Genetically modified microorganism capable of producing isoprene and method for producing isoprene using the same

Abstract

To provide a technique for producing isoprene from biomass being a renewable resource.SOLUTION: A genetically modified microorganism having isoprene producing ability has a methyl erythritol phosphoric acid (MEP) pathway and a mevalonic acid (MVA) pathway, and has phospho glucose isomerase (Pgi) activity which is reduced compared to an unmodified body, isoprene synthase (IspS) activity enhanced compared to an unmodified body, and 1-hydroxy-2- methyl-2-butenyl 4-diphosphoric acid reductase (IspH) activity and / or 1-hydroxy-2-methyl-2-butenyl 4-diphosphoric acid synthase (IspG) activity which are enhanced compared to an unmodified body.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a genetically modified microorganism capable of producing isoprene and a method for producing isoprene using the same, and more particularly to a genetically modified microorganism for producing isoprene from biomass, which is a renewable resource. [Background technology]

[0002] Polyisoprene rubber (IR) is attracting attention as a substitute for natural rubber, an important raw material for tires. Isoprene monomer (hereinafter simply referred to as "isoprene"), the raw material for polyisoprene rubber (IR), is extracted from the C5 fraction obtained by naphtha cracking. However, from the perspectives of resource conservation and environmental protection, there is a need for technology to produce isoprene from non-petroleum resources, especially from biomass, a renewable resource.

[0003] In relation to the present invention, for example, Non-Patent Document 1 proposes a method for producing isoprene using Escherichia coli in which the 1-deoxy-D-xylulose 5-phosphate synthase (Dxs) gene, the isopentyl diphosphate isomerase (Idi) gene, and the isoprene synthase (IspS) gene are overexpressed. Non-Patent Document 1 suggests that in this production method, the reaction catalyzed by 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase (IspG) in the methylerythritol phosphate (MEP) pathway is rate-limiting for isoprene production. However, it has been reported that simply overexpressing the IspG gene and the 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase (IspH) gene, which is located downstream of IspG in the MEP pathway, does not relieve the rate-limiting effect of the IspG reaction, and therefore does not increase the amount of isoprene produced. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] "Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis", Microbial Cell Factories, 2019, 18:192 Summary of the Invention [Problem to be solved by the invention]

[0005] A primary object of the present invention is to provide a technology for producing isoprene from biomass, which is a renewable resource. [Means for solving the problem]

[0006] In order to solve the above problems, the present invention provides the following [1]-

[13] . [1] It has the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway, Reduced phosphoglucose isomerase (Pgi) activity compared to the unmodified form; Enhanced isoprene synthase (IspS) activity compared to the unmodified strain; and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase (IspH) activity and / or 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase (IspG) activity that are enhanced compared to unmodified variants. A genetically modified microorganism capable of producing isoprene. [2] A genetically modified microorganism according to [1], in which at least one enzyme activity selected from the group consisting of 1-deoxy-D-xylulose 5-phosphate synthase (Dxs) activity, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) activity, and isopentyl diphosphate isomerase (Idi) activity is enhanced compared to an unmodified microorganism. [3] A genetically modified microorganism according to [1] or [2], in which citrate synthase (GltA) activity is reduced compared to an unmodified strain. [4] A genetically modified microorganism according to any one of [1] to [3], in which at least one enzyme activity selected from the group consisting of 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD) activity, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) activity, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) activity is enhanced compared to an unmodified strain. [5] A genetically modified microorganism according to any one of [1] to [4], in which at least one enzyme activity selected from the group consisting of acetyl-CoA acetyltransferase (MvaE) activity, 3-hydroxy-3-methylglutaryl-CoA synthase (MvaS) activity, 3-hydroxy-3-methylglutaryl-CoA reductase (MvaE) activity, mevalonate kinase (MVK) activity, phosphomevalonate kinase (PMK) activity, and diphosphomevalonate decarboxylase (MVD) activity is enhanced compared to an unmodified strain. [6] A genetically modified microorganism according to any one of [1] to [5], which is at least one selected from the group consisting of Escherichia coli, Corynebacterium, Bacillus, Pantoea, Enterobacter, and Pseudomonas.

[0007] [7] A method for producing isoprene, comprising a step of contacting a genetically modified microorganism capable of producing isoprene with an organic raw material in an aqueous medium, The genetically modified microorganism It has a methylerythritol phosphate (MEP) pathway and a mevalonate (MVA) pathway, Reduced phosphoglucose isomerase (Pgi) activity compared to the unmodified form; Enhanced isoprene synthase (IspS) activity compared to the unmodified strain; and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase (IspH) activity and / or 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase (IspG) activity that are enhanced compared to unmodified variants. Manufacturing method. [8] The method according to [7], wherein the organic raw material comprises xylose, arabinose, mannose, galactose, fructose, glucose, lactose, maltose, trehanose, cellobiose, sucrose, starch, dextrin, cellulose, hemicellulose, glycerol, and / or mannitol, and preferably comprises glucose and / or sucrose. [9] A method for producing [7] or [8], wherein the genetically modified microorganism has enhanced activity of at least one enzyme selected from the group consisting of 1-deoxy-D-xylulose 5-phosphate synthase (Dxs) activity, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) activity, and isopentyl diphosphate isomerase (Idi) activity compared to an unmodified microorganism.

[10] The method of any one of [7] to [9], wherein the genetically modified microorganism has reduced citrate synthase (GltA) activity compared to a non-modified strain.

[11] A method for producing any of [7] to

[10] , wherein the genetically modified microorganism has enhanced activity of at least one enzyme selected from the group consisting of 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD) activity, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) activity, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) activity compared to an unmodified strain.

[12] A production method according to any of [7] to

[11] , in which the genetically modified microorganism has enhanced activity of at least one enzyme selected from the group consisting of acetyl-CoA acetyltransferase (MvaE) activity, 3-hydroxy-3-methylglutaryl-CoA synthase (MvaS) activity, 3-hydroxy-3-methylglutaryl-CoA reductase (MvaE) activity, mevalonate kinase (MVK) activity, phosphomevalonate kinase (PMK) activity, and diphosphomevalonate decarboxylase (MVD) activity, compared to an unmodified strain.

[13] The manufacturing method of any of [7] to

[12] , wherein the genetically modified microorganism is at least one selected from the group consisting of Escherichia coli, Coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, and Pseudomonas bacteria. [Effects of the Invention]

[0008] The present invention provides a technology for producing isoprene from biomass, a renewable resource. DETAILED DESCRIPTION OF THE INVENTION

[0009] Preferred embodiments for carrying out the present invention will be described below. Note that the embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention should not be construed as being narrow.

[0010] [Genetically modified microorganisms] The genetically modified microorganism according to the present disclosure has the following characteristics: (1) It has the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway. (2) It has reduced phosphoglucose isomerase (Pgi) activity compared to the unmodified variant. (3) It has enhanced isoprene synthase (IspS) activity compared to the unmodified strain. (4) It has enhanced 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase (IspH) activity and / or 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase (IspG) activity compared to an unmodified strain. (5) It has the ability to produce isoprene.

