Methods for producing isoprene

Abstract

To improve the isoprene production rate in a method using mosses. [Solution] The isoprene production method according to the embodiment involves cultivating mosses belonging to the order Polytrichumales under conditions of a temperature of 17°C to 38°C and a light irradiation condition of 5 PPFD to 400 PPFD, thereby producing isoprene from the mosses.

Description

Technical Field

[0001] The present invention relates to a method for producing isoprene using a bryophyte. Background Art

[0002] In order to avoid procurement risks caused by diseases of Hevea brasiliensis and the like, studies on the production of isoprene or polyisoprene using various plants and microorganisms have been promoted. Specifically, production of natural rubber from guayule and Russian dandelion, and production of isoprene using genome-modified yeast have been studied. However, many problems such as productivity and yield still remain in these techniques, and it will take time for practical application, and a method capable of solving the problems of productivity and yield is required.

[0003] Non-Patent Document 1 discloses that in flowering plants, an increase in temperature increases the emission amount of isoprene. It is disclosed that an exponential increase in isoprene emission is observed when the leaf temperature is lower than 30°C, the emission amount continues to increase as the temperature rises until the maximum emission rate is reached at about 40°C, and thereafter the emission amount decreases rapidly.

[0004] Non-Patent Document 2 discloses that many mosses, which are bryophytes, produce isoprene, and among them, Polytrichales, Polytrichum commune and Atrichum undulatum produce large amounts of isoprene. Non-Patent Document 2 shows the isoprene production amount of Polytrichum juniperinum at a temperature of 30°C and a light intensity of 1600 PPFD, the isoprene production amount of Polytrichum piliferum at a temperature of 25°C and a light intensity of 500 PPFD, and the isoprene production amount of Atrichum undulatum at a temperature of 30°C and a light intensity of 1000 PPFD. Prior Art Documents Non-Patent Documents

[0005] Non-Patent Document 1 DAVID T. HANSON and 3 others “EVOLUTIONARY SIGNIFICANCE OF ISOPRENE EMISSION FROM MOSSES”, American Journal of Botany 86(5): 634-639. 1999. [Non-Patent Document 2] Alex B. Guenther, 4 others “Isoprene and monoterpene emission rate variability: Model evaluations and sensitivity analyses”, Journal of Geophysical Research: Atmospheres,<URL: https: / / agupubs.onlinelibrary.wiley.com / doi / 10.1029 / 93JD00527> [Overview of the project] [Problems that the invention aims to solve]

[0006] The inventors focused on mosses, particularly bryophytes, among plants known to produce isoprene. They specifically targeted mosses belonging to the order Polytrichumales and discovered that their production could be improved by controlling their cultivation conditions. In other words, the embodiment of the present invention aims to improve the isoprene production rate in a method using mosses. [Means for solving the problem]

[0007] The present invention includes embodiments shown below. [1] A method for producing isoprene, comprising cultivating mosses belonging to the order Polytrichumales under conditions of a temperature of 17°C to 38°C and a light intensity of 5 PPFD to 400 PPFD, and producing isoprene using the mosses. [2] The method for producing isoprene according to [1], wherein the moss plant is at least one species selected from the group consisting of mosses belonging to the genus Atrichum, mosses belonging to the genus Polytrichum, mosses belonging to the genus Polytrichastrum, and mosses belonging to the genus Pogonatum. [3] The method for producing isoprene according to [1], wherein the moss plant is a moss plant belonging to the genus Atrichum, and the conditions are a temperature of 17°C to 38°C, a light intensity of 5 PPFD to 400 PPFD, and a product of temperature (°C) and light intensity (PPFD) of 200 to 12000. [4] The method for producing isoprene according to [1], wherein the moss plant is a moss plant belonging to the genus Polytrichum, and the conditions are a temperature of 23°C to 38°C, a light intensity of 15 PPFD to 400 PPFD, and a product of temperature (°C) and light intensity (PPFD) of 350 to 12000. [Effects of the Invention]

[0008] According to embodiments of the present invention, the amount of isoprene produced can be improved in a method for producing isoprene using mosses. [Modes for carrying out the invention]

[0009] The method for producing isoprene in this embodiment involves cultivating mosses belonging to the order Polytrichumales under conditions of a temperature of 17 to 38°C and a light intensity of 5 to 400 PPFD, thereby allowing the mosses to produce isoprene. Here, cultivation refers to keeping the plants alive while they metabolize, and is a concept that includes growth, but does not necessarily require proliferation or growth.

