JP2020036576A - PANTOTHENATE KINASE THAT DOES NOT SUFFER FROM FEEDBACK INHIBITION BY COENZYME A(CoA) - Google Patents
PANTOTHENATE KINASE THAT DOES NOT SUFFER FROM FEEDBACK INHIBITION BY COENZYME A(CoA) Download PDFInfo
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- JP2020036576A JP2020036576A JP2019024126A JP2019024126A JP2020036576A JP 2020036576 A JP2020036576 A JP 2020036576A JP 2019024126 A JP2019024126 A JP 2019024126A JP 2019024126 A JP2019024126 A JP 2019024126A JP 2020036576 A JP2020036576 A JP 2020036576A
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- pantothenate kinase
- pantothenate
- kinase
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- coenzyme
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Abstract
Description
本発明は、好熱性細菌由来のコエンザイムＡ（CoA）の生成に関与するパントテン酸キナーゼおよび該酵素をコードする遺伝子に関する。 The present invention relates to a pantothenate kinase involved in the production of coenzyme A (CoA) derived from a thermophilic bacterium and a gene encoding the enzyme.
コエンザイムＡ（補酵素Ａ、CoA）は全ての生物に存在しており、生体内の多くの反応において補酵素として作用する。 Coenzyme A (coenzyme A, CoA) is present in all organisms and acts as a coenzyme in many reactions in the organism.
コエンザイムＡは、例えば酵母等の微生物から抽出、精製することにより製造することができるが、大量生産には適さず、製造コストも大きかった。 Coenzyme A can be produced, for example, by extracting and purifying it from a microorganism such as yeast, but is not suitable for mass production and its production cost is high.
CoA合成経路は、数段階の酵素反応からなり、パントテン酸を基質としてパントテン酸をリン酸化しホスホパントテン酸を生成する初発反応は、パントテン酸キナーゼ（PanK）が触媒し、CoA合成経路に関与する他の酵素と共役させることでパントテン酸からCoAを生産することも可能である。しかしながらほとんどのパントテン酸キナーゼは、CoAやアセチルCoAによるフィードバック阻害を受けるだけでなく（非特許文献１を参照）、高温では活性が低下してしまうためこのようなパントテン酸キナーゼを用いてCoAを効率的に生産することは困難であった。 The CoA synthesis pathway consists of several steps of enzymatic reactions. The initial reaction of phosphorylating pantothenic acid to generate phosphopantothenic acid using pantothenic acid as a substrate is catalyzed by pantothenate kinase (PanK) and involved in the CoA synthesis pathway CoA can be produced from pantothenic acid by conjugation with other enzymes. However, most pantothenate kinases are not only subject to feedback inhibition by CoA and acetyl-CoA (see Non-Patent Document 1), but their activity is reduced at high temperatures, so that CoA can be efficiently used using such pantothenate kinase. Production was difficult.
本発明は、CoAやアセチルCoAによるフィードバック阻害を受けず、なおかつ熱安定性に優れたパントテン酸キナーゼの提供を目的とする。 An object of the present invention is to provide a pantothenate kinase which is not subject to feedback inhibition by CoA or acetyl-CoA and has excellent thermostability.
本発明者らは、多くのパントテン酸キナーゼがCoAによるフィードバック阻害を受けることに鑑み、CoAによるフィードバック阻害を受けないパントテン酸キナーゼの開発を鋭意検討した。 In view of the fact that many pantothenate kinases are subject to feedback inhibition by CoA, the present inventors have intensively studied the development of pantothenate kinases that are not subject to feedback inhibition by CoA.
その結果、好熱性細菌であるGeobacillus sp.およびThermus thermophilusから単離したパントテン酸キナーゼがCoAおよびアセチルCoAによるフィードバック阻害を受けないことを見出した。さらに、これらのパントテン酸キナーゼが熱安定性に優れていることも見出し、本発明を完成させるに至った。 As a result, they found that pantothenate kinase isolated from the thermophilic bacteria Geobacillus sp. And Thermus thermophilus was not subject to feedback inhibition by CoA and acetyl-CoA. Furthermore, they have also found that these pantothenate kinases have excellent thermostability, and have completed the present invention.
すなわち、本発明は以下のとおりである。
[１] 以下の(a)または(b)のパントテン酸からコエンザイムＡを合成するためのパントテン酸キナーゼであって、コエンザイムＡのフィードバック阻害を受けないパントテン酸キナーゼ：
(a) 配列番号２で表されるアミノ酸配列からなるパントテン酸キナーゼ；
(b) 配列番号２で表されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を含み、かつパントテン酸からホスホパントテン酸の反応を触媒する活性を有するパントテン酸キナーゼ。
[２] Geobacillus sp.（ゲオバシラス・エスピー）30株由来であり熱安定性に優れている、[１]のパントテン酸キナーゼ。
[３] 以下の(a)または(b)のパントテン酸からコエンザイムＡを合成するためのパントテン酸キナーゼであって、コエンザイムＡのフィードバック阻害を受けないパントテン酸キナーゼをコードするDNA：
(a) 配列番号２で表されるアミノ酸配列からなるパントテン酸キナーゼ；
(b) 配列番号２で表されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を含み、かつパントテン酸からホスホパントテン酸の反応を触媒する活性を有するパントテン酸キナーゼ。
[４] パントテン酸からコエンザイムＡを合成するためのパントテン酸キナーゼであって、コエンザイムＡのフィードバック阻害を受けないパントテン酸キナーゼをコードする以下の(c)または(d)のDNA：
(c) 配列番号１で表される塩基配列を含むDNA
(d) 配列番号１で表される塩基配列を含むDNAと少なくとも90％以上の配列同一性を有し、かつパントテン酸からホスホパントテン酸の反応を触媒する活性を有するタンパク質をコードするDNA。
[５] Geobacillus sp.（ゲオバシラス・エスピー）30由来であり熱安定性に優れている、[３]または[４]のパントテン酸キナーゼをコードするDNA。
[６] 以下の(e)または(f)のパントテン酸からコエンザイムＡを合成するためのパントテン酸キナーゼであって、コエンザイムＡのフィードバック阻害を受けないパントテン酸キナーゼ：
(e) 配列番号４で表されるアミノ酸配列からなるパントテン酸キナーゼ；
(f) 配列番号４で表されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を含み、かつパントテン酸からホスホパントテン酸の反応を触媒する活性を有するパントテン酸キナーゼ。
[７] Thermus thermophilus（サーマス・サーモフィルス）HB8株由来であり熱安定性に優れている、[６]のパントテン酸キナーゼ。
[８] 以下の(e)または(f)のパントテン酸からコエンザイムＡを合成するためのパントテン酸キナーゼであって、コエンザイムＡのフィードバック阻害を受けないパントテン酸キナーゼをコードするDNA：
(e) 配列番号４で表されるアミノ酸配列からなるパントテン酸キナーゼ；
(f) 配列番号４で表されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を含み、かつパントテン酸からホスホパントテン酸の反応を触媒する活性を有するパントテン酸キナーゼ。
[９] パントテン酸からコエンザイムＡを合成するためのパントテン酸キナーゼであって、コエンザイムＡのフィードバック阻害を受けないパントテン酸キナーゼをコードする以下の(g)または(h)のDNA：
(g) 配列番号３で表される塩基配列を含むDNA
(h) 配列番号３で表される塩基配列を含むDNAと少なくとも90％以上の配列同一性を有し、かつパントテン酸からホスホパントテン酸の反応を触媒する活性を有するタンパク質をコードするDNA。
[１０] Thermus thermophilus（サーマス・サーモフィルス）HB8株由来であり熱安定性に優れている、[８]または[９]のパントテン酸キナーゼをコードするDNA。
[１１] [３]〜[５]のいずれかのDNAを含む発現ベクターを宿主細胞に導入し、該宿主細胞を培養することを含む、[１]または[２]のパントテン酸キナーゼを作製する方法。
[１２] [８]〜[１０]のいずれかのDNAを含む発現ベクターを宿主細胞に導入し、該宿主細胞を培養することを含む、[６]または[７]に記載のパントテン酸キナーゼを作製する方法。
[１３] [１]、[２]、[６]または[７]のパントテン酸キナーゼを用いて、パントテン酸からコエンザイムＡを生産する方法。
[１４] さらに、ホスホパントテン酸-システインリガーゼ（CoaB)、ホスホパントテノイルシステインデカルボキシラーゼ（CoaC）、パンテテインリン酸アデニリルトランスフェラーゼ（PA)及びデホスホCoAキナーゼ（DCK)を用いる、[１３]の方法。
That is, the present invention is as follows.
[1] A pantothenate kinase for synthesizing coenzyme A from the following pantothenic acid (a) or (b), which is not subjected to feedback inhibition of coenzyme A:
(a) a pantothenate kinase consisting of the amino acid sequence represented by SEQ ID NO: 2;
(b) pantothenic acid comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having an activity of catalyzing the reaction of pantothenic acid to phosphopantothenic acid Kinase.
[2] The pantothenate kinase according to [1], which is derived from 30 strains of Geobacillus sp. And has excellent thermostability.
[3] DNA encoding a pantothenate kinase for synthesizing coenzyme A from the following pantothenic acid (a) or (b), wherein the pantothenate kinase is not subject to feedback inhibition of coenzyme A:
(a) a pantothenate kinase consisting of the amino acid sequence represented by SEQ ID NO: 2;
(b) pantothenic acid comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having an activity of catalyzing the reaction of pantothenic acid to phosphopantothenic acid Kinase.
[4] DNA of the following (c) or (d) encoding a pantothenate kinase for synthesizing coenzyme A from pantothenate, which is not subject to feedback inhibition of coenzyme A:
(c) DNA containing the nucleotide sequence represented by SEQ ID NO: 1
(d) DNA encoding a protein having at least 90% or more sequence identity with DNA containing the base sequence represented by SEQ ID NO: 1, and having an activity of catalyzing the reaction of pantothenic acid to phosphopantothenic acid.
[5] A DNA encoding the pantothenate kinase of [3] or [4], which is derived from Geobacillus sp. 30 and has excellent thermostability.
[6] A pantothenate kinase for synthesizing coenzyme A from the following (e) or (f) pantothenate, which is not subject to feedback inhibition of coenzyme A:
(e) a pantothenate kinase consisting of the amino acid sequence represented by SEQ ID NO: 4;
(f) pantothenic acid comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and having an activity of catalyzing the reaction of pantothenic acid to phosphopantothenic acid Kinase.
[7] The pantothenate kinase of [6], which is derived from Thermus thermophilus HB8 strain and has excellent thermostability.
[8] A DNA encoding a pantothenate kinase for synthesizing coenzyme A from the following (e) or (f) pantothenate, which is not subject to feedback inhibition of coenzyme A:
(e) a pantothenate kinase consisting of the amino acid sequence represented by SEQ ID NO: 4;
(f) pantothenic acid comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and having an activity of catalyzing the reaction of pantothenic acid to phosphopantothenic acid Kinase.
