JP2013070675A - Method for culturing recombinant bacterium producing alkyl diol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing a target compound by culturing a microorganism while regulating the condition of oxidation-reduction potential so as to promote the consumption of a raw carbon source and the production of an alkyl diol in a culturing process using a gene-recombinant microorganism having the ability to produce a polyol compound, especially an alkyl diol (e.g., 1,3-propanediol).SOLUTION: The 1,3-propanediol can be produced more efficiently than conventional methods when the aeration condition in the culture is regulated so that the oxidation-reduction potential value in a culture solution is from -10 to 100 mV.

Description

    The present invention relates to a method for producing an alkyl diol using a genetically modified microorganism imparted with an alkyl diol production function. In particular, the present invention relates to a method for efficiently producing an alkyldiol using a carbon source in a medium as a raw material in the step of culturing a genetically modified microorganism imparted with an alkyldiol production function.

  1,3-propanediol is used as a monomer used in the production of polyesters and polyurethanes typified by polytrimethylene terephthalate, such as fibers, engineering plastics and films, and as a starting material for the synthesis of cyclic compounds. It is a compound with a wide range of uses.

Methods for producing 1,3-propanediol are roughly classified into a method by chemical synthesis and a method by fermentation using microorganisms. As a chemical synthesis method, a method of producing 1,3-propanediol by carrying out a hydration reaction using acrolein as a starting material (the following formula 3), and a synthesis gas (CO and H ₂ ) using ethyleneoxyside as a starting material. And a method for producing 1,3-propanediol (formula 4 below) by carrying out a reaction for hydroformylation in the presence of high temperature and high pressure.

(Formula 3)

(Formula 4)

  On the other hand, as a biological method, microorganisms capable of producing 1,3-propanediol from glycerol include, for example, the genus Klebsiella, the genus Clostridium, the genus Citrobacter, and the enterobacter. A method using an anaerobic bacterium belonging to the genus (Enterobacter) and the like can be mentioned. Another example is a method using genetically modified E. coli that is improved so that 1,3-propanediol can be produced from glycerol.

  As a method of utilizing anaerobic bacteria, as represented by Non-Patent Document 1, in a step of culturing Klebsiella pneumoniae, it grows under anaerobic conditions in which glycerol is added to block oxygen. There is a method in which 1,3-propanediol is obtained by converting glycerol into 1,3-propanediol. In addition, as represented by Non-Patent Document 2, parameters for monitoring the anaerobic state in an anaerobic fermentation process for producing 1,3-propanediol from glycerol in the step of culturing Klebsiella pneumoniae under anaerobic conditions. Describes a method by controlling the culture under anaerobic conditions using an oxidation-reduction potential (ORP) as an ORP value of -190 mV. In anaerobic bacteria capable of producing 1,3-propanediol from these glycerols, the enzyme involved in this reaction is converted to 1,3-propanediol in a two-step reaction via 3-hydroxypropionaldehyde. Glycerol dehydratase (EC 4.2.1.30) consisting of three subunits and 1,3-propanediol oxidoreductase (EC 1.1.1.202) have been disclosed. These genes involved in 1,3-propanediol production from glycerol have been shown to form an operon structure, and a glycerol dehydratase reactivating factor consisting of two subunits, adenosyltransferase (EC2. 5.1.17) and the like are included (Non-patent Document 3). As a result of the action of enzymes encoded by these groups of genes, 1,3-propanediol is produced from glycerol and accumulates in the culture medium. However, in the production of 1,3-propanediol from glycerol by an anaerobic fermentation process using these anaerobic bacteria, it is necessary to strictly block oxygen, which is not suitable for industrial production.

  As a method of using genetically modified E. coli improved so that 1,3-propanediol can be produced from glycerol, it was isolated from Saccharomyces cerevisiae as represented by Patent Document 1. DAR1, which is a gene encoding glycerol-3-phosphate dehydrogenase (G3PDH) that catalyzes the conversion of dihydroxyacetone phosphate to glycerol-3-phosphate, and catalyzes the conversion of glycerol-3-phosphate to glycerol A gene dhaB1 encoding three subunits of GPP2, which is a gene encoding G3P phosphatase, and glycerol dehydratase isolated from Klebsiella pneumoniae, Using an E. coli strain into which a plasmid containing genes orfX, orfZ, and orfW, orfY encoding two subunits of haB2, dhaB3, and glycerol dehydratase reactivation factor has been introduced, glycerol is produced using glucose as a raw material. A method for obtaining 1,3-propanediol by production and conversion to 1,3-propanediol is described. This method is a method for producing 1,3-propanediol using a saccharide such as glucose as a starting material, and culture is performed by adjusting the dissolved oxygen concentration (DO).

  Further, in Patent Document 2, a gene encoding a diol dehydratase derived from Klebsiella pneumoniae, or a gene encoding a diol dehydratase reactivation factor and a plasmid having a gene encoding a glycerol dehydratase is disclosed. Glycerol was dehydrated using the crude enzyme solution extracted from the cultured cells or the toluene-treated cells using an Escherichia coli strain into which the plasmid having the above was introduced to obtain 3-hydroxypropionaldehyde, and this 3-hydroxypropionaldehyde Describes a method for producing 1,3-propanediol by hydrogenation of a hydrogen atom by a chemical synthesis method using palladium carbon as a catalyst. That is, in the method described in Patent Document 2, it is necessary to treat bacterial cells in advance, and a two-step reaction is required, that is, a biological method and a chemical synthesis method.

  In recent years, from the viewpoint of fossil resource depletion problems and global warming problems, there has been an increasing social demand for establishing chemical and energy production systems from raw materials derived from biomass (plant resources) as renewable resources. . For example, biodiesel, which is a long-chain fatty acid methyl ester obtained by a transesterification reaction between a triglyceride derived from vegetable oil and methanol, is one of the energy derived from biomass. In this transesterification reaction, glycerol derived from triglyceride as a raw material is by-produced. Therefore, a new use of by-product glycerol is desired as a problem of biodiesel development as a biomass-derived fuel.

WO2004-033646 JP 2005-102533 A

Enzyme Microb. Tech. 20, 82-86 (1997) Appl. Microbiol. Biotechnol. 69,554-563 (2006) Biotechnol. Prog. 19, 263-272 (2003)

  The present invention relates to a redox potential condition that promotes consumption of a raw material carbon source and alkyldiol production in a culture process using a polyol compound, particularly a genetically modified microorganism having an ability to produce an alkyldiol (for example, 1,3-propanediol). It is an object of the present invention to provide a method for producing a target compound more efficiently by cultivating the microorganism after adjusting it downward.

  In order to solve the above-mentioned problems, the present inventor has intensively studied paying attention to the culture conditions of genetically modified microorganisms capable of producing alkyldiol.

  In order to produce alkyldiol using biomass (carbon source), which is a renewable resource, as a raw material, the present inventors first constructed a genetically modified microorganism having an alkyldiol-producing ability. An expression plasmid vector pSP-KGR1 into which a glycerol dehydratase gene, a glycerol dehydratase reactivation factor gene, and a 1,3-propanediol dehydrogenase gene derived from Klebsiella pneumoniae were constructed was constructed. Furthermore, a plasmid vector pMU-KAT1 into which an adenosyltransferase gene derived from Klebsiella oxytoca was introduced was constructed.

  Next, the present inventors adjusted the culture conditions by using E. coli W3110 strain transformed with the obtained plasmid vectors pSP-KGR1 and pMU-KAT1, thereby allowing more efficient 1,3 -Investigated propanediol production method. Specifically, in the culturing step for producing 1,3-propanediol using glycerol present in the medium as a raw material, the oxidation-reduction potential (ORP) of the culture solution shows good growth of the producing bacteria, Found to be an important factor for 3-propanediol production, adjusted the redox potential (ORP) value during culture, and confirmed the consumption status of raw carbon sources and the production status of alkyldiol under each condition went.

  As a result, the inventors of the present invention, when adjusting the oxidation-reduction potential at the time of culture so that the oxidation-reduction potential value in the culture solution becomes −10 to 100 mV (preferably −10 to 50 mV), than before. It has been found that 1,3-propanediol can be produced efficiently, and thus the present invention has been completed.

  That is, the present invention provides an efficient method for producing an alkyl diol using a genetically modified microorganism having an ability to produce an alkyl diol (for example, 1,3-propanediol).

