JPH1057068A - Pyruvate decarboxylase gene, recombinant vector and plasmid containing the same gene, cell of microalgae containing the same gene transduced thereinto and production of ethanol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a pyruvate decarboxylase gene capable of improving the conversion rate of intracellular accumulated polysaccharides in a microalga of the genus Chlamydomonas into ethanol by coding a specific amino acid sequence. SOLUTION: This pyruvate decarboxylase gene is capable of coding an amino acid sequence represented bpv the formula. Furthermore, ethanol is preferably produced by including the pyruvate decarboxylase gene in a host cell, providing a recombinant vector, transducing the resultant recombinant vector into a cell of microalgae, culturing the prepared recombinant cell of the microalga in a culture medium and producing ethanol from accumulated polysaccharides by metabolic reaction in the cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel pyruvate decarboxylase gene, a vector for introducing the pyruvate decarboxylase gene into microalgae, a transformant plasmid, a microalgal cell into which the gene has been introduced, and The present invention relates to a method for producing ethanol using microalgal cells.

[0002]

2. Description of the Related Art Conventionally, ethanol is produced by using fossil resources such as coal and petroleum as raw materials and chemically synthesizing via ethylene, or by using biomass resources such as sugar of sugarcane and starch of corn as raw materials to mold, yeast, etc. It is manufactured by a fermentation method using microorganisms.

[0003] Among biomass raw materials, microalgae, which are fine photosynthetic organisms represented by chlorella, donariella, chlamydomonas, scenedesmus, spirulina, etc., include:
It is known to contain a large amount (50% or more by dry weight) of polysaccharides such as starch and glycogen, which are raw materials for alcohol. Using these microalgae polysaccharides as raw materials, ethanol production by fermentation using microorganisms such as mold and yeast is known. However, extraction of polysaccharides from microalgae, microbial treatment, and production of polysaccharides by saccharifying enzymes are known. A process such as decomposition is required.

On the other hand, the present inventors do not need to extract polysaccharides from microalgae cells, and do not need to treat microorganisms or decompose polysaccharides by saccharifying enzymes or the like. In addition, the following method for efficiently producing ethanol by a simple process is disclosed in Japanese Patent Application No. Hei.
No. 239,845. (1) Microalgae are grown photoautotrophically by carbon assimilation by photosynthesis in the light, or heterotrophically grown in the dark by providing organic substances such as sugars and organic acids. (2) The proliferated microalgae is obtained by concentrating a culture solution containing algal cells storing polysaccharides in the cells, and keeping a slurry obtained in a dark and anaerobic atmosphere to produce ethanol from the microalgal cells.

A similar technique is disclosed in US Pat.
No. 70,175 describes a method for producing a soluble metabolite such as ethanol from a cultured large adherent algae. Here, as a means for improving ethanol productivity from a large adherent algae, yeast has a method. In order to introduce a known ethanol production pathway into algae, a yeast-derived pyruvate decarboxylase (hereinafter sometimes abbreviated as "PDC") gene and an alcohol dehydrogenase (hereinafter referred to as "AD
H) (in some cases, abbreviated as "H").

[0006]

The above-mentioned conventional method has the following problems. First, Japanese Patent Application No. 5--2398
In the method proposed in the specification of No. 45, the conversion rate of polysaccharides stored in cells to ethanol is only 40%, so that the energy cost per unit ethanol is large and the amount of waste is large. Further, in the method proposed in the above U.S. Patent Specification, in microalgae, the gene structure such as the usage of amino acid codons encoded by the genes is significantly different from existing yeast-derived genes. Even if it is introduced, it cannot function effectively in microalgae cells and cannot be used for process improvement.

[0007] Due to these problems, microalgae polysaccharides have many advantages over agricultural biomass such as sugarcane and corn, but have not yet been put to practical use on a large scale. An object of the present invention is to solve the above-mentioned problems of the prior art and to provide a method for efficiently producing ethanol from microalgae that produces ethanol at a high conversion rate from polysaccharides accumulated by photosynthesis.

[0008]

Means for Solving the Problems As means for solving the above problems, the present invention provides (1) a pyruvate decarboxylase gene encoding an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing; The pyruvate decarboxylase gene according to the above (1), which has a DNA having a nucleotide sequence represented by SEQ ID NO: 2 in the Sequence Listing. The present invention also includes (3) a host cell containing a DNA encoding the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing for improving pyruvate decarboxylase activity, and having a function of regulating the expression of the DNA. DN
(4) The recombinant vector according to (3), wherein the DNA encoding the amino acid sequence represented by SEQ ID NO: 1 is the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. (5) wherein the DNA having a function of regulating the expression of the DNA is a double-stranded DNA obtained by annealing the orionucleotides of SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing. 4) or (5) 5
A recombinant plasmid comprising the DNA of SEQ ID NO: 2 encoding the pyruvate decarboxylase gene and (6) the downstream of the promoter of the α-tubulin gene derived from Chlamydomonas;
A fragment obtained by double cutting RF3600 with restriction enzymes BamHI and SplI, plasmid pCRF4100 was converted to restriction enzymes NcoI and SplI.
Fragment and the plasmid pCRF5100 double-cleaved with the restriction enzymes BamHI and NcoI were subjected to T4 DN
A plasmid pCRPDC1 obtained from a transformant FERM P-1571, which was ligated with A ligase and introduced into E. coli strain JM109, is provided. Further, the present invention provides (7) a microalgal cell as a host cell,
(6) a recombinant microalgal cell having improved pyruvate decarboxylase activity, which is introduced by the recombinant vector according to (6), and (8) the host cell is an algal cell belonging to the genus Chlamydomonas. Or a recombinant microalgal cell according to the above (7). Still further, the present invention provides
(9) D encoding an amino acid represented by SEQ ID NO: 1 in the sequence listing for improving pyruvate decarboxylase activity in host cells
A recombinant microalgae cell containing NA and having an improved pyruvate decarboxylase activity transfected with a recombinant vector containing a DNA having a function of regulating the expression of the DNA is cultured in a medium, and the cells are cultured in a medium. (10) a method for producing ethanol by producing ethanol by a metabolic reaction in the cell, comprising (10) a DNA encoding an amino acid represented by SEQ ID NO: 1 in the sequence listing for improving pyruvate decarboxylase activity in a host cell; And culturing a recombinant microalgal cell having improved pyruvate decarboxylase activity transfected with a recombinant vector containing a DNA having a function of regulating the expression of the DNA in a medium, and metabolizing the cell in the cell. (11) A method for producing ethanol, which comprises producing ethanol from polysaccharides accumulated by the reaction. The method for producing ethanol according to the above (9) or (10), wherein the DNA encoding nonoic acid is represented by SEQ ID NO: 2, (12) the recombinant FERM P- The method for producing ethanol according to the above (10) or (11), which is a plasmid pCRPDC1 obtained from 15791.
And (13) The method for producing ethanol according to any of (10) to (12) above, wherein the host cell is an algal cell belonging to the genus Chlamydomonas.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention focuses on the metabolic pathway in microalgal cells, and based on the fact that a novel PDC gene that functions in microalgal cells has been identified, a vector containing the PDC gene has been developed. By using a transformed microalga possessed or having the PDC gene integrated into the chromosome, a method for producing ethanol with high yield has been realized.

