WO2015137352A1 - Gene related to full-dark heterotrophic ability in cyanobacteria, and use of same - Google Patents

Abstract

[Problem] To provide a gene related to full-dark heterotrophic ability that can be used to impart full-dark heterotrophic ability and to improve this trophic function in Cyanobacteria, and the use of this gene. [Solution] Cyanobacteria are transformed so as to inactivate the cytM gene that encodes cytochrome c_M protein. Inactivation of this gene imparts or potentiates full-dark heterotrophic ability in Cyanobacteria.

Description

Genes related to complete dark heterotrophic capacity and their utilization in cyanobacteria

The present specification relates to genes associated with complete dark heterotrophic ability in cyanobacteria and uses thereof.
This application is a related application of Japanese Patent Application No. 2014-046585, which is a Japanese patent application filed on March 10, 2014, and claims the priority based on this Japanese application. Is used.

Cyanobacteria are also called cyanobacteria (cyanobacterium) and are photoautotrophic bacteria. Utilizing the high photosynthetic ability of cyanobacteria, it is expected to be applied to the production of organic compounds such as biofuels and useful chemicals by genetic modification.

Cyanobacteria have high photosynthetic ability, but heterotrophic ability in the dark is not necessarily high. When natural light sources such as daylight are used to allow cyanobacteria to carry out photosynthesis, the growth rate decreases when there is not enough sunlight, etc. at night or under cloudy weather. There is a possibility that the production capacity of the substance may decrease. On the other hand, rather, the ability to produce useful substances may increase in the dark.

As a cyanobacteria, Synechocystissp. PCC 6803 is known as a standard strain. This cyanobacteria cannot grow when kept in a completely dark place and requires light irradiation for about 10 minutes a day (this phenomenon is also referred to as “Light-ActivatedActivHeterotrophic Growth” (LAHG)). On the other hand, there is Leptolyngbya boryana (L. boryana) (also called Plectonema boryanum) as a cyanobacteria. L. boryana belongs to the section III group of cyanobacteria, and is a type of cyanobacteria that does not differentiate into cells that specialize in nitrogen fixation called heterocysts. Unlike Synechocystis sp. PCC 6803, L. boryana is known to grow heterotrophically in the presence of sugar such as glucose even in a completely dark place. Moreover, a mutant strain having higher complete dark heterotrophic ability, that is, a faster growth in the dark has been isolated (Non-patent Document 1).

Many cyanobacteria do not have or are completely dark heterotrophic like the above-mentioned standard strains, and it is generally difficult to achieve both photoautotrophic and complete dark heterotrophic capacity in cyanobacteria. there were.

This specification provides a gene related to complete dark heterotrophic ability that can be used to impart complete dark heterotropy in cyanobacteria or to improve the nutrition, and uses thereof.

The present inventors have identified a mutation in the gene responsible for the mutation from a mutant strain of L. boryana excellent in complete dark heterosexuality. According to this specification, the following means are provided based on such knowledge.

(1) A cyanobacterial transformant comprising inactivation of a cytM gene encoding cytochrome c _M protein.
(2) The transformant according to (1), wherein the inactivation is the introduction of a mutation encoding the protein in which the cytM gene is disrupted or decreased in activity.
(3) The transformant according to (1) or (2), wherein the inactivation is a mutation that encodes a protein having the amino acid sequence represented by SEQ ID NO: 14 in the cytM gene.
(4) The cytM gene is any of the following proteins:
(A) a protein having the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and functionally associated with a cytochrome c _M protein Equivalent protein (c) A protein functionally equivalent to cytochrome c _M protein, having an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence represented by SEQ ID NO: 2 (D) a protein having an amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 1 (e) a protein encoded by a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 The transformant according to any one of (1) to (3), wherein the transformant encodes a protein functionally equivalent to the cytochrome c _M protein.
(5) The transformant according to any one of (1) to (4), which has photosynthetic autotrophic ability and complete dark heterotrophic ability.
(6) The transformant according to any one of (1) to (5), further comprising a foreign gene for producing a useful substance.
(7) A cyanobacteria having inactivation of the cytM gene encoding cytochrome c _M protein and having complete dark heterotrophic ability.
(8) The cyanobacteria according to (7), which is a cyanobacteria other than the L. boryana dg5 strain.
(9) The cyanobacteria according to (7) or (8), further comprising a foreign gene for producing a useful substance.
(10) A transformation vector for imparting complete dark heterotrophic ability to cyanobacteria, comprising a polynucleotide that imparts inactivation of the cytM gene encoding cytochrome c _M protein.
(11) The transformation vector according to (10), wherein the inactivation is introduction of a mutation encoding the protein in which the cytM gene is disrupted or decreased in activity.
(12) A method for producing the transformant according to any one of (1) to (6),
Transforming cyanobacteria with a polynucleotide conferring inactivation of the cytM gene encoding cytochrome c _M protein;
A manufacturing method comprising:
(13) The production method according to (12), wherein the inactivation is introduction of a mutation encoding the protein in which the cytM gene is disrupted or decreased in activity.
(14) The production method according to (12) or (13), wherein the transformation step introduces the polynucleotide into the cyanobacteria by electroporation.
(15) culturing the transformant according to any one of (1) to (6),
A method for producing a cyanobacterial culture product.
(16) The production method according to (15), wherein the culturing step includes culturing in a dark place.
(17) culturing the transformant according to any one of (1) to (6) in the dark,
A method for culturing cyanobacteria, comprising:

