JP7426052B2 - Screening method for squalene consuming enzyme and squalene-hopene cyclase - Google Patents
Screening method for squalene consuming enzyme and squalene-hopene cyclase Download PDFInfo
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Description
本発明は、スクアレン消費酵素のスクリーニング方法及びスクアレン-ホペン環化酵素に関する。 The present invention relates to a method for screening squalene-consuming enzymes and a squalene-hopene cyclase.
スクアレンは、C30の直鎖状炭化水素であり、化粧品原料、食油、燃料として注目されている。さらに、スクアレンを前駆体として生合成されるトリテルペン類も注目されている。
しかし、これらのスクアレンを起点とする有価化合物の合成活性をスクリーニングする系の開発が不十分であった。そのため、生合成経路の解明は限定的である。当然であるが、生合成経路が解明されていないので、スクアレンを起点とする有価化合物の生合成の試みもほとんどされていない状況である。
Squalene is a C30 linear hydrocarbon and is attracting attention as a raw material for cosmetics, edible oil, and fuel. Furthermore, triterpenes that are biosynthesized using squalene as a precursor are also attracting attention.
However, a system for screening the synthetic activity of these valuable compounds using squalene as a starting point has not been sufficiently developed. Therefore, the elucidation of the biosynthetic pathway is limited. Naturally, since the biosynthetic pathway has not been elucidated, very few attempts have been made to biosynthesize valuable compounds using squalene as a starting point.
スクアレン環化酵素である精製されたAlicyclobacillus由来のスクアレン/ホベンサイクラーゼに有機合成された人工基質を与えると、非天然のC35テルペン骨格が得られることが知られている(非特許文献1:Abe,JACS,124,14514(2002):Hoshino,Chem Eur;J.15,2091(2009))。このような新規機能の候補が無数に存在するが、それらの組織的な探索、あるいはそれらの特異性の改変は、スクリーニング系の不在により極めて困難であった。 It is known that an unnatural C35 terpene skeleton is obtained when an organically synthesized artificial substrate is provided to purified squalene/hobencyclase derived from Alicyclobacillus, which is a squalene cyclase (Non-patent Document 1: Abe, JACS, 124, 14514 (2002): Hoshino, Chem Eur; J. 15, 2091 (2009)). Although there are countless candidates for such new functions, systematic exploration of them or modification of their specificity has been extremely difficult due to the lack of screening systems.
スクアレン-ホペン環化酵素の変異型酵素(D377C、D377N、Y420H、Y420W等)を用いることによって、スクアレンから、単環性トリテルペンである3-デオキシアキレオールAを得る方法が知られている{非特許文献2: Biosci. Biotechnol. Biochem., (1999) Vol.63, pp. 2189-2198、非特許文献3:Biosci. Biotechnol. Biochem., (2001) Vol.65, pp.2233-2242、非特許文献4:Biosci. Biotechnol. Biochem., (2002) Vol.66, pp.1660-1670)}。
しかし、本発明の変異型スクアレン-ホペン環化酵素と前記変異型酵素と比較した場合、アミノ酸置換が明らかに異なる。
A method for obtaining 3-deoxyachileol A, a monocyclic triterpene, from squalene by using mutant squalene-hopene cyclase enzymes (D377C, D377N, Y420H, Y420W, etc.) is known. Patent document 2: Biosci. Biotechnol. Biochem., (1999) Vol.63, pp. 2189-2198, Non-patent document 3: Biosci. Biotechnol. Biochem., (2001) Vol.65, pp.2233-2242, Patent Document 4: Biosci. Biotechnol. Biochem., (2002) Vol. 66, pp. 1660-1670)}.
However, when the mutant squalene-hopene cyclase of the present invention is compared with the mutant enzyme, the amino acid substitutions are clearly different.
本発明者らは、「テルペン合成酵素変異体をコードする遺伝子であって、酵素活性及び/又はその発現が野生型酵素と比較して増強されたテルペン合成酵素変異体をコードする遺伝子を導入した細胞に、被検遺伝子を導入して培養し、細胞に導入した被検遺伝子のう
ち生存した細胞に導入した被検遺伝子を選択することを含む、テルペン合成酵素の遺伝子及び/又はその変異体のスクリーニング方法(参照:特許文献1)」を開示している。
しかし、前記スクリーニング方法は、本発明のスクアレン消費酵素及び/又はその変異体のスクリーニング方法とは原理が異なる。
The present inventors have introduced a gene encoding a terpene synthase mutant in which enzyme activity and/or expression thereof is enhanced compared to the wild-type enzyme. The process involves introducing a test gene into cells, culturing the cells, and selecting the test gene introduced into surviving cells from among the test genes introduced into the cells. Screening method (reference: Patent Document 1)' is disclosed.
However, the principle of the screening method is different from that of the present invention for screening squalene-consuming enzymes and/or variants thereof.
本発明者らは、「被検遺伝子を色素合成可能な細胞に導入し、色素合成量の低下を検出することを特徴とする、テルペン合成酵素の遺伝子及び/又はその変異体のスクリーニング方法(参照:特許文献2)」を開示している。
しかし、前記スクリーニング方法は、本発明のスクアレン消費酵素及び/又はその変異体のスクリーニング方法とはスクリーニングの対象が異なる。
The present inventors have developed a method for screening terpene synthase genes and/or variants thereof, which is characterized by introducing a test gene into cells capable of pigment synthesis and detecting a decrease in the amount of pigment synthesis (see : Patent Document 2)" is disclosed.
