JP2018014964A - Method for producing isoprene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing isoprene, which can easily recover the produced isoprene and has a high yield per unit volume. SOLUTION: A step of accommodating a liquid medium and algae transformed with a polynucleotide encoding isoprene synthase in a container, and a step of culturing diatoms in the liquid medium to obtain a culture solution. A step of collecting isoprene from the gas phase in a container, and a method for producing isoprene containing. [Selection diagram] None

Description

  The present invention relates to a method for producing isoprene.

  As an industrial production method of isoprene (2-methyl-1,3-butadiene) which is a raw material of polyisoprene rubber, it is known that extraction can be performed from a C5 fraction produced as a by-product when naphtha is pyrolyzed. However, since the amount of isoprene produced from the above-mentioned by-products is not always sufficient, and is easily affected by fluctuations in the price of oil as a raw material, in recent years, in order to stably secure isoprene, Development of a method for producing isoprene derived from petroleum resources, specifically, development of a method for producing isoprene using a transformant obtained by incorporating an isoprene synthase gene into animal cells or plant cells, etc. I came.

  Examples of the transformant include, for example, Patent Document 1, “(a) a polynucleotide encoding isoprene synthase, (b) a polynucleotide encoding 5-phosphomevalonate kinase, and (c) diphosphomevalonate deoxygenase”. A transformed plant that has been transformed with at least one selected from the group consisting of a polynucleotide encoding a carboxylase and that has increased the productivity of the useful part. "As a specific example, isoprene synthase An Arabidopsis thaliana gene expressing the gene is described.

JP 2008-035831 A

  The inventors of the present invention tried to produce isoprene using Arabidopsis thaliana in which the isoprene synthase gene was expressed as described in Patent Document 1. As a result, the recovery of the produced isoprene was complicated and low in efficiency. I found out. It was also found that cultivation of Arabidopsis requires a predetermined volume and the isoprene yield per unit volume is poor.

  This invention is made | formed in view of the said situation, and makes it a subject to collect | recover the produced | generated isoprene easily and to provide the manufacturing method of isoprene with a high yield per unit volume.

  As a result of intensive studies to achieve the above problems, the present inventors have found that the above problems can be solved by using algae transformed with a polynucleotide encoding isoprene synthase. Was completed.

[1] Step A of containing a liquid medium and an algae transformed with a polynucleotide encoding isoprene synthase in a container, and Step B of culturing the algae in a liquid medium to obtain a culture solution And step C for collecting isoprene from the gas phase in the container.
[2] The method for producing isoprene according to [1], wherein the algae is diatom.
[3] The method for producing isoprene according to [1] or [2], wherein the culture temperature in Step B is 10 to 40 ° C.

  ADVANTAGE OF THE INVENTION According to this invention, the production method of isoprene which can collect | recover the produced | generated isoprene easily and has a high yield per unit volume can be provided.

Hereinafter, the present invention will be described in detail based on embodiments.
In addition, description of the component requirement described below is made | formed based on the embodiment of this invention, and this invention is not limited to such an embodiment.
In the present specification, a numerical range expressed using “to” means a range including numerical values described before and after “to” as a lower limit value and an upper limit value.

[Method for producing isoprene]
The method for producing isoprene according to the above embodiment includes:
Step A: A step of containing a liquid medium and an algae transformed with a polynucleotide encoding isoprene synthase in a container Step B: a step of culturing the algae in a liquid medium to obtain a culture solution -Process C: The process of extract | collecting isoprene from the gaseous phase in a container is contained.