cytM gene shows a phylogenetic tree and alignment results with other cytochrome c class I protein cytochrome c _M protein closely related encoding. It is a diagram showing a mutation in the gene encoding cytochrome c _M protein in L. boryana IAM-M101 dg5 strain. It is a figure which shows the DNA construct for the homologous recombination with respect to the gene which may have a causal variation of complete dark heterotrophic ability. It is a figure which shows the evaluation result (agar medium) of the complete dark place heterotrophic ability after transformation. It is a figure which shows the evaluation result (liquid culture medium) of the complete dark place heterotrophic ability after transformation. It is a figure which shows the outline | summary of arrangement | positioning of the cytM gene on the genome of Synechocystis (sp) (PCC) 6803, and construction of (DELTA) cytM strain | stump | stock. It is a figure which shows the PCR result for confirmation of (DELTA) cytM strain | stump | stock. It is a figure which shows the growth comparison result of the wild strain (WT) and (DELTA) cytM strain | stump | stock on four growth conditions (agar medium). It is a figure which shows the dark place heterotrophic growth result of WT and (DELTA) cytM strain | stump | stock in a liquid medium. It is a figure which shows the growth comparison result on the short day and long day conditions in an agar medium.

The disclosure herein relates to genes associated with complete dark heterotrophic capacity in cyanobacteria and uses thereof. According to the disclosure herein, modification of cyanobacteria to provide for inactivation of the cytM gene encoding cytochrome c _M protein provides complete darkness to cyanobacteria with photoautotrophic properties against cyanobacteria. It can give a place dependent nutrition or enhance its complete dark place nutrition.

As a result, cyanobacteria use natural light energy such as solar energy and become mixed nutrients with complete dark heterotropy due to organic nutrition. Even in a suppressed state, it can continuously grow and produce useful substances efficiently. In addition, since the modified cyanobacteria can grow and produce useful substances even in the dark, it is possible to efficiently produce useful substances that are produced specifically in the dark.

Hereinafter, representative and non-limiting specific examples of the present disclosure will be described in detail with reference to the drawings as appropriate. This detailed description is intended merely to provide those skilled in the art with details for practicing the preferred embodiments of the present invention and is not intended to limit the scope of the present disclosure. Further, the additional features and inventions disclosed below can be used separately from or together with other features and inventions to provide further improved cyanobacteria and the like.

Further, the combinations of features and steps disclosed in the following detailed description are not essential in carrying out the present disclosure in the broadest sense, and are particularly only for explaining representative specific examples of the present disclosure. It is described. Moreover, various features of the representative embodiments described above and below, as well as those described in the independent and dependent claims, are described herein in providing additional and useful embodiments of the present disclosure. They do not have to be combined in the specific examples given or in the order listed.

All features described in this specification and / or claims, apart from the configuration of the features described in the examples and / or claims, are individually disclosed as limitations on the original disclosure and claimed specific matters. And are intended to be disclosed independently of each other. Further, all numerical ranges and group or group descriptions are intended to disclose intermediate configurations thereof as a limitation to the original disclosure and claimed subject matter.

Hereinafter, the disclosure of this specification will be described in detail.

(Cyanobacteria transformant)
The cyanobacterial transformants disclosed herein can comprise inactivation of the cytM gene encoding cytochrome c _M protein (also referred to as Cytochrome c _M ).

(Cyanobacteria)
In the present specification, cyanobacteria are gram-negative true bacteria and are prokaryotic photosynthetic microorganisms that perform oxygen-generating photosynthesis. Cyanobacteria herein refers to the ability of heterotrophic ability to grow or proliferate as a carbon source by organic compounds such as sugars, fatty acids and amino acids.

Also, in this specification, the word “dark place” includes a complete dark place and a limited dark place. Here, the complete dark place means a state in which light is blocked during the growth or multiplication period, except that it is placed under light irradiation for a time and a necessary amount necessary for artificial manipulation for growth or multiplication. . Light irradiation conditions that are unavoidable for artificial manipulation for growth or proliferation are, for example, conditions in which light irradiation is performed within 10 to 30 minutes, preferably 20 minutes or less once every 10 days to 2 weeks. is there. On the other hand, the limited dark place means a state in which light is blocked except that it is placed under light irradiation for about 10 to 20 minutes, preferably about 10 minutes per day.

Cyanobacteria cannot grow in complete darkness, but may exhibit dependent nutrition in limited darkness. This phenomenon is also called “Light-ActivatedActivHeterotrophic Growth” (LAHG). On the other hand, the case where it is possible to grow heterotrophically in a completely dark place is called complete dark place heterotrophic ability. The cyanobacteria may or may not have such dark heterotrophic ability.

Cyanobacteria are prokaryotes that carry out oxygen-generating photosynthesis, and are not particularly limited, and include, but are not limited to, the order of the order of the order Chroococcus, Pleurocapsa, Euremo, Nemoptera, Stigonema, Groeobacter. Furthermore, Synechococcus genus such as Synechococcus elongatus PCC 7942, Prochlorococcus genus such as Prochlorococcus marinus MED4, Anabaena genus such as Anabaena sp. PCC 7120, Synechocystis genus such as Synchocystis sp. Examples include the Gloeobacter genus such as Gloeobacter violaceus PCC 7421, the Arthrospira genus such as Arthrospira platensis, the Trichodesmium genus such as Trichodesmium erythraeum IMS101, and the Acaryochloris genus such as Acaryochloris marina MBIC11017.

(CytM gene)
The cytochrome c _M protein encoded by the cytM gene is thought to function as a Cu domain electron donor of cytochrome c oxidase, although its function is not necessarily clear (Bernroitner et al. 2009, Biochim Biophys Acta 1787 ( 3): 135-43).

In general, the base sequence of the cytM gene in cyanobacteria is already known. L. boryana IAM-M101 nucleotide sequence encoding a cytochrome c _M protein cytM gene is represented by SEQ ID NO: 1, The amino acid sequence of the protein shown in SEQ ID NO: 2. L. boryana IAM-M101 and its mutant L. boryana IAM-M101 dg5 are a part of the Graduate School of Bioagricultural Sciences, Nagoya University (No. 1 Fufu-cho, Chikusa-ku, Nagoya, Aichi, Japan). Is possible.

