JP2013005764A - Method for producing organic substance from mixed sugar in escherichia coli - Google Patents

Abstract

The present invention provides an E. coli mutant strain in which CCR useful for the production of useful substances has been released, and a useful substance production method using the mutant strain.
An E. coli strain in which carbon catabolite suppression (CCR) is released by introducing a gene mutation that overexpresses Mlc protein, and a method for producing a useful organic substance from a mixed sugar using the E. coli strain .
[Selection figure] None

Description

  The present invention relates to a method for efficiently producing an organic substance from mixed sugars in E. coli. Especially, it is related with the method of producing organic substances, such as alcohol and an organic acid, using the culture medium containing two or more types of sugars as a raw material.

  Alcohol and organic acids are used for various purposes as useful substances. These can be produced by metabolizing sugars to microorganisms, but sugars as raw materials are often derived from renewable organic resources and biomass made from animals and plants for reasons such as being inexpensive. Biomass-derived sugar is generally a mixed sugar in which other types of sugars such as glucose, xylose, and arabinose are mixed (Non-Patent Document 1). However, when a mixed sugar is given to a microorganism, only the most energy-efficient glucose is selectively consumed, and other sugars may not be metabolized until the glucose is consumed. This is a phenomenon that occurs because a genetic mechanism called carbon catabolite repression (CCR) exists in microorganisms (Non-Patent Document 2). As a result, among the sugar-derived mixed sugars, sugars other than glucose are not efficiently converted into the target substance and may be wasted (Non-Patent Document 3).

  In the CCR mechanism, the expression of genes involved in the consumption of sugars other than glucose is suppressed in the presence of glucose. For example, lacZYA of Escherichia coli is a gene operon required for lactose consumption and encodes β-galactosidase, β-galactoside permease, and galactoside acetyltransferase, respectively. When glucose and lactose coexist in the culture medium, lacI gene product (lactose repressor) and crp gene product (cAMP receptor protein) cooperate in the lacZYA expression regulatory region so that lacZYA mRNA is not transcribed. Negative control. However, when only lactose is present as a sugar in the culture solution, this negative control is released by allo- lactose in which the lactose incorporated into the cells has changed, and the lacZYA mRNA is actively transcribed. This causes lactose to be consumed by E. coli. Moreover, the gene group required for consumption of xylose and arabinose is also subjected to similar gene expression regulation (Non-patent Document 4).

  Escherichia coli is one of the most commonly used hosts for substance production by microorganisms, but has a CCR mechanism as described above. Therefore, a contrivance has been made to create a mutant strain in which CCR is released and to simultaneously metabolize various sugars. For example, strains in which the ptsG gene has been disrupted (DptsG) and strains that have point mutations in 3 base pairs in the crp gene (crp *) are known as CCR-released strains. There is an example used for substance production (Patent Document 1 and Non-Patent Documents 5 to 8).

  However, the ptsG gene is localized in the cell membrane and encodes a glucose permease responsible for the function of taking glucose into cells. In the DptsG strain, the glucose uptake efficiency is lowered, and the productivity of the target substance may be lowered. The crp gene encodes a cAMP receptor protein type transcription factor, but a considerable number of genes are under the control of Crp, and it is considered that the metabolic pathway is greatly changed in the crp * strain. Furthermore, the crp gene disruption strain (Dcrp) is known to be resistant to certain organic solvents, and conversely, the crp * strain is also expected to be sensitive to organic solvents (Non-Patent Documents). 9). Therefore, in the case of the crp * strain, when alcohol, which is a kind of organic solvent, is produced, productivity may decrease due to the toxicity problem.

U.S. Patent No. 6159738

van Maris et al., Antonie van Leeuwenhoek (2006) 90: 391-418 Deutscher, Curr. Opin. Microbiol. (2008) 11: 87-93 Kim et al., Appl. Microbiol. Biotechnol. (2010) 88: 1077-1085 Song and Park, J. Bacteriol. (1997) 179: 7025-7032 Cirino et al., Biotechnol. Bioeng. (2006) 95: 1167-1176 Khankal et al., J. Biol. Eng. (2009) 3:13 Dien et al., J. Ind. Microbiol. Biotechnol. (2002) 29: 221-227 Nichols et al., Appl. Microbiol. Biotechnol. (2001) 56: 120-125 Okochi et al., J. Biosci. Bioeng. (2008) 105: 389-394

  From the above, the present inventor decided to isolate a mutant strain from which a new CCR useful for production of useful substances was released. In addition, it was decided to actually demonstrate useful substance production using such mutant strains.

  The lacZYA operon of Escherichia coli is a gene that undergoes CCR as described above. To determine whether it is expressed, use 5-chloro-4-bromo-3-indolyl-β-D-galactose (x-gal). You can easily know by the blue-white selection. This is based on the fact that colorless and transparent x-gal exhibits a blue color when it is degraded by β-galactosidase, which is a lacZ gene product. That is, when wild-type Escherichia coli is cultured on an agar medium containing glucose, lactose, x-gal and other suitable nutrient sources, white colonies are formed. This is because glucose and lactose coexist in the medium, and transcription of the lacZ gene is suppressed by CCR. However, if a mutation causes abnormalities in CCR and the lacZ gene is transcribed even in the presence of glucose and lactose, a blue colony will be formed on the same medium.

  Therefore, the present inventor applied the wild-type E. coli MG1655 strain on an agar medium containing glucose, lactose, x-gal, and other appropriate nutrient sources, and induced mutations by irradiating ultraviolet rays, The color was determined. This should isolate mutants with CCR abnormalities (ie, CCR released).

  Subsequently, it was decided to identify the mutant gene of the isolated mutant so that CCR could be released in any strain. For this purpose, shotgun cloning is performed. At this stage, since it cannot be determined whether the mutated gene is dominant or recessive, two plasmid genome libraries prepared using the genome of the wild strain or the mutant strain are introduced into the mutant strain and the wild strain, respectively, and blue-white selection is performed. At this time, since many false positive clones are likely to be separated from the positive clones, secondary screening and base sequence determination are also performed to identify mutant genes. Once the mutant gene has been identified, the mutant gene is then artificially introduced into the wild type strain by homologous recombination. The mutant strain obtained by homologous recombination and the original mutant strain isolated by ultraviolet irradiation are compared in terms of sugar consumption rate, etc., and observed to show the same phenotype to confirm the final mutant gene.

  Furthermore, actual production of useful substances is carried out using the above mutant strains. Therefore, isobutanol (isobutyl alcohol) is produced from the mixed sugar, and it is demonstrated that the mutant strain of the present invention has better isobutanol productivity than the wild type, DptsG strain or crp * strain. In addition, isobutanol is a kind of alcohol that has recently attracted attention as an alternative fuel for gasoline or light oil. The method of isobutanol production uses a method via 2-keto acid, which has the highest production efficiency among currently known methods (Atsumi et al., Nature (2008) 451: 86-89).

