JP6622564B2 - Method for producing 1,4-butanediol - Google Patents

Description

  The present invention relates to a method for producing 1,4-butanediol using a genetically modified microorganism imparted with the ability to produce 1,4 butanediol. More specifically, the present invention relates to a microorganism imparted with a biosynthetic ability to synthesize 1,4-butanediol from glucose.

  1,4-Butanediol is used as a monomer used in the production of polyesters and polyurethanes represented by polyethylene terephthalate, such as fibers, engineering plastics and films, and as a starting material for the synthesis of cyclic compounds. Tetrahydrofuran and γ-butyrolactone The compound has a wide range of uses such as the production of

  As a method for producing 1,4-butanediol, a method by chemical synthesis is known. Examples include a method for producing 1,4-butanediol by synthesizing 1,4-butynediol from acetylene and hydrogenating it, and a method for producing butadiene from the raw material. Disadvantages are chemical synthesis methods that require high-temperature and high-pressure equipment and depend on fossil resources as raw materials, which may lead to rising raw materials due to resource depletion.

  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. .

  As a biological method using biomass-derived raw materials, a method of producing 1,4-butanediol via a CoA intermediate using a host-derived metabolic route from glucose by a genetically modified microorganism Is known (Patent Document 1).

  However, there have been few reports on the production of 1,4-butanediol from biomass-derived raw materials so far, and even in the method of Patent Document 1, the disadvantage is that it passes through a CoA intermediate through the use of a host-derived metabolic route, There are many reaction steps and production control is expected to be difficult. Therefore, a more efficient method for producing 1,4-butanediol from renewable resources such as new biomass has been desired.

WO2011 / 066076

  An object of the present invention is to provide a microorganism capable of producing 1,4-butanediol using a renewable resource of biomass (plant resource) as a raw material. Another object of the present invention is to provide a method capable of producing 1,4-butanediol using a microorganism having a target function.

（経路２）グルコース−6リン酸からmyo-イノシトール-3リン酸

（経路３）myo-イノシトール-3リン酸からmyo-イノシトール

（経路４）myo-イノシトールからグルクロン酸

（経路５）グルクロン酸からグルカル酸

（経路６）グルカル酸から5-ケト-4-デオキシ-グルカル酸

（経路７）5-ケト-4-デオキシ-グルカル酸から2-オキソグルタル酸セミアルデヒド

（経路８）2-オキソグルタル酸セミアルデヒドから2-オキソ-5-ヒドロキシ-ペンタン酸

（経路９）2-オキソ-5-ヒドロキシ-ペンタン酸から4-ヒドロキシ-ブチルアルデヒド

（経路１０）4-ヒドロキシ-ブチルアルデヒドから1,4-ブタンジオール

。 With the aim of developing a method for producing 1,4-butanediol from biomass, which is a renewable resource, the present inventors have conducted extensive research, and as a result, have found that the glucokinase gene, inositol-3-phosphorus Gene group with acid synthase gene, inositol phosphate phosphatase gene, myo-inositol oxygenase gene, uronic acid dehydrogenase gene, glucaric acid dehydratase gene, 5-keto-4-deoxy-glucaric acid dehydratase gene, alcohol dehydrogenase gene, decarboxylase gene A recombinant microorganism was produced, and 1,4-butanediol was successfully produced from glucose by an artificial metabolic pathway via a keto acid intermediate by fermentation using the genetically modified microorganism. Specifically, it succeeded in producing 1,4-butanediol from glucose through the route shown below.
(Route 1) Glucose to glucose-6 phosphate

(Route 2) Glucose-6 phosphate to myo-inositol-3 phosphate

(Route 3) myo-inositol from myo-inositol-3-phosphate

(Route 4) Myo-inositol to glucuronic acid

(Route 5) Glucuronic acid to glucaric acid

(Route 6) Glucaric acid to 5-keto-4-deoxy-glucaric acid

(Route 7) 5-Keto-4-deoxy-glucaric acid to 2-oxoglutaric acid semialdehyde

(Route 8) 2-oxo-5-hydroxy-pentanoic acid from 2-oxoglutaric acid semialdehyde

(Path 9) 2-Oxo-5-hydroxy-pentanoic acid to 4-hydroxy-butyraldehyde

(Route 10) 1,4-Butanediol from 4-hydroxy-butyraldehyde

.

  Based on the above knowledge, the present inventors have succeeded in producing 1,4-butanediol in a microorganism containing the above enzyme by utilizing an enzyme group which is a metabolite of the microorganism, thereby completing the present invention. It came to do.

〔２〕以下の反応の（経路１）から（経路１０）を触媒する国際酵素分類により特定された各酵素を発現し、グルコースから1,4-ブタンジオールを生成することができる、遺伝子組換え微生物：
（経路１）国際酵素分類においてEC: 2.7.1.-に分類されるグルコキナーゼ、
（経路２）国際酵素分類においてEC:5.5.1.4に分類されるシンターゼ、
（経路３）国際酵素分類においてEC:3.1.3.-に分類されるホスファターゼ、
（経路４）国際酵素分類においてEC:1.13.-.-に分類されるオキシゲナーゼ、
（経路５）国際酵素分類においてEC:1.1.1.-に分類されるデヒドロゲナーゼ又はレダクターゼ、
（経路６）国際酵素分類においてEC:4.2.1.-に分類されるデヒドラターゼ、
（経路７）国際酵素分類においてEC:4.2.1.-に分類されるデヒドラターゼ、
（経路８）国際酵素分類においてEC: 1.1.1.-に分類されるデヒドロゲナーゼ又はレダクターゼ、
（経路９）国際酵素分類においてEC: 4.1.1.-に分類されるデカルボキシラーゼ、及び
（経路１０）国際酵素分類においてEC: 1.1.1.-に分類されるデヒドロゲナーゼ又はレダクターゼ：
[反応]

。
〔３〕各酵素が、以下に特定される、〔２〕記載の遺伝子組換え微生物：
（経路１）国際酵素分類においてEC: 2.7.1.2に分類されるグルコキナーゼ、
（経路２）国際酵素分類においてEC:5.5.1.4に分類されるシンターゼ、
（経路３）国際酵素分類においてEC: 3.1.3.25に分類されるホスファターゼ、
（経路４）国際酵素分類においてEC: 1.13.99.1に分類されるオキシゲナーゼ、
（経路５）国際酵素分類においてEC: 1.1.1.203に分類されるデヒドロゲナーゼ、
（経路６）国際酵素分類においてEC:4.2.1.40に分類されるデヒドラターゼ、
（経路７）国際酵素分類においてEC: 4.2.1.41に分類されるデヒドラターゼ、
（経路８）国際酵素分類においてEC: 1.1.1.-に分類されるデヒドロゲナーゼ又はレダクターゼ、
（経路９）国際酵素分類においてEC: 4.1.1.74に分類されるデカルボキシラーゼ、及び
（経路１０）国際酵素分類においてEC: 1.1.1.-に分類されるデヒドロゲナーゼ又はレダクターゼ。
〔４〕以下の酵素１〜酵素１０を発現する、遺伝子組換え微生物：
グルコキナーゼ活性を有する酵素１が、以下からなる群から選択され、
（ａ）配列番号：1に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：1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からなる酵素、
（ｃ）配列番号：2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からなる酵素、
（ｄ）配列番号：2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からなる酵素、及び、
（ｅ）配列番号：1に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
イノシトール-3-リン酸シンターゼ活性を有する酵素２が、以下からなる群から選択され、
（ａ）配列番号：3に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：3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からなる酵素、
（ｃ）配列番号：4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からなる酵素、
（ｄ）配列番号：4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からなる酵素、及び、
（ｅ）配列番号：3に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
イノシトールリン酸ホスファターゼ活性を有する酵素３が、以下からなる群から選択され、
（ａ）配列番号：5に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：5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からなる酵素、
（ｃ）配列番号：6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からなる酵素、
（ｄ）配列番号：6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からなる酵素、及び、
（ｅ）配列番号：5に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
ミオイノシトールオキシゲナーゼ活性を有する酵素４が、以下からなる群から選択され、
（ａ）配列番号：7に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：7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からなる酵素、
（ｃ）配列番号：8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からなる酵素、
（ｄ）配列番号：8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からなる酵素、及び、
（ｅ）配列番号：7に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
ウロン酸デヒロドゲナーゼ活性を有する酵素５が、以下からなる群から選択され、
（ａ）配列番号：9に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：9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からなる酵素、
（ｃ）配列番号：10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からなる酵素、
（ｄ）配列番号：10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からなる酵素、及び、
（ｅ）配列番号：9に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
グルカル酸デヒドラターゼ活性を有する酵素６が、以下からなる群から選択され、
（ａ）配列番号：11に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：11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からなる酵素、
（ｃ）配列番号：12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からなる酵素、
（ｄ）配列番号：12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からなる酵素、及び、
（ｅ）配列番号：11に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
5-ケト-4-デオキシ-グルカル酸デヒドラターゼ活性を有する酵素７が、以下からなる群から選択され、
（ａ）配列番号：13に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：13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からなる酵素、
（ｃ）配列番号：14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からなる酵素、
（ｄ）配列番号：14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からなる酵素、及び、
（ｅ）配列番号：13に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
アルコールデヒドロゲナーゼ活性を有する酵素８が、以下からなる群から選択され、
（ａ）配列番号：15又は21に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：15又は21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からなる酵素、
（ｃ）配列番号：16又は22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からなる酵素、
（ｄ）配列番号：16又は22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からなる酵素、及び、
（ｅ）配列番号：15又は21に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
デカルボキシラーゼ活性を有する酵素９が、以下からなる群から選択され、
（ａ）配列番号：17又は19に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：17又は19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からなる酵素、
（ｃ）配列番号：18又は20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からなる酵素、
（ｄ）配列番号：18又は20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からなる酵素、又は、
（ｅ）配列番号：17又は19に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
アルコールデヒドロゲナーゼ活性を有する酵素１０が、以下からなる群から選択される：
（ａ）配列番号：15又は21に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：15又は21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からなる酵素、
（ｃ）配列番号：16又は22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からなる酵素、
（ｄ）配列番号：16又は22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からなる酵素、及び、
（ｅ）配列番号：15又は21に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体。
〔５〕微生物が、以下からなる群から選択される、〔１〕〜〔４〕のいずれか記載の微生物：
エシェリヒア（Escherichia）属、バチルス（Bacillus）属、シュードモナス（Pseudomonas）属、セラチア（Serratia）属、ブレビバクテリウム（Brevibacterium）属、コリネバクテリイウム（Corynebacterium）属、ストレプトコッカス（Streptococcus）属、ラクトバチルス（Lactobacillus）属、ロドコッカス（Rhodococcus）属、ストレプトマイセス（Streptomyces）属、サッカロマイセス（Saccharomyces）属、クライベロマイセス（Kluyveromyces）属、シゾサッカロマイセス（Schizosaccharomyces）属、チゴサッカロマイセス（Zygosaccharomyces）属、ヤロウイア（Yarrowia）属、トリコスポロン（Trichosporon）属、ロドスポリジウム（Rhodosporidium）属、ピキア（Pichia）属、キャンディダ（Candida）属、ノイロスポラ（Neurospora）属、アスペルギルス（Aspergillus）属、セファロスポリウム（Cephalosporium）属、及びトリコデルマ（Trichoderma）属。
〔６〕以下の工程からなる、グルコースから1,4-ブタンジオールを生産する方法：
（工程１）グルコースからグルコース−6リン酸を生成する工程：