（式中、Ｒは、1以上の水酸基を有する炭素数１から４個のアルキル基を表す。）
〔２〕遺伝子組換え微生物を好気条件下で培養した後に、〔１〕に記載の条件下で培養しアルキルジオールを生産させることを特徴とする、アルキルジオールの製造方法。
〔３〕遺伝子組換え微生物を好気条件下で培養し、培養液中の酸化還元電位が-10〜100 mVから選択される範囲であるときに、該前培養後に〔１〕に記載の条件下で培養しアルキルジオールを生産させることを特徴とするアルキルジオールの製造方法。
〔４〕前記培養液中の炭素源がグリセロール、グルコース、スクロース、フルクトース、マルトース、マンノース、キシロース、ガラクトース、酢酸、クエン酸、エタノール、メタノールから選択される少なくとも一つ以上の炭素源を含むことを特徴とする〔１〕〜〔３〕のいずれかに記載のアルキルジオールの製造方法。
〔５〕前記遺伝子組換え微生物の宿主がエシャリヒア（Escherichia）属の微生物であることを特徴とする〔１〕〜〔４〕のいずれかに記載のアルキルジオールの製造方法。
〔６〕前記アルキルジオールが1,3-プロパンジオールであることを特徴とする〔１〕〜〔５〕のいずれかに記載のアルキルジオールの製造方法。
〔７〕前記遺伝子組換え微生物が、下記(a)から(e)のいずれかに記載のタンパク質又は酵素を発現する遺伝子を少なくとも１つ以上有することを特徴とする、〔６〕に記載の1,3-プロパンジオールの製造方法；
（ａ）配列番号：２、４、６、８、１０、１２又は１４に記載されたアミノ酸配列を有するタンパク質、
（ｂ）配列番号：２、４、６、８、１０、１２又は１４に記載のアミノ酸配列において、1若しくは複数のアミノ酸が置換、欠失、挿入、もしくは付加されたアミノ酸配列を有する酵素、
（ｃ）配列番号：１、３、５、７、９、１１又は１３に記載された塩基配列によりコードされるアミノ酸配列を有する酵素、
（ｄ）配列番号：１、３、５、７、９、１１又は１３に記載された塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるアミノ酸配列を有する酵素、及び
（ｅ）配列番号：２、４、６、８、１０、１２又は１４に記載のアミノ酸配列と８５%以上の同一性を有する酵素。 More specifically, the present invention provides the following [1] to [7].
[1] A genetically modified microorganism modified so as to produce an alkyldiol represented by the following formula 1 using a carbon source in a culture solution has an oxidation-reduction potential value in the culture solution of -10 to 100 mV A method for producing an alkyl diol, comprising culturing under the conditions of
[Formula 1]

(In the formula, R represents an alkyl group having 1 to 4 carbon atoms having one or more hydroxyl groups.)
[2] A method for producing an alkyldiol, comprising culturing a genetically modified microorganism under an aerobic condition and then culturing the recombinant microorganism under the condition described in [1] to produce an alkyldiol.
[3] The condition described in [1] after the pre-culture when the genetically modified microorganism is cultured under aerobic conditions and the oxidation-reduction potential in the culture solution is in a range selected from −10 to 100 mV. A method for producing an alkyl diol, characterized by culturing under pressure to produce an alkyl diol.
[4] The carbon source in the culture medium contains at least one carbon source selected from glycerol, glucose, sucrose, fructose, maltose, mannose, xylose, galactose, acetic acid, citric acid, ethanol, and methanol. The method for producing an alkyl diol as described in any one of [1] to [3].
[5] The method for producing an alkyl diol according to any one of [1] to [4], wherein the host of the genetically modified microorganism is a microorganism belonging to the genus Escherichia.
[6] The method for producing an alkyl diol according to any one of [1] to [5], wherein the alkyl diol is 1,3-propanediol.
[7] The gene recombinant microorganism according to [6], wherein the genetically modified microorganism has at least one gene that expresses the protein or enzyme according to any of (a) to (e) below: A process for producing 1,3-propanediol;
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14,
(B) an enzyme having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14;
(C) an enzyme having an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13,
(D) an enzyme having an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13; and (E) an enzyme having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14;

  According to the present invention, a method for efficiently producing an alkyldiol (for example, 1,3-propanediol) from carbohydrate raw materials such as glucose and glycerol, which are raw materials derived from renewable resources, has been provided. The method of the present invention is industrially advantageous in that an alkyldiol can be produced using a less expensive raw material. The present invention makes it possible to produce alkyl diols using renewable resources as raw materials.

It is a figure which shows the structure of plasmid pSP-KGR1 and pMU-KAT1. It is a figure which shows the result of 1, 3- propanediol production using E. coli W3110 (pSP-KGR1 / pMU-KAT1). The vertical axis represents the amount of residual glycerol (g / L) and the amount of 1,3-propanediol produced (g / L), and the horizontal axis represents the culture time. It is a figure which shows the relationship between the oxidation reduction potential value (ORP value) when air ventilation is stopped, and the produced | generated 1, 3- propanediol. The vertical axis represents the final 1,3-propanediol accumulation concentration, and the horizontal axis represents the oxidation-reduction potential value (mV). Results of 1,3-propanediol production using E. coli W3110 (pSP-KGR1 / pMU-KAT1) when the redox potential (ORP value) is 12 mV, stirring is 200 rpm, and air aeration is stopped FIG. The vertical axis represents the amount of residual glycerol (g / L) and the amount of 1,3-propanediol produced (g / L), and the horizontal axis represents the culture time. It is a figure which shows the result of 1, 3- propanediol production using E. coli W3110 (pSP-KGR1 / pMU-KAT1). When culturing is continued under aerobic conditions (A), and after DO becomes 0%, stirring is set to 200 rpm with ORP of about 20 mV, air aeration is stopped, and further culturing is continued (B) The results are shown respectively. The vertical axis represents the amount of residual glycerol (g / L) and the amount of 1,3-propanediol produced (g / L), and the horizontal axis represents the culture time. It is a figure which shows the result of 1, 3- propanediol production using E. coli W3110 (pSP-KGR1 / pMU-KAT1). After culturing for 40 hours under aerobic conditions, continue culturing while maintaining ORP at -20 mV (A), and after culturing for 40 hours under aerobic conditions, adjust ORP to 10-50 mV (B) shows the results when the culture is continued while controlling DO to 2%. The vertical axis represents the amount of residual glycerol (g / L) and the amount of 1,3-propanediol produced (g / L), and the horizontal axis represents the culture time.

  The present invention relates to a method for producing an alkyl diol using a genetically modified microorganism having an ability to produce an alkyl diol (for example, 1,3-propanediol). More specifically, at least one active substance selected from the group consisting of a culture, a fungus body, and a processed product of a genetically modified microorganism that functionally expresses an enzyme activity related to alkyldiol production, and a carbon source It is related with the manufacturing method of alkyl diol including the process of contacting and the process of collect | recovering alkyl diol.

  In the present invention, the alkyl diol is preferably produced as an alkyl diol represented by (Formula 1) through a reaction represented by (Formula 2).

[Formula 2]

（式中、Ｒは、1以上の水酸基を有する炭素数１から４個のアルキル基を表す。） [Formula 1]

(In the formula, R represents an alkyl group having 1 to 4 carbon atoms having one or more hydroxyl groups.)

  In the present invention, in formula 1, the R group is an alkyl group having 1 to 4 carbon atoms having at least one hydroxyl group (methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group). , An isobutyl group or a tert-butyl group).

  Preferred alkyl diols produced in the present invention include those represented by the above formula 1 wherein the R group has at least one hydroxyl group and has 1 to 4 carbon atoms (methyl group, ethyl group, propyl group, or isopropyl group). And a compound which is a group).

  More specifically, as the alkyl diol produced in the present invention, 1,3-propanediol (when R is both hydroxymethyl in Formula 1 and Formula 2) can be mentioned as a preferred example. The optical activity of 1,3-propanediol produced in the present invention is not particularly limited, and the optical activity of (R) or (S) (R-form or S-form) at the 1st and 3rd positions of carbon. 1,3-propanediol is included in the 1,3-propanediol produced in the present invention.

  By contacting a genetically modified microorganism or the like that functionally expresses an enzyme activity related to alkyldiol production with a carbon source, by assimilating the carbon source by the genetically modified microorganism, An alkyl diol can be produced. In addition, as a contact form of a genetically modified microorganism and a fermentable substrate, the culture method in a culture solution can be illustrated, However, It is not limited to this. The carbon source is obtained by dissolving the genetically modified microorganism in a suitable solvent that assimilate the fermentable substrate and provides a desirable environment for the expression of the target enzyme activity.