In the method for producing isoprene, algae transformed with a polynucleotide encoding isoprene synthase is cultured in a container, and isoprene is obtained from a gas phase portion (for example, headspace) in the container. Collect.
Since algae has a higher culture efficiency per unit volume than terrestrial plants such as Arabidopsis thaliana, isoprene can be produced efficiently.
Algae has a high ability to fix carbon dioxide in the atmosphere, and dimethylallyl pyrophosphate described later can be efficiently produced by photosynthesis, so that isoprene can be produced efficiently.
In addition, since algae can be cultured in a closed container, the produced isoprene can be easily collected from the gas phase in the container.
Due to the above synergistic effect, it is presumed that the method for producing isoprene according to the above embodiment has achieved a desired effect.
Below, the aspect is demonstrated about each process.

[Process A]
Step A is a step of storing a liquid medium and algae transformed with a polynucleotide encoding isoprene synthase in a container.

<Liquid medium>
The liquid medium used in step A is not particularly limited, and a known liquid medium can be used.
As a liquid culture medium, the liquid culture medium containing seawater is mentioned, for example.
Examples of the liquid medium include a medium containing a nitrogen source, phosphorus, magnesium, silicon, potassium, sodium, calcium, vitamins, and the like.
Specific examples of the liquid medium include, for example, Daigo IMK culture medium supplemented with sea salt and a predetermined amount of Na ₂ SiO ₃ .

<Algae transformed with a polynucleotide encoding isoprene synthase>
The algae transformed with the polynucleotide encoding the isoprene synthase used in step A are not particularly limited, and algae transformed by a known method can be used.

(Isoprene synthase)
In this specification, the isoprene synthase is an enzyme that intends a protein having isoprene synthesizing activity and catalyzes a reaction in which dimethylallyl pyrophosphate (DMAPP) is oxidatively dephosphorylated.
Examples of the isoprene synthase include those derived from all species that produce isoprene synthase, among which, for example, those derived from higher plants or ferns are preferable, and in particular, broad-leaved trees, ferns, and conifers. Those derived from species having a large amount of released isoprene, such as, are preferred. In particular, the willow family (for example, poplar), the myrtaceae family (for example, eucalyptus), the beech family (for example, Quercus kunugi, the Quercus genus Naragashi), the fern family (for example, fern) and the like, which release a large amount of isoprene, are preferable.
Specific examples of isoprene synthase include isoprene synthase (Genbank accession number: BAD98243) of Populus alba (Populus alba).
The isoprene synthase is about 80% or more, preferably 85% or more, more preferably about 90% or more, more preferably 93%, more preferably about 96% or more with the amino acid sequence of the isoprene synthase according to the above specific example. More preferably, an isoprene synthase having an amino acid sequence having a homology of about 98% or more is included.

(Polynucleotide encoding isoprene synthase)
The polynucleotide encoding isoprene synthase is not particularly limited as long as it is a polynucleotide encoding isoprene synthase, and known polynucleotides can be used.
Specific examples of the polynucleotide encoding isoprene synthase include, for example, the base sequence of poplar isoprene synthase gene (Genbank accession number: AB198180) and the base sequence of kudzu isoprene synthase gene (Genbank accession number). : AY316691) and the like.
The polynucleotide encoding isoprene synthase has a nucleotide sequence of about 80% or more, preferably 85% or more, more preferably about 90% or more, more preferably 93%, more preferably about 96% or more, and still more preferably A polynucleotide having a base sequence having about 98% or more identity is included.

(Algae)
Examples of algae include algae belonging to the diatom class, hapto algae class, pingio algae class, golden algae class, and the like. Of these, algae belonging to the diatom net (hereinafter simply referred to as “diatom”) is preferable. The diatoms are classified into the genus Chaetoceros, Phaeodactylum, Cycloterra, Skeletonema, Odonterra, and the genus Nitzia. Among these, Chaetoceros is preferable, hornflower is more preferable, and Chaetoceros gracilis is still more preferable. The diatom may be a mutant.

(Method for producing algae transformed with a polynucleotide encoding isoprene synthase)
The method for producing algae transformed with a polynucleotide encoding isoprene synthase is not particularly limited, and known transformation methods can be used.
In particular, a method of introducing the polynucleotide into an algal cell as an expression plasmid linked to an expression vector is preferable.

