Patent Application
20080009039
The present invention is directed to a recombinant microorganism obtained by transferring, into a host microorganism capable of producing protein or polypeptide with increased productivity, a gene encoding a protein or polypeptide, and to a method for producing a protein or polypeptide by use of the recombinant microorganism.
The present inventors have conducted extensive studies on, among many different genes encoded on the genome of a microorganism, genes which are not needed in or which are detrimental to the production of useful proteins or polypeptides, and have found that, when a gene encoding a target protein or polypeptide is transferred to a microorganism such as Bacillus subtilis after a specific gene is deleted or knocked out from the genome of the microorganism, productivity of the target protein or polypeptide is enhanced as compared with the case before the deletion or knocking out.
In the microorganism of the present invention, since genes which are unnecessary in or detrimental to the production or a target protein or polypeptide are deleted or knocked out, waste of culture media, including energy loss, production of byproducts, and reduced specific production rate, is significantly reduced, and in addition, protein and polypeptide can be produced over a prolonged period, whereby a target product can be produced with high efficiency.
In the present invention, homology between amino acid sequences and that between nucleic acid sequences are both determined by use of the Lipman-Pearson method (Science, 227, 1435 (1985)). Specifically, calculation is performed by use of a homology analysis program (Search Homology) developed by genetic information processing software, Genetyx-Win (Software Development Co., Ltd.), with ktup (the unit size to be compared) being set 2.
No particular limitation is imposed on a parent microorganism for constructing the microorganism of the present invention, so long as it has a gene which is not necessary for producing a target protein or polypeptide; specifically, any of the Bacillus subtilis genes or genes functionally equivalent thereto as shown in Table 1, wherein the gene may be of wild-type or a mutant. Specific examples include Bacillus subtilis and similar microorganisms belonging to the genus Bacillus, microorganisms belonging to the genus Clostridium, and yeast. Inter alia, microorganisms belonging to the genus Bacillus are preferred. In particular, Bacillus subtilis is preferred, from the viewpoint that complete genomic information of this microorganism has already been obtained, and thus genetic engineering techniques and genomic engineering techniques have been established, and that the microorganism has ability to secrete the produced protein extracellularly.
Examples of the target protein or polypeptide to be produced by use of the microorganism of the present invention include enzymes, physiologically active substances, and other proteins and polypeptides which find utility in foods, pharmaceuticals, cosmetics, detergents, fiber-treating agents, clinical assay agents, etc.
Taking Bacillus subtilis, which is known to have 4,106 genes on the genome, as an example, one or more genes which are to be deleted or knocked out are any of the Bacillus subtilis genes shown in Table 1, or are selected from among the genes functionally equivalent thereto. The present inventors have found that such genes do not directly participate in production of the target protein or polypeptide and are unnecessary for the growth of microorganism in ordinary industrial production media.
The names, numbers, and functions of respective genes in the Tables contained herein conform with the Bacillus subtilis genome data reported in Nature, 390, 249-256 (1997) and made public by JAFAN (Japan Functional Analysis Network for Bacillus subtilis; BSORF DB) on the Internet (http://bacillus.genome.ad.jp/, renewed Jun. 17, 2003).
Genes derived from other microorganisms, preferably from bacteria belonging to the genus Bacillus, which have the same functions as any of the Bacillus subtilis genes shown in Table 1, or have 70% or more homology with the nucleotide sequence of any of the genes shown in Table 1, preferably 80% or more homology, more preferably 90% or more, further preferably 95% or more, yet more preferably 98% or more, should be interpreted to be functionally equivalent to the genes shown in Table 1, and thus to constitute the genes which are to be deleted or knocked out according to the present invention. In this connection, homology of nucleotides is computed by use of the Lipman-Pearson method (Science, 227, 1435, 1985).
Many of the genes shown in Table 1 which encode Bacillus subtilis are regulatory genes participating in activation or suppression of expression of a variety of genes, or genes deduced to be such regulatory genes. The present invention has been attained on the basis of this finding; i.e., the presence of regulatory genes unnecessary in or detrimental to production of protein or polypeptide has now been unveiled in the present invention.
