Methods of making various poly(ester amides)

Abstract

This invention relates to metabolically engineered recombinant microbial enzymes and methods for preparing poly(esteramides) using the same. The recombinant microorganisms and methods for preparing poly(esteramides) according to the invention can biosynthesize various poly(esteramide) polymers from various carbon sources such as glucose, amino acids, hydroxy acids, and carboxylic acids, and are therefore suitable for the industrial production of poly(esteramides).

Description

Technical Field

[0001] This invention relates to metabolically engineered recombinant microorganisms and methods for producing poly(esteramides) using the same, and more specifically, to recombinant microorganisms and methods for producing poly(esteramides) using the same, wherein genes encoding coenzyme A transferases using organic acids comprising amino acids as substrates and genes encoding PHA synthases are introduced or amplified in the recombinant microorganisms. Background Technology

[0002] Due to recent oil depletion and environmental concerns, there is significant interest in using microorganisms to sustainably produce a variety of value-added chemicals and polymers. In particular, research is underway into producing various types of biopolymers to replace petrochemical-based plastics; however, to date, there have been no successful cases of using recombinant microorganisms to produce various poly(esteramides).

[0003] Poly(esteramides) are polymers containing both ester and amide bonds. Due to these properties, poly(esteramides) possess the advantages of both polyesters and polyamides. Although there have been no successful cases of poly(esteramide) production to date, numerous studies have been reported on the use of metabolic engineering to produce naturally occurring polyesters called polyhydroxyalkanoates (CHoi et al., Adv. Mat. 32: 1-37, 2020). Additionally, there are reports on the biosynthesis of poly(lactic acid-random glycolic acid) and poly(3-hydroxybutyric acid-random phenyllactic acid) using microorganisms through systems metabolic engineering, which are polyesters not found in nature (CHoi et al., Nat. Biotech. 34: 435-440, 2016; Yang et al., 9:79, 2018).

[0004] Therefore, the inventors focused on developing a method for the efficient production of poly(esteramide) from various carbon sources using microorganisms. As a result, it was confirmed that poly(esteramide) can be biosynthesized using recombinant microorganisms. These recombinant microorganisms were infused with genes encoding enzymes that convert amino acids used as substrates into aminoacyl-CoA and enzymes that polymerize aminoacyl-CoA, thus completing the present invention. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant microorganism capable of producing poly(esteramide) from various carbon sources.

[0006] Another object of the present invention is to provide a method for producing poly(esteramide) using recombinant microorganisms.

[0007] To achieve the above objectives, the present invention provides a recombinant microorganism capable of producing poly(esteramide) from a carbon source, wherein genes encoding coenzyme A transferase and genes encoding PHA (polyhydroxyalkanoate) synthase are introduced or amplified.

[0008] The present invention also provides a method for producing poly(esteramide), comprising:

[0009] (a) Culturing the recombinant microorganisms of the present invention to produce poly(esteramide); and

[0010] (b) Recycle the produced poly(esteramide).

[0011] The present invention also provides a method for producing poly(esteramide), comprising:

[0012] (a) The recombinant microorganism of the present invention was cultured in a medium supplemented with organic acids as substrates for coenzyme A transferase to produce poly(esteramide); and

[0013] (b) Recycle the produced poly(esteramide).

[0014] The present invention also provides poly(esteramide) produced by the method according to the invention. Attached Figure Description

[0015] Figure 1 Metabolic pathways for the production of various poly(esteramides) and the amino acid monomers contained in these poly(esteramides) are illustrated. Red arrows indicate newly designed metabolic reactions for the biosynthesis of poly(esteramides) that do not exist in nature, black arrows indicate metabolic pathways for the biosynthesis of polyhydroxy fatty acid esters that exist in nature, gray boxes indicate amino acid monomers that can be polymerized via metabolic pathways, pink indicates amine and amide functional groups, and blue indicates hydroxyl and ester functional groups. The listed genes represent the following enzymes: act, β-alanine CoA transferase; pct540, engineered propionate CoA transferase; phaC, polyhydroxy fatty acid ester synthase. The compounds are abbreviated as follows: 3AB, 3-aminobutyric acid; 3AIB, 3-aminoisobutyric acid; 3AP, 3-aminopropionic acid; 3AV, 3-aminovaleric acid; 4AB, 4-aminobutyric acid; 5AV, 5-aminovaleric acid.

[0016] Figure 2 shows the analytical results confirming the biosynthesis of various poly(esteramides) including various amino acid monomers. 2a shows a schematic diagram of the PEA01 strain cultured in MR medium supplemented with (RS)-3HB and target amino acids, where multiple metabolic pathways are indicated by black dashed arrows. 2b shows the fractionation of amino acid monomers in polymers obtained by culturing PEA00 and PEA01 strains according to the schematic diagram shown in (2a). 2c shows the mass spectra of 3AP, (R)-3AB, 3AIB, (R)-3AV, 4AB, and 5AV monomers and their corresponding standard compounds. The listed genes represent the following enzymes: act, β-alanine CoA transferase; ldhA, (R)-lactate dehydrogenase; pct540, engineered propionate CoA transferase; phaC1437Ps6-19, engineered Pseudomonas sp. MBEL 6-19 polyhydroxy fatty acid ester synthase. The compounds are abbreviated as follows: 3AB, 3-aminobutyric acid; 3AIB, 3-aminoisobutyric acid; 3AP, 3-aminopropionic acid; 3AV, 3-aminovaleric acid; 3HB, 3-hydroxybutyric acid; 4AB, 4-aminobutyric acid; 5AV, 5-aminovaleric acid; 6AC, 6-aminohexanoic acid; 7AH, 7-aminoheptanoic acid.

[0017] Figure 3 The results of the analysis confirming the biosynthesis of poly(esteramides) containing multiple amino acid monomers are shown. When strain PEA01 was cultured in MR medium supplemented with (RS)-3HB and 3AP, 3AP / (RS)-3AB, or 3AP / (RS)-3AB / (RS)-3AV, monomer fractions of 3AP, (R)-3AB, or (R)-3AV were obtained. The fractions of 3AP, (R)-3AB, and (R)-3AV are shown in pink, yellow, and orange, respectively. The compounds are abbreviated as follows: 3AP, 3-aminopropionic acid; 3AV, 3-aminovaleric acid.

[0018] Figure 4 shows the results of reactivity analysis to various substrates using purified β-alanine coenzyme A transferase. 4a shows a schematic diagram of the in vitro assay of Act for various substrates classified as amino acids, hydroxy acids, or carboxylic acids. 4b shows the enzymatic activity of Act for amino acids, hydroxy acids, or carboxylic acids. 4c shows the chemical structures of the substrates to which Act exhibits activity. The compounds are abbreviated as follows: 2AB, 2-aminobutyric acid; 3AB, 3-aminobutyric acid; 3AIB, 3-aminoisobutyric acid; 3AP, 3-aminopropionic acid; 3AV, 3-aminovaleric acid; 3HB, 3-hydroxybutyric acid; 4AB, 4-aminobutyric acid; 5AV, 5-aminovaleric acid; 6AC, 6-aminohexanoic acid; 7AH, 7-aminoheptanoic acid; ALA, alanine; BPA, β-phenylalanine; GLY, glycine; PAB, para-aminobenzoic acid.

[0019] Figure 5 A schematic diagram of a strain producing poly(esteramide) using glucose as the sole carbon source, its analytical results, and the results of fed-batch fermentation for poly(esteramide) production are shown. a shows a schematic diagram of strain PEA06 cultured in MR medium. b shows the fractionation of 3AP monomers in the polymer obtained by culturing strain PEA06 according to the schematic diagram shown in (a). c shows a schematic diagram of strain PEA07 cultured in MR medium. d shows the fractionation of 4AB monomers in the polymer obtained by culturing strain PEA07 according to the schematic diagram shown in (c). In the schematic diagrams, multiple metabolic pathways are indicated by black dashed arrows, and inactive reactions are marked with red "X". e shows an overview of fed-batch fermentation of strain PEA06 when glucose is used as the sole carbon source, where cell concentration, polymer content, and glucose concentration are represented by gray, orange, white, and black circles. The compounds are abbreviated as follows: 3AP, 3-aminopropionic acid; 3HB, 3-hydroxybutyric acid; 4AB, 4-aminobutyric acid; AKG, α-ketoglutarate; PEP, phosphoenolpyruvate; LA, lactic acid.

[0020] Figure 6 Representative poly(esteramides), poly(3-hydroxybutyrate-random-3-aminopropionic acid), are shown, which can be biosynthesized using various polyhydroxy fatty acid ester synthases. The listed genes represent the following enzymes: wild-type class I PhaCs (PhaCCne, PhaCAca, PhaCAhy, and PhaCAsa) derived from *C. necator*, *Aeromonas caviae*, *Aeromonas hydrophila*, and *Aeromonas salmonicida*, respectively; and those derived from *Pseudomonas* sp. MBEL 6-19, *Pseudomonas* sp. 61-3, *Pseudomonas putida* KT2440, *Pseudomonas aeruginosa* PAO1, *Pseudomonas chlororaphis*, and *Pseudomonas* sp. Engineered class II PhaCs with E130D, S325T, S477G and Q481K mutations in resinovorans (PhaC1437Ps6-19, PhaC1437Ps61-3, PhaC1437Ppu, PhaC1437Pae, PhaC1437Pch, and PhaC1437Pre, respectively). Detailed Implementation

[0021] 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. Generally, the nomenclature used herein is well-known and typical in the art.