[9] DNA of the following (g) or (h) encoding a pantothenate kinase for synthesizing coenzyme A from pantothenate, which is not subject to feedback inhibition of coenzyme A:
(g) DNA containing the base sequence represented by SEQ ID NO: 3
(h) DNA encoding a protein having at least 90% or more sequence identity with DNA containing the nucleotide sequence represented by SEQ ID NO: 3, and having an activity of catalyzing the reaction of pantothenic acid to phosphopantothenic acid.
[10] A DNA encoding the pantothenate kinase of [8] or [9], which is derived from the Thermus thermophilus HB8 strain and has excellent thermostability.
[11] The pantothenate kinase of [1] or [2], which comprises introducing an expression vector containing the DNA of any one of [3] to [5] into a host cell and culturing the host cell. Method.
[12] The pantothenate kinase according to [6] or [7], which comprises introducing an expression vector containing the DNA of any one of [8] to [10] into a host cell and culturing the host cell. How to make.
[13] A method for producing coenzyme A from pantothenic acid using the pantothenate kinase of [1], [2], [6] or [7].
[14] Furthermore, using phosphopantothenate-cysteine ligase (CoaB), phosphopantothenoylcysteine decarboxylase (CoaC), pantetheine phosphate adenylyltransferase (PA) and dephospho-CoA kinase (DCK), [13] the method of.
好熱性細菌であるGeobacillus sp.およびThermus thermophilus由来のパントテン酸キナーゼは、CoAやアセチルCoAによりフィードバック阻害を受けない。また、好熱性細菌由来の酵素であるため熱安定性が高い。よって、本発明のパントテン酸キナーゼは、コエンザイムAの有用中間体製造の生体触媒として有用である。 Pantothenate kinases from thermophilic bacteria Geobacillus sp. And Thermus thermophilus are not feedback inhibited by CoA or acetyl-CoA. In addition, since it is an enzyme derived from a thermophilic bacterium, it has high thermostability. Therefore, the pantothenate kinase of the present invention is useful as a biocatalyst for producing a useful intermediate of coenzyme A.
以下、本発明を詳細に説明する。
本発明は、新規なパントテン酸キナーゼ（PanK)である。
本発明のパントテン酸キナーゼは、好熱性細菌から単離することができる。好ましくはGeobacillus sp.（ゲオバシラス・エスピー）30株またはThermus thermophilus (サーマス・サーモフィルス）HB8株から単離することができる。Geobacillus sp.はゲオバシラス属に属するグラム陽性の好熱菌である。T. thermophilusはサーマス属に属するグラム陰性の好気性真正細菌であり、約75℃に至適生育温度を有する高度好熱菌である。
Hereinafter, the present invention will be described in detail.
The present invention is a novel pantothenate kinase (PanK).
The pantothenate kinase of the present invention can be isolated from a thermophilic bacterium. Preferably, it can be isolated from 30 strains of Geobacillus sp. (Geobacillus sp.) Or HB8 strain of Thermus thermophilus (Thermus thermophilus). Geobacillus sp. Is a gram-positive thermophile belonging to the genus Geobacillus. T. thermophilus is a Gram-negative aerobic eubacterium belonging to the genus Thermus and is a highly thermophilic bacterium having an optimum growth temperature of about 75 ° C.
Geobacillus sp.（ゲオバシラス・エスピー）30株から単離されたパントテン酸キナーゼをGbPanKと呼び、T. thermophilus（サーマス・サーモフィルス）HB8株から単離されたパントテン酸キナーゼをTtPanKと呼ぶ。GbPanKをコードするDNAの塩基配列を配列番号１にアミノ酸配列を配列番号２に示す。また、TtPanKをコードするDNAの塩基配列を配列番号３にアミノ酸配列を配列番号４に示す。 The pantothenate kinase isolated from 30 strains of Geobacillus sp. (Geobacillus sp.) Is called GbPanK, and the pantothenate kinase isolated from T. thermophilus (B. thermophilus) HB8 strain is called TtPanK. The nucleotide sequence of the DNA encoding GbPanK is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The nucleotide sequence of TtPanK-encoding DNA is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.
本発明のパントテン酸キナーゼは、そのアミノ酸配列からなるタンパク質がパントテン酸キナーゼ活性を有する限り、当該アミノ酸配列において少なくとも１個、好ましくは１若しくは数個のアミノ酸に欠失、置換、付加等の変異を有していてもよい。 The pantothenate kinase of the present invention may be mutated to at least one, preferably one or several amino acids in the amino acid sequence, such as deletion, substitution or addition, as long as the protein comprising the amino acid sequence has pantothenate kinase activity. You may have.
例えば、配列番号２または４で表わされるアミノ酸配列の少なくとも１個、好ましくは１または数個（例えば１〜10個、好ましくは１〜５個、さらに好ましくは１〜３個、特に好ましくは１若しくは２個）のアミノ酸が欠失してもよく、配列番号２または４で表わされるアミノ酸配列に少なくとも１個、好ましくは１または数個（例えば１〜10個、好ましくは１〜５個、さらに好ましくは１〜３個、特に好ましくは１若しくは２個）のアミノ酸が付加してもよく、あるいは、配列番号２または４で表わされるアミノ酸配列の少なくとも１個、好ましくは１または数個（例えば１〜10個、好ましくは１〜５個、さらに好ましくは１〜３個、特に好ましくは１若しくは２個）のアミノ酸が他のアミノ酸に置換してもよい。また、配列番号２および４のアミノ酸配列において、１番目のMetはなくてもよい。 For example, at least one, preferably one or several (for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, particularly preferably 1 or Two amino acids may be deleted, and at least one, preferably one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1) amino acid sequence represented by SEQ ID NO: 2 or 4 may be deleted. May have 1 to 3, particularly preferably 1 or 2 amino acids added thereto, or at least one, preferably 1 or several amino acids (for example, 1 to 2) of the amino acid sequence represented by SEQ ID NO: 2 or 4. Ten, preferably one to five, more preferably one to three, particularly preferably one or two) amino acids may be replaced by other amino acids. In the amino acid sequences of SEQ ID NOs: 2 and 4, the first Met may not be present.
このような配列番号２または４のアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列として、配列番号２または４のアミノ酸配列と、BLAST（Basic Local Alignment Search Tool at the National Center for Biological Information（米国国立生物学情報センターの基本ローカルアラインメント検索ツール））等（例えば、デフォルトすなわち初期設定のパラメータ）を用いて計算したときに、少なくとも85％以上、好ましくは90％以上、さらに好ましくは95％以上、さらに好ましくは97％以上、さらに好ましくは98％以上、特に好ましくは98％以上の配列同一性を有しているものが挙げられる。 As such an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 or 4, the amino acid sequence of SEQ ID NO: 2 or 4 and BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information (Basic Local Alignment Search Tool of the United States National Biological Information Center)) calculated using (eg, default or default parameters), at least 85% or more, preferably 90% or more, More preferably, those having a sequence identity of 95% or more, more preferably 97% or more, further preferably 98% or more, particularly preferably 98% or more are exemplified.
このような配列番号２または４のアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換、若しくは付加されたアミノ酸配列を有するタンパク質は配列番号２または４のアミノ酸配列を有するタンパク質と実質的に同一である。 A protein having an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 2 or 4 is substantially the same as the protein having the amino acid sequence of SEQ ID NO: 2 or 4. It is.
また、配列番号１または３に表される塩基配列からなるDNAと相補的な配列からなるDNAと下記のストリンジェントな条件下でハイブリダイズすることができるDNAであってパントテン酸キナーゼ活性を有するタンパク質をコードするDNAも本発明のDNAに含まれる。すなわち、DNAを固定したフィルターを用いて、0.7〜1.0 MのNaCl存在下、68℃でハイブリダイゼーションを行った後、0.1〜2倍濃度のSSC溶液（1倍濃度のSSCとは150 mM NaCl、15 mM クエン酸ナトリウムからなる）を用い、68℃で洗浄することにより同定することができる条件をいう。あるいは、サザンブロッティング法によりニトロセルロース膜上にDNAを転写、固定後、ハイブリダイゼーション緩衝液〔50％ フォルムアミド、４×SSC、50 mM HEPES(pH7.0)、10×デンハルツ（Denhardt's）溶液、100μg/mlサケ精子DNA〕中で42℃で一晩反応させることによりハイブリッドを形成することができるDNAである。 A protein capable of hybridizing with a DNA consisting of a sequence complementary to the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3 under the following stringent conditions, wherein the protein has pantothenate kinase activity: Is also included in the DNA of the present invention. That is, after performing hybridization at 68 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter having DNA immobilized thereon, a 0.1 to 2 times concentration of SSC solution (1 times concentration of SSC is 150 mM NaCl, (Comprising 15 mM sodium citrate) and washing at 68 ° C. Alternatively, DNA is transferred onto a nitrocellulose membrane by Southern blotting and fixed, and then a hybridization buffer [50% formamide, 4 × SSC, 50 mM HEPES (pH 7.0), 10 × Denhardt's solution, 100 μg / ml salmon sperm DNA] at 42 ° C overnight to form a hybrid.
また、配列番号１または３に表される塩基配列からなるDNAとBLAST（Basic Local Alignment Search Tool at the National Center for Biological Information（米国国立生物学情報センターの基本ローカルアラインメント検索ツール））等（例えば、デフォルトすなわち初期設定のパラメータ）を用いて計算したときに、少なくとも85％以上、好ましくは90％以上、さらに好ましくは95％以上、さらに好ましくは97％以上、さらに好ましくは98％以上、特に好ましくは98％以上の配列同一性を有しているDNAであって、パントテン酸キナーゼ活性を有するタンパク質をコードするDNAも本発明のパントテン酸キナーゼをコードするDNAに包含される。 In addition, DNA consisting of the base sequence represented by SEQ ID NO: 1 or 3 and BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) and the like (for example, (Calculated using default or default parameters), at least 85% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, particularly preferably DNAs having a sequence identity of 98% or more and encoding a protein having pantothenate kinase activity are also included in the DNA encoding the pantothenate kinase of the present invention.
さらに、上記DNAに対するRNA、または該RNAとストリンジェントな条件下でハイブリダイズすることができるRNAであってパントテン酸キナーゼ活性を有するタンパク質をコードするRNAも本発明に含まれる。 Furthermore, the present invention also includes RNA against the above DNA, or RNA capable of hybridizing with the RNA under stringent conditions and encoding a protein having pantothenate kinase activity.
