Genetically modified microorganisms capable of producing 3-hydroxypropionic acid, and methods for producing 3-hydroxypropionic acid.

Abstract

This invention aims to develop microorganisms that can produce 3 HP more efficiently. [Solution] A genetically modified microorganism having the ability to produce 3-hydroxypropionic acid, comprising the following gene: (i) the gene encoding acryloyl-CoA reductase, and (ii) gene encoding crotonase The mutation includes, wherein the mutation results in the suppression of the expression of the genes (i) and (ii) above, or inactivation of the enzymes shown in (i) and (ii) above, or a reduction in enzyme activity compared to a control without the mutation. Furthermore, a microorganism into which a gene encoding malonyl-CoA reductase has been introduced.

Description

Technical Field

[0001] The present invention relates to a genetically modified microorganism having the ability to produce 3-hydroxypropionic acid, a method for producing 3-hydroxypropionic acid using the microorganism, and a method for producing the microorganism.

Background Art

[0002] 3-Hydroxypropionic acid (hereinafter also referred to as "3HP") is an organic acid that can be used as a raw material for biodegradable polyester and a scale inhibitor (a substance in which water-soluble metals or the like become solid and adhere, deposit, and solidify on pipes or the like). In addition, acrylic acid obtained by dehydrating 3HP is a chemical product in high demand as a raw material for superabsorbent resins, water distribution treatment agents, paints, and the like. Currently, acrylic acid is mainly produced by the oxidation of propylene, but production from biomass raw materials using microorganisms is expected as a sustainable production means.

[0003] As a 3HP biosynthesis pathway in microorganisms, there is a pathway in which 3HP is produced by the two-step reduction of malonyl-CoA, which is an intermediate of fatty acid biosynthesis, using acetyl-CoA derived from glucose or the like as a precursor (Non-Patent Documents 1 and 2), and a pathway in which 3HP is produced by the oxidation of 3-hydroxypropionaldehyde produced by the dehydration of glycerin (Non-Patent Documents 3 and 4). Microbial synthesis of 3HP using Escherichia coli or yeast incorporating the enzyme genes required for these pathways has been reported.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005] This invention aims to develop microorganisms that can produce 3 HP more efficiently. [Means for solving the problem]

[0006] The Cupriavidus necator (hereinafter also referred to as "C. necator") H16 strain is an efficient producer of poly(3-hydroxybutanoic acid) [hereinafter also referred to as "P(3HB)"], a biodegradable polyester. Since P(3HB) is biosynthesized using acetyl-CoA as a precursor, it has a high capacity to supply acetyl-CoA from various carbon sources. The inventors of this invention expected to enable 3HP production by the microorganism by introducing the 3HP biosynthesis pathway from acetyl-CoA via malonyl-CoA into the microorganism. On the other hand, if the microorganism has the ability to decompose and metabolize 3HP, it is necessary to block that decomposition pathway. Therefore, as a result of diligent research, the inventors succeeded in developing a microbial strain that can efficiently produce 3HP from carbohydrates by introducing the above-mentioned pathway for biosynthesis of 3HP from acetyl-CoA into the P(3HB)-producing bacterium Cupriavidus nekator, and by identifying and blocking the 3HP degradation pathway in the bacterium. Thus, the present invention was completed.

[0007] In other words, the present invention provides the following embodiments. [1] A genetically modified microorganism having the ability to produce 3-hydroxypropionic acid, wherein the following gene: (i) the gene encoding acryloyl-CoA reductase, and (ii) gene encoding crotonase The mutation includes, wherein the mutation results in the suppression of the expression of the genes (i) and (ii) above, or inactivation of the enzymes shown in (i) and (ii) above, or a reduction in enzyme activity compared to a control without the mutation. Furthermore, a microorganism into which a gene encoding malonyl-CoA reductase has been introduced. [2] The following genes: (iii) The gene encoding methylmalonate semialdehyde dehydrogenase, (iv) The gene encoding 3-hydroxypropionic acid dehydrogenase, (v) The gene encoding isobutanoate dehydrogenase, (vi) the gene encoding malonyl-CoA decarboxylase, and (vii) A gene encoding one or more enzymes selected from the group consisting of polyhydroxyalkanoate polymerase, β-ketothiolase, and acetoacetyl-CoA reductase. The microorganism according to [1] further comprises a mutation in one or more genes selected from the group consisting of, wherein the mutation results in repression of the expression of the genes (iii) to (vii) above, or inactivation of the enzymes shown in (iii) to (vii) above, or a reduction in enzyme activity compared to a control without the mutation. [3] The microorganism described in [1] or [2], wherein a gene encoding acetyl-CoA carboxylase has been further introduced. [4] The microorganism according to [2] or [3], wherein the methylmalonate semialdehyde dehydrogenase is MmsA1, or MmsA2, or MmsA1 and MmsA2. [5] A microorganism according to any one of [2] to [4], wherein the gene (vii) is a gene encoding polyhydroxyalkanoate polymerase, a gene encoding polyhydroxyalkanoate polymerase and β-ketothiolase, a gene encoding polyhydroxyalkanoate polymerase and acetoacetyl-CoA reductase, or a gene encoding polyhydroxyalkanoate polymerase, β-ketothiolase and acetoacetyl-CoA reductase. [6] The microorganism according to any one of [1] to [5], wherein the malonyl-CoA reductase comprises an amino acid sequence having a triple mutation corresponding to the substitution of asparagine at position 940 to valine, lysine at position 1106 to tryptophan, and serine at position 1114 to arginine in the amino acid sequence shown in SEQ ID NO: 32. [7] The microorganism according to any one of [1] to [7], wherein the mutation is a deletion of all or part of the base sequence of the genes (i) and (ii), an insertion or substitution of one or more bases, or a combination thereof. [8] The microorganism according to any one of [2] to [7], wherein the mutation is a deletion of all or part of the base sequence of the gene (iii) to (vii), an insertion or substitution of one or more bases, or a combination thereof. [9] The microorganism described in any of [1] to [8], wherein the microorganism is a modified strain of Cupriavidus necator. A method for producing 3-hydroxypropionic acid, comprising culturing a microorganism described in any of

[10] [1] to [8]. A method for producing 3-hydroxypropionic acid, comprising culturing the microorganisms described in

[11] [9].

[12] In microorganisms, the following genes: (i) the gene encoding acryloyl-CoA reductase, and (ii) gene encoding crotonase Introducing a mutation, wherein the mutation results in the suppression of the expression of the genes (i) and (ii) above, or inactivation of the enzymes shown in (i) and (ii) above, or a reduction in enzyme activity compared to a control without the mutation, Introducing the gene encoding malonyl-CoA reductase. A method for producing genetically modified microorganisms having the ability to produce 3-hydroxypropionic acid.

[13] In microorganisms, the following genes: (iii) The gene encoding methylmalonate semialdehyde dehydrogenase, (iv) The gene encoding 3-hydroxypropionic acid dehydrogenase, (v) The gene encoding isobutanoate dehydrogenase, (vi) the gene encoding malonyl-CoA decarboxylase, and (vii) A gene encoding one or more enzymes selected from the group consisting of polyhydroxyalkanoate polymerase, β-ketothiolase, and acetoacetyl-CoA reductase. The method according to [1], further comprising introducing a mutation into one or more genes selected from the group consisting of, wherein the mutation results in repression of the expression of the genes (iii) to (vii), or inactivation of the enzymes shown in (iii) to (vii), or a reduction in enzyme activity compared to a control without the mutation.

[14] The method according to

[12] or

[13] , further comprising introducing a gene encoding acetyl-CoA carboxylase.

[15] The method according to

[13] or

[14] , wherein the methylmalonate semialdehyde dehydrogenase is MmsA1, or MmsA2, or MmsA1 and MmsA2.

[16] The method according to any one of

[13] to

[15] , wherein the gene (vii) is a gene encoding polyhydroxyalkanoate polymerase, a gene encoding polyhydroxyalkanoate polymerase and β-ketothiolase, a gene encoding polyhydroxyalkanoate polymerase and acetoacetyl-CoA reductase, or a gene encoding polyhydroxyalkanoate polymerase, β-ketothiolase and acetoacetyl-CoA reductase.

