Recombinant microorganism capable of producing polyhydroxyalkanoate and method for producing polyhydroxyalkanoate using the same

Abstract

The present invention relates to a recombinant microorganism capable of producing polyhydroxyalkanoate and a method for producing polyhydroxyalkanoate, using same and, more specifically, to: a recombinant microorganism in which a gene encoding an enzyme for converting amino acids into 2-hydroxy acids, a gene encoding hydroxyisocaproate-CoA transferase, and a gene encoding polyhydroxyalkanoate synthase are introduced or amplified; and a method for producing polyhydroxyalkanoate, using the recombinant microorganism.

Description

Recombinant microorganism having polyhydroxyalkanoate production ability and method for producing polyhydroxyalkanoate using the same

[0001] The present invention relates to a recombinant microorganism having the ability to produce polyhydroxyalkanoate and a method for producing polyhydroxyalkanoate using the same, and more particularly, to a recombinant microorganism into which a gene encoding an enzyme for converting an amino acid into a 2-hydroxy acid, a gene encoding a hydroxyisocaproate-CoA transferase, and a gene encoding a polyhydroxyalkanoate synthase have been introduced or amplified, and a method for producing polyhydroxyalkanoate using the recombinant microorganism.

[0002]

[0003] Polyhydroxyalkanoates (PHAs) are biological polyesters synthesized by various microorganisms. These polymers are thermoplastics with biodegradable and biocompatible properties. Because they can exhibit properties similar to those of petroleum-based polymers, they have diverse industrial and biomedical applications and are produced from renewable resources (Lee, SYBiotechnol. Bioeng. 49:1, 1996).

[0004] Depending on the length of the side chain, PHAs are classified into SCL-PHA (short-chain-length PHA) with a short carbon number and MCL-PHA (medium-chain-length PHA) with a long carbon number. Various PHAs have been synthesized by cloning PHA synthetic genes derived from microorganisms such as Ralstonia eutropha, pseudomonas, and Bacillus to produce recombinant microorganisms (Qiet et al., FEMS Microbiol. Lett., 157:155, 1997; Qiet et al., FEMS Microbiol. Lett., 167:89, 1998; Langenbach et al., FEMS Microbiol. Lett., 150:303, 1997; WO 01 / 55436; US 6,143,952; WO 98 / 54329; WO 99 / 61624).

[0005] Numerous naturally occurring PHA-producing bacteria have been isolated, and genes encoding polymerases (PHA synthase) have been identified. Genes encoding metabolic enzymes and auxiliary proteins involved in PHA biosynthesis have been characterized at the molecular level. Various metabolic engineering approaches have been identified to develop PHA-producing bacteria with higher PHA titers and to increase productivity (GQ Chen, Chem. Soc. Rev. 2009, 38, 2434).

[0006] Under these technical backgrounds, the inventors of the present application have made great efforts to develop a method for biosynthesizing PHA containing 2-hydroxy acid as a monomer, and as a result, have confirmed that production of PHA is possible through 2-hydroxy acid converted from amino acids using 20 amino acids as substrates, thereby completing the present invention.

[0007]

[0008] Summary of the invention

[0009] The purpose of the present invention is to provide a recombinant microorganism having the ability to produce polyhydroxyalkanoate containing a 2-hydroxy acid converted from an amino acid as a monomer.

[0010] Another object of the present invention is to provide a method for producing polyhydroxyalkanoate containing a 2-hydroxy acid converted from an amino acid as a monomer using the recombinant microorganism.

[0011] To achieve the above object, the present invention provides a recombinant microorganism having the ability to produce polyhydroxyalkanoate, which comprises 2-hydroxyacid (2-HA) converted from amino acids as a monomer, wherein a gene encoding amino acid deaminase; a dehydrogenase encoding gene; a CoA transferase encoding gene; and a gene encoding polyhydroxyalkanoate synthase are introduced or amplified into a microorganism having the ability to produce acetyl-CoA from a carbon source.

[0012] The present invention also provides a method for producing polyhydroxyalkanoate comprising 2-hydroxyacid (2-HA) as a monomer converted from an amino acid, comprising the following steps:

[0013] (a) a step of culturing the recombinant microorganism to produce polyhydroxyalkanoate; and (b) a step of obtaining the polyhydroxyalkanoate.

[0014]

[0015] Figure 1 is an overview of the process for synthesizing an amino acid-based aliphatic 2-hydroxy acid-containing polymer.

[0016] Figure 2 shows the pathway for producing 3-hydroxybutyric acid in microorganisms and polymerizing it into a polymer monomer.

[0017] Figure 3 shows the process by which 2-HA is produced by deamination of 20 types of amino acids by amino acid deaminase, for example, the LAAD coding gene, and dehydrogenation by the dehydrogenase coding gene.

[0018] Figure 4 describes a group of enzyme candidates for the production of amino acid-based aliphatic 2-hydroxy acid-containing polymers.

[0019]

[0020] Detailed description of the invention and preferred embodiments

[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. In general, the nomenclature used herein is well known and commonly used in the art.

[0022] By adding a total of 20 amino acids to a recombinant microorganism, it was confirmed that various polymers containing 2-hydroxy acids with a hydroxyl group attached to the second carbon could be produced as monomers within the microorganism.

[0023] When amino acids are added during the cultivation of recombinant microorganisms, 2-ketoacids (2KA) are produced through the action of amino acid deaminase, such as LAAD, and the produced 2KA is converted into 2-hydroxyacid (2-HA) by dehydrogenase.

