JP4603171B2 - (S) -4-Halo-3-hydroxybutyric acid ester-encoding gene useful for enzyme, method for obtaining the same, and method for producing optically active alcohol using the same - Google Patents

Description

【００６２】
［実施例３］（4-ハロアセト酢酸エステル還元酵素遺伝子のコア領域のクローニング）
実施例２により増幅されたDNA断片を、フェノール−クロロホルム抽出後、エタノール沈殿によりDNAを回収した。得られたDNAをEcoRI, HindIIIにより2重消化し、アガロースゲル電気泳動により精製した。得られたDNA断片を、同じ制限酵素EcoRI, HindIIIにより制限酵素消化したpUC18とライゲーションし、目的とする550bp の挿入断片を有するプラスミドpBstKR-1を得た。
【００６３】
［実施例４］（4-ハロアセト酢酸エステル還元酵素遺伝子のコア領域の塩基配列の解析）
実施例３により得られたプラスミドpBstKR-1の挿入断片の塩基配列を解析した。DNA塩基配列の解析には、BigDye Terminator Cycle Sequencing ready Reaction Kit(アプライドバイオシステムズ製)を用いてPCRを行い、PRISM 310 Genetic Analyzer(アプライドバイオシステムズ製)により行った。決定されたコア領域の塩基配列を配列番号：５として示した。
【００６４】
［実施例５］（4-ハロアセト酢酸エステル還元酵素遺伝子のコア領域周辺のInverse PCR）
実施例１で調製した染色体DNAをHindIIIで消化し、自己環化反応後、SalI 消化して鋳型として用いた。まず、1st PCRとして、BST-N1NE（配列番号：６）、BST-C1NE（配列番号：８）をプライマーとしてPCRを行い、得られたPCR産物を精製後更に、BST-N2NE（配列番号：７）、BST-C2NE（配列番号：９）をプライマーとして2nd PCRを行った。PCRは、熱変性94℃, 1分、アニール55℃1分、伸長72℃3分のサイクルを30サイクル行った。
【００６５】
PCR終了後、反応液の一部をアガロース電気泳動した結果、特異的な約5kbの増幅断片が確認できた。

【００６６】
［実施例６］（4-ハロアセト酢酸エステル還元酵素遺伝子のコア周辺領域の塩基配列の解析）
Inverse PCRで得られたDNA断片の塩基配列を直接解析した。その結果、β-ケトアシルACP還元酵素 (fabG遺伝子) の全域が含まれており、同時に、fabG遺伝子の上流にアシルＣｏＡ脱水素酵素（fabD遺伝子）及び下流にアシルキャリアープロテイン（ACP）遺伝子の一部が見いだされた。
得られた4-ハロアセト酢酸エステル還元酵素遺伝子は、762bpからなり、254アミノ酸をコードしていた。ORF部分の配列を配列番号：１に示した。
【００６７】
［実施例７］（4-ハロアセト酢酸エステル還元酵素遺伝子発現プラスミドの構築）
実施例６で得られた4-ハロアセト酢酸エステル還元酵素のORFの塩基配列を元に、大腸菌における発現プラスミド構築のためのプライマーFABG-E（配列番号：１０）、FABG-H（配列番号：１１）を合成した。0.2mM dNTP、5%ジメチルスルホキシド(DMSO)、プライマーを各10pmol、染色体DNA 50ng、ExTaq用緩衝液、ExTaq 2.5Uを含む50μLの反応液を用い、変性（94℃1分）、アニール（55℃1分）、伸長（72℃3分）を１サイクルとして30サイクル行った。反応には、GeneAmp PCR System 2400 (パーキンエルマー製)を用いた。

得られたPCR産物をEcoRI, HindIIIで2重消化し、pTrc99A (ファルマシア製) のEcoRI, HindIII間にサブクローニングした。得られたプラスミドをpTrc-Bstとした。
pTrc-Bstのプラスミドマップを図２に示した。
【００６８】
［実施例８］（組換え4-ハロアセト酢酸エステル還元酵素の大腸菌による生産）
pTrc-Bstで形質転換した大腸菌JM109株を50mg/Lアンピシリンを含むLB培地（バクトトリプトン10g、バクト酵母エキス5g、塩化ナトリウム10g/1L、pH7.2）に植菌し、37℃で終夜培養した。約1日後に50mg/Lのアンピシリン及び119 mg/LのIPTG（イソプロピルチオガラクトピラノシド）を含む100mLのLB液体培地に植菌し、37℃で16〜20時間培養した。
【００６９】
培養した菌体を遠心分離により回収し、0.01% 2-メルカプトエタノールを含む100mMリン酸カリウム緩衝液(pH7.4) 3mLに懸濁して、超音波（30sec破砕、30sec
intervalを20回繰り返す）により菌体破砕した。
遠心分離により上清を得た後、0.01% 2-メルカプトエタノールを含む10mMリン酸カリウム緩衝液(pH7.4)に透析し、無細胞抽出液として利用した。
【００７０】
［実施例９］（組換え4-ハロアセト酢酸エステル還元酵素の熱処理と電気泳動）
実施例８で調製した無細胞抽出液を(lane 3), 無細胞抽出液30℃で10分熱処理したもの(lane 4)、30℃で20分処理したもの(lane 5)、30℃で40分処理したもの(lane 6)、60℃で10分処理したもの(lane 7)、60℃で20分処理したもの(lane 8)、60℃で40分処理したもの(lane 9)並びにベクターpTrc99Aのみで形質転換したJM109株より抽出した無細胞抽出液(lane 2)と分子量マーカー(lane 1)をドデシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳動(SDS-PAGE)した。
【００７１】
図３に示したように、ベクターpTrc99Aで形質転換した菌由来の無細胞抽出液には見られない27kDaのバンド（図３中ではBstKR1として示した）がpTrc-Bstで形質転換した大腸菌に由来する無細胞抽出液には見られた。この分子量は、DNA配列から予想されるアミノ酸配列からの計算値27kDaとよく一致した。
また、中等度好熱菌由来の4-ハロアセト酢酸エステル還元酵素のもつ耐熱性を利用して、熱処理のみで極めて効率的に酵素の精製が行えることが示された。
【００７２】
［実施例１０］（組換え4-ハロアセト酢酸エステル還元酵素の基質特異性）
実施例８で調製した組換え4-ハロアセト酢酸エステル還元酵素を用いて種々の基質に対する活性を測定した。補酵素としては、還元型ニコチンアミドアデニンジヌクレオチドリン酸（ＮＡＤＰＨ）のみを電子供与体として利用し、還元型ニコチンアミドアデニンジヌクレオチド（ＮＡＤＨ）は利用できなかった。還元反応の基質としては、４−クロロアセト酢酸エチルを効率的に還元したが、大腸菌や枯草菌のβ−ケトアシルACP還元酵素の還元基質となるアセトアセチル-CoAを全く還元しなかった。また、酸化型ニコチンアミドアデニンジヌクレオチドリン酸（ＮＡＤＰ^＋）を電子受容体とした（Ｒ）もしくは（Ｓ）−４−クロロ−３−ヒドロキシ酪酸エチルエステル酸化活性は全く有しなかった。
【００７３】
［実施例１１］（組換え4-ハロアセト酢酸エステル還元酵素の至適温度）
実施例８で調製した4-ハロアセト酢酸エステル還元酵素を用いて４−クロロアセト酢酸エチル還元反応の至適温度を測定した。
反応の至適温度は45℃であった。また、40℃から55℃の広い範囲で最大活性の80%以上の相対活性を図４に示した。なお反応の基質として用いた４−クロロアセト酢酸エチルは、高温では不安定な物質である。したがって、組換え4-ハロアセト酢酸エステル還元酵素を、高温で安定な他の基質化合物に対して作用させた場合には、酵素反応の至適温度が更に高くなる可能性がある。
【００７４】
［実施例１２］（組換え4-ハロアセト酢酸エステル還元酵素による（Ｓ）−４−クロロ−３−ヒドロキシ酪酸エチル生産）
実施例８で調製した4-ハロアセト酢酸エステル還元酵素を用いて４−クロロアセト酢酸エチルからの (S) -4-クロロ-3-ヒドロキシ酪酸エチル合成を行った。
200mMリン酸カリウム緩衝液(pH6.5)、2% 4-クロロアセト酢酸エチル、基質に対して2等量のグルコース、1mM ＮＡＤＰ^＋、38.4mUの4-ハロアセト酢酸エステル還元酵素、4-ハロアセト酢酸エステル還元酵素の2倍量のグルコース脱水素酵素（和光純薬）を含む反応液中で１晩反応させた。
【００７５】
反応終了液を用いて (S) -4-クロロ-3-ヒドロキシ酪酸エチルの光学純度の測定を以下の方法により行った。反応液から4-クロロ-3-ヒドロキシ酪酸エチルを酢酸エチルで抽出し、脱溶媒した後、光学分割カラムを用いた液体クロマトグラフィーにより分析した。光学分割カラムは、ダイセル化学工業株式会社製キラルセルOD（CHIRALCEL OD. 4.6mm × 25cm）を用い、n-ヘキサン：2-プロパノール＝93：7の溶離液、流速1mL/min、RI検出により行った。その結果、本発明によって生成した4-クロロ-3-ヒドロキシ酪酸エチルは、97.8%eeの（Ｓ）体であった。
