JP2007082476A - Method for producing 3-hydroxypropionic acid from glycerol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing 3-hydroxypropionic acid from glycerol by an enzymatic reaction. <P>SOLUTION: The method for producing the 3-hydroxypropionic acid from the glycerol by the enzymatic reaction is characterized by performing an enzymatic reaction with a glycerol dehydrase and/or a diol dehydrase and performing an enzymatic reaction with a hydroxyaldehyde dehydrogenase. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing 3-hydroxypropionic acid from glycerin by enzymatic reaction, a method for producing acrylic acid by dehydrating 3-hydroxypropionic acid obtained by the method, and a method for use in the method. It relates to recombinant microorganisms and enzyme active substances.

  In the surfactant industry and the biodiesel fuel industry, glycerin is discharged in large quantities as a by-product. Normally glycerin is used in the manufacture of nitroglycerin, other medicines and cosmetics, etc. However, since glycerin discharged as a by-product is in the state of an aqueous solution containing impurities, it cannot be used as it is for the above purpose and is discarded. This is the current situation. If glycerin as waste can be effectively used in this way, it is considered very preferable from the viewpoint of effective utilization of resources such as global environmental problems.

  3-Hydroxypropionic acid and its ester are compounds useful as raw materials for aliphatic polyesters, and polyesters synthesized from these are attracting attention as biodegradable and environmentally friendly polyesters. 3-Hydroxypropionic acid is usually produced by the addition of water to acrylic acid or by the reaction of ethylene chlorohydrin and sodium cyanide. Since the reaction of hydrating acrylic acid is an equilibrium reaction, there is a problem that the reaction rate is limited. In the case of the reaction of ethylene chlorohydrin, it is necessary to use a highly toxic substance, and an additional hydrolysis step must be added. In this case, there is also a problem that sodium chloride and ammonium salt are produced in large quantities.

  In Patent Document 1, glycerol is dehydrated using diol dehydratase and / or glycerol dehydratase and a microbial cell and / or a treated product containing the reactivation factor thereof to obtain 3-hydroxypropionaldehyde, which is oxidized. And a method for producing 3-hydroxypropionic acid. In the method described in Patent Document 1, only the step of dehydrating glycerin to obtain 3-hydroxypropionaldehyde is performed by an enzymatic reaction, and the oxidation to 3-hydroxypropionic acid is performed by a chemical method in a separate step. Therefore, before the oxidation reaction to 3-hydroxypropionic acid, 3-hydroxypropionaldehyde accumulates in the enzyme reaction system, and there is a problem that the cells are killed or the enzyme is deactivated.

  Moreover, 3-hydroxypropionic acid can manufacture acrylic acid by dehydrating. Acrylic acid is mainly used as an intermediate in the manufacture of acrylic esters, and acrylic esters are used in the manufacture of coating agents, finishes, paints, and adhesives, and in the manufacture of adsorbents and detergent additives. Has also been used. Most commercial acrylic acids are produced by the oxidation of propylene. As an alternative method for producing acrylic acid, hydrolysis of acrylonitrile with sulfuric acid is known. However, this process produces a large amount of ammonium sulfate waste and is not commercially implemented due to the associated costs.

JP 2005-102533 A

  The present invention is to provide a method for efficiently producing 3-hydroxypropionic acid from glycerin by an enzymatic reaction.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that glycerin is subjected to an enzymatic reaction with glycerol dehydratase and / or diol dehydratase and hydroxyaldehyde dehydratase without using a chemical method. From this, it was found that 3-hydroxypropionic acid can be produced, and the present invention has been completed.

That is, the present invention includes the following inventions.
(1) A method for producing 3-hydroxypropionic acid from glycerin by an enzymatic reaction, comprising performing an enzymatic reaction with glycerol dehydratase and / or diol dehydratase and performing an enzymatic reaction with hydroxyaldehyde dehydrogenase.
(2) The method according to (1), wherein the hydroxyaldehyde dehydrogenase is derived from E. coli.
(3) An enzyme reaction is carried out by bringing glycerol into contact with a microorganism that produces glycerol dehydratase and / or diol dehydratase, or a treated product thereof, and a microorganism that produces hydroxyaldehyde dehydrogenase or a treated product thereof, and glycerol. ) Or (2).
(4) The method according to (1) or (2), wherein the enzyme reaction is carried out by contacting glycerol with a microorganism that produces glycerol dehydratase and / or diol dehydratase and hydroxyaldehyde dehydrogenase or a processed product thereof.
(5) The method according to any one of (1) to (4), wherein the enzyme reaction is performed under aerobic conditions.
(6) A method for producing acrylic acid by dehydrating 3-hydroxypropionic acid produced by the method according to any one of (1) to (4).
(7) A recombinant microorganism containing a glycerol dehydratase gene and / or a diol dehydratase gene and a hydroxyaldehyde dehydrogenase gene.
(8) A composition for producing 3-hydroxypropionic acid from glycerin, comprising glycerol dehydratase and / or diol dehydratase and hydroxyaldehyde dehydrogenase.

  According to the present invention, 3-hydroxypropionic acid can be produced from glycerin by a series of enzymatic reactions without using a chemical technique.

  The present invention relates to a method for producing 3-hydroxypropionic acid from glycerin by an enzymatic reaction, and the enzymatic reaction includes both an enzymatic reaction with glycerol dehydratase and / or diol dehydratase and an enzymatic reaction with hydroxyaldehyde dehydrogenase.

  In the enzyme reaction of the present invention, glycerol dehydratase and / or diol dehydratase dehydrates glycerin and converts it to 3-hydroxypropionaldehyde and water, and 3-hydroxypropionaldehyde is dehydrogenated by hydroxyaldehyde dehydrogenase. Includes a reaction to produce hydroxypropionic acid. The enzyme reaction by glycerol dehydratase and / or diol dehydratase and the enzyme reaction by hydroxyaldehyde dehydrogenase may be performed simultaneously in the same reaction system or may be performed continuously in separate reaction systems, preferably Simultaneously in the same reaction system. By performing the enzymatic reaction with glycerol dehydratase and / or diol dehydratase and the enzymatic reaction with hydroxyaldehyde dehydrogenase in the same reaction system, damage to the enzyme-producing microorganisms due to accumulation of the intermediate 3-hydroxypropionaldehyde and loss of the enzyme Life can be prevented.

  Glycerol dehydratase and diol dehydratase both catalyze the reaction of dehydrating glycerin and converting it to 3-hydroxypropionaldehyde and water. In the present invention, glycerol dehydratase and / or diol dehydratase, Enzymatic reactions using only dehydratase, enzymatic reactions using only diol dehydratase, and enzymatic reactions using both glycerol dehydratase and diol dehydratase are included.

  Glycerol dehydratase and diol dehydratase are dependent on coenzyme B12 (also referred to as adenosylcobalamin), and the presence of coenzyme B12 is essential for the reaction of dehydrating glycerin into 3-hydroxypropionaldehyde and water. For this reason, coenzyme B12 is allowed to coexist in the enzyme reaction with glycerol dehydratase and / or diol dehydratase. Coenzyme B12 may be added to the enzyme reaction system, or its synthesis system such as coenzyme B12 synthase may be present. The amount of coenzyme B12 in the enzyme reaction system using glycerol dehydratase and / or diol dehydratase is such that the coenzyme B12 concentration is 1 nM or more, preferably 100 nM or more and 1 mM or less, more preferably 100 μM per 50 mM glycerol concentration as a substrate. The amount is preferably as follows.

