JP2015180609A - Methods for producing ribosome peptides derived from fungi - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for recombinantly producing ribosome peptides derived from fungi by identifying and using a gene cluster participating in biosynthesis of ribosome peptides derived from fungi.SOLUTION: The invention relates to a method for producing a ribosome peptide derived from a fungus comprising the steps of: a) forcibly expressing a gene cluster participating in ribosome peptide biosynthesis in a cell, where the gene cluster at least comprises: a gene encoding a ribosome peptide precursor of which amino acid sequence comprises an endoplasmic reticulum transport signal sequence and a repeated sequence; and from one to four genes encoding proteins participating in the ribosome peptide biosynthesis, of which base sequences have E-values less than 1e-4 for four specified base sequences respectively; and b) collecting ribosome peptides from the cell. By using this production method, novel ribosome peptides, secondary metabolites of fungi, can be provided with expectation for application to medicines and other useful materials.

Description

  The present invention relates to a method for producing a peptidic secondary metabolite using a fungal-specific biosynthetic gene cluster involved in a peptidic secondary metabolite produced by a fungus, and a protein whose expression is induced in the cluster.

  Secondary metabolites produced by microorganisms including fungi, actinomycetes and bacteria are used in various applications including pharmaceuticals or pharmaceutical raw materials.

  As a result of several genome analyzes of some fungi, typically those of the genus Aspergillus, the fungi contain polyketide compounds, non-ribosomal peptides (non-ribosomal peptide synthesis without going through ribosomes) It is clear that there is a gene cluster (biosynthetic gene cluster) involved in biosynthesis of various secondary metabolites including terpenes or alkaloids, which are synthesized from amino acids by enzymes) Has been.

  For example, in A. flavus, 55 secondary metabolite biosynthesis gene clusters are shown to be encoded on the chromosome. However, in reality, more than half of such gene clusters are hardly expressed in fungal cells, or are expressed only to such an extent that only a very small amount of secondary metabolites are produced.

  This means that the fungus has the potential to produce a large number of unknown secondary metabolites whose specific structures and functions have not yet been identified. Therefore, in order to demonstrate the ability to produce potential secondary metabolites of fungi, we tried to artificially induce the expression of secondary metabolite biosynthetic gene clusters and synthesize a sufficient amount of secondary metabolites. An attempt to do so has been proposed. One of them is a method of increasing the amount of secondary metabolite synthesis by overexpressing the transcription factor of the gene cluster contained in the target secondary metabolite biosynthesis gene cluster. The other is a method of synthesizing a secondary metabolite by constructing cDNA of a secondary metabolite biosynthetic gene cluster and highly expressing it using an appropriate host expression system. Particularly in the latter case, an attempt to synthesize a non-natural secondary metabolite by recombining the constituent genes of each cluster among a plurality of biosynthetic gene clusters has also been proposed.

  By the way, it is known that some actinomycetes produce a compound called a ribosomal peptide as a secondary metabolite. Ribosomal peptides are biosynthesized through ribosomes, the field of protein synthesis, but contain chemical features that are rarely found in normal constituent proteins and enzyme proteins, including cyclic structures, branched structures, and unnatural amino acids. Is a compound having a peptide bond. On the other hand, as described above, polyketide compounds, non-ribosomal peptides, terpenes or alkaloids are known as secondary metabolites produced by fungi, but fungal-derived ribosomal peptides and biosynthetic gene clusters related to their biosynthesis To date, there are only a few reports on what is derived from the genus Amanita (Non-patent Document 1).

  The present inventors have proposed a bioinformatics method for estimating the presence of secondary metabolite biosynthetic gene clusters based on base sequence information (Patent Document 1).

Hallen et al., 2007, Proc. Nati. Acad. Sci. USA, vol. 104, pages 19097-19101.

WO2012 / 039484

  An object of the present invention is to provide a method for identifying a gene cluster involved in the biosynthesis of a fungal ribosomal peptide and using this to recombinantly produce a fungal ribosomal peptide. The identification and utilization of fungal-derived ribosomal peptide biosynthetic gene clusters to elucidate the further existence of fungal-derived ribosomal peptide biosynthetic gene clusters and to enable their recombinant production It is important for effective utilization of peptides.

  In the course of research on the biosynthetic pathway of ustiloxin B, which is known as a secondary metabolite produced by rice mushroom fungus (Ustylaginoidea virens), We found that genes related to biosynthesis formed a gene cluster. Furthermore, a gene (ustA) encoding a precursor peptide of ustyroxin B, which includes an endoplasmic reticulum transition signal sequence and a repetitive sequence in which the amino acid sequence found in the chemical structure of ustyroxin B is repeated multiple times, is included in the cluster. It was found that a gene encoding a protein containing a fungal-specific amino acid sequence exists as a constituent gene of the cluster. Furthermore, it encodes a protein consisting of an amino acid sequence having a certain homology with an amino acid sequence of a protein containing the endoplasmic reticulum translocation signal sequence and a repetitive sequence, and a protein containing the fungus-specific amino acid sequence. A ribosomal peptide biosynthetic gene cluster containing genes that After confirming the presence of a large number in the genome of a plurality of filamentous fungi including flavus, the following inventions were completed.

(1) A method for producing a ribosomal peptide derived from a fungus,
Step a) Biosynthesis of a ribosomal peptide having an E value of less than 1e-4 for a gene encoding a ribosomal peptide precursor consisting of an amino acid sequence including an endoplasmic reticulum translocation signal sequence and a repetitive sequence, and the base sequence shown in SEQ ID NO: 1. A gene having a base sequence encoding a protein involved, a gene consisting of an amino acid sequence having an E value of less than 1e-4 with respect to the amino acid sequence shown in SEQ ID NO: 2, or a gene encoding a protein involved in ribosomal peptide biosynthesis, or SEQ ID NO: A gene encoding a protein involved in the biosynthesis of a ribosomal peptide consisting of an amino acid sequence having an E value of less than 1e-4 with respect to the amino acid sequence shown in Fig. 3, and / or an E value of 1e- with respect to the amino acid sequence shown in SEQ ID NO: 6 Ribosomal peptide consisting of less than 4 amino acid sequences Step of including at least ribosomal peptide biosynthetic gene cluster of genes encoding proteins involved in the synthesis is forcibly expressed in the cell, as well as step b) recovering the ribosomal peptide from the cells, the production method.
(2) genes encoding ribosomal peptide precursor, AspGD ID number AFL2G_08516, Acar5010_211921, Acar5010_131040, ACLA_063320, Aspbr1_0148406, An06g02130, Aspzo1_1501727, Acar5010_210441, Aspzo1_0132961, Aacu16872_042334, An03g05230, Asptu1_0047109, Aspfo1_0048058, Aspka1_0178058, Aspzo1_0673756, AFUB_096780, Aspwe1_0028756 Aspwe1_01775551, Aspfo1_0041528, Aspka1_0176174, Aspzo1_01 The production method according to (1), which is a gene selected from the group consisting of 38840 and Acu16887_044658.
(3) A gene encoding a protein involved in the biosynthesis of a ribosomal peptide consisting of an amino acid sequence having an E value of less than 1e-4 with respect to the amino acid sequence shown in SEQ ID NO: 2 has AspGD ID numbers of AFL2G — 08517, Acar5010 — 156521, Acar5010 — 131041, ACLA — 063300 , ACLA — 063310, Aspbr1 — 0073580, An06g02120, Aspzo1 — 0020615, Aspzo1 — 0147593, Acar5010 — 399409, Aspozo1 — 0097397, Acu168872 — 043333, An03g05220, Asp611sp0A1 The production method according to (1) or (2), which is a gene selected from the group consisting of B — 096790, Aspwe1 — 0041401, Aspwe1 — 0188242, Aspfo1 — 0203572, Aspka1 — 0176173, Aspzo1 — 0163562, and Acu168872 — 046657.
(4) The forced expression in step a) is achieved by recombining a promoter of a gene encoding a transcription factor contained in a ribosomal peptide biosynthesis gene cluster with a high expression promoter, any of (1) to (3) The manufacturing method of crab.
(5) The production method according to any one of (1) to (4), wherein the ribosomal peptide biosynthesis gene cluster further includes a gene encoding a protein having peptidase activity.
(6) The production method according to any one of (1) to (5), wherein the cell is a fungal cell.
(7) The production method according to (6), wherein the fungal cell is a cell of the genus Aspergillus.
(8) From the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence in which one to several amino acids of the amino acid sequence are deleted or substituted, or the amino acid sequence in which one to several amino acids are added to the amino acid sequence A protein involved in the biosynthesis of ribosomal peptides in fungal cells.
(9) cDNA encoding the protein according to (8).
(10) From the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence in which one to several amino acids of the amino acid sequence are deleted or substituted, or the amino acid sequence in which one to several amino acids are added to the amino acid sequence A protein involved in the biosynthesis of ribosomal peptides in fungal cells.
(11) cDNA encoding the protein according to (10).
(12) A method for producing a ribosomal peptide derived from a fungus,
Step a) A gene encoding a ribosomal peptide precursor consisting of an amino acid sequence including an endoplasmic reticulum transition signal sequence and a repetitive sequence, and 20% or more identical to the 81st to 225th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 3 20% or more identity with the 57th to 259th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 6 and / or the gene encoding the protein involved in the biosynthesis of ribosomal peptides A step of forcibly expressing a ribosomal peptide biosynthetic gene cluster in a cell including at least a gene encoding a protein involved in ribosomal peptide biosynthesis, and b) a step of recovering the ribosomal peptide from the cell. The manufacturing method.
(13) a gene encoding ribosomal peptide precursor, AspGD ID number AFL2G_08516, Acar5010_211921, Acar5010_131040, ACLA_063320, Aspbr1_0148406, An06g02130, Aspzo1_1501727, Acar5010_210441, Aspzo1_0132961, Aacu16872_042334, An03g05230, Asptu1_0047109, Aspfo1_0048058, Aspka1_0178058, Aspzo1_0673756, AFUB_096780, Aspwe1_0028756 , Aspwe1 — 0177551, Aspfo1 — 0041528, Aspka1 — 0176174, Aspzo1 — 0 The production method according to (12), which is a gene selected from the group consisting of 138840 and Acu16887_044658.
(14) A gene containing an amino acid sequence having 20% or more identity with the 81st to 225th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 3 and encoding a protein involved in ribosomal peptide biosynthesis is AspGD ID number is AFL2G_08517, Acar5010_156521, Acar5010_131041, ACLA_063300, ACLA_063310, Aspbr1_0073580, An06g02120, Aspzo1_0020615, Aspzo1_0147593, Acar5010_399409, Aspzo1_0097397, Aacu16872_042333, An03g05220, Asptu1_0047108, Aspfo1_0048061, Aspka1_0178059 , Aspzo1 — 0070555, AFUB — 096790, Aspwe1 — 0041401, Aspwe1 — 0188242, Aspfo1 — 0203572, Aspka1 — 0176563, Aspzo1 — 0163562 and Acu16887 — 046657.
(15) The forced expression in step a) is achieved by recombining a promoter of a gene encoding a transcription factor contained in a ribosomal peptide biosynthesis gene cluster with a high expression promoter, any of (12) to (14) The manufacturing method of crab.
(16) The production method according to any one of (12) to (15), wherein the ribosomal peptide biosynthesis gene cluster further comprises a gene encoding a protein having peptidase activity.
(17) The production method according to any one of (12) to (16), wherein the cell is a fungal cell.