[0011] The genetically modified microorganism according to the present disclosure may be Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, and the like. Furthermore, the genetically modified microorganism according to the present disclosure may be a yeast of the genus Saccharomyces, Candida, Shizosaccharomyces, or Pichia, or a filamentous fungus of the genus Aspergillus. The genetically modified microorganisms disclosed herein are not limited as long as they have the characteristics (1) to (5) above, but Escherichia coli is particularly preferred because it is simple to use and there are a wide variety of genetic modification tools available.

[0012] Genetically modified microorganisms can be produced using conventionally known genetic engineering techniques. Genetically modified microorganisms that have reduced activity of a target enzyme compared to unmodified microorganisms can be produced by, for example, disrupting the nucleic acid encoding the enzyme on the chromosome of the microorganism, replacing the nucleic acid encoding the enzyme with a weaker promoter, modifying the promoter of the nucleic acid encoding the enzyme or expression regulatory sequences such as the Shine-Dalgarno (SD) sequence to reduce expression levels, suppressing translation by expressing antisense RNA to inhibit ribosome binding to the target mRNA, suppressing transcription by CRISPR interference, or reducing the activity per enzyme molecule by introducing a mutation into the nucleic acid encoding the enzyme. Genetically modified microorganisms that have enhanced activity of a target enzyme compared to unmodified microorganisms can be produced by, for example, introducing a nucleic acid encoding the enzyme into a vector and transforming the microorganism with the vector system, introducing an expression cassette containing the nucleic acid encoding the enzyme into the chromosome of the microorganism, replacing the nucleic acid encoding the enzyme with a stronger promoter, modifying the promoter of the nucleic acid encoding the enzyme or an expression regulatory sequence such as a Shine-Dalgarno (SD) sequence to increase the expression level, or introducing a mutation into the nucleic acid encoding the enzyme to increase the activity per molecule of the enzyme.

[0013] [Methylerythritol phosphate (MEP) pathway / mevalonate (MVA) pathway] The MEP pathway is a biosynthetic pathway for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The MEP pathway involves the following genes in order from upstream to downstream: 1-Deoxy-D-xylulose 5-phosphate synthase (Dxs) 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) 4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase (IspG) 1-hydroxy-2-methyl-2-butenyl 4-bisphosphate reductase (IspH) Isopentyl diphosphate isomerase (Idi)

[0014] The MVA pathway is a biosynthetic pathway for isopentenyl diphosphate (IPP). The MVA pathway involves the following genes in order from upstream to downstream: Acetyl-CoA acetyltransferase (MvaE) 3-hydroxy-3-methylglutaryl-CoA synthase (MvaS) 3-hydroxy-3-methylglutaryl-CoA reductase (MvaE) Mevalonate kinase (MVK) Phosphomevalonate kinase (PMK) activity Diphosphomevalonate decarboxylase (MVD) activity MvaE has both acetyl-CoA acetyltransferase activity and 3-hydroxy-3-methylglutaryl-CoA reductase activity. Hereinafter, the term "MvaE activity" refers to both enzyme activities.

[0015] In the genetically modified microorganisms disclosed herein, the MEP pathway and the MVA pathway may be naturally present before genetic modification, or may be newly introduced by genetic modification. Although there are exceptions, the MEP pathway is present in many prokaryotes and plants (chloroplasts), and the MVA pathway is present in many eukaryotes and plants (cytoplasm).

[0016] [Pgi gene] The genetically modified microorganisms of the present disclosure have reduced Pgi activity compared to unmodified microorganisms. Pgi is an enzyme that converts glucose-6-phosphate to fructose-6-phosphate. In the present invention, an "unmodified organism" refers to a naturally occurring microorganism, but may also be a microorganism having other genetic modifications as long as it has not been subjected to the genetic modifications disclosed herein. "Activity reduced compared to the unmodified form" means that the amount of product per unit time of a reaction catalyzed by a genetically modified microorganism or an enzyme expressed by the genetically modified microorganism is less than that of the unmodified form or the enzyme expressed by the genetically modified microorganism, for example, 10% or more, 20% or more, preferably 30% or more, 40% or more, more preferably 50% or more, 60% or more, even more preferably 70% or more, 80% or more, most preferably 90% or more, 95% or more, and particularly preferably 99% or more, 99.5% or more. "Activity reduced compared to the unmodified form" also includes cases where enzyme activity is zero due to gene deletion. Enzyme activity can be measured by using the target enzyme and a reaction substrate for the reaction catalyzed by the enzyme, conducting the reaction under conditions that allow the reaction to proceed, and measuring the amount of reaction product produced from the reaction substrate.

[0017] [MEP pathway genes] The genetically modified microorganisms of the present disclosure have enhanced IspS activity, IspH activity, or IspG activity in the MEP pathway compared to unmodified microorganisms. More preferably, the genetically modified microorganisms of the present disclosure have enhanced IspS activity, IspH activity, and IspG activity compared to unmodified microorganisms. "Enhanced activity compared to an unmodified organism" means that the amount of product per unit time of a reaction catalyzed by a unit amount of a genetically modified microorganism or an enzyme expressed by it is greater than that of an unmodified organism or an enzyme expressed by it, for example, 50% or more, 60% or more, preferably 70% or more, 80% or more, more preferably 80% or more, 90% or more, even more preferably 100% or more, 150% or more, most preferably 200% or more, 300% or more, and particularly preferably 400% or more, 500% or more.

[0018] Furthermore, the genetically modified microorganisms according to the present disclosure preferably further have enhanced Dxs activity, Dxr activity, and / or Idi activity in the MEP pathway compared to unmodified microorganisms, and more preferably have enhanced Dxs activity, Dxr activity, and Idi activity compared to unmodified microorganisms.

[0019] The genetically modified microorganism according to the present disclosure can further have enhanced IspD activity, IspE activity, and / or IspF activity in the MEP pathway, more preferably compared to a non-modified microorganism.

[0020] [MVA pathway genes] The genetically modified microorganisms of the present disclosure have enhanced MvaE activity, MvaS activity, MVK activity, PMK activity, and / or MVD activity in the MVA pathway compared to unmodified microorganisms. More preferably, the genetically modified microorganisms of the present disclosure have enhanced MvaE activity, MvaS activity, MVK activity, PMK activity, and MVD activity compared to unmodified microorganisms.

[0021] [GltA gene] The genetically modified microorganisms of the present disclosure preferably have reduced GltA activity compared to unmodified microorganisms. GltA is an enzyme that synthesizes citrate from acetyl-CoA and oxaloacetate.

