JPWO2013137277A1 - Method for producing 3-hydroxypropionic acid, genetically modified microorganism, and method for producing acrylic acid, water-absorbing resin, acrylic ester, and acrylic ester resin using said method - Google Patents

Abstract

The present invention provides a method for efficiently producing 3-hydroxypropionic acid (3HP). In the present invention, a gene encoding an exogenous malonyl-CoA reductase or a gene encoding an exogenous malonate semialdehyde dehydrogenase and a gene encoding an exogenous 3-hydroxypropionate dehydrogenase are introduced into a microorganism having acid resistance. A gene encoding a foreign glycerin dehydratase and a gene encoding a glycerin dehydratase reactivating factor, or a gene encoding a foreign diol dehydratase and a diol dehydratase reactivation A method for producing 3-hydroxypropionic acid, comprising using a second genetically modified microorganism into which a gene encoding a factor and a gene encoding a foreign aldehyde dehydrogenase are introduced To.

Description

  The present invention relates to a method for producing 3-hydroxypropionic acid, a genetically modified microorganism, and a method for producing acrylic acid, a water absorbent resin, an acrylic ester, and an acrylic ester resin using the method.

  From the viewpoint of global warming prevention and environmental protection, attention has been focused on the use of biological resources that can be recycled as carbon sources as an alternative to conventional fossil raw materials. For example, biomass resources such as starch-based biomass such as corn and wheat, sugar-based biomass such as sugarcane, and cellulose-based biomass such as rapeseed residue and rice straw are used as raw materials for general-purpose chemical products, plastics, and fuel production. Attempts have been made to develop methods for use as raw materials. In addition to the use of biomass-derived saccharides, a method of using carbon monoxide and hydrogen obtained by gasifying woody biomass as fermentation raw materials and a method of gasifying woody biomass to synthesize methanol are also studied. ·It has been reported.

  3-Hydroxypropionic acid (3HP) and its ester are useful compounds as raw materials for aliphatic polyesters, and polyesters synthesized therefrom are attracting attention as biodegradable, earth-friendly polyesters. 3-Hydroxypropionic acid is usually produced by the addition of water to acrylic acid or by the reaction of ethylene chlorohydrin with sodium cyanide. Since the reaction of hydrating acrylic acid is an equilibrium reaction, there is a problem that the reaction rate is controlled. In the case 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.

  3-hydroxypropionic acid can produce acrylic acid by dehydration. 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. Moreover, a water absorbing resin can also be manufactured by partially neutralizing acrylic acid and copolymerizing with a crosslinkable monomer. 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.

  It has been reported that 3-hydroxypropionic acid can be produced by enzymatic reaction or fermentation. As a 3HP fermentation production route by the enzyme reaction, (1) a route by 3HP cycle (carbon fixation route) and (2) a route having β-alanine as an intermediate have been proposed.

  Helge Holo. , Arch Microbiol, 1989, 151: 252-256, describes the fermentation production of 3-HP using the route of (1) above. Specifically, 3HP is obtained as an intermediate metabolite of the 3HP cycle in the fermentation of Chloroflexus aurantiacus possessing the 3HP cycle. However, since 3HP in the produced fermentation broth is further converted into 3-hydroxypropionyl CoA and acryloyl CoA by the 3HP cycle, the amount of 3HP produced is very small.

  In addition, International Publication No. 2008/027742 describes a method for fermentative production of 3HP using the route (2). However, according to the route (2), the number of reaction stages essential from pyruvic acid as a raw material to 3HP becomes 4 or more, and it cannot be said that it is an efficient 3HP production method.

  As described above, the method using the routes (1) and (2) described above cannot efficiently produce 3HP. Therefore, in recent years, methods using other 3HP fermentation production routes have been studied. Examples of the other 3HP fermentation production pathway include (3) a pathway by malonyl CoA → 3HP, (4) a pathway by glycerin → 3-hydroxypropionaldehyde (3HPA) → 3HP, and the like. In the method using the pathways (3) and (4) described above, 3HP is efficiently produced mainly using a genetically modified microorganism in which a gene of a required enzyme is introduced into a microorganism such as Escherichia coli. To do. For example, in the method (3), a gene encoding malonyl CoA reductase or the like is introduced, and in the method (4), a microorganism into which a gene encoding glycerol dehydratase, aldehyde dehydrogenase or the like is introduced is used. .

  Specific examples include Appl Microbiol Biotechnol, 2009 Sep, 84 (4), p. No. 649-657 describes a method for producing 3HP by fermentation using the route (4). More specifically, a recombinant E. coli having a dhaB gene derived from Klebsiella pneumoniae encoding glycerol dehydratase and an aldH gene derived from E. coli encoding aldehyde dehydrogenase was prepared and cultured in the presence of glycerol. It is disclosed that 4.4 g / L of scale batch culture was produced in 48 hours, and 31 g / L (yield 35%) of 3HP was produced in 72 hours in fed-batch culture using a bioreactor.

  By the way, the fermentation production of 3HP is usually carried out by culturing microorganisms, but the pH of the culture medium becomes acidic with the production of 3HP. As a result, the growth of microorganisms is inhibited, and the production of 3HP can be suppressed. Then, for example, even if the amount of 3HP fermentation production can be theoretically increased using the routes (3) and (4) above, if the pH in the fermentation broth becomes acidic, the growth of microorganisms Since 3HP production is suppressed, the fermentation yield of 3HP actually obtained can be reduced.

  Therefore, in order to prevent such production suppression of 3HP accompanying the pH reduction of the culture medium, a method has been proposed in which fermentation is performed while adding an alkaline reagent to the culture medium and keeping the pH near neutral. However, when the fermentation is performed near a neutral pH, 3HP in the fermentation broth after completion of the fermentation process is obtained in the form of a salt (3HP salt). When the 3HP salt is purified from the fermentation broth, the recovery rate is low, and problems such as a decrease in yield may occur in the subsequent dehydration reaction of 3HP to acrylic acid.

  Therefore, as a method for efficiently purifying the 3HP salt, a method for purifying 3HP after converting the 3HP salt in the fermentation broth to 3HP has been proposed. For example, in International Publication No. 2002/090312, an amine solvent is added to a solution containing 3HP ammonium salt obtained by fermentation near neutrality, and the 3HP ammonium salt is converted to 3HP by heating. A method for extracting 3HP is disclosed. In addition, in International Publication No. 2005/073161, a tertiary amine solvent is added to a solution containing a 3HP calcium salt, and the 3HP calcium salt is converted into 3HP by feeding carbon dioxide, in a tertiary amine solvent. Discloses a method for extracting 3HP.

  The fermentation host used in the method for fermentative production of many 3HPs including the routes (3) and (4) above is usually E. coli whose growth is suppressed under acidic conditions. Thus, for example, Appl Microbiol Biotechnol, 2009 Sep, 84 (4), p. A method for the fermentation production of 3HP by combining the method for producing 3HP of 649-657 and the purification method of International Publication No. , Have a certain effect.

  However, there is a problem in that a reagent and a process for converting to 3HP after fermentation are required, and costs and additional steps are required, and a more efficient method for producing 3HP is required.

  Accordingly, an object of the present invention is to provide a method for efficiently producing 3-hydroxypropionic acid (3HP).

  As a result of intensive studies to solve the above problems, the present inventors have improved the amount of 3HP produced by using a genetically modified microorganism into which a gene encoding an enzyme required for an acid-resistant microorganism has been introduced. As a result, it was found that the produced 3HP can be easily purified from the fermentation broth, and the present invention has been completed. That is, the present invention introduces a gene encoding an exogenous malonyl-CoA reductase or a gene encoding an exogenous malonic semialdehyde dehydrogenase and a gene encoding an exogenous 3-hydroxypropionate dehydrogenase into an acid-resistant microorganism. A gene encoding a foreign glycerol dehydratase and a gene encoding a glycerin dehydratase reactivating factor or a gene encoding a foreign diol dehydratase and a diol dehydratase 3-hydroxypropionic acid comprising using a second genetically modified microorganism introduced with a gene encoding an activator and a gene encoding an exogenous aldehyde dehydrogenase Production method on.

It is a figure which shows notionally the method of this invention. It is a figure which shows the plasmid map of pADH. It is a figure which shows the plasmid map of pGPD. It is a figure which shows the plasmid map of pG418. It is a figure which shows the plasmid map of pHYG. It is a figure which shows the plasmid map of pAUR. It is a figure which shows the mass spectrum of the culture supernatant of 3HP, and the culture supernatant of S. pombe (mcr). It is the graph which compared the transition of 3HP production amount and pH transition of S.pombe (mcr, acc, gpd1) and S.pombe (mcr, acc) in Example 4.

  In the present invention, a gene encoding an exogenous malonyl-CoA reductase or a gene encoding an exogenous malonate semialdehyde dehydrogenase and a gene encoding an exogenous 3-hydroxypropionate dehydrogenase are introduced into a microorganism having acid resistance. A gene encoding a foreign glycerin dehydratase and a gene encoding a glycerin dehydratase reactivating factor, or a gene encoding a foreign diol dehydratase and a diol dehydratase reactivation Of 3-hydroxypropionic acid (3HP), comprising using a second genetically modified microorganism into which a gene encoding a factor and a gene encoding a foreign aldehyde dehydrogenase are introduced Production method on.

  According to the present invention, 3-hydroxypropionic acid (3HP) can be produced efficiently.

  Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In addition, the names of enzymes or enzyme genes that catalyze a desired reaction are described below. However, any enzyme or enzyme gene that can catalyze a desired reaction is related to the enzyme name, enzyme gene name, and EC number. It can be used in the same manner as the enzyme or enzyme gene described in this patent.

  The 3HP generation route according to the present invention can be any route as long as 3HP generation is possible. For example, (a) Glucose →→→ Pyruvate → Lactic acid → Lactyl CoA → Acrylyl CoA → 3-Hydroxypropionyl CoA → 3-HP through lactic acid 3HP production pathway via lactic acid, (b) Glucose →→→ Pyruvate → 3HP generation route via malonyl CoA that generates 3HP by the route of acetyl CoA → malonyl CoA → 3HP, (3) 3HP is generated by the route of (u) glucose →→→ pyruvic acid → acetyl CoA → malonyl CoA → malonic acid semialdehyde → 3HP 3HP formation route via malonyl CoA-malonic acid semialdehyde, (d) glucose →→→ pyruvic acid → oxaloacetic acid → aspartic acid → β alanine → β alanyl CoA → acrylyl CoA → 3-hydroxypropionyl CoA → 3HP 3HP production pathway via β-alanine-acrylyl-CoA to produce 3HP, β-alanine-malonic acid to produce 3HP by the route of (g) glucose →→→ pyruvic acid → oxaloacetate → aspartic acid → βalanine → malonic acid semialdehyde → 3HP 3HP production pathway via semialdehyde, (f) glucose →→→ pyruvic acid → α alanine → β alanine → malonic acid semialdehyde → 3HP production pathway via α alanine-β alanine that produces 3HP, (ki) glucose →→→ Pyruvic acid → α Alanine → β Alanine → β Alanyl CoA → Acrylyl CoA → 3-Hydroxypropionyl CoA → 3HP generation pathway via α-alanine-acrylyl CoA, (3) Glucose → → oxaloacetate → malonic acid semial 3HP generation via oxaloacetate that generates 3HP by the route of dehydr → 3HP, 3HP generation via 3HP via glycerol, which generates 3HP by the route of (g) glucose → glycerol → 3-hydroxypropionaldehyde → 3HP, etc. is possible Metabolic pathways are available. The 3HP generation paths (A) to (K) above may be used alone or in combination with a plurality of 3HP generation paths. Because of the small number of reaction stages up to 3HP and the large amount of 3HP precursor produced in the cell, (3) the 3HP production pathway via malonyl-CoA and / or (3) the 3HP production pathway via glycerin can be used. preferable. Therefore, in the following, a method for producing 3HP by (i) the 3HP production route via malonyl CoA and (3) the 3HP production route via glycerin will be described in detail. However, the present invention is not limited to the following form.

  FIG. 1 is a diagram conceptually illustrating a method according to an embodiment of the present invention.

  According to the first genetically modified microorganism, 3HP is produced by the route of (3) malonyl CoA → 3HP. At this time, the route of (3) includes (3-a) malonyl-CoA directly from 3HP, and (3-b) malonyl-CoA through the malonic acid semialdehyde intermediate 3HP. Is classified as a route to be manufactured. In the route (3-b), malonic acid semialdehyde is used. Further, according to the second genetically modified microorganism, 3HP is produced by the route of (4) glycerin → 3-hydroxypropionaldehyde (3HPA) → 3HP.

[First genetically modified microorganism]
The first genetically modified microorganism according to the present invention is (a) a gene having a gene encoding an exogenous malonyl-CoA reductase introduced into an acid-resistant microorganism; A gene encoding an acid semialdehyde dehydrogenase and a gene encoding an exogenous 3-hydroxypropionate dehydrogenase; or (c) a gene encoding an exogenous malonyl-CoA reductase to an acid-resistant microorganism, exogenous malon Any of a gene encoding acid semialdehyde dehydrogenase and a gene into which exogenous 3-hydroxypropionate dehydrogenase is introduced may be used.

(Microorganisms with acid resistance)
The microorganism having acid resistance is not particularly limited as long as it can grow on a medium having 3HP of 90 g / L or more, preferably 100 g / L or more, more preferably 100 to 300 g / L. Examples of the acid-resistant microorganism include bacteria and yeast.

  Examples of the acid-resistant bacteria include bacteria of the genus Acetobacter such as Acetobacter pasteurianus and Acetobacter acetic, bacteria of the genus Glucacetobacterium such as Gluconobacter oxydans, Gluconobacter genus Lactobacillus, Examples include bacteria and lactic acid bacteria such as Leuconostoc bacteria.

  Examples of the acid-resistant yeast include Schizosaccharomyces, Saccharomyces, Kluyveromyces, Pichia, Torulaspora, Zygosaccharomyces, and Candida. Specific examples include Schizosaccharomyces pombe, Saccharomyces cerevisiae, Kluyveromyces marxianus, Kluyveromyces thermotolans, Cluyveromyces lactis, Cluyveromyces lactis, Cluyverometics lactis, and Cluyveromyces.

  In addition to the above bacteria and yeast, genetically modified microorganisms modified to continue fermentation even at low pH, genetically modified microorganisms modified to suppress growth inhibition by 3HP, mutation treatment to continue fermentation even at low pH Any of the mutant strains that have been subjected to the above and the mutant strains that have improved resistance to growth inhibition by 3HP can be used.

  Among these, the acid-resistant microorganism is preferably a yeast, more preferably a genus Schizosaccharomyces, and still more preferably a Schizosaccharomyces pombe. Usually, when fermentation is performed using bacteria, acetic acid can be produced as a by-product together with 3HP. However, when fermentation is performed using yeast, ethanol is produced as a by-product and the amount of acetic acid produced as a by-product can be reduced. Acetic acid is difficult to separate from 3HP, and also when acrylic acid is produced using the produced 3HP, it is difficult to separate acrylic acid and acetic acid. It is preferable to use yeast that can be used.

(Gene encoding exogenous malonyl CoA reductase)
Malonyl CoA reductase is a protein having an enzyme activity that catalyzes a deCoA reaction and a reduction reaction using malonyl CoA as a substrate.

  A gene encoding a foreign malonyl CoA reductase that can be used is not particularly limited, and a known gene can be used. For example, Chloroflexus aurantiacus, Chloroflexus aggregans, Roseiflexus castenholzii, Roseiflexus sp. Erythrobacter sp. , Gamma proteobacterium, Roseflexussp. strain RS-1, Erythrobactersp. strain NAP1, Metallosphere sedura, Sulfolobus tokodaiii, Acidinus brisalelyi, Sulfobus metallicus, Acidibulus inferno, Acidibrius , A gene encoding malonyl CoA reductase derived from Styrobolus azoricos, Pyrolobus fumariii. Of these, Chloroflexus aurantiacus, Chloroflexus agregans, Roseiflexus castenholzi, Roseiflexus sp. Erythrobacter sp. , Gamma proteobacterium, Acidianus brierleyi, Sulfolobus metallicus, Metallospherea sedula, Acidianus ambivalens, Sulfolobus sp. It is preferable to use a gene encoding malonyl-CoA reductase derived from, and more preferable to use a gene encoding malonyl-CoA reductase derived from Chloroflexus aurantiacus. The gene encoding the malonyl CoA reductase may be used alone or in combination of two or more. A modified malonyl CoA reductase modified by adding a point mutation to the malonyl CoA reductase may be used.

  SEQ ID NO: 1 exemplifies the amino acid sequence of a malonyl CoA reductase derived from Chloroflexus aurantiacus, and SEQ ID NO: 2 exemplifies the base sequence of a malonyl CoA reductase gene derived from Chloroflexus aurantiacus. As long as the protein containing these amino acid sequences has malonyl-CoA reductase activity, mutations such as deletion, substitution, addition, etc. may occur in one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1.

  Moreover, it is also possible to generate 3HP more efficiently by using a modified malonyl CoA reductase modified to a NADH-dependent enzyme by a point mutation method or the like as the NADPH-dependent malonyl CoA reductase.

(Gene encoding exogenous malonic acid semialdehyde dehydrogenase)
Malonate semialdehyde dehydrogenase is a protein having an enzyme activity that catalyzes a reaction for producing malonate semialdehyde using malonyl CoA as a substrate.

  The gene encoding foreign malonic acid semialdehyde dehydrogenase that can be used is not limited, and a known gene can be used. For example, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas sp. Lactobacillus casei, Pseudomonas sp. , Chloroflexus aurantiacus, Chloroflexus aggregans, Metallosphaera sedula, Sulfolobus tokodaii, Acidianus brierleyi, Sulfolobus metallicus, Acidianus infernus, Acidianus brierleyi, Metallosphaera sedula, Acidianus ambivalens, Sulfolobus sp. A gene encoding malonic acid semialdehyde dehydrogenase derived from These genes encoding malonic acid semialdehyde dehydrogenase may be used alone or in combination of two or more. A modified malonate semialdehyde dehydrogenase modified by adding a point mutation to the malonate semialdehyde dehydrogenase may be used.

(Gene encoding exogenous 3-hydroxypropionate dehydrogenase)
3-Hydroxypropionate dehydrogenase is a protein having an enzyme activity that catalyzes a reaction of producing 3HP using malonic acid semialdehyde as a substrate.

  The gene encoding exogenous 3-hydroxypropionic acid dehydrogenase that can be used is not particularly limited, and a known gene can be used. For example, Candida catenulata, Candida gropengiesseri, Candida rugosa, Escherichia coli, Pichia haplophila, Propionibacterium shermanii, Rhodobacter sphaeroides, Pseudomonas aeruginosa, Alcaligenes faecalis, Pseudomonas putida, Escherichia coli, Chloroflexus aurantiacus, Chloroflexus aggregans, Metallosphaera sedula, Sulfolobus tokodaii, Acidianus brierleyi, ulfolobus metallicus, Acidianus infernus, Acidianus brierleyi, Metallosphaera sedula, Acidianus ambivalens, Sulfolobus sp. A gene encoding 3-hydroxypropionate dehydrogenase derived from Of these, a gene encoding 3-hydroxypropionate dehydrogenase derived from Rhodobacter sphaeroides, Pseudomonas aeruginosa, Alcaligenes faecalis, Pseudomonas putida, or Escherichia coli is preferably used. The gene encoding the 3-hydroxypropionic acid dehydrogenase may be used alone or in combination of two or more. Alternatively, modified 3-hydroxypropionate dehydrogenase modified by adding a point mutation to the 3-hydroxypropionate dehydrogenase may be used.

  The base sequence of 3-hydroxypropionate dehydrogenase of Alcaligenes faecalis is exemplified in SEQ ID NO: 3, and the amino acid sequence is exemplified in SEQ ID NO: 4. The base sequence of 3-hydroxypropionate dehydrogenase of Pseudomonas aeruginosa is exemplified in SEQ ID NO: 5 and the amino acid sequence is exemplified in SEQ ID NO: 6. The base sequence of 3-hydroxypropionate dehydrogenase of Pseudomonas putida is exemplified in SEQ ID NO: 7 and the amino acid sequence is exemplified in SEQ ID NO: 8. The base sequence of 3-hydroxypropionate dehydrogenase of Escherichia coli is exemplified in SEQ ID NO: 9, and the amino acid sequence is exemplified in SEQ ID NO: 10. As long as the protein comprising the amino acid sequence of SEQ ID NO: 4, 6, 8, or 10 has 3-hydroxypropionic acid dehydrogenase activity, one or a number in the amino acid sequence represented by SEQ ID NO: 4, 6, 8, or 10 Mutations such as deletion, substitution and addition may occur in one amino acid.

  In the above, the first genetically modified microorganism is introduced with a gene encoding an exogenous malonyl-CoA reductase, or encodes an exogenous malonate semialdehyde dehydrogenase and an exogenous 3-hydroxypropionate dehydrogenase Although a form in which a gene is introduced is described, other enzymes capable of generating 3HP from malonyl CoA may be used alone or in combination of two or more.

(Gene encoding foreign acetyl-CoA carboxylase)
A gene encoding an exogenous acetyl CoA carboxylase may be further introduced into the first genetically modified microorganism. Thereby, the first genetically modified microorganism can produce malonyl CoA in the first genetically modified microorganism using acetyl CoA as a raw material.

  As described above, acetyl CoA carboxylase is a protein having an enzyme activity that catalyzes a reaction of producing malonyl CoA using acetyl CoA as a substrate.

  A gene encoding a foreign acetyl-CoA carboxylase that can be used is not particularly limited, and a known gene can be used. For example, Corynebacterium glutamicum, Bacillus cereus, Bacillus subtilis, Bos taurus, Brevibacterium thiogenitalis, Candida lipolytica, Chloroflexus aurantiacus, Cryptosporidium parvum, Drosophila melanogaster, Escherichia coli, Homo sapiens, Hordeum vulgare, Lactobacillus plantarum ,, Mycobacterium avium, Mycobacterium phlei, Saccharomyces cerevisiae , Schizo accharomyces pombe, Setaria viridis, Solanum tuberosum, Spinacia oleracea, Staphylococcus aureus, Streptomyces coelicolor, Sulfolobus metallicus, Takifugu sp. , Candida catnutrata, Candida groupingseri, Candida rugosa, Pichia haplophila, Propionibacterium shermanii, Pseudomonas aeruginosa, Pseudomonas. , Chloroflexus aggregans, Metallosphaera sedula, Aspergillus clavatus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus oryzae, Aspergillus ochraceus, Aspergillus niger, Escherichia blattae, Candida sonorensis, Candida methanosorbosa, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Ittatchenkia orientalis, Candida curv ta, Rhodotorula glutinis, Rhodosporidium toruloides, Cryptococcus curvatus, Trichosporon cutaneum, include genes encoding acetyl CoA carboxylase derived from Lipomyces starkeyi. Among these, it is preferable to use a gene encoding acetyl CoA carboxylase derived from Corynebacterium glutamicum or Saccharomyces cerevisiae. The gene encoding the acetyl CoA carboxylase may be used alone or in combination of two or more. Alternatively, a modified acetyl CoA carboxylase obtained by modifying the acetyl CoA carboxylase by adding a point mutation may be used.

