Recombinant microorganism having ability to produce PHA copolymer, and method for producing PHA copolymer using same

Abstract

The present invention relates to a recombinant microorganism having the ability to produce a polyhydroxyalkanoate (PHA) copolymer, and a method for producing a PHA copolymer using same. More particularly, the present invention relates to a recombinant microorganism having the ability to produce a PHA copolymer containing 3-hydroxyalkanoate (3-HHx) as a monomer, in which a gene encoding a PHA synthase derived from an Aeromonas sp. strain is introduced or amplified, and to a method for producing a PHA copolymer using same.

Description

Recombinant microorganism having the ability to produce PHA copolymer and a method for producing PHA copolymer using the same

[0001] The present invention relates to a recombinant microorganism having the ability to produce polyhydroxyalkanoates (PHA) copolymers and a method for producing PHA copolymers using the same. More specifically, the invention relates to a recombinant microorganism having the ability to produce PHA copolymers comprising 3-hydroxyalkanoate (3-HHx) as a monomer, in which a gene encoding a PHA synthase derived from a strain of the genus Aeromonas is introduced or amplified, and a method for producing PHA copolymers using the same.

[0002]

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

[0004] Polyhydroxybutyrate (PHB) is the most characteristic PHA containing only 3-hydroxybutyrate (3-HB) as a component. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) consists of 3-hydroxybutyrate (HB) and 3-hydroxyhexanoate (HHx) as components.

[0005] These PHBHHx possess superior physical and mechanical properties, such as a lower melting point than PHB, enhanced flexibility, and excellent impact strength due to HHx. Furthermore, they exhibit better biocompatibility than PHB, demonstrating excellent material properties for tissue engineering (Yang XS, Zhao K, Chen GQ (2002) Effect of surface treatment on the biocompatibility of microbial polyhydroxyalkanoates. Biomaterials 23: 1391-1397).

[0006] In this regard, PHBHHx is considered a promising material in various fields.

[0007] PHBHHx has been synthesized by constructing a recombinant microorganism through the cloning of a microbial-derived PHA synthesis gene based on a carbon source (Lu XY, Wu Q, Zhang WJ, Zhang G, Chen GQ (2004) Molecular Cloning of polyhydroxyalkanoate synthesis operon from Aeromonas hydrophila and its expression in Escherichia coli. Biotechnol. Prog. 20: 1332-1336).

[0008] Genes encoding enzymes or proteins involved in the biosynthesis of PHBHHx have been identified at the molecular level, allowing for the consideration of various metabolic engineering approaches to develop PHBHHx-producing strains with superior characteristics.

[0009]

[0010] Summary of the Invention

[0011] The object of the present invention is to provide a recombinant microorganism having the ability to produce a PHA copolymer comprising 3-hydroxyalkanoate (3-HHx) as a monomer.

[0012] Another objective of the present invention is to provide a method for producing a PHA copolymer comprising 3-hydroxyalkanoate (3-HHx) as a monomer using the recombinant microorganism.

[0013] To achieve the above objective, the present invention provides a recombinant microorganism having the ability to produce a PHA copolymer comprising 3-hydroxyalkanoate (3-HHx) as a monomer, wherein a gene encoding a PHA synthase derived from an Aeromonas strain is introduced or amplified into a microorganism having a polyhydroxyalkanoate (PHA) biosynthetic pathway.

[0014] The present invention also provides a method for preparing a PHA copolymer comprising 3-hydroxyalkanoate (3-HHx) as a monomer, comprising the steps of: (a) culturing a recombinant microorganism to produce a PHA copolymer; and (b) obtaining the PHA copolymer.

[0015]

[0016] Figure 1 is an overview of the metabolic pathway for the biosynthesis of P(3HB-co-3HHx) from glucose in Pseudomonas putida KT2440.

[0017] Figure 2 shows Pseudomonas putida KT2440 used for the biosynthesis of P(3HB-co-3HHx).

[0018]

[0019] Specific details for implementing the invention

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0021] A representative type of polyhydroxyalkanoate (PHA) is P(3HB)[Poly(3-hydroxybutyrate)], which has the disadvantages of being hard, brittle, and difficult to process due to its high crystallinity.

[0022] However, these properties are improved by copolymerizing P(3HB) with mcl-PHA monomers (C6~C14), and in the case of P(3HB-co-3HHx), elasticity and elongation at break are increased and crystallinity is reduced. In this regard, for example, when the mole fraction of 3HHx is 17 mol %, it can have properties similar to LDPE (Low Density Polyethylene).

[0023] Pseudomonas putida KT2440 is a strain that efficiently produces mcl-PHA composed of C6 to C14 groups, and the metabolic pathway for mcl-PHA production (Malonyl-CoA, Fatty acid de novo, B-oxidation, PHA biosynthesis) is shown in Figure 1.

[0024] To produce P(3HB-co-3HHx), 3HB-CoA and 3HHx-CoA are required as precursors.

[0025] In a specific embodiment according to the present invention, since 3HB-CoA is produced in very small quantities within P. putida KT2440, the 3HB-CoA pool was increased by introducing PhaA and PhaB derived from Ralstonia eutropha, a P(3HB) producing strain.

[0026] 3HHx-CoA is one of the precursors of mcl-PHA and can be produced in P. putida KT2440 through fatty acid de novo and β-oxidation. We aimed to increase the 3HHx-CoA pool by manipulating genes ranging from Acetyl-CoA to 3HHx-CoA. Genes that have a positive effect on the increase of 3HHx-CoA in each pathway were manipulated and tested.

[0027] In particular, it was confirmed that P(3HB-co-3HHx) can be produced by deleting the intrinsic PhaC of P. putida KT2440 and introducing 3AZ2C derived from Aeromonas salmonicid asph. achromogenes, which accepts 3HB-CoA and 3HHx-CoA.