Notably, attention is drawn to the fact that many of the listed “unnecessary” or “detrimental” genes are regulatory genes participating in sugar intake or metabolism, as exemplified by the glcT gene, which acts as an anti-terminator for a glucose PTS intake operon; the licT gene, which acts as an anti-terminator for a lichenan hydrolysis operon; the treR gene, which acts as a repressor of trehalose intake and metabolism; and the hprK gene and ccpA gene, which relate to glucose catabolite repression.
Also, in addition to the regulatory genes involved in sugar intake and metabolism, the rocR gene participating in activation of arginine assimilation, and competence-related comA gene and sir gene, which are also regulatory genes, may be deleted or knocked out, to thereby improve productivity of protein or polypeptide.
The genes shown in Table 1 include the yhdK gene, and the rsiX gene encoding the anti-EC F sigma factor which suppresses expression of an ECF sigma factor, sigma x. The yhdK gene has been reported to participate in suppression of sigma M (Mol. Microbiol., 32, 41, 1999). The sigL gene, which encodes sigma L, is also included in the genes of Table 1. This suggests that expression of a gene under regulation by sigma X or sigma M is favorable for production of protein, and conversely, some gene expression under regulation by sigma L is unfavorable.
By deleting or knocking out one or more genes selected from the above-mentioned genes, expression which is unnecessary in or harmful to the production of protein or polypeptide can be prevented, leading to enhanced productivity in such production of protein or polypeptide.
The number of gene(s) to be deleted or knocked out is one or more, preferably two or more, more preferably three or more, even more preferably 5 or more. When a microorganism of the present invention is constructed, deletion or inactivation of a gene or genes other than those mentioned above is possible. In such a case, a more improved effect is expected. An alternative method for achieving the present invention is inactivation, or knocking out, of a target gene by inserting thereto a DNA fragment of another origin or introducing a mutation to the transcription/translation-initiation region of the gene. Preferably, however, the target genes are physically deleted.
In an example procedure for deleting or knocking out the genes, any of the target genes shown in Table 1 is deleted or knocked out according to a plan which has been set up in advance. Alternatively, randomized deletion of genes or mutation by way of knocking out is performed, followed by evaluation on protein productivity and gene analysis.
The target gene may be deleted or knocked out through homologous recombination. That is, a DNA fragment containing a portion of the target gene is cloned with an appropriate plasmid vector to thereby obtain a ring-shaped recombinant plasmid, and the resultant plasmid is transferred into cells of a parent microorganism. Thereafter, through homologous recombination effected in a partial region of the target gene, the target gene on the genome of the parent microorganism is cleaved, thereby completing inactivation of the target gene. Alternatively, the target gene is knocked out by substitution or insertion of a base, or a linear DNA fragment containing a region outside the target gene sequence but not containing the target gene may be constituted through PCR or a similar method, and the thus-engineered gene or fragment is transferred into a cell of a parent microorganism. At two sites outside the mutation within the target gene in the genome of the parent microorganism genome, or at two regions outside the target gene sequence, double crossing-over homologous recombination is caused to occur, to thereby attain substitution with a gene fragment in which the target gene on the genome is deleted or knocked out.
Particularly when the parent microorganism used to construct the microorganism of the present invention is Bacillus subtilis, since several reports have already described methods for deleting or knocking out the target gene (see, for example, Mol. Gen. Genet., 223, 268 1990), repetition of any of such methods may be followed, to thereby produce a host microorganism of the present invention.
Randomized gene deletion or inactivation may be performed through use of a method similar to the above-described method for inducing homologous recombination by use of a randomly cloned DNA fragment, or by way of irradiation of a parent microorganism with gamma rays or similar rays.
Next will be described in more detail a deletion method employing double crossing over by use of a DNA fragment designed for the deletion purpose, the DNA fragment being prepared through SOE-PCR (Gene, 77, 61, 1989). However, in the present invention, the method for deleting genes is not limited to only the below-described method.