本発明のパントテン酸キナーゼは、上記のGeobacillus sp.（ゲオバチルス・エスピー）30株またはT. thermophilus（サーマス・サーモフィルス）HB8株を培養し、該株に産生させ公知の方法で精製することにより得ることができる。本発明において、遺伝子の導入は、遺伝子の機能を有するポリヌクレオチドを組換え核酸として宿主に導入する全ての場合を含む。ポリヌクレオチドは、核酸、および１本鎖、２本鎖または３本鎖のDNAまたはRNAを包含する。遺伝子の導入には、組換えベクターによって導入する場合や、PCR等で合成した核酸を用いて相同組換えによって導入する場合が含まれる。 The pantothenate kinase of the present invention is obtained by culturing the above-described 30 strains of Geobacillus sp. (Geobacillus sp.) Or T. thermophilus (Thermus thermophilus) HB8 strain, producing the strain, and purifying the strain by a known method. be able to. In the present invention, introduction of a gene includes all cases where a polynucleotide having a gene function is introduced into a host as a recombinant nucleic acid. Polynucleotides include nucleic acids and single-, double- or triple-stranded DNA or RNA. Gene transfer includes a case where the gene is introduced by a recombinant vector and a case where the gene is introduced by homologous recombination using a nucleic acid synthesized by PCR or the like.
遺伝子を導入する宿主の種類は限定されず、細菌、真菌、各種の酵母などの単細胞真核生物、または動物もしくは植物の生細胞を任意に選択できるが、本発明においては、微生物が好ましく、特に大腸菌が好ましい。宿主大腸菌は通常遺伝子工学に用いられる大腸菌K-12株の中から適切なものを選択する。代表的なものとしてJM105やJM109が挙げられるが、DH5あるいは誘導型の発現系に用いられるBL21やBL21(DE3)などを使用してもよい。 The type of host into which the gene is introduced is not limited, and bacteria, fungi, unicellular eukaryotes such as various yeasts, or live cells of animals or plants can be arbitrarily selected.In the present invention, microorganisms are preferable, and in particular, microorganisms are preferable. E. coli is preferred. As the host Escherichia coli, an appropriate one is selected from Escherichia coli K-12 strains usually used for genetic engineering. Representative examples include JM105 and JM109, but DH5 or BL21 or BL21 (DE3) used in an inducible expression system may also be used.
遺伝子は、より好ましくは、遺伝子の発現を強化する発現ベクターによって導入される。発現ベクターは、導入しようとする遺伝子を、その発現を強化する種々のDNA断片またはRNA断片と融合させたものである。好ましくは、発現ベクターは、遺伝子を恒常的または誘導的に発現させるための転写プロモーター、転写ターミネーター、選択マーカーを含み得る。所望によりエンハンサーなどのシスエレメント、オペレーター、プロモーターを制御する遺伝子が含まれていてもよい。 The gene is more preferably introduced by an expression vector that enhances the expression of the gene. An expression vector is obtained by fusing a gene to be introduced with various DNA or RNA fragments that enhance its expression. Preferably, the expression vector may include a transcription promoter, a transcription terminator, and a selectable marker for constitutively or inducibly expressing the gene. If desired, the gene may control a cis element such as an enhancer, an operator, or a promoter.
ベクターとしては、限定はされないが、大腸菌を宿主とする場合によく利用されるプラスミド、pUC18、pUC19、pUC118、pUC119、pSC101、pBR322、pHSG298、pVC18、pVC19、pTrc99A、pMal-c2、pGEX2T、pTV118N、pTV119N、pTRP等を好ましく使用でき、その他にもS.cerevisiaeを宿主とする場合によく利用されるYep13、Yep24、YCp50、pRS414、pRS415、pRS404、pAUR101、pKG1等も利用でき、枯草菌を宿主とする場合によく利用されるプラスミドpUB110、pC194等も利用できる。更に、pBI122、pBI1101その他の各種のものも限定なく使用できる。 Examples of the vector include, but are not limited to, a plasmid often used when E. coli is used as a host, pUC18, pUC19, pUC118, pUC119, pSC101, pBR322, pHSG298, pVC18, pVC19, pTrc99A, pMal-c2, pGEX2T, pTV118N, pTV119N, pTRP, etc. can be preferably used, and Yep13, Yep24, YCp50, pRS414, pRS415, pRS404, pAUR101, pAUR101, pKG1, etc., which are often used when S. cerevisiae is used as a host, can also be used. In this case, plasmids pUB110, pC194, etc., which are often used, can also be used. Further, pBI122, pBI1101 and other various substances can be used without limitation.
大腸菌での発現用の転写プロモーターとして、例えばトリプトファン合成酵素(trp)、ラクトースオペロン(lac)、あるいはこれらを融合したtacおよびtrcプロモーター、λファージPLおよびPRプロモーター、T7ファージのプロモーター等が例として挙げられる。しかし、プロモーターが強力すぎると目的タンパク質の大腸菌内における発現が過多となり、その結果、目的タンパク質が封入体を形成しやすくなり、その後の分離および精製工程が困難となる。ゆえに可溶性画分への発現あるいは培養上清中へのタンパク質の分泌を可能にすべく最適なプロモーターを選択する必要があり、このような点を考慮するとトリプトファンプロモーター(trp)が望ましい。トリプトファンプロモーター(trp)を有する発現ベクターとしては、pTRP(Clinica Chimica Acta 237,43-58(1995))等が挙げられる。 Examples of transcription promoters for expression in Escherichia coli include, for example, tryptophan synthase (trp), lactose operon (lac), or tac and trc promoters obtained by fusing them, λ phage PL and PR promoters, and T7 phage promoters. Can be However, if the promoter is too strong, the expression of the target protein in Escherichia coli becomes excessive, and as a result, the target protein tends to form inclusion bodies, and the subsequent separation and purification steps become difficult. Therefore, it is necessary to select an optimal promoter so as to enable expression in the soluble fraction or secretion of the protein into the culture supernatant. In view of such points, the tryptophan promoter (trp) is preferable. Examples of an expression vector having a tryptophan promoter (trp) include pTRP (Clinica Chimica Acta 237, 43-58 (1995)) and the like.
選択マーカーの例としては、ホルムアルデヒド耐性マーカー、カナマイシン、アンピシリン、テトラサイクリン、クロラムフェニコールなどの薬剤耐性マーカー、ロイシン、ヒスチジン、リジン、メチオニン、アルギニン、トリプトファン、ウラシルなどの栄養要求性マーカーが挙げられるがこれに限定されない。 Examples of selectable markers include formaldehyde resistance markers, drug resistance markers such as kanamycin, ampicillin, tetracycline, chloramphenicol, and auxotrophic markers such as leucine, histidine, lysine, methionine, arginine, tryptophan, uracil. It is not limited to this.
一般的な組換えベクターの構築方法としては、例えば、PCR法等で調製した遺伝子断片を、適当な制限酵素とリガーゼを用いて組換えベクターに組み込む方法が挙げられる。好ましくは市販のライゲーションキット、例えばLigation high（東洋紡社製）を用いて、規定の条件にてライゲーション反応を行うことにより組換えベクターを得ることができる。また、これらのベクターを、必要であればボイル法、アルカリSDS法、磁性ビーズ法およびそれらの原理を使用した市販されているキット等により精製し、さらにエタノール沈殿法、ポリエチレングリコール沈殿法などの濃縮手段により濃縮することができる。 As a general method of constructing a recombinant vector, for example, a method of incorporating a gene fragment prepared by a PCR method or the like into a recombinant vector using an appropriate restriction enzyme and ligase can be mentioned. Preferably, a recombinant vector can be obtained by performing a ligation reaction under specified conditions using a commercially available ligation kit, for example, Ligation high (manufactured by Toyobo). Further, if necessary, these vectors are purified by a boil method, an alkaline SDS method, a magnetic bead method, or a commercially available kit using the principle thereof, and further concentrated by an ethanol precipitation method, a polyethylene glycol precipitation method, or the like. It can be concentrated by means.
遺伝子の導入方法としては、特に制限されないが、例えば、電気パルス法、コンピテントセル法、塩化カルシウム法、プロトプラスト法、パーティクルガン法、エレクトロポレーション法等を挙げることができる。具体的には、大腸菌への遺伝子の導入には、ハナハンの方法等を利用でき、酵母への遺伝子の導入には、リチウムイオン法等を利用できる。 The method for introducing the gene is not particularly limited, and examples thereof include an electric pulse method, a competent cell method, a calcium chloride method, a protoplast method, a particle gun method, and an electroporation method. Specifically, the Hanahan's method can be used to introduce a gene into Escherichia coli, and the lithium ion method can be used to introduce a gene into yeast.
相同組換えによってゲノム上の任意の位置に目的の遺伝子を挿入する方法は、ゲノム上の配列と相同な配列に目的遺伝子をプロモーターとともに挿入し、このDNA断片をエレクトロポレーションによって細胞内に導入して相同組換えを起こさせることにより実施できる。ゲノムへの導入の際には目的遺伝子と選択マーカー遺伝子を連結したDNA断片を用いると容易に相同組換えが起こった株を選抜することができる。また、薬剤耐性遺伝子と特定の条件下で致死的になる遺伝子を連結した遺伝子をゲノム上に上記の方法で相同組換えによって挿入し、その後、薬剤耐性遺伝子と特定の条件下で致死的になる遺伝子を置き換える形で目的遺伝子を相同組換えを利用して導入することもできる。 The method of inserting a gene of interest into an arbitrary position on the genome by homologous recombination involves inserting the gene of interest into a sequence homologous to a sequence on the genome together with a promoter, and introducing this DNA fragment into cells by electroporation. To cause homologous recombination. At the time of introduction into the genome, a strain in which homologous recombination has occurred can be easily selected by using a DNA fragment obtained by linking the target gene and the selectable marker gene. In addition, a gene obtained by linking a drug resistance gene and a gene that is lethal under specific conditions is inserted into the genome by homologous recombination by the above-described method, and then, the drug resistance gene and the gene that becomes lethal under specific conditions The target gene can be introduced using homologous recombination in a form that replaces the gene.
一般的に形質転換体を利用する目的物の生産方法において認められることであるが、本発明のパントテン酸キナーゼにおいても、導入遺伝子の選択、導入すべき宿主の選択、発現ベクターの導入手段とそれに適したDNAまたはRNAの構築方法の選択、培地もしくはこれに対する添加物の種類や濃度の選択、形質転換体の培養条件または生育条件の選択等の要因が、パントテン酸キナーゼの生産量に影響する場合がある。 As generally observed in a method for producing a target substance using a transformant, also in the pantothenate kinase of the present invention, selection of a transgene, selection of a host to be introduced, means for introducing an expression vector, and When factors such as selection of a suitable DNA or RNA construction method, selection of the type or concentration of a medium or an additive thereto, and selection of culture conditions or growth conditions of a transformant affect pantothenate kinase production. There is.
上記形質転換体を培地で培養する方法は、宿主の培養に用いられる通常の方法に従って行われる。大腸菌や酵母菌等の微生物を宿主として得られた形質転換体を培養する培地としては、微生物が資化し得る炭素源、窒素源、無機塩類等を含有し、形質転換体の培養を効率的に行うことができる培地であれば、天然培地、合成培地のいずれを用いてもよい。 The method for culturing the above transformant in a medium is performed according to a usual method used for culturing a host. The culture medium for culturing the transformant obtained by using a microorganism such as Escherichia coli or yeast as a host contains a carbon source, a nitrogen source, and inorganic salts that can be used by the microorganism to efficiently culture the transformant. Either a natural medium or a synthetic medium may be used as long as the medium can be performed.
