KR20110032376A - Mutant microorganisms and methods for producing the same, which can prevent the ability of the target substance to decrease due to the deletion of the giant plasmid - Google Patents

Abstract

PURPOSE: A mutant microorganism which prevents productivity reduction of a target material is provided to prevent degeneration of the microorganism regardless of medium and fermentation condition. CONSTITUTION: A mutant microorganism which prevents productivity reduction of a target material by megaplasmid deletion is prepared by a part of inserting essential chromosome DNA and complementary site to a microorganism with the megaplasmid. The part of the chromosome DNA is a gene encoding DNA polymerase, RNA polymerase, or ribosome.

Description

Transformed microorganisms and methods for preparing the same, which can prevent the ability of the target substance to decrease due to the deletion of a large plasmid {preparing method for non-degenerative microorganism by protecting megaplasmid lose and the microorganism}

The present invention relates to a mutant microorganism capable of preventing degradation of the target substance by the deletion of the giant plasmid in the microorganism having a large plasmid and a method for producing the same, and more specifically, repeated passage culture and continuous fermentation process The present invention relates to a mutant microorganism and a method for producing the same, which do not decrease the ability to produce a target substance.

Clostridium genus microorganisms are Gram-positive rod bacteria, fully anaerobic, have low GC content of chromosomal DNA, and have endogenous spores. This microorganism is also one of the largest taxa after Streptomyces genus (Durre, Handbook on Clostridia, Taylor & Francis, 2005). The genus Clostridium contains microorganisms having various characteristics such as pathogens, butyric acid fermentation bacteria, butanol fermentation bacteria, and ethanol fermentation bacteria, and thus are of high interest in medicine and biotechnology.

Butanol is one of typical oxo alcohols, and has been mainly used as a solvent for plastics and paints. Generally, these oxo alcohols are obtained by adding hydrogen and carbon monoxide to olefins to obtain aldehydes and adding hydrogen to aldehydes. Throughout these two processes, we call it oxo syntheis. In the case of butanol, butyraldehyde is obtained using propylene as a raw material, and is obtained through a hydrogenation reaction.

Recently, interest in microorganisms producing butanol has been increasing in relation to high oil prices. Butanol has the advantages of being similar to gasoline as a fuel and not exhibiting the hygroscopy of ethanol (Durre, Biotechnol. J., 2: 1525-1534, 2007). In addition, butanol synthesis requires non-renewable petrochemical products as raw materials and relatively high temperatures associated with expensive catalyst systems as well as multi-step and muti-reactor designs. Pressure is required. In addition, existing chemical synthesis pathways are not good for the environment because they are very toxic and flammable. Thus, as an alternative to the chemical production process, the production of butanol from renewable biomass-derived carbon source is required.

The most widely known butanol-producing microorganisms belong to the genus Clostridium (Jones and Woods, Microbiol. Rev., 50: 484-524, 1986). Butanol fermentation of these microorganisms is largely divided into two phases (phase). In the early stage of growth, it grows while producing organic acids such as acetic acid and butyric acid, and when the concentration of these acids becomes more than a certain level in the latter stage of growth, butanol and ethanol begin to be produced. At this time, acetic acid and butyric acid are absorbed into the cell and used to make ethanol butanol, and acetone is produced in the process of reusing the organic acid.

Traditionally, butanol fermentation using Clostridium spp. Microorganisms has been pointed out as a disadvantage in that solvents, including butanol, are degraded during long-term culturing such as repeated passages or continuous fermentations, and only later, produce organic acids (McCoy and Fred. , J. Bacteriol., 41: 90-91, 1941). Strains with reduced solvent generation ability due to long-term culture are said to be degeneration.

 It is known that in order to prevent the degeneration of Clostridium spp., Microorganisms must be inoculated with spores and then heated to kill growth-type cells (Cornillot et al., J. Bacteriol., 179: 5442-5447, 1997). In commercial fermentation, butanol production is fatal, so studies have been made to identify degenerated cells. Among them, a method of judging colony appearance in a solid medium (Adler and Crow, Appl. Environ. Microbiol., 53: 2496-2499, 1987), and a method of determining the presence or absence of a specific gene (Sabath ㅹ et. al., FEMS Microbiol. Lett., 210: 93-98, 2002).

Recently, with increasing interest in butanol, studies have been continued to discover the causes of such deterioration in butanol production biochemically and molecularly. Clostridium acetobutylicum , the most widely known butanol-producing microorganism, has a large plasmid pSOL1 in addition to chromosomal DNA, and the percentage of cells with pSOL1 decreases over a long period of time, resulting in reduced solvent production. Known (Cornillot et al., J. Bacteriol., 179: 5442-5447, 1997). Subsequent genome sequencing has revealed that pSOL1 contains genes involved in the production of acetone and butanol (Nolling et al., J. Bacteriol., 183: 4823-4838, 2001).

Another butanol producing microorganism, Clostridium beijerinckii , is known to degrade very rapidly during subculture without the addition of acetic acid in the medium (Chen and Blaschek, Appl. Environ. Microbiol. 65: 499-505, 1999). In the case of Clostridium saccobroperbutylacetonicum , it is known that the supernatant of a non-degenerated strain culture solution is taken into a degenerated strain and recovered from degeneration (Kosaka et al., Biosci. Bioteconol. , 71: 58-68., 2007). Unlike the Clostridium acetobutylicum in these two strains, genes related to solvent generation are normally present in the degenerate strains, and in the degenerate strains, these genes may not be properly expressed in the solvent generator. It is unknown.