[0022] For the above-mentioned MEP pathway genes and MVA pathway genes, depending on the type of microorganism, genes derived from the microorganism may be used, or genes derived from a different species may be used. For example, the IspS gene can be a gene derived from Mucuna bracteate (the nucleotide sequence is shown in positions 577-2232 of SEQ ID NO: 14). The IspH gene can be a gene derived from Escherichia coli (the nucleotide sequence is shown in positions 4-954 of SEQ ID NO: 18). The IspG gene can be a gene derived from Escherichia coli (the nucleotide sequence is shown in positions 4-1122 of SEQ ID NO: 17). The Dxs gene can be a gene derived from Rhizobium radiobacter (the nucleotide sequence is shown at positions 3-1922 of SEQ ID NO: 13). The Dxr gene can be a gene derived from Escherichia coli (the nucleotide sequence is shown in positions 1947-3143 of SEQ ID NO: 13). The Idi gene can be a gene derived from Escherichia coli (the nucleotide sequence is shown in positions 4-552 of SEQ ID NO: 14). The MvaE gene can be a gene derived from Enterococcus gallinarum (the nucleotide sequence is shown in positions 3-2450 of SEQ ID NO: 15). The MvaS gene can be a gene derived from Enterococcus gallinarum (the nucleotide sequence is shown at positions 2474-3628 of SEQ ID NO: 15). The MVK gene can be a gene derived from Methanococcoides burtonii (the nucleotide sequence is shown in positions 4-915 of SEQ ID NO: 16). The PMK gene can be a gene derived from Saccharomyces cerevisiae (the nucleotide sequence is shown in positions 939-2294 of SEQ ID NO: 16). The MVD gene can be a gene derived from Saccharomyces cerevisiae (the nucleotide sequence is shown at positions 2316-3506 of SEQ ID NO: 16). The enzyme encoded by the MvaE gene has both acetyl-CoA acetyltransferase activity and 3-hydroxy-3-methylglutaryl-CoA reductase activity, but instead of the MvaE gene, genes encoding enzymes that have each activity alone may be used separately. A partial sequence of a gene derived from Escherichia coli adjacent to a PAM sequence (the base sequence is shown in positions 6315-6334 of SEQ ID NO: 20) can be used as a guide RNA to suppress transcription of the GltA gene in E. coli using CRISPR interference.

[0023] The base sequence of each gene is not limited to the above base sequences, and may be a base sequence in which several or several bases have been deleted, inserted, substituted, and / or added to the above base sequences, as long as the enzyme consisting of the amino acid sequence encoded by the base sequence maintains its activity.

[0024] [Genes involved in electron transfer in the IspG and IspH reactions] The genetically modified microorganisms according to the present disclosure are preferably modified so that the expression levels of ferredoxin and / or flavodoxin are increased compared to non-modified strains. IspG and IspH in the MEP pathway are enzymes containing iron-sulfur clusters, and their reduction reactions proceed via electron transfer via ferredoxin or flavodoxin, respectively. Therefore, this modification can efficiently enhance IspG and / or IspH activity, which is expected to lead to improved isoprene production.

[0025] In addition, the genetically modified microorganism according to the present disclosure may further contain ferredoxin / flavodoxin-NADP + It is preferable that the strain is modified so that the reductase activity is enhanced compared to that of an unmodified strain. Ferredoxin / Flavodoxin-NADP + The reductase can convert ferredoxin and / or flavodoxin, which have been converted from reduced to oxidized forms as the enzymatic reaction of IspG and / or IspH proceeds, back to the reduced form. This modification can efficiently enhance IspG activity and / or IspH activity, and is expected to improve isoprene production.

[0026] [Isoprene manufacturing method] The genetically modified microorganisms disclosed herein have significant isoprene production ability and can therefore be used to produce isoprene by contacting them with an organic raw material in an aqueous medium.

[0027] In the method for producing isoprene according to the present disclosure, the organic feedstock can be biomass, a renewable resource, and may include, but is not limited to, xylose, arabinose, mannose, galactose, fructose, glucose, lactose, maltose, trehanose, cellobiose, sucrose, starch, dextrin, cellulose, hemicellulose, glycerol, mannitol, etc. Of these, glucose and / or sucrose are preferred.

[0028] The culture medium of the genetically modified microorganism may be used as is, or bacterial cells obtained from the culture medium by a cell collection procedure such as centrifugation, or a processed product thereof, etc. Examples of processed bacterial cells include immobilized bacterial cells obtained by immobilizing microbial cells with acrylamide, carrageenan, etc., disrupted bacterial cells, the supernatant obtained by centrifugation thereof, or a cell-free extract obtained by partially purifying the supernatant, as well as crude enzymes or purified enzymes obtained by extracting enzymes from these.

[0029] The step of contacting the genetically modified microorganism with the organic raw material may be carried out in an appropriate aqueous medium. The aqueous medium may be, for example, a culture medium for culturing the microorganism or a buffer solution such as a phosphate buffer. The aqueous medium is preferably an aqueous solution containing a nitrogen source, an inorganic salt, and the like. Examples of the nitrogen source include various organic and inorganic nitrogen compounds such as ammonium salts, nitrates, urea, soybean hydrolysates, casein hydrolysates, peptone, yeast extract, meat extract, and corn steep liquor. Examples of inorganic salts include various phosphates, sulfates, and metal salts such as magnesium, potassium, manganese, iron, and zinc. Furthermore, factors that promote microbial growth, such as vitamins such as biotin, thiamine, pantothenic acid, inositol, and nicotinic acid, nucleotides, and amino acids, may be added as needed. Furthermore, to suppress foaming during the reaction, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the reaction solution.

[0030] The process time, temperature, pH, and amounts of genetically modified microorganisms and organic raw materials added are not particularly limited and can be adjusted as appropriate. The temperature is preferably adjusted to a range that allows the activity of the microorganism to be most effectively exhibited depending on the type of microorganism used. Specifically, when E. coli is used, the temperature is usually in the range of 20-45°C, preferably 25-37°C. When yeast is used, the temperature is usually in the range of 20-45°C, preferably 27-40°C. The time is usually 1 to 168 hours, preferably 3 to 120 hours, and more preferably 6 to 72 hours. The pH condition is also preferably adjusted depending on the type of microorganism used so that its activity is most effectively exhibited. Specifically, when Escherichia coli is used, the pH is usually in the range of 4.5 to 9.0, preferably 5.5 to 8.0. When yeast is used, the pH is usually in the range of 2.5 to 8.5, preferably 3.5 to 7.5. The amount of genetically modified microorganisms to be added is usually 1 to 700 g / L, preferably 10 to 500 g / L, and more preferably 20 to 400 g / L in terms of wet cell weight. The concentration of the organic raw material used is typically 0.1-10% (W / V), preferably 0.2-5% (W / V), of the carbohydrates contained therein relative to the aqueous medium. Additional organic raw material may be added as the amount of organic raw material decreases with the progress of isoprene production. [Example]

[0031] [Example 1] The E. coli HMS174(DE3) strain was subjected to disruption of the pgi gene and enhanced expression of the dxs gene, dxr gene, ispG gene, idi gene, ispS gene, mvaE gene, mvaS gene, MVK gene, PMK gene, and MVD gene (see Table 1).