[0022] In one embodiment of the invention, it was confirmed that poly(esteramide) can be synthesized from various carbon sources (e.g., amino acids, sugars, organic acids, etc.) using recombinant microorganisms, wherein genes encoding coenzyme A transferases using various amino acids as substrates and genes encoding PHA (polyhydroxyalkanoate) synthases are introduced into the recombinant microorganisms.

[0023] In other embodiments of the invention, it has been confirmed that poly(esteramide) can be synthesized from glucose as a single carbon source using recombinant microorganisms, which are additionally introduced with genes capable of synthesizing hydroxy acids and amino acids from glucose.

[0024] Therefore, aspects of the present invention relate to recombinant microorganisms capable of synthesizing poly(esteramides), wherein genes encoding coenzyme A transferase and genes encoding PHA (polyhydroxyalkanoate) synthase are introduced or amplified.

[0025] In this invention, the recombinant microorganism may be characterized by the introduction of a gene encoding at least one coenzyme A transferase and a gene encoding at least one PHA (polyhydroxyalkanoate) synthase.

[0026] In embodiments of the present invention, recombinant microorganisms can synthesize poly(esteramides) comprising organic acids as monomers, wherein the organic acids include amino acids as precursors.

[0027] In other embodiments of the invention, recombinant microorganisms can synthesize poly(esteramides) including amino acids and hydroxy acids as precursors.

[0028] The i) coenzyme A transferase introduced or amplified in the recombinant microorganisms of the present invention can be used to catalyze the synthesis of acyl-CoA using organic acids including amino acids as precursors, and the ii) PHA synthase can be used to catalyze the synthesis of poly(esteramide) by polymerization of synthesized acyl-CoA.

[0029] As used herein, the term "coenzyme A transferase" refers to a transferase that catalyzes the transfer of the coenzyme A group from an acyl-CoA donor to a carboxylic acid.

[0030] In this invention, coenzyme A transferase can be used to catalyze the synthesis of aminoacyl-CoA using amino acids as substrates. In this invention, amino acids are used to refer to proteogenic amino acids and non-protein amino acids that constitute proteins.

[0031] In this invention, examples of proteinogenic amino acids may include, but are not limited to, 21 naturally occurring amino acids or their isomers found in eukaryotes.

[0032] In this invention, more preferably, the amino acid is a non-protein amino acid. In this invention, non-protein amino acid means all organic acids other than protein-derived amino acids, including an amino group (-NH2) and a carboxyl group (-COOH). For example, non-protein amino acids may be selected from, but are not limited to, the group consisting of: 2-aminobutyric acid, 3-aminopropionic acid, isoserine, 3-aminobutyric acid, 3-aminovaleric acid, β-phenylalanine (BPA), 4-aminobutyric acid, 4-amino-3-methylbutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 3-aminoisobutyric acid, para-aminobenzoic acid (PAB), and their isomers.

[0033] In this invention, coenzyme A transferase can be used to synthesize acyl-CoA using carboxylic acids other than amino acids as substrates.

[0034] In this invention, the carboxylic acid is preferably a hydroxy acid including a hydroxyl group. In this invention, hydroxy acids include all organic acids, excluding proteogenic amino acids, that include a hydroxyl group (-OH) and a carboxyl group (-COOH). For example, hydroxy acids can be selected from, but are not limited to, the group consisting of: glycolic acid, lactic acid, 2-hydroxybutyric acid, 3-hydroxypropionic acid, 3-hydroxybutyric acid, carboxylic acids including those with carbon chains longer than their carbon chains, and their isomers.

[0035] In this invention, the carboxylic acid may be selected from, but is not limited to, the group consisting of: propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, 2-methylbutyric acid, hexanoic acid, malonic acid, succinic acid, carboxylic acids having a carbon chain longer than or equal to their carbon chain and their isomers, as examples other than amino acids and hydroxy acids.

[0036] In this invention, isomers can be structural isomers or stereoisomers. In this invention, when the carboxylic acid is an amino acid or a hydroxy acid, the isomers include, but are not limited to, (R)-type or (S)-type stereoisomers.

[0037] In particular, in this invention, the isomer is preferably an (R) type stereoisomer.

[0038] In this invention, the coenzyme A transferase includes wild-type, naturally occurring variants, or artificially engineered variants. Preferably, the coenzyme A transferase is at least one selected from the group consisting of: β-alanine coenzyme A transferase, propionate coenzyme A transferase, and isocaprenyl coenzyme A:2-hydroxyisocaproate coenzyme A transferase.

[0039] In this invention, the gene for coenzyme A transferase can be derived from various organisms. More preferably, the gene is selected from at least one of the following groups: 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 not limited thereto.

[0040] In this invention, preferred examples of coenzyme A transferase include, but are not limited to, sequences selected from SEQ ID NO:1 to 3 or sequences 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% identity with them.

[0041] SEQ ID NO:1: Act protein sequence from Clostridium propionitum

[0042]

[0043] SEQ ID NO:2: Engineered Pct protein sequence from Clostridium propionitum (Pct540) (Jung et al., Biotechnol. Bioeng. 105:161-171 2010)

[0044]

[0045] SEQ ID NO:3: hadA gene from Clostridium difficile

[0046]

[0047] In this invention, the sequence of the gene encoding coenzyme A transferase can be readily obtained from databases widely used in the art or from the amino acid sequence of the aforementioned coenzyme A transferase. In this invention, the gene sequence encoding coenzyme A transferase can be codon-optimized using methods known in the art to suit the host microorganism.

[0048] In embodiments of the present invention, it has been demonstrated that various organic acids can be used as substrates for coenzyme A transferases encoded by genes including SEQ ID NO:1. For example, the substrate may be a carboxylic acid, preferably an amino acid or hydroxy acid, and most preferably selected from the group consisting of: glycine, alanine (preferably (S)-alanine), 2-aminobutyric acid, 3-aminobutyric acid, 3-aminoisobutyric acid, 3-aminovaleric acid, β-phenylalanine (BPA), 4-aminobutyric acid, para-aminobenzoic acid (PAB), 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, glycolic acid, lactic acid, 2-hydroxybutyric acid, 3-hydroxypropionic acid, 3-hydroxybutyric acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, 2-methylbutyric acid, hexanoic acid, malonic acid, succinic acid, and isomers thereof, but not limited thereto.

[0049] In this invention, the isomer can be a structural isomer or a stereoisomer. In this invention, when the carboxylic acid is an amino acid or a hydroxy acid, the isomer can be, for example, an (R) type or an (S) type stereoisomer, more preferably an (R) type isomer.

[0050] As used herein, the term "PHA (polyhydroxyalkanoate) synthase" refers to an enzyme that synthesizes polyhydroxyalkanoates from hydroxyacyl-CoA. In this invention, it has been confirmed that PHA synthase can use aminoacyl-CoA as a substrate to synthesize poly(esteramides).

[0051] In this invention, PHA synthases include wild-type, naturally occurring variants, or artificially engineered variants. In this invention, PHA synthases can be class I PhaC or class II PhaC, but are not limited thereto. In this invention, the gene sequence of engineered PHA synthases is described, for example, in Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010, but the invention is not limited thereto.

[0052] In this invention, the gene encoding PHA synthase can be the phaC gene or a variant thereof derived from various organisms.

[0053] In this invention, the gene encoding PHA synthase can be derived from various organisms. Specifically, the gene encoding PHA synthase can be derived from any microorganism with PHA synthetic activity, but is not limited to it. Preferred examples of genes encoding PHA synthase include, but are not limited to, the following: phaC genes derived from strains selected from the group consisting of: *Cupriavidus necator*, *Aeromonas guinea pigensis*, *Aeromonas hydrophila*, *Aeromonas salmonicidae*, *Pseudomonas* spp. MBEL6-19, *Pseudomonas* spp. 61-3, *Pseudomonas putidae* KT2440, *Pseudomonas aeruginosa* PAO1, *Pseudomonas aeruginosa*, and *Pseudomonas resinophilus*.

[0054] In this invention, preferred examples of the protein sequence of PHA synthase include, but are not limited to, sequences selected from SEQ ID NO:4 to 13 or sequences 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% identity with them.