L. boryana is a filamentous cyanobacteria. Cyanobacteria are classified into sections I to V because of their morphological characteristics, whereas L. boryana is classified into section III, which has the morphological characteristics of filamentous properties that do not form heterocysts. Section III is further subdivided according to the morphological characteristics of the filamentous body and the presence or absence of gas vesicles, but forms linear filaments (not branched) from cells of equal diameter, without gas vesicles, and between cells L. boryana, which has the characteristic of no constriction, belongs to LPP group B. L. boryana is a type that does not differentiate into cells specialized in nitrogen fixation called heterocysts.

L. boryana can grow heterotrophically in the presence of sugar such as glucose even in a completely dark place (complete dark place heterotrophic ability). In addition, L. 、 boryana can introduce DNA into cells by electroporation and obtain mutants in which a specific gene is destroyed by homologous recombination with the genome (Fujita et al. 1992, Plant Cell Physiology). , 33: 81-92).

In addition, as a cyanobacterial cytM gene, for example, a gene encoding a protein having the following amino acid sequence is known. SEQ ID NOs: 3 to 12 are cytochrome cM proteins. On the other hand, SEQ ID NO: 13 is a cytochrome c class I protein, but shows only about 30% identity (%) with the amino acid sequence represented by SEQ ID NO: 2, and is functionally considered as a cytochrome c6 protein.

 

The cytM gene to be inactivated in the present specification has an amino acid sequence having a certain identity with the amino acid sequence of a known cytochrome c _M protein in addition to those already disclosed in this way, and the cytochrome c _M protein It may encode proteins that are functionally equivalent. Such a gene to be inactivated preferably has, for example, 60% or more identity with the amino acid sequence represented by SEQ ID NO: 2, more preferably 65% or more identity, 70% or more identity, preferably 75% or more, more preferably 80% or more, still more preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 97% or more, More preferably, it has 98% or more, and most preferably 99% or more identity.

Further, for example, the cytM gene may be a protein encoding a protein that, when aligned, comprises a -NCXXCH-motif and a -TPPMP-motif. These two motifs are commonly observed in cytochrome c _M protein in this specification (Bernroitner et al Biochem Biophys Acta, 2009, 1787:... 135-143). For example, these motifs correspond to positions 36 to 41 and positions 74 to 78 in the amino acid sequence represented by SEQ ID NO: 2, respectively. Therefore, CytM gene, for example, when the amino acid sequence alignment of SEQ ID NO: 2, found these motifs at positions corresponding to these, moreover, cytochrome c _M protein functionally equivalent to a protein that is a May be encoded. FIG. 1 shows the phylogenetic tree and alignment results for cytochrome c _M protein encoded by the cytM gene and other related cytochrome c class I proteins.

In the present specification, “identity” in an amino acid sequence or a base sequence is used to mean the degree of coincidence of amino acid residues or bases constituting each sequence between sequences to be compared. At this time, regarding the amino acid sequence, the existence of a gap and the nature of the amino acid are considered (Wilbur, Proc. Natl. Acad. Sci. U.S.A. 80: 726-730 (1983)). For the calculation of identity, commercially available softwares such as BLAST (Altschul: J. Mol. Biol. 215: 403-410 (1990)), FASTA (Peasron: Methods in Enzymology 183: 63-69 (1990)), etc. Can be used. Any numerical value of “identity” may be a numerical value calculated using a homology search program known to those skilled in the art. For example, the homology algorithm BLAST (BasicＢlocal alignment) of the National Center for Biotechnology Information (NCBI) search)) http://www.ncbi.nlm.nih.gov/BLAST/, using default (initial setting) parameters.

Functionally equivalent to cytochrome c _M protein means maintaining substantially the same level of function to express or enhance complete dark heterotrophic capacity. The functional equivalence is determined by comparing the gene-disrupted cyanobacteria that have disrupted the gene in cyanobacteria with the non-disrupted cyanobacteria, where the complete dark heterotrophic ability is expressed or enhanced, or limited darkness. This can be determined by the fact that the place's heterotrophic capacity is enhanced to become complete dark place heterotrophic. That is, in gene-disrupted cyanobacteria, when the complete dark heterotrophic ability is expressed and enhanced or is completely dark heterotrophic, the protein encoded by the gene is functionally equivalent to the cytochrome c _M protein. Can be determined. In addition, the gene can be determined to be functionally equivalent to the cytM gene.

Further, CytM gene, the amino acid sequences of known cytochrome c _M protein, 1 or insertion of a plurality of amino acids, substitutions, cytochrome c _M protein functionally equivalent have deletions and additions in the amino acid sequence It may encode a protein. Such proteins have, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, more preferably 1 to 2 amino acid insertions, substitutions and deletions. And additions can be provided.

Such a protein has an amino acid sequence having a conservative substitution of one or several, preferably 1 to 10, more preferably 1 to 5, more preferably about 1 to 2 amino acids in the known cytochrome c _M protein. It is preferable that

Here, “conservative substitution” means substitution of one or more amino acid residues with another chemically similar amino acid residue so as not to substantially alter the function of the protein. For example, when a certain hydrophobic residue is substituted by another hydrophobic residue, a certain polar residue is substituted by another polar residue having the same charge, and the like. Functionally similar amino acids that can make such substitutions are known in the art for each amino acid. Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Furthermore, the cytM gene is a complementary DNA that hybridizes under stringent conditions with a DNA encoding a known cytochrome c _M protein, and encodes a protein functionally equivalent to the cytochrome c _M protein. It may be a thing.