[0024] Within the cell, CoA is attached to 2HA by CoA transferase to produce 2HA-CoA, and ultimately, polyhydroxyalkanoate synthase (PHA synthase) polymerizes the produced 2HA-CoA into a polymer monomer (Figure 1).

[0025] The manufactured polymer is a polymer in the form of P(3HB-Co-2HA) with 3-hydroxybutyric acid (3HB) as a backbone and 2-HA included.

[0026] Specifically, according to the present invention, it is a polymer having 3-hydroxybutyric acid (3HB) as a backbone and containing various 2-HAs at levels of 0.5 mol% to 20 mol%.

[0027] E. coli XL1-blue was used as the microbial species. The pathway for 3-hydroxybutyric acid production in microorganisms and polymerization into a polymer monomer is as shown in Figure 2. To produce P(3HB), PhaA and PhaB derived from Ralstonia eutropha are required, and PhaC was used as Pseudomonas sp. MBEL 6-19 phaC variant (PhaC1437; E130D, S325T, S477G, Q481K). The plasmid containing all three PhaA, PhaB, and PhaC coding genes was named p437ReAB.

[0028] Based on this, the present invention relates to a recombinant microorganism having the ability to produce polyhydroxyalkanoate, which comprises 2-hydroxyacid (2-HA) converted from an amino acid as a monomer, wherein a gene encoding an amino acid deaminase; a dehydrogenase encoding gene; a CoA transferase encoding gene; and a gene encoding a polyhydroxyalkanoate synthase are introduced or amplified into a microorganism having the ability to produce acetyl-CoA from a carbon source.

[0029] The carbon source may be, but is not limited to, carbohydrates such as glucose, fructose, sucrose, maltose, mannitol, sorbitol, etc.; alcohols such as sugar alcohols, glycerol, pyruvic acid, lactic acid, citric acid, etc.; organic acids, etc. The carbon source may be used alone or in combination of two or more, but is not limited thereto.

[0030] The amino acid may be, but is not limited to, one or more selected from the group consisting of glycine, alanine, valine, norvaline, leucine, norleucine, isoleucine, threonine, serine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, and proline.

[0031] The 20 types of amino acids used in the present invention can be deaminated by amino acid deaminase, for example, the LAAD coding gene, and dehydrogenated by the dehydrogenase coding gene to produce 2-HA (Fig. 3). Since each amino acid has an amine group and a sulfur group, it is believed that the production of an amine functional polymer or a sulfur functional polymer is possible.

[0032] The 20 types of amino acids and the specific types of 2-HA into which the amino acids can be converted are as shown in Table 1 below.

[0033] [Table 1]

[0034]

[0035]

[0036] The above amino acid deaminase is an enzyme for producing 2-keto acid from amino acids, and the amino acid deaminase coding gene may be, for example, as follows.

[0037] ilvA* from Escherchia coli (ilvA;F352A, R362F);

[0038] Type I LAAD from Proteus mirabilis (PmILAAD);

[0039] Type I LAAD from Proteus myxofaciens (PyxLAAD);

[0040] Type II LAAD from Proteus mirabilis (PmIILAAD); or

[0041] Type II LAAD from Proteus vulgaris (PvLAAD).

[0042]

[0043] Specifically, the amino acid deaminase coding gene may be at least one selected from the group consisting of an ilvA gene encoding an enzyme comprising a sequence of SEQ ID NO: 1, a PmILAAD gene encoding an enzyme comprising a sequence of SEQ ID NO: 2, a PmIILAAD gene encoding an enzyme comprising a sequence of SEQ ID NO: 3, a PvLAAD gene encoding an enzyme comprising a sequence of SEQ ID NO: 4, and a PyxLAAD gene encoding an enzyme comprising a sequence of SEQ ID NO: 5.

[0044] [Table 2]

[0045]

[0046]

[0047] The above dehydrogenase is an enzyme for producing 2-hydroxy acid from 2-keto acid, and the dehydrogenase coding gene may be, for example, as follows.

[0048] PanE fromLactococcus lactissubsp.lactisIl1403 (LlpanE);

[0049] ManDH from Enterococcus faecium (ManDH);

[0050] KIVR from Beauveria bassiana (KIVR)];

[0051] Lcppr from Lactobacillus sp. CGMCC 9967 (Lcppr);

[0052] Lcddh fromLacatobacillus casei (Lcddh); or

[0053] Paldh fromPediococcus acidilactici(Paldh).

[0054]

[0055] Specifically, the dehydrogenase coding gene may be at least one selected from the group consisting of the LlpanE gene encoding an enzyme comprising the sequence of SEQ ID NO: 6, the KIVR gene encoding an enzyme comprising the sequence of SEQ ID NO: 7, the ManDH gene encoding an enzyme comprising the sequence of SEQ ID NO: 8, the Lcppr gene encoding an enzyme comprising the sequence of SEQ ID NO: 9, the Lcddh gene encoding an enzyme comprising the sequence of SEQ ID NO: 10, and the Paldh gene encoding an enzyme comprising the sequence of SEQ ID NO: 11.

