JP6907478B2 - Isoprene manufacturing method - Google Patents

Description

The present invention relates to a method for producing isoprene.

It is known that isoprene (2-methyl-1,3-butadiene), which is a raw material of polyisoprene rubber, can be extracted from the C5 distillate produced as a by-product when naphtha is thermally decomposed as an industrial production method. However, the supply of isoprene produced from the above-mentioned by-products is not always sufficient, and it is easily affected by fluctuations in the price of petroleum as a raw material. 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 the isoprene synthase gene into animal cells or plant cells is being promoted. I came.

Examples of the transformant include, in Patent Document 1, "(a) a polynucleotide encoding isoprene synthase, (b) a polynucleotide encoding 5-phosphomevalonate kinase, and (c) diphosphomevalonate de. A transformed plant that has been transformed with at least one selected from the group consisting of a polynucleotide encoding a carboxylase and has an increased productivity of a useful portion. ”As a specific example, isoprene synthase. The white dogs that have expressed the gene are described.

Japanese Unexamined Patent Publication No. 2008-035831

The present inventors attempted to produce isoprene using Arabidopsis thaliana expressing the isoprene synthase gene described in Patent Document 1, and the recovery of the produced isoprene was complicated and low efficiency. Was found. It was also found that the cultivation of Arabidopsis thaliana requires a predetermined volume and the isoprene yield per unit volume is poor.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for producing isoprene, which can easily recover the produced isoprene and has a high yield per unit volume.

As a result of diligent 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, and the present invention has been made. Was completed.

[1] A step A in which a liquid medium and algae transformed with a polynucleotide encoding isoprene synthase are housed in a container, and a step B in which the algae are cultured in the liquid medium to obtain a culture solution. A method for producing isoprene, which comprises the step C of collecting isoprene from the gas phase in the container.
[2] The method for producing isoprene according to [1], wherein the algae are diatoms.
[3] The method for producing isoprene according to [1] or [2], wherein the culture temperature in step B is 10 to 40 ° C.

According to the present invention, it is possible to provide a method for producing isoprene, which can easily recover the produced isoprene and has a high yield per unit volume.

Hereinafter, the present invention will be described in detail based on the embodiments.
It should be noted that the description of the constituent elements described below is based on the embodiment of the present invention, and the present invention is not limited to such an embodiment.
In the present specification, the numerical range represented by using "~" means a range including the numerical values before and after "~" as the lower limit value and the upper limit value.

[Manufacturing method of isoprene]
The method for producing isoprene according to the above embodiment is
-Step A: A step of accommodating a liquid medium and 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. -Step C: Contains a step of collecting isoprene from the gas phase in the container.

In the above 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 have higher culture efficiency per unit volume than terrestrial plants such as Arabidopsis thaliana, isoprene can be efficiently produced.
In addition, algae have a high ability to fix carbon dioxide gas in the atmosphere, and dimethylallyl pyrophosphate, which will be described later, can be efficiently produced by photosynthesis, so that isoprene can be efficiently produced.
Moreover, since the algae can be cultured in a closed container, the produced isoprene can be easily collected from the gas phase in the container.
It is presumed that the method for producing isoprene according to the above embodiment was able to obtain a desired effect due to the above synergistic effect.
Hereinafter, aspects of each step will be described.

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

<Liquid medium>
The liquid medium used in the step A is not particularly limited, and a known liquid medium can be used.
Examples of the liquid medium include a liquid medium containing seawater.
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 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 is not particularly limited, and the algae transformed by a known method can be used.

(Isoprene synthase)
As used herein, the isoprene synthase is an enzyme that is intended to be a protein having isoprene synthase activity and catalyzes a reaction in which dimethylallyl pyrophosphate (DMAPP) is oxidatively dephosphoricated.
Examples of the isoprene synthase include those derived from all species that produce isoprene synthase, and among them, those derived from higher plants or pterophyta plants are preferable, and hardwoods, ferns, and conifers are particularly preferable. Those derived from species with a large amount of isoprene released, such as, are preferable. In particular, Salixaceae (eg, poplar), Myrtaceae (eg, Eucalyptus), Fagaceae (eg, Quercus acutissima, Quercus aliena), Dryopteridaceae (eg, Oakida), etc., which release a large amount of isoprene, are preferable.
Specific examples of the isoprene synthase include isoprene synthase (Genbank accession number: BAD98243) of Poplar (Populus alba) of the Salix family.
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 about 98% or more homology 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 the isoprene synthase gene of poplar (Genbank accession number: AB198180) and the base sequence of the isoprene synthase gene of kudzu (Genbank accession number). : AY316691) and the like.
The polynucleotide encoding 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, still more preferably about 80% or more, more preferably about 96% or more, with the above base sequence. A polynucleotide having a base sequence having about 98% or more identity is included.