However, the screening target of the above-described screening method is different from that of the screening method for squalene-consuming enzymes and/or variants thereof of the present invention.
本発明は、新規なスクアレン消費酵素のスクリーニング方法及びスクアレン-ホペン環化酵素を提供することである。 The present invention provides a novel method for screening squalene-consuming enzymes and squalene-hopene cyclizing enzymes.
本発明者らは鋭意検討した結果、スクアレン消費酵素遺伝子を、スクアレンを経由して合成される色素合成可能な細胞に導入し、該色素合成量の増加又は低下を指標とする、スクアレン消費酵素及び/又はその変異体のスクリーニング方法を完成し、さらに該方法により変異型スクアレン-ホペン環化酵素を得ることができて、本発明を完成した。 As a result of intensive studies, the present inventors introduced squalene consuming enzyme genes into cells capable of synthesizing pigments that are synthesized via squalene, and determined whether squalene consuming enzymes or The present invention has been completed by completing a method for screening / or a mutant thereof, and by using the method to obtain a mutant squalene-hopene cyclase.
すなわち、本発明は以下の通りである。
1.配列番号1で示されるアミノ酸配列における42位、61位、69位、71位、90位、107位、117位、120位、124位、128位、129位、131位、136位、142位、145位、153位、157位、159位、171位、181位、187位、195位、203位、207位、208位、211位、214位、219位、236位、251位、255位、257位、259位、265位、271位、285位、298位、332位、335位、348位、354位、357位、359位、365位、378位、390位、424位、437位、439位、440位、478位、497位、503位、514位、525位、574位、604位、及び623位からなる群より選択される少なくとも1つの部位にアミノ酸置換を有することを特徴とする、変異型スクアレン-ホペン環化酵素又はその誘導体。
2.前記変異型スクアレン-ホペン環化酵素は、野生型スクアレン-ホペン環化酵素と比較して、ホペン合成量が高いことを特徴とする、前項1に記載の変異型スクアレン-ホペン環化酵素又はその誘導体。
3.前記のアミノ酸配列番号1の部位で示されるアミノ酸置換は、以下の群より選択される少なくとも1以上からなる前項1又は2に記載の変異型スクアレン-ホペン環化酵素
又はその誘導体、
M42V、M42T、M61V、L69S、E71D、T90M、E107K、I117V、Q120R、E124G、F129L、V128L、V128M、R131Q、L136P、W142R、V145A、M153L、K157E、M159I、R171Q、M181V、M181R、M181T、F187L、Y195A、V203G、R207W、R208H、K211R、K211M、G214C、D219G、F236S、F236L、D251H、D255G、S257N、G259S、W265C、A271V、K285E、D298E、R332C、K335R、P348S、K354M、N357Y、K359M、F365L、T378A、L390S、N424D、F437L、E439G、V440L、E478G、T497S 、A503V、P514S、Q525R、A574T、D604G及びR623L。
4.前記アミノ酸置換は、以下のいずれから選択される1以上からなる前項1~3のいずれか1項に記載の変異型スクアレン-ホペン環化酵素又はその誘導体、
M181V、V128L、L136P、N424D、V440L。
5.前記アミノ酸置換は、以下の(1)~(24)のいずれから選択される1からなる前項1~3のいずれか1項に記載の変異型スクアレン-ホペン環化酵素又はその誘導体、
(1)R131Q、M159I、F437L
(2)F129L、F236L、P514S、R623L
(3)G259S、K335R、F437L、Q525R、A574T
(4)V128L
(5)L136P
(6)F236S、A503V、D604G
(7)V145A、E478G
(8)I117V、Q120R、R171Q、D251H
(9)V440L
(10)M153L、E439G
(11)E71D、Y195A、D219G、A271V、T378A
(12)W142R、K211R、K359M
(13)R208H、S257N、W265C
(14)M42V、V203G
(15)M181T、T497S
(16)M42T、M61V、L69S、K157E、D255G、K285E
(17)N424D
(18)T90M、E124G、P348S
(19)E107K、M181R、K354M、L390S
(20)G214C、D298E
(21)M181V
(22)R207W、K211M
(23)V128M、F187L、及び、
(24)R332C、N357Y、F365L。
6.配列番号1で示されるアミノ酸配列において、R131Q、M159I及びF437Lのアミノ酸置換を有することを特徴とする、変異型スクアレン-ホペン環化酵素又はその誘導体。
7.以下の(1)~(7)のいずれか1である遺伝子からなる変異型スクアレン-ホペン環化酵素遺伝子、
(1)配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドをコードする遺伝子、
(2)配列番号2~4のいずれか1に記載のアミノ酸配列において、1~20個のアミノ酸が置換、欠損、挿入及び/又は付加しており、かつ配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドが有するスクアレンを閉環して5環性のホペンを生成する活性と実質的同等の活性を有するポリペプチドをコードする遺伝子、
(3)配列番号2~4のいずれか1に記載のアミノ酸配列と90%以上の相同性を有し、かつ配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドが有するスクアレンを閉環して5環性のホペンを生成する活性と実質的同等の活性を有するポリペプチ
ドをコードする遺伝子、
(4)配列番号5~7のいずれか1に記載の塩基配列からなるDNAからなる遺伝子、
(5)配列番号5~7のいずれか1に記載の塩基配列からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドが有するスクアレンを閉環して5環性のホペンを生成する活性と実質的同等の活性を有するポリペプチドをコードする遺伝子、
(6)配列番号5~7のいずれか1に記載の塩基配列からなるDNAにおいて、1~50個の塩基配列が置換、欠損、挿入及び/又は付加しているDNAからなる遺伝子、及び、
(7)配列番号5~7のいずれか1に記載の塩基配列からなるDNAと相同性が90%以上のDNAからなる遺伝子。
8.以下の(1)~(3)のいずれか1であるアミノ酸配列からなる変異型スクアレン-ホペン環化酵素、