[0056] [Table 3]

[0057]

[0058]

[0059] The above CoA transferase is an enzyme for producing 2-HA CoA from 2-hydroxy acid, and the CoA transferase coding gene may be, for example, as follows:

[0060] 4HBS fromMetallosphaera sedula(4HBS);

[0061] Pct fromClostridium propionicumpct variant (pct540; V193A,silent mutations of T78C,T669C,A1125G,T1158C) ;

[0062] HadA from Clostridioides difficile (HadA);

[0063] AbfT from Clostridium aminobutyricum (AbfT);

[0064] Cat2 from Clostridium aminobutyricum (Cat2); or

[0065] PrpE from Cupriavidus necator (PrpE).

[0066] The CoA transferase coding gene according to the present invention may be specifically a hadA gene coding for an enzyme comprising the sequence of SEQ ID NO: 12.

[0067]

[0068] The CoA transferase coding gene according to the present invention may be at least one selected from the group consisting of a 4HBS gene encoding an enzyme comprising a sequence of SEQ ID NO: 16, an AbfT gene encoding an enzyme comprising a sequence of SEQ ID NO: 17, a Cat2 gene encoding an enzyme comprising a sequence of SEQ ID NO: 18, a Pct540 gene encoding an enzyme comprising a sequence of SEQ ID NO: 19, and a PrPE gene encoding an enzyme comprising a sequence of SEQ ID NO: 20.

[0069]

[0070]

[0071] Polymers containing 2-HA at various molar fractions were produced using the branched-chain amino acids leucine, isoleucine, valine, norleucine, and norvaline. These results confirmed that among the five types of CoA transferases, only HadA can accommodate 2-HA.

[0072] The 2-HA produced by the present invention can be converted into 2-HA-CoA by CoA transferase, and polymerized with 3HB-CoA produced by an enzyme selected from the group consisting of PHA synthetase, for example, PhaA, PhaB, and phaC, to produce a polymer in the form of P(3HB-Co-2HA) (Fig. 4).

[0073] The gene encoding the polyhydroxyalkanoate synthase may be, for example, Pseudomonassp. MBEL 6-19phaCvariant. Specifically, the gene encoding the polyhydroxyalkanoate synthase according to the present invention may be the PhaC gene encoding an enzyme comprising the sequence of SEQ ID NO: 13.

[0074]

[0075] Additionally, PhaA and PhaB may be included for polyhydroxyalkanoate synthesis. Specifically, each of the PhaA and PhaB genes may encode enzymes comprising the sequences of SEQ ID NOs: 14 and 15.

[0076]

[0077]

[0078] Microorganisms capable of producing acetyl-CoA from the above carbon source may include, for example, Escherichia coli, Alcaligenes genus, Pseudomonas genus, Escherichia genus, Ralstonia genus, Bacillus genus, or Corynebacterium, but any applicable microorganism may be used without limitation.

[0079] From another perspective, the present invention relates to a method for producing polyhydroxyalkanoate comprising 2-hydroxy acid (2-HA) converted from an amino acid as a monomer, the method comprising the steps of (a) culturing the recombinant microorganism to produce polyhydroxyalkanoate; and (b) obtaining the polyhydroxyalkanoate.

[0080] The amino acid may be, but is not limited to, one or more selected from the group consisting of glycine, alanine, valine, norvaline, leucine, norleucine, isoleucine, threonine, serine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, and proline.

[0081] The 20 types of amino acids used in the present invention can be deaminated by amino acid deaminase, for example, the LAAD coding gene, and dehydrogenated by the dehydrogenase coding gene to produce 2-HA (Fig. 3). Since each amino acid has an amine group and a sulfur group, it is believed that the production of an amine functional polymer or a sulfur functional polymer is possible.

[0082] The 20 types of amino acids and the specific types of 2-HA into which the amino acids can be converted are as described above.

[0083] The above amino acid deaminase coding gene may be, for example, as follows.

[0084] ilvA* from Escherchia coli (ilvA;F352A, R362F);

[0085] Type I LAAD from Proteus mirabilis (PmILAAD);

[0086] Type I LAAD from Proteus myxofaciens (PyxLAAD);

[0087] Type II LAAD from Proteus mirabilis (PmIILAAD); or

[0088] Type II LAAD from Proteus vulgaris (PvLAAD).

[0089]

[0090] Specifically, the amino acid deaminase coding gene may be at least one selected from the group consisting of an ilvA gene encoding an enzyme comprising a sequence of SEQ ID NO: 1, a PmILAAD gene encoding an enzyme comprising a sequence of SEQ ID NO: 2, a PmIILAAD gene encoding an enzyme comprising a sequence of SEQ ID NO: 3, a PvLAAD gene encoding an enzyme comprising a sequence of SEQ ID NO: 4, and a PyxLAAD gene encoding an enzyme comprising a sequence of SEQ ID NO: 5.

[0091] The above dehydrogenase coding gene may be, for example, as follows.

[0092] PanE fromLactococcus lactissubsp.lactisIl1403 (LlpanE);

[0093] ManDH from Enterococcus faecium (ManDH);

[0094] KIVR from Beauveria bassiana (KIVR)];

[0095] Lcppr from Lactobacillus sp. CGMCC 9967 (Lcppr);

[0096] Lcddh fromLacatobacillus casei (Lcddh); or

[0097] Paldh fromPediococcus acidilactici(Paldh).