“Stringent conditions” means that the reaction is carried out at a temperature of 40 ° C. to 70 ° C., preferably 60 ° C. to 65 ° C. in a hybridization buffer that can be usually used by those skilled in the art. It can be carried out according to a method of washing in a washing solution of 300 mmol / L, preferably 15 to 60 mmol / L. The temperature and salt concentration can be appropriately adjusted according to the length of the probe used. Furthermore, the conditions for washing the hybridized material can be 0.2 or 2 × SSC, 0.1% SDS, and a temperature of 20 ° C. to 68 ° C. Whether the conditions are stringent (high stringency) or mild (low stringency), a difference can be made depending on the salt concentration or temperature at the time of washing. When providing a difference in hybridization at the salt concentration, 0.2 × SSC as a stringent wash buffer (high バ ッ フ ァ ー stringency wash buffer), 0.1% SDS, 2 × SSC as a mild washbuffer (low stringency wash buffer), This can be done with 0.1% SDS. In addition, in the case of providing a difference in hybridization by temperature, 68 ° C for stringent, 42 ° C for moderate-stringency, and room temperature (20 ° C to 25 ° C) for mild May be performed with 0.2 × SSC and 0.1% SDS.

The cytM gene may have a base sequence having a certain identity with a base sequence encoding a known cytochrome c _M protein, and may encode a protein that is functionally equivalent to the cytochrome c _M protein. Such a gene to be inactivated preferably has, for example, 60% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 encoding a known cytochrome c _M protein, more preferably 65% or more. More preferably 70% or more, more preferably 75% or more, more preferably 80% or more, still more preferably 85% or more, more preferably 90% or more, and still more preferably 95%. More preferably, it has an identity of 97% or more, more preferably 98% or more, and most preferably 99% or more.

In the present specification, “the insertion, substitution or deletion of one or a plurality of amino acids, or addition to one or both ends thereof in the amino acid sequence” is a well-known site-directed mutagenesis method or the like. Or by substitution of a plurality of amino acids to the extent that can occur naturally.

(Inactivation of cytM gene)
It has inactivation of the cytM gene in cyanobacteria. Here, the inactivation of the gene may be anything as long as the activity of the protein encoded by the gene is reduced or lost as a result. Therefore, gene inactivation includes gene disruption, transcription inhibition, transcript degradation, translation inhibition, translation of inactive proteins, and the like.

Such gene inactivation is generally performed in the form of gene disruption, for example, insertion of foreign sequences into the cytM gene, replacement of all or part of the cytM gene by foreign sequences, cytM gene Can be done by deletion of all or part of The number of bases in the foreign sequence and the positions of substitutions, deletions and insertions are not particularly limited as long as the expression and activity of cytochrome c _M protein is substantially lost. In addition, from the viewpoint of selecting a gene recombinant, the foreign sequence is preferably a selection marker gene. The selectable marker gene can be appropriately selected from known ones and is preferably a drug resistance gene.

The cytM gene can be disrupted preferably by replacing all or part of the cytM gene with a foreign gene. The part of cytM gene to be replaced, for example, the form to replace the C-terminal side of the cytochrome c _M protein.

Further, when the cytM gene is disrupted, a protein with reduced activity may be produced by introducing a mutation into the protein encoded by the cytM gene. For example, it may be a frameshift mutation by inserting or deleting a base in the coding sequence, or a part of the protein may be substituted with a different amino acid, or a combination thereof. It may be a thing.

Gene disruption techniques are known to those skilled in the art, and those skilled in the art can perform gene disruption according to known methods. Gene disruption generally involves introducing into a cell a polynucleotide (preferably a selectable marker gene) in which the nucleotide sequence of the target gene has been modified, and causing homologous recombination between the introduced polynucleotide and the target gene. By selecting cells in which homologous recombination has occurred, mutations can be introduced into the target gene.

In addition, in order to inactivate the cytM gene, for example, it may be a variant which encodes a protein consisting of the amino acid represented by the cytochrome c _M protein in SEQ ID NO: 14.

The transformant disclosed in this specification is a cyanobacteria equipped with inactivation of the cytM gene. When an endogenous cytM gene is provided, inactivation of the cytM gene can be imparted, and dark heterotrophic ability can be imparted or enhanced to the cyanobacteria.

(Production of transformation vector and transformant)
In order to impart inactivation of the cytM gene to cyanobacteria, a transformation vector comprising a polynucleotide for imparting inactivation of various forms of the cytM gene already described can be used. Polynucleotides are generally DNA and RNA, but are typically DNA. This transformation vector can be used as a vector for imparting dark heterotrophic ability to cyanobacteria. In addition, the above-described polynucleotide (for example, a polynucleotide cassette having at least a homology region for destroying and replacing the target cytM gene) is itself a dark place for imparting dark heterotrophic ability to cyanobacteria. It can be used as a heterotrophic agent.

The polynucleotide on the transformation vector has, for example, an appropriate homologous recombination region capable of disrupting one or more cytM genes on the chromosome of cyanobacteria or introducing mutations, etc. for example, with or substituted region encoding a foreign gene or the like, a replacement area) for the mutation for the production of drug resistance marker region and inactive cytochrome c _M protein.

The transformation vector may further contain one or more enhancers or silencers, an operator, a terminator, a polyadenylation signal, one or more drug resistance genes, a cloning site for inserting a foreign gene, and the like.

Drug resistance genes include, but are not limited to, chloramphenicol resistance gene, neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, ampicillin resistance gene, spectinomycin resistance gene and the like.

The transformation vector can take various forms without particularly specifying the form. Although it may be a DNA fragment, it is typically a plasmid vector, but for example, non-plasmid vectors such as viral vectors can also be used. Also preferably, the transformation vector disclosed herein is a shuttle vector for adapting to multiple host cells. For example, vectors that can be introduced into any cell of E. coli and cyanobacteria and can express foreign genes in these host cells can be mentioned. Such shuttle vectors can be replicated in large quantities using E. coli.