  The present inventor has intensively studied to develop a method for producing an organic substance from a mixed sugar in E. coli, and has completed the present invention.

That is, the present invention is as follows.
[1] An Escherichia coli strain in which carbon catabolite repression (CCR) is released by overexpression of Mlc protein by gene mutation introduction or overexpression using an expression vector.
[2] An E. coli strain in which carbon catabolite repression (CCR) in [1] is released, wherein the gene mutation that overexpresses Mlc protein is a mutation in the mlc promoter.
[3] The carbon catabolite repression (CCR) in [1] or [2], in which the mutation in the mlc promoter is a substitution of the 31st c to t in the base sequence of the mlc promoter shown in SEQ ID NO: 34 has been released E. coli strain.
[4] An Escherichia coli strain in which carbon catabolite repression (CCR) is released by overexpression of one, two, three, four or five genes of anmK, slyB, slyA, ydhI or ydhJ.
[5] The E. coli strain according to any one of [1] to [4], wherein a gene involved in the synthesis of an organic substance is further introduced.
[6] The Escherichia coli strain according to [5], wherein an exogenous kivd gene, Adh2 gene, alsS gene, ilvC gene and ilvD gene are introduced.
[7] A method for releasing carbon catabolite repression (CCR) of an E. coli strain, which comprises introducing a gene mutation that overexpresses the Mlc protein into an E. coli strain, or overexpressing the Mlc protein using an expression vector.
[8] The method according to [7], wherein the gene mutation that overexpresses Mlc protein is a mutation in the mlc promoter.
[9] The method according to [7] or [8], wherein the mutation in the mlc promoter is substitution of the 31st c in the mlc promoter nucleotide sequence shown in SEQ ID NO: 34 with t.
[10] A method for releasing carbon catabolite repression (CCR) of an Escherichia coli strain, comprising overexpressing one, two, three, four or five genes of anmK, slyB, slyA, ydhI or ydhJ .
[11] The E. coli strain from which carbon catabolite repression (CCR) has been released in any one of [1] to [4] is cultured in the presence of a mixed sugar containing glucose and other sugars other than glucose. A method of producing organic substances using other sugars as raw materials.
[12] An E. coli strain from which carbon catabolite repression (CCR) of any one of [1] to [4] is released, and further introduced with an E. coli strain into which a gene involved in the synthesis of an organic substance has been introduced. A method of culturing in the presence of a mixed sugar containing other sugars other than glucose and producing the organic substance from glucose and other sugars other than glucose.
[13] The method according to [12], wherein the organic substance is an alcohol or an organic acid.
[14] The method according to [12] or [13], wherein the organic substance is isobutyl alcohol, and the mixed sugar contains glucose and xylose.
[15] The method according to [14], wherein an exogenous kivd gene, Adh2 gene, alsS gene, ilvC gene and ilvD gene are introduced into an E. coli strain.

As shown in the Examples, it can be seen that when a substance is produced in Escherichia coli using a mixed sugar as a raw material, it is more productive to use a CCR-released strain than to use a wild type strain. Among the CCR-released strains, those with mlc ^* may be advantageous. It has never been reported that mlc ^* or mlc gene overexpression releases CCR and consumes glucose and other sugars simultaneously. In addition, there is no example of producing organic substances using mlc ^* or a strain overexpressing the mlc gene.

It is a figure which shows the structure of the genome fragment of the gen strain | stump | stock cloned by the shotgun. FIG. 1a shows a fragment containing the mlc gene, and FIG. 1b shows a fragment containing the anmK, slyB, slyA, ydhI and ydhJ genes. In the figure, the arrow indicates the ORF, and the black line indicates the genomic fragment taken by shotgun cloning. The sequence of the mlc promoter part and the mutation site of mlc ^* are also shown. It is a figure which shows the plasmid map of pHN1234, pHN1532, and pHN1534. Chl ^r and Kan ^r indicate a chloramphenicol resistance gene and a kanamycin resistance gene, respectively. It is a figure which shows co-consumption of glucose and xylose in each strain | stump | stock. It is a figure which shows the productivity of isobutanol in each strain | stump | stock. FIG. 4a shows the result when glucose is used as the carbon source, FIG. 4b shows the result when glucose and xylose are used as the carbon source, and FIG. 4c shows the result when xylose is used as the carbon source. It is a figure which shows the productivity of the more optimized isobutanol. FIG. 5a shows the result when glucose is used as the carbon source, and FIG. 5b shows the result when glucose and xylose are used as the carbon source.

  Hereinafter, the present invention will be described in detail.

  The present invention is a method for producing an organic substance from a mixed sugar in E. coli, and a method for producing an organic substance using a mutant from which carbon catabolite repression (CCR) is released. . CCR is a mechanism that suppresses gene expression in the degradation system of other carbon sources until there is a preferentially metabolized carbon source such as glucose in the medium until it is consumed. In nature, there is an advantage that a carbon source can be used more efficiently, but there is a disadvantage that sugars other than glucose cannot be used when producing useful substances using microorganisms. In the present invention, a sugar that is preferentially metabolized under a culture condition using a mixed sugar in which glucose and other sugars coexist by releasing CCR of Escherichia coli, and other sugars such as glucose and glucose. Use of other sugars simultaneously makes it possible to produce useful substances. Here, the release of CCR is also referred to as CCR insufficiency or CCR abnormality. In E. coli, sugars other than glucose are used when cultured under conditions where sugars such as glucose and other sugars are mixed with other sugars. This means that not only glucose but also other sugars are simultaneously metabolized and can be used for the synthesis of organic substances in the Escherichia coli body.

  In the present invention, CCR can be released by, for example, overexpression of Mlc (making large colonies) protein. Mlc protein is a 44 kDa DNA-binding protein known to regulate the expression of several genes and operons as a regulator of hydrocarbon metabolism (Hosono, K., et al., Biosci, Biotechnol. Biochem., 59, 256-261 (1995)). As an example, it is known that the expression of the ptsG gene is negatively regulated by Mlc protein (Plumbridge, J. Curr. Opin. Microbiol. 5: 187-193 (2002)). Overexpression of Mlc protein causes PtsG protein deficiency and CCR is released.

  Overexpression of Mlc protein can be performed, for example, by introducing into Escherichia coli a vector into which mlc gene has been introduced and Mlc can be expressed. Further, a mutation that makes the mlc promoter highly active may be introduced into the base sequence of the mlc promoter DNA. As such a mutation, for example, a mutation from caccat to tattat of the -10 sequence of the mlc promoter can be mentioned. This mutation is a substitution of the 31st c in the mlc promoter sequence shown in SEQ ID NO: 34 to t.