The DNA fragment use for the deletion purpose is a fragment constructed such that a drug resistant marker gene is inserted between a ca. 0.5 to 3 kb upstream sequence which flanks and is upstream of the gene to be deleted, and a ca. 0.5 to 3 kb downstream sequence which flanks and is downstream of the same gene. In the first cycle of PCR, the following three fragments are prepared: the upstream and the downstream fragments, which are to be deleted, and the drug resistant marker gene. The primers to be used in this step may, for example, be those specifically designed so that an upstream 10-30 base pair sequence of a drug resistance gene is added to the lower end of the upstream fragment, and a downstream 10-30 base pair sequence of the drug resistance marker gene is added to the upper end of the downstream fragment (
Next, using three PCR fragments prepared in the first cycle as templates, the second cycle of PCR is performed by use of an upper primer of the upstream fragment and a lower primer of the downstream fragment. This step causes annealing with the drug resistance marker gene fragment in the sequence of the above-engineered drug resistance marker gene, and through PCR amplification, there can be obtained a DNA fragment with the drug resistance marker gene inserted between the upstream fragment and the downstream fragment (
When a chloramphenicol-resistant gene is employed as a drug resistance marker gene, a DNA fragment for deleting a gene can be obtained through SOE-PCR under typical conditions described in literature (see, for example, PCR Protocols. Current Methods and Applications, Edited by B. A. White, Humana Press, pp. 251 (1993), Gene, 77, 61, 1989), by use of a primer set such as that shown in Table 2 and a conventional enzyme kit for PCR (e.g., Pyrobest DNA Polymerase (product of Takara Shuzo)).
When the thus-obtained DNA fragment for effecting gene deletion is introduced into cells through the competent method or a similar method, intracellular genetic recombination occurs in homologous regions which are present upstream and downstream of the gene to be deleted. Thus, cells in which the target gene has been substituted by a drug resistance gene can be selectively separated through employment of a drug resistance marker (
Subsequently, when a gene encoding a target protein or polypeptide is transferred to a host mutant microorganism strain from which any of the Bacillus subtilis genes shown in Table 1, or one or more genes selected from among the genes corresponding thereto has been deleted or knocked out, the microorganism of the present invention can be obtained.
No particular limitation is imposed on the gene encoding the target protein or polypeptide. Examples of the protein and polypeptide include physiologically-active peptides and enzymes for industrial purposes such as detergents, foods, fibers, feeds, chemicals, medicine, and diagnostic agents. Industrial enzymes may be functionally grouped into oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases/synthetases. Preferably, hydrolases such as cellulase, α-amylase, and protease may be used. Specific examples include cellulase belonging to family 5 in the classification of hydrolase (Bioche M. J., 280, 309, 1991); in particular, cellulase derived from a microorganism, more particularly cellulase derived from the genus Bacillus. Other specific examples of the types of industrial enzymes include alkaline cellulase which is derived from the genus Bacillus and has an amino-acid of SEQ ID NOs: 2 or 4, and cellulase which has an another amino-acid sequence having 70% homology with said amino-acid sequence, preferably 80% homology, more preferably 90%, further preferably 95%, still further preferably 98% or more.
Specific examples of α-amylase include α-amylase derived from a microorganism, preferably liquefied amylase derived from the genus Bacillus. More specific examples include alkaline amylase which is derived from the genus Bacillus and has an amino-acid sequence of SEQ ID NO: 6, and amylase which has another amino-acid sequence having 70% homology with said amino-acid sequence, preferably 80% homology, more preferably 90%, further preferably 95%, particularly preferably 98% or more. The homology of the amino-acid sequence is calculated by the Lipman-Pearson method (Science, 227, 1435 (1985)). Specific examples of protease include serine protease and metallo-protease which are derived from microorganisms, particularly those belonging to the genus Bacillus.