[0055] SEQ ID NO:4: Wild-type type I PhaC from hookworm copper-loving fungus

[0056]

[0057] SEQ ID NO:5: Wild-type class I PhaC from Aeromonas vaginalis

[0058]

[0059] SEQ ID NO:6: Type I PhaC from Aeromonas hydrophila

[0060]

[0061] SEQ ID NO:7: Wild-type class I PhaC from Aeromonas salmonidae

[0062]

[0063] SEQ ID NO:8: Engineered class II PhaC from Pseudomonas MBEL 6-19 (Jung et al., Biotechnol. Bioeng. 105:161-171 2010) (phaC1437Ps6-19)

[0064]

[0065] SEQ ID NO:9: Engineered class II PhaC from Pseudomonas 61-3 (Yang et al., Appl. Microbiol. Biotech. 90: 603-614 2011) (phaC1437Ps613)

[0066]

[0067] SEQ ID NO:10: Engineered class II PhaC from *Pseudomonas putida* KT2440 (Yang et al., *Appl. Microbiol. Biotech.* 90: 603-614 2011)

[0068]

[0069] SEQ ID NO:11: Engineered class II PhaC from Pseudomonas aeruginosa PAO1 (Yang et al., Appl. Microbiol. Biotech. 90: 603-614 2011)

[0070]

[0071] SEQ ID NO:12: Engineered class II PhaC from *Pseudomonas aeruginosa* (Yang et al., *Appl. Microbiol. Biotech.* 90: 603-614 2011)

[0072]

[0073] SEQ ID NO:13: Engineered class II PhaC from Pseudomonas aeruginosa (Yang et al., Appl. Microbiol. Biotech. 90: 603-614 2011)

[0074]

[0075] In this invention, the sequence of the gene encoding PHA synthase can be readily obtained from databases widely used in the art or from the amino acid sequence of the aforementioned PHA synthase. In this invention, the gene sequence encoding PHA synthase can be codon-optimized using methods known in the art to suit the host microorganism.

[0076] As used herein, the term "poly(ester amide)" is used to refer to polymers having both ester and amide bonds, but is not limited thereto. The structures and uses of various poly(ester amides), including a variety of amino acid-based poly(ester amides), are well known in the art (e.g., Polym. Chem., 2016, 7, 7039-7046).

[0077] In this invention, poly(esteramide) may include an organic acid as a monomer, which is a substrate of coenzyme A transferase.

[0078] In this invention, poly(esteramide) may include amino acids that serve as substrates for coenzyme A transferase as monomers.

[0079] In this invention, poly(esteramide) is characterized by comprising amino acids and hydroxy acids as substrates for coenzyme A transferase as monomers.

[0080] In this invention, as a non-limiting example, poly(esteramide) may include, but is not limited to, structures represented by the following chemical formulas:

[0081]

[0082] R1 to R4 are each independently any chemical residue, but not limited to, preferably each independently hydrogen, alkyl, alcohol or amine, and x, y, n and m are each independently 0, or 1 or a larger integer.

[0083] (0 indicates that it does not exist)

[0084] In this invention, poly(esteramide) can be a polymer comprising one or more monomers.

[0085] In this invention, the monomers and their composition ratios in the poly(esteramide) synthesized by recombinant microorganisms can be controlled according to the level of organic acids (e.g., amino acids and hydroxy acids) in the monomers (i.e. precursors) used as poly(esteramide), which is obvious to those skilled in the art.

[0086] In embodiments of the present invention, for example, the monomers and their composition ratios in the poly(esteramide) synthesized by recombinant microorganisms can be controlled according to the concentration and ratio of the supplied carbon source (sugar or organic acid).

[0087] In other embodiments of the invention, for example, the monomers and their composition ratios 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 by, but not limited to, creating, strengthening, weakening or blocking the precursor biosynthesis pathway through genetic engineering.

[0088] In this invention, the host microorganisms in which the genes encoding coenzyme A transferase and PHA (polyhydroxyalkanoate) synthase are introduced or amplified can be selected from the group consisting of: *Escherichia coli*. *Rhizobium*, *Bifidobacterium*, *Rhodococcus*, *Candida*, *Erwinia*, *Enterobacter*, *Pasteurella*, *Mannheimia*, *Actinobacillus*, *Aggregatibacter*, *Xanthomonas*, *Vibrio*, *Pseudomonas*, *Azotobacter*, *Acinetobacter*, *Ralstonia*, *Agrobacterium* The genera *Aspergillus*, *Rhodobacter*, *Zymomonas*, *Bacillus*, *Staphylococcus*, *Lactococcus*, *Streptococcus*, *Lactobacillus*, *Clostridium*, *Corynebacterium*, *Streptomyces*, *Bifidobacterium*, *Cyanobacterium*, *Saccharomyces*, and *Cyclobacterium*, but not limited to these.

[0089] In this invention, for example, the host microorganism is preferably selected from the group consisting of: *Escherichia coli*, *Bacillus subtilis*, *Corynebacterium glutamicum*, *Lactobacillus brevis*, *Lactobacillus casei*, *Lactobacillus reuteri*, *Lactococcus lactis*, *Aspergillus niger*, *Saccharomyces cerevisiae*, and *Saccharomyces pombe*, but is not limited thereto.

[0090] Most preferably, the host microorganism can be Escherichia coli, but is not limited to this.

[0091] In embodiments of the present invention, it has been demonstrated that when various carbon sources (hydroxy acids, sugars, amino acids) are provided to recombinant microorganisms, the aforementioned carbon sources can be used as substrates to synthesize poly(esteramides), and when recombinant microorganisms are engineered through additional genetic modifications to have the ability to synthesize precursors (e.g., hydroxy acids and amino acids), poly(esteramides) can be synthesized from a single carbon source (e.g., glucose).

[0092] In embodiments of the present invention, the recombinant microorganisms may not have the ability to produce coenzyme A transferase or PHA synthase substrates. Poly(esteramide) can be synthesized by providing coenzyme A transferase or PHA synthase substrates externally.

[0093] In other embodiments of the invention, the recombinant microorganism may have 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 this invention, the organic acid may be, for example, an amino acid.

[0094] In this invention, examples of carbon sources may include, but are not limited to, sugars or organic acids.

[0095] In this invention, the sugar used as a carbon source can be selected from, but is not limited to, the group consisting of monosaccharides, disaccharides, and polysaccharides, including, for example, glucose, sucrose, galactose, maltose, xylose, glycerol, fructose, and sucrose.

[0096] In this invention, the organic acid can be an organic acid produced in the metabolic pathway of a carbon source (e.g., pyruvate, glutamic acid, etc.), but is not limited thereto.

[0097] As used in this article, the term "biosynthetic pathway" refers to a biological pathway that can produce target substances from organic acid precursors through enzymatic reactions.

[0098] In this invention, recombinant microorganisms can possess an inherent biosynthetic pathway of organic acids that serve as substrates for coenzyme A transferase.

[0099] In this invention, recombinant microorganisms can be engineered to have biosynthetic pathways for organic acids that are substrates of coenzyme A transferase. For example, biosynthetic pathways for organic acids that are substrates of coenzyme A transferase can be generated, enhanced, weakened, or blocked by introducing, amplifying, weakening, or deleting genes associated with the biosynthetic pathway.

[0100] For example, as shown in Table 2 of the embodiments of the present invention, recombinant microorganisms have enhanced production of hydroxyacyl-CoA and amino acids by additional engineering of at least one of the following: deletion or weakening of the ldhA gene; introduction or amplification of the phaAB gene; introduction or amplification of the davAB gene; and introduction or amplification of the panD gene or the gadB gene, but the present invention is not limited thereto.

[0101] As used herein, the term "host microorganism" means any microorganism capable of expressing functional genes and / or gene products derived from other cells or organs, and more specifically, it means the microorganism prior to the introduction or amplification of the gene encoding coenzyme A transferase and the gene encoding PHA synthase according to the present invention.

[0102] As used in this article, the term “recombination” refers to polynucleotides or proteins that are not naturally present in host cells.

[0103] In this invention, introduction means that the gene is either used as an extrachromosomal element or becomes replicable through chromosomal integration. The gene is transformed, and the transformed gene can be inserted into the chromosome of a microorganism or located extrachromosomally, as long as it can be expressed within the microorganism. Furthermore, the gene can include DNA and RNA, as a polynucleotide capable of encoding a protein.

[0104] In this paper, the term "gene" should be considered in the broadest sense and can encode structural or regulatory proteins. Therefore, regulatory proteins include transcription factors, heat shock proteins, or proteins involved in DNA / RNA replication, transcription, and / or translation.

[0105] As used herein, the terms “amplification” or “enhancement” are concepts that encompass the increase of the activity of a corresponding enzyme or thus the enhancement of pathway progression by mutating, substituting or deleting some bases of a gene, introducing some bases, or introducing a gene from another microorganism encoding the same enzyme.

[0106] In this invention, "attenuation" encompasses the concept of reducing the activity of an enzyme expressed by a gene by mutating, substituting, or deleting some bases of the gene or by introducing some bases, and includes all ways of blocking part or most of the biosynthetic pathways in which the enzyme of the gene participates.

[0107] In this invention, "deletion" encompasses the concept of preventing a gene from being expressed, or preventing a gene from exhibiting enzymatic activity even if it is expressed, by mutating, substituting, deleting, or introducing a portion or all of the bases of the gene, and includes all ways of blocking the biosynthetic pathways involving the enzymes of the gene.

[0108] In this invention, "vector" means a DNA construct containing a DNA sequence operatively ligated to a suitable regulatory sequence capable of expressing the DNA in a suitable host. The vector can 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, it can integrate into the genome itself. Since plasmids are currently the most commonly used form of vector, "plasmid" and "vector" are sometimes used interchangeably in the context of this invention. For the purposes of this invention, plasmid vectors are preferred. Typical plasmid vectors used for this purpose may have the following structures: (a) a replication origin that enables efficient replication, such that each host cell includes one to hundreds of plasmid vectors; (b) an antibiotic resistance gene that enables selection of host cells transformed with the plasmid vector; and (c) a restriction endonuclease cleavage site into which a foreign DNA fragment can be inserted. Even in the absence of a suitable restriction enzyme cleavage site, the vector and foreign DNA can be readily ligated using synthetic oligonucleotide aptamers or adapters according to typical methods. After ligation, the vector must be transformed into a suitable host cell. The transformation can be readily achieved using the calcium chloride method or electroporation (Neumann, et al. EMBO J., 1:841, 1982).