（工程２）グルコース−6リン酸からmyo-イノシトール-3リン酸を生成する工程：

（工程３）myo-イノシトール-3リン酸からmyo-イノシトールを生成する工程：

（工程４）myo-イノシトールからグルクロン酸を生成する工程：

（工程５）グルクロン酸からグルカル酸を生成する工程：

（工程６）グルカル酸から5-ケト-4-デオキシ-グルカル酸を生成する工程：

（工程７）5-ケト-4-デオキシ-グルカル酸から2-オキソグルタル酸セミアルデヒドを生成する工程：

（工程８）2-オキソグルタル酸セミアルデヒドから2-オキソ-5-ヒドロキシ-ペンタン酸を生成する工程：

（工程９）2-オキソ-5-ヒドロキシ-ペンタン酸から4-ヒドロキシ-ブチルアルデヒドを生成する工程：

（工程１０）4-ヒドロキシ-ブチルアルデヒドから1,4-ブタンジオールを生成する工程：

。
〔７〕〔６〕記載の（工程１）〜（工程１０）の各工程が、以下の酵素により触媒される、グルコースから1,4-ブタンジオールを生産する方法：
（工程１）国際酵素分類においてEC: 2.7.1.-に分類されるグルコキナーゼ、
（工程２）国際酵素分類においてEC:5.5.1.4に分類されるシンターゼ、
（工程３）国際酵素分類においてEC:3.1.3.-に分類されるホスファターゼ、
（工程４）国際酵素分類においてEC:1.13.-.-に分類されるオキシゲナーゼ、
（工程５）国際酵素分類においてEC:1.1.1.-に分類されるデヒドロゲナーゼ又はレダクターゼ、
（工程６）国際酵素分類においてEC:4.2.1.-に分類されるデヒドラターゼ、
（工程７）国際酵素分類においてEC:4.2.1.-に分類されるデヒドラターゼ、
（工程８）国際酵素分類においてEC: 1.1.1.-に分類されるデヒドロゲナーゼ又はレダクターゼ、
（工程９）国際酵素分類においてEC: 4.1.1.-に分類されるデカルボキシラーゼ、及び
（工程１０）国際酵素分類においてEC: 1.1.1.-に分類されるデヒドロゲナーゼ又はレダクターゼ。
〔８〕（工程１）〜（工程１０）の各工程が、以下の酵素により触媒される、〔７〕記載の方法：
（工程１）国際酵素分類においてEC: 2.7.1.2に分類されるグルコキナーゼ、
（工程２）国際酵素分類においてEC:5.5.1.4に分類されるシンターゼ、
（工程３）国際酵素分類においてEC: 3.1.3.25に分類されるホスファターゼ、
（工程４）国際酵素分類においてEC: 1.13.99.1に分類されるオキシゲナーゼ、
（工程５）国際酵素分類においてEC: 1.1.1.203に分類されるデヒドロゲナーゼ、
（工程６）国際酵素分類においてEC:4.2.1.40に分類されるデヒドラターゼ、
（工程７）国際酵素分類においてEC: 4.2.1.41に分類されるデヒドラターゼ、
（工程８）国際酵素分類においてEC: 1.1.1.-に分類されるデヒドロゲナーゼ又はレダクターゼ、
（工程９）国際酵素分類においてEC: 4.1.1.74に分類されるデカルボキシラーゼ、及び
（工程１０）国際酵素分類においてEC: 1.1.1.-に分類されるデヒドロゲナーゼ又はレダクターゼ。
〔９〕（工程１）〜（工程１０）を触媒する各酵素が、以下に特定されるものである、〔６〕記載の方法：
（工程１）を触媒する、グルコキナーゼ活性を有する酵素が、以下からなる群から選択され、
（ａ）配列番号：1に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：1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からなる酵素、
（ｃ）配列番号：2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からなる酵素、
（ｄ）配列番号：2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からなる酵素、及び、
（ｅ）配列番号：1に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
（工程２）を触媒する、イノシトール-3-リン酸シンターゼ活性を有する酵素が、以下からなる群から選択され、
（ａ）配列番号：3に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：3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からなる酵素、
（ｃ）配列番号：4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からなる酵素、
（ｄ）配列番号：4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からなる酵素、及び、
（ｅ）配列番号：3に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
（工程３）を触媒する、イノシトールリン酸ホスファターゼ活性を有する酵素が、以下からなる群から選択され、
（ａ）配列番号：5に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：5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からなる酵素、
（ｃ）配列番号：6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からなる酵素、
（ｄ）配列番号：6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からなる酵素、及び、
（ｅ）配列番号：5に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
（工程４）を触媒する、ミオイノシトールオキシゲナーゼ活性を有する酵素が、以下からなる群から選択され、
（ａ）配列番号：7に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：7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からなる酵素、
（ｃ）配列番号：8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からなる酵素、
（ｄ）配列番号：8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からなる酵素、及び、
（ｅ）配列番号：7に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
（工程５）を触媒する、ウロン酸デヒロドゲナーゼ活性を有する酵素が、以下からなる群から選択され、
（ａ）配列番号：9に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：9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からなる酵素、
（ｃ）配列番号：10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からなる酵素、
（ｄ）配列番号：10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からなる酵素、及び、
（ｅ）配列番号：9に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
（工程６）を触媒する、グルカル酸デヒドラターゼ活性を有する酵素が、以下からなる群から選択され、
（ａ）配列番号：11に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：11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からなる酵素、
（ｃ）配列番号：12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からなる酵素、
（ｄ）配列番号：12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からなる酵素、及び、
（ｅ）配列番号：11に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
（工程７）を触媒する、5-ケト-4-デオキシ-グルカル酸デヒドラターゼ活性を有する酵素が、以下からなる群から選択され、
（ａ）配列番号：13に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：13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からなる酵素、
（ｃ）配列番号：14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からなる酵素、
（ｄ）配列番号：14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からなる酵素、及び、
（ｅ）配列番号：13に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
（工程８）を触媒する、アルコールデヒドロゲナーゼ活性を有する酵素が、以下からなる群から選択され、
（ａ）配列番号：15又は21に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：15又は21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からなる酵素、
（ｃ）配列番号：16又は22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からなる酵素、
（ｄ）配列番号：16又は22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からなる酵素、及び、
（ｅ）配列番号：15又は21に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
（工程９）を触媒する、デカルボキシラーゼ活性を有する酵素が、以下からなる群から選択され、
（ａ）配列番号：17又は19に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：17又は19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からなる酵素、
（ｃ）配列番号：18又は20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からなる酵素、
（ｄ）配列番号：18又は20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からなる酵素、及び、
（ｅ）配列番号：17又は19に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体、
（工程１０）を触媒する、アルコールデヒドロゲナーゼ活性を有する酵素が、以下からなる群から選択される、
（ａ）配列番号：15又は21に記載されたアミノ酸配列からなるタンパク質、
（ｂ）配列番号：15又は21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からなる酵素、
（ｃ）配列番号：16又は22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からなる酵素、
（ｄ）配列番号：16又は22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からなる酵素、及び、
（ｅ）配列番号：15又は21に記載のアミノ酸配列と８５%以上の同一性を有するタンパク質誘導体。
〔１０〕グルコースと〔１〕〜〔５〕のいずれか記載の微生物とを混合し、該微生物を培養する工程を含む、1,4-ブタンジオールを生産する方法。 That is, the present invention provides a 1,4-butanediol-producing microorganism. Alternatively, the present invention provides an efficient production method of 1,4-butanediol using the microorganism, and specifically provides the following [1] to [10].
[1] A genetically modified microorganism capable of expressing each enzyme that catalyzes (Route 1) to (Route 10) of the following reaction and producing 1,4-butanediol from glucose:
(Route 1) Glucokinase,
(Route 2) Inositol-3-phosphate synthase,
(Route 3) Inositol phosphate phosphatase,
(Route 4) Myo-inositol oxygenase,
(Route 5) Uronic acid dehydrogenase,
(Route 6) Glucarate dehydratase,
(Route 7) 5-keto-4-deoxy-glucarate dehydratase,
(Route 8) Alcohol dehydrogenase,
(Route 9) Decarboxylase, and (Route 10) Alcohol dehydrogenase.
[reaction]

[2] Genetic recombination capable of expressing each enzyme specified by the international enzyme classification that catalyzes (Route 1) to (Route 10) of the following reaction and producing 1,4-butanediol from glucose Microorganisms:
(Route 1) Glucokinase classified as EC: 2.7.1.- in the international enzyme classification,
(Route 2) Synthase classified as EC: 5.5.1.4 in the international enzyme classification,
(Route 3) Phosphatase classified as EC: 3.1.3.- in the international enzyme classification,
(Route 4) Oxygenase classified as EC: 1.13 .-.- in the international enzyme classification,
(Route 5) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Route 6) Dehydratase classified as EC: 4.2.1.- in the international enzyme classification,
(Route 7) Dehydratase classified as EC: 4.2.1.- in the international enzyme classification,
(Route 8) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Route 9) Decarboxylase classified as EC: 4.1.1.- in the international enzyme classification, and (Path 10) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification:
[reaction]