[0010] The present inventors have studied the stages of the metabolic pathway of polysaccharides in microalgae cells. In microalgae, especially Chlamydomonas, after polysaccharides are degraded to pyruvate by a glycolytic system, pyruvate It was confirmed that the pathway of conversion to acetaldehyde by the action of acid decarboxylase (PDC) and conversion to ethanol by alcohol dehydrogenase (ADH) is the main pathway of ethanol production. Furthermore, in this pathway, it was found that the process of converting pyruvic acid to acetaldehyde was rate-limiting.

By the way, as shown in FIG. 7, in cells, pyruvic acid is converted to lactate by lactate dehydrogenase (LDH) in addition to metabolism to acetaldehyde by PDC.
Acetyl Co by pyruvate formate lyase (PFL)
It is metabolized to A. For this reason, when the metabolism of pyruvate to acetaldehyde by PDC is rate-limiting, as in conventional Chlamydomonas cells, a considerable amount of pyruvate is produced in LD.
It was found that H and PFL metabolized lactic acid and acetyl-CoA, which reduced the conversion rate of polysaccharides to ethanol.

Therefore, the amount of PDC in cells is increased,
We believe that enhancing the pathway of metabolizing pyruvic acid to acetaldehyde could improve the conversion efficiency of polysaccharides to ethanol, and as a result of intensive research efforts, we found that the microalga Chlamydomonas reinhardy Chlamydomonas
A PDC gene could be found from DNA from reinhardii. In addition, the PDC gene was successfully cloned. Furthermore, a transformed microalga into which this gene was introduced was prepared, and the alcohol conversion rate was successfully improved. Thus, the present invention was completed.

[0013] The term "DNA encoding pyruvate decarboxylase" in the present invention is composed of a DNA chain of a unit capable of expressing the PDC gene when the DNA is incorporated into an appropriate expression vector, and has substantially the same size. The concept encompasses all that encode the enzyme active substance. Specific examples include all DNA sequences encoding the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, and encoding such an amino acid sequence;
As long as the protein produced by the expression exhibits the PDC activity, it also includes those encoding additional amino acids together (for example, as a fusion protein). Specific examples of such a DNA sequence include a DNA sequence represented by SEQ ID NO: 2 in the sequence listing.

One embodiment of the gene of the present invention comprises a sequence from Met at amino acid number 1 to His at amino acid number 580 of SEQ ID NO: 1.
Up to amino acids. However, the Met at position 1 corresponding to the translation initiation codon may be removed during post-expression processing, and is not present when the target enzyme is expressed as a fusion protein with another peptide. Therefore, another embodiment of the gene of the present invention encodes a protein comprising an amino acid sequence from Ala of an amino acid number 2 of SEQ ID NO: 1 to His of amino acid number 580.

One specific example of this gene contains a nucleotide sequence from nucleotide number 1 or 4 to nucleotide number 1740 shown as SEQ ID NO: 2 in the sequence listing. In general, enzymes that have the same type of activity from a homologous or homologous microorganism, but that have slight differences in amino acid sequence due to, for example, spontaneous mutation, for example, nucleotide substitution, addition and / or deletion, are present. It is known. However, according to the method described herein, even a gene encoding an enzyme having an amino acid sequence that is not completely the same as the amino acid sequence described in SEQ ID NO: 1 can be cloned.
Such an enzyme has very high homology to the amino acid sequence shown in SEQ ID NO: 1, for example, 95% or more, and even 98%.
% Of homology. Therefore, SEQ ID NO: 1
Not only the gene encoding pyruvate decarboxylase having the amino acid sequence of SEQ ID NO: 1 but also 95% or more, for example 98%, of the amino acid sequence of SEQ ID NO: 1 derived from a microorganism belonging to Chlamydomonas reinhardy The gene encoding pyruvate decarboxylase having the amino acid sequence having the above homology also belongs to the present invention.

In eukaryotes including microalgae,
There may be a DNA sequence called an intron. In this case, the DNA sequence encoding the amino acid is not a continuous sequence but is divided into a plurality of frames by an intron sequence. Therefore, the pyruvate decarboxylase-encoding gene having the amino acid sequence shown in SEQ ID NO: 1 is not a continuous DNA sequence but contains one or more intron sequences between a series of gene sequences. Genes encoding carbonic enzymes also belong to the invention.

In one enzyme, a specific amino acid sequence is not necessarily essential in a region other than a so-called active center or active region and a portion which supports the active region three-dimensionally. It is known that modifications such as substitution, deletion and / or addition of one or a few amino acids can be made while maintaining the activity. The number of amino acids that can be modified as described above is within a range that can be easily modified by common gene modification techniques, for example, site-directed mutagenesis using a mutagenic probe,
For example, about 20 or less amino acids. Furthermore, substitution, deletion, and / or addition of an amino acid sequence can also be performed using a restriction enzyme and / or a ligation enzyme.

