JP6635535B1 - Escherichia coli expressing EFP protein and method for producing flavonoid compound using the same - Google Patents

Abstract

A flavonoid compound is produced by bioconversion using Escherichia coli. An Escherichia coli cell expressing 4-coumarate CoA ligase enzyme (4CL), chalcone synthase (CHS), and EFP protein is provided. There is also provided a method for producing a flavonoid compound by adding p-coumaric acid or caffeic acid to the composition containing the E. coli cells. [Selection diagram] Fig. 1

Description

  The present disclosure relates to the production of flavonoid compounds. More specifically, the present disclosure relates to producing flavonoid compounds using E. coli that expresses genes from plants.

  Flavonoid compounds are known to have various useful physiological activities, and also have industrial utility as dyes or precursors thereof. Flavonoids are produced by natural plants, and there are known examples in which flavonoids are produced by expressing microorganisms of plant enzymes involved in flavonoid biosynthesis in microorganisms. Non-Patent Document 1 expresses a phenylalanine ammonia lyase enzyme derived from Rhodotorula yeast, a 4-coumarate CoA ligase enzyme derived from Streptomyces bacteria, and a chalcone synthase derived from a Glycyrrhiza plant (Russian licorice) in Escherichia coli. To produce flavanone. Patent Document 1 describes that host microbial cells such as yeast and Escherichia coli express flavonoid biosynthetic enzymes derived from plants to synthesize flavonoid compounds.

  In another type of study, Non-Patent Document 2 describes the identification of an EFP (Enhancer of Flavonoid Production) gene that is involved in flavonoid production and flower coloring in plants. The EFP gene encodes a chalcone isomerase (CHI) -like protein, but lacks some amino acid residues involved in CHI enzyme activity and appears to be a non-enzymatic protein. Non-Patent Document 2 states: “The detailed molecular mechanism of how EFP enhances flavonoid production is unknown, but EFP is not enzyme-like without showing enzymatic activity. It appears to be a unique positive regulator protein that has a structure and enhances metabolic pathways involving related enzymes. "

US Patent Application Publication No. 2009/0239272

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2003, Vol. 69, No. 5, p. 2699-2706 The Plant Journal (2014) 78, 294-304

  An object of an embodiment of the present disclosure is to produce a flavonoid compound by bioconversion using Escherichia coli.

  The present inventors have found that by expressing a plant-derived EFP protein together with a flavonoid biosynthesis-related enzyme in Escherichia coli, it is possible to efficiently produce a target flavonoid compound while suppressing generation of by-products.

The present disclosure includes the following embodiments.
[1]
E. coli cells expressing 4-coumarate CoA ligase enzyme (4CL), chalcone synthase (CHS), and EFP protein.
[2]
The 4CL comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 derived from soybean,
The CHS comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 4 derived from snapdragon,
The EFP comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 5 derived from snapdragon.
The Escherichia coli cell according to the above [1].
[3]
The E. coli cell according to [1], wherein the 4CL, CHS, and EFP are expressed from genes on the same plasmid.
[4]
The 4CL comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 derived from soybean,
The CHS comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 2 derived from soybean,
The EFP comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 derived from soybean,
The E. coli cell according to the above [3].
[5]
The 4CL comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 derived from soybean,
The CHS comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 4 derived from snapdragon,
The EFP comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 5 derived from snapdragon.
The E. coli cell according to the above [3].
[6]
A composition for producing a flavonoid compound, comprising the Escherichia coli cell according to any one of [1] to [5].
[7]
A method for producing naringenin, comprising adding p-coumaric acid to the composition according to [6].
[8]
A method for producing eriodictyol, comprising adding caffeic acid to the composition according to [6].
[9]
A composition for producing naringenin, comprising the Escherichia coli cell according to any one of [1] to [5] and p-coumaric acid.
[10]
A composition for producing eriodictyol, comprising the Escherichia coli cell according to any one of [1] to [5] and caffeic acid.