  In addition, glycerol dehydratase and diol dehydratase, when catalyzing the reaction of dehydrating glycerol and converting it to 3-hydroxypropionaldehyde and water, crushes coenzyme B12 and deactivates the active center. By the activating factor and / or the diol dehydratase reactivating factor, the collapsed coenzyme B12 can be replaced with a new coenzyme B12 to reactivate glycerol dehydratase and diol dehydratase. Accordingly, the enzymatic reaction of the present invention preferably further comprises reactivating glycerol dehydratase and / or diol dehydratase with a glycerol dehydratase reactivating factor and / or diol dehydratase reactivating factor. In the enzyme reaction of the present invention, when an enzyme reaction by glycerol dehydratase is included, it is preferable to reactivate glycerol dehydratase by a glycerol dehydratase reactivating factor, and when performing an enzyme reaction by diol dehydratase, diol dehydratase reactivation is preferable. It is preferable to reactivate the diol dehydratase with an activator.

  Glycerol dehydratase is an enzyme composed of three subunits, a large subunit, a medium subunit, and a small subunit. In the present invention, glycerol dehydratase is not particularly limited as long as it is a protein having an enzyme activity that catalyzes a reaction of dehydrating glycerin and converting it to 3-hydroxypropionaldehyde and water, and a known one can be used. Specifically, microorganisms belonging to the genus Klebsiella, Citrobacter genus, Clostridium genus, Lactobacillus genus, Enterobacter genus, Caloramator genus, Salmonella genus and Listeria genus, and more specifically, Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Lactobacillus media , Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus brevis, Enterobacter agglomerans, Clostridium butyricum, Caloramator viterbensis, Lactobacillus collinoides, Lactobacillus hilgardii, Salmonella typhimurium, Listeria monocytogenes, Listeria monocytogenes de These glycerol dehydratases may be used alone or as a mixture of two or more.

  Diol dehydratase is also an enzyme composed of three subunits, a large subunit, a medium subunit, and a small subunit. In the present invention, the diol dehydratase is not particularly limited as long as it is a protein having an enzyme activity that catalyzes a reaction of dehydrating glycerol and converting it to 3-hydroxypropionaldehyde and water, and a known diol dehydratase can be used. Specifically, microorganisms belonging to the genus Klebsiella, Citrobacter genus, Clostridium genus, Lactobacillus genus, Enterobacter genus, Caloramator genus, Salmonella genus and Listeria genus, and more specifically, Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Lactobacillus media , Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus brevis, Enterobacter agglomerans, Clostridium butyricum, Caloramator viterbensis, Lactobacillus collinoides, Lactobacillus hilgardii, Salmonella typhimurium, Listeria monocytogenes, Listeria monocytogenes, diol These diol dehydratases may be used alone or as a mixture of two or more.

  In the present invention, the hydroxyaldehyde dehydrogenase is not particularly limited as long as it is a protein having an enzyme activity that catalyzes a dehydrogenation reaction that desorbs hydrogen from 3-hydroxypropionaldehyde and passes it to an electron acceptor, and a known one can be used. Specifically, Escherichia, Aerobacter, Agrobacterium, Alcaligenes, Arthrobacter, Bacillus, Corynebacterium, Flavobacterium, Klebsiella, Micrococcus, Protaminobacter, Proteus, Pseudomonas, Salmonella, Sarcina, Microorganisms belonging to the genus Staphylococcus, Shigella, Erwinia, Neisseria, more specifically Aerobacter aerogenes, Agrobacterium radiobacter, Agrobacterium tumefaciens, Alcaligenes viscolactis, Arthrobacter simplex, Bacillus licheniformis, Bacillus megaterium, F. Klebsiella pneumonia (locus name AB106869), Micrococcus glutamicus, Protaminobacter alboflavus, Proteus vulgaris, Pseudomonas fluorescens, Salmonella typhimurium, Sarcina lutea, Staphylococcus aureus, Shigella flexneri (eria AL162753), Neisseria gonorrhoeae (loc us name AE004969) (Morita, H .; Nishimura, T .; Tani, Y .; Ogata, K .; Yamada, H .; Agric. Biol. Chem. 43, 185- 186 (1979)). In the present invention, hydroxyaldehyde dehydrogenase derived from E. coli is preferably used as hydroxyaldehyde dehydrogenase, more preferably aldA or aldB. These enzymes have high substrate specificity for 3-hydroxypropionaldehyde obtained by dehydration of glycerin, and show high activity in the dehydrogenation reaction of 3-hydroxypropionaldehyde. Particularly preferably, aldA derived from E. coli is used.

  Glycerol dehydratase reactivation factor and diol dehydratase reactivation factor are composed of two subunits, a large subunit and a small subunit. In the present invention, the glycerol dehydratase reactivation factor and the diol dehydratase reactivation factor are complementary to the reaction center portion in glycerol dehydratase or diol dehydratase inactivated by catalyzing the conversion reaction of glycerin to 3-hydroxypropionaldehyde and water. The protein is not particularly limited as long as it has a role of replacing the enzyme B12 and regaining the activity, and a known protein can be used. Glycerol dehydratase reactivation factors include WO98 / 21341; Daniel et al., J. Bacteriol., 177, 2151 (1995); Toraya and Mori, J. Biol. Chem., 274, 3372 (1999); and Tobimatsu et al., J. Bacteriol. 181, 4110 (1999).

  A gene encoding a glycerol dehydratase reactivation factor or a diol dehydratase reactivation factor is generally present in a group of genes called dha regulon and pdu operon possessed by a group of bacteria capable of assimilating glycerin under anaerobic conditions. Examples include gdrA, gdrB, pduG, pduH, ddrA, ddrB, dhaF, dhaG, orfZ, and orfY. Specifically, derived from microorganisms belonging to the genus Lactobacillus, Klebsiella, Citrobacter, Clostridium, and Enterobacter. Examples include those derived from pasteurianum, Enterobacter agglomerans, and Clostridium butyricum. These glycerol dehydratase reactivating factor and diol dehydratase reactivating factor may be used alone or as a mixture of two or more.

  In one embodiment, the enzymatic reaction of the present invention is performed by contacting glycerol with an enzyme active substance containing a microorganism that produces glycerol dehydratase and / or diol dehydratase or a processed product thereof, and a microorganism that produces hydroxyaldehyde dehydrogenase or a processed product thereof. To implement.

  In this embodiment, the enzyme reaction by glycerol dehydratase and / or diol dehydratase and the enzyme reaction by hydroxyaldehyde dehydrogenase are performed in the same reaction system, and microorganism damage and enzyme due to accumulation of 3-hydroxypropionaldehyde as an intermediate Can be prevented from being deactivated.

  In the present invention, it is further preferable to use an enzyme active substance containing a microorganism that produces a glycerol dehydratase reactivation factor and / or a diol dehydratase reactivation factor or a processed product thereof. Alternatively, it is preferable to use a microorganism that also produces a glycerol dehydratase reactivation factor and / or a diol dehydratase reactivation factor as a microorganism that produces glycerol dehydratase and / or diol dehydratase.

  As microorganisms that produce glycerol dehydratase and / or diol dehydratase, and microorganisms that produce hydroxyaldehyde dehydrogenase, known microorganisms that produce each of the above enzymes, recombination into which a glycerol dehydratase gene and / or diol dehydratase gene has been introduced. A microorganism and a recombinant microorganism into which a hydroxyaldehyde dehydrogenase gene has been introduced can be used. Preferably, a recombinant microorganism into which each gene is introduced is used.

  The introduction of the glycerol dehydratase gene and the diol dehydratase gene is carried out by introducing genes encoding three kinds of large subunit, medium subunit and small subunit constituting each enzyme. In addition, the glycerol dehydratase reactivation factor gene and the diol dehydratase reactivation factor gene are introduced by introducing genes encoding the large subunit and the small subunit constituting each enzyme, respectively.