  Furthermore, partial or complete CCR release can also be achieved by overexpression of one, two, three, four or five genes of anmK, slyB, slyA, ydhI or ydhJ. Here, partial release means incomplete CCR release. However, even such cancellation of CCR is useful because it is possible to simultaneously use sugars that are preferentially metabolized and other sugars. In the present invention, CCR cancellation includes not only complete cancellation but also partial cancellation. Of the above five genes, overexpression of the slyA gene is particularly preferable. Overexpression of these genes can be performed by introducing a vector into which these genes are introduced and capable of expressing these genes into E. coli.

  Whether the mutation in the base sequence of the mlc promoter causes overexpression of the Mlc protein and cancels CCR, the Escherichia coli mlc promoter is subjected to physical mutagenesis such as UV irradiation and chemical mutagenesis using nitrosoguanidine. It is sufficient to artificially induce a mutation and examine whether the lacZ gene is expressed when the mutant is cultured in a medium in which glucose and lactose coexist. Furthermore, the expression of the lacZ gene may be examined by the same method to determine whether overexpression of the mlc gene or overexpression of the anmK, slyB, slyA, ydhI, or ydhJ gene releases CCR. In E. coli with normal CCR, the lacZ gene is not expressed, whereas in E. coli with CCR released, the lacZ gene is expressed. The expression of the lacZ gene may be performed by adding 5-chloro-4-bromo-3-indolyl-β-D-galactose (x-gal) to a medium in which glucose and lactose coexist, and performing blue-white selection. In Escherichia coli with CCR released, the lacZ gene is expressed even in the presence of glucose and lactose, β-galactosidase, which is the expression product of the lacZ gene, is produced, β-galactosidase degrades x-gal, and the colony is blue . Therefore, E. coli from which CCR is released can be isolated from the blue colonies.

  Thus, if a mutation causing overexpression of the Mlc protein is identified, the CCR of E. coli can be released by introducing the mutation into the mlc promoter of E. coli. In addition, mutation can be introduced into a specific site by site-specific mutagenesis or homologous recombination. As described above, E. coli having a mutation introduced at a specific site may be subjected to the above-described blue-white selection.

  Once a mutation in the mlc promoter that causes overexpression of the Mlc protein is identified, the mutation may be introduced into the mlc promoter of E. coli. The method for introducing the mutation of the mlc promoter in E. coli is not limited, but can be carried out by, for example, site-directed mutagenesis or homologous recombination.

  The present invention includes an E. coli mutant strain into which a gene mutation that excessively expresses Mlc protein is introduced and CCR is released. In addition, a strain in which wild-type Mlc protein is excessively expressed using an expression vector or the like is also included.

  By using E. coli from which CCR is released, a useful organic substance can be produced using a mixed sugar containing glucose and other sugars. Here, Escherichia coli strains used for production of organic substances after releasing CCR include all E. coli belonging to E. coli. For example, Escherichia coli K-12 strain, B strain, W strain and variants thereof can be mentioned. Examples of the mutant include MG1655 strain (ATCC (American Type Culture Collection) 47076), W3110 strain (ATCC27325) and the like. A mutation that overproduces the above Mlc protein may be introduced into these E. coli cells. Escherichia coli from which CCR is released can produce organic substances using mixed sugar as a raw material by the organic substance production system that it originally has. In addition, a transformed E. coli can be produced by introducing a gene involved in the synthesis of an organic substance into E. coli from which CCR has been released and used for the production of the organic substance. Furthermore, CCR of a transformed E. coli strain into which a gene involved in the synthesis of an organic substance has been introduced can be released and the E. coli can be used for the production of the organic substance. Examples of E. coli strains into which genes involved in the synthesis of organic substances have been introduced include KO11 strain (ATCC55124) that can produce ethanol.

  Moreover, when aiming at alcohol production, you may improve the alcohol tolerance of colon_bacillus | E._coli. For example, an alcohol-resistant strain can be obtained by culturing in a medium containing alcohol, or by inducing random mutation by ultraviolet irradiation or chemical mutagen treatment.

  Furthermore, a pathway for synthesizing an organic substance that is a metabolite from an intermediate of an organic substance to be produced by the method of the present invention and is not the target organic substance may be blocked. Blocking may be achieved by knocking out a gene encoding an enzyme related to the synthesis of an organic substance that attempts to block the synthetic pathway. Knockout can be performed, for example, by homologous recombination. For example, when alcohol is produced, the lactic acid synthesis pathway or succinic acid synthesis pathway may be blocked.

  A transformed Escherichia coli strain capable of producing an organic substance can be prepared by inserting a gene encoding an enzyme involved in the synthesis of the organic substance into an expression vector and transforming Escherichia coli using the expression vector. The expression vector to be used includes a promoter, a gene encoding the above enzyme, a terminator, a cis element such as an enhancer, if necessary, a splice donor site present at the 5 ′ end of the intron, and a splice present at the 3 ′ end of the intron. What contains a splicing signal consisting of a receptor site, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence) and the like may be operably linked. Examples of the selection marker include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like. To insert an enzyme-encoding gene into an expression vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, inserted into an appropriate vector DNA restriction enzyme site or multiple cloning site, and ligated to the expression vector. A method or the like may be used. In the case where genes encoding two kinds of enzymes are simultaneously inserted into one expression vector for co-expression, an expression vector containing a plurality of multiple cloning sites may be used.

  The method for introducing the recombinant vector into E. coli is not limited. For example, a method using calcium ions [Cohen, SNet al .: Proc. Natl. Acad. Sci., USA, 69: 2110 (1972)], electroporation For example. The vector to be used is not limited. For example, a known expression vector for Escherichia coli such as plasmid DNA or phage DNA can be used. Multiple genes may be inserted into separate vectors and introduced into E. coli, or multiple genes may be inserted into one vector and introduced into E. coli.

  Mixed sugars used as raw materials when producing organic substances using E. coli include monosaccharides such as arabinose, xylose, hexose, ribose, galactose, mannose, etc. in addition to glucose; lactose, malhatose And disaccharides such as sucrose; sugars containing at least one of various oligosaccharides. Monosaccharides remain as they are, disaccharides and oligosaccharides become monosaccharides in the body of E. coli, metabolized, and other organic substances are synthesized. Preferably, glucose and xylose and / or arabinose are used simultaneously. Biomass-derived sugar mixtures can also be used. The biomass-derived sugar mixture is sugar obtained from plants and animals, and includes various sugars including glucose. Since sugar derived from biomass can be obtained in large quantities at low cost, it can be suitably used as a raw material for producing an organic substance using E. coli having the CCR released of the present invention. Examples of plants and animals include corn, sugar cane, wheat and the like, which can be obtained from plant roots, stems, trunks, branches, leaves, flowers, seeds and the like. Also, woody biomass including wood chips and pulp can be used. Plant-derived biomass includes polysaccharides such as cellulose and hemicellulose, but these polysaccharides can be decomposed by alkali or acid, or enzymatically or microbially decomposed so that E. coli can be used for the production of organic substances. What is necessary is just to raise content of sugar.