[0028] To produce P(3HB), PhaA and PhaB derived from Ralstonia eutropha are required, and 3AZ2C derived from Aeromonas salmonicidasp. achromogenes was used for PhaC.

[0029] Based on this, the present invention relates, in one aspect, to a recombinant microorganism having the ability to produce a PHA copolymer comprising 3-hydroxyalkanoate (3-HHx) as a monomer, wherein a gene encoding a PHA synthase derived from an Aeromonas strain is introduced or amplified into a microorganism having a polyhydroxyalkanoate (PHA) biosynthetic pathway.

[0030] The above microorganisms are microorganisms having a PHA biosynthetic pathway and may include, for example, Escherichia coli, Alcaligenes, Pseudomonas, Escherichia, Ralstonia, Bacillus, or Corynebacterium.

[0031] In a specific embodiment according to the present invention, the microorganism may be Pseudomonas putida. Specifically, the microorganism may be Pseudomonas putida KT2440.

[0032] The above PHA biosynthetic pathway may include a carbon source conversion pathway; a malonyl-CoA pool generation pathway; a fatty acid synthesis pathway; a β-ogenesis pathway; and a PHA synthesis pathway.

[0033] The above carbon source may be carbohydrates such as glucose, fructose, sucrose, maltose, mannitol, sorbitol, etc.; alcohols such as sugar alcohols, glycerol, pyruvate, lactic acid, citric acid, etc.; organic acids, etc., but is not limited thereto. The above carbon source may be used alone or in combination of two or more types, but is not limited thereto.

[0034] The above-mentioned strain of the genus Aeromonas may be Aeromonas salmonicidasp. Accordingly, the gene encoding the above-mentioned PHA synthase may be of Aeromonas salmonicidasp.

[0035] The gene encoding the above PHA synthase may be, for example, a PhaC coding gene.

[0036] The gene encoding the above PHA synthase PhaC may be named 3AZ2C, and specifically, it may be a gene containing the sequence of SEQ ID NO. 1.

[0037] [Sequence No. 1]

[0038]

[0039] In relation to the introduction of the gene encoding the above PHA synthase, we intended to produce P(3HB-co-3HHx) by deleting PhaC contained in P. putida KT2440 and introducing 3AZ2C derived from Aeromonas salmonicida sp., which accepts 3HB-CoA and 3HHx-CoA.

[0040] The genome of P. putidaKT2440, which produces Mcl-PHA, contains PhaC1, PhaZ (PHA depolymerase), and PhaC2. A strain in which these were removed and 3AZ2C, PhaA, and PhaB were introduced was named P. putidaKT2440△PhaC1ZC2::3AZ2C-ReAB, and a strain in which only 3AZ2C was introduced was named P. putidaKT2440△PhaC1ZC2::3AZ2C.

[0041] Based on data on mcl-PHA production from glucose, two strains were used to manipulate genes that had a positive effect on mcl-PHA production from glucose to confirm the production of P(3HB-co-3HHx) from glucose.

[0042] Based on this, one or more genes coding for PHA synthase PhaC1, PHA depolymerase PhaZ, and PHA synthase PhaC2 among the above microorganisms may be deleted, and a gene 3AZ2C containing the sequence of SEQ ID NO. 1 coding for PHA synthase may be introduced.

[0043] The sequences of the genes encoding the deleted PhaC1, PhaZ, and PhaC2, respectively, among the microorganisms are as follows.

[0044] [Sequence No. 4] PhaC1 coding gene

[0045]

[0046] [Sequence No. 5] PhaZ coding gene

[0047]

[0048] [Sequence No. 6] PhaC2 coding gene

[0049]

[0050] To produce P(3HB), PhaA and PhaB derived from Ralstonia eutropha are required because they are produced in very small quantities within P. putida KT2440, and 3AZ2C derived from Aeromonas salmonicidasp. was used for PhaC.

[0051] In consideration of this, polyhydroxyalkanoate synthase PhaA and / or PhaB coding genes may be additionally introduced into the microorganism.

[0052] The above polyhydroxyalkanoate synthases PhaA and / or PhaB can introduce PhaA and PhaB derived from Ralstonia eutropha.

[0053] The above PhaA and PhaB coding genes may each include the sequences of SEQ ID NO. 2 and SEQ ID NO. 3.

[0054] [Sequence No. 2]

[0055]

[0056] [Sequence No. 3]

[0057]

[0058] The above PHA copolymer may include HB (hydroxybutyrate). The above PHA copolymer may be, for example, P(3HB-co-3HHx) (Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)).

[0059] In some cases, the recombinant microorganism according to the present invention may additionally include one or more gene variants selected from the group consisting of the following:

[0060] Genes coding for enzymes related to the glucose conversion pathway;

[0061] Genes coding for enzymes related to the Malonyl-CoA pool generation pathway;

[0062] Genes coding for enzymes related to fatty acid synthesis pathways;

[0063] β-oxidation pathway-related enzyme-coding genes; and

[0064] Genes coding for enzymes related to the PHA synthesis pathway.

[0065] In a specific embodiment, the gene variant coding for the enzyme related to the glucose conversion pathway may include a weakening or deletion of the glucose dehydrogenase coding gene.

[0066] The glucose dehydrogenase coding gene mentioned above may be, for example, the Gcd gene. The glucose dehydrogenase coding gene Gcd may be weakened or deleted.

[0067] The glucose dehydrogenase coding gene Gcd above may include the sequence of SEQ ID NO. 7.

[0068] [Sequence No. 7]

[0069]

[0070] The weakening or deletion of Gcd had a positive effect on mcl-PHA production. Accordingly, the deletion effect of Gcd was tested in P. putidaKT2440 βPhaC1ZC2::3AZ2C-ReAB and P. putidaKT2440 △PhaC1ZC2::3AZ2C.

