JP2014158456A - Transgenic plant cultured cell subjected to rational metabolism flow switching, and biosynthesis method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To rationally switch a metabolism flow of a secondary metabolite that a plant cultured cell inherently has to the production of a new exogenous secondary metabolite.SOLUTION: A transgenic plant cultured cell is introduced with an exogenous secondary metabolism biosynthetic gene comprising a metabolic intermediate of a secondary metabolite produced by a plant cultured cell as a substrate.

Description

  The present invention relates to genetically engineered plant cultured cells, and more particularly to a biosynthetic method obtained by switching the metabolic flow of secondary metabolites contained in the original plant cultured cells.

In recent years, research on biological material production methods that do not rely on chemical synthesis using a large amount of petroleum-derived solvents has been actively conducted.
Until now, as such a production method, a technique (synthetic biology) in which a biosynthetic gene (group) of a target substance is introduced by gene recombination has been a trend.
In this case, a microorganism is used as a host mainly because the gene recombination operation and culture are easy, and the growth of cells is fast.
However, many useful substances are derived from plants, and the biosynthetic genes (groups) of such useful substances are expressed in microorganisms as hosts to produce substances. There is a limit.
Thus, the present inventors have reached the present invention by applying the analysis of metabolic ability of plant culture cells and the gene recombination technique (Patent Document 1) established so far.
It should be noted that no related patent documents or academic papers were found in the range investigated by the present inventors.

Japanese Patent Application No. 2012-202138

  An object of the present invention is to rationally switch the metabolic flow of secondary metabolites inherent in plant cultured cells to production of new foreign secondary metabolites.

The present invention aims to rationally use the active metabolic flow inherent in plant cultured cells, and the transformed plant cultured cells according to the present invention are metabolized intermediates of secondary metabolites produced by plant cultured cells. It is characterized by introducing an exogenous secondary metabolic biosynthetic gene using the body as a substrate.
By using such a transformed plant culture cell, it is possible to utilize the intermediate production ability in the metabolic flow originally possessed by the original plant culture cell. Metabolites (foreign secondary metabolites) can be biosynthesized.
Specifically, an exogenous secondary metabolic biosynthetic gene that uses a metabolic intermediate of a secondary metabolite produced by a plant culture cell as a substrate is introduced and replaced with the secondary metabolite produced by the original plant culture cell. It is characterized by producing a secondary metabolite.
The plant cultured cells that can be used in the present invention are those obtained by culturing and growing plant tissue cells, and there are no particular restrictions on the type of plant or the site of the tissue. Is present in the metabolic flow.
Biosynthesis of a new secondary metabolite by introducing a gene involved in biosynthesis using the original plant cultured cell as a host and using an intermediate synthesized in the original metabolic flow as a substrate Can do.
At that time, the method for transforming cultured cells is not particularly limited.
The contents of Patent Document 1 previously proposed by the present inventors may be used as a method for inducing cultured cells from plant tissue and a method for transforming cultured cells.

  The transformed plant cultured cell according to the present invention and the biosynthetic method using rational metabolic flow switching using the transformed cell culture cell exhibit the ability to produce secondary metabolites inherent in the original plant cultured cell, thereby enabling Since a new secondary metabolite can be biosynthesized by a recombinant gene introduced from outside using a large amount of intermediate as a substrate, it is not necessary to supply the substrate from the outside.

Shown are HPLC chromatograms of H4 suspension cell extracts cultured in MS P680 Pic medium. The ¹ H-NMR (CD ₃ OD, 400 MHz) (top) and ¹³ C-NMR (CD ₃ OD, 100 MHz) (bottom) spectra of the main unknown compound purified from the H4 suspension cell extract are shown. The ¹ H-NMR (CD ₃ OD, 400 MHz) (top) and ¹³ C-NMR (CD ₃ OD, 100 MHz) (bottom) spectra of chemically synthesized feruloylputrescine are shown. The HPLC chromatogram of Hulosuspension cell extract (left) and compound synthesized feruloylputrescine (right) is shown. The HPLC chromatogram of the H4 suspension cell extract cultured with 1 / 2MS Free medium is shown. 1 shows HPLC of H4 suspension cell extract (left) and chemically synthesized p-coumaroylputrescine (right). Figure 3 shows the biosynthesis of hydroxycinnamic acid putrescine amide in H4 suspension cells and metabolic flow switching to hydroxycinnamic acid agmatine amide biosynthesis by introduction of ACT enzyme. Figure 2 shows the production of p-coumaroylagmatine and feruloylagmatine in ACT recombinant cell callus. Shown are HPLC chromatograms of ACT recombinant suspension cell extracts cultured in MS P680 Pic medium (top) and 1/2 MS Free medium (bottom). The growth curve in each culture condition of a H4 suspension cell wild type strain and an ACT recombinant strain is shown. The metabolite time-dependent change in a H4 suspension cell wild strain is shown. The metabolite time-dependent change in H4 suspension cell ACT recombinant strain # 22 is shown. The metabolite time course in H4 suspension cell ACT recombinant strain # 15 is shown.

