2O, 1 g CuSO4ㆍ5H2O, 0.1 g (NH4)6Mo7O per liter of distilled water 24ㆍ4H2O, 0.02g Na2B4O7ㆍ10H2O) is a medium composed of the following components. Unlike the previous examples, in the above composition, no amino acids or sodium (RS)-3-hydroxybutyrate were additionally supplied so that glucose could be used as the sole carbon source. Cultivation was performed in a shaking incubator (jSR, Korea) operating at 30℃ and 200 rpm for 96 hours. After cultivation, the culture solution was centrifuged at 13,200 rpm for 10 minutes, and the cells were collected and analyzed to confirm polyester amide production.

[0232] As a result, as disclosed in FIG. 5, it was confirmed that the recombinant microorganism according to the present invention produces a poly(ester amide) having 3-aminopropionate (FIGS. 5a,b) or 4-aminobutyrate (FIGS. 5c,d) as a monomer.

[0233] From these results, it was confirmed that the recombinant microorganism according to the present invention produces poly(ester amide) using glucose as the sole carbon source.

[0234]

[0235] Example 9: Confirmation of high-efficiency production of poly(ester amide) containing 3-aminopropionate as a monomer using fed-batch fermentation of recombinant microorganisms.

[0236] The recombinant microorganism (PEA06) manufactured in Example 2 was inoculated into 200 mL of LB medium and pre-cultured at 37°C for 8 hours. 200 mL of the pre-cultured culture was inoculated into 2 L of MR medium in a 6.6 L fermenter to perform fed-batch fermentation.

[0237] The composition of MR medium (pH 7.0) is as follows: 20 g glucose, 6.67 g KH2PO4, 4.0 g (NH4)2HPO4, 0.8 g citric acid, 0.8 g MgSO4ㆍ7H2O, 0.01 g CaCl2ㆍ2H2O, 5 mL trace metal solution (10 g FeSO4ㆍ7H2O, 2.2 g ZnSO4ㆍ4H2O, 0.58 g MnSO4ㆍ4H2O, 1 g CuSO4ㆍ5H2O, 0.1 g (NH4)6Mo7O per liter of distilled water 24 ㆍ4H2O, 0.02g Na2B4O7ㆍ10H2O) is a medium composed of components. Whenever glucose, the sole carbon source, was completely consumed, a feeding solution composed of 700g glucose, 8g MgSO4ㆍ7H2O per liter was additionally supplied. Cultivation was performed in a bioreactor (New Brunswick Scientific Co, NJ) operated to maintain 30℃ and dissolved oxygen level at 40% for 96 hours. After cultivation, the culture medium was centrifuged at 13,200 rpm for 10 minutes, and cells were collected and analyzed to confirm polyester amide production.

[0238] As a result, as disclosed in FIG. 5e, it was confirmed that the recombinant microorganism according to the present invention produces poly(ester amide) having 3-aminopropionate as a monomer at a high production concentration, productivity, and yield.

[0239] From these results, it was confirmed that the recombinant microorganism according to the present invention produces poly(ester amide) with high efficiency using fed-batch fermentation.

[0240]

[0241] Example 10: Confirmation of poly(ester amide) production using polyhydroxyalkanoates derived from various microorganisms.

[0242] The recombinant microorganisms (PEA08, PEA09, PEA10, PEA11, PEA04, PEA12, PEA13, PEA14, PEA15, PEA16) manufactured in Example 2 were inoculated into 10 mL of LB medium and pre-cultured at 37°C for 8 hours. 1.0 mL of the pre-cultured culture was inoculated into 100 mL of LB medium in a 350 mL flask and cultured.

[0243] The composition of LB medium is a medium composed of 10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter of distilled water. In the above composition, 3-aminopropionate, an amino acid known as a substrate for beta-alanine coenzyme A transferase, was supplied as an additional carbon source at a concentration of 2 g / L. In addition, 50 g / L of sodium (RS)-3-hydroxybutyrate was also supplied. Cultivation was performed for 96 h in a shaking incubator (jSR, Korea) operating at 30°C and 200 rpm. After cultivation, the culture medium was centrifuged at 13,200 rpm for 10 minutes, and cells were collected and analyzed to confirm polyester amide production.

[0244] As a result, as disclosed in FIG. 6, it was confirmed that all recombinant microorganisms according to the present invention have 3-aminopropionate as a monomer and produce poly(ester amides) having various molecular weights.

[0245] From these results, it was confirmed that the recombinant microorganisms according to the present invention produce poly(ester amide) having 3-aminopropionate as a monomer.