[0071] In a specific embodiment, the enzyme-coding gene variant associated with the malonyl-CoA pool generation pathway may comprise i) weakening or deletion of the isocitrate lyase-coding gene; and / or ii) amplification of the acetyl-CoA carboxylase A, B, C, and D-coding genes.

[0072] The above isocitrate lyase coding gene may be, for example, the AceA gene. The above isocitrate lyase coding gene AceA may be weakened or deleted.

[0073]

[0074] *69 The above isocystate lyase coding gene AceA may include the sequence of SEQ ID NO. 18.

[0075] [Sequence No. 18]

[0076]

[0077] The above acetyl-CoA carboxylase A, B, C, and D coding genes may be, for example, AccA, AccB, AccC, and AccD genes, respectively. The above acetyl-CoA carboxylase A, B, C, and D coding genes AccA, AccB, AccC, and AccD may be amplified.

[0078] The above-mentioned acetyl-CoA carboxylase A, B, C, and D coding genes AccA, AccB, AccC, and AccD may each include the sequences of SEQ ID NOs 8 to 11.

[0079] [Sequence No. 8]

[0080]

[0081] [Sequence No. 9]

[0082]

[0083] [Sequence No. 10]

[0084]

[0085] [Sequence No. 11]

[0086]

[0087] To increase mcl-PHA production, it is necessary to increase the Malonyl-CoA pool, which is the first substance of fatty acid de novo, and the deletion of AceA and the overexpression of AccABCD had a positive effect on mcl-PHA production from Malonyl-CoA. Accordingly, the effects of deletion of AceA and overexpression of AccABCD were tested in P. putidaKT2440 △PhaC1ZC2::3AZ2C-ReAB and P. putidaKT2440 △PhaC1ZC2::3AZ2C.

[0088] The enzyme-coding gene variant related to the fatty acid synthesis pathway mentioned above may include amplification of the acyl-CoA thioesterase I or acyl-CoA thioesterase II coding gene.

[0089] The above-mentioned acyl-CoA thioesterase I or acyl-CoA thioesterase II coding genes may be, for example, the TesA and TesB genes, respectively. The above-mentioned acyl-CoA thioesterase I or acyl-CoA thioesterase II coding genes TesA or TesB may be amplified.

[0090] The above acyl-CoA thioesterase I or acyl-CoA thioesterase II coding gene TesA or TesB may each include the sequence of SEQ ID NO. 12 or SEQ ID NO. 13.

[0091] [Sequence No. 12]

[0092]

[0093] [Sequence No. 13]

[0094]

[0095] Overexpression of TesAB had a positive effect on mcl-PHA production in the fatty acid de novo biosynthesis pathway. Accordingly, the effect of TesAB overexpression was tested in P. putidaKT2440 △PhaC1ZC2::3AZ2C-ReAB and P. putidaKT2440 △PhaC1ZC2::3AZ2C.

[0096] The above-mentioned β-oxidation pathway-related enzyme-coding gene variants may include i) amplification of the acyl-CoA thioesterase-coding gene; ii) amplification of the acyl-CoA dehydrogenase-coding gene; iii) weakening or deletion of the 3-ketoacyl-CoA thiolase-coding gene; iv) weakening or deletion of the enoyyl-CoA hydrolase / 3-hydroxyacyl-CoA dehydrogenase-coding gene; and / or v) weakening or deletion of the acyl-CoA thioesterase I-coding gene.

[0097] The above acyl-CoA thioesterase-coding gene may be, for example, the FadDI gene. The above acyl-CoA thioesterase-coding gene FadDI may be amplified.

[0098] The above acyl-CoA dehydrogenase coding gene may be, for example, the FadE gene. The above acyl-CoA dehydrogenase coding gene FadE may be amplified.

[0099] The above 3-ketoacyl-CoA thiolase-coding gene may be, for example, the FadA gene. The above 3-ketoacyl-CoA thiolase-coding gene FadA may be attenuated or deleted.

[0100] The above-mentioned enoyl-CoA hydrolase / 3-hydroxyacyl-CoA dehydrogenase coding gene may be, for example, FadBA1. The above-mentioned enoyl-CoA hydrolase / 3-hydroxyacyl-CoA dehydrogenase coding gene FadBA1 may be attenuated or deleted.

[0101] The above acyl-CoA thioesterase I coding gene may be, for example, TesA. The above acyl-CoA thioesterase I coding gene TesA may be weakened or deleted.

[0102] Each of the above i) acyl-CoA thioesterase coding gene FadDI; ii) acyl-CoA dehydrogenase coding gene FadE; iii) 3-ketoacyl-CoA thiolase coding gene FadA; iv) enoyl-CoA hydrolase / 3-hydroxyacyl-CoA dehydrogenase coding gene FadBA1; and v) acyl-CoA thioesterase I coding gene TesA may comprise the sequences of SEQ ID NOs 14 to 17 and SEQ ID NO. 12.

[0103] [Sequence No. 14]

[0104]

[0105] [Sequence No. 15]

[0106]

[0107] [Sequence No. 16]

[0108]

[0109] [Sequence No. 17]

[0110]

[0111]

[0112] The overexpression of FadDI, overexpression of FadE, deletion of FadA, deletion of FadAB, and deletion of TesA had a positive effect on mcl-PHA production in the above β-oxidation pathway. Accordingly, the effects of overexpression of FadDI, overexpression of FadE, deletion of FadA, deletion of FadAB, and deletion of TesA were tested in P. putidaKT2440 △PhaC1ZC2::3AZ2C-ReAB and P. putidaKT2440 △PhaC1ZC2::3AZ2C.

[0113] The above PHA synthesis pathway-related enzyme-coding gene variants may include i) amplification of the (R)-3-hydroxyacyl-ACP-CoA transferase-coding gene; ii) amplification of the lipid acyl-CoA synthase-coding gene; iii) attenuation or deletion of the acyl-CoA thioesterase II-coding gene; and / or iv) amplification of the enoyyl-coenzyme A (CoA) hydrolase-coding gene.