Despite these long studies, few strains have been produced that do not cause degeneration. In US Patent 5210032, Clostridium biyeringkiai NCIMB 8052 was prepared by a mutation method using a transposon did not degenerate. However, in this case, there is a disadvantage that it is difficult to determine exactly where the mutation occurred.

Accordingly, the present inventors have made diligent efforts to produce mutant microorganisms capable of preventing the ability of the target substance to decrease due to the deletion of the giant plasmid. As a result, (1) the homologous to a part of the chromosomal DNA essential for microbial growth in the giant plasmid Inserting the site and then deleting a portion of the chromosomal DNA; (2) inserting a selective marker into the macroplasmid; Or (3) inserting the entire macroplasmid into the chromosome of the microorganism or introducing a DNA having the same or similar function as the DNA essential for the production of the target substance in the macroplasmid to the microorganism. The present invention provides a mutant microorganism that is not lost, and thus does not degrade the ability to produce a target substance even in long-term culture, thereby completing the present invention.

It is an object of the present invention to provide a mutant microorganism and a method for producing the same, which can prevent the ability of producing a target substance to be lowered by the deletion of the giant plasmid in the microorganism having the macroplasmid.

In order to achieve the above object, the present invention is characterized by inserting a region homologous to a part of chromosomal DNA essential for the growth of the microorganism in the microplasma having a large plasmid, and then deleting a portion of the DNA from the chromosome. Provided is a method for producing a mutant microorganism, which can prevent the production ability of a target substance from being lowered due to deletion of a giant plasmid.

The present invention also relates to a macroplasmic plasmid characterized in that, in a microorganism having a large plasmid, a portion homologous to a portion of the chromosomal DNA essential for the growth of the microorganism is inserted in the large plasmid, and a portion of the DNA is deleted from the chromosome. It provides a mutant microorganism that can prevent the ability of the target material to decrease by the deletion of.

The present invention also provides a method for producing a mutant microorganism capable of preventing the production of a target substance from being lowered by deletion of a giant plasmid, wherein a selective marker is inserted into the giant plasmid in a microorganism having a giant plasmid. .

The present invention also provides a mutant microorganism capable of preventing degradation of the ability to produce a target substance by deletion of a giant plasmid, wherein a selective marker is inserted into the giant plasmid in a microorganism having a giant plasmid.

The present invention also provides a microorganism having a large plasmid inserting the entire large plasmid into the chromosome of the microorganism, or the DNA having the same or similar function as the DNA essential for the production of the target substance in the large plasmid is introduced into the microorganism. Provided is a method for producing a mutant microorganism, which can prevent the ability to produce a target substance from being lowered due to deletion of a giant plasmid.

The present invention also relates to a microorganism having a large plasmid, wherein the entire plasmid is inserted into the chromosome of the microorganism, or DNA having the same or similar function as the DNA essential for the production of a target substance in the large plasmid is introduced into the microorganism. The present invention provides a mutant microorganism capable of preventing the production of a target substance from being lowered due to the deletion of a giant plasmid.

According to the present invention, it is possible to produce a mutant microorganism that does not degrade, regardless of the medium or fermentation conditions, and does not decrease the ability to produce a target substance in repeated passage and continuous fermentation.

In the present invention, 'degeneration' refers to a phenomenon in which butanol producing ability gradually decreases due to certain factors when the microorganism producing butanol is cultured for a long time.

In the present invention, the term 'deletion' is a concept encompassing a mutation, substitution, or deletion of part or all of the bases of the gene, or introduction of some bases so that the gene is not expressed or does not exhibit enzymatic activity even when expressed. It includes everything that blocks the biosynthetic pathways involved in the enzymes of the gene.

In the present invention, 'homologous site' and 'recombination site' may be used in the same sense, and mean a nucleotide sequence capable of causing homologous recombination with DNA of another region having the same nucleotide sequence.

In the present invention, 'site specific recombinase system' refers to a system consisting of a single enzyme or a group of enzymes that recognizes a specific DNA sequence and causes recombination, and may cause rearrangement or deletion of DNA. Representative examples include flipppase (FLP), Cre recombinase and the like.

In the present invention, the "selective marker" refers to a product of a specific gene, and the microorganism including the product exhibits a special trait not appearing in a microorganism not included, and thus can be easily distinguished.

In the present invention, the 'screening' refers to the operation of distinguishing and selecting a specific microorganism, wherein the specific microorganism is a concept including having a specific phenotype or a specific DNA sequence that does not exist in other microorganisms.

In the present invention, 'homologous' or 'homologous' is applied to a site where recombination occurs, and means that two DNAs share the same or nearly identical nucleotide sequences with each other.

In the present invention, 'transformation' refers to introducing a specific foreign DNA strand into the cell from outside the cell. Host microbes, including the introduced DNA strands, are referred to as 'transformed microorganisms'.

In the present invention, 'restriction enzyme' refers to an enzyme that binds a sequence to a specific DNA nucleotide and cleaves the DNA. In the present invention, 'DNA cleavage enzyme' refers to an enzyme that non-specifically binds to DNA nucleotides to cleave DNA at both ends of the DNA or to cut DNA inside.

In the present invention, the "shuttle vector" means a vector capable of stably replicating in both heterologous microorganisms having at least one origin that can be replicated in both heterologous microorganisms, or having both origins capable of replicating only in each microorganism.

The present invention relates to a mutant microorganism and a method for producing the same, which can prevent the production of a target substance from being lowered by the deletion of the giant plasmid in a microorganism having a macroplasmid.