(Algae)
Examples of algae include diatoms, haptophytes, pingio algae, and algae belonging to the golden algae. Of these, algae belonging to the diatom net (hereinafter, simply referred to as "diatoms") are preferable. As diatoms, the genus Chaetoceros, the genus Phaeodactylum, the genus Cyclotella, the genus Skeletonema, the genus Odontella, and the genus Nitzschia are classified into the genus Nitzschia. Among them, the genus Chaetoceros is preferable, Chaetoceros is more preferable, and Chaetoceros glacilis is further 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 a known transformation method can be used.
Of these, a method of introducing the above-mentioned polynucleotide into an algae 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 algae cells, and examples thereof include a plasmid vector, a phage vector, and a cosmid vector. Of these, a plasmid vector is preferable.
In particular, those having a promoter at a position capable of autonomous replication or integration into a chromosome in a host and enabling transcription of a polynucleotide are preferably used. The 5'end of the polynucleotide amplified by PCR is linked in-frame to the 3'end of the promoter either directly or via a suitable sequence (eg, restriction enzyme recognition site). The expression vector may further contain a plant cell enhancer (eg, EL2 enhancer, CaMV35S enhancer, etc.), a selectable marker gene, a labeling tag, and the like. The stop codon is not always required, but is preferably located directly below the structural gene. In addition, the polynucleotide can be linked to an expression promoter, enhancer, start codon, etc. in-frame with reference to the amino acid sequence. 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 system for estimating the amino acid sequence based on the base sequence, an N-terminal analysis, a C-terminal analysis, or other conventional methods.

The promoter is not particularly limited as long as it promotes the expression of isoprene synthase in algae 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.

In addition, the above vector may contain other sequences contained in a general vector. For example, it may include so-called regulatory sequences such as an operator, a terminator, and an enhancer that regulate the expression of the isoprene synthase gene. Further, a promoter different from the above, a gene controlled by the promoter (for example, a drug regime gene) and the like may be included.

The vector can be prepared by a method well known to those skilled in the art. For example, it can be obtained by amplifying the promoter sequence with a primer provided with an appropriate restriction enzyme site and inserting it into a donor vector cleaved with a restriction enzyme. Alternatively, it can be prepared by a method using an "In-Fusion®" reaction.
The promoter sequence may be introduced into a donor vector as an expression cassette in which a terminator sequence is incorporated downstream thereof. The isoprene synthase gene may be integrated into the promoter sequence and the terminator sequence. In order to insert the isoprene synthase gene, it is preferable that the flow motor sequence and the terminator sequence have a multiple cloning site having a plurality of restriction enzyme cleavage sites.
The terminator sequence is not particularly limited as long as it functions in algae cells, and examples thereof include a terminator sequence of Cglhcr14 (Genbank accession number: AB981623): CgLhcr14ter.

The method for introducing the polynucleotide or the expression plasmid containing the polynucleotide into algae is not particularly limited, and a known method can be used.
Examples of the above-mentioned introduction method include lithium acetate method, electroporation method, Agrobacterium method, gene gun (particle gun) method, calcium phosphate method, protoplast method, Gene, 17, 107 (1982), Molecular & Genetic. Genetics, 168, 111 (1979) and Molecular Cloning 2nd ed. (Sambrook, J., Fritsch, EF, Maniatis, T. Cold Spring Laboratory Laboratory Press 1989), Photosynth Res. 123: 203-211. doi: 10.1007 / s11120-014-0048-y. , And the literature such as Plant Molecular Biological Manual (Stanton B. Gelvin and Robert A. Schilperort. Kruwer Academic Public Publics 1988), etc. are preferably used in the literature (these are preferred in this document, etc.). can.
In addition, individuals into which the expression plasmid has been introduced are usually selected. The individual into which the expression plasmid has been introduced can be selected by a conventional method, for example, selection by a drug-containing medium, selection by a hormone-containing medium, or selection by auxotrophy. Furthermore, the integration of the above polynucleotide into the plant cell chromosome can be confirmed by using conventional methods such as genome PCR, RT-PCR, Northern hybridization, and Western analysis.

Here, transformation generally refers to a genetic phenomenon in which DNA isolated from one cell is taken up by another cell and recombines with a cell chromosome (Tokyo Chemical Dominant Biochemistry Dictionary, 2nd Edition,). 416-417), which means that DNA molecules including plasmids and genes bound to them are directly introduced into cells (Iwanami Biology Dictionary, 4th edition, 380-381). As used herein, transformation means introducing a polynucleotide (eg, DNA, mRNA) into a cell, with or without the polynucleotide undergoing recombination with a cell chromosome. .. In order to stably express the phenotype and genotype, it is desirable that the oligonucleotide undergoes recombination with a cell chromosome, and further, it is desirable that the recombination with the cell chromosome is homozygous.