[0010] As mosses belonging to the order Polytrichales, plants belonging to the family Polytrichaceae are preferred, such as plants belonging to the genus Polytrichum, such as Polytrichum juniperinum, Polytrichum commune, Polytrichum piliferum, and Polytrichum sphaerothecium; plants belonging to the genus Atrichum, such as Atrichum angustatum and Atrichum undulatum; plants belonging to the genus Polytrichastrum, such as Polytrichastrum sp.; and plants belonging to the genus Pogonatum, such as Pogonatum sp. One of these mosses may be used, or two or more may be used in combination.

[0011] In one embodiment, as a moss belonging to the order Polytrichumales, at least one species selected from the group consisting of mosses belonging to the genus Atrichum, mosses belonging to the genus Polytrichum, mosses belonging to the genus Polytrichastrum, and mosses belonging to the genus Pogonatum is preferred from the viewpoint of isoprene production. More preferably, as a moss, at least one species selected from the group consisting of mosses belonging to the genus Atrichum, mosses belonging to the genus Polytrichum, and mosses belonging to the genus Polytrichastrum is preferred, and even more preferably, at least one species selected from the group consisting of mosses belonging to the genus Atrichum and mosses belonging to the genus Polytrichum.

[0012] The cultivation conditions for bryophytes are a thermal temperature of 17-38°C and a light intensity of 5-400 PPFD. By cultivating bryophytes belonging to the order Polytrichumales at these temperatures and light intensities, isoprene production can be improved. The bryophytes to be cultivated may be protonemas or fronds, but fronds are preferred.

[0013] Here, "PPFD," the unit of light intensity, stands for Photosynthetic Photon Flux Density, and is an indicator of the amount of light at wavelengths (400-700 nm) involved in plant photosynthesis. More specifically, PPFD is expressed as micromoles per square meter per second (μmol / m²). 2 s) indicates the amount of photons (particles of light) that are emitted per square meter per second.

[0014] The thermal temperature is more preferably 20-38°C, more preferably 23-38°C, and even more preferably 27-35°C.

[0015] The light intensity for the light irradiation conditions is more preferably 10 to 350 PPFD, more preferably 15 to 200 PPFD, more preferably 17 to 150 PPFD, and even more preferably 20 to 120 PPFD.

[0016] The preferred conditions for cultivating mosses are a product of temperature (°C) and light intensity (PPFD) of 200 to 12000, more preferably 250 to 10000, more preferably 350 to 8000, more preferably 400 to 5000, and even more preferably 500 to 3000.

[0017] Artificial white light is preferable as the irradiation light. Artificial white light differs from natural light (sunlight) in that its wavelength is in the range of approximately 400 to 700 nm and does not contain ultraviolet light (wavelength less than 400 nm) or far-red light (wavelength of 730 nm or more). Artificial white light can be produced using a light source such as an LED, for example, by blending light of three colors using a blue LED, a green LED, and a red LED.

[0018] There are no particular limitations on the respective intensities of blue, green, and red constituting artificial white light, but it is preferable that the ratio of blue:green:red in terms of PPFD is approximately 1:approximately 2:approximately 1.5. Specifically, when the blue light intensity (unit: PPFD) is taken as 1, it is preferable that the green light intensity is 1.8 to 2.2 (preferably 1.9 to 2.1), and the red light intensity is 1.3 to 1.7 (preferably 1.4 to 1.6). The wavelength of blue is 400 nm or more and less than 500 nm, and for example, a blue LED having a wavelength of 420 to 480 nm may be used. The wavelength of green is 500 nm or more and less than 600 nm, and for example, a green LED having a wavelength of 510 to 560 nm may be used. The wavelength of red is 600 nm or more and 700 nm or less, and for example, a red LED having a wavelength of 630 to 690 nm may be used.

[0019] There is no particular limitation on the culture time of bryophytes, that is, the time for treating bryophytes under the aforementioned temperature conditions and light irradiation conditions, as long as the bryophytes survive and produce isoprene. Usually, bryophytes produce isoprene as long as they are alive, and thus begin to release isoprene when culture starts.