In one aspect, the mutant microorganism according to the present invention can be prepared by inserting a region homologous to a part of the chromosomal DNA essential for the growth of the microorganism in the large plasmid, and then deleting a part of the DNA from the chromosome.

In the present invention, a part of the chromosomal DNA essential for the growth of the microorganism may be characterized in that the gene is indispensable for the growth of the microorganism. In this case, when the macroplasmic plasmid is deleted, the microorganisms are no longer able to grow, so that the microorganisms are naturally excluded from the fermentation process for the production of the target substance, thereby substantially preventing the production of the target substance. This is not an antibiotic resistance gene, but a gene used for growth as a selective marker. 1 is a schematic diagram illustrating a method for producing a mutant microorganism that does not degenerate without using a selective marker by inserting a specific gene into a large plasmid and removing a gene that performs the same function in chromosomal DNA.

In the present invention, the specific gene essential for cell growth may be a gene involved in functions:

(1) intracellular DNA replication

 (2) Transcription of RNA from Intracellular DNA

(3) translation of proteins from intracellular RNA

(4) Activation of Synthetic Pathway of Amino Acids for Synthesizing Protein

(5) activation of metabolic pathways for synthesizing cell membranes

(6) activation of metabolic pathways to synthesize cell walls

(7) Activation of the synthetic pathway of coenzymes responsible for other functions in the cell

In addition, the specific gene is preferably a gene that satisfies the following conditions in order not to affect the expression of other genes specific gene deletion in the chromosome later.

(a) not expressed as an operon but expressed alone

(b) no other genes with amino acid homology inside the microorganism

(c) present on chromosomal DNA and not on macroplasmids

In the present invention, certain genes are used to generate gene phospholipids or peptidoglycans that encode components of DNA polymerase, RNA polymerase or ribosome. It may be characterized in that the gene involved or NAD, NADP, FAD, or gene involved in Coenzyme A biosynthesis, but is not limited thereto.

In another aspect, the mutant microorganism according to the present invention can be prepared by inserting a selective marker into the large plasmid. In the present invention, the selective marker may be characterized in that the antibiotic resistance gene, the antibiotic resistance gene may be characterized in that the chloramphenicol resistance gene, erythromycin resistance gene, or tetracycline (tetracycline) resistance gene, but this It is not limited.

In another aspect, the mutant microorganism according to the present invention may be inserted into the microorganism by inserting the entire macroplasmid into the chromosome of the microorganism or by introducing a DNA having the same or similar function as the DNA essential for the production of a target substance in the macroplasmid. It can manufacture.

In the present invention, introducing the DNA into the microorganism may be performed by inserting the DNA into the chromosome of the microorganism or transforming the microorganism in the form of a recombinant vector. In the present invention, the DNA essential for the production of the target substance may be characterized by a gene, and the gene encompasses a gene derived from a large plasmid or from another microorganism.

In the present invention, the target material is butanol, the microorganism is Clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium beijerinckii, Clostridium saccharobutylicum (Clostridium saccharobutylicum ), Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Clostridium buty Clostridium butyricum, Clostridium butylicum, Clostridium kluyveri, Clostridium tyrobutylicum, and Clostridium tyrobutyricum tyrobutyricum) but may be characterized in that the butanol-producing microorganism selected from the group consisting of, but is not limited to no.

In one embodiment of the present invention, long-term cultivation using Clostridium acetobutylicum , which has a large plasmid pSOL1 and is known to reduce the rate of solvent production by decreasing the proportion of pSOL1-containing cells during long-term culture, Mutant microorganisms were produced in which the proportion of pSOL1-containing cells did not decrease, so that butanol production was not reduced.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these embodiments.

Particularly, in the following examples, only a method for preparing butanol-producing mutant microorganisms using a macroplasmid of Clostridium acetobutylicum was used, but using other microorganisms having a macroplasmid plasmid, butanol as a target substance It will also be apparent to those skilled in the art to apply to microorganisms having the ability to produce other substances.

Example 1 Search for Genes Essential for Growth in the Genes of Clostridium Acetobutylicum

In order to use genes other than antibiotic resistance genes as selective markers, genes essential for growth were searched. In order to grow cells, all of the following functions should be performed normally.

(1) intracellular DNA replication

(2) Transcription of RNA from Intracellular DNA

(3) translation of proteins from intracellular RNA

(4) Activation of Synthetic Pathway of Amino Acids for Synthesizing Protein

(5) activation of metabolic pathways for synthesizing cell membranes

(6) activation of metabolic pathways to synthesize cell walls

(7) Activation of the synthetic pathway of coenzymes responsible for other functions in the cell

In the present invention, the genes were searched by focusing on the items (5), (6) and (7) which are not significantly influenced by the composition of the medium without changing the cell traits extensively. However, it will be apparent to those skilled in the art that a strain which is not degraded even by the items corresponding to (1), (2), (3), and (4) can be produced.

In addition, it was confirmed that the following conditions were met in order not to affect the expression of other genes in the specific gene deletion in the future.

(a) not expressed as an operon but expressed alone

(b) no other genes with amino acid homology within Clostridium acetobutylicum

(c) present on chromosomal DNA and not on macroplasmids

Gene search was performed using KEGG ( http://www.genome.jp ), and checking whether the searched gene satisfies the above conditions was also performed using the 'Genome Map' function and the BLAST function in KEGG. DNA sequences used for gene searching are GenBank ID NC_003030 and NC_001988.

 Genes searched in this way are listed below.