【００７６】
また、生成された４−クロロ−３−ヒドロキシ酪酸エチルをガスクロマトグラフィーで定量し、出発原料である４−クロロアセト酢酸エチルに対する収率を求めた。ガスクロマトグラフィーの条件は以下のとおりである。すなわち、サーモン−３０００ ５%クロモソルブＷ ６０−８０（ＡＷ−ＤＭＣＳ）(Thermon-3000 5% Chromosorb W 60〜80 (AW-DMCS)、 信和化工株式会社)、3.2mm ×210cmを用い、カラム温度150℃とし、水素炎イオン化検出器(FID)を利用して分析した。その結果、反応の収率は約48%であった。
【００７７】
【発明の効果】
本発明は、（Ｓ）−４−ハロ−３−ヒドロキシ酪酸エステル誘導体などの光学活性アルコールの生産に有用な新規なβ−ケトアシルACP還元酵素及びそのホモログの取得方法を提供する。更に子の方法に基づいて、バチルス・ステアロサーモフィラス由来のβ-ケトアシルACP還元酵素遺伝子、これによりコードされる蛋白質が提供された。本酵素を利用することにより、光学純度の高い (S) -4-クロロ-3-ヒドロキシ酪酸エチルの効率的な生産方法が提供された。
【００７８】
【配列表】

【図面の簡単な説明】
【図１】fabGアミノ酸配列のアラインメントを示す図である。
【図２】pTrc-Bstのプラスミドマップ。
【図３】組換え4-ハロアセト酢酸エステル還元酵素及びその熱処理物のSDS-PAGEの結果を表す写真である。
【図４】組換え4-ハロアセト酢酸エステル還元酵素の至適温度を示すグラフである。縦軸は相対活性、横軸は反応温度を表す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel 4-haloacetoacetate reductase useful for the production of optically active alcohols, in particular, (S) -4-halo-3-hydroxybutyrate, DNA encoding the enzyme protein, and production of the enzyme The present invention relates to a method, and a method for producing an alcohol, particularly a (S) -4-chloro-3-hydroxybutyric acid ester derivative, using the enzyme.
[0002]
[Prior art]
Conventionally, the following methods are known as a manufacturing method of optically active (S) -4-halo-3-hydroxybutyric acid ester.
Asymmetric reduction using microorganisms such as baker's yeast
J. Am. Chem. Soc., 105, 5925-5926 (1983)
JP-A-61-146191 etc.
Asymmetric reduction method using enzymes such as 3α-hydroxysteroid dehydrogenase
Japanese Unexamined Patent Publication No. 1-277494
By cloning a gene of an enzyme that reduces 4-haloacetoacetate derived from a microorganism to produce (S) -4-halo-3-hydroxybutyrate, and recombinant E. coli that expresses the gene in E. coli, A method for producing (S) -4-halo-3-hydroxybutyrate by reducing 4-haloacetoacetate
WO 9835025
JP2000-236883
[0003]
Moreover, the present inventors reduced β-haloacetoacetate by β-ketoacyl-ACP reductase derived from Escherichia coli and Bacillus subtilis (hereinafter sometimes abbreviated as KR), It has been confirmed that (S) -4-halo-3-hydroxybutyrate is synthesized. And the (S) -4-halo-3-hydroxybutyric acid synthesis method using this enzyme was established. Among the fatty acid synthases, KR that constitutes type II fatty acid synthase (Neochemistry Laboratory 4 Lipid I, p34-37) has a subunit molecular weight of about 20,000-40,000 and a small molecular weight. Moreover, these KR has the characteristic that it is hard to be inhibited by SH reagent.
[0004]
Furthermore, the inventors of the present invention have reported that acetoacetyl-CoA reductase produced by Alkaligenes eutrophas or Zoogloea ramigera reduces 4-haloacetoacetate, and (S) -4-halo-3-hydroxy. It has been confirmed that it has an activity to synthesize butyrate esters (Japanese Patent Laid-Open No. 2000-189170). A gene encoding acetoacetyl-CoA reductase (hereinafter sometimes abbreviated as AR) is generally abbreviated as phbB or phaB. In general, the gene encoding KR is abbreviated as fabG. phbB or phaB was found by obtaining a polyβ-hydroxy acid synthase (PHS) gene having high homology to fabG. Alkaline Genes Eutrophas is now called Ralstonia eutropha.
However, all the methods using known enzymes have problems such as low enzyme stability, low product productivity, and low product accumulation concentration. In order to industrially carry out the enzymatic synthesis of (S) -4-halo-3-hydroxybutyric acid ester, improvement of these problems is desired.
[0005]
[Problems to be solved by the invention]
The present invention relates to a method for obtaining a DNA encoding a novel 4-haloacetoacetate reductase useful for the production of (S) -4-halo-3-hydroxybutyrate, and a novel 4-haloacetoacetate reductase It is an object to provide a method for producing (S) -4-halo-3-hydroxybutyric acid ester using the enzyme.