The origin of the parent vector of the transformation vector disclosed in the present specification is not particularly limited as long as the gene can be expressed efficiently with cyanobacteria. For example, pVZ321 (Zinchenko VV, Piven IV, Melnik VA, Shestakov SV (1999) Genetika 35: 291-296), pMB1 (Kreps S, Ferino F, Mosrin C, Mergeay C, Mergeay), vectors used in cyanobacteria Known vectors such as M, Thuriaux1990P (1990) Mol. Gen. Genet.133221: 129-133), RSF1010 (Meyer R (2009) Plasmid 62: 57-70) can be used. To insert each region into the vector, a ligase reaction or the like can be used as appropriate.

The transformation vector may further comprise a foreign gene to be expressed in cyanobacteria. The foreign gene may be a gene introduced from the outside by a transformation vector or the like, and may be a gene endogenous to cyanobacteria. The foreign gene may encode various useful substances or one or more enzymes for producing the useful substances. For example, proteins useful as pharmaceutical ingredients (eg, interferon, insulin, erythropoietin, human growth hormone, various cytokines), proteins useful as reagents (eg, DNA / RNA polymerase, protease, restriction enzyme), fluorescent proteins (eg, GFP, DsRed, Phycocyanin), bioplastic production related proteins (eg polyhydroxyalkanoic acid (PHA), polyhydroxybutyric acid (PHB) or polyhydroxyvaleric acid (PHV) synthase), amino acid synthases, biofuel related synthases (eg triglycerides, fatty acids) (Such as stearic acid or oleic acid) or hydrocarbon (alkane / alkene) synthase, hydrogen producing enzyme), antibacterial active substance synthase, functional substance synthase (for example, γ-linolenic acid, docosahexaene Acid (DHA), eicosapentaenoic acid, β-carotene, and astaxanthin) and the like, and biofuel-related synthase genes are particularly preferable. Also, thioesterase gene for fatty acid production, structural gene of nitrogenase, nifE, nifN, nifH, nifU, nifS, nifB, nifP, nifM nifV, nifZ, nifX, nifW, hesA, hesB, fdxN, fdxH, Examples include genes involved in metal-center biosynthesis of nitrogenase, hydrogenase structural genes, and genes involved in metal-center biosynthesis of hydrogenases such as hydE and hydG. A gene encoding such a useful substance is a known method such as a PCR method or a hybridization method using a primer or probe designed from information on the base sequence or amino acid sequence registered in an existing database such as GenBank. It can be obtained by.

A cyanobacterial transformant can be obtained by using a known transformation method for cyanobacteria using the transformation vector disclosed herein for cyanobacteria. As a transformation method of cyanobacteria, for example, natural transformation method and electroporation method can be used. Further, a junction transmission method can be used. The natural transformation method is a method in which gene introduction is possible only by mixing cyanobacteria and a transformant vector. The electroporation method is a method for introducing a gene by applying an electric pulse to a mixed solution of cyanobacteria and a transformant vector. The conjugation transfer method is a method in which a plasmid into which a gene to be introduced is inserted is transformed into E. coli, and then the E. coli and cyanobacteria are mixed to introduce the gene. Those skilled in the art can appropriately implement these methods. Moreover, in order to select a transformant, the drug resistance gene etc. which were hold | maintained at the transformation vector can be used.

The thus obtained transformant, cyanobacteria, can have the inherent photosynthetic autotrophic ability and the dark heterotrophic ability imparted for the first time or the enhanced dark heterotrophic ability. The presence or degree of heterotrophic ability in the dark can be determined by observing growth or proliferation when transformants are cultured under heterotrophic conditions using a medium containing organic nutrients such as glucose in the dark. Judgment can be made.

Since the transformant of the present disclosure has imparted or enhanced dark heterotrophic ability, it uses natural light energy such as sunlight and is in a depleted state (dark place) of the light energy. Because it can grow and proliferate in a heterotrophic manner, continuous growth and material production are possible throughout the day and night. That is, efficient and practical growth and material production are possible. In addition, the substances to be produced can be made different by growing them only in the dark dependent conditions or only in the presence of light energy as necessary.

The transformant of the present disclosure can further include a foreign gene for producing a useful substance. According to such a transformant, a useful substance can be efficiently and / or selectively produced using light energy and an organic compound.

In addition, according to the above indication, the manufacturing method of the transformant disclosed by this specification is also provided. Moreover, according to the above disclosure, a cyanobacteria that is not a transformant but has inactivation of the cytM gene and has dark heterotrophic ability is also provided. This is because screening for cyanobacteria based on the inactivation or absence of the cytM gene makes it possible to easily select cyanobacteria having excellent dark heterotrophic ability from natural cyanobacteria. As natural cyanobacteria, it is preferable to target cyanobacteria other than L. boryana. Moreover, a practical cyanobacteria can be obtained by introducing and transforming a foreign gene for producing a useful substance into the thus screened cyanobacteria according to the above method.

(Production method of cyanobacterial culture product)
According to the disclosure of the present specification, a method for producing a culture product of cyanobacteria is also provided. This production method may comprise a step of culturing a cyanobacteria or cyanobacterial transformant provided with inactivation of the cytM gene disclosed in the present specification. According to this production method, since the dark heterotrophic ability is imparted or enhanced, cyanobacteria can be cultured continuously, efficiently, or selectively, and the culture product can be obtained.

This production method preferably includes a culture step under heterotrophic conditions in the dark particularly because the complete dark heterotrophic ability is imparted / enhanced or modified in the complete dark heterotrophic capacity.