[0020] In one embodiment, when a bryophyte belonging to the genus *Physcomitrium* is used as the bryophyte, the culture conditions preferably satisfy the following (A1) and (A2), and more preferably satisfy (A1), (A2), and (A3). 17 ≦ temperature (°C) ≦ 38 (A1) 5 ≦ light intensity (PPFD) ≦ 400 (A2) 200 ≦ temperature (°C) × light intensity (PPFD) ≦ 12000 (A3) For formula (A1), the temperature is more preferably 23 to 38°C, more preferably 25 to 35°C, and still more preferably 27 to 33°C. For formula (A2), the light intensity is more preferably 10 to 200 PPFD, more preferably 15 to 150 PPFD, still more preferably 17 to 120 PPFD, and even more preferably 20 to 70 PPFD. For formula (A3), the product of temperature (°C) and light intensity (PPFD) is more preferably 250 to 11000, more preferably 300 to 10000, still more preferably 400 to 8000, even more preferably 500 to 5000, and most preferably 600 to 3500.

[0021] In one embodiment, when a bryophyte belonging to the genus *Sphagnum* is used as the bryophyte, the culture conditions preferably satisfy the following (B1) and (B2), and more preferably satisfy (B1), (B2) and (B3). 23 ≦ temperature (°C) ≦ 38 (B1) 15 ≦ light intensity (PPFD) ≦ 400 (B2) 350 ≦ temperature (°C) × light intensity (PPFD) ≦ 12000 (B3) For formula (B1), the temperature is more preferably 25 to 38°C, more preferably 27 to 38°C, and still more preferably 30 to 38°C. For formula (B2), the light intensity is more preferably 20 to 350 PPFD, more preferably 30 to 200 PPFD, still more preferably 35 to 150 PPFD, and even more preferably 40 to 145 PPFD. For formula (B3), the product of temperature (°C) and light intensity (PPFD) is more preferably 400 to 10000, more preferably 500 to 8000, still more preferably 600 to 5000, and even more preferably 700 to 3500.

[0022] In the present embodiment, the method for culturing bryophytes is not particularly limited except for the temperature and light intensity described above, and can be cultured by a known method. A medium may be used for culturing bryophytes, and the medium is not particularly limited, and examples thereof include agar media such as BCDAT agar medium, Murashige-Skoog medium and the like.

[0023] In this embodiment, bryophytes belonging to the order Polytrichumales are cultured under the above culture conditions to produce isoprene. The produced isoprene is released from the bryophytes as a gas, and isoprene can be obtained by recovering the released gas. The recovery method is not particularly limited; it may be batch production in which bryophytes are cultured in a sealed space and the gas in the space is recovered after cultivation, or it may be continuous production in which the gas in the culture space is continuously recovered while the bryophytes are being cultured. [Examples]

[0024] The following are examples of the present invention, but the present invention is not limited to these examples.

[0025] (1) Pre-experimental treatment Mosses were sterilized and cultured on BCDAT agar at 22°C for 1-2 months. Sterilization was performed to remove the influence of microorganisms attached to the mosses and to confirm the influence of the mosses themselves. The sterilization process employed a general sterilization method for plant materials (see "Sterilization Methods for Plant Tissue Culture Materials," Chemistry and Biology vol.26 No.3, pp. 173-175, The Japan Society for Bioscience, Biotechnology, and Agrochemistry). The composition of the BCDAT agar medium was as described in Setsuyuki Aoki and Mamoru Sugita, "Chapter 1: Acquisition, Cultivation, and Culture of Plant, Algae, and Bacteria Materials 5. Physcomitrella patens," Low Temperature Science vol.67, 31-33, 2009.

[0026] A low-temperature incubator (model LTE-510, manufactured by Tokyo Rikakikai Co., Ltd.) was used as the culture device. The light irradiation conditions were continuous white light at 40 PPFD.

[0027] Following the above culturing procedure, in order to conduct the isoprene production experiment under flat conditions, the cells were left undisturbed in the dark at 25°C for 24 hours to stop the isoprene synthesis pathway that is expressed under light conditions.

[0028] After standing in the dark, stems and leaves aged 2-3 months were removed from the obtained bryophytes. 0.1 g of fresh bryophytes were transferred to a 50 mL vial, and 3 mL of Milli-Q water prepared using a Merck ultrapure water system was added at the same time. The lid was closed, and Parafilm was wrapped around the lid and vial to seal it tightly and prevent air from entering.

[0029] (2) Experimental method for isoprene production The sealed vials described in (1) above were placed in a low-temperature incubator (model LTE-510, manufactured by Tokyo Rikakikai Co., Ltd.), and treated at a specified temperature and light intensity for 24 hours to cultivate mosses.

