JPWO2014104310A1 - Expression vector for cellulose production, transformant and method for producing cellulose - Google Patents

Abstract

Provided is a method for producing cellulose easily and inexpensively. 5 ' A cellulose production vector having an upstream sequence of a side gene is used. Cellulose can be biosynthesized by the transformant by culturing the transformant introduced with the cellulose production vector and co-expressing the vector and the dgc gene.

Description

  The present invention relates to an expression vector for producing cellulose, a transformant, and a method for producing cellulose.

  Cellulose has been used for many years as a useful raw material in various industries. Cellulose is a linear polymer in which glucose is polymerized, and can be classified into crystal structures such as type I and type II depending on the aggregation form of the molecular chain. Natural cellulose is called cellulose I having a crystal structure of type I, and is generally used as a material for paper and clothes. In addition, regenerated cellulose is called cellulose II having a type II crystal structure, and has a unique texture and high hydrophilicity, and thus has high added value and is generally used as a material for cellophane and rayon. However, since natural cellulose usually uses pulp obtained from natural wood as a raw material, development of a method using no pulp raw material is required from the viewpoint of preservation of natural resources such as forests. Furthermore, since the regenerated cellulose is produced by immersing the natural cellulose in a high-concentration alkaline solution, there is also a problem of high environmental load and cost.

  On the other hand, with the development of genetic engineering, as a method for producing cellulose, a method for synthesizing cellulose by a microorganism by a recombinant technique is being studied instead of preparation from natural products as described above. As a specific example, for example, a method has been reported in which a cellulose synthase gene bcsAB of acetic acid bacteria is introduced into cyanobacteria and cellulose is produced using the transformant (Non-patent Document 1). However, in this method, since the gene is introduced into the genomic DNA of cyanobacteria, there is a problem that labor and time are required and the yield is low. Therefore, provision of a method for producing cellulose easily and inexpensively is strongly demanded.

Transgenic expression of Gluconacetobacter xylinus strain ATCC 53582 cellulose synthase genes in the cyanobacterium Synechococcus leopoliensis strain UTCC 100 ”, Cellulose 15, 691-701 (2008)

  Then, this invention aims at provision of the method of manufacturing the cellulose which has various physical properties simply and cheaply.

In order to achieve the above object, the cellulose production vector of the present invention has a promoter, a CesA gene and a CesB gene,
It has an upstream sequence of the 5 ′ gene in an organism derived from the 5 ′ gene between the promoter and the start codon of the 5 ′ gene upstream of the CesA gene and the CesB gene. To do.

  The transformant of the present invention is characterized by including the above-described cellulose production vector of the present invention.

  The cellulose production method of the present invention is characterized by including a step of culturing a transformant containing the cellulose production vector of the present invention and a dgc gene.

  The cellulose of the present invention is obtained by the cellulose production method of the present invention.

  According to the present invention, cellulose can be easily produced by co-expressing the cellulose production vector and the dgc gene in a transformant obtained by introducing the cellulose production vector of the present invention into a host. Moreover, since cellulose can be produced by biosynthesis of the transformant, it is not necessary to use pulp as a raw material as in the prior art, and the environmental burden due to the use of natural raw materials can be avoided. Therefore, the present invention can be said to be a useful technique for various industries using cellulose as a raw material.

In Example 1, it is the schematic which shows the expression system structure of a CesABCD gene. In Example 1, it is a figure which shows the procedure of construction | assembly of a CesAB expression system. In Example 1, it is a figure which shows the procedure of construction | assembly of a dgc expression system. In Example 1, it is a photograph which shows the result of the negative dyeing | staining observation of a sample. In Example 1, it is a photograph which shows the result of the electron diffraction of a sample. In Example 1, it is a graph which shows the infrared spectrum of a sample. In Example 3, it is a photograph which shows the result of the negative dyeing | staining observation of a sample. In Example 4, it is a figure which shows the length of the upstream sequence and spacer sequence of a Ces expression system. In Example 4, it is a photograph which shows the result of Western blotting. In Example 5, it is a photograph which shows the result of Western blotting.

(1) Cellulose production vector The cellulose production vector of the present invention has a promoter, a CesA gene and a CesB gene as described above,
It has an upstream sequence of the 5 ′ gene in an organism derived from the 5 ′ gene between the promoter and the start codon of the 5 ′ gene upstream of the CesA gene and the CesB gene. To do. Hereinafter, the upstream gene of the CesA gene and the CesB gene is also referred to as a 5′-side gene, and the downstream gene is also referred to as a 3′-side gene.

  The lower limit of the length of the upstream sequence is, for example, 13 bases long, preferably 16 bases long, more preferably 21 bases long, and the upper limit is, for example, 100 bases long, preferably 70 bases long. It is a base length, More preferably, it is 24 base length, The range is 13-100 base length, for example, Preferably it is 13-70 base length, More preferably, it is 13-24 base length. In the present invention, description of a numerical range also means disclosure of values included in the range. That is, for example, 1 to 3 descriptions mean one, two and three disclosures.

  The upstream sequence may be linked directly to the initiation codon of the 5 'gene, for example, or indirectly via a spacer sequence, preferably the former. The upstream sequence may be directly linked to the 3 ′ end of the promoter, for example, or indirectly via a spacer sequence.

  As described above, the upstream sequence is not particularly limited as long as it is a sequence upstream of the 5 'gene in an organism derived from the 5' gene. The upstream sequence is, for example, an upstream sequence linked to the start codon of the 5 ′ gene in the organism derived from the 5 ′ gene, and specifically, an upstream sequence linked directly to the start codon. preferable.

  The upstream sequence may include, for example, a functional sequence involved in the expression of the CesA gene and the CesB gene. As a specific example, examples of the upstream sequence include a sequence having a ribosome binding site (RBS) or a partial sequence thereof. The upstream sequence may be, for example, a sequence consisting only of RBS, a sequence including RBS, a sequence consisting only of the partial sequence, or a sequence including the partial sequence.

  Examples of the partial sequence of RBS include the nucleotide sequence of SEQ ID NO: 16 (GGACGAGCTATT). The upstream sequence may be, for example, a sequence consisting of the base sequence of SEQ ID NO: 16 or a sequence containing the base sequence.

  When the upstream sequence includes, for example, the base sequence of SEQ ID NO: 16, the base sequence is preferably a 3 ′ region sequence in the upstream sequence. In this case, the upstream sequence is, for example, the base sequence. Is preferably linked to the start codon of the 5 ′ gene.

  The length between the promoter and the start codon is not particularly limited. The lower limit of the length is, for example, 13 bases, preferably 27 bases, more preferably 29 bases, and the upper limit of the length is, for example, 100 bases, preferably 86 The base length, more preferably 70 base length, still more preferably 30 base length, and the range of the length is, for example, 13 to 100 base length, preferably 13 to 86 base length, more preferably Is 13 to 86 bases long, more preferably 27 to 86 bases long or 29 to 86 bases long.

  In the case of having the spacer sequence between the promoter and the start codon, the length of the spacer sequence is not particularly limited, and the lower limit is, for example, 1 base length, preferably 6 bases, and the upper limit. Is, for example, 100 bases long, preferably 16 bases long, and the range is, for example, 1-100 bases long, preferably 6-16 bases long. When the spacer sequence is provided between the promoter and the upstream sequence, the length of the spacer sequence is, for example, in such a range.

In the present invention, the order of the CesA gene and the CesB gene is not particularly limited. The cellulose production vector of the present invention may have the both genes in the following order, for example, from the upstream side. [A] represents the CesA gene, and [B] represents the CesB gene (hereinafter the same).
[A]-[B]
[B]-[A]
In particular, the present invention preferably has [A]-[B], that is, the CesA gene-CesB gene in this order from the upstream. The CesA gene and the CesB gene may be linked directly or indirectly, for example. In the present invention, for example, it is preferred that the promoters located upstream are functionally linked so that the expression of both genes is controlled together. As a specific example, the CesA gene and the CesB gene are one An operon may be formed.