[0006]
[Means for Solving the Problems]
The present inventor has noted that KR constituting the type II fatty acid synthase is likely to be widely distributed in microorganisms, viruses, plants, and the like. Therefore, if β-ketoacyl-ACP reductase derived from various organisms, including microorganisms that do not grow with normal culture methods, can be obtained, it will be possible to find an enzyme with advantageous properties. It was. More specifically, it was thought that an enzyme excellent in organic solvent resistance, stability, stereoselectivity, or productivity by a recombinant microorganism could be obtained.
[0007]
Based on such predictions, the present inventors analyzed a known gene encoding KR (fabG) to find a relatively conserved region. A PCR primer corresponding to this part was synthesized, and PCR was performed using a microbial genomic DNA for which no fabG gene was reported as a template, and a part of the fabG gene could be obtained. Furthermore, the entire region of the fabG gene was cloned using the base sequence of the obtained gene and expressed using a host such as Escherichia coli. As a result, it was confirmed that the expressed enzyme reduced 4-haloacetoacetate to produce (S) -4-halo-3-hydroxybutyrate, thereby completing the present invention.
[0008]
That is, the present invention relates to a method for obtaining a 4-haloacetoacetate reductase gene useful for the production of (S) -4-halo-3-hydroxybutyrate, the obtained gene, and the 4-haloacetate encoded by the gene. The present invention relates to an acetate reductase and a method for producing an optically active alcohol using the same.
[1] A 4-haloacetoacetate ester comprising a combination of oligonucleotides comprising at least 15 consecutive base sequences complementary to the base sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4 or their complementary sequences A primer set for synthesizing a gene that encodes a protein having an enzyme activity that acts on the enzyme and produces (S) -4-halo-3-hydroxybutyric acid ester.
[2] A method for obtaining a gene encoding a protein having an enzymatic activity that acts on 4-haloacetoacetate and produces (S) -4-halo-3-hydroxybutyrate, including the following steps.
(A) a step of performing PCR using the primer set according to [1], using a polynucleotide derived from an organism as a template,
(B) collecting the amplification product of step (a),
(C) obtaining a gene encoding a protein comprising the base sequence of the amplification product and having a target enzyme activity;
[3] The method according to [2], wherein the organism is a thermophilic microorganism.
[4] A protein having an enzyme activity that acts on 4-haloacetoacetate and produces (S) -4-halo-3-hydroxybutyrate, according to any one of (a) to (e) below: Polynucleotide.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1,
(B) a polynucleotide encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2;
(C) a polynucleotide encoding a protein consisting of amino acids in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2;
(D) a polynucleotide that hybridizes with the polynucleotide comprising the base sequence set forth in SEQ ID NO: 1 under stringent conditions;
(E) a polynucleotide encoding an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 2;
[5] A protein encoded by the polynucleotide according to [4].
[6] A recombinant vector into which the polynucleotide according to [4] is inserted.
[7] The recombinant vector according to [6], into which a polynucleotide encoding a dehydrogenase capable of catalyzing a redox reaction using β-nicotinamide adenine dinucleotide as a coenzyme is inserted.
[8] A transformant capable of expressing the polynucleotide according to [4] or the vector according to [6].
[9] A protein having enzymatic activity for culturing the transformant according to [8] and acting on 4-haloacetoacetate to produce (S) -4-halo-3-hydroxybutyrate, or The method for producing a protein according to [5], which comprises a step of collecting the fraction containing the protein.
[10] Production of alcohol, characterized in that the protein according to [5], the microorganism producing the protein, or the processed product of the microorganism is allowed to act on the ketone, and the ketone is reduced to produce alcohol. Method.
[11] The method for producing an alcohol according to [10], wherein the microorganism is the transformant according to [8].
[12] The method for producing an alcohol according to [10], wherein the ketone is a 4-haloacetoacetic acid ester derivative and the alcohol is a (S) -4-halo-3-hydroxybutyric acid ester derivative.
[13] The method for producing an alcohol according to [12], wherein the 4-haloacetoacetic acid ester derivative is 4-chloroacetoacetic acid ethyl ester and the alcohol is (S) -4-chloro-3-hydroxybutyric acid ethyl ester.
[14] The method for producing an alcohol according to [10], further comprising a step of additionally converting oxidized β-nicotinamide adenine dinucleotide to a reduced form.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, an enzyme having an enzymatic activity that acts on 4-haloacetoacetate and produces (S) -4-halo-3-hydroxybutyrate is referred to as 4-haloacetoacetate reductase. The 4-haloacetoacetate reductase according to the present invention is characterized by reducing the 3-position carbonyl group of 4-haloacetoacetate to produce a hydroxyl group in the (S) configuration. In the present invention, 4-haloacetoacetate reductase activity is represented by a reduction activity with respect to ethyl 4-chloroacetoacetate, and can be confirmed as follows.
[0010]
Method for measuring reduction activity against ethyl 4-chloroacetoacetate:
100 mM potassium phosphate buffer (pH 6.5), 0.2 mM reduced nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADPH), 20 mM 4-chloroacetoacetate and the enzyme are reacted at 30 ° C. Measure the decrease in absorbance at 340 nm with decreasing NADPH. 1 U is the amount of enzyme that catalyzes the decrease of 1 μmol of NADPH per minute. Proteins are quantified by a dye binding method using a Bio-Rad protein assay kit.
[0011]
The present invention relates to a primer set for obtaining a gene encoding 4-haloacetoacetate reductase. The primer set in the present invention consists of a combination of oligonucleotides comprising a base sequence set forth in SEQ ID NO: 3 and SEQ ID NO: 4, or a continuous base sequence of at least 15 bases complementary to their complementary sequences. In the present invention, “complementary sequence” refers to a base sequence constituting the other strand of one strand of double-stranded DNA comprising A: T and G: C base pairs. Further, the term “complementary” is not limited to the case where the sequence is completely complementary in at least 15 consecutive nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95%. What is necessary is just to have the homology on the above base sequences. A known algorithm such as BLASTN may be used as an algorithm for determining homology.
[0012]
The present inventors performed alignment of β-ketoacyl ACP reductase gene (fabG) described in a known database (such as SWISS-PROT), and exceeded the species in the region surrounded by □ in FIG. And found that the amino acid sequence is conserved. The base sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 are degenerative sequences that encode this conserved amino acid sequence. The present inventors are able to synthesize genes encoding 4-haloacetoacetate reductase derived from a wide range of species by using degenerative primers set for this region. I found it.
[0013]
In the present invention, examples of 4-haloacetoacetate reductase include β-ketoacyl ACP reductase and acetoacetyl-CoA reductase. On the other hand, the biological species for obtaining the genes encoding these enzymes are not particularly limited. In particular, bacteria, viruses, plants, or the like for which the β-ketoacyl ACP reductase gene is not known are useful for obtaining a novel 4-haloacetoacetate reductase. Among them, an enzyme derived from a thermophilic microorganism is generally suitable as an enzyme supply source because it can be expected to have high stability. Enzyme proteins that are stable against heat are also advantageous in enzyme purification. That is, by heating the crudely purified enzyme, components contaminating the enzyme protein are denatured such as decomposition and formation of aggregates. At this time, the enzyme protein having heat stability is not denatured. As a result, the target enzyme protein can be easily purified.