Preferably, a gene coding for a target protein or polypeptide has, on its upstream region thereof, one or more regulatory regions relating to transcription, translation, or secretion of the gene (specially, one or more regions selected from among a transcription initiation regulatory region including a promoter and a transcription initiation site; a translation initiation region including a ribosome-binding site and a start codon; and a secretion signal peptide region) properly ligated thereto. Preferably, it is preferred that three regions consisting of the transcription initiation regulatory region, the translation initiation regulatory region, and the secretion signal region be ligated to the target gene. Further preferably, the secretion signal peptide region is one that originates from the cellulase gene of a microorganism belonging to the genus Bacillus, and the transcription initiation region and the translation initiation region is a 0.6 to 1 kb region upstream of the cellulase gene. In one preferred example, a transcription initiation regulatory region, a translation initiation region, and a secretion signal peptide region of a cellulase gene derived from a microorganism belonging to the genus Bacillus disclosed in, for example, Japanese Patent Application Laid-Open (kokai) Nos. 2000-210081 and 190793/1990; i.e., a cellulase gene derived from KSM-S237 strain (FERM BP-7875) or KSM-64 strain (FERM BP-2886), is properly ligated to a structural gene of the target protein or polypeptide. More specifically, preferred DNA fragments to be ligated include a nucleotide sequence of base numbers 1 to 659 of SEQ ID NO: 1; a nucleotide sequence of base numbers 1 to 696 of a cellulase gene of SEQ ID NO: 3; a DNA fragment having a nucleotide sequence having 70% homology with any one of said nucleotide sequences, preferably 80% homology, more preferably 90%, further preferably 95%, even more preferably 98% or more; or a DNA fragment having a nucleotide sequence lacking a portion of any one of said nucleotide sequences. Preferably, one of these DNA fragments is properly ligated to a structural gene of the target protein or polypeptide. As used herein, a DNA fragment having a nucleotide sequence lacking a portion of any one of the above-mentioned nucleotide sequences is intended to mean a DNA fragment which has functions relating to transcription, translation, and secretion of the gene, without having a portion of any one of the above-mentioned nucleotide sequences.
The recombinant microorganism of the present invention can be obtained by a conventional transformation technique in which a recombinant plasmid containing a DNA fragment which includes a gene encoding the target protein or polypeptide, and is ligated to a proper plasmid vector is transferred into a host microorganism cell. Alternatively, the recombinant microorganism may be obtained making use of a DNA fragment prepared by ligating the above DNA fragment to a proper region which is homologous with a certain portion of the host microorganism genome, and inserted directly into a host microorganism genome.
The target protein or polypeptide obtained by use of the recombinant microorganism of the present invention may be produced in such a manner that a corresponding cell strain is inoculated onto a culture medium containing assimilable carbon sources and nitrogen sources, and other essential components; the cell strain is cultured through a conventional microorganism culturing method; and subsequently, protein or polypeptide is collected and purified.
Through the aforementioned procedure, a host mutant microorganism strain in which any of the Bacillus subtilis genes shown in Table 1 or one or more genes selected from genes functionally equivalent thereto have been deleted or knocked out can be engineered. In addition, by use of such a mutant strain, a recombinant microorganism can be produced. Thus, a useful protein or polypeptide can be effectively produced through employment of the mutant strain or the recombinant microorganism.
The method for constructing a recombinant microorganism according to the present invention, and the method for producing cellurase and α-amylase by use of the recombinant microorganism will next be described in detail, centering on working examples for constructing recombinant strain belonging to Bacillus subtilis from which the ccpA gene (BG10376) of Bacillus subtilis has been deleted.
A genome DNA sample, serving as a template, extracted from Bacillus subtilis 168 strain and two primer sets (ccpA-AF and ccpA-A/CmR; and ccpA-B/CmF and ccpA-BR) shown in Table 2 were used to prepare a 0.6 kb fragment (A) flanking the upstream side of the ccpA gene on the genome and a 0.6 kb fragment (B) flanking the downstream side of the ccpA gene. A chloramphenicol-resistant gene of plasmid pC194 (J. Bacteriol. 150 (2), 815 (1982))) was inserted into the XbaI-BamHI cleavages site of plasmid pUC18, to thereby prepare a recombinant plasmid pCBB 31. The recombinant plasmid pCBB and a primer set consisting of CmF and CmR shown in Table 2 were used to prepare a 1 kb fragment (C) containing the chloramphenicol-resistant gene. Subsequently, SOE-PCR was performed by use of the primers ccpA-AF and ccpA-BR shown in Table 2, and by use of the thus-prepared three fragments (A), (B), and (C) in combination as templates, a 2.2 kb DNA fragment in which the fragments (A), (B), and (C) were ligated in this sequence was prepared (see
In a manner similar to that described in Example 1, sporulation gene-deleted strains into which a chloramphenicol-resistant gene had been introduced by way of substitution in place of the below-described deleted genes were separated through use of a DNA fragment for effecting deletion prepared from an adequate primer set selected from among various primer sets shown in Table 2; i.e., gene-AF, gene-A/CmR, gene-B/CmF, gene-BR, CmF, and CmR. The gene deleted from the genome was comA, yopO, treR, yvbA, yvaN, yttP, yurK, yozA, licR, sigL, mntR, glcT, ykvE, slr, rocR, yyaA, or rsiX.