[17] The method according to any one of

[12] to

[16] , wherein the malonyl-CoA reductase comprises an amino acid sequence having a triple mutation corresponding to the substitution of asparagine at position 940 to valine, lysine at position 1106 to tryptophan, and serine at position 1114 to arginine in the amino acid sequence shown in SEQ ID NO: 32.

[18] The method according to any one of

[12] to

[17] , wherein the mutation is a deletion of all or part of the base sequence of the genes (i) and (ii), an insertion or substitution of one or more bases, or a combination thereof.

[19] The method according to any one of

[13] to

[18] , wherein the mutation is a deletion of the whole or a part of the nucleotide sequence of the gene of (iii) to (vii) above, an insertion or substitution of one or more bases, or a combination thereof.

[20] The method according to any one of

[12] to

[19] , wherein the microorganism is a modified strain of Cupriavidus necator. [Advantages of the Invention]

[0008] In the present invention, it has been found that in C. necator, which is known as a P(3HB) producing bacterium, acryloyl-CoA reductase and crotonase are involved in an unknown 3HP degradation pathway. Furthermore, Crt2 was identified as such a crotonase in C. necator. Therefore, according to the present invention, in a microorganism, by inactivating the above two genes, that is, the gene encoding acryloyl-CoA reductase and the gene encoding crotonase, and introducing the gene encoding malonyl-CoA reductase as a 3HP biosynthesis pathway, the microorganism can be modified into a 3HP producing microorganism. The thus obtained genetically modified microorganism of the present invention can produce 3HP using a saccharide as a single carbon source. [Brief Description of the Drawings]

[0009] [Figure 1] [[ID=IS]]Shows the P(3HB) biosynthesis and 3HP metabolic pathways in C. necator. In the figure, an "×" is attached to the enzyme inactivated by the gene in the example. ACC and Mcr3m are the enzymes into which the gene was introduced in the example. [Figure 2] Shows the malonyl-CoA reductase mutant expression vector pBBad-mcr3m used in the example. [Figure 3] Shows the acetyl-CoA carboxylase expression vector pJBPP-acc used in the example. [Modes for Carrying Out the Invention]

[0010] (1) Construction of 3HP-producing microorganisms The construction of 3HP-producing microorganisms according to the present invention involves blocking or suppressing the 3HP degradation pathway in the microorganism and introducing the 3HP biosynthesis pathway.

[0011] As described below, the 3HP-producing microorganism of the present invention utilizes a pathway that generates 3HP from acetyl-CoA derived from carbohydrates. Therefore, any microorganism that utilizes acetyl-CoA as a metabolic intermediate or possesses acetyl-CoA carboxylase can be used as a microorganism (host) for constructing the 3HP-producing microorganism of the present invention. That is, since organisms generally produce acetyl-CoA as a metabolic intermediate and also possess acetyl-CoA carboxylase, the microorganism is not particularly limited. Furthermore, as described below, the 3HP-producing microorganism of the present invention has mutations introduced into the gene encoding acryloyl-CoA reductase and the gene encoding crotonase in order to block or suppress the 3HP degradation pathway. Therefore, the microorganism (host) for constructing the 3HP-producing microorganism is a microorganism that possesses the gene encoding acryloyl-CoA reductase and the gene encoding crotonase. According to the explosively increasing amount of microbial genome information in recent years, the gene encoding crotonase is present in almost all microorganisms, and there are also many microorganisms that possess the gene encoding acryloyl-CoA reductase, so the microorganism is not particularly limited. For example, bacteria, yeast, or fungi may be used as the microorganism. The microorganism may be one that has the ability to produce polyhydroxyalkanoates such as P(3HB) (hereinafter referred to as "PHA-producing ability"), or it may be one that does not have PHA-producing ability. Further examples of the microorganism include, but are not limited to, Escherichia coli, bacteria of the genus Corynebacterium, bacteria of the genus Cupriavidus, bacteria of the genus Ralstonia, bacteria of the genus Alcaligenes, bacteria of the genus Hydrogenomonas, bacteria of the genus Wautersia, bacteria of the genus Pseudomonas, bacteria of the genus Bacillus, yeasts of the genus Saccharomyces, and fungi of the genus Aspergillus.

[0012] <3HP biosynthesis pathway> One 3HP biosynthesis pathway in microorganisms involves the production of 3HP from acetyl-CoA derived from carbohydrates such as glucose, via malonyl-CoA. In this pathway, malonyl-CoA is produced by carboxylation of acetyl-CoA, and then 3HP is produced by a two-step reduction of malonyl-CoA. The conversion reaction from acetyl-CoA to malonyl-CoA is catalyzed by acetyl-CoA carboxylase (hereinafter also referred to as "ACC"). The two-step reduction from malonyl-CoA to 3HP via malonyl-semialdehyde is catalyzed by malonyl-CoA reductase (hereinafter also referred to as "Mcr") and malonyl-semialdehyde reductase. Some microbial species possess Mcr, a bifunctional enzyme that catalyzes a two-step reaction of reducing malonyl-CoA and malonic acid semialdehyde (see "Malonyl-coenzyme A reductase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO2 fixation", M. Huegler et al., J Bacteriol, vol 184, 2404-2410 (2002), DOI https: / / doi.org / 10.1128 / jb.184.9.2404-2410.2002). Since acetyl-CoA carboxylase is an enzyme commonly found in microorganisms, in this invention, at least a gene encoding malonyl-CoA reductase (hereinafter also referred to as "mcr") is introduced into the microorganism as part of the 3HP biosynthesis pathway.

[0013] Malonyl-CoA reductase can be derived from any organism, but preferably from a microorganism, such as any bacterium, yeast, or fungus. Malonyl-CoA reductases from various organisms are known, and information can be obtained from known databases such as GenBank. As an example, though not limited to, malonyl-CoA reductase from the green non-sulfur bacterium (Chloroflexus aurantiacus, hereinafter also referred to as "C. aurantiacus") may be used. Malonyl-CoA reductase from green non-sulfur bacterium is known to be a bifunctional enzyme. Sequence information of malonyl-CoA reductase from green non-sulfur bacterium can be obtained from known databases (e.g., nucleotide sequence: GeneBank Accession No. AY530019, coding region: nucleotides 79-3741, amino acid sequence: GeneBank Accession No. AAS20429). In this specification, an example of the amino acid sequence of malonyl-CoA reductase from green non-sulfur bacterium is shown in SEQ ID NO. 32.

[0014] Therefore, the gene encoding malonyl-CoA reductase to be introduced into a microorganism is, for example, (a) A protein containing the amino acid sequence shown in Sequence ID No. 32, (b) A protein having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in Sequence ID No. 32, and which retains malonyl-CoA reductase activity, or (c) A protein having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 32, and retaining malonyl-CoA reductase activity. It may also be a nucleic acid that codes for [something].

[0015] In a further example, the gene encoding malonyl-CoA reductase to be introduced into a microorganism is, (a') A protein consisting of the amino acid sequence shown in Sequence ID No. 32, (b') A protein having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in Sequence ID No. 32, and which retains malonyl-CoA reductase activity, or (c') A protein having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 32, and which retains malonyl-CoA reductase activity. It may also be a nucleic acid that codes for [something].