The cellulose production vector of the present invention may further have, for example, at least one of a CesC gene and a CesD gene, specifically, may have both or only one of them. Also good. The cellulose production vector of the present invention may have the genes in the following order, for example, from upstream. [C] represents the CesC gene, and [D] represents the CesD gene (hereinafter the same).
[A]-[B]-[C]
[A]-[B]-[C]-[D]
[A]-[B]-[D]
[A]-[B]-[D]-[C]
[B]-[A]-[C]
[B]-[A]-[C]-[D]
[B]-[A]-[D]
[B]-[A]-[D]-[C]
For example, the CesA gene, the CesB gene, the CesC gene, and the CesD gene may be linked directly or indirectly, respectively. In the present invention, for example, it is preferable that the genes are functionally linked so that expression of each gene is controlled by the promoter located upstream. As a specific example, each gene forms one operon. Also good.

  The CesA gene, the CesB gene, the CesC gene, and the CesD gene are known as cellulose synthase genes. For example, in bacteria such as acetic acid bacteria, these genes form one operon. It has been known. Hereinafter, the CesA gene, the CesB gene, the CesC gene, and the CesD gene are also referred to as Ces genes.

  In the present invention, the origin of the Ces gene is not limited. For example, all of the Ces genes may be derived from the same, or all or a part thereof may be derived from different sources. Examples of the origin include organisms such as bacteria, and examples of the bacteria include acetic acid bacteria, Salmonella bacteria, phytopathogenic bacteria, plant symbiotic bacteria, and photosynthetic bacteria. The acetic acid bacterium is, for example, Acetobacter genus, Acidiphyllium genus, Acidifera spp. , Paracoulococcus genus, Rhodopyra genus, Roseococcus genus, Lubritepida genus, Stella genus, Teicococcus genus or Zavardinia genus, and the like, preferably the gluconoacetobacter genus. The acetic acid bacteria belonging to the genus Gluconacetobacter are not particularly limited, and examples thereof include Gluconacetobacter xylinus.

  The Ces gene may be prepared by, for example, a genetic engineering technique or a chemical technique. In the former case, for example, it may be cloned from the derived organism or may be prepared by a gene amplification method such as PCR. In the latter case, it may be chemically synthesized. The preparation can be performed, for example, based on the base sequence information of each Ces gene or the amino acid sequence information of the protein encoded by each gene. In addition, as the Ces gene, for example, a polynucleotide having a nucleotide sequence that is modified in a range in which the encoded protein maintains an equivalent function in the nucleotide sequence of a known Ces gene can be used.

  As specific examples, the operon bases of CesA gene, CesB gene, CesC gene and CesD gene derived from Gluconacetobacter xylinus, the base sequence of each gene and the amino acid sequence of the encoded protein are shown below. In the present invention, the Ces gene is not limited to these sequences.

Ces gene operon (SEQ ID NO: 1, From-70)

CesA gene (SEQ ID NO: 2, positions 71 to 2335 in the operon)

CesA protein (SEQ ID NO: 3)

CesB gene (SEQ ID NO: 4, positions 2337-4745 in the operon)

CesB protein (SEQ ID NO: 5)

CesC gene (SEQ ID NO: 6, positions 4748-8728 in the operon)

CesC protein (SEQ ID NO: 7)

CesD gene (SEQ ID NO: 8, positions 8728-9198 in the operon)
atgacaactttgaacgcaaaaccggacttttcgcttttcctgcaggcactgtcctgggagatcgatgatcaggccgggatcgaggtcaggaatgacctgttgcgcgaggtcggccggggtatggctggtcgtttccagccgccgctgtgcaacaccatccaccagctccagatcgagctgaacgccctgctggccatgatcaactggggctacgtaaagctggacctgctggcggaagaacaggccatgcgcatcgtgcatgaagacctgccgcaggtgggcagcgcgggcgaacccgccggcacatggcttgccccggtgctggaagggctttatggccgctggatcacgtcgcagcccggcgccttcggtgattatgtcgtgacgcgtgatatcgacgcggaagacctgaactcggtcccggcccagacggtcatcctgtacatgcgcacccgcagcgccgcgacctga
CesD protein (SEQ ID NO: 9)
MTTLNAKPDFSLFLQALSWEIDDQAGIEVRNDLLREVGRGMAGRFQPPLCNTIHQLQIELNALLAMINWGYVKLDLLAEEQAMRIVHEDLPQVGSAGEPAGTWLAPVLEGLYGRWITSQPGAFGDYVVTRDIDAEDLNSVPAQTVILYMRTRSAAT

  The vector for producing cellulose of the present invention can be produced, for example, by inserting the Ces gene into a vector serving as a backbone (hereinafter also referred to as “basic vector”) so as to satisfy the above conditions. In the cellulose production vector of the present invention, the promoter may be, for example, a promoter of the basic vector, or may be a promoter inserted into the basic vector separately. In the former case, for example, the upstream sequence and the Ces gene may be inserted downstream of the promoter with respect to the basic vector.

  For example, the promoter is not particularly limited as long as it functions in a host into which the vector for producing cellulose of the present invention is introduced. Examples of the promoter include T5 promoter (PT5), trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, PSE promoter, SP01 promoter, SP02 promoter, penP promoter, TDH3p, PYK1p, PGK1p, TPI1p, GAL1p = GAL10p, ADH2p, PHO5p, CUP1p, MFα1p and the like. Examples of the high expression promoter include TDH3p, TPI1p, PGK1p, PGI1p, PYK1p, ADH1p and the like.

  The type of the basic vector is not particularly limited, and examples thereof include a plasmid derived from E. coli and a plasmid derived from yeast. The plasmid derived from E. coli is, for example, pQE system (pQE-80L, pQE-60 and pQE-30L etc.), pEX system, pBR system (pBR322 etc.), pUC system (pUC18, pUC19, pUC118 and pUC119 etc.), pBAD PGEX, pFLAG, pBluescript, pET vector, pCold vector, and the like. Examples of the yeast-derived plasmid include a multicopy type (YEp type), a single copy type (YCp type), a chromosome integration type (YIp type), and the like. The YEp type vector is, for example, YEp24 (JR Broach et al., Experimental Manipulation of Gene Expression, Academic Press, New York, 83, 1983), and the YCp type vector is, for example, YCp50 (MD Rose et al., Gene, 60, 237, 1987), and YIp type vectors include, for example, YIp5 (K. Struhl et al., Proc. Natl. Acad. Sci. USA, 76, 1035, 1979), etc. It has been reported. In addition, for example, pYE22m (Biosci. Biotech. Biochem., 59, 1221-2228, 1995), YIp1 (X70480) and the like can be mentioned. Above all, the basic vector is preferably a plasmid derived from E. coli, more preferably the pQE plasmid, and further preferably pQE-80L.

  The cellulose production vector of the present invention may further have a regulatory sequence that regulates the expression of the Ces gene, for example. The regulatory sequence only needs to function in the host into which the cellulose production vector of the present invention is introduced. Examples thereof include a terminator, an enhancer, a polyadenylation signal sequence, and an origin of replication sequence (ori). As the regulatory sequence, for example, a sequence provided in advance in the basic vector may be used, or the regulatory sequence may be further inserted into the basic vector, or the regulatory sequence provided in the basic vector may be changed to another sequence. It may be replaced with a regulatory sequence. In the cellulose production vector of the present invention, the arrangement of the regulatory sequence is not particularly limited as long as it is arranged so that the expression of the Ces gene can be functionally controlled, and is performed based on a known method. Can do.

