Patent Application
20190352622
The present invention relates to a method for producing polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) monomer. More particularly, the present invention relates to a recombinant microorganism which is introduced with a gene encoding a 2-hydroxyisocaproate-CoA transferase and a gene encoding polyhydroxyalkanoate synthase and is capable of producing polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) monomer, and a method for producing polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-HA monomer using the recombinant microorganism.
Polyhydroxyalkanoates (PHAs) are biological polyesters synthesized by a variety of microorganisms. These polymers are biodegradable and biocompatible thermoplastic materials, can be utilized in a variety of industrial biomedical applications because the properties thereof are similar to petroleum-based polymers, and are produced from renewable sources (Lee, S. Y. Biotechnol. Bioeng. 49:1 1996).
PHAs are classified into short-chain-length PHAs having a short carbon number and medium-chain-length PHAs having a long carbon number depending on the length of the side chain.
Various PHAs have been synthesized through recombinant microorganisms produced by cloning PHA synthetic genes derived from microorganisms such as Ralstonia eutropha, Pseudomonas and Bacillus (Qi et al., FEMS Microbiol. Lett., 157:155, 1997; Qi et al., FEMS Microbiol. Lett., 167:89, 1998; Langenbach et al., FEMS Microbiol. Lett., 150:303, 1997; WO 01/55436; U.S. Pat. No. 6,143,952; WO 98/54329; WO 99/61624).
PHAs having short side chains such as PHBs, which are homopolymers of R-3-hydroxy butyric acid, are thermoplastic materials of crystals and are readily broken due to the low elasticity thereof. On the other hand, MCL-PHAs with long side chains have higher elasticity. PHBs first became known about 70 years ago (Lemoigne & Roukhelman, 1925). On the other hand, MCL-PHAs relatively recently became known (deSmet et al., J. Bacteriol. 154:870-78 1983). These copolymers can be represented by poly(3HB-co-3-HX), wherein X represents 3-hydroxyalkanoate, or alkanoate or alkenoate having 6 or more carbon atoms. A particular example of a copolymer of two particular monomers is poly(3HB-co-3-hydroxyhexanoate) (Brandl et al., Int. J. Biol. Macromol. 11:49, 1989; Amos & McInerney, Arch. Microbiol., 155:103, 1991; U.S. Pat. No. 5,292,860).
The biosynthesis of PHAs includes converting hydroxyl acid into hydroxyacyl-CoA through CoA-transferase or CoA-ligase and polymerizing the converted hydroxyacyl-CoA using a PHA synthase. In the case of natural PHA synthase, the activity for 2-hydroxyacyl-CoA is much lower than the activity for 3-hydroxyacyl-CoA. However, recently, the present inventors have developed a genetically engineered PHA synthase (PhaClps6-19) of Pseudomonas sp. 6-19 so as to use lactyl-CoA, which is a kind of 2-hydroxyacyl-CoA, as a substrate (WO 08/062996; Yang et al., Biotechnol. Bioeng., 105:150, 2010; Jung et al., Biotechnol. Bioeng., 105:161, 2010). PhaClps6-19 has a wide variety of substrate specificities, and can use lactyl-CoA, which is one kind of 2-hydroxyacyl-CoA, as a substrate. Thus, synthesis of new PHAs containing different types of 2-hydroxy acids will be made possible by developing a system for converting various kinds of 2-hydroxy acid into 2-hydroxyacyl-CoA. Accordingly, the present inventors have made intensive efforts to develop a novel method for biosynthesizing PHAs containing 2-hydroxy acid. As a result, the present inventors have found that, when screening an enzyme that converts 2-hydroxy acid into 2-hydroxyacyl-CoA using acetyl-CoA and using the enzyme, various kinds of 2-hydroxyacyl-CoA can be produced under in vitro conditions and various PHAs can be produced using the same. Based on this finding, the present invention has been completed.
Therefore, it is one object of the present invention to provide a recombinant microorganism capable of producing polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) monomer.
It is another object of the present invention to provide a method for producing polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-HA monomer using the recombinant microorganism.
In accordance with one aspect of the present invention, provided is a recombinant microorganism obtained by introducing a gene encoding a 2-hydroxyisocaproate-CoA transferase and a gene encoding polyhydroxyalkanoate synthase into a microorganism capable of producing acetyl-CoA from a carbon source, wherein the recombinant microorganism is capable of producing polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) monomer.
In accordance with another aspect of the present invention, provided is a method for producing polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) monomer including (a) culturing the recombinant microorganism to produce polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) monomer; and (b) recovering the produced polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) monomer.
In accordance with another aspect of the present invention, provided is a recombinant microorganism obtained by amplifying a gene encoding a 2-hydroxyisocaproate-CoA transferase, a gene encoding a polyhydroxyalkanoate synthase, a gene encoding a 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, a gene encoding a chorismate mutase/prephenate dehydrogenase, and a gene encoding a D-lactate dehydrogenase in a microorganism capable of producing acetyl-CoA from a carbon source, wherein the recombinant microorganism is capable of producing polyhydroxyalkanoate having phenyllactate as a monomer.
In accordance with another aspect of the present invention, provided is a method for producing polyhydroxyalkanoate having phenyllactate as a monomer including: (a) culturing the recombinant microorganism to produce polyhydroxyalkanoate having phenyllactate as a monomer; and (b) recovering the produced polyhydroxyalkanoate having phenyllactate as a monomer.
In accordance with another aspect of the present invention, provided is a recombinant microorganism obtained by amplifying a gene encoding a 2-hydroxyisocaproate-CoA transferase, a gene encoding a polyhydroxyalkanoate synthase, a gene encoding a 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, a gene encoding a chorismate mutase/prephenate dehydrogenase, a gene encoding a D-lactate dehydrogenase, a gene encoding a hydroxymandelate synthase, a gene encoding a hydroxymandelate oxidase, and a gene encoding a D-mandelate dehydrogenase in a microorganism capable of producing acetyl-CoA from a carbon source, wherein the recombinant microorganism is capable of producing polyhydroxyalkanoate having mandelate as a monomer.
In accordance with another aspect of the present invention, provided is a method for producing polyhydroxyalkanoate having mandelate as a monomer including: (a) culturing the recombinant microorganism to produce polyhydroxyalkanoate having mandelate as a monomer; and (b) recovering the produced polyhydroxyalkanoate having mandelate as a monomer.
Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.
PhaC1437, without external addition of 3HB.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as appreciated by those skilled in the field to which the present invention pertains. In general, the nomenclature used herein is well-known in the art and is ordinarily used.
Aromatic polyesters are essential plastics which are mainly produced from petroleum. The present invention establishes a method of producing a polymer having aromatic polyester or long-chain 2-hydroxyalkanoate as a monomer from glucose in one step through metabolically engineered E. coli expressing a polyhydroxyalkanoate (PHA) synthase and coenzyme A (CoA) transferases that are active toward aromatic monomer.
In one embodiment of the present invention, in order to produce PHA containing phenyllactate as an aromatic polyester, cinnamoyl-CoA:phenyllactate CoA-transferase (FldA) and 4-coumarate:CoA ligase (4CL), which were found to have activity through in vitro analysis, were expressed together with a PHA synthase in the D-phenyllactate-producing E. coli strain. The strain prepared a poly(l6.8 mol % D-lactate-co-80.8 mol % 3HB-co-1.6 mol % D-phenyllactate-co-0.8 mol % D-4-hydroxyphenyllactate) polymer in an amount of 12.8 wt % of the dry cell weight using in-vivo-produced cinnamoyl-CoA as a CoA donor.