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

[Step 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 culturing 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 in which algae are added to a medium and culture is performed while controlling the culture conditions. In the batch culture method, the algae go from a mild induction phase to a logarithmic growth phase and finally to a stationary phase in which the growth rate decreases or stops. Isoprenes are often produced by transformants in the logarithmic and / or stationary phase.

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

The continuous culture method is a culture method in which a predetermined amount of 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 medium are mainly in the logarithmic growth phase.
By replacing a part or all of the medium as appropriate, nutrients can be supplemented, and the accumulation of metabolic by-products and dead cells that may adversely affect the growth of algae can be prevented.

The culture conditions for algae are not particularly limited as long as the isoprene synthase gene can be expressed, and standard cell culture conditions can be used. The culture temperature is preferably, for example, 10 to 40 ° C, more preferably 20 to 25 ° C.
As for the gas composition, the CO ₂ concentration is preferably 0.04 to 10%, and the pH is preferably 4 to 10.
Among them, when diatoms are used as algae, the culture temperature is preferably 15 to 35 ° C, more preferably 20 to 25 ° C. The gas composition _{preferably has a CO 2} concentration of 0.04 to 5% and a pH of 7 to 9. By culturing the transformed diatom at the above culture temperature, the expression of the isoprene synthase gene is further promoted.

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

According to the above-mentioned method for producing isoprene, isoprene can be efficiently produced from dimethylallyl diphosphate (DMAPP). In the above method for producing isoprene, 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 the algae can be collected. DMAPP, which is a substrate of isoprene synthase, is synthesized by the mevalonate pathway or the methylerythritol phosphate pathway in the host based on the carbon source in the medium.

Since algae (among others, diatoms) _{have high light-collecting ability and CO 2} fixing ability, the above-mentioned DMAPP can be efficiently produced, so that the isoprene production efficiency is increased.
Further, since algae can be cultivated in a large amount in a container as described above, the amount of isoprene produced per unit volume is higher than that in the case of using Arabidopsis thaliana or the like.
Further, since the algae can be cultured in the closed system container as described above, the produced isoprene can be easily recovered from the head space of the closed system 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. Moreover, the above-mentioned method for producing isoprene has a high yield per unit volume.

The present invention will be described in more detail below based on examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be appropriately changed as long as they do not deviate from the gist of the present invention. Therefore, the scope of the present invention should not be construed as limiting by the examples shown below.

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

[Extraction of total RNA from Populus alba]
Extraction of Total RNA was performed according to the protocol of RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). The sample is P.I. 130 mg of alba (about 5 cm plant height) green leaves were sampled and frozen in liquid nitrogen.

[P. Reverse transcription reaction using alba total RNA]
2.5 μg of Total RNA ^{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 (volumetric flask with RNase free water)
50 μM oligo (dT) ₂₀ 1 μl
10 mM dNTP Mix 1 μl
14 μl in total

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

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

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

10 x KOD plus buffer 5 μl
2 mM dNTP 5 μl
25 mM sulfonyl ₄ 2 μl
50 μM primer (Fw.1, Rv.1) 1 μl each RT product 1 μl
1 U / μl KOD-Plus-DNA polymerase 1 μl
Female up the above-mentioned up to 50μl 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 ℃ 5min
# 4: 4 ℃ ∞

After confirming that a band of the desired size was amplified by agarose gel electrophoresis, GAT
After subcloning to pDONR221 by BPrecombination using the EWAY TM system (Invitrogen), the sequence was confirmed by sequence. The obtained construct was designated as 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.

[Preparation 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 portion of the pCgNRp plasmid (GenBank accession number: AB981622) with the promoter sequence of the Fcp gene (Cg_lhcf4) (GenBank accession number: AB981630). The expression vector obtained as described above was designated as an L4fPaIS vector (SEQ ID NO: 1).

[Preparation of L4fP31PaIS vector]
A modified PaIspS gene encoding a fusion protein in which the original chloroplast transfer sequence of the PaIspS gene was replaced with the chloroplast transfer sequence of the diatom CgPsb31 gene (Genbank accession number: AB373992) was prepared by the In_Fusion reaction, and pCgLhcf4p. The L4fP31PaIS vector was obtained by inserting into the BamHI-PstI site of plasmid. The expression vector obtained as described above was designated as an L4fP31PaIS vector (SEQ ID NO: 3).

[Preparation of NRPOPAIS vector]
The pCgNRp plasmid (GenBank accession number: AB981622) was modified to add a chloroplast transfer signal. 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 / PsbOptp vector.
Next, the PaIspS gene (the sequence excluding the region encoding the chloroplast translocation sequence) to which the sequence required for cloning was added to both ends was amplified by PCR, and AflII-designed in advance in the pCgNRp / PsbOtp vector was added. It was inserted into the XbaI site by the In_Fusion reaction. This was designated as the NRPOPAIS vector (SEQ ID NO: 5).