The culture product can include cultured cells and culture supernatant, but may be a part thereof. When a foreign gene is introduced in cyanobacteria and a useful substance based on the expression of the foreign gene is produced, the useful substance is also included in the culture product.

As the culture process, natural light energy conditions such as solar energy, heterotrophic conditions and the like can be appropriately combined. As other culture conditions, known culture conditions for cyanobacteria can be employed.

According to the above disclosure, a method for culturing cyanobacteria is also provided. This culture method can comprise a step of culturing cyanobacteria or cyanobacteria transformants having inactivation of the cytM gene in the dark. According to this culture method, cyanobacteria can be grown and propagated without light energy. In addition, cyanobacteria can grow and proliferate even under autotrophic conditions under light energy, enabling efficient culture.

(Isolation of dg5)
The natural mutant dg5 of Leptolyngbya boryana (formerly Plectonema boryanum) IAM-M101 with improved complete dark heterotrophic ability was isolated from a wild strain by Fujita in 1991 (Fujita et al. 1996, Plant and Cell Physiology 37: 313-323).

(Redetermining the genome sequence of the wild strain)
The genome sequence of the wild strain (WTo) was determined as described below, and compared with a known dg5 genome sequence.

Genomic DNA was extracted using the Wizard Genomic DNA Purification kit (Promega) as shown below and partially modified from the manual. About 20 mg of WTo cells were suspended in 450 μl of a buffer solution (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 5 mM NaCl, EDTA), and 50 μL of lysozyme (2.5 mg / 50 μl) was added. Then, it left still at 37 degreeC for 1 hour. The sample was centrifuged (15,000 rpm, 2 minutes, room temperature), and the supernatant was removed. From the procedure of adding NucleileLysis Solution 0.6 ml to the subsequent precipitate, the procedure was followed according to the Wizard Genomic DNA Purification kit (Promega) manual.

As a genome sequencing method, one of the high-speed sequencers, SOLiD system 5500, was used. A paired-end genomic library was created according to the manual and sequenced. As a result, sequence data from both sides of 4,802,686 library DNA fragments were obtained. At this time, as a control, sequence data was obtained using SOLiD system 5500 in the same manner as the genomic sequence of dg5. The data was mapped using the analysis software BioScope® (Applied® Biosystems) with the dg5 genomic sequence as a reference, and then single nucleotide polymorphism (SNP) and insertion deletion (Indel) analysis was performed. The initial setting was used for the setting at that time.

As a result of re-determining the genome sequence, there were four differences between dg5 and WTo genomes (Table 2). However, the genome sequences of other mutants derived from dg5 were also redetermined in the same way, and for one of the four sites, the mutant derived from dg5 had the same sequence as WTo, so this mutation was dg5 After being isolated, it was considered a recently fixed mutation and was excluded from the candidates. As a result, the region containing the corresponding site for the remaining 3 sites was amplified by colony direct PCR using WTo cells with the primers shown in Table 3, and the nucleotide sequence of the fragment was determined using the Sanger method. It was confirmed that the sequence of WTo is different from the sequence of dg5. The primers used in the sequencing by the Sanger method are Pb1305262-r, Pb3151287-f, and Pb5175165-f.

FIG. 2 shows an overview of mutations at dg5 in the cytM gene. It was found that truncation of the protein was caused by the frameshift mutation.

(Construction of plasmid used to introduce dg5 mutation into WTo by transformation)
In order to determine which of the three genetic differences between dg5 and WTo identified in Example 2 promotes complete dark heterotrophic viability, each dg5-type nucleotide sequence was individually introduced into WTo.

As a method, three fragments having the dg5 mutation were ligated using overlap PCR, and this was cloned into the BamHI or BamHI and KpnI sites of pUC19, which is a high copy plasmid. At this time, Escherichia coli JM105 strain was used for plasmid construction. The base sequences of the primers used are shown in Table 4, and the annealing sites in the target DNA fragment are shown in FIG. Two fragments serving as scaffolds for homologous double recombination were subjected to PCR using the combination of primer-f1 and primer-r1 and the combination of primer-f3 and primer-r3 using the dg5 genome as a template. The kanamycin resistance cassette was amplified using primer-f2 and primer-r2 using pUCK121 as a template. Each fragment has the same sequence at the end of 30 bp between the fragments to be ligated by base addition to the primer used for amplification. By performing secondary PCR using primer-f1 and primer-r3 using these three fragments as templates, the target DNA fragment in which the three fragments were ligated was obtained. Primer-f1 and primer-r3 are designed so that this fragment has a restriction enzyme site. The restriction enzyme was treated with BamHI or BamHI and KpnI, and cloned into the corresponding cloning site of pUC19.

In addition, regarding the mutation of Chr; 5175165, it was assumed that the mutation caused a frame shift and caused a loss of function of the gene. Therefore, a plasmid for preparing a completely defective strain excluding the entire coding region of the gene was similarly prepared.

For the preparation of the control strain, a plasmid in which only the drug resistance marker not having each dg5 mutation was inserted was also prepared. For this purpose, PCR was performed using the WTo genome instead of the dg5 genome as a template at the stage of PCR amplification of only the three DNA fragments that retained the dg5 mutation, and the others followed the procedure for constructing the plasmid described above. .

Double-homologous recombination into WTo was performed by electroporation (Fujita et al. 1992, Plant Cell Physiology, 33: 81-92). For the transformation, PCR fragments obtained using primer-f1 and primer-r3 using the prepared plasmid as a template were used. For the WTo culture, an agar medium containing BG-11BG (20 M HEPES-KOH; containing pH 7.5, hereinafter referred to as BG-11H) and agar (BactoAgar, Difco) 15% (w / v) was used. In addition, a white fluorescent lamp (FLR40SW / M / 36-B, Sakai Hitachi) was used as a culture light source. WTo cells that have been photosynthesised on agar at 30 ° C are collected in water, aspirated on 150 mL Bottle Top Vacuum Filter, 0.22 μm Pore (Corning), resuspended in sterile cold water, and then aspirated. The water was removed. The salt was removed from the cell suspension by performing this operation three times in total. The cells were resuspended in 0.5-2.0 ml of chilled sterilized water and used as a competent cell for electroporation.