However, since the utilization range of aromatic substrate of the phenyllactate CoA-transferase (FldA) is very narrow, 2-isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase (HadA) that can produce a variety of kinds of aromatic hydroxyacyl CoA using acetyl-CoA as a CoA donor was identified, selected and used for the production of aromatic PHA. In order to mass-produce aromatic PHAs containing a high mole fraction of D-phenyllactate, an optimal metabolic pathway was first designed to over-produce the D-phenyllactate monomer.
In one embodiment of the present invention, in order to produce a metabolically engineered E. coli having a metabolic pathway optimal for the production of D-phenyllactate, the feedback-resistant aroG, pheA and fldH genes in tyrR-deficient E. coli were overexpressed, the competitive metabolic pathways (pflB, poxB, adhE and frdB) were deleted, and the tyrB and aspC genes were further deleted according to in-silico genomic scale metabolic flux analysis. The metabolically engineered E. coli produced 1.62 g/L of D-phenyllactate. When HadA and PHA synthases were expressed in the D-phenyllactate-overproducing strain, poly(52.1 mol % 3HB-co-47.9 mol % D-phenyllactate) was produced in an amount of 15.8 wt % of the dry cell weight.
Also, the potential of preparing various aromatic polyesters was confirmed by preparing polyesters including 4-hydroxyphenyllactate, mandelate and 3-hydroxy-3-phenylpropionate. Therefore, in one aspect, the present invention is directed to a recombinant microorganism obtained by introducing a gene encoding a 2-hydroxyisocaproate-CoA transferase and a gene encoding polyhydroxyalkanoate synthase into a microorganism capable of producing acetyl-CoA from a carbon source, wherein the recombinant microorganism is capable of producing polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) monomer.
In the present invention, long-chain 2-HA means 2-hydroxyalkanoate having 6 to 8 carbon atoms.
In the present invention, the aromatic monomer or long chain 2-HA monomer is selected from the group consisting of 2-hydroxyisocaproate, 2-hydroxyhexanoate, 2-hydroxyoctanoate, phenyllactate, 2-hydroxy-4-phenylbutyrate, 3-hydroxy-3-phenylpropionate, 4-hydroxybenzoic acid and mandelate.
In the present invention, the polyhydroxyalkanoate synthase is a PHA synthase derived from a strain selected from the group consisting of Ralstonia eutropha, Pseudomonas, Bacillus and Pseudomonas sp. 6-19, or a mutant enzyme of a PHA synthase having an amino acid sequence selected from the following:
an amino acid sequence having at least one mutation selected from the group consisting of E130D, S325T, L412M, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in the amino acid sequence of SEQ ID NO: 2;
an amino acid sequence (C1335) having mutations of E130D, S325T, L412M, S477G and Q481M in the amino acid sequence of SEQ ID NO: 2;
an amino acid sequence (C1310) having mutations of E130D, S477F and Q481K in the amino acid sequence of SEQ ID NO: 2; and
an amino acid sequence (C1312) having mutations of E130D, S477F and Q481R in the amino acid sequence of SEQ ID NO: 2.
In the present invention, the 2-hydroxyisocaproate-CoA transferase may be hadA derived from Clostridium difficile 630.
In the present invention, the 2-hydroxyisocaproate-CoA transferase may use acetyl-CoA as a CoA donor.
The microorganism of the present invention may be further introduced with a gene encoding a β-ketothiolase involved in 3-hydroxybutyryl-CoA biosynthesis and a gene encoding an acetoacetyl-CoA reductase in order for the microorganism to produce a polymer even without the supply of 3HB from the outside.
In another aspect, the present invention is directed to a method for producing polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) monomer including: (a) culturing the recombinant microorganism to produce polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) monomer; and (b) recovering the produced polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-hydroxyalkanoate (2-HA) monomer.
In one embodiment of the present invention, it was confirmed whether or not Pct (propionyl-CoA transferase) used for the synthesis of polyhydroxyalkanoate is capable of activating phenyllactate and mandelate with phenyllactyl-CoA and mandelyl-CoA, respectively. The mutant of Pctcp (Pct540) has been successfully applied to the in-vivo production of polyesters including various hydroxy acids such as glycolic acid, lactic acid, 2-hydroxybutyric acid, 2-hydroxyisovalerate and 2-hydroxy acid. For this reason, it can be considered that Pct540 has a broad substrate spectrum with respect to the carbon number and the hydroxyl group position. However, it has been confirmed that Pct540 does not have catalytic activity for phenyllactate and mandelate. Thus, in the present invention, an attempt was made to find a novel CoA-transferase capable of activating CoA derivatives corresponding to aromatic compounds for the production of aromatic copolymers.
The cinnamoyl-CoA:phenyllactate CoA-transferase (F1dA) of Clostridium sporogenes has been reported to be able to convert phenyllactate into phenyllactyl-CoA using cinnamoyl-CoA as a CoA donor (Dickert, S. et al., Eur. J. Biochem. 267: 3874, 2000). Since cinnamoyl-CoA is a non-natural metabolite of E. coli, FldA derived from Clostridium botulinum A str. ATCC 3502, which has 99.0% homology with FldA of C. sporogenes, was tested in order to confirm whether or not acetyl-CoA, which is a metabolite abundant in cells, is used as a CoA donor. However, FldA of C. botulinum A str. ATCC 3502 was found to have no catalytic activity to produce phenyllactyl-CoA using acetyl-CoA as a CoA donor.
Meanwhile, it is known that Streptomyces coelicolor 4-coumarate:CoA ligase (4CL) plays a key role in the metabolism of phenylpropanoids which produce precursors of secondary metabolites of plants such as lignin, flavonoids and phytoalexins (Kaneko, M. et al., J. Bacteriol., 185:20, 2003). Therefore, in one embodiment of the present invention, a biosynthetic pathway for synthesizing cinnamoyl-CoA from cinnamate was designed by introducing 4CL. 4CL mutants were used to convert cinnamate into cinnamoyl-CoA, and cinnamoyl-CoA was used as a CoA donor for FldA to form phenyllactyl-CoA. As a result, phenyllactyl-CoA was successfully synthesized through successive in-vitro reactions of 4CL and FldA. These results demonstrated that 4CL and FldA could be used for the production of phenyllactyl-CoA and the production of aromatic polyesters. Similarly, it could be confirmed that another promising aromatic monomer, 4-hydroxyphenyllactate, was also converted into 4-hydroxyphenyllactyl-CoA by successive in-vitro reactions of 4CL variants with FldA.
In the production of non-natural polyesters, it is important to select mutants of the PHA synthase for polymerization of the corresponding CoA substrate. Therefore, in order to investigate the performance of various PHA synthases, Pseudomonas sp. MBEL 6-19 PHA synthase (PhaCPs6-19) mutants were expressed in E. coli XL1-Blue overexpressing AroGfbr, PAL, 4CL, FldA and Pct540. The prepared recombinant strains were cultured in a MR medium supplemented with 20 g/L of glucose, 1 g/L of D-phenyllactate and 1 g/L of sodium 3-hydroxybutyrate (3HB). Sodium 3-hydroxybutyrate (3HB) was converted through Pct540 into 3HB-CoA, which is a preferred substrate of PhaC, and was added to enhance the production of the polymer since it allowed the production of sufficient amounts of PHAs. E. coli XL1-Blue expressing other PHA synthase mutants can produce various amounts of poly(D-lactate-co-3HB-co-D-phenyllactate) having different monomer compositions.