  The nucleotide sequence of the gene encoding the large subunit of glycerol dehydratase is SEQ ID NO: 1, the nucleotide sequence of the gene encoding the medium subunit is SEQ ID NO: 3, and the nucleotide sequence of the gene encoding the small subunit is SEQ ID NO: 5. Illustrate. The base sequence of the gene encoding the large subunit of diol dehydratase is SEQ ID NO: 7, the base sequence of the gene encoding the medium subunit is SEQ ID NO: 9, and the base sequence of the gene encoding the small subunit is SEQ ID NO: 11 illustrates. Further, the base sequence of the gene encoding the large subunit of the glycerol dehydratase reactivation factor is shown in SEQ ID NO: 13, the base sequence of the gene encoding the small subunit is shown in SEQ ID NO: 15, and the large subunit of the diol dehydratase reactivation factor is shown. The base sequence of the gene encoding the unit is exemplified in SEQ ID NO: 17, and the base sequence of the gene encoding the small subunit is exemplified in SEQ ID NO: 19.

  Here, the gene encoding the large subunit of glycerol dehydratase, the gene encoding the medium subunit, and the gene encoding the small subunit are genes comprising the nucleotide sequences represented by SEQ ID NOs: 1, 3 and 5, respectively. And functionally equivalent genes are included. The same applies to the gene encoding each subunit of diol dehydratase. “Functionally equivalent gene” means that the protein encoded by the target gene has the same biological function and biochemical properties as the protein encoded by the gene consisting of the base sequence represented by each SEQ ID NO. It means having a function.

  Methods well known to those skilled in the art for preparing genes encoding proteins that are functionally equivalent to a protein include hybridization techniques (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring). Harbor Lab.press (1989)).

  For example, in the full length of the base sequence represented by SEQ ID NO: 1, by various artificial treatments such as site-directed mutagenesis, random mutagenesis by mutagen treatment, mutation, deletion, ligation, etc. , Even if the sequence is partially changed, these mutant genes hybridize under stringent conditions with a gene consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1, The activity of the large subunit of glycerol dehydratase, i.e. included in the gene encoding the large subunit of glycerol dehydratase, as long as it encodes a protein that exhibits glycerol dehydratase activity together with the medium subunit and small subunit of glycerol dehydratase It is. The same applies to genes encoding other subunits and genes encoding the subunits of diol dehydratase, glycerol dehydratase reactivation factor and diol dehydratase reactivation factor.

  Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed, that is, conditions in which DNA having high homology to each gene hybridizes. More specifically, such conditions include hybridization at 42-68 ° C. in the presence of 0.5-1 M NaCl, 42 ° C. in the presence of 50% formamide, or 65-68 ° C. in an aqueous solution. Thereafter, the filter can be washed at room temperature to 68 ° C. using a SSC (saline sodium citrate) solution having a concentration of 0.1 to 2 times.

  Under stringent conditions as described above, at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, even more preferably at least 99% identity with the subject base sequence. A gene consisting of a base sequence having identity can hybridize with a gene consisting of a base sequence complementary to the target base sequence.

  The gene encoding the large subunit of glycerol dehydratase includes the gene encoding the protein consisting of the amino acid sequence of SEQ ID NO: 2, and the gene encoding the medium subunit includes the protein consisting of the amino acid sequence of SEQ ID NO: 4. The gene encoding the small subunit includes the gene encoding the protein consisting of the amino acid sequence of SEQ ID NO: 6. The gene encoding the large subunit of diol dehydratase includes the gene encoding the protein consisting of the amino acid sequence of SEQ ID NO: 8, and the gene encoding the medium subunit encodes the protein consisting of the amino acid sequence of SEQ ID NO: 10. The gene encoding the small subunit includes the gene encoding the protein consisting of the amino acid sequence of SEQ ID NO: 12. The gene encoding the large subunit of the glycerol dehydrator reactivation factor includes a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 14, and the gene encoding the small subunit includes the amino acid sequence of SEQ ID NO: 16. A gene encoding a protein consisting of The gene encoding the large subunit of the diol dehydratase reactivation factor includes a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 18, and the gene encoding the small subunit is derived from the amino acid sequence of SEQ ID NO: 20. The gene encoding the protein is included.

  Each gene includes a gene encoding a protein that is functionally equivalent to a protein comprising each amino acid sequence. For example, a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2 consists of an amino acid sequence in which one or several amino acids are deleted, added, inserted or substituted in the amino acid sequence of SEQ ID NO: 2. A protein that exhibits the activity of glycerol dehydratase together with the medium subunit and small subunit of glycerol dehydratase.

  Here, the side chains of amino acids that constitute protein components differ in hydrophobicity, charge, size, etc., but substantially affect the three-dimensional structure (also referred to as a three-dimensional structure) of the entire protein. Some relationships that are highly conserved in the sense of being absent are known empirically and by physicochemical measurements. For example, examples of conservative substitution between different amino acid residues include glycine (Gly) and proline (Pro), glycine and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid ( Amino acids such as Glu) and glutamine (Gln), aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), threonine and serine (Ser) or alanine, lysine (Lys) and arginine (Arg) The substitution between is known.

  Therefore, even in the case of a mutant protein consisting of an amino acid sequence obtained as a result of deletion, addition, insertion or substitution of one or several amino acids in the amino acid sequence of SEQ ID NO: 2, the mutation is changed to SEQ ID NO: 2. If the mutation is highly conserved in the three-dimensional structure of the described amino acid sequence and the mutant protein has the activity of the large subunit of glycerol dehydratase, the genes encoding these mutant proteins also It is also included in the gene encoding the large subunit of glycerol dehydratase. Here, the term “several” means usually 2 to 5, preferably 2 to 3.

  In addition, the gene encoding the large subunit of glycerol dehydratase has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, even more preferably, with the amino acid sequence of SEQ ID NO: 2. A gene encoding a protein consisting of an amino acid sequence having at least 99% identity and having the activity of the large subunit of glycerol dehydratase is also included.

  The same applies to genes encoding other subunits and genes encoding the subunits of diol dehydratase, glycerol dehydratase reactivation factor and diol dehydratase reactivation factor.

  As long as the gene encoding each subunit is expressed in the same host, it may be introduced into the genome, introduced into the same vector, or transformed, or introduced into separate vectors. Conversion may be performed. Also. Each subunit is preferably derived from the same species or the same strain.

  The nucleotide sequence of the hydroxyaldehyde dehydrogenase gene is exemplified in SEQ ID NOs: 21 and 23. Here, the hydroxyaldehyde dehydrogenase gene includes a gene functionally equivalent to the gene consisting of the base sequence represented by SEQ ID NO: 21 or 23. That is, in the full length of the base sequence represented by SEQ ID NO: 21 or 23, various artificial treatments such as site-directed mutagenesis, random mutation by mutagen treatment, mutation, deletion, ligation of nucleic acid fragments by restriction enzyme cleavage Even if the sequence is partially changed by the above, these mutant genes are under stringent conditions with a gene consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 21 or 23. As long as it hybridizes and encodes a protein that exhibits hydroxyaldehyde dehydrogenase activity, it is included in the hydroxyaldehyde dehydrogenase gene. The stringent conditions are as described above.