The expression vector is not particularly limited as long as it can be introduced into algal cells, and examples thereof include a plasmid vector, a phage vector, and a cosmid vector. Of these, plasmid vectors are preferred.
In particular, those that are capable of autonomous replication in a host or that can be integrated into a chromosome and that contain a promoter at a position that allows transcription of the polynucleotide are preferably used. The 5 ′ end of the polynucleotide amplified by PCR is linked to the 3 ′ end of the promoter directly or in-frame via an appropriate sequence (for example, a restriction enzyme recognition site). The expression vector may further contain a plant cell enhancer (for example, EL2 enhancer, CaMV35S enhancer, etc.), a selection marker gene, a labeling tag, and the like. A stop codon is not necessarily required, but is preferably arranged immediately below the structural gene. In addition, with reference to the amino acid sequence, the polynucleotide can be linked in-frame to an expression promoter, enhancer, initiation codon, and the like. Various base sequences and amino acid sequences can be obtained from search systems such as Genbank and EMBL. The amino acid sequence can also be determined by a conventional method such as a system that estimates an amino acid sequence based on a base sequence, N-terminal analysis, C-terminal analysis, or the like.

The promoter is not particularly limited as long as it promotes the expression of isoprene synthase in algal cells, and a known promoter can be used.
Examples of the promoter include EL2 promoter, CaMV35S promoter, Cab promoter, RuBisCo promoter, PR1 promoter, NR promoter, ubiquitin promoter, and modified ones thereof.

  Moreover, the said vector may also contain the other arrangement | sequence contained in a general vector. For example, a so-called regulatory sequence such as an operator, terminator, and enhancer that regulates the expression of the isoprene synthase gene may be included. Furthermore, a promoter different from the above and a gene controlled by the promoter (for example, drug system gene) may be included.

The above vector can be prepared by methods well known to those skilled in the art. For example, it can be obtained by amplifying the promoter sequence using a primer provided with an appropriate restriction enzyme site and inserting it into a donor vector cut with a restriction enzyme. Alternatively, it can be prepared by a method using an “In-Fusion (registered trademark)” reaction.
The promoter sequence may be introduced into the donor vector as an expression cassette in which a terminator sequence is incorporated downstream thereof. An isoprene synthase gene may be incorporated between the promoter sequence and the terminator sequence. In order to insert the isoprene synthase gene, it is preferable that a multiple cloning site having a plurality of restriction enzyme cleavage sites exists in the between the floor motor sequence and the terminator sequence.
The terminator sequence is not particularly limited as long as it functions in algal cells. For example, the terminator sequence of Cglhcr14 (Genbank accession number: AB981633) is CgLhcr14ter.

A method for introducing a polynucleotide or an expression plasmid containing the polynucleotide into algae is not particularly limited, and a known method can be used.
Examples of the introduction method include lithium acetate method, electroporation method, Agrobacterium method, gene gun (particle gun) method, calcium phosphate method, protoplast method, and Gene, 17, 107 (1982), Molecular & General. Genetics, 168, 111 (1979) and Molecular Cloning 2nd ed. (Sambrook, J., Fritsch, EF, Maniatis, T. Cold Spring Harbor Laboratory Press 1989), Photosynthesis Res. 123: 203-211. doi: 10.1007 / s11120-014-0048-y. And a document such as Plant Molecular Biology Manual (Stanton B. Gelvin and Robert A. Stilperort. Kluwer Academic Publishers 1988), which is incorporated herein by reference in its entirety. it can.
Furthermore, an individual into which an expression plasmid has been introduced is usually selected. Selection of an individual into which an expression plasmid has been introduced can be performed by a conventional method, for example, selection by a drug-containing medium, selection by a hormone-containing medium, or selection by auxotrophy. Furthermore, incorporation of the polynucleotide into the plant cell chromosome can be confirmed using conventional methods such as genomic PCR, RT-PCR, Northern hybridization, and Western analysis.