.
[3] The genetically modified microorganism according to [2], wherein each enzyme is specified below:
(Route 1) Glucokinase classified as EC: 2.7.1.2 in the international enzyme classification,
(Route 2) Synthase classified as EC: 5.5.1.4 in the international enzyme classification,
(Route 3) Phosphatase classified as EC: 3.1.3.25 in the international enzyme classification,
(Route 4) Oxygenase classified as EC: 1.13.99.1 in the international enzyme classification,
(Route 5) Dehydrogenase classified as EC: 1.1.1.203 in the international enzyme classification,
(Route 6) Dehydratase classified as EC: 4.2.1.40 in the international enzyme classification,
(Route 7) Dehydratase classified as EC: 4.2.1.41 in the international enzyme classification,
(Route 8) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Route 9) Decarboxylase classified as EC: 4.1.1.74 in the international enzyme classification, and (Path 10) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification.
[4] Genetically modified microorganisms that express the following enzymes 1 to 10:
The enzyme 1 having glucokinase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 1;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 2;
(D) an enzyme consisting of 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: 2, and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 1;
The enzyme 2 having inositol-3-phosphate synthase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) in the amino acid sequence set forth in SEQ ID NO: 3, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence described in SEQ ID NO: 4,
(D) an enzyme consisting of 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: 4; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 3;
The enzyme 3 having inositol phosphate phosphatase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 5;
(B) in the amino acid sequence set forth in SEQ ID NO: 5, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 6;
(D) an enzyme consisting of 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: 6; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 5;
The enzyme 4 having myo-inositol oxygenase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 7;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 7;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 8;
(D) an enzyme consisting of 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: 8; and
(E) a protein derivative having 85% or more identity with the amino acid sequence of SEQ ID NO: 7,
The enzyme 5 having uronic acid dehydrogenase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 9;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 9;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 10;
(D) an enzyme consisting of 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: 10; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 9;
The enzyme 6 having glucarate dehydratase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 11;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 11;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 12;
(D) an enzyme comprising an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 12, and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 11;
Enzyme 7 having 5-keto-4-deoxy-glucarate dehydratase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 13;
(B) in the amino acid sequence set forth in SEQ ID NO: 13, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 14;
(D) an enzyme consisting of 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: 14; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 13;
The enzyme 8 having alcohol dehydrogenase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 21,
(B) in the amino acid sequence set forth in SEQ ID NO: 15 or 21, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence described in SEQ ID NO: 16 or 22,
(D) an enzyme consisting of 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: 16 or 22, and
(E) a protein derivative having 85% or more identity with the amino acid sequence of SEQ ID NO: 15 or 21;
The enzyme 9 having decarboxylase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19,
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 17 or 19;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 18 or 20,
(D) an enzyme consisting of 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: 18 or 20, or
(E) a protein derivative having 85% or more identity with the amino acid sequence of SEQ ID NO: 17 or 19,
The enzyme 10 having alcohol dehydrogenase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 21,
(B) in the amino acid sequence set forth in SEQ ID NO: 15 or 21, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence described in SEQ ID NO: 16 or 22,
(D) an enzyme consisting of 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: 16 or 22, and
(E) A protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 15 or 21.
[5] The microorganism according to any one of [1] to [4], wherein the microorganism is selected from the group consisting of:
Escherichia genus, Bacillus genus, Pseudomonas genus, Serratia genus, Brevibacterium genus, Corynebacterium genus, Streptococcus genus, Lactobacillus ( Lactobacillus genus, Rhodococcus genus, Streptomyces genus, Saccharomyces genus, Kluyveromyces genus, Schizosaccharomyces genus, Zygosaccharomyces genus Yarrowia, Trichosporon, Rhodosporidium, Pichia, Candida, Neurospora, Aspergillus, Cephalosporium And Trichoderma (Trichoderma) sp.
[6] A method for producing 1,4-butanediol from glucose, comprising the following steps:
(Step 1) Step of producing glucose-6 phosphate from glucose:

(Step 2) Step of producing myo-inositol-3 phosphate from glucose-6 phosphate:

(Step 3) Step of producing myo-inositol from myo-inositol-3-phosphate:

(Step 4) Step of generating glucuronic acid from myo-inositol:

(Step 5) Step of generating glucaric acid from glucuronic acid:

(Step 6) Step of producing 5-keto-4-deoxy-glucaric acid from glucaric acid:

(Step 7) Step of producing 2-oxoglutaric acid semialdehyde from 5-keto-4-deoxy-glucaric acid:

(Step 8) Step of producing 2-oxo-5-hydroxy-pentanoic acid from 2-oxoglutarate semialdehyde:

(Step 9) Step of producing 4-hydroxy-butyraldehyde from 2-oxo-5-hydroxy-pentanoic acid:

(Step 10) Step of producing 1,4-butanediol from 4-hydroxy-butyraldehyde:

.
[7] A method for producing 1,4-butanediol from glucose, wherein each step of (Step 1) to (Step 10) described in [6] is catalyzed by the following enzyme:
(Process 1) Glucokinase classified as EC: 2.7.1.- in the international enzyme classification,
(Process 2) Synthase classified as EC: 5.5.1.4 in the international enzyme classification,
(Process 3) Phosphatase classified as EC: 3.1.3.- in the international enzyme classification,
(Step 4) Oxygenase classified as EC: 1.13 .-.- in the international enzyme classification,
(Step 5) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Step 6) Dehydratase classified as EC: 4.2.1.- in the international enzyme classification,
(Step 7) Dehydratase classified as EC: 4.2.1.- in the international enzyme classification,
(Step 8) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Step 9) Decarboxylase classified as EC: 4.1.1.- in the international enzyme classification; and (Step 10) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification.
[8] The method according to [7], wherein each step of (Step 1) to (Step 10) is catalyzed by the following enzyme:
(Process 1) Glucokinase classified as EC: 2.7.1.2 in the international enzyme classification,
(Process 2) Synthase classified as EC: 5.5.1.4 in the international enzyme classification,
(Process 3) Phosphatase classified as EC: 3.1.3.25 in the international enzyme classification,
(Process 4) Oxygenase classified in EC: 1.13.99.1 in the international enzyme classification,
(Step 5) Dehydrogenase classified as EC: 1.1.1.203 in the international enzyme classification,
(Step 6) Dehydratase classified as EC: 4.2.1.40 in the international enzyme classification,
(Step 7) Dehydratase classified as EC: 4.2.1.41 in the international enzyme classification,
(Step 8) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Step 9) Decarboxylase classified as EC: 4.1.1.74 in the international enzyme classification, and (Step 10) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification.
[9] The method according to [6], wherein each enzyme that catalyzes (Step 1) to (Step 10) is specified below:
The enzyme having glucokinase activity that catalyzes (Step 1) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 1;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 2;
(D) an enzyme consisting of 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: 2, and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 1;
The enzyme having inositol-3-phosphate synthase activity that catalyzes (step 2) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) in the amino acid sequence set forth in SEQ ID NO: 3, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence described in SEQ ID NO: 4,
(D) an enzyme consisting of 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: 4; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 3;
The enzyme having inositol phosphate phosphatase activity that catalyzes (Step 3) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 5;
(B) in the amino acid sequence set forth in SEQ ID NO: 5, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 6;
(D) an enzyme consisting of 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: 6; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 5;
The enzyme having myo-inositol oxygenase activity that catalyzes (Step 4) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 7;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 7;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 8;
(D) an enzyme consisting of 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: 8; and
(E) a protein derivative having 85% or more identity with the amino acid sequence of SEQ ID NO: 7,
The enzyme having uronic acid dehydrogenase activity that catalyzes (Step 5) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 9;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 9;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 10;
(D) an enzyme consisting of 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: 10; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 9;
The enzyme having glucarate dehydratase activity that catalyzes (Step 6) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 11;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 11;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 12;
(D) an enzyme comprising an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 12, and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 11;
The enzyme having 5-keto-4-deoxy-glucarate dehydratase activity that catalyzes (Step 7) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 13;
(B) in the amino acid sequence set forth in SEQ ID NO: 13, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 14;
(D) an enzyme consisting of 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: 14; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 13;
The enzyme having alcohol dehydrogenase activity that catalyzes (Step 8) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 21,
(B) in the amino acid sequence set forth in SEQ ID NO: 15 or 21, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence described in SEQ ID NO: 16 or 22,
(D) an enzyme consisting of 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: 16 or 22, and
(E) a protein derivative having 85% or more identity with the amino acid sequence of SEQ ID NO: 15 or 21;
The enzyme having decarboxylase activity that catalyzes (Step 9) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19,
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 17 or 19;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 18 or 20,
(D) an enzyme consisting of 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: 18 or 20, and
(E) a protein derivative having 85% or more identity with the amino acid sequence of SEQ ID NO: 17 or 19,
The enzyme having alcohol dehydrogenase activity that catalyzes (step 10) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 21,
(B) in the amino acid sequence set forth in SEQ ID NO: 15 or 21, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence described in SEQ ID NO: 16 or 22,
(D) an enzyme consisting of 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: 16 or 22, and
(E) A protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 15 or 21.
[10] A method for producing 1,4-butanediol, comprising a step of mixing glucose and the microorganism according to any one of [1] to [5] and culturing the microorganism.

  In the above [3] and [8], (route 8) and (route 10) and (step 8) and (step 10) may be either dehydrogenase or reductase or both. This is because the enzyme responsible for the pathway has sufficient activity to be able to advance the aldehyde reduction reaction. Therefore, in the said path | route, this invention can be implemented by either (or both) of a dehydrogenase or a reductase. Therefore, it is not necessary to specify the international enzyme classification to the minor classification.