[0014]
The present invention also relates to a method for obtaining a gene encoding 4-haloacetoacetate reductase using the primer set. In the method of the present invention, a part of the fabG gene (core region) can be obtained by performing PCR using a gene derived from an appropriate organism as a template. The gene used as a template is not particularly limited. For example, the above-mentioned genes such as bacteria, viruses or plants can be used. As the gene, genomic DNA, genomic RNA, mRNA, or cDNA can be used. When a gene is interrupted by an intron, such as a plant genome, it is advantageous to use cDNA as a template. For PCR, Taq DNA polymerase (Takara Shuzo), KOD DNA polymerase (Toyobo), Pfu DNA polymerase (Strategene), etc. can be used, and the reaction is performed under the standard conditions described in the instructions attached to the product. Just do it.
[0015]
If a specific product could not be obtained by PCR, the annealing temperature was lowered to 37 ° C, the temperature was gradually raised from the annealing temperature to the extension temperature of 60 ° C by 10 ° C per minute, and the extension reaction was performed at 60 ° C. After that, 5 cycles of heat treatment are performed. After that, the annealing temperature is raised to 45 ° C., the elongation reaction is performed at 70 ° C., and then the heat treatment is performed for 25 cycles to amplify specific bands. be able to. When a non-specific PCR product is obtained, a specific PCR product can be obtained by a method such as Nested PCR (BioTechniques 18, 609-610 (1995)).
[0016]
The gene synthesized in this way is usually a fragment (core) that lacks the 5′-side and 3′-side end sequences of genes encoding 4-haloacetoacetate reductase. A method for obtaining the full length of a target gene based on the nucleotide sequence information of a fragment of a gene is known.
For example, based on inverse PCR (Genetics 120, 621-623 (1988)), a region having an unknown base sequence adjacent to both sides of a fragment can be obtained. In inverse PCR, DNA that has been self-circulated after digestion of chromosomal DNA with an appropriate restriction enzyme is used as a template. If PCR is performed using the circularized DNA as a template and PCR primers for extending outside the known DNA, a region with an unknown base sequence can be amplified. It is also possible to obtain the full length of the gene by the RACE method (Rapid Amplification of cDNA End, “PCR Experiment Manual” p25-33, HBJ Publishing Bureau).
In addition, clones containing the target gene can also be selected by hybrid screening of genomic libraries and cDNA libraries using PCR amplification products using the primers as probes.
[0017]
The present invention relates to a protein having an enzymatic activity that acts on 4-haloacetoacetate and produces (S) -4-halo-3-hydroxybutyrate as described in any of (a) to (e) below. It relates to the encoding polynucleotide.
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1,
(B) a polynucleotide encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2;
(C) a polynucleotide encoding a protein consisting of amino acids in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2;
(D) a polynucleotide that hybridizes with the polynucleotide comprising the base sequence set forth in SEQ ID NO: 1 under stringent conditions;
(E) a polynucleotide encoding an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 2;
In contrast to the polynucleotides described in (a) and (b), the polynucleotides described in (c) to (e) are particularly referred to as homologs in the present invention.
The polynucleotide of the present invention can include DNA, RNA, a chimera of both, or an artificial nucleotide derivative. The polynucleotide having the base sequence set forth in SEQ ID NO: 1 is the base sequence of the 4-haloacetoacetate reductase gene obtained from Bacillus stearothermophilus IFO 12550 strain, which is a thermophilic bacterium. A polynucleotide containing the base sequence described in SEQ ID NO: 1 can be obtained from the same strain by PCR or hybridization based on the base sequence information described in SEQ ID NO: 1. Alternatively, it can be synthesized completely artificially. The base sequence described in SEQ ID NO: 1 encodes the amino acid sequence described in SEQ ID NO: 2. The polynucleotide of the present invention includes any polynucleotide that can encode the amino acid sequence set forth in SEQ ID NO: 2.
Furthermore, the present invention relates to a homologue of the polynucleotide of the present invention. Those skilled in the art will be able to introduce site-directed mutagenesis into the DNA described in SEQ ID NO: 1 (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., to obtain homologs by appropriately introducing substitutions, deletions, insertions, and / or addition mutations It is possible.
[0018]
The homologue of the present invention is a polynucleotide capable of hybridizing under stringent conditions with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1, and reducing 4-haloacetoacetate, (S)- Also included are polynucleotides that encode proteins having activity to produce 4-halo-3-hydroxybutyric acid esters.
The polynucleotide capable of hybridizing under stringent conditions is one or more selected from any at least 20, preferably at least 30, for example 40, 60 or 100 consecutive sequences in SEQ ID NO: 1. The probe DNA is used as a probe DNA, for example, using ECL direct nucleic acid labeling and detection system (Amersham Pharmaica Biotech) under the conditions described in the manual (wash: 42 ° C, primary wash buffer containing 0.5x SSC). It refers to a polynucleotide that soys.
[0019]
Furthermore, the homologue of the present invention comprises a polynucleotide encoding a protein having at least 80%, preferably at least 90%, more preferably 95% or more homology with the amino acid sequence shown in SEQ ID NO: 2. Protein homology searches include databases related to amino acid sequences of proteins such as SWISS-PROT and PIR, databases related to DNA sequences such as DDBJ, EMBL, and Gene-Bank, and databases related to predicted amino acid sequences based on DNA sequences. , BLAST, FASTA, etc. can be used, for example, via the Internet.
[0020]
As a result of homology search using BLAST program for SWISS-PROT, PIR and other protein databases using the amino acid sequence shown in SEQ ID NO: 2, the highest homology among the known proteins was shown. , A putative short chain oxidoreductase derived from Streptomyces coelicolor A3 (2), a predicted ORF derived from genomic analysis of unknown function, with an identity of 58% and a positive of 72%. The homology of 80% or more of the present invention represents, for example, the value of Positive homology using the BLAST program.
The polynucleotide of the present invention is useful for producing a protein having 4-haloacetoacetate reductase activity. Alternatively, the polynucleotide of the present invention is useful for producing an optically active alcohol using a protein having 4-haloacetoacetate reductase activity.
[0021]
The present invention relates to a protein encoded by the polynucleotide. The protein comprising the amino acid sequence shown in SEQ ID NO: 2 of the present invention constitutes a preferred embodiment of the protein of the present invention. The amino acid sequence shown in SEQ ID NO: 2 is encoded by the base sequence described in SEQ ID NO: 1. On the other hand, in the present invention, the protein encoded by the polynucleotide described in (c) to (e) above is referred to as a homolog of the protein of the present invention.
[0022]
The protein of the present invention can be obtained by introducing and expressing the polynucleotide described in (a) to (e) above in a host. Furthermore, the homologue of the protein of the present invention includes a protein having at least 80%, preferably at least 90%, more preferably 95% or more homology with the amino acid sequence shown in SEQ ID NO: 2. Protein homology searches include databases related to amino acid sequences of proteins such as SWISS-PROT and PIR, databases related to DNA sequences such as DDBJ, EMBL, and Gene-Bank, and databases related to predicted amino acid sequences based on DNA sequences. , BLAST, FASTA, etc. can be used, for example, via the Internet.