炭素源としては資化可能な炭素化合物であればよく、例えば、グリセリンなどのポリオール類、またはピルビン酸、コハク酸もしくはクエン酸等の有機酸類を使用することができる。また、窒素源としては利用可能な窒素化合物であればよく、例えば、ペプトン、肉エキス、酵母エキス、カゼイン加水分解物、大豆粕アルカリ抽出物、またはアンモニアもしくはその塩などを使用することができる。その他、リン酸塩、炭酸塩、硫酸塩、マグネシウム、カルシウム、カリウム、鉄、マンガン、亜鉛などの塩類、特定のアミノ酸、特定のビタミン、消泡剤なども必要に応じて使用してもよい。また、イソプロピル−β−Ｄ−チオガラクトピラノシドなどのタンパク質発現誘導剤を必要に応じて培地に添加してもよい。 The carbon source may be any carbon compound that can be assimilated, and for example, a polyol such as glycerin or an organic acid such as pyruvic acid, succinic acid or citric acid can be used. The nitrogen source may be any nitrogen compound that can be used, and examples thereof include peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkaline extract, and ammonia or a salt thereof. In addition, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, antifoaming agents, and the like may be used as necessary. Further, a protein expression inducer such as isopropyl-β-D-thiogalactopyranoside may be added to the medium as needed.
培養は、通常、振盪培養または通気攪拌培養などの好気的条件下、好ましくは0〜40℃、より好ましくは10〜37℃、特に好ましくは15〜37℃で培養を行う。培養期間中、培地のpHは宿主の発育が可能で、生産されたパントテン酸キナーゼの活性が損なわれない範囲で適宜変更することができるが、好ましくはpH 4〜8程度の範囲である。pHの調整は、無機または有機酸、アルカリ溶液等を用いて行う。培養中は必要に応じてアンピシリンやテトラサイクリン等の抗生物質を培地に添加してもよい。 The cultivation is usually performed under aerobic conditions such as shaking cultivation or aeration and stirring cultivation, preferably at 0 to 40 ° C, more preferably 10 to 37 ° C, particularly preferably 15 to 37 ° C. During the culture period, the pH of the medium can be appropriately changed within a range in which the host can grow and the activity of the produced pantothenate kinase is not impaired, but is preferably in a range of about pH 4 to 8. Adjustment of the pH is performed using an inorganic or organic acid, an alkaline solution or the like. During culture, antibiotics such as ampicillin and tetracycline may be added to the medium as needed.
続いて、形質転換体の培養により可溶化発現したパントテン酸キナーゼを、培養物から採取する。培養物には、培養液、培養上清、培養細胞、培養菌体、細胞または菌体の破砕物が包含される。採取方法は、通常行われる細胞または菌体の破砕物からの抽出だけでなく、場合によっては、適当な抽出溶媒を用いて培養液からも直接抽出できる。利用する宿主の種類によっては、パントテン酸キナーゼの少なくとも一部が宿主細胞内または細胞表面に止まる場合があるが、細胞膜または細胞壁の破壊や、適宜な抽出溶媒による抽出等の公知の各種操作を経て、採取することができる。 Subsequently, the pantothenate kinase solubilized and expressed by culturing the transformant is collected from the culture. The culture includes a culture solution, a culture supernatant, a cultured cell, a cultured microbial cell, and a crushed cell or microbial cell. The method of collection is not limited to extraction from a crushed cell or cells, which is usually performed, and in some cases, can be directly extracted from a culture solution using an appropriate extraction solvent. Depending on the type of the host used, at least a part of the pantothenate kinase may be stopped in the host cell or on the cell surface, but through various known operations such as destruction of the cell membrane or cell wall and extraction with an appropriate extraction solvent. , Can be collected.
宿主の対数増殖期を過ぎたときに上記温度範囲に設定することでパントテン酸キナーゼ遺伝子が発現し、宿主内に非常に高い比活性を示すパントテン酸キナーゼを製造することができる。培養後、目的のパントテン酸キナーゼが宿主内に生産されるため菌体または細胞を破砕し、粗酵素懸濁液を調製する。この粗酵素懸濁液には、非常に多くのパントテン酸キナーゼが含まれる。したがって、得られた粗酵素懸濁液をそのまま利用してもよい。なお、得られた粗酵素懸濁液から熱処理によってパントテン酸キナーゼを簡易精製することもできる。また、タンパク質の単離精製に用いられる一般的な生化学的方法、例えば、硫酸アンモニウム沈殿、ゲルクロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー等を単独でまたは適宜組み合わせても単離精製することができる。単離精製されたパントテン酸キナーゼは、所定のpHの緩衝液等に懸濁された状態で利用することができる。 When the temperature is set within the above-mentioned temperature range after the logarithmic growth phase of the host, the pantothenate kinase gene is expressed, and pantothenate kinase having extremely high specific activity in the host can be produced. After the culture, the cells or cells are disrupted to produce the desired pantothenate kinase in the host, thereby preparing a crude enzyme suspension. This crude enzyme suspension contains a great deal of pantothenate kinase. Therefore, the obtained crude enzyme suspension may be used as it is. In addition, pantothenate kinase can be easily purified from the obtained crude enzyme suspension by heat treatment. Further, general biochemical methods used for isolation and purification of proteins, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., can be used alone or in combination with one another as appropriate. it can. The isolated and purified pantothenate kinase can be used in a state of being suspended in a buffer having a predetermined pH or the like.
本発明のパントテン酸キナーゼ(GbPanKおよびTtPanK)は、パントテン酸(pantothenate)をホスホパントテン酸(phosphopantothenate)に変換する反応を触媒する。パントテン酸からCoAに至る反応経路の他の反応を触媒する酵素と組合せて用いることにより、本発明のパントテン酸キナーゼを用いてCoA等のパントテン酸誘導体を生産することができる。パントテン酸からCoAに至る反応経路を図１に示す。パントテン酸誘導体として、ホスホパントテン酸（phosphopantothenate）、ホスホパントテノイルシステイン（phosphopantothenoyl-cysteine)、ホスホパンテテイン（phosphopantetheine）、デホスホCoA（dephospho CoA）、コエンザイムＡが挙げられる。ホスホパントテン酸をホスホパントテノイルシステインに変換する酵素として、ホスホパントテン酸-システインリガーゼ（phosphopantothenate-cysteine ligase：CoaB）が挙げられ、ホスホパントテノイルシステインをホスホパンテテインに変換する酵素として、ホスホパントテノイルシステインデカルボキシラーゼ（phosphopantothenoylcysteine decarboxylase：CoaC）が挙げられ、ホスホパンテテインをデホスホCoAに変換する酵素として、パンテテインリン酸アデニリルトランスフェラーゼ（pantetheine-phosphate adenylyltransferase）(PA)が挙げられ、デホスホCoAをCoAに変換する酵素として、デホスホCoAキナーゼ（dephospho-CoA kinase） (DCK)が挙げられる。すなわち、本発明のパントテン酸キナーゼGbPanKまたはTtPanKとこれらの酵素を組合せて用いることにより、CoA等のパントテン酸誘導体を生産することができる。すなわち、図７に示すように、D-パントテン酸をPanKによりD-ホスホパントテン酸に変換し、次いでホスホパントテン酸-システインリガーゼ（CoaB)によりD-ホスホパントテノイル-L-システインに変換し、次いでホスホパントテノイルシステインデカルボキシラーゼ（CoaC）により、D-ホスホパンテテインに変換し、次いでパンテテインリン酸アデニリルトランスフェラーゼ（PA)によりデホスホCoAに変換し、次いでデホスホCoAキナーゼ（DCK)によりCoAに変換する。 The pantothenate kinases (GbPanK and TtPanK) of the present invention catalyze the reaction of converting pantothenate to phosphopantothenate. When used in combination with an enzyme that catalyzes another reaction in the reaction pathway from pantothenic acid to CoA, a pantothenic acid derivative such as CoA can be produced using the pantothenate kinase of the present invention. The reaction route from pantothenic acid to CoA is shown in FIG. Examples of the pantothenic acid derivative include phosphopantothenic acid, phosphopantothenoyl-cysteine, phosphopantetheine, dephospho CoA, and coenzyme A. Examples of an enzyme that converts phosphopantothenic acid to phosphopantothenoyl cysteine include phosphopantothenate-cysteine ligase (CoaB), and an enzyme that converts phosphopantothenoyl cysteine to phosphopantethein is Enzymes that convert phosphopantethein to dephosphoCoA include pantetheine-phosphate adenylyltransferase (PA), and dephosphoCoA. Enzymes that convert phosphopantethein to dephosphoCoA include, for example, totenoylcysteine decarboxylase (CoaC). Dephospho-CoA kinase (DCK) is an example of an enzyme that converts A to CoA. That is, a pantothenic acid derivative such as CoA can be produced by using the pantothenate kinase GbPanK or TtPanK of the present invention in combination with these enzymes. That is, as shown in FIG. 7, D-pantothenic acid is converted to D-phosphopantotenoic acid by PanK, and then to D-phosphopantotenoyl-L-cysteine by phosphopantothenate-cysteine ligase (CoaB), It is then converted to D-phosphopantethein by phosphopantotenoyl cysteine decarboxylase (CoaC), then to dephospho-CoA by pantetheine phosphate adenylyltransferase (PA), and then to CoA by dephospho-CoA kinase (DCK). Convert to
この際、ホスホパントテン酸-システインリガーゼ（CoaB)及びホスホパントテノイルシステインデカルボキシラーゼ（CoaC）として、Methanocaldococcus jannaschii（メタノカルドコックス・ヤンナスキイ）由来のものを用いることができる。また、パンテテインリン酸アデニリルトランスフェラーゼ（PA)及びデホスホCoAキナーゼ（DCK)として、T. thermophilus（サーマス・サーモフィルス）HB8株由来のものを用いることができる。 At this time, those derived from Methanocaldococcus jannaschii (Methanocardococcus jannaschii) can be used as phosphopantothenic acid-cysteine ligase (CoaB) and phosphopantothenoyl cysteine decarboxylase (CoaC). Further, as the pantetheine phosphate adenylyltransferase (PA) and dephospho-CoA kinase (DCK), those derived from the T. thermophilus (Thermus thermophilus) HB8 strain can be used.
用いる酵素は、上記の微生物から精製して用いてもよいし、大腸菌や動物細胞により作製したリコンビナント酵素を用いてもよい。M. jannaschiiは本来、CoaB、CoaCの2つの機能ドメインがひとつのポリペプチド鎖としてコードされた融合遺伝子を有するが、CoaB（C末側）及びCoaC（N末側）として、それぞれのドメインのリコンビナントタンパク質を用いることができる。また、大腸菌を用いてリコンビナント酵素を作製する場合、コドンを大腸菌用に最適化することが好ましい。 The enzyme to be used may be purified from the above-mentioned microorganism and used, or a recombinant enzyme produced by Escherichia coli or animal cells may be used. M. jannaschii originally has a fusion gene in which two functional domains, CoaB and CoaC, are encoded as a single polypeptide chain, but as a CoaB (C-terminal) and CoaC (N-terminal), the respective domains are recombinant. Proteins can be used. When a recombinant enzyme is prepared using Escherichia coli, it is preferable to optimize codons for Escherichia coli.