  SEQ ID NO: 13 shows the base sequence of the acetyl CoA carboxylase (accBC) gene derived from Corynebacterium glutamicum, SEQ ID NO: 14 shows the base sequence of the acetyl CoA carboxylase (dtsR1) gene derived from Corynebacterium glutamicum, and SEQ ID NO: 29 shows the base sequence of Saccharevices. The nucleotide sequence of the acetyl-CoA carboxylase (acc1) gene of Saccharomyces cerevisiae is exemplified in SEQ ID NO: 30 as the gene sequence of Biotin: apoprotein ligase (bpl1). As long as the protein containing an amino acid sequence derived from these base sequences has acetyl CoA carboxylase activity, the base sequence represented by SEQ ID NOs: 13, 14, 29, and 30 can be used to change one or several amino acids in the corresponding amino acid sequence. Mutations such as deletion, substitution and addition may occur in amino acids.

(Modification of acid-resistant microorganisms and genes encoding enzymes)
In order to increase the amount of 3HP fermentation produced by the first genetically modified microorganism, a gene encoding an acid-resistant microorganism or enzyme may be modified. Specifically, modification of a gene related to at least one selected from the group consisting of glucose, acetyl CoA, malonyl CoA, malonic acid semialdehyde, 3HP, and by-products (acetic acid, ethanol, etc.) obtained in the fermentation process Is mentioned.

  As specific modifications, examples of modification of acetyl CoA carboxylase, modification of increasing intracellular malonyl-CoA amount, modification of 3HP metabolism suppression, and modification of increasing the amount of acetyl CoA are given below.

  As for acetyl CoA carboxylase, there is a possibility that it is subject to feedback inhibition by the produced malonyl CoA, 3HP and the like. Therefore, a microorganism having acid resistance or a gene encoding acetyl CoA carboxylase can be modified to have an effect of increasing the biosynthesis amount of malonyl CoA. The modification method is not particularly limited, but (a) a method for highly expressing an acetyl CoA carboxylase gene originally possessed by the host, (b) a method for introducing acetyl CoA carboxylase derived from an organism different from the host, (c And a method of introducing a mutation into the acetyl CoA carboxylase gene so that control by intracellular acetyl CoA and malonyl CoA does not occur.

  The method for highly expressing the acetyl CoA carboxylase gene originally possessed by the host in (a) is not particularly limited. For example, a fragment in which the acetyl CoA carboxylase gene is inserted downstream of the high expression promoter is placed on the chromosome of the host microorganism. A method of introducing multiple copies (for example, 1 to 10 copies); a method of transforming an expression vector having multiple copies (for example, 1 to 10 copies) of an acetyl CoA carboxylase gene into the same host; and a high expression of acetyl CoA carboxylase A method of culturing under such culture conditions; a method of using a mutant strain in which acetyl CoA carboxylase is highly expressed by a mutation treatment; N-methyl-N′-nitro-N-nitrosoguanidine (NTG), ethylmethanesulfonic acid By treatment with mutagen such as (EMS) A method using malonyl CoA and / or acetyl-CoA biosynthesis large amount since mutations organisms like. Further, since acetyl CoA carboxylase is a biotin-dependent enzyme, a genetically modified organism or a mutant organism with improved biotin biosynthesis ability may be used.

  Further, the method for introducing acetyl-CoA carboxylase derived from an organism different from the host in (b) is not particularly limited as long as the gene encoding acetyl-CoA carboxylase derived from the above-described microorganism can be introduced into another microorganism. For example, one or more copies (for example, 1 to 200 copies) of the acetyl CoA carboxylase gene are transformed into a host (for example, a microorganism) belonging to a different species from the host (for example, a microorganism) that originally has the acetyl CoA carboxylase. The method of doing is mentioned. Since the introduced acetyl-CoA carboxylase can be stably retained in the microorganism by inserting it into the chromosome of the host microorganism, it is preferably inserted into the chromosome.

  The method for introducing a mutation into the acetyl CoA carboxylase gene so as not to cause control by intracellular acetyl CoA or malonyl CoA in (c) is not particularly limited, but for example, it is responsible for feedback control etc. in the acetyl CoA carboxylase gene Examples include a method of using a mutated acetyl CoA carboxylase modified by introducing a mutation into an amino acid so as not to be subjected to feedback control or the like.

  The methods (a) to (c) may be applied singly or in combination of two or more. Of these, the method (a) and the method (b) are preferably used, and the method (b) is more preferably used. That is, the effect of increasing the biosynthetic amount of malonyl CoA is preferably obtained by transforming acetyl CoA carboxylase into a microorganism belonging to a species different from the microorganism originally having the acetyl CoA carboxylase. Here, the form of acetyl CoA carboxylase is not particularly limited, and any known form can be applied.

  Further, the modification that increases the amount of malonyl-CoA in the cell can be carried out by suppressing or destroying the activity of an enzyme gene that catalyzes a reaction (such as a conversion reaction to fatty acid) using malonyl-CoA as a substrate. For example, activity suppression or destruction of a fatty acid synthase gene that catalyzes a fatty acid synthesis reaction from malonyl CoA, synthesis of malonyl-ACP using malonyl CoA as a substrate, destruction of a malonyl-CoA-ACP transacylase gene, β-ketoacyl-ACP A method of suppressing the conversion of malonyl-CoA to a fatty acid by highly expressing the fabF gene encoding synthase II can be used. It is also possible to increase the amount of malonyl CoA in the cell by modifying the malonyl CoA reductase gene to be highly expressed or to introduce and highly express an exogenous malonyl CoA reductase gene.

  Further, the modification of 3HP metabolism suppression includes a method of modifying an enzyme gene that catalyzes a reaction using 3HP as a substrate. Specifically, by destroying the enzyme gene that catalyzes the redox reaction using propionyl CoA synthase, 3-hydroxypropionyl-CoA synthetase or 3HP as a substrate, it becomes possible to suppress the degradation of 3HP in culture. Can be improved.

  As a modification method for increasing the amount of acetyl CoA, modification to an enzyme gene that catalyzes a reaction for producing acetyl CoA, for example, introduction or modification of a foreign gene having an effect of increasing the amount of acetyl CoA production, acetyl CoA Examples include introduction or modification of a foreign gene having an effect of reducing consumption. Specifically, use of a genetically modified strain that highly expresses a pyruvate dehydrogenase complex gene that generates pyracetylate as a substrate and acetyl CoA, or a host that uses a pyruvate dehydrogenase complex gene derived from an organism other than the organism used as the host. Introduction and high expression method, method of using a gene-disrupted strain in which the PdhR gene is disrupted that controls the expression of the pyruvate dehydrogenase complex gene, or gene modification in which the amount of expression of the pyruvate dehydrogenase complex is increased due to a mutation in the PdhR gene. A method of using stocks can be considered. In addition, a method of highly expressing an acetyl-CoA synthetase (acetyl CoA ligase) gene that synthesizes acetyl CoA using acetic acid as a substrate can be used. According to the modification, since the amount of acetic acid consumed can be improved, the amount of acetic acid produced as a by-product can be reduced. Furthermore, a method of introducing phosphoketolase such as D-xylose-5-phosphate phosphoketolase and / or fructose-6-phosphate phosphoketolase and phosphotransacetylase can also be used. . D-xylose-5-phosphate phosphoketolase is an enzyme that catalyzes the reaction of converting xylulose-5-phosphate into acetyl phosphate and glyceraldehyde-3-phosphate. Further, fructose-6-phosphate phosphoketolase is an enzyme that catalyzes a reaction for converting fructose-6-phosphate into erythrose-4-phosphate and acetylphosphate. Phosphotransacetylase is an enzyme that catalyzes the reaction of converting acetyl phosphate and CoA to acetyl CoA. Therefore, according to the modification, acetyl phosphate is synthesized by phosphoketolase (D-xylose-5-phosphate phosphoketolase and / or fructose-6-phosphate phosphoketolase), and the obtained acetyl phosphate is used as a raw material. Acetyl-CoA can be synthesized by transacetylase. Thereby, the amount of acetyl CoA can be increased. In addition, the amount of acetyl CoA can be further increased by destroying an enzyme that catalyzes the pathway of acetaldehyde → ethanol, for example, an alcohol dehydrogenase gene. Furthermore, an enzyme gene that catalyzes a reaction using pyruvate, which is a precursor of acetyl CoA, as a substrate, for example, lactate dehydrogenase that catalyzes the reaction of pyruvate → lactic acid, alanine dehydrogenase that catalyzes the reaction of pyruvate → L alanine, and pyrubin. By destroying enzyme genes such as pyruvate carboxylase that catalyzes the reaction of acid → oxaloacetate, pyruvate decarboxylase that catalyzes the reaction of pyruvate → acetaldehyde, and pyruvate oxidase that catalyzes the reaction of pyruvate → acetyl phosphate The amount of pyruvic acid consumed in other metabolic pathways can be reduced, and as a result, the amount of intracellular acetyl CoA produced can be increased. It is also possible to increase the amount of acetyl CoA in the cell by destroying or weakening other metabolic pathways that consume acetyl CoA. For example, phosphocoylase gene that produces acetyl phosphate using acetyl CoA as a substrate, citrate synthase gene that synthesizes citric acid using acetyl CoA and oxaloacetate as substrates, and acetyl CoA such as acetaldehyde dehydrogenase gene that produces acetaldehyde using acetyl CoA as a substrate Examples include a method using a gene disruption strain obtained by disrupting an enzyme gene that catalyzes an enzyme reaction using a substrate, or a mutant microorganism having a reduced enzyme activity using acetyl CoA as a substrate. Further, modification to the citric acid cycle (TCA cycle), which is the metabolic pathway in which acetyl CoA is most utilized, is also effective in increasing the amount of malonyl CoA produced. Specifically, (1) use of genetically modified strains and mutants in which each enzyme activity constituting the TCA cycle is lowered, (2) change of culture conditions such that each enzyme activity constituting the TCA cycle is lowered, etc. Is mentioned. As the method (2), for example, the amount of acetyl CoA flowing in the TCA cycle can be reduced by culturing under anaerobic conditions.

  According to one embodiment of the present invention, the acid-resistant microorganism is preferably S. Pombe. Therefore, according to the present invention, a gene encoding an exogenous malonyl-CoA reductase, a gene encoding an exogenous acetyl-CoA carboxylase, and a gene encoding an exogenous gene having an effect of increasing the amount of acetyl-CoA generated are added to Schizosaccharomyces pombe. A genetically modified microorganism into which is introduced is provided. The foreign gene having an effect of increasing the amount of acetyl CoA produced is preferably a combination of a gene encoding phosphoketolase and a gene encoding phosphotransacetylase. In this case, the phosphoketolase is preferably D-xylose-5-phosphate phosphoketolase and / or fructose-6-phosphate phosphoketolase, and D-xylose-5-phosphate phosphoketolase and Since it has both activities of fructose-6-phosphate phosphoketolase, it is more preferably a phosphoketolase derived from a bacterium belonging to the genus Bifidobacterium. According to an embodiment of the present invention, the foreign gene having an effect of increasing the amount of acetyl CoA produced is a gene encoding fructose-6-phosphate phosphoketolase and a phosphotransacetylase. It is preferably a gene encoding (phosphotransacetylase).

  Even if the gene has been modified as described above, for example, a gene in which acetyl-CoA carboxylase has been modified, acetyl-CoA carboxylase obtained from the gene can contain malonyl-CoA using acetyl-CoA as a substrate. As long as it has an enzyme activity that catalyzes the reaction to be generated, it is a gene functionally equivalent to acetyl CoA carboxylase, and the functionally equivalent gene is also encompassed in the present invention. “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 a gene encoding a protein functionally equivalent to a protein include hybridization techniques (Sambrook, Jetal., 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: 2, various artificial treatments such as site-directed mutagenesis, random mutation by mutagenesis treatment, mutation, deletion, ligation of nucleic acid fragments by restriction enzyme cleavage, etc. Thus, 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: 2. However, as long as it has the activity of malonyl CoA reductase, that is, the enzymatic activity of producing 3HP using malonyl CoA as a substrate, it is included in the malonyl CoA reductase gene.

  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, or 42 ° C. in the presence of 50% formamide, or 65-68 ° C. in an aqueous solution. After that, the filter can be washed at room temperature (20 ° C.) 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.

  Each gene also includes a gene encoding a protein functionally equivalent to a protein consisting of each amino acid sequence. For example, as a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 1, 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: 1 And a protein exhibiting the activity of malonyl-CoA reductase.

  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, conservative substitutions 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 if the mutant protein consists 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: 1, the mutation is SEQ ID NO: If the mutation is highly conserved in the three-dimensional structure of the amino acid sequence described in 1, and the mutant protein has an enzymatic activity to generate 3HP using malonyl CoA as a substrate, these mutant proteins The gene encoding is also encompassed by the gene encoding malonyl CoA reductase. Here, the term “several” means usually 2 to 5, preferably 2 to 3.

  Also, the gene encoding malonyl CoA reductase has at least 80% identity, preferably at least 90% identity, more preferably at least 95% identity, more preferably at least at least with the amino acid sequence of SEQ ID NO: 1. A gene encoding a protein consisting of an amino acid sequence having 99% identity and having an activity of generating 3HP using malonyl-CoA as a substrate is also included. The same applies to the other genes encoding malonate semialdehyde dehydrogenase, 3-hydroxypropionate dehydrogenase, and acetyl CoA carboxylase.

  Among the first genetically modified microorganisms described above, Schizosaccharomyces pombe NSH-2 (reception number: FERM ABP-11527) (S. pombe (mcr)), Schizosaccharomyces pombe NSH-3 (reception number: FER15 B-1) It is particularly preferable to use (S. pombe (mcr, acc)).

[Second genetically modified microorganism]
The second genetically modified microorganism according to the present invention encodes (a) an acid-resistant microorganism into a gene encoding a foreign glycerol dehydratase, a gene encoding a glycerol dehydratase reactivating factor, and a foreign aldehyde dehydrogenase. (B) A gene encoding an exogenous diol dehydratase, a gene encoding a diol dehydratase reactivating factor, and a gene encoding an exogenous aldehyde dehydrogenase into a microorganism having acid resistance Or (c) a gene encoding an exogenous glycerin dehydratase, a gene encoding a glycerin dehydratase reactivating factor, a gene encoding an exogenous diol dehydratase, a diol dehydratase Obtained by introducing a gene encoding a gene coding for a sex factor, and foreign aldehyde dehydrogenase may be either.

(Microorganisms with acid resistance)
Since the acid-resistant microorganism is the same as the first genetically modified microorganism, description thereof is omitted here.

  Schizosaccharomyces pombe itself has a metabolic pathway from glucose to glycerin, and glucose can be used for the production of 3HP, and the amount of glycerin produced is also a general genetically modified microorganism such as glycerin produced. The amount of glycerin produced is higher than that of genetically modified Escherichia coli to which glycerin-producing ability is imparted by introducing glycerol-3-phosphate dehydrogenase gene and glycerol-3-phosphatase gene derived from Saccharomyces cerevisiae into E. coli that does not have the ability To preferred.

(Gene encoding foreign glycerin dehydratase)
Glycerin dehydratase (GD) is a protein having an enzyme activity that catalyzes a reaction of producing 3-hydroxypropionaldehyde (3HPA) using glycerin as a substrate.

  A gene encoding a foreign glycerin dehydratase that can be used is not particularly limited, and a known gene can be used. For example, genes derived from microorganisms belonging to the genus Klebsiella, Citrobacter, Clostridium, Lactobacillus, Enterobacter, Calorator, Salmonella, Escherichia, and Listeria can be used. More specifically, Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Lactobacillus leichmannii, Citrobacter intermedium, Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus brevis, Enterobacter agglomerans, Clostridium butyricum, Caloramator viterbensis, Lactobacillus collinoides, Lactobacillus hilgardii, Salmonella typhimurium, Esc and a gene encoding glycerin dehydratase derived from Herichia brattatae, Listeria monocytogenes, and Listeria innocua. The gene encoding glycerin dehydratase may be used alone or in combination of two or more. Moreover, you may utilize the modified glycerol dehydratase which added the point mutation to the said glycerol dehydratase and modified | changed. In addition to the above, Clostridium butyricum-derived GD, which is GD independent of vitamin B12, can also be used.

(Gene encoding glycerin dehydratase reactivation factor)
Glycerin dehydratase reactivation factor (GDR) replaces coenzyme B12 in the reaction center of glycerin dehydratase inactivated by catalyzing the conversion reaction from glycerin to 3-hydroxypropionaldehyde to regain activity. Have a role. Therefore, when GDR is not introduced, the inactivated glycerol dehydratase is not reactivated, and the glycerol dehydration activity cannot be maintained, so that the glycerol dehydration activity becomes very low. In some cases, enzyme activity may not be observed. Therefore, by introducing a gene encoding a glycerin dehydratase reactivating factor together with a gene encoding glycerin dehydratase, a suitable enzyme activity can be obtained when 3HP is produced by fermentation. Thereby, the conversion of glycerin → 3HPA is suitably performed, and as a result, the yield of 3HP can be improved. Therefore, in one embodiment, the second genetically modified microorganism essentially includes a glycerin dehydratase reactivating factor together with glycerin dehydratase. Similarly to the above-mentioned GD derived from Clostridium butyricum, which is independent of vitamin B12, glycerol dehydratase activation enzyme (GD-AE), which plays an important role in maintaining glycerin dehydration activity, suitably converts glycerin to 3HPA. In order to do so, it is essential.

  The gene encoding a glycerin dehydratase reactivating factor that can be used is not particularly limited, and a known gene can be used. For example, WO 98/21341; Daniel et al. , J .; Bacteriol. 177, 2151 (1995); Toraya and Mori, J. et al. Biol. Chem. , 274, 3372 (1999); and Tobimatsu et al. , J .; Bacteriol. 181, 4110 (1999). In addition, a glycerin dehydratase gene derived from a microorganism belonging to the genus Clostridium, specifically, a glycerin dehydratase gene derived from Clostridium perfringens may be used. The gene encoding the glycerin dehydratase reactivating factor may be used alone or in combination of two or more. Moreover, you may utilize the glycerol dehydratase reactivation factor modified by adding a point mutation to the said glycerol dehydratase reactivation factor.

(Gene encoding exogenous diol dehydratase)
Diol dehydratase (DD) is a protein having an enzyme activity that catalyzes a reaction of producing 3-hydroxypropionaldehyde (3HPA) using glycerin as a substrate.

  The gene encoding a foreign diol dehydratase that can be used is not particularly limited, and a known gene can be used. Examples thereof include genes encoding diol dehydratase derived from microorganisms belonging to the genera Klebsiella, Citrobacter, Clostridium, Lactobacillus, Enterobacter, Caloratorator, Salmonella, and Listeria. More specifically, Lactobacillus reuteri, Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Lactobacillu sleichmannii, Citrobacter intermedium, Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus brevis, Enterobacter agglomerans, Clostridium butyricum, Caloramator viterbensis, Lactobacillus collinoides, Lactobacillus hilgardii, Salm Examples include genes encoding diol dehydratase derived from onella typhimurium, Listeria monocytogenes, and Listeria innocua. The gene encoding the diol dehydratase may be used alone or in combination of two or more. Alternatively, a diol dehydratase modified by adding a point mutation to the diol dehydratase may be used.

(Gene encoding diol dehydratase reactivation factor)
Diol dehydratase reactivation factor (DDR) replaces coenzyme B12 at the reaction center in diol dehydratase inactivated by catalyzing the conversion reaction from glycerin to 3-hydroxypropionaldehyde to regain activity. Have a role. Therefore, when DDR is not introduced, the inactivated diol dehydratase is not reactivated, and the glycerin dehydrating activity cannot be maintained, so that the dehydrating activity of glycerin becomes very low. In some cases, enzyme activity may not be observed. Therefore, by introducing a gene encoding a diol dehydratase reactivating factor together with a gene encoding diol dehydratase, a suitable enzyme activity can be obtained when 3HP is produced by fermentation. Thereby, the conversion of glycerin → 3HPA is suitably performed, and as a result, the yield of 3HP can be improved. Therefore, in one embodiment, the second genetically modified microorganism essentially includes a diol dehydratase reactivating factor together with the diol dehydratase.

  The gene encoding the diol dehydratase reactivating factor that can be used is not particularly limited, and known genes can be used. For example, Kajiura H. et al. et al. , J .; Biol. Chem. , 276, 36514 (2001).

  Among the genes encoding enzymes that catalyze the dehydration reaction of glycerin exemplified above, in this embodiment, a GD-GDR gene derived from a microorganism belonging to the genus Clostridium and a DD-DDR gene derived from a microorganism belonging to the genus Lactobacillus are used. The GD-GDR gene derived from Clostridium perfringens and the DD-DDR gene derived from Lactobacillus reuteri are more preferable.

(Gene encoding foreign aldehyde dehydrogenase)
Aldehyde dehydrogenase is a protein having an enzyme activity that catalyzes a reaction of producing 3-hydroxypropionic acid (3HP) using 3HPA as a substrate.

  The gene encoding a foreign aldehyde dehydrogenase that can be used is not particularly limited, and a known gene can be used. For example, Escherichia genus, Aerobacter genus, Agrobacterium genus, Alcaligenes genus, Arthrobacter genus, Bacillus genus, Corynebacterium genus, Flavobacterium genus, Klebsiella genus, Micrococcus genus, Micrococcus genus Examples include genes encoding aldehyde dehydrogenases derived from microorganisms belonging to the genus Shigella, Erwinia, Neisseria, and Lactobacillus. More specifically, Escherichia coli, Aerobacter aerogenes, Agrobacterium radiobacter, Agrobacterium tumefaciens, Alcaligenes viscolactis, Arthrobacter simplex, Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis, Corynebacterium equi, Flavobacterium sp. , Klebsiella pneumonia, Micrococcus glutamicus, Protaminobacter alboflavus, Proteus vulgaris, Pseudomonas fluorescens, Salmonella typhimurium, Sarcina lutea, Staphylococcus aureus, Shigella flexneri, Erwinia carotovora, Neisseria meningitides, Neisseria gonorr hoeae, Lactobacillus reuteri, Schizosaccharomyces pombe, from Azospirillum brasilense Arudehidodehi Gene encoding Rogenaze the like. Among these, it is preferable to use an enzyme gene derived from Escherichia coli, Schizosaccharomyces pombe, or Azospirillum brasilense, and it is more preferable to use an aldH gene derived from Escherichia coli.