  According to the present invention, a genetically modified microorganism capable of efficiently producing 1,4-butanediol from a carbohydrate raw material such as glucose which is a raw material derived from a renewable resource is provided. Further, a method for producing 1,4-butanediol using the microorganism was provided. The method of the present invention is industrially advantageous in that 1,4-butanediol can be produced using a less expensive raw material. According to the present invention, 1,4-butanediol can be produced from renewable resources.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a series of enzyme reactions for producing 1,4-butanediol from glucose as a raw material via inositol in the present invention. It is a figure which shows 1, 4- butanediol production using a crude enzyme liquid. It is a figure which shows the 1, 4- butanediol production by the microorganisms of this invention.

  The present invention includes inositol-3-phosphate synthase gene, inositol phosphate phosphatase gene, myo-inositol oxygenase gene, uronic acid dehydrogenase gene, glucaric acid dehydratase gene, 5-keto-4-deoxy-glucaric acid dehydratase gene, alcohol dehydrogenase gene This can be solved by preparing a transformant carrying the decarboxylase gene.

。 The present invention provides a genetically modified microorganism capable of expressing each enzyme that catalyzes (Route 1) to (Route 10) of the following reaction and producing 1,4-butanediol from glucose:
(Route 1) Glucokinase,
(Route 2) Inositol-3-phosphate synthase,
(Route 3) Inositol phosphate phosphatase,
(Route 4) Myo-inositol oxygenase,
(Route 5) Uronic acid dehydrogenase,
(Route 6) Glucarate dehydratase,
(Route 7) 5-keto-4-deoxy-glucarate dehydratase,
(Route 8) Alcohol dehydrogenase,
(Route 9) Decarboxylase, and (Route 10) Alcohol dehydrogenase:
[reaction]

.

。 Further, the present invention expresses each enzyme specified by the international enzyme classification catalyzing (Route 1) to (Route 10) of the following reaction, and can produce 1,4-butanediol from glucose. Provide a genetically modified microorganism:
(Route 1) Glucokinase classified as EC: 2.7.1.- in the international enzyme classification,
(Route 2) Synthase classified as EC: 5.5.1.4 in the international enzyme classification,
(Route 3) Phosphatase classified as EC: 3.1.3.- in the international enzyme classification,
(Route 4) Oxygenase classified as EC: 1.13 .-.- in the international enzyme classification,
(Route 5) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Route 6) Dehydratase classified as EC: 4.2.1.- in the international enzyme classification,
(Route 7) Dehydratase classified as EC: 4.2.1.- in the international enzyme classification,
(Route 8) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Route 9) Decarboxylase classified as EC: 4.1.1.- in the international enzyme classification, and (Path 10) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification:
[reaction]

.

In a more preferred embodiment, the microorganism of the present invention is a genetically modified microorganism, wherein each enzyme is specified below:
(Route 1) Glucokinase classified as EC: 2.7.1.2 in the international enzyme classification,
(Route 2) Synthase classified as EC: 5.5.1.4 in the international enzyme classification,
(Route 3) Phosphatase classified as EC: 3.1.3.25 in the international enzyme classification,
(Route 4) Oxygenase classified as EC: 1.13.99.1 in the international enzyme classification,
(Route 5) Dehydrogenase classified as EC: 1.1.1.203 in the international enzyme classification,
(Route 6) Dehydratase classified as EC: 4.2.1.40 in the international enzyme classification,
(Route 7) Dehydratase classified as EC: 4.2.1.41 in the international enzyme classification,
(Route 8) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Route 9) Decarboxylase classified as EC: 4.1.1.74 in the international enzyme classification, and (Path 10) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification.

Furthermore, as one aspect of the present invention, a recombinant microorganism that expresses the following enzymes 1 to 10 is provided:
The enzyme 1 having glucokinase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 1;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 2;
(D) an enzyme consisting of 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: 2, and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 1;
The enzyme 2 having inositol-3-phosphate synthase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) in the amino acid sequence set forth in SEQ ID NO: 3, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence described in SEQ ID NO: 4,
(D) an enzyme consisting of 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: 4; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 3;
The enzyme 3 having inositol phosphate phosphatase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 5;
(B) in the amino acid sequence set forth in SEQ ID NO: 5, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 6;
(D) an enzyme consisting of 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: 6; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 5;
The enzyme 4 having myo-inositol oxygenase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 7;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 7;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 8;
(D) an enzyme consisting of 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: 8; and
(E) a protein derivative having 85% or more identity with the amino acid sequence of SEQ ID NO: 7,
The enzyme 5 having uronic acid dehydrogenase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 9;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 9;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 10;
(D) an enzyme consisting of 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: 10; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 9;
The enzyme 6 having glucarate dehydratase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 11;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 11;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 12;
(D) an enzyme comprising an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 12, and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 11;
Enzyme 7 having 5-keto-4-deoxy-glucarate dehydratase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 13;
(B) in the amino acid sequence set forth in SEQ ID NO: 13, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 14;
(D) an enzyme consisting of 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: 14; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 13;
The enzyme 8 having alcohol dehydrogenase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 21,
(B) in the amino acid sequence set forth in SEQ ID NO: 15 or 21, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence described in SEQ ID NO: 16 or 22,
(D) an enzyme consisting of 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: 16 or 22, and
(E) a protein derivative having 85% or more identity with the amino acid sequence of SEQ ID NO: 15 or 21;
The enzyme 9 having decarboxylase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19,
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 17 or 19;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 18 or 20,
(D) an enzyme consisting of 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: 18 or 20, or
(E) a protein derivative having 85% or more identity with the amino acid sequence of SEQ ID NO: 17 or 19,
The enzyme 10 having alcohol dehydrogenase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 21,
(B) in the amino acid sequence set forth in SEQ ID NO: 15 or 21, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence described in SEQ ID NO: 16 or 22,
(D) an enzyme consisting of 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: 16 or 22, and
(E) A protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 15 or 21.

The transformant of the present invention can be prepared using various host microbial cells. In particular, using a prokaryotic microorganism as a host makes it possible to construct a new 1,4-butanediol biosynthetic pathway in the host cell, which is very attractive in the application of synthetic biological techniques. In order to express 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 can express each enzyme activity. If it is a living thing, there will be no restriction | limiting in particular. Examples of usable microorganisms 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 Molds that have been developed for host vector systems such as 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”, particularly regarding yeast, Adv. Biochem. Eng. 43, 75-102 (1990), Yeast 8, 423-488 (1992). In the genus Escherichia, particularly Escherichia coli, for example, plasmids such as pBR and pUC can be used, 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, Rhodococcus
Plasmid vectors isolated from rhodochrous are available (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 is possible. 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. 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.

  As a fermentable substrate used as a raw material in the method for producing 1,4-butanediol according to the present invention, in addition to sugars such as glucose, microorganisms such as other sugars and glycerol can catabolize and metabolize depending on the host microorganism used. Any metabolite that can produce myo-inositol, glucaric acid and the like can be preferably 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.

  Although there is no restriction | limiting in particular in the density | concentration of the fermentable substrate which is a raw material for producing 1, 4- butanediol, Usually, about 0.1-30%, Preferably it is 0.5-15%, More preferably, it is 1-10. % Concentration is used.

  In the present invention, “%” means “weight / volume (w / v)”.

  The raw materials can be added all at once at the start of fermentation, but can also be added continuously or intermittently to the fermentation broth.

  In addition, the genetically modified microorganism of the present invention can be preferably used a genetically modified Escherichia coli whose activity is enhanced using Escherichia coli as a host after isolating an enzyme gene that catalyzes the reaction represented by Formula 1. . 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 1,4-butanediol production. it can.

  The genetically modified microorganism of the present invention used for 1,4-butanediol production produces 1,4-butanediol while growing in a 1,4-butanediol production fermentation solution containing the following fermentable substrate. It is also possible to use a culture solution that has been cultured and proliferated in advance, or a recovered microbial cell. The amount of the culture solution that has been cultured and grown in advance or the amount of the collected cells is usually 0.1 to 100%, preferably 1 to 100% of the fermentation solution for producing 1,4-butanediol containing a fermentable substrate. Can be used in an amount of about 0.5 to 50%, more preferably about 1 to 20% (here, “%” indicates the inoculation rate with respect to 1,4-butanediol-producing fermentation broth, and “[[ Preliminarily grown culture solution] / [fermented solution for producing 1,4-butanediol] (v / v) ”).

  In addition to the fermentable substrate for producing 1,4-butanediol, fermented liquor for 1,4-butanediol production includes those that promote the production of 1,4-butanediol as necessary. Preferably, it may contain a medium component that serves as a nutrient source for the recombinant E. coli used for this purpose. 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), M9 medium (6.8 g / L Na2HPO4, 3.0 g / L KH2PO4, 0.5 g / L) NaCl, 1.0 g / L NH4Cl, 0.493 g / L MgSO4 · 7H2O, 14.7 mg / L CaCl2 · 2H2O, pH 7.5). When a sufficient amount of cells are cultured in advance and used in the fermentation liquid for 1,4-butanediol production, 1,4-butanediol can be produced in the presence of a higher concentration of fermentable substrate, The medium can also be removed. Further, if necessary, components for maintaining the pH during fermentation at a pH suitable for 1,4-butanediol production, such as a buffering agent, 10 mM to 800 mM, preferably 50 to 500 mM, more preferably 100 to 250 mM. Specific examples include MOPS buffer, HEPES buffer, MES buffer, Tris buffer, phosphate buffer, and the like. The fermentation temperature is a temperature at which the genetically modified microorganism of the present invention can express the ability to assimilate the fermentable substrate, can express the enzyme activity that catalyzes the reaction represented by Formula 1, and can produce 1,4-butanediol. What is necessary is just to be 5-60 degreeC normally, Preferably it is 10-50 degreeC, More preferably, it can carry out at 20-40 degreeC. The pH may be any pH as long as the enzyme activity catalyzing the reaction represented by the formula 1 can be expressed and 1,4-butanediol can be generated. Usually, pH 4 to 12, preferably pH 5 to 11, more preferably. It can be carried out at pH 6-9. Moreover, fermentation can be performed under stirring or standing still. In order to more efficiently convert the fermentable substrate into 1,4-butanediol, aerobic conditions with a sufficient amount of oxygen supplied, microaerobic conditions with a limited oxygen supply, or The reaction can be performed in a reaction medium under anaerobic conditions in which oxygen is not supplied.