[0114] The above (R)-3-hydroxyacyl-ACP-CoA transferase-coding gene may be, for example, PhaG. The above (R)-3-hydroxyacyl-ACP-CoA transferase-coding gene PhaG may be amplified.

[0115] The above-mentioned lipid acyl-CoA synthase coding gene may be, for example, AlkK. The above-mentioned lipid acyl-CoA synthase coding gene AlkK may be amplified.

[0116] The above acyl-CoA thioesterase II coding gene may be, for example, TesB or TesBII. The above acyl-CoA thioesterase II coding gene TesB or TesBII may be attenuated or deleted.

[0117] The above-mentioned enoyl-coenzyme A (CoA) hydrolase-coding gene may be, for example, PhaJ1. The above-mentioned enoyl-coenzyme A (CoA) hydrolase-coding gene PhaJ1 may be amplified.

[0118] Each of the above i) (R)-3-hydroxyacyl-ACP-CoA transferase coding gene PhaG; ii) lipid acyl-CoA synthase coding gene AlkK; iii) acyl-CoA thioesterase II coding gene TesB or TesBII; and iv) enoyyl-coenzyme A(CoA) hydrolase coding gene PhaJ1 may include the sequences of SEQ ID NOs 19 to 20, SEQ ID NOs 13, SEQ ID NOs 21 and 22.

[0119] [Sequence No. 19]

[0120]

[0121] [Sequence No. 20]

[0122]

[0123] [Sequence No. 21]

[0124]

[0125] [Sequence No. 22]

[0126]

[0127] In the PHA biosynthesis pathway, the positive effects on mcl-PHA production were the overexpression of PhaG, the overexpression of PhaG and AlkK, the deletion of TesB, the deletion of TesBII, and the overexpression of PhaJ1. Accordingly, the effects of overexpression of PhaG, overexpression of PhaG and AlkK, the deletion of TesB, the deletion of TesBII, and the overexpression of PhaJ1 were tested in P. putidaKT2440 △PhaC1ZC2::3AZ2C-ReAB and P. putidaKT2440 △PhaC1ZC2::3AZ2C.

[0128] In the present invention, "weakening" may mean removing or reducing the intracellular activity of one or more enzymes encoded by the corresponding DNA in a microbial strain.

[0129] In the present invention, "deletion" may refer to a form in which part or all of the nucleotide sequence region from the start codon corresponding to the target gene to the stop codon, or the nucleotide sequence of the regulatory region, is removed from within the chromosome.

[0130] In the present invention, "recombination" means including polynucleotides or proteins that do not occur naturally within a host cell.

[0131] In the present invention, introduction means that a gene becomes replicable as an extrachromosomal factor or through the completion of chromosomal integration. The gene is transformed, and the transformed gene may include either one inserted into the chromosome of the microorganism or one located extrachromosomally, as long as it can be expressed within the microorganism. Furthermore, the gene may include DNA and RNA as a polynucleotide capable of encoding a protein.

[0132] Any form in which it is introduced and expressed within a microorganism is acceptable. For example, the gene may be introduced into the microorganism in the form of an expression cassette, which is a polynucleotide structure containing all the elements necessary for self-expression.

[0133] In the present invention, "overexpression" refers to a level of expression higher than the level at which a corresponding gene is expressed within a cell under normal conditions, and is a concept that includes methods such as replacing the promoter of a gene existing on the genome with a potent promoter, or increasing the expression amount by cloning the corresponding gene into an expression vector and transforming it into a cell.

[0134] In the present invention, "vector" refers to a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA within a suitable host.

[0135] Specifically, the vector may be a plasmid, a phage particle, or simply a potential genomic insert. When transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, be incorporated into the genome itself. Since plasmids are the most commonly used form of vectors currently, "plasmid" and "vector" are sometimes used interchangeably in this specification.

[0136] For the purposes of the present invention, it is preferable to use a plasmid vector. A typical plasmid vector that can be used for this purpose has a structure comprising (a) a replication initiation site that enables efficient replication to include hundreds of plasmid vectors per host cell, (b) an antibiotic resistance gene that enables selection of host cells transformed with the plasmid vector, and (c) a restriction enzyme cleavage site into which an exogenous DNA fragment can be inserted.

[0137] Even if a suitable restriction enzyme cleavage site is not present, the vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adapter or linker according to conventional methods.

[0138] 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, may also be used.

[0139] In addition to the aforementioned E. coli, strains of the genus Agrobacterium such as Agrobacterium A4, bacilli such as Bacillus subtilis, other intestinal bacteria such as Salmonella typhimurium or Serratia marcescens, and various strains of the genus Pseudomonas can be used as host cells.

[0140] Nucleic acids are "operably linked" when arranged in a functional relationship with other nucleic acid sequences. This can be a gene and regulatory sequence(s) linked in such a way that an appropriate molecule (e.g., a transcription-activating protein) enables gene expression when it binds to the regulatory sequence(s).

[0141] For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a pre-protein participating in the secretion of a polypeptide; a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of a sequence; or a ribosome binding site is operably linked to a coding sequence when it affects the transcription of a sequence; or a ribosome binding site is operably linked to a coding sequence when it is positioned to facilitate translation.

[0142] "Operably linked" means that the linked DNA sequences are in contact, and in the case of a secretion reader, are in contact and exist within the reading frame. However, enhancers do not require contact. The linkage of these sequences is performed by ligation at a convenient restriction enzyme site. If such a site is not present, a synthetic oligonucleotide adapter or linker is used according to conventional methods.

[0143] As used in this specification, the term "expression vector" generally refers to a recombinant carrier into which a fragment of heterogeneous DNA is inserted, typically a fragment of double-stranded DNA. Here, heterogeneous DNA refers to DNA that is not naturally found in host cells. Once inside a host cell, the expression vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (heterogeneous) DNA may be produced.

