WO2015182719A1 - Method for improving yield of substance from micro-organism, and kit using said method - Google Patents

Abstract

　Provided are: a modified host cell formed by introducing into a micro-organism host cell a polynucleotide that codes tRNA having a high usage frequency relative to the amount supplied; a method for manufacturing said modified host cell; a nucleic acid molecule containing said polynucleotide for modifying the host cell; a vector and kit; and a method for using the polynucleotide to improve the yield of a substance from a micro-organism. The introduction of the gene that codes the tRNA having a high usage frequency makes it possible to increase the expression of proteins in the micro-organism host cells.

Description

Method for improving substance productivity of microorganisms and kit used for the method

The present invention relates to a method for improving substance productivity by microorganisms, and a modified host cell and kit used in the method.

Material production techniques such as recombinant protein expression using microorganisms are used to produce various useful substances. In order to industrially mass-produce target substances using microorganisms, it is required to increase the expression efficiency of target proteins themselves or enzyme proteins involved in biosynthesis of target compounds.

In the recombinant expression of a protein using a microorganism as a host cell, a decrease in expression efficiency due to a gene encoding the protein containing a so-called rare codon that is not frequently used in the host cell can be a problem. As means for solving such problems, (1) a method in which a modified gene in which a rare codon present in a nucleotide sequence of a gene encoding a protein is replaced with a synonymous codon frequently used in a host cell is used for protein expression (so-called codon) (Optimization) (Patent Document 2), (2) A method of introducing a gene encoding a tRNA corresponding to a rare codon into a host cell and increasing the supply amount of the tRNA is known (Patent Document 1, Patent Document) 3, Non-Patent Documents 1 to 3).

However, the above-mentioned codon optimization requires a lot of time and effort to modify the coding gene for each protein when there are many kinds of target proteins. In addition, when different host cells are used, codon optimization is required again. Furthermore, when the target protein is a homologous protein (a protein derived from the same species as the microorganism used as the host cell), the nucleotide sequence encoding the protein is usually adapted to the codon usage of the microorganism, and a rare codon is used. In many cases, the amount of protein expression cannot be increased by the above-mentioned methods because they are not included.

Therefore, there is a demand for the development of a new protein expression improvement technique that can improve the production efficiency of various proteins in microorganisms.

International Publication No. 00/44926 International Publication No. 2004/024915 European Patent No. 2027268

The object of the present invention is to provide a method capable of increasing the expression level of a protein in a microorganism regardless of the type of protein or the species of origin. Another object of the present invention is to provide a method for improving the productivity of a compound in a microorganism.

The present inventors relate to the production of a protein using a microorganism as a host cell, and the tRNA possessed by the host cell is frequently used in the translation of the entire protein coding sequence in the host cell relative to the supply amount in the host cell. It was found that tRNA exists and that the expression level of the protein in the host cell can be increased by introducing and expressing a gene encoding such a frequently used tRNA in the host cell.

The present invention relates to a modified host cell for protein expression, a method for producing the modified host cell based on supplementing a frequently used tRNA (hereinafter referred to as “highly active tRNA”) with respect to the supply amount in the host cell, Nucleic acid molecules, vectors and kits for modifying host cells, methods for improving the productivity of compounds by microorganisms, and the like are provided.

More specifically, the present invention provides the embodiments described below:
(1) a modified host cell for protein expression,
(A) a polynucleotide encoding at least one tRNA having a deviation value of availability factor A defined by the following formula of 70 or more, and / or (b) a deviation of availability factor B defined by the formula below A modified host cell into which a polynucleotide encoding at least one tRNA having a value of 70 or more has been introduced (first embodiment);

(2) The modified host cell according to (1), which is used for expression of a protein derived from a biological species to which the host cell belongs;
(3) The modified host cell according to (1) or (2), further introduced with a polynucleotide encoding tRNA, which is not present in the genome of the unmodified host cell;
(4) A method for producing a modified host cell for protein expression, comprising the following steps (second embodiment):
(I) providing a host cell; and (ii) a polynucleotide encoding at least one tRNA having a deviation in operating rate A as defined in (1) of 70 or more and / or defined in (1) A step of transforming the host cell with a nucleic acid molecule or vector comprising a polynucleotide encoding at least one tRNA having a deviation value of availability factor B of 70 or more;
(5) A nucleic acid molecule or vector comprising the following, which is used to modify a host cell (third aspect):
(A) a polynucleotide encoding at least one tRNA having a deviation value of utilization rate A defined in (1) of 70 or more, and / or (B) deviation of utilization rate B defined in (1) A polynucleotide encoding at least one tRNA having a value of 70 or greater;
(6) A kit comprising the following, which is used for modifying a host cell (fourth embodiment):
(A) A polynucleotide encoding at least one tRNA having a deviation value of availability factor A defined in (1) of 70 or more and / or a deviation value of availability factor B defined in (1) is 70. A polynucleotide encoding at least one tRNA as described above; and (B) a vector into which the polynucleotide can be incorporated, the vector being used to transform a host cell;
(7) A kit comprising the following for producing a modified host cell for protein expression (fifth aspect):
(A) a host cell; and (B) a polynucleotide encoding at least one tRNA having a deviation value of the availability A defined in (1) of 70 or more and / or the availability defined in (1). A nucleic acid molecule or vector comprising a polynucleotide encoding at least one tRNA having a B deviation value of 70 or greater;
(8) A kit comprising the following for producing a modified host cell for protein expression (sixth aspect):
(A) a host cell;
(B) A polynucleotide encoding at least one tRNA having a deviation value of availability A defined in (1) of 70 or more and / or a deviation value of availability B defined in (1) of 70 or more. A polynucleotide encoding at least one tRNA which is; and (C) a vector into which the polynucleotide can be incorporated, the vector being used to transform a host cell;
(9) A method for improving the productivity of a compound by a microorganism, comprising the following steps (seventh aspect):
(I) A polynucleotide encoding at least one tRNA having a deviation value of availability factor A defined in (1) of 70 or more and / or a deviation value of availability factor B defined in (1) is 70 or more. Providing a nucleic acid molecule or vector comprising a polynucleotide encoding at least one tRNA, and (ii) transforming a microorganism with the nucleic acid molecule or vector;
(10) A modified microorganism obtained by the method of (9), which has an increased compound productivity as compared with an unmodified microorganism of the same species (eighth aspect);
(11) A method for producing a compound comprising the following steps (ninth aspect):
(I) (a) a polynucleotide encoding an enzyme that catalyzes the synthesis reaction of the compound, and (b) encoding at least one tRNA having a deviation in operating rate A defined in (1) of 70 or more. (Ii) providing a host cell into which a polynucleotide and / or a polynucleotide encoding at least one tRNA that has a deviation value of availability B as defined in (c) of (1) is 70 or more; and (ii) ) Culturing the host cell; and (12) a method for producing a compound comprising the following steps (tenth embodiment):
(I) providing a nucleic acid molecule or vector comprising a polynucleotide encoding an enzyme that catalyzes a synthesis reaction of the compound;
(Ii) A polynucleotide encoding at least one tRNA having a deviation value of availability A defined in (1) of 70 or more and / or a deviation value of availability B defined in (1) of 70 or more. Providing a nucleic acid molecule or vector comprising a polynucleotide encoding at least one tRNA which is
(Iii) transforming a host cell with the nucleic acid molecule or vector of (i) and (ii); and (iv) culturing the transformed host cell.

In the modified host cell of the present invention, the supply amount of the tRNA is increased by the introduction of a gene encoding a high-performance tRNA. As a result, the modified host cell of the present invention can achieve an increased protein expression level for various proteins compared to the unmodified host cell. In addition, since the introduction of a gene encoding a highly active tRNA according to the present invention can increase the expression of all proteins including biosynthetic enzymes of compounds possessed by microorganisms, production of useful substances such as secondary metabolites by microorganisms It can also improve the performance.