As a result of the above experiment, among the PhaC mutants, PhaC1437 having four amino acid substitutions (E130D, S325T, S477G and Q481K) produced poly(l8.3 mol % D-lactate-co-76.9 mol % 3HB-co-4.8 mol % D-phenyllactate) in an amount of 7.8% by weight of dry cell weight, which means that PhaC1437 is the most suitable PhaC mutant.
Next, E. coli was engineered in vivo to produce D-phenyllactate from glucose. The biosynthesis of aromatic compounds begins with the synthesis of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP), which is produced by condensation between phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) by DAHP synthase. The produced DAHP is converted into phenylpyruvate (PPA) which is then converted into D-phenyllactate by D-lactate dehydrogenase (FldH) (
In the present invention, feedback-inhibiting resistant mutants, AroGfbr [AroG (D146N)] and PheAfbr [PheA (T326P)], were constructed to release feedback inhibition by L-phenylalanine (Zhou, H. Y. et al., Bioresour. Technol. 101:4151, 2010; Kikuchi, Y. et al., Appl. Environ. Microbiol. 63:761, 1997). E. coli XL1-Blue expressing AroGfbr, PheAfbr and FldH of C. botulinum A str. ATCC 3502 produced 0.372 g/L of D-phenyllactate from 15.2 g/L of glucose. The overexpression of PAL, 4CL, FldA, Pct540 and PhaC1437 of the strain increased poly(l6.8 mol % D-lactate-co-80.8 mol % 3HB-co-1.6 mol % D-phenyllactate-co-0.8 mol % D-4-hydroxyphenyllactate) to 12.8 wt % of the dry cell weight.
There are two problems in producing an aromatic PHA containing D-phenyllactate. The first problem is that the efficiency of polymer synthesis and the content of aromatic monomer are very low. This is considered to be due to inefficiency of FldA using cinnamoyl-CoA as a CoA donor. The second problem is that the monomer spectrum of aromatic PHA is very narrow. The results of in vitro enzymatic analysis showed that FldA can transfer CoA to phenyllactate and 4-hydroxyphenyllactate, but cannot transfer the same to substrates such as mandelate, 2-hydroxy-4-phenylbutyrate, 3-hydroxy-3-phenylpropionate and 4-hydroxybenzoic acid.
In order to solve these problems, the present invention uses acetyl-CoA as a CoA donor to find an enzyme having a broad aromatic substrate spectrum. Sequence similarity analysis was performed to identify homologous enzymes for FldA, and 2-isocaprenoyl-CoA:2-hydroxyisocapronate CoA-transferase (HadA) of Clostridium difficile having an amino acid sequence identity of 48% or more with FldA among various FldAs having different origins was screened (
In addition, LC-MS analysis after in vitro assays showed that HadA can convert mandelate, 4-hydroxymandelate, phenyllactate, 4-hydroxyphenyllactate, 2-hydroxy-4-phenylbutyrate, 3-hydroxy-3-phenylpropionate and 4-hydoxybenzoic acid to the corresponding CoA derivatives (
Next, in order to increase the production amount of aromatic monomers by metabolic engineering, the yield was increased by metabolically engineering an E. coli XL1-Blue strain expressing AroGfbr, PheAfbr and FldH producing a small amount (0.372 g/L) of D-phenyllactate from glucose. The E. coli XBT strain expressing AroGfbr, PheAfbr and FldH, which deleted TyrR, which is a double transcriptional regulatory factor that performs regulation to inhibit aromatic amino acid biosynthesis, was constructed and the E. coli XBT strain produced 0.5 g/L of D-phenyllactate from 16.4 g/L of glucose and thus showed 30% higher productivity than the E. coli XL1-Blue strain which did not delete TyrR. In order to remove the pathway competing with D-phenyllactate biosynthesis, E. coli XB201T was constructed by deleting poxB (a gene encoding a pyruvate oxidase), pflB (a gene encoding a pyruvate formate lyase), adhE (a gene encoding an acetaldehyde dehydrogenase/alcohol dehydrogenase) and frdB (a gene encoding a fumarate reductase) from E. coli XBT. The E. coli strain XB201T expressing AroGfbr, PheAfbr and FldH produced 0.55 g/L of D-phenyllactate from 15.7 g/L of glucose, which indicates a yield 10% higher than that of E. coli XBT.
In addition, metabolic engineering analysis according to in-silico genome scale metabolism flux analysis was performed to further increase the production of D-phenyllactate (
When E. coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA were cultured in a medium containing 20 g/L glucose and 1 g/L sodium 3HB, poly(52.1 mol % 3HB-co-47.9 mol % D-phenyllactate) was produced in an amount of 15.8 wt % of the dry cell weight (
In another aspect, the present invention is directed to a recombinant microorganism obtained by introducing a gene encoding a 2-hydroxyisocaproate-CoA transferase, a gene encoding a polyhydroxyalkanoate synthase, a gene encoding a DAHP (3-deoxy-D-arabino-heptulosonate-7-phosphate) synthase, a gene encoding a chorismate mutase/prephenate dehydrogenase, and a gene encoding a D-lactate dehydrogenase into a microorganism capable of producing acetyl-CoA from a carbon source, wherein the recombinant microorganism is capable of producing polyhydroxyalkanoate having phenyllactate as a monomer.
In the present invention, the 2-hydroxyisocaproate-CoA transferase may be HadA derived from Clostridium difficile 630 and the polyhydroxyalkanoate synthase is a PHA synthase derived from a strain selected from the group consisting of Ralstonia eutropha, Pseudomonas, Bacillus and Pseudomonas sp. 6-19, or a mutant enzyme of a PHA synthase having an amino acid sequence selected from the following:
an amino acid sequence having at least one mutation selected from the group consisting of E130D, S325T, L412M, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in an amino acid sequence of SEQ ID NO: 2;
an amino acid sequence (C1335) having mutations of E130D, S325T, L412M, S477G and Q481M in the amino acid sequence of SEQ ID NO: 2;
an amino acid sequence (C1310) having mutations of E130D, S477F and Q481K in the amino acid sequence of SEQ ID
NO: 2; and
an amino acid sequence (C1312) having mutations of E130D, S477F and Q481R in the amino acid sequence of SEQ ID NO: 2.
In the present invention, the gene encoding the DAHP (3-deoxy-D-arabino-heptulosonate-7-phosphate) synthase is a gene encoding the amino acid sequence represented by SEQ ID NO: 8, the gene encoding chorismate mutase/prephenate dehydrogenase may be a gene encoding the amino acid sequence represented by SEQ ID NO: 9, and the gene encoding D-lactate dehydrogenase may be a gene encoding the amino acid sequence represented by SEQ ID NO: 10.
In the present invention, the introduced gene encoding D-lactate dehydrogenase may be a fldH gene which replaces the ldhA gene.