[0144] 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 operably linked to transcriptional and translational expression regulatory sequences that are functional within the selected expression host. Preferably, the expression regulatory sequences and the gene are contained within a single expression vector that includes both a bacterial selection marker and a replication origin. If the host cell is a eukaryotic cell, the expression vector must additionally include expression markers useful within the eukaryotic expression host.

[0145] Host cells transformed or transfected by the expression vector described above constitute another aspect of the present invention. As used herein, the term "transformation" means the introduction of DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or through chromosomal integration completion. As used herein, the term "transfection" means the acceptance of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed.

[0146] It must be understood that the vector and the expression regulatory sequence do not all function equally in expressing the DNA sequence of the present invention. Likewise, not all hosts function equally for the same expression system.

[0147] However, a person skilled in the art can make an appropriate selection among various vectors, expression control sequences, and hosts without departing from the scope of the present invention and without excessive experimental burden. 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 also be considered.

[0148] For example, considerations should be given to the relative strength of the sequence, modulation capabilities, and compatibility with the DNA sequence of the present invention, particularly regarding possible secondary structures. A unicellular host must be selected by considering factors such as the selected vector, the toxicity and secretory characteristics of the product encoded by the DNA sequence of the present invention, the ability to accurately fold the protein, culture and fermentation requirements, and the ease of purifying the product encoded by the DNA sequence of the present invention from the host. Within the range of these variables, a person skilled in the art may select various vector / expression control sequence / host combinations capable of expressing the DNA sequence of the present invention in fermentation or culture.

[0149] Binding methods, panning methods, film emulsion methods, etc., may be applied as screening methods when attempting to clone the cDNA of a protein according to the present invention by expression cloning.

[0150] As demonstrated in various embodiments of the present invention, such a system can be used for the production of various aromatic polymers. According to the present invention, it can contribute to establishing a bioprocess for the production of aromatic polyesters from renewable non-food biomass.

[0151] Based on this, the present invention relates, from another perspective, to a method for producing a polyhydroxyalkanoate comprising a monomer of 3-hydroxyalkanoate (3-hydroxyalkanoate: 3-HHx), comprising the steps of: (a) culturing a recombinant microorganism to produce a polyhydroxyalkanoate; and (b) obtaining the polyhydroxyalkanoate.

[0152] Specifically, the medium used in the step of culturing the recombinant microorganism may include, for example, LB medium and MR-N medium. The LB medium included in the medium may contain 10 g / L tryptone, 5 g / L NaCl, and 5 g / L yeast extract. The MR-N medium included in the medium contains 6.67 g KH2PO4, 4 g Na2HPO4, and 0.8 g MgSO4· per 1L. It contains 7H2O, 0.8 g citric acid, and 5 ml trace metal solution, wherein per 1 L, the trace metal solution comprises: 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 It contains ·H2O, and 0.02 g Na2B4O7·10H2O.

[0153] The above recombinant microorganism may be characterized by being cultured at a temperature of 30°C to 37°C, but is not limited thereto. Polyhydroxyalkanoates can be efficiently obtained under such temperature conditions.

[0154] The above culture may be characterized by maintaining the pH at 6.8 to 7.2, but is not limited thereto. By maintaining a pH within this range, the growth rate of the recombinant microorganism according to the present invention can be improved.

[0155] Examples

[0156] The present invention will be described in more detail below through examples. These examples are solely for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0157] Example 1. Recombinant Pseudomonas putida expression vector and strain construction

[0158] To replace the PhaC1ZC2 gene of Pseudomonas putida KT2440 with the 3AZ2C and 3AZ2C-ReAB genes, 500 bp DNA fragments upstream and downstream of the phaC1ZC2 gene were amplified using primer 1 / primer 2 and primer 3 / primer 4, respectively. The PCR products were cleaved with HindIII / SbfI and BamHI / EcoRI, respectively, and then inserted into pk19mobsacB to construct pk19-△phaC1ZC2. Then, the 3AZ2C and 3AZ2C-ReAB genes under the promoter were amplified using primer 5 / primer 6 and primer 5 / primer 7. The PCR products were cleaved with SbfI / BamHI and BamHI, respectively, and then inserted into pk19-△phaC1ZC2 to construct pk19-△phaC1ZC2::3AZ2C and pk19-△phaC1ZC2::3AZ2C-ReAB.

[0159] The constructed phaC1ZC2::3AZ2C and pk19-△phaC1ZC2::3AZ2C-ReAB plasmids were inserted into wild-type Pseudomonas putida KT2440, respectively, using electroporation. Pseudomonas putida KT2440 possessing the respective pk19-△phaC1ZC2::3AZ2C and pk19-△phaC1ZC2::3AZ2C-ReAB plasmids were screened in LB medium supplemented with 10% sucrose, and strains in which the sacB gene and kanamycin resistance gene were removed and the respective target genes were deleted were selected. Pseudomonas putida KT2440 with each gene deleted through a series of processes were named P. putida KT2440 PhaC1ZC2::3AZ2C and P. putida KT2440 PhaC1ZC2::3AZ2C-ReAB.