  In addition, γ-glutamyl-γ-aminobutyraldehyde dehydrogenase, α-ketoglutarate semialdehyde dehydrogenase and the like can also be used.

(Modification of acid-resistant microorganisms and genes encoding enzymes)
In order to increase the amount of 3HP fermentation produced by the second genetically modified microorganism, a gene encoding an acid-resistant microorganism or enzyme may be modified. Specifically, the modification | change of the gene relevant to at least 1 selected from the group which consists of glucose, glycerol, 3HPA, and 3HP is mentioned.

  About the said modification | change, it can carry out by the method similar to the method described in the 1st genetically modified microorganism.

  Of the second genetically modified microorganisms described above, it is particularly preferable to use Schizosaccharomyces pombe NSH-1 (S. Pombe OB1 (GD, GDR, aldH).

[Method of constructing genetically modified microorganisms]
Known methods can be used for the construction of the first and second genetically modified microorganisms. For example, a gene encoding an enzyme may be introduced into the genome, may be introduced into the same vector for transformation, or may be introduced into a separate vector for transformation.

  For introduction of a gene into an acid-resistant microorganism used as a host, the above gene or a part thereof is linked to an appropriate vector, and the resulting recombinant vector is introduced into the host so that the target gene can be expressed. Or by inserting the gene of interest or a part thereof at any position on the genome by homologous recombination. “Part” refers to a part of each gene that can express a protein encoded by each gene or a protein having a desired enzyme activity 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 the known genes, the genes encoding the above-mentioned enzymes are registered in a public database such as GenBank by means of a hybridization method, a PCR method, etc. using appropriately designed synthetic primers based on the nucleotide sequences of known genes. It is also possible to obtain genes that have not been performed.

  Methods for preparing a genomic DNA library used for gene cloning, hybridization, PCR, plasmid preparation, DNA cutting 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. For example, plasmids, phages, cosmids, Escherichia coli artificial chromosomes (BAC), etc. used for introducing foreign genes can be mentioned. Examples of plasmids used when introducing foreign genes into E. coli include, for example, pHSG398, pUC18, pBR322, pSC101, pUC19, pUC118, pUC119, pACYC117, pAUR224, pGPD, pBluescript II SK (+), pETDuduet-1p 1, pCDFDuet-1, pRSFDuet-1, pCOLADuet-1, PinPoint Xa-1 (Promega) and the like. Examples of phages include λgt10, Charon 4A, EMBL-, M13mp18, M13mp19, and the like. In addition, plasmids used when introducing foreign genes into yeast include any promoter, terminator, and origin of replication responsible for plasmid replication in yeast. Is available. In addition, a 5'-untranslated region and a 3'-untranslated region may be included, and a marker gene such as an auxotrophic complementary marker and an antibiotic resistance gene may be included. Specific examples include pART, pSM, REP3X, pSLF101, pAUR224, pDUAL, and pDUAL2.

  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 a person skilled in the art may appropriately select a promoter that enables efficient gene expression under the culture conditions suitable for the host to be used and 3HP fermentation production. For example, in addition to T7 promoter, lac promoter, trp promoter, trc promoter, λ-PL promoter, tac promoter, T7 promoter, etc. that are generally used for foreign gene expression in E. coli, it is involved in nitrate respiration from E. coli. The Nar promoter region of the nitrate reduction gene narGHJI operon and the promoter region of the Frd gene which is a nitrate reductase gene of Escherichia coli can also be used. The Nar promoter region and the Frd promoter are promoters that undergo gene expression induction under anaerobic conditions. Furthermore, promoter regions such as glyceraldehyde 3-phosphate dehydrogenase gene and 6-phosphofructokinase gene involved in glucose metabolism, and promoter regions such as glutamate decarboxylase gene involved in acid resistance mechanism in E. coli are also used. be able to. In addition, promoters used for foreign gene expression in yeast include nmt1 promoter, nmt41 promoter, nmt81 promoter, fbp1 promoter, inv1 promoter, ctr4 promoter, CaMV35S promoter, adh1 promoter, SV40 promoter, urg1 promoter, cytomegalovirus (CMV ) Promoters, efa1a-c promoter, tif51 promoter, cam1 promoter, promoter regions such as glyceraldehyde 3-phosphate dehydrogenase gene and 6-phosphofructokinase gene involved in glucose metabolism, and the like can be used.

  A known method can be used as the gene disruption method. Specifically, a method of destroying the gene using a vector (targeting vector) that causes homologous recombination at an arbitrary position of the target gene (gene targeting method), or a trap vector (promoter at an arbitrary position of the target gene). Knockout cells, transgenic animals (including knockout animals), etc. in this technical field, such as methods that insert a reporter gene that does not possess) and destroy the gene to lose its function (gene trap method), methods that combine them, etc. A method used for manufacturing can be used. In addition, a method of introducing a vector expressing an antisense cDNA of a gene desired to be disrupted or a method of introducing a vector expressing a double-stranded RNA of a gene desired to be disrupted into a cell can be used. Such vectors include viral vectors, plasmid vectors, etc., and are based on conventional genetic engineering techniques, for example, Sambrook, J et al. , Molecular Cloning 2nd ed. , 9.47-9.58, Cold Spring Harbor Lab. It can be produced according to a basic book such as press (1989). In addition, 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. As a method for disrupting the yeast gene, Yeast, 1999, 15, p. 1419-1427, Nature Protocols, 2006, 1 (5), p. By performing gene disruption according to 2457-64, a desired gene disrupted yeast can be produced.

  The position where homologous substitution occurs or the position where the trap vector is inserted is not particularly limited as long as it causes a mutation that eliminates the expression of the target gene to be destroyed.

  Moreover, if a gene disruption system (Red system) by homologous recombination using a recombination protein sold by Gene Bridge is used, destruction of Escherichia, Salmonella, Shigella, Yersinia, Serratia, or Citrobacter bacteria It is possible to construct a gene-disrupted strain in which only a desired gene is selectively disrupted. Furthermore, using the TargetTron Gene Knockout System, a gene disruption system using a group 2 intron sold by Sigma-aldrich, Escherichia, Staphylococcus, Clostridium, Lactococcus, Shigcellis, Lactococcus, Gene disruption of Pseudomonas and Agrobacterium is also possible.

  The method for introducing the vector into the host may be selected depending on the host used, and is not particularly limited. For example, a method using calcium ions generally used for vector introduction, a protoplast method, an electroporation method, a lithium acetate method and the like can be used.

  The method of inserting a gene of interest at an arbitrary position on the genome by homologous recombination is to insert the gene of interest together with a promoter into a sequence homologous to the sequence on the genome, and this nucleic acid fragment is inserted into cells by electroporation or lithium acetate method. It can be carried out by introducing the DNA into the DNA and 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 functions lethally under specific conditions is inserted into the genome by homologous recombination as described above, and then the drug resistance gene is lethal under specific conditions. The target gene can also be introduced by 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 technique that can easily select only a recombinant microorganism into which a target gene has been introduced is preferable.

[Method for producing 3HP]
The 3HP solution is produced by culturing the first genetically modified microorganism or the second genetically modified microorganism in the presence of malonyl CoA or glycerin, respectively, and producing the microorganism assimilating malonyl CoA or glycerin. Can be carried out by accumulating 3HP in the culture medium. The first genetically modified microorganism and the second genetically modified microorganism may be used in combination.

(Malonyl CoA and glycerin)
Malonyl CoA or glycerin used as a raw material for 3HP may be prepared separately and added to the medium, or sugar or the like using microorganisms (including the first genetically modified microorganism and the second genetically modified microorganism). It may be prepared from

  The method for preparing malonyl CoA and glycerin using a microorganism is not particularly limited, and a known method can be used.

  Malonyl CoA is an important precursor in fatty acid synthesis and can be synthesized by many microorganisms. Further, if necessary, the gene encoding acetyl CoA carboxylase described above can be introduced to prepare malonyl CoA using acetyl CoA as a substrate. Acetyl CoA is an important metabolic intermediate located at the entrance of an important TCA cycle for living organisms and produced in large amounts as a metabolic intermediate in the metabolism of various compounds. Therefore, malonyl CoA can be made from sugar alcohol, alcohol, fatty acid and carboxylic acid.

  The microorganism that can be used for the preparation of malonyl-CoA is not particularly limited, and is not limited to the genus Escherichia, Lactobacillus, Salmonella, Klebsiella, Propionibacterium, Genus Bacillus, D, Corynebacterium, Genus, Corynebacterium, Rhodobacter genus, Rhodopseudomonas genus, Streptomyces genus, Synechococcus genus, Sulfolobus genus, Thermoplasma genus, Acetobacterium genus, Moorella genus, Chloroflexus genus, Chloroflexus genus, Chroroflexus genus nus spp., Alcaligenes sp., Thermomicrobium genus, Rhodobaca genus, Rhodospirillum spp., Saccharomyces spp., Schizosaccharomyces spp., and a microorganism belonging to the genus Pichia.

  Moreover, there is no restriction | limiting in particular as microorganisms which fermentate-produce glycerol, (1) Microorganism which originally has the capability to produce | generate glycerol from saccharides, (2) Glycerin production of the microorganism which originally has the capability to produce glycerol from sugar Selected from the group consisting of genes that encode enzymes required to produce glycerin from sugars for microorganisms whose ability is enhanced by genetic techniques, and (3) host microorganisms that do not have the ability to produce glycerin from sugars Any of the microorganisms into which at least one selected from the above is introduced can be used.

  Microorganisms originally having the ability to produce glycerin from sugars in (1) and (2) above are not particularly limited, for example, Schizosaccharomyces genus, Saccharomyces genus, Kluyveromyces genus, Pichia genus, Torulaspora genus, Zygosaccharomyces, Zygosaccharomyces, Yeast belonging to the above can be used. Of these microorganisms, Saccharomyces cerevisiae and Schizosaccharomyces pombe are more preferable, and Schizosaccharomyces pombe is more preferable.

  In (2) above, there is no particular limitation on the genetic technique for increasing the ability of microorganisms to produce glycerin, and any technique used in this technical field can be adopted as appropriate. For example, (2a) a method for increasing the expression of an enzyme that catalyzes at least one of the reactions from sugar to glycerol; (2b) a method for destroying an enzyme gene that catalyzes a reaction that metabolizes glycerol; (2c) Examples thereof include a method of culturing under high osmotic pressure conditions.

  (2a) As a method for enhancing the expression of an enzyme that catalyzes at least one of the reactions from sugar to glycerin, specifically, in the case of a high expression promoter such as yeast A method of changing to the GAP promoter, CMV promoter or nmt1 promoter, a method of increasing the number of copies of an enzyme gene in a cell by cloning and introducing it into a plasmid that can exist in multiple copies in the cell, an enzyme gene in the host chromosome For example, a method of increasing the copy number of the enzyme gene by inserting it at a plurality of positions above.

  As a method for destroying the enzyme gene that catalyzes the reaction of metabolizing glycerol (2b), specifically, a glycerol-3-phosphate dehydrogenase and / or glycerol-3-phosphatase gene is disrupted, or glycerol-3 -The method of substituting the promoter of a phosphate dehydrogenase and / or a glycerol-3-phosphatase gene with a promoter with a low expression level is mentioned. Moreover, the method of destroying a dihydroxyacetone kinase and / or a glycerol dehydrogenase gene, or the method of substituting the promoter of a dihydroxyacetone kinase and / or a glycerol dehydrogenase gene with a promoter with a low expression level etc. are mentioned.

  Specific examples of the method of culturing under the above (2c) high osmotic pressure conditions include a method of culturing using a medium having a high sugar concentration or salt concentration.

  The host microorganism that does not have the ability to produce glycerin from the sugar in (3) is not particularly limited, and a microorganism that can be used for the preparation of malonyl-CoA can be used.

  In the above (3), the enzyme necessary for producing glycerin from sugar is not particularly limited. For example, glycerol-3-phosphate dehydrogenase (GDP), which catalyzes a reaction for converting dihydroxyacetone phosphate generated in glycolysis to glycerol-3-phosphate, and glycerol-3-phosphate to glycerol Glycerol-3-phosphatase (GPP) that catalyzes the reaction of dihydroxyacetone, and dihydroxyacetone phosphatase that catalyzes the reaction of converting dihydroxyacetone phosphate generated in the glycolysis to dihydroxyacetone, and glyceraldehyde to glycerin And glycerol dehydrogenase that catalyzes the conversion reaction.

  A gene encoding an enzyme necessary for producing glycerin from the above-mentioned sugar is not particularly limited, and examples of a gene encoding GDP include a gene derived from a microorganism belonging to the genus Saccharomyces. Among these, it is preferable to use a gene derived from Saccharomyces cerevisiae.

  Examples of the gene encoding GPP include genes derived from microorganisms belonging to the genus Saccharomyces. Among these, it is preferable to use a gene derived from Saccharomyces cerevisiae.

  The malonyl-CoA and glycerin are produced by one or more microorganisms that have been brought into contact with the first genetically modified microorganism and / or the second genetically modified microorganism that perform 3HP fermentation production, even if added directly to the medium. However, it is preferable to impart malonyl CoA and / or glycerin producing ability to the first genetically modified microorganism and / or the second genetically modified microorganism. The contact includes culturing a microorganism or a processed product thereof in the presence of a compound used as a raw material, and performing a reaction using the processed product of the microorganism. 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.

(sugar)
As described above, malonyl CoA and glycerin can be derived from sugar.

  As sugars that can be used, common sugars that can be used in culture in the art can be used without limitation. Specific examples of sugars in this embodiment include sugars derived from biomass resources, hexoses such as glucose, mannose, galactose, fructose, sorbose, and tagatose, and pentoses such as arabinose, xylose, ribose, xylulose, and ribulose. Derived from disaccharides and polysaccharides such as maltose, sucrose, lactose, trehalose, starch and cellulose, sugar alcohols such as mannitol, xylitol and ribitol (excluding glycerin), saccharified biomass such as cellulose and lignocellulose Examples include sugar. Among these, it is preferable to use glucose and / or xylose. These sugars may be used alone or in combination of two or more, and are selected as appropriate depending on the microorganism species used.

(Production of 3HP by the first genetically modified microorganism)
When the reaction from malonyl CoA to 3HP is performed in the presence of malonyl CoA reductase using the first genetically modified microorganism, the reaction is preferably performed under conditions of a high NADPH amount. At this time, the first genetically modified microorganism may be introduced with another foreign gene, for example, a gene encoding acetyl CoA carboxylase. This condition is suitably applied particularly when NADPH-dependent malonyl CoA reductase is used. At this time, the gene encoding NADPH-dependent malonyl CoA reductase is not particularly limited, and examples thereof include genes encoding malonyl CoA reductase derived from Chloroflexus aurantiacus and Sulfolobus tokodaiii.

  Examples of the method for carrying out the reaction from malonyl CoA to 3HP under the condition of a high NADPH amount include, for example, microorganisms capable of producing more NADPH on a metabolic pathway utilizing a raw material compound (such as glucose), and more A method using a genetically modified organism that has been metabolically modified to produce NADPH is preferred. Examples of microorganisms capable of producing more NADPH include microorganisms that highly express genes having an effect of increasing intracellular NADPH amount. In this case, the gene having an effect of increasing the amount of NADPH in the cell is not particularly limited, but is glucose-6-phosphate dehydrogenase gene, 6-phosphogluconate dehydrogenase gene, NADP-dependent glyceraldehyde-3- Examples thereof include phosphate dehydrogenase (glyceraldehyde 3-phosphate dehydrogenase, EC.1.2.1.9, EC.1.2.1.13, EC.1.2.1.59, etc.) genes. In addition, a genetically modified organism into which the above three genes have been introduced can also be used as a genetically modified organism that has been metabolically modified so that more NADPH is produced.

  The glucose-6-phosphate dehydrogenase gene and the 6-phosphogluconate dehydrogenase gene are enzyme genes that catalyze NADP-dependent reactions in the pentose phosphate pathway. Glyceraldehyde-3-phosphate dehydrogenase is an enzyme gene that catalyzes NADP-dependent reactions in glycolysis, gluconeogenesis, carbon fixation, and the like. By introducing or highly expressing these genes, the amount of NADPH produced in sugar metabolism and the like can be increased, and 3HP can be fermented and produced more efficiently. That is, when the reaction from malonyl CoA to 3HP is carried out in the presence of malonyl CoA reductase, the reaction from malonyl CoA to 3HP is carried out using glucose-6-phosphate dehydrogenase gene, 6-phosphogluconate dehydrogenase gene, NADP-dependent It is preferable to use a microorganism that highly expresses soluble glyceraldehyde-3-phosphate dehydrogenase. Or the following reaction:

By introducing or highly expressing a transhydrogenase gene that catalyzes oxidization, the intracellular redox balance can be controlled, and efficient 3HP fermentation production can also be performed.

  Here, glucose-6-phosphate dehydrogenase has the following reaction:

As long as the protein has an enzyme activity that catalyzes, any known protein can be used. The glucose-6-phosphate dehydrogenase gene to be used is glucose originally possessed by the organism used as the host because of the ease of expression of the gene and the ease of synthesis of the enzyme protein synthesized by the expression of the gene. It is preferable to highly express the -6-phosphate dehydrogenase gene. For example, when using Escherichia coli as a host, a glucose-6-phosphate dehydrogenase gene derived from Escherichia coli is preferable.

  SEQ ID NO: 19 contains E.I. The base sequence of the E. coli-derived glucose-6-phosphate dehydrogenase gene is exemplified. As long as the protein containing an amino acid sequence derived from these base sequences has glucose-6-phosphate dehydrogenase activity, the base sequence represented by SEQ ID NO: 19 is changed to one or several amino acids in the corresponding amino acid sequence. Mutations such as deletion, substitution and addition may occur.

  In addition, 6-phosphogluconate dehydrogenase has the following reaction:

As long as the protein has an enzyme activity that catalyzes, any known protein can be used. The 6-phosphogluconate dehydrogenase gene to be used is inherently possessed by the organism used as the host due to the ease of expression of the gene and the ease of synthesis of the enzyme protein synthesized by the expression of the gene. It is preferable to highly express the phosphogluconate dehydrogenase gene. For example, when E. coli is used as a host, 6-phosphogluconate dehydrogenase derived from Escherichia coli is preferable.

  SEQ ID NO: 20 contains E.I. The base sequence of the 6-phosphogluconate dehydrogenase gene derived from E. coli is exemplified. As long as a protein containing an amino acid sequence derived from these base sequences has 6-phosphogluconate dehydrogenase activity, a change in the base sequence represented by SEQ ID NO: 20 results in a loss of one or several amino acids in the corresponding amino acid sequence. Mutations such as loss, substitution, and addition may occur.

  Furthermore, the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase has the following reaction:

As long as the protein has an enzyme activity that catalyzes, any known protein can be used. Specific examples of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase include, for example, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase derived from Kluyveromyces lactis (glyceraldehyde 3-phosphate dehydrogenase). The gene encoding the NADP-dependent glyceraldehyde 3-phosphate dehydrogenase derived from Kluyveromyces lactis may be introduced into a microorganism such as S. pombe as a foreign gene. At this time, as long as the expressed protein has NADP-dependent glyceraldehyde-3-phosphate dehydrogenase activity, mutations such as deletion, substitution, addition, etc. have occurred in one or several amino acids in the base sequence of the gene. Also good.

  The above glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase may be used alone or as a mixture of two or more. Alternatively, one or more of glucose-6-phosphate dehydrogenase, one or more of 6-phosphogluconate dehydrogenase, and one or more of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase may be used.

  The form of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase used in the method of the present invention is not particularly limited, and any known form Is also applicable. Specifically, the enzyme protein itself purified from the cell may be used, or the glucose-6-phosphate dehydrogenase gene, 6-phosphogluconate dehydrogenase gene, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene in the cell. Cells containing enzymes synthesized by the expression of, and immobilized enzymes of the above enzymes can be used. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase can be converted into one or several amino acids in the amino acid sequence as long as the enzyme activity is exhibited. Mutations such as deletion, substitution and addition may occur.

  In addition, an organism using a glucose-6-phosphate dehydrogenase gene, a 6-phosphogluconate dehydrogenase gene, and a NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene as a genetic recombination host is acetyl-CoA. Any organism capable of biosynthesizing malonyl-CoA may be used, but microorganisms are preferred in view of ease of operation, versatility of a usable host, growth rate of the organism, and the like. The host may be selected depending on whether the compound used as a raw material for 3HP has assimilability. Further, it may be used by imparting the assimilability of a compound used as a raw material to an organism having advantageous traits in 3HP fermentation production such as high growth rate and high acid resistance. In addition, a microorganism having a 3HP cycle may be used as a genetic recombination host. The gene encoding malonyl-CoA reductase and the glucose-6-phosphate dehydrogenase gene, 6-phosphogluconate dehydrogenase gene and / or NADP-dependent glyceraldehyde-3-phosphate dehydrogenase are the same host (first May be transformed into one genetically modified microorganism) or transformed into a different host, but is preferably transformed into the same host. By transforming into a single host in this way, the reaction of acetyl CoA → malonyl CoA → 3HP is carried out in one host (for example, the first genetically modified microorganism), and thus the above reaction proceeds efficiently. In addition, since it is not necessary to consider the difference in the culture conditions between the hosts, the transformed host can be cultured under the optimal culture conditions. In addition to the above, by carrying out the enzyme reaction by each enzyme gene in one cell, it is possible to efficiently transfer the oxidizing power (NAD, NADP) and reducing power (NADH, NADPH) required for each enzyme reaction. There is an advantage that 3HP productivity can be improved.

  In this way, specific examples of metabolic modifications that produce more NADPH or genetic modifications to control intracellular redox balance in metabolic pathways that assimilate compounds as raw materials have been described However, any metabolically modified or mutant strain that can achieve the same effect can be used as a genetically modified host for the method of the present invention.

  According to one embodiment of the present invention, the acid-resistant microorganism is preferably S. Pombe. Therefore, according to the present invention, Schizosaccharomyces pombe encodes a gene encoding a foreign malonyl CoA reductase, a gene encoding a foreign acetyl CoA carboxylase, and a foreign gene having an effect of increasing the amount of NADPH in the cell. A genetically modified microorganism into which a gene has been introduced is provided. The foreign genes having an effect of increasing the amount of NADPH in the cells are glucose-6-phosphate dehydrogenase gene, 6-phosphogluconate dehydrogenase gene, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene Is more preferable, and it is more preferably a NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene, and further preferably a gene encoding NADP-dependent glyceraldehyde 3-phosphate dehydrogenase derived from Kluyveromyces lactis.