  The hydroxyaldehyde dehydrogenase gene includes a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 22 or 24. And the gene which codes a protein functionally equivalent to the protein which consists of an amino acid sequence of sequence number 22 or 24 is included. Therefore, as a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 22 or 24, one or several amino acids are deleted, added, inserted or substituted in the amino acid sequence of SEQ ID NO: 22 or 24 A protein having an amino acid sequence, which shows the activity of hydroxyaldehyde dehydrogenase. Here, the term “several” means usually 2 to 5, preferably 2 to 3. Also, the hydroxyaldehyde dehydrogenase gene has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 99% identity with the amino acid sequence of SEQ ID NO: 22 or 24. Also included is a gene that encodes a protein comprising an amino acid sequence having identity and exhibiting the activity of hydroxyaldehyde dehydrogenase.

  The gene can be introduced into a microorganism by linking the above gene or a part thereof to an appropriate vector and introducing the obtained recombinant vector into a host so that the target gene can be expressed, or by homologous recombination. Can be carried out by inserting the gene of interest or a part thereof at an arbitrary position on the genome. “Part” refers to a part of each gene capable of expressing the protein encoded by each gene when introduced into a host. In the present invention, the gene includes DNA and RNA, preferably DNA.

  A method for obtaining a desired gene from the genome of a microorganism by cloning is well known in the field of molecular biology. For example, when the gene sequence is known, a suitable genomic library can be prepared by restriction endonuclease digestion and screened using a probe complementary to the desired gene sequence. Once the sequence has been isolated, the DNA is amplified using standard amplification methods such as the polymerase chain reaction (PCR) (US Pat. No. 4,683,202) to obtain a quantity suitable for transformation. Can do. In addition to known genes, genes such as glycerol dehydratase gene, diol dehydratase gene, and hydroxyaldehyde dehydrogenase gene are hybridization methods, PCR methods, etc. using synthetic primers appropriately designed based on the base sequences of known genes Can also be obtained.

  Methods such as preparation of genomic DNA library used for gene cloning, hybridization, PCR, plasmid preparation, DNA cleavage and ligation, transformation and the like are described in Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58. , Cold Spring Harbor Lab.press (1989).

  The vector to which the gene is linked is not particularly limited as long as it can replicate in the host, and examples thereof include plasmids, phages and cosmids. Examples of the plasmid include pHSG398, pUC18, pBR322, pSC101, pUC19, pUC118, pUC119, pACYC117, pBluescript II SK (+), pETDuet-1, pACYCDuet-1, and the like. , EMBL-, M13mp18, M13mp19 and the like.

  In the above vector, in order to ensure that the inserted gene is expressed, an appropriate expression promoter is connected upstream of the gene. The expression promoter to be used is not particularly limited, and may be appropriately selected by those skilled in the art depending on the host. For example, when the host is Escherichia coli, T7 promoter, lac promoter, trp promoter, λ-PL promoter and the like can be used, and when the host is a Bacillus bacterium, an SPO promoter or the like can be used.

  The host is not particularly limited as long as it can express the target gene.For example, Ralstonia, Pseudomonas, Bacillus, Escherichia, Propionibacterium, Lactobacillus, Salmonella, Klebsiella, Acetobacterium, Flavobacterium Genus, Citrobacter, Agrobacterium, Anabaena, Bradyrhizobium, Brucella, Chlorobium, Clostridium, Corynebacterium, Fusobacterium, Geobacter, Gloeobacter, Leptospira, Mycobacterium, Mycobacterium, Porphydus, Porphydus Prochlorococcus sp , Microorganisms belonging to the genus Sulfolobus and Thermoplasma, specifically, Escherichia coli, Acetobacte rium sp., Citrobacter freundii, Flavobacterium sp., Ralstonia solanacearum, Ralstonia eutropha, Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas denitrificans, Bacillus subtilis, Bacillus megaterium, Propionibacterium acidipropionici, Propines Propionibacterium jensenii, Propionibacterium microaephilum, Propionibacterium propionicum, Propionibacterium thoenii, Propionibacterium freudenreichii, Agrobacterium tumefaciens, Anabaena sp., Bradyrhizobium japonicum, Brucella melitensis, Brucella suis, Chlorobium tritridium, Loslotritridium, sulfurreducens, Gloeobacter violaceus, Leptospira interrogans, Mycobacterium bovis, Mycobacterium tuberculosis, Photorhabdus luminescens, Porphyromonas gingivalis, P rochlorococcus marinus, Rhodobacter capsulatus, Rhodopseudomonas palustris, Sinorhizobium meliloti, Streptomyces avermitilis, Streptomyces coelicolor, Synechococcus sp., Thermosynechococcus elongatus, Treponema denticola, Archaeoglobus fulgidus, Halobacterium sp, Mesorhizobium loti, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Methanopyrus kandleri, Methanosarcina acetivorans, Methanosarcina mazei, Pyrobaculum aerophilum, Sulfolobus solfataricus, Sulfolobus tokodaii, Thermoplasma acidophilum, Thermoplasma volcanium.

  In addition, yeast belonging to the genus Saccharomyces such as Saccharomyces cerevisiae, yeast belonging to the genus Candida such as Candida maltosa, animal cells such as COS cells, CHO cells, mouse L cells, rat GH3 and human FL cells, insect cells such as SF9 cells, etc. Can be used.

  In one embodiment, it is preferable to use a host having a coenzyme B12 synthesis system. For example, Lactobacillus, Salmonella, Klebsiella, Propionibacterium, Agrobacterium, Anabaena, Bacillus, Bradyrhizobium, Brucella, Chlorobium, Clostridium, Corynebacterium, Fusobacterium, Geobacter, Gloepobacter, Mycobacterium genus Mycobacterium genus Photorhabdus genus Porphyromonas genus Prochlorococcus genus Pseudomonas genus Ralstonia genus Rhodobacter genus Rhodopseudomonas genus A microorganism belonging to the genus Methanobacterium, Methanococcus, Methanopyrus, Methanosarcina, Methanosarcina, Pyrobaculum, Sulfolobus, or Thermoplasma is used as a host. Alternatively, a host into which a coenzyme B12 synthetic gene has been introduced by recombination may be used.

  As host cells, it is preferable to use host cells that do not express glycerol dehydrogenase, that is, cells that do not have a glycerol dehydrogenase gene and cells in which a glycerol dehydrogenase gene has been knocked out. By using these, the pathway in which glycerin is oxidized and converted to dihydroxyacetone can be blocked, and 3-hydroxypropionic acid can be produced in a higher yield. As a method for knocking out the glycerol dehydrogenase gene, a known method can be used. Specifically, the knockout bacterium uses a glycerol dehydrogenase gene in a cell as a target gene and destroys the gene using a vector (targeting vector) that causes homologous recombination at an arbitrary position of the target gene (gene targeting). Method), a method of destroying the gene by inserting a trap vector (reporter gene without a promoter) at an arbitrary position of the target gene and losing its function (gene trap method), a method combining them, etc. It is produced by disrupting the glycerol dehydrogenase gene in the cells using a method used for producing knockout cells, transgenic animals (including knockout animals) and the like in the technical field. The position at which homologous substitution occurs or the position at which the trap vector is inserted is not particularly limited as long as it causes a mutation that results in loss of expression of the glycerol dehydrogenase gene, but it preferably replaces the transcriptional regulatory region, more preferably the second exon. . Other methods for knocking out the glycerol dehydrogenase gene include a method of introducing a vector expressing an antisense cDNA of the glycerol dehydrogenase gene and a method of introducing a vector expressing a double-stranded RNA of the glycerol dehydrogenase gene into a cell. It is done. Such vectors include viral vectors, plasmid vectors, etc., and based on conventional genetic engineering techniques, for example, Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab.press (1989 ) And other basic documents. Alternatively, a commercially available vector can be cleaved with an arbitrary restriction enzyme, and a desired gene or the like can be incorporated and semi-synthesized.