It is a graph which shows the calculation result of the deviation value of the operation rate (operation rate A) of tRNA based on the whole protein coding sequence of Streptomyces coelicolor A3 (2). The horizontal axis label represents the type of tRNA by the anticodon sequence (3 'to 5' direction). It is a graph which shows the calculation result of the deviation value of the operation rate (operation rate B) of tRNA based on the coding sequence of highly expressed protein in Streptomyces coelicolor A3 (2). The horizontal axis label represents the type of tRNA by the anticodon sequence (3 'to 5' direction). It is a figure which shows the structure of the tRNA expression vector of Streptomyces coelicolor (A3 (2)). It is a figure which shows the fixed_quantity | quantitative_assay result of the tRNA expression level corresponding to codon GCC (Ala) in Streptomyces lividans 1326 strain transformed with the tRNA expression vector. The left is an unmodified strain, and the right is a strain into which a tRNA gene corresponding to the codon GCC (Ala) is introduced. It is a figure which shows the structure of a protein expression vector. Various promoters correspond to the “promoter” position, and protein-coding genes used for protein expression experiments correspond to the “target protein” position. It is a graph which shows the expression level of various proteins in the tRNA high expression strain obtained by introduce | transducing any of tRNA expression vector pBuT04, pBuT02, pRaT02, and pSH04. The “original strain” is a protein expression strain into which a tRNA expression vector has not been introduced. The horizontal axis label indicates the type of protein expressed and the promoter used. It is a figure which shows the structure of the expression vector which integrated the tRNA gene corresponding to codon GCC (Ala). It is a figure which shows the structure of the vector which integrated the melC operon area | region. It is a figure which shows the calculation result of the deviation value of the operation rate (operation rate A) of tRNA of Escherichia coli K12 (Escherichia coli K12). It is a figure which shows the structure of tRNA expression vector pBuT04E, pRaT02E, and pSH04E.

As used herein, “host cell” refers to a cell into which a genetic factor (typically a recombinant nucleic acid) is introduced from the outside for the purpose of, for example, recombinant expression of a protein or gene cloning. Examples of the types of host cells that can be used include microbial cells, animal cells (for example, mammalian cells and insect cells), plant cells, and the like. In a preferred embodiment of the present invention, microbial cells are used as host cells.

In the first to sixth and ninth and tenth aspects of the present invention, the host cell is preferably Escherichia, Corynebacterium, Brevibacterium, Bacillus, Brevibacillus, Microbacterium, Pseudomonas, Aspergillus, Arthrobacter, Arthrobacter Examples include microorganisms belonging to the genus, genus Rhodobacter, genus Rhodococcus, genus Streptomyces, genus Zymomonas, genus Novosphigobium, genus Saccharomyces, genus Schizosaccharomyces, genus Zymobacter and the like. Specific examples of the microorganism include, for example, Aspergillus niger, Aspergillus oryzae, Aspergillus phoenicis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus subtilis, Corynebacterium ammoniagenes, Corynebacterium PPichimicus, Corynebacterium PPichimicus, pombe, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces lividans, Streptomyces griseus, Streptomyces venezuelae, Zymobacter palmaem, Zymomonas mobilis and the like. In one embodiment, the host cell is a microorganism of the genus Streptomyces or Escherichia. As a microorganism belonging to the genus Streptomyces, for example, Streptomyces lividans (for example, Sterptomyces lividans 1326 strain) can be used. Further, not limited to Streptomyces lividans, species or strains whose properties are analyzed in detail among the genus Streptomyces can be used. As a microorganism belonging to the genus Escherichia, for example, Escherichia coli (for example, Escherichia coli K12 strain) can be used. Moreover, not only Escherichia coli, but also a species or strain whose properties are analyzed in detail among the genus Escherichia can be used. In one embodiment, the host cell is Streptomyces lividans or Escherichia coli.

In the present specification, “codon usage frequency” means the frequency of appearance of each codon in the protein coding sequence present in the genome of the host cell. In the present specification, it is represented by the number of appearances per certain number of codons (for example, XX times / 1,000 codons). As used herein, the term “genome” includes not only chromosomes but also extrachromosomal genetic elements such as plasmids. Therefore, the “coding sequence of all proteins encoded in the genome” includes protein coding sequences present in extrachromosomal genetic elements such as plasmids in addition to protein coding sequences present in chromosomes.

In the present specification, the “frequency of use of tRNA” is the frequency of use of a tRNA gene that encodes a tRNA corresponding to a specific codon, and is expressed as the frequency of appearance of each corresponding codon, as with the frequency of codon usage (for example, XX times / 1,000 codons).

In the present specification, the “protein coding sequence” refers to a nucleic acid sequence that encodes an amino acid sequence of a protein, and corresponds to an open reading frame (ORF) or a coding sequence (CDS). Such a nucleic acid sequence is usually a DNA or RNA sequence.

In the present invention, a polynucleotide (tRNA gene, protein-encoding gene) introduced into a host cell or a microorganism is various regulatory sequences involved in the regulation of transcription or translation (for example, promoter, enhancer, 5 ′ untranslated region, 3 ′ non-translated region). It may be accompanied by a translation area or the like. These regulatory sequences can be selected as appropriate depending on, for example, the protein to be expressed, but are not limited to specific sequences. For example, a polynucleotide in which a desired protein coding sequence is linked downstream of a desired promoter sequence can be introduced into a host cell.

The first aspect of the present invention is a modified host cell into which a polynucleotide encoding a frequently used tRNA in the host cell is introduced.
The present invention is based on the discovery of the presence of tRNAs that are very frequently used in the translation of protein coding sequences in host cells and are in short supply for demand. The present invention is characterized by improving the protein translation efficiency and increasing the expression level of the protein in the host cell by overexpressing such tRNA in the host cell.

In the present specification, the ratio of the usage frequency of the tRNA in the translation of the protein coding sequence to the supply amount of the tRNA is referred to as “operation rate”. Such a high availability tRNA is referred to as a “high availability tRNA”.

The supply amount of a specific tRNA is usually considered to reflect the number of genes encoding the specific tRNA in the genome. In addition, the distribution of the frequency of use of each codon in the coding sequence of a protein highly expressed in a certain species is considered to match the distribution of the frequency of use of tRNA corresponding to each codon in the species. . Therefore, in this specification, the “operating rate” and the deviation value (T score) for a specific tRNA in the host cell are calculated by the following formula and used as an index of the high operating tRNA.

Or

In the first aspect of the present invention, the polynucleotide to be introduced into the host cell is (a) a polynucleotide encoding a tRNA having a deviation value of availability A defined by the above formula of 70 or more, and / or (B) A polynucleotide that encodes a tRNA having a deviation in operating rate B defined by the above formula of 70 or more. The deviation value of the operating rate A is preferably 72.5 or more, more preferably 75 or more. The deviation value of the operation rate B is preferably 71 or more, more preferably 72 or more. In one embodiment, the deviation value of the operation rate B may be 78 or more.

The host cell according to the first aspect of the present invention includes at least one tRNA (which encodes the tRNA) that satisfies any criterion that the deviation value of the operation rate A is 70 or more or the deviation value of the operation rate B is 70 or more. It is sufficient that the polynucleotide is introduced. Optionally, two or more tRNAs that meet such criteria, eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more tRNAs are introduced into the host cell. May be.

The polynucleotide encoding the tRNA introduced into the host cell may be retained as an extrachromosomal genetic factor such as a plasmid, or may be integrated into the genome by non-homologous recombination or homologous recombination.

The “one or more species of the same genus as the host species to which the host cell belongs” in the formula of the operating rate A and the operating rate B is preferably one species of the same genus as the host species to which the host cell belongs. More preferably, it is the same species as the species to which the host cell belongs.

“A coding sequence of all proteins encoded in the genome” of a biological species means all open reading frames (ORFs) present in the genome of the biological species. However, the amount and accuracy of the genome information obtained varies depending on the species. Therefore, the “all” protein coding sequences of the species used to identify highly active tRNAs in the present invention are usually all open reading frames to the extent that information can be obtained from various genome databases and the like.