[0160] For the deletion of the AceA gene, 500 bp DNA fragments upstream and downstream of the aceA gene were amplified using primers 8 / primer 9 and 10 / primer 11, respectively. The PCR products were cleaved with HindIII / SbfI and BamHI / EcoRI, respectively, and then inserted into pk19mobsacB to construct pk19-△aceA. For the deletion of the Gcd gene, 500 bp DNA fragments upstream and downstream of the gcd gene were amplified using primers 12 / primer 13 and 14 / primer 15, respectively. The PCR products were cleaved with HindIII / SbfI and BamHI / EcoRI, respectively, and then inserted into pk19mobsacB to construct pk19-△gcd. For the deletion of the TesA gene, 500 bp DNA fragments upstream and downstream of the tesA gene were amplified using primers 16 / primer 17 and 18 / primer 19, respectively. The PCR products were cleaved with HindIII / SbfI and BamHI / EcoRI, respectively, and then inserted into pk19mobsacB to construct pk19mobsacB pk19-△tesA. For the deletion of the TesB gene, 500 bp DNA fragments upstream and downstream of the tesB gene were amplified using primers 20 / primer 21 and 22 / primer 23, respectively. The PCR products were cleaved with HindIII / SbfI and BamHI / EcoRI, respectively, and then inserted into pk19mobsacB to construct pk19-△tesB. To delete the TesBII gene, 500 bp DNA fragments upstream and downstream of the TesBII gene were amplified using primers 24 / primer 25 and 26 / primer 27, respectively. The PCR products were cleaved with HindIII / SbfI and BamHI / EcoRI, respectively, and then inserted into pk19mobsacB to construct pk19-△tesBII.For the deletion of the FadA gene, 500 bp DNA fragments upstream and downstream of the FadA gene were amplified using primers 28 / primer 29 and 30 / primer 31, respectively. The PCR products were cleaved with HindIII / SbfI and BamHI / EcoRI, respectively, and then inserted into pk19mobsacB to construct pk19-△fadA. For the deletion of the FadBA1 gene, 500 bp DNA fragments upstream and downstream of the FadBA1 gene were amplified using primers 32 / primer 33 and 34 / primer 35, respectively. The PCR products were cleaved with HindIII / SbfI and BamHI / EcoRI, respectively, and then inserted into pk19mobsacB to construct pk19-△fadBA1.

[0161] The constructed pk19mobsacB plasmid was inserted into P. putida KT2440 PhaC1ZC2::3AZ2C and P. putida KT2440 PhaC1ZC2::3AZ2C-ReAB, respectively, using electroporation. Pseudomonas putida KT2440s possessing each pk19mobsacB plasmid were screened in LB medium supplemented with 10% sucrose, and strains in which the sacB gene and kanamycin resistance gene were removed and the respective target genes were deleted were selected. Through this series of processes, the Pseudomonas putida KT2440s with deleted genes were named as shown in Table X.

[0162] The PhaG gene was amplified by PCR using primers 36 and 37, and the PCR product was cleaved with EcoRI and KpnI and inserted into pSEVA631pt-MCS to construct pSEVA631pt PhaG. The AlkK gene was amplified by PCR using primers 38 and 39, and the PCR product was cleaved with KpnI and BamHI and inserted into pSEVA631pt PhaG to construct pSEVA631pt PhaG AlkK. The FadDI gene was amplified by PCR using primers 40 and 41, and the PCR product was cleaved with MfeI and BamHI and inserted into pSEVA631pt-MCS to construct pSEVA631pt FadDI. The FadE gene was amplified by PCR using primers 42 and 43, and the PCR product was cleaved with MfeI and BamHI and inserted into pSEVA631pt-MCS to construct pSEVA631pt FadE. The PhaJ1 gene was amplified by PCR using primers 44 and 45, and the PCR product was cleaved with EcoRI and KpnI and inserted into pSEVA631pt-MCS to construct pSEVA631pt PhaJ1. The AccA gene was amplified by PCR using primers 46 and 47, and the PCR product was cleaved with MfeI and SacI and inserted into pSEVA631pt-MCS to construct pSEVA631pt AccA. The AccB gene was amplified by PCR using primers 48 and 49, and the PCR product was cleaved with SacI and BamHI and inserted into pSEVA631pt AccA to construct pSEVA631pt AccAB. The AccC gene was amplified by PCR using primers 50 and 51, and the PCR product was cleaved with XbaI and SbfI and inserted into pSEVA631pt AccAB to construct pSEVA631pt AccABC.The AccD gene was amplified by PCR using primers 52 and 53, and the PCR product was cleaved with SbfI and HindIII and inserted into pSEVA631pt AccABC to construct pSEVA631pt AccABCD. The TesA gene was amplified by PCR using primers 54 and 55, and the PCR product was cleaved with EcoRI and KpnI and inserted into pSEVA631pt-MCS to construct pSEVA631pt TesA. The TesB gene was amplified by PCR using primers 56 and 57, and the PCR product was cleaved with KpnI and BamHI and inserted into pSEVA631pt TesA to construct pSEVA631pt TesAB.

[0163] The constructed pSEVA631pt plasmid was inserted into P. putida KT2440 PhaC1ZC2::3AZ2C and P. putida KT2440 PhaC1ZC2::3AZ2C-ReAB, respectively, using electroporation. Through a series of processes, P. putida KT2440 PhaC1ZC2::3AZ2C and P. putida KT2440 PhaC1ZC2::3AZ2C-ReAB, into which the respective plasmids were inserted, were named as shown in Tables 4 and 5.

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172] Example 2. Production of P(3HB-co-3HHx) from glucose in P. putidaKT2440 △PhaC1ZC2::3AZ2C

[0173] <PHA 생산을 위한 플라스크 배양>

[0174] To produce P(3HB-co-3HHx) from glucose using the PH0 strain and strains derived from the PH0 strain (Table 4), a two-stage flask culture consisting of a cell growth phase and a PHA production phase was conducted. For the flask culture, recombinant Pseudomonas putida strains were first prepared by incubating overnight in a 15 ml tube containing 5 ml of LB medium at 30°C and 230 rpm. Then, the seed culture was inoculated at a concentration of 1% into a 250 ml flask containing 100 ml of LB medium and incubated at 30°C and 230 rpm for 24 hours. Afterward, the 100 ml of culture medium was centrifuged at 4°C and 4000 xg for 25 minutes, and the supernatant was discarded. The collected cell pellet was transferred to a 250 ml flask containing 100 ml of MR-N medium containing 20 g / L glucose and cultured for 72 hours at 30°C and 230 rpm.