  Here, transformation is generally a genetic phenomenon in which DNA isolated from one cell is taken up by another cell and recombines with the cell chromosome (Tokyo Chemical Dojin Biochemistry Dictionary 2nd edition, 416-417), currently means that DNA molecules including plasmids and genes bound thereto are directly introduced into cells (Iwanami Biology Dictionary, 4th edition, pages 380-381). In the specification, transformation means introduction of a polynucleotide (eg, DNA, mRNA) into a cell, and at this time, the polynucleotide may or may not cause recombination with a cell chromosome. . In order to stably express the phenotype and the genotype, it is desirable that the oligonucleotide undergoes recombination with the cell chromosome, and that the recombination with the cell chromosome is desirably homozygous.

<Container>
The container for containing the liquid medium and transformed algae is not particularly limited, and a known container can be used.
Examples of the container include a plate culture container, a tube culture container, an air dome culture container, and a hollow cylinder container. The container may be a sealed container.

[Process B]
Step B is a step of culturing algae in a liquid medium to obtain a culture solution.
The method for culturing algae in a liquid medium is not particularly limited, and a known method can be used.

Examples of the culture method include a method of culturing algae using a known fermentation method such as a batch culture method, a fed-batch culture method, and a continuous culture method.
The batch culture method is a method of culturing while adding algae to a medium and controlling the culture conditions. In the batch culture method, the algae go from a gentle induction phase to a logarithmic growth phase and finally to a stationary phase where the growth rate decreases or stops. Isoprene is often produced by logarithmic and / or stationary phase transformants.

  The fed-batch culture method is a method in which a carbon source is gradually added as the fermentation process proceeds in addition to the batch method described above. The fed-batch culture method is effective when the metabolism of algae tends to be suppressed by catabolite suppression, and it is preferable to limit the amount of the carbon source in the medium.

The continuous culture method is a culture method in which a predetermined amount of culture medium is continuously supplied to a bioreactor at a constant rate, and at the same time, the same amount of culture medium is withdrawn. In the continuous culture method, the culture can be kept at a constant high density, and the algae in the culture solution are mainly in the logarithmic growth phase.
By appropriately replacing part or all of the medium, nutrients can be supplied, and accumulation of metabolic byproducts and dead cells that may adversely affect the growth of algae can be prevented.

Algal culture conditions are not particularly limited as long as the isoprene synthase gene can be expressed, and standard cell culture conditions can be used. As culture | cultivation temperature, 10-40 degreeC is preferable, for example, and 20-25 degreeC is more preferable.
Further, as the gas composition, it is preferable that the CO ₂ concentration is .04 to 10%, pH is preferably 4 to 10.
Especially, when using diatom as algae, as culture temperature, 15-35 degreeC is preferable and 20-25 degreeC is more preferable. Further, as the gas composition, it is preferable that the CO ₂ concentration is 0.04 to 5%, pH is preferably 7 to 9. By culturing the transformed diatom at the above culture temperature, the expression of the isoprene synthase gene is further promoted.

[Process C]
Step C is a step of collecting isoprene from the gas phase in the container. Examples of the method for collecting isoprene produced by the above method include gas stripping, fractional distillation, heat of isoprene monomer adsorbed on the solid phase, desorption from the solid phase by vacuum, and extraction with a solvent. .
For example, in gas stripping, isoprene gas is continuously removed from the outgas. Such isoprene gas can be removed by various methods, such as adsorption to a solid phase, separation into a liquid phase, or a method of directly condensing isoprene gas.

  According to the method for producing isoprene, isoprene can be efficiently produced from dimethylallyl diphosphate (DMAPP). In the isoprene production method, DMAPP is efficiently supplied from a carbon source in the medium by algae. Since algae produce isoprene mainly as outgas from a carbon source in the medium, isoprene generated from algae can be collected. Note that DMAPP, which is a substrate for isoprene synthase, is synthesized by the mevalonate pathway or methylerythritol phosphate pathway in the host, using the carbon source in the medium as the basis.