[0032] (A) Construction of lambda red expression plasmid To obtain a DNA fragment containing the sequence required for autonomous replication of the plasmid and the ampicillin resistance marker sequence, PCR was performed using the sSN1012 and sSN1013 primers (SEQ ID NO: 1 and SEQ ID NO: 2) and the pHN1009 plasmid (Nakashima et al., Nucleic Acids Res. (2006) 34: e138) as a template. One end of the resulting DNA fragment was treated with the restriction enzyme NsiI and purified. To obtain the sequence of the doxycycline-inducible promoter and the sequence of the expression control protein of that promoter, a PCR reaction was performed using the sSN1175 and sSN1011 primers (SEQ ID NO: 3 and SEQ ID NO: 4) and the pHN1271 plasmid (JP 2014-209872) as a template. Prior to PCR, the 5' end of sSN1011 was phosphorylated with T4 DNA kinase. One end of the resulting DNA fragment was treated with the restriction enzyme PstI and purified. These DNA fragments were ligated with T4 DNA ligase to obtain the plasmid pHN1340, which contains an autonomous replication origin derived from pBR322, an ampicillin resistance gene, a doxycycline-inducible promoter, and a sequence of the expression control protein (TetR) of the doxycycline-inducible promoter.

[0033] To obtain a DNA fragment containing the gam-bet-exo gene sequence derived from lambda red, PCR was performed using the sSNredN and sSN1099 primers (SEQ ID NO: 5 and SEQ ID NO: 6) and the pKD46 plasmid (obtained from CGSG: https: / / cgsc.biology.yale.edu / Site.php?ID=64672) as a template. The ends of the resulting DNA fragment were digested with the restriction enzymes NcoI and XhoI and purified. This fragment was inserted into the NcoI and XhoI restriction sites of pHN1092 (JP 2013-005764) to obtain plasmid pHN1102. Furthermore, pHN1102 was digested with the restriction enzymes NcoI and SpeI, and the purified DNA fragment (containing the gam-bet-exo gene derived from lambda red) was inserted into the NcoI and SpeI restriction sites of pHN1340 to obtain plasmid pHN1755.

[0034] To obtain a DNA fragment containing the arabinose-inducible promoter sequence, the expression control protein (AraC) of the arabinose-inducible promoter, and the toxin Maz sequence, PCR was performed using the sSN1583 and sSN1482 primers (SEQ ID NO: 7 and SEQ ID NO: 8) and the pHN1629 plasmid (JP 2013-005764) as a template. The ends of the resulting DNA fragment were digested with the restriction enzymes NheI and SpeI and purified. This fragment was inserted into the NheI restriction enzyme site of pHN1755 to obtain plasmid pHN1757. This pHN1757 contains an autonomous replication origin derived from pBR322, an ampicillin resistance gene, a doxycycline-inducible promoter, an expression control protein (TetR) of the doxycycline-inducible promoter, the gam-bet-exo gene downstream of the doxycycline-inducible promoter, the arabinose-inducible promoter sequence, the expression control protein (AraC) of the arabinose-inducible promoter, and the mazF gene downstream of the arabinose-inducible promoter. The gam-bet-exo genes can confer homologous recombination ability to E. coli strains lacking the recA gene, and the mazF can be used for curing the plasmid.

[0035] (B) Construction of pgi disruption plasmid To obtain a DNA fragment containing a portion of the pgi gene and the upstream sequence of the pgi gene, PCR was performed using the sSN1593 and sSN1684 primers (SEQ ID NO: 9 and SEQ ID NO: 10) and the genomic DNA of E. coli MG1655 as a template. The ends of the resulting DNA fragment were digested with the restriction enzymes PstI and SpeI and purified. This fragment was inserted into the PstI and XbaI restriction enzyme sites of pHN1234 (JP 2014-209872) to obtain the plasmid pHN1892. To obtain a DNA fragment containing a portion of the pgi gene and the downstream sequence of the pgi gene, PCR was performed using the sSN1682R2 and sSN1683R primers (SEQ ID NO: 11 and SEQ ID NO: 12) and the genomic DNA of E. coli MG1655 as a template. The ends of the resulting DNA fragment were digested with the restriction enzymes NsiI and PciI and purified. This fragment was inserted into the NsiI and NcoI restriction enzyme sites of pHN1892 to obtain plasmid pHN1922, which contains the sacB gene, the temperature-sensitive autonomous replication origin derived from pSC101, the chloramphenicol resistance gene, a portion of the pgi gene, and sequences upstream and downstream of the pgi gene.

[0036] (C) Construction of pgi disruptant The pgi gene in E. coli HMS174(DE3) was disrupted according to the method described previously (Nakashima and Miyazaki, Int J Mol Sci. 2014 15:2773-2793). E. coli HMS174(DE3) was transformed with the plasmids pHN1757 and pHN1922 constructed in Examples 1(A) and (B). To express the gam-bet-exo genes required for homologous recombination, gene disruption was performed by constantly adding 20 ng / mL doxycycline hydrochloride to the LB medium. After gene disruption, pHN1757 was cured by culturing it at 37°C in LB medium containing 0.02% L-arabinose. The resulting pgi gene-disrupted strain was designated HMS174(DE3) / Δpgi.

[0037] (D) Construction of dxs, dxr, idi, and ispS-enhancing plasmids The dxs gene from Rhizobium radiobacter and the dxr gene from Escherichia coli were synthesized by adding the recognition sequence for the restriction enzyme BspHI at the 5' end and the recognition sequence for the restriction enzyme BamHI at the 3' end (SEQ ID NO: 13). The synthesized DNA was inserted into the restriction enzyme sites NcoI and BamHI of the E. coli expression vector pCOLADuet-1 (Novagen) and ligated downstream of the T7 promoter. The constructed plasmid was named pCOLA_P T7 It was named -dxs-dxr.

[0038] The idi gene from Escherichia coli and the ispS gene from Mucuna bracteate were synthesized by adding the recognition sequence for the restriction enzyme NdeI at the 5' end and the recognition sequence for the restriction enzyme BglII at the 3' end (SEQ ID NO: 14). The chloroplast targeting signal of the ispS gene was truncated, and the codons were optimized for efficient expression in E. coli. Furthermore, the lysine at position 481 was mutated to glutamic acid. The synthesized DNA was inserted into the pCOLA_P construct described above. T7 The plasmid was inserted into the NdeI and BglII restriction enzyme sites of -dxs-dxr and ligated downstream of the T7 promoter. T7 -dxs-dxr_P T7 It was named -idi-ispS.

[0039] (E) Construction of mvaE, mvaS, MVK, PMK, and MVD-enhancing plasmids The mvaE and mvaS genes from Enterococcus gallinarum were synthesized by adding the recognition sequence for the restriction enzyme NcoI at the 5' end and the recognition sequence for the restriction enzyme BamHI at the 3' end (SEQ ID NO: 15). The mvaE and mvaS genes were designed with codon-optimized sequences for efficient expression in E. coli. The synthesized DNA was inserted into the NcoI and BamHI restriction enzyme sites of the E. coli expression vector pACYCDuet-1 (Novagen) and ligated downstream of the T7 promoter. The constructed plasmid was named pACYC_P T7 They named it -mvaE-mvaS.