  According to the present invention, a novel fungal-derived ribosomal peptide that has not been reported so far can be produced. This makes it possible to provide a ribosomal peptide that is a secondary metabolite derived from a fungus expected to be applied to pharmaceuticals and other useful substances.

It is a general formula showing the chemical structure of Ustyroxin B. A. deletion of the constituent genes of the Uustyroxin B biosynthetic gene cluster It is a graph which shows the ustyroxin B production ability of a flavus variant. Panel A shows a chart of LC-MS analysis of ustyroxin B contained in the culture medium of each mutant. Panel B is a graph showing the results of quantifying ustyroxin B in the culture solution. The arrangement | positioning of each structural gene in the austyroxin B biosynthesis gene cluster is shown. The figure which contrasted the position of ORF with respect to a genome sequence about each of the estimated amino acid sequences deduced from the genome sequence of the protein involved in the biosynthesis of ustyroxin B actually translated from mRNA and the ustyroxin B biosynthesis gene cluster ( Upstream). In the figure, dark gray indicates the ORF deduced from the genome sequence, and light gray indicates the ORF of the amino acid sequence translated from mRNA. The figure which contrasted the position of ORF with respect to a genome sequence about each of the estimated amino acid sequences deduced from the genome sequence of the protein involved in the biosynthesis of ustyroxin B actually translated from mRNA and the ustyroxin B biosynthesis gene cluster ( Downstream). In the figure, dark gray indicates the ORF deduced from the genome sequence, and light gray indicates the ORF of the amino acid sequence translated from mRNA. A. flavus and U.S. It is a figure which shows the comparison of arrangement | positioning of each structural gene within the virenus ustyroxine biosynthesis gene cluster. (A) A. It is a figure which shows the structure of the ustyroxine precursor which flavus produces. (B) U.I. It is a figure which shows the structure of the ustyroxin precursor which virens produces. It is a figure which shows the chemical structure of a ustyroxine F. It is a figure which shows the chemical structure of compound F (DELTA) Me from which the methyl group was removed from the ustyroxine F. A. transformed with a gene encoding a point mutant of the HXXHC motif of ustYa. It is a chart of LC-MS analysis which shows that flavus loses the production ability of ustyrokin B. In addition to the austyroxin B biosynthetic gene cluster by the method described in Patent Document 1, A. It is a figure which shows the gene cluster area | region involved in biosynthesis of the ribosomal peptide of molecular weight 1019 discovered by flavus. A part of the biosynthetic gene cluster of a ribosomal peptide having a molecular weight of 1019 was destroyed. It is a LC-MS analysis chart which shows that a flavus mutant loses the production ability of the ribosomal peptide of molecular weight 1019. In addition to the austyroxin B biosynthetic gene cluster by the method described in Patent Document 1, A. It is a figure which shows the gene cluster area | region involved in biosynthesis of the ribosome peptide of molecular weight 809 discovered by flavus. A part of the biosynthetic gene cluster of a ribosomal peptide having a molecular weight of 809 was destroyed. It is a LC-MS analysis chart which shows that a flavus mutant loses the production ability of the ribosomal peptide of molecular weight 809.

  The present invention is a method for producing a ribosomal peptide derived from a fungus, which is represented by step a) a gene encoding a ribosomal peptide precursor consisting of an amino acid sequence including an endoplasmic reticulum translocation signal sequence and a repetitive sequence, and SEQ ID NO: 1. A gene having a base sequence encoding a protein involved in ribosomal peptide biosynthesis with an E value of less than 1e-4 for the base sequence, and an amino acid sequence having an E value of less than 1e-4 for the amino acid sequence shown in SEQ ID NO: 2 A gene encoding a protein involved in ribosomal peptide biosynthesis, or a gene encoding a protein involved in ribosomal peptide biosynthesis consisting of an amino acid sequence having an E value of less than 1e-4 with respect to the amino acid sequence shown in SEQ ID NO: 3, And / or against the amino acid sequence shown in SEQ ID NO: 6 A step of forcibly expressing a ribosomal peptide biosynthetic gene cluster comprising at least a gene comprising an amino acid sequence having an E value of less than 1e-4 and encoding a protein involved in ribosomal peptide biosynthesis; and b) from the cell to the ribosome The production method includes the step of recovering the peptide.

  The present inventors have further developed a bioinformatics method for estimating the presence of the secondary metabolite biosynthesis gene cluster described in Patent Document 1, and have developed a MIDDAS-M (Motif-Independent Detective Algorithm for Secondary). Metagene Gene clusters (Umemura et al., 2013, PLOS ONE, Vol. 8, e84028, http://www.ncbi.nlm.nih.gov/PMC/articles/PMC3877130.pdf02pond.pdf02pond. ). Using this MIDDAS-M, A. Presence of 480 secondary metabolite biosynthetic gene clusters on the genome base sequences of three types of molds including flavus, The ustyroxin B produced by flavus is a ribosomal peptide, NCBI (http://www.ncbi.nlm.nih.gov/gene) gene ID that provides a database of genetic information, and is composed of AFLA_094940 to AFLA_095110 genes It was clarified that it is produced by the function of the gene cluster.

  Ustyroxine B is a substance represented by the general formula shown in FIG. 1 and is known as a secondary metabolite produced by rice Koji disease fungus (Ustilaginidea virens). Ustyroxine B has a basic structure in which tyrosine (Tyr), alanine (Ala), isoleucine (Ile) and glycine (Gly) are linked by a peptide bond, and a cyclic structure between Tyr and Ile. It has a structure in which norvaline is bonded to the ring through a sulfur atom.

<Gene encoding ribosomal peptide precursor>
The ribosomal peptide biosynthesis gene cluster in the present invention includes a gene encoding a ribosomal peptide precursor (hereinafter referred to as a ribosomal peptide precursor gene) consisting of an amino acid sequence including an endoplasmic reticulum translocation signal sequence and a repetitive sequence. Here, the endoplasmic reticulum migration signal sequence is an amino acid sequence generally recognized as an amino acid sequence composed of positively charged amino acids, regions rich in hydrophobic amino acids, and polar amino acids located at the N-terminus of the protein molecule. It is well known to those skilled in the art that there are many specific amino acid sequences.

  Unlike non-ribosomal peptides, ribosomal peptides are biosynthesized via the ribosome, which is the site of protein synthesis. However, ribosomal peptides include cyclic structures, branched structures, or unnatural amino acids, and so on. A compound having a peptide bond with chemical characteristics that are rarely seen. The present invention is a method for producing a ribosomal peptide derived from a fungus, which is biosynthesized from a ribosomal peptide precursor encoded in the fungal genome.

  When the constituent genes of the biosynthesis gene cluster of ustyroxin B were analyzed in detail, the gene whose NCBI gene ID was AFLA_094980, whose function was unidentified, was found to be 4 amino acids ( It was confirmed that Tyr, Ala, Ile, and Gly) encode an amino acid sequence including a repetitive sequence obtained by repeating a sequence arranged in this order 16 times.

  From the results of the above analysis, AFLA — 0949980 is a gene encoding a ustyroxin B precursor that has an endoplasmic reticulum transfer ability and is converted to ustyroxin B through a peptide synthesis pathway in the ER, that is, a ribosomal peptide precursor gene. It is judged that.

  In addition, AspGD (http://www.aspgd.org/, Cerqueira et al., 2013, Nucleic Acids Res., 42, 705-710) is a database of molecular biological information of fungi belonging to the genus Aspergillus. In contrast, using the Smith-Waterman algorithm (Smith et al., 1981, J. Mol. Biol., 147, 195-197), including repetitive sequences having locally similar sequences within the self-sequence ProteinP 4.1 (Nielsen et al., 1997, Protein Eng., Vol. 10, pages 1-6 and Petersen et al., 2011, Nat. Methods, Vol. 8, 785- 786 pages ) Was to confirm the presence or absence of a signal peptide using a ribosomal peptide precursor gene was found in fungi of a plurality of the genus Aspergillus, as shown in Table 1 below. These genes constitute a suitable example as a fungal-derived ribosome peptide precursor gene in the present invention.

<UstYa homolog>
The ribosomal peptide biosynthesis gene cluster in the present invention is a gene having a base sequence encoding a protein involved in ribosomal peptide biosynthesis with an E value of less than 1e-4 with respect to the base sequence shown in SEQ ID NO: 1, A gene that encodes a protein involved in the biosynthesis of a ribosomal peptide consisting of an amino acid sequence with an E value of less than 1e-4 for the amino acid sequence shown, or an amino acid with an E value of less than 1e-4 for the amino acid sequence shown in SEQ ID NO: 3 It contains a gene that encodes a protein consisting of a sequence and involved in the biosynthesis of ribosomal peptides.

  The ribosomal peptide biosynthetic gene cluster in the present invention is involved in the biosynthesis of ribosomal peptides having a bit score of 50 or more, preferably 60 or more, more preferably 75 or more with respect to the base sequence shown in SEQ ID NO: 1. A gene having a base sequence encoding a protein to be encoded, and an amino acid sequence having a bit score of 50 or more, preferably 60 or more, more preferably 75 or more with respect to the amino acid sequence shown in SEQ ID NO: 2, and is involved in the biosynthesis of ribosomal peptides A protein encoding a protein or a protein involved in the biosynthesis of a ribosomal peptide consisting of an amino acid sequence having a bit score of 50 or more, preferably 60 or more, more preferably 75 or more with respect to the amino acid sequence shown in SEQ ID NO: 3 Gene may include a that.

  Furthermore, the ribosomal peptide biosynthetic gene cluster in the present invention is 20% or more, preferably 40% or more, more preferably 60% or more, even more preferably, the amino acid sequence of amino acid sequence 81 to 225 of SEQ ID NO: 3. May comprise an amino acid sequence having an identity of 80% or more, more preferably 90% or more, and most preferably 95% or more, and a gene encoding a protein involved in ribosomal peptide biosynthesis.

The ribosomal peptide biosynthesis gene cluster in the present invention is
(I) a gene having a base sequence encoding a protein involved in biosynthesis of a ribosomal peptide having an E value of less than 1e-4 with respect to the base sequence shown in SEQ ID NO: 1, and an E value for the amino acid sequence shown in SEQ ID NO: 2 Biosynthesis of a ribosomal peptide comprising an amino acid sequence comprising an amino acid sequence of less than 1e-4 and encoding a protein involved in ribosomal peptide biosynthesis, or an amino acid sequence having an E value of less than 1e-4 relative to the amino acid sequence shown in SEQ ID NO: 3 A gene encoding a protein involved in
(Ii) a gene having a base sequence encoding a protein involved in biosynthesis of a ribosomal peptide having a bit score of 50 or more, preferably 60 or more, more preferably 75 or more with respect to the base sequence shown in SEQ ID NO: 1, SEQ ID NO: 2. A gene encoding a protein involved in the biosynthesis of a ribosomal peptide consisting of an amino acid sequence having a bit score of 50 or more, preferably 60 or more, more preferably 75 or more with respect to the amino acid sequence shown in 2, or shown in SEQ ID NO: 3 A gene encoding a protein involved in the biosynthesis of a ribosomal peptide, comprising an amino acid sequence having a bit score of 50 or more, preferably 60 or more, more preferably 75 or more, relative to the amino acid sequence;
Or (iii) 20% or more, preferably 40% or more, more preferably 60% or more, still more preferably 80% or more, and more preferably, the amino acid sequence of amino acid sequence 81 to 225 of SEQ ID NO: 3 A gene encoding a protein involved in ribosomal peptide biosynthesis, comprising an amino acid sequence having 90% or more, most preferably 95% or more identity, and having at least one binuclear metal capture motif It may contain a gene encoding a protein. A preferred amino acid sequence of the binuclear metal capture motif is His-Xxx-Xxx-His-Cys (HXXHC).