[0098]

[0099] Specifically, the dehydrogenase coding gene may be at least one selected from the group consisting of the LlpanE gene encoding an enzyme comprising the sequence of SEQ ID NO: 6, the KIVR gene encoding an enzyme comprising the sequence of SEQ ID NO: 7, the ManDH gene encoding an enzyme comprising the sequence of SEQ ID NO: 8, the Lcppr gene encoding an enzyme comprising the sequence of SEQ ID NO: 9, the Lcddh gene encoding an enzyme comprising the sequence of SEQ ID NO: 10, and the Paldh gene encoding an enzyme comprising the sequence of SEQ ID NO: 11.

[0100] The above CoA transferase coding gene may be, for example, as follows:

[0101] 4HBS fromMetallosphaera sedula(4HBS);

[0102] Pct fromClostridium propionicumpct variant (pct540; V193A,silent mutations of T78C,T669C,A1125G,T1158C) ;

[0103] HadA from Clostridioides difficile (HadA);

[0104] AbfT from Clostridium aminobutyricum (AbfT);

[0105] Cat2 from Clostridium aminobutyricum (Cat2); or

[0106] PrpE from Cupriavidus necator (PrpE).

[0107]

[0108] The CoA transferase coding gene according to the present invention may be specifically a hadA gene coding for an enzyme comprising the sequence of SEQ ID NO: 12.

[0109]

[0110] The CoA transferase coding gene according to the present invention may be at least one selected from the group consisting of a 4HBS gene encoding an enzyme comprising a sequence of SEQ ID NO: 16, an AbfT gene encoding an enzyme comprising a sequence of SEQ ID NO: 17, a Cat2 gene encoding an enzyme comprising a sequence of SEQ ID NO: 18, a Pct540 gene encoding an enzyme comprising a sequence of SEQ ID NO: 19, and a PrPE gene encoding an enzyme comprising a sequence of SEQ ID NO: 20.

[0111]

[0112] Polymers containing 2-HA at various molar fractions were produced using the branched-chain amino acids leucine, isoleucine, valine, norleucine, and norvaline. These results confirmed that among the five types of CoA transferases, only HadA can accommodate 2-HA.

[0113] The 2-HA produced by the present invention can be converted into 2-HA-CoA by CoA transferase, and polymerized with 3HB-CoA produced through PhaA and PhaB by PHA synthetase to produce a polymer in the form of P(3HB-Co-2HA).

[0114] The gene encoding the polyhydroxyalkanoate synthase may be, for example, Pseudomonassp. MBEL 6-19phaCvariant. Specifically, the gene encoding the polyhydroxyalkanoate synthase according to the present invention may be the PhaC gene encoding an enzyme comprising the sequence of SEQ ID NO: 13.

[0115]

[0116] Additionally, PhaA and PhaB may be included for polyhydroxyalkanoate synthesis. Specifically, each of the PhaA and PhaB genes may encode enzymes comprising the sequences of SEQ ID NOs: 14 and 15.

[0117]

[0118]

[0119] Microorganisms capable of producing acetyl-CoA from the above carbon source may include, for example, Escherichia coli, Alcaligenes genus, Pseudomonas genus, Escherichia genus, Ralstonia genus, Bacillus genus, or Corynebacterium, but any applicable microorganism may be used without limitation.

[0120] In the present invention, “recombinant” means including a polynucleotide or protein that does not occur naturally within a host cell.

[0121] 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.

[0122] Any gene can be introduced into a microorganism and expressed in any form. For example, the gene can be introduced into the microorganism in the form of an expression cassette, a polynucleotide structure containing all the elements necessary for its self-expression.

[0123] In the present invention, "overexpression" refers to a level of expression higher than the level at which the corresponding gene is expressed in a cell under normal conditions, and is a concept that includes increasing the amount of expression by replacing the promoter of a gene existing in the genome with a strong promoter or cloning the corresponding gene into an expression vector and transforming the cell.

[0124] In the present invention, "vector" means a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host.

[0125] Specifically, 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.

[0126] For the purposes of the present invention, it is preferred to use a plasmid vector. A typical plasmid vector that can be used for this purpose has a structure that includes (a) an origin of replication that allows efficient replication to include hundreds of plasmid vectors per host cell, (b) an antibiotic resistance gene that allows 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.

[0127] Even if an appropriate restriction enzyme cleavage site does not exist, the vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adaptor or linker according to conventional methods.

[0128] After ligation, the vector must be transformed into a suitable host cell. The host cell is a prokaryotic cell. Suitable prokaryotic host cells include E. coli DH5α, E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL-1Blue (Stratagene), E. coli B, E. coli B21, etc. However, E. coli strains such as FMB101, NM522, NM538, and NM539, as well as other species and genera of prokaryotes, can also be used.

[0129] In addition to the aforementioned E. coli, strains of the genus Agrobacterium such as Agrobacterium A4, bacilli such as Bacillus subtilis, other enterobacteria such as Salmonella typhimurium or Serratia marcescens, and various strains of the genus Pseudomonas can be used as host cells.

[0130] A nucleic acid is "operably linked" when it is placed into 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, when an appropriate molecule (e.g., a transcriptional activating protein) binds to the regulatory sequence(s), gene expression is enabled.

[0131] 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 affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it affects 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.

[0132] "Operably linked" means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and within the reading frame. However, enhancers do not need to be in contact. Linking of these sequences is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, conventionally synthesized oligonucleotide adapters or linkers are used.

[0133] 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.

[0134] 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 host cell is eukaryotic, the expression vector must further include an expression marker useful in the eukaryotic expression host.