[0109] In this invention, a base sequence is "operably linked" when it is functionally related to another nucleic acid sequence. This can be a gene and one or more regulatory sequences linked in such a way that the gene can be expressed when a suitable molecule (e.g., a transcription activator protein) binds to one or more regulatory sequences. For example, the DNA of the current sequence or secretion leader sequence is operably linked to the DNA of the polypeptide when it is expressed as a precursor protein involved in polypeptide secretion; the promoter or enhancer is operably linked to the coding sequence when it affects the transcription of the sequence; the ribosome binding site is operably linked to the coding sequence when it affects the transcription of the sequence; or the ribosome binding site is operably linked to the coding sequence when it is localized to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretion leader sequence, they are contiguous and present within the reading frame. However, enhancers do not necessarily have to be contiguous. These sequences are linked by ligation at convenient restriction endonuclease sites. When such sites are missing, synthetic oligonucleotide adaptors or linkers are used according to typical methods.

[0110] In this invention, any gene can be introduced into a host microorganism along with a suitable promoter. For example, in this invention, a gene can be introduced along 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 the invention is not limited thereto.

[0111] Other aspects of the invention relate to a method for producing poly(esteramide), comprising: (a) culturing the recombinant microorganism of the invention to produce poly(esteramide) and (b) recovering the produced poly(esteramide).

[0112] In step (a) according to the invention, poly(esteramide) can be produced by culturing in a medium supplemented with an external carbon source. In this invention, the carbon source can be at least one selected from the group consisting of sugars, amino acids, and hydroxy acids, but is not limited thereto.

[0113] In this invention, as described above regarding recombinant microorganisms, when the host microorganism naturally or through additional genetic engineering possesses a biosynthetic pathway for organic acids (e.g., hydroxy acids or amino acids) that serve as substrates for the coenzyme A transferase of this invention, poly(esteramide) can be produced by culturing the recombinant microorganisms of this invention without adding a single carbon source or without adding a carbon source separately from the outside.

[0114] Another aspect of the present invention relates to a method for producing poly(esteramide), comprising:

[0115] (a) The recombinant microorganism of the present invention was cultured in a medium supplemented with organic acids as substrates for coenzyme A transferase to produce poly(esteramide); and

[0116] (b) Recycle the produced poly(esteramide).

[0117] In this invention, known culture methods can be used to culture recombinant microorganisms. In addition to the specific culture media and specific culture methods used in the examples of this invention, saccharified liquids such as whey, CSL (corn steep liquor) and other culture media can also be used, and various methods such as fed-batch culture and continuous culture can be employed (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] As used herein, the term "culture" means culturing microorganisms to achieve a desired effect, and in this invention, the desired effect means the production of poly(esteramide). Here, culturing can typically be carried out in an incubator equipped with a suitable culture medium optimized for the microorganisms used and containing at least one simple energy source and, if necessary, a co-substrate.

[0119] In this article, the term “culture” can be used interchangeably with “fermentation” in the sense of producing poly(esteramide) through microbial culture.

[0120] In this invention, the production of poly(esteramide) by culturing recombinant microorganisms is characterized by being carried out through fed-batch fermentation.

[0121] As used herein, the terms “feed-batch culture” or “feed-batch fermentation” refer to a culture / fermentation in which the concentration of the culture medium is controlled by intermittently supplying additional culture medium.

[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 potassium dihydrogen phosphate or dipotassium hydrogen phosphate; trace elements (e.g., metal salts) such as magnesium salts, cobalt salts, and / or manganese salts; and nutrients essential or beneficial to cell maintenance and / or growth, such as growth factors, like amino acids and vitamins; yeast extracts, antibiotics, etc. In this invention, cultivation can be carried out within a suitable temperature range for the production of poly(esteramide), for example, 20°C to 40°C, preferably 25°C to 35°C, but the invention is not limited thereto and can be carried out under different conditions depending on the type of host microorganism, the material being expressed, etc.

[0123] In this document, the term "vector" means a DNA construct containing a DNA sequence operatively linked to a suitable regulatory sequence capable of expressing that DNA in a suitable host. A vector can 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, it can integrate into the genome itself. Because plasmids are currently the most commonly used form of vector, "plasmid" and "vector" are sometimes used interchangeably in the context of this invention. However, this invention includes other forms of vectors having the same functions as those known or to be known in the art. Typical expression vectors for expression in mammalian cell cultures are based on, for example, pRK5 (EP 307,247), pSV16B (WO 91 / 08291), and pVL1392 (Pharmingen).

[0124] The term "expression control sequence" refers to the DNA sequence necessary for the expression of a coding sequence operablely linked to it in a specific host organism. This control sequence includes promoters for transcription, operon sequences for regulating such transcription, sequences encoding appropriate mRNA ribosome binding sites, and sequences regulating transcription and translation termination. For example, control sequences suitable for prokaryotes include promoters, any operon sequences, and ribosome binding sites. In eukaryotic cells, they include promoters, polyadenylation signals, and enhancers. The promoter is the most influential factor on gene expression levels in plasmids. SRα promoters and promoters derived from cytomegalovirus are preferred as promoters for high expression.

[0125] To express the DNA sequence of the present invention, any of a variety of expression control sequences can be used in the vector. Examples of useful expression control sequences include 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 operon and promoter regions of bacteriophage λ, the regulatory region of fd-encoded proteins, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of phosphatases such as Pho5, promoters of yeast α-mating system, other conformations and inductions known for regulating gene expression in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. The T7 RNA polymerase promoter Φ10 can be used to express the protein NSP in Escherichia coli.

[0126] When a nucleic acid is functionally related to other nucleic acid sequences, it is "operably linked." This can be a gene and one or more regulatory sequences linked in such a way that the gene can be expressed when a suitable molecule (e.g., a transcription activator protein) binds to one or more regulatory sequences. For example, when expressed as a pre-protein involved in polypeptide secretion, the DNA of the pre-sequence or secretory leader sequence is operably linked to the DNA of the polypeptide; when influencing the transcription of the sequence, a promoter or enhancer is operably linked to the coding sequence; when influencing the transcription of the sequence, a ribosome binding site is operably linked to the coding sequence; or when the ribosome binding site is positioned to promote translation, it is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, they are contiguous and present within the reading frame. However, enhancers do not need to be contiguous. These sequences are linked by ligation at convenient restriction endonuclease sites. When such sites are missing, synthetic oligonucleotide adaptors or linkers according to typical methods are used.

[0127] As used herein, the term "expression vector" generally refers to a fragment of double-stranded DNA that serves as a recombinant vector, into which a foreign DNA fragment is inserted. Here, "foreign DNA" is foreign DNA that is not naturally present in the host cell. Once in the host cell, the expression vector can replicate independently of the host chromosomal DNA and can produce copies of the vector and its inserted (foreign) DNA.

[0128] As is well known in the art, in order to increase the expression level of a transfected gene in a host cell, the gene must be operatively linked to a transcriptional and translational expression control sequence that functions in the selected expression host. Preferably, the expression control sequence and the corresponding gene are contained in a single expression vector comprising both a bacterial selection marker and an origin of replication. When the expression host is a eukaryotic cell, the expression vector must further include a useful expression marker in the eukaryotic expression host.

[0129] Host cells transformed or transfected with the above-described expression vectors constitute other aspects of the present invention. As used herein, the term "transformation" means the introduction of DNA into a host such that the DNA becomes reproducible as an extrachromosomal element or through the completion of chromosomal integration. As used herein, the term "transfection" means that the expression vector is accommodated in a host cell, regardless of whether any coding sequence is actually expressed.

[0130] The host cells of this invention can be prokaryotic or eukaryotic cells. Additionally, hosts with high DNA importation efficiency and high expression efficiency of the imported DNA are typically used. Examples of host cells that can be used include well-known eukaryotic and prokaryotic hosts such as *Escherichia 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 human cells cultured from tissue. When cloning cDNA encoding the enzymes of this invention (coenzyme A transferase and PHA synthase), animal cells are preferred as the host. When using COS cells, since the SV40 large T antigen is expressed in COS cells, plasmids with the SV40 origin of replication are present in the cells, and higher expression than usual can be expected. The imported DNA sequence can be obtained from the same species as the host cell, from a different species than the host cell, or can be a hybrid DNA sequence that includes any heterologous or homologous DNA.

[0131] It should also be understood that not all vectors and expression control sequences function sufficiently identically when expressing the DNA sequences of the present invention. Similarly, not all hosts function identically for the same expression system. However, those skilled in the art can make appropriate selections from a variety of vectors, expression control sequences, and hosts without incurring excessive experimental burden or departing from the scope of the invention. For example, when selecting a vector, the host must be considered, as the vector must replicate within it. The copy number of the vector, the ability to control the copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. When selecting an expression control sequence, various factors must be considered. For example, the relative strength, controllability, and compatibility with the DNA sequences of the present invention, particularly aspects related to possible secondary structures, must be considered. Factors such as the selected vector, the toxicity of the product encoded by the DNA sequences of the present invention, secretion characteristics, the ability to accurately fold proteins, culture and fermentation factors, and the ease of purifying the product encoded by the DNA sequences of the present invention from the host must be considered. Within these variables, those skilled in the art can select various vector / expression control sequence / host combinations capable of expressing the DNA sequences of the present invention in fermentation or large-scale animal culture. When attempting to clone the cDNA of the NSP protein through expression cloning, methods such as binding, panning, and membrane emulsion can be used as screening methods.