  The gene consisting of the base sequence shown in SEQ ID NO: 1 (hereinafter referred to as ustYa) is an unidentified gene whose NCBI gene ID is AFLA_094990. According to the studies by the present inventors, it was confirmed that a strain in which ustYa was disrupted on the genome of A. flavus capable of biosynthesizing ustyroxin B does not produce ustyroxin B. That is, ustYa is a gene involved in biosynthesis of ustyroxin B, which is a ribosomal peptide. Further, the position on the genome is adjacent to the AFLA_094980 encoding the precursor of ustyroxin B, and is one of the constituent genes of the biosynthesis gene cluster of ustyroxin B.

  SEQ ID NO: 2 is an amino acid sequence registered in the NCBI database as an amino acid sequence deduced from the base sequence of SEQ ID NO: 1. When a Blastp search was performed on the AspGD and UniProtKB (version 2013 — 08, Magrane et al., 2011, Database bar009) as of December 16, 2013 based on this amino acid sequence, the E value was less than 1e-4. As shown in Table 2 below, a gene encoding a protein consisting of the amino acid sequences of Aspergillus was found in a plurality of fungi of the genus Aspergillus. Furthermore, all of these genes are adjacent to the ribosomal peptide precursor gene in each fungus or within a range of 25 Kb at the maximum (in terms of the number of genes, there is a maximum of 10 genes between the ribosomal peptide precursor gene and each gene). It was also confirmed that it is located in the vicinity. These genes are constituent genes of fungal-derived ribosomal peptide biosynthesis gene clusters together with ribosomal peptide precursor genes, and suitable examples as genes encoding proteins involved in ribosomal peptide biosynthesis from the ribosomal peptide precursors. Configure. The AspGD ID number in the table is an ID number of a registered gene used in the AspGD.

  As will be described later, the amino acid sequence shown in SEQ ID NO: 3 is an amino acid sequence of a protein that is actually biosynthesized by transcription and translation from a gene having the base sequence of SEQ ID NO: 1 (hereinafter referred to as a ustYa protein). It is.

  The E value (E-value, extraction value) is one of indices for evaluating the homology between a certain sequence (query sequence) and another sequence, and is a random sequence database for a certain query sequence. Is the expected value of the number of alignments that becomes a value equal to or higher than that score. In particular, in the present invention, homology search using any of the blastx, tblastx, tblastn, blastp, or psi-blast homology search programs (hereinafter collectively referred to as the Blast method) as a search means for the sequence database. The E value when executed is adopted. In particular, the E value is preferred when the homology search is executed under the default parameter conditions in the Blast method. Therefore, for the entire sequence information of a microorganism of a certain species, the base sequence shown in SEQ ID NO: 1 is used as a query sequence, and the E value is less than 1e-4 as a result of homology search using the Blast method. A gene containing a sequence, or an amino acid sequence having an E value of less than 1e-4 as a result of a homology search using the Blast method using the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 3 as a query sequence A ribosomal peptide biosynthetic gene cluster containing a gene coding for and the like is a subject of the present invention. Examples of databases to be searched include UniProt or AspGD, or UniProtKB / Swiss-Prot (http://web.expasy.org/docs/swiss-prot_guideline.html).

  The bit score is preferably a value obtained when a homology search is executed under the default parameter conditions in the Blast method.

  Further, in the present invention, the amino acid sequence of amino acids 81 to 225 of SEQ ID NO: 3 is 20% or more, preferably 40% or more, more preferably 60% or more, still more preferably 80% or more, even more preferably. Also included are ribosomal peptide biosynthetic gene clusters comprising amino acid sequences having 90% or more identity, most preferably 95% or more identity, and including genes encoding proteins involved in ribosomal peptide biosynthesis. The 81st to 225th amino acid sequences are regions C-terminal to the transmembrane (TM) helix of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3.

  In the present invention, a gene exhibiting the above-mentioned certain homology with ustYa is represented as a UstYa homolog gene.

  The combination of the ribosome precursor peptide and the ustYa homolog gene referred to in the present invention includes 22 types of genes encoding the ribosome precursor peptide shown in Table 1 and 22 types of ustYa homolog genes shown in Table 2. As of the 16th of the month, the present inventors have confirmed that a large number of bacteria exist in the genus Aspergillus registered in AspGD. Tables 3 to 6 show combinations of these two genes. Ribosomal peptide biosynthetic gene clusters containing these combinations are examples of those that can be used in the production method of the present invention.

<UstYb homolog>
The ribosomal peptide biosynthetic gene cluster in the present invention consists of an amino acid sequence having an E value of less than 1e-4 with respect to the amino acid sequence shown in SEQ ID NO: 6 together with or in place of the above ustYa homolog gene and is involved in the biosynthesis of ribosomal peptides. It may contain a gene encoding the protein to be processed.

  The ribosomal peptide biosynthetic gene cluster in the present invention has a bit score of 50 or more, preferably 60 or more, more preferably 75 or more with respect to the amino acid sequence shown in SEQ ID NO: 6 together with or instead of the above ustYa homolog gene. It may contain an amino acid sequence and a gene encoding a protein involved in ribosomal peptide biosynthesis.

  The ribosomal peptide biosynthetic gene cluster in the present invention is 20% or more, preferably 40% of the 57th to 259th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 6, together with or in place of the above ustYa homolog gene. Or more, more preferably 60% or more, still more preferably 80% or more, more preferably 90% or more, most preferably 95% or more of an amino acid sequence having an identity, and a protein involved in ribosomal peptide biosynthesis It may contain a gene to be encoded.

  The ribosomal peptide biosynthetic gene cluster in the present invention has an E value of less than 1e-4 for the amino acid sequence shown in SEQ ID NO: 6, and a bit score of 50 or more, preferably 60 for the amino acid sequence shown in SEQ ID NO: 6. More preferably, the amino acid sequence is 75 or more, or the 57th to 259th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 6 and 20% or more, preferably 40% or more, more preferably 60% or more, and even more Preferably, the gene comprises a gene encoding a protein having an amino acid sequence having an identity of 80% or more, more preferably 90% or more, most preferably 95% or more, and having at least one binuclear metal capture motif. There may be. A preferred amino acid sequence of the binuclear metal capture motif is His-Xxx-Xxx-His-Cys (HXXHC).

  The gene consisting of the base sequence shown in SEQ ID NO: 4 is a gene whose function was unidentified whose NCBI gene ID is AFLA — 095020. According to the study by the present inventors, it was confirmed that a strain in which the gene AFLA — 095020 was deleted on the genome of A. flavus capable of biosynthesizing ustyroxin B hardly produces ustyroxin B. It was done. That is, gene AFLA — 095020 is a gene involved in biosynthesis of ustyroxin B, which is a ribosomal peptide. In addition, the position on the genome is in the vicinity of the AFLA_0949980 encoding the precursor of ustyroxin B and within a range of about 2 kb, and is one of the constituent genes of the biosynthesis gene cluster of ustyroxin B. is there.

  On the other hand, the amino acid sequence of the protein deduced from the gene AFLA — 095020 and registered in NCBI is shown in SEQ ID NO: 5, but the amino acid sequence of the protein that is actually transcribed and translated from the genomic region containing the gene AFLA — 095020 is It was confirmed that it was shown in SEQ ID NO: 6. Therefore, it was clarified that the protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (hereinafter referred to as ustYb protein) is involved in the ustyroxin B biosynthetic pathway.

  The present invention provides a gene encoding a protein involved in the biosynthesis of a ribosomal peptide (hereinafter referred to as a ustYb homolog gene), comprising the amino acid sequence having an E value of less than 1e-4 described above for the ribosomal peptide precursor gene and the ustYb protein. A ribosomal peptide biosynthetic gene cluster including

  Here, the E value, the bit score, the search condition of the Blast method, the target database, and the like are as described above. Therefore, for the amino acid sequence information registered in the database, the amino acid sequence shown in SEQ ID NO: 6 is used as a query sequence, and as a result of homology search using the Blast method, an amino acid sequence having an E value of less than 1e-4 or A gene encoding an amino acid sequence having a bit score of 50 or more, preferably 60 or more, more preferably 75 or more, and a ribosome peptide biosynthesis gene cluster including the ribosome peptide precursor gene are a subject of the present invention.

  In the present invention, the 57-259th amino acid sequence of the ribosomal peptide precursor gene and the amino acid sequence shown in SEQ ID NO: 6 is 20% or more, preferably 40% or more, more preferably 60% or more, and even more preferably. Is a ribosomal peptide biosynthesis gene cluster comprising an amino acid sequence having an identity of 80% or more, more preferably 90% or more, most preferably 95% or more, and a gene encoding a protein involved in ribosomal peptide biosynthesis. It is included in the subject of the present invention. The 57th to 259th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 6 are regions on the C-terminal side of the transmembrane (TM) helix of the protein consisting of the amino acid sequence shown in SEQ ID NO: 6.

  In the present invention, a gene showing a certain homology with ustYb is represented as a UstYb homolog gene.

<Forced expression>
The present invention includes a step a) of forcibly expressing in a cell a ribosomal peptide biosynthetic gene cluster containing at least a gene encoding the above-mentioned ribosomal peptide precursor and a ustYa homolog gene and / or a ustYb homolog gene.

  There are no reports on ribosomal peptide biosynthetic gene clusters having the above-mentioned characteristics in fungi other than Non-Patent Document 1, and an attempt is made to produce fungal ribosomal peptides by forcibly expressing such ribosomal peptide biosynthetic gene clusters in cells. No attempt was made. The present invention provides a method for producing a novel fungal-derived ribosomal peptide that has not been reported so far.

  A ribosomal peptide biosynthetic gene cluster including at least a gene encoding a ribosomal peptide precursor and a ustYa homolog gene and / or a ustYb homolog gene may be identified on the genome sequence by using the MIDDAS-M algorithm. In addition, when sequence information of ribosomal peptides derived from fungi is accumulated, SMURF (J. Craig Venter Institute, http://jcvi.org/smurf/index.php) or anti-SMASH (Antibiotics & Secondary Metals / Secondary Metals: Also known secondary metabolic gene cluster prediction algorithms such as /antismash.secondarymetabolites.org/) are available. Alternatively, even if each homologous gene is searched based on the amino acid sequence or base sequence shown in SEQ ID NOs: 1 to 6 and a cluster containing a ribosomal peptide precursor gene in the vicinity of the gene is specified based on the result, Good. The gene encoding the ribosomal peptide precursor and the ustYa homolog gene and / or the ustYb homolog gene are adjacent to each other or located within a range of 25 Kb at the maximum, or the ribosomal peptide precursor gene and the ustYa homolog A ribosomal peptide biosynthetic gene cluster located within a range where there are at most 10 genes between the gene and / or the ustYb homolog gene is the subject of the present invention.