The microorganism of the present invention may be further introduced with a gene encoding a β-ketothiolase and a gene encoding an acetoacetyl-CoA reductase involved in 3-hydroxybutyryl-CoA biosynthesis in order for the microorganism to produce a polymer even without external supply of sodium 3HB.
When the expression amounts of the gene (phaA) encoding a β-ketothiolase and the gene (phaB) encoding an acetoacetyl-CoA reductase, which are introduced in the present invention, are regulated through the strength (intensity) of the promoter, the mole fraction of the D-phenyllactate monomer contained in PHA can be controlled.
In one embodiment of the present invention, five different plasmids which express phaA and phaB with five types of promoters having different strengths were constructed and introduced into XB201TBAL strains expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA. It was confirmed that, as phaA and phaB expression decreased, the mole fraction of the phenyllactate monomer increased (
In the present invention, the recombinant microorganism has a deletion of at least one gene selected from the group consisting of a tyrR gene, a gene encoding a pyruvate oxidase, a gene encoding a pyruvate formate lyase, a gene encoding an acetaldehyde dehydrogenase, a gene encoding a fumarate reductase, a gene encoding a tyrosine aminotransferase, and a gene encoding an aspartic acid aminotransferase.
In another aspect, the present invention is directed to a method for producing polyhydroxyalkanoate having phenyllactate as a monomer including: (a) culturing the recombinant microorganism to produce polyhydroxyalkanoate having phenyllactate as a monomer; and (b) recovering the produced polyhydroxyalkanoate having phenyllactate as a monomer.
In order to identify whether or not the method described above can be used for the preparation of various aromatic polymers, experimentation was conducted using mandelate as a monomer. The reason for this is that polymandelate, which is a homopolymer of mandelate, is a pyrolysis-resistant polymer having a relatively high Tg of 100° C., and has properties similar to those of polystyrene. Polymandelate is chemically synthesized through ring-opening polymerization of a cyclic dimer of mandelate produced in the petroleum industry. When E. coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA was cultured in a medium containing 1 g/L of sodium 3HB and 0.5 g/L of D-mandelate, poly(55.2 mol % 3HB-co-43 mol % D-phenyllactate-co-1.8 mol % D-mandelate) was produced in an amount of 11.6 wt % of the dry cell weight (
In the present invention, an aromatic copolymer containing D-mandelate was successfully prepared using D-mandelate as a substrate, and then D-mandelate was prepared in vivo by metabolic engineering. A hydroxymandelate synthase (HmaS) derived from Amycolatopsis orientalis, a hydroxymandelate oxidase (Hmo) of S. coelicolor and a D-mandelate dehydrogenase (Dmd) of Rhodotorula graminis were expressed in E. coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA in order to produce an aromatic copolymer containing D-mandelate from glucose. When the engineered strains were cultured in a medium containing 20 g/L of glucose and 1 g/L of sodium 3HB, poly(92.9 mol % of 3HB-co-6.3 mol % D-phenyllactate-co-0.8 mol % D-mandelate) was prepared in an amount of 16.4 wt % of the dry cell weight.
In another aspect, the present invention is directed to a recombinant microorganism obtained by introducing a gene encoding a 2-hydroxyisocaproate-CoA transferase, a gene encoding a polyhydroxyalkanoate synthase, a gene encoding a DAHP (3-deoxy-D-arabino-heptuloonate-7-phosphate) synthase, a gene encoding a chorismate mutase/prephenate dehydrogenase, a gene encoding a D-lactate dehydrogenase, a gene encoding a hydroxymandelate synthase, a gene encoding a hydroxymandelate oxidase, and a gene encoding a D-mandelate dehydrogenase into a microorganism capable of producing acetyl-CoA from a carbon source, wherein the recombinant microorganism is capable of producing polyhydroxyalkanoate having mandelate as a monomer.
In the present invention, the 2-hydroxyisocaproate-CoA transferase may be hadA derived from Clostridium difficile 630 and the polyhydroxyalkanoate synthase may be a PHA synthase derived from a strain selected from the group consisting of Ralstonia eutropha, Pseudomonas, Bacillus and Pseudomonas sp. 6-19, or a mutant enzyme of a PHA synthase having an amino acid sequence selected from the following:
an amino acid sequence having at least one mutation selected from the group consisting of E130D, S325T, L412M, S477R, S477H, S477F, S477Y, S477G, Q481M, Q481K and Q481R in an amino acid sequence of SEQ ID NO: 2;
an amino acid sequence (C1335) having mutations of E130D, S325T, L412M, S477G and Q481M in the amino acid sequence of SEQ ID NO: 2;
an amino acid sequence (C1310) having mutations of E130D, S477F and Q481K in the amino acid sequence of SEQ ID NO: 2; and
an amino acid sequence (C1312) having mutations of E130D, S477F and Q481R in the amino acid sequence of SEQ ID NO: 2.
In the present invention, the gene encoding the DAHP (3-deoxy-D-arabino-heptulosonate-7-phosphate) synthase may be a gene encoding the amino acid sequence represented by SEQ ID NO: 8, the gene encoding chorismate mutase/prephenate dehydrogenase may be a gene encoding the amino acid sequence represented by SEQ ID NO: 9, and the gene encoding D-lactate dehydrogenase may be a gene encoding the amino acid sequence represented by SEQ ID NO: 10.
In the present invention, the gene encoding hydroxymandelate synthase may be a gene encoding the amino acid sequence represented by SEQ ID NO: 11, the gene encoding hydroxymandelate oxidase may be a gene encoding the amino acid sequence represented by SEQ ID NO: 12, and the gene encoding D-mandelate dehydrogenase may be a gene encoding the amino acid sequence represented by SEQ ID NO: 13.
The microorganism of the present invention may be further introduced with genes encoding a β-ketothiolase and a gene encoding an acetoacetyl-CoA reductase involved in 3-hydroxybutyryl-CoA biosynthesis in order for the microorganism to produce a polymer even without the external supplementation of sodium 3HB.
In another aspect, the present invention is directed to a method for producing polyhydroxyalkanoate having mandelate as a monomer including: (a) culturing the recombinant microorganism to produce polyhydroxyalkanoate having mandelate as a monomer; and (b) recovering the produced polyhydroxyalkanoate having mandelate as a monomer.
Further, in the present invention, in order to confirm the possibility of the production of polyhydroxyalkanoate containing various long-chain 2-HA using the recombinant strain of the present invention, polymer productivity was identified using long-chain 2-HA monomers such as 2-hydroxyisocaproate (2HIC), 2-hydroxyhexanoate (2HH) and 2-hydroxyoctanoate (2HO) as monomers. As a result, it was identified that copolymers containing 2-hydroxyisocaproate, 2-hydroxyhexanoate or 2-hydroxyoctanoate were produced and that, as the concentration of 2-HA contained in the medium increased, the mole fraction of the monomer contained in the copolymer increased (Tables 4, 5 and 6).
Accordingly, in another aspect, the present invention is directed to a method for producing polyhydroxyalkanoate having, as a monomer, a compound selected from the group consisting of 2-hydroxyisocaproate, 2-hydroxyhexanoate and 2-hydroxyoctanoate, including: (a) culturing the recombinant microorganism capable of producing polyhydroxyalkanoate having an aromatic monomer or a long-chain 2-HA monomer in a medium containing a compound selected from the group consisting of 2-hydroxyisocaproate, 2-hydroxyhexanoate and 2-hydroxyoctanoate; and (b) recovering polyhydroxyalkanoate containing, as a monomer, a compound selected from the group consisting of 2-hydroxyisocaproate, 2-hydroxyhexanoate and 2-hydroxyoctanoate.