[0132] In the context of this invention, "substantially pure" means that the polypeptide according to the invention and the DNA sequence encoding the polypeptide substantially do not contain other proteins derived from bacteria.

[0133] Prokaryotic cells, such as *Escherichia coli* and *Bacillus subtilis*, have been widely used as host cells for recombinant protein expression due to their ability to be cultured at high density in a short time, ease of genetic manipulation, and well-defined genetic and physiological characteristics. However, to address issues related to post-translational modifications, secretion processes, the three-dimensional structure of active forms, and the active state of proteins, recently, single-celled eukaryotic cells from higher organisms, such as yeast species (*Pichia pastoris*, *Saccharomyces cerevisiae*, *Hansenula polymorpha*, etc.), filamentous fungi, insect cells, plant cells, and mammalian cells, have been used as host cells for recombinant protein production. Therefore, not only *Escherichia coli* as illustrated in the examples, but other host cells can also be readily used, as will be apparent to those skilled in the art.

[0134] Another aspect of the present invention relates to poly(esteramide) produced by the method of the present invention.

[0135] [Example]

[0136] A better understanding of the present invention can be obtained through the following embodiments. These embodiments are set forth merely as illustrative of the invention and should not be construed as limiting the scope of the invention, as will be apparent to those skilled in the art.

[0137] Example 1: Construction of vectors for genetic engineering

[0138] Example 1-1: pTac15k_davAB vector

[0139] Using the pKE112_davAB vector (Park et al., Metab. Eng. 16:42-47 2013) as a DNA template and primers for SEQ ID NO:14 and 15, the davAB gene fragment encoding 5-aminopentanamide amide hydrolase and L-lysine monooxygenase was constructed by PCR.

[0140] [SEQ ID NO: 14] P1:

[0141] 5'-AGACAGGAATTCATGCGCATCGCTCTGTACC-3'

[0142] [SEQ ID NO: 15] P2:

[0143] 5'-AGACAGGGATCCTCAATCCGCCAG-3'

[0144] The recombinant plasmid pTac15k_davAB was then constructed by treating the davAB fragment and the pTac15k plasmid with strong gene expression via the tac promoter using restriction endonucleases (EcoRI and BamHI) (US Patent 20110269183), followed by treatment with T4 DNA ligase to ligate the restriction endonuclease-cleaved davAB fragment and the pTac15k plasmid.

[0145] Examples 1-2: pTac15k_act_davAB vector

[0146] Using the pKE112_davAB vector (Park et al., Metab. Eng. 16:42-47 2013) as a DNA template and primers for SEQ ID NO:16 and 17, the davAB gene fragment encoding 5-aminopentanamide amide hydrolase and L-lysine monooxygenase was constructed by PCR.

[0147] [SEQ ID NO: 16] P3:

[0148] 5'-AGACAGGAGCTCTTTCACACAGGAAACAATGCGCATCGCTCTGTACC-3'

[0149] [SEQ ID NO: 17] P4:

[0150] 5'-AGACAGCTGCAGTCAATCCGCCAGGGCG-3'

[0151] The recombinant plasmid pTac15k_act_davAB was then constructed as follows: the davAB fragment and the pTac15k_act vector with strong gene expression via the tac promoter were treated with restriction endonucleases (SacI and PstI) (Chae et al., Metab. Eng. 41: 82-91, 2017), followed by treatment with T4 DNA ligase to ligate the restriction endonuclease-cleaved davAB fragment and the pTac15k_act vector.

[0152] Examples 1-3: pTac15k_act_panD vector

[0153] Using chromosomal DNA from Corynebacterium glutamicum strain as a template and primers SEQ ID NO:18 and 19, the panD gene fragment encoding aspartate decarboxylase was constructed by PCR.

[0154] [SEQ ID NO: 18] P5:

[0155] 5'-AGACAGGAGCTCTTTCACACAGGAAACAATGCTGCGCACCATCC-3'

[0156] [SEQ ID NO: 19] P6:

[0157] 5'-AGACAGGGTACCCTAAATGCTTCTCGACGTCAAAAG-3'

[0158] The recombinant plasmid pTac15k_act_panD was then constructed as follows: the panD fragment and the pTac15k_act plasmid were treated with restriction endonucleases (SacI and KpnI) (Chae et al., Metab. Eng. 41: 82-91, 2017), followed by treatment with T4 DNA ligase to ligate the restriction endonuclease-cleaved panD fragment and the pTac15k_act plasmid.

[0159] Examples 1-4: pPs619C1437PhaAB vector

[0160] Using chromosomal DNA from hookworm Copper-loving bacteria as a template and primers SEQ ID NO:20 and 21, the phaAB gene fragment encoding acetyl-CoA acetyltransferase and acetyl-CoA reductase was constructed by PCR.

[0161] [SEQ ID NO: 20] P7:

[0162] 5'-AGACAGTTCGAATAGTGACGGCAGAGAGACAATCAAATC-3'

[0163] [SEQ ID NO: 21] P8:

[0164] 5'-AGACAGCCTGCAGGCCTGCCG-3'

[0165] The recombinant plasmid pPs619C1437PhaAB was then constructed as follows: the phaAB fragment and the pPs619C1437Pct540 plasmid were treated with restriction endonucleases (BstBI and SbfI) (Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010), followed by treatment with T4 DNA ligase to ligate the restriction endonuclease-digested phaAB fragment and the pPs619C1437Pct540 plasmid.

[0166] Examples 1-5: pCneCPct540 vector

[0167] Using chromosomal DNA from hookworm copper-loving bacteria as a template and primers SEQ ID NO:22 and 23, the phaCCne gene fragment encoding polyhydroxy fatty acid ester synthase was constructed by PCR.

[0168] [SEQ ID NO: 22] P9:

[0169] 5'-AGACAGTTCGAATAGTGACGGCAGAGAGACAATCAAATCATGGCGACCGGCAAAG-3'

[0170] [SEQ ID NO: 23] P10:

[0171] 5'-AGACAGCCTGCAGGCCTGCCGGCGCCGTGCATGACGCACGCCGGCACTCATGCAAGCGTCATGCCTTGGCTTTGACG-3'

[0172] The recombinant plasmid pCneCPct540 was then constructed as follows: the phaCCne fragment and the pPs619C1437Pct540 plasmid were treated with restriction endonucleases (BstBI and SbfI) (Jung et al., Biotechnol. Bioeng., 105:161-171, 2010), followed by treatment with T4 DNA ligase to ligate the restriction endonuclease-digested phaCCne fragment and the pPs619C1437Pct540 plasmid.

[0173] Examples 1-6: pAcaCPct540, pAhyCPct540 and pAsaCPct540 vectors

[0174] Using chromosomal DNA from *Aeromonas guinea pigensis*, *Aeromonas hydrophila*, and *Aeromonas salmonicidae* strains as templates and primers SEQ ID NO:24 and 25, gene fragments encoding polyhydroxy fatty acid ester synthases, namely PhaCAca, phaCAhy, and phaCAsa, were constructed by PCR.

[0175] [SEQ ID NO: 24] P11:

[0176] 5'-AGACAGTTCGAATAGTGACGGCAGAGAGACAATCAAATCATGAGCCAACCATCTTATGGC-3'

[0177] [SEQ ID NO: 25] P12:

[0178] 5'-AGACAGCCTGCAGGCCTGCCGGCGCCGTGCATGACGCACGCCGGCACTCATGCAAGCGTCATGCGGCGTCCTCCT-3'

[0179] Then, the recombinant plasmids pAcaCPct540, pAhyCPct540, and pAsaCPct540 were constructed as follows: the phaCAca, phaCAhy, and phaCAsa gene fragments and the pPs619C1437Pct540 plasmid were treated with restriction endonucleases (BstBI and SbfI) (Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010), followed by treatment with T4 DNA ligase to ligate the restriction endonuclease-digested phaCAca, phaCAhy, and phaCAsa gene fragments and the pPs619C1437Pct540 plasmid.

[0180] Information regarding the vectors constructed in Examples 1-1 to 1-6 is shown in Table 1 below.

[0181] [Table 1]

[0182] name Basic carrier Imported genes Remark pTac15k_davAB pTac15k davAB import Example 1-1 pTac15k_act_davAB pTac15k_act <![CDATA[ davAB Import]]> Examples 1-2 pTac15k_act_panD pTac15k_act <![CDATA[ panD Import]]> Examples 1-3 pPs619C1437PhaAB pPs619C1437Pct540 <![CDATA[ Pct540 Import]]> Examples 1-4 pCneCPct540 pPs619C1437Pct540 <![CDATA[ PhaCCne Import]]> Examples 1-5 pAcaCPct540 pPs619C1437Pct540 <![CDATA[ PhaCAca Import]]> Examples 1-6 pAhyCPct540 pPs619C1437Pct540 <![CDATA[ PhaCAhy Import]]> Examples 1-6 pAsaCPct540 pPs619C1437Pct540 <![CDATA[ PhaCAsa Import]]> Examples 1-6

[0183] Example 2: Production of Recombinant Microorganisms

[0184] The recombinant microorganisms shown in Table 2 below were generated using the vector constructed in Example 1.