[0023]
A homology search using the BLAST program for databases that have accumulated amino acid sequences such as SWISS-PROT and PIR using the amino acid sequence set forth in SEQ ID NO: 2 shows the highest homology among known proteins. It was putative short chain oxidoreductase derived from Streptomyces coelicolor A3 (2). This amino acid sequence was a predicted ORF derived from genomic analysis of unknown function, with an identity of 58% and a positive of 72%. The homology of 80% or more of the present invention represents, for example, the value of Positive homology using the BLAST program.
[0024]
By inserting the polynucleotide of the present invention into a known expression vector, a 4-haloacetoacetate reductase expression vector is provided. Further, the 4-haloacetoacetate reductase of the present invention can be obtained as a recombinant by culturing a transformant transformed with this expression vector.
[0025]
In order to express 4-haloacetoacetate reductase in the present invention, a microorganism to be transformed is transformed with a recombinant vector containing a polynucleotide encoding a polypeptide having 4-haloacetoacetate reductase activity. Any organism that can express 4-haloacetoacetate reductase activity is not particularly limited. Examples of the microorganisms that can be used include the following microorganisms.
Escherichia
Genus Bacillus
Pseudomonas genus
Serratia genus
Brevibacterium genus
Corynebacterium genus
Streptococcus genus
Bacteria for which host vector systems such as Lactobacillus are developed
Rhodococcus genus
Streptomyces genus Streptomyces and other host vector systems being developed
The genus Saccharomyces
Kluyveromyces genus
The genus Schizosaccharomyces
Genus Zygosaccharomyces
The genus Yarrowia
Genus Trichosporon
Rhodosporidium genus
Pichia genus
Yeasts for which host vector systems such as Candida are being developed
Genus Neurospora
Aspergillus genus
Cephalosporium genus
Molds for which host vector systems such as Trichoderma are developed
[0026]
A procedure for producing a transformant and 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 the 4-haloacetoacetate reductase gene of the present invention in a microorganism or the like, first, the DNA is introduced into a plasmid vector or a phage vector stably existing in the microorganism, and the genetic information is transcribed and transferred. Need to translate. 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, it is necessary to use a promoter and terminator known to function in a microorganism used as a host. “Microbiology Basic Course 8 Genetic Engineering / Kyoritsu Publishing” regarding vectors, promoters, terminators, and the like that can be used in these various microorganisms, especially regarding yeast, Adv. Biochem. Eng. 43, 75-102 (1990), Yeast 8 , 423-488 (1992), and the like.
[0027]
For example, in the genus Escherichia, particularly Escherichia coli, pBR and pUC plasmids can be used as plasmid vectors, and lac (β-galactosidase), trp (tryptophan operon), tac, trc (lac, trp Fusion), promoters derived from λ phage PL, PR, etc. can be used. In addition, as a terminator, a terminator derived from trpA, phage, or rrnB ribosomal RNA can be used. Among these, the vector pSE420D (described in JP-A-2000-189170) obtained by partially modifying the multicloning site of commercially available pSE420 (manufactured by Invitrogen) can be suitably used.
[0028]
In the genus Bacillus, pUB110 series plasmids, pC194 series plasmids and the like can be used as vectors, and can be integrated into chromosomes. Further, apr (alkaline protease), npr (neutral protease), amy (α-amylase) and the like can be used as promoters and terminators.
[0029]
In the genus Pseudomonas, host vector systems have been developed for Pseudomonas putida, Pseudomonas cepacia, and the like. A broad host range vector (including genes necessary for autonomous replication derived from RSF1010) pKT240 based on the plasmid TOL plasmid, which is involved in the degradation of toluene compounds, can be used, and a lipase (specialized as a promoter and terminator) can be used. Kaihei 5-284973) Genes can be used.
[0030]
In the genus Brevibacterium, particularly in Brevibacterium lactofermentum, plasmid vectors such as pAJ43 (Gene 39, 281 (1985)) can be used. As the promoter and terminator, promoters and terminators used in Escherichia coli can be used as they are.
[0031]
In the genus Corynebacterium, especially Corynebacterium glutamicum, plasmid vectors such as pCS11 (JP 57-183799) and pCB101 (Mol.Gen.Genet.196, 175 (1984) are available. is there.
[0032]
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.
[0033]
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. .
[0034]
In the genus Rhodococcus, a plasmid vector isolated from Rhodococcus rhodochrous can be used (J. Gen. Microbiol. 138, 1003 (1992)).
[0035]
In the genus Streptomyces, a plasmid 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. A similar plasmid can also be used in Streptomyces virginiae (Actinomycetol. 11, 46-53 (1997)).
[0036]
In the genus Saccharomyces (especially Saccharomyces cerevisiae), YRp, YEp, YCp, and YIp plasmids can be used, and homologous recombination with ribosomal DNA present in multiple copies 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) Promoters and terminators such as are available.
[0037]
In the genus Klayveromyces, especially Kluyveromyces lactis, 2 μm-type plasmid derived from Saccharomyces cerevisiae, pKD1-type plasmid (J. Bacteriol. 145, 382-390 (1981)), involved in killer activity A plasmid derived from pGKl1, an autonomously proliferating gene KARS system 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.
[0038]
In the genus Schizosaccharomyces, plasmid vectors containing ARS (genes involved in autonomous replication) derived from Schizosaccharomyces pombe and selectable markers that complement the 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.
[0039]
In the genus Zygosaccharomyces, plasmid vectors derived from pSB3 (Nucleic Acids Res. 13, 4267 (1985)) derived from Zygosaccharomyces rouxii are available, and the PHO5 promoter derived from Saccharomyces cerevisiae Alternatively, the promoter of GAP-Zr (glyceraldehyde-3-phosphate dehydrogenase) derived from Tigosaccharomyces rouxi (Agri. Biol. Chem. 54, 2521 (1990)) can be used.
[0040]
In the genus Pichia, a host vector system has been developed in Pichia Angusta (formerly Hansenula polymorpha). As vectors, genes involved in autonomous replication derived from Pichia Angusta (HARS1, HARS2) can be used, but they are relatively unstable, so multicopy integration into the chromosome is effective (Yeast 7, 431- 443 (1991)). In addition, AOX (alcohol oxidase) and FDH (formate dehydrogenase) promoters induced by methanol and the like can be used. In addition, Pichia pastoris and other host vector systems using genes involved in autonomous replication from Pichia (PARS1, PARS2) and multi-copy integration vectors to chromosomes (commercially available as Pichia Expression Kit from Invitrogen) Has been developed (Mol. Cell. Biol. 5, 3376 (1985)), and strong promoters such as AOX that can be induced with high-concentration culture and methanol can be used (Nucleic Acids Res. 15,3859 (1987)) .
[0041]
In the genus Candida, the host vector system 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 has been developed for the chromosome integration type vector (Japanese Patent Laid-Open No. 08-173170).
[0042]
In the genus Aspergillus, Aspergillus niger, Aspergillus oryzae, etc. are the most studied among molds, and integration into plasmids and chromosomes is available. Promoters derived from external proteases, amylases and amidases are available (Trends in Biotechnology 7, 283-287 (1989)).
[0043]
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)).
[0044]
In addition to microorganisms, various host and vector systems have been developed for plants and animals, especially in insects using moths (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes and other plants. A system for expressing a large amount of heterologous protein has been developed and can be suitably used.