  The ribosomal peptide biosynthesis gene cluster in the present invention is preferably specified so as to include a gene encoding a protein having peptidase activity. Proteins having peptidase activity are involved in the biosynthesis of ribosomal peptides, such as by cleaving repetitive sequences of ribosomal peptide precursors. A gene encoding a protein having peptidase activity can be identified as a gene encoding a known peptidase domain sequence deduced from the base sequence of the genome or an amino acid sequence similar thereto.

  The forced expression of a ribosomal peptide biosynthesis gene cluster may be performed in a fungal cell having such a ribosomal peptide biosynthesis gene cluster on its genome, or ribosomal peptide biosynthesis using a genetic recombination technique known to those skilled in the art. The entire gene cluster may be cloned into an appropriate expression vector and introduced into heterologous or homologous fungal cells, yeast, bacteria or other host cells and then forced to express. When fungal cells are used as a host, A. niger, A.M. nidulans, A.N. flavus, A.M. It is preferable to use oryzae and other Aspergillus fungal cells.

  The forced expression of the ribosomal peptide biosynthetic gene cluster identified by the above algorithm or homology search induces the transcription of the gene encoding the transcription factor of the ribosomal peptide biosynthetic gene cluster itself (hereinafter referred to as C6 type TF gene). This can be done. The transcription of the C6 type TF gene may be performed by the promoter of the C6 type TF gene itself, or may be induced by replacing the promoter of the C6 type TF gene with another promoter that functions in the cell. Alternatively, transcription of the C6 type TF gene may be induced by linking another promoter that functions in the cell to the promoter of the C6 type TF gene. Alternatively, the entire C6 type TF gene may be replaced with another TF gene having a promoter that functions in the cell to induce expression of the entire ribosome biosynthesis gene cluster. Further, in accordance with the above method, the entire ribosome peptide biosynthesis gene cluster may be forcibly expressed by inducing the expression of each gene constituting the ribosome peptide biosynthesis gene cluster.

  Another promoter that functions in the cell is appropriately selected according to the type of cell for which the ribosomal peptide biosynthesis gene cluster is to be forcibly expressed, and recombined on the genome or on the vector by genetic recombination methods known to those skilled in the art. Can. Examples of promoters suitable for forced expression of ribosomal peptide biosynthetic gene clusters in fungal cells include the tef1 gene promoter from Aspergillus (Kitamoto et al., 1998, Appl. Microbiol. Biotechnol., 50, 85-92), gpdA gene promoter (Punt et al., J Biotechnol. 1991, 17, 19-33), β-xylosidase xlnD promoter, alcohol dehydrogenase (alcA) promoter (Waring et al., Gene, 1989, 79, 119-130), promoter of α-amylase gene (Tada et al., Mol. Gene. Genet., 1991, 229, 301-306), hiA promoter (Shoji et al., FEMS Microbiol. Lett., 2005, 244, 41-46), Escherichia coli β-glucuronidase gene promoter (Roberts et al., Curr. Genet., 1989, 15, 177-). 180)) and a promoter of glucoamylase gene (glaA).

  The forced expression of a ribosomal peptide biosynthesis gene cluster in a cell can be performed by culturing a cell having the cluster in a medium suitable for the cell. Examples of the medium suitable for fungal culture include Sabouraud dextrose medium, potato dextrose medium, mycocell medium, brain heart infusion medium, vegetable juice medium, yeast extract medium, tryptic soy medium, and the like. It may be a medium or a solid medium such as an agar medium. The culture may be aerobically cultured under conditions common to fungi, for example, at a temperature around 30 ° C.

<Recovery>
The present invention includes step b) of recovering the ribosomal peptide from the cell. The ribosomal peptide can be recovered from the cells by a generally known technique from a cell culture medium cultured under conditions capable of producing the ribosomal peptide. Further, when ribosomal peptides are accumulated inside the cells, the cells may be collected by a normal method after being destroyed by an appropriate method. The recovered ribosomal peptide may be stored and used after dissolving or suspending in water, a suitable buffer solution or an organic solvent. Moreover, you may dry by freeze drying and other suitable methods.

<UstYa protein>
The present invention further includes the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence in which one to several amino acids of the amino acid sequence are deleted or substituted, or the amino acid sequence in which one to several amino acids are added. A protein comprising a sequence and involved in the biosynthesis of a ribosomal peptide in a fungal cell, and a cDNA encoding the same are provided.

  The protein consisting of the amino acid sequence shown in SEQ ID NO: 3 is a protein that is transcriptionally translated from the gene consisting of the base sequence shown in SEQ ID NO: 1, ie, ustYa (NCBI gene ID AFLA — 094990), and biosynthesized. The analysis of the base sequence of ustYa in consideration of the start codon and the reading frame shows the presence of a plurality of introns and exons and a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 as the ustYa gene product. However, A. As a result of experimental determination of the base sequence of mRNA transcribed in flavus cells, it was confirmed that the amino acid sequence of the ustYa protein is as shown in SEQ ID NO: 3, which is different from that of SEQ ID NO: 2 deduced. It was done. Thus, the protein consisting of the amino acid sequence shown in SEQ ID NO: 3 is a novel substance.

  As will be shown later in the examples, A.I. By replacing the ustYa gene on the flavus genome with a foreign gene, the function of the ustYa gene is lost. The ability to produce flavus ustyroxine B and ustyroxine F is lost. That is, the ustYa protein is a protein involved in biosynthesis of ustyroxin B and ustyroxine F. The ustYa protein includes a motif sequence region involved in binding to metal ions, particularly zinc ions, and a region similar to the WH1 domain. Protein transport during translation of the ribosomal peptide precursor, between tyr and Ile It is presumed that it is involved in the biosynthesis of austyroxin B through functions such as the formation of a cyclic structure, redox activity, GTPase activity, ATP binding ability, gene expression or transcription control ability.

  In addition, when a blast search was performed on the UniProtKB, which is a protein sequence database, based on the amino acid sequence shown in SEQ ID NO: 3, no sequence that was found to be homologous in species other than fungi (Fungi) was detected.

  The amino acid sequence shown in SEQ ID NO: 3 consists of an amino acid sequence in which one to several amino acids are deleted or substituted, or an amino acid sequence in which one to several amino acids are added to the amino acid sequence. A protein involved in ribosomal peptide biosynthesis is also an embodiment of the present invention.

  It has been empirically recognized that the amino acid sequence of a protein can tolerate highly conservative mutations in physicochemical properties such as charge, size, and hydrophobicity of amino acid residues. For example, for substitution of amino acid residues, glycine (Gly) and proline (Pro), Gly and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln) ), Aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), Thr and serine (Ser) or Ala, lysine (Lys) and arginine (Arg), and the like. It is also experienced by those skilled in the art that even when the above-described conservation is exceeded, mutations that do not lose the essential function of the protein can exist.

  Therefore, a protein comprising an amino acid sequence in which one to several amino acids of the amino acid sequence shown in SEQ ID NO: 3 has been deleted or substituted, or an amino acid sequence in which one to several amino acids have been added to the amino acid sequence, Proteins involved in the biosynthesis of fungal ribosomal peptides, in particular proteins involved in protein transport in the translation process of ribosomal peptide precursors or the formation of a cyclic structure between Tyr and Ile, are understood as one aspect of the present invention. The

<UstYb protein>
The present invention further includes an amino acid sequence represented by SEQ ID NO: 6, an amino acid sequence in which one to several amino acids of the amino acid sequence are deleted or substituted, or an amino acid in which one to several amino acids are added to the amino acid sequence A protein comprising a sequence and involved in the biosynthesis of a ribosomal peptide in a fungal cell, and a cDNA encoding the same are provided.

  The protein consisting of the amino acid sequence shown in SEQ ID NO: 6 is a protein that is transcriptionally translated from a peripheral region partially including the gene consisting of the base sequence shown in SEQ ID NO: 4 (NCBI gene ID AFLA — 095020) and biosynthesized. The amino acid sequence of the protein deduced from the gene AFLA — 095020 and registered in NCBI is shown in SEQ ID NO: 5, but the amino acid sequence of the protein actually transcribed and translated from the genomic region containing the gene AFLA — 095020 is SEQ ID NO: 6 was confirmed. Thus, the protein consisting of the amino acid sequence shown in SEQ ID NO: 6 is a novel substance.

  As will be shown later in the examples, A.I. by replacing the gene AFLA — 095020 on the flavus genome with a foreign gene and deleting it. The ability of flavus to produce ustyroxine is lost. That is, the ustYb protein is a protein involved in the biosynthesis of ustyroxin B. The ustYb protein has two motif sequence regions involved in binding to metal ions, particularly zinc ions, and a histidine-rich region. Protein transport during the translation process of the ribosomal peptide precursor, between tyr and Ile It is presumed to be involved in the biosynthesis of ustyroxin B through functions such as the formation of a cyclic structure, redox activity, GTPase activity, ATP binding ability, gene expression or transcriptional control ability.

  In addition, when a blast search was performed on the UniProtKB, which is a protein sequence database, based on the amino acid sequence shown in SEQ ID NO: 6, no sequence that was found to be homologous in any species other than fungi (Fungi) was detected.

  The amino acid sequence shown in SEQ ID NO: 6 consists of an amino acid sequence in which one to several amino acids are deleted or substituted, or an amino acid sequence in which one to several amino acids are added to the amino acid sequence. A protein involved in ribosomal peptide biosynthesis is also an embodiment of the present invention.

  As described above, it has been empirically recognized that the amino acid sequence of a protein can tolerate highly conservative mutations in physicochemical properties such as the charge, size, and hydrophobicity of amino acid residues.

  Therefore, a protein consisting of an amino acid sequence in which one to several amino acids of the amino acid sequence shown in SEQ ID NO: 6 are deleted or substituted, or an amino acid sequence in which one to several amino acids are added to the amino acid sequence, Proteins involved in the biosynthesis of ribosomal peptides derived from fungi, in particular proteins involved in protein transport in the translation process of ribosomal peptide precursors or the formation of a cyclic structure between tyr and Ile are understood as one aspect of the present invention. The

<CDNA>
The present invention further provides a cDNA encoding the ustYa protein or ustYb protein. The cDNA herein is a DNA molecule comprising a base sequence in which a part of the base sequence on the genome is modified, which is suitable for recombinantly producing the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6. Means. Suitable examples thereof include cDNA encoding the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6 as a series of open reading frames (ORFs), or 1 to the number of amino acid sequences shown in SEQ ID NO: 3 or SEQ ID NO: 6. A cDNA encoding an amino acid sequence in which one or more amino acids have been deleted or substituted, or an amino acid sequence in which one to several amino acids have been added to the amino acid sequence as a series of ORFs. As a different embodiment, a cDNA containing a part of an intron sequence in the base sequence on the genome is also included in the scope of the present invention.

  The cDNA of the present invention may be a single strand or a double strand or triple strand bound to a nucleic acid having a complementary sequence thereto. Further, it may be labeled with an enzyme such as horseradish peroxidase (HRPO), a radioisotope, a fluorescent substance, a chemiluminescent substance, or the like.