  When producing an alkyldiol by contacting a carbon source with a genetically modified microorganism that functionally expresses an enzyme activity related to the production of alkyldiol, the growth status of the recombinant microorganism and the production of alkyldiol are considered. Conditions favorable for such enzyme activity can be selected.

  The present invention relates to a method for producing an alkyldiol, characterized by culturing a genetically modified microorganism modified so as to produce an alkyldiol under conditions where the oxidation-reduction potential value in the culture solution is -10 to 100 mV. About.

  In the present invention, the oxidation-reduction potential value in the culture solution is not particularly limited as long as it is in the range of −10 to 100 mV, but is preferably in the range of −10 to 50 mV.

  In the present invention, the oxidation-reduction potential value in the culture solution can be adjusted by changing the air aeration rate, the ratio of gases such as oxygen and nitrogen, the stirring speed, and the like. The oxidation-reduction potential value in the culture solution may be adjusted by adding an oxidizing agent or a reducing agent.

  In the present invention, the genetically modified microorganism may be cultured under aerobic conditions to proliferate the genetically modified microorganism before the alkyldiol is produced by the genetically modified microorganism under the above culture conditions. In the previous stage of culture, the oxidation-reduction potential value in the culture solution may be adjusted to −10 to 100 mV.

  In the present invention, the “carbon source” which is a raw material for producing alkyldiol means any carbon source that can be metabolized by a microorganism whose substrate contains at least one carbon atom. The type is not limited, but preferably sugars such as glycerol, glucose, sucrose, fructose, maltose, mannose, galactose, starch hydrolysate, molasses, and organic acids such as acetic acid and citric acid, ethanol, etc. Alcohols, more preferably glycerol can be mentioned. These carbon sources may be a single type or a plurality of types.

  In the present invention, the concentration of the carbon source that is a raw material for producing the alkyl diol is not particularly limited, but is usually about 0.1 to 30%, preferably 0.5 to 15%, more preferably 1 to 10%. Is used.

  The carbon source can be added all at once at the start of culture, but can also be added continuously or intermittently to the culture solution.

  As a genetically modified microorganism suitably used in the above method, a transformant that functionally expresses a suitable enzyme that catalyzes the reaction represented by Formula 2 can be raised.

  In order to introduce one or more genes into a host, a method of transforming the host with a recombinant vector in which genes are separately introduced into a plurality of vectors different from the origin of replication to avoid incompatibility, or a single vector A method of introducing each gene into the gene, a method of introducing one or more genes into the chromosome, and the like can be used.

  When introducing multiple genes into a single vector, methods such as linking promoter- and terminator-related regions related to expression control to each gene, or expressing as an operon containing multiple cistrons such as the lactose operon. Is possible.

  In the transformant treated with a suitable enzyme activity that catalyzes the reaction represented by Formula 2 in the present invention, specifically, the permeability of the cell membrane is changed by treatment with an organic solvent such as a surfactant or toluene. Microorganisms, dried cells prepared by freeze-drying or spray-drying, cell-free extracts obtained by crushing cells by glass beads or enzyme treatment, partially purified products, purified enzymes, transformants and enzymes Examples include immobilized immobilized enzymes and immobilized microorganisms.

  In the genetically modified microorganism of the present invention, preferably, an enzyme gene that catalyzes the reaction represented by Formula 2 is isolated, and then the activity is enhanced using a microorganism belonging to the genus Escherichia (for example, Escherichia coli) as a host. Recombinant E. coli can be used. The genetically modified E. coli used for this purpose can be cultured in a medium generally used for culturing E. coli, and can be highly expressed by inducing expression by a known method. For example, E. coli having enhanced enzyme activity is cultured in 2 × YT medium (2.0% bacto-tryptone, 1.0% bacto-yeast extract, 1.0% sodium chloride, pH 7.2), and isopropyl After induction of expression with -thio-β-D-galactopyranoside (IPTG) and sufficient growth, the culture solution can be used as it is, or the cells can be recovered and used for alkyldiol production.

  The genetically modified microorganism of the present invention used for alkyldiol production may produce alkyldiol while growing in a culture solution for producing alkyldiol containing a carbon source, or a culture solution grown in advance by culture, or You may use the collect | recovered microbial cell.

  The amount of the culture broth that has been cultured and proliferated in advance, or the amount of the collected cells is usually 0.1 to 100%, preferably 0. 0% with respect to the amount of the culture broth for producing alkyldiol containing the fermentable substrate. 5 to 50%, more preferably about 1 to 20% can be used ("%" mentioned here indicates the inoculation rate relative to the alkyldiol-producing culture solution, and "[previously grown culture solution] / [Fermented liquor for producing alkyldiol] (v / v) ”).

It is preferable that the medium for producing alkyl diol contains a substance that promotes the production of alkyl diol as necessary, in addition to the carbon source for producing alkyl diol, and the gene used for this purpose. A medium component that serves as a nutrient source for the recombinant Escherichia coli may be included. Specifically, LB (1.0% bacto-tryptone, 0.5% bacto-yeast extract, 1.0% sodium chloride, pH 7.2), which is a medium used for culturing Escherichia coli, 2 × YT (2 0.0% bacto-tryptone, 1.0% bacto-yeast extract, 1.0% sodium chloride, pH 7.2) with a carbon source added to produce alkyldiol, or M9G medium (60 g / L glycerol) 6.8 g / L Na ₂ HPO ₄ , 3.0 g / L KH ₂ PO ₄ , 0.5 g / L NaCl, 1.0 g / L NH ₄ Cl, 0.493 g / L MgSO ₄ .7H ₂ O, 14 0.7 mg / L CaCl ₂ · 2H ₂ O, pH 7.5) and the like, and a medium of a preferable carbon source type and amount can be appropriately used. When a sufficient amount of cells are cultured in advance and used in a medium for producing alkyldiol, the alkyldiol can be produced in the presence of a higher concentration of fermentable substrate, and the medium can be removed. Further, it contains 10 mM to 800 mM, preferably 50 to 500 mM, more preferably 100 to 250 mM of a component for maintaining the pH in the culture medium at a pH suitable for alkyldiol production, if necessary. Specific examples include a MOPS buffer, a HEPES buffer, a MES buffer, a Tris buffer, and a phosphate buffer. The fermentation temperature may be any temperature at which the genetically modified microorganism of the present invention can express the ability to assimilate the carbon source, can express the enzyme activity that catalyzes the reaction represented by Formula 2, and can produce an alkyldiol. The reaction can be performed at 5 to 60 ° C, preferably 10 to 50 ° C, more preferably 20 to 40 ° C. Moreover, pH should just be pH which can express the enzyme activity which catalyzes reaction shown by Formula 2, and can produce | generate alkyldiol, Usually, pH 4-12, Preferably it is pH 5-11, More preferably, it is pH 6-9. It can be carried out. Moreover, fermentation can be performed under stirring or standing still.

  The production of the alkyl dial of the present invention is an organic solvent that is difficult to dissolve in water or water, for example, an organic solvent such as ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, methyl isobutyl ketone, methyl tertiary butyl ether, and diisopropyl ether. It can be carried out in a medium or in a two-phase system with an aqueous medium, or in a mixed system with an organic solvent that dissolves in water, for example, methanol, ethanol, isopropyl alcohol, acetonitrile, acetone, dimethyl sulfoxide and the like. The reaction of the present invention can be carried out by any of batch, fed-batch, and continuous production methods, and can also be carried out using immobilized cells, immobilized enzymes, membrane reactors, and the like. .

  Purification of the alkyldiol produced by the reaction includes separation by centrifugation or filtration, extraction with an organic solvent, various chromatographies such as ion exchange chromatography, adsorption with an adsorbent, dehydration or agglomeration with a dehydrating agent, crystallization , Distillation, etc., can be performed as appropriate.

  For example, after removing microbial cells and removing proteins from a fermentation broth containing microbial cells by centrifugation or membrane filtration, alkyldiol can be purified from an aqueous solution by a known method such as concentration or distillation. .

  The genetically modified microorganism of the present invention has a gene that expresses an enzyme related to alkyldiol production, that is, a suitable enzyme that catalyzes the reaction represented by Formula 2.