The frequency of use of various codons in various species can be obtained from Kazusa DNA Research Institute's Codon Usage Database (http://www.kazusa.or.jp/codon/). Alternatively, the base sequence of the genome (preferably the entire genome) of the desired species is obtained from various genome databases, etc., and Glimmer3 (http://ccb.jhu.edu/software/glimmer/index.shtml), GeneMark (http : //opal.biology.gatech.edu/), PRODIGAL (http://prodigal.ornl.gov/), etc. are used to extract the ORF from the base sequence and count the number of occurrences of each codon The frequency of codon usage can also be specified by such a method. Calculation of the codon usage frequency in a desired base sequence can be easily performed using, for example, the “Countcodon” program (http://www.kazusa.or.jp/codon/countcodon.html) provided by Kazusa DNA Research Institute. it can. Alternatively, an analysis program can be created using a programming language such as Perl, and the codon usage frequency can be calculated using the program.

The number of genes encoding a specific tRNA in the genome of the desired species can be determined using the tRNAdb database (http://trna.bioinf.uni-leipzig.de/DataOutput/), Genomic tRNA Database (http: // lowelab. ucsc.edu/GtRNAdb/) etc. Alternatively, the base sequence of the genome of the desired species (preferably the entire genome) is obtained from various genome databases, etc., and analysis software such as tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/) is used. The number of genes encoding tRNA can also be specified by a method of extracting a gene encoding tRNA from the base sequence.

When the species to be used as the host cell is not registered in the database, the utilization rate A and / or the data of another species closely related to the species, for example, another species of the same genus as the species is used. Alternatively, the operating rate B can be calculated. Data of one species belonging to the same species as the species to which the host cell belongs may be used, or data of a plurality of species belonging to the same genus may be pooled. If data on the species closely related to the species to which the host cell belongs are not available, genome analysis of one or more species of the same genus as the species to which the host cell belongs is performed using known methods. In addition, the frequency of codon usage in the ORF and information on the tRNA gene can be obtained.

In the present specification, in particular, with respect to the above-described expression of the operation rate B, “a protein highly expressed” in a certain biological species means (a) a reliable spot in two-dimensional electrophoresis of all proteins in the biological species. A protein to be detected, or (b) a protein having an expression level equal to or higher than that of one or more proteins selected from the group consisting of the proteins listed in Table 1 and their homologs in the biological species. .
Regarding the protein (a) above, for example, SWICZ (http://proteom.biomed.cas.cz/), SWISS-2DPAGE (http://world-2dpage.expasy.org/swiss-2dpage/), Proteome 2D-PAGE Database (http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi), DOGAN (http: //www.bio.nite.go. jp / dogan / top) and other proteome databases such as Streptomyces coelicolor, Streptomyces granaticolor (more SWICZ), Escherichia coli, Saccharomyces cerevisiae (more SWISS-2DPAGE), Bacillus amyloliquefaciens, Bacillus anthracis (more Proteome 2D-PAGE oryz), Asperae A list of proteins identified based on the results of proteomic analysis (two-dimensional electrophoresis) of various species including Rhodococcus opacus (DOGAN) can be obtained, and these proteins are expressed in the present invention. Can be used to calculate the “operation rate B”.
In addition, in the case of a biological species for which a database for proteome analysis is not available, it is possible to identify the protein detected as a reliable spot by performing two-dimensional electrophoresis of all the proteins of the biological species by a known method It is. In this case, the selection of a reliable spot is determined by, for example, the “spot quality” value defined in Garrels et. Al, J. Biol. Chem., 1989, vol. 264, No. 9, pp. 5269-5282. This can be done by selecting a spot having a value of 50 or more.

The modified host cell of the present invention is, for example, a polynucleotide encoding at least one tRNA having a deviation of 70 or more in operation rate A and / or at least one tRNA having a deviation of 70 or more in operation rate B. Can be obtained by transforming the host cell with a nucleic acid molecule or vector comprising a polynucleotide encoding. In addition, the modified host cell of the present invention is obtained by other modification methods such as cell fusion as long as a polynucleotide encoding tRNA that satisfies the above criteria of deviation in operating rate can be introduced. Also good.

The modified host cell of the present invention can be suitably used for protein expression. By introducing a polynucleotide encoding a desired protein into the modified host cell of the present invention and culturing the host cell, the protein can be produced with high efficiency. The protein to be expressed may be derived from a biological species belonging to the same species as the biological species to which the host cell belongs, or may be derived from a biological species belonging to a genus different from the biological species to which the host cell belongs. . Further, the gene encoding the protein to be expressed may be a modified gene or an artificial gene that has been subjected to codon optimization or the like in accordance with the codon usage frequency in the host cell. For example, when expressing a protein derived from a species different from the species to which the host cell belongs or a species of a different genus, using such a modified gene or an artificial gene can be an effective means for efficient expression. Genetic modification such as codon optimization, synthesis of an artificial gene having a desired (for example, codon optimized) sequence, and the like can be performed using methods known in the art. In one embodiment, the modified host cell of the present invention is used for the expression of a protein derived from the same species as the species to which the host cell belongs, preferably the same species. In another embodiment, the modified host cell of the present invention is used for the expression of a protein derived from a species different from or different from the species to which the host cell belongs.

In one embodiment, the modified host cell of the invention includes a polynucleotide that encodes a tRNA that is not present in the genome of the unmodified host cell (also referred to herein as a “absent tRNA gene”). May be further introduced. As a result, tRNA that is not encoded in the genome of the unmodified host cell and is not present at all in the host cell is newly supplied, and the expression of the protein that includes the codon corresponding to the tRNA in the coding sequence Efficiency can be improved. A non-existing tRNA gene in a desired host cell is a tRNA gene having an anticodon corresponding to all types of codons excluding a stop codon in the tRNAdb database as a tRNA gene not possessed by the species to which the host cell belongs. Can be identified.

For example, in Streptomyces coelicolor A3 (2), the frequency of use of codon GCC (Ala) in all ORFs is 78.4 times / 1000 codons (from Kazusa DNA Research Institute Codon U Usage Database) There are two genes (from tRNAdb) encoding the tRNA corresponding to the codon GCC (Ala) (anticodon is CGG (3 ′ to 5 ′ direction)) in the genome of Streptomyces coelicolor. Therefore, the “operation rate A” of tRNA corresponding to the codon GCC (Ala) is 78.4 ÷ 2 = 39.2. Further, when the operation rate A is similarly determined for all tRNAs encoded in the Streptomyces coelicolor genome, the average value of the operation rate A is 10.0 and the standard deviation is 10.5. Using the average value and the standard deviation thus obtained, the deviation value of the availability A of tRNA corresponding to the codon GCC (Ala) is obtained by the above formula, and the deviation value is 77.8. Therefore, when using a species of the genus Streptomyces as a host cell, for example, Streptomyces coelicolor, Streptomyces evamethyls, Streptomyces griseus, Streptomyces lividans, etc., the tRNA corresponding to the codon GCC (Ala) is: Satisfies the high-activity tRNA standard in the present invention.

The second aspect of the present invention is a method for producing a modified host cell with improved protein expression efficiency by transforming the host cell with a nucleic acid molecule or vector containing a polynucleotide encoding a highly active tRNA. .

The polynucleotide encoding a high-activity tRNA used in the second aspect is a polynucleotide encoding at least one tRNA having a deviation value of the operation rate A defined as described above of 70 or more, and / or as described above. It is a polynucleotide that encodes at least one tRNA having a defined deviation rate of availability B of 70 or more. About the preferable deviation value of the operation rate A and the deviation value of the operation rate B, it is the same as that described in the first aspect.

When two or more highly active tRNAs are introduced into a host cell, the polynucleotides encoding the tRNAs may be contained in a single nucleic acid molecule or vector, and are separately contained in multiple nucleic acid molecules or vectors. It may be.