  Based on the sequence information shown in SEQ ID NOs: 1 to 6, the cDNA of the present invention is cloned using a genetic engineering technique such as hybridization or PCR, or a chemical synthesis technique such as a phosphoramidite method. Can be easily manufactured by those skilled in the art. Moreover, the cDNA of the present invention can be used after being recombined into various vectors that are commercially available or generally available to those skilled in the art. The vector may have other base sequences as necessary. Examples of other base sequences include enhancer sequences, promoter sequences, ribosome binding sequences, base sequences used for the purpose of copy number amplification, base sequences encoding signal peptides, base sequences encoding other proteins, poly sequences A additional sequence, splicing sequence, replication origin, base sequence of gene serving as a selection marker, and the like.

  The cDNA of the present invention, a recombinant vector containing the cDNA, and a host cell transformed with the vector can be used for recombinant production of ustYa protein and ustYb protein. In order to recombinantly produce the protein of the present invention, host cells transformed with the cDNA of the present invention or a vector containing the cDNA are cultured under appropriate conditions, and the culture mixture is recovered, and the protein of the present invention is recovered. It may be purified as necessary. As a method for purifying the protein of the present invention from the culture mixture, an appropriate method can be appropriately selected from methods usually used for the purification of proteins and low molecular compounds. That is, salting-out method, ultrafiltration method, isoelectric point precipitation method, gel filtration method, electrophoresis method, various affinity chromatography such as ion exchange chromatography, hydrophobic chromatography and antibody chromatography, chromatofocusing method, adsorption An appropriate method may be appropriately selected from commonly used methods such as chromatography and reverse phase chromatography, and if necessary, purification may be performed in an appropriate order using an HPLC system or the like.

  The protein of the present invention can also be expressed as a fusion protein with other proteins and tags (eg, glutathione S transferase, protein A, histidine tag, FLAG tag, etc.). Such a fusion protein is also an embodiment of the present invention. These fusion proteins can be excised using an appropriate protease (eg, thrombin or the like), and the protein can be prepared more advantageously.

  As described above, the protein of the present invention can be prepared in the form of a single protein alone or in the form of a fusion protein with another type of protein. However, the present invention is not limited to these. It is also possible to convert it into a form. For example, various chemical modifications to proteins, binding to polymers such as polyethylene glycol, binding to insoluble carriers, encapsulation in liposomes, and the like can be performed by various techniques known to those skilled in the art.

  The fungal-derived ribosomal peptide synthesis gene cluster identified through the present invention includes genes encoding proteins involved in various modification reactions in addition to the ribosomal peptide precursor gene and the ustYa homolog gene and / or ustYb homolog gene. These proteins are presumed to be proteins optimized for the biosynthesis of the respective ribosomal peptides. For this reason, non-natural ribosomal peptides with structures and physiological activities different from those produced in nature can be produced by recombining the constituent genes of each cluster with each other or arbitrarily combining multiple constituent genes. Is also possible.

  In addition, when the specified ribosomal peptide synthesis gene cluster lacks a gene encoding a regulatory factor, or a part of the ustYa gene homolog and / or ustYb gene homolog is deleted, etc. It is also possible to produce a new ribosomal peptide by supplementing a gene having a corresponding function or another ustYa gene homolog and / or ustYb gene homolog from the genes constituting the ribosomal peptide synthesis gene cluster.

  For the genetic recombination technique, cell culture, and protein chemistry technique in the present invention, various experimental handbooks, manuals, textbooks known to those skilled in the art, or techniques described in various academic literatures and patent literatures are appropriately selected and adopted. can do. For example, J. et al. Examples include Sambrook et al. (Molecular Cloning, a Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989) and other methods described in experimental operation manuals.

  The following examples further illustrate the present invention.

<Example 1> Identification of a Ustyroxine B biosynthetic gene cluster (1) Secondary metabolite using MIDDAS-M method based on whole genome information of Aspergillus flavus registered as GenBankEQ963472-EQ966232 Synthetic gene cluster analysis was performed. As a result, 18 genes from NCLA gene IDs AFLA — 095040 to AFLA — 095110 were estimated as candidate genes constituting the cluster.

(2) According to the method of Min et al. (2007, Process Biochemistry, Vol. 42, 729-733), deletion was performed by replacing the above genes one by one with the selection marker gene using the pyrG gene as a selection marker. A total of 18 types of A.P. A flavus mutant was created. The specific work is as follows.

  First, PCR was performed for each of the 18 target genes using a primer set designed to amplify the genomic region including 1 kb upstream and downstream thereof, and amplified fragment 1 of the genomic region was prepared. On the other hand, A. PCR was performed using a primer set designed to amplify an approximately 1.8 kb genomic region containing the nidulans pyrG gene, and an amplified fragment 2 of the genomic region containing the pyrG gene was prepared. Furthermore, PCR was performed using the amplified fragments 1 and 2 as a template and a primer set designed to amplify the nucleic acid to which both fragments were linked to prepare a fusion PCR amplified fragment 3 of both genomic regions.

1. Add 24 g / L Difco potato dextrose medium containing 12 mg / mL uracil to The conidia of flavus (CA14 strain, Δku70 ΔpyrG ΔniaD) were suspended (10 ⁶ / mL) and cultured at 37 ° C. for 2 days. The cells were collected, suspended in a buffer containing a lytic enzyme (lysing enzyme from Sigma, Yatalase from TaKaRa, cellulase from Yakult), and incubated at 30 ° C. for 3 hours to prepare a protoplast. After suspending this protoplast in an isotonic solution, the 18 kinds of fusion PCR amplified fragments 3 (1 μg) were added separately for transformation, and incubated in the presence of polyethylene glycol. After incubation, the protoplasts were placed on the regeneration medium and cultured for 3-5 days to regenerate the fungal cells. A nucleic acid fragment obtained by amplifying by PCR using the recombined genomic region as a template that the regenerated 18 mutant strains have a genomic structure in which each target gene has been deleted by substitution with the introduced pyrG gene. This was confirmed by sequence analysis.

(3) Each of the 18 mutants was treated in 30 mL of V8 juice medium (containing 20% Campbell V8 vegetable juice and 0.3% calcium carbonate) at 30 ° C. for 5 days, then 23 mL of acetone was added at room temperature. Incubated for 1 hour. Acetone is evaporated from the extract from which the mycelium has been removed with a filter, an equivalent amount of ethyl acetate is added to the remaining concentrated solution, and the mixture is allowed to stand for 1 hour. The aqueous phase is passed through a filter having a pore size of 0.22 μm, and LC- A sample for MS was prepared.

LC-MS samples were fractionated using Ultimate 3000 HPLC (Dionex) with Develosil XG-C18M-5 reverse phase column (Nomura Chemical) and water / acetonitrile gradient (0% -100%) solvent. . The content of ustyroxin B in the eluted fraction was quantified using micrOTOFII KIK2 MS (Bruker). Quantification was carried out by calculating the peak area of EICs [M−H] − at m / z 644.2 ± 0.1 at RT = 9 minutes. The results of the LC-MS are shown in FIG.

  Among the above genes, deletion of ustH (AFLA — 095030, γ-glutamyl transpeptidase) did not affect the regenerative ability of ustyroxin B. It is inferred that another gene (AFLA — 074100) encoding a protein having the same physiological activity exists in the flavus genome, and this supplements the function of the deleted ustH.

  All of the mutant strains lacking ustF1 (AFLA — 094950) and ustP2 (AFLA — 095010) showed the ability to produce a small amount of ustyroxin B. A. There is another gene having high homology with these genes in the flavus genome, and it is speculated that these supplement the function of the deleted gene.

  For ustP1 (AFLA — 095000) and ustYb (AFLA — 095020), other highly homologous genes do not exist in the genome, but mutant strains lacking ustP1 and ustYb, respectively, also showed the ability to produce small amounts of ustyroxin B. . As will be described later, the amino acid sequence of the protein expressed from each of ustP1 and ustYb determined by the RNA sequence was different from the amino acid sequence annotated in the NCBI database. Although not constrained by speculation, as a result, replacement with the pyrG gene allows the expression of mutants in which a part of the protein expressed from each of ustP1 and ustYb is deleted. It is presumed that this has brought about the production ability of.

  According to the results of the Blastp search for UniProtKB, ustP1 encodes a sequence corresponding to the N-terminal region of the S41 peptidase family and ustP2 encodes a sequence corresponding to the active region of the same S41 peptidase family, and both genes encode one protein. It was inferred that

  As a result of searching against the Blast database, it was confirmed that ustYa (AFLA — 0949990) and ustYb are genes specific to fungi.

  ustS (AFLA — 095110, glutathione S-transferase) is an enzyme that catalyzes the reaction that transfers S-norvaline to the aromatic ring of the tyrosine residue that constitutes ustyroxin, which is an essential reaction step in the biosynthesis of ustyroxin B. . However, deletion of ustS did not affect the ability to produce usthyroxine. This is presumed to be because the function of ustS lacking the homologous gene AFLA — 087240 existing outside the gene cluster is compensated.

The amino acid sequences encoded by AFLA — 095080 and AFLA — 095090, respectively, completely retained the fungal-specific transcription factor domain (pfam domain) and the Zn2Cys6 binucure cluster DNA binding domain. The N-terminal and C-terminal amino acid sequences of the oryzae-derived transcription factor (gene name: AO090113000035) were identical. It was confirmed by quantitative real-time PCR that the region connecting AFLA — 095080 and AFLA — 095090 was amplified as cDNA. From the above, it was confirmed that AFLA — 095080 and AFLA — 095090 were continuously transcribed to form one transcription factor (ustR).

  From the results of the analysis of the above mutants, a gene whose production of ustyroxin B disappears due to the deletion of the gene, and a gene that plays an important role in the biosynthetic pathway even though the production ability of ustyroxin B is maintained even if the deletion The genes from ustO (AFLA — 094940) to ustS (AFLA — 095110) were determined to be the ustyroxin B biosynthesis gene cluster that is substantially involved in the biosynthesis of ustyroxin B. Table 7 shows NCBI ID numbers, estimated functions, and sizes (number of base pairs) of these genes, and FIG. 3 shows the arrangement on the genome.

<Example 2> Enhancement of ustB production by overexpression of ustR Amplified fragment prepared using primers designed so that the promoter region of the flavus tef1 gene (Kitamoto et al., 1998, Appl. Microbiol. Biotechnol., 50, 85-92) can be obtained as an amplified fragment. 4 and the amplified fragment 5 prepared using a primer designed so that the ustR promoter region in the nucleic acid encoding the ustyroxin B biosynthetic gene cluster can be obtained as an amplified fragment. An amplified fragment 6 in which the tef1 gene promoter was replaced was prepared by a fusion PCR method. Using this amplified fragment 6, A. The flavus CA14 strain (Δku70 ΔpyrG ΔniaD) was transformed to obtain a ustR overexpression mutant ustR ^OE .

ustR ^OE was inoculated into 100 mL of V8 juice medium (containing 20% Campbell V8 vegetable juice and 0.3% calcium carbonate), stirred and cultured at 30 ° C. for 4 days, and the cells were collected and ISOGEN (Nippon Gene) The total RNA was extracted from the cells using a high capacity RNA-to-cDNA kit (Applied Biosystems). On the OneOnePlus Real-Time PCR System (Applied Biosystems), the quantitative analysis of the expression of ustR was performed using qPCR with Power SYRB Green PCR master mix (Applied Biosystems). The expression level was standardized based on the expression level of tubulin β subunit (AFLA — 051840).