  The EFP protein cooperates with the 4CL enzyme and the CHS enzyme in Escherichia coli live cells to improve the production amount of the target flavonoid compound and / or to reduce by-products generated in parallel with the target flavonoid compound. It is possible to provide an in vivo flavonoid compound production system that achieves suppression of the production rate and is characterized by sustainability, simplicity, low cost, and the like.

FIG. 1 shows a composition comprising strain D (empty vector control), strain E (expressing Gm4CL and AmCHS), and strain F (expressing Gm4CL, AmCHS and AmEFP), respectively, added to p-coumaric acid. The result of the detection of naringenin (*) is shown. The presence of by-product CTAL (**) is also shown. FIG. 2 shows that naringenin (Nar) when different concentrations of p-coumaric acid were added to compositions containing strain H (expressing Gm4CL and GmCHS) and strain I (expressing Gm4CL, GmCHS and GmEFP), respectively. ) And CTAL production concentration. The left part of FIG. 3 shows the composition produced by adding caffeic acid to a composition containing strain G (empty vector control), strain H (expressing Gm4CL and GmCHS), and strain I (expressing Gm4CL, GmCHS and GmEFP), respectively. 3 shows the results of detection of eriodictyol (#). The presence of CTAL-like by-product Compound 5 (##) is also shown. The right side of FIG. 3 shows the production ratio of compound 5 and eriodictyol obtained in the compositions containing strain H and strain I, respectively.

  As described above, Patent Document 1 and Non-Patent Document 1 describe that flavonoids can be produced by expressing a flavonoid biosynthesis-related enzyme in a heterologous microorganism host. Theoretically, if individual enzyme activities are present in the cytoplasm, it is thought that the target product can be produced at a constant rate even by simple diffusion of the substrate. However, non-enzymatic proteins such as EFPs are introduced into bacteria from plants and are involved in the synthesis of flavonoids in the bacterial live cell environment (ie, in an in vivo environment, rather than an in vitro environment where high concentrations of purified protein are mixed). Until now, no idea has been found.

  The present inventors have discovered that plant-derived EFP proteins can function in vivo when expressed in E. coli cells with 4-coumarate CoA ligase enzyme (4CL) and chalcone synthase (CHS). What kind of function and where on the molecular level the EFP protein, which appears not to be an enzyme, and what kind of molecular state is required for its function The presence or absence of binding, functional dependence on other molecules, presence or absence of inhibition by other molecules, particularly resistance to heterogeneous intracellular environments in which various heterogeneous molecules coexist, etc., has not been elucidated. It was unexpected that such unidentified plant proteins could function in the in vivo environment in E. coli cells in conjunction with the combination of the enzymes 4CL and CHS and contribute to bioconversion.

  By expressing EFP protein in addition to 4CL enzyme and CHS enzyme in Escherichia coli, the ratio between the production of the target flavonoid compound and the production of by-products occurring in parallel with it is shifted toward the former. Was found to be possible. That is, the ratio of the flavonoid compound product to the by-product can be increased. This can lead to an increase in the production amount of the target flavonoid compound, and can also mean that the separation of the product can be further simplified. For example, when naringenin is produced using p-coumaric acid as a substrate, the ratio of coumaroyltriacetic acid lactone (CTAL), which is branched off from the naringenin production reaction pathway, can be reduced.

  In addition to the plant-derived 4CL, CHS, and EFP, other genes may be introduced into the Escherichia coli of the present embodiment. For example, a plasmid vector is usually associated with an antibiotic resistance gene. In addition, a gene for a lyase enzyme such as tyrosine ammonia lyase (TAL) and phenylalanine ammonia lyase (PAL) may be introduced. This may enable flavonoid synthesis using amino acids such as tyrosine and phenylalanine as starting substrates.