[0040] The MVK gene from Methanococcoides burtonii, the PMK gene from Saccharomyces cerevisiae, and the MVD gene were synthesized with the recognition sequences for the restriction enzyme NdeI at the 5' end and BglII at the 3' end (SEQ ID NO: 16). The MVK, PMK, and MVD genes were designed with codon-optimized sequences for efficient expression in E. coli. The synthesized DNA was inserted into the pACYC_P vector constructed above. T7 The plasmid was constructed as pACYC_P. T7 -mvaE-mvaS_P T7 -It was named MVK-PMK-MVD.

[0041] (F) Construction of ispG-enhancing plasmid The ispG gene from Escherichia coli was synthesized by adding the recognition sequence for the restriction enzyme NdeI at the 5' end and the recognition sequence for the restriction enzyme XhoI at the 3' end (SEQ ID NO: 17). The synthesized DNA was inserted into the NdeI and XhoI restriction enzyme sites of the E. coli expression vector pCDFDuet-1 (Novagen) and ligated downstream of the T7 promoter. The constructed plasmid was named pCDF_P T7 -ispG.

[0042] (G) Construction of pgi-disrupted, dxs-, dxr-, ispG-, idi-, ispS-, mvaE-, mvaS-, MVK-, PMK-, and MVD-enhanced strains The HMS174(DE3) / Δpgi strain prepared in Example 1(C) was transfected with the plasmid pCOLA_P constructed in Examples 1(D), (E), and (F). T7 -dxs-dxr_P T7 -idi-ispS, pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD, and pCDF_P T7 -ispG, and the resulting strain was named HMS174(DE3) / Δpgi / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD / pCDF_P T7 -ispG.

[0043] [Table 1]

[0044] [Example 2] The E. coli HMS174(DE3) strain was subjected to disruption of the pgi gene and enhancement of expression of the dxs gene, dxr gene, ispH gene, idi gene, ispS gene, mvaE gene, mvaS gene, MVK gene, PMK gene, and MVD gene (see Table 1).

[0045] (A) Construction of the ispH-enhancing plasmid The ispH gene from Escherichia coli was synthesized by adding the recognition sequence for the restriction enzyme NdeI at the 5' end and the recognition sequence for the restriction enzyme XhoI at the 3' end (SEQ ID NO: 18). The synthesized DNA was inserted into the NdeI and XhoI restriction enzyme sites of the E. coli expression vector pCDFDuet-1 (Novagen) and ligated downstream of the T7 promoter. The constructed plasmid was named pCDF_PT7 It was named -ispH.

[0046] (B) Construction of pgi-disrupted, dxs-, dxr-, ispH-, idi-, ispS-, mvaE-, mvaS-, MVK-, PMK-, and MVD-enhanced strains. The HMS174(DE3) / Δpgi strain prepared in Example 1(C) was transfected with the plasmid pCOLA_P constructed in Examples 1(D) and 1(E) and Example 2(A). T7 -dxs-dxr_P T7 -idi-ispS, pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD, and pCDF_P T7 -ispH, and the resulting strain was named HMS174(DE3) / Δpgi / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD / pCDF_P T7 -ispH.

[0047] [Example 3] The E. coli HMS174(DE3) strain was subjected to disruption of the pgi gene and enhanced expression of the dxs gene, dxr gene, ispG gene, ispH gene, idi gene, ispS gene, mvaE gene, mvaS gene, MVK gene, PMK gene, and MVD gene (see Table 1).

[0048] (A) Construction of ispG and ispH-enhancing plasmids The ispG gene and ispH gene from Escherichia coli were synthesized with the recognition sequence for the restriction enzyme NdeI at the 5' end and the recognition sequence for the restriction enzyme XhoI at the 3' end (SEQ ID NO: 19). The synthesized DNA was inserted into the NdeI and XhoI restriction enzyme sites of the E. coli expression vector pCDFDuet-1 (Novagen) and ligated downstream of the T7 promoter. The constructed plasmid was named pCDF_P T7 -ispG-ispH.

[0049] (B) Construction of strains with pgi disruption, dxs, dxr, ispG, ispH, idi, ispS, mvaE, mvaS, MVK, PMK, and MVD amplified. The HMS174(DE3) / Δpgi strain prepared in Example 1(C) was transfected with the plasmid pCOLA_P constructed in Examples 1(D) and 1(E) and Example 3(A). T7 -dxs-dxr_P T7 -idi-ispS, pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD, and pCDF_P T7 -ispG-ispH, and the resulting strain was HMS174(DE3) / Δpgi / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD / pCDF_P T7 -ispG-ispH.

[0050] [Example 4] For the E. coli HMS174(DE3) strain, we performed disruption of the pgi gene, enhanced expression of the dxs gene, dxr gene, ispG gene, ispH gene, idi gene, ispS gene, mvaE gene, mvaS gene, MVK gene, PMK gene, and MVD gene, and reduced expression of the GltA gene (see Table 1).

[0051] (A) Construction of the GltA-reducing plasmid The Cas9 gene from Streptococcus pyogenes linked to a rhamnose-inducible promoter and the gltA gene from Escherichia coli linked to the constitutive promoter J23119 were targeted by guide RNA sequences, which were inserted at the 5' end by the restriction enzyme PstI. The recognition sequence for the restriction enzyme SpeI was added to the 3'-end of the DNA (SEQ ID NO: 20). In order to eliminate nuclease activity, the Cas9 gene was designed with the 10th aspartic acid mutated to alanine and the 840th histidine mutated to alanine (dCas9). The synthesized DNA was inserted into the PstI and SpeI restriction enzyme sites of the E. coli expression vector pRK2 (SEQ ID NO: 21). The constructed plasmid was named pRK2_P rha -dC as9_P J23119 -gRNA-gltA.

[0052] (B) Construction of a strain with pgi disruption, dxs, dxr, ispG, ispH, idi, ispS, mvaE, mvaS, MVK, PMK, and MVD enhancement, and GltA reduction. HMS174(DE3) / Δpgi / pCOLA_P prepared in Example 3(B) T7 -dxs-dxr_P T7 -idi-ispS / pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD / pCDF_P T7 The ispG-ispH strain was transformed with the plasmid pRK2_P constructed in Example 4(A). rha -dCas9_P J23119 -gRNA-gltA, and the resulting strain was named HMS174(DE3) / Δpgi / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD / pCDF_P T7 -ispG-ispH / pRK2_P rha -dCas9_P J23119 -gRNA-gltA.

[0053] [Comparative Example 1] The expression of the dxs gene, dxr gene, idi gene, and ispS gene was enhanced in the E. coli HMS174(DE3) strain (see Table 1).

[0054] E. coli HMS174(DE3) strain (Novagen) was transformed with the plasmid pCOLA_P constructed in Example 1(D). T7 -dxs-dxr_P T7 -idi-ispS, and the resulting strain was transformed into HMS174(DE3) / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS.