  The method for introducing the vector into the host is not particularly limited, and examples thereof include an electric pulse method, a method using calcium ions, a protoplast method, and an electroporation method.

  In the method of inserting a target gene at an arbitrary position on the genome by homologous recombination, the target gene is inserted into a sequence homologous to the sequence on the genome together with a promoter, and this nucleic acid fragment is introduced into the cell by electroporation. Can be carried out by causing homologous recombination. At the time of introduction into the genome, a strain in which homologous recombination has occurred can be easily selected by using a nucleic acid fragment in which a target gene and a drug resistance gene are linked. In addition, a gene linked to a drug resistance gene and a gene that becomes lethal under specific conditions is inserted into the genome by homologous recombination by the above method, and then becomes lethal under specific conditions with the drug resistance gene. The target gene can also be introduced using homologous recombination in the form of replacing the gene.

  A method for selecting a recombinant microorganism into which a target gene has been introduced is not particularly limited, but a method by which only a recombinant microorganism into which a target gene has been introduced can be easily selected is preferable.

  In one embodiment, the enzymatic reaction of the present invention is carried out by contacting glycerol with a microorganism that produces glycerol dehydratase and / or diol dehydratase and hydroxyaldehyde dehydrogenase or a processed product thereof. As such a microorganism, a recombinant microorganism containing a glycerol dehydratase gene and / or a diol dehydratase gene and a hydroxyaldehyde dehydrogenase gene can be used. The microorganism preferably further contains a glycerol dehydratase reactivation factor gene and / or a diol dehydratase reactivation factor gene. Alternatively, in addition to a microorganism that produces glycerol dehydratase and / or diol dehydratase and hydroxyaldehyde dehydrogenase or a processed product thereof, a microorganism that contains a glycerol dehydratase reactivating factor gene and / or a diol dehydratase reactivating factor gene or a processed product thereof The enzyme reaction may be carried out by contacting the enzyme active substance with glycerin. The gene introduction method and the method for producing a recombinant microorganism such as a host have already been described.

  By carrying out an enzymatic reaction using such a microorganism or a processed product thereof, the enzymatic reaction by glycerol dehydratase and / or diol dehydratase and the enzymatic reaction by hydroxyaldehyde dehydrogenase are carried out in the same reaction system, Damage to enzyme-producing microorganisms due to accumulation of certain 3-hydroxypropionaldehyde and inactivation of the enzyme can be prevented.

  In the present invention, 3-hydroxypropionic acid is produced by bringing the above-mentioned microorganism or a processed product thereof, or an enzyme active substance containing a plurality of microorganisms or a processed product thereof into contact with glycerin, and 3-hydroxypropionic acid in the reaction product Can be accumulated and 3-hydroxypropionic acid can be collected.

  The contact of the microorganism or the processed product thereof or the enzyme active substance containing them with glycerin means culturing the microorganism or the processed product in the presence of glycerin, or reacting with the processed product of the microorganism of the present invention. To include. Examples of the treated products include cells treated with acetone, toluene, etc., fungus bodies, freeze-dried cells, disrupted cells, cell-free extracts obtained by disrupting cells, crude enzyme solutions obtained by extracting enzymes from these, purification An enzyme etc. are mentioned. Moreover, the microbial cell fixed to the support | carrier by the conventional method, this processed material, an enzyme, etc. can also be used.

  The culture medium and culture conditions used for the culture can be appropriately set according to the type of microorganism to be used, but a normal medium containing a nitrogen source, inorganic ions and other organic micronutrients as necessary, for example, 2 culture media Etc. can be used. The culture is performed under conditions suitable for the growth of the microorganism to be used. Usually, it is carried out at a culture temperature of 10 ° C to 45 ° C for 16 to 96 hours. In addition, when a thermophilic bacterium is used as a host, it can be cultured at a culture temperature of 42 ° C to 60 ° C. When the culture is continuously performed, the culture is performed for 1 week to 3 months.

The enzymatic reaction by hydroxyaldehyde dehydrogenase is a nicotinamide nucleotide (NAD ⁺ ) -dependent reaction, but by performing culture under aerobic conditions, an oxidation reaction of NADH + H ⁺ → NAD ⁺ is performed in the respiratory chain, The enzymatic reaction with hydroxyaldehyde dehydrogenase can be carried out continuously (see FIG. 1).

It is also possible to carry out fermentation by aerobic culture, increasing the amount of cells and then culturing under anaerobic conditions, giving glycerin. In that case, the oxidation reaction of NADH + H ⁺ → NAD ⁺ in the respiratory chain is not carried out, but by carrying out the reduction reaction of 3-hydroxypropionaldehyde to 1,3-propanediol in parallel with alcohol dehydrogenase, The enzymatic reaction with aldehyde dehydrogenase can be carried out continuously (see FIG. 2).

  The reduction reaction of 3-hydroxypropionaldehyde to 1,3-propanediol by alcohol dehydrogenase can be carried out using a microorganism that produces alcohol dehydrogenase or a processed product thereof. Microorganisms used in the enzyme reaction of the present invention, that is, microorganisms producing glycerol dehydratase and / or diol dehydratase, microorganisms producing hydroxyaldehyde dehydrogenase, or glycerol dehydratase gene and / or diol dehydratase gene and hydroxyaldehyde dehydrogenase gene An alcohol dehydrogenase gene may be introduced into the recombinant microorganism. Moreover, you may use the microorganisms which produce alcohol dehydrogenase, and the thing which introduce | transduced the alcohol dehydrogenase gene as a host of recombinant microorganisms.

  The alcohol dehydrogenase is not particularly limited as long as it is a protein that catalyzes the reduction reaction of 3-hydroxypropionaldehyde to 1,3-propanediol, and a known one can be used. Specific examples include those derived from bacteria belonging to the genus Lactobacillus, Citrobacter, Clostridium, Klebsiella, Enterobacter, Caloramator, Salmonella, Listeria and the like. These alcohol dehydrogenases may be used alone or as a mixture of two or more. Preferably, propanol dehydrogenase is used. The base sequence of SEQ ID NO: 25 is exemplified as the base sequence of the propanol dehydrogenase gene. The propanol dehydrogenase gene also includes a gene functionally equivalent to the gene consisting of the nucleotide sequence of SEQ ID NO: 25.

  By carrying out the enzyme reaction with alcohol dehydrogenase in parallel under anaerobic conditions as described above, both 3-hydroxypropionaldehyde and 1,3-propanediol can be produced. 1,3-propanediol is a useful monomer used in the production of polyester fibers and in the production of polyurethane and cyclic compounds. According to this embodiment, two useful substances can be efficiently produced.

In biological systems, glycerin is converted to 1,3-propanediol via a two-stage enzyme-catalyzed reaction, and in the first stage, glycerol dehydratase converts glycerin to 3-hydroxypropionaldehyde and water, In the stage, 3-hydroxypropionaldehyde is reduced to 1,3-propanediol by NAD ⁺ -linked oxidoreductase. However, since the production of 1,3-propanediol from glycerin in biological systems is generally performed under anaerobic conditions with glycerin as the sole carbon source and in the absence of other exogenous reducing equivalent acceptors, Initially, a parallel pathway for glycerin, which is the oxidation of glycerin to dihydroxyacetone (DHA) by NAD ⁺ -(or NADP ⁺ -)-linked glycerol dehydrogenase (glycerin + NAD ⁺ → DHA + NADH + H) works, and DHA is dihydroxyacetone phosphorylated by DHA kinase. In the conventional method for producing 1,3-propanediol using microorganisms, half of the glycerin as a raw material is consumed in the parallel pathway because it is phosphorylated into an acid and used for biosynthesis and ATP production. , Product yield relative to raw glycerin However, there was a problem in efficiency and cost. However, by adopting the above embodiment, it is possible to reduce the loss of raw material glycerin and produce 3-hydroxypropionic acid together with 1,3-propanediol.