  In the present disclosure, "expression" of an enzyme or protein means that transcription and translation occur from gene DNA encoding the enzyme or protein, and a polypeptide as a gene product is present in a cell. I do. However, the promoter for causing transcription is not limited to a constitutive promoter, but may be an inducible promoter. Thus, “E. coli that expresses a gene product” should be understood to mean E. coli that is not always expressing the gene product but has the ability to express the gene product.

  Those skilled in the art can appropriately select a promoter suitable for the E. coli expression system. Examples include, but are not limited to, the T7 promoter, SP6 promoter, T3 promoter, PBAD promoter, tac promoter, cspA promoter, and the like. The T7 promoter is a particularly preferred promoter. It is more preferable that the 4CL gene, the CHS gene, and the EFP gene each have a promoter than the operon type in which a plurality of genes are transcribed from one promoter. It is preferable that the promoters of these three genes are the same type of promoter. It is particularly preferred that each of these three genes has a T7 promoter.

  As means for assisting the expression of a heterologous gene in Escherichia coli, those known to those skilled in the art may be appropriately used. Such means can be, for example, one or more of including an RBS (ribosome binding site) sequence, codon optimization of the coding sequence for E. coli, and the use of a host strain with an additional tRNA gene. Various techniques for introducing a foreign gene with a promoter or the like into E. coli cells are known to those skilled in the art, and can be used to prepare E. coli of the present embodiment. It is preferable that these genes are introduced into Escherichia coli cells in a state contained in one or more plasmid vectors.

  In particular, when a plurality of heterologous enzymes are to be expressed in a specific microorganism (for example, Escherichia coli), how and in what combination enzymes from any species can be important. For example, in Non-Patent Document 1, the use of a 4-coumarate CoA ligase (4CL) enzyme derived from a bacterium belonging to the genus Streptomyces in combination with another enzyme derived from another species is a key to the successful synthesis of flavonoids in E. coli. The demonstration of flavonoid synthesis in Patent Literature 1 has been performed using a specific 4CL enzyme derived from a plant species of Petroselinum crispum.

  In fact, in an early experiment by the present inventors, the gene for the 4CL enzyme from soybean (Glycine max) was inserted into a plasmid vector, and the gene for the CHS enzyme from soybean was inserted into another plasmid vector. Plasmids were introduced into Escherichia coli, and the use of multiple types of Escherichia coli strains was attempted, but flavonoids could not be efficiently produced. However, the inventor has found that two broad approaches can dramatically improve the efficiency of flavonoid production by E. coli expressing 4CL, CHS, and EFP.

  In the first approach, a 4CL enzyme containing an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 derived from soybean, and the amino acid sequence of SEQ ID NO: 4 derived from Antirrhinum majus A CHS enzyme containing an amino acid sequence having 98% or more sequence identity to an EFP protein containing an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 5 derived from snapdragon The combination is expressed in E. coli.

  Each of these sequence identities is more preferably 99% or more, and even more preferably 100%. Sequence identity means that a query sequence (eg, the amino acid sequence of SEQ ID NO: 1) and a target sequence are aligned and aligned over the entire length of the query sequence to form pairs of amino acid residues that correspond in position. (This is commonly referred to as an alignment), which is a percentage of the total number of pairs that indicates how many pairs of exactly matching amino acid types can be found. In cases involving insertion or deletion of amino acid residues, it may also include a pair of an amino acid in the query sequence and a blank in the target sequence (or vice versa). Sequence identity can be conveniently calculated using software known to those skilled in the art, such as, for example, BLAST. The sequence identity between the snapdragon CHS (SEQ ID NO: 4) and the soybean CHS (SEQ ID NO: 2) is only 88%. Also, the sequence identity between the snapdragon EFP (SEQ ID NO: 5) and the soybean EFP (SEQ ID NO: 3) is only 67%.