In the present invention, production of an aromatic polymer was identified using 3-hydroxy-3-phenylpropionate (3HPh) as another aromatic monomer capable of producing PHA. When the E. coli strain XB201TBA was cultured in a medium containing 20 g/L of glucose, 0.5 g/L of 3-hydroxy-3-phenylpropionic acid and 1 g/L of sodium 3HB, poly(33.3 mol % 3HB-co-18 mol % D-phenyllactate-co-48.7 mol % 3HPh) was produced in an amount of 14.7 wt % of dry cell weight (
Finally, the physical properties of aromatic PHAs produced by metabolically engineered E. coli were investigated. The poly(52.1 mol % 3HB-co-47.9 mol % D-phenyllactate) was amorphous, and, as the mole fraction of D-phenyllactate in the copolymer increased, the Tg increased significantly to 23.86° C., although the molecular weight was decreased. Also, the copolymer containing an aromatic compound in the polymer had decreased crystallinity. It is considered that the aromatic ring of the polymer interferes with the crystallization of P(3HB). P(3HB) has high brittleness due to the strong crystallinity thereof, whereas the resulting copolymer has improved mechanical toughness due to decreased crystallinity and increased Tg.
In the present invention, a bacterial platform system was developed for the production of various aromatic polyesters. The aromatic polymer production system of the present invention identified a novel CoA-transferase having a wide range of substrates for activating an aromatic compound into a CoA derivative thereof and established PHA synthase mutants capable of polymerizing aromatic CoA derivatives thereof and a pathway to over-produce aromatic monomers in vivo through the design and optimization of metabolisms.
As evidenced using several aromatic monomers in various embodiments of the present invention, such a system can be used in the preparation of various aromatic polymers. For example, according to the present invention, HadA (or related enzymes) and PHA synthases can be engineered to accommodate the desired aromatic monomers. The bacterial platform system developed in the present invention can contribute to establishment of a bioprocess for the production of aromatic polyesters from renewable non-food biomass.
As used herein, the term “vector” means a DNA product containing a DNA sequence operably linked to a control sequence capable of expressing DNA in a suitable host. The vector may be a plasmid, a phage particle or a simple potential genome insert. Once the vector is transformed with an appropriate host, it may replicate and function independently of the genome of the host, or may often be integrated with the genome itself. Since the plasmid is the most commonly used type of vector, the terms “plasmid” and “vector” are sometimes used interchangeably throughout the specification of the present invention. For the purpose of the present invention, a plasmid vector is preferably used. A typical plasmid vector that can be used for this purpose includes (a) a replication origin to efficiently conduct replication so as to include several to several hundred plasmid vectors per host cell, (b) an antibiotic resistance gene to screen a host cell transformed with the plasmid vector, and (c) a restriction enzyme cleavage site into which a foreign DNA fragment is inserted. Even if an appropriate restriction enzyme cleavage site is not present, the vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adapter or a linker according to a conventional method.
After ligation, the vector should be transformed into an appropriate host cell. In the present invention, the preferred host cells are prokaryotic cells. Suitable prokaryotic host cells include E. coli DH5a, E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL-1 Blue (Stratagene), E. coli B, E. coli B21 and the like. However, E. coli strains such as FMB101, NM522, NM538 and NM539, as well as the species and genera of other prokaryotes, and the like, can also be used. In addition to the E. coli mentioned above, the genus Agrobacterium strains such as Agrobacterium A4, Bacillus strains such as Bacillus subtilis, other enterobacteria such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas genus strains can be used as host cells.
Transformation of prokaryotic cells can be easily carried out using a calcium chloride method described in the section 1.82 of Sambrook et al., supra. Alternatively, electroporation (Neumann, et al., EMBO J., 1: 841, 1982) can be used for transformation of these cells.
The vector used for overexpression of the gene according to the present invention may be any expression vector known in the art and is preferably a pET-based vector (Novagen). When cloning is performed using the pET-based vector, histidine groups are bonded to the ends of the expressed protein, so that the protein can be effectively purified. The expressed protein can be isolated from the cloned gene through a general method known in the art and can be specifically isolated using a chromatographic method using Ni-NTA His-conjugated resin (Novagen). In the present invention, the recombinant vector may be pET-SLTI66, and the host cell may be E. coli or Agrobacterium.
As used herein, the term “expression control sequence” means a DNA sequence essential for the expression of a coding sequence operably linked to a particular host organism. Such a control sequence includes promoters for conducting transcription, any operator sequences for controlling such transcription, sequences for encoding suitable mRNA ribosome-binding sites, and sequences for controlling the termination of transcription and translation. For example, control sequences suitable for prokaryotes include promoters, optionally operator sequences and ribosome binding sites. Eukaryotic cells include promoters, polyadenylation signals and enhancers. The factor that has the greatest impact on the expression level of the gene in the plasmid is a promoter. SRa promoters, cytomegalovirus-derived promoters and the like are preferably used as promoters for high expression. Any of a wide variety of expression control sequences may be used for the vector in order to express the DNA sequences of the present invention. Useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, T3 and T7 promoters, the major operator and promoter regions of phage lambda, control regions of fd code proteins, promoters of 3-phosphoglycerate kinase or other glycol lyases, promoters of the phosphatase, such as Pho5, promoters of yeast alpha-mating systems and other sequences known to control gene expression of prokaryotic or eukaryotic cells or viruses and various combinations thereof. The T7 promoter may be useful for expressing proteins of the present invention in E. coli.
When a nucleic acid sequence is aligned with another nucleic acid sequence based on a functional relationship, it is “operably linked” thereto. This may be gene(s) and control sequence(s) linked in such a way so as to enable gene expression when a suitable molecule (e.g., a transcriptional activator protein) is linked to the control sequence(s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide, when expressed as a pre-protein involved in the secretion of the polypeptide; and a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; or a ribosome-binding site is operably linked to a coding sequence when it affects the transcription of the sequence; or the ribosome-binding site is operably linked to a coding sequence when positioned to facilitate translation. Generally, “operably linked” means that the linked DNA sequence is in contact therewith, or that a secretory leader is in contact therewith and is present in the reading frame. However, the enhancer need not be in contact therewith. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction enzyme sites. When no such site exists, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.
As used herein, the term “expression vector” commonly refers to a recombinant carrier, into which a fragment of heterologous DNA is inserted, and generally means a fragment of double-stranded DNA. Herein, the heterologous DNA means exogenous DNA that is not naturally found in the host cell. Once an expression vector is present in a host cell, it can replicate independently of the host chromosomal DNA, and several copies of the vector and inserted (heterologous) DNA thereof can be produced.
As is well known in the art, in order to increase the expression level of a transgene in a host cell, the gene should be operably linked to a transcriptional/translational expression control sequence that functions in a selected expression host. Preferably, the expression control sequence and the corresponding gene are included in one recombinant vector containing both a bacterial selection marker and a replication origin. When the host cell is a eukaryotic cell, the recombinant vector should further include a useful expression marker in the eukaryotic expression host.