(1)配列番号2~4のいずれか1に記載のアミノ酸配列、
(2)配列番号2~4のいずれか1に記載のアミノ酸配列において、1~20個のアミノ酸が置換、欠損、挿入及び/又は付加しており、かつ配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドが有するスクアレンを閉環して5環性のホペンを生成する活性を有するアミノ酸配列、及び、
(3)配列番号2~4のいずれか1に記載のアミノ酸配列と90%以上の相同性を有し、かつ配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドが有するスクアレンを閉環して5環性のホペンを生成する活性を有するアミノ酸配列。
9.スクアレン消費酵素遺伝子を、スクアレンを経由して合成される色素合成可能な細胞に導入し、該色素合成量の増加又は低下を指標とする、スクアレン消費酵素及び/又はその変異体のスクリーニング方法。
10.前記色素合成量の低下が、色素合成に関与する又は競合する酵素の基質がスクアレン消費酵素により消費されることによる色素合成量の低下である、前項9に記載のスクリーニング方法。
11.前記細胞が大腸菌又は酵母である、前項9又は10に記載のスクリーニング方法。
12.前記細胞がスクアレンを経由して合成される色素合成に関与する若しくは競合する遺伝子又はその変異体により形質転換された細胞である、前項9~11のいずれか1項に記載のスクリーニング方法。
13.スクアレン消費酵素がスクアレン-ホペン環化酵素であり、スクアレンを経由して合成される色素合成に関与する遺伝子がスクアレン不飽和化酵素(CrtN)遺伝子である、前項9~12のいずれか1項に記載のスクリーニング方法。
14.スクアレン消費酵素がスクアレン不飽和化酵素であり、スクアレンを経由して合成される色素合成に競合する遺伝子がスクアレン-ホペン環化酵素遺伝子である、前項9~12のいずれか1項に記載のスクリーニング方法。
That is, the present invention is as follows.
1. 42nd, 61st, 69th, 71st, 90th, 107th, 117th, 120th, 124th, 128th, 129th, 131st, 136th, 142nd in the amino acid sequence shown in SEQ ID NO: 1 , 145th, 153rd, 157th, 159th, 171st, 181st, 187th, 195th, 203rd, 207th, 208th, 211th, 214th, 219th, 236th, 251st, 255th 257th, 259th, 265th, 271st, 285th, 298th, 332nd, 335th, 348th, 354th, 357th, 359th, 365th, 378th, 390th, 424th, Having an amino acid substitution at at least one site selected from the group consisting of positions 437, 439, 440, 478, 497, 503, 514, 525, 574, 604, and 623. A mutant squalene-hopene cyclase or a derivative thereof, characterized by:
2. The mutant squalene-hopene cyclase is the mutant squalene-hopene cyclase according to item 1 above, or the mutant squalene-hopene cyclase according to item 1, wherein the mutant squalene-hopene cyclase synthesizes a higher amount of hopene than the wild-type squalene-hopene cyclase. derivative.
3. The amino acid substitution shown at the site of amino acid sequence number 1 is the mutant squalene-hopene cyclase or derivative thereof according to item 1 or 2 above, consisting of at least one selected from the following group;
M42V, M42T, M61V, L69S, E71D, T90M, E107K, I117V, Q120R, E124G, F129L, V128L, V128M, R131Q, L136P, W142R, V145A, M153L, K157E, M159I, R171Q, M181 V, M181R, M181T, F187L, Y195A, V203G, R207W, R208H, K211R, K211M, G214C, D219G, F236S, F236L, D251H, D255G, S257N, G259S, W265C, A271V, K285E, D298E, R332C, K335R, P34 8S, K354M, N357Y, K359M, F365L, T378A, L390S, N424D, F437L, E439G, V440L, E478G, T497S, A503V, P514S, Q525R, A574T, D604G and R623L.
4. The amino acid substitution is a mutant squalene-hopene cyclase or a derivative thereof according to any one of items 1 to 3 above, consisting of one or more selected from the following:
M181V, V128L, L136P, N424D, V440L.
5. The amino acid substitution is a mutant squalene-hopene cyclase or a derivative thereof according to any one of items 1 to 3 above, consisting of 1 selected from any of the following (1) to (24);
(1) R131Q, M159I, F437L
(2) F129L, F236L, P514S, R623L
(3) G259S, K335R, F437L, Q525R, A574T
(4) V128L
(5) L136P
(6) F236S, A503V, D604G
(7) V145A, E478G
(8) I117V, Q120R, R171Q, D251H
(9) V440L
(10) M153L, E439G
(11) E71D, Y195A, D219G, A271V, T378A
(12) W142R, K211R, K359M
(13) R208H, S257N, W265C
(14) M42V, V203G
(15) M181T, T497S
(16) M42T, M61V, L69S, K157E, D255G, K285E
(17) N424D
(18) T90M, E124G, P348S
(19) E107K, M181R, K354M, L390S
(20) G214C, D298E
(21) M181V
(22) R207W, K211M
(23) V128M, F187L, and
(24) R332C, N357Y, F365L.