CAC2937 (ketopantoate reductase PanE / ApbA; NCBI GeneID: 1119120)

CAC2895 (D-alanine-D-alanine ligase; NCBI GeneID: 1119078)

CAC3225 (UDP-N-acetylmuramate--L-alanine ligase; NCBI GeneID: 1119407)

Example 2 Construction of a Vector for Inserting a Gene into pSOL1

PSOL1 used in this example refers to a large plasmid present in Clostridium acetobutylicum ATCC 824 strain (GenBank ID: NC_001988). In order to insert the gene searched in Example 1 into the large plasmid, homologous recombination was used. In the present embodiment, in order to reduce acetone production while maintaining butanol production ability, an ORF inside of adc (NCBI GeneID: 1116170), which is a gene encoding acetoacetate decarboxylase, was determined as an insertion position. However, it will be apparent to those skilled in the art that other sites of the macroplasmid can also be designated as insertion sites.

First, in order to amplify the first homologous sequence, genomic DNA of Clostridium acetobutylicum (including chromosomal DNA and pSOL1) was used as a template, and PCR was performed using primers of SEQ ID NOs: 1 and 2.

SEQ ID NO: 5'-ATATAAGCTTTATTTATGGAGCTTGTAAGGGC-3 '

SEQ ID NO: 5'-ATATGCATGCCGTATCATGCATTGCCATAA-3 '

The amplified DNA strand (574bp) and pUC18 (high-copy plasmid GenBank ID: L08752 that can be replicated in Escherichia coli) were digested with restriction enzyme HindIII / SphI, and then conjugated using T4 DNA ligase to prepare pUC18-adcRH1.

In order to insert the gene searched for in Example 1, a gene that performs the same function as that of the gene was amplified and inserted in Clostridium bieringkiai NCIMB 8052. At this time, after confirming that the gene is not composed of the operon like the homologous gene of Clostridium acetobutylicum, it was amplified to the promoter region. That is, the gene having the same function as CAC2937 is Cbei_1960 (NCBI GeneID: 5293174), which was amplified using the primers of SEQ ID NOs: 3 and 4. The gene having the same function as CAC2895 is Cbei_0581 (NCBI GeneID: 5291812), which was amplified using primers SEQ ID NOs: 5 and 6. Finally, the gene having the same function as CAC3225 is Cbei_0078 (NCBI GeneID: 5291312), which was amplified using primers SEQ ID NOs: 7 and 8.

SEQ ID NO: 5'-ATATCTGCAGTGGACACTGTGAGAAAGACTTT-3 '

SEQ ID NO: 5'-ATATCTGCAGTGTGTTTCAGAATATCTTACACAGG-3 '

SEQ ID NO: 5'-ATATCTGCAGTGTGGGGAATGTCCAATAAA-3 '

SEQ ID NO: 5'-ATATCTGCAGCTATCTGCTTATTTTCAATGAATCC-3 '

SEQ ID NO: 5'-ATATCTGCAGCACACGAATTTTTTTTATATTAAATC-3 '

SEQ ID NO: 5'-ATATCTGCAGAATTAAGTATTTAGGCTTTTAGGTATTT-3 '

The amplified DNA strand and pUC18-adcRH1 were digested with Pst I. Then, pUC18-adcRH1 prevented the self-conjugation of the vector using Calf intestine alkaline phosphatase, and prepared pJLa1-1960, pJLa1-0581 and cpJLa1-0078, respectively, by using T4 DNA ligase.

To insert a second homologous sequence, genomic DNA of C. acetobutylicum ATCC 824 was used as a template, and PCR was performed using primers of SEQ ID NOs: 9 and 10.

SEQ ID NO: 5'-ATATCCCGGGCTATGGAAAACTTAGAGTTGCG-3 '

SEQ ID NO: 10'-ATATGAGCTCGGCAAAGTCTCAAGCAAATCTA-3 '

The amplified DNA strands (588 bp) and pJLa1-1960, pJLa1-0581 and pJLa1-0078 were digested with restriction enzymes Xma I / Sac I, and then conjugated using T4 DNA ligase to pJLa12-1960, pJLa12-0581 and pJLa12. -0078 was produced.

Then, to insert a chloramphenicol marker, modified pECmulox (Kim et al., FEMS Microbiol. Lett., 278: 78-85, containing a loxP site and expressing the cat gene, chloramphenicol resistance gene, with the thiolase promoter of C. acetobutylicum) 2008) as a template and PCR was performed using the primers of SEQ ID NOs: 11 and 12.

SEQ ID NO: 5'-ATATTCTAGACACGACGTTGTAAAACGACG-3 '

SEQ ID NO: 12'-ATATCCCGGGCACACAGGAAACAGCTATGACC-3 '

The amplified DNA strands (1298bp), pJLa12-1960 and pJLa12-0078 were digested with restriction enzymes Xba I / Xma I, and then conjugated using T4 DNA ligase to prepare pJLadcI-1960 and pJLadcI-0078.

In the case of pJLa12-0581, since there is an Xba I site inside the Cbei_0581 gene, it is difficult to use Xba I, and PCR was performed using primers of SEQ ID NOs: 13 and 12.

SEQ ID NO: 5'-ATATCCCGGGCACGACGTTGTAAAACGACG-3 '

The amplified DNA strand (1298bp) and pJLa12-0581 were digested with restriction enzyme Xma I and the vector was treated with Calf Intestine Alkaline Phosphatase to prevent autoconjugation, and then conjugated with T4 DNA ligase to prepare pJLadcI-0581. .

Example 3: Gene insertion by introducing pJLadcI-1960 into a large plasmid

In the present Example, pJLadcI-1960 prepared in Example 2 was transformed into Clostridium acetobutylicum to prepare a strain in which the gene was inserted into a large plasmid. Although only pJLadcI-1960 is described in this embodiment, it will be apparent to those skilled in the art that the same can be applied to transformation of the remaining vectors.