  Further, in Schizosaccharomyces pombe, a gene encoding an exogenous malonyl CoA reductase, a gene encoding an exogenous acetyl-CoA carboxylase, a gene encoding an exogenous gene having an effect of increasing intracellular NADPH amount, and intracellular acetyl A genetically modified microorganism into which a gene encoding a foreign gene having an effect of increasing the amount of CoA produced is introduced can also be used. Specifically, a gene encoding a foreign malonyl CoA reductase, a gene encoding a foreign acetyl CoA carboxylase, a foreign NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, a foreign fructose More preferably, it is a microorganism into which a gene encoding -6-phosphate phosphoketolase and a gene encoding a foreign phosphotransacetylase are introduced.

(Medium and culture conditions)
The culture medium and culture conditions used for the culture may be selected according to the growth conditions of the microorganism, and in addition to the above-mentioned sugar, use a normal medium containing a nitrogen source, inorganic ions, and other organic micronutrients as necessary. There is no particular limitation. For example, when Escherichia coli is used as the host microorganism, LB medium can be exemplified, and when yeast is used as the host microorganism, YPD medium, YES medium, EMM medium and the like can be exemplified.

  The culture is not particularly limited as long as it is performed under conditions suitable for the growth of microorganisms. Considering the production efficiency of 3HP, as a preferred embodiment when Escherichia coli is used as a host microorganism, the culture temperature is more than 20 ° C. and less than 37 ° C., more preferably 22 to 35 ° C., further preferably 25-30 ° C. When a genetically modified microorganism using Escherichia coli as a host microorganism is cultured in a temperature range lower than the general culture temperature, the production efficiency of 3HP can be improved. The experimental result which uses colon_bacillus | E._coli as a host microorganism in the below-mentioned Example is shown. It can be seen that the production efficiency of 3HP is higher in the case of 25 ° C., which is lower than in the case where the culture temperature is the optimum temperature of E. coli (37 ° C.). In addition, when yeast is used as a host, the preferred embodiment is 20 ° C. to 42 ° C. in consideration of the production efficiency of 3HP. The culture time is not particularly limited, but is preferably 10 to 100 hours, more preferably 15 to 60 hours.

  The pH in the culture of microorganisms is not particularly limited as long as 3HP can be efficiently produced by fermentation. In the present invention, since the production of fermentation products continues even at low pH, it is possible to culture without adjusting the pH to near neutrality by adding an alkaline reagent. For example, when Schizosaccharomyces pombe is used as a host, the culture starts from the pH of the medium to be used (near neutral) at the start of the culture, but the pH of the medium gradually decreases as 3HP is produced. The growth of the host is suppressed when the pH of the medium is lowered to around pH 3, but even if the pH of the medium is lowered to pH 3 or lower, for example, around pH 1, the carbon source in the medium is assimilated and 3HP production continues. When the culture is terminated when the concentration of 3HP in the medium exceeds 100 g / L, the pH of the medium becomes less than 4. Thus, for example, about 3HP having a growth inhibitory activity of microorganisms about 1.4 times higher than lactic acid, culturing without adding an alkaline reagent in the fermentation step, and obtaining a 3HP fermentation solution having a pH of less than 4, When used in a process after the fermentation process, for example, a 3HP recovery process from the fermentation broth, reduction of raw materials, simplification of the process, and improvement of the utility can be expected. The pH may be adjusted to an appropriate range in consideration of microbial growth, 3HP pKa (4.5), and the like. As a method for adjusting the pH, there are a method of adding an alkaline substance to a culture solution in a timely manner during fermentation, a method of using a medium having a buffering action, and the like. When alkaline substances are added to the culture solution in a timely manner, alkaline reagents such as aqueous sodium hydroxide, aqueous potassium hydroxide, aqueous ammonium hydroxide, aqueous calcium hydroxide, aqueous potassium carbonate, aqueous sodium carbonate, aqueous potassium acetate, etc. Any alkaline reagent can be used. In addition, as a method of using a buffer medium, calcium hydroxide, calcium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, ammonium carbonate, ammonia, hydroxide are previously added to the medium used for fermentation. The method of using the culture medium which added ammonium etc. is mentioned. Although the above was described as the method of pH adjustment, the method of performing 3HP fermentation, without substantially performing pH adjustment as mentioned above is more preferable.

  The nitrogen source is not particularly limited as long as a nitrogen source suitable for the growth of the microorganism to be used is selected. For example, in addition to ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, use of peptone, meat extract, yeast extract, corn steep liquor and the like can be mentioned. Similarly, the inorganic material is not particularly limited as long as a nitrogen source suitable for the growth of microorganisms is selected. For example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride and the like can be mentioned.

  During the culture, antibiotics such as kanamycin, ampicillin, streptomycin, tetracycline, chloramphenicol, erythromycin, G418, hygromycin B, aureobasidin A, streptoslysin, blasticidin, phleomycin 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), arabinose, lactose and the like can be added to the medium.

  Alternatively, cells are collected from the culture of the organism 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 malonyl CoA and / or acetyl CoA and performing a reaction in the bacterial cell. 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. The number is 5 or more and 10.0 or less, preferably 9.7 or less, but is not particularly limited as long as it is a reaction condition suitable for 3HP generation. When the reaction is carried out continuously, the reaction is carried out for 1 week to 3 months.

  The 3HP-containing solution to be subjected to the 3HP purification step is preferably a fermentation solution recovered from the luminescent layer when the 3HP concentration has reached 90 g / L or more, more preferably 100 g / L or more, and even more preferably 100 to 300 g / L. It is preferable to use it.

  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 performance liquid chromatography (HPLC) analysis.

  As described above, according to the production method of the present invention, the production amount of 3-hydroxypropionic acid can be improved by using the first and / or second genetically modified microorganisms. Further, since 3-hydroxypropionic acid is obtained in the acid form, 3-hydroxypropionic acid can be obtained more easily and in a higher yield as compared with the conventional method of adding an alkaline reagent obtained in the form of 3HP salt. Can be purified.

[Method for producing acrylic acid]
The 3-hydroxypropionic acid produced by the method of the present invention can be used in various fields. In one embodiment, acrylic acid can be produced by dehydrating 3-hydroxypropionic acid obtained by the above method. That is, the present invention also provides a method for producing acrylic acid, which comprises dehydrating 3-hydroxypropionic acid produced by the method of the present invention. Here, dehydration of 3-hydroxypropionic acid can be carried out by a known reaction. For example, as described in US Pat. No. 2,469,701, 3-hydroxypropionic acid can be easily converted to acrylic acid by distillation under reduced pressure in the presence of a catalyst.

  Moreover, the composition containing acrylic acid obtained by the method of the present invention may be purified. When purification is performed, a crystallization step is preferably used. Therefore, this invention also provides the manufacturing method which refine | purifies acrylic acid manufactured by the method of this invention by refinement | purification processes, such as crystallization, and obtains purified acrylic acid.

  The crystallization step is a step of obtaining purified acrylic acid by supplying a composition containing acrylic acid to a crystallization apparatus to cause crystallization. The crystallization method may be a conventionally known crystallization method and is not particularly limited. For example, the crystallization may be performed using a continuous or batch crystallization apparatus. It can be carried out in stages or in two or more stages. The obtained acrylic acid crystals can be further purified by washing, sweating, or the like, if necessary, to obtain purified acrylic acid with higher purity.

  Examples of the continuous crystallizer include a crystallizer in which a crystallization part, a solid-liquid separation part and a crystal purification part are integrated (for example, a BMC (Backmixing Column Crystallizer) apparatus manufactured by Nippon Steel Chemical Co., Ltd., Tsukishima Continuous melt purification system manufactured by Kikai Co., Ltd., crystallization unit (eg, CDC (Cooling Disk Crystallizer) device manufactured by GMF GOUDA), solid-liquid separation unit (eg, centrifuge, belt filter) and crystal purification unit ( For example, a crystallizer combined with KCP (Kureha Crystal Purifier) manufactured by Kureha Techno Engineering Co., Ltd. can be used.

  As the batch crystallization apparatus, for example, a layer crystallization apparatus (dynamic crystallization apparatus) manufactured by Sulzer Chemtech, a static crystallization apparatus manufactured by BEFS PROKEM, or the like can be used.

  Dynamic crystallization is, for example, a tubular crystallizer equipped with a temperature control mechanism for performing crystallization, sweating and melting, a tank for collecting the mother liquor after sweating, and supplying crude acrylic acid to the crystallizer. This is a method in which crystallization is performed using a dynamic crystallization apparatus that includes a circulation pump and can transfer crude acrylic acid from a reservoir provided at the lower part of the crystallizer to the upper part of the crystallizer tube. Static crystallization is, for example, a tubular crystallizer equipped with a temperature control mechanism for crystallization, sweating, and melting, and a crystallizer having an extraction valve at the bottom and a mother liquor after sweating are collected. The crystallization is carried out using a static crystallization apparatus equipped with a tank to be used.

  Specifically, crude acrylic acid is introduced into the crystallizer as a liquid phase, and acrylic acid in the liquid phase is solidified and generated on the cooling surface (tube wall surface). As soon as the mass of the solid phase generated on the cooling surface becomes 10 to 90% by mass, more preferably 20 to 80% by mass, based on the crude acrylic acid introduced into the crystallizer, the liquid phase is immediately converted into the crystallizer. The solid phase and the liquid phase are separated. The liquid phase may be discharged by either a pumping method (dynamic crystallization) or a discharging method from a crystallizer (static crystallization). On the other hand, after the solid phase is taken out from the crystallizer, purification such as washing and sweating may be performed in order to further improve the purity.

  When dynamic crystallization or static crystallization is performed in multiple stages, it can be advantageously carried out by adopting the countercurrent principle. At this time, the acrylic acid crystallized in each stage is separated from the residual mother liquor and supplied to the stage where acrylic acid having higher purity is generated. On the other hand, the residual mother liquor is fed to the stage where acrylic acid with lower purity is produced.

  In dynamic crystallization, if the purity of acrylic acid is low, crystallization becomes difficult, but in static crystallization, the time for the residual mother liquor to contact the cooling surface is longer than in dynamic crystallization, In addition, since the influence of temperature is easily transmitted, crystallization is easy even if the purity of acrylic acid is lowered. Therefore, in order to improve the recovery rate of acrylic acid, the final residual mother liquor in dynamic crystallization may be subjected to static crystallization and further crystallization may be performed.

  The number of crystallization stages required depends on the degree of purity required, but the number of stages required to obtain high purity acrylic acid is usually 1 to 6 purification steps (dynamic crystallization). The stripping step (dynamic crystallization and / or static crystallization) is usually 0 to 5 times, preferably 0 to 3 times, preferably 2 to 5 times, more preferably 2 to 4 times. In general, all stages where acrylic acid having a higher purity than the crude acrylic acid supplied is obtained are purification stages, and all other stages are stripping stages. The stripping step is performed to recover acrylic acid contained in the residual mother liquor from the purification step. Note that the stripping step is not necessarily provided. For example, when the low boiling point component is separated from the residual mother liquor of the crystallizer using a distillation column, the stripping step may be omitted.

  Whether using dynamic crystallization or static crystallization, the acrylic acid crystals obtained in the crystallization process may be used directly as products, or if necessary, such as washing and sweating. It may be a product after purification. On the other hand, the residual mother liquor discharged in the crystallization step may be taken out of the system.

[Method for producing water-absorbing resin]
Since acrylic acid produced by the above method is known to be usable as a raw material for acrylic acid derivatives such as polyacrylic acid, the production method for acrylic acid is used in the production method for acrylic acid derivatives. It is also possible to use an acrylic acid production process. That is, in one embodiment, partially neutralized acrylic acid obtained by the above method to produce partially neutralized acrylic acid, and if necessary, (co) polymerize with other monomers to absorb water. Resin can be produced. Accordingly, the present invention provides a water absorption, comprising partially neutralizing acrylic acid produced by the method of the present invention to produce partially neutralized acrylic acid, and copolymerizing the partially neutralized acrylic acid with a crosslinkable monomer. A method for producing a conductive resin is also provided. Here, the partial neutralization and (co) polymerization can be carried out by a known reaction. For example, acrylic acid and / or a salt thereof obtained by the production method of the present invention is used as the main component of the monomer component. (Preferably 70 mol% or more, more preferably 90 mol% or more), and further a crosslinking agent of about 0.001 to 5 mol% (value relative to acrylic acid), 0.001 to 2 mol% (based on the monomer component A water-absorbent resin can be obtained by carrying out cross-linking polymerization using a radical polymerization initiator of value) and then drying and pulverizing.

  Here, the water-absorbing resin is a water-swellable, water-insoluble polyacrylic acid having a cross-linked structure, and absorbs pure water or physiological saline 3 times or more, preferably 10 to 1,000 times its own weight. In addition, it means polyacrylic acid that forms a water-insoluble hydrogel having a water-soluble component (water-soluble component) of preferably 25% by mass or less, more preferably 10% by mass or less. Specific examples of such water-absorbing resins and methods for measuring physical properties are described in, for example, US Pat. No. 6,107,358, US Pat. No. 6,174,978, US Pat. No. 6,241,928, and the like. ing.

  Further, from the viewpoint of improving productivity, preferred production methods include, for example, US Pat. No. 6,867,269, US Pat. No. 6,906,159, US Pat. No. 7,091,253, International Publication No. 01/253, No. 038402, International Publication No. 2006/034806, and the like.

  A series of steps for producing a water-absorbing resin by neutralization, polymerization, drying, etc. using acrylic acid as a starting material is as follows, for example.

  A part of acrylic acid obtained by the production method of the present invention is supplied to the production process of the water-absorbent resin via a line. In the manufacturing process of the absorbent resin, acrylic acid is introduced into the neutralization step, the polymerization step, and the drying step, and a desired treatment is performed to manufacture the water-absorbent resin. For the purpose of improving various physical properties, a desired treatment may be performed. For example, a crosslinking step may be interposed during or after the polymerization.

  The neutralization step is an optional step. For example, a method of mixing a predetermined amount of a basic substance powder or aqueous solution with acrylic acid or polyacrylic acid (salt) is exemplified. It may be employed and is not particularly limited. The neutralization step may be performed either before or after polymerization, or may be performed both before and after polymerization. As a basic substance used for neutralization of acrylic acid or polyacrylic acid (salt), for example, a conventionally known basic substance such as a carbonic acid (hydrogen) salt, an alkali metal hydroxide, ammonia, an organic amine or the like is appropriately used. Use it. Moreover, the neutralization rate of polyacrylic acid is not specifically limited, What is necessary is just to adjust so that it may become arbitrary neutralization rates (for example, arbitrary values in the range of 30-100 mol%).

  The polymerization method in the polymerization step is not particularly limited, and a conventionally known polymerization method such as polymerization with a radical polymerization initiator, radiation polymerization, polymerization by irradiation with an electron beam or active energy ray, ultraviolet polymerization with a photosensitizer, etc. May be used. Various conditions such as a polymerization initiator and polymerization conditions can be arbitrarily selected. Of course, if necessary, conventionally known additives such as a crosslinking agent and other monomers, and further a water-soluble chain transfer agent and a hydrophilic polymer may be added.

  The acrylate polymer after polymerization (namely, water-absorbing resin) is subjected to a drying step. The drying method is not particularly limited, and using a conventionally known drying means such as a hot air dryer, a fluidized bed dryer, a nauter dryer, etc., a desired drying temperature, preferably 70 to 230 ° C., What is necessary is just to dry suitably.

  The water-absorbent resin obtained through the drying step may be used as it is, or may be used after granulation / pulverization and surface cross-linking into a desired shape, and conventionally known reducing agents, perfumes, binders, etc. You may use it, after giving post-processing according to a use, such as adding an additive.

[Method for producing acrylic ester and acrylic ester resin]
In one embodiment of the present invention, acrylic acid ester can be produced by reacting acrylic acid produced by the above-described method with alcohol to esterify. Accordingly, the present invention provides a method for producing an acrylate ester comprising esterifying acrylic acid produced by the method of the present invention.

  The esterification is a reaction in which a carboxy group of acrylic acid is dehydrated and condensed with a hydroxyl group of alcohol. The esterification reaction is not particularly limited, and a known technique can be appropriately employed. However, it is preferable to perform the esterification reaction in the presence of a catalyst.

  The alcohol can be appropriately selected according to a desired acrylic ester. Specific examples of the alcohol include lower aliphatic alcohols having 1 to 4 carbon atoms and alicyclic alcohols having 3 to 6 carbon atoms.

  Although it does not restrict | limit especially as said catalyst, Strong acid cation exchange resin etc. are mentioned. At this time, as specific examples of the strong acid cation exchange resin, C-26C (manufactured by Duolite), PK-208, PK-216, PK-228 (all manufactured by Mitsubishi Chemical Corporation), MSC-1 , 88 (manufactured by Dow), Amberlisto 16 (manufactured by Rohm and Haas), SPC-108, SPC-112 (both manufactured by Bayer), and the like.

  In the esterification reaction, an additive such as a polymerization inhibitor may be added.

  The polymerization inhibitor is not particularly limited, but quinones such as hydroquinone and methoquinone (p-methoxyphenol); phenothiazine, bis- (α-methylbenzyl) phenothiazine, 3,7-dioctylphenothiazine, bis- (α- Phenothiazines such as (dimethylbenzyl) phenothiazine; 2,2,6,6-tetramethylpiperidinooxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidinooxyl, 4,4 ′, 4 ″ N-oxyl compounds such as tris- (2,2,6,6-tetramethylpiperidinooxyl) phosphite; copper dialkyldithiocarbamate, copper acetate, copper naphthenate, copper acrylate, copper sulfate, copper nitrate, Copper salt compounds such as copper chloride; manganese dialkyldithiocarbamate, diphenyldithiocarbamate Manganese salt compounds such as manganese, formate manganese, manganese acetate, octanoate, manganese naphthenate, manganese permanganate, manganese salt of ethylenediaminetetraacetic acid; N-nitrosophenylhydroxylamine and its salts, p-nitrosophenol, N- Examples thereof include nitroso compounds such as nitrosodiphenylamine and salts thereof, etc. Among these, quinones such as hydroquinone and methoquinone are preferably used as the polymerization inhibitor, and these polymerization inhibitors may be used alone. Alternatively, two or more kinds may be used in combination.

  Esterification can usually be carried out in the liquid phase. Moreover, as reaction temperature of esterification, although it changes also with reaction, it is the range of 50 to 110 degreeC normally.

  As a specific method for producing an acrylate ester by esterification, for example, a method disclosed in Japanese Patent Publication No. 6-86406 can be used.

  Moreover, in one Embodiment of this invention, acrylic ester resin can be manufactured by performing the polymerization reaction which uses the acrylic ester manufactured above as a monomer component. Therefore, this invention also provides the manufacturing method of acrylic ester resin which has superposing | polymerizing the monomer component containing the acrylic ester manufactured by the method of this invention.

  The acrylic ester resin can be produced by polymerizing a monomer component containing an acrylic ester and any other monomer by a known polymerization method.

  Examples of the other monomers include (meth) acrylic acid and esters and amides thereof. By polymerizing these monomers in combination, a desired polymer can be obtained.

  The polymerization method is not particularly limited, and known methods such as solution polymerization, suspension polymerization, emulsion polymerization, and bulk polymerization can be used.

  As with normal (meth) acrylic polymers, acrylic ester resins having various properties are obtained by designing based on the FOX equation using the glass transition temperature of the homopolymer of each monomer component as an index. be able to. The acrylate resin thus obtained can be used for various applications such as pressure-sensitive adhesives, dispersants, adhesives, films, sheets, paints and the like.

  The present invention also relates to a composition for producing 3-hydroxypropionic acid from malonyl CoA and / or acetyl CoA comprising malonyl CoA reductase activity and / or acetyl CoA carboxylase activity. The said composition can be manufactured from the organism which produces these enzymes, or its processed material.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

(Example 1: Acid resistance test of Schizosaccharomyces pombe OB2)
Using an acidic medium (3-HP added YES liquid medium) containing 3-hydroxypropionic acid (3HP), it was examined whether S. pombe OB2 contains 3HP and whether metabolites can be produced under acidic conditions. .

  First, S. pombe OB2 was cultured. More specifically, S. pombe OB2 (h + leu1-32 ura4-D18) (distributed from Shimane University, Faculty of Bioresource Sciences, Professor Makoto Kawamukai), 5 mL of YES liquid medium (yeast extract = 5 g / L, glucose = 30 g) / L, leucine = 0.225 g / L, uracil = 0.225 g / L, adenine = 0.225 g / L, histidine = 0.225 g / L), and cultured with shaking at 30 ° C. for 72 hours. Centrifugation was performed at 3000 rpm for 3 minutes using the obtained culture solution to obtain bacterial cell pellets.

  Next, an acidic medium (3HP-added YES liquid medium) containing 3HP was prepared as follows. Specifically, 3-hydroxypropionic acid (3HP) reagent (manufactured by Tokyo Chemical Industry Co., Ltd.) is added to the YES liquid medium so that the final concentration is 120 g / L, and the pH 2.6 2.6-added YES liquid medium (3HP concentration of 3HP reagent was converted to 200 g / L).

  And it was examined by the following method whether S. pombe OB2 can produce a metabolite in an acidic medium containing 3HP. That is, the whole amount of the cell pellet obtained above was suspended in 5 mL of 3HP-added YES liquid medium, and cultured with shaking at 30 ° C. for 96 hours. The obtained culture broth was centrifuged at 15000 rpm for 5 minutes, and the culture supernatant was collected. The supernatant was diluted 10-fold with 5 mM sulfuric acid and then passed through a 0.45 μm filter to obtain a liquid chromatography (LC) analysis sample. Using the sample, LC analysis was performed under the following conditions.

Column used: Aminex HPX-87Hlon exclusion column 300mm × 7.8mm (BIO-RAD)
Mobile phase: 5 mM sulfuric acid Flow rate: 0.5 mL / min
Column temperature: 20 ° C
Detection: RI
Injection volume: 10 μL.

  From the LC analysis, the glucose concentration and ethanol concentration in the culture supernatant were calculated. The analysis results are shown in Table 1. Consumption of glucose and production of ethanol were confirmed in the culture solution of S. pombe OB2 strain using 3HP-added YES liquid medium. Also, no decrease in 3HP was observed. This suggested that S. pombe OB2 can sufficiently produce metabolites even in an acidic medium containing 3HP. That is, it was suggested that S. pombe OB2 can continue metabolism without neutralization.

(Example 2: Acid resistance test of Kluyveromyces marxianus ATCC52486)
Using an acidic medium (3-HP-added YM liquid medium) containing 3-hydroxypropionic acid (3HP), whether or not K. marxianus ATCC52486 contains 3HP and can be grown under acidic conditions was examined.