6. A mutant squalene-hopene cyclase or a derivative thereof, which is characterized by having R131Q, M159I, and F437L amino acid substitutions in the amino acid sequence shown in SEQ ID NO: 1.
7. A mutant squalene-hopene cyclase gene consisting of a gene that is any one of the following (1) to (7),
(1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4,
(2) In the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4, 1 to 20 amino acids are substituted, deleted, inserted, and/or added, and A gene encoding a polypeptide having substantially the same activity as the activity of ring-closing squalene to produce pentacyclic hopene, which the polypeptide consisting of the amino acid sequence described above has;
(3) Squalene possessed by a polypeptide having 90% or more homology with the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4 and consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4. A gene encoding a polypeptide having an activity substantially equivalent to the activity of ring-closing to produce pentacyclic hopene,
(4) a gene consisting of DNA consisting of the base sequence set forth in any one of SEQ ID NOS: 5 to 7;
(5) Hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence set forth in any one of SEQ ID NOs: 5 to 7, and any one of SEQ ID NOS: 2 to 4 A gene encoding a polypeptide having substantially the same activity as the activity of ring-closing squalene to produce pentacyclic hopene, which the polypeptide consisting of the amino acid sequence described in
(6) A gene consisting of a DNA consisting of the base sequence set forth in any one of SEQ ID NOs: 5 to 7, in which 1 to 50 base sequences have been substituted, deleted, inserted, and/or added, and
(7) A gene consisting of DNA having 90% or more homology to the DNA consisting of the base sequence set forth in any one of SEQ ID NOs: 5 to 7.
8. A mutant squalene-hopene cyclase consisting of an amino acid sequence that is any one of the following (1) to (3),
(1) the amino acid sequence according to any one of SEQ ID NOs: 2 to 4;
(2) In the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4, 1 to 20 amino acids are substituted, deleted, inserted, and/or added, and An amino acid sequence having the activity of ring-closing squalene to produce a pentacyclic hopene, which a polypeptide consisting of the amino acid sequence described above has, and
(3) Squalene possessed by a polypeptide having 90% or more homology with the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4 and consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4. An amino acid sequence having the activity of ring-closing to produce pentacyclic hopene.
9. A method of screening for a squalene consuming enzyme and/or its mutants, which comprises introducing a squalene consuming enzyme gene into cells capable of synthesizing a pigment synthesized via squalene, and using an increase or decrease in the amount of pigment synthesis as an indicator.
10. 10. The screening method according to item 9, wherein the decrease in the amount of pigment synthesis is a decrease in the amount of pigment synthesis due to a squalene-consuming enzyme consuming a substrate of an enzyme that participates in or competes with pigment synthesis.
11. 11. The screening method according to item 9 or 10, wherein the cell is E. coli or yeast.
12. 12. The screening method according to any one of items 9 to 11 above, wherein the cell is a cell transformed with a gene involved in or competitive with pigment synthesis synthesized via squalene, or a mutant thereof.
13. In any one of the preceding paragraphs 9 to 12, the squalene consuming enzyme is squalene-hopene cyclase, and the gene involved in pigment synthesis synthesized via squalene is the squalene desaturase (CrtN) gene. Screening method described.
14. The screen according to any one of the preceding items 9 to 12, wherein the squalene consuming enzyme is squalene desaturase, and the gene that competes with the synthesis of pigment synthesized via squalene is a squalene-hopene cyclase gene. Method.
本発明は、野生型スクアレン-ホペン環化酵素と比較してホペン合成量が高い変異型スクアレン-ホペン環化酵素並びに該酵素のスクリーニング方法を提供することができた。 The present invention was able to provide a mutant squalene-hopene cyclase that synthesizes a higher amount of hopene than the wild-type squalene-hopene cyclase, and a method for screening the enzyme.
本発明は、「変異型スクアレン-ホペン環化酵素又はその変異体」、並びに、「スクアレン消費酵素及び/又はその変異体のスクリーニング方法」に関する。 The present invention relates to a "mutant squalene-hopene cyclase or a mutant thereof" and a "method for screening a squalene-consuming enzyme and/or a mutant thereof."
(変異型スクアレン-ホペン環化酵素又はその誘導体)
本発明の変異型スクアレン-ホペン環化酵素又はその誘導体は、野生型スクアレン-ホペン環化酵素と比較して、アミノ酸又は塩基が1以上異なる酵素を意味する。
野生型スクアレン-ホペン環化酵素は、Alicyclobacillus属、Zymomonas属、Bradyrhizobium属、Methylococcus属等の細菌で産出され、スクアレンを閉環して、5環性のホペン又はホパノールを生成する酵素として知られている。
特に、本発明の変異型スクアレン-ホペン環化酵素又はその誘導体は、野生型スクアレン-ホペン環化酵素と比較して、ホペン合成量が高い(合成活性が高い)ことを特徴とする。
また、本発明の誘導体とは、変異型スクアレン-ホペン環化酵素の保護化誘導体、糖鎖修飾体、アシル化誘導体、若しくはアセチル化誘導体を対象とする。
なお、保護化誘導体、糖鎖修飾体、アシル化誘導体、若しくはアセチル化誘導体は、自体公知の方法で得ることができる。
(Mutant squalene-hopene cyclase or derivative thereof)
The mutant squalene-hopene cyclase or derivative thereof of the present invention refers to an enzyme that differs from the wild-type squalene-hopene cyclase in one or more amino acids or bases.