The host cell transfected or transformed by the recombinant vector described above constitutes another aspect of the present invention. As used herein, the term “transfection” means introducing DNA into a host and making the DNA replicable by an extrachromosomal factor or chromosomal integration. As used herein, the term “transformation” means that an expression vector is accommodated by the host cell, regardless of whether or not any coding sequence is actually expressed.
It should be understood that not all vectors function identically in expressing the DNA sequences of the present invention. Likewise, not all hosts function identically for the same expression system. However, those skilled in the art will be able to make appropriate selections from among a variety of vectors, expression control sequences and hosts without excessive burden of experimentation and without departing from the scope of the present invention. For example, selection of a vector should be carried out in consideration of a host because the vector should be replicated therein. The number of replications of the vector, the ability to control the number of replications, and the expression of other proteins encoded by the corresponding vector, such as the expression of antibiotic markers, should also be considered. In selecting the expression control sequence, a number of factors should be considered. For example, the relative strength of the sequence, controllability, and compatibility with the DNA sequences of the present invention should be considered, particularly in relation to possible secondary structures. The single cell host may be selected in consideration of factors such as the selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, secretion characteristics, the ability to accurately fold proteins, culture and fermentation factors, and ease of purification of the product encoded by the DNA sequence according to the present invention. Within the scope of these factors, those skilled in the art can select various vector/expression control sequences/host combinations capable of expressing the DNA sequences of the present invention in fermentation or large animal cultures. As a screening method for cloning the cDNA of the protein according to the present invention through expression cloning, a binding method, a panning method, a film emulsion method or the like can be applied.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, it will be obvious to those skilled in the art that the following examples are provided only for illustration of the present invention and should not be construed as limiting the scope of the present invention.
In the following examples, Escherichia coli was used as a recombinant microorganism. However, any microorganism can be used without limitation so long as it is capable of producing acetyl-CoA from a carbon source. Examples of the microorganism include the genera Alcaligenes, Pseudomonas, Escherichia, Ralstonia, Bacillus and Corynebacterium and the like.
The recombinant strains, plasmids and primers used or produced in the present invention are shown in Tables 1 to 3.
1
2
3
4
5
R. eutropha PHA biosynthesis operon; ApR
6
5
5
5
5
5
7
C. propionicum pct540 gene; ApR
maritimus PALgenes; KmR
R. eutropha PHA biosynthesis operon promoter,
R. eutropha phaA and phaB genes; KmR
C. botulinum A str. ATCC 3502 fldH gene; CmR
S. coelicolor (A294G) gene; CmR
S. coelicolor (A294G) and the C. botulinum A str.
orientalis hmaS gene; KmR
7
7
lactis Il1403 panE gene; CmR
1 Datsenko, K. A. & Wanner, B. P Natl Acad Sci USA 97:6640, 2000.
2 Lee, K. H. et al., Molecular Systems Biology 3, doi:ARTN 149 10.1038/msb4100196, 2007.
3 Palmeros, B. et al. Gene 247:255, 2000.
4 Park, S. J. et al., Metab Eng 20, 20, 2013.
5 Yang, T. H. et al. Biotechnol Bioeng 105:150, 2010.
6 Yang, T. H. et al., Appl Microbiol Biotechnol 90:603, 2011.
7 Choi, S. Y. et al., Nat Biotechnol 34:435, 2016.
8 Knobloch, K. H. & Hahlbrock, K., Archives of Biochemistry and Biophysics 184: 237, 1977.
9 Kaneko, M. et al., J Bacteriol 185:20, 2003.
An enzyme using acetyl-CoA as a CoA donor and having a broad spectrum of aromatic substrates was found. Sequence similarity analysis was performed to identify homologous enzymes for FldA, and 2-isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase of Clostridium difficile (HadA, SEQ ID NO: 1), which has an amino acid sequence identity of 48% or more with FldA, was screened from various FldAs having different origins (
In order to produce a recombinant vector containing a gene encoding HadA, PCR was performed using the chromosomal DNA of Clostridium difficile 630 strain as a template, and HadA-hisF and HadA-hisR as primers to produce a his_HadA gene fragment encoding a 2-hydroxyisocaproate-CoA transferase having a his-tag at the C terminus thereof.
Next, the his HadA fragment thus produced and the pET22b plasmid, which conducted strong gene expression of the T7 promoter, were treated with restriction enzymes (NdeI and NotI) and then the his HadA fragment cleaved with the restriction enzyme was ligated with the pET22b plasmid using a T4 DNA ligase to produce pET22b hisHadA as a recombinant plasmid (
The pET22b_hisHadA was introduced into E. coli XL1-Blue (Stratagene Cloning Systems, USA), cultured and added with IPTG to induce HadA expression. Then, HadA was purified in the culture medium using His-tag in a Ni-NTA spin kit (Quiagen, Germany) (
In order to identify whether or not HadA is capable of using acetyl-CoA as a donor, in-vitro assays were performed using HadA prepared in Example 1.
10 μg of HadA was added to 50 mM phosphate buffer (pH 7.5) containing 0.1 mM acetyl-CoA and a 10 mM substrate, and the reaction was carried out at 30° C. for 10 minutes. After the reaction, 0.1 mM oxaloacetic acid, 5 μg of citrate synthase and 0.5 mM 5.5′-dithiobis-(2-nitrobenzoic acid) (DTNB) were added. Then, the amount of released CoA was analyzed by measuring the absorbance at 412 nm (
Analysis of the resulting aliphatic and aromatic acyl-CoA was performed on LC-MS (Agilent 1100 series and LC/MSD VL, Agilent) equipped with an Eclipse XDB-C18 column (5 μm, 4.6×150 mm, Agilent).
As a result, as can be seen from
E. coli was engineered to produce D-phenyllactate from glucose in vivo. The biosynthesis of aromatic compounds begins with the synthesis of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP), which is synthesized by the condensation of phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P). The produced DAHP is converted into phenylpyruvate (PPA), which is then converted into D-phenyllactate through D-lactate dehydrogenase (FldH) (
In the present invention, feedback-inhibition-resistant mutants, AroGfbr [AroG (D146N)] and PheAfbr [PheA (T326P)], were constructed to release the feedback inhibition by L-phenylalanine (Zhou, H. Y. et al., Bioresour. Technol. 101:4151, 2010; Kikuchi, Y. et al., Appl. Environ. Microbiol. 63:761, 1997). E. coli XL1-Blue expressing AroGfbr, PheAfbr and FldH of C. botulinum A str. ATCC 3502 was produced.
In order to construct pKM212-AroGfbr, a PCR product was obtained from a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase gene (aroG), which is a feedback-inhibiting resistance mutant, using AroG-F and AroG-R as primers and using pTyr-a plasmid as a template (Na, D. et al., Nature Biotechnol. 31: 170, 2013), and the PCR product was ligated to pKM212-MCS with restriction enzymes site (EcoRI/HidIII) (Park, S J et al., Metab. Eng. 20:20, 2013) to produce pKM212-AroGfbr.
The pKM212-AroGfbrPheAfbr plasmid was constructed as follows. First, a DNA fragment of 991 bp was amplified by PCR from the genomic DNA of E. coli using PheA-F and PheAmut-R as primers of a single mutated nucleotide (T976G). Second, a DNA fragment of 200 bp was amplified from the genomic DNA of E. coli using PheA-R and PheAmut-F as primers of a single mutated nucleotide (A976C).