In the present specification, the “nucleic acid molecule comprising a polynucleotide” that can be used for transformation of a host cell includes a nucleic acid molecule comprising only the polynucleotide, in addition to a nucleic acid molecule comprising the polynucleotide and another nucleotide sequence such as an adapter. Include molecules. Such nucleic acid molecules can be, for example, in the form of linear DNA.

Examples of vectors that can be used for transformation of host cells include, but are not limited to, plasmid vectors and viral vectors, as well as vectors using transposons and transposable elements such as insertion sequences (IS).

As a host cell transformation method, methods known in the art such as electroporation, heat shock method, lithium acetate method, Agrobacterium method, protoplast-PEG method and the like can be used.

A third aspect of the present invention is a nucleic acid molecule or vector comprising a polynucleotide encoding a highly efficient tRNA used to modify a host cell.
The polynucleotide encoding the high-efficiency tRNA, and the nucleic acid molecule and vector containing the polynucleotide are the same as in the second embodiment. A preferable range of the deviation value of the operation rate A and the deviation value of the operation rate B, which is an index of the high operation tRNA, is the same as that described in the first aspect.

A fourth aspect of the present invention is a kit comprising a polynucleotide encoding a high-efficiency tRNA used for modifying a host cell and a transformation vector capable of incorporating the polynucleotide.
The polynucleotide encoding the high-efficiency tRNA and the vector that can be used for transformation of the host cell are the same as in the second embodiment. A preferable range of the deviation value of the operation rate A and the deviation value of the operation rate B, which is an index of the high operation tRNA, is the same as that described in the first aspect.

A fifth aspect of the present invention is a kit comprising a nucleic acid molecule or vector comprising a host cell and a polynucleotide encoding a high-efficiency tRNA for producing a modified host cell for protein expression.
The polynucleotide encoding the high-efficiency tRNA, and the nucleic acid molecule and vector containing the polynucleotide are the same as in the second embodiment. A preferable range of the deviation value of the operation rate A and the deviation value of the operation rate B, which is an index of the high operation tRNA, is the same as that described in the first aspect.

A sixth aspect of the present invention is a kit comprising a host cell, a polynucleotide encoding a high-efficiency tRNA, and a transformation vector capable of incorporating the polynucleotide, for producing a modified host cell for protein expression. It is.
The polynucleotide encoding the high-efficiency tRNA and the vector that can be used for transformation of the host cell are the same as in the second embodiment. A preferable range of the deviation value of the operation rate A and the deviation value of the operation rate B, which is an index of the high operation tRNA, is the same as that described in the first aspect.

A seventh aspect of the present invention is a method for improving the productivity of a compound by a microorganism, comprising the step of transforming the microorganism with a nucleic acid molecule or a vector containing a polynucleotide encoding a highly active tRNA.
The polynucleotide encoding the high-efficiency tRNA, the nucleic acid molecule and vector containing the polynucleotide, and the transformation method are the same as in the second embodiment. A preferable range of the deviation value of the operation rate A and the deviation value of the operation rate B, which is an index of the high operation tRNA, is the same as that described in the first aspect.

The eighth aspect of the present invention is a modified microorganism obtained by the method described as the seventh aspect of the present invention, which has an increased compound productivity as compared with an unmodified microorganism of the same species. .

The kind of microorganism that can be used in the seventh and eighth aspects of the present invention is not particularly limited. In one embodiment, the microorganism is preferably Escherichia, Corynebacterium, Brevibacterium, Bacillus, Brevibacillus, Microbacterium, Pseudomonas, Aspergillus, Arthrobacter, Rhodobacter, Rhodococcus, Streptomyces, Zymomonas And microorganisms belonging to the genus Novosphigobium, the genus Saccharomyces, the genus Schizosaccharomyces, the genus Zymobacter, and the like.
In the seventh and eighth aspects of the present invention, specific examples of the microorganism include, for example, Aspergillus niger, Aspergillus oryzae, Aspergillus phoenicis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus subtilis, Brevibacillus choshinensis, Corynebacterium ammoniagenes, Corynebacterium chemobacterium, E. coli, Psudomonas putida, Rhodococcus rhodochrous, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces lividans, Streptomyces griseus, Streptomyces venezuelae, Zymobacter palmem, etc. In one embodiment, the host cell is a microorganism of the genus Streptomyces or Escherichia. As a microorganism belonging to the genus Streptomyces, for example, Streptomyces lividans (for example, Sterptomyces lividans 1326 strain) can be used. Further, not limited to Streptomyces lividans, species or strains whose properties are analyzed in detail among the genus Streptomyces can be used. As a microorganism belonging to the genus Escherichia, for example, Escherichia coli (for example, Escherichia coli K12 strain) can be used. Moreover, not only Escherichia coli but the seed | species or strain | stump | stock whose property is analyzed in detail among the genus Escherichia can be used. In one embodiment, the host cell is Streptomyces lividans or Escherichia coli.

The introduction of a highly operational tRNA into a microorganism can improve the production efficiency of all proteins expressed by the microorganism. Accordingly, the compounds capable of improving productivity according to the seventh and eighth aspects of the present invention are biosynthesized in microorganisms or produced by reactions catalyzed by enzymes produced in microorganisms. As long as it is a thing, it will not specifically limit. For example, an enzyme that catalyzes a synthesis reaction of a compound may be one that is produced by a microorganism, secreted outside the cell, and catalyzes the reaction outside the cell.

In one embodiment, the compound whose productivity in the microorganism is improved by the method of the present invention may be a secondary metabolite of the microorganism, such as an amino acid derivative, an isoprenoid, an antibiotic, a pigment, a polyketide and the like. For example, when the microorganism modified by the method of the present invention is actinomycetes (for example, Streptomyces lividans), the productivity of diosmin, actinorhodin, etc. can be improved.

A ninth aspect of the present invention is a method for producing a compound by culturing a host cell into which a polynucleotide encoding an enzyme that catalyzes a compound synthesis reaction and a polynucleotide encoding a highly active tRNA are introduced. .
The polynucleotide encoding the high-performance tRNA is the same as in the second embodiment. A preferable range of the deviation value of the operation rate A and the deviation value of the operation rate B, which is an index of the high operation tRNA, is the same as that described in the first aspect.

According to a tenth aspect of the present invention, a nucleic acid molecule or vector containing a polynucleotide encoding an enzyme that catalyzes a compound synthesis reaction and a nucleic acid molecule or vector containing a polynucleotide encoding a high-efficiency tRNA were transformed. A method for producing a compound by culturing host cells.
The polynucleotide encoding the high-efficiency tRNA, the nucleic acid molecule and vector containing the polynucleotide, and the transformation method are the same as in the second embodiment. A preferable range of the deviation value of the operation rate A and the deviation value of the operation rate B, which is an index of the high operation tRNA, is the same as that described in the first aspect.

In the ninth and tenth aspects of the present invention, the compound produced by a host cell into which a polynucleotide encoding a highly active tRNA has been introduced is not particularly limited. Any desired compound can be produced as long as the selection of an enzyme that catalyzes the synthesis reaction of the target compound is appropriate, and a compound serving as a substrate for the enzyme is provided. In one embodiment, the compound produced by the host cell can be an amino acid derivative such as melanin, porphyra-334, and the like. In another embodiment, the compound produced by the host cell can be an isoprenoid, such as α-serinene, β-carotene, and the like. For example, α-pinene can be obtained by culturing a host cell into which a polynucleotide encoding a highly active tRNA and a polynucleotide encoding α-pinene synthase have been introduced.

A compound serving as a substrate for an enzyme that catalyzes a synthesis reaction of a target compound (hereinafter also simply referred to as “synthetic enzyme”) can be produced by a host cell. In this case, the target compound can be obtained by culturing host cells into which the polynucleotide encoding the synthase has been introduced in any medium used in the art.

In another embodiment, production of a target compound can be achieved by culturing a host cell into which a polynucleotide encoding a synthase has been introduced in the presence of a compound that is a substrate for the synthase. In this case, the compound serving as the substrate for the synthetic enzyme may be present in the medium for culturing the host cell.