  The amino acid sequence of soybean 4CL (SEQ ID NO: 1) is registered under the accession number NP_001237270.1 in the NCBI (National Center for Biotechnology Information) database. The amino acid sequence of soybean CHS (SEQ ID NO: 2) is registered in the NCBI database under accession number NP_001337038.1. The amino acid sequence of soybean EFP (SEQ ID NO: 3) is registered in the NCBI database under accession number NP_001236782.1. The amino acid sequence of Snapdragon CHS (SEQ ID NO: 4) is registered under the accession number P06515.1 in the NCBI database. The amino acid sequence of snapdragon EFP (SEQ ID NO: 5) is registered in the NCBI database under accession number BAO32071.1.

  The polypeptide in the present embodiment may consist of only the indicated amino acid sequence, respectively, or may have a peptide tag added to the N-terminus and / or the C-terminus. Peptide tags are well known to those skilled in the art, and examples include His tag (HHHHHH), HA tag (YPYDVPDYA), FLAG tag (DYKDDDDK), Myc tag (EQKLISEEDL), and S tag (KETAAAKFERQHMDS). It is not limited to. The length of one peptide tag is preferably 25 amino acids or less, more preferably 20 amino acids or less, even more preferably 15 amino acids or less, and particularly preferably 10 amino acids or less. As known to those skilled in the art, these peptide tags are usually added to facilitate purification and detection of the polypeptide, etc. (However, peptide tags are not intended for such specific use. The function of the polypeptide may not be impaired).

  A second approach to dramatically improving the efficiency of flavonoid production by E. coli expressing 4CL, CHS, and EFP is to include these plant-derived genes together in one plasmid. This means that the three genes together inserted into one plasmid vector are introduced into E. coli cells. While it is relatively easy to clone a plurality of foreign genes into a plurality of plasmids and maintain the plurality of plasmids in Escherichia coli in the presence of an appropriate antibiotic, it is relatively easy to clone three foreign genes into one. Cloning into a plasmid requires extra effort. However, it was found that by doing this, the production of flavonoids by the cooperation of the 4CL enzyme, CHS enzyme and EFP protein was significantly improved. In particular, a combination of soybean 4CL, soybean CHS, and soybean EFP, or a combination of soybean 4CL, snapdragon CHS, and snapdragon EFP, can be expressed from the corresponding gene on the same plasmid. Specific plasmid vectors used in the present disclosure can be appropriately selected by those skilled in the art, and those having a pBR322 replication origin or a ColE1-type replication origin or a ColA replication origin, such as a pET plasmid vector, a pUC plasmid vector, and a pCOLA plasmid Vectors and pCold plasmid vectors are particularly preferred.

  Here, for the soybean 4CL, soybean CHS, soybean EFP or soybean 4CL, snapdragon CHS, snapdragon EFP to be inserted into the plasmid vector, the description of the sequence identity described above is applied as it is. That is, these polypeptides include an amino acid sequence having 98% or more, preferably 99% or more, more preferably 100% sequence identity with the amino acid sequence described in the corresponding SEQ ID NO.

In another aspect, the present disclosure provides a composition for producing a flavonoid, comprising the above-mentioned live E. coli cell. This composition preferably contains at least water in addition to the living cells of Escherichia coli, and more preferably further contains a medium component for ensuring the survival of Escherichia coli. Numerous media components capable of surviving or growing E. coli are known to those of skill in the art and can be included in the composition in this embodiment. Such a medium component usually contains at least an inorganic salt and a carbon source. As an example, the composition may include at least the composition of the M9 medium. The composition of the M9 medium is 48 mM Na ₂ HPO _4, 22 mM KH ₂ PO ₄ , 19 mM NH ₄ Cl, 8.6 mM NaCl, 0.4% glucose (w / v), 1 mM MgSO ₄ , and 100 μM CaCl ₂ . "At least may include" means that higher concentrations of any of these components are included. From the viewpoint of increasing the purity of the product, it may be advantageous to use only glucose as a carbon source. The composition can include an antibiotic to promote selective growth of cells that have taken up the plasmid vector. Examples of antibiotics include, but are not limited to, ampicillin, kanamycin, streptomycin, and chloramphenicol.