[0045]
The present invention also relates to a method for producing an alcohol by reducing a ketone of the 4-haloacetoacetate reductase. Examples of the alcohol include (S) -4-halo-3-hydroxybutyric acid ester derivatives. The enzyme reaction of the present invention can be performed by bringing a transformant such as an enzyme molecule, a processed product thereof, a culture containing the enzyme molecule, or a microorganism producing the enzyme into contact with the reaction solution in a living state. The contact form between the enzyme and the reaction solution is not limited to these specific examples.
[0046]
In the present invention, the reaction solution is obtained by dissolving or mixing and suspending a substrate and a coenzyme necessary for an enzyme reaction in an appropriate solvent that provides a desirable environment for the expression of enzyme activity. As the coenzyme, a coenzyme that can be used by 4-haloacetoacetate reductase that catalyzes the reaction is used. For example, the enzyme consisting of the amino acid sequence of SEQ ID NO: 2 requires reduced nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADPH) as a coenzyme. On the other hand, a substrate compound that is converted into a target compound by an enzymatic reaction is used as the substrate. The relationship between the product alcohol and the substrate compound will be specifically described later.
[0047]
The microorganism treated product containing 4-haloacetoacetate reductase in the present invention is specifically a microorganism whose cell membrane permeability has been changed by treatment with an organic solvent such as a surfactant or toluene, or a glass bead or enzyme treatment. Cell-free extract obtained by crushing bacterial cells by the above, or a partially purified product thereof.
[0048]
As the ketone in the method for producing an alcohol according to the present invention, a 4-haloacetoacetic acid ester derivative is preferably used. Examples of the halogen include chlorine, bromine, iodine atom and the like, and chlorine atom is particularly preferable. Examples of the ester include lower alkyl esters such as methyl ester, ethyl ester, propyl ester, isopropyl ester, and butyl ester, and ethyl ester is particularly desirable. Further, the 4-halogenated acetoacetate derivative includes a derivative having a lower alkyl group which may contain a halogen substitution at the β-position, a halogen or the like.
[0049]
The oxidized coenzyme produced accompanying the reduction reaction can be regenerated again as a reduced form by the enzymatic reaction. For example, oxidized nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP) produced from NADPH. ⁺ Can be regenerated enzymatically to NADPH. Regeneration to NADPH is the NADP of microorganisms ⁺ It can be carried out using the reducing ability (glycolytic system, methylotrophic C1 compound utilization pathway, etc.). These NADP ⁺ The reducing ability can be enhanced by adding glucose, ethanol, formic acid or the like to the reaction system. NADP ⁺ It can also be carried out by adding to the reaction system a microorganism having the ability to produce NADPH, a processed product thereof, or an enzyme.
[0050]
For example, glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malate dehydrogenase etc.), hydrogenase (Alcaligenes genus, Clostridium genus etc.) Produces hydrogen as an electron donor NADP ⁺ Using microorganisms including enzymes (Biotechnol.Bioeng. 27, 1277-1281 (1985), Biocatal.Bioteransform. 15, 281-296 (1997)), etc. that perform the reduction reaction, treated products, and partially purified or purified enzymes The coenzyme can be regenerated. The components constituting the reaction necessary for such regeneration are added to the reaction system for producing the alcohol according to the present invention, added in an immobilized state, or contacted through a membrane capable of exchanging coenzymes. Can be made.
[0051]
In addition, when a microorganism transformed with a recombinant vector containing the polynucleotide of the present invention is used as a host in the alcohol production method in the state of living cells, an additional reaction system for coenzyme regeneration is used. It may be unnecessary. That is, by using a microorganism having a high coenzyme regeneration activity, an efficient reaction can be performed in the reduction reaction using the transformant without adding an enzyme for coenzyme regeneration.
Furthermore, a reduction reaction can be carried out more efficiently by simultaneously introducing DNA encoding an enzyme that can be used for coenzyme regeneration and DNA encoding the NADPH-dependent 4-haloacetoacetate reductase of the present invention into a host. It is also possible to perform. Examples of enzymes that can be used for coenzyme regeneration include glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, or organic acid dehydrogenase (malate dehydrogenase, etc.) Can do.
To introduce these two or more genes into the host, there is a method in which the host is transformed with a recombinant vector in which the genes are separately introduced into a plurality of vectors different from the origin of replication in order to avoid incompatibility. Used. In addition, a method of introducing both genes into a single vector, a method of introducing one or both genes into a chromosome, and the like can also be used.
[0052]
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.
[0053]
The reduction reaction using the enzyme of the present invention can be performed in a mixed system of water and an organic solvent having compatibility with water. Examples of the solvent having compatibility with water include methanol, ethanol, isopropanol, acetone, and acetonitrile. In addition, the enzyme reaction for the present invention can be carried out in an organic solvent that is hardly soluble in water or in a two-phase system of an organic solvent that is hardly soluble in water and an aqueous medium. As an organic solvent that is difficult to dissolve in water, for example, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, cyclohexane, octane, or 1-octanol can be used.
[0054]
The enzyme constituting the enzyme reaction of the present invention can be immobilized and used. The method for immobilizing the enzyme is not particularly limited. For example, a method for immobilizing an enzyme using glutaraldehyde, acrylamide, κ-carrageenan, calcium alginate, ion exchange resin, celite, or the like is known. The reaction of the present invention can also be performed using a membrane reactor or the like. Examples of membranes that can constitute a membrane reactor include ultrafiltration membranes, hydrophobic membranes, cationic membranes, and nanofiltration membranes (J. Ferment. Bioeng. 83, 54-58 (1997)). it can.
[0055]
The reaction of the present invention can be carried out at a reaction temperature of 4-70 ° C., preferably 15-50 ° C., pH 3-11, preferably pH 6-8, and a substrate concentration of 0.01-90%, preferably 0.1-30%. In the reaction system, if necessary, coenzyme NADP ⁺ Alternatively, NADPH can be added in an amount of 0.001 mM to 100 mM, preferably 0.01 to 10 mM. The substrate can be added all at once at the start of the reaction, but it is desirable to add it continuously or discontinuously so that the substrate concentration in the reaction solution does not become too high.
[0056]
The compound added to the reaction system for NADPH regeneration can be added in a molar ratio of 0.1-20, preferably 1-5 times with respect to the substrate ketone. As a compound for NADPH regeneration, for example, glucose when using glucose dehydrogenase, formic acid when using formate dehydrogenase, ethanol or isopropanol when using alcohol dehydrogenase, and the like are added. On the other hand, the enzyme constituting the reaction for NADPH regeneration can be added in an amount of 0.1 to 100 times, preferably about 0.5 to 20 times in terms of enzyme activity, compared to the NADPH-dependent 4-haloacetoacetate reductase of the present invention. .
[0057]
The alcohol produced by the reduction of the ketone of the present invention can be purified by appropriately combining microbial cells, protein centrifugation, separation by membrane treatment, solvent extraction, distillation and the like.