As a result, it was confirmed that the expression level of ustR in ustR ^OE increased 15 times compared to the control. In addition, when the amount of ustyroxin B was quantified in the same manner as in Example 1, the production amount of ustyroxine B was increased 4.8 times.

<Example 3> Re-annotation of translation region of ustB cluster gene by RNA sequencing A revertant strain in which the pyrG gene was restored was prepared using the flavus CA14 strain (Δku70 ΔpyrG ΔniaD) as a parent strain. The revertant strain was inoculated into 100 mL of V8 juice medium (containing 20% Campbell V8 vegetable juice and 0.3% calcium carbonate), and cultured with stirring at 30 ° C. for 4 days. The cells recovered after the cultivation were frozen with liquid nitrogen and sufficiently pulverized in a mortar. The crushed cells were added to 15 mL of ISOGEN (Nippon Gene), mixed well, and placed on ice. The supernatant after centrifugation was added to 3 mL of chloroform and mixed well, and then allowed to stand at room temperature for 3 minutes. The aqueous layer recovered by centrifugation again was added to 7.5 mL of isopropanol and allowed to stand at room temperature for 10 minutes. The pellet obtained by centrifugation again was washed with ethanol to obtain an RNA sample.

  A fragment library was created using the SOLiD Total RNA-Seq kit (Applied Biosystems) based on the manufacturer's instructions. RNA sequencing was performed using a next-generation sequencer system SOLiD 5500xl (Applied Biosystems) according to the protocol.

  The XSQ file calculated from the SOLiD system was expanded to obtain a fastq file corresponding to each sample. This data and the read data registered in the public sequence read database NCBI SRA (http://www.ncbi.nlm.nih.gov/SRA) are SRR283857, SRR283858, SRR495778, SRR495942, SRR4594431, SRR544487, SRR544872, The following operations were performed using the SRR 610531, the SRR 610532, the SRR 610533, the SRR 610534, the SRR 610535, the SRR 610537, and the SRR 610538. Mapping work was performed using a lead having a length of 30 bases or more after trimming bases that do not contain unidentified bases and have a Q value (Quality value) of less than 20 consecutive 7 bases on average. The software used was bowtie and tophat. The work was carried out on a computer (DELL: CPU Intel Xeon E52643, memory 256 GB, hard disk 40 TB, OS: Ubuntu Linux (registered trademark) 13.10)). Display the mapping result with the genome information visualization tool, visually check the corresponding gene region (gene AFLA_0994940-AFLA_095110 on scaffold GenBank EQ963480), and identify the region that is consistent with the start codon and codon reading frame as the CDS region did. The results are shown in Tables 8-12. In addition, for each of the obtained results, that is, each protein actually translated from mRNA and the deduced amino acid sequence deduced from the genome sequence, the positions of the ORFs relative to the genome sequence are compared in FIGS. 4 and 5. Show.

<Example 4> Identification of Ustylaginidea virens ustyroxine biosynthetic gene cluster Ustylaginoide virens (U. virens) reported by Zhang et al. (Nat. Commun. 2014, Vol. 5, page 3849) and registered in NCBI. Of the genomic base sequence UV-8b (NCBI Accession Nos. JHTR01000001-JHTR01000449) shown in Tables 8-12 above. Of the flavus ustyroxin B biosynthetic gene cluster, each base sequence of the gene excluding ustU (base sequence corresponding to the CDS region encoding each amino acid sequence shown in the table) is used as a query, and the E value is 1e. The tBLASTx search was performed with the homology cutoff value being less than −4. After the gene annotation by the pipeline using the ALN method and GlimerHMM method is performed again for the detected region, the amino acid sequence information encoded by the annotated gene is used as a query. A blastp search was performed for each gene constituting the flavus ustyroxin B biosynthesis gene cluster, and the homology was evaluated. In addition, with a similar query, a Blastp search was performed on the UniProtKB database to perform function prediction. Based on these analyses, U.I. Thirteen genes shown in Table 13 were identified in the scaffold of the virens genome.

  In addition to UV8b — 7484 to UV8b — 7494 annotated by Zhang et al., Three genes (uv_ustT, uv_ustC and uv_ustYb) were newly annotated by the inventors. All of these 13 genes are A. The E value for each gene constituting the flavus ustyroxin B biosynthetic gene cluster was less than 1e-4, confirming high homology. In the above search conditions, A. The genes homologous to ustP and ustH contained in the flavus ustyroxin B biosynthesis gene cluster are described in U.S. Pat. It was not found from the range of the above Virens scaffold. The gene order is A. It was different from that of flavus (FIG. 6).

By analysis of the amino acid sequence encoded by each gene, uv_ustA is an endoplasmic reticulum transition signal sequence and a repetitive sequence in which Uyroxin A amino acid residues Tyr, Val, Ile, and Gly are arranged in this order, and ustyroxin It was confirmed that the amino acid residues constituting B, Tyr, Ala, Ile and Gly, encoded a repetitive sequence arranged in this order (FIG. 7B).
From this, it was speculated that uv ustA is a gene encoding a ustyroxin A precursor and a ustyroxine B precursor. A. uv_ustYa and uv_ustYb were found as genes having high homology with flavus ustYa and ustYb (E value less than 1e-4 and bit score of 50 or more), respectively. These correspond to the ustYa homolog gene and ustYb homolog gene according to the present invention, respectively.

  Furthermore, it was confirmed that the 13 genes shown in Table 13 existed together within a range of about 30 kb in the scaffold. Furthermore, the distance between uv_ustA and uv_ustYa and between uv_ustA and uv_ustYb was 1.4 Kb and 3.0 kb, respectively.

  From the above, the 13 genes confirmed this time in Uviresns are the U.S. genes capable of synthesizing ustyroxins A and B. It was inferred that it constitutes a biosynthetic gene cluster of virenes, ustyroxin A and B.

<Example 5> Analysis of each gene disruption strain in the ustyroxine biosynthesis gene cluster LC-MS analysis was performed on the culture supernatant of each of the 15 gene disruption strains in the ustyroxine biosynthesis gene cluster specified in Example 1. When performed, in addition to the MS peak of m / z 644.2 [M−H] ⁻ corresponding to ustyroxine B, two MS peaks (m / z 465. 2 [M−H] ⁻ and m / z 451.2 [M−H] ⁻ ) were found. These peaks were separated and subjected to structure determination by NMR. As a result, ustyroxin F from which S-norvaline was removed from ustyroxin B (FIG. 8) and compound FΔMe from which the methyl group was further removed (FIG. 9) were obtained. Was identified.

Moreover, it was confirmed that in the disrupted strains of each gene of ustA, ustR, ustYa, ustYb and ustQ, the peaks of the cyclic peptides of ustyroxin B, ustyroxine F and compound FΔMe all disappeared. Since ustA is a ribosomal peptide precursor protein gene, and ustR is a transcription factor that induces gene cluster expression, ustYa, ustYb, and ustQ circulate each of the above compounds and use peptidases such as the Golgi apparatus. It was presumed to play a role in protecting against degradation.
<Example 6> Function identification of ustYa

  For HXXHC motifs, which are two binuclear metal capture motifs included in ustYa (AFLA — 094990), the respective HCs (168-169th HC and 195-196th HC in SEQ ID NO: 3) are AS-AS, AS An expression vector containing genes encoding four types of ustYa mutants mutated to HC, HS-HC and HC-HS was constructed as follows.

  The base sequence of ustYa containing 145 bp upstream and 443 bp downstream was introduced into a plasmid vector by TA cloning using TTarget Clone-Plus- (Toyobo, TAK-201) with a restriction enzyme site SmaI added to both ends. After confirming the sequence, two HCs in the motif become HC-HC (control), AS-AS, AS-HC, HS-HC, HC-HS using PrimeSTA Mutagenesis Basal Kit (TaKaRa, R046A). Five types of mutated cloning vectors were prepared. The codons used at this time are A: GCC, S: TCC, and H: CAT.

  For each of the five types, after confirming the sequence of the mutation site on the plasmid vector, the DNA construct excised by the restriction enzyme SmaI was used as a ustP sequence adjacent to ustYa and a DNA construct that is downstream and also the remaining upstream part of ustYa, A DNA construct having the Aspergillus nidulans pyrG marker linked to the Cre-loxP sequence, which is a marker recycling system, was fused with the fusion PCR method to prepare a DNA construct of less than 7 kb.

  This DNA construct was prepared by A.P. The flavus CA14 strain was transformed, PCR confirmation and base sequence confirmation were performed by amplification of the construct on the genome sequence, and the four types of point mutant strains (two AS-AS strains, respectively) in which two types of HXXHC motifs of ustYa were changed. AS-HC strain, HS-HC strain, HC-HS strain) and control strain (HC-HC strain) were obtained.

The obtained four types of point mutants and HC-HC strains were cultured in a medium containing 1% xylose to remove the Cre-loxP sequence, and then cultured on a corn solid medium. The aqueous layer after extraction with ethyl acetate was analyzed by LC-MS. As a result, in a control strain HC-HC strain, ^[M-H] of m / z 644.2 and m / z 465.2 corresponding to the mouse thyroxine B and mouse thyroxine F - but MS peaks were confirmed In the remaining 4 point mutants, these peaks disappeared (FIG. 10).

  From the above, it was presumed that ustYa and its homologue ustYb are enzymes having an essential activity for the production of ustyroxine, and the active center is a binuclear metal capture motif part centered on two HXXHC motifs.

<Example 7> Identification of a ribosomal peptide biosynthetic gene cluster that biosynthesizes a peptide compound having a molecular weight of 1019 Based on whole genome information of Aspergillus flavus registered as GenBank EQ963472-EQ966232, an endoplasmic reticulum migration signal sequence A gene encoding a ribosomal peptide precursor consisting of an amino acid sequence including a repetitive sequence and a gene encoding a protein consisting of an amino acid sequence having an E value of less than 1e-4 with respect to the amino acid sequence shown in SEQ ID NO: 3 (UstYa), By the Smith-Waterman algorithm, signalP 4.1 and the Blast method. As a result, five genes having a base sequence encoding a protein consisting of an amino acid sequence having an E value of 1e-11 with respect to the gene (AFLA — 063260) encoding a ribosomal peptide precursor and the amino acid sequence (UstYa) represented by SEQ ID NO: 3 ( AFLA_063270 to AFLA_0631010) were found to be adjacent.

  Furthermore, expression information obtained under 28 conditions including culture conditions that produce a lot of secondary metabolites registered in a public database (NCBI GEO http://www.ncbi.nlm.nih.gov/geo/) Using (GSE15435) and the above-mentioned MIDDAS-M, the gene region including the above gene and co-expressed was examined.

  As a result (FIG. 11) and gene function annotation, at least a region consisting of NCBI gene IDs AFLA — 063170 to AFLA — 063330 was estimated as a novel ribosomal peptide biosynthesis gene cluster.

  Further, in the above cluster region, all seven types of AFA_06260 to AFLA_06320, each of AFLA_063280 and AFLA_0631010 were deleted by substituting each with a selectable marker gene (pyrG). A flavus mutant was created. The specific work is as follows.