The present invention takes advantage of the production ability of secondary metabolites inherent to cultured cells derived from plant tissues, and will be described below by taking, as an example, a cultured cell derived from a bamboo plant, but is limited to this cultured cell. is not.
<Identification of major secondary metabolites in Hachiku H4 suspension cultured cells>
H4 suspension cells derived from beech (Phyllostachys nigra) bamboo shoots were added to MS (Murashige and Skoog) medium with 3% sucrose and 10 μM Picram, and the KH ₂ PO ₄ concentration was changed to 680 mg / l, which is 4 times the normal concentration. Using the prepared MS liquid medium (hereinafter abbreviated as MS P680 Pic), the cells were cultured at 25 ° C. under dark conditions at 110 rpm.
1 ml of methanol / 2% acetic acid was added to about 100 mg of H4 suspension cells on the 8th day of culture and subjected to ultrasonic extraction for 10 minutes. The centrifuged supernatant was subjected to HPLC analysis.
As a result, as shown in the chromatogram of FIG. 1, a main unknown compound detected at a retention time of 5.4 minutes was found.

Next, this unknown compound was isolated and its structure was determined.
H4 suspension cells were cultured under the conditions described above, and cells having a wet weight of 97.4 g were recovered from 1 L of the culture solution on the sixth day of culture by suction filtration.
This was subjected to ultrasonic extraction in 1 L of methanol / 2% acetic acid at room temperature for 1 hour. The extract from which cell debris was removed by suction filtration was concentrated, and then defatted with hexane three times.
The combined aqueous layer was concentrated, and the filtered solution was subjected to purification by preparative HPLC.
The HPLC conditions are as follows (column: TSKgel ODS-80Ts 5 μm (20 × 250 mm), solvent: 14% acetonitrile / 0.1% TFA, flow rate: 5 ml / min, detection wavelength: 280 nm).
The collected fraction was concentrated and freeze-dried to obtain 58.5 mg of purified product.
Since a molecular ion peak of m / z = 265 [M + H] ⁺ was detected in the mass spectrum analysis, the molecular weight of this compound was 264, and the characteristics of the UV absorption spectrum (λ _max : 217 nm, 233 nm, 294 nm, 319 nm) From the mass spectrum fragment ion (m / z = 177), it was estimated that this compound had a ferulic acid unit which is a derivative of cinnamic acid.
Furthermore, based on the NMR spectrum shown in FIG. 2, the structure of this compound was estimated to be feruloylputrescine.

Next, feruloylputrescine was chemically synthesized to prove the predicted structure.
Feruloicputrescine was obtained by reacting Ferulic acid with Boc-putrescine in the presence of N, N'-dicyclohexylcarbodiimide (DCC), a dehydrating condensing agent, to form Boc-feruloylputrescine, followed by deprotection under strongly acidic conditions. .
The NMR spectrum of this product is shown in FIG.

^{In both 1} H- and ¹³ C-NMR, the spectral data of the purified product from the cell extract (FIG. 2) and the synthetic sample (FIG. 3) were completely consistent.
Furthermore, the mass spectrum (m / z = 265 [M + H] ⁺ ), UV absorption spectrum (λ _max : 218 nm, 234 nm, 293 nm, 319 nm), and retention time on the HPLC chromatogram (FIG. 4) are consistent. As a result, the structure of the main unknown compound found in H4 suspension cells was identified as feruloylputrescine.