[0246] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0247]

[0248] The recombinant microorganism of the present invention is capable of biosynthesizing poly(ester amide) polymers from various carbon sources such as glucose, amino acids, hydroxy acids, carboxylic acids, etc., and is therefore useful for the industrial production of poly(ester amide).

[0249]

[0250] Electronic file attached.

Claims

1. A recombinant microorganism having the ability to produce poly(ester amide), in which a gene encoding coenzyme A transferase and a gene encoding polyhydroxyalkanoate synthase are introduced or amplified into a host microorganism.

2. A recombinant microorganism characterized in that it has a biosynthetic pathway for an organic acid that serves as a substrate for the coenzyme A transferase from a carbon source in the first paragraph.

3. A recombinant microorganism according to claim 2, characterized in that the carbon source is a sugar or an organic acid.

4. A recombinant microorganism, characterized in that in the second paragraph, the organic acid that serves as a substrate for the coenzyme A transferase is at least one of an amino acid and a hydroxy acid.

5. A recombinant microorganism according to claim 1, characterized in that the coenzyme A transferase uses an amino acid as a substrate.

6. In the fifth paragraph, the amino acid is selected from the group consisting of glycine, alanine, 2-aminobutyrate, 3-aminopropionate, isoserine, 3-aminobutyrate, 3-aminovalerate, beta-phenylalanine (BPA), 4-aminobutyrate, 4-amino-3-methylbutyrate, 5-aminovalerate, 6-aminocaproate, 7-aminoheptanoate, 3-aminoisobutyrate, para-aminobenzoate (PAB) and isomers thereof. A recombinant microorganism characterized by being selected.

7. A recombinant microorganism according to claim 1, characterized in that the coenzyme A transferase is beta-alanine coenzyme A transferase.

8. A recombinant microorganism according to claim 1, characterized in that the gene encoding the coenzyme A transferase is an act gene derived from Clostridium propionicum.

9. A recombinant microorganism according to claim 1, characterized in that the coenzyme A transferase comprises a sequence selected from sequence numbers 1 to 3.

10. A recombinant microorganism according to claim 1, characterized in that the PHA synthase is PhaC.

11. In the first paragraph, the gene encoding the PHA synthase is selected from the group consisting of Cupriavidus necator, Aeromonas caviae, Aeromonas hydrophila, Aeromonas salmonicida, Pseudomonas MBEL 6-19, Pseudomonas sp. MBEL 6-19, Pseudomonas 61-3, Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1, Pseudomonas chlororaphis, and Pseudomonas resinovorens. A recombinant microorganism characterized by a phaC gene derived from a strain selected from the group consisting of spp. resinovorans.

12. A recombinant microorganism according to claim 1, characterized in that the PHA synthase comprises a sequence selected from the group consisting of sequence numbers 4 to 13.

13. A recombinant microorganism characterized in that, in the first paragraph, the poly(ester amide) contains an organic acid as a monomer that serves as a substrate for the coenzyme A transferase.

14. In the 13th paragraph, the organic acid that is the substrate of the coenzyme A transferase is glycine, alanine, 2-aminobutyrate, 3-aminopropionate, isoserine, 3-aminobutyrate, 3-aminoisobutyrate, 3-aminovalerate, beta-phenylalanine (BPA), 4-aminobutyrate, 4-amino-3-methylbutyrate, para-aminobenzoate (PAB), 5-aminovalerate, 6-aminocaproate, A recombinant microorganism characterized by being selected from the group consisting of 7-aminoheptanoate, glycolate, lactate, 2-hydroxybutyrate, 3-hydroxypropionate, 3-hydroxybutyrate, propionate, butyrate, isobutyrate, valerate, isovalerate, 2-methylbutyrate, caproate, malonate, succinate, and isomers thereof.

15. In the first paragraph, the host microorganism is a recombinant microorganism, characterized in that the recombinant microorganism is selected from the group consisting of bacteria, yeast, and mold.

16. A method for producing poly(ester amide) comprising the following steps: (a) a step of producing poly(ester amide) by culturing a recombinant microorganism of any one of claims 1 to 15; and (b) A step of recovering the poly(ester amide) produced above.

17. A method for producing poly(ester amide) comprising the following steps: (a) a step of producing poly(ester amide) by culturing a recombinant microorganism of any one of claims 1 to 15 in a medium containing an organic acid that serves as a substrate for coenzyme A transferase; and (b) A step of recovering the poly(ester amide) produced above.

18. Poly(ester amide) manufactured by the method of Article 16.

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