[0055] Comparative Example 2 The expression of the dxs gene, dxr gene, ispG gene, idi gene, and ispS gene was enhanced in the E. coli HMS174(DE3) strain (see Table 1).

[0056] E. coli HMS174(DE3) strain (Novagen) was used, and the plasmid pCOLA_P constructed in Example 1 (D) and (F) was used. T7 -dxs-dxr_P T7 -idi-ispS, and pCDF_P T7 -ispG, and the resulting strain was transformed into HMS174(DE3) / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pCDF_P T7 -ispG.

[0057] Comparative Example 3 The expression of the dxs gene, dxr gene, ispH gene, idi gene, and ispS gene was enhanced in the E. coli HMS174(DE3) strain (see Table 1).

[0058] The E. coli HMS174(DE3) strain (Novagen) was cultured with the plasmid pCOLA_P constructed in Example 1(D) and Example 2(A). T7 -dxs-dxr_P T7 -idi-ispS, and pCDF_P T7 -ispH, and the resulting strain was transformed with HMS174(DE3) / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pCDF_P T7 -ispH.

[0059] Comparative Example 4 The expression of the dxs gene, dxr gene, ispG gene, ispH gene, idi gene, and ispS gene was enhanced in the E. coli HMS174(DE3) strain (see Table 1).

[0060] The E. coli HMS174(DE3) strain (Novagen) was used, and the plasmid pCOLA_P constructed in Example 1(D) and Example 3(A) was used. T7 -dxs-dxr_P T7 -idi-ispS, and pCDF_P T7 -ispG-ispH, and the resulting strain was transformed into HMS174(DE3) / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pCDF_P T7 -ispG-ispH.

[0061] Comparative Example 5 The E. coli HMS174(DE3) strain was subjected to disruption of the pgi gene and enhancement of expression of the dxs gene, dxr gene, idi gene, and ispS gene (see Table 1).

[0062] The HMS174(DE3) / Δpgi strain prepared in Example 1(C) was transfected with the plasmid pCOLA_P constructed in Example 1(D). T7 -dxs-dxr_P T7 -idi-ispS, and the resulting strain was named HMS174(DE3) / Δpgi / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS.

[0063] Comparative Example 6 The E. coli HMS174(DE3) strain was subjected to disruption of the pgi gene and enhancement of expression of the dxs gene, dxr gene, ispG gene, idi gene, and ispS gene (see Table 1).

[0064] The HMS174(DE3) / Δpgi strain prepared in Example 1(C) was transfected with the plasmid pCOLA_P constructed in Examples 1(D) and (F). T7 -dxs-dxr_P T7 -idi-ispS, and pCDF_P T7 -ispG, and the resulting strain was named HMS174(DE3) / Δpgi / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pCDF_P T7 -ispG.

[0065] Comparative Example 7 The E. coli HMS174(DE3) strain was subjected to disruption of the pgi gene and enhancement of expression of the dxs gene, dxr gene, ispH gene, idi gene, and ispS gene (see Table 1).

[0066] The HMS174(DE3) / Δpgi strain prepared in Example 1(C) was transfected with the plasmid pCOLA_P constructed in Example 1(D) and Example 2(A). T7 -dxs-dxr_P T7 -idi-ispS, and pCDF_P T7 -ispH, and the resulting strain was named HMS174(DE3) / Δpgi / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pCDF_P T7 -ispH.

[0067] [Comparative Example 8] The E. coli HMS174(DE3) strain was subjected to disruption of the pgi gene and enhancement of expression of the dxs gene, dxr gene, ispG gene, ispH gene, idi gene, and ispS gene (see Table 1).

[0068] The HMS174(DE3) / Δpgi strain prepared in Example 1(C) was transfected with the plasmid pCOLA_P constructed in Example 1(D) and Example 3(A). T7 -dxs-dxr_P T7 -idi-ispS, and pCDF_P T7-ispG-ispH, and the resulting strain was HMS174(DE3) / Δpgi / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pCDF_P T7 -ispG-ispH.

[0069] Comparative Example 9 The expression of the dxs gene, dxr gene, idi gene, ispS gene, mvaE gene, mvaS gene, MVK gene, PMK gene, and MVD gene was enhanced in the E. coli HMS174(DE3) strain (see Table 1).

[0070] The E. coli HMS174(DE3) strain (Novagen) was cultured with the plasmid pCOLA_P constructed in Examples 1(D), (E), and 3(A). T7 -dxs-dxr_P T7 -idi-ispS, and pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD, and the resulting strain was transformed into HMS174(DE3) / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD.

[0071] [Comparative Example 10] For the E. coli HMS174(DE3) strain, expression of the dxs gene, dxr gene, ispG gene, ispH gene, idi gene, ispS gene, mvaE gene, mvaS gene, MVK gene, PMK gene, and MVD gene was enhanced (see Table 1).

[0072] The E. coli HMS174(DE3) strain (Novagen) was cultured with the plasmid pCOLA_P constructed in Examples 1(D), (E), and 3(A). T7 -dxs-dxr_P T7 -idi-ispS, pACYC_P T7 -mvaE-mvaS_PT7 -MVK-PMK-MVD, and pCDF_P T7 -ispG-ispH, and the resulting strain was transformed into HMS174(DE3) / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD / pCDF_P T7 -ispG-ispH.

[0073] [Comparative Example 11] The E. coli HMS174(DE3) strain was subjected to disruption of the pgi gene and enhancement of expression of the dxs gene, dxr gene, idi gene, ispS gene, mvaE gene, mvaS gene, MVK gene, PMK gene, and MVD gene (see Table 1).

[0074] The HMS174(DE3) / Δpgi strain prepared in Example 1(C) was transfected with the plasmid pCOLA_P constructed in Examples 1(D) and (E). T7 -dxs-dxr_P T7 -idi-ispS, and pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD, and the resulting strain was transformed into HMS174(DE3) / Δpgi / pCOLA_P T7 -dxs-dxr_P T7 -idi-ispS / pACYC_P T7 -mvaE-mvaS_P T7 -MVK-PMK-MVD.

[0075] [Test example] The genetically modified strains prepared in Example 1-3 and Comparative Example 1-11 were evaluated for isoprene production.

[0076] Each genetically modified strain was cultured overnight in LB medium (16 g / L Bactotrypton, 10 g / L Yeast extract, 5 g / L NaCl) containing 50 μg / mL kanamycin, 34 μg / mL chloramphenicol, 50 μg / mL spectinomycin, and 50 μg / mL apramycin (the appropriate combination of antibiotics is selected depending on the strain used). Ten mL of M9+YE medium (6 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 2 mM MgSO4 7H2O, 0.1 mM CaCl2 2H2O, 10 mg / L thiamine hydrochloride, 8.3 mg / L FeSO4 7H2O, 5 g / L yeast extract) containing 0.5% glucose, 50 μg / mL kanamycin, 34 μg / mL chloramphenicol, 50 μg / mL spectinomycin, and 50 μg / mL apramycin (1 mM L-rhamnose was added if using a strain that induces dCas9 gene expression). The culture solution in the LB medium was inoculated at a 1 / 40 dilution and cultured at 180 rpm and 30 °C for 16 hours with shaking. 20 mL of M9+YE medium containing 0.5% glucose, 50 μg / mL kanamycin, 34 μg / mL chloramphenicol, 50 μg / mL spectinomycin, and 0.5 mM IPTG was added to a 50 mL flask, and the culture in the M9+YE medium was grown at OD. 600 The bacteria were inoculated so that the saturation concentration was 0.6, and the mixture was cultured with shaking at 180 rpm at 30°C for 4 hours.