In the ninth and tenth aspects of the present invention, the conditions for culturing a host cell are not particularly limited as long as a synthetic enzyme and a high-activity tRNA are produced from a polynucleotide introduced into the host cell. The culture conditions can be appropriately determined according to the host cell used, the synthetic enzyme, the substrate of the synthetic enzyme, and the like.

Hereinafter, the present invention will be described more specifically by way of examples. However, the scope of the present invention is not limited to the examples.

1. Identification of highly efficient tRNA in actinomycetes (1) Calculation of availability based on total protein coding sequence and identification of highly efficient tRNA Streptomyces coelicolor A3 (2) genome (GenBank No. NC_003888) and plasmid Information on codon usage in all ORFs included in (GenBank No. NC — 003903, NC — 003904) was obtained from the Codon Usage Database of Kazusa DNA Laboratory. The number of tRNA genes in the genome of Streptomyces coelicolor is registered as possessed by Streptomyces coelicolor in tRNAdb (http://trna.bioinf.uni-leipzig.de/DataOutput/), a tRNA database. A value obtained by correcting the number of tRNA genes in consideration of recognition of a plurality of codons by the wobble rule ([number of genes encoding tRNA having an anticodon corresponding to a specific codon] + [specific codon by the wobble rule) The number of genes encoding other tRNAs that recognize X × 0.6]).
Based on the codon usage frequency and the number of tRNA genes, the availability A of tRNA was calculated using the following formula. The calculation result of the operation rate A for each tRNA is shown in FIG.

Next, based on the average value and the standard deviation of the operation rate A obtained for all tRNAs, a tRNA having a deviation value of the operation rate A calculated using the following formula of 70 or more was determined as a high operation tRNA.

As a result of calculation, in Streptomyces coelicolor A3 (2), the average value of the operation rate A was 10.0 and the standard deviation was 10.5. TRNA corresponding to codon GCC (Ala) (operation rate A was 39.2, deviation value was 77.8), and tRNA corresponding to codon CTG (Leu) (operation rate A was 38.1, deviation value) Was identified as a highly operational tRNA and used in subsequent experiments. In addition, the codon CGG satisfies the condition of a deviation value of 70 or more (operation rate A is 31.9, deviation value is 70.9), but the deviation value of operation rate B (deviation value 72.2) is higher. Therefore, in subsequent experiments, it was handled as a tRNA having a high operation rate B.

(2) Calculation of availability based on highly expressed protein coding sequences and identification of highly active tRNAs Using the proteome database SWICZ: Swiss-Czech Proteomics server (http://proteom.biomed.cas.cz/) A protein highly expressed in the fungus body was selected. The protein identified as a spot in the two-dimensional electrophoresis image (Master Gel) of Streptomyces coelicolor registered in SWICZ is used as the “highly expressed protein” in the present invention to calculate the operation rate. It was. A list of such highly expressed proteins is shown in Table 1. (Each of these proteins has a spot quality value of 50 or more as defined in Garrels et. Al, J. Biol. Chem., 1989, vol. 264, No. 9, pp. 5269-5282. Corresponding.)

Based on the codon usage in the coding sequence of the protein shown in Table 1 and the codon usage in the entire ORF of Streptomyces coelicolor that was also used in the calculation of the availability A, using the following formula: The operating rate B of tRNA was calculated. The calculation result of the operation rate B for each tRNA is shown in FIG.

Next, based on the average value and standard deviation of the utilization rate B obtained for the tRNA corresponding to the codon whose frequency of use in the protein coding sequence shown in Table 1 is 10 times / 1000 codons or more, the following formula is used. A tRNA having a deviation value of 70 or more calculated operation rate B was determined to be a high operation tRNA.

As a result of calculation, in Streptomyces coelicolor A3 (2), the average value of the operation rate B was 1.02 and the standard deviation was 0.29. TRNA corresponding to codon CGG (Arg) (operation rate B was 1.66, deviation value was 72.2), and tRNA corresponding to codon GGG (Gly) (operation rate B was 1.91, deviation value) Was 80.7) and was identified as a highly operational tRNA and used in subsequent experiments.

(3) Identification of non-existing tRNA gene From the information of the tRNA gene registered in the above-described tRNAdb, the genome of Streptomyces coelicolor A3 (2) contains codons CGC (Arg), CGA ( Arg) and ATA (Ile) were found to lack a gene encoding a tRNA. These tRNAs were also used in subsequent experiments (host cell modification).

2. Construction of high-performance tRNA expression strain (modified host cell) (1-1) Cloning of tRNA gene 1
Primers for amplifying the tRNA gene were designed based on the genomic information (GenBank no. NC — 003888) of Streptomyces coelicolor A (3) 2 (Table 2). Streptomyces lividans TK64 (Streptomyces lividans TK64) genomic DNA as a template, designed primers as forward primer and reverse primer, PCR reaction using PrimeSTAR ^{(registered trademark)} GXL DNA Polymerase kit from Takara Bio Inc. The tRNA gene was amplified. For example, the PCR reaction for obtaining the GCC fragment is 1 cycle after (2 minutes at 98 ° C.), followed by 30 cycles (30 seconds at 98 ° C., 30 seconds at 55 ° C., 30 seconds at 68 ° C.). It went on condition of. The sequences of tRNA genes (including upstream and downstream regions) corresponding to GCC (Ala), CTG (Leu), CGG (Arg), GGG (Gly) and ATC (Ile) obtained by such amplification are respectively shown in SEQ ID NOs: Corresponding to 17, 18, 19, 20 and 21. Thereafter, each gene was introduced into a plasmid having a replication origin of pSG5.

(1-2) Cloning of tRNA gene 2
A tRNA gene which does not exist in the Streptomyces coelicolor genome was prepared by a point mutation introduction method. The primers shown in Table 3 were designed for mutagenesis. PCR was performed using the designed primer and PrimeSTAR ^{(registered trademark)} GXL DNA Polymerase kit of Takara Bio Inc. as a template with a plasmid in which the tRNA gene before point mutation was introduced. For example, the PCR reaction was carried out under the conditions of 20 cycles (after 98 minutes at 98 ° C.) and after 1 cycle (30 seconds at 98 ° C., 30 seconds at 55 ° C., 420 seconds at 68 ° C.). Thereafter, the amplified product is treated with DpnI, thereby including tRNA genes (including upstream and downstream regions corresponding to codons CGC (Arg), CGA (Arg) and ATA (Ile), respectively, in SEQ ID NOs: 22, 23 and 24, respectively. Obtained).

(2) Preparation of tRNA expression vector The tRNA gene obtained as described above was linked to a vector having a pSG5 replication origin to prepare a tRNA expression vector. The name of the prepared tRNA expression vector and the incorporated tRNA gene are shown in Table 4, and the structure of the tRNA expression vector is shown in FIG.

(3) Transformation of actinomycetes
Streptomyces lividans 1326 strain (National Institute of Technology and Evaluation, Biotechnology Headquarters, Biogenetic Resource Division (NBRC): NBRC No. 15675) was transformed with each tRNA expression vector. Transformation of actinomycetes is a method described in “Genetic Manipulation of Streptomyces” (Hopwood, DA et al., 1985, Genetic Manipulation of Streptomyces: a Laboratory Manual, the John Innes Foundation, Norwich) on genetic engineering techniques for actinomycetes. Went according to.