[0016] The malonyl-CoA reductase used in this application may be a wild-type enzyme or a mutant enzyme. For Mcr derived from C. aurantiacus, it has been shown to have low reducing activity of malonate semialdehyde in the second reduction step, and a triple mutation (N940V-K1106W-S1114R) that improves this activity has been reported (see "Functional balance between enzymes in malonyl-CoA pathway for 3-hydroxypropionate biosynthesis", C. Liu et al., Metab Eng, vol 34, 104-111 (2016), DOI https: / / doi.org / 10.1016 / j.ymben.2016.01.001). Therefore, preferably, nucleic acids encoding the mutant enzyme described below are used. (d) A protein containing an amino acid sequence in the amino acid sequence shown in Sequence ID No. 32 that includes a triple mutation (hereinafter referred to as "N940V-K1106W-S1114R") consisting of a substitution of asparagine at position 940 to valine, a substitution of lysine at position 1106 to tryptophan, and a substitution of serine at position 1114 to arginine. (e) A protein that contains an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in Sequence ID No. 32, and which includes an amino acid sequence containing a triple mutation corresponding to N940V-K1106W-S1114R, and which retains malonyl-CoA reductase activity. (f) An amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 32, which includes an amino acid sequence containing a triple mutation corresponding to N940V-K1106W-S1114R, and which retains malonyl-CoA reductase activity. (g) A protein containing an amino acid sequence in which the amino acid sequence of a malonyl-CoA reductase protein derived from a microorganism other than green non-sulfur bacteria contains a triple mutation corresponding to N940V-K1106W-S1114R in the amino acid sequence shown in SEQ ID NO: 32, (d') A protein having an amino acid sequence in which the amino acid sequence shown in Sequence ID No. 32 contains a triple mutation (hereinafter referred to as "N940V-K1106W-S1114R") consisting of a substitution of asparagine at position 940 to valine, a substitution of lysine at position 1106 to tryptophan, and a substitution of serine at position 1114 to arginine. (e') In the amino acid sequence shown in Sequence ID No. 32, one or more amino acids are deleted, substituted, inserted, or added, and the amino acid sequence consists of an amino acid sequence containing a triple mutation corresponding to N940V-K1106W-S1114R, and the protein retains malonyl-CoA reductase activity. (f') A protein having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 32, comprising an amino acid sequence containing a triple mutation corresponding to N940V-K1106W-S1114R, and retaining malonyl-CoA reductase activity, or (g') A protein consisting of an amino acid sequence containing a triple mutation corresponding to N940V-K1106W-S1114R in the amino acid sequence of malonyl-CoA reductase derived from microorganisms other than green non-sulfur bacteria, as shown in SEQ ID NO: 32.

[0017] In this specification, "triple mutation corresponding to N940V-K1106W-S1114R" means a triple mutation in malonyl-CoA reductase containing the amino acid sequence shown in SEQ ID NO: 32, consisting of a substitution of asparagine at position 940 to valine, a substitution of lysine at position 1106 to tryptophan, and a substitution of serine at position 1114 to arginine. This refers to an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added to the amino acid sequence shown in SEQ ID NO: 32, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence shown in SEQ ID NO: 32. For malonyl-CoA reductase containing an amino acid sequence with %, 98%, or 99% sequence identity, it means that the amino acids at positions 940, 1106, and 1114 in the amino acid sequence of SEQ ID NO: 32 are substituted with valine, tryptophan, and arginine, respectively. For malonyl-CoA reductase derived from microorganisms other than green non-sulfur bacteria, it means that the amino acids at positions 940, 1106, and 1114 in the amino acid sequence of SEQ ID NO: 32 are substituted with valine, tryptophan, and arginine, respectively.

[0018] In addition to the gene encoding malonyl-CoA reductase, the host microorganism may also have a gene encoding malonate semialdehyde reductase introduced. If the malonyl-CoA reductase is not a bifunctional enzyme that catalyzes a two-step reaction of reduction of malonyl-CoA and reduction of malonate semialdehyde, it is preferable to introduce a gene encoding malonate semialdehyde reductase in addition to the gene encoding malonyl-CoA reductase. Malonate semialdehyde reductase may be of any biological origin, but preferably it may be an enzyme derived from a microorganism, such as any bacterium, yeast, or fungus. Malonate semialdehyde reductases from various biological origins are known, and information can be obtained from known databases such as GenBank.

[0019] The host microorganism may also have a gene encoding acetyl-CoA carboxylase introduced into it. As mentioned above, microorganisms generally possess acetyl-CoA carboxylase, but by introducing exogenous acetyl-CoA carboxylase, the carbon fixation reaction from acetyl-CoA to malonyl-CoA can be enhanced, ultimately increasing the 3HP production.

[0020] Acetyl-CoA carboxylase can be derived from any organism, but preferably from a microorganism, such as any bacterium, yeast, or fungus. Various acetyl-CoA carboxylases from different organisms are known, and information can be obtained from known databases such as GenBank. Acetyl-CoA carboxylase is known to contain multiple subunits or domains. Therefore, it is sufficient to introduce genes encoding at least the subunits and domains involved in ACC enzyme activity into the host microorganism.

[0021] For example, though not limited to, acetyl-CoA carboxylase derived from bacteria of the genus Corynebacterium may be used, one example being acetyl-CoA carboxylase derived from Corynebacterium glutamicum. Acetyl-CoA carboxylase derived from Corynebacterium glutamicum is composed of subunits DtsR1 (also called AccD1), AccBC, and AccE. Therefore, as an example, the genes dtsR1, accBC, and accE encoding DtsR1, AccBC, and AccE may be introduced into the host microorganism. For example, dtsR1, accBC, and accE derived from Corynebacterium glutamicum strain ATCC13032 have been identified as Cgl0708, Cgl0700, and Cgl0706, respectively. In this specification, the amino acid sequences encoded by the acetyl-CoA carboxylase constituent genes dtsR1, accBC, and accE from the Corynebacterium glutamicum ATCC13032 strain are shown as SEQ ID NOs. 33, SEQ ID NOs. 34, and SEQ ID NOs. 35, respectively.

[0022] Therefore, the gene encoding acetyl-CoA carboxylase to be introduced into a microorganism is, for example, (a) Three polypeptides comprising the amino acid sequences shown in SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively (b) Three polypeptides comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in one or more of the amino acid sequences shown in SEQ ID NOs: 33, SEQ ID NOs: 34, and SEQ ID NOs: 35, and which retain ACC enzyme activity, or (c) Three polypeptides that contain amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NOs. 33, SEQ ID NOs. 34, and SEQ ID NOs. 35, respectively, and that retain malonyl-CoA reductase activity. It may also be a nucleic acid that codes for [something].

[0023] The acetyl-CoA carboxylase used in the present invention may be a wild-type enzyme or a mutant enzyme. The mutant enzyme may contain one or more amino acid mutations or nucleotide mutations in the amino acid sequence of the wild-type enzyme or the base sequence encoding said amino acid sequence, within the range that retains ACC enzyme activity.

[0024] The genes encoding malonyl-CoA reductase, acetyl-CoA carboxylase, and malonate semialdehyde reductase can be obtained by known methods, for example, by amplification from template chromosome DNA using appropriate primers designed based on the sequence information of the genes, or by chemical synthesis.

[0025] The genes encoding malonyl-CoA reductase and acetyl-CoA carboxylase can be introduced into microorganisms by conventional methods. For example, these genes may be introduced into microorganisms by insertion into a suitable vector. Examples of vectors include plasmid vectors and phage vectors, and a vector suitable for the host should be selected. For example, although not limited to these, a vector capable of autonomous replication in the host may be used. Specific examples of vectors include, but are not limited to, broad-host-range vectors that autonomously replicate in a wide range of Gram-negative bacteria, such as pBBR1-MCS2 (GenBank Accession No. U23751), pJRD215 (M16198), pHRP311, and pBBad. Plasmids for E. coli include, but are not limited to, pBAD24 (GenBank Accession No. X81837), pDONR201, pBluescript, pUC118, pUC119, pUC18, pUC19, and pBR322. Gene insertion into vectors can be carried out using methods known in the field, and various vectors and vector kits are commercially available.

[0026] Furthermore, an appropriate promoter sequence may be ligated upstream of the gene introduced into the host. The promoter can be any one capable of regulating gene transcription within the host cell and can be selected according to the type of host used. For example, an inducible promoter that induces gene expression under specific conditions (e.g., the presence of an inducing substance, dryness, temperature, etc.) can be used. In addition, the vector may be ligated with enhancer sequences, terminator sequences, operator sequences, splicing signals, poly(A) addition signals, ribosome binding sequences, selection marker genes, regions necessary for conjugation, etc., as needed.