  First, K. marxianus ATCC52486 was cultured. More specifically, K. marxianus ATCC52486 is inoculated into 5 mL of YM liquid medium (yeast extract = 3 g / L, Malt extract = 3 g / L, glucose = 10 g / L, peptone = 5 g / L) at 30 ° C. The shaking culture was performed for 72 hours. Centrifugation was performed at 3000 rpm for 3 minutes using the obtained culture solution to obtain bacterial cell pellets.

  Next, an acidic medium (3HP-added YM liquid medium) containing 3HP was prepared as follows. Specifically, 3HP reagent (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the YM liquid medium so that the final concentration was 120 g / L to obtain a 3HP-added YM liquid medium having a pH of 2.4 (of 3HP reagent). 3HP concentration is calculated as 200 g / L).

  And it was examined by the following method whether K. marxianus ATCC52486 can be grown in an acidic medium containing 3HP. That is, the whole cell pellet obtained above was suspended in 5 mL of 3 HP-added YM liquid medium, and cultured with shaking at 30 ° C. for 96 hours. The obtained culture broth was centrifuged at 15000 rpm for 5 minutes, and the culture supernatant was collected. The supernatant was diluted 10-fold with 5 mM sulfuric acid and then passed through a 0.45 μm filter to obtain a liquid chromatography (LC) analysis sample. Using the sample, LC analysis was performed under the following conditions.

Column used: Aminex HPX-87Hlon exclusion column 300mm × 7.8mm (BIO-RAD)
Mobile phase: 5 mM sulfuric acid Flow rate: 0.5 mL / min
Column temperature: 20 ° C
Detection: RI
Injection volume: 10 μL.

  From the LC analysis, the glucose concentration and ethanol concentration in the culture supernatant were calculated. The analysis results are shown in Table 1. Consumption of glucose and production of ethanol were not confirmed in the culture solution of K. marxianus ATCC52486 strain using 3HP-added YES liquid medium. Also, no decrease in 3HP was observed. This suggests that K. marxianus ATCC52486 cannot be metabolized in an acidic medium containing 3HP, and neutralization may be essential.

(Example 3: Construction of various plasmids)
(Example 3-1: Construction of pADH)
A plasmid pADH (G418R-adh1-nmt1 / pUC18) in which the adh1 fragment, the G418R fragment, and the nmt1 fragment were introduced into pUC18 was constructed. Details are shown below. In addition, the plasmid map of pADH is shown in FIG.

  Using S. pombe OB2 genomic DNA as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain an Alchol dehydrogenase peripheral region fragment (adh1 fragment).

  Using the plasmid DNA of pUC18 (Takara) as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pUC18 fragment.

  Using the adh1 fragment and pUC18 fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct adh1 / pUC18.

  PCR amplification was performed using the plasmid DNA of adh1 / pUC18 as a template and the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain an adh1 / pUC18 fragment.

  Using the plasmid DNA of pMK38 (obtained from National BioResource Project, Yeast, NBRP ID: BYP6739) as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes), and the G418 resistance gene fragment (G418R fragment) was obtained.

  Using the adh1 / pUC18 fragment and G418R fragment obtained by the above PCR amplification, cloning was performed according to the protocol of In-Fusion PCR cloning kit (Takara) to construct G418R-adh1 / pUC18.

  PCR amplification was performed using G418R-adh1 / pUC18 plasmid DNA as a template and the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a G418R-adh1 / pUC18 fragment.

  PCR amplification was carried out using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) using plasmid DNA of REP3X (ATCC87603) as a template to obtain an nmt1 fragment.

  Using the G418R-adh1 / pUC18 fragment and nmt1 fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct G418R-adh1-nmt1 / pUC18.

(Example 3-2: Construction of pGPD)
A plasmid pGPD (G418R-gpd1-nmt1 / pUC18) in which the gpd1 fragment, the G418R fragment, and the nmt1 fragment were introduced into pUC18 was constructed. Details are shown below. The plasmid map of pGPD is shown in FIG.

  Using the genomic DNA of S. pombe OB2 strain as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes). Glycerol-3-phophate dehydrogenase peripheral region fragment (gpd1 fragment) Obtained.

  Using the plasmid DNA of pUC18 (Takara) as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pUC18 fragment.

  Using the gpd1 fragment and the pUC18 fragment obtained by the above PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct gpd1 / pUC18.

  PCR amplification was performed on the plasmid DNA of gpd1 / pUC18 using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a gpd1 / pUC18 fragment.

  Using the plasmid DNA of pMK38 (obtained from National BioResource Project, Yeast, NBRP ID: BYP6739) as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes), and the G418 resistance gene fragment (G418R fragment) was obtained.

  Using the gpd1 / pUC18 fragment and G418R fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct G418R-gpd1 / pUC18.

  PCR amplification was performed using plasmid DNA of G418R-gpd1 / pUC18 as a template and the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a G418R-gpd1 / pUC18 fragment.

  PCR amplification was carried out using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) using plasmid DNA of REP3X (ATCC87603) as a template to obtain an nmt1 fragment.

  Using the G418R-gpd1 / pUC18 fragment and nmt1 fragment obtained by the PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct G418R-gpd1-nmt1 / pUC18.

(Example 3-3: Construction of pG418)
A G418R fragment, a HygR fragment (constructed by introducing a HygR_ORF fragment into pAUR224), and a plasmid pG418 (G418R-HygR-nmt1 / pUC18) in which the nmtl fragment was introduced into pUC18 were constructed. Details are shown below. In addition, the plasmid map of pG418 is shown in FIG.

  Using the plasmid DNA of pUC18 (Takara) as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pUC18 fragment.

  Using the plasmid DNA of pMK38 (obtained from National BioResource Project, Yeast, NBRP ID: BYP6739) as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes), and the G418 resistance gene fragment (G418R fragment) was obtained.

  Using the pUC18 fragment and G418R fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of In-Fusion PCR cloning kit (Takara) to construct G418R / pUC18.

  PCR amplification was performed using plasmid DNA of G418R / pUC18 as a template and the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a G418R / pUC18 fragment.

  Using the plasmid DNA of pAUR224 (Takara) as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pAUR224 fragment.

  Using plasmid DNA of pEBMulti-Hyg (Wako Pure Chemical Industries) as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes), and hygromycin B resistant structural gene fragment (HygR_ORF fragment) )

  Using the pAUR224 fragment and HygR_ORF fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara), and the hygromycin B resistance gene was found downstream of the cytomegalovirus (CMV) promoter. The inserted HygR / pAUR224 was constructed.

  Using the plasmid DNA of HygR / pAUR224 as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a hygromycin B resistant gene fragment (HygR fragment).

  Using the G418R / pUC18 fragment and HygR fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct G418R-HygR / pUC18.

  PCR amplification was performed using plasmid DNA of G418R-HygR / pUC18 as a template and the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a G418R-HygR / pUC18 fragment.

  PCR amplification was carried out using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) using plasmid DNA of REP3X (ATCC87603) as a template to obtain an nmt1 fragment.

  Using the G418R-HyR / pUC18 fragment and nmt1 fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct G418R-HygR-nmt1 / pUC18.

(Example 3-4: Construction of pHYG)
Plasmid pHYG (HygR-AbAR-nmt1 / pUC18) in which the HygR fragment, AbAR fragment, and nmt1 fragment were introduced into pUC18 was constructed. Details are shown below. The pHYG plasmid map is shown in FIG.

  Using the plasmid DNA of pUC18 (Takara) as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pUC18 fragment.

  Using the plasmid DAN of HygR / pAUR224 as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a hygromycin B resistant gene fragment (HygR fragment).

  Using the pUC18 fragment and HygR fragment obtained by the above PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct HygR / pUC18.

  Using the plasmid DNA of HygR / pUC18 as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a HygR / pUC18 fragment.

  Using the plasmid DNA of pAUR224 (Takara) as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain an aureobasidin A resistance gene fragment (AbAR fragment). .

  Using the HygR / pUC18 fragment and AbAR fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct HygR-AbAR / pUC18.

  Using the plasmid DNA of HygR-AbAR / pUC18 as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a HygR-AbAR / pUC18 fragment.

  PCR amplification was carried out using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) using plasmid DNA of REP3X (ATCC87603) as a template to obtain an nmt1 fragment.

  Using the HygR-AbAR / pUC18 fragment and nmt1 fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct HygR-AbAR-nmt1 / pUC18.

(Example 3-5: Construction of pAUR)
A plasmid pAUR (leu2-AbAR-nmt1 / pUC18) in which the AbAR fragment, leu2 fragment, and nmt1 fragment were introduced into pUC18 was constructed. Details are shown below. The plasmid map of pAUR is shown in FIG.

  Using the plasmid DNA of pAUR224 (Takara) as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain an aureobasidin A resistance gene fragment (AbAR fragment). .

  Using the plasmid DNA of pUC18 (Takara) as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pUC18 fragment.

  Using the AbAR fragment and pUC18 fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct AbAR / pUC18.

  Using the plasmid DNA of AbAR / pUC18 as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain an AbAR / pUC18 fragment.

  PCR amplification was performed using plasmid DNA of REP3X (ATCC87603) as a template and the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a beta-isopropyl-malate dehydrogenase fragment (leu2 fragment).

  Using the AbAR / pUC18 fragment and leu2 fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct leu2-AbAR / pUC18.

  Using leu2-AbAR / pUC18 plasmid DNA as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a leu2-AbAR / pUC18 fragment.

  PCR amplification was carried out using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) using plasmid DNA of REP3X (ATCC87603) as a template to obtain an nmt1 fragment.

  Using the leu2-AbAR / pUC18 fragment and nmt1 fragment obtained by the PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct leu2-AbAR-nmt1 / pUC18.

(Example 4: Cloning of foreign genes into various vectors)
(Example 4-1: Construction of CP_GD_α / pADH)
The α subunit of glycerol dehydrogenase (GD) derived from Clostridium perfiringens ATCC13124 (CP) strain was introduced into pADH to construct CP_GD_α / pADH. Details are shown below.

  Using the genomic DNA of the CP strain as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain an α subunit fragment of GD (CP_GD_α fragment).

  PCR amplification was performed using plasmid DNA of pADH as a template and the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pADH fragment.

  Using the CP_GD_α fragment and the pADH fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct CP_GD_α / pADH in which the CP_GD_α fragment was inserted downstream of the nmt1 promoter. .

(Example 4-2: Construction of CP_GD_β / pADH)
The β subunit of glycerol dehydrogenase (GD) derived from Clostridium perfiringens ATCC13124 (CP) strain was introduced into pADH to construct CP_GD_β / pADH. Details are shown below.

  Using the genomic DNA of the CP strain as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a GD β subunit fragment (CP_GD_β fragment).

  PCR amplification was performed using plasmid DNA of pADH as a template and the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pADH fragment.

  Using the CP_GD_β fragment and the pADH fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct CP_GD_β / pADH in which the CP_GD_β fragment was inserted downstream of the nmt1 promoter. .

(Example 4-3: Construction of CP_GD_γ / pG418)
A glycerol subunit of glycerol dehydrogenase (GD) derived from Clostridium perfiringens ATCC13124 (CP) was introduced into pG418 to construct CP_GD_γ / pG418. Details are shown below.

  Using the genomic DNA of the CP strain as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a GD γ subunit fragment (CP_GD_γ fragment).

  Using the plasmid DNA of pG418 as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pG418 fragment.

  Using the CP_GD_γ fragment and the pG418 fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct CP_GD_γ / pG418 in which the CP_GD_γ fragment was inserted downstream of the nmt1 promoter. .

(Example 4-4: Construction of CP_GD_βandγ / pG418)
The β subunit and γ subunit of glycerol dehydrogenase (GD) derived from Clostridium perfiringens ATCC13124 (CP) strain were introduced into pG418 to construct CP_GD_βandγ / pG418. Details are shown below.

  Using the plasmid DNA of CP_GD_β / pADH as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a Glycerol dehydratase β subunit expression cassette fragment (CP_GD_β expression cassette fragment) It was.

  Using the plasmid DNA of CP_GD_γ / pG418 as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a CP_GD_γ / pG418 necessary region fragment.

  Using the CP_GD_β expression cassette fragment and the CP_GD_γ / pG418 necessary region fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct CP_GD_βandγ / pG418.

(Example 4-5: Construction of CP_GDR_L / pHYG)
CP_GDR_L / pHYG was constructed by introducing L (Large) subunit of glycerol dehydrogenase reactivating factor (GDR) derived from Clostridium perfiringens ATCC13124 (CP) strain into pHYG. Details are shown below.

  Using the genomic DNA of the CP strain as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain an L subunit fragment (CP_GDR_L fragment) of GDR.

  Using the pHYG plasmid DNA as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pHYG fragment.

  Using the CP_GDR_L fragment and the pHYG fragment obtained by the above PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct CP_GDR_L / pHYG in which the CP_GDR_L fragment was inserted downstream of the nmt1 promoter. .

(Example 4-6: Construction of CP_GDR_S / pADH)
CP_GDR_S / pADH was constructed by introducing S (Small) subunit of glycerol dehydrogenase reactivating factor (GDR) derived from Clostridium perfiringens ATCC13124 (CP) strain into pHYG. Details are shown below.

  Using the genomic DNA of the CP strain as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a GDR S subunit fragment (CP_GDR_S fragment).

  PCR amplification was performed using plasmid DNA of pADH as a template and the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pADH fragment.

  Using the CP_GD_S fragment and the pADH fragment obtained by the above PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct CP_GDR_S / pADH in which the CP_GDR_S fragment was inserted downstream of the nmt1 promoter. .

(Example 4-7: Construction of CP_GDR_LandS / pHYG)
CP_GDR_LandS / pHYG was constructed by introducing L (Large) subunit and S (Small) subunit of glycerol dehydrogenase reactivating factor (GDR) from Clostridium perfiringens ATCC13124 (CP) strain into pHYG. Details are shown below.

  Using the plasmid DNA of CP_GDR_S / pADH as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes), and a Glycerol dehydratase reactivating factor Small subunit fragment expression cassette fragment (CP_GDR_S expression cassette fragment) )

  Using the plasmid DNA of CP_GDR_L / pHYG as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a CP_GDR_L / pHYG necessary region fragment.

  Using the CP_GDR_S expression cassette fragment and the CP_GDR_L / pHYG necessary region fragment obtained by PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct CP_GDR_LandS / pHYG.

(Example 4-8: Construction of aldH / pAUR)
γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (aldH) derived from Escherichia coli W3110 (E. coli) strain was introduced into pAUR to construct aldH / pAUR. Details are shown below.

  PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) using the genomic DNA of E. coli strain as a template to obtain an aldH fragment.

  PCR amplification was performed using plasmid DNA of pAUR as a template and the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pAUR fragment.

  Using the aldH fragment and the pAUR fragment obtained by the above PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct aldH / pAUR in which the aldH fragment was inserted downstream of the nmt1 promoter. .

(Example 5: Preparation of DNA fragment used for transformation)
(Example 5-1: Production of G418R-nmt1 promoter (GD_α))
The G418R-nmt1 promoter was prepared from CP-GD_α / pADH.

  Specifically, using the plasmid DNA of CP-GD_α / pADH as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain the G418R-nmt1 promoter (GD_α). It was.

(Example 5-2: Production of HygR-nmt1 promoter (GD_βandγ))
The G418R-nmt1 promoter was prepared from CP-GD_βandγ / pG418.

  Specifically, using the plasmid DNA of CP-GD_βandγ / pG418 as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain the HygR-nmt1 promoter (GD_βandγ). It was.

(Example 5-3: Production of AbAR-nmt1 promoter (GDR_S))
AbAR-nmt1 promoter was prepared from CP-GDR_LandS / pHYG.

  Specifically, using the plasmid DNA of CP-GDR_LandS / pHYG as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzyme Finnzyme) to obtain an AbAR-nmt1 promoter (GDR_LandS). It was.

(Example 5-4: Production of Leu2-nmt1 promoter (aldH))
The Leu2-nmt1 promoter was prepared from aldH / pAUR.

  Specifically, using the plasmid DNA of aldH / pAUR as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain a Leu2-nmt1 promoter (aldH).

(Example 6: Transformation)
(Example 6-1: Production of S. pombe OB1 (GD_α))
The G418R-nmt1 promoter (GD_α) prepared in Example 5-1 was transformed into Schizosaccharomyces pombe OB1 to prepare S. pombe OB1 (GD_α). Details are shown below.

First, S. pombe OB1 was cultured. More specifically, S. pombe OB1 (h-leu1-32 ura4-D18) (distributed by Shimane University, Faculty of Bioresource Sciences, Professor Makoto Kawamukai) in YES liquid medium (yeast extract = 5 g / L, glucose = 30 g / L, histidine = 0.225 g / L, adenine = 0.225 g / L, leucine = 0.225 g / L, uracil = 0.225 g / L), and shaking culture at 30 ° C. until 1 × 10 ⁷ cells / mL Went. Centrifugation was performed at 3000 rpm for 1 minute using the obtained culture solution to obtain a cell pellet.

  And G418R-nmt1 promoter (GD_ (alpha)) was introduce | transduced into cultured S. pombe OB1. More specifically, a 0.1 M LiAc solution (containing 100 mM Litium Acetate, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and acetic acid) adjusted to pH 4.9 is added to the cell pellet obtained above. 10 mL was added to suspend the cell pellet. Centrifugation was performed at 3000 rpm for 1 minute, and the obtained bacterial cell pellet was suspended in 300 μL of a 0.1 M LiAc solution. To 100 μL of the cell suspension, 2 μg of G418R-nmt1 promoter (GD_α) and 290 μL of 50% PEG solution (polyethylene glycol 3345 = 500 g / L) were added and mixed, and incubated at 30 ° C. for 1 hour. Centrifugation was performed at 3000 rpm for 1 minute, and 500 μL of sterilized water was added to the obtained cell pellet to suspend the cell pellet. Centrifugation is performed at 3000 rpm for 1 minute, the cell pellet is suspended in 300 μL of sterilized water, and then YES agar medium (yeast extract = 5 g / L, glucose = 30 g / L, agar = 20 g / L, histidine = 0.225 g / L, adenine = 0.225 g / L, leucine = 0.225 g / L, uracil = 0.225 g / L) and incubated at 30 ° C. for 24 hours. The replica agar plate was inoculated into a YES agar medium supplemented with G418 to a final concentration of 150 μg / mL and incubated at 30 ° C. for 72 hours. The obtained single colony was inoculated in a YES liquid medium and subjected to shaking culture at 30 ° C. Subsequently, centrifugation was performed at 3000 rpm for 1 minute, and the microbial cell pellet was obtained. Using the obtained bacterial cell pellet, genomic DNA was extracted according to the protocol of Puregene Yeast / Bact kit B (Qiagen) to obtain G418-resistant S. pombe genomic DNA. Using the obtained genomic DNA as a template, PCR amplification was performed using the following primer set (1) and Phusion High-Fidelity DNA Polymerase (Finnzyme).

  When it was confirmed whether the G418R-nmt1 promoter (GD_α) was introduced into S. pombe, a PCR amplified fragment of about 3.8 kb that was observed only when the G418R-nmt1 promoter (GD_α) was inserted was confirmed. From this, it was confirmed that the G418R-nmt1 promoter (GD_α) was inserted into the chromosome of S. pombe.

(Example 6-2: Production of S. pombe OB1 (GD))
The HygR-nmt1 promoter (GD_βandγ) prepared in Example 5-2 was transformed into S. pombe OB1 (GD_α) prepared in Example 6-1 to prepare S. pombe OB1 (GD). Details are shown below.

First, S. pombe OB1 (GD_α) was cultured. More specifically, S. pombe OB1 (GD_α) was inoculated into a YES liquid medium, and shake-cultured at 30 ° C. until 1 × 10 ⁷ cells / mL. Centrifugation was performed at 3000 rpm for 1 minute using the obtained culture solution to obtain a cell pellet.

  Then, the HygR-nmt1 promoter (GD_βandγ) was introduced into the cultured S. pombe OB1 (GD_α). More specifically, 10 mL of 0.1 M LiAc solution was added to the cell pellet obtained above, and the cell pellet was suspended. Centrifugation was performed at 3000 rpm for 1 minute, and the obtained bacterial cell pellet was suspended in 300 μL of a 0.1 M LiAc solution. To 100 μL of the cell suspension, 2 μg of HygR-nmt1 promoter (GD_βandγ) and 290 μL of 50% PEG solution were added and mixed, and incubated at 30 ° C. for 1 hour. Centrifugation was performed at 3000 rpm for 1 minute, and 500 μL of sterilized water was added to the obtained cell pellet to suspend the cell pellet. Centrifugation was performed at 3000 rpm for 1 minute, the bacterial cell pellet was suspended in 300 μL of sterilized water, then applied to a YES agar medium and incubated at 30 ° C. for 24 hours. The replica agar plate was inoculated into a YES agar medium supplemented with Hygromycin B (Hyg) so that the final concentration was 150 μg / mL, and incubated at 30 ° C. for 72 hours. The obtained single colony was inoculated in a YES liquid medium and subjected to shaking culture at 30 ° C. Subsequently, centrifugation was performed at 3000 rpm for 1 minute, and the microbial cell pellet was obtained. Using the obtained bacterial cell pellet, genomic DNA was extracted according to the protocol of Puregene Yeast / Bact kit B (Qiagen) to obtain Hyg-resistant S. pombe genomic DNA. Using the obtained genomic DNA as a template, PCR amplification was performed using the following primer set (2), primer set (3), and Phusion High-Fidelity DNA Polymerase (Finnzyme).

  When it was confirmed whether HygR-nmt1 promoter (GD_βandγ) was introduced into S. pombe OB1 (GD_α), it was observed only when HygR-nmt1 promoter (GD_βandγ) was inserted. Since a 2 kb PCR amplified fragment was confirmed, it was confirmed that the HygR-nmt1 promoter (GD_βandγ) was inserted into the chromosome of S. pombe OB1 (GD_α).

(Example 6-3: Production of S. pombe OB1 (GD, GDR))
The AbAR-nmt1 promoter (GDR_LandS) prepared in Example 5-3 was transformed into S. pombe OB1 (GD) prepared in Example 6-2 to prepare S. pombe OB1 (GD, GDR). Details are shown below.

First, S. pombe OB1 (GD) was cultured. More specifically, S. pombe OB1 (GD) was inoculated in a YES liquid medium, and cultured with shaking at 30 ° C. until 1 × 10 ⁷ cells / mL. Centrifugation was performed at 3000 rpm for 1 minute using the obtained culture solution to obtain a cell pellet.