Compared to MS P680 Pic medium that promotes active cell growth, 1/2 MS medium (hereinafter abbreviated as 1 / 2MS Free) or 10 μM, which does not contain plant hormones, and only the inorganic salt concentration in the medium is changed to 1/2. It has been found that culturing H4 suspension cells in 1 / 2MS medium supplemented with 6-benzyladenine (BA) (hereinafter abbreviated as 1 / 2MSBA) promotes ligninization of the cells ( Shinjiro Ogita, Taiji Nomura, Takao Kishimoto and Yasuo Kato (2012) A Novel xylogenic suspension culture model for exploring lignifications in Phyllostachys bamboo. Plant Methods, 8, 40 (1-9)).
Then, the extract of the H4 suspension cell cultured on the lignin promotion conditions was analyzed, and the presence or absence of main secondary metabolites other than feruloylputrescine was investigated.
When the suspension cell extract on the 8th day of culture in ½ MS Free medium was subjected to HPLC analysis, a peak of retention time of 4.8 minutes was added to feruloylputrescine as shown in FIG. ) Was observed to be significantly increased.

Rt. In the mass spectrum analysis of 4.8, a molecular ion peak of m / z = 235 [M + H] ⁺ was detected, and thus the molecular weight of this compound was 234, and the UV absorption spectrum (λ _max : 223 nm, 291 nm) From the characteristic and mass spectrum fragment ions (m / z = 147), the compound was estimated to be p-coumaroylputrescine.
In order to prove the predicted structure, p-coumaroylputrescine was chemically synthesized using p-coumaric acid as a raw material by the same method as the synthesis of feruloylputrescine.
The mass spectrum (m / z = 235 [M + H] ⁺ ), UV absorption spectrum (λ _max : 223 nm, 291 nm) and the retention time on the HPLC chromatogram shown in FIG. Since it agreed with 4.8, the structure of this compound was identified as p-coumaroylputrescine.

<Transformation of H4 cells>
Next, switching of metabolic flow was examined.
Hydroxycinnamic acid amides, p-coumaroylputrescine and feruloylputrescine, have been identified as the major secondary metabolites in H4 suspension cells. In general, hydroxycinnamic amides are obtained by enzymatic condensation of hydroxycinnamic acid CoA ester with an amine compound. Since it is biosynthesized, it is considered that the phenylpropanoid metabolic system and the polyamine metabolic system are very active in H4 suspension cells as shown in the metabolic flow in FIG.
Therefore, an exogenous secondary metabolic biosynthetic enzyme using these biosynthetic intermediates as a substrate was introduced to try to modify the metabolic pathway of H4 cells.
HvACT1 gene of barley encoding agmatine coumaroyltransferase (ACT) catalyzing the condensation reaction of hydroxycinnamic acid CoA ester and putrescine biosynthesis intermediate agmatine (Taiji Nomura, Akihiro Ishizuka, Kazunori Kishida, AKM Rafiqul) Islam, Takashi R. Endo, Hajime Iwamura and Atsushi Ishihara (2007) Chromosome arm location of the genes for the biosynthesis of hordatines in barley. Genes & Genetic Systems, 82: 455-464).
The estimated switching flow is shown in FIG.

In order to highly express the HvACT1 gene under the control of the cauliflower mosaic virus 35S promoter, the pIG121-Hm plasmid (Shozo Ohta, Satoru Mita, Tsukaho Hattori and Kenzo Nakamura (1990) Construction and expression in tobacco of a β-glucuronidase (GUS) reporter HvACT1 / pIG121Hm plasmid in which the GUS gene region of Plant and Cell Physiology, 31: 805-813) is replaced with the HvACT1 gene, and the particle gun method using gold particles as a carrier by MS In P680 Pic medium, H4 suspension cells on the 9th to 10th day of culture were transformed.
Culture after transformation was carried out in MS P680 Pic / 0.3% gellan gum solid medium containing 50-100 mg / l hygromycin B after 1 to 3 weeks depending on the growth state of the cells. Were transplanted as appropriate.
Cells that showed good growth in a solid selection medium supplemented with antibiotics were selected as primary selection strains, and then subcultured every 3 to 4 weeks, followed by secondary selection from strains that showed stable growth. This was carried out to obtain 10 transformed callus strains (# 2, 3, 8, 13, 15, 17, 22, 24, 31, 32).