In the present invention, as shown in Formula 2, as an enzyme that produces hydroxyalkyl aldehyde from hydroxyalkyl diol, glycerol dehydratase (EC) that catalyzes a dehydration reaction by IUBMB (INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY). And enzymes such as diol dehydratase (EC 4.2.1.28) ( http://www.chem.qmul.ac.uk/iubmb/ ).

  The EC number is a number consisting of four sets of numbers for systematically classifying enzymes according to the reaction format, and is an enzyme number defined by the Enzyme Committee of the International Union of Biochemical and Molecular Biology. Specific examples include enzymes that catalyze the production of 3-hydroxypropionaldehyde from glycerol (for example, glycerol dehydratase).

  More specifically, an enzyme derived from a bacterium belonging to the genus Klebsiella can be mentioned, for example, a glycerol dehydratase gene product derived from Klebsiella pneumoniae or the like.

In the present invention, examples of the enzyme having glycerol dehydratase activity include the enzymes described in any one of (a) to (e) below.
(A) a protein having the amino acid sequence described in any one of SEQ ID NOs: 2, 4, or 6;
(B) In the amino acid sequence of SEQ ID NO: 2, 4 or 6, one or more (2 or more, preferably 2 to 20, more preferably 2 to 5) amino acids are substituted, deleted or inserted. Or an enzyme having an added amino acid sequence,
(C) an enzyme having an amino acid sequence encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 1, 3 and 5;
(D) an enzyme having an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence described in SEQ ID NO: 1, 3 or 5;
(E) A protein having 85% or more identity with the amino acid sequence of SEQ ID NO: 2, 4 or 6.

  In the present invention, a homologue of “an enzyme consisting of an amino acid sequence described in a specific SEQ ID NO” is one or more (two or more, preferably 2-20 in the amino acid sequence described in a specific SEQ ID NO. , More preferably 2 to 5) an enzyme having an amino acid sequence substituted, deleted, inserted or added. The homolog means a protein functionally equivalent to an enzyme consisting of the amino acid sequence described in a specific SEQ ID NO. In the present invention, “functionally equivalent” means having the same function as the enzyme activity (catalytic reaction, chemical reaction, etc.) of each enzyme described in the present specification.

In the amino acid sequence described in a specific SEQ ID NO, for example, mutation of amino acid residues of 100 or less, usually 50 or less, preferably 30 or less, more preferably 15 or less, more preferably 10 or less, or 5 or less is allowed. . In general, in order to maintain the function of a protein, the amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution. Such substitution of amino acid residues is called conservative substitution. For example, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp are all classified as nonpolar amino acids, and thus have similar properties to each other. Further, examples of the non-chargeability include Gly, Ser, Thr, Cys, Tyr, Asn, and Gln. Moreover, Asp and Glu are mentioned as an acidic amino acid. Examples of basic amino acids include Lys, Arg, and His. Amino acid substitutions within each of these groups are allowed.

  Those skilled in the art will be able to introduce site-directed mutagenesis into the DNA of each enzyme comprising the nucleotide sequence disclosed in the present specification (Nucleic Acid Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning 2ndEdt., Cold Spring Harbor Laboratory Press (1989), PCR A Practical Approach IRL Press pp.200 (1991)), etc., as appropriate substitution, deletion, insertion, and / or addition mutation A polynucleotide encoding a homologue of each enzyme can be obtained. By introducing a polynucleotide encoding the homologue of each enzyme into a host and expressing it, it is possible to obtain a homologue of each enzyme.

  Furthermore, the homologue of each enzyme of the present invention is at least 50%, preferably at least 70%, more preferably 80%, more preferably 85%, more preferably the amino acid sequence shown in SEQ ID NO: corresponding to each enzyme. A protein having 90%, even more preferably 95% or more (eg, 95%, 96%, 97%, 98%, or 99% or more) identity. Protein identity searches include databases related to amino acid sequences of proteins such as SWISS-PROT, PIR, and DAD, databases related to DNA sequences such as DDBJ, EMBL, and Gene-Bank, and databases related to predicted amino acid sequences based on DNA sequences. For example, it can be performed through the Internet using programs such as BLAST and FASTA.

  Each enzyme described in the present invention can bind an additional amino acid sequence as long as it has a functionally equivalent activity to the enzyme. For example, tag sequences such as histidine tags and HA tags can be added. Alternatively, it can be a fusion protein with another protein. Each enzyme of the present invention or a homologue thereof may be a fragment as long as it has an activity functionally equivalent to each enzyme.

  The polynucleotide encoding each enzyme described in the present invention can be isolated by the following method. For example, a primer for PCR is designed based on the base sequence represented by the sequence number corresponding to each enzyme, and the DNA of the present invention is obtained by performing PCR using the chromosomal DNA or cDNA library of the enzyme production strain as a template. be able to. Furthermore, using the obtained DNA fragment as a probe, restriction enzyme digests of chromosomal DNA of enzyme production strains are introduced into phages, plasmids, etc., and E. coli is transformed to obtain libraries and cDNA libraries. The polynucleotide of each enzyme can be obtained by colony hybridization, plaque hybridization, or the like.

  In addition, the base sequence of the DNA fragment obtained by PCR is analyzed, and from the obtained sequence, a PCR primer for extending outside the known DNA is designed, and the chromosomal DNA of the enzyme production strain is expressed with an appropriate restriction enzyme. After digestion, reverse PCR is performed using DNA as a template by self-cyclization reaction (Genetics 120, 621-623 (1988)). Also, RACE method (Rapid Amplification of cDNA End, “PCR Experiment Manual” p25-33, It is also possible to obtain polynucleotides of each enzyme by HBJ Publishing Bureau).

  In the present invention, the polynucleotide of each enzyme includes genomic DNA cloned by the method as described above, or cDNA, as well as DNA obtained by synthesis.

  In hybridization, the target nucleic acid is hybridized using a nucleic acid (DNA or RNA) consisting of a complementary sequence of the base sequence indicated by the sequence number corresponding to each enzyme or a partial sequence thereof as a probe, and stringent. After washing under various conditions, it is confirmed whether the probe is significantly hybridized to the target nucleic acid. The length of the probe is, for example, 20 consecutive bases or more, preferably 25 bases or more, more preferably 30 bases or more, more preferably 40 bases or more, more preferably 80 bases or more, more preferably 100 bases or more (for example, for each enzyme). The total length of the base sequence shown in the corresponding SEQ ID NO) is used. If the probe contains an unrelated sequence (such as a vector-derived sequence) other than the base sequence shown in the SEQ ID NO corresponding to each enzyme or its complementary sequence, only that sequence is used as a negative control as a negative control. Hybridization may be performed to confirm that the probe does not significantly hybridize to the target sequence after washing under the same conditions. Hybridization can be performed by a conventional method using a nitrocellulose membrane or nylon membrane (Sambrook et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratories; Ausubel, FM et al. (1994) Current. Protocols in Molecular Biology, Greene Publisher Associates / John Wiley and Sons, New York. NY).

  Specific examples of hybridization stringent conditions include, for example, 6 × SSC, 0.5% (W / V) SDS, 100 μg / ml denatured salmon sperm DNA, 5 × Denhardt solution (1 × Denhardt solution is 0.2% poly In a solution containing vinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% ficoll), hybridization is performed overnight at 45 ° C, preferably 55 ° C, more preferably 60 ° C, and even more preferably 65 ° C. The subsequent washing is performed under the same conditions as in hybridization, 4 × SSC, 0.5% SDS, and 20 minutes three times. More preferably, the conditions are such that washing after hybridization is performed twice at 4 × SSC, 0.5% SDS, 20 minutes, and once at 2 × SSC, 0.5% SDS, 20 minutes at the same temperature as hybridization. More preferably, the conditions are such that washing after hybridization is performed twice at 4 × SSC, 0.5% SDS, 20 minutes, followed by 1 × SSC, 0.5% SDS, 20 minutes at the same temperature as hybridization. . More preferably, post-hybridization washing is performed at the same temperature as the hybridization, 2 × SSC, 0.5% SDS, 20 minutes once, followed by 1 × SSC, 0.5% SDS, 20 minutes once, then 0.5 × SSC, 0.5% SDS, 20 minutes once. More preferably, post-hybridization washing is performed at the same temperature as the hybridization, 2 × SSC, 0.5% SDS, 20 minutes once, followed by 1 × SSC, 0.5% SDS, 20 minutes once, then 0.5 This is a condition in which × SSC, 0.5% SDS, 20 minutes once, followed by 0.1 × SSC, 0.5% SDS, 20 minutes once.

