JP6222643B2 - New strain of the genus Botryococcus brownie - Google Patents

Description

  The present invention relates to a novel strain belonging to Botryococcus braunii that produces linear saturated hydrocarbons.

  In recent years, technology for reducing carbon dioxide in the atmosphere has been actively developed as a countermeasure against global warming. In addition, the development of renewable energy is being promoted because of a sense of crisis against the fossil fuel depletion. Photovoltaic power generation and wind power generation have been put to practical use as renewable energies, but the use of photosynthetic organisms that convert water and carbon dioxide into hydrocarbons by light energy is also attracting attention.

  Algae are mentioned as photosynthetic organisms that are attracting attention as energy resources, among which green algae and diatoms are attracting attention. Ordinary green algae are composed of 15-17% lipids in their constituents, which are largely free of neutral lipids (30%), glycolipids (37%), phospholipids (26%) and fatty acids. Divided into lipids (7%).

  In recent years, Botryococcus braunii has attracted attention as an oil-producing green algae. The main component of the lipid produced by Botryococcus brownie is a hydrocarbon composed of carbon and hydrogen, and is known to accumulate hydrocarbons such as linear alkenes and triterpenes inside and outside the cell.

Based on the structural characteristics of the hydrocarbons produced, Botryococcus brownies are classified into three types: Race-A, Race-B, and Race-L. Race-A is a group that produces hydrocarbons having an odd number of carbon atoms of 25-31, linear, and having 2 or 3 double bonds in the molecule, Race-B is C _n H A group producing a hydrocarbon having a triterpene structure represented by _2n-10 (n = 30 to 37), Race-L produces a hydrocarbon having a tetraterpene lycopadiene (C ₄₀ H ₇₈ ) structure. Group.

  For the hydrocarbon content of Botryococcus brownies belonging to each group, reference can be made to published literature. Race-A varies depending on the strain and is reported to be in the range of 0.4 to 61.0% (weight of hydrocarbon relative to dry alga body weight) (Non-patent Document 1). In Race-B, many produce 30-40% hydrocarbons per weight, but strains producing only about 9% have also been reported (Non-patent Document 2). Race-L is reported to be 0.1% for Indian stocks and 8.0% for Thai stocks (Non-Patent Document 3), which is known to be less than Race-B. The composition of these hydrocarbons is a mixture of those having various carbon chain lengths and / or structures.

  Most of these hydrocarbons have a double bond, but the double bond is unstable and easily oxidized. For example, when a hydrocarbon double bond generally produced by Botryococcus is oxidized, it becomes an epoxide, and when it is also oxidized, it becomes a diol. In this process, if there is another epoxide in the vicinity, it polymerizes to form an ether bridge, and the other becomes alcohol or radical carbon. Moreover, if water is added to the epoxide, it becomes alcohol, and if another epoxide is bonded to the village bond, an ether bridge is also formed here, and polymerization occurs.

  On the other hand, hydrocarbons containing double bonds need to be set at a higher temperature during cracking when compared with petroleum or the like. This is not preferable from the viewpoint of industrial cost and energy consumption.

  So far, prokaryotic cyanobacteria have been reported as organisms producing saturated hydrocarbons (Patent Document 2). In this research, attention is paid to genes that are thought to control the mechanism of producing saturated hydrocarbons. However, there are many eukaryotic algae whose mass culture systems are generally established, and whether or not the prokaryotic genes reported in the above research are applied to eukaryotic algae as they are. It is unknown.

  Therefore, if there are eukaryotic algae that produce saturated hydrocarbons, research on production mechanisms and gene systems that control production is expected.

JP-A-9-9953 WO2009140695A1

Metzger, P., Berkaloff, C., Coute, A., Casadevall, E. (1985) Alakdiene- and botryococcene- producing Races of wild strains of Botryococcus braunii.Phytochemistry 24, 2305-2312. Okada, S., Murakami, M., Yamaguchi, K. (1995) Hydrocarbon composition of newly isolated strains of green alga Botryococcus braunii. J. Appl. Phycol. 7, 555-559. Metzger, P., Pouet, Y., Summons, S. (1997) Chemotaxonomic evidence for similarity between Botryococcus braunii L Race and Botryococcus neglectus.Phytochemistry 24, 2305-2312.

  In view of the above situation, an object of the present invention is to provide a new strain of Botryococcus brownie that produces saturated hydrocarbons.

  The present inventors collected Botryococcus brownie from nature, found a strain that solves the above problems from among a genus having a large number of systematically different subgroups, isolated this, and completed the present invention I came to let you.

  (1) The present invention provides a strain belonging to Botryococcus brownie that produces a linear saturated hydrocarbon.