[0027] The introduction (transformation) of the vector into host cells can be carried out by methods known in the field. Examples of such introduction methods, though not limited to them, include the protoplast method, competent method, calcium chloride method, conjugation method, calcium phosphate method, electroporation method, spheroplast method, and lithium acetate method. The gene may be introduced into the host chromosome or into the extrachromosome.

[0028] In this specification, "several" means, for example, 2 to 15, preferably 2 to 10, more preferably 2, 3, 4, 5, 6, 7, 8, or 9. In this specification, "mutation," when used in reference to an amino acid sequence, means the deletion, substitution, insertion, addition, or combination thereof of one or more amino acids, and when used in reference to a base sequence, means the deletion, substitution, insertion, addition, or combination thereof of one or more nucleosides. "Sequence identity" can be determined by methods known in the art based on a comparison of two sequences. In this specification, "nucleic acid" may be either DNA or RNA, but DNA is preferred.

[0029] <3HP Decomposition Pathway> One pathway for 3HP degradation in microorganisms is the conversion of malonic acid semialdehyde, produced by the oxidation of 3HP, to acetyl-CoA through oxidative decarboxylation. In C. necator, there are three types of (methyl)malonic acid semialdehyde dehydrogenases, MmsA1, MmsA2, and MmsA3, that catalyze this oxidative decarboxylation. Of these, MmsA1 and MmsA2 have been reported to contribute to 3HP degradation (see "The genetic basis of 3-hydroxypropanoate metabolism in Cupriavidus necator H16", C. Arenas-Loepez et al., Biotechnology for Biofuels, volume 12, Article number: 150 (2019), DOI https: / / doi.org / 10.1186 / s13068-019-1489-5). The oxidation of 3HP to malonyl semialdehyde involves two dehydrogenases: 3-hydroxypropionic acid dehydrogenase and isobutanoic acid dehydrogenase. Furthermore, there is a pathway that converts malonyl-CoA to acetyl-CoA via decarboxylation, a reaction catalyzed by malonyl-CoA decarboxylase. The genome of C. necator contains the gene for Mcd (mcd), which is presumed to be malonyl-CoA decarboxylase. Since these enzymes degrade 3HP and its intermediate metabolites, malonyl semialdehyde and malonyl-CoA, the 3HP degradation pathway can be blocked or inhibited by inactivating one or more of these enzymes or reducing their activity.

[0030] Furthermore, in C. necator, the methylcitric acid cycle exists as a degradation pathway for propionic acid, an organic acid containing three carbon atoms, similar to 3HP (see "The methylcitric acid pathway in Ralstonia eutropha: new genes identified involved in propionate metabolism", CO. Braemer and A. Steinbuechel., Microbiol, vol 147, 2203-2214 (2001), DOI https: / / doi.org / 10.1099 / 00221287-147-8-2203). Therefore, it was hypothesized that 3HP is converted to 3HP-CoA through CoA conversion, then acryloyl-CoA is produced by dehydration, and the double bond of this acryloyl-CoA is reduced to produce propionyl-CoA, which is then metabolized in the methylcitric acid cycle (see Figure 1). The genome of C. necator contains the gene for the putative acryloyl-CoA reductase YhdH (yhdH). Furthermore, in the genome of C. necator, the inventors have previously identified Crt2 as a crotonase that dehydrates (S)-3-hydroxybutanoic acid, which contains four carbon atoms and has a carbon chain length one longer than 3HP. Therefore, in this invention, as described later in the examples, the inventors considered the possibility of conversion of 3HP to propionyl-CoA by YhdH and Crt2 and degradation by the methyl-citric acid cycle, and created bacterial strains in which the gene yhdH, which is a putative acryloyl-CoA reductase, and the gene crt2, which encodes the crotonase Crt2, were deleted by homologous recombination in C. necator. As a result, an increase in 3HP production was observed in these deletion strains. Until now, no 3HP degradation pathway other than the oxidative degradation pathway carried out by MmsA1 and MmsA2 was known in C. necator. Therefore, this invention reveals the existence of a new 3HP degradation pathway by YhdH, Crt2, and the methyl-citric acid cycle. Since homologs of crt2 are present in almost all microorganisms, and homologs of yhdH are also found in many microorganisms, the novel 3HP degradation pathway described above is not limited to C. necator but is present in a variety of microorganisms.

[0031] Therefore, in this invention, mutations are introduced into the gene encoding acryloyl-CoA reductase and the gene encoding crotonase in microorganisms in order to block or suppress the 3HP degradation pathway. Sequence information of these genes in various microorganisms is available from public databases such as GenBank. Any of the aforementioned genes may be gene regions that are presumed to be the enzyme in question.

[0032] For example, the gene yhdH, presumed to be acryloyl-CoA reductase, in the C. necator H16 strain has been identified as H16_A3330 [location 3605624-3604620 on chromosome 1 (NCBI Reference Sequence: NC_008313)]. The gene crt2, encoding crotonase Crt2, in the C. necator H16 strain has been identified as H16_A3307 (location 3581726-3580950 on chromosome 1). The aforementioned genes in other strains of C. necator or other microorganisms can be identified by searching known databases or by homology searches such as BLAST searches.

[0033] Furthermore, in order to block or suppress another 3HP degradation pathway, mutations may be introduced in the genes encoding methylmalonate semialdehyde dehydrogenase, 3-hydroxypropionic acid dehydrogenase, isobutanoic acid dehydrogenase, and / or malonyl-CoA decarboxylase in microorganisms. Methylmalonate semialdehyde dehydrogenase may be either MmsA1 or MmsA2, or both MmsA1 and MmsA2. Sequence information for these genes in various microorganisms is available from public databases such as GenBank. Any of the aforementioned genes may be gene regions that are presumed to be the enzyme in question.

[0034] For example, in the C. necator H16 strain, the genes mmsA1 and mmsA2 encoding methylmalonate semialdehyde dehydrogenase have been identified as H16_A0273 (location 284579-286114 on chromosome 1) and H16_A3664 (location 3945851-3947377 on chromosome 1), respectively. In the C. necator H16 strain, the gene hpdH encoding 3-hydroxypropionic acid dehydrogenase has been identified as H16_A3663 (location 3945823-3944156 on chromosome 1). In the C. necator H16 strain, the gene hbdH encoding isobutanoic acid dehydrogenase has been identified as H16_B1190 [location 1344917-1344012 on chromosome 2 (NC_008314)]. In the C. necator H16 strain, the gene mcd encoding malonyl-CoA decarboxylase has been identified as H16_A2981 (location 3225600-3227057 on chromosome 1). The aforementioned gene in other strains of C. necator or other microorganisms can be identified by searching known databases or by homology searches such as BLAST searches.

[0035] Furthermore, when using a microorganism capable of producing PHA as a host, mutations may be introduced into the genes encoding enzymes involved in PHA biosynthesis in the microorganism to prevent or reduce the consumption of acetyl-CoA for PHA production, which competes with 3HP biosynthesis. Examples of such enzymes include, but are not limited to, β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), polyhydroxyalkanoate polymerase (also known as PHA synthase) (PhaC), and propionyl-CoA transferase (PCT). Two or more genes encoding two or more enzymes involved in PHA biosynthesis may exist as a cluster. For example, mutations may be introduced into one or more genes selected from the group consisting of a gene encoding PhaC, a gene encoding PhaA, and a gene encoding PhaC, which function in P(3HB) biosynthesis. Preferably, the genes encoding PhaC, PhaC, and PhaA, PhaC, and PhaB, or PhaC, PhaA, and PhaB, and more preferably, the genes encoding PhaC, PhaA, and PhaB. Sequence information of these genes in various microorganisms is available from public databases such as GenBank. Any of the genes may be gene regions that are presumed to be the enzyme in question.