For example, in ethyl (S) -4-chloro-3-hydroxybutyrate, a reaction solution containing microbial cells is centrifuged, and after removing the microbial cells, a solvent such as ethyl acetate or methyl isobutyl ketone is added to the supernatant. To extract ethyl (S) -4-chloro-3-hydroxybutyrate into the solvent layer. By purifying this after phase separation, high purity ethyl (S) -4-chloro-3-hydroxybutyrate can be purified. Purified ethyl (S) -4-chloro-3-hydroxybutyrate is useful as a raw material for pharmaceuticals. According to the present invention, an optically active alcohol can be obtained with high purity and efficiency. Optical isomers often have low physiological activity and compounds that cause side effects. Therefore, in pharmaceutical raw materials, optical purity greatly affects the quality of raw materials.
[0058]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail, this invention is not limited to this.
[Example 1] (Preparation of chromosomal DNA from Bacillus stearothermophilus)
Bacillus stearothermophilus IFO 12550 strain in M-64 medium (polypeptone 10g, yeast extract 2.5g, meat extract 2g, glycerol 2g, sodium chloride 2g, K ₂ HPO _Four 2g, KH ₂ PO _Four 2g, MgSO _Four ・ 7H ₂ O 0.1 g, biotin 0.4 μg / L, pH 7.2) and cultured at 55 ° C., and wet cells were obtained by centrifugation.
Chromosomal DNA was purified from the obtained wet cells using Qiagen Genome-Tip.
[0059]
[Example 2] (Amplification by PCR of core region of 4-haloacetoacetate reductase gene)
PCR was carried out using the primer fabG-N (SEQ ID NO: 3) and primer fabG-C (SEQ ID NO: 4) as a template and the chromosomal DNA prepared in Example 1. PCR was performed in 50 μL of a reaction solution containing 0.2 mM dNTP, 50 pmol of primer, 1 U of Taq DNA polymerase, and 50 ng or 125 ng of template DNA.
[0060]
The PCR conditions were as follows: 5 cycles of the following step 1 and 25 cycles of step 2.
(Step 1)
Denaturation 94 ℃ 30 seconds
Annealing 37 ℃ 30 seconds
Temperature rise at a rate of 10 ° C per minute
Elongation 60 ℃ 2 minutes
(Step 2)
Denaturation 94 ℃ 30 seconds
Annealing 45 ℃ 30 seconds
Elongation 70 ℃ 1 minute
[0061]
After completion of PCR, a part was analyzed by agarose gel electrophoresis. As a result, a specific band was detected around 550 bp regardless of the amount of template DNA.

[0062]
[Example 3] (Cloning of core region of 4-haloacetoacetate reductase gene)
The DNA fragment amplified in Example 2 was extracted with phenol-chloroform, and then DNA was collected by ethanol precipitation. The obtained DNA was double-digested with EcoRI and HindIII and purified by agarose gel electrophoresis. The obtained DNA fragment was ligated with pUC18 digested with the same restriction enzymes EcoRI and HindIII to obtain a plasmid pBstKR-1 having a target 550 bp insert fragment.
[0063]
[Example 4] (Analysis of base sequence of core region of 4-haloacetoacetate reductase gene)
The nucleotide sequence of the inserted fragment of plasmid pBstKR-1 obtained in Example 3 was analyzed. The DNA base sequence was analyzed by PCR using BigDye Terminator Cycle Sequencing ready Reaction Kit (Applied Biosystems) and PRISM 310 Genetic Analyzer (Applied Biosystems). The determined nucleotide sequence of the core region is shown as SEQ ID NO: 5.
[0064]
[Example 5] (Inverse PCR around the core region of the 4-haloacetoacetate reductase gene)
The chromosomal DNA prepared in Example 1 was digested with HindIII, and after self-cyclization reaction, it was digested with SalI and used as a template. First, as 1st PCR, PCR was performed using BST-N1NE (SEQ ID NO: 6) and BST-C1NE (SEQ ID NO: 8) as primers, and the resulting PCR product was purified and further BST-N2NE (SEQ ID NO: 7). 2nd PCR was performed using BST-C2NE (SEQ ID NO: 9) as a primer. PCR was performed by heat denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute, and extension at 72 ° C for 3 minutes for 30 cycles.
[0065]
After the completion of PCR, a part of the reaction solution was subjected to agarose electrophoresis, and a specific amplified fragment of about 5 kb was confirmed.

[0066]
[Example 6] (Analysis of base sequence of core peripheral region of 4-haloacetoacetate reductase gene)
The base sequence of the DNA fragment obtained by Inverse PCR was directly analyzed. As a result, the entire region of β-ketoacyl ACP reductase (fabG gene) is included, and at the same time, acyl CoA dehydrogenase (fabD gene) is upstream of fabG gene and part of acyl carrier protein (ACP) gene is downstream. Was found.
The resulting 4-haloacetoacetate reductase gene consisted of 762 bp and encoded 254 amino acids. The sequence of the ORF part is shown in SEQ ID NO: 1.
[0067]
[Example 7] (Construction of 4-haloacetoacetate reductase gene expression plasmid)
Based on the ORF base sequence of 4-haloacetoacetate reductase obtained in Example 6, primers FABG-E (SEQ ID NO: 10) and FABG-H (SEQ ID NO: 11) for constructing an expression plasmid in Escherichia coli. ) Was synthesized. Using a reaction solution of 0.2 mM dNTP, 5% dimethyl sulfoxide (DMSO), 10 pmol of each primer, 50 ng of chromosomal DNA, ExTaq buffer solution, ExTaq 2.5U, denaturation (94 ° C for 1 minute), annealing (55 ° C 1 minute) and elongation (72 ° C. for 3 minutes) was performed for 30 cycles. For the reaction, GeneAmp PCR System 2400 (manufactured by PerkinElmer) was used.

The obtained PCR product was double-digested with EcoRI and HindIII, and subcloned between EcoRI and HindIII of pTrc99A (Pharmacia). The obtained plasmid was designated as pTrc-Bst.
The plasmid map of pTrc-Bst is shown in FIG.
[0068]
[Example 8] (Production of recombinant 4-haloacetoacetate reductase by E. coli)
Escherichia coli JM109 transformed with pTrc-Bst is inoculated into LB medium (10g bactotryptone, 5g bacto yeast extract, 10g / 1L sodium chloride, pH7.2) containing 50mg / L ampicillin and cultured at 37 ° C overnight. did. About 1 day later, 100 mL of LB liquid medium containing 50 mg / L ampicillin and 119 mg / L IPTG (isopropylthiogalactopyranoside) was inoculated and cultured at 37 ° C. for 16 to 20 hours.
[0069]
The cultured cells are collected by centrifugation, suspended in 3 mL of 100 mM potassium phosphate buffer (pH 7.4) containing 0.01% 2-mercaptoethanol, and ultrasonicated (30 sec disruption, 30 sec
The cells were disrupted by repeating the interval 20 times.
After the supernatant was obtained by centrifugation, it was dialyzed against 10 mM potassium phosphate buffer (pH 7.4) containing 0.01% 2-mercaptoethanol and used as a cell-free extract.