First, Clostridium acetobutylicum contains the restriction enzyme Cac 824I. Since cleavage does not occur when the cleavage site (5'-GCNGC-3 ') of Cac 824I is methylated, it contains a pAN3 vector that expresses a methyl transfer enzyme that methylates pJL-adcI-1 to the 5'-GCNGC-3' site. E. coli TOP10 strain was transformed. The strain was cultured to purify plasmid DNA in large quantities and used for transformation of Clostridium acetobutylicum. PAN3 used in the present invention is a plasmid capable of replicating only in Escherichia coli, a plasmid 'pAN1' expressing the f3TI methyl transferase of Bacillus subtilis phage phage (Mermelstein and Papoutsakis, Appl. Environ.Microbiol. 59: 1077-1081. , 1993) is a vector of a kanamycin resistance gene replacement of the chloramphenicol resistance gene. pAN3 is a low copy plasmid that is small in Escherichia coli and does not replicate in Clostridium acetobutylicum and therefore does not significantly affect the transformation of Clostridium acetobutylicum.

To transform Clostridium acetobutylicum, first endocrine spore suspension was inoculated into a tube containing 10 mL CGM medium (Sillers et al., Biotechnol. Bioeng., 102: 38-49, 2009) To remove them, the mixture was heated to 80 ° C. for 10 minutes, cooled, and then anaerobicly cultured at 37 ° C. After this process, everything except centrifugation was performed in the anaerobic chamber. At an optical density of 0.6 at 600 nm, 200 mL 2x YTG (16 g / L Bacto Tryptone, 10 g / L Bacto Yeast Extract, 4 g / L NaCl, 5 g / L glucose) stored for one day in an anaerobic chamber , pH 5.2) 10 mL of the culture was inoculated into the flask containing the medium. At this time, the flask was stored in an incubator in an anaerobic chamber beforehand to 37 ° C. It was incubated at 37 ° C. until the optical density at 600 nm was 1.1. The flask was then placed on ice and cooled for 30 minutes, then centrifuged at 3000 g. After centrifugation, the supernatant was discarded and the remaining cell mass was suspended in 50 mL of electroporation buffer (270 mM sucrose, 5 mM NaH ₂ PO ₄ , pH 7.4) previously stored on ice. Centrifuge this suspension again at 3000 g and suspend the cell mass in 2.3 mL of electroporation buffer. 0.5 mL of each was added to a 0.4 mm gap electroporation cuvette, and 20 μg of the purified pTHL2-LR / pAN1 DNA was added thereto. It was electroporated under the conditions of 2.5 kV, 25 λF, and 8O. Immediately after electroporation, 0.75 mL of 2x YTG medium was added to the cuvette, mixed well, and then transferred to a 2 mL Eppendorf tube and incubated at 37 ° C. After 5 hours, 0.2 mL of the culture was plated in 2 × YTG solid medium (pH 5.8) containing 30 μg / mL of chloramphenicol. Since pJL-adcI-1 does not normally replicate in Clostridium acetobutylicum, the colony with chloramphenicol resistance is very likely to have the gene inserted into a large plasmid through homologous recombination. The colonies were inserted through colony PCR. Primers used for PCR are SEQ ID NOs: 4 and 14. The final identified strain was named C. acetobutylicum Δ adc :: Cbei_1960-Cm.

SEQ ID NO: 14'-TCTTGTCTAAACTGGTTAAGGC-3 '

Example 4 Preparation of Cre recombinase Expression Vector and Removal of Cm Marker Using the Same

For further strain manipulation it is necessary to remove Cm resistance markers from C. acetobutylicum Δ adc :: Cbei_1960-Cm. Mutant loxP sites are included on both sides of the Cm-resistant markers, so that only Cm-resistant markers can be selectively removed by expressing Cre recombinase.

To express Cre recombinase, the thiolase promoter of Clostridium acetobutylicum and the Nco I site in pIMP1 (E. coli- Clostridium acetobutylicum shuttle vector Mermelstein et al., Biotechnology, 10: 190-195, 1992) The shuttle vector pTHL2 prepared by inserting was used. Amplification of Cre recombinase was based on pJW168 (Palmeros et al., Gene, 247: 255-264, 2000) as a template, and primers of SEQ ID NOs: 15 and 16 were used.

SEQ ID NO: 15'-ATATCCATGGATGTCCAATTTACTGACCGTACA-3 '

SEQ ID NO: 5'-ATATCCCGGGCTAATCGCCATCTTCCAGCAGG-3 '

The amplified DNA strand (1052bp) and pTHL2 were digested with Nco I / Xma I restriction enzymes, and then conjugated with T4 DNA ligase to complete pTHL2-Cre. pTHL2-Cre was methylated with pAN3 vector and transformed into C. acetobutylicum Δ adc :: Cbei_1960-Cm as in Example 3. Transformed strains were selected in 2 × YTG solid medium (pH 5.8) containing 40 μg / mL of erythromycin. Each colony was identified by colony PCR using primers of SEQ ID NOs: 11 and 12 to remove Cm resistance markers. The final identified strain was at least 30 times in 2x YTGS medium (16 g / L Bacto Tryptone, 10 g / L Bacto Yeast Extract, 4 g / L NaCl, 2 g / L Glucose, 15 g / L Soluble starch, pH 6.8) Subculture was confirmed whether pTHL2-Cre was removed. The colonies from which pTHL2-Cre was removed were confirmed to have not lost pSOL1 involved in solvent generation through degeneration tests (Scotcher and Bennett, J. Bacteriol., 187 (6): 1930-1936, 2005). The final identified strain was named C. acetobutylicum Δ adc :: Cbei_1960.