Algae (especially diatoms) have high condensing ability and CO ₂ fixing ability, so that the above-mentioned DMAPP can be produced efficiently, and the production efficiency of isoprene is increased.
Moreover, since algae can be cultured in a large amount in a container as described above, the amount of isoprene produced per unit volume is higher than when using Arabidopsis thaliana or the like.
In addition, since algae can be cultured in a closed container as described above, the produced isoprene can be easily recovered from the head space of the closed container.
As described above, according to the method for producing isoprene according to the embodiment of the present invention, the produced isoprene can be easily recovered. The isoprene production method has a high yield per unit volume.

  Hereinafter, the present invention will be described in more detail based on examples. The materials, amounts used, ratios, processing details, processing procedures, and the like shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the following examples.

[Cloning of isoprene synthase gene from Gindro]
[Plant material and growth conditions]
The isoprene synthase gene was cloned from Populus alba tree by PCR using the isoprene synthase gene sequence.
(Populus alba) was cultured by adding 3% sucrose (pH 5.7) to Linsmeier-Skoog agar medium (1%, mass / volume) to a 900 mL cell culture flask. The culture conditions were 25 ° C. and long-day conditions (16 hours light period (120 μmol / m ² s), 8 hours dark period).

[Extraction of total RNA from Populus alba]
Total RNA was extracted according to the protocol of RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). The sample is P.I. Samples of alba (about 5 cm plant height) green leaves sampled and frozen in liquid nitrogen were used.

[P. reverse transcription reaction using alba total RNA]
Total RNA 2.5 μg was subjected to reverse transcription reaction using SuperScript III RNase H ^- reverse transcriptase Kit (Invitrogen, Carlsbad, CA, USA).

Total RNA (2.5 μg) 12 μl (up with RNase free water)
50 μM oligo (dT) ₂₀ 1 μl
1 mM 10 mM dNTP Mix
14 μl total

  The above reaction mixture was heated at 65 ° C. for 5 minutes, then transferred to ice, and 5 × First-Strand Buffer 4 μl, 0.1 M DTT 1 μl, RNaseOUT ™ RNase inhibitor 1 μl, SuperScript ™ III reverse transfer at 60 ° C. for 60 minutes. Then, reverse transcription reaction was performed. At that time, the reaction solution was first warmed up in a hot water bath and transferred to an air incubator to prevent the reaction solution from evaporating. Thereafter, the enzyme was inactivated by heating in a heat block (70 ° C.) for 15 minutes. After returning to room temperature, 1 μl of RNase H was added to the reaction solution and incubated at 37 ° C. for 20 minutes to decompose the template RNA. It was confirmed that β-actin (beta-actin) as a positive control was amplified using the reverse transcription reaction product as a template.

[P. isolation of alba isoprene synthase (PaIspS) gene]
Using a primer pair (Fw.1, Rv.1) designed based on the hybrid poplar (P. tremula x P. alba) isoprene synthase gene sequence, the reverse transcription product as a template, and KOD-Plus-DNA RT-PCR was performed by polymerase (TOYOBO, Osaka, Japan).

Fw.1: 5'- ggggacaagtttgtacaaaaaagcaggcttc atggcaactgaattattgtgcttgc -3 '
Rv.1: 5'- ggggaccactttgtacaagaaagctgggtc ttatctctcaaagggtagaataggctctg -3 '
(Underlined sequence is GATEWAY system a for subcloning.
ttB site sequence)

10 x KOD plus buffer 5 μl
2 mM dNTP 5 μl
25 mM MgSO ₄ 2 μl
50 μM primer (Fw.1, Rv.1) 1 μl each for RT product 1 μl
1 U / μl KOD-Plus-DNA polymerase 1 μl
Measure up to 50 μl with H ₂ O

PCR program
# 1: 95 ° C 4min
# 2: 95 ° C for 1 min
53 ° C 30 sec
68 ℃ 2min
(30 cycles)
# 3: 68 ° C 5min
# 4: 4 ° C ∞

After confirming that a band of the desired size was amplified by agarose electrophoresis, GAT
After subcloning into pDONR221 by BPrecombination using EWAY ™ system (Invitrogen), the sequence was confirmed by sequencing. The resulting construct was designated pDONR221-PaIspS (Genbank accession number: AB198180) (hereinafter referred to as “PaIspS gene”).