  The type of organic substance produced by the method of the present invention is not limited, but it is not limited to organic substances originally produced by natural E. coli strains, but may be newly produced by genetic recombination techniques or fungal breeding. Also included are organic substances. Examples of organic substances include alcohols, organic acids, proteins, amino acids and the like. Examples of the alcohol include ethanol, 1-butanol, isobutanol (also referred to as isobutyl alcohol), 2-methyl-1-butanol, 3-methyl-1-butanol, propanol, isopropanol and the like. Examples of the organic acid include lactic acid, succinic acid, butyric acid, propionic acid, and acetic acid.

  When introducing a gene involved in the production of a desired organic substance into an Escherichia coli strain, the above-mentioned genes involved in metabolic pathways and synthesis in vivo such as alcohols and organic acids are known, and those skilled in the art can use appropriate foreign By introducing this gene into E. coli, an organic substance synthesis system can be introduced into E. coli. Moreover, the gene to be introduced is preferably codon optimized for expression in E. coli. For example, when ethanol is produced, genes encoding pyruvate dehydrogenase (PDC) and alcohol dehydrogenase (ADH) may be introduced into E. coli. When producing isobutanol, branched α-keto acid decarboxylase (KIVD), alcohol dehydrogenase (ADH2), acetolactate synthase (ALSS), ketolate reductoisomerase (ILVC), dihydroxy acid dehydratase ( A gene encoding ILVD) may be introduced. In this case, isobutanol can be efficiently produced via 2-keto acid.

  By culturing Escherichia coli from which CCR has been released in a medium containing the above mixed sugar, a plurality of types of sugars contained in the mixed sugar can be simultaneously metabolized to effectively produce useful substances.

  An organic substance can be produced by culturing E. coli obtained by the above-described release of CCR or E. coli introduced with an organic substance synthesis system after the release of CCR in a medium supplemented with mixed sugar.

  E. coli can be cultured according to a known method. For example, DMEM, MEM, RPMI1640, IMDM, or the like can be used as a culture solution, and a serum supplement such as fetal calf serum (FCS), other field supplement reagents, or the like may be added. Furthermore, the transformed E. coli can be immobilized to produce organic substances. The immobilization can be carried out by a comprehensive method, a crosslinking method, a carrier binding method, etc., and as the immobilization carrier used for immobilization, glass beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid, agar, There are gelatin and the like.

  When organic substances are generated and accumulated outside the Escherichia coli cells, after completion of the culture, precipitates such as the cells are removed from the culture, and the organic substances are removed from the culture by a known method alone or in combination. It can be isolated and purified. In addition, when organic substances are produced and accumulated in E. coli cells, after culturing, after collecting the cells from the culture, the cells are disrupted by an appropriate method such as a mechanical or chemical method, An organic substance can be isolated and purified from the crushed cells by known methods alone or in combination. When the organic substance is alcohol, it can be recovered, for example, by distillation.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1]
Experimental Method In the examples described below, the cultivation of E. coli, gene recombination, protein activity measurement, etc., were conducted according to the papers and patents of the present inventors (Nakashima and Tamura, Biotechnol. Bioeng. (2004) 86: 136- 148, Nakashima and Tamura, Appl. Environ. Microbiol. (2004) 70: 5557-5568, Nakashima et al., Nucleic Acids Res. (2006) 34: e138, Nakashima and Tamura, Nucleic Acids Res. (2009) 37: e103, Japanese Patent No. 3793812, Japanese Patent No. 3944577). Unless otherwise specified, E. coli was cultured at 37 ° C. using LB (1% Difco Bacto Tryptone, 0.5% Difco Yeast Extract, 1% sodium chloride). In the case of culturing on an agar medium, the agar concentration was 1.7%, and the one in a 9 cm diameter plastic dish was used. In addition, when culturing Escherichia coli containing a plasmid, the corresponding antibiotic was added. The final concentrations of antibiotics are ampicillin: 50 μg / mL, chloramphenicol: 24 μg / mL (stock solution made at a concentration of 34 mg / mL with 100% ethanol as solvent), kanamycin: 15 μg / mL, apramycin: 35 μg / mL. The composition of the medium other than LB is as follows. M9YM: 17 mg / mL Na ₂ HPO ₄ -12H ₂ O, 3 mg / mL KH ₂ PO ₄ , 0.5 mg / mL NaCl, 1 mg / mL NH ₄ Cl, 2 mM MgSO ₄ , 0.1 mM CaCl ₂ , 0.25% Difco Yeast Extract, 50 mM MOPS buffer (pH 7.0), M9Y: 17 mg / mL Na ₂ HPO ₄ -12H ₂ O, 3 mg / mL KH ₂ PO ₄ , 0.5 mg / mL NaCl, 1 mg / mL NH ₄ Cl , 2 mM MgSO ₄ , 0.1 mM CaCl ₂ , 0.5% Difco Yeast Extract. A list of strains used is shown in Table 1.

[Example 2]
Isolation of CCR-cancelled mutant strain Wild-type Escherichia coli, MG1655 strain was cultured overnight. This was diluted 5000 times with fresh LB, and 20 μL was applied to 50 LB agar media containing 10 g / L glucose, 8 g / L lactose, and 0.125 mg / mL x-gal. Immediately afterwards, ultraviolet rays of 10-12 mJ / cm ² were irradiated using XL-1000 UV crosslinker (Spectronics). When these were cultured overnight, a total of about 30,000 colonies appeared, of which only one colony was blue. This mutant strain was provisionally named gen strain (glucose effect not observed).

Next, the following experiment was performed as a secondary screening. Wild strains and gen strains are cultured in a 4 mL liquid medium containing 10 g / L glucose or 10 g / L glucose and 8 g / L lactose or 8 g / L lactose until the mid-logarithmic growth phase. Was collected by a centrifuge. The cells are suspended in 500 μL of PBS (0.14 M NaCl, 2.7 mM KCl, 10.1 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ ), and 0.1 g of glass beads with a diameter of 0.1 mm (manufactured by Yasui Kikai Co., Ltd.) are added thereto. Mixed. This was subjected to a bead shocker manufactured by Yasui Machinery Co., Ltd., and the cells were crushed, and then centrifuged at 20000 × g, 4 ° C. for 15 minutes to obtain a supernatant. This becomes a crude protein solution. When the activity of β-galactosidase was measured using the crude protein solution (Nakashima et al., Nucleic Acids Res. (2006) 34: e138), the wild type showed activity only in the lactose-containing medium. On the other hand, in the gen strain, almost the same activity was observed in the glucose-containing lactose medium and the lactose-containing medium. Even in the case of the gen strain, no activity was observed in the glucose-containing medium. From this result, it was suggested that the gen strain may be a mutant with CCR released as expected.