Wild-type squalene-hopene cyclase is produced by bacteria such as Alicyclobacillus, Zymomonas, Bradyrhizobium, and Methylococcus, and is known as an enzyme that generates pentacyclic hopene or hopanol by ring-closing squalene. .
In particular, the mutant squalene-hopene cyclase of the present invention or its derivative is characterized by a higher amount of hopene synthesis (higher synthesis activity) than the wild-type squalene-hopene cyclase.
Further, the derivative of the present invention refers to a protected derivative, a sugar chain modified product, an acylated derivative, or an acetylated derivative of a mutant squalene-hopene cyclase.
Note that the protected derivative, sugar chain modified product, acylated derivative, or acetylated derivative can be obtained by a method known per se.
本発明の変異型スクアレン-ホペン環化酵素は、Alicyclobacillus acidocaldarius由来のスクアレン-ホペン環化酵素(配列番号1)と比較して、アミノ酸配列に以下で示される置換を有する。
42位、61位、69位、71位、90位、107位、117位、120位、124位、128位、129位、131位、136位、142位、145位、153位、157位、159位、171位、181位、187位、195位、203位、207位、208位、211位、214位、219位、236位、251位、255位、257位、259位、265位、271位、285位、298位、332位、335位、348位、354位、357位、359位、365位、378位、390位、424位、437位、439位、440位、478位、497位、503位、514位、525位、574位、604位、及び/又は623位。
また、本発明の変異型スクアレン-ホペン環化酵素は、Alicyclobacillus acidocaldarius由来のスクアレン-ホペン環化酵素(配列番号1)と比較して、アミノ酸配列に以下で示される置換を有する。
M42V、M42T、M61V、L69S、E71D、T90M、E107K、I117V、Q120R、E124G、F129L、V128L、V128M、R131Q、L136P、W142R、V145A、M153L、K157E、M159I、R171Q、M181V、M181R、M181T、F187L、Y195A、V203G、R207W、R208H、K211R、K211M、G214C、D219G、F236S、F236L、D251H、D255G、S257N、G259S、W265C、A271V、K285E、D298E、R332C、K335R、P348S、K354M、N357Y、K359M、F36
5L、T378A、L390S、N424D、F437L、E439G、V440L、E478G、T497S 、A503V、P514S、Q525R、A574T、D604G及び/又はR623L。
The mutant squalene-hopene cyclase of the present invention has the following substitutions in the amino acid sequence compared to the squalene-hopene cyclase derived from Alicyclobacillus acidocaldarius (SEQ ID NO: 1).
42nd, 61st, 69th, 71st, 90th, 107th, 117th, 120th, 124th, 128th, 129th, 131st, 136th, 142nd, 145th, 153rd, 157th , 159th, 171st, 181st, 187th, 195th, 203rd, 207th, 208th, 211th, 214th, 219th, 236th, 251st, 255th, 257th, 259th, 265th 271st, 285th, 298th, 332nd, 335th, 348th, 354th, 357th, 359th, 365th, 378th, 390th, 424th, 437th, 439th, 440th, 478th, 497th, 503rd, 514th, 525th, 574th, 604th, and/or 623rd.
Furthermore, the mutant squalene-hopene cyclase of the present invention has the following substitutions in the amino acid sequence, compared to the squalene-hopene cyclase derived from Alicyclobacillus acidocaldarius (SEQ ID NO: 1).
M42V, M42T, M61V, L69S, E71D, T90M, E107K, I117V, Q120R, E124G, F129L, V128L, V128M, R131Q, L136P, W142R, V145A, M153L, K157E, M159I, R171Q, M181 V, M181R, M181T, F187L, Y195A, V203G, R207W, R208H, K211R, K211M, G214C, D219G, F236S, F236L, D251H, D255G, S257N, G259S, W265C, A271V, K285E, D298E, R332C, K335R, P34 8S, K354M, N357Y, K359M, F36
5L, T378A, L390S, N424D, F437L, E439G, V440L, E478G, T497S, A503V, P514S, Q525R, A574T, D604G and/or R623L.
本発明の変異型スクアレン-ホペン環化酵素は、Alicyclobacillus acidocaldarius由来のスクアレン-ホペン環化酵素(配列番号1)と比較して、アミノ酸配列に以下の(1)~(24)のいずれか1で示される置換を有する。
(1)R131Q、M159I、F437L(配列番号2、配列番号5)
(2)F129L、F236L、P514S、R623L(配列番号3、配列番号6)
(3)G259S、K335R、F437L、Q525R、A574T(配列番号4、配列番号7)
(4)V128L
(5)L136P
(6)F236S、A503V、D604G
(7)V145A、E478G
(8)I117V、Q120R、R171Q、D251H
(9)V440L
(10)M153L、E439G
(11)E71D、Y195A、D219G、A271V、T378A
(12)W142R、K211R、K359M
(13)R208H、S257N、W265C
(14)M42V、V203G
(15)M181T、T497S
(16)M42T、M61V、L69S、K157E、D255G、K285E
(17)N424D
(18)T90M、E124G、P348S
(19)E107K、M181R、K354M、L390S
(20)G214C、D298E
(21)M181V
(22)R207W、K211M
(23)V128M、F187L
(24)R332C、N357Y、F365L
The mutant squalene-hopene cyclase of the present invention has any one of the following (1) to (24) in the amino acid sequence, compared to the squalene-hopene cyclase derived from Alicyclobacillus acidocaldarius (SEQ ID NO: 1). with the indicated substitutions.