  The cellulose production vector of the present invention may further have, for example, a selection marker coding sequence. Examples of the selection marker include an auxotrophic marker, a resistance marker such as drug resistance, a fluorescent protein marker, an enzyme marker, and a cell surface receptor marker. Examples of the auxotrophic marker include URA3 gene and LEU2 gene. Examples of the drug resistance marker include markers such as hygromycin resistance, zeocin resistance, and geneticin resistance. In addition, for example, a copper resistance gene (CUP1) (Marin et al., Proc. Natl. Acad. Sci. USA, 81, 337 1984), and a cerulenin resistance gene, the fas2m gene (Yoshigai et al., Biochemistry) 64, 660, 1992), PDR4 gene (Hussain et al., Gene, 101, 149, 1991) and the like can be used.

(2) Transformant As described above, the transformant of the present invention includes the cellulose production vector of the present invention. The transformant of the present invention is not particularly limited as long as the cellulose production vector of the present invention is introduced so that the Ces gene can be expressed.

  The transformant of the present invention can easily produce cellulose by expressing the cellulose production vector of the present invention together with the dgc gene. Therefore, the transformant of the present invention preferably further has a dgc gene, and specifically, preferably further includes a vector having the dgc gene. The dgc gene may be, for example, included in the cellulose production vector of the present invention, but is preferably included in a vector independent of the cellulose production vector of the present invention. Hereinafter, the vector having the dgc gene is also referred to as a dgc gene expression vector.

  The dgc gene is a gene known as a cyclic-di-guanylmonophosphate (c-di-GMP) synthase gene. In the present invention, the organism from which the dgc gene is derived is not particularly limited. Examples of the organism include archaea and eubacteria, thermophilic bacteria belonging to any of these. Examples of the eubacteria include Escherichia coli, Salmonella, Vibrio cholerae and the like. The thermophile is not particularly limited, and examples thereof include Pyrococcus genus, Methanopyrus genus, Thermococcus genus, Thermotoga genus, Pyrolovus genus, Brucanisaeta genus, Haloacula genus, Methanocera genus, Thermus genus, etc., preferably The genus Thermotoga. The thermophile belonging to the genus Thermotoga is not particularly limited, and examples thereof include Thermotoga maritima.

As specific examples, the base sequence of the dgc gene and the amino acid sequence of the protein encoded by these are shown in SEQ ID NOs: 10 to 15. In the present invention, the dgc gene is not limited to this sequence. The SoDGC shown below is a c-di-GMP synthase derived from Shewanella oneidensis , and tDGC is a c-di-GMP synthase derived from Thermotoga maritima , tDGC / K82 + R158A. Is a tDGC lacking the N-terminal 1-81th amino acid and having a point mutation of R158A.

SoDGC gene (SEQ ID NO: 10)

SoDGC protein (SEQ ID NO: 11)
MSRDLLQTAALNLKKAVPLMLKHQIPTTPINYALWYTYVGEQNLALNAQLDTVIAHYNTCPPVSSELLYRQYVADPVELNVRDMRLNLEAMATELSQSIKDTSLDTTEFQTCINSNFSKLNGIEEGALSVEQVLDLVRNLVKESDNIRSSTEFLTTQLQKAQTEIDALKQKLEKTEKDVLYDALTGCLNRRAFDADIAGMIAQAPEGTCLILVDIDHFKAFNDNYGHQLGDQVLKAVAKRLQEACRDGSKLYRFGGEEFAIIVPKSQLRITRQLAEAMRRGLEKLCLKDRRKDQLVDNITASFGVAQWQERQSVVQLIEKTDKLLYEAKRLGRNRVMPINN

tDGC gene (SEQ ID NO: 12)

tDGC protein (SEQ ID NO: 13)
MKVSGGEVPPFFKEGFFFTSEELTHLINVCSSTSAIISVLKDSKYRRSLVLYLKRPLSKDALLLIQTLLTTLEREDLSFGVKELEYMAYHDPLTGLPNRRYFFELGNRYLDLAKREGKKVFVYVDLAGFKAINDTYGHLSGDEVLKTVSKRILDRVRRSDVKTP

tDGC / K82 + R158A-gene (SEQ ID NO: 14)
aaagaactggaatacatggcctatcacgatccactgacagggcttccaaacaggcgttatttctttgaactgggaaacagatatctggacttagcaaagcgcgagggaaaaaaggtattcgttcttttcgtagatcttgctgggtttaaggcgataaatgacacatacggccatctgtcaggtgatgaggtattgaagacagtttcaaaaaggattctggacagggttgcaagaagcgatgtggtggctcgatacggcggtgatgagtttaccattctcctctacgatatgaaagaagagtatctgaaatccctcctggaaaggattctttccaccttcagagaacccgtcagagtggaaaataaacacttatctgtcacaccgaacataggagttgccagattccctgaagatggggaaaatcttgaagaacttctgaaagtagcggatatgaggatgtacaaagcaaaggaaatgaaagtgccttatttcagtctctcataa
tDGC / K82 + R158A-protein (SEQ ID NO: 15)
KELEYMAYHDPLTGLPNRRYFFELGNRYLDLAKREGKKVFVLFVDLAGFKAINDTYGHLSGDEVLKTVSKRILDRVARSDVVARYGGDEFTILLYDMKEEYLKSLLERILSTFREPVRVENKHLSVTPNIGVARFPEDGENLEELLKVADMRMYKAKEMKVPYFSLS

  The transformant of the present invention can be obtained, for example, by introducing the cellulose production vector of the present invention into a host. The transformant of the present invention can be obtained, for example, by introducing the dgc gene, specifically, the dgc gene expression vector. The order in which the cellulose production vector of the present invention and the dgc gene expression vector are introduced is not particularly limited, and either may be the first or simultaneous.

The host is not particularly limited and can be appropriately selected. Examples of the host include microorganisms, animal cells, insect cells, plant cells, or cultured cells thereof. Examples of the microorganism include prokaryotes and eukaryotes. The prokaryotes, for example, E. coli (Escherichia coli) Escherichia genus such as Bacillus subtilis (Bacillus subtilis) Bacillus such as, Pseudomonas such as Pseudomonas putida (Pseudomonas putida), Rhizobium meliloti (Rhizobium meliloti), such as Examples include bacteria of the genus Rhizobium. Examples of the eukaryote include yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe . Examples of the animal cells include COS cells and CHO cells, and examples of the insect cells include Sf9 cells and Sf21 cells. Said host is preferably E. coli.

  In the present invention, the method for introducing a vector or gene into the host is not particularly limited, and can be performed by a known method. The introduction method can be appropriately set depending on, for example, the type of the host.

  Examples of the introduction method include introduction method using a gene gun such as particle gun, calcium phosphate method, polyethylene glycol method, lipofection method using liposome, electroporation method, ultrasonic nucleic acid introduction method, DEAE-dextran method, micro glass tube, etc. Examples include a direct injection method using a hydrogel, a hydrodynamic method, a cationic liposome method, a method using an introduction aid, and a method using Agrobacterium. Examples of the liposome include lipofectamine and cationic liposome, and examples of the introduction aid include atelocollagen, nanoparticles, and polymers.

  In the present invention, a method for confirming whether or not the vector has been introduced into the host is not particularly limited, and a known method can be used. Known methods include, for example, a method using each of the selection markers possessed by the vector, a PCR method, a Southern hybridization method, a Northern hybridization method, and the like.

(3) Method for Producing Cellulose As described above, the method for producing cellulose of the present invention includes a step of culturing a transformant containing the vector for producing cellulose of the present invention and the dgc gene. The present invention is characterized by using the transformant of the present invention, and other steps and conditions are not particularly limited. According to the present invention, for example, cellulose can be easily synthesized without using a special substrate.

  In the present invention, the cellulose to be produced is not particularly limited, and examples thereof include cellulose I, cellulose II, and cellulose III, and cellulose II is preferable.

  In the present invention, the transformant of the present invention described above can be used as the transformant, and specifically includes a transformant having the cellulose production vector of the present invention and the dgc gene. In the transformant, the dgc gene may be introduced as the dgc gene expression vector as described above.

  In the culturing step, the type of the medium is not particularly limited, and can be appropriately selected according to the host of the transformant. When the host is Escherichia coli, examples thereof include media such as LB, 2xYT, and TB. In addition, the transformant of the present invention can synthesize cellulose even under a condition where a substrate such as glucose is not added as a foreign substrate.