[Introduction of expression vector into diatom cells]
Introduction into the diatom cell of the expression vector, "Ifuku K, Yan D, Miyahara M, Inoue-Kashino N, Yamamoto YY, Kashino Y. (2015) A stable and efficient nuclear transformation system for the diatom Chaetoceros gracilis. Photosynth Res. 123: 203-211. Doi: 10.1007 / s11120-014-0048-y. ”. Specifically, it was carried out by the following method.
First, the diatomaceous logarithmic growth phase (diatom cells were cultured at 23 ° C. in 10% IMK medium, intended state was about ^{1.8 × 10 6 cell / ml.} ) Grown to the proliferated diatom cells Recovered using centrifugation (conditions: 7000 g, 4 minutes). The recovered diatom cells were washed with 0.77 M mannitol (diluted with 10% IMK medium). After washing, the collected diatom cells were resuspended in 0.15 mL of IMK medium, and then 5 μg of linear plasmid DNA (the above-mentioned “L4fPaIS vector”, “L4fP31PaIS vector”, and “NRPOPaIS vector” was added to HindIII. A mixed solution was obtained by mixing with the one cut in the above to make it linear. The above mixture was placed in a cuvette for electroporation (gap 0.2 cm). Next, the mixed solution contained in the cuvette was electroporated under the following conditions using Neppagene.

300V (pulse width 5msec; 9 pulses; interval 50msec; 10% reduction rate), then transfer pulse 8V (pulse length 50msec; 40 pulses; interval 50msec; 40% reduction rate).
The above operation was performed at room temperature. After electroporation, diatom cells were transferred to 4 ml IMK medium and recovered in medium without selection pressure (20 ° C., 16-20 hours, normal light irradiation).
The diatom cells were then collected by centrifugation (conditions: 700 g, 4 minutes) and resuspended in 0.2 ml IMK medium. Transformed cells were selected in the presence of IMK plates (1% agar, 400 μg / ml noursetricin).

<Analysis of transgene expression status>
Colony PCR confirmed the presence of the transgene in drug-resistant colonies. After liquid culture of some clones, algae were collected by centrifugation to isolate total RNA, and the expression of mRNA 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 (manufactured by Wako)
-Sodium silicate 56.2 μg (final concentration 0.2 mM)
The above was dissolved in distilled water to make 1 L.

The transformed diatom was cultured and isoprene was recovered using the following device (reactor). As the device, a device in which a pump, activated carbon, a flask having a capacity of 300 mL, a filter, and a collecting tube were connected by tubes in this order was used.
The transformed diatom is cultured in an air environment at a temperature of 25 ° C. by adding a medium to the transformed diatom precultured in 5 mL of the above medium to make 100 mL, and storing the transformed diatom in a flask having a capacity of 300 mL. rice field.

The following parts were used in the reactor.
-Pump: non-noise S100 (manufactured by Artem Co., Ltd.)
・ Activated carbon: Organic matter removal pipe (with activated carbon) (manufactured by GL Sciences)
-Filter: LABODISCR Disposable Membrane Filter Unit

-Collection tube PerkinElmer's stainless steel tube for TurboMatrix (outer diameter 6.35 mm, length 89 mm)
The collection tube is 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 isoprene gas collected in the collection tube was analyzed using ATD-400 manufactured by Perkin-Elmer and GCFID-GC17A manufactured by Shimadzu Corporation. The analysis conditions are as follows.
-Heat desorption (ATD-400 manufactured by Perkin-Elmer) Analytical conditions Resolution: 280 ° C, 12 mL He / min, 10 min
Cold Trap Temp: -10 ° C
-GCFID (GCFID-GC17A manufactured by Shimadzu Corporation) Analytical conditions Volume: Spellco SPB-1 (0.25 mm x 60 m, 1 um)
Carrier Gas: He, 2.0 mL / min
Spirit Ratio: 5: 1
Volume Temp. : 35 ° C (maintain 5 min), 5 ° C / min up to 200 ° C, 200 ° C to 10 ° C / min 250 ° C (maintain 10 min)

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 the diatom culture solution.

[Examples 2 and 3]
When 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 the expression vector, 1.5 ng and 10 ng of isoprene were produced, respectively. Results were obtained in the same manner as in Example 1 except that it was observed.

Claims (2)

Step A in which the liquid medium and the diatom transformed with the polynucleotide encoding isoprene synthase are contained in a container.
Step B of culturing the diatom in the liquid medium to obtain a culture solution,
The step C of collecting isoprene from the gas phase in the container is included .
A method for producing isoprene _{, wherein the CO 2} concentration is 0.04 to 5% of the gas composition in the step B. The method for producing isoprene according to claim 1, wherein the culture temperature in the step B is 10 to 40 ° C.