[0030] The temperature conditions were controlled using the low-temperature constant-temperature incubator described above to set the temperature inside the chamber to the predetermined level. The lighting conditions were controlled using an LED lighting device installed inside the chamber (manufactured by Nippon Medical Instruments Co., Ltd., lighting unit "Multi-Specs" (registered trademark), model number PF5-75T) to set the intensity of the artificial white light inside the chamber. The combinations of artificial white light inside the chamber are shown in Table 1 below.

[0031] [Table 1]

[0032] (3) Method for analyzing isoprene To analyze the amount of isoprene inside the vial, the gas inside the vial was recovered using SPME (SPELCO, catalog number 57344-U). Subsequently, isoprene was quantified by GC-MS. In the GC-MS, the column was an Agilent J&W DB-624 (30m, inner diameter 0.25mm, film thickness 1.40μm), the carrier gas was He (flow rate 1mL / min), the inlet temperature was 180℃, the ion source temperature was 230℃, the oven temperature was 40℃, and the analysis time was 8 minutes.

[0033] After drying the bryophytes inside the vial at 75°C for 24 hours, the dry mass of the bryophytes was measured. The isoprene release per gram of bryophyte (mg / g(DW)) was determined by dividing the isoprene mass, which was obtained by GC-MS, by the dry mass of the bryophytes.

[0034] [First Experimental Example] The isoprene release levels of the 11 moss species shown in Table 2 were compared by performing the above steps (1) to (3). The culture conditions for the mosses were 25°C and 40 PPFD. The results are shown in Table 2.

[0035] [Table 2]

[0036] As shown in Table 2, when we investigated the isoprene production (release) of mosses, we confirmed isoprene production in bryophytes of the order Polytrichales, and found that isoprene production was particularly high in bryophytes of the genera Polytrichastrum, Polytrichas, and Polytrichastrum.

[0037] [Second Experimental Example] In the first experimental example, the amount of isoprene released was particularly high for the moss *Hygrophorus spp.*. The amount of isoprene released was measured by combining a temperature of 25-40°C and a light intensity of 0-65 PPFD as culture conditions and performing the above steps (1)-(3). The results are shown in Table 3.

[0038] [Table 3]

[0039] As shown in Table 3, isoprene production in *Hypnum cupressiforme* peaked at 30°C and decreased thereafter. Regarding light intensity, relatively high production was observed even at 20 PPFD, peaking at 40 PPFD and remaining nearly constant above that.

[0040] [Third experimental example] For the variegated moss *Hypnum cupressiforme*, the temperature was fixed at 30°C and the light intensity was increased compared to the second experimental example, and steps (1) to (3) above were carried out. The results, along with those from the second experimental example, are shown in Table 4.

[0041] [Table 4]

[0042] As shown in Table 4, the isoprene release of *Hypnum cupressiforme* was relatively high, at 5.7 mg / g (DW) at 10 PPFD and 8.4 mg / g (DW) at 350 PPFD. This indicates that high isoprene production occurs in the light intensity range of 5 to 400 PPFD.

[0043] [Fourth experimental example] For *Hypnum cupressiforme*, the light irradiation conditions were fixed at 40 PPFD, and the temperature (thermal conditions) was lower than in the second experimental example, and steps (1) to (3) above were carried out. The results, along with those from the second experimental example, are shown in Table 5.

[0044] [Table 5]

[0045] As shown in Table 5, the isoprene release of *Hygrophorus spp.* was relatively high, at 5.4 mg / g (DW) at 20°C and 7.2 mg / g (DW) at 35°C. This indicates that high isoprene production occurs in the temperature range of 17 to 38°C.

[0046] From the results of the second to fourth experiments described above, it can be seen that bryophytes belonging to the genus Atrichum, such as Atrichum commune, have high isoprene production when cultured under conditions of a temperature of 17-38°C, a light intensity of 5-400 PPFD, and a temperature (°C) × light intensity (PPFD) of 200-12000.

[0047] [Experimental Example 5] In the first experimental example, the amount of isoprene released was particularly high in the moss *Polytrichum commune*. The amount of isoprene released was measured by combining a culture condition of 25-40°C and a light intensity of 0-65 PPFD, and carrying out the above steps (1) to (3). The results are shown in Table 6.

[0048] [Table 6]

[0049] The results are shown in Table 6. It was found that isoprene production in *Polytrichum commune* peaked at 35°C and decreased thereafter. Regarding light intensity, it was found to peak at 40 PPFD.

[0050] [Experimental Example #6] For the moss *Polytrichum commune*, the temperature was fixed at 30°C, and the light intensity was increased compared to the fifth experiment, and steps (1) to (3) above were carried out. The results, along with those of the fifth experiment, are shown in Table 8 below.