  In the culturing step, biomass can also be used as a raw material for the medium.

  The culture conditions in the culture step are not particularly limited and can be appropriately selected according to the host. When the host is Escherichia coli, for example, known conditions for culturing Escherichia coli can be employed. The culture temperature is, for example, 4 to 40 ° C, preferably 4 to 30 ° C. The culture time is not particularly limited, and is, for example, 2 to 24 hours, preferably 5 to 12 hours.

  The method for producing cellulose of the present invention may further include an extraction step of extracting cellulose from the culture obtained by the culture step. The extraction step may include, for example, a separation step of a solid fraction and a liquid fraction, a purification step of cellulose from the solid fraction or the liquid fraction, and the like. When the cellulose synthesized by culturing the transformant of the present invention is insoluble, for example, in the purification step, it is preferable to purify the cellulose using the solid fraction, and in the case of water solubility, the purification step In the above, it is preferable to purify cellulose using the liquid fraction. In addition, the synthesized cellulose is, for example, released to the outside of the cells, but the cellulose retained in the cells is recovered and purified by destroying the transformed transformant contained in the solid fraction. May be.

  The separation step can be performed, for example, by centrifugation, filtration, or the like. Examples of the purification step include a step of acid-treating the solid fraction collected in the separation step. Moreover, the said refinement | purification process may also include the process of further alkali-treating the solid fraction after the said acid treatment, for example.

  In addition, for example, the present invention is expected to be used as a platform that enables the production of cellulose having completely different properties, such as cellulose having extremely high hydrophobicity, by using the above-described cellulose production vector. it can.

(4) Cellulose The cellulose of the present invention is obtained by the method for producing cellulose of the present invention as described above. The cellulose of the present invention is characterized by being obtained by the method for producing cellulose of the present invention, and other configurations are not particularly limited.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to the aspect described in the Example.

[Example 1]
(1) CesA gene, CeSb gene, from subcloning RIKEN BRC of CesC gene and CesD genes were obtained Gluconacetobacter xylinus (Gluconacetobacter xylinus) JCM9730 strain. The strain corresponds to the BPR2001 strain whose sequence of the cellulose synthase gene group has been identified (Nakai T .; Moriya A .; Tonouchi N .; Tsuchida T .; Yoshinaga F .; Horinouchi S .; Sone Y. Mori H .; Sakai F .; Hayashi T., "Control of expression by the cellulose synthase (bcsA) promoter region from Acetobacter xylinum BPR 2001" Gene 213, 93-100 (1998)).

  The strain is cultured in SH medium (Schramm M .; Hestrin S., “Factors affecting production of cellulose at the air / liquid interface of a culture of Acetobacter xylinum” J. Gen. Microbiol. 11, 123-129 (1954)). Then, genomic DNA was extracted from the obtained cells using a commercially available kit (manufactured by Invivogene). Using the genomic DNA as a template, the target gene was amplified by PCR using a kit. As the kit, AmpliTaqGold (Applied Biosystems), LA Taq (Takara) or PrimeStar GXL (Takara) was used. The obtained amplification product was inserted into the pGEM-T easy vector (Promega) by TA-ligation. When PrimeStar GXL was used as the kit, TA-ligation was performed after A-tailing with Taq (Takara).

The primers used for the PCR are shown below.
# A1 (SEQ ID NO: 17)
5'-ATATAAGGACGAGCTATTGATGTCAGAGGTTCAGTCGCCAGTACCC-3 '
# A2 (SEQ ID NO: 18)
5'-ATAGGATCCCGAGGCCGCACGGCTGATCTTTCC-3 '
# A3 (SEQ ID NO: 19)
5'-ATGAGCTCATGTCAGAGGTTCAGTCG-3 '
# A4 (SEQ ID NO: 20)
5'-GGATCCCAGGGCGATCTTGCGGTCAGC-3 '
# A5 (SEQ ID NO: 21)
5'-GCTAGCCACCATGTCAGAGGTTCAGTCG-3 '
# A6 (SEQ ID NO: 22)
5'-GGTACCTCATCACGAGGCCGCACGGCTGATCTT-3 '
# A7 (SEQ ID NO: 23)
5'-ACCGGTTGAGCGCAGGGAGCC-3 '
# A8 (SEQ ID NO: 24)
5'-CATATGGAACGTGCTCGGGCC-3 '
# A9 (SEQ ID NO: 25)
5'-GCCACCATGGCAATGGTGTCCCTGATCG-3 '
# A10 (SEQ ID NO: 26)
5'-CATATGGAACGTGCTCGG-3 '
# A11 (SEQ ID NO: 27)
5'-CTCGAGGCCTATCCGCTGCGT-3 '
# A12 (SEQ ID NO: 28)
5'-AGATCTCGTTCTCTGCCTTTCTTCCTG-3 '
# A13 (SEQ ID NO: 29)
5'-GGTACCTCACGTTCTCTGCCTTTCTTC-3 '
# A14 (SEQ ID NO: 30)
5'-ATGGATCCCTTGCGCGCTGAATCCGATG-3 '
# A15 (SEQ ID NO: 31)
5'-GCCACCATGGCAAGGCGATACGCCCTTTC-3 '
# A16 (SEQ ID NO: 32)
5'-GGTGCGCGTCGAGGCGATAC-3 '
# A17 (SEQ ID NO: 33)
5'-CATATGGGTTACCAGCAGCTCAACG-3 '
# A18 (SEQ ID NO: 34)
5'-AAGGATCCCGTGCCATAACGCGGCACATC-3 '
# A19 (SEQ ID NO: 35)
5'-AAGGATCCCAGGCCGACCCGGAGGCGAC-3 '
# A20 (SEQ ID NO: 36)
5'-GGATCCCTGGTCCATAATAAGATAG-3 '
# A21 (SEQ ID NO: 37)
5'-ATGAGCTCCCGGTCGCGGCGCTGCGGGTG-3 '
# A22 (SEQ ID NO: 38)
5'-ATAACCATGGCAACTTTGAACGCAAAAC-3 '
# A23 (SEQ ID NO: 39)
5'-ATAAGCTTCAGGTCGCGGCGCTGCGGGTG-3 '
# A24 (SEQ ID NO: 40)
5'-AAGCTTAATGATGATGATGATGATGGGTCGCGGCGCTGCG-3 '
# A25 (SEQ ID NO: 41)
5'-ATAAAAGCTTACTGGTCCATAATAAGATAG-3 '

  Table 1 below shows the relationship between the primers used for PCR and the prepared subcloning vector. Vector # a13 was prepared by ligating fragments from vector # a10 and vector # a12 to vector # a8. FIG. 1 shows the region of the CesABCD gene derived from glucone Acetobacter xylinus JCM9730 introduced into each vector.

  The DNA sequence of the target gene was confirmed, and the DNA fragment was excised from the subcloning vector by restriction enzyme treatment and inserted into an expression vector by ligation reaction. Subsequently, agarose gel electrophoresis is performed after the restriction treatment, the gel is stained with Mupid Blue (advance), a band of a target size is cut out, and a DNA extraction kit (Qiagen) is used to obtain a DNA fragment of the target gene. Isolated. The ligation reaction of the obtained DNA fragment was performed using DNA Ligation kit ver. 2.1 (Takara) or DNA ligation kit LONG (Takara) was used.

(2) Construction of Ces expression system (2-1) Construction of CesA expression system pQE-80L (Qiagen) was treated with SacI and KpnI, and SacI-EcoRI fragment (about 600 bp) of vector # a2 and vector The EcoRI-KpnI fragment of # a3 (about 1700 bp) was ligated simultaneously. Thereby, the CesA gene (SEQ ID NO: 2) was introduced, and a CesA expression system EV # 20 that expresses CesA with a His tag (His6 tag) at the N-terminus was prepared.