Example 5: CAC2937 deletion in chromosome after gene insertion

In this example, the strain C. acetobutylicum Δ adc :: Cbei_1960 produced in Example 4 was deleted so that pSOL1 is not lost by deleting the CAC2937 so that the inserted Cbei_1960 gene plays an essential role in growth. In the present embodiment, only the strain of C. acetobutylicum Δ adc :: Cbei_1960 is described, but it will be apparent to those skilled in the art that the same applies to strains prepared through the same process as the above-described examples.

In order to insert a Cm resistance marker in CAC2937, DNA strands having a sequence homologous to CAC2937 on both Cm resistance markers were prepared by overlapping PCR. First, the first homologous sequence was SEQ ID NO: 17 and 18, the second homologous sequence using the primers of SEQ ID NO: 19 and 20, and amplified by PCR using Clostridium acetobutylicum genomic DNA as a template. On the other hand, Cm resistance marker was amplified using the primers of SEQ ID NO: 21 and 22 as a template with modified pECmulox.

SEQ ID NO: 17'-ATATCCCGGGTATTGTATGATCCACAGAATAGCCG-3 '

SEQ ID NO: 18'-TTTTACAACGTCGTGGTGTGGAATCGCTGAATAAGTTTAG-3 '

SEQ ID NO: 19'5'-GCTGTTTCCTGTGTG GTTTAAAGCTTGCTCCTTAAAGACC-3 '

SEQ ID NO: 20'-ATATCCCGGGGCCAAGGTATAGAGTTTGATTATGG-3 '

SEQ ID NO: 21'-TCAGCGATTCCACAC CACGACGTTGTAAAACGACG-3 '

SEQ ID NO: 22'-GAGCAAGCTTTAAAC CACACAGGAAACAGCTATGACC-3 '

By amplifying the three amplified DNA products together and performing overlap PCR using primers of SEQ ID NOs: 17 and 20, DNA strands having homologous sequences bound to CAC2937 can be obtained with both Cm resistance markers. The DNA strand and pUC18 were digested with Xma I, and the vector was treated with Calf Intestine Alkaline Phosphatase to prevent self-conjugation, followed by T4 DNA ligase. The prepared vector was subjected to the same procedure as in Example 3 to confirm that CAC2937 was deleted, and then the inserted Cm resistance marker was removed through the same procedure as in Example 4. After the Cm resistance marker is removed, the protein coding sequence of CAC2937 is damaged and cannot function normally.

Example 6: Clostridium Acetobutylicum Chromosome DNA adhE1  Gene insertion

In this embodiment, adhE1 present in the claw cast large plasmids of lithium acetoacetate portion Tilikum ATCC 824: shows the step of inserting (NCBI GeneID 1116167) gene on the chromosome. In this embodiment, buk the insertion site Yes (NCBI GeneID 1119258) an internal ORF of the gene, adhE1 is however inserted by including the native promoter can also equally be applied that to express by using the site or other promoters of different chromosomes per It will be obvious to the supplier.

The basic vector production process is the same as in Example 2. However, in Example 2, a region homologous to the adc gene was replaced with a homology region of the buk gene. The first homologous region was amplified by PCR with primers SEQ ID NOs: 23 and 24.

SEQ ID NO: 23'-ATATAAGCTTAGTTGCTGGAAAAGCTGATATC-3 '

SEQ ID NO: 24'-ATATGCATGCTTGATTTACTGCATAAGTTCCACT-3 '

The amplified DNA strand (525bp) and pUC18 were digested with the restriction enzyme Hind III / Sph I, and then conjugated using T4 DNA ligase to prepare pUC18-bukRH1.

In order to insert adhE1 , Clostridium acetobutylicum genomic DNA was used as a template and amplified by primers of SEQ ID NOs: 25 and 26.

SEQ ID NO: 25'-ATATCTGCAGAAAGAAGCTATATCTTAATTCAAAA-3 '

SEQ ID NO: 26'-ATATCTGCAGTTAAGGTTGTTTTTTAAAACAATTTA-3 '

The amplified DNA strand (3278 bp) and pUC18-bukRH1 were cleaved with Pst I using the primers of the sequence. Then, pUC18-bukRH1 was treated with Calf intestine alkaline phosphatase to prevent the vector from self-conjugation, and pJLb1-adhE1 was produced by using T4 DNA ligase.

To insert a second homologous sequence, genomic DNA of C. acetobutylicum ATCC 824 was used as a template, and PCR was performed using primers of SEQ ID NOs: 27 and 28.

SEQ ID NO: 27'-ATATTCTAGACTATGGAAAACTTAGAGTTGCG-3 '

SEQ ID NO: 28'-ATATCCCGGGGGCAAAGTCTCAAGCAAATCTA-3 '

The amplified DNA strand (569 bp) and pJLb1-adhE1 were digested with restriction enzymes Xba I / Xma I and conjugated with T4 DNA ligase to prepare pJLb12-adhE1.

Then modified pECmulox (Kim et al., FEMS Microbiol. Lett., 278: 78-85, 2008) containing a loxP site to insert a chloramphenicol marker and expressing the cat gene, a chloramphenicol resistant gene, with the thiolase promoter of C. acetobutylicum . ) As a template and PCR was performed using primers SEQ ID NOs: 29 and 30.