In a manner similar to that described in Example 2, a DNA fragment for deletion was prepared by use of an adequate primer set selected from among the gene-AF, gene-A/Cm2R, gene-B/Cm2F, gene-BR, Cm2F, and Cm2R, which are shown in Table 2. By use of the thus-prepared DNA fragment, sporulation gene-deleted strains into which a chloramphenicol-resistant gene had been introduced by way of substitution in place of the below-described deleted genes were separated. The gene deleted from the genome was cspB, yvdE, yaaT, yycH, or ylbO.
In a manner similar to that described in Example 2, a DNA fragment for effecting deletion was prepared from an adequate primer set selected from among the gene-AF, gene-A/Cm4R, gene-B/Cm4F, gene-BR, Cm4F, and Cm4R, which are shown in Table 2. By use of the thus-prepared DNA fragment, sporulation gene-deleted strains into which a chloramphenicol-resistant gene had been introduced by way of substitution in place of deleted genes; yacP, hprK, and yhdK, were separated.
To each of the gene-deleted strains obtained in Examples 1 to 4 and to Bacillus subtilis 168 strain serving as a control, a recombinant plasmid pHY-S237 was introduced through the protoplast transformation method. The recombinant plasmid pHY-S237 was prepared by inserting a DNA fragment (3.1 kb) encoding an alkaline cellulase derived from Bacillus sp. KSM-S237 strain (Japanese Patent Application Laid-Open (kokai) No. 2000-210081) into the restriction enzyme BamHI cleavage site of a shuttle vector pHY300 PLK. Each of the thus-obtained cell strains was shake-cultured in an LB medium (5 mL) overnight at 30° C. The culture broth (0.03 mL) was inoculated to a 2× L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate, and 15 ppm tetracycline), followed by shake culturing at 30° C. for three days. After completion of culturing, cells were removed through centrifugation, and alkaline cellulase activity of the supernatant obtained from the culture was determined, thereby calculating the amount of the alkaline cellulase secreted from the cells during culturing; i.e., the amount of the extracellularly produced alkaline cellulase. As is clear from Table 3, more effective production, or secretion, of alkaline cellulase has been confirmed in the case where a gene-deleted strain was employed as a host, as compared with the control 168 strain (wild type strain).
To each of the gene-deleted strains obtained in Examples 1 to 4 and to Bacillus subtilis 168 strain serving as a control, recombinant plasmid pHSP-K38 was introduced through the protoplast transformation method. The recombinant plasmid pHSP-K38 was prepared by inserting, into the restriction enzyme BagII-XbaI cleavage site of a shuttle vector pHY300 PLK, a 2.1 kb fragment (SEQ ID No: 5) prepared by ligating an upstream 0.6 kb fragment (SEQ ID NO: 3) including portions of a promoter region and a signal sequence region of an alkaline cellulase gene with an upstream side of a DNA fragment (1.5 kb) encoding a mature enzyme region (Aspl-Gln480) of an alkaline amylase gene derived from Bacillus sp. KSM-K38 strain (Japanese Patent Application Laid-Open (kokai) No. 2000-1884882, Eur. J. Biochem., 268, 2974 (2001)). Each of the thus-obtained cell strains was shake-cultured in an LB medium (5 mL) overnight at 30° C. The culture broth (0.03 mL) was inoculated to a 2× L-maltose medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5 ppm manganese sulfate 4-5 hydrate, and 15 ppm tetracycline), followed by shake culturing at 30° C. for three to six days. After completion of culturing, cells were removed through centrifugation, and alkaline amylase activity of the supernatant obtained from the culture was determined, thereby calculating the amount of the alkaline amylase secreted from the cells during culturing; i.e., the amount of the extracellularly produced alkaline amylase. As is clear from Table 3, more effective production, or secretion, of alkaline amylase has been confirmed in the case where a gene-deleted strain was employed as a host, as compared with the control 168 strain (wild type strain).
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003-379167 | Nov 2003 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP04/16891 | 11/5/2004 | WO | 00 | 3/12/2007 |