Then, a DNA fragment of 1161 bp was amplified by overlap PCR using PheA-F and PheA-R as primers and two mixed fragments as templates. The PCR product was ligated to the pKM212-AroGfbr produced above using a restriction enzyme (HindIII).
D-lactate dehydrogenase (fldH) of C. botulinum A str. ATCC 3502 was used for construction of pACYC-FldH. The codon usage of fldH genes was optimized for E. coli and the fldH gene optimized for E. coli was amplified using FldH-F and FldH-R as primers and pUC57-FldHopt (GenScript, Piscataway, N.J., USA) as a template.
The PCR product was ligated with pTrc99A (Pharmacia, Biotech, Sweden) using restriction enzymes (BamHI/HindIII) to construct pTrc-FldH. Next, the fldH gene combined with the trc promoter and the rrnB terminator was amplified through PCR using Trc-F and Ter-R as primers and pTrc-FldH as a template. The amplified PCR product was ligated with pACYC184KS (Korean Patent Laid-Open No. 2015-0142304) using restriction enzymes (XhoI/SacI) to obtain pACYC-FldH.
The pKM212-AroGfbrPheAfbr and pACYC-FldH thus produced were introduced into E. coli XL1-Blue to produce a recombinant E. coli expressing AroGfbr, PheAfbr and FldH.
The E. coli produced 0.372 g/L of D-phenyllactate when cultured in MR medium containing 15.2 g/L of glucose.
The MR medium contained 6.67 g of KH2PO4, 4 g of (NH4)2HPO4, 0.8 g of MgSO4.7H2O, 0.8 g of citrate and 5 ml of a trace metal solution per liter, and the trace metal solution contained 0.5 M HCl: 10 g of FeSO4.7H2O, 2 g of CaCl2, 2.2 g of ZnSO4.7H2O, 0.5 g of MnSO4.4H2O, 1 g of CuSO4.5H2O, 0.1 g of (NH4)6Mo7O24.4H2O and 0.02 g of Na2B4O7.10H2O.
In order to increase the production amount of aromatic monomers through metabolic engineering, the yields of E. coli XL1-Blue strains expressing AroGfbr, PheAfbr and FldH, producing small amounts of D-phenyllactate (0.372 g/L) from glucose, were increased through metabolic engineering. E. coli XBT strains expressing AroGfbr, PheAfbr and FldH were produced by deleting TyrR, which is a double transcription regulator that performs regulation to inhibit aromatic amino acid biosynthesis.
The deletion of the tyrR gene in E. coli expressing AroGfbr, PheAfbr and FldH was carried out using a one-step inactivation method (Datsenko, K. A. et al., Proc. Natl Acad Sci. USA 97: 6640, 2000).
The E. coli XBT strain was cultured in an MR medium containing 16.4 g/L of glucose. As a result, the strain produced 0.5 g/L of D-phenyllactate, which corresponded to productivity of 30% higher than the E. coli XL1-blue strain from which tyrR was not deleted.
In order to remove the pathway colliding with D-phenyllactate synthesis, E. coli XB201T was constructed by deleting poxB (a gene encoding a pyruvate oxidase), pflB (a gene encoding a pyruvate formate lyase), adhE (a gene encoding an acetaldehyde dehydrogenase/alcohol dehydrogenase) and frdB (a gene encoding a fumarate reductase) from E. coli XBT.
The E. coli XB201T strain produced 0.55 g/L of D-phenyllactate from 15.7 g/L of glucose, which corresponded to a yield 10% higher than that of E. coli XBT. In addition, metabolic engineering analysis according to in-silico genome scale metabolism flux analysis was performed to further increase the production of D-phenyllactate.
For in-silico flux response analysis, the E. coli iJO1366 genome scale model, which consists of 2251 metabolism reactions and 1135 metabolites, was used, and the effects of central and aromatic amino acid biosynthesis on the production of D-phenyllactate were investigated. In order to reflect the same in the XB201T strain of the present invention, a heterologous metabolic reaction of D-phenyllactate biosynthesis (fldH gene) was further added to the model and the flux was fixed to zero to reflect the gene knockout in the model. The rate of D-phenyllactate production was maximized to a target function, while the central amino acid and aromatic amino acid biosynthesis reaction flux values were gradually increased from a minimum value to a maximum value. During the simulation, the glucose reaction rate was set at 10 mmol per 1 g of the dry cell weight on an hourly basis. All simulations were run in a Python environment using the Gurobi Optimizer 6.0 and the GurobiPy package (Gurobi Optimization, Inc. Houston, Tex.). Reading, writing and execution of COBRA-compliant SBML files were conducted using COBRApy32.
As a result, as shown in
The E. coli strain XB201TBA prepared, as a result of the in-silico flux response analysis, was found to produce 1.62 g/L of D-phenyllactate from 18.5 g/L of glucose, resulting in a great increase in yield, namely 4.35 times higher than the D-phenyllactate production of the E. coli XL1-blue strain expressing AroGfbr, PheAfbr and FldH.
In order to prevent the formation of D-lactate in XB201TBA, the ldhA gene was further deleted to prepare XB201TBAL strain. In order to prepare polyhydroxyalkanoate containing aromatic monomers, PhaC1437 and HadA were expressed in the E. coli XB201TBAL strain.
In order to prepare a recombinant vector containing genes encoding PhaC1437 and HadA, PCR was performed using HadA-sbF and HadA-ndR as primers and the chromosomal DNA of Clostridium difficile 630 strain as a template, to produce a hadA gene fragment encoding a hydroxyisocaproate-CoA transferase. The amplified PCR product was ligated with p619C1437-pct540 (Yang, T. H. et al. Biotechnol. Bioeng. 105: 150, 2010) using restriction enzymes (SbfI/NdeI) to obtain p619C1437-HadA. The obtained p619C1437-HadA was introduced into E. coli XB201TBAL to prepare recombinant E. coli expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA. The E. coli was cultured in MR medium containing 20 g/L of glucose and 1 g/L of sodium 3HB to obtain poly(52.1 mol % 3HB-co-47.9 mol % D-phenyllactate) in an amount of 15.8 wt % of the dry cell weight (
In order to identify whether or not the system using E. coli XB201TBAL can be used for the preparation of various aromatic copolymers, experiments were conducted using mandelate as a monomer.
E. coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA were cultured in an MR medium containing 1 g/L of sodium 3HB and 0.5 g/L of D-mandelate. As a result, poly(55.2 mol % 3HB-co-43.0 mol % D-phenyllactate-co-1.8 mol % D-mandelate) was produced in an amount of 11.6 wt % of the dry cell weight (
Next, D-mandelate was prepared in vivo through metabolic engineering. A hydroxymandelate synthase (HmaS) derived from Amycolatopsis orientalis, a hydroxymandelate oxidase (Hmo) of S. coelicolor, and D-mandelate dehydrogenase (Dmd) of Rhodotorula graminis were expressed in E. coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA in order to produce D-mandelate from glucose.
For construction of pKM212-HmaS, the plasmid pUC57-HmaSopt was prepared by using the hydroxymandelate synthase gene (hmaS) of A. orientalis and cloning the codon into a synthetic vector in E. coli (GenScript, Piscataway, N.J., USA).