[0135] A host cell transformed or transfected with the expression vector described above constitutes another aspect of the present invention. The term "transformation" as used herein 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. The term "transfection" as used herein refers to the uptake of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed.

[0136] It is important to understand that not all vectors and expression control sequences function equally well in expressing the DNA sequences of the present invention. Similarly, not all hosts function equally well with the same expression system.

[0137] However, those skilled in the art can appropriately select from among various vectors, expression control sequences, and hosts without undue experimental burden or deviating from the scope of the present invention. For example, when selecting a vector, consideration should be given to the host within which the vector must replicate. 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. The selection of an expression control sequence also requires consideration of various factors.

[0138] For example, the relative strength of the sequence, its modulability, and its compatibility with the DNA sequence of the present invention should be considered, particularly with respect to possible secondary structures. The unicellular host should be selected taking into account factors such as the selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, its secretion properties, its ability to fold the protein accurately, its 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, one of ordinary skill 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 culture.

[0139] When attempting to clone cDNA of a protein according to the present invention by expression cloning, a binding method, a panning method, a film emulsion method, etc. can be applied as a screening method.

[0140] As demonstrated by the use of 20 amino acids in various embodiments of the present invention, this system can be used to produce a variety of aromatic polymers. The present invention can contribute to the establishment of a bioprocess for producing aromatic polyesters from renewable, non-food biomass.

[0141] Specifically, the medium used in the step of culturing the recombinant microorganism may include, for example, LB medium and MR medium. The LB medium included in the medium may include a concentration of 10 g / L tryptone, 5 g / L NaCl, and 5 g / L yeast extract. The MR medium included in the medium may include, per 1 L, 6.67 g KH2PO4, 4 g (NH4)2HPO4, 0.8 g MgSO4· Contains 7H2O, 0.8 g citric acid and 5 ml trace metal solution, wherein the trace metal solution contains per 1 L, 0.5 M HCl: 10 g FeSO4·H2O, 2 g CaCl2, 2.2 g ZnSO4·H2O, 0.5 g MnSO4·H2O, 1 g CuSO4·H2O, 0.1 g (NH4)6Mo7O 24 ·H2O, and 0.02 g Na2B4O7·10H2O.

[0142] The recombinant microorganism may be characterized by being cultured at a temperature of 30°C to 37°C, but is not limited thereto. Polyhydroxyalkanoate can be efficiently obtained under the above temperature conditions.

[0143] The pH during the above culturing may be maintained at a range of 6.8 to 7.2, but is not limited thereto. By maintaining the pH within this range, the growth rate of the recombinant microorganism according to the present invention may be improved.

[0144]

[0145] Example

[0146] 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.

[0147] In the examples below, Escherichia coli was used as a recombinant microorganism, but any applicable microorganism can be used without limitation, and examples thereof include Alcaligenes genus, Pseudomonas genus, Escherichia genus, Ralstonia genus, Bacillus genus, and Corynebacterium genus.

[0148]

[0149] Example 1. Plasmid construction and strain and cultivation method of genes involved in PHA production including 2HA

[0150] In this experiment, Escherichia coli XL1-Blue (Stratagene, San Diego, CA) was used as the host strain for plasmid construction and maintenance. Recombinant E. coli strains were grown on LB or LB agar plates (2.0% w / v) containing 10 g / L tryptone, 5 g / L NaCl, and 5 g / L yeast extract. Appropriate antibiotics (50 μg / mL ampicillin, 40 μg / mL kanamycin, or 34 μg / mL chloramphenicol) were prepared and supplemented to the medium. The strains, plasmids, and primers used in the examples below are listed in Tables 1 and 2.

[0151]

[0152] 1-1 Construction of plasmid p437ReAB

[0153] To produce P(3HB) according to the pathway of Figure 2, which produces 3-HB (3-hydroxybutyric acid) in microorganisms and polymerizes it into a polymer monomer, PhaA and PhaB derived from Ralstonia eutropha are required, and here, PhaC was used as Pseudomonas sp. MBEL 6-19phaC variant (PhaC1437; E130D, S325T, S477G, Q481K). The plasmid containing all three genes was named p437ReAB. Plasmid p437ReAB is constructed under the PHA synthase operon promoter of Ralstonia eutropha and under the PHA synthase operon promoter of Pseudomonas sp. PhaC1437 containing four mutations, E130D, S325T, S477G and Q481K, in the PHA synthase of 6-19 and a recombinant plasmid expressing PhaA and PhaB from Ralstonia eutropha (Parket et al., Metab. Eng., 20, 20-28, 2013).

[0154]

[0155] 1-2 Construction of plasmid pKM212_ amino acid deaminase and dehydrogenase

[0156] As shown in Fig. 3, 20 kinds of amino acids were used, and the corresponding amino acids were deaminated by the LAAD gene and dehydrogenated by the dehydrogenase gene to produce 2-HA. Since each amino acid has an amine group and a sulfur group, it seems possible to produce amine functional polymers or sulfur functional polymers. To this end, first, ilvA, which encodes deaminase, a gene that deaminates amino acids, was synthesized from the chromosome of an E. coli strain through a codon optimization process for the sequences of PmILAAD, PmIILAAD, PvLAAD, and PyxLAAD, and PCR was performed using a C1000 Thermocycler (Bio-Rad, Hercules, CA). First, to construct the deamination modules, plasmids pKM212 ilvA, pKM212 PmILAAD, pKM212 PmIILAAD, pKM212 PvLAAD, and pKM212 PyxLAAD, plasmid pKM212 was digested with EcoRI / KpnI, respectively. The corresponding ilvA was digested with MfeI / KpnI, and the PmILAAD, PmIILAAD, PvLAAD, and PyxLAAD genes were digested with EcoRI / KpnI, respectively, and ligated into plasmid pKM212. Next, to construct a dehydrogenation-only module, pKM212 was digested using EcoRI / SbfI, and the corresponding LlpanE, KIVR, ManDH, Lcddh, Lcppr, and Paldh genes were digested using EcoRI / SbfI, respectively, and ligated into the five plasmids.