(1) R131Q, M159I, F437L (Sequence number 2, Sequence number 5)
(2) F129L, F236L, P514S, R623L (Sequence number 3, Sequence number 6)
(3) G259S, K335R, F437L, Q525R, A574T (SEQ ID NO: 4, SEQ ID NO: 7)
(4) V128L
(5) L136P
(6) F236S, A503V, D604G
(7) V145A, E478G
(8) I117V, Q120R, R171Q, D251H
(9) V440L
(10) M153L, E439G
(11) E71D, Y195A, D219G, A271V, T378A
(12) W142R, K211R, K359M
(13) R208H, S257N, W265C
(14) M42V, V203G
(15) M181T, T497S
(16) M42T, M61V, L69S, K157E, D255G, K285E
(17) N424D
(18) T90M, E124G, P348S
(19) E107K, M181R, K354M, L390S
(20) G214C, D298E
(21) M181V
(22) R207W, K211M
(23) V128M, F187L
(24) R332C, N357Y, F365L
本発明の変異型スクアレン-ホペン環化酵素は、Alicyclobacillus acidocaldarius由来のスクアレン-ホペン環化酵素(配列番号1)と比較して、以下のいずれから1以上のアミノ酸置換を有することにより、野生型スクアレン-ホペン環化酵素と比較して、ホペン合成量が高いことを下記の実施例で確認している。
M181V、V128L、L136P、N424D及びV440L。
The mutant squalene-hopene cyclase of the present invention has one or more amino acid substitutions from any of the following, compared to the squalene-hopene cyclase derived from Alicyclobacillus acidocaldarius (SEQ ID NO: 1). - It has been confirmed in the following example that the amount of hopene synthesized is higher than that of hopene cyclase.
M181V, V128L, L136P, N424D and V440L.
ホペン合成量(ホペン合成活性)が高いとは、Alicyclobacillus acidocaldarius由来の野生型スクアレン-ホペン環化酵素(配列番号1)と比較して、1.1倍以上、1.5倍以上、2.0倍以上、3.0倍以上、約4.0倍(以上)、約5.0倍(以上)、約6.0倍(以上)、約7.0倍(以上)、約8.0倍(以上)、約9.0倍(以上)、約10倍(以上)、約11倍(以上)、約12倍(以上)、約13倍(以上)、約14倍(以上)、約15倍(以上)、約16倍(以上)、約17倍(以上)、約18倍(以上)、約19倍(以上)、約20倍(以上)、約21倍(以上)、約22倍(以上)、約23倍(以上)、約24倍(以上)、約25倍(以上)、約26倍(以上)、約27倍(以上)、約28倍(以上)、約29倍(以上)、約30倍(以上)、約31倍(以上)、約35倍(以上)、約40倍(以上)のホペン合成量を例示することができる。 A high amount of hopene synthesis (hopene synthesis activity) means 1.1 times or more, 1.5 times or more, 2.0 times or more, 3.0 times or more compared to the wild type squalene-hopene cyclase derived from Alicyclobacillus acidocaldarius (SEQ ID NO: 1). , approximately 4.0 times (or more), approximately 5.0 times (or more), approximately 6.0 times (or more), approximately 7.0 times (or more), approximately 8.0 times (or more), approximately 9.0 times (or more), approximately 10 times (or more), Approximately 11 times (or more), approximately 12 times (or more), approximately 13 times (or more), approximately 14 times (or more), approximately 15 times (or more), approximately 16 times (or more), approximately 17 times (or more), approximately 18 times (or more), approximately 19 times (or more), approximately 20 times (or more), approximately 21 times (or more), approximately 22 times (or more), approximately 23 times (or more), approximately 24 times (or more), approximately 25 26x (or more), 27x (or more), 28x (or more), 29x (or more), 30x (or more), 31x (or more), 35x (or more), about 40 times (or more) the amount of hopene synthesized.