(4) Confirmation of tRNA gene expression Whether or not the tRNA gene was overexpressed in the host cell was confirmed by a quantitative PCR (qPCR) method. Streptomyces lividans transformed with the tRNA expression vector were cultured in a test tube (28 ° C., 48 hours) in 5 mL of SSMP medium containing kanamycin (50 μg / mL). 1 mL of the culture solution was collected, and the cells were collected from the culture solution by centrifugation (14,000 rpm, 20 minutes). After purifying small RNAs using Invitrogen's PureLink ^™ miRNA Isokation Kit, cDNA was prepared using ReverTraAce ^™ qPCR RT Kit from TOYOBO. Next, the expression level of the target tRNA was quantified using THUNDERBIRD ^{(registered trademark)} SYBR ^{(registered trademark)} qPCR Mix kit of TOYOBO. For detection of tRNA, primers shown in Table 5 were designed and used as a primer pair for detection. For quantitative PCR reaction, Rotorgene 3000 manufactured by Corvette Research was used. The PCR reaction was carried out under the conditions of 40 cycles (95 ° C for 60 seconds) followed by 1 cycle (95 ° C for 15 seconds, 55 ° C for 10 seconds, 72 ° C for 30 seconds).

Unmodified Streptomyces lividans strain 1326 and Streptomyces lividans strain 1326 transformed by protoplast-PEG method using an expression vector incorporating a tRNA gene corresponding to codon GCC (Ala) The amount of tRNA expression corresponding to the codon GCC (Ala) was quantified. As a control experiment, the expression level of tRNA corresponding to codon GAC (Asp) was quantified.

Quantitative results of tRNA are shown in FIG. In order to eliminate the difference between strains, the expression level of tRNA corresponding to codon GCC was corrected with the expression level of tRNA corresponding to codon GAC, and the relative expression level was calculated. With the introduction of the tRNA expression vector, the expression level of tRNA corresponding to the codon GCC increased 2.8 times. From these results, it was confirmed that tRNA was expressed from the tRNA expression vector.

3. Protein expression using modified host cells (1) Construction of protein expression vector and creation of modified host cells Four types of promoters (Streptomyces sunnamoneus-derived phossolipase D gene (PLD) promoter (sequence) No. 39) (disclosed in Japanese Patent Application Laid-Open No. 2002-51780), Streptomyces septatus-derived metalloendoprotease (SSMP) promoter (SEQ ID NO: 38) (disclosed in Japanese Patent Application Laid-Open No. 2009-65837), Streptomyces evamethylus-derived xylose isomerase xylA) promoter (SEQ ID NO: 40) (disclosed in J. Microbiol. Biotechnol. 2008 May; 18 (5): 837-44)) and Streptomyces evamethyls-derived metalloprotease gene promoter (SAV promoter) (SEQ ID NO: 8) 0) was used. Along with these promoters, the proteins listed in Table 6 were linked to a vector having the replication origin of pIJ101 to obtain a protein expression vector. The structure of the protein expression vector is shown in FIG. Table 7 shows combinations of the protein coding gene and the promoter introduced into the expression vector.
Using these protein expression vectors, the Streptomyces lividans strain 1326 was transformed to obtain a protein expression strain. Furthermore, these strains were transformed with the tRNA expression vectors shown in Table 4 above to obtain high-tRNA expression strains. All transformations were performed by the protoplast-PEG method.

(2) Culturing method The tRNA high-expressing strain prepared as described above was first used as a seed culture for TSB medium (Bacto ^(trademark) manufactured by Becton Dickinson) containing thiostrepton (50 μg / mL) and kanamycin (50 μg / mL ^). Tube culture (72 hours at 28 ° C.) in 5 ml of Tryptic Soy Broth (Soybean-Casein Digest Medium, product number: 211825) After that, 3% (150 μL) of the inoculum culture solution was inoculated into 5 ml of SSMP medium. (However, only the SAV — 6372 expression strain was cultured for 48 hours.) When the xylA promoter was used, 500 μL of a 10% xylose solution was added 48 hours after the start of the culture. Table 8 shows the composition.

(3) Evaluation of expression level After completion of the culture, extracellular proteins (SAV — 6208, SGR — 5506, SAV — 967, SCO7576, AAS68222, SCO3222, SAV — 5268, SAV — 6139, and SCO5912) were collected from 1 ml of the cell culture solution and centrifuged (14,000 rpm). 20 minutes), and the supernatant was recovered. Cellular proteins (SCO1352, SAV — 6372, SCO2431) were subjected to ultrasonic disruption after separating 1 ml of the cell culture solution. Next, the obtained sample was subjected to SDS-PAGE analysis. From the SDS-PAGE data, using the Bio Rad Gel Doc ™ EZ System and the attached analysis software (Image Lab ^™) , the target protein band was quantified and the expression level was quantified, and the protein expression level was compared.

(4) Comparison of protein expression level The protein expression level was evaluated as the ratio of the protein expression level in the tRNA high expression strain to the protein expression level in the strain before introduction of the tRNA expression vector. The results are shown in FIG. 6 and Tables 9 and 10. In any of the tRNA high-expression strains prepared, the protein expression level was significantly increased compared to the strain before the introduction of the tRNA expression vector. From these results, it was shown that the introduction of the gene encoding the highly active tRNA identified in the present invention has an effect of increasing the protein expression level in the host cell without depending on the promoter.

4). tRNA utilization rate and protein expression level increasing effect The effect of tRNA having a deviation value of the utilization rate A defined in the present specification of less than 70 and tRNA having the deviation value of 70 or more was compared. Specifically, in Streptomyces coelicolor A3 (2), tRNA corresponding to codon ATC (Ile) (operation rate A = 27.5, deviation value = 66.7) and codon GCC ( A tRNA gene encoding each of tRNA corresponding to Ala) (operation rate A = 39.2, deviation value = 77.8) was introduced into a vector having a replication origin of pSG5 to obtain a tRNA expression vector. FIG. 7 shows the structure of an expression vector into which a tRNA gene corresponding to the codon GCC (Ala) has been introduced.
Using the tRNA expression vector, a protein expression strain was constructed, the expression strain was cultured, and the protein expression level was evaluated in the same manner as in “3. Protein expression using modified host cells” above. The host cell is Streptomyces lividans strain 1326, the expressed protein is transglutaminase, and the promoter used is the SSMP promoter.

Table 11 shows the evaluation results of the protein expression level. In the strain into which the tRNA gene corresponding to the codon ATC was introduced, no increase in the protein expression level was observed, whereas in the strain into which the tRNA gene corresponding to the codon GCC was introduced, an increase in the protein expression level was confirmed. From these results, it was confirmed that an effect of increasing the protein expression level can be obtained by introducing at least one tRNA having a high operation rate, for example, a tRNA having a deviation of 70 or more in the operation rate A into the host cell. . That is, the deviation value of the operation rate defined in the present specification effectively functions as an index for identifying a high-operation tRNA that causes an increase in protein expression level by introduction into a host cell.

5. Compound production using modified host cells (1) Production of amino acid derivatives i) Construction of melanin production strain The melC operon region of Streptomyces antibioticus (SEQ ID NO: 43) present in plasmid pIJ702 was cloned using the primers shown in Table 12. And incorporated into a pK18mob cloning vector. The structure of the obtained vector is shown in FIG. The melC operon was introduced into the chromosome of Streptomyces lividans by transformation using this vector. Further, this strain was transformed with the four types of tRNA expression vectors (pBuT04, pBuT02, pRaT02 and pSH04) prepared in “2. Production of high-efficiency tRNA expression strain (modified host cell)”, and high expression of tRNA was performed. It was a stock. All transformations were performed by the protoplast-PEG method.

ii) Culture method The obtained tRNA high expression strain was cultured as a seed culture in 5 ml of TSB medium containing thiostrepton (50 μg / mL) and kanamycin (50 μg / mL) in a test tube culture (at 28 ° C. for 72 hours). )did. Thereafter, 3% (150 μL) of the inoculum culture solution was inoculated into 5 ml of TSB medium, and cultured with shaking at 28 ° C. for 96 hours.

iii) Determination of melanin After completion of the culture, 1 ml of the bacterial cell culture solution was recovered and separated by centrifugation (14,000 rpm, 20 minutes). The supernatant was collected and the absorbance at 405 nm was measured. Specifically, the ratio of the absorbance at 405 nm of the high tRNA expression strain to the absorbance at 405 nm of the strain before the introduction of the tRNA gene was calculated to evaluate the change in the amount of melanin production due to the introduction of the tRNA gene. The results are shown in Table 13. From the results shown in the table, it was shown that introduction of a gene encoding a highly active tRNA into a host cell contributes to an increase in the production amount of amino acid derivatives such as melanin.