<Metabolite analysis in ACT recombinant cells>
In order to examine the production of p-coumaroylagmatine and feruloylagmatine, which are ACT reaction products, from the selected 10 transformed cells, chemical analysis of these analytical samples was performed.
Reacting p-coumaric acid or ferulic acid with N-hydroxysccinimide in the presence of DCC to prepare active esters (p-coumaroyl-N-hydroxysuccinimide ester, feruloyl-N-hydroxysuccinimide ester) and reacting them with agmatine Thus, p-coumaroylagmatine and feruloylagmatine were obtained.
Callus cells (100 to 150 mg) were placed in an Eppendorf tube, 1 ml of methanol / 2% acetic acid was added per 100 mg of cells and subjected to ultrasonic extraction for 10 minutes, and the supernatant after centrifugation was subjected to HPLC analysis.
As shown in FIG. 8, p-coumaroylagmatine and feruloylagmatine were quantified using the coincidence of the retention time with the analytical sample as an index. In 6 strains of ACT recombinant strains # 2, 3, 8, 15, 22, and 31, Significant production of these compounds was observed.
At that time, p-coumaroylagmatine was mainly produced, and the amount of feruloylagmatine produced was extremely small.
This result was consistent with the substrate specificity of the HvACT1 enzyme.
That is, it is considered that p-coumaroylagmatine was preferentially synthesized because the HvACT1 enzyme uses p-coumaroyl-CoA as a better substrate than feruloyl-CoA.

<Metabolite analysis in ACT recombinant suspension cells>
The above 6 strains, in which the production of p-coumaroylagmatine was confirmed in callus, were transferred to suspension culture using MS P680 Pic liquid medium and subcultured in the same medium every 2 weeks.
Among them, the # 22 strain in which stable growth was observed was cultured under growth promotion conditions (MS P680 Pic) and lignin promotion conditions (1/2 MS Free), and the cell extract on the 8th day of culture was subjected to HPLC analysis. Provided.
The results are shown in FIG.

Compared with the wild type H4 suspension cell line (FIGS. 1 and 5), p-coumaroylputrescine and feruloylputrescine decreased in any culture condition, and peaks of retention time of 7.0 minutes and 7.8 / 7.9 minutes were observed. Newly detected.
In particular, an increase in both peaks was observed under the conditions for promoting lignin (1/2 MS Free).
Rt. 7.0 is p-coumaroylagmatine, Rt. 7.8 / 7.9 confirmed that the retention time was the same as that of feruloylagmatine in the analysis of the above callus cell extract, but for the sake of accuracy, the mass spectrum and UV absorption spectrum were measured. A comparison was made.
Rt. The mass spectrum analysis of 7.0 detected the same molecular ion peak as that of the p-coumaroylagmatine sample with m / z = 277 [M + H] ⁺ , and the UV absorption spectrum (λ _max : 224 nm, 293 nm) was also the same as the sample. there were.
Rt. In the mass spectral analysis of 7.8 / 7.9, the same molecular ion peak as that of the feruloylagmatine sample at m / z = 307 [M + H] ⁺ was detected, and the UV absorption spectrum (λ _max : 218 nm, 234 nm, 293 nm, 317 nm) Was the same as the standard.
From the above results, Rt. 7.0 is p-coumaroylagmatine, Rt. 7.8 / 7.9 was identified as feruloylagmatine.

<Antral analysis of metabolite accumulation in H4 suspension cell wild-type strain and ACT recombinant strain>
As a result of the above experiments, it was suggested that the metabolite composition differs between the growth promotion condition and the lignin promotion condition.
In addition, since H4 suspension cells were subcultured every two weeks, the change in the amount of accumulated metabolite during a series of culture periods under each growth / lignin culture condition was examined over time.
MS P680 Pic medium was used as the growth promotion condition, and 1/2 MS Free medium and 1/2 MS BA medium were used as the lignin promotion conditions.
In addition to the above-mentioned stable growth strain # 22, # 15 was also subjected to analysis as an ACT recombinant strain.
The cell density (in this case, sedimented cell) of the wild-type H4 suspension cells and the ACT recombinant strains (# 15, 22) subcultured for 2 weeks in MS P680 Pic medium, 1/2 MS Free medium, and 1/2 MS BA medium, respectively. (volume = SCV: using the amount of precipitated cells) was adjusted to 2.5%, and culture was started in a 300 ml Erlenmeyer flask with a medium volume of 100 ml (each for 8 conditions).
One flask was collected every other day from day 0 to day 16 and subjected to SCV measurement and quantification of major metabolites in the cell extract by HPLC.
The cell growth curve based on SCV is shown in FIG.