  The pH in the culture of microorganisms is adjusted using a reagent that does not interfere with the growth of the host and does not interfere with the separation of the acid from the culture solution. For the pH adjustment, an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used. Sodium carbonate, ammonia, a sodium ion source such as sodium chloride may be added. Moreover, you may use common alkali reagents, such as sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, ammonium hydroxide aqueous solution, calcium hydroxide aqueous solution, potassium carbonate aqueous solution, sodium carbonate aqueous solution, potassium acetate aqueous solution. During the culture period, the pH is 5.0 or more, preferably 5.5 or more and 10.0 or less, preferably 9.7 or less.

  Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, as well as peptone, meat extract, yeast extract, corn steep liquor, and the like. Examples of inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.

  During the culture, antibiotics such as kanamycin, ampicillin, and tetracycline may be added to the medium. When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer can be added to the medium. For example, isopropyl-β-D-thiogalactopyranoside (IPTG), indoleacetic acid (IAA), or the like can be added to the medium.

  Alternatively, cells are collected from the microorganism culture obtained above by centrifugation or the like, and suspended in an appropriate buffer or medium. 3-hydroxypropionic acid can also be produced by suspending this bacterial cell suspension in a buffer containing glycerin and carrying out the reaction. The reaction conditions are, for example, a reaction temperature of 10 to 80 ° C., preferably 15 to 50 ° C., a reaction time of 5 minutes to 96 hours, preferably 10 minutes to 72 hours, and a pH of 5.0 or more, preferably 5. It is 5 or more and 10.0 or less, preferably 9.7 or less. When the reaction is carried out continuously, the reaction is carried out for 1 week to 3 months.

  Methods for purifying 3-hydroxypropionic acid are well known in the art. For example, 3-hydroxypropionic acid can be obtained from the culture medium by subjecting the reaction mixture to extraction with an organic solvent, distillation and column chromatography (US Pat. No. 5,356,812). Further, it is preferable to concentrate the fermentation broth with an ultrafiltration membrane or a zeolite separation membrane that allows only low molecules such as water to pass through. By concentrating, the energy for evaporating water can be reduced. 3-hydroxypropionic acid can also be identified directly by subjecting the medium to high pressure liquid chromatography (HPLC) analysis. The same applies to the purification of 1,3-propanediol.

  In one embodiment, the present invention relates to a method for producing acrylic acid by dehydrating 3-hydroxypropionic acid obtained by the above method. 3-hydroxypropionic acid can be dehydrated by a known reaction. For example, as described in US2469701, 3-hydroxypropionic acid can be easily converted to acrylic acid by distillation under reduced pressure in the presence of a catalyst.

  The present invention also relates to a composition for producing 3-hydroxypropionic acid from glycerin comprising glycerol dehydratase and / or diol dehydratase and hydroxyaldehyde dehydrogenase. The said composition can be manufactured from the microorganisms which produce these enzymes, or its processed material.

  Glycerin aqueous solution containing impurities discharged as a by-product in the surfactant industry and biodiesel fuel industry has a problem as a raw material for chemical synthesis, but there is no problem when used as a raw material for enzyme reaction. The present invention capable of producing a useful substance using as a raw material is also excellent from the viewpoint of effective use of waste.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, the scope of the present invention is not limited to the range of an Example.

Example 1 Cloning of Klebsiella pneumoniae Glycerol Dehydratase Gene The glycerol dehydratase gene (GD gene) was amplified by PCR using the Klebsiella pneumoniae genome as a template and the following two primers. The ends of the fragments were cleaved with restriction enzymes NdeI and BglII, and the cleaved fragments were recovered by electrophoresis.

Forward primer:
5'-GGGGGGCCATATGAAAAGATCAAAACGATTTGCAGTACTG-3 '
Reverse primer:
5'-CCCCGGATCCTTAGCTTCCTTTACGCAGCTTATGCCGCTGC-3 '

  The vector sequence was amplified with the following two primers using the pHSG398 plasmid as a template, and a DNA fragment having an NdeI site behind the lac promoter of the pHSG398 plasmid and a BamHI site at the start codon position of the lacZ gene was amplified.

Forward primer:
5'-CCCCCCCATATGTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAATATACGAGCC-3 '
Reverse primer:
5'-CCCCGGATCCTTAGTTAAGCCAGCCCCGACACCCGCCAACACC-3 '

  The amplified fragment was cleaved with restriction enzymes BamHI and NdeI, and the cleaved fragment was separated and recovered by electrophoresis. These two DNA fragments were ligated, introduced into E. coli TOP10 competent cells, spread on ampicillin-containing plates and cultured. A plasmid was extracted from the obtained transformant, cleaved with the restriction enzyme NdeI, and the size of the cleaved fragment was confirmed by electrophoresis. The size was the same as the total size of the original two DNA fragments used for ligation. I understood it. One of the transformants was named GD / pHSG398 / TOP10.

(Example 2) Preparation of crude glycerol dehydratase enzyme solution of Klebella pneumoniae The above transformant was cultured at 37 ° C for 16 hours in 5 ml of a liquid medium containing 50 ppm of chloramphenicol in LB medium, and chloramphenicol was cultured in LB medium. The whole amount was inoculated into 50 ml of a medium supplemented with 50 ppm and IPTG 1 mM, and cultured at 37 ° C. for 12 hours. The culture was centrifuged and the cells were collected as a pellet. The obtained microbial cell was about 0.5 g.

The cells were suspended in 0.1 M potassium phosphate buffer (pH 8.0) so that the absorption of OD ₆₆₀ was 10, and the cells were treated with an ultrasonic crusher for 5 minutes to disrupt the cells. The crushed material was centrifuged, and the supernatant was collected to obtain a glycerol dehydratase crude enzyme solution.

(Example 3) Cloning of aldA gene of Escherichia coli K-12 strain Using the genome of Escherichia coli K-12 W3110 strain as a template, the aldA gene was amplified by PCR using the following two primers, and the ends of the amplified fragments were restricted. Cleaved with enzymes NdeI and BglII, and the cleaved fragments were recovered by electrophoresis.

Forward primer:
5'-GGGGGGCCATATGTCAGTACCCGTTCAACATCCTATG-3 '
Reverse primer:
5'-CCCCAGATCTTTAAGACTGTAAATAAACCACCTGGGTC-3 '

  The vector sequence was amplified with the following two primers using the pUC18 plasmid as a template, and a DNA fragment having an NdeI site behind the lac promoter of the pUC18 plasmid and a BamHI site at the position of the start codon of the lacZ gene was amplified.

Forward primer:
5'-CCCCCCCATATGTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAATATACGAGCC-3 '
Reverse primer:
5'-CCCCGGATCCTTAGTTAAGCCAGCCCCGACACCCGCCAACACC-3 '

  The amplified fragment was cleaved with restriction enzymes BamHI and NdeI, and the cleaved fragment was separated and recovered by electrophoresis. These two DNA fragments were ligated, introduced into E. coli TOP10 competent cells, spread on ampicillin-containing plates and cultured. A plasmid was extracted from the obtained transformant, cleaved with the restriction enzyme NdeI, and the size of the cleaved fragment was confirmed by electrophoresis. As a result, 1 of the same size as the original two DNA fragments (total) used for ligation was obtained. The band of the book was able to be confirmed. One of the transformants was named aldA / pUC18 / TOP10.