SEQ ID NO: 29'-ATATTCTAGACACGACGTTGTAAAACGACG-3 '

SEQ ID NO: 30'-ATATTCTAGACACACAGGAAACAGCTATGACC-3 '

The amplified DNA strand (1298bp) and pJLb12-adhE1 were digested with restriction enzyme Xba I, the vector was treated with Calf Intestine Alkaline Phosphatase, and conjugated with T4 DNA ligase to prepare pJLbukI-adhE1. The orientation of the inserted site was confirmed using Sac I, and the first homologous site was located in the 5 'direction of the Cm resistance marker protein coding sequence and the second homologous site was located in the 3' direction.

pJLbukI-adhE1 was inserted into the chromosomal DNA of Clostridium acetobutylicum ATCC 824 strain through the same procedure as illustrated in Example 4. Insertion of the Cm resistance marker was confirmed by colony PCR and the primers used were SEQ ID NOs: 31 and 30.

SEQ ID NO: 31'5-CCTAAAATGCCATCAACACTTG-3 '

1 is a schematic diagram illustrating a method for producing a microorganism which does not degenerate by inserting a portion homologous to a portion of chromosomal DNA essential for microbial growth in a large plasmid.

FIG. 2 is a schematic diagram illustrating a method for producing microorganisms that are not degraded by inserting a selective marker into a large plasmid.

FIG. 3 illustrates a method for producing a microorganism which is not degraded by inserting the entire macroplasmid into the chromosome of the microorganism or by introducing DNA having the same or similar function as the DNA essential for the production of a target substance in the macroplasmid to the microorganism. It is a schematic diagram.

<110> Korea Advanced Institute of Science and Technology          GS Caltex Corporation          Biofuelchem <120> Preparing Method for non-degenerative microorganism by protecting          megaplasmid lose and the microorganism <130> P09-B180 <160> 31 <170> KopatentIn 1.71 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 atataagctt tatttatgga gcttgtaagg gc 32 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 atatgcatgc cgtatcatgc attgccataa 30 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atatctgcag tggacactgt gagaaagact tt 32 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 atatctgcag tgtgtttcag aatatcttac acagg 35 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atatctgcag tgtggggaat gtccaataaa 30 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atatctgcag ctatctgctt attttcaatg aatcc 35 <210> 7 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 atatctgcag cacacgaatt ttttttatat taaatc 36 <210> 8 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 atatctgcag aattaagtat ttaggctttt aggtattt 38 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 atatcccggg ctatggaaaa cttagagttg cg 32 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 atatgagctc ggcaaagtct caagcaaatc ta 32 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 atattctaga cacgacgttg taaaacgacg 30 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 atatcccggg cacacaggaa acagctatga cc 32 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 atatcccggg cacgacgttg taaaacgacg 30 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tcttgtctaa actggttaag gc 22 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 atatccatgg atgtccaatt tactgaccgt aca 33 <210> 16 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 atatcccggg ctaatcgcca tcttccagca gg 32 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 atatcccggg tattgtatga tccacagaat agccg 35 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ttttacaacg tcgtggtgtg gaatcgctga ataagtttag 40 <210> 19 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gctgtttcct gtgtggttta aagcttgctc cttaaagacc 40 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 atatcccggg gccaaggtat agagtttgat tatgg 35 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 tcagcgattc cacaccacga cgttgtaaaa cgacg 35 <210> 22 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gagcaagctt taaaccacac aggaaacagc tatgacc 37 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 atataagctt agttgctgga aaagctgata tc 32 <210> 24 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 atatgcatgc ttgatttact gcataagttc cact 34 <210> 25 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 atatctgcag aaagaagcta tatcttaatt caaaa 35 <210> 26 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 atatctgcag ttaaggttgt tttttaaaac aattta 36 <210> 27 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 atattctaga ctatggaaaa cttagagttg cg 32 <210> 28 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 atatcccggg ggcaaagtct caagcaaatc ta 32 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 atattctaga cacgacgttg taaaacgacg 30 <210> 30 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 atattctaga cacacaggaa acagctatga cc 32 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 cctaaaatgc catcaacact tg 22  

Claims (32)