A 50 μl competent cell and 10 μl of each PCR fragment were mixed and subjected to an attenuation pulse treatment at a voltage of 1410 V, a resistance of 250Ω, a capacitance of 25 μF, and a distance between electrodes of 1 mm. The pulsed cells 350 [mu] l of BG-11 (20 mM HEPES- KOH; pH8.2 containing) was recovered by the addition of a nylon membrane ^{(Amersham Hybond TM -N +, GE} Healthcare) on an agar medium BG-11H bearing the Spread out. After growing for 2 days at 30 ° C, light intensity 2 μmol _photon m ^-2 s ^-1 , transferred to agar medium BG-11H supplemented with kanamycin (final concentration 15 mg / L) with nylon membrane, light intensity 50 μmol _photon Drug-resistant strains were selected under irradiation with m ⁻² s ⁻¹ .

Two weeks later, the individual dg5 mutant strains selected as kanamycin resistant strains were dm1 (having Chr; 1305262 mutation), dm2 (having Chr; 3151287), dm3 (having Chr; 5175165), dm3n (Chr; 5175165 And a gene (ORF No. LBDG_45050) completely deficient strain). In order to confirm the introduction of dg5 mutation in these mutant strains, DNA fragments were directly amplified from the cells by PCR using primer-f1 and primer-r3 shown in Table 3. At the same time, it was confirmed that the kanamycin resistance cassette was inserted into the genome by double recombination.

Further, the DNA fragment was purified from an agarose gel and used as a template, Chr; 1305262 was a combination of Pb1305262-f and primer-r2, Chr; 3151287 was a combination of Pb3151287-f and primer-r2, Chr: 5175165 In contrast, secondary PCR was performed with a combination of primer-f2 and Pb5175165-r, and the resulting DNA fragment was sequenced by the Sanger method to confirm the introduction of the dg5 mutation. The primers used for sequencing by the Sanger method are Pb1305262-f, Pb3151287-f, and Pb5175165-r shown in Table 1.

(Comparison of complete dark heterotrophic growth of transformants)
Growth under complete heterotrophic conditions using the four mutant strains obtained in Example 3 and their control strains (dm1c, dm2c, dm3c; strains that are resistant to kanamycin but have no corresponding mutation introduced) A comparison was made.

Growth conditions of dg5, WTo and the above mutants on agar medium BG-11H with and without 30 mM glucose in the dark or light (light intensity 50 μmol _photon m ^-2 s ^-1 ) Growth was confirmed. The plants were grown for 14 days under completely dark heterotrophic conditions and for 3 days under photosynthesis conditions. A white fluorescent lamp (FLR40SW / M / 36-B, Hitachi) was used as a culture light source. The results are shown in FIG.

In addition, for the four strains WT, dg5, dm3, and dm3c, dark heterotrophic growth and photosynthetic independent growth were evaluated by liquid culture. As pre-culture, the _cells were grown for 3 days photosynthesis at 30 ° C. and light intensity of 60 μmol _photon s ⁻¹ m ⁻² . At this time, 25 cm ² Tissue Culture Flask (IWAKI) was used as a culture vessel, and shaking was performed with ROTARY SHAKER NR-20 (TAITEC) (rotation speed 140 rpm). A preculture with a turbidity (OD ₇₃₀ ) of approximately 0.4 was used as an inoculum, inoculated into a new liquid medium so that the initial turbidity OD ₇₃₀ = 0.05, and growth was monitored under each condition. For evaluation of dark heterotrophic growth, 15 ml BG-11 medium with a final concentration of 30 mM glucose was added to 25 cm ² Tissue Culture Flask (IWAKI) shielded from aluminum foil, and cultured with shaking at a rotational speed of 140 rpm. In addition, for the evaluation of photosynthetic autotrophic growth, 15 ml BG-11 medium without glucose was used, and the light intensity was 60 μmol _photon s ^-1 m ^-2 without shading, and the other conditions were cultured in the same manner . A white fluorescent lamp (FLR40SW / M / 36-B, Hitachi) was used as a culture light source. The results are shown in FIG.

As shown in FIG. 4, the photosynthetic growth in the light place is not significantly different in all strains regardless of the presence or absence of glucose, but in the dark place, only dm3 and dm3n are remarkably good like dg5. Showed growth. This is presumed that the mutation (single nucleotide insertion) of Chr; 5175165 to dg5 caused a frame shift in LBDG_45050 and caused gene deletion. LBDG_45050 the result of homology search, it was found that encodes a cytochrome c _M. Cytochrome c _M deficiency is a causal mutation that promotes dg5's complete dark heterotrophic viability.

In addition, as shown in FIG. 5, even in the liquid medium, no significant difference was observed in all the strains in the light place, but only dm3 and dm3n showed significantly good growth like dg5 in the dark place. . dg5 and dm3 grew under the light autotrophic growth conditions in the same way as WT and dm3c, but under dark heterotrophic growth conditions, the growth rate and final turbidity were significantly higher.

In this example, a cytochrome cM-deficient strain of cyanobacteria (Synechocystis sp. PCC 6803) was produced and evaluated for improvement in heterotrophic growth. That is, as shown in FIG. 6, the ΔcytM strain was isolated by replacing the entire coding region of cytM (sll1245) with a gentamicin resistance cassette. In FIG. 6, the black bar indicates the genomic region used for the plasmid constructed for isolation of ΔcytM.