(Example 4) Preparation of aldA crude enzyme solution of Escherichia coli K-12 strain The above transformant was cultured at 37 ° C. for 16 hours in 5 ml of a liquid medium containing 100 ppm of ampicillin in LB medium, and 100 ppm of ampicillin and IPTG of 1 mM were added to the LB medium. The whole amount was inoculated into 50 ml of the prepared medium and cultured at 37 ° C. for 12 hours. The culture was centrifuged and the cells were collected as a pellet. The obtained microbial cell was about 0.5 g.

The cells were suspended in 0.1 M potassium phosphate buffer (pH 8.0) so that the absorption of OD ₆₆₀ was 10, and the cells were treated with an ultrasonic crusher for 5 minutes to disrupt the cells. The crushed material was centrifuged and the supernatant was recovered to obtain an aldA crude enzyme solution.

(Example 5) Cloning of aldB gene of Escherichia coli K-12 strain Using the genome of Escherichia coli K-12 W3110 as a template, the aldB gene was amplified by PCR using the following two primers, and the ends of the amplified fragments were restricted. Cleaved with enzymes NdeI and BamHI, and the cleaved fragments were recovered by electrophoresis.

Forward primer:
5'-GGGGGGCCATATGACCAATAATCCCCCTTCAGCACAG-3 '
Reverse primer:
5'-CCCCGGATCCTCAGAACAGCCCCAACGGTTTATCC-3 '

  Similarly to the cloning of the aldA gene, a PCR-amplified DNA fragment using pUC18 as a template and the aldB gene fragment were ligated, introduced into an E. coli TOP10 competent cell, spread on an ampicillin-containing plate and cultured. A plasmid was extracted from the obtained transformant, cleaved with restriction enzymes BamHI and NdeI, and the size of the cleaved fragment was confirmed by electrophoresis. As a result, two DNA fragments of the same size as the original two DNA fragments used for ligation were obtained. I was able to confirm the band. One of the transformants was named aldB / pUC18 / TOP10.

(Example 6) Preparation of aldB crude enzyme solution of Escherichia coli K-12 strain The above transformant was cultured at 37 ° C. for 16 hours in 5 ml of liquid medium containing 100 ppm of ampicillin in LB medium, and 100 ppm of ampicillin and 1 mM of IPTG were added to LB medium. The whole amount was inoculated into 50 ml of the prepared medium and cultured at 37 ° C. for 12 hours. The culture was centrifuged and the cells were collected as a pellet. The obtained microbial cell was about 0.5 g.

The cells were suspended in 0.1 M potassium phosphate buffer (pH 8.0) so that the absorption of OD ₆₆₀ was 10, and the cells were treated with an ultrasonic crusher for 5 minutes to disrupt the cells. The crushed material was centrifuged, and the supernatant was recovered to obtain an aldB crude enzyme solution.

(Example 7) Measurement of enzyme activity of glycerol dehydratase (1) The activity of glycerol dehydratase was confirmed using 1.2-propanediol.
First, the following reagents were prepared, and samples were prepared with the reaction solution compositions described in the following table.
(a) Buffer A: 50 mM potassium phosphate buffer (pH 8.0)
(b) 1M 1.2-propanediol (1M PDO)
(c) 0.5M KCl
(d) Adenosylcobalamin 38 mg / 10 ml (AdpB12)
(Weighed in a dark room, dissolved in water, dispensed 1.5 ml each and stored frozen)
(e) 0.1M potassium citrate buffer (pH 3.5, for stopping reaction)
(f) 1M glycerin
(g) 0.1% MBTH aqueous solution (3-methyl-2-benzosiazolinone hydrazone hydrochloride, for coloring)

  (V) other than (V) was added to a 1.5 ml tube, mixed, moved to a dark room, and (V) was added. The tube was shielded from light with aluminum foil, placed in a 37 ° C. bath, and allowed to react for 10 minutes. The reaction was stopped for 1 minute on ice and 1 ml of 0.1 M potassium citrate buffer was added. 0.2 ml of this reaction solution was diluted to 2 ml with 0.1 M potassium citrate buffer.

  To the obtained diluted solution, 0.5 ml of 0.1% MBTH solution was added, and color was developed by heating at 37 ° C. for 15 minutes. 0.5 ml of this color developing solution was diluted to 5 ml with water, and absorption at 305 nm was measured. The results are shown in the following table and FIG.

  As shown in FIG. 3, in the reaction using 1.2-propanediol as a substrate, the amount of enzyme and the absorbance are in a linear relationship, and the crude enzyme solution prepared from glycerol dehydratase-expressing cells is dehydrated using 1.2-propanediol as a substrate. It was confirmed that the reaction was catalyzed and that the amount of enzyme and aldehyde formation were in a clean linear relationship.

(2) Activity confirmation with glycerol The activity of glycerol dehydratase was similarly measured using glycerol instead of 1.2-propanediol.

  The results are shown in the following table and FIG.

  As shown in FIG. 4, a clean linear relationship is obtained even when glycerol is used as a substrate. The crude enzyme solution prepared from glycerol dehydratase-expressing cells catalyzes the dehydration reaction using glycerol as a substrate, and the amount of enzyme and aldehyde production are clean. It was confirmed that

(Example 8) Confirmation of expression of aldA and aldB and measurement of enzyme activity Confirmation of enzyme activity of aldA and aldB should be performed with 3-hydroxypropionaldehyde, but since there is no commercial preparation of 3-hydroxypropionaldehyde, aldA The activity was confirmed using glycol aldehyde, which has been confirmed to be oxidizable as a substrate, as a substrate. Moreover, since there is no literature which measured the activity of aldB of E. coli, both were evaluated by the method for measuring the activity of aldA.

Preparation of crude enzyme solution:
About 0.9 g of the cryopreserved cells were suspended in 0.1 M glycine Na buffer (pH 9.5) to make 10 ml. Each 0.33 ml was dispensed into an Eppendorf tube and pulverized at a sonication of 30 seconds / 5 seconds interval × 15 minutes. The supernatant was collected as a crude enzyme solution by centrifugation at 15000 rpm for 10 minutes.

Preparation of reaction solution:
A reaction solution was prepared by adding 1/100 amount of 0.1M glycol aldehyde and 0.25M NAD to 0.1M glycine Na buffer (pH 9.5) at room temperature. 2 ml of the reaction solution was placed in the spectrophotometer cell, and an appropriate amount of enzyme solution was added to start the reaction.

  Generation of NADH was performed by following the absorption at 340 nm every 10 seconds for 2 minutes. The blank used water instead of the enzyme solution. The result of aldA is shown in FIG. 5, and the result of aldB is shown in FIG. In FIG. 6, “2 ml + 50 μl” refers to a system in which 50 μl of enzyme solution is added to 2 ml of reaction solution.

  In the crude enzyme solution prepared from the aldA-expressing cells, the reaction rate increased by about 2 times when the amount of the enzyme was increased, and it was confirmed that NADH was produced along with the oxidation of glycolaldehyde. Therefore, it was confirmed that aldA caused oxidation reaction of aldehyde. AldB was also less active than aldA, but aldehyde oxidation was confirmed. aldA was considered more suitable for the oxidation of 3-hydroxypropionaldehyde than aldB.

(Example 9) Production of 3-hydroxypropionic acid from glycerin A crude enzyme solution was prepared by sonication from glycerol dehydratase-expressing cells and aldA-expressing cells, each of which has been confirmed to have activity, and 3-hydroxypropion from glycerol The acid was produced.