  In another aspect, the present disclosure provides a method for producing naringenin, comprising adding p-coumaric acid to the composition.

  The 4CL enzyme activity converts p-coumaric acid to p-coumaroyl-CoA, and the CHS enzyme activity is thought to produce naringenin chalcone using one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA. ing. Naringenin chalcone can be spontaneously converted to naringenin (a flavonoid compound). However, the possibility that unidentified factors present in E. coli promote the conversion to naringenin is not excluded.

  The intermediate that occurs from p-coumaroyl-CoA and malonyl-CoA to naringenin chalcone can be lactonized instead of naringenin chalcone to give the tetraketide lactone, coumaroyyl triacetate lactone (CTAL). CTAL has the same molecular weight as naringenin, and is a by-product that can reduce the efficiency of naringenin production and separation. However, it has been found that the composition of the present embodiment containing E. coli expressing the EFP protein can reduce the generation ratio of this by-product, as compared with the composition containing E. coli not having the EFP protein. . For example, the production weight of CTAL with respect to the production weight of naringenin can be suppressed to 10% or less, preferably 5% or less, more preferably 3% or less.

  In another aspect, the present disclosure provides a method of making eriodictyol, comprising adding caffeic acid to the composition. Because caffeic acid is present in virtually all plants, it may be suitable as a substrate for industrial production of flavonoids. Eriodictyol is a further flavonoid compound different from naringenin. Eriocitrin, a glycoside of eriodictyol, has a strong antioxidant effect, and has been suggested to be effective in preventing diabetic complications and hypertension. The composition of this embodiment not only produces eriodictyol from caffeic acid, but also, as in the case of naringenin, yields the corresponding tetraketide lactone by-product compared to a composition lacking EFP expression. Has been found to be able to be reduced. This by-product of tetraketide lactone has the same molecular weight as eriodictyol, and can reduce the production efficiency and separation efficiency of eriodictyol.

  The effect of the composition of this embodiment on biosynthesis from a caffeic acid substrate was not always predictable, even based on the results using the p-coumaric acid substrate. The only substrates and products of metabolic pathways involving EFPs in plants are p-coumaric acid and naringenin, and it is completely unknown what effect the EFP protein may have on caffeic acid metabolism in E. coli. For example, Non-Patent Literature 2 describes an efp-1 mutant plant (Asagao) lacking EFP expression as follows: "In contrast to (anthocyanins, flavonols and chalcone reductions), caffeic acid Levels of derivatives, chlorogenic acid and 1-O-caffeoylglucoside, were significantly increased in the petals of efp-1 (FIG. S4b), and these caffeic acid derivatives share a common with the CHS substrate p-coumaroyl-CoA. The biosynthetic pathway is partially shared (FIG. 1a). " That is, in the biological context in which EFPs naturally function, the caffeic acid pathway is understood to cross the biosynthetic pathway from p-coumaric acid to flavonoids, but diverge in a completely different direction.

  Adding p-coumaric acid or caffeic acid to the composition of this embodiment can substantially mean including p-coumaric acid or caffeic acid in the culture medium. The concentration of p-coumaric acid or caffeic acid after being added to the composition is preferably less than 10 mM, more preferably 5 mM or less, still more preferably 3 mM or less, and particularly preferably 2 mM or less. As a specific form of the composition for producing a flavonoid described above, a composition for producing naringenin containing the Escherichia coli cells of the above embodiment and p-coumaric acid, and a production of eriodictyol containing the E. coli cells and caffeic acid of the above embodiments It is understood that compositions for use are also provided. These compositions can have the above substrate concentrations. After the addition of p-coumaric acid or caffeic acid, shaking, aeration, temperature control, and the like can be performed during the period for causing bioconversion, as in the usual E. coli culture. The temperature can be, for example, 15-42 ° C, preferably 18-38 ° C, more preferably 20-37 ° C, even more preferably 25-32 ° C. The temperature may be varied over time within these ranges. Those skilled in the art can appropriately determine the temperature conditions under which E. coli can survive continuously.