配列番号９にCoaB、CoaCの2つの機能ドメインがひとつのポリペプチド鎖としてコードされた融合遺伝子の発現産物のアミノ酸配列を、配列番号１０にCoaCドメイン（N末端）のアミノ酸配列を、配列番号１１にCoaBドメイン（C末端）のアミノ酸配列を示す。また、配列番号１２に本来、CoaB、CoaCの2つの機能ドメインをひとつのポリペプチド鎖としてコードするM. jannaschiiのゲノムDNAの配列を示し、配列番号１３にCoaCドメインをコードするゲノムDNAの配列を示し、配列番号１４にCoaBドメインをコードするゲノムDNAの配列を示す。さらに、配列番号１５にCoaB、CoaCの2つの機能ドメインをひとつのポリペプチド鎖としてコードするDNAを大腸菌用にコドンを最適化した配列を示し、配列番号１６にCoaCドメインをコードするDNAを大腸菌用にコドンを最適化した配列を示し、配列番号１７にCoaBドメインをコードするDNAを大腸菌用にコドンを最適化した配列を示す。さらに、配列番号１８及び１９に、それぞれパンテテインリン酸アデニリルトランスフェラーゼ（PA)のDNA配列及びアミノ酸配列を示し、配列番号２０及び２１に、それぞれデホスホCoAキナーゼ（DCK)のDNA配列及びアミノ酸配列を示す。 SEQ ID NO: 9 shows the amino acid sequence of the expression product of the fusion gene in which two functional domains of CoaB and CoaC are encoded as one polypeptide chain, SEQ ID NO: 10 shows the amino acid sequence of the CoaC domain (N-terminal), and SEQ ID NO: 11 Shows the amino acid sequence of the CoaB domain (C-terminal). In addition, SEQ ID NO: 12 shows the sequence of genomic DNA of M. jannaschii which originally encodes two functional domains of CoaB and CoaC as one polypeptide chain, and SEQ ID NO: 13 shows the sequence of genomic DNA encoding the CoaC domain. SEQ ID NO: 14 shows the sequence of genomic DNA encoding the CoaB domain. Further, SEQ ID NO: 15 shows a sequence obtained by codon-optimizing DNA encoding two functional domains of CoaB and CoaC as one polypeptide chain, and SEQ ID NO: 16 shows DNA encoding a CoaC domain for E. coli. Shows a sequence obtained by optimizing a codon, and SEQ ID NO: 17 shows a sequence obtained by optimizing a DNA encoding a CoaB domain for Escherichia coli. Further, SEQ ID NOs: 18 and 19 show the DNA sequence and amino acid sequence of pantetheine phosphate adenylyltransferase (PA), respectively, and SEQ ID NOs: 20 and 21 show the DNA sequence and amino acid sequence of dephospho-CoA kinase (DCK), respectively Is shown.
本発明のパントテン酸キナーゼは、熱安定性に優れている。例えば、本発明のパントテン酸キナーゼを60℃で２時間インキュベートした場合であっても、インキュベート前の活性に対して、80％以上、好ましくは90％以上、さらに好ましくは93％以上、特に好ましくは95％以上の活性を有している。また、70℃で２時間インキュベートした場合であっても、インキュベート前の活性に対して、85％以上、好ましくは90％以上、さらに好ましくは93％以上、特に好ましくは95％以上の活性を有している。さらに、80℃で２時間インキュベートした場合であっても、インキュベート前の活性に対して、85％以上、好ましくは90％以上、さらに好ましくは93％以上、特に好ましくは95％以上の活性を有している。 The pantothenate kinase of the present invention is excellent in thermostability. For example, even when the pantothenate kinase of the present invention is incubated at 60 ° C. for 2 hours, its activity before incubation is at least 80%, preferably at least 90%, more preferably at least 93%, particularly preferably at least 93%. Has over 95% activity. In addition, even when incubated at 70 ° C. for 2 hours, it has an activity of 85% or more, preferably 90% or more, more preferably 93% or more, and particularly preferably 95% or more of the activity before incubation. are doing. Furthermore, even when incubated at 80 ° C. for 2 hours, it has an activity of 85% or more, preferably 90% or more, more preferably 93% or more, and particularly preferably 95% or more, of the activity before incubation. are doing.
なお、パントテン酸キナーゼの活性は、例えば、D-パントテン酸のリン酸化に伴うATP消費量とADP蓄積量をHPLCにて定量することにより測定することができる。 The activity of pantothenate kinase can be measured, for example, by quantifying ATP consumption and ADP accumulation due to phosphorylation of D-pantothenic acid by HPLC.
通常、パントテン酸キナーゼの活性は、上記経路の最終生産物であるCoAによりフィードバック阻害されるが、本発明のGbPanKおよびTtPanKは、CoAのフィードバック阻害を受けない。従って、PanKおよびCoA生産に関わる他の酵素を混合して用いてCoAを生産する場合であっても、CoAによるフィードバック阻害を受けないので、CoAを高率的に生産することができる。また、本発明のGbPanKおよびTtPanKは、アセチルCoAによってもフィードバック阻害を受けない。 Normally, the activity of pantothenate kinase is feedback inhibited by CoA, the final product of the above pathway, but GbPanK and TtPanK of the present invention are not affected by feedback inhibition of CoA. Therefore, even when CoA is produced using a mixture of PanK and other enzymes involved in CoA production, CoA can be produced at a high rate because feedback is not inhibited by CoA. GbPanK and TtPanK of the present invention are not subject to feedback inhibition even by acetyl-CoA.
本発明は、本発明のパントテン酸キナーゼを用いて、パントテン酸誘導体を生産する方法を包含する。さらに、CoA合成経路に関与する他の酵素と組合せてCoAを生産する方法を包含する。 The present invention includes a method for producing a pantothenic acid derivative using the pantothenic acid kinase of the present invention. Additionally, it encompasses methods of producing CoA in combination with other enzymes involved in the CoA synthesis pathway.
この際、好熱性細菌から単離したGbPanKまたはTtPanKを基質と混合して酵素反応を起こさせてもよい。また、GbPanKまたはTtPanKをコードするDNAを発現ベクターに挿入し、該発現ベクターを宿主細胞に導入し、該宿主細胞を基質と混合し、GbPanKまたはTtPanKを発現させて、酵素反応を起こさせてもよい。この際、基質としては、パントテン酸の他、ATPやL-システインも適宜混合する。 At this time, GbPanK or TtPanK isolated from a thermophilic bacterium may be mixed with a substrate to cause an enzymatic reaction. Alternatively, the DNA encoding GbPanK or TtPanK may be inserted into an expression vector, the expression vector may be introduced into a host cell, the host cell may be mixed with a substrate, and GbPanK or TtPanK may be expressed to cause an enzymatic reaction. Good. At this time, as a substrate, ATP and L-cysteine are appropriately mixed in addition to pantothenic acid.
さらに、CoAの生産に関与する他の酵素をコードするDNAを宿主細胞に導入し、それらの酵素を共発現させ、図１に示す一連の反応を起こさせ、CoAを生産することもできる。
生産したCoAは、公知のクロマトグラフィー等の手法により精製することができる。
In addition, DNA encoding other enzymes involved in the production of CoA can be introduced into host cells, the enzymes can be co-expressed, and a series of reactions shown in FIG. 1 can be performed to produce CoA.
The produced CoA can be purified by a known technique such as chromatography.
本発明を以下の実施例によって具体的に説明するが、本発明はこれらの実施例によって限定されるものではない。 The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
実施例１ 好熱性細菌からのPanKの単離
［方法］
（使用微生物とプラスミド)
H. pylori、P. aeruginosa (Brand and Strauss, J Biol Chem 280:20185-20188頁, 2005年)およびT. maritima (Shimosaka等, J Bacteriol 198:1993-2000頁, 2016年)由来の既知のtype III PanKのアミノ酸配列をクエリとしたBLAST検索により、本発明者等が保有する好熱菌のうち、Geobacillus sp. 30株(Tsuji等, Appl Microbiol Biotechnol 98:5925-5935頁, 2014年)およびThermus thermophilus HB8株(Oshima等, Microbiol. 76:18-27頁, 1974年)よりtype III PanKと推定されるホモログ酵素（それぞれGbPanK、TtPanKとする）遺伝子を見出した。
Example 1 Isolation of PanK from a thermophilic bacterium [Method]
(Microorganism used and plasmid)
Known types from H. pylori, P. aeruginosa (Brand and Strauss, J Biol Chem 280: 20185-20188, 2005) and T. maritima (Shimosaka et al., J Bacteriol 198: 1993-2000, 2016) By BLAST search using the amino acid sequence of III PanK as a query, among the thermophiles owned by the present inventors, Geobacillus sp. 30 strain (Tsuji et al., Appl Microbiol Biotechnol 98: 5925-5935, 2014) and Thermus From the thermophilus HB8 strain (Oshima et al., Microbiol. 76: 18-27, 1974), homologous enzyme genes (GbPanK and TtPanK, respectively) putatively identified as type III PanK were found.
GbPanK遺伝子は、Geobacillus sp. 30株のゲノムDNAをテンプレートとし、AGTCTACATATGATTTTTGTGTTAGATGTT（配列番号５）(下線はNdeI認識配列）およびACGGTCCTCGAGTCATTGTTTTTTATCTACATT（配列番号６）（下線はXhoI認識配列）に示すプライマーを用いたPCRにより取得した。これらのプライマーの５’末端には制限酵素NdeI、XhoIの認識配列が付加されている。得られたPCR産物をNdeI、XhoI消化し、pET21aの同サイトに連結した。 The GbPanK gene was prepared using the genomic DNA of the Geobacillus sp. Strain 30 as a template and primers shown in AGTCTA CATATG ATTTTTGTGTTAGATGTT (SEQ ID NO: 5) (underlined NdeI recognition sequence) and ACGGTC CTCGAG TCATTGTTTTTTATCTACATT (SEQ ID NO: 6) (underlined XhoI recognition sequence) Was obtained by PCR using Recognition sequences for restriction enzymes NdeI and XhoI are added to the 5 'end of these primers. The obtained PCR product was digested with NdeI and XhoI and ligated to the same site of pET21a.
TtPanK発現ベクターは、T. thermophilus HB8株のゲノムDNAをテンプレートとし、AGTCTACATATGCTCCTCGCGGTGGACATC（配列番号７）（下線はNdeI認識配列）およびACGGGATCCTCACCCTCGCCCCAGGTG（配列番号８）（下線はBamHI認識配列）に示すプライマーを用いたPCRにより取得した。これらのプライマーの５’末端には制限酵素NdeI、BamHIの認識配列が付加されている。得られたPCR産物をNdeI、BamHI消化し、pET21aの同サイトに連結した。 The TtPanK expression vector is represented by AGTCTA CATATG CTCCTCGCGGTGGACATC (SEQ ID NO: 7) (underlined NdeI recognition sequence) and ACG GGATCC TCACCCTCGCCCCCCAGGTG (underlined BamHI recognition sequence) using genomic DNA of T. thermophilus HB8 as a template. Obtained by PCR using primers. Recognition sequences for restriction enzymes NdeI and BamHI are added to the 5 'end of these primers. The obtained PCR product was digested with NdeI and BamHI and ligated to the same site of pET21a.