  Furthermore, in the gen strain, it was examined whether sugars other than lactose show similar CCR release. First, a pip gene (encoding proline iminopeptidase) serving as a reporter was excised from plasmid pHN171 (Nakashima and Tamura, Biotechnol. Bioeng. (2004) 86: 136-148) with NcoI and SpeI, and the pHN540u vector (Nakashima et al., (Nucleic Acids Res. (2006) 34: e138). This plasmid was named pHN1325. In pHN1325, the pip gene is linked downstream of the L-arabinose-inducible bad promoter (Pbad). Transcription from Pbad is induced by L-arabinose, but transcription hardly occurs when L-arabinose and glucose coexist. In other words, it means that CCR is working normally. pHN1325 was introduced into wild and gen strains respectively, and transformants containing 10 g / L glucose or 10 g / L glucose and 8 g / L L-arabinose or 8 g / L L-arabinose, respectively. Cultured in mL liquid medium. From the cultured cells, a crude protein solution was prepared in the same manner as for β-galactosidase, and the activity of PIP was measured. The measurement was performed as follows. First, a mixture of 2 μL of crude protein solution and 56 mL of PBS was held at 60 ° C. for 10 minutes, and then 2 mL of 10 mM H-Pro-AMC (manufactured by Bachem Holding AG) was added and held there for 10 minutes. . Of this, 50 μL was taken out, 25 μL of 10% sodium dodecyl sulfate solution was added thereto, and after mixing, it was placed in a black 96-well plate (Costar, product no. 3915). Excitation, 380 nm; emission, 460 nm; excitation and emission bandwidths, 12 nm; gain, 80; number of flashes, 10; Z-position 11019 mm; and integration time, 500μs Measured with the setting.

As a result, the wild strain showed activity only in the L-arabinose medium, while the gen strain had almost the same activity in the glucose-containing L-arabinose medium and the L-arabinose medium. It was. Even in the case of the gen strain, no activity was observed in the glucose-containing medium.
From the above, it was shown that the gen strain is a mutant in which CCR is released.

Example 3
Mutant gene cloning of gen strain
If the mutation occurring in the gen strain that cancels CCR is a dominant mutation, the following experiment should show the mutated gene. First, the following work was performed to create a gen strain genomic library. The gen strain was cultured overnight, and genomic DNA was purified using it (Nakashima et al., Nucleic Acids Res. (2006) 34: e138). Genomic DNA was partially digested with Sau3AI and subjected to agarose electrophoresis, and a fragment of about 10-6 kb was recovered from an agarose gel. It was purified and ligated into the BamHI site of vector pSPT18 (Roche Applied Science). The ligation product was transformed into E. coli XL1-blue to obtain a genomic library consisting of about 30000 colonies. Subsequently, in order to reduce false positive clones as much as possible, the following plasmid pHN1629 was prepared. Two oligonucleotide primers, sSN1481 (aaccatggtaagccgatacgtacccgata (SEQ ID NO: 1)) and sSN1482 (aaactagtctacccaatcagtacgttaatt (SEQ ID NO: 2)) were prepared, and PCR reaction was performed using the genome of the wild type MG1655 strain as a template. As a result, the ORF of a gene called mazF gene is amplified (0.3 kb), and it is known that when this gene is overexpressed, E. coli cannot grow. Both ends of this PCR fragment were cleaved with NcoI and SpeI and cloned into the same site of pHN540u vector. This plasmid was named pHN1629. In pHN1629, the mazF gene is linked downstream of Pbad. Therefore, even if pHN1629 is introduced into a wild strain and cultured in a medium containing both glucose and L-arabinose, the fungus grows, but the strain does not grow in the strain in which CCR is released. Actually, the gen strain was transformed with pHN1629, and when it was cultured on an agar medium containing only glucose, it was confirmed that it grew on an agar medium containing both glucose and L-arabinose.

  First, the wild type MG1655 strain was transformed with pHN1629, and then further double transformed with the above-described gen strain genomic library. When the double transformant was grown on an agar medium containing 10 g / L glucose, 8 g / L lactose, and 0.125 mg / mL x-gal, 9 out of about 40,000 colonies were blue. . Furthermore, 9 colonies were grown on an agar medium containing 10 g / L glucose and 2 g / L L-arabinose. As a result, the growth of 6 colonies was significantly inhibited as compared with the double transformant of control pSTP18 (empty vector) and pHN1629.

  For the gen strain genomic library of these 6 colonies, the plasmid was recovered and purified, and the DNA sequence was partially determined. As a result, 4 overlapping fragments (Fig. 1a) containing the mlc gene, 2 anmK, slyB, slyA, This was a plasmid having overlapping fragments (FIG. 1b) containing ydhI and ydhJ genes.

  First, both of the two plasmids were reintroduced into the wild-type MG1655 strain, cultured in a liquid medium in the same manner as in Example 2, and then the activity of β-galactosidase was measured. However, in any of the plasmids, no activity was observed when cultured in a glucose-containing lactose medium, and the activity was observed only in a lactose-containing medium. Furthermore, when all the DNA sequences of the genomic fragments in the plasmid were determined and compared with the published DNA sequence of MG1655 strain (GenBank accession number U00096.2), they were completely identical. Therefore, it was concluded that these were false positive clones or clones that caused incomplete CCR release due to overexpression.

  Next, the activity of β-galactosidase was also measured for the four former plasmids. Then, substantially the same activity was recognized at the time of culture | cultivation in a glucose-containing lactose culture medium and a lactose-containing culture medium. That is, it was suggested that there is a high possibility that the gen strain-derived genomic fragment contained in this plasmid contains a mutation. As a result of determining the entire DNA sequence of the shortest fragment among these fragments, it was found that there was a mutation at one base as compared with those derived from the wild type. The mutation site is shown in FIG. 1a. This mutation was present within the predicted site of the mlc gene, not within any open reading frame (ORF).

  In the E. coli promoter, there is a common sequence called -10 sequence, which is considered to be the most efficient promoter when it is tataat (de Boer et al., Proc. Natl. Acad. Sci. USA (1983) 80: 21-25). In the wild type mlc gene, this is caccat, which is much less homologous to the consensus sequence. However, the mutants obtained by the present inventor contain mutations that increase homology with taccat and common sequences. Therefore, it is predicted that the promoter is highly active in this mutant mlc gene. As a result, it is predicted that the Mlc protein is overexpressed.