Accordingly, the present invention relates to a pyruvate deprotection having an amino acid sequence modified by substitution, deletion and / or addition of 20 or less, for example, 10 or less amino acids with respect to the amino acid sequence shown in SEQ ID NO: 1. The present invention relates to a gene encoding a carbonic enzyme. The DNA of the present invention can be prepared from Chlamydomonas reinhardy, which is a known microalgal strain available from various microorganism depositors, by a method known per se as described in detail below.

Hereinafter, the present invention will be described in detail. Examples of the microalgal strain used in the present invention include, for example, Chlamydomonas
Reinhardy CW15nit1-305 (Chlamydo
monas reinhardii CW15nit1-305) [Kallen Kindle (Section of Biochemistry, Molecular and Cell Biology and Plant Science Center, Cornell University)
(Karen L. Kindle (Section of Biochemistry, Molecular
and Cell Biology and Plant Science Center, Cornel
l University)).

The PDC gene of the present invention can be obtained by the following steps. (1) Extract DNA from Chlamydomonas reinhardy. (2) DN of known PDC gene containing DNA sequence region conserved in known PDC genes of various organisms
The fragment A is used as a probe for Southern hybridization. (3) cutting the DNA obtained in (1) with an appropriate restriction enzyme,
On the other hand, Southern hybridization is performed using the probe obtained in (2). (4) The DNA fragment of (1) is cleaved with a restriction enzyme having an appropriate positive signal obtained in (3), and a fragment having a chain length corresponding to the positive signal is recovered by electrophoresis and ligated to a vector. The gene is introduced into a fungus to obtain a Chlamydomonas gene library. (5) The library obtained in (4) is subjected to colony hybridization using the probe obtained in (2), and a host colony in which a positive signal is obtained is separated.
The NA is recovered, and the sequence of the Chlamydomonas-derived DNA bound to the vector is read. (6) The DNA sequence obtained in (5) and the encoded amino acid sequence are compared with the DNA sequence of a known PDC gene and the amino acid sequence of PDC, and the Chlamydomonas-derived PDC gene contained in the sequence of (5) is compared. Clarify the sequence part of the structural region. (7) Using a DNA fragment containing only the sequence of the Chlamydomonas-derived PDC gene structural region determined from the results of (6) as a probe, Southern hybridization was performed on the DNA obtained by cutting the DNA of (1) with an appropriate restriction enzyme. Then, colonies of host bacteria that gave a positive signal were separated,
The NA is recovered, and the sequence of the Chlamydomonas-derived DNA fragment bound to the vector is read. (8) Repeat (6) to (7) to obtain a group of DNA fragments covering the entire region encoding PDC. (9) Based on the results of (8), the DNA of (1) is cut with a restriction enzyme capable of obtaining a fragment containing the entire region encoding PDC, and ligated to a vector. The vector-ligated DNA fragment is introduced into a host bacterium, a transformant containing the target DNA fragment is selected, and a fragment containing the entire region encoding PDC is recovered. (10) As an alternative to (9), the fragments obtained in (8) may be cleaved with appropriate restriction enzymes and ligated to construct and obtain a fragment containing the entire region encoding PDC. it can. (11) The DNA fragment obtained in (9) or (10) or a DNA fragment obtained by ligating a DNA having a function of regulating expression to the fragment is introduced into Chlamydomonas, the transformant is cultured, and the intracellular PDC is cultured. Confirm the improvement of activity.

[0021]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples. [1] Chlamydomonas reinhardy chromosome DNA
Preparation: Chlamydomonas reinhardy CW15n
it1-305 (Chlamydomonasreinhardii CW15nit1-3
05) at 25 ° C under fluorescent lighting (2000-4000lu)
In x), shaking culture was performed in the SGII culture medium shown in Table 1.
8 m of algal cells obtained from 750 ml of the culture solution that has reached a steady state
1 A buffer (0.1 M NaCl, 50 mM EDTA, 20 mM Tris-HCl,
pH 8.0) and 100 μl of proteinase K solution
(10 mg / ml), and after mixing, 0.5 ml of 20% S
The DS solution was added, and the mixture was left at 50 ° C. for 45 minutes. 10 more
After adding 0 μl of proteinase K solution and left at 50 ° C. for 45 minutes, 1000 μl of proteinase K solution was added and left at 50 ° C. for 50 minutes. After phenol extraction, ethanol precipitation was performed to obtain a dry crude purified DNA. 2. DNA was treated with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
After suspending to 8 ml and dissolving 4.14 g of cesium chloride, 0.5 ml of ethidium bromide solution (700 μl) was dissolved.
g / ml) and mixed. Chromosomal DNA was separated by parallel density gradient fractionation using an ultracentrifuge, and ethidium bromide was removed by extraction with sodium chloride saturated isopropyl alcohol, followed by ethanol precipitation to recover DNA. By the above operation, about 20 μg of DNA was obtained.

[0022]

[Table 1]

[2] Preparation of probe using known PDC sequence: rice-derived PDC gene fragment sequence (RICS77 [D1
[0413] Based on the above, an oligonucleotide of SEQ ID NO: 3 in the sequence listing was synthesized. Also, an oligonucleotide of SEQ ID NO: 4 that anneals to the 3 'end of the oligonucleotide of SEQ ID NO: 3 was synthesized. 200 pmol of the oligonucleotide of SEQ ID NO: 3
Is used as a template, and oligonucleotide 200p of SEQ ID NO: 4 is used.
Using the mol as a primer, a digoxigenin-labeled probe, Probe 1, was obtained with a DIG DNA labeling kit (Boehringer Mannheim).

As the template DNA, in addition to synthetic oligonucleotides, those obtained by fragmenting PDC gene DNA derived from plants such as rice or maize, or microorganisms such as yeast or Zymomonas with restriction enzymes can be used. It is possible. The labeling method also depends on the positive signal detection method used in the subsequent Southern hybridization, in addition to digoxigenin, biotin, or
It is possible to use a fluorescent dye or a radioisotope. These hybridization methods are described in Moleculer cloning (J. Sambrook. E, F. Fritsch, T .;
Maniatis's Cold Spring Harbor Laboratory Press) and reagent manufacturers' catalogs.