Focusing on the growth promotion conditions, in the wild strain, as shown in FIG. 10a, the SCV reached about 80% on the 14th day of culture and the growth stopped, but in the ACT recombinant strain, as shown in FIGS. 10b and 10c. In both # 15 and # 22, the rise of growth was slower than that of the wild strain, and the growth continued even after the 16th day of culture, and the growth cycle seemed to be longer than that of the wild strain.
In addition, among the conditions for promoting lignin, there was no difference between the wild strain and the ACT recombinant strain in the 1 / 2MS Free medium, but the growth of the ACT recombinant strain was suppressed in the 1 / 2MS BA medium compared to the wild strain. I found out.
Changes with time in the accumulated amount of major metabolites are shown in FIGS.

In the wild strain, as shown in FIG. 11, the accumulation of feruloylputrescine was only slightly observed under the growth promotion conditions, but under the conditions of lignin promotion, both ½ MS Free and ½ MS BA significantly increased with the culture. Increased feruloylputrescine was observed.
On the other hand, as shown in FIG. 12, in the ACT recombinant strain # 22, feruloylputrescine, which was the main metabolite of the wild strain, was significantly reduced under any culture condition, and p-coumaroylagmatine, which is an ACT reaction product, was replaced instead. High accumulation was observed, and the accumulation amount was particularly high under the 1/2 MS BA condition.
At that time, as in the case of the above-mentioned ACT recombinant callus cells, the production of feruloylagmatine was also observed, although the accumulated amount was lower than that of p-coumaroylagmatine.
This result shows that the biosynthesis of hydroxycinnamic acid putrescine amide was carried out by introducing ACT enzyme using agmatine as a substrate into cells that are active in the phenylpropanoid metabolic pathway and polyamine metabolic pathway that produces putrescine from arginine via agmatine. This is thought to be because the system was switched to the hydroxycinnamic acid agmatine amide biosynthetic system (FIG. 7).
As shown in FIG. 13, high accumulation of p-coumaroylagmatine, which is an ACT reaction product, was also observed in ACT recombinant strain # 15, but generation of p-coumaroylagmatine was accompanied by a decrease in feruloylputrescine in strain # 22. On the other hand, the # 15 strain did not decrease feruloylputrescine, but was still accumulated.
In the # 15 strain, the amount of p-coumaroylagmatine produced is about twice as high as that in the # 22 strain under any culture conditions, but this is because the polyamine metabolic pathway of the # 15 strain is more active than the # 22 strain and supplies the ACT enzyme. This is thought to be due to the large amount of agmatine produced.
At that time, it is presumed that agmatine that could not be treated with the ACT enzyme headed for putrescine biosynthesis, leading to the production of feruloylputrescine.
Table 1 shows the results of determining the amount of intracellular compounds accumulated in 1 L of the culture solution on the number of days of culture when the amount of accumulated major metabolites reached the maximum for each culture condition for each cell line.
The maximum production of feruloylputrescine, which is the main metabolite in the wild type, is 208 mg in 1 / 2MS Free, which is the lignin promoting condition, and the maximum production of p-coumaroylagmatine, which became the main metabolite in place of feruloylputrescine, in the ACT recombinant strain In the # 15 strain, the production amount reached 359 mg under the lignin promoting condition 1/2 MS BA, and the # 22 strain reached 173 mg under the lignin promoting condition 1/2 MS Free.
Thus, it was found that rational metabolic flow switching using plant cultured cells as a host is extremely useful in establishing an efficient production system for exogenous secondary metabolites.

  In the present invention, if a biosynthetic enzyme gene of an exogenous secondary metabolite intended for biosynthesis has been identified, this biosynthetic gene can be introduced into cultured plant cells in which the metabolic pathway of the biosynthetic precursor is active. Since it is good, it is highly versatile and has a wide range of applications in the field of biosynthesis.

Claims (2)

  A transformed plant cultured cell characterized by introducing an exogenous secondary metabolic biosynthetic gene using a metabolic intermediate of a secondary metabolite produced by the plant cultured cell as a substrate. Introducing an exogenous secondary metabolic biosynthetic gene that uses the metabolic intermediate of a secondary metabolite produced by plant culture cells as a substrate,
A biosynthetic method using plant culture cells by rational metabolic flow switching, wherein a new secondary metabolite is produced instead of the secondary metabolite produced by the original plant culture cell.