In the present invention, as shown in formula 2, NADH and / or NADPH is used as a coenzyme to reduce 3-hydroxyalkyl aldehyde to produce alkyldiol (1,3-alkyldiol). UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY), 1,3-propanediol dehydrogenase (gene name dhaT, EC1.1.1.202) or alcohol dehydrogenase (gene name is yqhD, EC1.1.-) .-) and the like ( http://www.chem.qmul.ac.uk/iubmb/ ).

  Specific examples include enzymes that catalyze the production of 1,3-propanediol from 3-hydroxypropionaldehyde (for example, 1,3-propanediol dehydrogenase). Examples of the gene encoding this enzyme or an enzyme that catalyzes a similar reaction include dhaT and yqhD.

  More specifically, an enzyme derived from a bacterium belonging to the genus Klebsiella can be mentioned, for example, a 1,3-propanediol dehydrogenase gene product derived from Klebsiella pneumoniae or the like.

In the present invention, examples of the enzyme having 1,3-propanediol dehydrogenase activity include the enzymes described in any of (a) to (e) below.
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 12;
(B) In the amino acid sequence set forth in SEQ ID NO: 12, one or more (2 or more, preferably 2 to 20, more preferably 2 to 5) amino acids are substituted, deleted, inserted or added. Having an enzyme,
(C) an enzyme having an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 11;
(D) an enzyme having an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence set forth in SEQ ID NO: 11;
(E) a protein having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 12.

  Further, a transformant that functionally expresses a suitable enzyme that catalyzes the reaction represented by the formula 2, a glycerol dehydratase reactivation factor gene or a diol dehydratase reactivation factor (accession No. AF017781 gene), and / or Alternatively, more efficient alkyldiol production can be achieved by simultaneously introducing an adenosyltransferase gene (several types of enzymes classified into EC2.5.1.17).

  More specifically, examples of the glycerol dehydratase reactivating factor gene include enzymes derived from bacteria belonging to the genus Klebsiella, such as a glycerol dehydratase reactivating factor gene derived from Klebsiella pneumoniae. .

In the present invention, examples of the enzyme having glycerol dehydratase reactivation activity include the enzymes described in any of (a) to (e) below.
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 8 or 10,
(B) In the amino acid sequence described in SEQ ID NO: 8 or 10, one or more (two or more, preferably 2 to 20, more preferably 2 to 5) amino acids are substituted, deleted, inserted, or added. An enzyme having an amino acid sequence,
(C) an enzyme having an amino acid sequence encoded by the base sequence described in SEQ ID NO: 7 or 9,
(D) an enzyme having an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 7 or 9;
(E) a protein having 85% or more identity with the amino acid sequence of SEQ ID NO: 8 or 10.

  More specifically, examples of the adenosyltransferase gene include enzymes derived from bacteria belonging to the genus Klebsiella, such as an adenosyltransferase gene derived from Klebsiella oxytoca.

Examples of the enzyme having adenosyltransferase activity in the present invention include the enzymes described in any of (a) to (e) below.
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 14;
(B) In the amino acid sequence set forth in SEQ ID NO: 14, one or more (2 or more, preferably 2 to 20, more preferably 2 to 5) amino acids are substituted, deleted, inserted or added. Having an enzyme,
(C) an enzyme having an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 13;
(D) an enzyme having an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence set forth in SEQ ID NO: 13;
(E) a protein having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 14.

In order to express at least each enzyme in the present invention, a microorganism to be transformed is transformed with a recombinant vector containing a polynucleotide encoding a polypeptide having each enzyme, and expresses each enzyme activity. There is no particular limitation as long as it is a living organism. Examples of the microorganisms that can be used include the following microorganisms.
Host vector system such as Escherichia genus Bacillus genus Pseudomonas genus Serratia genus Brevibacterium genus Corynebacterium genus Streptococcus genus Lactobacillus genus Bacteria Rhodococcus spp. Streptomyces spp. Host vector systems such as Streptomyces spp. Saccharomyces spp. Kluyveromyces spp. Schizosaccharomyces spp. Saccharomyces (Zygosaccharomyces) genus Yarrowia genus Trichosporon genus Rhodosporidium genus Pichia genus Candida genus Yeast neurospora (Neur Ospora, Aspergillus, Cephalosporium, and Trichoderma

  The procedure for producing transformants and the construction of a recombinant vector suitable for the host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Sambrook et al., Molecular cloning, Cold Spring Harbor Laboratories).

  In order to express each enzyme gene of the present invention in a microorganism or the like, first, the DNA of the present invention is introduced into a plasmid vector or a phage vector stably existing in the microorganism, and the genetic information is transcribed and translated. For that purpose, a promoter corresponding to a unit controlling transcription / translation may be incorporated upstream of the 5′-side of the DNA strand of the present invention, more preferably a terminator downstream of the 3′-side. As the promoter and terminator, a promoter and terminator known to function in a microorganism used as a host is used. Regarding vectors, promoters, terminators and the like that can be used in these various microorganisms, for example, “Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Shuppan”, especially regarding yeast, Adv. Biochem. Eng. 43, 75-102 (1990) , Yeast 8, 423-488 (1992), etc.

  In the genus Escherichia, especially Escherichia coli, for example, pBR and pUC plasmids can be used as plasmid vectors, and lac (β-galactosidase), trp (tryptophan operon), tac, trc (lac, trp) Fusion), promoters derived from λ phage PL, PR, etc. can be used. Moreover, as the terminator, a terminator derived from trpA, phage, or rrnB ribosomal RNA can be used.

  In the genus Bacillus, pUB110 series plasmids, pC194 series plasmids and the like can be used as vectors, and can be integrated into chromosomes. Moreover, apr (alkaline protease), npr (neutral protease), amy (α-amylase), or the like can be used as a promoter or terminator.

  In the genus Pseudomonas, host vector systems such as Pseudomonas putida and Pseudomonas cepacia have been developed. A broad host range vector (including genes necessary for autonomous replication derived from RSF1010) pKT240 based on the plasmid TOL plasmid involved in the degradation of toluene compounds can be used. As the promoter or terminator, a lipase (Japanese Patent Laid-Open No. 5-284973) gene or the like can be used.

  In the genus Brevibacterium, especially Brevibacterium lactofermentum, plasmid vectors such as pAJ43 (Gene 39, 281 (1985)) can be used. As the promoter or terminator, promoters and terminators used in E. coli can be used as they are.

  In the genus Corynebacterium, especially Corynebacterium glutamicum, plasmid vectors such as pCS11 (Japanese Patent Laid-Open No. 57-183799) and pCB101 (Mol. Gen. Genet. 196, 175 (1984) are available. is there.

  In the genus Streptococcus, pHV1301 (FEMS Microbiol. Lett. 26, 239 (1985), pGK1 (Appl. Environ. Microbiol. 50, 94 (1985)), etc. can be used as a plasmid vector.

  In the genus Lactobacillus, pAMβ1 (J. Bacteriol. 137, 614 (1979)) developed for Streptococcus can be used, and those used in Escherichia coli as promoters can be used. .

  In the genus Rhodococcus, plasmid vectors isolated from Rhodococcus rhodochrous can be used (J. Gen. Microbiol. 138, 1003 (1992)).

  In the genus Streptomyces, plasmids can be constructed according to the method described in Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory Manual Cold Spring Harbor Laboratories (1985). In particular, in Streptomyces lividans, pIJ486 (Mol. Gen. Genet. 203, 468-478, 1986), pKC1064 (Gene 103,97-99 (1991)), pUWL-KS (Gene 165,149) -150 (1995)) can be used. The same plasmid can also be used in Streptomyces virginiae (Actinomycetol. 11, 46-53 (1997)).

  In the genus Saccharomyces (especially Saccharomyces cerevisiae), YRp, YEp, YCp, and YIp plasmids can be used, and homologous recombination with multiple copies of ribosomal DNA in the chromosome can be performed. The integration vector used (EP 537456, etc.) is extremely useful because it can introduce a gene in multiple copies and can stably hold the gene. ADH (alcohol dehydrogenase), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), PHO (acid phosphatase), GAL (β-galactosidase), PGK (phosphoglycerate kinase), ENO (enolase) And other promoters and terminators are available.