[3] Search for PDC gene by genomic southern hybridization: 2 µg of the chromosomal DNA prepared in the above [1] was digested with a restriction enzyme EcoRV and subjected to electrophoresis. Then DN on agarose gel
A was transferred to a nylon membrane, and Southern hybridization was performed using the probe 1 prepared in the above [2]. Hybridization conditions are 50% formamide
5 × SSC, 0.1% lauroyl sarcosine, 0.02
Performed at 42 ° C. for 12 hours in% SDS, 2% blocking reagent (Boehringer Mannheim). Using a Nucleic Acid Detection Kit (Boehringer Mannheim), DNA hybridized with Probe 1 was detected. As a result, a positive signal was detected at a position of about 4.1 kb in length.

[4] Cloning of DNA fragment containing PDC gene part: chromosomal DNA prepared in [1] above
5 μg was digested with restriction enzyme EcoRV, electrophoresed, and DNA
DNA having a chain length of about 4.1 kb was recovered from the agarose gel. Plasmid pUC18 was digested with restriction enzyme SmaI, treated with alkaline phosphatase, prepared, mixed with the recovered fragment, ligated with T4 DNA ligase, and ligated with circular DNA.
I got From the transformant population obtained by introducing this DNA into E. coli SURE2 (trade name, manufactured by Stratagene) strain, using the probe 1 prepared in Example [2],
Colony hybridization was performed to recover the target transformant. The plasmid was recovered from the selected transformant and named pCRF4100. The DNA sequence is read and the amino acid sequence of the known PDC and the PDC gene DN
By comparing the homology with the A sequence, it was confirmed that a part of the region encoding the amino acid sequence of PDC and a sequence considered to be an intron region were present. FIG. 1 shows a DN according to the present invention.
FIG. 4 shows a plasmid map of plasmid pCRF4100 containing a portion of the A sequence.

[5] Preparation of a probe using the sequence of the Chlamydomonas-derived PDC gene: Based on the DNA sequence encoding the amino acid sequence of Chlamydomonas-derived PDC identified in the above [4], SEQ ID NO: 5 and the sequence Number 6
Were synthesized. Subsequently, an oligonucleotide of SEQ ID NO: 7 that anneals to the 3 'end of the oligonucleotide of SEQ ID NO: 5 and an oligonucleotide of SEQ ID NO: 8 that anneals to the 3' end of the oligonucleotide of SEQ ID NO: 6 were synthesized. DIG DN
A labeling kit (Boehringer Mannheim)
200 pmo of the oligonucleotide of SEQ ID NO: 5
1 as a template, oligonucleotide 200 pm of SEQ ID NO: 7
ol as a primer, digoxigenin-labeled probe of probe 2, oligonucleotide 200p of SEQ ID NO: 6
mol as a template, oligonucleotide 200 of SEQ ID NO: 8
Using pmol as a primer, a digoxigenin-labeled probe of probe 3 was obtained.

[6] Cloning of a fragment group covering the entire sequence of the PDC gene: 2 μg of the chromosomal DNA prepared in the above [1] is digested with the restriction enzyme NdeI, subjected to electrophoresis, and then the DNA on the agarose gel is subjected to electrophoresis. Transferred to nylon membrane. Two of these were prepared, and Southern hybridization was performed using the probe 2 or the probe 3 prepared in the above [5]. Hybridization conditions were 50% formamide, 5 × SSC, 0.1%
% Lauroyl sarcosine, 0.02% SDS, 2% blocking reagent (Boehringer Mannheim) at 42 ° C. for 6 hours. Nucleic Acid
Detection kit (Boehringer Mannheim)
Was used to detect DNA hybridized with probe 2 or probe 3. As a result, by hybridization with probe 2, a position having a chain length of about 3.4 kb,
And a positive signal was detected at a position of a chain length of about 5.1 kb.

Therefore, the chromosomal DNA prepared in the above [1]
5 μg was digested with restriction enzyme NdeI, electrophoresed, and chain length was 3.
DNA around 4 kb and around 5.1 kb were recovered from agarose gel. Plasmid pUC18 was digested with NdeI, treated with alkaline phosphatase, prepared, mixed with each recovered fragment, and ligated with T4 DNA ligase to obtain a circular DNA. From a population of transformants obtained by introducing this DNA into Escherichia coli SURE2 (trade name, manufactured by Stratagene) strain, the target transformant was obtained by colony hybridization using probe 2 and probe 3, respectively. Collected. The plasmid was recovered from the transformant detected by the probe 2, and named pCRF3400. The plasmid was recovered from the transformant detected by the probe 3, and named pCRF5100.

The DNA sequences of pCRF3400 and pCRF5100 were read, and a part of each was read from plasmid pCRF3.
It was confirmed to overlap with 4100 sequences. FIG. 2 shows a plasmid map of pCRF3400 containing a part of the DNA sequence of the present invention, and FIG. 3 shows a plasmid map of pCRF3400 containing a part of the DNA sequence of the present invention.
Fig. 4 shows a plasmid map of CRF5100. The overlapping sequence portions were overlapped and ligated to obtain a sequence of about 8.8 kb. This sequence, the amino acid sequence of the known PDC and the PDC gene D
As a result of comparison of the homology with the NA sequence, about 8.8 kb of the present sequence, the sequence of SEQ ID NO: 2 representing the region encoding the amino acid sequence of Chlamydomonas-derived PDC shown in SEQ ID NO: 1 and separated by the intron sequence, It was confirmed that the DNA sequence was contained.