以上のとおり作製されたTtPanK、GbPanK発現用プラスミドをそれぞれpET-TtPanK、pET-GbPanKとした。 The TtPanK and GbPanK expression plasmids prepared as described above were designated as pET-TtPanK and pET-GbPanK, respectively.
次いで、GbPanK、TtPanKのそれぞれのN末端にヘキサヒスチジン(His6)タグを付した融合タンパク質の発現ベクターを作製した。具体的には、GbPanKについては配列番号５および６のプライマー（GbPanK-F、GbPanK-R）を、およびTtPanKについては配列番号７および配列番号８のプライマー（TtPanK-F、TtPanK-R)を用いたPCRによりGbPanK遺伝子およびTtPanK遺伝子を増幅した。GbPanK遺伝子は、NdeI、XhoIで、TtPanK遺伝子はNdeI、BamHIで消化し、pET19bの該当するサイトに連結した。得られたプラスミドをそれぞれpET-His6-GbPanK、pET-His6-TtPanKとする。 Next, an expression vector for a fusion protein in which a hexahistidine (His 6 ) tag was added to the N-terminus of each of GbPanK and TtPanK was prepared. Specifically, primers of SEQ ID NOS: 5 and 6 (GbPanK-F, GbPanK-R) are used for GbPanK, and primers (TtPanK-F, TtPanK-R) of SEQ ID NOs: 7 and 8 are used for TtPanK. GbPanK gene and TtPanK gene were amplified by PCR. The GbPanK gene was digested with NdeI and XhoI, and the TtPanK gene was digested with NdeI and BamHI, and ligated to the corresponding site of pET19b. The resulting plasmids are referred to as pET-His 6 -GbPanK and pET-His 6 -TtPanK, respectively.
構築した各プラスミドは、Escherichia coli Rosetta 2 (DE3)に導入し、発現株を作製した。組換え大腸菌の培養は、100μg mL-1アンピシリン、30μg mL-1クロラムフェニコールを含むLuria Bertani (LB) 培地を使用し、37℃で好気的に行った。対数増殖後期の培養液に終濃度0.2 mMのisopropyl β-D-1-thiogalactopyranoside (IPTG)を添加することで目的遺伝子の発現を誘導し、さらに3時間の培養を行った。 Each of the constructed plasmids was introduced into Escherichia coli Rosetta 2 (DE3) to prepare an expression strain. The recombinant Escherichia coli was cultured aerobically at 37 ° C. using a Luria Bertani (LB) medium containing 100 μg mL- 1 ampicillin and 30 μg mL- 1 chloramphenicol. The expression of the target gene was induced by adding 0.2 mM final concentration of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture medium at the late stage of logarithmic growth, and the cells were further cultured for 3 hours.
（酵素調製)
培養後の組換え大腸菌を8,000 rpm、2分間の遠心分離にて回収した。得られた湿菌体を200 mg wet cells mL-1程度になるように50 mM Tris-HCl (pH7.5)に再懸濁し、超音波破砕を行った。破砕後、15,000 rpm、4℃、5分間の遠心分離を行い、得られた上清を70℃、30分間の熱処理に供した。再度、15,000 rpm、4℃、5分間の遠心分離を行い、変性タンパク質を取り除き、得られた上清を熱処理粗酵素液として得た。
(Enzyme preparation)
The cultured E. coli was recovered by centrifugation at 8,000 rpm for 2 minutes. The obtained wet cells were resuspended in 50 mM Tris-HCl (pH 7.5) to about 200 mg wet cells mL −1 and sonicated. After crushing, centrifugation was performed at 15,000 rpm at 4 ° C. for 5 minutes, and the obtained supernatant was subjected to a heat treatment at 70 ° C. for 30 minutes. Again, centrifugation was performed at 15,000 rpm at 4 ° C. for 5 minutes to remove denatured proteins, and the obtained supernatant was obtained as a heat-treated crude enzyme solution.
His6タグを付加したPanKはAKTA purifierを用いたニッケル（Ni）アフィニティカラムクロマトグラフィーによる精製に供した。HisTrap HP (1 mL)カラムを2 mLの500 mM NaCl、30 mMイミダゾールを含む50 mM Tris-HCl (pH7.5)で平衡化し、熱処理を行った酵素液をマウントした。上記の溶離液5 CV(カラム体積）で非吸着タンパク質を洗い流した後、20 CVの溶離液中でイミダゾール濃度を30 mMから500 mMまで直線的にあげるグラジエント溶出で目的タンパク質を回収した。 PanK with His 6 tag added was subjected to purification by nickel (Ni) affinity column chromatography using AKTA purifier. The HisTrap HP (1 mL) column was equilibrated with 2 mL of 50 mM Tris-HCl (pH 7.5) containing 500 mM NaCl and 30 mM imidazole, and the heat-treated enzyme solution was mounted. After the non-adsorbed protein was washed away with 5 CV (column volume) of the above eluent, the target protein was recovered by gradient elution in which the imidazole concentration was linearly increased from 30 mM to 500 mM in the eluent of 20 CV.
[結果と考察]
GbPanK、TtPanK遺伝子のそれぞれをpETベクターのT7プロモーター制御下に組み込み、大腸菌内で発現させた。この際、IPTGによる発現誘導後の培養温度を20℃、37℃の2通りで行い、SDS-PAGEにて可溶性画分中の目的酵素生産量の比較を行った。His6タグ付加のないPanKの場合、由来と培養温度の別によらず目的酵素の推定分子量（約28 kDa）付近に目立ったバンドは見られず酵素の発現を確認することができなかった。これに対し、N 末端にHis6タグを付した場合、いずれのケースにおいても28 kDa付近に空ベクター保持株（Null）には見られないバンドが確認できた。以上の結果から、GbPanK、TtPanKの大腸菌内での発現にはN末端へのHis6タグの付加が有効であることが示された。
[Results and discussion]
Each of the GbPanK and TtPanK genes was incorporated under the control of the T7 promoter of a pET vector, and expressed in Escherichia coli. At this time, the culture temperature after expression induction by IPTG was performed in two ways, 20 ° C. and 37 ° C., and the production amount of the target enzyme in the soluble fraction was compared by SDS-PAGE. In the case of PanK without His 6 tag addition, no remarkable band was observed near the estimated molecular weight (about 28 kDa) of the target enzyme regardless of the origin and the culture temperature, and the expression of the enzyme could not be confirmed. On the other hand, when the His 6 tag was added to the N-terminus, a band not found in the empty vector-holding strain (Null) was confirmed at around 28 kDa in each case. From the above results, it was shown that the addition of a His 6 tag to the N-terminus is effective for the expression of GbPanK and TtPanK in E. coli.
実施例２ PanKの酵素学的性能評価
［方法］
酵素活性測定は、50 mM Tris-HCl (pH7.5)、5 mM ATP、10 mM MgCl2、60 mM NH4Cl、60 mM KCl、10 mM pantothenate calcium、および酵素液からなる反応液（100μL）を用いて行った。酵素液を含まない反応液を60℃で約2分間プレインキュベートした後、適量の酵素液を加え反応を開始した。酵素液添加後、60℃で30分間のインキュベートを行い、1 M HCl (Wako Pure Chemical) 20μLを加えることで反応を停止させた。反応液を15,000 rpm、4℃、5分間の遠心分離に供し、上清中のATPおよびADPをHPLCにて分析した。HPLC分析には5C18-AR-II (Nacalai Tesque, φ4.6 × 150 mm)カラムを使用し、溶出にはA溶液；20 mM tert-butyl amine (pH6.8)、B溶液; 10% (v/v) methanol in 20 mM tert-butyl amine (pH6.8)を用いた。カラム温度40℃、溶離液流速1.0 mL min-1で、B溶液の濃度を10分間で20% (v/v)から100% (v/v)に直線的に増加させるグラジエント溶出を行った。検出は溶離液の254 nmの吸収を測定することで行った。
Example 2 Evaluation of Enzymatic Performance of PanK [Method]
Enzyme activity was measured using a reaction solution (100 μL) consisting of 50 mM Tris-HCl (pH 7.5), 5 mM ATP, 10 mM MgCl 2 , 60 mM NH 4 Cl, 60 mM KCl, 10 mM pantothenate calcium, and enzyme solution. This was performed using After pre-incubating the reaction solution containing no enzyme solution at 60 ° C. for about 2 minutes, an appropriate amount of the enzyme solution was added to start the reaction. After the addition of the enzyme solution, incubation was performed at 60 ° C. for 30 minutes, and the reaction was stopped by adding 20 μL of 1 M HCl (Wako Pure Chemical). The reaction solution was subjected to centrifugation at 15,000 rpm at 4 ° C. for 5 minutes, and ATP and ADP in the supernatant were analyzed by HPLC. For HPLC analysis, a 5C18-AR-II (Nacalai Tesque, φ4.6 × 150 mm) column was used. For elution, A solution; 20 mM tert-butyl amine (pH 6.8), B solution; 10% (v / v) methanol in 20 mM tert-butyl amine (pH 6.8) was used. At a column temperature of 40 ° C. and an eluent flow rate of 1.0 mL min −1 , a gradient elution was performed in which the concentration of the B solution was linearly increased from 20% (v / v) to 100% (v / v) in 10 minutes. Detection was performed by measuring the absorption at 254 nm of the eluent.
[結果と考察]
（比活性)
PanK活性測定は、パントテン酸を基質とした際のADP生成量（およびATP消費量）をHPLCで定量することにより行った。アッセイには、カラム精製後のGbPanK、TtPanKを使用した。反応に供する酵素量を適宜調整し、ADP生成速度に直線性が見られる範囲で反応の初速度を求めた。また精製酵素標品中のタンパク質濃度をBradford法で定量し、各酵素の比活性を算出した。この結果、60℃、pH7.5におけるGbPanK、TtPanKの比活性はそれぞれ3.97、2.78μmol min-1 mg-1 enzymeと求められた。
[Results and discussion]
(Specific activity)
The measurement of PanK activity was performed by quantifying the amount of ADP produced (and the amount of ATP consumed) when pantothenic acid was used as a substrate by HPLC. For the assay, GbPanK and TtPanK after column purification were used. The amount of the enzyme to be subjected to the reaction was appropriately adjusted, and the initial rate of the reaction was determined in a range where the ADP generation rate was linear. The protein concentration in the purified enzyme preparation was quantified by the Bradford method, and the specific activity of each enzyme was calculated. As a result, the specific activities of GbPanK and TtPanK at 60 ° C. and pH 7.5 were determined to be 3.97 and 2.78 μmol min −1 mg −1 enzyme, respectively.