[0077] The resulting culture was collected by centrifugation at 3000 × g for 5 minutes and cultured in M9 medium (6 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 2 mM MgSO4 7H2O, 0.1 mM CaCl2 2H2O, 10 mg / L thiamine hydrochloride, 8.3 mg / L FeSO4 7H2O) containing 0.5% glucose, 50 μg / mL kanamycin, 34 μg / mL chloramphenicol, 50 μg / mL spectinomycin, 50 μg / mL apramycin, and 0.5 mM IPTG (plus 1 mM L-rhamnose if using a strain that induces dCas9 gene expression). 600 The cells were suspended so that the β-glucan concentration was 1 / 2. One mL of the cell suspension was added to a 20 mL headspace vial (Shimadzu GLC), sealed with a butyl rubber septum cap (Shimadzu GLC), and cultured with shaking at 180 rpm and 30°C for 19 hours. After the culture was completed, the isoprene concentration in the headspace of the vial was measured using a GC-MS (Shimadzu Corporation: GCMS-TQ8040NX). The residual glucose concentration in the culture medium was also measured using a glucose analyzer (Oji Scientific Instruments: BF-5). The amount of isoprene produced, the residual glucose concentration, and the isoprene yield relative to sugar consumption are shown in Table 2.

[0078] The isoprene concentration was measured using a GC-MS (Shimadzu Corporation: GCMS-TQ8040NX) under the following analytical conditions. A GL Sciences TC-70 column (0.25 mm internal diameter, 30 m length) was used in the gas chromatography section, with helium as the carrier gas. The gas chromatography mass spectrometer settings were: sample inlet temperature 220 °C, column oven temperature 80 °C (constant), splitless injection, pressure 100 kPa, linear velocity 44.5 cm / s (constant control), mass spectrometer interface temperature 80 °C, ion source temperature 200 °C, scan measurement mode, mass range (m / z) 60 to 70, and analysis time 3 min. Quantitation was performed based on the isoprene peak appearing approximately between 1.175 and 1.180 min. Wako Pure Chemical Industries, Ltd.'s Wako First-Class Isoprene was used as the isoprene standard solution. An appropriate amount was mixed with M9 medium and then fed to the gas chromatography mass spectrometer to create a calibration curve.

[0079] [Table 2]

[0080] The amount of isoprene produced by the genetically modified strain in which expression of the dxs gene, dxr gene, idi gene, and ispS gene was enhanced (Comparative Example 1) was 26.4 mg / L. Furthermore, when expression of the ispG gene was enhanced (Comparative Example 2), when expression of the ispH gene was enhanced (Comparative Example 3), and when expression of both the ispG gene and the ispH gene was enhanced (Comparative Example 4), the amounts of isoprene produced were 1.4 mg / L, 1.7 mg / L, and 1.6 mg / L, respectively, which did not increase compared to Comparative Example 1, but rather decreased. This result is consistent with Non-Patent Document 1, which reported that overexpression of the ispG and ispH genes in E. coli overexpressing the dxs, idi, and ispS genes did not increase isoprene production.

[0081] In addition to enhanced expression of the dxs gene, dxr gene, idi gene, and ispS gene (see Comparative Example 1), genetically modified strains (Examples 1-3) in which the pgi gene was disrupted, expression of the ispG gene and / or ispH gene was enhanced, and expression of genes in the MVA pathway was enhanced showed a significant increase in isoprene production. The genetically modified strain (Example 1) in which the pgi gene was disrupted and the expression of the ispG gene and the expression of the genes in the MVA pathway were enhanced produced 87.4 mg / L of isoprene. The amount of isoprene produced by the genetically modified strain (Example 2) in which the pgi gene was disrupted and the expression of the ispH gene and the gene in the MVA pathway was enhanced was 185.6 mg / L. The genetically modified strain (Example 3) in which the pgi gene was disrupted and the expression of the ispG gene and ispH gene was enhanced, as well as the expression of the genes in the MVA pathway, produced 211.7 mg / L of isoprene.

[0082] When the expression of the dxs, dxr, idi, and ispS genes was enhanced (see Comparative Example 1), and the pgi gene alone was disrupted without enhancing the expression of any of the genes in the MVA pathway, there was almost no increase in isoprene production (Comparative Example 5). Furthermore, when the expression of the dxs, dxr, idi, and ispS genes was enhanced, and the pgi gene alone was disrupted without enhancing the expression of any of the genes in the MVA pathway (see Comparative Example 5), enhancing the expression of the ispG and / or ispH genes actually reduced the amount of isoprene produced (Comparative Examples 6-8). Furthermore, in addition to enhancing the expression of the dxs gene, dxr gene, idi gene, and ispS gene (see Comparative Example 1), the amount of isoprene produced remained relatively low even when the expression of the MVA pathway genes was enhanced without disrupting the pgi gene (Comparative Examples 9 and 10). Furthermore, as described above, in Comparative Examples 1-4, no improvement in isoprene production was observed due to enhanced expression of the ispG gene and / or the ispH gene. Based on the results of these Comparative Examples 1-10, the significant increase in isoprene production observed in Examples 1-3 can be said to be a completely unexpected effect brought about by the synergistic action of the disruption of the pgi gene, the enhanced expression of genes in the MVA pathway, and the enhanced expression of the ispG gene and / or ispH gene.

[0083] The yields of isoprene relative to consumed sugars in Comparative Examples 2, 3, and 4 were significantly lower, at 0.052 times, 0.064 times, and 0.060 times that of Comparative Example 1, respectively. These results demonstrate that when isoprene is produced via the MEP pathway without introducing the MVA pathway and with enhanced Dxs and Dxr expression, the isoprene yield relative to consumed sugars decreases even when IspG or IspH is enhanced. The yields of isoprene relative to consumed sugars in Comparative Examples 6, 7, and 8 were significantly lower, at 0.053 times, 0.082 times, and 0.076 times that of Comparative Example 5, respectively. These results demonstrate that the same tendency was observed in the Pgi-disrupted strain when the MVA pathway was not introduced, Dxs and Dxr were enhanced, and isoprene was produced via the MEP pathway. Furthermore, the isoprene yield relative to consumed sugars was significantly reduced even when IspG or IspH was enhanced. Furthermore, the yield of isoprene relative to consumed sugar in Comparative Example 10 was 1.03 times that of Comparative Example 9, which was almost the same. These results demonstrate that when the MVA pathway is introduced, Dxs and Dxr are enhanced, and isoprene is produced via the MEP pathway, the isoprene yield relative to consumed sugars remains almost unchanged even when IspG and IspH are enhanced. However, the yields of isoprene relative to consumed sugars in Examples 1, 2, and 3 were significantly improved to 1.5 times, 2.3 times, and 2.4 times that of Comparative Example 11, respectively. These results demonstrate that when the MVA pathway is introduced into a Pgi-disrupted strain, Dxs and Dxr are enhanced, and isoprene is produced via the MEP pathway, enhancing IspG and IspH significantly improves the isoprene yield relative to consumed sugars.