[0051] As a comparative example, an isoprene production experiment was conducted outdoors to confirm the isoprene production volume under high light irradiation conditions exceeding 1000 PPFD. The experiment was conducted outdoors because the light irradiation limit with the above-mentioned LED lighting device is 800 PPFD. The outdoor test was carried out as follows.

[0052] (Field condition testing) On October 2, 2024, moss plants were cultured under natural light by placing a sealed vial in a sunny location for 5 hours, in accordance with (1) above. The measured PPFD values ​​and temperature from the start of the experiment to 5 hours later are shown in Table 7 below. The light intensity was approximately 1300-1700 PPFD, and the temperature was approximately 30°C. Note that natural sunlight outdoors contains ultraviolet light in the 10-400 nm range and far-red light above 730 nm, but the photosynthetic photon flux density (PPFD) analyzer used in this study cannot measure the amount of photons in this range, and therefore these are not included in the PPFD values ​​in Table 7.

[0053] [Table 7]

[0054] After the experiment was completed, the amount of isoprene inside the vial was analyzed according to (3) above. An Agilent J&W DB-1 column was used. The result showed that the isoprene release after 5 hours of incubation was 1.31 mg / g (DW). To match the 24-hour incubation time of the example, this was multiplied by 4.8, resulting in an isoprene release of approximately 6.3 mg / g (DW) under field conditions. In Table 8 below, the temperature × light intensity under field conditions was calculated by multiplying the average values ​​of temperature and light intensity from Table 7 (temperature: 31°C, light intensity: 1417 PPFD).

[0055] [Table 8]

[0056] As shown in Table 8, the isoprene release of Polytrichum commune was 10.4 mg / g (DW) at 20 PPFD and 8.6 mg / g (DW) at 350 PPFD, which were higher than those observed under field conditions with 800 PPFD or higher PPFD values. This indicates that high isoprene production occurs in the light intensity range of 15 to 400 PPFD.

[0057] [Experimental Example 7] For the moss *Hypnum cupressiforme*, the light intensity was fixed at 40 PPFD, and the temperature was lower than in Experiment 5, and steps (1) to (3) above were carried out. The results, along with those of Experiment 5, are shown in Table 9.

[0058] [Table 9]

[0059] As shown in Table 9, the isoprene release of Polytrichum commune increased with rising temperature, reaching 11.2 mg / g (DW) at 25°C, peaking at 35°C, and decreasing thereafter. This indicates that high isoprene production is observed in the temperature range of 23 to 38°C.

[0060] From the results of the 5th to 7th experiments described above, it can be seen that bryophytes belonging to the genus Polytrichum, such as Polytrichum polyblepharum, have high isoprene production when cultured under conditions of a temperature of 23 to 38°C, a light intensity of 15 to 400 PPFD, and a temperature (°C) × light intensity (PPFD) ratio of 350 to 12000.

[0061] As described above, according to this embodiment, isoprene production can be improved by cultivating mosses belonging to the order Polytrichumales under significantly lower light irradiation conditions than those previously reported.

[0062] Furthermore, the various numerical ranges described in this specification can be any combination of their upper and lower limits, and all such combinations are described herein as preferred numerical ranges. Also, the description of a numerical range as "X~Y" means X or greater and Y or less.

Claims

1. A method for producing isoprene, comprising cultivating mosses belonging to the order Polytrichumales under conditions of a temperature of 17°C to 38°C and a light intensity of 5 PPFD to 400 PPFD, and producing isoprene using the mosses.

2. The method for producing isoprene according to claim 1, wherein the moss plant is at least one species selected from the group consisting of mosses belonging to the genus Atrichum, mosses belonging to the genus Polytrichum, mosses belonging to the genus Polytrichastrum, and mosses belonging to the genus Pogonatum.

3. The method for producing isoprene according to claim 1, wherein the moss plant is a moss plant belonging to the genus Atrichum, and the conditions are a temperature of 17°C or higher and 38°C or lower, a light intensity of 5 PPFD or higher and 400 PPFD or lower, and the product of temperature (°C) and light intensity (PPFD) is 200 or higher and 12000 or lower.

4. The method for producing isoprene according to claim 1, wherein the moss plant is a moss plant belonging to the genus Polytrichum, and the conditions are a temperature of 23°C or higher and 38°C or lower, a light intensity of 15 PPFD or higher and 400 PPFD or lower, and the product of temperature (°C) and light intensity (PPFD) is 350 or higher and 12000 or lower.

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