[0036] For example, in the C. necator H16 strain, the gene phaC encoding PhaC has been identified as H16_A1437 (location 1557353-1559122 on chromosome 1), the gene phaA encoding PhaA has been identified as H16_A1438 (location 1559207-1560388 on chromosome 1), and the gene phaB encoding PhaB has been identified as H16_A1439 (location 1560463-1561203 on chromosome 1). The aforementioned genes in other strains of C. necator or other microorganisms can be identified by searching known databases or by homology searches such as BLAST searches.

[0037] Thus, a 3HP-producing microorganism capable of producing 3HP from a single carbon source is constructed. Furthermore, according to the present invention, a method for producing a 3HP-producing microorganism is provided, which includes blocking or suppressing the 3HP degradation pathway and introducing a 3HP biosynthesis pathway in the microorganism, as described above.

[0038] In this specification, homolog means a gene that is identical or similar in structure and function between different species or strains.

[0039] The mutation in the gene is either a mutation that results in inactivation of the enzyme, or a mutation that results in a reduction of the enzyme activity compared to a control strain that does not contain the mutation. Furthermore, "inactivation" includes loss, suppression, and reduction of the enzyme activity, that is, loss, suppression, and reduction of the expression of the enzyme, as well as loss, suppression, and reduction of the function of the gene encoding the enzyme. Examples of such mutations, but not limited to, include deletion of all or part of the base sequence of the gene encoding the enzyme, insertion or substitution of one or more bases in the base sequence, or a combination thereof, insertion of a stop codon in the gene sequence, insertion or deletion of one or more bases to introduce a frameshift mutation, or introduction of one or more point mutations in the gene sequence. The introduction of mutations into a gene can be carried out by methods known in the field, such as homologous recombination or the CRISPR-Cas9 method.

[0040] (2) Culturing of 3HP-producing microorganisms 3HP can be produced by culturing the 3HP-producing microorganism of the present invention. 3HP accumulates in cultured cells (e.g., in bacterial cells) or in the culture (e.g., in the culture medium). Culturing can be carried out according to methods and conditions normally used for culturing host microorganisms.

[0041] The 3HP-producing microorganism of the present invention can produce 3HP using carbohydrates as a single carbon source. Examples of such carbon sources, though not limited to them, include glucose, galactose, fructose, sucrose, maltose, gluconic acid, N-acetylglucosamine, glucosamine, arabinose, xylose, starch, cellulose, chitin, and chitosan. Furthermore, when using hydrogen bacteria such as C. necator as the host microorganism, carbon dioxide or carbonic acid can also be used as a carbon source.

[0042] The culture medium may be further supplemented with nitrogen sources, inorganic substances, trace metals, etc., as needed. Examples of nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, urea, peptone, and yeast extract. Examples of inorganic substances include monopotassium phosphate, dipotassium phosphate, sodium chloride, magnesium sulfate, and calcium chloride. For example, culture may be performed in phosphorus-restricted medium, although this is not limited to these. For example, MB medium, MOPS medium, etc., with the above carbon sources added can be used. Furthermore, gene expression inducers such as arabinose, indole-acrylic acid (IAA), and isopropyl-β-D thiogalactopyranoside (IPTG) may be added to the culture medium as appropriate. Furthermore, antibiotics such as kanamycin and ampicillin may be added to the culture medium as appropriate. Those skilled in the art can appropriately select the culture conditions necessary for desired gene expression.

[0043] Culturing is typically carried out under aerobic conditions at approximately 25°C to 37°C in a liquid medium by shaking, but is not limited to this. Appropriate culture conditions suitable for the host microorganism used should be selected. The culture period is also not limited. A culture period that produces the desired amount of 3 HP should be selected; for example, it may be cultured for 1 day to 1 week.

[0044] The 3HP thus produced can be recovered by methods known in the field. For example, cells (bacterial cells) can be recovered from the culture medium by centrifugation, washed and dried, extracted with chloroform or the like, and then purified.

[0045] The obtained product can be identified by methods such as high-performance liquid chromatography, NMR (nuclear magnetic resonance), and gas chromatography. [Examples]

[0046] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

[0047] Example 1: Preparation of Cupriavidus necatol H16_4ΔM strain In the C. necator H16 strain, a quintuple deletion strain, H16_4ΔM, was created by deleting the 3-hydroxypropionic acid dehydrogenase gene hpdH and the 3-hydroxyisobutanoic acid dehydrogenase gene hbdH, which oxidize 3HP to malonic acid semialdehyde; the methylmalonic acid semialdehyde dehydrogenase genes mmsA1 and mmsA2, which oxidatively decarboxylate malonic acid semialdehyde to produce acetyl-CoA; and the malonyl-CoA decarboxylase gene mcd, which decarboxylates malonyl-CoA to acetyl-CoA.

[0048] hpdH and hbdH have been identified as H16_A3663 (locations 3945823-3944156 on chromosome 1 (NC_008313)) and H16_B1190 (locations 1344917-1344012 on chromosome 2 (NC_008314)), respectively; mmsA1 and mmsA2 as H16_A0273 (locations 284579-286114 on chromosome 1) and H16_A3664 (locations 3945851-3947377 on chromosome 1), respectively; and mcd as H16_A2981 (locations 3225600-3227057 on chromosome 1).

[0049] (Example 1-1) Preparation of a vector for MMSA1 destruction Using the genomic DNA of the Cupriavidus nekator H16 strain as a template, the mmsA1 region and the region containing approximately 1 kbp upstream and downstream were amplified by PCR using oligonucleotides consisting of the sequences shown in SEQ ID NOs: 1 and 2 as primers. PCR was performed using KOD Plus (Toyo Bo Co., Ltd.) as the DNA polymerase, with one cycle consisting of 20 seconds at 98°C, 15 seconds at 65°C, and 3 minutes at 68°C, and this was repeated 30 times. The amplified fragments were purified using a DNA purification kit (Promega Corporation).

[0050] The obtained fragments were digested with the restriction enzyme XbaI and purified by isopropanol precipitation. pK18mobsacB, a widely used homologous recombination vector, was treated with XbaI, its 5' end was dephosphorylated by alkaline phosphatase (Toyobo Co., Ltd.), and purified by isopropanol precipitation. The XbaI-treated amplified fragment and the vector fragment were ligated using Ligation High (Toyobo Co., Ltd.) to obtain a plasmid in which the target gene and its upstream and downstream regions were inserted into pK18mobsacB. Using the obtained plasmid as a template, DNA fragments consisting of homologous regions other than the target gene and the plasmid backbone were amplified by inverse PCR using the oligonucleotides of Sequence ID No. 3 and Sequence ID No. 4 as primers. The 5' end of this amplified fragment was phosphorylated with T4 kinase (Toyobo Co., Ltd.), and the mmsA1 disruption vector pK18ms-ΔmmsA1 was obtained by self-ligation using Ligation High.

[0051] [Table 1]

[0052] (Examples 1-2) Preparation of a vector for disrupting mmsA2-hpdH In the Cupriavidus necatol H16 strain, mmsA2 and hpdH are adjacent on chromosome 1 as H16_A3664-H16_A3663, so both gene regions were deleted simultaneously. Targeting mmsA2-hpdH, the same procedure as described in Example 1-1 was performed to obtain the mmsA2-hpdH disruption vector pK18ms-ΔmmsA2-hpdH. The primers used are shown below.

[0053] [Table 2]

[0054] (Examples 1-3) Preparation of HBDH disruption vectors The hpdH region was targeted, and the same procedure as described in Example 1-1 was performed to obtain the hpdH disruption vector pK18ms-ΔhbdH. The primers used are shown below.

[0055] [Table 3]

[0056] (Examples 1-4) Preparation of vectors for MCD disruption The mcd region was targeted, and the same procedure as described in Example 1-1 was performed to obtain the mcd disruption vector pK18ms-Δmcd. The primers used are shown below.