In a microorganism having a large plasmid, a region homologous to a portion of the chromosomal DNA essential for the growth of the microorganism is inserted into the large plasmid, and then a portion of the DNA is deleted from the chromosome. Method for producing a mutant microorganism that can prevent the production capacity of the deterioration. The method of claim 1, wherein a part of the chromosomal DNA is a specific gene. The method of claim 2, wherein the specific gene is a gene involved in a function selected from the group consisting of: (1) replication of DNA in cells; (2) transcription of RNA from intracellular DNA; (3) translation of proteins from intracellular RNA; (4) activation of synthetic pathways of amino acids to synthesize proteins; (5) activation of metabolic pathways for synthesizing cell membranes; (6) activation of metabolic pathways to synthesize cell walls; And (7) Activation of the synthetic pathway of coenzymes responsible for other functions in the cell. The method of claim 3, wherein the specific gene is a gene satisfying the following conditions:  (a) expressed alone, not as an operon; (b) no other genes with amino acid homology inside the microorganism; And (c) Be on chromosomal DNA rather than macroplasmids. The method of claim 2, wherein the specific gene is a gene encoding a component of a DNA polymerase, an RNA polymerase, or a ribosome. The method of claim 2, wherein the specific gene is a gene involved in the production of phospholipids or peptidoglycans. The method of claim 2, wherein the specific gene is a gene involved in NAD, NADP, FAD, or Coenzyme A biosynthesis. The method of claim 1, wherein the microorganism is Clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium beijerinckii, Clostridium saccharobutylicum, Clostridium saccha Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Clostridium butyricum, In the group consisting of Clostridium butylicum, Clostridium kluyveri, Clostridium tyrobutylicum, and Clostridium tyrobutyricum Method for producing a mutant microorganism, characterized in that the selected butanol-producing microorganism. In a microorganism having a large plasmid, a region homologous to a part of chromosomal DNA essential for the growth of the microorganism is inserted in the large plasmid, and a portion of the DNA is deleted from the chromosome by deletion of the large plasmid. Mutant microorganisms that can prevent degradation of the ability to produce a target substance. The mutant microorganism according to claim 9, wherein a part of the chromosomal DNA is a specific gene. The mutant microorganism of claim 10, wherein the specific gene is a gene involved in a function selected from the group consisting of: (1) replication of DNA in cells; (2) transcription of RNA from intracellular DNA; (3) translation of proteins from intracellular RNA; (4) activation of synthetic pathways of amino acids to synthesize proteins; (5) activation of metabolic pathways for synthesizing cell membranes; (6) activation of metabolic pathways to synthesize cell walls; And (7) Activation of the synthetic pathway of coenzymes responsible for other functions in the cell. The mutant microorganism according to claim 11, wherein the specific gene is a gene satisfying the following conditions:  (a) expressed alone, not as an operon; (b) no other genes with amino acid homology inside the microorganism; And (c) Be on chromosomal DNA rather than macroplasmids. The mutant microorganism of claim 10, wherein the specific gene is a gene encoding a component of a DNA polymerase, an RNA polymerase, or a ribosome. The mutant microorganism according to claim 10, wherein the specific gene is a gene involved in the production of phospholipids or peptidoglycans. The mutant microorganism of claim 10, wherein the specific gene is a gene involved in NAD, NADP, FAD, or Coenzyme A biosynthesis. The method of claim 9, wherein the microorganism is Clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium beijerinckii, Clostridium saccharobutylicum, Clostridium saca Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Clostridium butyricum, In the group consisting of Clostridium butylicum, Clostridium kluyveri, Clostridium tyrobutylicum, and Clostridium tyrobutyricum Mutant microorganism, characterized in that the selected butanol producing microorganism. A method for producing a mutant microorganism capable of preventing the production of a target substance from being lowered by the deletion of a giant plasmid, wherein a selective marker is inserted into the giant plasmid in a microorganism having a giant plasmid. 18. The method of claim 17, wherein the selective marker is an antibiotic resistance gene. 19. The method of claim 18, wherein the antibiotic resistance gene is a chloramphenicol resistance gene, an erythromycin resistance gene or a tetracycline resistance gene. 18. The method of claim 17, wherein the microorganism is Clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium beijerinckii, Clostridium saccharobutylicum, Clostridium saccha Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Clostridium butyricum, In the group consisting of Clostridium butylicum, Clostridium kluyveri, Clostridium tyrobutylicum, and Clostridium tyrobutyricum Method for producing a mutant microorganism, characterized in that the selected butanol-producing microorganism. In a microorganism having a large plasmid, a selective microorganism having a selective marker is inserted into the large plasmid. The mutant microorganism of claim 21, wherein the selective marker is an antibiotic resistance gene. The mutant microorganism according to claim 22, wherein the antibiotic resistance gene is a chloramphenicol resistance gene, an erythromycin resistance gene or a tetratracycline resistance gene. 22. The method of claim 21, wherein the microorganism is Clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium beijerinckii, Clostridium saccharobutylicum, Clostridium saccha Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Clostridium butyricum, In the group consisting of Clostridium butylicum, Clostridium kluyveri, Clostridium tyrobutylicum, and Clostridium tyrobutyricum Mutant microorganism, characterized in that the selected butanol producing microorganism. In a microorganism having a large plasmid, the entire large plasmid is inserted into a chromosome of the microorganism, or the large plasmid is characterized by introducing DNA having a function identical or similar to that of DNA necessary for the production of a target substance in the large plasmid. Method for producing a mutant microorganism, which can prevent the ability of the target material to decrease by the deletion of. The method of claim 25, wherein the DNA essential for the production of the target substance is a specific gene. The method of claim 26, wherein the target substance is butanol and the specific gene is adhE1 . The method of claim 25, wherein the microorganism is Clostridium acetobutylicum (Clostridium acetobutylicum), Clostridium beijerinckii, Clostridium saccharobutylicum, Clostridium saca Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Clostridium butyricum, In the group consisting of Clostridium butylicum, Clostridium kluyveri, Clostridium tyrobutylicum, and Clostridium tyrobutyricum Method for producing a mutant microorganism, characterized in that the selected butanol-producing microorganism. In the microorganism having a large plasmid, the entire large plasmid is inserted into the chromosome of the microorganism, or the DNA having the same or similar function as the DNA essential for the production of the target substance in the large plasmid is introduced into the microorganism. The mutant microorganism which can prevent the production | generation of the target substance falls by deletion of a large plasmid. The mutant microorganism according to claim 29, wherein the DNA essential for the production of the target substance is a specific gene. The mutant microorganism according to claim 30, wherein the target substance is butanol and the specific gene is adhE1 . The method of claim 29, wherein the microorganisms are Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, Clostridium saccharin Clostridium saccharoperbutylacetonicum, Clostridium perfringens, Clostridium tetani, Clostridium difficile, Clostridium butyricum, In the group consisting of Clostridium butylicum, Clostridium kluyveri, Clostridium tyrobutylicum, and Clostridium tyrobutyricum Mutant microorganism, characterized in that the selected butanol producing microorganism.

Images