  And AbAR-nmt1 promoter (GDR_LandS) was introduce | transduced into cultured S. pombe OB1 (GD). More specifically, 10 mL of 0.1 M LiAc solution was added to the cell pellet obtained above, and the cell pellet was suspended. Centrifugation was performed at 3000 rpm for 1 minute, and the obtained bacterial cell pellet was suspended in 300 μL of a 0.1 M LiAc solution. To 100 μL of the cell suspension, 2 μg of AbAR-nmt1 promoter (GDR_LandS) and 290 μL of 50% PEG solution were added and mixed, and incubated at 30 ° C. for 1 hour. Centrifugation was performed at 3000 rpm for 1 minute, and 500 μL of sterilized water was added to the obtained cell pellet to suspend the cell pellet. Centrifugation was performed at 3,000 rpm for 1 minute, the bacterial cell pellet was suspended in 300 μL of sterilized water, then applied to a YES agar medium and incubated at 30 ° C. for 24 hours. The replica agar plate was inoculated into a YES agar medium supplemented with Aureobasidin A (AbA) to a final concentration of 0.5 μg / mL and incubated at 30 ° C. for 72 hours. The obtained single colony was inoculated in a YES liquid medium and subjected to shaking culture at 30 ° C. Subsequently, centrifugation was performed at 3000 rpm for 1 minute, and the microbial cell pellet was obtained. Using the obtained bacterial cell pellet, genomic DNA was extracted according to the protocol of Puregene Yeast / Bact kit B (Qiagen) to obtain AbA-resistant S. pombe genomic DNA. Using the obtained genomic DNA as a template, PCR amplification was performed using the following primer set (4), primer set (5), and Phusion High-Fidelity DNA Polymerase (Finnzyme).

  When it was confirmed whether the AbAR-nmt1 promoter (GDR_LandS) was introduced into S. pombe OB1 (GD), it was observed only when the AbAR-nmt1 promoter (GDR_LandS) was inserted. Since a 5 kb PCR amplified fragment was confirmed, it was confirmed that the AbAR-nmt1 promoter (GDR_LandS) was inserted into the chromosome of S. pombe OB1 (GD).

(Example 6-4: Preparation of cultured cell crude enzyme solution of S. pombe OB1 (GD, GDR))
Colonies of S. pombe OB1 (GD, GDR) were inoculated into an EMM liquid medium (MP Biomedicals) supplemented with thiamine so as to have a final concentration of 15 μM, and cultured at 30 ° C. with shaking. 100 μL of the obtained culture solution was inoculated into a 15 μM thiamine-added EMM liquid medium, followed by shaking culture at 30 ° C. 100 μL of the obtained culture solution was inoculated into an EMM liquid medium supplemented with 15 μM thiamine and subjected to shaking culture at 30 ° C. until 1 × 10 ⁷ cells / mL. The microbial cell washing was performed 3 times in the EMM liquid medium, and the obtained microbial cell pellet was suspended in the EMM liquid medium to obtain a microbial cell suspension. The bacterial cell suspension was inoculated into an EMM liquid medium so as to be 2.21 × 10 ⁵ cells / mL, followed by shaking culture at 30 ° C. for 48 hours. The obtained culture solution was centrifuged at 12,000 rpm for 10 minutes, and then the supernatant was discarded to obtain cultured cells. The obtained cultured cells were treated with YeastBuster Protein Extraction Reacgent (Novagen) to obtain a crude enzyme solution.

(Example 6-5: Measurement of glycerol dehydration activity of S. pombe (GD, GDR) cultured bacterial crude enzyme solution)
100 mM Glycerol, 15 μM Ado-Cbl, 50 mM KCl, 3 mM MgCl ₂ , 3 mM ATP, the crude enzyme 0.56 mg / mL, 20 mM Imidazole (pH 7.0) were mixed at 37 ° C. in the dark. Incubated. The sampled reaction solution was added to 0.1 M citrate K buffer (pH 3.5) in the same amount as the reaction solution to stop the reaction. A 1 / 4-fold amount of 0.1% 3-methyl-2-benzothiazolinone hydrazone was added to a solution obtained by diluting the obtained reaction solution 10 times with distilled water, and incubated at 37 ° C. for 15 minutes. This was diluted 10 times, and the value of OD305 was measured. It was confirmed that 0.1 mM aldehyde considered to be produced by dehydration of glycerin was contained in the reaction solution after stopping the reaction 60 minutes after the start of the reaction. Thus, the above results revealed that S. pombe (GD, GDR) has glycerol dehydrating activity.

(Example 6-6: Production of S. pombe OB1 (GD, GDR, aldH))
The Leu2-nmt1 promoter (aldH) prepared in Example 5-4 was transformed into S. pombe OB1 (GD, GDR) prepared in Example 6-3, and S. pombe OB1 (GD, GDR, aldH) was transformed. Produced. Details are shown below.

First, S. pombe OB1 (GD, GDR) was cultured. More specifically, S. pombe OB1 (GD, GDR) was inoculated into a YES liquid medium, and cultured with shaking at 30 ° C. until 1 × 10 ⁷ cells / mL. The obtained culture solution was centrifuged at 3000 rpm for 1 minute to obtain a cell pellet.

  Then, the Leu2-nmt1 promoter (aldH) was introduced into the cultured S. pombe OB1 (GD, GDR). More specifically, 10 mL of 0.1 M LiAc solution was added to the cell pellet obtained above, and the cell pellet was suspended. Centrifugation was performed at 3000 rpm for 1 minute, and the obtained bacterial cell pellet was suspended in 300 μL of a 0.1 M LiAc solution. To 100 μL of the cell suspension, 2 μg of Leu2-nmt1 promoter (aldH) and 290 μL of 50% PEG solution were added and mixed, and incubated at 30 ° C. for 1 hour. Centrifugation was performed at 3000 rpm for 1 minute, and 500 μL of sterilized water was added to the obtained cell pellet to suspend the cell pellet. Centrifugation is performed at 3000 rpm for 1 minute, and the cell pellet is suspended in 300 μL of sterilized water. Then, leucine-free EMM agar medium (MP Biomedicals reagent, agar = 20 g / L, histidine = 0.225 g / L, adenine = 0.225 g / L, uracil = 0.225 g / L added) and incubated at 30 ° C. for 96 hours. The obtained single colony was inoculated in a YES liquid medium and subjected to shaking culture at 30 ° C. Subsequently, centrifugation was performed at 3000 rpm for 1 minute, and the microbial cell pellet was obtained. Using the obtained bacterial cell pellet, genomic DNA was extracted according to the protocol of Puregene Yeast / Bact kit B (Qiagen) to obtain leucine-requiring complementary S. pombe genomic DNA. Using the obtained genomic DNA as a template, PCR amplification was performed using the following primer set (6) and Phusion High-Fidelity DNA Polymerase (Finnzyme).

  When it was confirmed whether the Leu2-nmt1 promoter (aldH) was introduced into S. pombe OB1 (GD, GDR), about 4.7 kb PCR observed only when the Leu2-nmt1 promoter (aldH) was inserted. Since the amplified fragment was confirmed, it was confirmed that the Leu2-nmt1 promoter (aldH) was inserted into the chromosome of S. pombe OB1 (GD, GDR).

  A genetically modified microorganism into which a gene encoding a foreign glycerin dehydratase, a gene encoding a glycerin dehydratase reactivating factor, and a gene encoding a foreign aldehyde dehydrase obtained in this way, that is, Schizosaccharomyces pombe OB01 (GD, GDR, aldH) was named Schizosaccharomyces pombe NSH-1.

(Example 1: 3HP production by S. pombe OB1 (GD, GDR, aldH))
It was investigated whether 3HP was produced using S. pombe OB1 (GD, GDR, aldH): Schizosaccharomyces pombe NSH-1 prepared in Example 6-3.

First, S. pombe OB1 (GD, GDR, aldH) was cultured. Specifically, colonies of S. pombe OB1 (GD, GDR, aldH) were inoculated into an EMM liquid medium (MP Biomedicals) supplemented with thiamine so that the final concentration was 15 μM, and cultured at 30 ° C. with shaking. Went. 100 μL of the obtained culture solution was inoculated into a 15 μM thiamine-added EMM liquid medium, followed by shaking culture at 30 ° C. 100 μL of the obtained culture solution was inoculated into a 15 μM thiamine-added EMM liquid medium and subjected to shaking culture at 30 ° C. until 1 × 10 ⁷ cells / mL. The microbial cell was washed three times with the EMM liquid medium, and the obtained microbial cell pellet was suspended in the EMM liquid medium to obtain a microbial cell suspension.

Then, it was examined whether 3HP was produced. Specifically, the bacterial cell suspension was inoculated into an EMM liquid medium supplemented with 10 g / L glycerin and 0.8 mM adenosylcobalamin so as to be 1.56 × 10 ⁵ cells / mL, followed by shaking culture at 30 ° C. went. The obtained culture broth was centrifuged at 15,000 rpm for 10 minutes to obtain a culture supernatant. 200 μL of an internal standard solution was added to 100 μL of a diluted solution obtained by diluting the obtained supernatant 10-fold with 0.25 M sulfuric acid. Next, 200 μL of reagent A solution and 200 μL of reagent B solution of a hydroxycarboxylic acid labeling reagent (YMC) were added and mixed well, followed by treatment at 60 ° C. for 20 minutes. After cooling to room temperature, it was passed through a 0.45 mm filter to obtain an LC analysis sample. Using the sample, LC analysis was performed under the following conditions.

Column used: YMC-pACK FA (YMC)
Flow rate: 0.2ml / min
Injection volume: 10μl
Eluent: methanol / acetonitrile / H ₂ O = 40/5/55 (V / V / V)
Internal standard: 2-Hydroxy-2-methyl-n-butyric acid
Detection: UV 400nm.

  As a result, in the LC analysis sample 112 hours after the start of the culture, a peak coincident with the 3HP peak was confirmed at a position 39 minutes after the injection.

  Moreover, the production | generation of acetic acid was confirmed using the said supernatant. Specifically, the supernatant was diluted 10-fold with a 5 mM sulfuric acid solution and then passed through a 0.45 mm filter to obtain an LC analysis sample. Using the sample, LC analysis was performed under the following conditions.

Column used: Aminex HPX-87Hlon exclusion column 300mμ × 7.8mμ (Bio-Rad)
Flow rate: 0.5ml / min
Injection volume: 10μl
Mobile phase: 5 mM sulfuric acid solution Detection: RI.

  As a result, the acetic acid production amount 0.4 g / L at 64 hours after the start of the culture was the maximum. In addition, the acetic acid production amount of S. pombe OB1 (GD, GDR, aldH) is the same as that of E. coli fusion blue (TacP-GDP-GPP / pCDF, TacP-LR_DD-DDR / pACYC, TacP-aldH / pUC18) described later. In contrast to the amount of acetic acid produced, it was confirmed that the amount of acetic acid produced was lower when S. pombe was used as a host (Table 2).

(Example 7: Construction of various plasmids)
(Example 7-1: Construction of TacP-GDP-GPP / pCDF)
A GDP-GPP fragment (constructed by introducing a GDP fragment and a GPP fragment into pUC18) and a plasmid TacP-GDP-GPP / pCDF in which a TacP fragment was introduced into pCDF were constructed.

  PCR amplification was performed using plasmid DNA of pUC18 (Takara) as a template and the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain a pUC18 fragment.

  Using the genomic DNA of Saccharomyces cerevisiae ATCC204508 as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain a glycerol-3-phosphate dehydrogenase gene fragment (GDP fragment).

  Using the genomic DNA of Saccharomyces cerevisiae ATCC204508 as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain a glycerol-3-3 phosphatase gene fragment (GPP fragment).

  Using the pUC18 fragment, GDP fragment, and GPP fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara), and the GDP gene and GPP gene derived from S. cerevisiae were GDP-GPP / pUC18 cloned downstream of the promoter was constructed.

  Using a plasmid DNA of GDP-GPP / pUC18 as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain a GDP-GPP fragment.

  PCR amplification was performed using plasmid DNA of pCDFDuet-1 (Novagen) as a template and the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain a pCDF fragment.

  Using pCDF fragment and GDP-GPP fragment obtained by PCR amplification, cloning was performed according to the protocol of In-Fusion PCR cloning kit (Takara) to construct GDP-GPP / pCDF.

  Using a plasmid DNA of GDP-GPP / pCDF as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain a GDP-GPP / pCDF fragment.

  PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) using the plasmid DNA of PinPoint Xa-1 (Invitrogen) as a template to obtain a TacP fragment.

  Using the GDP-GPP / pCDF fragment and TacP fragment obtained by PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara), and the S. cerevisiae-derived GDP gene and GPP gene were located downstream of the Tac promoter. Cloned TacP-GDP-GPP / pCDF was constructed.

(Example 7-2: Construction of TacP-LR-DD-DDR / pACYC)
A plasmid TacP-LR-DD-DDR / pACYC was constructed by introducing a TacP-LR-DD-DDR fragment (constructed by introducing the LR-DD-DDR fragment into PinPoint Xa-1) into pACYC.

  The region containing the diol dehydratase gene (DD gene) and the diol dehydratase reactivation factor (DDR gene) using the genomic DNA of Lactobacillus reuteri JCM1112 as a template, the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) ) Was used to obtain an LR-DD-DDR fragment.

  PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) using the plasmid DNA of PinPoint Xa-1 (Invitrogen) as a template to obtain a PinPoint Xa-1 fragment.

  Using the LR-DD-DDR fragment and PinPoint Xa-1 fragment obtained by PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara), and DD-derived from L. reuteri downstream of the Tac promoter. LR-DD-DDR / PinPoint Xa-1 in which the DDR gene was inserted was constructed.

  Using TacP-LR-DD-DDR / PinPoint Xa-1 plasmid DNA as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finzyme), and TacP-LR-DD-DDR fragment was obtained. Obtained.

  PCR amplification was performed using plasmid DNA of pACYCDuet-1 (Novagen) as a template and the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain a pACYC fragment.

  Using the TacP-LR-DD-DDR fragment and pACYC fragment obtained by PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct TacP-LR-DD-DDR / pACYC. .

(Example 7-3: Construction of TacP-aldH / pUC18)
Plasmid TacP-aldH / pUC18 was constructed by introducing a TacP-aldH fragment (constructed by introducing an aldH fragment into PinPoint Xa-1) into pUC18.

  Using the genomic DNA of Escherichia coli W3110 as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain a γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (aldH) fragment. .

  PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) using the plasmid DNA of PinPoint Xa-1 (Invitrogen) as a template to obtain a PinPoint Xa-1 fragment.

  Using the aldH fragment and pinpoint Xa-1 fragment obtained by PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara), and the aldH gene derived from E. coli was inserted downstream of the Tac promoter. TacP-aldH / pinpoint Xa-1 was constructed.

  PCR amplification was performed using TacP-aldH / pinpoint Xa-1 plasmid DNA as a template and the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain a TacP-aldH fragment.

  PCR amplification was performed using plasmid DNA of pUC18 (Takara) as a template and the following primers and Phusion High-Fidelity DNA Polymerase (Finnzyme) to obtain a pUC18 fragment.

  Using the TacP-aldH fragment and pUC18 fragment obtained by PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct TacP-aldH / pUC18.

(Example 8: Transformation)
TacP-GDP-GPP / pCDF, TacP-LR-DD-DDR / pACYC, and TacP-aldH / pUC18 prepared in Example 7-1 to Example 7-3 were introduced into Escherichia coli. Details are shown below.

  Using Fusion-Blue Competent Cells (Takara), TacP-GDP-GPP / pCDF, TacP-LP_DD-DDR / pACYC, and TacP-aldH / pUC18 were introduced by the heat shock method, and E. coli fusion blue (TacP -GDP-GPP / pCDF, TacP-LP_DD-DDR / pACYC, TacP-aldH / pUC18). The heat shock method was performed according to the protocol attached to Fusion-Blue Competent Cells (Takara), and the transformants were selected for chloramphenicol at a final concentration of 100 μg / mL, streptomycin at a final concentration of 50 μg / mL, and ampicillin. It was carried out using an agar medium prepared by adding to LB medium (Bacto-tryptone = 10 g, Bacto-yeast extract = 5 g, NaCl = 5 g) so as to have a final concentration of 100 μg / mL.

(Example 9: Conversion of glucose to glycerin by E. coli fusion blue (TacP-GDP-GPP / pCDF, TacP-LP_DD-DDR / pACYC, TacP-aldH / pUC18))
Using E. coli fusion blue (TacP-GDP-GPP / pCDF, TacP-LP_DD-DDR / pACYC, TacP-aldH / pUC18), a genetically modified microorganism prepared as described above, to glucose glycerin The conversion of was examined.

  Colonies of E. coli fusion blue (TacP-GDP-GPP / pCDF, TacP-LP_DD-DDR / pACYC, TacP-aldH / pUC18) have final concentrations of 100 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. Inoculated into an LB liquid medium supplemented with chloramphenicol, streptomycin, and ampicillin as described above, followed by shaking culture at 37 ° C. for 24 hours. The obtained culture solution was inoculated so that the value of OD660 at the start of the culture was 0.018, and shaking culture was started at 25 ° C., which is a temperature suitable for producing glycerin from glucose. After the start of culture, when the value of OD660 exceeded 1, IPTG and Ado-Cbl were added so that the final concentrations were 0.1 M and 0.8 mM, respectively, and the culture was continued at 25 ° C. The obtained culture broth was centrifuged at 15,000 rpm for 10 minutes to obtain a culture supernatant. 1 mL of the obtained culture supernatant was diluted 10-fold with a 5 mM sulfuric acid solution, and then passed through a 0.45 mm filter to obtain an LC analysis sample. Using the LC analysis sample, high performance liquid chromatography (LC) analysis was performed under the following conditions.

Column used: Aminex HPX-87H lon exclusion column 300mm x 7.8mm (Bio-Rad)
Flow rate: 0.5ml / min
Injection volume: 10μl
Mobile phase: 5 mM sulfuric acid solution Detection: RI
As a result, the glycerin production amount 5.8 g / L 144 hours after the start of the culture was the maximum accumulation amount, and thereafter decreased to 3 g / L or less. In addition, the amount of glycerin produced by E. coli fusion blue (TacP-GDP-GPP / pCDF, TacP-LP_DD-DDR / pACYC, TacP-aldH / pUC18), glycerin of S. pombe ATCC26189 and S. pombe OB1 described later The comparison with the production amount is shown in Table 3 below.

  The amount of acetic acid produced was also quantified. As a result, the amount of acetic acid produced after 144 hours from the start of culture was 2.6 g / L, and the amount of acetic acid produced after 216 hours from the start of culture was 4.3 g / L. The contrast of the amount of acetic acid produced with S. pombe OB1 (GD, GDR, aldH) is as shown in Table 2 above.

(Example 10: Conversion of glucose to glycerin by S. pombe)
Using S. pombe, the conversion of glucose to glycerin was examined. As S. pombe, S. pombe ATCC26189 (972h-) and S. pombe OB1 were used.

Colonies of S. pombe ATCC26189 (972h-) and S. pombe OB1 were inoculated into a YES liquid medium and cultured at 30 ° C. with shaking. 100 μL of the obtained culture solution was inoculated into a YES liquid medium, and shaking culture was performed at 30 ° C. The resulting culture broth was added to a glucose-added YES liquid medium (yeast extract = 5 g / L, glucose = 255 g / L, histidine = 0.225 g / L, adenine = 0.225 g / L, leucine = 0.225 g / L, uracil = 0.225. g / L) was inoculated to be 3.13 × 10 ⁴ cells / mL, and cultured at 30 ° C. with shaking. The obtained culture broth was centrifuged at 15,000 rpm for 10 minutes to obtain a culture supernatant. 1 mL of the obtained culture supernatant was diluted 10-fold with a 5 mM sulfuric acid solution, and then passed through a 0.45 mm filter to obtain an LC analysis sample. Using the LC sample, high performance liquid chromatography (LC) analysis was performed under the following conditions.

Column used: Aminex HPX-87Hlon exclusion column 300mm x 7.8mm (Bio-Rad)
Flow rate: 0.5ml / min
Injection volume: 10μl
Mobile phase: 5 mM sulfuric acid solution Detection: RI
As a result, the amount of glycerin produced 72 hours after the start of culture reached 10.3 g / L for S. pombe ATCC26189 and 11.3 g / L for S. pombe OB1. Table 3 shows the amount of glycerin produced by S. pombe ATCC26189 and S. pombe OB1 and the E. coli fusion blue (TacP-GDP-GPP / pCDF, TacP-LP_DD-DDR / pACYC, TacP-aldH / pUC18). The comparison with the amount of glycerin produced is shown in Table 3 below.

  According to Table 3, when S. pombe was used as a host, it was confirmed that glycerin can be produced without genetic modification and a large amount of glycerin can be obtained.

(Example 11: Construction of mcr / pGPD plasmid)
Plasmid mcr / pGPD was constructed by introducing the mcr fragment into pGPD.

  Using the genomic DNA of Chloroflexus aurantiacus ATCC29365 strain as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a malonyl CoA reductase (mcr) gene fragment.

  Using the plasmid DNA of pGPD as a template, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pGPD fragment.

  Using the mcr fragment and pGPD fragment obtained by the above PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara), and the mcr gene derived from C. aurantiacus was cloned downstream of the nmt1 promoter. / PGPD was constructed.

(Example 12: Preparation of G418R-nmt1 promoter (mcr) fragment)
A G418R-nmt1 promoter fragment was prepared from mcr / pGPD.

  Specifically, PCR amplification was performed using the following two primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) using mcr / pGPD plasmid DNA as a template to obtain a G418R-nmt1 promoter (mcr) fragment. .

(Example 13: Transformation)
The G418R-nmt1 promoter (mcr) fragment prepared in Example 12 above was transformed into Schizosaccharomyces pombe OB2 to prepare S. pombe OB2 (mcr). Details are shown below.

First, S. pombe OB2 was cultured. More specifically, S. pombe OB2 was added to a YES liquid medium (yeast extract = 5 g / L, glucose = 30 g / L, histidine = 0.225 g / L, adenine = 0.225 g / L, leucine = 0.225 g / L, uracil = 0.225 g / L), and shaking culture was performed at 30 ° C. until 1 × 10 ⁷ cells / mL. Centrifugation was performed at 3000 rpm for 1 minute using the obtained culture solution to obtain a cell pellet.