(2-2) Construction of CesB Expression System pQE-60 was treated with NcoI and BglII, and this was treated with an NcoI-XhoI fragment (about 1350 bp) of vector # a5 and an XhoI-BglII fragment (about 1050 bp) of vector # a6. And ligated at the same time. As a result, a CesB gene (SEQ ID NO: 4) was introduced, and a CesB expression system EV # 32 that expresses CesB with a His tag at the C-terminus was prepared.

(2-3) Construction of CesC expression system pQE-60 was treated with NcoI and BamHI, and this was treated with an NcoI-KpnI fragment of vector # a9 (about 500 bp) and a KpnI-AgeI fragment of vector # a10 (about 2200 bp). And the AgeI-BamHI fragment of vector # 11 (about 1350 bp) were simultaneously ligated. Thereby, the CesC gene (SEQ ID NO: 6) was introduced, and a CesC expression system EV # 59 that expresses CesC was produced.

(2-4) Construction of CesAB Expression System FIG. 2 shows a procedure for constructing a CesAB expression system that expresses CesA and CesB. EV # 20 was treated with AgeI and KpnI, and the AgeI-XhoI fragment of vector # a4 (about 1400 bp) and the XhoI-KpnI fragment of vector # a7 (about 1050 bp) were ligated simultaneously. Thereby, CesAB expression system EV # 21 expressing CesA and CesB was produced.

  EV # 21 was treated with EcoRI, the resulting vector fragment (about 9000 bp) was dephosphorylated by alkaline phosphatase treatment, and the EcoRI fragment (about 600 bp) of vector # a1 was ligated thereto. This produced CesAB expression system EV # 51. Thereby, the CesA gene (SEQ ID NO: 2) and the CesB gene (SEQ ID NO: 4) were introduced, and a CesAB expression system EV # 51 expressing CesA and CesB was produced. The fragment insertion direction was confirmed by PCR.

  EV # 51 was treated with BamHI and HindIII, and the BamHI-HindIII fragment (about 500 bp) of EV # 32 was ligated to the resulting vector fragment (about 9000 bp). As a result, the CesA gene (SEQ ID NO: 2) and the CesB gene (SEQ ID NO: 4) were introduced, and a CesAB expression system EV # 53 that expresses CesA and CesB with a His tag at the C-terminus was prepared.

  In the CesAB expression system EV # 53, the CesA gene was located upstream of the CesB gene, and the length between the TQ promoter derived from pQE-80L and the CesA gene was 29 bases long. The sequence from the promoter to the 5 'region of the CesA gene is shown below. In the following sequence, the underlined portion on the 5 ′ side is the promoter region, and the ununderlined region is a sequence derived from pGEM-T easy brought from the EcoRI site and the subcloning vector # a1, The enclosed region is the upstream sequence (13 base length) upstream of the start codon of CesA gene derived from gluconacetobacter xylinus, and the underlined portion on the 3 ′ side is the start codon of CesA gene.

(2-5) Construction of CesABC expression system Vector # a8 was treated with XmaI and SpeI. To this, XmaI-AgeI fragment (about 1600 bp) of vector # a10 and AgeI-SpeI fragment of vector # 12 (about 1800 bp) And ligated at the same time. Thereby, vector # a13 was produced. Vector # a13 contains from the 3 ′ region of CesB to the full length of CesC and the 3 ′ end of CesD. Subsequently, EV # 53 was treated with BamHI and HindIII, and the resulting vector fragment (about 9000 bp) was combined with the BamHI-SacII fragment (about 2150 bp) of vector # a13 and the SacII-HindIII fragment (about 2300 bp) of EV # 59. And ligated at the same time. Thereby, the CesA gene (SEQ ID NO: 2), the CesB gene (SEQ ID NO: 4), and the CesC gene (SEQ ID NO: 6) are introduced, and CesABC expression system that expresses CesA, CesB, and CesC with His tag at the C-terminus EV # 63 was produced.

  Further, instead of the BamHI-SacII fragment of the vector a # 13, the BamHI-XhoI fragment (about 2700 bp) of the vector a # 13, and the XhoI-HindIII fragment of the vector a # 16 instead of the SacII-HindIII fragment of EV # 59 ( Ligation was performed in the same manner except that about 1700 bp) was used. Thereby, the CesA gene (SEQ ID NO: 2), the CesB gene (SEQ ID NO: 4), and the CesC gene (SEQ ID NO: 6) are introduced, and the CesABC expression system EV # 100 that expresses CesA, CesB, and CesC without a His tag. Was made.

  In the CesABC expression system EV # 63, the CesA gene, the CesB gene, and the CesC gene were arranged from the upstream side, and the length between the pQE-80L-derived T5 promoter and the CesA gene was 29 bases long. The sequence from the promoter to the 5 'region of the CesA gene is the same as in EV # 53.

(2-6) Construction of CesABCD Expression System EV # 53 was treated with BamHI and HindIII, and the resulting vector fragment (about 9000 bp) was added to the BamHI-SacII fragment (about 2400 bp) of vector # a13 and vector # a13. The SacII-EagI fragment (about 2150 bp) and the EagI-HindIII fragment (about 400 bp) of vector # a14 were ligated simultaneously. Thereby, the CesA gene (SEQ ID NO: 2), the CesB gene (SEQ ID NO: 4), the CesC gene (SEQ ID NO: 6) and the CesD gene (SEQ ID NO: 8) are introduced, and CesABCD expressing CesA, CesB, CesC and CesD An expression system EV # 79 was produced.

  Further, ligation was carried out in the same manner except that vector # a15 was used instead of vector # a14. Thereby, the CesA gene (SEQ ID NO: 2), the CesB gene (SEQ ID NO: 4), the CesC gene (SEQ ID NO: 6), and the CesD gene (SEQ ID NO: 8) are introduced, and the His tag at the CesA, CesB, CesC, and C-terminus. A CesABCD expression system EV # 80 that expresses CesD marked with is prepared.

  In the CesABC expression systems EV # 63 and # 80, the CesA gene, CesB gene, CesC gene and CesD gene are arranged from the upstream side, and the length between the T5 promoter derived from pQE-80L and the CesA gene is 29 bases long It became. The sequence from the promoter to the 5 'region of the CesA gene is the same as in EV # 53.

(3) Cloning of dgc gene Thermotoga maritime MSB8 (NBRC 100826) was obtained from NBRC (NITE, Japan). The dgc gene encoding DGC (Diguanyl cyclase) was amplified by PCR using AmpliTaq Gold (Applied Biosystems) using the aqueous suspension of the cells as a template. The dgc gene has the accession number TM1788 (Gene ID: 897742) (Rao F .; Pasunooti S .; Ng Y .; Zhuo W .; Lim L .; Liu A.W .; Liang Z.-., “Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase "Anal. Biochem. 389, 138-142 (2009)). Then, the obtained amplification product was inserted into the pGEM-T easy vector by TA-ligation to prepare vector # b1. Furthermore, using vector # b1 as a template, point mutation R158A was introduced by PCR (using Takara, PrimeStar GXL) using the QuikChange (Qiagen) protocol to prepare vector # b2 into which the dgc gene (SEQ ID NO: 14) was introduced.

The primers used for the PCR are shown below.
# B1 (SEQ ID NO: 43)
5'-GCTAGCAAAGAACTGGAATACATGG-3 '
# B2 (SEQ ID NO: 44)
5'-GAATTCTTATGAGAGACTGAAATAAGGC-3 '
# B3 (SEQ ID NO: 45)
5'-CTGGACAGGGTTGCAAGAAGCGATGTGG-3 '
# B4 (SEQ ID NO: 46)
5'-CCACATCGCTTCTTGCAACCCTGTCCAG-3 '

The relationship between the primers used for PCR and the prepared vector is shown in Table 3 below.