[0070]
[Example 9] (Heat treatment and electrophoresis of recombinant 4-haloacetoacetate reductase)
Cell-free extract prepared in Example 8 (lane 3), cell-free extract heat treated at 30 ° C. for 10 minutes (lane 4), treated at 30 ° C. for 20 minutes (lane 5), 40 at 30 ° C. Fraction treated (lane 6), treated at 60 ° C for 10 minutes (lane 7), treated at 60 ° C for 20 minutes (lane 8), treated at 60 ° C for 40 minutes (lane 9), and vector pTrc99A The cell-free extract (lane 2) and molecular weight marker (lane 1) extracted from JM109 strain transformed only with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
[0071]
As shown in FIG. 3, a 27 kDa band (shown as BstKR1 in FIG. 3) not found in the cell-free extract derived from the bacteria transformed with the vector pTrc99A is derived from E. coli transformed with pTrc-Bst. Found in cell-free extracts. This molecular weight agreed well with the calculated value of 27 kDa from the amino acid sequence predicted from the DNA sequence.
Moreover, it was shown that the enzyme can be purified very efficiently only by heat treatment, utilizing the heat resistance of 4-haloacetoacetate reductase derived from moderately thermophilic bacteria.
[0072]
[Example 10] (Substrate specificity of recombinant 4-haloacetoacetate reductase)
The activity against various substrates was measured using the recombinant 4-haloacetoacetate reductase prepared in Example 8. As a coenzyme, only reduced nicotinamide adenine dinucleotide phosphate (NADPH) was used as an electron donor, and reduced nicotinamide adenine dinucleotide (NADH) could not be used. As a substrate for the reduction reaction, ethyl 4-chloroacetoacetate was efficiently reduced, but acetoacetyl-CoA, which is a reducing substrate for β-ketoacyl ACP reductase of Escherichia coli and Bacillus subtilis, was not reduced at all. In addition, oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ (R) or (S) -4-chloro-3-hydroxybutyric acid ethyl ester oxidation activity using) as an electron acceptor.
[0073]
[Example 11] (Optimum temperature of recombinant 4-haloacetoacetate reductase)
Using the 4-haloacetoacetate reductase prepared in Example 8, the optimal temperature for the ethyl 4-chloroacetoacetate reduction reaction was measured.
The optimum temperature for the reaction was 45 ° C. Moreover, the relative activity of 80% or more of the maximum activity in a wide range from 40 ° C. to 55 ° C. is shown in FIG. Note that ethyl 4-chloroacetoacetate used as a reaction substrate is an unstable substance at high temperatures. Therefore, when the recombinant 4-haloacetoacetate reductase is allowed to act on other substrate compounds that are stable at high temperatures, the optimum temperature for the enzyme reaction may be further increased.
[0074]
[Example 12] (Production of (S) -4-chloro-3-hydroxybutyrate by recombinant 4-haloacetoacetate reductase)
Using the 4-haloacetoacetate reductase prepared in Example 8, ethyl (S) -4-chloro-3-hydroxybutyrate was synthesized from ethyl 4-chloroacetoacetate.
200 mM potassium phosphate buffer (pH 6.5), 2% ethyl 4-chloroacetoacetate, 2 equivalents of glucose to substrate, 1 mM NADP ⁺ The reaction was carried out overnight in a reaction solution containing 38.4 mU of 4-haloacetoacetate reductase and glucose dehydrogenase (Wako Pure Chemical Industries) twice as much as 4-haloacetoacetate reductase.
[0075]
Using the reaction completed solution, the optical purity of ethyl (S) -4-chloro-3-hydroxybutyrate was measured by the following method. From the reaction solution, ethyl 4-chloro-3-hydroxybutyrate was extracted with ethyl acetate, desolvated, and analyzed by liquid chromatography using an optical resolution column. The optical resolution column was Chiral Cell OD (CHIRALCEL OD. 4.6 mm × 25 cm) manufactured by Daicel Chemical Industries, Ltd., and the eluent was n-hexane: 2-propanol = 93: 7, flow rate was 1 mL / min, and RI detection was performed. . As a result, ethyl 4-chloro-3-hydroxybutyrate produced according to the present invention was a 97.8% ee (S) isomer.
[0076]
The produced ethyl 4-chloro-3-hydroxybutyrate was quantified by gas chromatography, and the yield relative to the starting material, ethyl 4-chloroacetoacetate, was determined. The conditions for gas chromatography are as follows. That is, using Salmon-3000 5% Chromosorb W 60-80 (AW-DMCS) (Thermon-3000 5% Chromosorb W 60-80 (AW-DMCS), Shinwa Chemical Co., Ltd.), 3.2 mm × 210 cm, column temperature 150 The analysis was carried out using a flame ionization detector (FID). As a result, the yield of the reaction was about 48%.
[0077]
【The invention's effect】
The present invention provides a novel β-ketoacyl ACP reductase useful for production of optically active alcohols such as (S) -4-halo-3-hydroxybutyric acid ester derivatives and a method for obtaining homologues thereof. Furthermore, a β-ketoacyl ACP reductase gene derived from Bacillus stearothermophilus and a protein encoded thereby were provided based on the method of the child. By using this enzyme, an efficient method for producing ethyl (S) -4-chloro-3-hydroxybutyrate having high optical purity was provided.
[0078]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows an alignment of fabG amino acid sequences.
FIG. 2: Plasmid map of pTrc-Bst.
FIG. 3 is a photograph showing the results of SDS-PAGE of recombinant 4-haloacetoacetate reductase and its heat-treated product.
FIG. 4 is a graph showing the optimum temperature of recombinant 4-haloacetoacetate reductase. The vertical axis represents relative activity, and the horizontal axis represents the reaction temperature.

Claims (10)

The polynucleotide encoding a protein having an enzymatic activity that acts on 4-haloacetoacetate and produces (S) -4-halo-3-hydroxybutyrate, according to any one of (a) to (e) below: .
(A) a polynucleotide comprising the base sequence set forth in SEQ ID NO: 1,
(B) a polynucleotide encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2;
(C) a polynucleotide encoding a protein consisting of amino acids in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2;
(D) a polynucleotide that hybridizes with the polynucleotide comprising the base sequence set forth in SEQ ID NO: 1 under stringent conditions;
(E) a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 2; A protein encoded by the polynucleotide of claim 1 . A recombinant vector into which the polynucleotide according to claim 1 is inserted. The recombinant vector according to claim 3 , further comprising a polynucleotide encoding a dehydrogenase capable of catalyzing a redox reaction using β-nicotinamide adenine dinucleotide as a coenzyme. Transformants expressible hold the vectors described polynucleotide or claim 3, according to claim 1. A protein having an enzymatic activity for culturing the transformant according to claim 5 and acting on 4-haloacetoacetate to produce (S) -4-halo-3-hydroxybutyrate, or a fraction containing the same The manufacturing method of the protein of Claim 2 including the process of collect | recovering. A cell-free extract obtained by crushing the protein according to claim 2 or the transformant according to claim 5 is allowed to act on a ketone, and the ketone is reduced to produce an alcohol. A method for producing alcohol. The method for producing an alcohol according to claim 7 , wherein the ketone is a 4-haloacetoacetic acid ester derivative and the alcohol is a (S) -4-halo-3-hydroxybutyric acid ester derivative. The method for producing an alcohol according to claim 8 , wherein the 4-haloacetoacetic acid ester derivative is 4-chloroacetoacetic acid ethyl ester, and the alcohol is (S) -4-chloro-3-hydroxybutyric acid ethyl ester. The method for producing an alcohol according to claim 7 , further comprising a step of additionally converting oxidized β-nicotinamide adenine dinucleotide to a reduced form.

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