[0157]

[0158] 1-3 Construction of plasmid pKA312_ CoA transferase

[0159] 2-HA is converted to 2HA-CoA by CoA transferase, and polymerized with 3HB-CoA produced through PhaA and PhaB by PHA synthase to produce a polymer in the form of P(3HB-Co-2HA) (Fig. 4). To use CoA transferase, the sequences of 4HBS, AbfT, Cat2, HadA, Pct540, and PrpE encoding CoA transferase were synthesized through a codon optimization process, and PCR was performed using a C1000 thermocycler (Bio-Rad, Hercules, CA). To construct the CoA transferase modules pKA312 4HBS, pKA312 AbfT, pKA312 Cat2, pKA312 HadA, pKA312 PrpE, and pKA312 Pct540, plasmid pKA312 was digested with EcoRI / KpnI, respectively. The corresponding 4HBS, AbfT, Cat2, HadA, Pct540, and PrpE genes were digested using EcoRI / KpnI and ligated into plasmid pKA312.

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] Example 2. Confirmation of production of 2HA-containing polymer in recombinant E. coli

[0175] In an embodiment of the present invention, the MR medium used to produce PHA including 2HA in recombinant E. coli contains, per 1 L, 6.67 g KH2PO4, 4 g (NH4)2HPO4, 0.8 g MgSO4· Contains 7H2O, 0.8 g citric acid and 5 ml trace metal solution, wherein the trace metal solution contains per 1 L, 0.5 M HCl: 10 g FeSO4·H2O, 2 g CaCl2, 2.2 g ZnSO4·H2O, 0.5 g MnSO4·H2O, 1 g CuSO4·H2O, 0.1 g (NH4)6Mo7O 24 ·H2O, and 0.02 g Na2B4O7·10H2O.

[0176] For each strain culture, the seed culture was shaken overnight at 30°C in a 25-mL tube containing 5 mL of LB medium, and 1 mL of the culture was inoculated into a 250-mL flask containing 100 mL of MR medium containing 20 g / L glucose and 2 g / L amino acids, and shaken at 30°C for 96 hours. If necessary, 2 g / L of 3HB was additionally supplied.

[0177] The concentration of PHA and the composition of PHA monomer were analyzed by gas chromatography (6890N GC System, Agilent Technologies, Palo Alto, CA, USA) equipped with a fused silica capillary column (Supelco SPBTM-5, 30 m × 0.32 mm ID 0.25 mm film, Bellefonte, PA, USA) using benzoic acid methyl ester as an internal standard. Dry cell weight per liter of culture broth was measured using a previously reported method. PHA content (wt%) is defined as the ratio of dry cell weight to PHA concentration.

[0178]

[0179] 2-1 L-amino acid deaminase screening

[0180] In the selection of amino acid deaminase enzymes showing different activities for each amino acid, tests were conducted on ilvA, PmILAAD, PmIILAAD, PvLAAD, and PyxLAAD, which produce poly(2HA-co-3HB) copolymers with high 2HA fractions. In this case, a strain that does not express the foreign deaminase gene and uses the deaminase existing in E. coli itself was used as a control. The dehydrogenase used here was LlpanE, the PHA synthase was PhaC1437 (E130D, S325T, S477G, and Q481K), and the CoA transferase was fixed to HadA. In this example, a recombinant E. coli XB-l strain, a host cell with ldhA deleted, was used to suppress the incorporation of lactate monomers into high molecular weight copolymers. The results of the analysis of polymers produced from the transformed recombinant E. coli XB-l strain cultured in the above medium are shown in Table 3.

[0181]

[0182] : When leucine was added externally, it was confirmed that among the above deaminase enzymes, PyxLAAD produced a polymer with a relatively highest 2HIC content.

[0183]

[0184] : When isoleucine was added externally, it was confirmed that a polymer with a relatively highest 2H3MP content was produced when the E. coli deaminase itself was used without expressing the deaminase enzyme.

[0185]

[0186] : When valine was added externally, it was confirmed that among the above deaminase enzymes, PmILAAD and PmIILAAD, and E. coli's own deaminase were used to produce a polymer with the highest 2HIV content.

[0187]

[0188] : When norvaline was added externally, it was confirmed that a polymer with a relatively highest 2HV content was produced when the E. coli deaminase itself was used without expressing the deaminase enzyme.

[0189]

[0190] : When norleucine was added externally, it was confirmed that a polymer with a relatively highest 2HHx content was produced when the E. coli deaminase itself was used without expressing the deaminase enzyme.

[0191]

[0192] : When methionine was added externally, it was confirmed that among the above deaminase enzymes, PmILAAD and E. coli's own deaminase were used to produce a polymer with the highest TB content.