(4) Construction of dgc expression system FIG. 3 shows a procedure for constructing a dgc expression system that expresses DGC. PCR (using Takara, PrimeStar GXL) was performed using the vector # b2 as a template and the following primers and the primer # b2. The obtained amplification product was inserted into pBAD202 / D-TOPO (Invitrogen) by TOPO cloning reaction to prepare pBAD202 / D-tDGC.
Primer (SEQ ID NO: 47)
5'-CACCAAAGAACTGGAATACATGG-3 '

Two oligonucleotides shown below were inserted into the pBAD202 / D-TOPO by TOPO reaction to obtain pBAD202 / R. Then, pBAD33 distributed from BBRP (NIG, Japan) was treated with MluI and HindIII, and the resulting vector fragment was pBAD202 / R MluI-HindIII fragment (about 600 bp) and DNA ligation kit ver2.1. And ligated. This produced pBAD33 / 202R.
Oligonucleotide
5'-CACCGAATTCGTCGACGGTACCGCTAGC-3 '(SEQ ID NO: 48)
5'-GCTAGCGGTACCGTCGACGAATTC-3 '(SEQ ID NO: 49)

  The pBAD33 / 202R was treated with MluI and SacI, and the resulting vector fragment was ligated with the MluI-SacI fragment (about 1100 bp) of pBAD202 / D-tDGC using Takara DNA ligation kit ver2.1. Thereby, the dgc gene (SEQ ID NO: 14) was introduced, and pBAD33 / 202-tDGC expressing DGC was prepared.

(5) Cellulose synthesis The obtained Ces expression system vector (CesAB expression system EV # 53, CesABC expression system EV # 63 or CesABCD expression system EV # 80) and a dgc expression system vector (pBAD33 / 202-tDGC), By introducing it into E. coli XL1-Blue strain by the rubidium method, a transformant co-expressing both the expression vectors was produced.

  Then, each transformant was cultured in a 2 × YT medium (pH 7) medium containing 0.2% L-arabinose and 0.4 mmol / L isopropylthiogalactoside (IPTG) at a temperature of 28 ° C. for 5 hours. .

  About the obtained culture solution, the following processes were given, the sample was prepared, and the cellulose analysis mentioned later was performed. First, the culture solution was centrifuged under conditions of 7,500 g, and the precipitate containing the bacterial cells was collected. After suspending the precipitate in water, a 4-fold volume of a mixed solution of acetic acid 8: nitric acid 1 was added to the suspension and treated at 100 ° C. for 30 minutes. This treatment solution was centrifuged to collect the precipitate, and the precipitate was washed until the supernatant after centrifugation became neutral.

  Cellulose analysis was performed on the sample by negative staining observation and electron diffraction imaging with 2% uranium acetate using a transmission electron microscope (official name JEM-2000EXII) and infrared spectrum using Spectrum One (Perkin Elmer). Performed by measurement. The infrared spectrum was analyzed together with standard products of type I cellulose and type II cellulose. As a representative of these results, the results of the samples obtained from the transformants into which the CesAB expression system EV # 53 was introduced as the Ces expression system vector are shown in FIG. 4, FIG. 5 and FIG.

  FIG. 4 shows the results of negative staining observation, (A) shows the results of low magnification observation, and (B) shows the results of high magnification observation. In (A), the scale bar indicates 10 μm, and in (B), the scale bar indicates 100 nm. 5 is a photograph of an electron diffraction pattern, FIG. 6 is an infrared spectrum, Native BC is a cellulose I type standard sample, Mercerized BC is a cellulose II type standard sample, and EC-Synthesized is an example. The result of the sample is shown. In FIG. 4, an aggregate having a size of 10 to 20 nm was confirmed. Moreover, in FIG. 5, the diffraction rings of 110 and 020 characteristic to the cellulose II crystal were confirmed. Furthermore, as shown in FIG. 6, the same pattern as the type II cellulose (Mercerized BC) was confirmed in the sample (EC-Synthesized). From these results, it was confirmed that type II cellulose was contained in the sample, that is, type II cellulose was biosynthesized by culturing the transformant. Similar results were obtained for samples obtained from transformants into which other Ces expression vectors were introduced. It was also found that when the expression vector into which the Ces gene was inserted did not satisfy the conditions of the present invention, protein expression could hardly be confirmed, so that cellulose synthesis could not be performed.

[Example 2]
In the present Example, it confirmed that the transformant which introduce | transduced the said CesAB expression system is expressing the cellulose.

  In the same manner as in Example 1, a transformant into which the Ces expression system vector (CesAB expression system EV # 51) and the dgc expression system vector (pBAD33 / 202-tDGC) were introduced was produced.

  The transformant was cultured using a 2 × YT medium (pH 7) medium at a temperature of 28 ° C. Next, L-arabinose and IPTG were added so that it might become 0.2% L-arabinose and 0.4 mmol / L IPTG, and also it culture | cultivated on the conditions of the temperature of 28 degreeC for 6-7 hours. After the culture, a precipitate containing the cells was collected at 5000 g. And the liquid mixture whose volume ratio is acetic acid 8: nitric acid 1 was added to the precipitation containing the said microbial cell, and it processed at 100 degreeC for 30 minutes. The treatment liquid was centrifuged to recover the cellulose precipitate, and the cellulose precipitate was washed until the centrifuged supernatant became neutral. After the washing, the cellulose precipitate was freeze-dried and the presence or absence of cellulose was visually confirmed.

  Control 1 was the same except that the CesA expression system EV # 98 was used instead of the CesAB expression system EV # 51, and Control 2 was the same except that only the dgc expression system vector was used. The presence or absence of cellulose was confirmed visually. The CesA expression system EV # 98 was prepared by treating EV # 20 with EcoRI and ligating the EcoRI fragment (about 600 bp) of the vector a # 01.

  In the sample derived from the transformant into which the CesAB expression system was introduced, cellulose was confirmed, whereas in Control 1 and Control 2, cellulose was not confirmed. From these results, it was found that the CesAB expression system is important for cellulose synthesis for cellulose synthesis.

[Example 3]
In this example, the culture solution of the transformant introduced with the CesAB expression system was subjected to cellulase treatment, and it was confirmed that cellulose was synthesized.

  In the same manner as in Example 1, a culture solution of the transformant was produced. Next, the culture solution was used as a sample and mounted on a nickel grid covered with a carbon film. The nickel grid after mounting was washed with TBS (Tris buffered Saline) and water, and dried for 5 minutes. Furthermore, the nickel grid was floated on a buffer solution (50 mmol / L MES-NaOH, pH 5.6) containing 0.5% cellulase (trade name: Celluclast 1.5 L, Novozyme), and the mixture was incubated at 45-50 ° C. for 1 hour. Processed. The nickel grid after the treatment was washed with water, and negative staining observation with 2% uranium acetate was performed in the same manner as in Example 1. The same sample was subjected to negative staining observation in the same manner except that the buffer solution containing no cellulase was used instead of the buffer solution containing the cellulase.

  These results are shown in FIG. FIG. 7 shows the result of negative staining observation. In FIG. 7, the upper row is the result of the cellulase-untreated sample, the lower row is the result of the cellulase-treated sample, the left row is the result of low magnification observation, and the right row is the result of high magnification observation. is there. In the left photograph, the scale bar indicates 2000 nm, and in the right photograph, the scale bar indicates 200 nm. In FIG. 7, as shown in the upper photograph, the cellulose fiber structure was observed at any magnification in the cellulase-untreated sample, while on the other hand, as shown in the lower photograph, at any magnification, the cellulase-treated sample. Also, the fiber structure of cellulose was not observed. From these results, it was found that cellulose was synthesized by culturing the transformant.

[Example 4]
In this example, CesAB expression systems having different upstream sequence base lengths and spacer sequence base lengths were prepared, and it was confirmed that upstream sequences were required for the expression of CesA protein and CesB protein.