  After the addition of p-coumaric acid or caffeic acid, for example, 3 hours or more, preferably 10 hours or more, more preferably 20 hours or more, naringenin or eriodictyol is accumulated in the composition. During this bioconversion period, media components and / or substrates may be supplemented as appropriate. Normally, most of the naringenin or eriodictyol is collected extracellularly, ie, in the supernatant after centrifugation, but naringenin or eriodictyol may also be contained intracellularly, ie, in the solids fraction.

  In the following Examples, the sequence derived from soybean (Glycine max) is denoted as Gm, and the sequence derived from snapdragon (Antirrhinum majus) is denoted as Am.

[Preparation of plasmid for expression of Gm4CL]
There are four soybean 4CLs whose functions have been analyzed (4CL1 to 4CL4). According to the evolutionary phylogenetic tree analysis, 4CL1 and 4CL2 are included in the clades of enzymes involved in phenylpropanoid biosynthesis, and 4CL3 and 4CL4 are included in the clades of enzymes involved in flavonoid biosynthesis. Therefore, 4CL3 having a higher expression level in soybean was selected from the two belonging to the latter clade. The coding sequence of 4CL3 was amplified by PCR using cDNA prepared from mRNA obtained from the lateral root of soybean (Enray variety) as a template. Hereinafter, this soybean-derived 4CL3 is referred to as Gm4CL. This coding sequence was inserted into a pET15b vector to obtain a plasmid pET15b-Gm4CL. From this plasmid, Gm4CL having a His tag at the N-terminus is expressed by the T7 promoter. A T7 terminator is present downstream of the Gm4CL gene.

[Preparation of plasmid for co-expression of GmCHS and GmEFP or AmCHS and AmEFP]
In a similar manner, the coding sequences of GmCHS and GmEFP were sequentially inserted into the pCOLADuet-1 vector to obtain pCOLA-GmCHS-GmEFP. In this plasmid, GmCHS having a His tag at the N-terminus and GmEFP having an S-tag at the C-terminus are arranged in tandem with a T7 promoter. Although a T7 terminator exists downstream of the GmEFP gene, a terminator sequence is not inserted between the GmCHS gene and the GmEFP gene. In a similar manner, pCOLA-AmCHS-AmEFP in which the soybean gene was replaced by the snapdragon gene was also obtained. In addition, pCOLA-GmCHS and pCOLA-AmCHS lacking the EFP gene were also prepared.

[Preparation of plasmid for co-expression of Gm4CL, GmCHS, and GmEFP]
A GmCHS-GmEFP expression cassette containing a promoter and a terminator was PCR-amplified from pCOLA-GmCHS-GmEFP, and this was inserted into the EagI site of pET15b-Gm4CL to prepare pET15b-Gm4CL-GmCHS-GmEFP. In addition, pET15b-Gm4CL-GmCHS lacking the EFP gene was also prepared.

[Introduction of plasmid into E. coli]
The above plasmid was introduced into Escherichia coli (Rosetta2 (DE3) strain) which was made into competent cells according to a conventional method to obtain a transformed Escherichia coli strain. When a plurality of plasmids were to be introduced, they were sequentially introduced while selecting each with an appropriate antibiotic. Experimental E. coli strains shown in the table below were prepared.