[0193]

[0194] : When phenylalanine was added externally, it was confirmed that among the above deaminase enzymes, PmILAAD and PyxLAAD, and E. coli's own deaminase were used to produce a polymer with the highest PhP content.

[0195]

[0196] a For each strain, it was cultured for 96 hours in MR medium supplemented with 3HB.

[0197] b 3HB: 3-hydroxybutyrate; LA: lactate; 2HIC: 2-hydroxyisocaproate; 2H3MP: 2-hydroxy-3-methylpentanoate; 2HIV: 2-hydroxyisovalerate; 2HV: 2-hydroxyvalerate; TB: 2-hydroxy-4-(methylthio)butyrate; PhP (PhLA): 2-hydroxy-3-phenylpropionate

[0198]

[0199] : Through the above results, it was confirmed that the deaminase supporting high 2HA molar fraction for each amino acid is different.

[0200]

[0201] 2-2 Dehydrogenase screening

[0202] In the selection of dehydrogenases that convert 2KA to 2HA, tests were conducted on LlpanE, KIVR, ManDH, Lcddh, Lcppr, and Paldh that produce poly(2HA-co-3HB) copolymers with high 2HA fractions. Different types of deaminase were selected for each amino acid, and PHA synthase was fixed to PhaC1437 (E130D, S325T, S477G, and Q481K) and CoA transferase was fixed to HadA. The results of analysis of the polymers produced from the transformed recombinant E. coli XL1-Blue strain cultured in the above medium are shown in Table 10.

[0203]

[0204] : When leucine was added externally, it was confirmed that among the above dehydrogenase enzymes, LlpanE produced a polymer with a relatively highest 2HIC content.

[0205]

[0206] : When isoleucine was added externally, it was confirmed that among the dehydrogenase enzymes, LlpanE produced a polymer with a relatively highest 2H3MP content.

[0207]

[0208] : When valine was added externally, it was confirmed that among the above dehydrogenase enzymes, LlpanE and Paldh produced polymers with relatively the highest 2HIV content.

[0209]

[0210] : When norleucine was added externally, it was confirmed that among the above dehydrogenase enzymes, LlpanE and Paldh produced polymers with relatively the highest 2HHx content.

[0211]

[0212] : When norvaline was added externally, it was confirmed that among the above dehydrogenase enzymes, LlpanE and Paldh produced polymers with relatively the highest 2HV content.

[0213]

[0214] : When methionine was added externally, it was confirmed that among the dehydrogenase enzymes, Lcddh and Lcpph produced polymers with relatively the highest TB content.

[0215]

[0216] : When phenylalanine was added externally, it was confirmed that among the dehydrogenase enzymes, Lcpph was used to produce a polymer with the highest PhP content.

[0217]

[0218] a For each strain, it was cultured in MR medium for 96 hours.

[0219] b 3HB: 3-hydroxybutyrate; LA: lactate; 2HIC: 2-hydroxyisocaproate; 2H3MP: 2-hydroxy-3-methylpentanoate; 2HIV: 2-hydroxyisovalerate; 2HV: 2-hydroxyvalerate; TB: 2-hydroxy-4-(methylthio)butyrate; PhP (PhLA): 2-hydroxy-3-phenylpropionate

[0220]

[0221] : Through the above results, it was confirmed that the dehydrogenase supporting high 2HA molar fractions for each amino acid is different.

[0222]

[0223] 2-3 CoA transferase screening

[0224] In the selection of CoA transferase that converts 2HA to 2HA-CoA, tests were conducted on 4HBS, AbfT, Cat2, Pct540, PrpE, and HadA, which produce poly(2HA-co-3HB) copolymers with high 2HA fractions. Different types of deaminase were selected for each amino acid, and the dehydrogenase was fixed to LlpanE and the PHA synthase was PhaC1437 (E130D, S325T, S477G, and Q481K). The results of the analysis of the polymer produced from the transformed recombinant E. coli XL1-Blue strain cultured in the above medium are shown in Table 16.

[0225]

[0226] : When leucine was added externally, it was confirmed that among the CoA transferase enzymes, HadA produced a polymer with a relatively highest 2HIC content.

[0227]

[0228] : When isoleucine was added externally, it was confirmed that among the CoA transferase enzymes, HadA produced a polymer with a relatively highest 2H3MP content.

[0229]

[0230] : When valine was added externally, it was confirmed that among the CoA transferase enzymes, HadA produced a polymer with a relatively highest 2HIV content.

[0231]

[0232] : When norleucine was added externally, it was confirmed that among the CoA transferase enzymes, HadA produced a polymer with a relatively highest 2HHx content.

[0233]

[0234] : When norvaline was added externally, it was confirmed that among the CoA transferase enzymes, HadA produced a polymer with a relatively highest 2HIV content.

[0235]

[0236] : When methionine was added externally, it was confirmed that among the dehydrogenase enzymes, Lcddh and Lcpph produced polymers with relatively the highest TB content.

[0237]

[0238] : When phenylalanine was added externally, it was confirmed that a polymer with a relatively highest PhP content was produced when HadA was used among the CoA transferase enzymes.

[0239]

[0240] a For each strain, it was cultured in MR medium for 96 hours.