(2) The present invention provides a strain belonging to Botryococcus brownie that produces a linear saturated hydrocarbon having the molecular formula C ₂₀ H ₄₂ .

  (3) The present invention relates to Botryococcus brownie tsukuba-2 strain (reception number: FERM AP-22238, trust organization: National Institute of Technology and Evaluation, Patent Biological Deposit Center, receipt date: May 22, 2012) Day).

  (4) In the present invention, the base sequence of the gene encoding 18S rRNA has the base sequence shown in SEQ ID NO: 1, or has a base sequence showing 90% or more homology with the sequence (1) to ( 3) A strain according to any one of the above is provided.

  (5) The present invention provides a method for producing a linear saturated hydrocarbon from a strain belonging to Botryococcus brownie according to any one of (1) to (4).

  The novel strain of Botryococcus brownie of the present invention can produce linear saturated hydrocarbons.

The 18S rRNA gene base sequence of Botryococcus braunii Tsukuba-2 is shown.

1. Separation of Botryococcus Algae The new strain of Botryococcus brownies of the present invention can be isolated from a sample containing Botryococcus algae collected from lakes and ponds using the following method.

  The sample containing the algae of the genus Botryococcus can be collected from, for example, a lake or a pond using a plankton net having a mesh size of 30 to 100 μm.

  Effective chlorine is allowed to act on the sample containing the algae of the genus Botryococcus to sterilize microorganisms other than the genus Botryococcus. In this case, the sample to be treated may be cultured in advance in an appropriate medium to grow algae. Examples of the medium include CHU medium, JM medium, MDM medium, AF-6 medium, and the like. However, the medium is not particularly limited as long as it is suitable as a medium for Botryococcus algae. Moreover, before making this effective chlorine act, colonies of the genus Botryococcus may be separated by means such as centrifugation, filtration, or using a micropipette under a microscope.

  After treating the sample with available chlorine as described above, the sample may be used as it is, but it is washed by repeating the operation of separating the algal bodies by filtration or centrifugation, etc., and suspending them in the culture solution. Is preferred.

Subsequently, the sample treated with the effective chlorine is applied to a medium suitable for culturing Botryococcus algae, for example, a plate medium such as CHU medium, JM medium, MDM medium, AF-6 medium, and the like. Do. The pH of the medium can be pH 1-14, preferably pH 2-13, more preferably pH 3-11, and even more preferably pH 4-10. The culture temperature can be generally 0-50 ° C, preferably 5-40 ° C, more preferably 10-30 ° C. The culture is performed under light irradiation using a fluorescent lamp or the like. This light irradiation may be performed continuously or may be performed for a certain period of time at intervals. The time interval can be selected from 1 to 72 hours, preferably 1 to 24 hours, more preferably 1 to 12 hours. The illuminance is usually 0 to 300 μE / m ² / s, preferably 5 to 100 μE / m ² / s. The culture period is usually 1 to 60 days, preferably 1 to 30 days.

  Then, a single strain of Botryococcus algae is obtained by collecting a single colony of Botryococcus algae generated on the plate medium. When collecting a single colony, it may be performed under a microscope.

Morphological features of the strain belonging to Botryococcus brownie of the present invention are as follows. Colonies that can be seen with the naked eye are spherical and yellow-green to green. When crushed under a microscope, it is observed as a tufted colony of grapes. The average colony size is 30-100 μm, and the maximum reaches 500 μm. The cells are club-shaped and have a cell wall, but can form an outer shell called a socket wall around the cell wall. The size of the cells is about 8 μm for the short axis and about 14 μm for the long axis, and the ratio is about 1.8. Hydrocarbons secreted from the cell can accumulate between the cell and the outer shell.
It grows by photosynthesis using CO2 as a carbon source.

The strain belonging to Botryococcus brownie of the present invention produces a linear saturated hydrocarbon, preferably a hydrocarbon of molecular formula C ₂₀ H ₄₂ , and the base sequence of the gene encoding 18S rRNA is shown in SEQ ID NO: 3. Have a base sequence or high homology to the sequence, for example at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% It can have a base sequence showing homology. Alternatively, the strain belonging to Botryococcus brownie of the present invention produces a linear saturated hydrocarbon, preferably a hydrocarbon of molecular formula C ₂₀ H ₄₂ , and the base sequence of the gene encoding 18S rRNA is SEQ ID NO: 3 It may have a base sequence capable of hybridizing under the stringent conditions to the base sequence shown in FIG.

  For example, high stringency conditions. Examples of the “stringent conditions” include hybridization conditions at 42 ° C., 2 × SSC and 0.1% SDS, preferably 65 ° C., 0.1 × SSC and 0.1% SDS. Good.