  Buffers containing aldA-expressing cells (aldA2 strain cultured in 2 medium + Ap + IPTG for 10 hours) and glycerol dehydratase expressing cells (GD-GDR-BglII-15 strain cultured in 2 media + Ap + IPTG for 10 hours) at a concentration of about OD10 It was suspended in A, dispensed 1 ml at a time, and pulverized for 5 minutes with an sonicator. The treated product was centrifuged at 15000 rpm for 10 minutes, and the supernatant was used as a crude enzyme solution.

  After adding 1/20 amount of 150 μM NAD to buffer A and adding the substrate, enzyme solution, etc., the reaction was started by adding adenosylcobalamin under dark conditions. As a control, glycerol dehydratase was not added, and conditions under which no aldehyde was produced were used.

  The addition amount of adenosylcobalamin was set to 1/10 of the concentration used for measuring the activity of glycerol dehydratase from the result of spectrum check in the control. The reaction solution composition is shown below.

  Absorption at 340 nm was measured every 10 seconds for up to 4 minutes. The results are shown in FIG. Absorption started to increase after the start of the reaction, and a curve started from about one minute later. It can be seen that the aldehyde dehydrogenation progressed as the aldehyde was produced. The slope of the rising portion of the curve was 1.377 A340 / min. It can be seen that NAD was reduced to NADH by dehydrogenation of the aldehyde. When the product was confirmed under the following conditions, the peak of 3-hydroxypropionaldehyde which is a reaction intermediate is located at 4.5 minutes, and the peak of 3-hydroxypropionic acid which is a product is located at 5.2 minutes. I was able to confirm.

Analytical conditions for high performance liquid chromatography:
Column: Shiseido Capsule Pack C-18 UG120 4.6mm diameter x 150mm
Mobile phase: 0.1% trifluoroacetic acid Flow rate: 0.5 ml / min

  From the above results, it was shown that 3-hydroxypropionic acid can be synthesized from glycerin using glycerol dehydratase (GD) and hydroxyaldehyde dehydrogenase (aldA).

Example 10 Reaction Using Crude Enzyme Solution Prepared from Cells Introduced with Glycerol Dehydratase Gene and aldA Gene into Escherichia coli The GD / pHSG398 plasmid prepared in Example 1 was introduced into aldA / pUC18 / TOP10 strain, and ampicillin Thus, Escherichia coli recombinant GD / pHSG398-aldA / pUC18 / TOP10 strain which became chloramphenicol resistant was obtained.

  This strain was cultured at 37 ° C. for 16 hours in 5 ml of a liquid medium containing 100 ppm ampicillin and 50 ppm chloramphenicol in an LB medium, and the whole amount was inoculated into 50 ml of medium containing 100 ppm ampicillin, 50 ppm chloramphenicol and 1 mM IPTG. And cultured at 37 ° C. for 12 hours. The culture was centrifuged and the cells were collected as a pellet. The obtained microbial cell was about 0.5 g.

The cells were suspended in 0.1 M potassium phosphate buffer (pH 8.0) so that the absorption of OD ₆₆₀ was 10, and the cells were treated with an ultrasonic crusher for 5 minutes to disrupt the cells. The crushed material was centrifuged, and the supernatant was collected to obtain a glycerol dehydratase-aldA crude enzyme solution.

  Glycerin was used under the same conditions as in Example 9 except that 0.4 ml of a crude enzyme solution prepared from cells expressing glycerol dehydratase and aldA at the same time was used instead of 0.2 ml of each crude enzyme solution of glycerol dehydratase and aldA. The production reaction of 3-hydroxypropionic acid was carried out.

  As a result, the absorption at 340 nm accompanying the generation of NADH began to increase after the start of the reaction, and it was shown that the aldehyde dehydrogenation proceeded according to the generation of aldehyde.

  When the product was confirmed in the same manner as in Example 9, the peak of 3-hydroxypropionaldehyde as a reaction intermediate was 4.5 minutes, and the peak of 3-hydroxypropionic acid as a product was 5.2. We were able to confirm at the position of the minute.

Example 11 Reaction Using Escherichia coli Bacteria Introduced with Glycerol Dehydratase Gene and aldA Gene E. coli recombinant GD / pHSG398-aldA / pUC18 / TOP10 strain prepared in Example 10 was added to ampicillin 100 ppm, The cells were cultured at 37 ° C. for 16 hours in 5 ml of a liquid medium containing 50 ppm of lamphenicol, and the whole amount was inoculated into 50 ml of a medium obtained by adding 100 ppm of ampicillin, 50 ppm of chloramphenicol and 1 mM IPTG to the LB medium, and cultured at 37 ° C. for 12 hours. The culture was centrifuged and the cells were collected as a pellet. The obtained microbial cell was about 0.5 g.

The cells were suspended in 0.1 M potassium phosphate buffer (pH 8.0) so that the absorption of OD ₆₆₀ was 10.

  The same conditions as in Example 9 except that 0.4 ml of the cell suspension prepared from cells expressing glycerol dehydratase and aldA at the same time was used instead of 0.2 ml of each crude enzyme solution of glycerol dehydratase and aldA. Then, the formation reaction of 3-hydroxypropionic acid from glycerin was performed.

  When the product was confirmed in the same manner as in Example 9, the peak of 3-hydroxypropionaldehyde as a reaction intermediate was 4.5 minutes, and the peak of 3-hydroxypropionic acid as a product was 5.2. We were able to confirm at the position of the minute.

2 shows the enzymatic reaction of the present invention under aerobic conditions. 2 shows the enzymatic reaction of the present invention under anaerobic conditions. The result of measuring the enzyme activity of glycerol dehydratase using 1,2-propanediol as a substrate is shown. The result of having measured the enzyme activity of glycerol dehydratase using glycerol as a substrate is shown. The result of having measured the enzyme activity of hydroxy aldehyde dehydrogenase (aldA) using glycol aldehyde as a substrate is shown. The result of having measured the enzyme activity of hydroxy aldehyde dehydrogenase (aldB) using glycol aldehyde as a substrate is shown. The result of producing 3-hydroxypropionic acid from glycerin using a crude enzyme solution prepared from glycerol dehydratase-expressing cells and aldA-expressing cells is shown.

Claims (8)

  A method for producing 3-hydroxypropionic acid from glycerin by enzymatic reaction, comprising performing an enzymatic reaction with glycerol dehydratase and / or diol dehydratase and an enzymatic reaction with hydroxyaldehyde dehydrogenase.   The method according to claim 1, wherein the hydroxyaldehyde dehydrogenase is derived from E. coli.   The enzyme reaction is carried out by bringing glycerol into contact with a microorganism that produces glycerol dehydratase and / or diol dehydratase or a processed product thereof, and an enzyme active substance containing a microorganism that produces hydroxyaldehyde dehydrogenase or a processed product thereof. The method described.   The method according to claim 1 or 2, wherein the enzymatic reaction is carried out by contacting glycerol with a microorganism that produces glycerol dehydratase and / or diol dehydratase and hydroxyaldehyde dehydrogenase or a processed product thereof.   The method according to any one of claims 1 to 4, wherein the enzymatic reaction is carried out under aerobic conditions.   A method for producing acrylic acid by dehydrating 3-hydroxypropionic acid produced by the method according to claim 1.   A recombinant microorganism comprising a glycerol dehydratase gene and / or a diol dehydratase gene and a hydroxyaldehyde dehydrogenase gene.   A composition for producing 3-hydroxypropionic acid from glycerol, comprising glycerol dehydratase and / or diol dehydratase and hydroxyaldehyde dehydrogenase.

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