[Preparation of expression vector]
Each expression vector was prepared by the following method.

[Production of L4fPaIS vector]
The PaIspS gene encoding the full-length protein including the chloroplast translocation sequence was inserted into the BamHI-PstI site of pCgLhcf4p plasmid to obtain an L4fPaIS vector. The pCgLhcf4p plasmid was obtained by replacing the NR promoter part of pCgNRp plasmid (GenBank accession number: AB981622) with the promoter sequence of the Fcp gene (Cg_lhcf4) (GenBank accession number: AB981630). The expression vector obtained above was designated as an L4fPaIS vector (SEQ ID NO: 1).

[Production of L4fP31PaIS vector]
A modified PaIspS gene encoding a fusion protein in which the original chloroplast translocation sequence of the PaIspS gene was replaced with a chloroplast translocation sequence of the diatom CgPsb31 gene (Genbank accession number: AB373996) was prepared by In_Fusion reaction, and pCgLhcf4p The L4fP31PaIS vector was obtained by inserting into the BamHI-PstI site of plasmid. The expression vector obtained above was designated as an L4fP31PaIS vector (SEQ ID NO: 3).

[Production of NRPOPaIS vector]
pCgNRp plasmid (GenBank accession number: AB981622) was modified to a chloroplast transition signal addition type. C. From the gracilis genomic DNA, the region encoding the chloroplast translocation sequence of the CgPsbO (Genbank accession number: AB373993) gene was amplified by PCR and inserted into the BamHI-XbaI site of the pCgNRp vector. As a result, the obtained plasmid was used as a pCgNRp / PsbOtp vector.
Next, the PaIspS gene (sequence excluding the region encoding the chloroplast translocation sequence) added with sequences necessary for cloning at both ends was amplified by PCR, and the AflII− designed in advance in the pCgNRp / PsbOtp vector. Inserted into the XbaI site by In_Fusion reaction. This was designated as an NRPOPaIS vector (SEQ ID NO: 5).

[Introduction of expression vector into diatom cells]
The introduction of the expression vector into diatom cells is described in “Ifuku K, Yan D, Miyahara M, Inoue-Kashino N. Yamamoto YY, Kashino Y. (2015) A stable and efficient nucleate. 123: 203-211. Doi: 10.1007 / s11120-014-0048-y. " Specifically, the following method was used.
First, diatoms are grown to the logarithmic growth phase (intended to be a state in which diatom cells are cultured at 23 ° C. in 10% IMK medium at about 1.8 × 10 ⁶ cells / ml). It was recovered using centrifugation (conditions: 7000 g, 4 minutes). The collected diatom cells were washed with 0.77 M mannitol (diluted with 10% IMK medium). After washing, the recovered diatom cells were resuspended in 0.15 mL of IMK medium, and 5 μg of linear plasmid DNA (the above “L4fPaIS vector”, “L4fP31PaIS vector”, and “NRPOPaIS vector” were replaced with Hind III). And the mixture was made into a linear form) to obtain a mixed solution. The above mixed solution was placed in an electroporation cuvette (gap 0.2 cm). Next, electroporation was performed on the mixed solution in the cuvette using a nepagene under the following conditions.