[0185] [Table 2]

[0186] name Host microorganism Imported carrier Engineering features Remark PEA00 (Control) XL1 - Blue pPs619C1437Pct540 (Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010), pTac15k (US Patent 20110269183) pct540 and phaC1437Ps6-19 gene introduction Examples 3 and 6 PEA01 XL1 - Blue pPs619C1437Pct540 (Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010), pTac15k_act (Chae et al., Metab. Eng. 41: 82-91, 2017) pct540, phaC1437Ps6-19, and act gene introduction Examples 3, 6, and 7 PEA02 (control) XL1 - Blue pPs619C1437Pct540 (Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010), pTac15k_davAB (Example 1-1) pct540, phaC1437Ps6-19, and davAB gene introduction Example 4 PEA03 XL1 - Blue pPs619C1437Pct540 (Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010), pTac15k_act_davAB (Examples 1-2) pct540, phaC1437Ps6-19, act, and davAB gene introduction Example 4 PEA04 XL1-Blue ΔldhA pPs619C1437Pct540 (Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010), pTac15k_act (Chae et al., Metab. Eng. 41: 82-91, 2017) pct540, phaC1437Ps6-19, act gene introduction, ldhA gene deletion Example 8 PEA05 XL1-Blue ΔldhA pPs619C1437PhaAB (Examples 1-4), pTac15k-act (Chae et al., Metab.Eng. 41: 82-91, 2017) phaAB, phaC1437Ps6-19, act gene introduction, ldhA gene deletion Example 8 PEA06 XL1-Blue ΔldhA pPs619C1437PhaAB (Examples 1-4), pTac15k-act_panD (Examples 1-3) phaAB, phaC1437Ps6-19, act, panD gene introduction, ldhA gene deletion Examples 8 and 9 PEA07 XL1-Blue ΔldhA pPs619C1437PhaAB (Examples 1-4), pTac15k_act_gadB_mut (Chae et al., Metab. Eng. 41: 82-91, 2017) phaAB, phaC1437Ps6-19, act, gadB_mut gene introduction, ldhA gene deletion Examples 8 and 9 PEA08 XL1-Blue ΔldhA pCneCPct540 (Examples 1-5), pTac15k_act (Chae et al., Metab. Eng. 41:82-91, 2017) pct540, phaCCne, act gene introduction, ldhA gene deletion Example 10 PEA09 XL1-Blue ΔldhA pAcaCPct540 (Examples 1-6), pTac15k_act (Chae et al., Metab. Eng. 41:82-91, 2017) pct540, phaCAca, act gene introduction, ldhA gene deletion Example 10 PEA10 XL1-Blue ΔldhA pAhyCPct540 (Examples 1-6), pTac15k_act (Chae et al., Metab. Eng. 41:82-91, 2017) pct540, phaCAhy, act gene introduction, ldhA gene deletion Example 10 PEA11 XL1-Blue ΔldhA pAsaCPct540 (Examples 1-6), pTac15k_act (Chae et al., Metab. Eng. 41:82-91, 2017) pct540, phaCAsa, act gene introduction, ldhA gene deletion Example 10 PEA12 XL1-Blue ΔldhA pPs613C1437Pct540 (Yang et al., Appl. Microbiol. Biotechnol. 90:603-614, 2011), pTac15k_act (Chae et al., Metab. Eng. 41: 82-91, 2017) pct540, phaC1437Ps613, act gene introduction, ldhA gene deletion Example 10 PEA13 XL1-Blue ΔldhA pPpuC1437Pct540 (Yang et al., Appl. Microbiol. Biotechnol. 90:603-614, 2011), pTac15k_act (Chae et al., Metab. Eng. 41: 82-91, 2017) pct540, phaC1437Ppu, act gene introduction, ldhA gene deletion Example 10 PEA14 XL1-Blue ΔldhA pPaeC1437Pct540 (Yang et al., Appl. Microbiol. Biotechnol. 90:603-614, 2011), pTac15k_act (Chae et al., Metab. Eng. 41: 82-91, 2017) pct540, phaC1437Pae, act gene introduction, ldhA gene deletion Example 10 PEA15 XL1-Blue ΔldhA pPchC1437Pct540 (Yang et al., Appl. Microbiol. Biotechnol. 90:603-614, 2011), pTac15k_act (Chae et al., Metab. Eng. 41: 82-91, 2017) pct540, phaC1437Pch, act gene introduction, ldhA gene deletion Example 10 PEA16 XL1-Blue ΔldhA pPreC1437Pct540 (Yang et al., Appl. Microbiol. Biotechnol. 90:603-614, 2011), pTac15k_act (Chae et al., Metab. Eng. 41: 82-91, 2017) pct540, phaC1437Pre, act gene introduction, ldhA gene deletion Example 10

[0187] Recombinant microorganisms (PEA01) were produced by introducing the pTac15k_act plasmid (Chae et al., Metab. Eng. 41: 82-91, 2017) which expresses the act gene encoding β-alanine coenzyme A transferase in microorganisms and the pPs619C1437Pct540 plasmid (Jung et al., Biotechnol. Bioeng., 105: 161-171, 2010) which expresses the phaC1437Ps6-19 gene encoding engineered polyhydroxy fatty acid ester synthase and the pct540 gene encoding engineered propionate coenzyme A transferase into Escherichia coli XL1-Blue (Stratagene). Escherichia coli introduced with the empty vector pTac15k (PEA00) was used as a control strain.

[0188] In addition, to verify the possibility of production from various carbon sources, the pTac15k_act_davAB vector constructed in Examples 1-2 to express the act gene encoding β-alanine coenzyme A transferase, the davA gene encoding 5-aminopentanamide aminohydrolase, and the davB gene encoding L-lysine monooxygenase in microorganisms was introduced into Escherichia coli XL1-Blue / pPs619C1437Pct540 to produce recombinant microorganism (PEA03), and Escherichia coli (PEA02) introduced with pTac15k_davAB constructed in Example 1-1 was used as a control strain.

[0189] In addition, to verify the feasibility of production using glucose as the sole carbon source and efficient production through fed-batch fermentation, recombinant microorganisms (XL1-Blue ΔldhA / pPs619C1437PhaAB) were produced by introducing the pPs619C1437PhaAB plasmid constructed in Example 1 into Escherichia coli XL1-BlueΔldhA. This plasmid was used to express the phaC1437Ps6-19 gene encoding engineered polyhydroxy fatty acid ester synthase, the phaA gene encoding acetyl-CoA acetyltransferase, and the phaB gene encoding acetoacetyl-CoA reductase. E. coli strains (PEA06 and PEA07) carrying either the pTac15k_act_panD vector constructed in Example 1 to express the act gene encoding β-alanine coenzyme A transferase and the panD gene encoding aspartate decarboxylase, or the pTac15k_act_gadB_mut vector (Chae et al., Metab. Eng. 41: 82-91, 2017) carrying the act gene encoding β-alanine coenzyme A transferase 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) that did not express the panD or gadB_mut genes was introduced into the XL1-Blue ΔldhA / pPs619C1437PhaAB strain was used.

[0190] In addition, to confirm the possibility of utilizing polyhydroxyalkanoate synthases derived from various microorganisms, the vectors pCneCPct540, pAcaCPct540, pAhyCPct540, and pAsaCPct540 constructed in Example 1 (these vectors allow expression of the phaC gene encoding polyhydroxyalkanoate synthase derived from hookworm *Aeromonas gargarizans*, *Aeromonas vulgaris*, *Aeromonas hydrophila*, and *Aeromonas salmonicidae*, respectively), as well as the previously reported vectors pPs619C1437Pct540, pPs613C1437Pct540, pPpuC1437Pct540, pPaeC1437Pct540, pPchC1437Pct540, and pPreC1437Pct540 (Yang et al., Appl. Microbiol. Biotechnol. 90: 603-614), will be used. (2011) These vectors allow the expression of the phaC gene encoding polyhydroxy fatty acid ester synthase, derived from Pseudomonas MBEL 6-19, Pseudomonas 61-3, Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa and Pseudomonas resinophilus, respectively, and were co-introduced into Escherichia coli XL1-Blue ΔldhA / pTac15k_act to prepare recombinant microorganisms (PEA08, PEA09, PEA10, PEA11, PEA04, PEA12, PEA13, PEA14, PEA15 and PEA16).

[0191] Example 3: Confirmation of the production of poly(esteramide) using recombinant microorganisms including 3-aminopropionic acid and 4-aminobutyric acid as monomers

[0192] Each of the recombinant microorganisms (PEA00 and PEA01) produced 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 medium was then inoculated into 100 ml of MR medium in a 350 ml flask and cultured.