  Therefore, the wild-type mlc promoter and the mutant-type mlc promoter were each separated by PCR, and a plasmid was prepared by cloning the pip gene downstream thereof. Subsequently, these plasmids were introduced into the MG1655 strain, cultured, and each Pip activity was measured. The mutant mlc promoter was 17 times higher. That is, as predicted from the sequence information, it was suggested that the mutant mlc promoter is highly active.

  In view of the mechanism, this mlc gene mutation is considered to be dominant. In fact, only false positive clones were isolated in experiments in which a wild-type genomic library was introduced into a gen strain, assuming that this mutation was recessive.

From the above results, it can be said that the mutation causing the cancellation of the CCR mechanism in the gen strain is a highly active promoter mutation of the mlc gene, and hereinafter the gen gene mutation will be referred to as the mlc ^* mutation.

When wild-type mlc and mutant-type mlc ^* genes were overexpressed in wild-type MG1655 strain using plasmid pHN1242 (Nakashima and Tamura, Nucleic Acids Res. (2009) 37: e103), CCR was canceled in both cases I found out. In this case, however, CCR was almost completely released by the mutant mlc ^* gene, whereas CCR was partially released by wild-type mlc. This indicates that even if the mlc gene or its promoter is not mutated, as long as the Mlc protein is overexpressed, CCR is at least partially released.

Example 4
Introduction of mlc ^* mutation into wild type strain In order to introduce the mlc ^* gene into the original MG1655 strain using homologous recombination, the following plasmid was prepared.

  Using pMW118 (manufactured by Nippon Gene) as a template, sSN1097 (taagctagctatggacagttttccctttgatatgta (SEQ ID NO: 5)) and sSN1097R (gatgatgaacatcagtagggaaaatgc (SEQ ID NO: 6)) were used as primers to amplify a part of the pSC101 ori sequence. Prior to this PCR, sSN1097R was phosphorylated at the 5 'end using T4 DNA kinase (New England Biolab). On the other hand, another portion of the pSC101 ori sequence was PCR amplified using pMW118 as a template, sSN1098 (aaactgcaggttgatgataccgctgccttactg (SEQ ID NO: 7)) and sSN1098R (tgaacgtattggttataagtgaacgatacc (SEQ ID NO: 8)) as primers. The former fragment was cleaved with NheI and the latter fragment with PstI, and both fragments were simultaneously cloned into the NheI and PstI sites of pHN540u. The resulting plasmid was named pHN1092. pHN1092 is a plasmid with pSC101 ori, but it is a temperature-sensitive plasmid due to a point mutation (GenBank accession number X01654, the 5439th base changed from cytosine to thymine), which exceeds 37 ° C. When cultured at temperature, it is not maintained intracellularly as a plasmid (Armstrong et al., J. Mol. Biol. (1984) 175: 331-347). Thus, at temperatures above 37 ° C, the plasmid either drops off or is inserted into the E. coli genome. Using this property and the natural homologous recombination ability of Escherichia coli, a specific site of the genome is manipulated (Hamilton et al., J. Bacteriol. (1989) 171: 4617-4622, Emmerson et al., Biol. Proced. Online (2006) 8: 153-162).

  The temperature sensitive pSC101 ori sequence and the chloramphenicol resistance gene were PCR amplified using pHN1092 as a template and sSN1098 (described above) and sSN1173 (ggggtacctgagcaaactggcctca (SEQ ID NO: 9)) as primers. Prior to this PCR, sSN1173 was phosphorylated at the 5 'end using T4 DNA kinase. On the other hand, sSN1172 (aactgcagttctagaaggcctggatccccatggccgttcactattatttagtgaaatgag (sequence number 10)) and sSN1108 (aaagctagcattttctctttttgcgtttttatttgcactt (sequence number 11) and PCR using sSN1108 (sequence number 11) and PCR as sg1) Prior to this PCR, sSN1108 was phosphorylated at the 5 'end using T4 DNA kinase. Both amplified fragments were ligated with T4 DNA ligase to obtain pHN1234.

  A kanamycin resistance gene (1.4 kb) excised from pHN267 (Nakashima and Tamura, Appl. Environ. Microbiol. (2004) 70: 5557-5568) with XbaI and SpeI was subcloned into the NheI site of pHN1234. This was named pHN1516. PCR amplification was performed using pHN1516 as a template, sSN1415 (tccatggggatcctcgcgaggccgttcactattatttagtgaaatgag (SEQ ID NO: 12)) and sSN1421 (tatgcattctagagatatcttagttgctctccaggtgacccaggtgacc (SEQ ID NO: 13)) as primers. Prior to this PCR, sSN1415 and sSN1421 were phosphorylated at the 5 'end using T4 DNA kinase. On the other hand, PCR amplification was performed using pHN1234 as a template and sSN1173 (described above) and sSN1098 (described above) as primers. Both amplified fragments were ligated with T4 DNA ligase to obtain pHN1534.

  PCR amplification was performed using pHN1234 as a template, sSN1417 (cccatggggatccaggccttctagaact (SEQ ID NO: 14)) and sSN1173 (described above) as primers. Prior to this PCR, sSN1417 was phosphorylated at the 5 'end using T4 DNA kinase. The amplified fragment was self-circulated with T4 DNA ligase to obtain pHN1532.

  The plasmid structures of pHN1234, pHN1534, and pHN1532 are shown in FIG. pHN1534 serves as a gene disruption plasmid and pHN1532 serves as a point mutation introduction plasmid (described later).

  PCR amplification was performed using E. coli MG1655 genomic DNA as a template and sSN1522 (atactgcagttcgatggttgaaacactctg (SEQ ID NO: 15)) and sSN1513 (gcctgcaggtgagggaaatttcagcgaaaa (SEQ ID NO: 16)) as primers. The amplified fragment was digested with PstI and ScaI and cloned into the NsiI and EcoRV sites of pHN1534. The resulting plasmid was named pHN1677. Next, PCR amplification was performed using E. coli MG1655 genomic DNA as a template, sSN1523 (agggatccaatacgtgtcgtcaaatttttacttagg (SEQ ID NO: 17)) and sSN1524 (ggccatgggattgctgcgtaacacagcgt (SEQ ID NO: 18)), and the ends were cleaved with BamHI and NcoI. It was cloned into the BamHI and NcoI sites of pHN1677. The resulting plasmid was named pHN1678. This is the mlc gene disruption plasmid.

PCR amplification was performed using the genomic DNA of the gen strain described in Example 2 as a template and sSN1522 and sSN1524 (both described above) as primers. The amplified fragment was cleaved with PstI and NcoI, cloned into the PstI and NcoI sites of pHN1532, and the resulting plasmid was named pHN1693. This becomes a plasmid for exchanging with mlc ^* after disrupting the mlc gene with pHN1678.