The pUC57-HmaSopt was ligated to pKM212-MCS using a restriction enzyme (EcoRI/KpnI). In order to construct pKM212-HmaSHmo, the codon-optimized hmo gene (GenScript, Piscataway, N.J., USA) was synthesized using the hydroxymandelate oxidase gene (hmo) of S. coelicolor and was amplified through PCR using Hmo-F and Hmo-R as primers. The PCR product was ligated with pKM212-HmaS using restriction enzymes (KpnI/BamHI) to construct pKM212-HmaSHmo.
In order to construct pKM212-HmaSHmoDmd, pUC57-Dmd containing the E. coli codon-optimized dmd gene was synthesized (GenScript, Piscataway, N.J., USA) and an E. coli codon-optimized R. graminis D-mandelate dehydrogenase gene (dmd) was amplified through PCR using Dmd-F and Dmd-R as primers. The PCR product was ligated with pKM212-HmaSHmo using restriction enzymes (BamHI/SbfI) to prepare pKM2l2-HmaSHmoDmd.
The prepared pKM212-HmaSHmoDmd was introduced into E. coli XB201TBAL expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA to construct a recombinant strain having the potential to produce mandelate.
The constructed recombinant strain having the potential to produce mandelate was cultured in a medium containing 20 g/L of glucose and 1 g/L of sodium 3HB. As a result, poly(92.9 mol % 3HB-co-6.3 mol % D-phenyllactate-co-0.8 mol % D-mandelate) was produced in an amount of 16.4 wt % of the dry cell weight.
The production of an aromatic polymer using 3-hydroxy-3-phenylpropionate (3HPh) as another aromatic monomer was identified. When the E. coli strain XB201TBAL was cultured in a medium containing 20 g/L of glucose, 0.5 g/L of 3-hydroxy-3-phenylpropionic acid and 1 g/L of sodium 3HB, poly(33.3 mol % 3HB-co-18 mol % D-phenyllactate-co-48.7 mol % 3HPh) was produced in an amount of 14.7% by weight of dry cell weight (
In order to identify whether or not the system using 2-hydroxyisocaproate-CoA transferase of the present invention can be used for the production of polyhydroxyalkanoates containing various long-chain 2-HA, polymer productivity was confirmed using various long-chain 2-HA monomers [2-hydroxyisocaproate (2HIC), 2-hydroxyhexanoate (2HH) and 2-hydroxyoctanoate (2HO)]. E. coli XL1-Blue expressing PhaC1437 and HadA was cultured in an MR medium containing 1 g/L of 3HB, 20 g/L of glucose and different concentrations (0.25, 0.5 and 1 g/L) of long-chain 2-HA. As a result, a copolymer containing hydroxyisocaproate, 2-hydroxyhexanoate or 2-hydroxyoctanoate was prepared. In addition, it was confirmed that, as the concentration of 2-HA contained in the medium increased, the mole fraction of the monomer contained in the copolymer increased (Tables 4, 5 and 6).
In order to produce a strain producing aromatic PHA without the supply of 3HB from the outside, R. eutropha R-ketothiolase (PhaA) and acetoacetyl-CoA reductase (PhaB) were further expressed in an XB201TBAL strain, and whether or not aromatic PHA was prepared from glucose without the external supplementation of 3HB was identified.
As a result, as expected, the XB201TBAL strain expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA produced poly(86.2 mol % 3HB-co-13.8 mol % D-phenyllactate) from 20 g/L of glucose in an amount of 18.0 wt % of the dry cell weight. In addition, the production of aromatic PHAs having various monomer mole fractions important for industrial applications was attempted by controlling the metabolic flux catalyzed by PhaAB using the synthetic Anderson promoter (http://parts.igem.org/). Five different plasmids expressing PhaAB with five different promoters of different strength (SEQ ID NOS: 89-93) were prepared and introduced into XB201TBAL strains expressing AroGfbr, PheAfbr, FldH, PhaC1437 and HadA.
As PhaAB expression decreased, the mole fraction of D-phenyllactate monomers increased; copolymers having 11.0 mol %, 15.8 mol %, 20.0 mol %, 70.8 mol % and 84.5 mol % of D-phenyllactate could be prepared (
In this example, pH-stat culturing of the E. coli strain XB201TBAL expressing AroGfbr, PheAfbr, FldH, HadA, PhaC1437 and PhaAB under the promoter BBa_J23114 was performed without the supply of 3HB. After 96 hours of culture, poly(67.6 mol % 3HB-co-32.4 mol % D-phenyllactate) with a polymer content of 43.8 wt % of the dry cell weight was produced at 2.5 g/L (
Also, in order to further improve the production of aromatic polyhydroxyalkanoate, the gene expression system was optimized by replacing the ldhA gene of the E. coli XB201TBA chromosome with the fldH gene. In addition, the expression of the fldH gene was increased by replacing the natural promoter of the ldhA gene with a strong trc promoter. Fed-batch fermentation including feeding glucose using a pulse feeding method was performed. The E. coli strain XB201TBAF expressing AroGfbr, PheAfbr, FldH, HadA, PhaC1437 and PhaAB under the promoter BBa_J23114 produced 13.9 g/L of poly(69.1 mol % 3HB-co-38.1 mol % D-phenyllactate) with a polymer content of 55.0 wt % of the dry cell weight through fed-batch fermentation (
Finally, the physical properties of aromatic PHAs produced by metabolically engineered E. coli were investigated.
The polyhydroxyalkanoate (PHA) content and monomer composition were determined through GC or GC-MS. The collected cells were washed three times with distilled water and lyophilized for 24 hours, and the PHA of the lyophilized cells was converted to the corresponding hydroxymethylester through acid-catalyzed methanolysis. The resulting methylester was purified using a GC apparatus (Agilent 6890N, Agilent, USA) equipped with an Agilent 7683 automatic injector, a frame ionization detector and a fused silica capillary column (ATTM-Wax, 30 m, ID 0.53 mm, thickness 1.20 μm, Alltech, USA). The polymer was extracted through chloroform extraction and purified in cells using solvent extraction. The structure, molecular weight and thermal properties of the polymer were measured using nuclear magnetic resonance (NMR), gel permeation chromatography (GPC) and differential scanning calorimetry (DSC).
As a result,
The poly(52.1 mol % 3HB-co-47.9 mol % D-phenyllactate) was amorphous, and, as the mole fraction of D-phenyllactate in the copolymer increased, the Tg increased significantly to 23.86° C., in spite of the decreased molecular weight thereof. Also, the copolymer containing an aromatic compound in the polymer had decreased crystallinity. It is considered that the aromatic ring of the polymer interferes with the crystallization of P(3HB) (induced by stereochemistry). P(3HB) exhibited high brittleness due to strong crystallinity, whereas the resulting copolymer caused improved mechanical toughness due to lowered crystallinity and increased Tg.
Although the present invention have been described in detail with reference to specific configurations, those skilled in the art will appreciate that this description relates to preferred embodiments and should not be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the accompanying filed claims and equivalents thereto.
According to the present invention, a biodegradable polymer containing an aromatic monomer or a long-chain 2-HA monomer can be prepared.
Although specific configurations of the present invention have been described in detail, those skilled in the art will appreciate that this detailed description is provided as preferred embodiments and should not be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the accompanying filed claims and equivalents thereto.
An electronic file is attached.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2017-0026266 | Feb 2017 | KR | national |
| 10-2017-0172899 | Dec 2017 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2018/002305 | 2/26/2018 | WO | 00 |