Next, the strain isolated as described above is cultured in a liquid medium. As the type of the liquid medium, for example, a CHU medium, a JM medium, an MDM medium, an AF-6 medium, or a modified medium thereof can be used. The pH of the medium can be pH 1-14, preferably pH 2-13, more preferably pH 3-11, and even more preferably pH 4-10. The culture temperature can be generally 0-60 ° C, preferably 5-50 ° C, more preferably 10-40 ° C. The culture is performed under light irradiation using a fluorescent lamp or the like. This light irradiation may be performed continuously or may be performed for a certain period of time at intervals. The time interval can be selected from 1 to 72 hours, preferably 1 to 24 hours, more preferably 1 to 12 hours. The illuminance is usually 0 to 300 μE / m ² / s, preferably 5 to 100 μE / m ² / s. The culture period is usually 1 to 5 months, preferably 1 to 3 months. Further, during culturing, it may be left aerated or not aerated, but it is preferable to leave without aeration.

2. Extraction and analysis of hydrocarbons produced by Botryococcus algae Hydrocarbons produced by Botryococcus algae can be extracted and analyzed by methods known to those skilled in the art. For example, algae of the genus Botryococcus is cultured and proliferated, and wet algal bodies recovered from the obtained culture solution by filtration or the like are dried by freeze drying or drying by heating. Thereafter, hydrocarbons can be extracted from the dried alga using an organic solvent. The extraction may be performed twice or more using different organic solvents. As the organic solvent, n-hexane, chloroform: methanol mixture (for example, 1: 1, 1: 2) or the like can be used. Preferably, after extraction with a chloroform: methanol mixture, for example, it is concentrated to dryness under a stream of nitrogen and extracted again with n-hexane. The obtained extract is analyzed by NMR, IR, gas chromatography, GC / MS and the like.

  By analyzing the hydrocarbons produced from Botryococcus obtained by the above procedure by the above procedure and screening Botryococcus producing hydrocarbons having a molecular weight of 282 with respect to the total hydrocarbons, The Botryococcus brownie strain of the present invention is obtained.

3. Hydrocarbon The hydrocarbon obtained from the Botryococcus brownie strain of the present invention has the following characteristics.

  The total hydrocarbon produced is 10 to 70%, preferably 20 to 65% by weight, more preferably 25 to 50% by weight, and even more preferably 25 to 40% by weight, based on the alga body. possible.

  In the total hydrocarbons produced, the proportion of linear saturated hydrocarbons is 25% or more, preferably 30% or more, more preferably 40% or more, more preferably 50% or more, more preferably 60% or more, Most preferably it is present at 65% or more.

  The hydrocarbons other than the linear saturated hydrocarbon may include isomers thereof, for example, geometric isomers. Further, unsaturated hydrocarbons and non-linear hydrocarbons may be included.

The linear saturated hydrocarbon is a hydrocarbon having a molecular weight of 282 and the molecular formula is C ₂₀ H ₄₂ .

4). Use of Hydrocarbon Obtained in the Present Invention The hydrocarbon (Eicosane) obtained in the present invention can be used in a wide range such as direct fuel and waterproofing agent. It belongs to paraffin with a melting point of 36.7 ° C and is solid at room temperature. On the other hand, when used as a fuel for an internal combustion engine, treatment such as thermal decomposition or cracking using a catalyst is required.

  In the following, the isolation of the Botryococcus brownie strain of the present invention and the analysis of the produced hydrocarbons are shown as examples, but the scope of the claims of the present invention is not limited by these examples.

1. Culture and separation Samples containing Botryococcus were collected from lakes and ponds in various parts of Japan using a plankton net having a mesh size of 30 to 100 μm. Botryococcus colonies were isolated from this sample using a micropipette under a microscope, and immersed in AF-6 medium added to an effective chlorine concentration of about 0.1%, and microorganisms other than Botryococcus were removed. After sterilization, the cells were washed 3 times with AF-6 medium (Table 1), and cultured under light irradiation at 22 ° C. for 12 hours. Thereafter, a strain cultured with 200 ml of Chu modified medium (Table 2) was added to the Erlenmeyer flask, and cultured for 2 to 3 months under light irradiation at 20 ° C. for 12 hours.

2. Analysis of hydrocarbon The obtained culture solution of Botryococcus was concentrated with a filter having a pore size of 5 μm, freeze-dried, total lipid was extracted using chloroform / methanol (2: 1, V / V), and concentrated and dried. After solidification, re-extraction with n-hexane was performed. This sample was analyzed by GC / MS according to the following procedure.