Hereinafter, the process of introducing mlc ^* into the wild type MG1655 strain will be described, which is based on the method of Emmerson et al. (Emmerson et al., Biol. Proced. Online (2006) 8: 153-162). First, MG1655 was transformed with pHN1678 and the mlc gene was disrupted by causing two homologous recombination at the mlc gene locus. The disrupted strain was then transformed with pHN1693 and replaced with mlc ^* by similarly causing two homologous recombination. Hereinafter, in order to avoid confusion, the strain created by this homologous recombination will be referred to as mlc ^* HR, and the gen strain isolated in Example 2 (the entity is mlc ^* mutant) will be referred to as mlc ^* UV. By DNA sequence analysis, it was confirmed that the same single base mutation as mlc ^* UV was contained in the mlc ^* HR strain genome as compared with the wild type MG1655 strain.

Example 5
Wild-type strain, mlc ^* UV strain, compared wild strain of mlc ^* HR strain, mlc ^* UV strain, using the mlc ^* HR strain, as in Example 2, blue-white colony determination experiment by LB agar medium containing x-gal Went. As expected, the wild strains formed blue colonies in the lactose-containing medium and white colonies in the glucose-containing lactose medium, whereas the mlc ^* UV strain and mlc ^* HR were both on the lactose-containing and glucose-containing lactose media. A blue colony formed. All strains formed white colonies on the glucose-containing medium.

Furthermore, in order to analyze CCR for sugars other than lactose, wild strains, mlc ^* UV strains, and mlc ^* HR strains were cultured in a medium in which 8 g / L each of glucose and xylose was added to M9YM. The amount of glucose and xylose remaining in these media was measured over time using high performance liquid chromatography (D-2000 LaChrom Elite, manufactured by Hitachi). The analysis conditions are as follows. Separation column: aminex HPX-74H (Bio-Rad), guard column: Cation H micro-guard cartridge (Bio-Rad), separation solvent: 4 mM H ₂ SO ₄ , flow rate: 0.5 mL / min, detection Apparatus: UV detector (detected at 210 nm by L-2400, manufactured by Hitachi) and differential refractive index detector (L-2490, manufactured by Hitachi), column temperature: 45 ° C. As a result (FIG. 3), the wild strain did not consume xylose at all under this culture condition, whereas the mlc ^* UV strain and mlc ^* HR strain consumed glucose and xylose at the same rate.

From the above results, it was concluded that CCR was canceled in the mlc ^* UV strain only due to the mlc ^* mutation, and mutations in other genes were not related to CCR, if any. The From then on, only mlc ^* HR was used in the experiment without worrying about unexpected mutations.

Example 6
Expression plasmid construction for isobutanol production E. coli naturally produces little isobutanol. Therefore, foreign genes must be introduced and related metabolic pathways must be strengthened. A plasmid required for this purpose was prepared as follows.

PCR amplification using pTrc99a (manufactured by Amersham Biosciences) as a template, sSN1283 (gctatgcatcgactgcacggtgcaccaat (SEQ ID NO: 19)) and sSN1284 (aaactagtctcgagaagcttgcatgcctgcaggtcgact (SEQ ID NO: 20)) as an IPTG-inducible trc lac ^O), to obtain a fragment containing the multiple cloning site sequence. Both ends of the fragment were treated with NsiI and SpeI and cloned into the NsiI and SpeI sites of pHN1257 (NAR). This plasmid was named pHN1344. On the other hand, PCR amplification was performed using pTrc99a as a template and sSN1283 (gctatgcatcgactgcacggtgcaccaat (SEQ ID NO: 19)) and sSN1345 (aatctagaggggaattgttatccgctcacaattccacac (SEQ ID NO: 22)) to obtain a fragment containing Ptrc and lac ^O sequences. The fragment was treated with XbaI and cloned into the XbaI and EcoRV sites of pET28a (Novagen). This plasmid was named pHN1420. From PHN1420, excised sequence comprising the Ptrc and lac ^O by NsiI and NcoI, NsiI of PHN1344, was subcloned into the NcoI site. The resulting plasmid was named pHN1431. This is the original vector for expressing the foreign gene.

We requested Genscript to synthesize Lactococcus lactis-derived kivd gene (GenBank accession number, AJ746364) artificially. This artificial kivd gene has a codon optimized for expression in E. coli, and has been cloned into the pUC57 vector (GenBank accession number, Y14837), and its sequence is shown in the sequence listing. On the other hand, the Adh2 gene of the budding yeast Saccharomyces cerevisiae, the genome of S. cerevisiae W303 strain (Takeuchi and Tamura, FEBS Lett. (2004) 565: 1-3: 39-42) as a template, sSN1287 (aaagcatgcaggagatataccatgtctattccagaaactcaaaaagc (SEQ ID NO: 24) ) And sSN1288 (gcctcgagttatttagaagtgtcaacaacgtat (SEQ ID NO: 25)) were used as primers for PCR amplification. The NcoI-SphI fragment (1.6 kb) of the kivd gene and the SphI-XhoI fragment (1.0 kb) of the adh2 gene were simultaneously cloned into the NcoI and XhoI sites of pHN1431. The resulting plasmid was named pHN1435. pHN1435 has lac ^O downstream of Ptrc, and further has kivd gene and adh2 gene as operons downstream thereof.

PCR amplification of a sequence containing Ptrc was performed using pTrc99a as a template and sSN1283 (gctatgcatcgactgcacggtgcaccaat (SEQ ID NO: 21)) and sSN1004 (cgaattccatggtctgtttcctgtg (SEQ ID NO: 27)) as primers. The amplified fragment was digested with NsiI and NcoI and cloned into the NsiI and NcoI sites of pHN1270 (Nakashima and Tamura, Nucleic Acids Res. (2009) 37: e103). The resulting plasmid was named pHN1375. PCR amplification of the alsS gene was carried out using the genomic DNA of Bacillus subtilis 168 strain (Table 1) as a template and sSN1343R (aaccatggtgacaaaagcaacaaaagaacaaaaatcccttgtga (SEQ ID NO: 28)) and sSN1344 (aaaggatcctagagagctttcgttttcatgagt (SEQ ID NO: 29)) as primers. The amplified fragment was digested with NcoI and BamHI and cloned into the NcoI and BamHI sites of pHN1375. The resulting plasmid was named pHN1447. PCR amplification was performed using the genomic DNA of MG1655 strain as a template and sSN1327 (aaggatccgaggaatcaccatggctaactacttca (SEQ ID NO: 30)) and sSN1328 (gcctcgagttaacccgcaacagcaatacgtttcat (SEQ ID NO: 31)) to obtain a fragment containing the ilvC gene. Both ends of the fragment were treated with BamHI and XhoI. On the other hand, PCR amplification was performed using MG1655 strain genomic DNA as a template, sSN1329 (aaagtcgacaggagatataccatgcctaagtaccgttccgccacca (SEQ ID NO: 32)) and sSN1330 (agactagtttaaccccccagtttcgatttatcgcg (SEQ ID NO: 33)) to obtain a fragment containing the ilvD gene. The fragment was treated with SalI and SpeI. These ilvC gene fragment and ilvD gene fragment were simultaneously cloned into the BamHI and SpeI sites of pBluscript SKII + (Stratagene). This plasmid was named pHN1406. A sequence containing the ilvC gene and the ilvD gene was excised from pHN1406 with BamHI and SpeI and subcloned into the same site of pHN1447. This plasmid was named pHN1451. pHN1451 has lac ^O downstream of Ptrc, and further has alsS, ilvC, and ilvD genes as operons downstream thereof.