[7] DNA having a function of regulating expression
Based on the promoter region sequence (CRTBA1G [K01805]) of the α-tubulin gene derived from Chlamydomonas, SEQ ID NO: 9 in the sequence listing
Were synthesized. In addition, an oligonucleotide of SEQ ID NO: 10, which is a complementary strand of SEQ ID NO: 9, was synthesized. Both were annealed to obtain a double-stranded DNA whose expression was effectively regulated in Chlamydomonas cells. The ends of the double-stranded DNA were ligated with the plasmid pC obtained in [6] above.
It has an overhang at the 3 'end of 3 bases "G, C, G", which can be ligated to a fragment obtained by cutting RF3400 with the restriction enzyme DraIII.

[8] DNA having a function of regulating expression
And the PDC gene portion: the plasmid pCRF3400 obtained in the above [6] is cut with the restriction enzyme DraIII, the DNA having the expression regulating function obtained in the above [7] and T4DN
The DNA was ligated with A ligase to obtain a circular DNA. The restriction enzyme Dra III cleavage point disappears due to the ligation of both, but when the plasmid pCRF3400 self-closes, the Dra III cleavage point is maintained.
Plasmid pCRF3400 is self-cyclized and converted to linear DN
A was used to reduce the transformation efficiency, and the plasmid to which the expression control DNA was ligated was transformed preferentially. Several strains were isolated from a transformant population obtained by introducing this DNA sample into Escherichia coli strain JM109, and plasmid DNA was recovered. Read the sequence of each DNA, and connect the expression control region to the target plasmid DN.
A was selected and named pCRP3600. FIG. 4 shows the PDC gene expression control sequence and the DN of the PDC gene according to the present invention.
Figure 5 shows a plasmid map of plasmid pCRP3600 containing the A sequence.

[0033]

[9] Construction of PDC gene: Plasmid pCRP3600 obtained in [8] above was replaced with restriction enzymes BamHI and Spl.
The fragment was double cut with I, electrophoresed, and a fragment of about 5.7 kb was recovered from the agarose gel. Plasmid pCRF4100 was double cut with restriction enzymes NcoI and SplI, electrophoresed, and subjected to about 1.7.
A kb fragment was recovered from the agarose gel. Plasmid p
CRF5100 is double cut with restriction enzymes BamHI and NcoI,
After electrophoresis, a fragment of about 4.2 kb was recovered from the agarose gel. These three fragments were mixed and ligated with T4 DNA ligase. Next, several types of clones were taken from the population of transformants obtained by introducing the strain into Escherichia coli JM109 strain, and plasmids were respectively recovered. The sequence of each DNA was read, and a transformant containing a plasmid (designated pCRPDC1) in which the three fragments were ligated as desired was selected. This transformant was identified by JM-109-PCRP
DC1 and deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology as FERM P-15791 on August 20, 1996.
Was commissioned on the day. The transformant was cultured again, and plasmid pCRPDC1 was obtained from the cultured cells. FIG. 5 shows the PDC
FIG. 1 shows a plasmid map of a plasmid pCRPDC1 containing a gene expression control sequence and a DNA sequence (of the PDC gene) according to the present invention.

The scientific properties of FERM P-15791:
It is an Escherichia coli K12 strain carrying a plasmid containing the pyruvate decarboxylase gene of Chlamydomonas reinhardii and the promoter sequence of the α-tubulin gene. E. coli harboring this plasmid is Ampicillin
It is resistant. Taxonomic position of FERM P-15791: Escheric
hia coli K12 strain

[10] Introduction of PDC gene into Chlamydomonas cells: Chlamydomonas reinhardy CW1
5nit1-305 (Chlamydomonas reinhardii CW15n
it1-305) at 25 ° C under fluorescent lighting (2000-400)
(0 lux) with shaking culture in SGII medium. Algal cells were obtained from the culture solution 200ml of the logarithmic growth phase, SGII the composition shown in Table 2 (NH ₄ -) was washed four times with each culture 10 ml, SGII as the cell concentration becomes 10 ⁸ cells / ml
(NH ₄ −) was suspended in a culture solution.

[0036]

[Table 2]

[0037]

[Table 3]

Diameter 0.5 previously cleaned and dry-heat sterilized
0.3 g of a glass bead was placed in a micro-sampling tube, and 300 μl of algae suspension, 20% PE
400 μl of G6000 solution, plasmid pMN24 containing a gene for nitrate reductase as a selectable marker [Kallen Kindle (Section of Biochemistry, Molecular and Cell Biology and Plant Science Center, Cornell University)]
(Karen L. Kindle (Section of Biochemistry, Molecular
and Cell Biology and Plant Science Center, Cornel
l University))] 10 μg, plasmid pCR
Mix 50μg of PDC1 and vortex mixer
After vigorous shaking for 30 seconds, the HS (N
a NO ₃ ) Spread on plate medium, continuous light conditions (2000lux)
Cultured at 25 ° C. After culturing for 14 days, the formed transformant colonies were separated and chromosomal DNA was isolated from the transformants.
Was prepared, digested with the restriction enzyme PvuI, and electrophoresed.
Southern hybridization was performed using probe 3. The chromosomal DNA of the non-transformed strain is about 25
In contrast to the positive signal at one kb, the transformant showed several positive signals other than about 25 kb,
It was confirmed that the C gene had been introduced. This strain was deposited on August 20, 1996 with the National Institute of Bioscience and Human Technology, but was rejected for the reason "because it is a green algae".

[0039]

[Table 4]

[0040]

[Table 5]

[11] Production of alcohol by PDC gene-introduced Chlamydomonas: Chlamydomonas reinhardy CW15nit1-305 (Ch
lamydomonas reinhardii CW15nit1-305) plasmid
Transformants with pCRPDC1 were incubated at 25 ° C. under fluorescent lamp illumination (15000 lux) in SGII broth at 50 ml / min.
The cells were cultured with air aeration containing 5% CO ₂ . Algae obtained from 1000 ml of the culture medium at the end of logarithmic growth were transferred to a universal buffer (H ₃
After washing with PO ₃ -acetic acid-boric acid-NaOH pH 7.5), the solution was concentrated by a centrifugal sedimentation method to obtain a dried algal body slurry sample of 90 mg / ml. A volume of 50 ml of this sample
ml, and nitrogen gas was injected into the slurry for a short time to remove oxygen from the container.
The mixture was shaken while maintaining the temperature at ℃ to produce ethanol. On the other hand, normal Chlamydomonas
Using Reinhardy CW15nit1-305,
Ethanol was produced under exactly the same conditions.