（基礎性能評価)
（１）反応温度、pHが及ぼす影響の検証
反応温度とpHがGbPanKおよびTtPanKの活性に及ぼす影響を検証した。図２に示す各温度で30分間の反応を行いADP生成量より活性を求めた。相対活性は90℃で反応を行ったときの活性値を100%として算出した。また、図３のpH活性については、反応液のpH調整には50 mM リン酸ナトリウム緩衝液（pH 5〜8、四角シンボル）、もしくは50 mM 炭酸ナトリウム緩衝液（pH 9〜10、丸シンボル）を用いた。GbPanKはpH 6、TtPanKはpH 7で反応させたときの酵素活性を100%として相対活性を算出した。図２に温度の影響を示し、図３にpHの影響を示す。図２に示すとおり、GbPanKおよびTtPanKともに60〜90℃の範囲で優れた活性を示した。また、図３に示すとおり、pHに関しては中性付近が好適であることが認められた。
(Basic performance evaluation)
(1) Verification of effects of reaction temperature and pH The effects of reaction temperature and pH on the activity of GbPanK and TtPanK were verified. The reaction was performed for 30 minutes at each temperature shown in FIG. The relative activity was calculated assuming that the activity value when the reaction was performed at 90 ° C was 100%. Regarding the pH activity in FIG. 3, the pH of the reaction solution was adjusted using a 50 mM sodium phosphate buffer (pH 5 to 8, square symbols) or a 50 mM sodium carbonate buffer (pH 9 to 10, circle symbols). Was used. GbPanK was reacted at pH 6 and TtPanK was reacted at pH 7, and the relative activity was calculated assuming that the enzyme activity was 100%. FIG. 2 shows the effect of temperature, and FIG. 3 shows the effect of pH. As shown in FIG. 2, both GbPanK and TtPanK exhibited excellent activity in the range of 60 to 90 ° C. Further, as shown in FIG. 3, it was recognized that the pH was preferably around neutral.
（２）熱安定性の評価
図４Ａおよび図４Ｂは、それぞれ、GbPanKおよびTtPanKの熱安定性を示す。His-tag付加した各酵素をニッケルアフィニティカラムにて精製後、図４に示した温度で50 mM Tris-HCl (pH7.5)中で2時間インキュベートし、その残存活性を評価した。活性測定はD-パントテン酸のリン酸化に伴うATP消費量とADP蓄積量をHPLCにて定量することで行った。残存活性は、氷上（4℃）で同時間インキュベートした酵素の残存活性を100%とした相対残存活性で表した。図４Ａおよび図４Ｂに示すように、いずれの酵素も優れた熱安定性を有し、GbPanKでは60℃、TtPanKでは80℃までの範囲で90%以上の活性を保持できることがわかった。これはGeobacillus sp. 30が50〜60℃の範囲で最もよく生育できることに対し、Thermus thermophilus HB8は70〜75℃付近に最適生育温度を有する事実ともよく合致する。
(2) Evaluation of thermal stability FIGS. 4A and 4B show the thermal stability of GbPanK and TtPanK, respectively. After purifying the His-tagged enzyme with a nickel affinity column, the enzyme was incubated in 50 mM Tris-HCl (pH 7.5) for 2 hours at the temperature shown in FIG. 4 to evaluate the residual activity. The activity was measured by quantifying ATP consumption and ADP accumulation due to phosphorylation of D-pantothenic acid by HPLC. The residual activity was expressed as a relative residual activity when the residual activity of the enzyme incubated on ice (4 ° C.) for the same time was taken as 100%. As shown in FIG. 4A and FIG. 4B, it was found that both enzymes have excellent thermostability, and can maintain 90% or more activity in the range of 60 ° C. for GbPanK and 80 ° C. for TtPanK. This is in good agreement with the fact that Geobacillus sp. 30 can grow best in the range of 50-60 ° C, whereas Thermus thermophilus HB8 has an optimum growth temperature around 70-75 ° C.
（３）CoAおよびアセチルCoAが酵素活性に及ぼす影響の評価
図５Ａおよび図５Ｂは、それぞれ、CoAがGbPanKおよびTtPanKの活性に及ぼす影響を示し、図６Ａおよび図６Ｂは、それぞれ、アセチルCoAがGbPanKおよびTtPanKの活性に及ぼす影響を示す。His-tag付加した各酵素をニッケルアフィニティカラムにて精製後、図５および図６に示した濃度のCoA、アセチルCoA存在下での活性を評価した。相対活性はCoA、アセチルCoAをともに含まない反応液中での活性に対するパーセンテージで表した。図５および図６に示すように、GbPanKおよびTtPanKともに活性はCoAおよびアセチルCoAにより影響を受けなかった。このことは、CoAおよびアセチルCoAによるフィードバック阻害を受けなかったことを示す。
(3) Evaluation of the Effect of CoA and Acetyl-CoA on Enzyme Activity FIGS. 5A and 5B show the effect of CoA on the activity of GbPanK and TtPanK, respectively. FIGS. 6A and 6B show the effect of acetyl-CoA on GbPanK, respectively. And the effect on TtPanK activity. After purifying each of the His-tagged enzymes with a nickel affinity column, the activity in the presence of CoA and acetyl-CoA at the concentrations shown in FIGS. 5 and 6 was evaluated. The relative activity was expressed as a percentage of the activity in a reaction solution containing neither CoA nor acetyl-CoA. As shown in FIGS. 5 and 6, the activity of both GbPanK and TtPanK was not affected by CoA and acetyl-CoA. This indicates that there was no feedback inhibition by CoA and acetyl-CoA.
実施例３ コエンザイムA(CoA)のin vitro合成
１．酵素の調製
（１）CoAのin vitro合成に用いた酵素を表１に示す。
Example 3 In Vitro Synthesis of Coenzyme A (CoA) Preparation of enzymes (1) The enzymes used for the in vitro synthesis of CoA are shown in Table 1.
（２）PanKの取得
PanK発現株は実施例１と同様に、pET19bを用いてN末端にHisタグを付加したベクターを作製し、E. coli Rosetta 2 (DE3)を宿主とした。
(2) PanK acquisition
For the PanK expression strain, as in Example 1, a vector having a His tag added to the N-terminus using pET19b was prepared, and E. coli Rosetta 2 (DE3) was used as a host.
（３）Phosphopantothenate-cysteine ligase（ホスホパントテン酸-システインリガーゼ） (CoaB), phosphopantothenoylcysteine decarboxylase（ホスホパントテン酸-システインデカルボキシラーゼ）(CoaC)の取得方法
Thomas K., Wolfgang S., J Biol Chem (2006) 281: 5435-5444を参考にMethanocaldococcus jannaschii由来のCoaBCを、CoaB及びCoaCの各ドメインごとに別々にクローニングし、大腸菌内で発現させた。遺伝子は両端に制限酵素NdeIとBamHI認識配列を付加した合成DNA（大腸菌用にコドン最適化）を用いた。ベクターはpET21aを用い、宿主としてはE. coli BL21 (DE3)を用いた。
(3) Method for obtaining Phosphopantothenate-cysteine ligase (CoaB), phosphopantothenoylcysteine decarboxylase (CoaC)
CoaBC derived from Methanocaldococcus jannaschii was separately cloned for each domain of CoaB and CoaC with reference to Thomas K., Wolfgang S., J Biol Chem (2006) 281: 5435-5444, and expressed in Escherichia coli. As the gene, a synthetic DNA (codon optimized for Escherichia coli) having restriction enzymes NdeI and BamHI recognition sequences added to both ends was used. PET21a was used as a vector, and E. coli BL21 (DE3) was used as a host.
（前培養)
5 mLのLB培地（100μg/mLアンピシリン、PanK発現株にはさらに30μg/mLクロラムフェニコールを添加）を含む試験管内に、それぞれの組換え株のコロニーを植菌し、37℃で一晩振とう培養した。
(Pre-culture)
Inoculate the colonies of each recombinant strain in a test tube containing 5 mL of LB medium (100 μg / mL ampicillin, and 30 μg / mL chloramphenicol for PanK expression strain) and incubate at 37 ° C. overnight. The cells were cultured with shaking.
（本培養)
500 mLフラスコ内に100 mL LB培地（100μg/mLアンピシリン、PanK発現株にはさらに30 g/mLクロラムフェニコールを添加）。上述の前培養液を5 mL添加し、3時間、37℃で振とう培養した。その後100 mM IPTGを100μL加え、再度37℃で3時間インキュベートした。集菌は8,000 rpm、5分間の遠心分離により行った。
(Main culture)
100 mL LB medium (100 µg / mL ampicillin, 30 g / mL chloramphenicol added to PanK expression strain) in a 500 mL flask. 5 mL of the above preculture was added, and the cells were cultured with shaking at 37 ° C. for 3 hours. Thereafter, 100 μL of 100 mM IPTG was added, and the mixture was again incubated at 37 ° C. for 3 hours. The cells were collected by centrifugation at 8,000 rpm for 5 minutes.
(超音波破砕・熱処理)
得られた湿菌体を200 mg wet cells/mLとなるように50 mM Tris-HCl (pH8)に懸濁し、超音波破砕を行った。15,000 rpmで8分間遠心分離した後の上清を70℃、30分熱処理した。再度遠心分離により変性したタンパク質を取り除き、上清を熱処理酵素液として用いた。調製した酵素液は1 mLずつエッペンドルフチューブに分注し、-20℃で保管した。
(Ultrasonic crushing and heat treatment)
The obtained wet cells were suspended in 50 mM Tris-HCl (pH 8) at a concentration of 200 mg wet cells / mL and sonicated. The supernatant after centrifugation at 15,000 rpm for 8 minutes was heat-treated at 70 ° C. for 30 minutes. The denatured protein was removed again by centrifugation, and the supernatant was used as a heat-treated enzyme solution. The prepared enzyme solution was dispensed into Eppendorf tubes in 1 mL portions and stored at -20 ° C.
（４）パンテテインリン酸アデニリルトランスフェラーゼ(PA)及びデホスホCoAキナーゼ(DCK)の取得
（発現系構築と培養)
Thermus thermophilus由来のパンテテインリン酸アデニリルトランスフェラーゼ(PA)遺伝子を化学合成し、EcoRIおよびSalIで切断後、pTRP発現ベクターに導入した。E. coli JM101を形質転換して得られたコロニーをLB培地 (2 mL)で種培養したのち、25 mL LB/ 100 mLフラスコに0.5 mLを接種し、30℃で培養した。その後、得られた菌体を20 mM Tris-HCl (pH 8.0)バッファーに懸濁したのち、超音波破砕を行なった。次いで、遠心分離により破砕液を清澄化したのち、SDS-PAGEにより目的蛋白質の発現を確認した。尚、デホスホCoAキナーゼ(DCK)もPAと同様の方法で目的蛋白質の発現を確認した。
(4) Obtaining pantetheine phosphate adenylyltransferase (PA) and dephospho-CoA kinase (DCK) (expression system construction and culture)
The pantethein phosphate adenylyltransferase (PA) gene from Thermus thermophilus was chemically synthesized, digested with EcoRI and SalI, and then introduced into a pTRP expression vector. After colony obtained by transforming E. coli JM101 was seed-cultured in LB medium (2 mL), 0.5 mL was inoculated into a 25 mL LB / 100 mL flask, and cultured at 30 ° C. Thereafter, the obtained cells were suspended in a 20 mM Tris-HCl (pH 8.0) buffer, followed by sonication. Next, the crushed liquid was clarified by centrifugation, and the expression of the target protein was confirmed by SDS-PAGE. The expression of the target protein was also confirmed for dephospho-CoA kinase (DCK) in the same manner as for PA.