[0057] [Table 4]

[0058] (Examples 1-5) Transformation of Cupriavidus necatol H16 strain Using the pK18ms-ΔmmsA1 obtained in Example 1-1, C. necator H16 strain was transformed by conjugation. First, the vector was introduced into E. coli S17-1 strain by the calcium chloride method. Next, this recombinant E. coli was cultured overnight at 37°C in 3.0 ml of LB medium (1 wt% tryptone, 1 wt% sodium chloride, 0.5 wt% yeast extract, pH 7.2). In parallel, the host Cupriavidus nekator strain was cultured overnight at 30°C in 3.0 ml of NR medium (1 wt% fish extract, 1 wt% polypeptone, 0.2 wt% yeast extract). Subsequently, 0.1 ml of the host Cupriavidus nekator strain culture was mixed with 0.2 ml of the E. coli culture and cultured at 30°C for 6 hours. This bacterial cell mixture was spread onto Simmons Citrate agar (DIFCO) supplemented with 0.2 mg / ml kanamycin and cultured at 30°C for 3 days. Recombinant E. coli vectors were transmitted to Cupriavidus nekator and incorporated into the chromosome by homologous recombination, resulting in kanamycin resistance. On the other hand, recombinant E. coli cannot grow on Simmons Citrate agar; therefore, the colonies that grew on the above medium were C. necator transformants (pop-in strains) in which pK18ms-ΔmmsA1 was incorporated into the chromosome from recombinant E. coli. Furthermore, the pop-in strains were cultured overnight at 30°C in NR medium, then spread onto NR medium supplemented with 10% by weight sucrose and cultured at 30°C for 3 days. The levans sucrase encoded by sacB on pK18mobsacB uses sucrose as a substrate to accumulate toxic polysaccharides in the cell. Therefore, in a medium supplemented with 10% sucrose, only strains with the plasmid region removed (pop-out strains) could grow. From these colonies, clones lacking the mmsA1 region on the chromosome were selected by PCR.

[0059] Using the H16_ΔmmsA1 strain obtained in this manner as the host and the pK18ms-ΔmmsA2-hpdH obtained in Example 1-2 as the conjugate vector, the same procedure as described in Example 1-5 was performed to obtain the triple deletion strain H16_ΔmmsA1ΔmmsA2-hpdH strain, which further deleted the mmsA2-hpdH region.

[0060] Using the thus obtained H16_ΔmmsA1ΔmmsA2-hpdH strain as the host and pK18ms-ΔhbdH obtained in Example 1-3 as the conjugate vector, the same procedure as described in Example 1-5 was performed to obtain the quadruple deletion strain H16_ΔmmsA1ΔmmsA2-hpdHΔhbdH strain, which further deleted the hbdH region.

[0061] Thus, using the obtained H16_ΔmmsA1ΔmmsA2-hpdHΔhbdH strain as the host and the pK18ms-Δmcd obtained in Example 1-4 as the conjugate vector, the same procedure as described in Example 1-5 was performed to obtain the quintuple deletion strain H16_ΔmmsA1ΔmmsA2-hpdHΔhbdHΔmcd strain, which further deleted the mcd region. This strain was referred to as the H16_4ΔM strain.

[0062] Example 2: Preparation of Cupriavidus necatol H16_4ΔMΔCAB strain Gene knockout strains lacking P(3HB) biosynthesis ability were created by deleting the β-ketothiolase gene phaA, the acetoacetyl-CoA reductase gene phaB, and the PHA synthase gene phaC, all of which are functional in the biosynthesis of the biopolyester P(3HB), as described below.

[0063] phaC has been identified as H16_A1437 (location 1557353-1559122 on chromosome 1), phaA as H16_A1438 (location 1559207-1560388 on chromosome 1), and phaB as H16_A1439 (location 1560463-1561203 on chromosome 1). In the C. necator H16 strain, these genes exist as a cluster of phaC1-phaA-phaB1, so these three gene regions were deleted together.

[0064] (Example 2-1) Preparation of a vector for disrupting phaCAB pK18msC'R, previously produced in the inventors' laboratory, contains a region ligated with an approximately 1 kbp upstream phaC region, an Aeromonas caviae-derived PHA synthase gene, and an approximately 1 kbp downstream phaB region. It is a homologous recombination vector that replaces phaC with an Aeromonas caviae-derived PHA synthase gene while deleting the phaA-phaB region on chromosome 1 of the C. necator H16 strain ("Engineering of pha operon on Cupriavidus necator chromosome for efficient biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil", J. Mifune et al., Polym Degrad Stab, vol 95, 1305-1312 (2010), DOI https: / / doi.org / 10.1016 / j.polymdegradstab.2010.02.026). By cleaving pK18msC'R with restriction enzymes Csp45I and NdeI, the Aeromonas caviae-derived PHA synthase gene was excised, yielding a DNA fragment in which approximately 1 kbp of the phaC upstream region and approximately 1 kbp of the phaB downstream region were ligated on both sides of the plasmid sequence. This DNA fragment was blunted at the ends, and the phaCAB disruption vector pK18ms-ΔphaCAB was obtained by a self-ligation reaction using Ligation High.

[0065] (Example 2-2) Transformation of Cupriavidus necatol H16_4ΔM strain Using the H16_4ΔM strain obtained in Example 1 as the host and the pK18ms-ΔphaCAB obtained in Example 2-1 as the conjugate vector, the same procedure as described in Example 1-5 was performed to obtain the H16_4ΔMΔCAB strain in which the phaC-phaA-phaB region was further deleted.

[0066] Example 3: Preparation of Cupriavidus necatol H16_4ΔMΔCABΔyhdHΔcrt2 strain Gene knockout strains were created in C. necator H16 strain by deleting the yhdH gene, which is presumed to be the acryloyl-CoA reductase gene, and the crt2 gene, which is the crotonase gene, as follows. yhdH was identified as H16_A3330 (location 3605624-3604620 on chromosome 1), and crt2 as H16_A3307 (location 3581726-3580950 on chromosome 1).

[0067] (Example 3-1) Preparation of a vector for yhdH disruption The yhdH region was targeted, and the same procedure as described in Example 1-1 was performed to obtain the disruption vector pK18ms-ΔyhdH. The primers used are shown below.

[0068] [Table 5]

[0069] (Example 3-2) Preparation of a vector for CRT2 disruption The same procedure as described in Example 1-1 was performed on the crt2 region to obtain the disruption vector pK18ms-Δcrt2. The primers used are shown below. However, restriction enzymes XbaI and HindIII were used to cleave the amplified fragment and the vector.

[0070] [Table 6]

[0071] (Example 3-3) Transformation of Cupriavidus necatol H16_4ΔMΔCAB strain Using the H16_4ΔMΔCAB strain obtained in Example 2-2 as the host and the pK18ms-ΔyhdH obtained in Example 3-1 as the conjugate vector, the same procedure as described in Example 1-5 was performed to obtain the H16_4ΔMΔCABΔyhdH strain, in which the yhdH region was further deleted.

[0072] Using the H16_4ΔMΔCABΔyhdH strain obtained in this manner as the host and pK18ms-Δcrt2 obtained in Example 3-2 as the conjugate vector, the same procedure as described in Example 1-5 was performed to obtain the H16_4ΔMPΔyhdHΔcrt2 strain, which further deleted the crt2 region.

[0073] Example 4: Preparation of Cupriavidus necatol malonyl-CoA reductase mutant / acetyl-CoA carboxylase expressing strain

[0074] (Example 4-1) Preparation of the malonyl-CoA reductase mutant expression vector pBBad-mcr3m pBBad-mcr is a vector in which the malonyl-CoA reductase gene (mcr) derived from the green sulfur bacterium Chloroflexus aurantiacus is inserted into the pBBad expression vector, which has an arabinose-inducible BAD promoter and autologously replicates in a wide range of Gram-negative bacteria. The Cupriavidus necator strain, which carries this vector, expresses Mcr upon the addition of arabinose (see, for example, Japanese Patent Publication No. 5103619 and “Microbial synthesis of poly((R)-3-hydroxybutyrate-co-3-hydroxypropionate) from unrelated carbon sources by engineered Cupriavidus necator”, T. Fukui et al., Biomacromolecules, vol 10, 700-706 (2009), DOI https: / / doi.org / 10.1021 / bm801391j).