300 V (pulse width 5 msec; 9 pulses; interval 50 msec; 10% decrease rate), then transfer pulse 8 V (pulse length 50 msec; 40 pulses; interval 50 msec; 40% decrease rate).
The above operation was performed at room temperature. After electroporation, the diatom cells were transferred to 4 ml of IMK medium, and recovered with a medium not subjected to selection pressure (20 ° C., 16-20 hours, normal light irradiation).
Next, diatom cells were collected using centrifugation (conditions: 700 g, 4 minutes) and resuspended in 0.2 ml IMK medium. The transformed cells were selected in the presence of an IMK plate (1% agar, 400 μg / ml nurserytricin).

<Analysis of transgene expression status>
Colony PCR confirmed the presence of the transgene in drug resistant colonies. About some clones, after liquid culture, algae were collected by centrifugation, total RNA was isolated, and mRNA expression was confirmed by RT-PCR.

[Example 1]
Isoprene was produced by the following method using diatoms transformed with the L4fPaIS vector.

The composition of the medium used is as follows.
・ Sea salts 40 g (manufactured by Sigma-Aldrich)
-Daigo IMK medium 0.252 g (Wako)
・ Sodium silicate 56.2 μg (final concentration 0.2 mM)
The above was dissolved in distilled water to make 1 L.

Culture of the transformed diatom and recovery of isoprene were performed using the following apparatus (reactor). As a device, a device in which a pump, activated carbon, a flask with a capacity of 300 mL, a filter, and a collection tube were connected in this order by a tube was used.
The transformed diatom is cultured in a flask with a capacity of 300 mL by adding a medium to the transformed diatom pre-cultured with 5 mL of the above medium in a 300 mL volumetric flask at 25 ° C. in an atmospheric environment. It was.

The following were used for each part in the reactor.
・ Pump: non-noise S100 (manufactured by Artem)
・ Activated carbon: Organic substance removal tube (with activated carbon) (manufactured by GL Sciences Inc.)
・ Filter: LABODSCR disposable membrane filter unit

・ Collecting tube PerkinElmer's TurboMatrix stainless steel tube (outer diameter 6.35mm, length 89mm)
The collection tube was filled with weakly polar porous polymer beads (Tenax TA) based on 2.6-Diphenyl-p-phenylene Oxide and non-porous activated carbon (Carbopack B).

The analysis of the isoprene gas collected in the collection tube was performed using Perkin-Elmer ATD-400 and Shimadzu GCFID-GC17A. The analysis conditions are as follows.
Heat desorption (ATD-400 manufactured by Perkin-Elmer) Analysis conditions Desorption: 280 ° C., 12 mL He / min, 10 min
Cold Trap Temp: -10 ° C
GCFID (Shimadzu GCFID-GC17A) Analysis condition Column: Spelco SPB-1 (0.25 mm × 60 m, 1 um)
Carrier Gas: He, 2.0 mL / min
Split Ratio: 5: 1
Column Temp. : 35 ° C. (5 min maintenance), 5 ° C./min up to 200 ° C., 200 ° C. to 10 ° C./min 250 ° C. (10 min maintenance)

  From the above results, in the method for producing isoprene of Example 1, a maximum of about 1.2 ng of isoprene was produced per hour from 100 ml of diatom culture.

[Examples 2 and 3]
Isoprene was produced in the same manner as in Example 1 except that the L4fP31PaIS vector (Example 2) or the NRPOPaIS vector (Example 3) was used as an expression vector. As a result, 1.5 ng and 10 ng of isoprene were produced. The results were obtained in the same manner as in Example 1 except that it was recognized.

Claims (3)

A step A in which a liquid medium and an algae transformed with a polynucleotide encoding isoprene synthase are contained in a container;
Step B of culturing the algae in the liquid medium to obtain a culture solution;
A process C for collecting isoprene from the gas phase in the container, and a method for producing isoprene.   The method for producing isoprene according to claim 1, wherein the algae is diatom.   The method for producing isoprene according to claim 1 or 2, wherein the culture temperature in Step B is 10 to 40 ° C.