[Bioconversion using transformed E. coli]
At least three replica experiments were performed for each of the E. coli strains shown in Table 1 above. First, these strains were inoculated into 3 mL of LB medium containing appropriate antibiotics (kanamycin, ampicillin, chloramphenicol for strains A to F; ampicillin, chloramphenicol for strains G to I), Preculture was performed overnight at 37 ° C. and 120 rpm. Thereafter, 200 μL of the preculture was added to 20 mL of LB medium containing an appropriate antibiotic, and main culture was performed at 37 ° C. and 120 rpm. When the OD ₆₀₀ reached 0.4 to 0.5, IPTG (isopropyl β-D-1-thiogalactopyranoside) was added at a final concentration of 1 mM, and the cells were cultured at 18 ° C. and 180 rpm for 20 hours. At this stage, the expression of the polypeptide shown in Table 1 was induced.

The total amount of the culture solution after 20 hours was centrifuged at 4 ° C. and 10,000 × g for 5 minutes, and the cells were collected as a pellet. The pellet was resuspended in 1 mL of M9 medium and washed by centrifugation again. This washing was repeated twice. The pellet after washing was resuspended in 1 mL of M9 medium, prepared in M9 medium so that OD ₆₀₀ = 5.0, and 200 μL of the obtained composition was used in the following experiments.

  A substrate (p-coumaric acid or caffeic acid, final concentration 1 to 10 mM) was added to the above composition, and the mixture was incubated at 30 ° C for 3 hours. Thereafter, the supernatant (S) and the cells (I) were separated by centrifugation at 4 ° C. and 15000 × g for 10 minutes. The I fraction was resuspended in 200 μL of water. 600 μL of methanol: chloroform (ratio 2: 1, further containing 1 μM Apigenin-7-O-glucoside (A7G) as an internal standard for LC-MS analysis) was added to each of the S and I fractions. Was extracted. Thereafter, 200 μL each of methanol and water was added and mixed, and centrifuged at 15000 × g at 4 ° C. for 10 minutes. The product was detected by subjecting the supernatant after centrifugation to LC-MS.

[Mass spectrometry of product]
LC-MS analysis conditions are as follows.
Control software: Lab Solutions LCMS (SHIMADZU)
Column: CAPCELL CORE C18 (2.1 mm ID x 100 mm, SHISEIDO)
Column temperature: 40 ° C
Flow rate: 0.2 mL / min
Analysis mode: Selected ion detection (SIM), negative mode developing solvent: A solution (0.05% formic acid aqueous solution), B solution (100% CH ₃ CN)
Deployment conditions:
Equilibration, 20% B, 2 min
Gradient, 20% B-35% B, 18 min
Isocratic, 35% B, 5 min
Gradient, 35% B-90% B, 1 min
Equilibration, 20% B, 10 min

  The m / z corresponding to naringenin, CTAL, eriodictyol, and the deprotonated form of compound 5 ([M-H]-) analyzed in this experiment are 271, 271, 287, and 287, respectively. Each product was quantified based on the peak area in LC-MS.

[result]
Under the specific experimental conditions described above, neither naringenin nor CTAL was detected from compositions containing strains B and C with the addition of p-coumaric acid. These compositions appeared to require increased polypeptide expression induction and / or increased bioconversion time. However, under the same experimental conditions, it was confirmed that naringenin was efficiently produced from the composition containing strain E to which p-coumaric acid was added (FIG. 1). Strain E differs from strain B only in that the origin of the CHS enzyme is snapdragon (Am) instead of soybean (Gm). The results illustrate that certain combinations of enzymes from different plant species can significantly alter the efficiency of reactant production in E. coli.

  Compositions containing strain E expressing Gm4CL and AmCHS also produced significant amounts of CTAL by-products (**) in addition to the target flavonoid compound naringenin (*) (FIG. 1). In contrast, the composition containing strain F expressing Gm4CL, AmCHS, and AmEFP significantly suppressed the production of CTAL while maintaining a high production of naringenin (FIG. 1). The ratio of the amount of CTAL product to naringenin product was 14.1% ± 1.0% for strain E and 2.0% ± 0.2% for strain F (mean ± SD, n = 3). ).