  In Kluyveromyces genus, especially Kluyveromyces lactis, 2μm plasmid derived from Saccharomyces cerevisiae, pKD1 plasmid (J. Bacteriol. 145, 382-390 (1981)), involved in killer activity A plasmid derived from pGKl1, an autonomously proliferating gene KARS plasmid in the genus Kleberomyces, a vector plasmid (EP 537456 etc.) that can be integrated into the chromosome by homologous recombination with ribosomal DNA and the like can be used. In addition, promoters and terminators derived from ADH, PGK and the like can be used.

  In the genus Schizosaccharomyces, plasmid vectors containing ARS (genes involved in autonomous replication) derived from Schizosaccharomyces pombe and selection markers that complement auxotrophy derived from Saccharomyces cerevisiae are available. (Mol. Cell. Biol. 6, 80 (1986)). Moreover, the ADH promoter derived from Schizosaccharomyces pombe can be used (EMBO J. 6, 729 (1987)). In particular, pAUR224 is commercially available from Takara Shuzo and can be easily used.

  In the genus Zygosaccharomyces, plasmid vectors derived from pSB3 (Nucleic Acids Res. 13, 4267 (1985)) derived from Zygosaccharomyces rouxii are available, and PHO5 derived from Saccharomyces cerevisiae Promoters and GAP-Zr (Glyceraldehyde-3-phosphate dehydrogenase) promoters (Agri. Biol. Chem. 54, 2521 (1990)) derived from Tigo Saccharomyces rouxi can be used.

  In the genus Pichia, a host vector system has been developed in Pichia angusta (former name: Hansenula polymorpha). As vectors, genes involved in autonomous replication derived from Pichia Angusta (HARS1, HARS2) can be used, but because they are relatively unstable, multicopy integration into the chromosome is effective (Yeast 7, 431- 443 (1991)). In addition, AOX (alcohol oxidase), FDH (formic acid dehydrogenase) promoters induced by methanol, etc. can be used. In addition, host vector systems using Pichia pastoris and other genes involved in Pichia-derived autonomous replication (PARS1, PARS2) have been developed (Mol. Cell. Biol. 5, 3376 (1985)). Strong promoters such as high concentration culture and methanol-inducible AOX can be used (Nucleic Acids Res. 15, 3859 (1987)).

  In the genus Candida, host vector systems in Candida maltosa, Candida albicans, Candida tropicalis, Candida utilis, etc. Has been developed. In Candida maltosa, ARS derived from Candida maltosa has been cloned (Agri. Biol. Chem. 51, 51, 1587 (1987)), and vectors using this have been developed. In Candida utilis, a strong promoter for a chromosome integration type vector has been developed (Japanese Patent Laid-Open No. 08-173170).

  In the genus Aspergillus, Aspergillus niger, Aspergillus oryzae, etc. are the most studied among molds, and integration into plasmids and chromosomes is possible, Promoters derived from extracellular proteases or amylases are available (Trends in Biotechnology 7, 283-287 (1989)).

  In the genus Trichoderma, a host vector system using Trichoderma reesei has been developed, and an extracellular cellulase gene-derived promoter or the like can be used (Biotechnology 7, 596-603 (1989)).

  In addition to microorganisms, various host and vector systems have been developed in plants and animals, especially in plants such as insects using moths (Nature 315, 592-594 (1985)), rapeseed, corn, or potatoes. A system for expressing a large amount of heterologous protein has been developed and can be suitably used.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to this.

[Example 1] Construction of glycerol dehydratase gene high expression plasmid pSU-KGD1 Genomic DNA was purified from Klebsiella pneumoniae according to the method of Nucleic. Acids Res. 8, 4321 (1980).
Based on the nucleotide sequence of the glycerol dehydratase gene derived from Klebsiella pneumoniae, the corresponding oligonucleotide primer KpGLDA-A1 (5'-gtctcATGAAACGTTCAAAACGTTTTGCAGTACTGGC-3 ', SEQ ID NO: 15) is synthesized from the upstream site of the gldA gene. The corresponding oligonucleotide primer KpGLDC-T1 (5′-gactctagaTTAGCTTCCTTTACGCAGCTTATGACGCTGCTG-3 ′, SEQ ID NO: 16) was synthesized from the downstream site of the gldC gene. Using Klebsiella pneumoniae genomic DNA as a template, 10 pmol of each primer set, 0.2 mM dNTP, AmpliTaq DNA polymerase buffer (PerkinElmer), 2.5 U AmpliTaq DNA polymerase (PerkinElmer) PCR reaction cycle (BIO-RAD) using a 50 μL reaction solution containing 30 cycles of denaturation (94 ° C, 30 seconds), annealing (50 ° C, 30 seconds), extension (72 ° C, 1 minute). PCR was performed using The amplified DNA fragment was double-digested using restriction enzymes BspHI and XbaI, and ligated using Mighty Mix Ligation Kit (manufactured by Takara Bio) to pSE420U (WO 2006-132145) double-digested using NcoI and XbaI. Escherichia coli JM109 was transformed with the ligated DNA, grown in LB medium containing ampicillin (50 mg / L), and the plasmid was purified from the resulting transformant by QIAprep spin Miniprep Kit (manufactured by QIAGEN). As a result, pSU-KGD1, which is a plasmid having a glycerol dehydratase gene, was obtained.

[Example 2] Construction of glycerol dehydratase reactivation factor gene high expression plasmid pSU-KGR1 Upstream site of orf2b (gdrB) gene based on the base sequence of glycerol dehydratase reactivation factor gene derived from Klebsiella pneumoniae And a corresponding oligonucleotide primer KpGDRB-A1 (5′-gaggaattcatacATGTCACTTTCACCTCCAGGTGTACGC-3 ′, SEQ ID NO: 17) and primer KpGDRB-T1 (5′-tttagaattcctctagaTCAGTTTCTCTCACTTAACGGCAGGAC-3 ′, SEQ ID NO: 18), respectively. From the upstream and downstream sites of the dhaB4 (gdrA) gene, the corresponding oligonucleotide primer KpGDRA-A1 (5'-tctagaggaattctaaaATGCCGTTAATAGCCGGGATTGATATC-3 ', SEQ ID NO: 19) and primer KpGDRA-T1 (5'-gacaagcttctagaTTAATTAGCTTGACCAGCCAGTAG SEQ ID NO: 20) was synthesized. Using Klebsiella pneumoniae genomic DNA as a template, 10 pmol of each primer set, 0.2 mM dNTP, AmpliTaq DNA polymerase buffer (PerkinElmer), 2.5 U AmpliTaq DNA polymerase (PerkinElmer) PCR reaction cycle (BIO-RAD) using a 50 μL reaction solution containing 30 cycles of denaturation (94 ° C, 30 seconds), annealing (50 ° C, 30 seconds), extension (72 ° C, 1 minute). And a DNA fragment containing orf2b (gdrB) and dhaB4 (gdrA) was prepared. Furthermore, PCR was similarly performed using the primer sets of Primer KpGdrB-A1 and Primer KpGdrA-T1 using these DNA fragments to obtain DNA fragments containing a glycerol dehydratase reactivation factor gene. The amplified DNA fragment was double-digested using restriction enzymes EcoRI and HindIII, and ligated using the Mighty Mix Ligation Kit (manufactured by Takara Bio Inc.) in the same double-digested pSE420U (WO 2006-132145). Escherichia coli JM109 was transformed with the ligated DNA, grown in LB medium containing ampicillin (50 mg / L), and the plasmid was purified from the resulting transformant by QIAprep spin Miniprep Kit (manufactured by QIAGEN). As a result, pSU-KGR1, which is a plasmid having a glycerol dehydratase reactivation factor gene, was obtained.

[Example 3] Construction of 1,3-propanediol dehydrogenase gene high expression plasmid pSU-KPD1 Based on the nucleotide sequence of 1,3-propanediol dehydrogenase gene derived from Klebsiella pneumoniae, From the downstream site, corresponding oligonucleotide primers KpPDH-A1 (5′-ctgcatATGTCATATCGTATGTTTGATTATCTGGTG-3 ′, SEQ ID NO: 21) and primer KpPDH-T1 (5′-gtcttaaTTAAAATGCTTGACGGAAAATCGCGGCAA-3 ′, SEQ ID NO: 22) were synthesized. Using Klebsiella pneumoniae genomic DNA as a template, 10 pmol of each primer set, 0.2 mM dNTP, AmpliTaq DNA polymerase buffer (PerkinElmer), 2.5 U AmpliTaq DNA polymerase (PerkinElmer) PCR reaction cycle (BIO-RAD) using a 50 μL reaction solution containing 30 cycles of denaturation (94 ° C, 30 seconds), annealing (50 ° C, 30 seconds), extension (72 ° C, 1 minute). PCR was performed using The amplified DNA fragment was double-digested using restriction enzymes NdeI and PacI, and ligated to pSE420U (WO 2006-132145), which was similarly double-digested, using Mighty Mix Ligation Kit (manufactured by Takara Bio). Escherichia coli JM109 was transformed with the ligated DNA, grown in LB medium containing ampicillin (50 mg / L), and the plasmid was purified from the resulting transformant by QIAprep spin Miniprep Kit (manufactured by QIAGEN). As a result, pSU-KPD1, which is a plasmid having a 1,3-propanediol dehydrogenase gene, was obtained.