  The production of 1,4-butanediol of the present invention can be carried out in water or an organic solvent that is difficult to dissolve in water, such as ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, methyl isobutyl ketone, methyl tertiary butyl ether, diisopropyl ether. Or a two-phase system with an aqueous medium, or a mixed system with an organic solvent that dissolves in water, for example, methanol, ethanol, isopropyl alcohol, acetonitrile, acetone, dimethyl sulfoxide, or 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 1,4-butanediol produced by the reaction includes separation by centrifugation or filtration, extraction by organic solvent, various chromatography such as ion exchange chromatography, adsorption by adsorbent, flocculant, dehydration by dehydrating agent or Aggregation, crystallization, distillation, and the like can be combined as appropriate.

  For example, fermentation liquid containing microbial cells is removed by centrifugation, membrane filtration, etc., microbial cells are removed, proteins are removed, and 1,4-butanediol is purified from the aqueous solution by known methods such as concentration and distillation. can do.

Examples of the enzyme shown in FIG. 1 of the present invention include the enzymes described in any of (a) to (e) below.
(A) a protein having an amino acid sequence described in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21;
(B) In the amino acid sequence according to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, one or more (two or more, preferably 2 to 20, More preferably, an enzyme having an amino acid sequence in which 2 to 5) amino acids are substituted, deleted, inserted or added (c) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, A protein having 85% or more identity with the amino acid sequence of any one of 19 and 21

  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 2-5) 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 (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. 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.

  Specific examples of “stringent conditions” for hybridization in the present invention include 6 × SSC, 0.5% (W / V) SDS, 100 μg / ml denatured salmon sperm DNA, 5 × Denhardt's solution (1 X Denhardt's solution in a solution containing 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% ficoll) at 45 ° C, preferably 55 ° C, more preferably 60 ° C, more preferably 65 ° C overnight. The conditions are such that hybridization is performed, and washing after hybridization is performed 3 times at 4 × 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, 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 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.

Method for measuring glucokinase activity-1
After equilibrating a composition solution consisting of 100 mM potassium phosphate buffer (pH 7.5), 10 mM glucose, 5 mM magnesium chloride, 0.3 mM ATP, 0.3 mM NADP for 3 minutes at 25 ° C., glucokinase and glucose-6-phosphate Add cell-free extract containing dehydrogenase. Glucose is phosphorylated by glucokinase to produce glucose-6-phosphate, and 6-phospho-glucono-1,5-lactone is produced by glucose-6-phosphate dehydrogenase. Measure the increase in absorbance at 340 nm with increasing NADPH in the oxidation of glucose-6-phosphate. The amount of enzyme that catalyzes the increase of 1 μmol NADPH per minute can be 1 U. The protein is quantified by a dye binding method using Biovine protein assay kit using Bovine serum albumin as a standard protein.

Inositol-3-phosphate synthase activity measurement method-2
1 ml 50 mM TrisHCl buffer (pH 8.0), 5 mM glucose 6 phosphate, 0.2 mM DTT, 2 mM NH ₄ Cl, 1 mM NAD ⁺ The cell extract is added and allowed to react at 37 ° C. for 60 minutes, and 100 μl 20% trichloroacetic acid is added to stop the enzyme reaction. Centrifuge at 10000xg for 15 minutes at 4 ° C and collect the supernatant. Mix 0.5 ml supernatant solution and 0.1 ml 0.2 M NaIO ₄ and react at 37 ° C. for 60 minutes. By this operation, glucose 6-phosphate is oxidized and phosphate is liberated. Enzymatic activity is determined by measuring free phosphate using a malachite green phosphate assay kit manufactured by BioAssay system. Mix according to the instructions in the kit and measure the absorbance at 620 nm of the dye. A calibration curve is prepared from a 0.1 mM KH ₂ PO ₄ standard solution and quantified. The amount of enzyme that catalyzes 1 μmol of substrate per minute can be 1 U. To avoid fading, these operations should be performed within 2 hours after staining.

Inositol phosphate phosphatase (SuhB) activity measurement method-3
1 ml 50 mM TrisHCl buffer (pH 8.0), 0.7 mM d-myo-inositol-1-phosphate, 10 mM MgCl ₂ , 250 mM KCl equilibrated at 37 ° C for 3 minutes, and cell extract containing SuhB Is added and allowed to react at 37 ° C for 2 hours, and 100 µl 20% trichloroacetic acid is added to stop the enzyme reaction. Centrifuge at 10000xg for 15 minutes at 4 ℃ and collect the supernatant. As with Ino1, the enzyme activity is measured for free phosphate using a malachite green phosphate assay kit manufactured by BioAssay system. Mix according to the instructions in the kit and measure the absorbance at 620 nm of the dye. A calibration curve is prepared from a 0.1 mM KH ₂ PO ₄ standard solution and quantified. The amount of enzyme that catalyzes 1 μmol of substrate per minute can be 1 U. To avoid fading, these operations should be performed within 2 hours after staining.

Myoinositol oxygenase (MIOX) activity measurement method-4
A composition consisting of 50 mM TrisHCl buffer (pH 8.0), 2 mM L-cysteine, 1 mM Fe (NH ₄ ) (SO ₄ ) ₂ , 60 mM myo-inositol is equilibrated at 30 ° C. for 10 minutes and contains MIOX The cell extract is added and reacted at 30 ° C. for 60 minutes, and 1/10 amount of 20% trichloroacetic acid is added to stop the enzyme reaction. Centrifuge at 10000xg for 15 minutes at 4 ℃ and collect the supernatant. The activity is measured by mixing the sample and orcinol solution = 1: 2, reacting at 98 ° C. for 30 minutes, cooling at room temperature, and measuring the increase in absorbance at 670 nm. A calibration curve is prepared from a standard solution of known concentration of glucuronic acid and quantified. The amount of enzyme that catalyzes the increase of 1 μmol of NADH per minute can be 1 U.
Orcinol solution composition:
10 ml 5 M HC1 mixed with 40 mg orcinol and 5.4 mg FeCl ₃

Uronic acid dehydrogenase (Udh) activity assay-5
A composition solution consisting of 100 mM potassium phosphate buffer (pH 8.0), 10 mM glucuronic acid, and 1 mM NAD ⁺ was equilibrated at 25 ° C. for 3 minutes, and then a cell-free extract containing uronic acid dehydrogenase was added. Measure the increase in absorbance at 340 nm with increasing NADH in oxidation. The amount of enzyme that catalyzes the increase of 1 μmol of NADH per minute can be 1 U. The protein is quantified by a dye binding method using Biovine protein assay kit using Bovine serum albumin as a standard protein.

Glucarate Dehydratase (GudD) Activity Measurement Method-6
A composition solution consisting of 100 mM potassium phosphate buffer (pH 7.5), 10 mM glucaric acid, 5 mM magnesium chloride was equilibrated at 30 ° C. for 3 minutes, and then a cell-free extract containing glucaric acid dehydratase was added to Dehydration produces 5-keto-4-deoxy-glucaric acid. After the reaction, an equal amount of 1% sodium acetate / 1% semicarbazide hydrochloride solution is added to the composition solution, and the mixture is kept at 30 ° C. for 1 hour to form a semicarbazone derivative, and the increase in absorbance at 250 nm is measured. The amount of enzyme that catalyzes the increase of 1 μmol of NADH per minute can be 1 U. The protein is quantified by a dye binding method using Biovine protein assay kit using Bovine serum albumin as a standard protein.

5-Keto-4-deoxy-glucarate dehydratase (KdgD) activity assay-7
A composition solution consisting of 100 mM potassium phosphate buffer (pH 7.5), 10 mM 5-keto-4-deoxy-glucaric acid, 5 mM magnesium chloride, 0.3 mM NADPH was equilibrated at 25 ° C. for 3 minutes, and then 5-keto- A cell-free extract containing 4-deoxy-glucarate dehydratase and alcohol dehydrogenase (yjgB) is added. 5-keto-4-deoxy-glucaric acid dehydratase dehydrates 5-keto-4-deoxy-glucaric acid to produce 2-oxoglutaric acid semialdehyde, which is reduced by alcohol dehydrogenase to give 2-oxo-5-hydroxy- Pentanoic acid is produced. Measure the decrease in absorbance at 340 nm with NADPH reduction in the reduction of 2-oxoglutarate semialdehyde. The amount of enzyme that catalyzes the increase of 1 μmol of NADH per minute can be 1 U. The protein is quantified by a dye binding method using Biovine protein assay kit using Bovine serum albumin as a standard protein.

Alcohol dehydrogenase (YjgB) activity measurement method-8
A composition solution consisting of 100 mM potassium phosphate buffer (pH 7.5), 5 mM pentanal, and 0.3 mM NADPH is equilibrated at 25 ° C. for 3 minutes, and then a cell-free extract containing alcohol dehydrogenase is added to the NADPH by reduction of pentanal. Measure the increase in absorbance at 340 nm with increasing. The amount of enzyme that catalyzes the increase of 1 μmol of NADH per minute can be 1 U. The protein is quantified by a dye binding method using Biovine protein assay kit using Bovine serum albumin as a standard protein.

Decarboxylase (KivD / KdcA) activity measurement method-9
After equilibrating a composition solution consisting of 100 mM potassium phosphate buffer (pH 6.5), 10 mM 3-methyl-2-oxobutyric acid, 5 mM magnesium chloride, 1 mM thiamine pyrophosphate, 0.3 mM NADPH for 3 minutes at 25 ° C., A cell-free extract containing carboxylase and alcohol dehydrogenase (yjgB) is added. Decarboxylase decarboxylates 3-methyl-2-oxobutyric acid to produce 2-methyl-propanal, and reduction with alcohol dehydrogenase produces 2-methyl-propanol. Measure the decrease in absorbance at 340 nm with the decrease in NADPH in the reduction of 2-methyl-propanal. The amount of enzyme that catalyzes the increase of 1 μmol of NADH per minute can be 1 U. The protein is quantified by a dye binding method using Biovine protein assay kit using Bovine serum albumin as a standard protein.