[0175] LB medium consists of 10 g / L of tryptone, 5 g / L of yeast extract, and 5 g / L of NaCl. The MR-N medium (pH 7.0) consists of 6.67 g / L of KH2PO4, 4 g / L of Na2HPO4, 0.8 g / L of MgSO4·7H2O, 0.8 g / L of citric acid, and 5 mL of trace metal solution, and the trace metal solution consists of 10 g / L of FeSO4·7H2O, 2 g / L of CaCl2, 2.2 g / L of ZnSO4·7H2O, 0.5 g / L of MnSO4·4H2O, 1 g / L of CuSO4·5H2O, 0.1 g / L of (NH4)6Mo7O24·4H2O, 0.02 g / L of Na2B4O7·10H2O, and 0.5 M of HCl. MgSO4·7H2O and glucose were each sterilized. In addition, 40 µg / ml of kanamycin or 25 µg / ml of gentamicin was added to each medium as needed to select bacteria, culture, and prevent curing of the plasmid.

[0176] LC Analysis for Glucose Concentration Measurement

[0177] The concentration of glucose consumed during the PHA production step was measured using HPLC. The culture medium taken at 0 and 72h during the PHA production step was centrifuged at 4℃ at 4000 xg for 25 minutes, and

[0178] Only the supernatant was collected, diluted 10-fold with distilled water, and filtered through a 0.22 μm syringe filter (hydrophilic) to be used as a sample for HPLC analysis. For HPLC analysis, 5 μl of the sample was injected, and 5 mM sulfuric acid was flowed as the mobile phase at a rate of 0.8 ml / min. An Aminex HPX-87H Ion Exclusion Column (Bio-Rad) was used at 50 °C, and a Refractive Index Detector (RID) was used as the detector.

[0179] <PHA 생산 측정을 위한 GC 분석>

[0180] The content of the produced P(3HB-co-3HHx) and the mole fraction of the monomer were measured using GC.

[0181] After the 2-stage flask culture was completed, 100 ml of the culture medium was centrifuged at 4000 xg at 4°C for 25 minutes, the supernatant was discarded, and the cell pellet was dried overnight in a 65°C oven. DCW was measured, and 1 ml of PHA solution and 2 ml of chloroform were added to 50 mg of the dried cells and mixed, then reacted overnight in a 95°C oven. After the reaction was complete, 1 ml of distilled water was added, and once layer separation occurred, the sample from the lower layer was collected using a syringe and used as the sample for GC analysis. A DB-WAX column (Agilent J&W, 30 m × 0.32 mm × 0.25 μm column) was used, and a flame ionization detector (FID) was used as the detector.

[0182]

[0183] The performance of the PH0 strain and strains derived from it (Table 4) for P(3HB-co-3HHx) production from glucose was investigated through flask culture. The Content and 3HHx mole fraction of the PH11 strain were 17.425 wt% and 12.847 mo%, respectively, confirming that both the 3HHx mole fraction and Content were improved compared to PH0 (Control). (Table 6)

[0184]

[0185] Example 3. Production of P(3HB-co-3HHx) from glucose in P. putida KT2440 △PhaC1ZC2::3AZ2C-ReAB

[0186] <PHA 생산을 위한 플라스크 배양>

[0187] To produce P(3HB-co-3HHx) from glucose using the PBH0 strain and strains derived from it (Table 5), a two-stage flask culture consisting of a cell growth phase and a PHA production phase was conducted. For the flask culture, recombinant Pseudomonas putida strains were first prepared by incubating overnight in a 15 ml tube containing 5 ml of LB medium at 30°C and 230 rpm. Then, the seed culture was inoculated at a concentration of 1% into a 250 ml flask containing 100 ml of LB medium and incubated at 30°C and 230 rpm for 24 hours. Afterward, the 100 ml of culture was centrifuged at 4°C and 4000 xg for 25 minutes, and the supernatant was discarded. The collected cell pellet was transferred to a 250 ml flask containing 100 ml of MR-N medium containing 20 g / L glucose and cultured for 72 hours at 30°C and 230 rpm.

[0188] LB medium consists of 10 g / L of tryptone, 5 g / L of yeast extract, and 5 g / L of NaCl. The MR-N medium (pH 7.0) consists of 6.67 g / L of KH2PO4, 4 g / L of Na2HPO4, 0.8 g / L of MgSO4·7H2O, 0.8 g / L of citric acid, and 5 mL of trace metal solution, and the trace metal solution consists of 10 g / L of FeSO4·7H2O, 2 g / L of CaCl2, 2.2 g / L of ZnSO4·7H2O, 0.5 g / L of MnSO4·4H2O, 1 g / L of CuSO4·5H2O, 0.1 g / L of (NH4)6Mo7O24·4H2O, 0.02 g / L of Na2B4O7·10H2O, and 0.5 M of HCl. MgSO4·7H2O and glucose were each sterilized. In addition, 40 µg / ml of kanamycin or 25 µg / ml of gentamicin was added to each medium as needed to select bacteria, culture, and prevent curing of the plasmid.

[0189] LC Analysis for Glucose Concentration Measurement

[0190] The concentration of glucose consumed during the PHA production step was measured using HPLC. Culture samples taken at 0 and 72 h during the PHA production step were centrifuged at 4°C at 4000 xg for 25 minutes. Only the supernatant was collected, diluted 10-fold with distilled water, and filtered through a 0.22 µm syringe filter (hydrophilic) to be used as the sample for HPLC analysis. For HPLC analysis, 5 µl of the sample was injected, and 5 mM sulfuric acid was flowed as the mobile phase at a rate of 0.8 ml / min. An Aminex HPX-87H Ion Exclusion Column (Bio-Rad) was used at 50°C, and a Refractive Index Detector (RID) was used as the detector.