[Example 4] Construction of glycerol dehydratase and glycerol dehydratase reactivator gene high expression plasmid pSD-KGR1 Plasmid pSU-KGR1 obtained in Example 2 was double digested with restriction enzymes EcoRI and HindIII, and ethanol precipitated. Thereafter, agarose electrophoresis was performed, and a band of about 2.2 bp containing the KpGDR gene was excised and purified and recovered by GFX PCR DNA and Gel Band Purification Kit (manufactured by GE Healthcare). Using the Mighty Mix Ligation Kit (manufactured by Takara Bio), the obtained DNA fragment and the plasmid pSU-KGD1 (Example 1) digested with the same restriction enzymes, phenol extracted, phenol-chloroform extracted, chloroform extracted and ethanol precipitated were used. Ligated. Escherichia coli JM109 was transformed with the ligated DNA, grown in LB medium containing ampicillin (50 mg / L), and the plasmid was purified from the resulting transformant by QIAprep spin Miniprep Kit (manufactured by QIAGEN). As a result, pSD-KGR1, which is a plasmid capable of simultaneously expressing glycerol dehydratase and glycerol dehydratase reactivation factor, was obtained.

[Example 5] Construction of glycerol dehydratase, glycerol dehydratase reactivating factor and 1,3-propanediol dehydrogenase gene high expression plasmid pSP-KGR1 Plasmid pSU-KPD1 obtained in Example 3 was used with restriction enzymes SacI and PacI. Then, after ethanol digestion and agarose electrophoresis, a band of about 1.3 bp containing the KpPDH gene was excised and purified and collected by GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). Using the Mighty Mix Ligation Kit (manufactured by Takara Bio), the obtained DNA fragment and the plasmid pSD-KGR1 (Example 4) digested with the same restriction enzymes, phenol extracted, phenol-chloroform extracted, chloroform extracted and ethanol precipitated were used. Ligated. Escherichia coli JM109 was transformed with the ligated DNA, grown in LB medium containing ampicillin (50 mg / L), and the plasmid was purified from the resulting transformant by QIAprep spin Miniprep Kit (manufactured by QIAGEN). As a result, pSP-KGR1 (FIG. 1), which is a plasmid capable of simultaneously expressing glycerol dehydratase, glycerol dehydratase reactivating factor and 1,3-propanediol dehydrogenase, was obtained.

[Example 6] Construction of high-expression plasmid pMU-KAT1 of adenosyltransferase gene Based on the base sequence of the adenosyltransferase gene derived from Klebsiella oxytoca, the corresponding oligos were respectively detected from the upstream and downstream sites of the pduO gene. The nucleotide primer KoADT-A1 (5′-agtacatATGGCaATTTATACCCGtACGGGCGACGC-3 ′, SEQ ID NO: 23) and the primer KoADT-T1 (5′-atgattaattaaTtATTGATGAGTTCCCACGTTAATGG-3 ′, SEQ ID NO: 24) were synthesized. Using Klebsiella oxytoca genomic DNA as a template, each 10 pmol primer set, 0.2 mM dNTP, AmpliTaq DNA polymerase buffer (Perkin Elmer), 2.5 U AmpliTaq DNA polymerase (Perkin Elmer) PCR reaction cycle (BIO-RAD) using a 50 μL reaction solution containing 30 cycles of denaturation (94 ° C, 30 seconds), annealing (50 ° C, 30 seconds), extension (72 ° C, 1 minute). PCR was performed using

  Oligonucleotide primer SE420U-F (5′-CGTGAGCTATGAGAAGTCGACGTTTTTTGCGCCGACATCATAACG-3 ′, SEQ ID NO: 25) and primer SE420U-R (5′-GGAAAACTGTCCATAACGCGTAAGAGTTTGTAGAAACGCAAAAAGGCCA-3 ′, SEQ ID NO: 26) were synthesized and pSE420U (WO 2006-132) Using 50 μL of each reaction solution containing 10 pmol of each primer set, 0.2 mM dNTP, AmpliTaq DNA polymerase buffer (Perkin Elmer), 2.5 U AmpliTaq DNA polymerase (Perkin Elmer) PCR was performed using denaturation (94 ° C., 30 seconds), annealing (50 ° C., 30 seconds), extension (72 ° C., 1 minute) for 30 cycles and a PCR iCycle thermal cycler (manufactured by BIO-RAD). Furthermore, oligonucleotide primer MW219-F (5′-TTCTCATAGCTCACGCTGTAGGTA-3 ′, SEQ ID NO: 27) and primer MW219-R (5′-TATGGACAGTTTTCCCTTTGATATGTAACG-3 ′, SEQ ID NO: 28) were synthesized and pMW219 (manufactured by Nippon gene) )) As a template, 50 μL of the reaction solution containing 10 pmol of each primer set, 0.2 mM dNTP, AmpliTaq DNA polymerase buffer (Perkin Elmer), 2.5 U AmpliTaq DNA polymerase (Perkin Elmer) Denaturation (94 ° C, 30 seconds), annealing (50 ° C, 30 seconds), extension (72 ° C, 1 minute) for 30 cycles, PCR was performed using PCR iCycle Thermal Cycler (BIO-RAD) . Plasmid pMW219U was constructed by ligating the DNA fragment amplified from pME420U and the DNA fragment amplified using pMW219 as a template.

  A DNA fragment amplified by PCR from the genomic DNA of Klebsiella oxytoca was double-digested using restriction enzymes NdeI and PacI, and ligated using the Mighty Mix Ligation Kit (manufactured by Takara Bio Inc.) to pMW219U that was also double-digested. did. Escherichia coli JM109 was transformed with the ligated DNA, grown in LB medium containing ampicillin (50 mg / L), and the plasmid was purified from the resulting transformant by QIAprep spin Miniprep Kit (manufactured by QIAGEN). As a result, pMU-KAT1 (FIG. 1), which is a plasmid having an adenosyltransferase gene, was obtained.

[Example 7] Production of 1,3-propanediol using E. coli W3110 (pSP-KGR1 / pMU-KAT1) -1
The E. coli W3110 strain transformed with the plasmids pSP-KGR1 and pMU-KAT1 obtained in Examples 5 and 6 was added to a liquid LB medium containing ampicillin (50 mg / L) and kanamycin (50 mg / L). 1.0% bacto-tryptone, 0.5% bacto-yeast extract, 1.0% sodium chloride, pH 7.2) and inoculated overnight at 33 ° C. in a test tube. 14 mL of this cultured microbial cell was added to 1.2 L of M9G medium (60 g / L glycerol, 6. g) containing ampicillin (50 mg / L), kanamycin (50 mg / L), 0.02 mM IPTG, and 20 mg / L CN-Cbl. 8 g / L Na ₂ HPO ₄ , 3.0 g / L KH ₂ PO ₄ , 0.5 g / L NaCl, 1.0 g / L NH ₄ Cl, 0.493 g / L MgSO ₄ .7H ₂ O, 14.7 mg / L CaCl ₂ · 2H ₂ O (pH 7.5) was inoculated, maintained at 33 ° C. and pH 7.5, and cultured under aerobic conditions by stirring at 580 rpm under air aeration at 0.5 vvm. During the culture, when DO (dissolved oxygen) became 0% and OD600 reached about 10, stirring was performed at 200 rpm, air aeration was stopped, and the culture was further continued for a total of 136 hours. Glycerol and 1,3-propanediol in the culture supernatant were analyzed using HPLC under the following conditions. As a result, the residual glycerol was 22.5 g / L, the produced 1,3-propanediol was 6.62 g / L, the glycerol conversion was 62.5%, and the 1,3-propanediol molar yield was 13.4% (FIG. 2). .