FIG. 6 shows the time-dependent changes in the ethanol concentration in the slurry during the two ethanol production steps. In FIG. 6, the solid line shows the result when the transformed algal slurry of the present invention was used, and the broken line shows the result when the ordinary algal slurry was used. Thus, when the normal Chlamydomonas alga was used, the ethanol concentration was only about 4500 mg / L, whereas when the transformed Chlamydomonas alga was used, the ethanol concentration reached 5500 mg / L, and the PDC increased. It was found that introduction of the gene into algal cells increased the production of ethanol.

[0043]

Industrial Applicability According to the present invention, PDC gene DNA having a function of improving the conversion rate of polysaccharide accumulated in cells of the microalga Chlamydomonas into ethanol, a recombinant vector containing the DNA, regulation of the DNA and expression of the DNA A plasmid obtained from E. coli into which a functional plasmid was introduced was provided. In addition, by introducing the DNA of the present invention, a transformed microalga having an improved conversion rate of polysaccharide accumulated in cells to ethanol was provided. Furthermore, a method for producing ethanol more efficiently than before has been provided by using the transformed microalgae.

[0044]

[Sequence list] SEQ ID NO: 1 Sequence length: 580 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Origin: Chlamydomo
nas reinhardii) Sequence: Met Ala Thr Thr Val Ser Pro Ala Asp Ala Asn Leu Gly Leu His Ile 5 10 15 Ala Asn Arg Leu Val Glu Ile Gly Cys Thr Ser Cys Phe Ala Val Pro 20 25 30 Gly Glu Phe Asn Leu Leu Leu Leu Asp Gln Leu Leu Lys Gln Pro Glu 35 40 45 Leu Ser Leu Val Trp Cys Cys Asn Glu Leu Asn Ala Gly Tyr Ala Ala 50 55 60 Asp Gly Tyr Ala Arg Lys Arg Gly Val Gly Cys Leu Cys Val Thr Phe 65 70 75 80 Cys Val Gly Gly Phe Ser Ala Leu Asn Ala Val Gly Gly Ala Tyr Ser 85 90 95 Glu Asp Leu Pro Leu Ile Val Ile Ser Gly Gly Pro Asn Ser Gln Asp 100 105 110 His Ala Ser Asn Arg Ile Leu His His Thr Thr Gly Ala Asn Glu Tyr 115 120 125 Gly Gln Gln Leu Arg Ala Phe Arg Glu Val Thr Cys Cys Gln Val Val 130 135 140 Ile Gln His Ile Glu Asp Ala His Met Leu Leu Asp Thr Ala Ile Ser 145 150 155 160 Glu Ala Met Leu Lys Arg Lys Pro Val Tyr Ile Glu Val Ala Cys Asn 165 170 175 Leu Ala Asp Leu Thr His Pro Ser Phe Ala Arg Pro Pro Val Pro Tyr 180 185 190 Ala Leu Ala Ser Ser His Thr Asn Ala Ala Ser Leu Glu Ala Ala Val 195 200 20 5 Glu Ala Ala Val Glu Trp Leu Gly Gly Gly Val Lys Pro Leu Leu Leu 210 215 220 Ala Gly Val Arg Thr Arg Pro Pro Ala Ala Arg Lys Ala Met Leu Ala 225 230 235 240 Leu Ala Glu Ala Ser Arg Tyr Pro Val Ala Val Met Pro Asp Ala Lys 245 250 255 Gly Met Phe Pro Glu Asp His Glu Gln Tyr Ile Gly Met Tyr Trp Gly 260 265 270 Pro Val Ser Thr Pro Cys Val Cys Glu Val Val Glu Ser Ser Asp Ile 275 280 285 Val Leu Cys Val Gly Gly Val Trp Thr Asp Tyr Ser Thr Ala Gly Tyr 290 295 300 Ser Leu Leu Leu Lys Pro Glu Lys Met Leu Arg Val Asp Asn Asn Arg 305 310 315 320 Val Thr Leu Gly Asn Gly Pro Thr Phe Gly Cys Ile Val Met Thr Asp 325 330 335 Phe Leu Glu Ala Leu Ala Lys Arg Val Ala Pro Asn Asp Thr Gly His 340 345 350 Val Ile Tyr Lys Arg Met Ala Leu Pro Pro Ser Glu Pro Pro Pro Gln 355 360 365 Ala Glu Gly Glu Leu Leu Arg Thr Asn Val Leu Phe Lys His Ile Gln 370 375 380 His Met leu Thr Pro Ser Thr Ser Leu Ile Ser Glu Val Gly Asp Ser 385 390 395 400 Trp Phe Asn Thr Leu Lys Leu Lys Leu Pro Ala Gly Cys Glu Tyr Glu 405 410 41 5 Leu Gln Met Arg Tyr Gly Ser Ile Gly Trp Ser Val Gly Ala Val Leu 420 425 430 Gly Tyr Gly Val Ala Glu Arg Gln Thr Ala Pro Asp Arg Arg Val Val 435 440 445 Ala Cys Ile Gly Asp Gly Ser Phe Gln Met Thr Ala Gln Glu Val Ser 450 455 460 Thr Met Leu Arg Tyr Gly Leu Asp Pro Ile Ile Phe Leu Ile Asn Asn 465 470 475 480 480 Gly Gly Tly Thr Ile Glu Val Glu Ile His Asp Gly Pro Tyr Asn Val 485 490 495 Ile Lys Asn Trp Asp Tyr Pro Gly Met Val Arg Ala Leu His Asn Gly 500 505 510 Gln Gly Lys Leu Trp Thr Ala Glu Ala Arg Thr Glu Pro Glu Leu Gln 515 520 525 Ala Ala Val Ala Glu Ala Val Gln Arg Arg Gly Glu Leu Cys Phe Ile 530 535 540 Met Val Val Thr His Arg Asp Asp Cys Ser Lys Glu Leu Leu Glu Trp 545 550 555 560 Gly Ser Arg Val Ala Ala Ala Asn Ser Arg Lys Pro Pro Thr Thr Gly 565 570 575 Tyr Gly Gly His 580