  As described above, in strains B and C, even if a plurality of soybean-derived polypeptides were expressed from separate plasmids, no detectable amount of naringenin could be produced in a bioconversion time of 3 hours. However, in strains H and I that co-express the same set of soybean-derived polypeptides from one and the same plasmid, as in strains E and F that combine a snapdragon-derived polypeptide, p-coumaric acid is highly efficient. Naringenin could be produced (FIG. 2). Again, as compared to strain H, strain I to which GmEFP was added significantly suppressed the production of by-product CTAL (FIG. 2). In this example, a remarkable improvement in the yield of naringenin was also obtained. The ratio of the amount of CTAL product to naringenin product was 20.2% ± 1.3% for strain H and 4.7% ± 0.3% for strain I (mean ± SD, n = 3). ). As shown in FIG. 2, the yield tended to decrease with increasing p-coumaric acid concentration, which may be due to the toxicity of p-coumaric acid itself to E. coli.

  Compositions containing these strains capable of producing naringenin from a p-coumaric acid substrate were also found to be useful for producing the flavonoid compound eriodictyol using caffeic acid as a substrate (FIG. 3). In FIG. 3, in the composition of strain H lacking EFP, compound 5 (##), which is a by-product, is produced in an amount larger than that of eriodictyol (#). Compound 5 is a CTAL-like tetraketide lactone having a caffeoyl group instead of a cumaroyl group. However, in the composition of strain I to which EFP was added, the production ratio of eriodictyol could be almost equal to that of compound 5 (FIG. 3, right).

  In another experiment, pET15b-Gm4CL-GmCHS-GmEFP- was inserted into the SapI site of pET15b-Gm4CL-GmCHS-GmEFP in which a tyrosine ammonia lyase (TAL) gene derived from a Herpetosiphon aurantiacus bacterium was linked to the T7 promoter. HaTAL was prepared. To a composition containing Escherichia coli transformed with this plasmid in an M9 medium, L-tyrosine as an amino acid substrate was added at a final concentration of 0 mM, 0.04 mM, 0.5 mM, or 1 mM, and the mixture was incubated at 30 ° C. for 3 hours. However, naringenin could be obtained with high efficiency. In particular, at a L-tyrosine concentration of 0.5 mM, naringenin exceeding 500 nM was obtained, and the ratio of CTAL to naringenin was less than 2%.

Claims (8)

An E. coli cell expressing 4-coumarate CoA ligase enzyme (4CL), chalcone synthase (CHS), and EFP protein,
The 4CL comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 derived from soybean,
The CHS comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 4 derived from snapdragon,
The EFP comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 5 derived from snapdragon.
E. coli cells. An E. coli cell expressing 4-coumarate CoA ligase enzyme (4CL), chalcone synthase (CHS), and EFP protein,
The 4CL, CHS, and EFP are expressed from genes on the same plasmid,
The 4CL comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 derived from soybean,
The CHS comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 2 derived from soybean,
The EFP comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 derived from soybean,
E. coli cells. An E. coli cell expressing 4-coumarate CoA ligase enzyme (4CL), chalcone synthase (CHS), and EFP protein,
The 4CL, CHS, and EFP are expressed from genes on the same plasmid,
The 4CL comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 derived from soybean,
The CHS comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 4 derived from snapdragon,
The EFP comprises an amino acid sequence having 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 5 derived from snapdragon.
E. coli cells. A composition for producing a flavonoid compound, comprising the Escherichia coli cell according to any one of claims 1 to 3 . A method for producing naringenin, comprising adding p-coumaric acid to the composition according to claim 4 . A method for producing eriodictyol, comprising adding caffeic acid to the composition of claim 4 . A composition for producing naringenin, comprising the Escherichia coli cell according to any one of claims 1 to 3 and p-coumaric acid. A composition for producing eriodictyol, comprising the Escherichia coli cell according to any one of claims 1 to 3 and caffeic acid.