  For each of the above three types of target gene regions, PCR was performed using a primer set designed to amplify a genomic region containing 1 kb upstream and downstream thereof, thereby producing an amplified fragment 1 of the genomic region. On the other hand, A. PCR was performed using a primer set designed to amplify an approximately 1.8 kb genomic region containing the nidulans pyrG gene, and an amplified fragment 2 of the genomic region containing the pyrG gene was prepared. Furthermore, PCR was performed using the amplified fragments 1 and 2 as a template and a primer set designed to amplify the nucleic acid to which both fragments were linked to prepare a fusion PCR amplified fragment 3 of both genomic regions.

1. Add 24 g / L Difco potato dextrose medium containing 12 mg / mL uracil to The conidia of flavus (CA14 strain, Δku70 ΔpyrG ΔniaD) were suspended (10 ⁶ / mL) and cultured at 37 ° C. for 2 days. The cells were collected, suspended in a buffer containing a lytic enzyme (lysing enzyme from Sigma, Yatalase from TaKaRa, cellulase from Yakult), and incubated at 30 ° C. for 3 hours to prepare a protoplast. After suspending this protoplast in an isotonic solution, the three kinds of fusion PCR amplified fragments 3 (1 μg) were separately added for transformation, and incubated in the presence of polyethylene glycol. After incubation, the protoplasts were placed on the regeneration medium and cultured for 3-5 days to regenerate the fungal cells. A nucleic acid fragment obtained by PCR amplification using the recombined genomic region as a template, indicating that each of the three regenerated mutant strains has a genomic structure in which each target gene has been deleted by substitution with the introduced pyrG gene. This was confirmed by sequence analysis.

  Each of the three mutants was sterilized by adding 1.2 mL of distilled water, cultivated on 2.5 g of ground corn for 6 days at 30 ° C., then added with 10 mL of 80% acetone and incubated at room temperature for 1 hour. did. Acetone is evaporated from the extract from which the bacterial cells and solid medium have been removed, and an equivalent amount of ethyl acetate is added to the remaining concentrated solution. After reacting for 1 hour, the aqueous phase is passed through a filter having a pore size of 0.22 μm. A sample for MS was prepared. This sample was fractionated using Ultimate 3000 HPLC (Dionex) equipped with a Develosil XG-C18M-5 reverse phase column (Nomura Chemical Co.) and a water / acetonitrile gradient (0% -100%) solvent.

When the patterns of m / z values corresponding to the retention time and molecular weight were compared between the three types of disrupted strains and the control strain (pyrG marker-reverted strain), it was present in the control strain, but the ribosomal peptide biosynthesis gene cluster An MS peak of m / z 1018.4 [M−H] ⁻ (m / z 1020.4 [M + H] ⁺ with a cation) disappeared only in the corresponding three types of disrupted strains was found at a retention time of 12 minutes. (FIG. 12).

  The above peaks are collected and the types of constituent amino acids are analyzed by acidolysis, and this compound is a peptide compound having a molecular weight of 1019 (hereinafter referred to as PMW1019) whose core peptide sequence is a novel ribosomal peptide precursor protein. I identified it.

From the analysis results of the above mutants, 17 genes AFLA — 063170 to AFLA — 063330 were determined to be constituent genes of the biosynthetic gene cluster that is substantially involved in PMW1019 biosynthesis. Table 14 shows the NCBI ID numbers, estimated functions, and sizes (number of base pairs) of these genes.

<Example 8> Enhancement of production amount of PMW1019 by overexpression of transcription factor NCBI gene AFLA_063180 contained in the biosynthetic gene cluster specified in Example 6 is homologous to a protein containing the zinc finger C2H2 motif contained in the transcription factor It was revealed by amino acid sequence analysis. Therefore, A. Amplified fragment prepared using primers designed so that the promoter region of the flavus tef1 gene (Kitamoto et al., 1998, Appl. Microbiol. Biotechnol., 50, 85-92) can be obtained as an amplified fragment. 4 and an amplified fragment 5 prepared using a primer designed so that the translated region of AFLA — 063180 can be obtained as an amplified fragment, the amplified fragment 6 in which the promoter region of AFLA — 063180 is replaced with the tef1 gene promoter Prepared by the fusion PCR method. Using this amplified fragment 6, A. The flavus CA14 strain (Δku70 ΔpyrG ΔniaD) was transformed to obtain an overexpressing mutant of AFLA — 063180.

AFLA — 063180 overexpressing mutant strain was cultured for 6 days at 30 ° C. on 2.5 g of ground corn that had been sterilized by adding 1.2 mL of distilled water, and then 10 mL of 80% acetone was added to the medium at room temperature. And incubated for 1 hour. Acetone is evaporated from the extract from which the bacterial cells and solid medium have been removed, and an equivalent amount of ethyl acetate is added to the remaining concentrated solution. After reacting for 1 hour, the aqueous phase is passed through a filter having a pore size of 0.22 μm to perform LC-MS. Samples for measurement were prepared and fractionated using Ultimate 3000 HPLC (Dionex) equipped with Develosil XG-C18M-5 reverse phase column (Nomura Chemical Co.) and water / acetonitrile gradient (0% -100%) solvent. . The content of PMW1019 in the elution fraction was quantified using a microOTOFII KIK2 MS (Bruker). Quantification was performed by calculating the peak area of EICs [M−H] ⁻ at m / z 1018.4 ± 0.1 at RT = 12 minutes.
As a result, it was confirmed that the production amount of PMW1019 of the AFLA_063180 overexpressing mutant increased 1.8 times compared to the wild-type control strain.

<Example 9> Identification of constituent amino acids of PMW1019 The cultured cells were extracted with 80% acetone, filtered and concentrated. The relevant fraction dissolved in water was separated using an adsorption resin, and the adsorption fraction was purified using medium pressure chromatography and an ODS column. Furthermore, the relevant fraction was separated with an aqueous methanol solution using high performance liquid chromatography and an ODS column, and again separated and purified with an aqueous acetonitrile solution using high performance liquid chromatography and an ODS column. The purified peptide compound having a molecular weight of 1019 was subjected to acid hydrolysis. The resulting amino acid was derivatized with FDAA and analyzed using ultra high performance liquid chromatography and an ODS column. As a result, glutamic acid, alanine, and glycine were detected. These are the constituent amino acids of the core peptide sequence of the relevant ribosomal peptide precursor protein.

Example 10 Re-annotation of gene translation region of PMW1019 biosynthesis gene cluster by RNA sequencing Read data registered in public sequence read database NCBI SRA (http://www.ncbi.nlm.nih.gov/SRA) SRR 283857, SRR 283858, SRR 495778, SRR 495842, SRR 495932, SRR 544873, SRR 54472, SRR 544873, SRR 610531, SRR 610532, SRR 610533, SRR 610534, SRR 610535, SRR 610537 and SRR 610538 are used.

  Mapping work was performed using a lead having a length of 30 bases or more after trimming bases that do not contain unidentified bases and have a Q value (Quality value) of less than 20 consecutive 7 bases on average. The software used was bowtie and tophat. The work was carried out on a computer (DELL: CPU Intel Xeon E52643, memory 256 GB, hard disk 40 TB, OS: Ubuntu Linux (registered trademark) 13.10)). After displaying the mapping result with a genome information visualization tool, the corresponding gene region (genes AFLA_063120 to AFLA_0632020 on the scaffold GenBank EQ963477) is visually confirmed, and the region consistent with the reading frame of the start codon and codon is identified as the CDS region. did.

  As a result of sequence analysis, the protein consisting of the two amino acid sequences shown in SEQ ID NOs: 50 and 51 from AFLA_063230, the protein consisting of the amino acid sequence shown in SEQ ID NO: 52 from AFLA_063270, and the amino acid sequence shown in SEQ ID NO: 53 from AFLA_063280 It was confirmed that the protein consisting of the protein consisting of the amino acid sequence shown in SEQ ID NO: 54 from AFLA — 063320 and the protein consisting of the amino acid sequence shown in SEQ ID NO: 55 from AFLA — 0633330 were each translated and translated. Moreover, it was confirmed that the amino acid sequences of the proteins that are transcribed and translated from genes other than the above among the genes shown in Table 14 are the same as the amino acid sequences deduced and estimated by the NCBI gene ID of each gene.

<Example 11> Identification of a ribosomal peptide biosynthetic gene cluster that biosynthesizes a peptide compound having a molecular weight of 809 Based on whole genome information of A. flavus registered as GenBank EQ963472-EQ966232, an endoplasmic reticulum migration signal A gene encoding a ribosomal peptide precursor consisting of an amino acid sequence comprising a sequence and a repetitive sequence, and a gene encoding a protein consisting of an amino acid sequence having an E value of less than 1e-4 with respect to the amino acid sequence shown in SEQ ID NO: 3 (UstYa), The search was performed by the aforementioned Smith-Waterman algorithm, signalP 4.1 and the Blast method. As a result, a gene (AFLA — 041390) having a base sequence encoding a protein consisting of an amino acid sequence having an E value of less than 1e-48 relative to the gene (AFLA — 041400) encoding a ribosomal peptide precursor and the amino acid sequence (UstYa) shown in SEQ ID NO: 3 ) Was found adjacent.

  Furthermore, expression information obtained under 28 conditions including culture conditions that produce a lot of secondary metabolites registered in a public database (NCBI GEO http://www.ncbi.nlm.nih.gov/geo/) Using (GSE15435), the gene region including the above two genes and co-expressed was examined.

  From the results (FIG. 13) and gene function annotation, a total of 18 genes of NCBI genes AFLA — 043120 to AFLA — 041490 were estimated as novel ribosomal peptide biosynthesis gene clusters. Table 15 shows the NCBI ID numbers, estimated functions, and sizes (base pairs) of these genes.

  Furthermore, all 18 genes of AFLA_041320 to AFLA_041490, and AFLA_041330 (C6 type transcription factor), AFLA_041390 (UstYa homolog) and AFLA_041400 (precursor protein) were deleted by substituting each with a selectable marker gene, for a total of 4 Types of A. A flavus mutant was created. The specific work is as follows.

  For each of the above four types of target gene regions, PCR was performed using a primer set designed to amplify a genomic region containing 1 kb upstream and downstream thereof, thereby producing an amplified fragment 1 of the genomic region. On the other hand, A. PCR was performed using a primer set designed to amplify an approximately 1.8 kb genomic region containing the nidulans pyrG gene, and an amplified fragment 2 of the genomic region containing the pyrG gene was prepared. Furthermore, PCR was performed using the amplified fragments 1 and 2 as a template and a primer set designed to amplify the nucleic acid to which both fragments were linked to prepare a fusion PCR amplified fragment 3 of both genomic regions.

1. Add 24 g / L Difco potato dextrose medium containing 12 mg / mL uracil to The conidia of flavus (CA14 strain, Δku70 ΔpyrG ΔniaD) were suspended (10 ⁶ / mL) and cultured at 37 ° C. for 2 days. The cells were collected, suspended in a buffer containing a lytic enzyme (lysing enzyme from Sigma, Yatalase from TaKaRa, cellulase from Yakult), and incubated at 30 ° C. for 3 hours to prepare a protoplast. After suspending this protoplast in an isotonic solution, the four types of fusion PCR amplified fragments 3 (1 μg) were separately added for transformation, and incubated in the presence of polyethylene glycol. After incubation, the protoplasts were placed on the regeneration medium and cultured for 3-5 days to regenerate the fungal cells. Nucleic acid fragments obtained by PCR amplification using the recombined genomic region as a template, indicating that each of the four regenerated mutant strains has a genomic structure in which each target gene has been deleted by substitution with the introduced pyrG gene This was confirmed by sequence analysis.