(1) Construction of Ces expression system FIG. 8 shows the Ces expression system used in this example. As shown in FIG. 8, the control expression system EV # 43 is a comparative expression vector having no upstream sequence, and the CesAB expression system EV # 49-EV # 52 is an expression vector of the example having an upstream sequence. It is. The lengths of the upstream sequences of the control expression system EV # 43 and the CesAB expression systems EV # 49-EV # 52 are 0, 21, 24, 13, and 70 bases, respectively, and the spacer sequences are respectively long. The lengths are 27, 6, 6, 16, 16 bases, and the lengths between the promoter and the CesA gene (start codon) are 27, 27, 30, 29, and 86 bases, respectively. In this example, control expression system EV # 43 and CesAB expression systems EV # 49, EV # 50 and EV # 52 were prepared. The CesAB expression system EV # 51 of Example 1 was used as the CesAB expression system EV # 51.

A DNA fragment amplified by PCR was ligated to pGEM-T easy in the same manner as in Example 1 (1) except that the primer # a4 and the following primer # a26-29 were used, and vector # a17- # a20 was produced. The primers used for PCR are shown below.
# A26 (SEQ ID NO: 50)
5'-GAATTCATTAAAGAGGAGAAATTACATATGTCAGAGGTTCAGTCGCCAGTA-3 '
# A27 (SEQ ID NO: 51)
5'-GAATTCTTATTATCGGACGAGCTATTGATG-3 '
# A28 (SEQ ID NO: 52)
5'-GAATTCTATTTATTATCGGACGAGCTATTGATG-3 '
# A29 (SEQ ID NO: 53)
5'-ATGCGTCTGACGGTTTTCTTTTGAATATAT-3 '

  Table 4 below shows the relationship between the primers used in PCR and the prepared subcloning vector.

  A control expression system EV # 43 was prepared in the same manner as in Example 1 (2-4) except that the EcoRI fragment (about 600 bp) of vector # a17- # a20 was used instead of the EcoRI fragment of vector # a1. And CesAB expression system EV # 49, 50 and 52 were produced.

  In the control expression system EV # 43 and the CesAB expression systems EV # 49, EV # 50, EV # 51 and EV # 52 in Example 1, the CesA gene is located upstream of the CesB gene, and T5 derived from pQE-80L. The length between the promoter and the CesA gene was 27, 27, 30, 29 and 86 bases in length, respectively. The sequence from the promoter to the 5 'region of the CesA gene is shown below. In the following sequence, the underlined portion on the 5 ′ side is the promoter region, and the ununderlined region is the pQE-80L vector, EcoRI site, or EcoRI site and pGEM brought from the subcloning vector pGEM-T easy -A sequence derived from Teasy, and the region surrounded by a square is an upstream sequence (length of 0, 21, 24, 13 and 70 bases, respectively) upstream of the start codon of the CesA gene derived from Gluconacetobacter xylinus, The underlined part on the 3 ′ side is the start codon of the CesA gene.

(2) Confirmation of expression of CesA protein and CesB protein Except for using Ces expression system vectors (control expression system EV # 43 and CesAB expression systems EV # 49, EV # 50, EV # 51 and EV # 52), respectively. A transformant was prepared in the same manner as in Example 1 (5). Each transformant was cultured under the condition of a temperature of 28 ° C. using 2 × YT medium. Furthermore, IPTG was added so that it might become 0.5 mmol / L, and it culture | cultivated on the conditions of the temperature of 28 degreeC for 5 hours.

The cultured transformant is collected and suspended in a lysis buffer (10 mmol / L Tris-HCl (pH 8), 5 mmol / L EDTA, 0.02% NaN ₃ , 50 μg / mL chloramphenicol). SDS-PAGE sample buffer was added to prepare a sample for SDS-PAGE having a final concentration of 50 mM Tris-HCl (pH 8), 10% glycerin and 0.5% SDS, and this was subjected to SDS-PAGE. After the electrophoresis, CesA protein and CesB protein were detected using the polyclonal anti-CesA antibody and the polyclonal anti-CesB antibody described in Reference 1, respectively. Moreover, the negative control detected CesA protein and CesB protein similarly except collect | recovering each transformant before IPTG addition.
Reference 1: Carbohydrate Research 346, 2760-2768 (2011)

  These results are shown in FIG. FIG. 9 is a photograph of Western blotting showing the expression of CesB protein. In FIG. 9, the left side of the photograph shows the molecular weight of the molecular weight marker, and the upper side of the photograph shows the expression vector used for transformation and the type of sample.

  As shown in FIG. 9, no band showing the expression of CesB protein was observed in any negative control (before expression induction). As shown in FIG. 9, in the CesAB expression system (EV # 49, EV # 50, EV # 51 and EV # 52) having an upstream sequence, a band indicating normal CesB protein expression is observed around 85 kDa. It was. However, in the control expression system EV # 43 having no upstream sequence, a band showing the expression of CesB protein was observed around 100 kDa. This is because the CesB protein failed to form the correct folding, as will be described later. Although not shown, no band showing CesA protein expression was observed in any negative control. Moreover, in the CesAB expression system (EV # 49, EV # 50, EV # 51 and EV # 52) having an upstream sequence and the control expression system EV # 43 having no upstream sequence, all of them are normal CesA protein around 70 kDa. A band indicating the expression of was observed. From these results, it was found that normal CesA protein and normal CesB protein are expressed by having the upstream sequence of the 5'-side gene between the promoter and the start codon of the 5'-side gene. In addition, since normal CesA protein and normal CesB protein are expressed to synthesize cellulose, it can be said that the upstream sequence of the 5 'gene is necessary for cellulose synthesis.

[Example 5]
In this example, a CesB expression system, a control expression system, and a CesAB expression system are used. For normal CesB protein expression, co-expression of CesA protein and CesB protein and the upstream sequence of the 5′-side gene are required. It was confirmed.

  A sample (-βME sample) was prepared in the same manner as in Example 4 (2) except that only the CesAB expression system # 51 was used as the Ces expression system vector, and IPTG was added to 0.2 mmol / L. . Further, β mercaptoethanol was added to the sample to 1% to prepare a + βME sample in which disulfide bonds were reduced. And the expression of CesB protein was detected like the said Example 4 (2). Control 1 uses acetic acid bacteria that express normal CesA protein and normal CesB protein instead of transformants, and control 2 uses CesB expression system # 32 instead of CesAB expression system # 51. The control 3 was prepared by using the control expression system EV # 43 instead of the CesAB expression system # 51, and preparing a -βME sample and a + βME sample in the same manner. Detected.

  These results are shown in FIG. FIG. 10 is a photograph of Western blotting, (A) is a photograph showing the expression of CesB protein in the −βME sample, and (B) is a photograph showing the expression of CesB protein in the + βME sample. 10 (A) and (B), the left side of the photograph shows the molecular weight of the molecular weight marker, and each lane shows, from the left, acetic acid bacteria, CesB expression system # 32, control expression system EV # 43, CesAB expression system #. 51 results are shown.

  As shown in FIG. 10 (A), in the -βME sample of acetic acid bacteria and CesAB expression system # 51, a band showing the expression of CesB protein having normal folding is observed near 100 KDa, whereas CesB expression system In the -βME sample of # 32 or control expression system EV # 43, a band indicating the expression of CesB protein was observed in the vicinity of 90 KDa. In addition, as shown in (B), in the + βME sample of acetic acid bacteria and CesAB expression system # 51, a band showing the expression of CesB protein with a disulfide bond reduced is observed in the vicinity of 85 KDa, whereas CesB expression system In the + βME sample of # 32 or control expression system EV # 43, a band indicating the expression of CesB protein was observed around 90 KDa. From these results, it was found that the CesB protein observed in the CesB expression system # 32 or the control expression system EV # 43 is a misfolded CesB protein. From the above results, it was found that co-expression of CesA protein and CesB protein and the upstream sequence of the 5'-side gene were required for the expression of CesB protein having normal folding.

  Although the present invention has been described with reference to the exemplary embodiments and examples, the present invention is not limited to the above exemplary embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

  This application claims the priority on the basis of Japanese application Japanese Patent Application No. 2012-288335 for which it applied on December 28, 2012, and takes in those the indications of all here.