Analysis conditions: GC (gas chromatography) conditions are as follows: column length: 30 m, inner diameter: 0.25 φmm, Df: 0.25 μm, column: DB-5MS, split ratio: splitless, carrier gas: He, flow rate: 1. It was performed under the conditions of 0 ml / min, injection temperature: 280 ° C., column temperature: 60 ° C. (2 minutes) → 280 ° C. (5 ° C./min, 5 minutes maintained). Separator temperature: 280 ° C. MS (gravimetric analysis) conditions are model: Mstation MS-700KII, ion source (EI and CI, positive for CI, gas: isobutene) ionization current: 200 microA, ion source vacuum: 4 × 10 ⁻⁴ Pa, ionization Energy: 38 eV, analysis tube vacuum: 1.0 × 10 ⁻⁵ Pa, acceleration voltage: 8.0 kV, chamber temperature: 200 ° C., ion Multi. 1.0 kV, magnetic field: HS.

Of the strains obtained from this, attention was paid to strains producing hydrocarbons having a molecular weight of 282. The molecular weight 282 satisfies the composition formula of C ₂₀ H ₄₂ or C ₁₉ H ₃₈ O. Exact m / z was measured from high resolution GC / EI-MS. Was measured, and it was found that the composition formula was C ₂₀ H ₄₂ . Moreover, it was confirmed by GC using a standard product of linear C ₂₀ H ₄₂ that the obtained hydrocarbon was linear C ₂₀ H ₄₂ .

  This strain was deposited as the Botryococcus brownie tsukuba-2 strain: Deposited with the Patent Biological Depositary Center of the National Institute of Technology and Evaluation (Receipt number: FERM AP-22238, date of receipt: May 22, 2012) ).

Determination of the base sequence of the gene encoding 18SrRNA DNA was extracted from a part of freeze-dried alga bodies using a DNA extraction kit (DNeasy Plant Mini Kit, QIAGEN) according to a standard protocol. The 18S rRNA gene fragment was obtained from Takara's thermal cycler using the original primer set, 63F (5'-ACGCTTGTCTCAAAGATTA-3 ') (SEQ ID NO: 1) and 1818R (5'-ACGGAAACCTTGTTACGA-3') (SEQ ID NO: 2). (PERSONAL, TAKARA) was amplified under the following conditions.

PCR reaction solution: 0.2 mM dNTP, 0.5 mM primer set, 10XEx taq buffer
0.25 unit Ex taq DNA polymerase (TaKaRa)

  PCR conditions: Heat denaturation treatment at 94 ° C for 10 minutes, followed by 30 cycles of 94 ° C for 1 minute, 55 ° C for 45 seconds, 72 ° C for 30 seconds, and final extension at 72 ° C for 5 minutes

  After confirming the amplification of the DNA fragment by electrophoresis, it was purified using the QIAquik PCR Purification Kit (Qiagen) according to the standard protocol, and then directly using the DNA sequencer (CEQ8000, Beckman Coulter) according to the standard protocol of the same model. The nucleotide sequence was determined by the sequencing method. The result is shown in FIG.

  Obtained using 18S rDNA sequence information (14 strains) of other Botryococcus conserved strains uniquely determined by the same method and Genebank BLAST search (http://blast.ncbi.nlm.nih.gov/) The systematic position of BOT7 was confirmed by molecular phylogenetic analysis using the registered sequence information of Botryococcus (4 strains) and related green algae (9 strains). For molecular phylogenetic analysis, 18S rDNA sequence data was aligned using ClustalW ver.1.81, manually corrected and shaped, then trimmed at both ends of the sequence using Gene Doc ver.2.6, and then PHYLIP ver. Using 3.66, Neighbor-joining method, maximum parsimony method, maximum likelihood method were used. As a result, in the 18S rDNA phylogenetic tree, BOT7 forms a single clade with high bootstrap support (100%) with other Botryococcus, among which the Botryococcus with registration number BBR581911 (AJ581911) It turned out to be the closest (bootstrap support rate 100%). BBR581911 Botryococcus is an L-race strain collected from Songkhla, Thailand. The following shows the name of the most closely related species on the DNA database (BLAST) and the homology (%) information with the surrounding related species.

  FERM AP-22238 (receipt number)

Claims (3)

Botryococcus brownie tsukuba-2 strain (Reception number: FERM AP-22238) , which produces a linear saturated hydrocarbon and has the base sequence of the gene encoding 18SrRNA shown in SEQ ID NO: 3 . The strain of claim 1, wherein the linear saturated hydrocarbon produces a hydrocarbon having the molecular formula C ₂₀ H ₄₂ . Using strains belonging to Botryococcus Brownie according to claim 2, producing a hydrocarbon having a molecular formula C ₂₀ H _42.

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