  Then, a G418R-nmt1 promoter (mcr) fragment was introduced into the cultured S. pombe OB2. More specifically, a 0.1M LiAc solution (100 mM Litium Acetate, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, acetic acid) adjusted to pH 4.9 is added to the cell pellet obtained above. 10 mL was added to suspend the cell pellet. Centrifugation was performed at 3000 rpm for 1 minute, and the obtained bacterial cell pellet was suspended in 300 μL of a 0.1 M LiAc solution. 1 μg of G418R-nmt1 promoter (mcr) fragment and 290 μL of 50% PEG solution (polyethylene glycol 3345 = 500 g / L) were added to and mixed with 100 μL of the cell suspension, and incubated at 30 ° C. for 1 hour. Centrifugation was performed at 3000 rpm for 1 minute, and 500 μL of sterilized water was added to the obtained cell pellet to suspend the cell pellet. Centrifugation is performed at 3000 rpm for 1 minute, the cell pellet is suspended in 400 μL of sterilized water, and then YES agar medium (yeast extract = 5 g / L, glucose = 30 g / L, agar = 20 g / L, histidine = 0.225 g / L, adenine = 0.225 g / L, leucine = 0.225 g / L, uracil = 0.225 g / L) and incubated at 30 ° C. for 20 hours. After 20 hours of culturing, the cells were inoculated on a YES agar medium supplemented with G418 to a final concentration of 150 μg / mL, and incubated at 30 ° C. for 72 hours. The obtained single colony was inoculated into a YES liquid medium and cultured with shaking at 30 ° C. Subsequently, centrifugation was performed at 3,000 rpm for 5 minutes to obtain a cell pellet. Using the obtained bacterial cell pellet, genomic DNA was extracted according to the protocol of Puregene Yeast / Bact kit B (Qiagen) to obtain G418-resistant S. pombe genomic DNA. Using the obtained genomic DNA as a template, PCR amplification was performed using the following primer set (7) and Phusion High-Fidelity DNA Polymerase (Finnzymes).

  When it was confirmed whether the G418R-nmt1 promoter (mcr) was introduced into S. pombe, a PCR amplified fragment of about 7.3 kb that was observed only when the G418R-nmt1 promoter (mcr) was inserted was confirmed. Therefore, it was confirmed that the G418R-nmt1 promoter (mcr) fragment was inserted into S. pombe.

  The genetically modified microorganism introduced with the gene encoding the exogenous malonyl-CoA reductase thus obtained, ie, Schizosaccharomyces pombe (mcr), was named Schizosaccharomyces pombe NSH-2 and was dated March 9, 2012. Deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Tsukuba Center Ibaraki 1-1-1 Tsukuba Center Chuo No. 6) with the deposit number FERM P-2227 on February 27, 2013 , Transferred to international deposit. The receipt number is FERM ABP-11527.

(Example 2: 3HP production by S. pombe (mcr))
Using S. pombe (mcr): Schizosaccharomyces pombe NSH-2 (reception number: FERM ABP-11527) prepared in Example 13 above, it was examined whether 3HP was produced.

S. pombe (mcr) was inoculated into an EMM liquid medium (MP Biomedicals) supplemented with thiamine so that the final concentration was 15 μM, and cultured at 30 ° C. with shaking. 100 μL of the obtained culture solution was inoculated into a 15 μM thiamine-added EMM liquid medium, followed by shaking culture at 30 ° C. 100 μL of the obtained culture solution was inoculated into a 15 μM thiamine-added EMM liquid medium and subjected to shaking culture at 30 ° C. until 1 × 10 ⁷ cells / mL. The microbial cell was washed three times with the EMM liquid medium, and the obtained microbial cell pellet was suspended in the EMM liquid medium to obtain a microbial cell suspension. The bacterial cell suspension was inoculated into an EMM liquid medium and cultured at 30 ° C. for 24 hours to obtain a culture solution of 1 × 10 ⁷ cells / mL. Centrifugation was performed at 3000 rpm for 1 minute using the obtained culture solution, and suspended in an EMM liquid medium to obtain a cell suspension. The obtained cell suspension was used to inoculate an EMM liquid medium at a cell concentration of 1 × 10 ⁸ cells / mL, followed by shaking culture at 30 ° C. for 70 hours. The obtained culture solution was centrifuged at 12,000 rpm for 5 minutes to obtain a culture supernatant. 200 μL of an internal standard solution was added to 100 μL of a diluted solution obtained by diluting the obtained culture supernatant 10-fold with 0.25 M sulfuric acid. Next, 200 μL of reagent A solution and 200 μL of reagent B solution of a hydroxycarboxylic acid labeling reagent (YMC) were added and mixed well, followed by treatment at 60 ° C. for 20 minutes. Further, 200 μL of a reagent C solution of a hydroxycarboxylic acid labeling reagent (YMC) was added and mixed well, followed by treatment at 60 ° C. for 15 minutes. After cooling to room temperature, it was passed through a 0.45 μm filter to obtain an LC analysis sample. Using the sample, LC analysis was performed under the following conditions.

Column used: YMC-pACK FA (YMC)
Flow rate: 0.2 ml / min
Injection volume: 10μl
Eluent: methanol / acetonitrile / H ₂ O = 40/5/55 (V / V / V)
Internal standard: 2-Hydroxy-2-methyl-n-butyric acid
Detection: UV 400nm
As a result, a peak coincident with the 3HP peak was confirmed at a position 39 minutes after the injection.

  Moreover, LC-MS analysis was performed on the following conditions using the culture supernatant obtained by the said culture | cultivation.

Column used: TSKgel Amide-80
Column oven temperature: 40 ° C
Eluent: 0.1% formic acid Flow rate: 0.1 mL / min
Mass spectrometry: 3200 Q TRAP LC / MS / MS system (Applied Biosystems)
Mode: MRM
Polarity: Positive
Ion source: Turbo V source (ESI)
Temperature: 350 ° C
Ion Spray voltave: 5000V.

  As a result, m / z = 89 that matches 3HP was detected. FIG. 7 shows the mass spectrum of 3HP and the mass spectrum of the culture supernatant of S. pombe (mcr).

  Therefore, it was revealed that 3HP generation ability can be imparted by introducing the mcr gene into S. pombe.

(Example 14: Construction of plasmid nmt1P-acc1-bpl1 / pG418)
The plasmid nmt1P-acc1-bpl1 / pG418 was constructed by introducing the nmt1P-acc1 fragment and the nmt1P-bpl1 fragment (constructed by introducing the bpl1 fragment into pHYG) into pG418.

  Using the genomic DNA of Saccharomyces cerevisiae ATCC204508 as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain an acetyl CoA carboxylase fragment (acc1 fragment).

  Using the plasmid DNA of pG418 as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pG418 fragment.

  Cloning was performed according to the protocol of In-Fusion PCR cloning kit (Takara) using the acc1 fragment and pG418 fragment obtained by the PCR amplification, and the acc1 gene derived from S. cerevisiae was cloned downstream of the nmt1 promoter. nmt1P-acc1 / pG418 was constructed.

  Using the genomic DNA of Saccharomyces cerevisiae ATCC204508 as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a biotin apoprotein ligase fragment (bpl1 fragment).

  Using the pHYG plasmid DNA as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pHYG fragment.

  Using the Bpl1 fragment and pHYG fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara), and the bp11 gene derived from S. cerevisiae was cloned downstream of the nmt1 promoter. nmt1P-bpl1 / pHYG was constructed.

  Using the plasmid DNA of nmt1P-acc1 / pG418 as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain the nmt1P-acc1 / pG418 fragment.

  PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) using the plasmid DNA of nmt1P-bpl1 / pHYG as a template to obtain a nmt1P-bpl1 fragment.

  Using the nmt1P-acc1 / pG418 fragment and nmt1P-bpl1 fragment obtained by the PCR amplification described above, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct nmt1P-acc1-bpl1 / pG418. .

(Example 15: Preparation of HygR-nmt1P (acc1-bpl1) fragment)
A HygR-nmt1P (acc1-bpl1) fragment was prepared from nmt1P-acc1-bpl1 / pG418.

  Specifically, PCR amplification was performed using nmt1P-acc1-bpl1 / pG418 plasmid DNA as a template, the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes), and the HygR-nmt1P promoter (acc1-bpl1) fragment Got.

(Example 16: Transformation)
The HygR-nmt1P promoter (acc1-bpl1) fragment prepared in 15 above was transformed into S. pombe OB2 (mcr) to prepare S. pombe OB2 (mcr, acc1). Details are shown below.

First, S. pombe (mcr) was cultured. More specifically, S. pombe (mcr) was inoculated into a YES liquid medium, and shake culture was performed at 30 ° C. until 1 × 10 ⁷ cells / mL. Centrifugation was performed at 3000 rpm for 1 minute using the obtained culture solution to obtain a cell pellet.

  Then, the HygR-nmt1P promoter (acc1-bpl1) fragment was introduced into the cultured S. pombe OB2 (mcr). More specifically, 10 mL of 0.1 M LiAc solution was added to the cell pellet obtained above, and the cell pellet was suspended. Centrifugation was performed at 3000 rpm for 1 minute, and the obtained bacterial cell pellet was suspended in 300 μL of a 0.1 M LiAc solution. To 100 μL of the cell suspension, 1 μg of HygR-nmt1P promoter (acc1-bpl1) fragment and 290 μL of 50% PEG solution were added and mixed, and incubated at 30 ° C. for 1 hour. Centrifugation was performed at 3000 rpm for 1 minute, and 500 μL of sterilized water was added to the obtained cell pellet to suspend the cell pellet. Centrifugation was performed at 3000 rpm for 1 minute, the bacterial cell pellet was suspended in 400 μL of sterilized water, applied to a YES agar medium, and incubated at 30 ° C. for 20 hours. S. pombe (mcr, acc) was obtained by applying to a YES agar medium supplemented with Hygromycin B to a final concentration of 150 μg / mL and incubating at 30 ° C. for 72 hours.

  The thus obtained gene encoding the exogenous malonyl CoA reductase and the gene recombinant microorganism into which the gene encoding the exogenous acetyl-CoA carboxylase was introduced, ie, Schizosaccharomyces pombe (mcr, acc), were converted into Schizosaccharomyces pombe NSH- No. 3 and the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1-1 Tsukuba Center, Tsukuba, Chuo No. 6), National Institute of Advanced Industrial Science and Technology, on March 9, 2012, the accession number FERM P-2228 And transferred to an international deposit on February 27, 2013. The receipt number is FERM ABP-11528.

(Example 3: 3HP production by S. pombe (mcr, acc))
Using S. pombe (mcr, acc): Schizosaccharomyces pombe NSH-3 (reception number: FERM ABP-11528) produced in Example 16 above, it was examined whether 3HP was produced.

Colonies of S. pombe (mcr-acc) were inoculated into an EMM liquid medium (MP Biomedicals) to which thiamine was added so that the final concentration was 15 μM, and shaking culture was performed at 30 ° C. 100 μL of the obtained culture solution was inoculated into a 15 μM thiamine-added EMM liquid medium, followed by shaking culture at 30 ° C. 100 μL of the obtained culture solution was inoculated into an EMM liquid medium supplemented with 15 μM thiamine and subjected to shaking culture at 30 ° C. until 1 × 10 ⁷ cells / mL. The microbial cell was washed three times with the EMM liquid medium, and the obtained microbial cell pellet was suspended in the EMM liquid medium to obtain a microbial cell suspension. The bacterial cell suspension was inoculated into an EMM liquid medium and cultured at 30 ° C. for 24 hours to obtain a culture solution of 1 × 10 ⁷ cells / mL. Centrifugation was performed at 3,000 rpm for 1 minute using the obtained culture solution, and suspended in an EMM liquid medium to obtain a cell suspension. The obtained cell suspension was used to inoculate an EMM liquid medium at a cell concentration of 1 × 10 ⁸ cells / mL, followed by shaking culture at 30 ° C. for 120 hours. The obtained culture broth was centrifuged at 12000 rpm for 10 minutes to obtain a culture supernatant. The obtained culture supernatant was added with 200 μL of an internal standard solution to 100 μL of a 10-fold diluted diluted solution with 0.25 M sulfuric acid. Next, 200 μL of reagent A solution and 200 μL of reagent B solution of a hydroxycarboxylic acid labeling reagent (YMC) were added and mixed well, followed by treatment at 60 ° C. for 20 minutes. Further, 200 μL of a reagent C solution of a hydroxycarboxylic acid labeling reagent (YMC) was added and mixed well, followed by treatment at 60 ° C. for 15 minutes. After cooling to room temperature, it was passed through a 0.45 μm filter to obtain an LC analysis sample. Using the sample, LC analysis was performed under the following conditions.

Column used: YMC-pACK FA (YMC)
Flow rate: 0.2 ml / min
Injection volume: 10μl
Eluent: methanol / acetonitrile / H ₂ O = 40/5/55 (V / V / V)
Internal standard: 2-Hydroxy-2-methyl-n-butyric acid
Detection: UV 400nm.

  As a result, it was confirmed that 0.05 g / L of 3HP was produced. At this time, the pH of the culture solution was 2.8. In addition, since the production amount of 3HP of S. pombe (mcr) in Example 2 was 0.01 g / L or less, by introducing the acc1 gene and bpl1 gene derived from S. cerevisiae into S. pombe (mcr), It was revealed that the amount of 3HP produced was improved.

(Example 17: Construction of CMV-gpd1 / pAUR224)
CMV-gpd1 / pAUR224 was constructed by introducing the gpd1 fragment into pAUR224 (having CMV as a promoter).

  Genomic DNA of Kluyveromyces lactis NBRC1267 (purchased from Biological Resource Center, NITE) was extracted using Puregene Yeast / Bact kit (Qiagen). Using the extracted DNA as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (gpd1 fragment).

  Using the plasmid DNA of pAUR224 (Takara Bio Inc.) as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pAUR224 fragment.

  Using the gpd1 fragment and the pAUR224 fragment obtained by the above PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara), and gpd1 derived from Kluyveromyces lactis was cloned downstream of the CMV promoter. gpd1 / pAUR224 was constructed.

(Example 18: Production of CMV-gpd1 / pAUR224 fragment)
An AbaR-CMV-gpd1 fragment was prepared from CMV-gpd1 / pAUR224.

  Specifically, using the plasmid DNA of CMV-gpd1 / pAUR224 as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain an AbaR-CMV-gpd1 fragment.

(Example 19: Transformation)
The AbaR-CMV-gpd1 fragment prepared in Example 18 above was transformed into S. pombe OB2 (mcr, acc): Schizosaccharomyces pombe NSH-3 (reception number: FERM ABP-11528) prepared in Example 16 above. pombe OB2 (mcr, acc, gpd1) was prepared. Details are shown below.

First, S. pombe (mcr, acc) was cultured. More specifically, S. pombe (mcr, acc) was inoculated into a YES liquid medium, and shake-cultured at 30 ° C. until 1 × 10 ⁷ cells / mL. Centrifugation was performed at 3000 rpm for 1 minute using the obtained culture solution to obtain a cell pellet.

  Then, the AbaR-CMV-gpd1 fragment was introduced into the cultured S. pombe OB2 (mcr, acc). More specifically, 10 mL of 0.1 M LiAc solution was added to the cell pellet obtained above, and the cell pellet was suspended. Centrifugation was performed at 3000 rpm for 1 minute, and the obtained bacterial cell pellet was suspended in 300 μL of a 0.1 M LiAc solution. To 100 μL of the cell suspension, 1 μg of AbaR-CMV-gpd1 fragment and 290 μL of 50% PEG solution were added and mixed, and incubated at 30 ° C. for 1 hour. Centrifugation was performed at 3000 rpm for 1 minute, and 500 μL of sterilized water was added to the obtained cell pellet to suspend the cell pellet. Centrifugation was performed at 3000 rpm for 1 minute, the bacterial cell pellet was suspended in 400 μL of sterilized water, applied to a YES agar medium, and incubated at 30 ° C. for 20 hours. Spread on agar with YES agar supplemented with Aureobasidin A (Takara Bio) to a final concentration of 0.5 μg / mL, incubate at 30 ° C. for 96 hours, and add S. pombe (mcr, acc, gpd1) Obtained.

(Example 4: 3HP production by S. pombe (mcr, acc, gpd1))
Using S. pombe (mcr, acc, gpd1) produced in Example 19, a 3HP production test was conducted.

Colonies of S. pombe (mcr, acc, gpd1) were inoculated into an EMM liquid medium (MP Biomedicals) supplemented with thiamine so that the final concentration was 15 μM, and cultured at 30 ° C. with shaking. 100 μL of the obtained culture solution was inoculated into a 15 μM thiamine-added EMM liquid medium, followed by shaking culture at 30 ° C. 100 μL of the obtained culture solution was inoculated into an EMM liquid medium containing 15 μM thiamine, and shake-cultured at 30 ° C. until 1 × 10 7 cells / mL. The microbial cell was washed three times with the EMM liquid medium, and the obtained microbial cell pellet was suspended in the EMM liquid medium to obtain a microbial cell suspension. The cell suspension is inoculated into an AMM liquid medium having the composition shown in Table 4 below so that the cell suspension becomes 3.1 × 10 ⁵ cells / mL, cultured at 30 ° C., and the culture solution is added every 24 hours. Sampling.

  The sampled culture solution was centrifuged at 12000 rpm for 5 minutes to obtain a culture supernatant. The obtained culture supernatant was diluted 10-fold with 0.25 M sulfuric acid to obtain 100 μL of a diluted solution. 200 μL of the internal standard solution was added to the diluted solution, 200 μL of the reagent A solution of the hydroxycarboxylic acid labeling reagent (YMC) and 200 μL of the reagent B solution were mixed well, and then treated at 60 ° C. for 20 minutes. Next, 200 μL of a reagent C solution of a hydroxycarboxylic acid labeling reagent (YMC) was added and mixed well, followed by treatment at 60 ° C. for 15 minutes. After cooling to room temperature, it was passed through a 0.45 μm filter to obtain an LC analysis sample. Using the sample, LC analysis was performed under the following conditions. The obtained results are shown in FIG.

Column used: YMC-pACK FA (YMC)
Flow rate: 0.2 ml / min
Injection volume: 10μl
Eluent: methanol / acetonitrile / H ₂ O = 40/5/55 (V / V / V)
Internal standard: 2-Hydroxy-2-methyl-n-butyric acid
Detection: UV 400nm.

  As is clear from FIG. 8, it was confirmed that S.pombe (mcr, acc, gpd1) has a high 3HP production amount as compared with S.pombe (mcr, acc). Further, as is clear from Table 5, it is confirmed that 3HP can be selectively generated according to S.pombe (mcr, acc, gpd1) as compared with S.pombe (mcr, acc). It was done. Thus, it was confirmed that 3HP production can be improved by genetic modification that improves intracellular NADPH production.

(Example 20: Construction of CMV-pkt · pta / pAUR)
CMV-pkt · pta / pAUR224 was constructed by introducing the pkt fragment and the pta fragment into pAUR224 (having CMV as a promoter).

  First, the gene sequence of Fructose-6-phosphate phosphoketolase (pkt) (EC 4.1.2.22) derived from Bifidobacterium animalis (GenBank accession number: AB264557.1), the gene sequence of phosphotransacetylase derived from Methanosarcina thermophila (pta) (GenBank accession number: Based on the codon table of Schizosaccharomyces pombe described in the L23147.1) and Codon Usage Database (homepage address: http://www.kazusa.or.jp/codon/), the codon suitable for S.pombe is pkt Gene and pta gene optimization was performed. As a result, the pkt gene fragment of SEQ ID NO: 215 and the pta gene fragment of SEQ ID NO: 216 were selected as codons suitable for S. pombe. The DNA fragment having the above sequence was synthesized by gene synthesis (requested from Wako Pure Chemical Industries, Ltd.).

  Using the pkt gene fragment of SEQ ID NO: 215 as a DNA template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pkt fragment.

  Further, using the pka gene fragment of SEQ ID NO: 216 as a DNA template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pta fragment.

  Next, PCR amplification was performed using plasmid DNA of pAUR224 (Takara Bio Inc.) as a template and the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a pAUR224 fragment.

  Using the pkt fragment and the pAUR224 fragment obtained by the above PCR amplification, cloning was performed according to the protocol of the In-Fusion PCR cloning kit (Takara), and CMV-pkt / pAUR224 was cloned downstream of the CMV promoter. It was constructed.

  Similarly, cloning was performed using the pta fragment and the pAUR224 fragment according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct CMV-pta / pAUR224 in which pta was cloned downstream of the CMV promoter.

  Using the CMV-pkt / pAUR224 plasmid DNA as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a CMV-pkt fragment.

  Similarly, PCR amplification was performed using the CMV-pta / pAUR224 plasmid DNA as a template and the primers of SEQ ID NOS: 223 and 224 and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a CMV-pka fragment. .

  Using the plasmid pAUR (leu2-AbAR-nmt1 / pUC18) constructed in Example 3-5 as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes), and AbaR_arm_leu2 / A pUC18 fragment was obtained.

  Next, the CMV-pkt fragment and the AbaR_arm_leu2 / pUC18 fragment obtained by the PCR amplification were used for cloning according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct CMV-pkt / pAUR.

  Similarly, the CMV-pka fragment and the AbaR_arm_leu2 / pUC18 fragment were cloned according to the protocol of the In-Fusion PCR cloning kit (Takara) to construct CMV-pta / pAUR.

  Using CMV-pkt / pAUR plasmid DNA as a template, PCR amplification was performed using the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a CMV-pkt fragment.

  In addition, PCR amplification was performed using CMV-pta / pAUR plasmid DNA as a template and the following primers and Phusion High-Fidelity DNA Polymerase (Finnzymes) to obtain a leu_CMV-pta / pAUR fragment.

  Using CMV-pkt fragment and leu_CMV-pta / pAUR fragment obtained by PCR amplification, cloning was performed according to the protocol of In-Fusion PCR cloning kit (Takara) to construct CMV-pkt / pta / pAUR. .

(Example 21: Preparation of leu_CMV-pta · pta fragment)
A leu_CMV-pta · pta fragment was prepared from CMV-pkt · pta / pAUR.

  Specifically, PCR amplification was performed using the CMV-pkt · pta / pAUR plasmid DNA as a template, the primers of SEQ ID NOS: 93 and 94 and KOD-FX (Toyobo), and leu_CMV-pta · pta A fragment was obtained.

(Example 22: Transformation)
The leu_CMV-pta · pta fragment prepared in Example 21 was transformed into S. pombe OB2 (mcr, acc): Schizosaccharomyces pombe NSH-3 (reception number: FERM ABP-11528) prepared in Example 16 above. pombe OB2 (mcr, acc, pkt · pta) was prepared. Details are shown below.

First, S. pombe (mcr, acc) was cultured. More specifically, S. pombe (mcr, acc) was inoculated into a YES liquid medium, and shake-cultured at 30 ° C. until 1 × 10 ⁷ cells / mL. Centrifugation was performed at 3000 rpm for 1 minute using the obtained culture solution to obtain a cell pellet.