[0075] For Mcr, which consists of 1,219 amino acids, base substitutions corresponding to amino acid mutations—replacing asparagine at position 940 with valine (N940V), lysine at position 1,106 with tryptophan (K1106W), and serine at position 1,114 with arginine (S1114R)—were introduced into the mcr region of pBBad-mcr by site-directed mutagenesis using the following procedure.

[0076] Using pBBad-mcr as a template, a cycle replication reaction was performed using a primer set designed so that the mutation site was approximately in the center, and a heat-stable DNA polymerase to introduce a base mutation into the elongated strand. For the cycle replication reaction, a 50 μL reaction solution consisting of sterile water, 0.02-0.2 ng / μL of template plasmid DNA, 0.15 pmol / μL of each primer, and 1x KOD One PCR Master Mix was used. The reaction was performed for 16 cycles, with one cycle consisting of 10 seconds at 98°C, 5 seconds at 58.4°C, and 100 seconds at 68°C. The template plasmid was digested by treating the reaction product with the restriction enzyme DpnI, and the amplified DNA was purified using a DNA purification kit (Promega). Since the complementary amplified DNA strand takes on a circular structure with a gap, this circular DNA solution was diluted as appropriate and used to transform E. coli. Plasmid clones with correctly introduced site-directed mutations were selected from the obtained transformants. By sequentially repeating this operation with primer pairs corresponding to N940V, K1106W, and S1114R, we obtained pBBad-mcr3m, which expresses a Mcr mutant enzyme with three amino acid substitutions. The sequences of the primer pairs used are shown below.

[0077] [Table 7]

[0078] (Example 4-2) Preparation of the acetyl-CoA carboxylase expression vector pJBPP-acc We constructed pJBPP-acc by ligating it to a constitutive strong promoter of a broad-host-range vector. pBPP is an expression vector modified from the conjugative plasmid pBBR1-MCS2, which autonomously replicates in a wide range of Gram-negative bacteria, and contains the Cupriavidus necatol-derived phaP promoter region (P phaP )-Multi-cloning site (MCS)-Escherichia coli-derived rrnB terminator region (T rrnB) (see “Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16”, T. Fukui et al., Appl Microbiol Biotechnol, vol 89, 1527-1536 (2011), DOI https: / / doi.org / 10.1007 / s00253-011-3100-2). Furthermore, pJB866 is a broad-host-range expression plasmid that has a different origin of replication and tetracycline resistance marker than pBBR1 plasmids, and it contains the transcription factor xylS-pWWO promoter (Pm) region (see "Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in Gram-negative bacteria", JM Blatny et al., Plasmid, vol 38, 35-51 (1997), DOI https: / / doi.org / 10.1006 / plas.1997.1294).

[0079] In Corynebacterium glutamicum, the genes encoding DtsR1, AccBC, and AccE, which constitute acetyl-CoA carboxylase, were subjected to conventional genetic engineering techniques such as PCR and restriction enzyme digestion / ligation, and P was added to pBPP. phaP -dtsR1-P phaP -accBC-T rrnB This region was constructed. This region was excised using the restriction enzyme SspI and replaced with the xylS-Pm region of pJB866. Furthermore, accE was inserted downstream of accBC to construct pJBPP-acc. The complete sequence is shown as sequence number 31.

[0080] Furthermore, dtsR1, accBC, and accE derived from Corynebacterium glutamicum ATCC13032 strain have been identified as Cgl0708, Cgl0700, and Cgl0706, respectively.

[0081] (Examples 4-3) Transformation of Cupriavidus necatol strain using Mcr mutant expression vector and acetyl-CoA carboxylase expression vector The prepared pBBad-mcr3m and pJBPP-acc were introduced into Escherichia coli S17-1 strain, respectively, using the calcium chloride method.

[0082] Using Escherichia coli strain S17-1 containing pBBad-mcr3m as the donor and Cupriavidus nekator strains H16_4ΔMΔCAB and H16_4ΔMΔyhdHΔcrt2 as the recipient strains, transformation by conjugation was performed in the same manner as described in Examples 1-5. Cupriavidus nekator transformants exhibiting kanamycin resistance upon vector transfer were selected on Simmons Citrate agar medium supplemented with kanamycin.

[0083] Similarly, pJBPP-acc was introduced via conjugation using the Escherichia coli S17-1 strain as the donor and the Cupriavidus nekator H16_4ΔMΔyhdHΔcrt2 / pBBad-mcr3m strain as the receptor, as described in Examples 1-5. pJBPP-acc can coexist with pBBad-mcr3m within the same cell. Transformants exhibiting dual resistance to kanamycin / tetracycline were selected on Simmons Citrate agar medium supplemented with kanamycin / tetracycline.

[0084] Example 5: 3HP biosynthesis by modified Cupriavidus necatol strain Recombinant Cupriavidus nekator strains, pre-cultured in NR medium (as described above), were inoculated into 100 ml of MB medium (0.9 wt% disodium hydrogen phosphate dodecahydrate, 0.15 wt% potassium dihydrogen phosphate, 0.05 wt% ammonium chloride, 0.02 wt% magnesium sulfate, 0.1 vol% trace metal solution) and cultured in a Sakaguchi flask at 30°C for 144 hours with shaking. 1 wt% fructose was used as the carbon source. To maintain the expression vector pBBad-mcr3m, kanamycin was added at 0.1 mg / ml, and in pJBPP-acc-introduced strains, tetracycline was added at an additional 0.0125 mg / ml. At the start of culture, 0.1 wt% arabinose, a gene expression inducer, was added to the medium.

[0085] If necessary, the concentration of ammonium chloride added was set to 0.1% by weight. In addition, the nitrogen-limited medium, MB medium, was changed to the phosphorus-limited medium, MOPS medium [40 mM 3-morpholinopropanesulfonic acid (MOPS) buffer (pH 7.2), 0.1% by weight ammonium chloride, 0.087% by weight dipotassium hydrogen phosphate, 0.02% by weight magnesium sulfate, 0.1% by volume trace metal solution].

[0086] Example 6: 3HP analysis of culture supernatant One mL of culture medium sampled at any given time was centrifuged at 8,000 rpm at 4°C for 5 minutes, and the supernatant was transferred to a microcentrifuge tube and stored at -20°C. The sample, thawed on ice, was filtered using a Minisart RC filter (4 mm diameter, 0.2 μm pore size, Sartorius). 900 μL of filtered supernatant was transferred to a 1.5 mL screw vial (GL Sciences) or a 750 μL snap vial (LaboLabo Company) for analysis, and 100 μL of either a 50 mM propionic acid solution or a 100 mM n-butanoic acid solution (both filtered) was added as an internal standard and mixed to prepare the analytical sample (final concentration: 5 mM propionic acid or 10 mM n-butanoic acid). Calibration curves were prepared using 3HP solutions of known concentrations. If dilution of the supernatant was necessary, it was diluted with LC / MS ultrapure water (Fujifilm Wako Pure Chemical Industries) as appropriate.

[0087] The 3HP concentration in the culture supernatant was calculated by high-performance liquid chromatography (HPLC) using the pH buffering-electrical conductivity detection method. A Nexera X2 multi-system (Shimadzu Corporation) was used for the HPLC analysis, and two Shim-pack SCR-102H 300mm×8mm columns were used in series along with a Shim-pack guard column 50mm×6mm. The analytical conditions were as follows: Mobile phase: 5mM p-toluenesulfonic acid, flow rate: 0.8 mL / min, column temperature: 40°C, post-column mixed buffer: 5mM p-toluenesulfonic acid, 20mM Bis-Tris, 0.1mM EDTA 4H (Shimadzu standard pH buffer), buffer flow rate: 0.8 mL / min, detector: CDD-10Avp electroelectric detector, sample injection volume: 10 μL. The obtained results are shown in Table 8.