SEQ ID NO: 2 Sequence length: 1743 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: open reading frame (ORF) Origin: Chlamydomon
as reinhardii) Sequence: ATG GCC ACC ACC GTG TCG CCC GCA GAC GCC AAC CTC GGC CTG CAC ATT 48 GCC AAC CGG CTT GTT GAG ATC GGC TGC ACC AGC TGC TTC GCG GTG CCC 96 GGC GAA TTC AAC CTG CTG CTG CTG GAC CATG CTC AAG CAG CCC GAG 144 CTG TCC CTG GTG TGG TGC TGC AAC GAG CTG AAT GCG GGC TAC GCG GCG 192 GAC GGC TAC GCC CGC AAG CGC GGC GTG GGC TGC CTG TGC GTG ACC TTC 240 TGT GTG GGA GGC TTC TCC GCC CTG ACT GTG GGC GGT GCC TAC AGT 288 GAG GAC CTG CCG CTC ATC GTC ATC AGC GGG GGG CCC AAC TCG CAG GAC 336 CAC GCC TCC AAC CGC ATC CTG CAC CAC ACC ACG GGC GCC AAC GAG TAC 384 GGC CAG CAG CTG CGC GCC TTC AGC GTG ACC TGC TGC CAG GTG GTC 432 ATC CAG CAC ATC GAG GAC GCG CAC ATG CTG CTG GAT ACG GCC ATC AGT 480 GAG GCG ATG CTG AAG CGC AAG CCC GTG TAC ATC GAG GTG GCA TGC AAC 528 CTG GCG GAC CTG ACC CACC TTC GCC CGC CCC CCG GTG CCG TAC 576 GCG CTG GCC TCC TCC CAC ACC AAC GCC GCC AGC CTG GAG GCC GCG GTG 624 GAG GCG GCG GTG GAG TGG CTG GGC GGT GGC GTG AAG CCG CTG CTG CTG 672 GCG GGC GTG CGC AGC CG CCC GCC GCG CGC AAG GCG ATG CTG GCC 720 CTG GCG GAG GCC AGC CGC TAC CCC GTG GCC GTG ATG CCG GAC GCC AAG 768 GGC ATG TTC CCC GAG GAC CAC GAG CAG TAC ATC GGC ATG TAC TGG GGC 816 CCG GTG TCC ACC TGT GTG TGC GAG GTG GTG GAG AGC AGC GAC ATC 864 GTG CTG TGC GTG GGA GGA GTG TGG ACG GAC TAC TCC ACT GCC GGC TAC 912 TCG CTG CTG CTC AAG CCC GAG AAG ATG CTG CGC GTG GAC AAC AAC CGC 960 GTC ACA TGC AAC GGA CCG ACG TTT GGC TGC ATC GTG ATG ACC GAC 1008 TTC CTG GAG GCC CTG GCC AAG CGG GTG GCG CCC AAC GAC ACC GGC CAC 1056 GTC ATC TAC AAG CGC ATG GCT CTG CCG CCC TCG GAG CCG CCG CCG CAG 1GC GCC GAG GAA CTG CTG CGC ACC AAC GTG CTG TTC AAA CAC ATC CAG 1152 CAC ATG CTG ACT CCC TCC ACC AGC CTC ATC AGC GAG GTG GGC GAC TCC 1200 TGG TTC AAC ACA CTC AAG CTC AAG CTG CCC GCC GGC TGC GAG TAC GAG 1248 CTG CAG ATG CGC TAC GGC TCC ATT GGC TGG AGT GTG GGC GCG GTG CTG 1296 GGC TAC GGC GTG GCG GAG CGG CAG ACG GCG CCC GAC CGC CGC GTG GTG 1344 GCG TGC ATC GGC GAC GGC TCC TTC CAG ATG ACC GCA CAG GAG GTG AGC 392 ACC ATG CTG CGC TAC GGC CTG GAC CCC ATC ATC TTC CTC ATC AAC AAC 1440 GGC GGC TAC ACC ATC GAG GTG GAG ATC CAC GAC GGC CCC TAC AAC GTG 1488 ATC AAG AAC TGG GAC TAC CCC GGT ATG GTG CGC GCG CTG CAC GGC 1536 CAG GGC AAG CTG TGG ACC GCC GAG GCC CGC ACC GAG CCC GAG CTG CAG 1584 GCC GCC GTG GCC GAG GCT GTG CAG CGG CGC GGC GAG CTG TGC TTC ATC 1632 ATG GTG GTG ACT CAC CGT GAC GAC TGC AGC AAG GTG CGC GAG TGG 1680 GGC AGC CGC GTG GCG GCG GCC AAC AGC CGC AAG CCG CCC ACC ACC GGC 1728 TAC GGC GGC CAC TGA 1743

SEQ ID NO: 3 Sequence length: 154 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic Sequence: ACG CTC GGA TAC CCCAGGGC CCAAGGACAA GCGCTGTCAT CTCTGCATCG GCGACGGAAG 60 CTTCCAGATG ACGGCGCAGG ACGTGTCGAC GATGCTGCGC TGCGGGCAGA AGAGCATCAT 120 CTTCCTCATC AACAACGGCG GGTACACCAT CGAG 154

SEQ ID NO: 4 Sequence length: 21 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: synthetic Sequence: CTCGATGGTG TACCCGCCGT T 21

SEQ ID NO: 5 Sequence length: 135 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic Sequence: AAC CTG GCG GAC CTGACCCA CCCCAGCTTC GCCCGCCCCC CGGTGCCGTA CGCGCTGGCC 60 TCCTCCCACA CCAACGCCGC CAGCCTGGAG GCCGCGGTGG AGGCGGCGGT GGAGTGGCTG 120 GGCGGTGGCG TGAAG 135