[0241] b 3HB: 3-hydroxybutyrate; LA: lactate; 2HIC: 2-hydroxyisocaproate; 2H3MP: 2-hydroxy-3-methylpentanoate; 2HIV: 2-hydroxyisovalerate; 2HV: 2-hydroxyvalerate; TB: 2-hydroxy-4-(methylthio)butyrate; PhP (PhLA): 2-hydroxy-3-phenylpropionate

[0242]

[0243] : Through the above results, it was confirmed that HadA is an enzyme that supports a high 2HA molar fraction in all amino acids.

[0244]

[0245] Example 3. Production of P(3HB-Co-2HA)

[0246] 2-HA becomes 2HA-CoA by CoA transferase, and polymerizes with 3HB-CoA produced through PhaA and PhaB by PHA synthase to produce a polymer in the form of P(3HB-Co-2HA) (Fig. 4).

[0247] As described in Examples 1 and 2, when amino acids were supplied externally, polymers having various combinations of monomers and compositions were produced.

[0248]

[0249] A biodegradable polymer containing an aromatic 2-hydroxy acid (2-hydroxyacid: 2-HA) converted from an amino acid as a monomer can be produced through a recombinant microorganism having the ability to produce polyhydroxyalkanoate according to the present invention.

[0250] In addition, according to the present invention, a polymer containing 2-hydroxy acid in various molar fractions depending on the type of amino acid can be manufactured.

[0251]

[0252] 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.

[0253]

[0254] Electronic file attached.

Claims

1. A gene encoding amino acid deaminase; a gene encoding dehydrogenase; for a microorganism having the ability to produce acetyl-CoA from a carbon source; A recombinant microorganism having the ability to produce polyhydroxyalkanoate, which comprises 2-hydroxyacid (2-HA) converted from an amino acid as a monomer, wherein a gene encoding CoA transferase and a gene encoding polyhydroxyalkanoate synthase are introduced or amplified.

2. A recombinant microorganism in claim 1, wherein the amino acid is at least one selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, and proline.

3. A recombinant microorganism in the first paragraph, wherein the amino acid deaminase coding gene is at least one selected from the group consisting of an ilvA gene coding for an enzyme comprising a sequence of SEQ ID NO: 1, a PmILAAD gene coding for an enzyme comprising a sequence of SEQ ID NO: 2, a PmIILAAD gene coding for an enzyme comprising a sequence of SEQ ID NO: 3, a PvLAAD gene coding for an enzyme comprising a sequence of SEQ ID NO: 4, and a PyxLAAD gene coding for an enzyme comprising a sequence of SEQ ID NO:

5.

4. A recombinant microorganism in the first paragraph, wherein the dehydrogenase coding gene is at least one selected from the group consisting of a LlpanE gene coding for an enzyme comprising a sequence of SEQ ID NO: 6, a KIVR gene coding for an enzyme comprising a sequence of SEQ ID NO: 7, a ManDH gene coding for an enzyme comprising a sequence of SEQ ID NO: 8, a Lcppr gene coding for an enzyme comprising a sequence of SEQ ID NO: 9, a Lcddh gene coding for an enzyme comprising a sequence of SEQ ID NO: 10, and a Paldh gene coding for an enzyme comprising a sequence of SEQ ID NO:

11.

5. A recombinant microorganism in the first paragraph, wherein the CoA transferase coding gene is a hadA gene encoding an enzyme having a sequence of SEQ ID NO:

12.

6. A recombinant microorganism in the first paragraph, wherein the gene encoding the polyhydroxyalkanoate synthase is a PhaC gene encoding an enzyme having a sequence of SEQ ID NO:

13.

7. A method for producing polyhydroxyalkanoate comprising 2-hydroxy acid (2-HA) as a monomer converted from an amino acid comprising the following steps: (a) a step of culturing a recombinant microorganism of any one of claims 1 to 6 to produce polyhydroxyalkanoate; and (b) A step of obtaining the above polyhydroxyalkanoate.

8. A manufacturing method in claim 7, wherein the amino acid is at least one selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, and proline.

9. A manufacturing method in claim 7, wherein the amino acid deaminase coding gene is at least one selected from the group consisting of an ilvA gene coding for an enzyme comprising a sequence of SEQ ID NO: 1, a PmILAAD gene coding for an enzyme comprising a sequence of SEQ ID NO: 2, a PmIILAAD gene coding for an enzyme comprising a sequence of SEQ ID NO: 3, a PvLAAD gene coding for an enzyme comprising a sequence of SEQ ID NO: 4, and a PyxLAAD gene coding for an enzyme comprising a sequence of SEQ ID NO:

5.

10. A manufacturing method in claim 7, wherein the dehydrogenase coding gene is at least one selected from the group consisting of a LlpanE gene coding for an enzyme comprising a sequence of SEQ ID NO: 6, a KIVR gene coding for an enzyme comprising a sequence of SEQ ID NO: 7, a ManDH gene coding for an enzyme comprising a sequence of SEQ ID NO: 8, a Lcppr gene coding for an enzyme comprising a sequence of SEQ ID NO: 9, a Lcddh gene coding for an enzyme comprising a sequence of SEQ ID NO: 10, and a Paldh gene coding for an enzyme comprising a sequence of SEQ ID NO:

11.

11. A manufacturing method in claim 7, wherein the CoA transferase coding gene is a hadA gene coding for an enzyme having a sequence of SEQ ID NO:

12.

12. A manufacturing method in claim 7, wherein the gene encoding the polyhydroxyalkanoate synthase is a PhaC gene encoding an enzyme having a sequence of SEQ ID NO: 13.

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