HPLC analysis conditions:
Column: Shodex Ionpak KC-811 (8.0 mm x 300 mm) (made by Showa Denko)
Eluent: 10 mM sulfuric acid aqueous solution Flow rate: 0.7 mL / min
Column temperature: 40 ° C
Detection: RI

[Example 8] 1,3-propanediol production using E. coli W3110 (pSP-KGR1 / pMU-KAT1) -2
E. coli W3110 (pSP-KGR1 / pMU-KAT1) obtained by the same method as in Example 7 was added to a liquid LB medium (1.0% Bacto) containing ampicillin (50 mg / L) and kanamycin (50 mg / L). -Tryptone, 0.5% Bacto-yeast extract, 1.0% sodium chloride, pH 7.2) and inoculated in a test tube at 33 ° C overnight. 14 mL of this cultured bacterial cell was inoculated into 1.2 L of M9G medium containing ampicillin (50 mg / L), kanamycin (50 mg / L), 0.02 mM IPTG, 20 mg / L CN-Cbl, Cultivation was started under aerobic conditions by maintaining at pH 7.5 and stirring at 580 rpm under air aeration at 0.5 vvm. During the culture, the agitation was stopped at 200 rpm before and after the DO (dissolved oxygen) became 0%, the air aeration was stopped, and the culture was further continued for a total of 136 hours. In the same manner as in Example 7, glycerol and 1,3-propanediol were analyzed in the supernatant of the culture broth. As a result, the relationship between the redox potential value (ORP value) when stirring was 200 rpm and air aeration was stopped and the generated 1,3-propanediol was shown in FIG. As shown in FIG. 4, 1,3-propanediol was produced at the highest concentration when stirring was 200 rpm with ORP 12 mV and air aeration was stopped, the residual glycerol was 17.6 g / L, and the production 1,3- Propanediol was 13.6 g / L, glycerol conversion was 70.7%, and 1,3-propanediol molar yield was 27.6%.

[Example 9] 1,3-propanediol production using E. coli W3110 (pSP-KGR1 / pMU-KAT1) -3
E. coli W3110 (pSP-KGR1 / pMU-KAT1) obtained by the same method as in Example 7 was added to a liquid LB medium (1.0% Bacto) containing ampicillin (50 mg / L) and kanamycin (50 mg / L). -Tryptone, 0.5% Bacto-yeast extract, 1.0% sodium chloride, pH 7.2) and inoculated in a test tube at 33 ° C overnight. 14 mL of this cultured bacterial cell was inoculated into 1.2 L of M9G medium containing ampicillin (50 mg / L), kanamycin (50 mg / L), 0.02 mM IPTG, 20 mg / L CN-Cbl, Cultivation was started under aerobic conditions by maintaining at pH 7.5 and stirring at 580 rpm under air aeration at 0.5 vvm. Continued cultivation under aerobic conditions (ie, ORP is sufficiently higher than 100 mV) to produce 1,3-propanediol and cultured for 62 hours. As a result, 27.9 g / L of residual glycerol was produced. The 1,3-propanediol was 3.59 g / L, the glycerol conversion was 53.5%, and the 1,3-PD molar yield was 7.24% (FIG. 5A). On the other hand, stirring was carried out at 200 rpm with an ORP of about 20 mV, air aeration was stopped, and the culture was further continued to produce 1,3-propanediol. In the same manner as in Example 7, glycerol and 1,3-propanediol were analyzed in the supernatant of the culture broth. As a result, the residual glycerol was 31.7 g / L, the produced 1,3-propanediol was 5.76 g / L, the glycerol conversion was 47.2%, and the 1,3-PD molar yield was 11.6% (FIG. 5B).

[Example 10] Production of 1,3-propanediol using E. coli W3110 (pSP-KGR1 / pMU-KAT1) -4
E. coli W3110 (pSP-KGR1 / pMU-KAT1) obtained by the same method as in Example 7 was added to a liquid LB medium (1.0% Bacto) containing ampicillin (50 mg / L) and kanamycin (50 mg / L). -Tryptone, 0.5% Bacto-yeast extract, 1.0% sodium chloride, pH 7.2) and inoculated in a test tube at 33 ° C overnight. 14 mL of this cultured bacterial cell was inoculated into 1.2 L of M9G medium containing ampicillin (50 mg / L), kanamycin (50 mg / L), 0.02 mM IPTG, 20 mg / L CN-Cbl, Cultivation was started under aerobic conditions by maintaining at pH 7.5 and stirring at 580 rpm under air aeration at 0.5 vvm. The culture was continued under aerobic conditions, and during the culture, after each DO (dissolved oxygen) reached a predetermined value, the culture was continued while maintaining DO at 2%, 10%, and 20%. In the same manner as in Example 7, glycerol and 1,3-propanediol were analyzed in the supernatant of the culture broth. Table 1 shows the residual glycerol after 86 hours, the produced 1,3-propanediol, the glycerol conversion, and the 1,3-PD molar yield. The ORP was 33 mV when DO was controlled at 2%. On the other hand, ORP when DO was controlled at 10% and 20% was higher than 100 mV.

[Example 11] 1,3-PD production-4 (anaerobic side version) using E. coli W3110 (pSP-KGR1 / pMU-KAT1)
E. coli W3110 (pSP-KGR1 / pMU-KAT1) obtained by the same method as in Example 7 was added to a liquid LB medium (1.0% Bacto) containing ampicillin (50 mg / L) and kanamycin (50 mg / L). -Tryptone, 0.5% Bacto-yeast extract, 1.0% sodium chloride, pH 7.2) and inoculated in a test tube at 33 ° C overnight. 14 mL of this cultured bacterial cell was inoculated into 1.2 L of M9G medium containing ampicillin (50 mg / L), kanamycin (50 mg / L), 0.02 mM IPTG, 20 mg / L CN-Cbl, Cultivation was started under aerobic conditions by maintaining at pH 7.5 and stirring at 580 rpm under air aeration at 0.5 vvm. After culturing for 40 hours under aerobic conditions, the culture was continued while maintaining ORP at -20 mV. In the same manner as in Example 7, glycerol and 1,3-propanediol were analyzed in the supernatant of the culture broth. As a result, growth, glycerol consumption, and 1,3-propanediol production stopped, and the total glycerol was 42.1 g / L, and the resulting 1,3-propanediol was 5.0 g / L. The conversion rate was 29.8%, and the 1,3-PD molar yield was 10.1% (FIG. 6A). On the other hand, after culturing for 40 hours under aerobic conditions, culturing was continued while controlling 2% of DO to adjust ORP to 0-50 mV, and as a result of culturing for a total of 86 hours, glycerol was completely consumed, The produced 1,3-propanediol was 12.0 g / L, the glycerol conversion was 100%, and the 1,3-PD molar yield was 24.2% (FIG. 6B).

Claims (7)

A genetically modified microorganism modified so as to produce an alkyldiol represented by the following formula 1 using a carbon source in a culture solution under a condition that the redox potential value in the culture solution is -10 to 100 mV A method for producing an alkyl diol characterized by comprising culturing in a cell.
[Formula 1]

(In the formula, R represents an alkyl group having 1 to 4 carbon atoms having one or more hydroxyl groups.)   A method for producing an alkyldiol, comprising culturing a genetically modified microorganism under an aerobic condition and then culturing the recombinant microorganism under the condition according to claim 1 to produce an alkyldiol.   When the genetically modified microorganism is cultured under aerobic conditions, and the oxidation-reduction potential in the culture solution is in a range selected from -10 to 100 mV, the microorganisms are cultured under the conditions according to claim 1 after the preculture. A method for producing an alkyl diol characterized by producing an alkyl diol.   The carbon source in the culture solution contains at least one carbon source selected from glycerol, glucose, sucrose, fructose, maltose, mannose, xylose, galactose, acetic acid, citric acid, ethanol, and methanol. The manufacturing method of the alkyldiol in any one of Claims 1-3.   The method for producing an alkyldiol according to any one of claims 1 to 4, wherein the host of the genetically modified microorganism is a microorganism belonging to the genus Escherichia.   6. The method for producing an alkyl diol according to claim 1, wherein the alkyl diol is 1,3-propanediol. The 1,3-gene according to claim 6, wherein the genetically modified microorganism has at least one gene that expresses the protein or enzyme according to any one of (a) to (e) below. A method for producing propanediol;
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14,
(B) an enzyme having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14;
(C) an enzyme having an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13,
(D) an enzyme having an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13; and (E) an enzyme having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14;

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