[0088] Table 8. 3HP production by recombinant C. necator strain. [Table 8] MB medium or MOPS medium, 1% by weight fructose, 0.1% (inducer) arabinose, 30°C

[0089] In addition to the deletion of PhaC-PhaA-PhaB, which leads to a loss of P(3HB) biosynthesis ability, the deletion of HpdH and HbdH, which oxidize 3HP, and MmsA1 and MmsA2, which oxidize malonyl semialdehyde, which suppresses 3HP degradation. Furthermore, the deletion of Mcr, which degrades malonyl-CoA, a precursor of 3HP biosynthesis, was also modified. In this strain (No. 1), the expression vector pBBad-mcr3m of the malonyl-CoA reductase mutant was introduced, resulting in a very low 3HP production in the culture supernatant of 0.0058 g / L (96 hours).

[0090] On the other hand, in strain No. 2, which was introduced with pBBad-mcr3m using H16_4ΔMΔCABΔyhdHΔcrt2, a host cell that further lacked double deletions of acryloyl-CoA reductase YhdH and crotonase Crt2, both presumed to be involved in the conversion of 3HP-CoA to propionyl-CoA, a starting material for the methylcitric acid cycle, 3HP production increased to 0.032 g / L (144 hours), demonstrating the effect of YhdH and Crt2 deletion. Furthermore, in strain No. 3, which was introduced with the expression vector pJBPP-acc, an acetyl-CoA carboxylase that produces malonyl-CoA from acetyl-CoA, production increased to 0.051 g / L. In this strain, when the concentration of ammonium chloride was increased to 1 g / L, bacterial growth (OD600) doubled, and the 3HP concentration increased to 0.13 g / L (No. 4). Furthermore, when the culture medium was changed to MOPS medium, a phosphate-restricted medium, and the amount of ammonium chloride added as a nitrogen source was 1 g / L, the production of 3 HP increased to 0.28 g / L (No. 5).

[0091] Sequence ID 31: CAAATTGGGAGATATATCATGAAAGGCTGGCTTTTTCTTGTTATCGCAATAGTTGGCGAAGTAATCGCAACATCCGCATTAAAATCTAGCGAGGGCTTTACTAAGCTGATC

[0092] Sequence ID 32(1219aa):

[0093] Sequence ID 33: Cgl0708(543aa) MTISSPLIDVANLPDINTTAGKIADLKARRAEAHFPMGEKAVEKVHAAGRLTARERLDYLLDEGSFIETDQLARHRTTAFGLGAKRPATDGIVTGWGTIDGREVCIFSQDGTVFGGALGEVYGEKMIKIMELAID TGRPLIGLYEGAGARIQDGAVSLDFISQTFYQNIQASGVIPQISVIMGACAGGNAYGPALTDFVVMVDKTSKMFVTGPDVIKTVTGEEITQEELGGATTHMVTAGNSHYTAATDEEALDWVQDLVSFLPSNNRSYA PMEDFDEEEGGVEENITADDLKLDEIIPDSATVPYDVRDVIECLTDDGEYLEIQADRAENVVIAFGRIEGQSVGFVANQPTQFAGCLDIDSSEKAARFVRTCDAFNIPIVMLVDVPGFLPGAGQEYGGILRRGAKL LYAYGEATVPKITVTMRKAYGGAYCVMGSKGLGSDINLAWPTAQIAVMGAAGAVGFIYRKELMAADAKGLDTVALAKSFEREYEDHMLNPYHAAERGLIDAVILPSETRGQISRNLRLLKHKNVTRPARKHGNMPL

[0094] Sequence ID 34: Cgl0700(591aa) MSVETRKITKVLVANRGEIAIRVFRAARDEGIGSVAVYAEPDADAPFVSYADEAFALGGQTSAESYLVIDKIIDAARKSGADAIHPGYGFLAENADFAEAVINEGLIWIGPSPESIRSLGDKVTARHIADTAKAPMAPGTKEPVKDA AEVVAFAEEFGLPIAIKAAFGGGGRGMKVAYKMEEVADLFESATREATAAFGRGECFVERYLDKARHVEAQVIADKHGNVVVAGTRDCSLQRRFQKLVEEAPAPFLTDDQRERLHSSAKAICKEAGYYGAGTVEYLVGSDGLISFLEV NTRLQVEHPVTEETTGIDLVREMFRIAEGHELSIKEDPAPRGHAFEFRINGEDAGSNFMPAPGKITSYREPQGPGVRMDSGVVEGSEISGQFDSMLAKLIVWGDTREQALQRSRRALAEYVVEGMPTVIPFHQHIVENPAFVGNDEGF EIYTKWIEEVWDNPIAPYVDASELDEDEDKTPAQKVVVEINGRRVEVALPGDLALGGTAGPKKKAKKRRAGGAKAGVSGDAVAAPMQGTVIKVNVEEGAEVNEGDTVVVLEAMKMENPVKAHKSGTVTGLTVAAGEGVNKGVVLLEIK

[0095] Sequence ID 35: Cgl0706(82aa) MSEETTQDTKAAEKPFLQIVSGNPTDQEVAALTVVFAGLAKAAAQQMVSASKDRNNWGNLDERLSRPNTFNPSAFQNVNFF [Industrial applicability]

[0096] This invention makes it possible to produce 3HP, an important chemical product, from renewable raw materials such as carbohydrates and carbon dioxide, which is a contributing factor to global warming.

Claims

1. A genetically modified microorganism capable of producing 3-hydroxypropionic acid, comprising the following gene: (i) a gene encoding acryloyl-CoA reductase, and (ii) gene encoding crotonase The mutations include, wherein the mutations result in the suppression of the expression of the genes (i) and (ii) above, or inactivation of the enzymes shown in (i) and (ii) above, or a reduction in enzyme activity compared to a control without the mutations. Furthermore, a microorganism into which a gene encoding malonyl-CoA reductase has been introduced.

2. The following genes: (iii) The gene encoding methylmalonate semialdehyde dehydrogenase, (iv) The gene encoding 3-hydroxypropionic acid dehydrogenase, (v) The gene encoding isobutanoic acid dehydrogenase, (vi) The gene encoding malonyl-CoA decarboxylase, and (vii) A gene encoding one or more enzymes selected from the group consisting of polyhydroxyalkanoate polymerase, β-ketothiolase, and acetoacetyl-CoA reductase. The microorganism according to claim 1, further comprising a mutation in one or more genes selected from the group consisting of, wherein the mutation results in suppression of the expression of the genes (iii) to (vii), or inactivation of the enzymes shown in (iii) to (vii), or a reduction in enzyme activity compared to a control without the mutation.

3. The microorganism according to claim 1, further comprising a gene encoding acetyl-CoA carboxylase.

4. The microorganism according to claim 2, wherein the methylmalonate semialdehyde dehydrogenase is MmsA1, or MmsA2, or MmsA1 and MmsA2.

5. The microorganism according to claim 2, wherein the gene (vii) is a gene encoding polyhydroxyalkanoate polymerase, a gene encoding polyhydroxyalkanoate polymerase and β-ketothiolase, a gene encoding polyhydroxyalkanoate polymerase and acetoacetyl-CoA reductase, or a gene encoding polyhydroxyalkanoate polymerase, β-ketothiolase and acetoacetyl-CoA reductase.

6. The microorganism according to claim 1, wherein the malonyl-CoA reductase comprises an amino acid sequence containing a triple mutation corresponding to the substitution of asparagine at position 940 to valine, the substitution of lysine at position 1106 to tryptophan, and the substitution of serine at position 1114 to arginine in the amino acid sequence shown in SEQ ID NO:

32.

7. The microorganism according to claim 1, wherein the mutation is a deletion of all or part of the base sequence of the genes (i) and (ii), an insertion or substitution of one or more bases, or a combination thereof.

8. The microorganism according to claim 2, wherein the mutation is a deletion of all or part of the base sequence of the genes (iii) to (vii), an insertion or substitution of one or more bases, or a combination thereof.

9. The microorganism according to any one of claims 1 to 8, wherein the microorganism is a modified strain of Cupriavidus necator.

10. A method for producing 3-hydroxypropionic acid, comprising culturing a microorganism according to any one of claims 1 to 8.

11. A method for producing 3-hydroxypropionic acid, comprising culturing the microorganism described in claim 9.

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