(変異型スクアレン-ホペン環化酵素遺伝子)
本発明の変異型スクアレン-ホペン環化酵素遺伝子は、(1)配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドをコードする遺伝子、(2)配列番号2
~4のいずれか1に記載のアミノ酸配列において、1~20個のアミノ酸、好ましくは1~15個、より好ましくは1~10個、最も好ましくは1~5個が置換、欠損、挿入及び/又は付加しており、かつ配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドが有するスクアレンを閉環して5環性のホペンを生成する活性と実質的同等の活性を有するポリペプチドをコードする遺伝子、(3)配列番号2~4のいずれか1に記載のアミノ酸配列と90%以上の相同性を有し、かつ配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドが有するスクアレンを閉環して5環性のホペンを生成する活性と実質的同等の活性を有するポリペプチドをコードする遺伝子、(4)配列番号5~7のいずれか1に記載の塩基配列からなるDNAからなる遺伝子、(5)配列番号5~7のいずれか1に記載の塩基配列からなるDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドが有するスクアレンを閉環して5環性のホペンを生成する活性と実質的同等の活性を有するポリペプチドをコードする遺伝子、(6)配列番号5~7のいずれか1に記載の塩基配列からなるDNAにおいて、1~50個の塩基配列が置換、欠損、挿入及び/又は付加しているDNAからなる遺伝子、(7)配列番号5~7のいずれか1に記載の塩基配列からなるDNAと90%以上の相同性を有するDNAからなる遺伝子を、含む。
上記(1)の遺伝子は、本発明のスクアレン消費酵素及び/又はその変異体のスクリーニング方法から得られたスクアレン-ホペン環化酵素をコードする遺伝子である。該スクアレン-ホペン環化酵素は、スクアレンを閉環して5環性のホペンを生成する活性を持つ。
上記(2)の遺伝子は、本発明の変異型スクアレン-ホペン環化酵素に対して酵素活性を失わせない程度の変異を導入したポリペプチドをコードする遺伝子である。このような変異は、自然界において生じる変異のほかに、人為的な変異をも含む。人為的変異を生じさせる手段としては、部位特異的変異誘発法(Nucleic Acids Res. 10, 6487-6500, 1982)などを挙げることができる。変異したアミノ酸の数は、通常は、20アミノ酸以内であり、好ましくは10アミノ酸以内であり、更に好ましくは5アミノ酸以内であり、最も好ましくは3アミノ酸である。変異を導入したポリペプチドが酵素活性を保持しているかどうかは、例えば、変異を導入したポリペプチドをコードする遺伝子を大腸菌等に導入し、発現させ、その大腸菌等がスクアレンを閉環して5環性のホペンを生成することができるかどうか調べることによりわかる。特に、本実施例により、Alicyclobacillus acidocaldarius由来のスクアレン-ホペン環化酵素のアミノ酸配列において、酵素活性に重要なアミノ酸の位置であるM181、V128、L136、N424及びV440を特定している。これらのアミノ酸の位置・部位に変異が導入されると酵素活性が失われる可能性が高いので、変異はこれらのアミノ酸以外のアミノ酸に導入されることが好ましい。
上記(3)の遺伝子に関し、「配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドが有するスクアレンを閉環して5環性のホペンを生成する活性と実質的同等の活性」とは、その活性程度が、配列番号2~4のいずれか1に記載のアミノ酸配列のスクアレンを閉環して5環性のホペンを生成する活性と比較して強くても弱くてもよい。例えば、0.1倍、0.2倍、0.3倍、0.4倍、0.5倍、0.6倍、0.7倍、0.8倍、0.9倍、1.0倍、1.1倍、1.2倍、1.5倍、2.0倍、2.5倍、3.0倍、4.0倍、5.0倍、10倍、15倍、20倍、30倍、40倍、50倍の活性を例示することができる。
また、相同性は、BLAST(Basic Local Alignment Search Tool at the National Center forBiological Information)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)を用いて計算することができる。
上記(5)の遺伝子は、DNA同士のハイブリダイゼーションを利用することにより得られる遺伝子である。この遺伝子における「ストリンジェントな条件」とは、特異的なハイブリダイゼーションのみが起き、非特異的なハイブリダイゼーションが起きないような条件をいう。このような条件は、通常、5×SSC、1%SDSを含む緩衝液中の37℃でのハイブリダイゼーション及び1×SSC、0.1%SDSを含む緩衝液による37℃での洗浄処理といった条件であり、好ましくは、5×SSC、1%SDSを含む緩衝液中の42℃でのハイブリダイゼーション
及び0.5×SSC、0.1%SDSを含む緩衝液による42℃での洗浄処理といった条件であり、更に好ましくは、5×SSC、1%SDSを含む緩衝液中の65℃でのハイブリダイゼーション及び0.2×SSC、0.1%SDSを含む緩衝液による65℃での洗浄処理といった条件である。ハイブリダイゼーションを利用することにより得られたDNAが、活性を有するポリペプチドをコードするかどうかは、例えば、そのDNAを大腸菌等に導入し、発現させ、その大腸菌等がスクアレンを閉環して5環性のホペンを生成することができるかどうか調べることによりわかる。ハイブリダイゼーションにより得られるDNAは、上記(4)の遺伝子(配列番号5~7のいずれか1)と通常、高い相同性を有する。高い相同性とは、90%以上の相同性、好ましくは95%以上の相同性、更に好ましくは98%以上の相同性を指す。
上記(6)の遺伝子は、配列番号5~7のいずれか1に記載の塩基配列からなるDNAにおいて、1~50個、好ましくは1~30個、より好ましくは1~20個、特に好ましくは1~10個、最も好ましくは1~5個の塩基配列が置換、欠損、挿入及び/又は付加しているDNAからなる遺伝子である。
上記(7)の遺伝子は、配列番号5~7のいずれか1に記載の塩基配列からなるDNAと90%以上、好ましくは93%以上、より好ましくは95%以上、最も好ましくは98%以上の相同性を有するDNAからなる遺伝子を、含む。
(Mutant squalene-hopene cyclase gene)
The mutant squalene-hopene cyclase gene of the present invention includes (1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4; (2) SEQ ID NO: 2
In the amino acid sequence according to any one of 4 to 4, 1 to 20 amino acids, preferably 1 to 15 amino acids, more preferably 1 to 10 amino acids, most preferably 1 to 5 amino acids are substituted, deleted, inserted and/or or a polypeptide having substantially the same activity as the activity of ring-closing squalene to produce pentacyclic hopene, which the polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4 has. A gene encoding a peptide, (3) having 90% or more homology with the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4, and from the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4. (4) a base according to any one of SEQ ID NOs: 5 to 7; (5) A gene that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence set forth in any one of SEQ ID NOS: 5 to 7, and A gene encoding a polypeptide having substantially the same activity as the activity of ring-closing squalene to produce pentacyclic hopene, which the polypeptide consisting of the amino acid sequence according to any one of Nos. 2 to 4 has (6 ) A gene consisting of DNA in which 1 to 50 base sequences have been substituted, deleted, inserted, and/or added to the DNA consisting of the base sequence set forth in any one of SEQ ID NOs: 5 to 7, (7) SEQ ID NO: It includes a gene consisting of DNA having 90% or more homology with the DNA consisting of the base sequence described in any one of items 5 to 7.