[0193] The MR medium (pH 7.0) consists of the following per liter of distilled water: 20 g glucose, 6.67 g KH₂PO₄, 4.0 g (NH₄)₂HPO₄, 0.8 g citric acid, 0.8 g MgSO₄·7H₂O, 0.01 g CaCl₂·2H₂O, and 5 ml trace metal solution (per liter of distilled water: 10 g FeSO₄·7H₂O, 2.2 g ZnSO₄·4H₂O, 0.58 g MnSO₄·4H₂O, 1 g CuSO₄·5H₂O, 0.1 g (NH₄)₆Mo₇O). 24• 4H2O and 0.02 g of Na2B4O7·10H2O). Amino acids, commonly known as substrates of β-alanine coenzyme A transferase, were provided to the composition at a concentration of 2 g / L as an additional carbon source. These amino acids are generally known to be substrates of β-alanine coenzyme A transferase, namely 3-aminopropionic acid, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, and 7-aminoheptanoic acid. Additionally, 2 g / L of sodium (RS)-3-hydroxybutyrate was provided. Cultures were performed in a shaking incubator (JSR, Korea) at 30°C and 200 rpm for 96 hours. After culture, the culture was centrifuged at 13,200 rpm for 10 minutes, and cells were collected and analyzed to confirm the production of poly(esteramide).

[0194] As a result, as shown in Figure 2, it was confirmed that the recombinant microorganisms according to the present invention produced poly(esteramides) with 3-aminopropionic acid and 4-aminobutyric acid as monomers.

[0195] Based on these results, it was confirmed that the recombinant microorganisms according to the present invention produce poly(esteramides) with 3-aminopropionic acid and 4-aminobutyric acid as monomers.

[0196] Example 4: Confirmation of the production of poly(esteramide) containing 5-aminovaleric acid as a monomer using recombinant microorganisms.

[0197] Each of the recombinant microorganisms (PEA02 and PEA03) produced 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 medium was inoculated into 100 ml of MR medium in a 350 ml flask and cultured.

[0198] The MR medium (pH 7.0) consists of the following per liter of distilled water: 20 g glucose, 6.67 g KH₂PO₄, 4.0 g (NH₄)₂HPO₄, 0.8 g citric acid, 0.8 g MgSO₄·7H₂O, 0.01 g CaCl₂·2H₂O, and 5 ml trace metal solution (per liter of distilled water: 10 g FeSO₄·7H₂O, 2.2 g ZnSO₄·4H₂O, 0.58 g MnSO₄·4H₂O, 1 g CuSO₄·5H₂O, 0.1 g (NH₄)₆Mo₇O). 24• 4H2O and 0.02 g of Na2B4O7·10H2O). An additional carbon source of 2 g / L L-lysine, which can be converted to 5-aminovaleric acid via the davAB gene, was provided to the composition. Additionally, 2 g / L of sodium (RS)-3-hydroxybutyrate was provided. Culture was performed in a shaking incubator (JSR, Korea) at 30°C and 200 rpm for 96 hours. After culture, the culture medium was centrifuged at 13,200 rpm for 10 minutes, and cells were collected and analyzed to confirm the production of poly(esteramide).

[0199] As a result, as shown in Figure 2, it was confirmed that the recombinant microorganism according to the present invention produced poly(esteramide) with 5-aminovaleric acid as a monomer using L-lysine as a carbon source.

[0200] Based on these results, it is confirmed that the recombinant microorganisms according to the present invention successfully produced poly(esteramide) with 5-aminovaleric acid as a monomer.

[0201] Example 5: In vitro assay for identifying additional substrates for β-alanine coenzyme A transferase

[0202] To further expand the types of amino acid monomers of poly(esteramide), in vitro assays were performed to identify substrates on which β-alanine coenzyme A transferase exhibits activity, in addition to conventionally known substrates. To purify β-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) (a F-ompT hsdSB(rB-mB-)gal dcm (DE3) prophage carrying the T7 RNA polymerase gene) (New England Biolabs, USA).

[0203] The enzyme assay was performed in 50 mM potassium phosphate buffer (pH 7.5). The substrate and enzyme required for the assay were added as follows: 10 mM target substrate, 1 mM acetyl-CoA, and 2.5 μg of purified β-alanine coenzyme A transferase, and the reaction was carried out at 30 °C for 2 hours.

[0204] As shown in Figure 4, the enzyme was confirmed to be active not only on the commonly known amino acids 3-aminopropionic acid, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, and 7-aminoheptanoic acid, but also on amino acid substrates such as (RS)-3-aminobutyric acid, 3-aminoisobutyric acid, and (RS)-3-aminovaleric acid. Furthermore, the enzyme was confirmed to be active on five non-amino acid hydroxy acids (glycolic acid, (RS)-lactic acid, (RS)-2-hydroxybutyric acid, 3-hydroxypropionic acid, and (RS)-3-hydroxybutyric acid) and seven carboxylic acids (propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, 2-methylbutyric acid, and hexanoic acid).

[0205] Based on these results, it was confirmed that the β-alanine coenzyme A transferase according to the present invention can successfully convert various amino acids, hydroxy acids and carboxylic acids into coenzyme A derivatives in addition to conventionally known substrates.

[0206] Example 6: Confirmation of the production of poly(esteramide) using recombinant microorganisms including (R)-3-aminobutyric acid, 3-aminoisobutyric acid, and (R)-3-aminovaleric acid as monomers

[0207] Each of the recombinant microorganisms (PEA00 and PEA01) produced 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 medium was inoculated into 100 ml of MR medium in a 350 ml flask and cultured.

[0208] The MR medium (pH 7.0) consists of the following per liter of distilled water: 20 g glucose, 6.67 g KH₂PO₄, 4.0 g (NH₄)₂HPO₄, 0.8 g citric acid, 0.8 g MgSO₄·7H₂O, 0.01 g CaCl₂·2H₂O, and 5 ml trace metal solution (per liter of distilled water: 10 g FeSO₄·7H₂O, 2.2 g ZnSO₄·4H₂O, 0.58 g MnSO₄·4H₂O, 1 g CuSO₄·5H₂O, 0.1 g (NH₄)₆Mo₇O). 24• 4H2O and 0.02 g of Na2B4O7·10H2O). Each of the amino acids recently known as substrates of β-alanine coenzyme A transferase, as described in Example 5, was provided to the composition as an additional carbon source, namely (RS)-3-aminobutyric acid, 3-aminoisobutyric acid, and (RS)-3-aminovaleric acid. Additionally, 2 g / L of sodium (RS)-3-hydroxybutyrate was provided. Culture was performed in a shaking incubator (JSR, Korea) at 30°C and 200 rpm for 96 hours. After culture, the culture was centrifuged at 13,200 rpm for 10 minutes, and cells were collected and analyzed to confirm the production of poly(esteramide).

[0209] As a result, as shown in Figure 2, it was confirmed that poly(esteramide) with (R)-3-aminobutyric acid, 3-aminoisobutyric acid or (R)-3-aminovaleric acid as monomers was produced by the recombinant microorganism according to the present invention.

[0210] These results confirm that the recombinant microorganisms according to the present invention can produce poly(esteramides) having (R)-3-aminobutyric acid, 3-aminoisobutyric acid or (R)-3-aminovaleric acid as monomers.

[0211] Example 7: Confirmation of the production of poly(esteramide) comprising multiple amino acids as monomers using recombinant microorganisms

[0212] Each of the recombinant microorganisms (PEA00 and PEA01) produced 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 medium was inoculated into 100 ml of MR medium in a 350 ml flask and cultured.

[0213] The MR medium (pH 7.0) consists of the following per liter of distilled water: 20 g glucose, 6.67 g KH₂PO₄, 4.0 g (NH₄)₂HPO₄, 0.8 g citric acid, 0.8 g MgSO₄·7H₂O, 0.01 g CaCl₂·2H₂O, and 5 ml trace metal solution (per liter of distilled water: 10 g FeSO₄·7H₂O, 2.2 g ZnSO₄·4H₂O, 0.58 g MnSO₄·4H₂O, 1 g CuSO₄·5H₂O, 0.1 g (NH₄)₆Mo₇O). 24• 4H2O and 0.02 g of Na2B4O7·10H2O. An additional carbon source was provided to the composition by a combination of two (3-aminopropionic acid / (RS)-3-aminobutyric acid) or three (3-aminopropionic acid / (RS)-3-aminobutyric acid / (RS)-3-aminoisobutyric acid) amino acids commonly known as substrates of β-alanine coenzyme A transferase. Additionally, 2 g / L of sodium (RS)-3-hydroxybutyrate was provided. Cultures were performed in a shaking incubator (JSR, Korea) at 30°C and 200 rpm for 96 hours. After culture, the culture was centrifuged at 13,200 rpm for 10 minutes, and cells were collected and analyzed to confirm the production of poly(esteramide).

[0214] As a result, Figure 3 As shown, it has been confirmed that the recombinant microorganisms according to the present invention simultaneously produce poly(esteramides) having two (3-aminopropionic acid and (R)-3-aminobutyric acid) monomers or three (3-aminopropionic acid, (R)-3-aminobutyric acid and (R)-3-aminoisobutyric acid) monomers.

[0215] Based on these results, it was confirmed that the recombinant microorganisms according to the present invention produce poly(esteramide) having multiple amino acids as monomers.

[0216] Example 8: Confirmation of the production of poly(esteramide) using recombinant microorganisms, with glucose as the sole carbon source.