[0191] <PHA 생산 측정을 위한 GC 분석>

[0192] The content of the produced P(3HB-co-3HHx) and the mole fraction of the monomer were measured using GC.

[0193] After the 2-stage flask culture was completed, 100 ml of the culture medium was centrifuged at 4000 xg at 4°C for 25 minutes, the supernatant was discarded, and the cell pellet was dried overnight in a 65°C oven. DCW was measured, and 1 ml of PHA solution and 2 ml of chloroform were added to 50 mg of the dried cells and mixed, then reacted overnight in a 95°C oven. After the reaction was complete, 1 ml of distilled water was added, and once layer separation occurred, the sample from the lower layer was collected using a syringe and used as the sample for GC analysis. A DB-WAX column (Agilent J&W, 30 m × 0.32 mm × 0.25 μm column) was used, and a flame ionization detector (FID) was used as the detector.

[0194]

[0195] The performance of the PBH0 strain and strains based on it (Table 5) for P(3HB-co-3HHx) production from glucose was investigated through flask culture. The Content and 3HHx mole fraction of the PBH11 strain were 71.779 wt% and 2.749 mo%, respectively; it was confirmed that the Content did not differ significantly from PBH0 (Control) while the 3HHx mole fraction was improved. (Table 7)

[0196]

[0197] Through the recombinant microorganism according to the present invention, a PHA copolymer biodegradable polymer comprising 3-hydroxyalkanoate (3-HHx) as a monomer can be produced.

[0198] In addition, according to the present invention, PHA copolymers containing 3-hydroxyalkanoate included in the polymer in various mole fractions can be prepared.

[0199]

[0200]

[0201] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0202]

[0203] I have attached the electronic file.

Claims

1. A recombinant microorganism having the ability to produce a PHA copolymer containing 3-hydroxyalkanoate (3-HHx) as a monomer, wherein a gene encoding a PHA synthase derived from an Aeromonas strain is introduced or amplified into a microorganism having a polyhydroxyalkanoate (PHA) biosynthetic pathway.

2. A recombinant microorganism according to claim 1, characterized in that the microorganism is Pseudomonas putida.

3. The recombinant microorganism according to claim 1, characterized in that one or more genes coding for PHA synthase PhaC1, PHA depolymerase PhaZ, and PHA synthase PhaC2 among the microorganisms are deleted.

4. A recombinant microorganism according to claim 1, characterized in that the Aeromonas genus strain is Aeromonas salmonicidasp.

5. A recombinant microorganism according to claim 1, characterized in that the gene encoding the PHA synthase is a PhaC coding gene.

6. A recombinant microorganism characterized by including the sequence of SEQ ID NO. 1 in claim 5.

7. The recombinant microorganism according to claim 1, characterized in that a polyhydroxyalkanoate synthase PhaA and / or PhaB coding gene is additionally introduced.

8. A recombinant microorganism according to claim 7, characterized in that a gene comprising the sequence of SEQ ID NO. 2 and / or SEQ ID NO. 3 is additionally introduced.

9. A recombinant microorganism according to claim 1, characterized in that the PHA copolymer comprises HB (hydroxybutyrate).

10. A recombinant microorganism according to claim 9, characterized in that the PHA copolymer is P(3HB-co-3HHx) (Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)).

11. The recombinant microorganism of paragraph 1, further comprising one or more gene variants selected from the group consisting of the following: Genes coding for enzymes related to the glucose conversion pathway; Genes coding for enzymes related to the Malonyl-CoA pool generation pathway; Genes coding for enzymes related to fatty acid synthesis pathways; β-oxidation pathway-related enzyme-coding genes; and Genes coding for enzymes related to the PHA synthesis pathway.

12. A recombinant microorganism according to claim 11, characterized in that the glucose conversion pathway-related enzyme-coding gene variant comprises a weakening or deletion of a glucose dehydrogenase-coding gene.

13. The recombinant microorganism according to claim 11, characterized in that the enzyme-coding gene variant associated with the malonyl-CoA pool generation pathway comprises i) weakening or deletion of the isocitrate lyase-coding gene; and / or ii) amplification of the acetyl-CoA carboxylase A, B, C, and D-coding gene.

14. A recombinant microorganism according to claim 11, characterized in that the enzyme-coding gene variant related to the fatty acid synthesis pathway comprises an amplification of the acyl-CoA thioesterase I or acyl-CoA thioesterase II coding gene.

15. In paragraph 11, the gene variant coding for the enzyme related to the β-oxidation pathway is i) Amplification of the acyl-CoA thioesterase-coding gene; ii) Amplification of acyl-CoA dehydrogenase coding gene; iii) Weakening or deletion of the 3-ketoacyl-CoA thiolase-coding gene; iv) Weakening or deletion of the enoyl-CoA hydrolase / 3-hydroxyacyl-CoA dehydrogenase coding gene; and / or v) A recombinant microorganism characterized by comprising an attenuation or deletion of an acyl-CoA thioesterase I coding gene.

16. In paragraph 11, the enzyme-coding gene variant related to the PHA synthesis pathway is i) Amplification of the (R)-3-hydroxyacyl-ACP-CoA transferase-coding gene; ii) Amplification of the lipid acyl-CoA synthase-coding gene; iii) Weakening or deletion of the acyl-CoA thioesterase II coding gene; and / or iv) A recombinant microorganism characterized by including an amplification of an enoyyl-coenzyme A (CoA) hydrolase-coding gene.

17. A method for preparing a PHA copolymer comprising 3-hydroxyalkanoate (3-HHx) as a monomer, comprising the following steps: (a) a step of producing a PHA copolymer by culturing a recombinant microorganism of any one of claims 1 to 16; and (b) A step of obtaining the above PHA copolymer.

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