Alcohol dehydrogenase (YqhD) activity measurement method-10
A composition solution consisting of 100 mM potassium phosphate buffer (pH 7.5), 5 mM butanal, 0.3 mM NADPH was equilibrated at 25 ° C. for 3 minutes. Measure the increase in absorbance at 340 nm with increasing. The amount of enzyme that catalyzes the increase of 1 μmol of NADH per minute can be 1 U. The protein is quantified by a dye binding method using Biovine protein assay kit using Bovine serum albumin as a standard protein.

（工程２）グルコース−6リン酸からmyo-イノシトール-3リン酸を生成する工程：

（工程３）myo-イノシトール-3リン酸からmyo-イノシトールを生成する工程：

（工程４）myo-イノシトールからグルクロン酸を生成する工程：

（工程５）グルクロン酸からグルカル酸を生成する工程：

（工程６）グルカル酸から5-ケト-4-デオキシ-グルカル酸を生成する工程：

（工程７）5-ケト-4-デオキシ-グルカル酸から2-オキソグルタル酸セミアルデヒドを生成する工程：

（工程８）2-オキソグルタル酸セミアルデヒドから2-オキソ-5-ヒドロキシ-ペンタン酸を生成する工程：

（工程９）2-オキソ-5-ヒドロキシ-ペンタン酸から4-ヒドロキシ-ブチルアルデヒドを生成する工程：

（工程１０）4-ヒドロキシ-ブチルアルデヒドから1,4-ブタンジオールを生成する工程：

。 The present invention also provides a method for producing 1,4-butanediol from glucose, comprising the following steps:
(Step 1) Step of producing glucose-6 phosphate from glucose:

(Step 2) Step of producing myo-inositol-3 phosphate from glucose-6 phosphate:

(Step 3) Step of producing myo-inositol from myo-inositol-3-phosphate:

(Step 4) Step of generating glucuronic acid from myo-inositol:

(Step 5) Step of generating glucaric acid from glucuronic acid:

(Step 6) Step of producing 5-keto-4-deoxy-glucaric acid from glucaric acid:

(Step 7) Step of producing 2-oxoglutaric acid semialdehyde from 5-keto-4-deoxy-glucaric acid:

(Step 8) Step of producing 2-oxo-5-hydroxy-pentanoic acid from 2-oxoglutarate semialdehyde:

(Step 9) Step of producing 4-hydroxy-butyraldehyde from 2-oxo-5-hydroxy-pentanoic acid:

(Step 10) Step of producing 1,4-butanediol from 4-hydroxy-butyraldehyde:

.

Furthermore, the present invention provides a method for producing 1,4-butanediol from glucose, in which each of the above-mentioned (Step 1) to (Step 10) is catalyzed by the following enzyme:
(Process 1) Glucokinase classified as EC: 2.7.1.- in the international enzyme classification,
(Process 2) Synthase classified as EC: 5.5.1.4 in the international enzyme classification,
(Process 3) Phosphatase classified as EC: 3.1.3.- in the international enzyme classification,
(Step 4) Oxygenase classified as EC: 1.13 .-.- in the international enzyme classification,
(Step 5) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Step 6) Dehydratase classified as EC: 4.2.1.- in the international enzyme classification,
(Step 7) Dehydratase classified as EC: 4.2.1.- in the international enzyme classification,
(Step 8) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Step 9) Decarboxylase classified as EC: 4.1.1.- in the international enzyme classification; and (Step 10) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification.

In a more preferred embodiment of the present invention, each of the steps (step 1) to (step 10) described above is catalyzed by the following enzymes:
(Process 1) Glucokinase classified as EC: 2.7.1.2 in the international enzyme classification,
(Process 2) Synthase classified as EC: 5.5.1.4 in the international enzyme classification,
(Process 3) Phosphatase classified as EC: 3.1.3.25 in the international enzyme classification,
(Process 4) Oxygenase classified in EC: 1.13.99.1 in the international enzyme classification,
(Step 5) Dehydrogenase classified as EC: 1.1.1.203 in the international enzyme classification,
(Step 6) Dehydratase classified as EC: 4.2.1.40 in the international enzyme classification,
(Step 7) Dehydratase classified as EC: 4.2.1.41 in the international enzyme classification,
(Step 8) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Step 9) Decarboxylase classified as EC: 4.1.1.74 in the international enzyme classification, and (Step 10) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification.

In addition, as an aspect of the present invention, an aspect in which each enzyme that catalyzes (Step 1) to (Step 10) is an enzyme specified below:
The enzyme having glucokinase activity that catalyzes (Step 1) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 1;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 2;
(D) an enzyme consisting of 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: 2, and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 1;
The enzyme having inositol-3-phosphate synthase activity that catalyzes (step 2) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) in the amino acid sequence set forth in SEQ ID NO: 3, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence described in SEQ ID NO: 4,
(D) an enzyme consisting of 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: 4; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 3;
The enzyme having inositol phosphate phosphatase activity that catalyzes (Step 3) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 5;
(B) in the amino acid sequence set forth in SEQ ID NO: 5, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 6;
(D) an enzyme consisting of 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: 6; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 5;
The enzyme having myo-inositol oxygenase activity that catalyzes (Step 4) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 7;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 7;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 8;
(D) an enzyme consisting of 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: 8; and
(E) a protein derivative having 85% or more identity with the amino acid sequence of SEQ ID NO: 7,
The enzyme having uronic acid dehydrogenase activity that catalyzes (Step 5) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 9;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 9;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 10;
(D) an enzyme consisting of 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: 10; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 9;
The enzyme having glucarate dehydratase activity that catalyzes (Step 6) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 11;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 11;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 12;
(D) an enzyme comprising an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA comprising the base sequence set forth in SEQ ID NO: 12, and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 11;
The enzyme having 5-keto-4-deoxy-glucarate dehydratase activity that catalyzes (Step 7) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 13;
(B) in the amino acid sequence set forth in SEQ ID NO: 13, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 14;
(D) an enzyme consisting of 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: 14; and
(E) a protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 13;
The enzyme having alcohol dehydrogenase activity that catalyzes (Step 8) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 21,
(B) in the amino acid sequence set forth in SEQ ID NO: 15 or 21, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence described in SEQ ID NO: 16 or 22,
(D) an enzyme consisting of 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: 16 or 22, and
(E) a protein derivative having 85% or more identity with the amino acid sequence of SEQ ID NO: 15 or 21;
The enzyme having decarboxylase activity that catalyzes (Step 9) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19,
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 17 or 19;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 18 or 20,
(D) an enzyme consisting of 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: 18 or 20, and
(E) a protein derivative having 85% or more identity with the amino acid sequence of SEQ ID NO: 17 or 19,
The enzyme having alcohol dehydrogenase activity that catalyzes (step 10) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 21,
(B) in the amino acid sequence set forth in SEQ ID NO: 15 or 21, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence described in SEQ ID NO: 16 or 22,
(D) an enzyme consisting of 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: 16 or 22, and
(E) A protein derivative having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 15 or 21.

  The present invention also provides a method for producing 1,4-butanediol, which comprises the steps of mixing glucose and the microorganism of the present invention and culturing the microorganism.

  The concentration of the fermentable substrate such as glucose that is a raw material for producing 1,4-butanediol is not particularly limited, but is usually about 0.1 to 30%, preferably 0.5 to 15%, more preferably A concentration of 1-10% can be used.

  The raw materials can be added all at once at the start of fermentation, but can also be added continuously or intermittently to the fermentation broth.

  In addition, the genetically modified microorganism of the present invention is preferably a genetically modified Escherichia coli whose activity is enhanced using Escherichia coli as a host after isolating the enzyme gene that catalyzes the reaction shown in FIG. . 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 1,4-butanediol production. it can.

  The genetically modified microorganism of the present invention used for 1,4-butanediol production produces 1,4-butanediol while growing in a 1,4-butanediol production fermentation solution containing the following fermentable substrate. It is also possible to use a culture solution that has been cultured and proliferated in advance, or a recovered microbial cell. The amount of the culture solution that has been cultured and grown in advance or the amount of the collected cells is usually 0.1 to 100%, preferably 1 to 100% of the fermentation solution for producing 1,4-butanediol containing a fermentable substrate. Can be used in an amount of about 0.5 to 50%, more preferably about 1 to 20% (here, “%” indicates the inoculation rate with respect to 1,4-butanediol-producing fermentation broth, and “[[ Preliminarily grown culture solution] / [fermented solution for producing 1,4-butanediol] (v / v) ”).

  In addition to the fermentable substrate for producing 1,4-butanediol, fermented liquor for 1,4-butanediol production includes those that promote the production of 1,4-butanediol as necessary. Preferably, it may contain a medium component that serves as a nutrient source for the recombinant E. coli used for this purpose. 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), M9 medium (6.8 g / L Na2HPO4, 3.0 g / L KH2PO4, 0.5 g / L) NaCl, 1.0 g / L NH4Cl, 0.493 g / L MgSO4 · 7H2O, 14.7 mg / L CaCl2 · 2H2O, pH 7.5). When a sufficient amount of cells are cultured in advance and used for 1,4-butanediol-producing fermentation broth, 1,4-butanediol can be produced in the presence of a higher concentration of fermentable substrate, The medium can also be removed. Further, if necessary, components for maintaining the pH during fermentation at a pH suitable for 1,4-butanediol production, for example, a buffering agent of 10 mM to 800 mM, preferably 50 to 500 mM, more preferably 100 to 250 mM. Specific examples include MOPS buffer, HEPES buffer, MES buffer, Tris buffer, phosphate buffer, and the like. The fermentation temperature is a temperature at which the genetically modified microorganism of the present invention can express the ability to assimilate the fermentable substrate, can express the enzyme activity that catalyzes the reaction represented by Formula 1, and can produce 1,4-butanediol. What is necessary is just to be 5-60 degreeC normally, Preferably it is 10-50 degreeC, More preferably, it can carry out at 20-40 degreeC. The pH may be any pH as long as the enzyme activity catalyzing the reaction represented by the formula 1 can be expressed and 1,4-butanediol can be generated. Usually, pH 4 to 12, preferably pH 5 to 11, more preferably. It can be carried out at pH 6-9. Moreover, fermentation can be performed under stirring or standing still. In order to more efficiently convert the fermentable substrate into 1,4-butanediol, aerobic conditions with a sufficient amount of oxygen supplied, microaerobic conditions with a limited oxygen supply, or The reaction can be performed in a reaction medium under anaerobic conditions in which oxygen is not supplied.