Each of the four mutants was sterilized by adding 1.2 mL of distilled water and sterilized on 2.5 g of ground corn for 6 days at 30 ° C., and then added with 10 mL of 80% acetone and incubated at room temperature for 1 hour. Acetone is evaporated from the extract from which the bacterial cells and solid medium have been removed, and an equivalent amount of ethyl acetate is added to the remaining concentrated solution. After reacting for 1 hour, the aqueous phase is passed through a filter having a pore size of 0.22 μm to perform LC-MS. A sample was prepared. The sample was fractionated using an Ultimate 3000 HPLC (Dionex) with a Develosil XG-C18M-5 reverse phase column (Nomura Chemical) and a water / acetonitrile gradient (0% -100%) solvent.

When the patterns of m / z values corresponding to the retention time and molecular weight were compared between the four types of disrupted strains and the control strain (pyrG marker-reverted strain), AFLA_041320-AFLA_041490, AFLA_041390 (UstYa) existed in the control strain. Homolog), MS of m / z 808.3 [M−H] ⁻ (m / z 810.3 [M + H] ^{+ in} cation) disappeared only in three types of disrupted strains that destroyed AFLA — 041400 (precursor protein) A peak was found at a retention time of 13.8 minutes (FIG. 14).

  As a result of separating the peak and analyzing the structure by NMR, the present compound is a novel peptide compound having a molecular weight of 809 (hereinafter referred to as PMW809) having a core peptide sequence of a novel ribosomal peptide precursor protein as a constituent element. It was confirmed that there was. On the other hand, even when AFLA — 041330, which is a C6 transcription factor, was disrupted or overexpression of the C6 transcription factor using the tef1 promoter, there was no change in the productivity of PMW809.

  From the above, the four genes (Table 16) of NCBI genes AFLA — 041370 to AFLA — 041400 were determined to be biosynthetic gene clusters that are substantially involved in the biosynthesis of PMW809, a novel ribosomal peptide.

<Example 12> Identification of constituent amino acids of PMW809 The cultured cells were extracted with 80% acetone, filtered and concentrated. The relevant fraction dissolved in butanol was purified using medium pressure chromatography and an ODS column. Furthermore, the relevant fraction was separated with an aqueous methanol solution using high performance liquid chromatography and an ODS column, and again separated and purified with an aqueous acetonitrile solution using high performance liquid chromatography and an ODS column. The structural analysis of the purified peptide compound having a molecular weight of 809 was performed by high resolution ESI mass spectrometry and nuclear magnetic resonance spectrum. First, the molecular formula was determined as C ₄₂ H ₄₃ N ₅ O ₁₂ by high resolution mass spectrometry. Further, from the ¹³ C-nuclear magnetic resonance spectrum and 1 H-nuclear magnetic resonance spectrum, it was found that the present compound contained phenylalanine, three tyrosine, threonine, and glycine. These are amino acids contained in the core peptide sequence of the corresponding ribosomal peptide precursor protein.

<Example 13> Re-annotation of gene translation region of PMW809 biosynthesis gene cluster by RNA sequencing As read data registered in public sequence read database NCBI SRA (http://www.ncbi.nlm.nih.gov/SRA) , SRR 283857, SRR 283858, SRR 495778, SRR 495842, SRR 495932, SRR 544971, SRR 54472, SRR 544873, SRR 610531, SRR 610532, SRR 610533, SRR 610534, SRR 610535, SRR 610537 and SRR 610537 are used. Mapping work was performed using a lead having a length of 30 bases or more after trimming bases that do not contain unidentified bases and have a Q value (Quality value) of less than 20 consecutive 7 bases on average. The software used was bowtie and tophat. The work was carried out on a computer (DELL: CPU Intel Xeon E52643, memory 256 GB, hard disk 40 TB, OS: Ubuntu Linux (registered trademark) 13.10)). After displaying the mapping result with the genome information visualization tool, the corresponding gene region (genes AFLA_041370 to AFLA_041400 on the scaffold GenBank EQ963476) is visually confirmed, and the region that is consistent with the reading frame of the start codon and codon is identified as the CDS region. did.

  According to the present invention, a novel fungal-derived ribosomal peptide that has not been reported so far can be produced. This makes it possible to provide a ribosomal peptide that is a secondary metabolite derived from a fungus expected to be applied to pharmaceuticals and other useful substances. In addition, by recombining the constituent genes of each cluster with each other or arbitrarily combining multiple constituent genes, non-natural ribosomal peptides with structures and physiological activities different from those produced in nature can be produced. Is also possible.

Claims (17)

A method for producing a ribosomal peptide derived from a fungus, comprising:
Step a) Biosynthesis of a ribosomal peptide having an E value of less than 1e-4 for a gene encoding a ribosomal peptide precursor consisting of an amino acid sequence including an endoplasmic reticulum translocation signal sequence and a repetitive sequence, and the base sequence shown in SEQ ID NO: 1. A gene having a base sequence encoding the protein involved,
The E value for the amino acid sequence shown in SEQ ID NO: 2 consists of an amino acid sequence of less than 1e-4 and encodes a protein involved in the biosynthesis of a ribosomal peptide or the E value for the amino acid sequence shown in SEQ ID NO: 3 is 1e-4 A gene encoding a protein consisting of less than the amino acid sequence and involved in ribosomal peptide biosynthesis,
And / or a ribosomal peptide biosynthesis gene cluster comprising at least a gene consisting of an amino acid sequence having an E value of less than 1e-4 with respect to the amino acid sequence represented by SEQ ID NO: 6 and encoding a protein involved in ribosomal peptide biosynthesis, The said manufacturing method including the process of forcedly expressing, and the process b) collect | recovering ribosome peptides from a cell.   Genes encoding ribosomal peptide precursor, AspGD ID number AFL2G_08516, Acar5010_211921, Acar5010_131040, ACLA_063320, Aspbr1_0148406, An06g02130, Aspzo1_1501727, Acar5010_210441, Aspzo1_0132961, Aacu16872_042334, An03g05230, Asptu1_0047109, Aspfo1_0048058, Aspka1_0178058, Aspzo1_0673756, AFUB_096780, Aspwe1_0028756, Aspwe1_0177551, Aspfo1_0041528, Aspka1_0176174, Aspzo1_0138 A gene selected from the group consisting of gene 40 and Aacu16872_044658, The process according to claim 1.   A gene encoding a protein consisting of an amino acid sequence having an E value of less than 1e-4 with respect to the amino acid sequence shown in SEQ ID NO: 2 and involved in the biosynthesis of a ribosomal peptide has an AspGD ID number of AFL2G — 08517, Acar5010 — 156521, Acar5010 — 131041, ACLA — 063300, ACLA — 063310, Aspbr1_0073580, An06g02120, Aspzo1_0020615, Aspzo1_0147453, Acar5010_399409, Aspzo1_0097397, Acu16887_042333, An03g05220, Asptu1_00471A87 96790, Aspwe1_0041401, Aspwe1_0188242, Aspfo1_0203572, Aspka1_0176173, a gene selected from the group consisting of genes that are Aspzo1_0163562 and Aacu16872_044657, The process according to claim 1 or claim 2.   The production according to any one of claims 1 to 3, wherein the forced expression in step a) is achieved by recombination of a promoter of a gene encoding a transcription factor contained in a ribosomal peptide biosynthesis gene cluster with a high expression promoter. Method.   The production method according to any one of claims 1 to 4, wherein the ribosomal peptide biosynthesis gene cluster further comprises a gene encoding a protein having peptidase activity.   The manufacturing method in any one of Claims 1-5 whose cell is a fungal cell.   The production method according to claim 6, wherein the fungal cell is a cell of the genus Aspergillus.   Consisting of the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence in which one to several amino acids of the amino acid sequence are deleted or substituted, or an amino acid sequence in which one to several amino acids are added to the amino acid sequence, A protein involved in the biosynthesis of ribosomal peptides in cells.   A cDNA encoding the protein according to claim 8.   Comprising the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence in which one to several amino acids of the amino acid sequence are deleted or substituted, or the amino acid sequence in which one to several amino acids are added to the amino acid sequence; A protein involved in the biosynthesis of ribosomal peptides in cells.   A cDNA encoding the protein according to claim 10. A method for producing a ribosomal peptide derived from a fungus, comprising:
Step a) A gene encoding a ribosomal peptide precursor consisting of an amino acid sequence including an endoplasmic reticulum transition signal sequence and a repetitive sequence, and 20% or more identical to the 81st to 225th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 3 20% or more identity with the 57th to 259th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 6 and / or the gene encoding the protein involved in the biosynthesis of ribosomal peptides A step of forcibly expressing a ribosomal peptide biosynthetic gene cluster in a cell including at least a gene encoding a protein involved in ribosomal peptide biosynthesis, and b) a step of recovering the ribosomal peptide from the cell. The manufacturing method.   Genes encoding ribosomal peptide precursor, AspGD ID number AFL2G_08516, Acar5010_211921, Acar5010_131040, ACLA_063320, Aspbr1_0148406, An06g02130, Aspzo1_1501727, Acar5010_210441, Aspzo1_0132961, Aacu16872_042334, An03g05230, Asptu1_0047109, Aspfo1_0048058, Aspka1_0178058, Aspzo1_0673756, AFUB_096780, Aspwe1_0028756, Aspwe1_0177551, Aspfo1_0041528, Aspka1_0176174, Aspzo1_0138 A gene selected from the group consisting of gene 40 and Aacu16872_044658, The method according to claim 12.   A gene that includes an amino acid sequence having 20% or more identity with the 81st to 225th amino acid sequences of the amino acid sequence shown in SEQ ID NO: 3 and that encodes a protein involved in ribosomal peptide biosynthesis has an AspGD ID number of AFL2G_08517, Acar5010_156521, Acar5010_131041, ACLA_063300, ACLA_063310, Aspbr1_0073580, An06g02120, Aspzo1_0020615, Aspzo1_0147593, Acar5010_399409, Aspzo1_0097397, Aacu16872_042333, An03g05220, Asptu1_0047108, Aspfo1_0048061, Aspka1_0178059, As zo1_0070555, AFUB_096790, Aspwe1_0041401, Aspwe1_0188242, Aspfo1_0203572, Aspka1_0176173, a gene selected from the group consisting of genes that are Aspzo1_0163562 and Aacu16872_044657, The method according to claim 12 or claim 13.   The production according to any one of claims 12 to 14, wherein the forced expression in step a) is achieved by recombination of a promoter of a gene encoding a transcription factor contained in a ribosomal peptide biosynthesis gene cluster with a high expression promoter. Method.   The production method according to any one of claims 12 to 15, wherein the ribosomal peptide biosynthesis gene cluster further comprises a gene encoding a protein having peptidase activity.   The production method according to any one of claims 12 to 16, wherein the cell is a fungal cell.

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