[0084] In a genetically modified strain (Example 4) in which the expression of the gltA gene was reduced in addition to disruption of the pgi gene and enhanced expression of the dxs, dxr, idi, ispS, ispG, and ispH genes, as well as enhanced expression of genes in the MVA pathway (see Example 3), further increased isoprene production was observed, reaching 481.8 mg / L. The yield of isoprene relative to consumed sugar in Example 4 was significantly improved, by 3.8 times that of Comparative Example 11. [Sequence List Free Text]

[0085] SEQ ID NO: 1: Nucleotide sequence of sSN1012 primer SEQ ID NO: 2: Nucleotide sequence of sSN1013 primer SEQ ID NO: 3: Nucleotide sequence of sSN1175 primer SEQ ID NO: 4: Nucleotide sequence of sSN1011 primer SEQ ID NO: 5: Nucleotide sequence of sSNredN primer SEQ ID NO: 6: Nucleotide sequence of sSN1099 primer SEQ ID NO: 7: Nucleotide sequence of sSN1583 primer SEQ ID NO: 8: Nucleotide sequence of sSN1482 primer SEQ ID NO: 9: Nucleotide sequence of sSN1593 primer SEQ ID NO: 10: Nucleotide sequence of sSN1684 primer SEQ ID NO: 11: Nucleotide sequence of sSN1682R2 primer SEQ ID NO: 12: Nucleotide sequence of sSN1683R primer SEQ ID NO: 13: Nucleotide sequences of the dxs gene derived from Rhizobium radiobacter and the dxr gene derived from Escherichia coli (containing the recognition sequence for the restriction enzyme BspHI at the 5' end and the recognition sequence for the restriction enzyme BamHI at the 3' end) SEQ ID NO: 14: Nucleotide sequences of the idi gene derived from Escherichia coli and the ispS gene derived from Mucuna bracteate (containing the recognition sequence for the restriction enzyme NdeI at the 5' end and the recognition sequence for the restriction enzyme BglII at the 3' end) SEQ ID NO: 15: Nucleotide sequences of the mvaE gene and mvaS gene derived from Enterococcus gallinarum (containing the recognition sequence for the restriction enzyme NcoI at the 5' end and the recognition sequence for the restriction enzyme BamHI at the 3' end) SEQ ID NO: 16: Nucleotide sequences of the MVK gene derived from Methanococcoides burtonii, the PMK gene derived from Saccharomyces cerevisiae, and the MVD gene (containing the recognition sequence for the restriction enzyme NdeI at the 5' end and the recognition sequence for the restriction enzyme BglII at the 3' end) SEQ ID NO: 17: Nucleotide sequence of the ispG gene derived from Escherichia coli (containing the recognition sequence for the restriction enzyme NdeI at the 5' end and the recognition sequence for the restriction enzyme XhoI at the 3' end) SEQ ID NO: 18: Nucleotide sequence of the ispH gene derived from Escherichia coli (containing the recognition sequence for the restriction enzyme NdeI at the 5' end and the recognition sequence for the restriction enzyme XhoI at the 3' end) SEQ ID NO: 19: Nucleotide sequences of the ispG gene and ispH gene derived from Escherichia coli (containing the recognition sequence for the restriction enzyme NdeI at the 5' end and the recognition sequence for the restriction enzyme XhoI at the 3' end) SEQ ID NO: 20: Nucleotide sequence of guide RNA targeting the Streptococcus pyogenes Cas9 gene linked to a rhamnose-inducible promoter and the Escherichia coli gltA gene linked to the constitutive promoter J23119 (containing the recognition sequence for the restriction enzyme PstI at the 5' end and the recognition sequence for the restriction enzyme SpeI at the 3' end). SEQ ID NO: 21: Sequence of E. coli expression vector pRK2

Claims

1. It has a methylerythritol phosphate (MEP) pathway and a mevalonate (MVA) pathway, Reduced phosphoglucose isomerase (Pgi) activity compared to the unmodified form; Reduced citrate synthase (GltA) activity compared to a non-modified strain; Enhanced isoprene synthase (IspS) activity compared to the unmodified strain; and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase (IspH) activity and / or 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase (IspG) activity that are enhanced compared to an unmodified strain. A genetically modified microorganism capable of producing isoprene.

2. The genetically modified microorganism according to claim 1, wherein at least one enzyme activity selected from the group consisting of 1-deoxy-D-xylulose 5-phosphate synthase (Dxs) activity, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) activity, and isopentyl diphosphate isomerase (Idi) activity is enhanced compared to an unmodified microorganism.

3. 3. The genetically modified microorganism according to claim 1, wherein at least one enzyme activity selected from the group consisting of 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD) activity, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) activity, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) activity is enhanced compared to an unmodified strain.

4. The genetically modified microorganism according to any one of claims 1 to 3, wherein at least one enzyme activity selected from the group consisting of acetyl-CoA acetyltransferase (MvaE) activity, 3-hydroxy-3-methylglutaryl-CoA synthase (MvaS) activity, 3-hydroxy-3-methylglutaryl-CoA reductase (MvaE) activity, mevalonate kinase (MVK) activity, phosphomevalonate kinase (PMK) activity, and diphosphomevalonate decarboxylase (MVD) activity is enhanced compared to an unmodified strain.

5. The genetically modified microorganism according to any one of claims 1 to 4, which is at least one selected from the group consisting of Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, and Pseudomonas bacteria.

6. 1. A method for producing isoprene, comprising a step of contacting a genetically modified microorganism capable of producing isoprene with an organic raw material in an aqueous medium, The genetically modified microorganism It has a methylerythritol phosphate (MEP) pathway and a mevalonate (MVA) pathway, Reduced phosphoglucose isomerase (Pgi) activity compared to the unmodified form; Reduced citrate synthase (GltA) activity compared to a non-modified strain; Enhanced isoprene synthase (IspS) activity compared to the unmodified strain; and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase (IspH) activity and / or 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase (IspG) activity that are enhanced compared to an unmodified strain. Manufacturing method.

7. 7. The method of claim 6, wherein the organic raw material comprises xylose, arabinose, mannose, galactose, fructose, glucose, lactose, maltose, trehanose, cellobiose, sucrose, starch, dextrin, cellulose, hemicellulose, glycerol, and / or mannitol.

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