  The production of 1,4-butanediol of the present invention can be carried out in water or an organic solvent that is difficult to dissolve in water, for example, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, methyl isobutyl ketone, methyl tertiary butyl ether, diisopropyl ether. Or a two-phase system with an aqueous medium, or a mixed system with an organic solvent that dissolves in water, for example, methanol, ethanol, isopropyl alcohol, acetonitrile, acetone, dimethyl sulfoxide, or 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 1,4-butanediol produced by the reaction includes separation by centrifugation or filtration, extraction by organic solvent, various chromatography such as ion exchange chromatography, adsorption by adsorbent, flocculant, dehydration by dehydrating agent or Aggregation, crystallization, distillation, and the like can be combined as appropriate.

  For example, fermentation liquid containing microbial cells is removed by centrifugation, membrane filtration, etc., microbial cells are removed, proteins are removed, and 1,4-butanediol is purified from the aqueous solution by known methods such as concentration and distillation. can do.

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

Example 1: Construction of a plasmid
1) Construction of inositol-3-phosphate synthase gene expression plasmid pSUSCIN1 Based on the amino acid sequence of Saccharomyces cerevisiae inositol-3-phosphate synthase gene (SEQ ID NO: 3), Escherichia coli JM109 The base sequence (SEQ ID NO: 4) of the ino1 gene optimized for expression of the strain was artificially synthesized and introduced into the NdeI site and the XhoI site of pSE420U (WO 2006-132145). Escherichia coli JM109 strain transformed with the obtained pSUSCIN1 plasmid was grown in LB medium containing ampicillin (50 mg / L) and protein expression was performed using the lac operon expression system. High expression of -3-phosphate synthase was confirmed.

2) Construction of inositol phosphate phosphatase gene expression plasmid pSUECSB1 Based on the amino acid sequence of the inositol phosphate phosphatase gene derived from Eschericha coli (SEQ ID NO: 5), it was optimized for the expression of E. coli (Eschericha coli JM109 strain). The base sequence of the suhB gene (SEQ ID NO: 6) was artificially synthesized and introduced into the NdeI site and XhoI site of pSE420U (WO 2006-132145). E. coli strain JM109 transformed with the obtained pSUECSB1 plasmid was grown in LB medium containing ampicillin (50 mg / L), and protein expression was performed using the lac operon expression system.Inositol in the soluble fraction of the E. coli High expression of phosphate phosphatase was confirmed.

3) Construction of myo-inositol oxygenase gene expression plasmid pSUECSB1 miox optimized for expression of Escherichia coli JM109 in the amino acid sequence (SEQ ID NO: 7) of the mouse (Mus musculus) myo-inositol oxygenase gene The base sequence of the gene (SEQ ID NO: 8) was artificially synthesized and introduced into the NdeI site and XhoI site of pSE420U (WO 2006-132145). The obtained pSUECSB1 plasmid transformed E. coli strain JM109 was grown in LB medium containing ampicillin (50 mg / L) and protein expression was performed using the lac operon expression system. High expression of inositol oxygenase was confirmed.

4) Construction of uronic acid dehydrogenase gene expression plasmid pSUAUDH1 Based on the amino acid sequence of uronate dehydrogenase derived from Agrobacterium tumefaciens (SEQ ID NO: 9), PCR was performed from the genome of Agrobacterium tumefaciens (agrobacterium tumefaciens). The base sequence of the udh gene (SEQ ID NO: 10) was synthesized and introduced into the EcoRI site and HindIII site of pSE420U (WO 2006-132145). E. coli strain JM109 transformed with the obtained pSUAUDH1 plasmid was grown in LB medium containing ampicillin (50 mg / L), and protein expression was performed using the lac operon expression system. High expression of acid dehydrogenase was confirmed.

5) Construction of glucaric acid dehydratase gene expression plasmid pSUECGD1 Based on the amino acid sequence of the glucaric acid dehydratase gene derived from Escherichia coli (SEQ ID NO: 11), the gudD gene was cloned from the genome of Escherichia coli W3110 by PCR. A base sequence (SEQ ID NO: 12) was synthesized and introduced into the EcoRI site and HindIII site of pSE420U (WO 2006-132145). Escherichia coli JM109 transformed with the obtained pSUECGD1 plasmid was grown in LB medium containing ampicillin (50 mg / L), and protein expression was performed using the lac operon expression system. High expression of acid dehydratase was confirmed.

6) Construction of 5-keto-4-deoxy-glucarate dehydratase gene expression plasmid pSUABKD1 Based on the amino acid sequence (SEQ ID NO: 13) of 5-keto-4-deoxy-glucarate dehydratase gene derived from Acinetobacter baylyi A kdgD base sequence (SEQ ID NO: 14) optimized for expression of E. coli (Eschericha coli JM109) was artificially synthesized and introduced into the NdeI site and XhoI site of pSE420U (WO 2006-132145). Escherichia coli JM109 transformed with the obtained pSUABKD1 plasmid was grown in LB medium containing ampicillin (50 mg / L), and protein expression was performed using the lac operon expression system. High expression of -keto-4-deoxy-glucarate dehydratase was confirmed.

7) Construction of the alcohol dehydrogenase gene expression plasmid pSUECYB1 Based on the amino acid sequence of the alcohol dehydrogenase gene from Escherichia coli (SEQ ID NO: 15), the base sequence of the yjgB gene from the genome of Escherichia coli W3110 by PCR. (SEQ ID NO: 16) was synthesized and introduced into the EcoRI site and HindIII site of pSE420U (WO 2006-132145). Escherichia coli JM109 strain transformed with the obtained pSUECYB1 plasmid was grown in LB medium containing ampicillin (50 mg / L), and protein expression was performed using the lac operon expression system. High expression of dehydrogenase was confirmed.

8) Construction of decarboxylase gene expression plasmids pSULLDC1 and pSULLKA1 Based on the amino acid sequence (SEQ ID NO: 17 and 19) of the decarboxylase gene (kivD, kdcA gene) derived from Lactococcus lactis, Escherichia coli JM109 strain ) Artificially synthesized base sequences (SEQ ID NOs: 18, 20) of the kivD gene (for pSULLDC1) or kdcA gene (for pSULLKA1) optimized for the expression of pSE420U (WO 2006-132145) NdeI site and PacI site Introduced. E. coli strain JM109 transformed with the obtained pSULLDC1 and pSULLKA1 plasmids was grown in LB medium containing ampicillin (50 mg / L), and protein expression was performed using the lac operon expression system. The activity was confirmed by measuring the enzyme activity used.

9) Alcohol dehydrogenase gene expression plasmid pSU-EPD1
Based on the amino acid sequence of the alcohol dehydrogenase gene derived from Escherichia coli (SEQ ID NO: 21), the base sequence of the yjgB gene (SEQ ID NO: 22) was synthesized from the genome of Escherichia coli W3110 by PCR. It was introduced into the NdeI site and PacI site of pSE420U (WO 2006-132145). Escherichia coli JM109 strain transformed with the obtained pSU-EPD1 plasmid was grown in LB medium containing ampicillin (50 mg / L), and protein expression was performed using the lac operon expression system. High expression of alcohol dehydrogenase was confirmed.

  Since Escherichia coli JM109 strain endogenously expresses glucokinase (the amino acid sequence is shown in SEQ ID NO: 1 and the base sequence is shown in SEQ ID NO: 2), a plasmid for transformation was not prepared.

Example 2: Production of 1,4-butanediol by enzymatic reaction E. coli (JM109 strain) transformed with each plasmid obtained in Example 1 was added to a liquid LB medium (1.0%) containing ampicillin (50 mg / L). Tryptone, 0.5% yeast extract, 1.0% sodium chloride, pH 7.2) was inoculated and cultured in a test tube at 30 ° C. overnight. Inoculate 1.2 mL of each cultured cell in a fermenter containing 1.2 L of liquid LB medium (1.0% tryptone, 0.5% yeast extract, 1.0% sodium chloride, pH 7.2) containing ampicillin (50 mg / L). Then, the culture was started under aerobic conditions by maintaining at 30 ° C. and pH 7.2, and stirring at 600 rpm under air aeration at 0.5 vvm. After the culture, when OD600 reached about 0.4, 2.4 mL of 50 mM isopropyl-β-thiogalactopyranoside (IPTG) was added, and the operation of the fermenter was stopped 24 hours after the start of the culture.

  After stopping the operation, the cells were collected from 300 mL of the culture solution, dissolved in 5 mL of 50 mM KPB (pH 7.5), and disrupted with a sealed ultrasonic crusher (Run 30 seconds, Stop 60 seconds, Cycle 16 ). The disrupted solution was centrifuged, and the supernatant was extracted as a crude enzyme solution.

  Based on each enzyme solution prepared above, the following reaction solution was prepared, an enzyme reaction at 30 ° C. was performed, 200 μl of the reaction solution was periodically sampled, and the 6-fold diluted reaction solution was analyzed by HPLC.

<HPLC分析条件>
カラム：ULTRON PS-80H (8.0 mm×300 mm) (信和化工製)
溶離液：0.1% ギ酸水溶液
流速：0.7 mL/min
カラム温度：40℃
検出：RI / UV (210nm)

<HPLC analysis conditions>
Column: ULTRON PS-80H (8.0 mm x 300 mm) (manufactured by Shinwa Kako)
Eluent: 0.1% formic acid aqueous solution Flow rate: 0.7 mL / min
Column temperature: 40 ° C
Detection: RI / UV (210nm)

  The peak of 1,4-butanediol was confirmed from the 6th hour after the start of the enzyme reaction, and it was confirmed that the maximum accumulation of 1,4-butanediol concentration was 14.8 mM (FIG. 2).