In the ΔcytM strain, the cytM gene was completely replaced with the gentamicin resistance cassette (gen) 、, and colonies of WT (lane 1) and ΔcytM strain (lane 2) were obtained with primers f4 and r4 specific to this cassette. It was confirmed by PCR. The results are shown in FIG. Lane 3 shows the results of PCR using a plasmid used for gene disruption as a template. From these results, it was found that the cytM gene was surely disrupted in the ΔcytM strain.

Next, WT and ΔcytM strains were compared in terms of growth under photosynthetic autotrophic conditions (lane 1), photosynthetic mixed nutrient conditions (lane 2), LAHG conditions (lane 3), and dark heterotrophic conditions (lane 4). 5.0 μl of a cell suspension with an OD ₇₃₀ of 0.1 was spotted. In FIG. 8, lanes 1 and 2 were cultured in a BG-11H medium for 3 days, and lanes 3 and 4 were cultured in a glucose-containing BG-11H medium for 10 days. In addition, LAHG (Light-activated heterotrophic growth photoactivated heterotrophic) conditions indicate that light irradiation was performed every 24 hours for 15 minutes, and otherwise, the culture was performed in the dark. The results are shown in FIG.

As shown in FIG. 8, ΔcytM was not different from WT under the photosynthetic condition (lane 1), but growth was significantly promoted under WT in the mixed nutrient condition with glucose (lane 2). Furthermore, the difference was most noticeable under LAHG conditions (lane 3). The WT ("YF") used in this study is a strain that does not grow well under LAHG conditions. Furthermore, under dark heterotrophic conditions (lane 4), WT did not grow at all, but ΔcytM showed clear growth.

Next, cover the culture bottle of WT and ΔcytM with aluminum foil in a glucose-containing BG-11H liquid medium (15 ml), completely shield the light, and perform a rotating culture at 140 rpm (Shaker NR-20, Tytech). Evaluation was performed by OD ₇₃₀ (spectrophotometer UV-1700, Shimadzu). The results are shown in FIG.

As shown in FIG. 9, in the liquid medium, the growth of WT almost ceases after 24 hours, but the ΔcytM strain grows at a generation time of about 33 hours (logarithmic growth phase from 24 to 92 hours), and an agar medium. It was confirmed that the proliferation was clearly promoted from the results of (1).

Further, WT and ΔcytM strains of Synechocystis sp. PCC 6803, photosynthesised on agar medium BG-11H for 6 days, were suspended in sterilized water, and turbidity (OD ₇₃₀ ) was adjusted to 0.1. 0 μl was spotted on an agar medium BG-11H (photosynthesis autotroph) and glucose (5 mM) -containing BG-11H (photosynthesis mixed nutrition) agar medium. Under short-day conditions (8L16D), the cells were cultured for 4 days under 8 hours light conditions-16 hours dark conditions, and under long days conditions (16L8D) under 16 hours light conditions-8 hours dark conditions. The results are shown in FIG.

As shown in FIG. 10, there is no difference in the growth of the WT and ΔcytM strains on both the short and long days under the photosynthetic conditions. However, under the mixed nutrient conditions in the presence of glucose, the growth of ΔcytM is observed under the short day conditions. You can see that it promotes more. Also, ΔcytM growth appears significantly better than WT even under long day conditions. From this result, it was found that the ΔcytM strain exhibited better growth in the presence of glucose even in an environment with a light-dark cycle.

SEQ ID NOs: 15-42: Primers

Claims (14)

1. A cyanobacterial transformant comprising inactivation of the cytM gene encoding cytochrome c _M protein.
2. The transformant according to claim 1, wherein the inactivation is introduction of a mutation encoding the protein in which the cytM gene is disrupted or decreased in activity.
3. The transformant according to claim 1 or 2, wherein the inactivation is a mutation that encodes a protein having the amino acid sequence represented by SEQ ID NO: 3 in the cytM gene.
4. The cytM gene is a protein according to any of the following:
   (A) a protein having the amino acid sequence represented by SEQ ID NO: 2 (b) an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and functionally associated with a cytochrome c _M protein Equivalent protein (c) A protein functionally equivalent to cytochrome c _M protein, having an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence represented by SEQ ID NO: 2 (D) a protein having an amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 1 (e) a protein encoded by a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 The transformant according to any one of claims 1 to 3, which encodes a protein functionally equivalent to the cytochrome c _M protein.
5. The transformant according to any one of claims 1 to 4, which has photosynthetic autotrophic ability and complete dark place heterotrophic ability.
6. The transformant according to any one of claims 1 to 5, further comprising a foreign gene for producing a useful substance.
7. A transformation vector for imparting heterotrophic ability to cyanobacteria, comprising a polynucleotide that imparts inactivation of a cytM gene encoding cytochrome c _M protein.
8. The transformation vector according to claim 7, wherein the inactivation is introduction of a mutation encoding the protein in which the cytM gene is disrupted or decreased in activity.
9. A method for producing the transformant according to any one of claims 1 to 6,
   Transforming cyanobacteria with a polynucleotide conferring inactivation of the cytM gene encoding cytochrome c _M protein;
   A manufacturing method comprising:
10. The production method according to claim 9, wherein the inactivation is the introduction of a mutation encoding the protein whose cytM gene has been disrupted or reduced in activity.
11. The method according to claim 9 or 10, wherein in the transformation step, the polynucleotide is introduced into the cyanobacteria by electroporation.
12. Culturing the transformant according to any one of claims 1 to 6,
    A method for producing a cyanobacterial culture product.
13. The production method according to claim 12, wherein the culturing step includes culturing in a dark place.
14. A step of culturing the transformant according to any one of claims 1 to 6 in a dark place;
    A method for culturing cyanobacteria, comprising:

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