(2) Production of isoprenoids (terpenes) i) Construction of α-serinen production strains Using artificial gene synthesis technology based on the information of the α-selinene synthesis gene (GenBank Gene ID: 5734860) derived from Herpetosiphon aurantiacus DSM 785 An artificial gene (SEQ ID NO: 44) having a codon usage optimized for bacteria was prepared. In addition, Streptomyces avermitilis MA-4680 genomic DNA was used as a template and Streptomyces avermitilis MA-4680-derived ptlB gene (GenBank Gene ID: 1210633, SEQ ID NO: 47) was cloned using the primers shown in Table 14. These two genes were introduced into a vector having an origin of replication of plasmid pIJ101 to obtain an α-serinen expression vector. When this vector corresponds to FIG. 5, the Streptomyces evamethyls-derived metalloprotease gene promoter (SAV promoter) (SEQ ID NO: 80) is located at the “promoter” position, and the α-serinen synthetic gene and the “target protein” are located. It has a structure in which the ptlB gene is incorporated.
Streptomyces lividans was transformed using this α-selinen expression vector, and this was used as an α-selinen expression strain. Further, this strain was transformed with the three types of tRNA expression vectors (pBuT04, pBuT02 and pRaT02) prepared in “2. Production of high-performance tRNA expression strain (modified host cell)” above, did. All transformations were performed by the protoplast-PEG method.

ii) Culture method The obtained tRNA high-expression strain was cultured as a seed inoculum in 5 ml of TSB medium containing thiostrepton (50 μg / mL) and kanamycin (50 μg / mL) at 28 ° C. for 72 hours. . Thereafter, 1% (50 μL) of the inoculum culture solution was inoculated into 5 ml of AVM semi-synthetic medium and cultured with shaking at 28 ° C. for 120 hours. The composition of the AVM semi-synthetic medium used is shown in Table 15.

iii) Quantification of α-selinen After completion of the culture, 2 ml of the bacterial cell culture solution was collected and then separated by centrifugation (14,000 rpm, 20 minutes) to collect the bacterial cells. The collected cells were dispersed with 1.5 ml of methanol, and shaken for 5 minutes to extract α-selinene. Subsequently, 1 ml of supernatant methanol was collected by centrifugation (14,000 rpm, 20 minutes), 0.25 ml distilled water and 0.5 ml hexane were added and mixed by shaking. After further centrifugation, 150 μl of the upper hexane layer was used as a sample, and the amount of α-selinene in this hexane solution was determined by a gas chromatography using Shimadzu Corporation's InertCap 5 column at a rate of 20 ° C./min. Elution was performed with a temperature gradient of 50 ° C. (2 minutes hold) to 280 ° C. (6.5 minutes hold), and quantified. Under these conditions, α-selinene is eluted at around 10.1 minutes. The amount of α-selinene is calculated from a calibration curve of a β-caryophyllene standard product in the chromatogram.
The results are shown in Table 16. The increase in production amount by pRaT02 was the largest, but an increase in the production amount of α-linenene was also confirmed when a highly operational tRNA set (pBuT04, pBuT02) was introduced.

6). Expression of heterologous proteins using actinomycete-modified host cells Based on the examples of Japanese Patent Application Laid-Open No. 2007-124922, 3-quinuclidinone asymmetric reductase derived from Rhodotolura rubra JCM3782 (hereinafter also simply referred to as “quinuclidinone”) CDNA (SEQ ID NO: 50) was cloned. This gene was introduced into a vector having the replication origin of pIJ101 to obtain a quinuclidinone expression vector. When this vector corresponds to FIG. 5, the Streptomyces evamethyls-derived metalloprotease gene promoter (SAV promoter) (SEQ ID NO: 80) was incorporated at the “promoter” position, and the quinuclidinone gene was incorporated at the “target protein” position. It has a structure.

Streptomyces lividans was transformed with this quinuclidinone expression vector, and this was used as a quinuclidinone expression strain. Further, this strain was transformed with the four types of tRNA expression vectors (pBuT04, pBuT02, pRaT02 and pSH04) prepared in “2. Production of high-performance tRNA expression strain (modified host cell)”, and a tRNA high-expression strain was obtained. It was. All transformations were performed by the protoplast-PEG method.

The culture method was performed in the same manner as in “3. Protein expression using modified host cells (2) Culture method”. Further, the expression level was evaluated by the same method as that for the intracellular protein in “3. Protein expression using modified host cells (3) Expression level evaluation”. Comparison of expression levels was performed in the same manner as "3. Protein expression using modified host cells (4) Comparison of protein expression levels". The results are shown in Table 17. The expression level of the protein was significantly increased in the tRNA high expression strain that was frequently used compared to the strain before the introduction of the tRNA expression vector. From this result, it was shown that high expression of high-activity tRNA has an effect of increasing the expression level of protein not only in genes derived from host cells but also in expression of genes derived from different species.

7). Identification of high- efficiency tRNA in E. coli (1) Calculation of operation rate A based on the total protein coding sequence and identification of high-performance tRNA gene Escherichia coli (Escherichia coli) tRNA operation on the genome of K12 (GenBank No. NC_000913) The rate A was calculated according to the same method as in “1. Identification of high-activity tRNA in actinomycetes (1) Calculation of availability based on all protein coding sequences and identification of high-activity tRNA”. As a result of the calculation (FIG. 9), in Escherichia coli K12, the average value of the operation rate A was 9.28, and the standard deviation was 5.29. TRNA corresponding to codon CAT (His) (operating rate 26.3, deviation value 82.2), tRNA corresponding to codon GCG (Ala) (operating rate A 21.4, deviation value 72.9) , And tRNA corresponding to codon GAT (Asp) (operating rate A is 21.1, deviation value is 72.3) was identified as a high operating rate tRNA. However, tRNA genes corresponding to these codons could not be found on the E. coli genome. Therefore, in accordance with the wobble rule, tRNAs that recognize these codons, that is, tRNAs corresponding to codon CAC (His), codon GCA (Ala), and codon GAC (Asp) were used as high availability tRNAs in the subsequent experiments.

(2) Identification of non-existing tRNA gene From the information of tRNA gene registered in tRNAdb, the codons CGC (Arg), CGA (Arg) and ATA (Ile) are contained in the genome of Escherichia coli K12 (Escherichia coli). There was no gene encoding the tRNA corresponding to. These tRNAs were also used in subsequent experiments.

8). Preparation of high-performance tRNA expression strain (Escherichia coli) Based on the genomic information of Escherichia coli K12, primers for amplifying the tRNA gene were designed (Table 18). In the same manner as in “2. Production of a high-efficiency tRNA expression strain (modified host cell) (1-1) Cloning of tRNA gene 1”, Escherichia coli K12 genomic DNA was used as a template, and the designed primer and Takara Bio Inc. PCR reaction was performed using PrimeSTAR ^{(registered trademark)} GXL DNA Polymerase kit to amplify the target tRNA gene. The PCR reaction conditions are also the same as in “2. (1) Cloning of tRNA gene 1 in preparation of high-performance tRNA expression strain (modified host cell)”. The sequences of tRNA genes (including upstream and downstream regions) corresponding to GAC (Asp), GCA (Ala), CAC (His), AGG (Arg), and ATC (Ile) obtained by such amplification are shown in SEQ ID NOs: Corresponding to 67, 68, 69, 70 and 71. Thereafter, each gene was introduced into a plasmid having pMB1 as a replication origin.