Example 7
Demonstration of whether mlc ^* mutation increases isobutanol production Next, it was verified how much isobutanol could be produced by mlc ^* mutation. At the same time, the DptsG strain and the crp ^* strain prepared from the wild type MG1655 strain were also used for comparison. In addition, in order to increase the productivity of isobutanol, the strains used here were ldhA (encoding lactate dehydrogenase), adhE (fused acetaldehyde-CoA dehydrogenase / iron-dependent alcohol dehydrogenase / pyruvate- The formate-lyase inactivating enzyme and pflB (encoding pyruvate-formate-lyase) genes were disrupted. These strains were prepared in the same manner as in Example 4. The genotypes of strains (LAF, LAFmlc ^* , LAFDptsG, LAFcrp ^* ) used for isobutanol production are shown in Table 1.

Four strains of LAF, LAFmlc ^* , LAFDptsG, and LAFcrp ^* were co-transformed with pHN1435 and pHN1451, and precultured first. The main culture was started by adding one-hundredth amount of these to a medium in which 40 g / L glucose, or 20 g / L glucose and 20 g / L xylose, or 40 g / L xylose was added to M9Y. The main culture was performed at 4 ° C. in a 4 mL culture solution at 30 ° C. using a 50 mL Erlenmeyer flask with a silicon stopper. In order to express genes from pHN1435 and pHN1451, IPTG was added to 1 mM when the absorbance at 600 nm reached 0.8.

The culture solution was collected at regular time intervals, the cells were removed by centrifugation, and the supernatant was analyzed by high performance liquid chromatography. The analysis was the residual glucose concentration, the residual xylose concentration, and the produced isobutanol concentration, and the analysis conditions are the same as in Example 5. As a result (FIG. 4), when 20 g / L glucose and 20 g / L xylose were used as carbon sources, the isobutanol productivity of both LAFmlc ^* and LAFDptsG strains was significantly higher than that of MG1655. In addition, when 40 g / L glucose or 40 g / L xylose was used as the carbon source, the productivity of LAFmlc ^* strain was the highest. From the above, it was shown that the LAFmlc ^* strain is effective for isobutanol production by E. coli.

Further, in order to enhance the isobutanol productivity, it was created LAFCmlc ^* strains by further destroying ackA-pta gene in LAFmlc ^* strains. Furthermore, the culture conditions for the main culture were also optimized. The conditions are as follows.In a 250 mL Erlenmeyer flask with a screw cap, 20 mL of M9Y supplemented with 40 g / L glucose or 20 g / L glucose and 20 g / L xylose is added, and pre-culture is 100 One minute is added and cultured at 30 ° C. When cultured with 40 g / L of glucose, 40 g / L of glucose was added when glucose was depleted, and when cultured with 20 g / L of glucose and 20 g / L of xylose, At the time of depletion, 20 g / L glucose and 20 g / L xylose were added. The addition of IPTG is the same as above. The result of producing isobutanol is shown in FIG. 80 g / L glucose produced 19 g / L and 40 g / L glucose and 40 g / L xylose produced 11 g / L isobutanol.

SEQ ID NO: 1-22, 24-33 Primer SEQ ID NO: 23 Synthesis

Claims (15)

  An Escherichia coli strain in which carbon catabolite repression (CCR) is released by overexpression of Mlc protein by gene mutation introduction or overexpression using an expression vector.   2. The Escherichia coli strain released from carbon catabolite repression (CCR) according to claim 1, wherein the gene mutation that overexpresses the Mlc protein is a mutation in the mlc promoter.   The Escherichia coli strain released from carbon catabolite repression (CCR) according to claim 1 or 2, wherein the mutation in the mlc promoter is substitution of the 31st c to t in the base sequence of the mlc promoter shown in SEQ ID NO: 34.   An E. coli strain in which carbon catabolite repression (CCR) has been released by overexpression of one, two, three, four or five genes of anmK, slyB, slyA, ydhI or ydhJ.   Furthermore, the colon_bacillus | E._coli strain | stump | stock of any one of Claims 1-4 into which the gene involved in the synthesis | combination of an organic substance is introduce | transduced.   The Escherichia coli strain according to claim 5, wherein an exogenous kivd gene, Adh2 gene, alsS gene, ilvC gene and ilvD gene have been introduced.   A method for releasing carbon catabolite repression (CCR) of an E. coli strain, comprising introducing a gene mutation that overexpresses the Mlc protein into an E. coli strain, or overexpressing the Mlc protein using an expression vector.   The method according to claim 7, wherein the gene mutation that overexpresses the Mlc protein is a mutation in the mlc promoter.   The method according to claim 7 or 8, wherein the mutation in the mlc promoter is a substitution of the 31st c in the base sequence of the mlc promoter shown in SEQ ID NO: 34 to t.   A method for releasing carbon catabolite repression (CCR) of an E. coli strain, comprising overexpressing one, two, three, four or five genes of anmK, slyB, slyA, ydhI or ydhJ.   The Escherichia coli strain from which carbon catabolite suppression (CCR) according to any one of claims 1 to 4 is released is cultured in the presence of a mixed sugar containing glucose and sugars other than glucose, and other than glucose and glucose. A method of producing organic substances from other sugars.   An Escherichia coli strain from which carbon catabolite repression (CCR) according to any one of claims 1 to 4 has been released, further introduced with a gene involved in the synthesis of an organic substance other than glucose and glucose. A method of culturing in the presence of a mixed sugar containing other sugars and producing the organic substance from glucose and other sugars other than glucose.   The method of claim 12, wherein the organic material is an alcohol or an organic acid.   The method according to claim 12 or 13, wherein the organic substance is isobutyl alcohol and the mixed sugar comprises glucose and xylose.   The method according to claim 14, wherein an exogenous kivd gene, Adh2 gene, alsS gene, ilvC gene, and ilvD gene are introduced into the E. coli strain.

Images