Example 3: Production of 1,4-butanediol by enzymatic reaction E. coli (JM109 strain) transformed with a plasmid ligated with the enzyme group obtained in Example 1 was transformed into liquid LB containing ampicillin (50 mg / L). The medium (1.0% tryptone, 0.5% yeast extract, 1.0% sodium chloride, pH 7.2) was inoculated and cultured in a test tube at 37 ° C. with shaking overnight. 0.5 mL of each cultured cell is added to 10 ml of liquid DM medium (0.2% glucose, 0.7% K ₂ HPO ₄ , 0.3% KH ₂ PO ₄ , 0.1% (NH ₄ ) SO ₄ , containing ampicillin (50 mg / L), 0.01% MgSO ₄ , pH 7.2) +1 ml / L Inoculated into a test tube mixed with tap water, and cultured under aerobic conditions by shaking at 37 ° C. After culturing, when OD ₆₆₀ reaches about 0.8, isopropyl-β-thiogalactopyranoside (IPTG) is added to a final concentration of 0.1 mM, periodically sampled, and the filtered reaction solution is subjected to HPLC. And analyzed.
<HPLC analysis conditions>
Column: ULTRON PS-80H (8.0 mm x 300 mm) (manufactured by Shinwa Processing)
Eluent: 0.1% formic acid aqueous solution Flow rate: 0.7 mL / min
Column temperature: 40 ° C
Detection: RI / UV (210nm)

  Direct production of 1,4-butanediol from glucose was confirmed, and it was confirmed that accumulation up to a maximum 1,4-butanediol concentration of 1.8 mM was achieved under the current culture conditions (FIG. 3).

  The method of the present invention is industrially advantageous in that 1,4-butanediol can be produced without depending on depletion of fossil resources and rising raw materials. According to the present invention, 1,4-butanediol can be produced from renewable resources.

Claims (7)

A genetically modified microorganism capable of expressing each enzyme that catalyzes (Route 1) to (Route 10) of the following reaction and producing 1,4-butanediol from glucose:
(Route 1) Glucokinase,
(Route 2) Inositol-3-phosphate synthase,
(Route 3) Inositol phosphate phosphatase,
(Route 4) Myo-inositol oxygenase,
(Route 5) Uronic acid dehydrogenase,
(Route 6) Glucarate dehydratase,
(Route 7) 5-keto-4-deoxy-glucarate dehydratase,
(Route 8) Alcohol dehydrogenase,
(Route 9) Decarboxylase, and (Route 10) Alcohol dehydrogenase:
[reaction]

. A genetically modified microorganism capable of expressing each enzyme specified by the international enzyme classification catalyzing (Route 1) to (Route 10) of the following reaction and producing 1,4-butanediol from glucose:
(Route 1) Glucokinase classified as EC: 2.7.1.2 in the international enzyme classification,
(Route 2) Synthase classified as EC: 5.5.1.4 in the international enzyme classification,
(Route 3) Phosphatase classified as EC: 3.1.3.25 in the international enzyme classification,
(Route 4) Oxygenase classified as EC: 1.13.99.1 in the international enzyme classification,
(Route 5) Dehydrogenase classified as EC: 1.1.1.203 in the international enzyme classification,
(Route 6) Dehydratase classified as EC: 4.2.1.40 in the international enzyme classification,
(Route 7) Dehydratase classified as EC: 4.2.1.41 in the international enzyme classification,
(Route 8) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Route 9) Decarboxylase classified as EC: 4.1.1.74 in the international enzyme classification, and
(Route 10) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification:
[reaction]

. A genetically modified microorganism that expresses the following enzymes 1 to 10:
The enzyme 1 having glucokinase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 1;
(C) an enzyme consisting of an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 2, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence of SEQ ID NO: 1,
The enzyme 2 having inositol-3-phosphate synthase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) in the amino acid sequence set forth in SEQ ID NO: 3, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme consisting of an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 4, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 3;
The enzyme 3 having inositol phosphate phosphatase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 5;
(B) in the amino acid sequence set forth in SEQ ID NO: 5, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 6, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 5,
The enzyme 4 having myo-inositol oxygenase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 7;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 7;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 8, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 7;
The enzyme 5 having uronic acid dehydrogenase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 9;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 9;
(C) an enzyme consisting of an amino acid sequence encoded by the base sequence described in SEQ ID NO: 10, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 9;
The enzyme 6 having glucarate dehydratase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 11;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 11;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 12, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 11;
Enzyme 7 having 5-keto-4-deoxy-glucarate dehydratase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 13;
(B) in the amino acid sequence set forth in SEQ ID NO: 13, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme consisting of an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 14, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 13;
The enzyme 8 having alcohol dehydrogenase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 21,
(B) in the amino acid sequence set forth in SEQ ID NO: 15 or 21, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 16 or 22, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence of SEQ ID NO: 15 or 21;
The enzyme 9 having decarboxylase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19,
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 17 or 19;
(C) an enzyme consisting of an amino acid sequence encoded by the base sequence described in SEQ ID NO: 18 or 20, or
( D ) a protein derivative having 90 % or more identity with the amino acid sequence of SEQ ID NO: 17 or 19,
The enzyme 10 having alcohol dehydrogenase activity is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 21,
(B) in the amino acid sequence set forth in SEQ ID NO: 15 or 21, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 16 or 22, and
( D ) A protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 15 or 21. The microorganism according to any one of claims 1 to 3 , wherein the microorganism is selected from the group consisting of:
Escherichia genus, Bacillus genus, Pseudomonas genus, Serratia genus, Brevibacterium genus, Corynebacterium genus, Streptococcus genus, Lactobacillus ( Lactobacillus genus, Rhodococcus genus, Streptomyces genus, Saccharomyces genus, Kluyveromyces genus, Schizosaccharomyces genus, Zygosaccharomyces genus Yarrowia, Trichosporon, Rhodosporidium, Pichia, Candida, Neurospora, Aspergillus, Cephalosporium And Trichoderma (Trichoderma) sp. (Step 1) Step of producing glucose-6 phosphate from glucose:

(Step 2) Step of producing myo-inositol-3 phosphate from glucose-6 phosphate:

(Step 3) Step of producing myo-inositol from myo-inositol-3-phosphate:

(Step 4) Step of generating glucuronic acid from myo-inositol:

(Step 5) Step of generating glucaric acid from glucuronic acid:

(Step 6) Step of producing 5-keto-4-deoxy-glucaric acid from glucaric acid:

(Step 7) Step of producing 2-oxoglutaric acid semialdehyde from 5-keto-4-deoxy-glucaric acid:

(Step 8) Step of producing 2-oxo-5-hydroxy-pentanoic acid from 2-oxoglutarate semialdehyde:

(Step 9) Step of producing 4-hydroxy-butyraldehyde from 2-oxo-5-hydroxy-pentanoic acid:

(Step 10) Step of producing 1,4-butanediol from 4-hydroxy-butyraldehyde:

A process for producing 1,4-butanediol from glucose, wherein each step of (Step 1) to (Step 10) is catalyzed by the following enzymes:
(Process 1) Glucokinase classified as EC: 2.7.1.2 in the international enzyme classification,
(Process 2) Synthase classified as EC: 5.5.1.4 in the international enzyme classification,
(Process 3) Phosphatase classified as EC: 3.1.3.25 in the international enzyme classification,
(Process 4) Oxygenase classified in EC: 1.13.99.1 in the international enzyme classification,
(Step 5) Dehydrogenase classified as EC: 1.1.1.203 in the international enzyme classification,
(Step 6) Dehydratase classified as EC: 4.2.1.40 in the international enzyme classification,
(Step 7) Dehydratase classified as EC: 4.2.1.41 in the international enzyme classification,
(Step 8) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification,
(Step 9) Decarboxylase classified as EC: 4.1.1.74 in the international enzyme classification, and
(Step 10) Dehydrogenase or reductase classified as EC: 1.1.1.- in the international enzyme classification . The method according to claim 5 , wherein each enzyme that catalyzes (Step 1) to (Step 10) is specified as follows:
The enzyme having glucokinase activity that catalyzes (Step 1) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 1;
(C) an enzyme consisting of an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 2, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence of SEQ ID NO: 1,
The enzyme having inositol-3-phosphate synthase activity that catalyzes (step 2) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 3;
(B) in the amino acid sequence set forth in SEQ ID NO: 3, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme consisting of an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 4, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 3;
The enzyme having inositol phosphate phosphatase activity that catalyzes (Step 3) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 5;
(B) in the amino acid sequence set forth in SEQ ID NO: 5, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 6, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 5,
The enzyme having myo-inositol oxygenase activity that catalyzes (Step 4) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 7;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 7;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 8, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 7;
The enzyme having uronic acid dehydrogenase activity that catalyzes (Step 5) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 9;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 9;
(C) an enzyme consisting of an amino acid sequence encoded by the base sequence described in SEQ ID NO: 10, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 9;
The enzyme having glucarate dehydratase activity that catalyzes (Step 6) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 11;
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 11;
(C) an enzyme comprising an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 12, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 11;
The enzyme having 5-keto-4-deoxy-glucarate dehydratase activity that catalyzes (Step 7) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 13;
(B) in the amino acid sequence set forth in SEQ ID NO: 13, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme consisting of an amino acid sequence encoded by the base sequence set forth in SEQ ID NO: 14, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 13;
The enzyme having alcohol dehydrogenase activity that catalyzes (Step 8) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 21,
(B) in the amino acid sequence set forth in SEQ ID NO: 15 or 21, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 16 or 22, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence of SEQ ID NO: 15 or 21;
The enzyme having decarboxylase activity that catalyzes (Step 9) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19,
(B) an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 17 or 19;
(C) an enzyme comprising an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 18 or 20, and
( D ) a protein derivative having 90 % or more identity with the amino acid sequence of SEQ ID NO: 17 or 19,
The enzyme having alcohol dehydrogenase activity that catalyzes (step 10) is selected from the group consisting of:
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 21,
(B) in the amino acid sequence set forth in SEQ ID NO: 15 or 21, an enzyme comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added;
(C) an enzyme comprising an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 16 or 22, and
( D ) A protein derivative having 90 % or more identity with the amino acid sequence set forth in SEQ ID NO: 15 or 21. A method for producing 1,4-butanediol, comprising a step of mixing glucose and the microorganism according to any one of claims 1 to 4 and culturing the microorganism.

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