[0217] Each of the recombinant microorganisms (PEA05, PEA06, or PEA07) prepared 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-culture was then inoculated into 100 mL of MR medium in a 350 mL flask and cultured. PEA06 and PEA07 were strains in which the panD and gadB_mut genes were overexpressed, respectively, to increase the metabolic flux of 3-aminopropionic acid and 4-aminobutyric acid biosynthesis. Additionally, both strains were inoculated with the phaAB gene to enable the biosynthesis of 3-hydroxybutyryl-CoA from glucose, thus providing hydroxy acid monomers without the introduction of external hydroxy acids. Furthermore, to prevent the polymerization of lactic acid (a hydroxy acid monomer different from the target 3-hydroxybutyric acid), a strain lacking the ldhA gene required for lactic acid biosynthesis was used.

[0218] The MR medium (pH 7.0) consists of the following per liter of distilled water: 20 g glucose, 6.67 g KH₂PO₄, 4.0 g (NH₄)₂HPO₄, 0.8 g citric acid, 0.8 g MgSO₄·7H₂O, 0.01 g CaCl₂·2H₂O, and 5 ml trace metal solution (per liter of distilled water: 10 g FeSO₄·7H₂O, 2.2 g ZnSO₄·4H₂O, 0.58 g MnSO₄·4H₂O, 1 g CuSO₄·5H₂O, 0.1 g (NH₄)₆Mo₇O). 24 • 4H2O and 0.02 g of Na2B4O7·10H2O). Unlike the previous examples, no additional amino acids or sodium (RS)-3-hydroxybutyrate were supplied to the composition, and therefore, glucose was used as the sole carbon source. Cultures were performed in a shaking incubator (JSR, Korea) at 30°C and 200 rpm for 96 hours. After culture, the culture was centrifuged at 13,200 rpm for 10 minutes, and cells were collected and analyzed to confirm the production of poly(esteramide).

[0219] As a result, Figure 5 As shown, the recombinant microorganisms according to the present invention produced a product containing 3-aminopropionic acid (…). Figure 5 a and b) or 4-aminobutyric acid ( Figure 5 c and d) are poly(esteramides) as monomers.

[0220] These results confirm that the recombinant microorganisms according to the present invention use glucose as the sole carbon source to produce poly(esteramide).

[0221] Example 9: Confirmation of efficient production of poly(esteramide) with 3-aminopropionic acid as a monomer using fed-batch fermentation of recombinant microorganisms

[0222] The recombinant microorganism (PEA06) produced 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 medium was then inoculated into 2 L of MR medium in a 6.6 L fermenter, followed by fed-batch fermentation.

[0223] The MR medium (pH 7.0) consists of the following per liter of distilled water: 20 g glucose, 6.67 g KH₂PO₄, 4.0 g (NH₄)₂HPO₄, 0.8 g citric acid, 0.8 g MgSO₄·7H₂O, 0.01 g CaCl₂·2H₂O, and 5 ml trace metal solution (per liter of distilled water: 10 g FeSO₄·7H₂O, 2.2 g ZnSO₄·4H₂O, 0.58 g MnSO₄·4H₂O, 1 g CuSO₄·5H₂O, 0.1 g (NH₄)₆Mo₇O). 24 • 4H2O and 0.02 g Na2B4O7·10H2O). When the sole carbon source—glucose—was completely consumed, an additional feed solution containing 700 g of glucose and 8 g of MgSO4·7H2O per liter was supplied. Cultures were carried out for 96 hours in a bioreactor (New Brunswick Scientific Co., NJ) operated to maintain a dissolved oxygen level of 40% at 30°C. After culture, the culture was centrifuged at 13,200 rpm for 10 minutes, and cells were collected and analyzed to confirm the production of poly(esteramide).

[0224] As a result, Figure 5 As shown in e, it was confirmed that the recombinant microorganisms according to the present invention produce poly(esteramide) with 3-aminopropionic acid as a monomer at high production concentrations, productivity and yield.

[0225] These results confirm that the recombinant microorganisms according to the present invention can efficiently produce poly(esteramide) using fed-batch fermentation.

[0226] Example 10: Confirmation of the production of poly(esteramide) using polyhydroxy fatty acid esters derived from various microorganisms

[0227] Each of the recombinant microorganisms (PEA08, PEA09, PEA10, PEA11, PEA04, PEA12, PEA13, PEA14, PEA15, and PEA16) produced 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 medium was then inoculated into 100 ml of LB medium in a 350 ml flask and cultured.

[0228] LB medium consisted of 10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter of distilled water. 3-aminopropionic acid (an amino acid known as a substrate of β-alanine coenzyme A transferase) was provided as an additional carbon source at a concentration of 2 g / L. Additionally, 50 g / L of sodium (RS)-3-hydroxybutyrate was provided. Cultures were performed in a shaking incubator (JSR, South Korea) at 30°C and 200 rpm for 96 hours. After culture, the culture was centrifuged at 13,200 rpm for 10 minutes, and cells were collected and analyzed to confirm the production of poly(esteramide).

[0229] As a result, Figure 6 As shown, it is confirmed that all recombinant microorganisms according to the present invention produce a variety of poly(esteramides) with 3-aminopropionic acid as a monomer and various molecular weights.

[0230] These results confirm that the recombinant microorganisms according to the present invention produce poly(esteramides) with 3-aminopropionic acid as a monomer.

[0231] Having described certain parts of the invention in detail above, those skilled in the art will understand that these specific descriptions are merely preferred embodiments, and the scope of the invention is not limited thereto. Therefore, the essential scope of the invention will be defined by the appended claims and their equivalents.

[0232] Industrial applicability

[0233] The recombinant microorganisms according to the present invention can biosynthesize poly(esteramide) polymers from various carbon sources such as glucose, amino acids, hydroxy acids, and carboxylic acids, and are therefore used in the industrial production of poly(esteramide).

[0234] sequence list

[0235] Electronic files have been attached.

Claims

1. A recombinant microorganism capable of producing poly(esteramide), wherein, Genes encoding coenzyme A transferase and genes encoding PHA (polyhydroxyalkanoate) synthase are introduced into or amplified in host microorganisms.

2. The recombinant microorganism according to claim 1 has a biosynthetic pathway for synthesizing organic acids from a carbon source, wherein the organic acids are substrates of the coenzyme A transferase.

3. The recombinant microorganism according to claim 2, wherein, The carbon source is sugar or organic acid.

4. The recombinant microorganism according to claim 2, wherein, The organic acid that serves as the substrate for the coenzyme A transferase is at least one of an amino acid or a hydroxy acid.

5. The recombinant microorganism according to claim 1, wherein, The coenzyme A transferase acts on the amino acids that serve as substrates.

6. The recombinant microorganism according to claim 5, wherein, The amino acids are selected from the group consisting of: glycine, alanine, 2-aminobutyric acid, 3-aminopropionic acid, isoserine, 3-aminobutyric acid, 3-aminovaleric acid, β-phenylalanine (BPA), 4-aminobutyric acid, 4-amino-3-methylbutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, 3-aminoisobutyric acid, para-aminobenzoic acid (PAB) and their isomers.

7. The recombinant microorganism according to claim 1, wherein, The coenzyme A transferase is β-alanine coenzyme A transferase.

8. The recombinant microorganism according to claim 1, wherein, The gene encoding the coenzyme A transferase is the act gene derived from Clostridium propionicum.

9. The recombinant microorganism according to claim 1, wherein, The coenzyme A transferase includes sequences selected from SEQ ID NO:1 to 3.

10. The recombinant microorganism according to claim 1, wherein, The PHA synthase is PhaC.

11. The recombinant microorganism according to claim 1, wherein, The gene encoding the PHA synthase is derived from the phaC gene of strains selected from the following groups: *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 resinovorans*.

12. The recombinant microorganism according to claim 1, wherein, The PHA synthase comprises a sequence selected from the group consisting of SEQ ID NO: 4 to 13.

13. The recombinant microorganism according to claim 1, wherein, The poly(esteramide) includes an organic acid as a monomer, which is a substrate of the coenzyme A transferase.

14. The recombinant microorganism according to claim 13, wherein, The organic acid serving as the substrate for said coenzyme A transferase is selected from the group consisting of: glycine, alanine, 2-aminobutyric acid, 3-aminopropionic acid, isoserine, 3-aminobutyric acid, 3-aminoisobutyric acid, 3-aminovaleric acid, β-phenylalanine (BPA), 4-aminobutyric acid, 4-amino-3-methylbutyric acid, para-aminobenzoic acid (PAB), 5-aminovaleric acid, 6-aminohexanoic acid, 7-aminoheptanoic acid, glycolic acid, lactic acid, 2-hydroxybutyric acid, 3-hydroxypropionic acid, 3-hydroxybutyric acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, 2-methylbutyric acid, hexanoic acid, malonic acid, succinic acid, and their isomers.

15. The recombinant microorganism according to claim 1, wherein, The host microorganism is selected from the group consisting of bacteria, yeast and fungi.

16. A method for producing poly(esteramide), comprising: (a) Cultivating the recombinant microorganism according to claim 1 to produce poly(esteramide); and (b) Recycle the produced poly(esteramide).

17. A method for producing poly(esteramide), comprising: (a) To produce poly(esteramide) by culturing the recombinant microorganism according to claim 1 in a culture medium supplemented with organic acids as substrates for coenzyme A transferase; and (b) Recycle the produced poly(esteramide).

18. A poly(esteramide) produced by the method according to claim 16.

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