A tRNA that does not exist in the Escherichia coli K12 genome was prepared by a point mutagenesis method. The primers shown in Table 19 were designed. In the same manner as in “2. (1) Cloning of tRNA gene 2 in preparation of high-efficiency tRNA expression strain (modified host cell)” above, a plasmid in which the tRNA gene before introduction of the point mutation was incorporated was used as a template. AGG (Arg) and ATC (Ile) serving as templates cloned the tRNA gene by the method described in the above item. After performing PCR reaction using the template and the designed primer and PrimeSTAR ^{(registered trademark)} GXL DNA Polymerase kit of Takara Bio Inc., it was converted to codons CGC (Arg), CGA (Arg) and ATA (Ile) by DpnI treatment, respectively. Corresponding genes (including upstream and downstream regions, corresponding to SEQ ID NOs: 72, 73 and 74, respectively) were obtained. The PCR reaction conditions are also the same as those described in “2. Production of high-performance tRNA expression strain (modified host cell) (1-2) Cloning of tRNA gene 2”.

The tRNA gene obtained as described above was ligated to a vector having the pACYC replication origin to prepare a tRNA expression vector. The name of the prepared tRNA expression vector and the incorporated tRNA gene are shown in Table 20, and the structure of the tRNA expression vector is shown in FIG.

9. Protein expression using E. coli modified host cells (1) Construction of protein expression vector and preparation of modified host cells In order to highly express three types of proteins, pKK233-3 vector (Pharmacia Biotex) was used. The lipase is Actinobacter calcaceticus subsp. The organism-derived lipase gene (SEQ ID NO: 77) was cloned using the genomic DNA of antiratus as a template and the primers shown in Table 21. The Eubus caballus-derived ferritin L subunit gene (SEQ ID NO: 78, GenBak Gene ID: 167621434) and the Listeria innocua-derived DPS gene (SEQ ID NO: 79, GenBak Gene ID: 26185792) were totally synthesized using an artificial gene synthesis technique. These genes together with the promoter were ligated to a vector having the pBR322 origin of replication to obtain a protein expression vector.
Using these protein expression vectors and the tRNA expression vector shown in Table 20, E. coli W3110 strain was transformed into a tRNA high expression strain. Transformation was performed by the heat shock method.

(2) Culture method The tRNA high-expression strain prepared as described above was first used as an inoculum culture for LB medium (Nacalai Tesque LB medium, Miller) containing ampicillin (50 μg / mL) and chloramphenicol (15 μg / mL). , Product number: 20068-75) 3 mL of the tube was cultured (at 37 ° C. for 24 hours). After that, inoculate the inoculum culture solution into 3 mL of 2 × YT medium (Nacalai Tesque, LB medium, Lennox, product number: 20066-95 used at double concentration) to which the lactose solution was added to a final concentration of 1%. .1% (3 μL) was inoculated and cultured in a test tube (72 ° C. for 72 hours).

(3) Expression level evaluation After completion of the culture, 10 μL of the bacterial cell culture solution was collected, and an equal amount of SDS buffer was added thereto, followed by vortexing. After heat treatment at 99 ° C. for 5 minutes, the obtained sample was analyzed by SDS-PAGE. From the SDS-PAGE data, using the Bio Rad Gel Doc ™ EZ System and the attached analysis software (Image Lab ^™) , the target protein band was quantified and the expression level was quantified, and the protein expression level was compared.
The protein expression level was evaluated in the same manner as in “3. Protein expression using modified host cells (4) Comparison of protein expression level”. The results are shown in Table 22. In any of the tRNA high-expression strains prepared, the protein expression level was significantly increased compared to the strain before the introduction of the tRNA expression vector. From these results, it can be seen that high expression of highly active tRNA has the effect of increasing the protein expression level even when the host cell is Escherichia coli, and does not depend on the host cell. It has been shown to have an effect of increasing the amount.

The present invention provides a modified host cell for expressing a protein, wherein a modified host cell into which a gene encoding a tRNA having high availability is introduced. The modified host cell expresses a protein with higher efficiency than a host cell into which the tRNA-encoding gene has not been introduced, and can be suitably used for recombinant expression of proteins and production of useful compounds.

Claims (12)

1. A modified host cell for protein expression comprising:
   (A) a polynucleotide encoding at least one tRNA having a deviation value of availability factor 70 defined by the following formula of 70 or more, and / or (b) a deviation of availability factor B defined by the formula A modified host cell into which a polynucleotide encoding at least one tRNA having a value of 70 or more has been introduced.
   

   

   
   

   
2. The modified host cell according to claim 1, which is used for expression of a protein derived from a biological species to which the host cell belongs.
3. The modified host cell according to claim 1 or 2, further introduced with a polynucleotide encoding tRNA, which is not present in the genome of the unmodified host cell.
4. A method for producing a modified host cell for protein expression, comprising the following steps:
   (I) providing a host cell; and (ii) a polynucleotide encoding at least one tRNA having a deviation in operating rate A as defined in claim 1 of 70 or more and / or as defined in claim 1. A step of transforming the host cell with a nucleic acid molecule or vector comprising a polynucleotide encoding at least one tRNA having a deviation value of availability ratio B of 70 or more.
5. A nucleic acid molecule or vector used to modify a host cell comprising:
   (A) A polynucleotide encoding at least one tRNA having a deviation value of availability A defined in claim 1 of 70 or more, and / or (B) a deviation of availability B defined in claim 1. A polynucleotide encoding at least one tRNA having a value of 70 or greater.
6. A kit comprising the following for use in modifying a host cell:
   (A) A polynucleotide encoding at least one tRNA having a deviation value of availability factor 70 defined in claim 1 of 70 or more, and / or a deviation value of availability factor B defined in claim 1 is 70. A polynucleotide encoding at least one tRNA as described above; and (B) a vector into which the polynucleotide can be incorporated, the vector being used to transform a host cell.
7. A kit comprising the following for producing a modified host cell for protein expression:
   (A) a host cell; and (B) a polynucleotide encoding at least one tRNA having a deviation value of the availability A defined in claim 1 of 70 or more and / or the availability defined in claim 1. A nucleic acid molecule or vector comprising a polynucleotide encoding at least one tRNA whose B deviation value is 70 or more.
8. A kit comprising the following for producing a modified host cell for protein expression:
   (A) a host cell;
   (B) A polynucleotide encoding at least one tRNA having a deviation value of utilization rate A defined in claim 1 of 70 or more and / or a deviation value of utilization rate B defined in claim 1 of 70 or more. A polynucleotide encoding at least one tRNA that is; and (C) a vector into which the polynucleotide can be incorporated, wherein the vector is used to transform a host cell.
9. A method for improving the productivity of a compound by a microorganism comprising the following steps:
   (I) A polynucleotide encoding at least one tRNA having a deviation value of availability A defined in claim 1 of 70 or more and / or a deviation value of availability B defined in claim 1 of 70 or more. Providing a nucleic acid molecule or vector comprising a polynucleotide encoding at least one tRNA, and (ii) transforming a microorganism with the nucleic acid molecule or vector.
10. A modified microorganism obtained by the method of claim 9, wherein the microorganism has increased compound productivity as compared with an unmodified microorganism of the same species.
11. A method for producing a compound comprising the following steps:
    (I) (a) a polynucleotide that encodes an enzyme that catalyzes the synthesis reaction of the compound, and (b) encodes at least one tRNA having a deviation value of availability factor A defined in claim 1 of 70 or more. And (ii) providing a host cell into which a polynucleotide and / or (c) a polynucleotide encoding at least one tRNA having a deviation value of availability B as defined in claim 1 of 70 or more is introduced; and (ii) ) Culturing the host cell.
12. A method for producing a compound comprising the following steps:
    (I) providing a nucleic acid molecule or vector comprising a polynucleotide encoding an enzyme that catalyzes a synthesis reaction of the compound;
    (Ii) A polynucleotide encoding at least one tRNA having a deviation value of availability A defined in claim 1 of 70 or more and / or a deviation value of availability B defined in claim 1 of 70 or more. Providing a nucleic acid molecule or vector comprising a polynucleotide encoding at least one tRNA which is
    (Iii) transforming host cells with the nucleic acid molecules or vectors of (i) and (ii); and (iv) culturing the transformed host cells.

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