  Then, the leu_CMV-pta · pta fragment was introduced into the cultured S. pombe OB2 (mcr, acc). More specifically, 10 mL of 0.1 M LiAc solution was added to the cell pellet obtained above, and the cell pellet was suspended. Centrifugation was performed at 3000 rpm for 1 minute, and the obtained bacterial cell pellet was suspended in 300 μL of a 0.1 M LiAc solution. To 100 μL of the cell suspension, 1 μg of leu_CMV-pta · pta fragment and 290 μL of 50% PEG solution were added and mixed, and incubated at 30 ° C. for 1 hour. Centrifugation was performed at 3000 rpm for 1 minute, and 500 μL of sterilized water was added to the obtained cell pellet to suspend the cell pellet. Centrifugation is performed at 3000 rpm for 1 minute, the bacterial cell pellet is suspended in 400 μL of sterilized water, applied to EMM medium without leucine, incubated at 30 ° C. for 96 hours, and S. pombe (mcr, acc, pkt · pta) Got.

(Example 5: 3HP production by S. pombe (mcr, acc, pkt · pta))
Using the S. pombe (mcr, acc, pkt · pta) prepared above, a 3HP generation test was conducted.

Colonies of S. pombe (mcr, acc, pkt · pta) were inoculated into EMM liquid medium (MP Biomedicals) supplemented with thiamine so that the final concentration was 15 μM, and cultured at 30 ° C. with shaking. 100 μL of the obtained culture solution was inoculated into a 15 μM thiamine-added EMM liquid medium, followed by shaking culture at 30 ° C. 100 μL of the obtained culture solution was inoculated into an EMM liquid medium supplemented with 15 μM thiamine and subjected to shaking culture at 30 ° C. until 1 × 10 ⁷ cells / mL. The microbial cell was washed three times with the EMM liquid medium, and the obtained microbial cell pellet was suspended in the EMM liquid medium to obtain a microbial cell suspension. The cell suspension is inoculated into an AMM liquid medium having the composition shown in Table 4 so that the cell suspension becomes 3.1 × 10 ⁵ cells / mL, cultured at 30 ° C., and the culture solution is added every 24 hours. Sampling.

  The sampled culture solution was centrifuged at 12000 rpm for 5 minutes to obtain a culture supernatant. The obtained culture supernatant was diluted 10-fold with 0.25 M sulfuric acid to obtain 100 μL of a diluted solution. 200 μL of the internal standard solution was added to the diluted solution, 200 μL of the reagent A solution of the hydroxycarboxylic acid labeling reagent (YMC) and 200 μL of the reagent B solution were mixed well, and then treated at 60 ° C. for 20 minutes. Next, 200 μL of a reagent C solution of a hydroxycarboxylic acid labeling reagent (YMC) was added and mixed well, followed by treatment at 60 ° C. for 15 minutes. After cooling to room temperature, it was passed through a 0.45 μm filter to obtain an LC analysis sample. Using the sample, LC analysis was performed under the following conditions. The obtained results are shown in Table 6 below.

Column used: YMC-pACK FA (YMC)
Flow rate: 0.2 ml / min
Injection volume: 10μl
Eluent: methanol / acetonitrile / H ₂ O = 40/5/55 (V / V / V)
Internal standard: 2-Hydroxy-2-methyl-n-butyric acid
Detection: UV 400nm.

  As is clear from Table 6, it was confirmed that S. pombe (mcr, acc, pkt · pta) had a high 3HP production amount as compared with S. pombe (mcr, acc). Therefore, it was confirmed that 3HP productivity can be improved by genetic modification that improves the amount of acetyl CoA produced.

(Example 23: Production of 3HP by Escherichia coli introduced with a gene encoding exogenous malonyl-CoA reductase)
Based on the base sequence (SEQ ID NO: 2) of the malonyl CoA reductase (mcr) gene of Chloroflexus aurantiacus (ATCC29365) strain, a primer that amplifies the structural gene region of malonyl CoA reductase (primer for amplification of mcr derived from Chloroflexus aurantiacus; SEQ ID NO: 11, 12) was designed. Using the designed primers, PCR amplification was performed using the genomic DNA of Chloroflexus aurantiacus ATCC29365 as a template to obtain the malonyl CoA reductase gene of Chloroflexus aurantiacus ATCC29365.

  The obtained malonyl CoA reductase gene was cloned downstream of the Tac promoter of the PinPoint Xa-1 vector (Promega) to construct mcr / PinPoint Xa-1. In accordance with the protocol of Escherichia coli fusion blue (Takara), the constructed mcr / PinPoint Xa-1 was introduced by the heat shock method. coli (mcr / PinPoint Xa-1) was obtained.

  E. E. coli (mcr / PinPoint Xa-1) was inoculated into M9 medium (manufactured by Difco) supplemented with IPTG (final concentration 1 mM) 2 wt / vol% glucose, and cultured at 37 ° C. for 48 hours. 200 μL of an internal standard solution was added to 100 μL of the culture supernatant. 200 μL of reagent A solution and 200 μL of reagent B solution of a hydroxycarboxylic acid labeling reagent (YMC) were added and mixed well, followed by treatment at 60 ° C. for 20 minutes. 200 μL of reagent C solution of a hydroxycarboxylic acid labeling reagent (YMC) was added and mixed well. After being treated at 60 ° C. for 15 minutes and cooled to room temperature, it was passed through a 0.45 μm filter to obtain an LC analysis sample. Using the sample, high performance liquid chromatography (LC) analysis was performed under the following conditions.

Column used: YMC-pACK FA (YMC)
Flow rate: 1 ml / min
Injection volume: 10μl
Eluent: methanol / acetonitrile / H ₂ O = 40/5/55 (V / V / V)
Internal standard: 2-Hydroxy-2-methyl-n-butyric acid
Detection: UV 400nm.

  As a result, a peak was confirmed at a position of 7.9 minutes, which coincided with the peak of 3-hydroxypropionic acid. Therefore, E.I. It was confirmed that 3HP was produced from glucose by fermentation using Escherichia coli (mcr / PinPoint Xa-1).

  In addition, E.E. E. coli (mcr / PinPoint Xa-1) was inoculated into IPTG-added (final concentration 1 mM) 4 wt / vol% glycerin-added M9 medium (Difco) and cultured at 37 ° C. for 48 hours. When the obtained culture solution was subjected to analysis by high performance liquid chromatography in the same manner as described above, a peak was confirmed at a position of 7.9 minutes, which coincided with the peak of 3-hydroxypropionic acid. Therefore, E.I. It was confirmed that 3HP was produced from glycerin by fermentation using Escherichia coli (mcr / PinPoint Xa-1).

(Example 24: Production of 3HP by Escherichia coli introduced with a gene encoding a foreign malonyl-CoA reductase and a gene encoding a foreign acetyl-CoA carboxylase)
Primer for amplifying accBC gene and dtsR1 gene based on accBC gene and dtsR1 gene base sequences (SEQ ID NOs: 13 and 14) encoding acetyl CoA carboxylase (acc) of Corynebacterium glutamicum ATCC13032 strain (Primer for accBC amplification derived from Corynebacterium glutamicum) SEQ ID NOs: 15 and 16 and primers for dtsR1 amplification derived from Corynebacterium glutamicum; SEQ ID NOs: 17 and 18) were designed. Using the designed primer, PCR amplification was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, and the accBC gene and dtsR1 gene of Corynebacterium glutamicum ATCC13032 were obtained.

  The obtained accBC gene and dtsR1 gene were cloned downstream of the mcr gene of mcr / PinPoint Xa-1 described in Example 23 to construct mcr-acc / PinPoint Xa-1. According to the protocol of Escherichia coli fusion blue (Takara), the constructed mcr-acc / PinPoint Xa-1 was introduced by the heat shock method. coli (mcr-acc / PinPoint Xa-1) was obtained.

  E. Escherichia coli (mcr-acc / PinPoint Xa-1) was inoculated into IP9-added (final concentration 1 mM) 2 wt / vol% glucose-added M9 medium (manufactured by Difco) and cultured at 37 ° C. for 48 hours. When the obtained culture broth was subjected to analysis by high performance liquid chromatography in the same manner as in Example 23, a peak at a position of 7.9 minutes corresponding to the peak of 3-hydroxypropionic acid was found compared with Example 23. Increased to the advantage. Therefore, it is confirmed that by using malonyl CoA reductase in combination with acetyl CoA carboxylase derived from an organism different from the organism used as the host, 3HP production can be performed more efficiently than when malonyl CoA reductase is used alone. It was done. In the culture using glycerin as a carbon source, a 3HP peak significantly increased from the 3HP peak described in Example 23 was also confirmed.

(Example 25: Production of 3HP by Escherichia coli introduced with a gene encoding exogenous malonyl-CoA reductase, which has been genetically modified to increase the availability of intracellular NADPH)
E. Based on the nucleotide sequences of the glucose-6-phosphate dehydrogenase (zwf) gene and 6-phosphogluconate dehydrogenase (gnd) gene of Escherichia coli (SEQ ID NOs: 19, 20), primers for amplifying the zwf and gnd genes (E. Primers for amplifying glucose-6-phosphate dehydrogenase derived from Escherichia coli; SEQ ID NOs: 21 and 22, and primers for amplifying 6-phosphogluconate dehydrogenase derived from E. coli; SEQ ID NOs: 23 and 24) were designed. Using the designed primers, E.I. PCR amplification was performed using the genomic DNA of E. coli K12 as a template. The zwf gene and gnd gene derived from Escherichia coli were obtained. The obtained zwf gene and gnd gene were cloned downstream of the T7 promoter of pCDFDuet-1 (Takara) to construct zwf-gnd / pCDF.

  As described in Example 23. zwf-gnd / pCDF is introduced into E. coli (mcr / PinPoint). coli (mcr / PinPoint Xa-1, zwf-gnd / pCDF) was obtained. E. coli (mcr / PinPoint Xa-1, zwf-gnd / pCDF) is inoculated into IPTG-added (final concentration 1 mM) 2 wt / vol% glucose-added M9 medium (Difco) and cultured at 37 ° C. for 48 hours went. The obtained culture broth was subjected to analysis by high performance liquid chromatography in the same manner as in Example 23. As a result, compared with Example 23, the peak was at the position of 7.9 minutes, which coincided with the peak of 3-hydroxypropionic acid. Increased to an advantage. Therefore, it was confirmed that 3HP productivity can be improved by performing genetic modification to increase the availability of intracellular NADPH and reacting malonyl-CoA reductase under high NADPH conditions.

(Example 26: Production of 3HP by Escherichia coli into which a gene encoding an exogenous malonyl-CoA reductase and a gene encoding an exogenous acetyl-CoA carboxylase have been genetically modified to enhance the availability of intracellular NADPH)
Into E. coli described in Example 24 (mcr-acc / PinPoint Xa-1), zwf-gnd / pCDF described in Example 25 was introduced, and E. coli (mcr-acc / PinPoint Xa-1, zwf-gnd / pCDF). E. coli (mcr-acc / PinPoint Xa-1, zwf-gnd / pCDF) was inoculated into IP9-added (final concentration 1 mM) 2 wt / vol% glucose-added M9 medium (Difco) for 48 hours at 37 ° C. Culture was performed. When the obtained culture broth was subjected to analysis by high performance liquid chromatography in the same manner as in Example 23, the peak at a position of 7.9 minutes corresponding to the peak of 3HP as compared with Example 23 and Examples 24 and 25 was obtained. Increased to an advantage. Therefore, in addition to the combined use of malonyl CoA reductase and acetyl CoA carboxylase derived from an organism different from the host to be used, genetic modification to increase the availability of intracellular NADPH can be performed, so that malonyl CoA reductase alone or malonyl CoA reductase and It was confirmed that 3HP production can be performed more efficiently than when acetyl CoA carboxylase is used in combination.

(Example 27: Synthesis of acrylic acid and superabsorbent resin using 3HP fermented from sugar)
100 g of the fermentation broth containing 3HP obtained in Examples 1, 2, and 3 and Examples 24, 25, and 26 was centrifuged at 6000 rpm for 20 minutes, respectively, and the culture supernatant was collected. 100 g of tri-n-octylamine was added to the collected culture supernatant and mixed gently at room temperature (25 ° C.) for 24 hours using a stir bar.

  Next, after mixing for a predetermined time, the mixed solution was allowed to stand to separate into two phases, and the organic phase was recovered. 50 g of aluminum oxide was added to the organic phase containing the reaction mixture, and the mixture was heated and distilled at 170 ° C. for 1 hour. The obtained distillate was collected and cooled to room temperature, and then gradually heated from 100 ° C. to 130 ° C. to remove the distillate. Thereafter, the pressure was reduced, and the system was gradually heated to 200 ° C. while maintaining the pressure in the system at 20 kPa, and the distillate was collected to obtain a solution containing acrylic acid (acrylic acid solution).

  To the resulting acrylic acid solution, 60 mass ppm (relative to acrylic acid) of hydroquinone was added as a polymerization inhibitor. Separately, by adding the polymerization inhibitor-added acrylic acid solution under cooling (liquid temperature 35 ° C.) to an aqueous NaOH solution obtained from caustic soda containing 0.2 mass ppm of iron, acrylic acid 75 mol% Neutralization was performed. By dissolving 0.05 mol% of polyethylene glycol diacrylate (value relative to sodium acrylate) as an internal cross-linking agent in the obtained sodium acrylate aqueous solution having a neutralization rate of 75 mol% and a concentration of 35 mass%, a monomer was obtained. Ingredients were obtained. 350 g of this monomer component was placed in a cylindrical container having a volume of 1 L, and nitrogen was blown at a rate of 2 L / min to deaerate for 20 minutes. Then, an aqueous solution of sodium persulfate 0.12 g / mol (value relative to the monomer component) and L-ascorbic acid 0.005 g / mol (value relative to the monomer component) was added under stirring with a stirrer to initiate polymerization. I let you. Stirring was stopped after the initiation of polymerization, and a standing aqueous solution polymerization was performed. After the temperature of the monomer component showed a peak polymerization temperature of 108 ° C. after about 15 minutes (polymerization peak time), the polymerization was allowed to proceed for 30 minutes. Thereafter, the polymer was taken out from the cylindrical container to obtain a hydrogel crosslinked polymer.

  The obtained hydrogel crosslinked polymer was subdivided at 45 ° C. with a meat chopper (pore diameter: 8 mm) and then heat-dried with a hot air dryer at 170 ° C. for 20 minutes. Further, the dried polymer (solid content: about 95%) is pulverized with a roll mill and classified to a particle size of 600 to 300 μm with a JIS standard sieve to obtain a polyacrylic acid-based water absorbent resin (neutralization rate: 75%). Obtained.

  Polymerizability of acrylic acid obtained by the production method of the present invention was equivalent to that of acrylic acid obtained by the production method of acrylic acid using propylene as a raw material.

  In addition, the water-absorbent resin obtained by the production method of the present invention had no odor and had the same physical properties as the water-absorbent resin produced from propylene.

(Example 28: Synthesis of acrylic ester using acrylic acid synthesized in Example 27)
The acrylic acid solution obtained in Example 27 was purified by crystallization. The obtained purified acrylic acid was subjected to a transesterification reaction at 65 ° C. using 3 parts by mass of n-butanol as a raw material and a strongly acidic cation exchange resin as a catalyst. By extracting the obtained reaction liquid with water, unreacted acrylic acid contained in the reaction liquid and water generated by the reaction were removed. Next, the extract was introduced into a distillation column and distilled to obtain n-butyl acrylate having a purity of 99.8% by mass or more from the bottom of the column. In addition, n-butanol containing n-butyl acrylate distilled from the top of the column was reused in the transesterification reaction.

(Example 29: Synthesis of acrylic ester resin using acrylic ester synthesized in Example 28 and application to adhesive)
A monomer component containing 3 parts by mass of the purified acrylic acid obtained in Example 28, 96 parts by mass of n-butyl acrylate synthesized in Example 28, and 1 part by mass of 2-hydroxyethyl acrylate was used as a polymerization initiator. Using 0.2 parts by mass of azobisisobutyronitrile, solution polymerization was performed under reflux in a mixed solution of toluene and ethyl acetate (toluene / ethyl acetate (mass ratio) = 1/1). After 8 hours, an acrylic ester resin solution having a nonvolatile content of 50% by mass and a weight average molecular weight of 500,000 was obtained.

  By adding 1 part by mass of Coronate L-55E (manufactured by Nippon Polyurethane Industry Co., Ltd.) that is an isocyanate-based crosslinking agent to 100 parts by mass of the obtained acrylic ester resin solution, stirring and mixing to be uniform, An adhesive was prepared.

  Next, performance evaluation about the obtained adhesive was performed. Specifically, the adhesive was applied on a polyethylene terephthalate (PET) film (manufactured by Toray Industries, Inc., thickness 25 μm) so that the thickness after drying was 30 μm, and then dried at 80 ° C. for 5 minutes. . Thereafter, release paper K-80HS (manufactured by Sanei Kaken Co., Ltd.) was attached to the surface of the adhesive to protect it, and then cured for 7 days in an atmosphere of 23 ° C. and relative humidity of 65%. The cured adhesive film was cut into a predetermined size to produce a test piece.

A holding power test was performed on the obtained test piece. Specifically, the test piece from which the release paper was peeled was attached to a SUS304 stainless steel plate, and the test piece was pressed against the stainless steel plate by reciprocating a 2 kg rubber roller in an atmosphere of 23 ° C. and a relative humidity of 65%. At this time, the pasting area was 25 mm × 25 mm. After being left for 25 minutes, it was suspended vertically in a holding force tester set at 80 ° C. and left for 20 minutes. Thereafter, a weight of 1 kg was applied to the sample. The load at this time was 1.568 N / cm ² . When the time from when the weight was applied to when the test piece dropped from the stainless steel plate was measured, the stuck test piece did not fall even after 24 hours had passed, and the acrylate resin obtained above was an adhesive. As a result, it was confirmed that sufficient performance was exhibited.

Among the first genetically modified microorganisms described above, Schizosaccharomyces pombe NSH-2 ( Accession number : FERM BP-11527 ) (S. pombe (mcr)), Schizosaccharomyces pombe NSH-3 ( Accession number : P28 : P28 ) It is particularly preferable to use (S. pombe (mcr, acc)).

The genetically modified microorganism introduced with the gene encoding the exogenous malonyl-CoA reductase thus obtained, ie, Schizosaccharomyces pombe (mcr), was named Schizosaccharomyces pombe NSH-2 and was dated March 9, 2012. Deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Tsukuba Center Ibaraki 1-1-1 Tsukuba Center Chuo No. 6) with the deposit number FERM P-2227 on February 27, 2013 , Transferred to international deposit. The accession number is FERM BP-11527 .

(Example 2: 3HP production by S. pombe (mcr))
Whether SHP was produced using S. pombe (mcr): Schizosaccharomyces pombe NSH-2 ( Accession number : FERM BP-11527 ) produced in Example 13 above was examined.

The thus obtained gene encoding the exogenous malonyl CoA reductase and the gene recombinant microorganism into which the gene encoding the exogenous acetyl-CoA carboxylase was introduced, ie, Schizosaccharomyces pombe (mcr, acc), were converted into Schizosaccharomyces pombe NSH- No. 3 and the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1-1 Tsukuba Center, Tsukuba, Chuo No. 6), National Institute of Advanced Industrial Science and Technology, on March 9, 2012, the accession number FERM P-2228 And transferred to an international deposit on February 27, 2013. The accession number is FERM BP-11528 .

(Example 3: 3HP production by S. pombe (mcr, acc))
Using S. pombe (mcr, acc): Schizosaccharomyces pombe NSH-3 ( accession number : FERM BP-11528 ) produced in Example 16 above, it was examined whether 3HP was produced.

(Example 19: Transformation)
The AbaR-CMV-gpd1 fragment prepared in Example 18 above was transformed into S. pombe OB2 (mcr, acc): Schizosaccharomyces pombe NSH-3 ( Accession No . : FERM BP-11528 ) prepared in Example 16 above. pombe OB2 (mcr, acc, gpd1) was prepared. Details are shown below.

(Example 22: Transformation)
The leu_CMV-pta · pta fragment prepared in Example 21 above was transformed into S. pombe OB2 (mcr, acc): Schizosaccharomyces pombe NSH-3 ( accession number : FERM BP-11528 ) prepared in Example 16 above. pombe OB2 (mcr, acc, pkt · pta) was prepared. Details are shown below.

Claims (12)

First gene set in which a gene encoding an exogenous malonyl CoA reductase or a gene encoding an exogenous malonate semialdehyde dehydrogenase and a gene encoding an exogenous 3-hydroxypropionate dehydrogenase are introduced into a microorganism having acid resistance A gene that encodes a foreign glycerin dehydratase and a gene that encodes a glycerin dehydratase reactivation factor or a gene that encodes a foreign diol dehydratase and a gene that encodes a diol dehydratase reactivation factor And a method for producing 3-hydroxypropionic acid, comprising using a second genetically modified microorganism into which a gene encoding a foreign aldehyde dehydrogenase is introduced.   A genetically modified microorganism in which a gene encoding an exogenous malonyl-CoA reductase is introduced into Schizosaccharomyces pombe.   A genetically modified microorganism in which a gene encoding an exogenous malonyl CoA reductase and a gene encoding an exogenous acetyl CoA carboxylase are introduced into Schizosaccharomyces pombe.   A genetically modified microorganism in which a gene encoding an exogenous malonyl CoA reductase, a gene encoding an exogenous acetyl CoA carboxylase, and an exogenous gene having an effect of increasing intracellular NADPH amount are introduced into Schizosaccharomyces pombe.   The genetically modified microorganism according to claim 4, wherein the foreign gene having an effect of increasing the amount of NADPH in the cell is a gene encoding NADP-dependent glyceraldehyde-3-phosphate dehydrogenase derived from Kluyveromyces lactis. .   A genetically modified microorganism, wherein a gene encoding an exogenous malonyl-CoA reductase, a gene encoding an exogenous acetyl-CoA carboxylase, and an exogenous gene having an effect of increasing acetyl-CoA production are introduced into Schizosaccharomyces pombe.   The genetically modified microorganism according to claim 6, wherein the foreign gene having an effect of increasing the amount of acetyl CoA is a gene encoding fructose-6-phosphate phosphoketolase and a gene encoding phosphotransacetylase.   A genetically modified microorganism in which a gene encoding a foreign glycerin dehydratase, a gene encoding a glycerol dehydratase reactivating factor, and a gene encoding a foreign aldehyde dehydrase are introduced into Schizosaccharomyces pombe.   A method for producing acrylic acid, comprising dehydrating 3-hydroxypropionic acid produced by the method according to claim 1.   A partially neutralized acrylic acid produced by the method according to claim 9 to produce a partially neutralized acrylic acid, and the partially neutralized acrylic acid is copolymerized with a crosslinkable monomer. Production method.   The manufacturing method of acrylic ester which has esterifying the acrylic acid manufactured by the method of Claim 9.   The manufacturing method of acrylic ester resin which has superposing | polymerizing the monomer component containing the acrylic ester produced by the method of Claim 11.