  As described above, according to the present invention, cellulose is easily produced by co-expressing the cellulose production vector and the dgc gene in a transformant obtained by introducing the cellulose production vector of the present invention into a host. it can. Moreover, since cellulose can be produced by biosynthesis of the transformant, it is not necessary to use pulp as a raw material as in the prior art, and the environmental burden due to the use of natural raw materials can be avoided. Therefore, the present invention can be said to be a useful technique for various industries using cellulose as a raw material.

In Example 1, it is the schematic which shows the expression system structure of a CesABCD gene. In Example 1, it is a figure which shows the procedure of construction | assembly of a CesAB expression system. In Example 1, it is a figure which shows the procedure of construction | assembly of a dgc expression system. In Example 1, it is a photograph which shows the result of the negative dyeing | staining observation of a sample. In Example 1, it is a photograph which shows the result of the electron diffraction of a sample. In Example 1, it is a graph which shows the infrared spectrum of a sample . In Example 4, it is a figure which shows the length of the upstream sequence and spacer sequence of a Ces expression system. In Example 4, it is a photograph which shows the result of Western blotting. In Example 5, it is a photograph which shows the result of Western blotting.

Se The cellulase untreated samples, the cellulose fiber structure in any of the magnification observation, while in cell cellulase treated sample, the fiber structure of the cellulose was not observed in any magnification. From these results, it was found that cellulose was synthesized by culturing the transformant.

(1) shown in Figure 7. Ces expression system used in constructing this embodiment of Ces expression systems. As shown in FIG. 7 , the control expression system EV # 43 is a comparative expression vector having no upstream sequence, and the CesAB expression system EV # 49-EV # 52 is an example expression vector having an upstream sequence. It is. The lengths of the upstream sequences of the control expression system EV # 43 and the CesAB expression systems EV # 49-EV # 52 are 0, 21, 24, 13, and 70 bases, respectively, and the spacer sequences are respectively long. The lengths are 27, 6, 6, 16, 16 bases, and the lengths between the promoter and the CesA gene (start codon) are 27, 27, 30, 29, and 86 bases, respectively. In this example, control expression system EV # 43 and CesAB expression systems EV # 49, EV # 50 and EV # 52 were prepared. The CesAB expression system EV # 51 of Example 1 was used as the CesAB expression system EV # 51.

The results are shown in Figure 8. FIG. 8 is a photograph of Western blotting showing the expression of CesB protein. In FIG. 8 , the left side of the photograph shows the molecular weight of the molecular weight marker, and the upper side of the photograph shows the expression vector used for transformation and the type of sample.

As shown in FIG. 8 , no band showing the expression of CesB protein was observed in any negative control (before expression induction). As shown in FIG. 8 , in the CesAB expression system (EV # 49, EV # 50, EV # 51 and EV # 52) having an upstream sequence, a band indicating normal CesB protein expression is observed around 85 kDa. It was. However, in the control expression system EV # 43 having no upstream sequence, a band showing the expression of CesB protein was observed around 100 kDa. This is because the CesB protein failed to form the correct folding, as will be described later. Although not shown, no band showing CesA protein expression was observed in any negative control. Moreover, in the CesAB expression system (EV # 49, EV # 50, EV # 51 and EV # 52) having an upstream sequence and the control expression system EV # 43 having no upstream sequence, all of them are normal CesA protein around 70 kDa. A band indicating the expression of was observed. From these results, it was found that normal CesA protein and normal CesB protein are expressed by having the upstream sequence of the 5 ′ gene between the promoter and the start codon of the 5 ′ gene. In addition, since normal CesA protein and normal CesB protein are expressed to synthesize cellulose, it can be said that the upstream sequence of the 5 ′ gene is necessary for cellulose synthesis.

The results are shown in Figure 9. FIG. 9 is a photograph of Western blotting, (A) is a photograph showing the expression of CesB protein in the −βME sample, and (B) is a photograph showing the expression of CesB protein in the + βME sample. 9 (A) and (B), the left side of the photograph shows the molecular weight of the molecular weight marker, and each lane shows, from the left, acetic acid bacteria, CesB expression system # 32, control expression system EV # 43, CesAB expression system #. 51 results are shown.

As shown in FIG. 9 (A), in the -βME sample of acetic acid bacteria and CesAB expression system # 51, a band showing the expression of CesB protein having normal folding in the vicinity of 100 KDa is observed, whereas the CesB expression system In the -βME sample of # 32 or control expression system EV # 43, a band indicating the expression of CesB protein was observed in the vicinity of 90 KDa. In addition, as shown in (B), in the + βME sample of acetic acid bacteria and CesAB expression system # 51, a band showing the expression of CesB protein with a disulfide bond reduced is observed in the vicinity of 85 KDa, whereas CesB expression system In the + βME sample of # 32 or control expression system EV # 43, a band indicating the expression of CesB protein was observed around 90 KDa. From these results, it was found that the CesB protein observed in the CesB expression system # 32 or the control expression system EV # 43 is a misfolded CesB protein. From the above results, it was found that co-expression of CesA protein and CesB protein and the upstream sequence of the 5 ′ gene are required for the expression of CesB protein having normal folding.

Claims (22)

Having a promoter, a CesA gene and a CesB gene;
It has an upstream sequence of the 5 ′ gene in an organism derived from the 5 ′ gene between the promoter and the start codon of the 5 ′ gene upstream of the CesA gene and the CesB gene. Cellulose production vector. The vector for cellulose production according to claim 1, wherein the upstream sequence has a length of 13 to 100 bases. The cellulose production vector according to claim 1 or 2, wherein the upstream sequence is linked to a start codon of the 5 'gene. The vector for producing cellulose according to any one of claims 1 to 3, wherein the upstream sequence is an upstream sequence linked to a start codon of the 5 'gene in an organism derived from the 5' gene. The cellulose production vector according to any one of claims 1 to 4, wherein the upstream sequence has a ribosome binding site (RBS) or a partial sequence thereof. The vector for producing cellulose according to any one of claims 1 to 5, wherein the upstream sequence comprises the nucleotide sequence of SEQ ID NO: 16 (GGACGAGCTATT). The vector for producing cellulose according to claim 6, wherein the base sequence of SEQ ID NO: 16 is the sequence of the 3 'region in the upstream sequence, and the 3' end thereof is linked to the start codon of the 5 'side gene. . The vector for cellulose production according to any one of claims 1 to 7, wherein a length between the promoter and the initiation codon is 13 to 100 bases in length. The vector for producing cellulose according to any one of claims 1 to 8, which has the CesA gene and the CesB gene in this order from the upstream side. Furthermore, the vector for cellulose manufacture as described in any one of Claim 1 to 9 which has at least one of a CesC gene and a CesD gene. The CesA gene and the CesB gene are derived from acetic acid bacteria. The vector for producing cellulose according to any one of claims 1 to 10. The vector for cellulose production according to claim 11, wherein the acetic acid bacterium belongs to the genus Gluconacetobacter. The vector for producing cellulose according to claim 12, wherein the acetic acid bacterium is gluconacetobacter xylinus. The vector for producing cellulose according to any one of claims 1 to 13, wherein the promoter is a promoter of a pQE vector. The vector for cellulose production according to any one of claims 1 to 14, wherein the promoter is a phage T5 promoter. A transformant comprising the vector for producing cellulose according to any one of claims 1 to 15. Furthermore, the transformant of Claim 16 containing the vector which has a dgc gene. The transformant according to claim 16 or 17, wherein the dgc gene is derived from Thermotoga maritima. The transformant according to any one of claims 16 to 18, wherein the transformant is Escherichia coli. A method for producing cellulose, comprising a step of culturing a transformant comprising the vector for producing cellulose according to any one of claims 1 to 15 and a dgc gene. The production method according to claim 20, wherein the cellulose is cellulose I or cellulose II. A cellulose obtained by the method for producing cellulose according to claim 20 or 21.