SEQ ID NO: 6 Sequence length: 97 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic Sequence: CACCCGTCGT GGATCTCCAC CTCGATGGTG TAGCCGCCGT TGTTGATGAG GAAGATGATG 60 GGGTCCAGGC CGTAGCGCAG CATGGTGCTC ACCTCCT 97

SEQ ID NO: 7 Sequence length: 26 Sequence type: number of nucleic acid chains: single strand Topology: linear Sequence type: synthetic Sequence: CTTCACGCCA CCGCCCAGCC ACTCCA 26

SEQ ID NO: 8 Sequence length: 22 Sequence type: number of nucleic acid chains: single strand Topology: linear Sequence type: synthetic Sequence: AG GAG GTG AGC ACCATGCTG CG 22

SEQ ID NO: 9 Sequence length: 197 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic Sequence: AATTACGTCA GCACGGAATG CATCACGCCT GCAAGCGCGC TCGAAGGCTC GAAGTCGGCA 60 TCGTACAGCC CCATTCCGGG GCGGGGGAGT GCTTATAAGCATTCGCC 120 GCATTCACAG CTCTGCTTTT ACTCCCCTTA ACCTTACATT ATCATCCCAG TTGGGACGCG 180 GGTCATTACC CCTCGCG 197

SEQ ID NO: 10 Sequence length: 197 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic Sequence: GAGGGGTAAT GACCCGCGTC CCAACTGGGA TGATAATGTA AGGTTAAGGG GAGTAAAAGC 60 AGAGCTGTGA ATGCCTGGCA AATCGGGCCC CGTTGCCCCCAG 120 ATGGGGCTGT ACGATGCCGA CTTCGAGCCT TCGAGCGCGC TTGCAGGCGT GATGCATTCC 180 GTGCTGACGT AATTGC 197

[Brief description of the drawings]

FIG. 1 shows a plasmid containing a part of the DNA sequence of the present invention.
It is a figure which shows the plasmid map of pCRF4100.

FIG. 2 shows a plasmid containing a part of the DNA sequence of the present invention.
It is a figure which shows the plasmid map of pCRF3400.

FIG. 3 shows a plasmid containing a part of the DNA sequence of the present invention.
It is a figure which shows the plasmid map of pCRF5100.

FIG. 4 is a diagram showing a plasmid map of a plasmid pCRP3600 containing a gene expression control sequence and a part of the DNA sequence of the present invention.

FIG. 5 is a diagram showing a plasmid map of a plasmid pCRPDC1 containing a gene expression control sequence and a part of the DNA sequence of the present invention.

FIG. 6 is a graph showing a change over time in the concentration of ethanol in a slurry solution during a step of producing ethanol by a recombinant algal cell into which the DNA of the present invention has been introduced. In the figure, the solid line shows the result when the transformed algal slurry was used, and the broken line shows the result when the normal algal slurry was used as a control.

FIG. 7 is a schematic diagram illustrating a part of the metabolic pathway of pyruvate.

Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication location // (C12N 1/12 C12R 1:89) (C12P 7/06 C12R 1:89)

Claims (13)

[Claims] 1. A pyruvate decarboxylase gene encoding the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 2. The pyruvate decarboxylase gene according to claim 1, which has a DNA having a nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing. 3. A host cell containing a DNA encoding the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing for improving pyruvate decarboxylase activity, and containing a DNA having a function of regulating the expression of the DNA. Recombinant vector. 4. The recombinant vector according to claim 3, wherein the DNA encoding the amino acid sequence represented by SEQ ID NO: 1 is the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. 5. A double-stranded DNA obtained by annealing said DNA having a function of regulating the expression of said DNA with the orionucleotides of SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing.
The recombinant vector according to claim 3 or 4, wherein 6. A recombinant plasmid comprising a pyruvate decarboxylase gene encoding the DNA of SEQ ID NO: 2 downstream of the promoter of the Chlamydomonas-derived α-tubulin gene, wherein the plasmid pCRF3600 is replaced with the restriction enzyme BamHI, Fragment double-cut with SplI, plasmid pC
A fragment obtained by double cutting RF4100 with restriction enzymes NcoI and SplI, and a plasmid pCRF5100 were digested with restriction enzymes BamHI and BamHI.
The fragment double-cut with NcoI was ligated with T4 DNA ligase and transformed into E. coli strain JM109 (FE
RM P-15791)
PDC1. 7. A recombinant microalgae cell having improved pyruvate decarboxylase activity, wherein the microalgae cell is used as a host cell and the gene is introduced by the recombinant vector according to claim 3 or 6. 8. The recombinant microalgal cell according to claim 7, wherein the host cell is an algal cell belonging to the genus Chlamydomonas. 9. The host cell contains a DNA encoding an amino acid represented by SEQ ID NO: 1 in the sequence listing for improving pyruvate decarboxylase activity and a DNA having a function of regulating the expression of the DNA. A method for producing ethanol, comprising culturing a recombinant microalgal cell having improved pyruvate decarboxylase activity, which has been transfected with a recombinant vector, in a medium, and producing ethanol by a metabolic reaction in the cell. 10. A host cell containing a DNA encoding an amino acid represented by SEQ ID NO: 1 in the sequence listing for improving pyruvate decarboxylase activity, and containing a DNA having a function of regulating the expression of the DNA. Ethanol characterized by culturing a recombinant microalgal cell having improved pyruvate decarboxylase activity introduced by a recombinant vector in a medium and producing ethanol from polysaccharide accumulated by a metabolic reaction in the cell. Manufacturing method. 11. The method for producing ethanol according to claim 9, wherein the DNA encoding the amino acid represented by SEQ ID NO: 1 in the sequence listing is represented by SEQ ID NO: 2. 12. The transformant (FE) is a recombinant vector (FE).
RM P-15791)
The PDC1 is a PDC1.
0. The method for producing ethanol according to 0. 13. The method for producing ethanol according to claim 9, wherein the host cell is an algal cell belonging to the genus Chlamydomonas.