（熱処理による簡易精製)
本酵素は好熱菌由来であるため、熱処理により目的蛋白質を簡易的に精製することができる。そこで、60-80℃で熱処理を行なうことで、CoA合成能評価のための粗精製品を調製した。コロニーを20 mL LB/ 100 mLフラスコに接種し、30℃で一晩培養した。次いで、1 L LB培地/ 2 Lフラスコに種培養(全量)を接種し、30℃で培養を行なった。遠心分離により菌体を回収したのち、20 mM Tris-HCl (pH 8.0)に懸濁した。その後、超音波破砕を行ない、遠心分離により清澄化した。それぞれの酵素において熱処理条件を検討した結果、70℃で30 minの熱処理を行なうことで、SDS-PAGEにおいてほぼ単一のバンドに精製することができた。
(Simple purification by heat treatment)
Since the present enzyme is derived from a thermophilic bacterium, the target protein can be easily purified by heat treatment. Thus, a crude product for evaluating the ability to synthesize CoA was prepared by performing a heat treatment at 60-80 ° C. The colony was inoculated into a 20 mL LB / 100 mL flask and cultured at 30 ° C. overnight. Next, a seed culture (total amount) was inoculated into a 1 L LB medium / 2 L flask and cultured at 30 ° C. After the cells were collected by centrifugation, they were suspended in 20 mM Tris-HCl (pH 8.0). Thereafter, the cells were sonicated and clarified by centrifugation. As a result of examining the heat treatment conditions for each enzyme, heat treatment at 70 ° C. for 30 min was able to purify almost a single band on SDS-PAGE.
２．酵素反応
反応液を100μlずつ分注し、サーマルサイクラーのグラジエント機能を用いて、30, 50, 70℃の各温度で、24時間インキュベートした。インキュベート終了後、各サンプルに1M HCl 20μlを加えてクエンチングを行った。15,000 rpm, 5分間遠心分離し、上清をHPLC分析に供した。
酵素反応サンプル液組成は、表２に示すとおりである。
2. Enzyme reaction 100 μl of the reaction solution was dispensed, and the mixture was incubated at 30, 50, and 70 ° C. for 24 hours using a gradient function of a thermal cycler. After incubation, each sample was quenched by adding 20 μl of 1M HCl. After centrifugation at 15,000 rpm for 5 minutes, the supernatant was subjected to HPLC analysis.
The composition of the enzyme reaction sample solution is as shown in Table 2.
３．HPLC分析条件
カラム：5C18-AR-II (ナカライテスク社製, φ4.6 × 250mm)
溶離液：A. 50 mM リン酸Na緩衝液 pH 5.0
B. 50 mM リン酸Na緩衝液 pH 5.0 + 20% アセトニトリル
検出：UV210 nm
インジェクションボリューム：2 μl
なお、HPLC分析は表３に示すプログラムで実施した。
3. HPLC analysis condition column: 5C18-AR-II (Nakarai Tesque, φ4.6 × 250mm)
Eluent: A. 50 mM Na phosphate buffer pH 5.0
B. 50 mM Na phosphate buffer pH 5.0 + 20% acetonitrile detection: UV210 nm
Injection volume: 2 μl
In addition, HPLC analysis was performed by the program shown in Table 3.
４．結果
構築したCoA生産経路を図７に示す。上記スキームに則り、CoAおよび同HPLC分析条件で検出が可能なdephospho CoAについて反応液中の濃度を定量した。実験は2回実施し、得られたデータの平均値を結果とした。CoA生合成に関わる耐熱性酵素を発現させた大腸菌の熱処理粗酵素液のほか、コントロールとして、耐熱性酵素遺伝子を含まない空ベクター(pET19b)を形質転換した大腸菌より調製した熱処理粗酵素液を用いた試験を行った。結果は図８に示す。コントロール（Ａ）では、極めて微量のdephospho CoA、CoAが検出されたが、これらは大腸菌の細胞に由来するものと考えられる。一方、耐熱性酵素を加えたサンプル（Ｂ）では、反応温度が高くなるほどCoAの蓄積量は増加していき、70℃において、0.96 mMに達した。今回の試験では、5 mMのパントテン酸を基質とし、補酵素として15 mM ATP、5 mM CTPを用いて行っているため、本試験で得られた生産物収率は19.3% (mol/mol)と求められる。
4. Results The constructed CoA production pathway is shown in FIG. In accordance with the above scheme, the concentration of CoA and dephospho CoA detectable under the same HPLC analysis conditions in the reaction solution was quantified. The experiment was performed twice, and the average value of the obtained data was used as the result. In addition to the heat-treated crude enzyme solution of Escherichia coli expressing a heat-resistant enzyme involved in CoA biosynthesis, as a control, a heat-treated crude enzyme solution prepared from Escherichia coli transformed with an empty vector (pET19b) containing no heat-resistant enzyme gene was used. Tests were performed. The results are shown in FIG. In control (A), very small amounts of dephospho-CoA and CoA were detected, which are considered to be derived from E. coli cells. On the other hand, in the sample (B) to which the thermostable enzyme was added, the accumulated amount of CoA increased as the reaction temperature increased, reaching 0.96 mM at 70 ° C. In this test, 5 mM pantothenic acid was used as a substrate, and 15 mM ATP and 5 mM CTP were used as coenzymes, so the product yield obtained in this test was 19.3% (mol / mol). Is required.
本発明のパントテン酸キナーゼは、コエンザイムAの有用中間体製造の生体触媒として利用することができる。 The pantothenate kinase of the present invention can be used as a biocatalyst for producing a useful intermediate of coenzyme A.
配列番号５：プライマー（GbPanK-F)
配列番号６：プライマー（GbPanK-R)
配列番号７：プライマー（TtPanK-F)
配列番号８：プライマー（TtPanK-R)
SEQ ID NO: 5: Primer (GbPanK-F)
SEQ ID NO: 6: Primer (GbPanK-R)
SEQ ID NO: 7: primer (TtPanK-F)
SEQ ID NO: 8: primer (TtPanK-R)
Claims (14)
(a) 配列番号２で表されるアミノ酸配列からなるパントテン酸キナーゼ；
(b) 配列番号２で表されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を含み、かつパントテン酸からホスホパントテン酸の反応を触媒する活性を有するパントテン酸キナーゼ。 A pantothenate kinase for synthesizing coenzyme A from the following (a) or (b) pantothenate, wherein the pantothenate kinase is not subject to feedback inhibition of coenzyme A:
(a) a pantothenate kinase consisting of the amino acid sequence represented by SEQ ID NO: 2;
(b) pantothenic acid comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having an activity of catalyzing the reaction of pantothenic acid to phosphopantothenic acid Kinase.
(a) 配列番号２で表されるアミノ酸配列からなるパントテン酸キナーゼ；
(b) 配列番号２で表されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を含み、かつパントテン酸からホスホパントテン酸の反応を触媒する活性を有するパントテン酸キナーゼ。 A DNA encoding a pantothenate kinase for synthesizing coenzyme A from the following pantothenic acid (a) or (b), wherein the pantothenate kinase is not subject to feedback inhibition of coenzyme A:
(a) a pantothenate kinase consisting of the amino acid sequence represented by SEQ ID NO: 2;
(b) pantothenic acid comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having an activity of catalyzing the reaction of pantothenic acid to phosphopantothenic acid Kinase.
(c) 配列番号１で表される塩基配列を含むDNA
(d) 配列番号１で表される塩基配列を含むDNAと少なくとも90％以上の配列同一性を有し、かつパントテン酸からホスホパントテン酸の反応を触媒する活性を有するタンパク質をコードするDNA。 DNA of the following (c) or (d) encoding a pantothenate kinase for synthesizing coenzyme A from pantothenic acid, wherein the pantothenate kinase is not subject to feedback inhibition of coenzyme A:
(c) DNA containing the nucleotide sequence represented by SEQ ID NO: 1
(d) DNA encoding a protein having at least 90% or more sequence identity with DNA containing the base sequence represented by SEQ ID NO: 1, and having an activity of catalyzing the reaction of pantothenic acid to phosphopantothenic acid.
(e) 配列番号４で表されるアミノ酸配列からなるパントテン酸キナーゼ；
(f) 配列番号４で表されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を含み、かつパントテン酸からホスホパントテン酸の反応を触媒する活性を有するパントテン酸キナーゼ。 A pantothenate kinase for synthesizing coenzyme A from the following (e) or (f) pantothenate, wherein the pantothenate kinase is not subject to feedback inhibition of coenzyme A:
(e) a pantothenate kinase consisting of the amino acid sequence represented by SEQ ID NO: 4;
(f) pantothenic acid comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and having an activity of catalyzing the reaction of pantothenic acid to phosphopantothenic acid Kinase.
(e) 配列番号４で表されるアミノ酸配列からなるパントテン酸キナーゼ；
(f) 配列番号４で表されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列を含み、かつパントテン酸からホスホパントテン酸の反応を触媒する活性を有するパントテン酸キナーゼ。 A DNA encoding a pantothenate kinase for synthesizing coenzyme A from the following (e) or (f) pantothenate, which is not subject to feedback inhibition of coenzyme A:
(e) a pantothenate kinase consisting of the amino acid sequence represented by SEQ ID NO: 4;
(f) pantothenic acid comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4, and having an activity of catalyzing the reaction of pantothenic acid to phosphopantothenic acid Kinase.
(g) 配列番号３で表される塩基配列を含むDNA
(h) 配列番号３で表される塩基配列を含むDNAと少なくとも90％以上の配列同一性を有し、かつパントテン酸からホスホパントテン酸の反応を触媒する活性を有するタンパク質をコードするDNA。 The following (g) or (h) DNA encoding a pantothenate kinase for synthesizing coenzyme A from pantothenate, which is not subject to feedback inhibition of coenzyme A:
(g) DNA containing the base sequence represented by SEQ ID NO: 3
(h) DNA encoding a protein having at least 90% or more sequence identity with DNA containing the nucleotide sequence represented by SEQ ID NO: 3, and having an activity of catalyzing the reaction of pantothenic acid to phosphopantothenic acid.
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| CN116926092A (en) * | 2022-10-28 | 2023-10-24 | 昆明理工大学 | Pantothenate kinase gene RkPank and application thereof |
| CN116926092B (en) * | 2022-10-28 | 2024-04-26 | 昆明理工大学 | Pantothenate kinase gene RkPank and application thereof |
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