The gene (1) above is a gene encoding a squalene-hopene cyclase obtained from the method of screening for squalene-consuming enzymes and/or variants thereof of the present invention. The squalene-hopene cyclase has the activity of ring-closing squalene to produce pentacyclic hopene.
The gene (2) above is a gene encoding a polypeptide into which a mutation has been introduced into the mutant squalene-hopene cyclase of the present invention to the extent that the enzymatic activity is not lost. Such mutations include not only mutations that occur in nature but also artificial mutations. Examples of means for producing artificial mutations include site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1982). The number of mutated amino acids is usually within 20 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably 3 amino acids. Whether or not a mutated polypeptide retains enzymatic activity can be determined by, for example, introducing a gene encoding a mutated polypeptide into E. coli, etc., expressing it, and the E. coli etc. closing squalene to form a 5-ring compound. This can be determined by examining whether it is possible to generate sexual hopene. In particular, according to this example, amino acid positions M181, V128, L136, N424, and V440, which are important for enzyme activity, are identified in the amino acid sequence of squalene-hopene cyclase derived from Alicyclobacillus acidocaldarius. If mutations are introduced into the positions/sites of these amino acids, there is a high possibility that enzyme activity will be lost, so mutations are preferably introduced into amino acids other than these amino acids.
Regarding the gene (3) above, "an activity substantially equivalent to the activity of ring-closing squalene to produce pentacyclic hopene, which the polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4 has" The activity may be stronger or weaker than the activity of ring-closing squalene having the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4 to produce pentacyclic hopene. For example, 0.1x, 0.2x, 0.3x, 0.4x, 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, 1.0x, 1.1x, 1.2x, 1.5x, 2.0x, 2.5x, 3.0x, Examples include 4.0-fold, 5.0-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, and 50-fold activity.
Homology can also be calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (eg, using default or default parameters).
The gene (5) above is a gene obtained by utilizing hybridization between DNAs. "Stringent conditions" for this gene refer to conditions under which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions typically include hybridization at 37°C in a buffer containing 5x SSC, 1% SDS and washing at 37°C with a buffer containing 1x SSC, 0.1% SDS. The conditions are preferably hybridization at 42°C in a buffer containing 5x SSC and 1% SDS and washing at 42°C with a buffer containing 0.5x SSC and 0.1% SDS, and more preferably. , hybridization at 65°C in a buffer containing 5x SSC and 1% SDS, and washing at 65°C with a buffer containing 0.2x SSC and 0.1% SDS. Whether or not the DNA obtained by hybridization encodes an active polypeptide can be determined by, for example, introducing the DNA into E. coli, etc., expressing it, and the E. coli, etc. closing the squalene to form a 5-ring ring. This can be determined by examining whether it is possible to generate sexual hopene. The DNA obtained by hybridization usually has high homology to the gene (4) above (any one of SEQ ID NOs: 5 to 7). High homology refers to 90% or more homology, preferably 95% or more homology, more preferably 98% or more homology.
There are 1 to 50 genes, preferably 1 to 30 genes, more preferably 1 to 20 genes, particularly preferably A gene consisting of DNA in which 1 to 10 base sequences, most preferably 1 to 5 base sequences, have been substituted, deleted, inserted, and/or added.
The gene (7) above has a DNA consisting of the base sequence set forth in any one of SEQ ID NOs: 5 to 7 at least 90%, preferably at least 93%, more preferably at least 95%, and most preferably at least 98%. Contains genes consisting of DNA with homology.
(変異型スクアレン-ホペン環化酵素)
本発明の変異型スクアレン-ホペン環化酵素は、(1)配列番号2~4のいずれか1に記載のアミノ酸配列、(2)配列番号2~4のいずれか1に記載のアミノ酸配列において、1~20個、好ましくは1~15個、より好ましくは1~10個、最も好ましくは1~5個のアミノ酸が置換、欠損、挿入及び/又は付加しており、かつ配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドが有するスクアレンを閉環して5環性のホペンを生成する活性を有するアミノ酸配列、(3)配列番号2~4のいずれか1に記載のアミノ酸配列と90%以上の相同性を有し、かつ配列番号2~4のいずれか1に記載のアミノ酸配列からなるポリペプチドが有するスクアレンを閉環して5環性のホペンを生成する活性を有するアミノ酸配列を、含む。
なお、ペプチドの変異の導入において、当該ペプチドの基本的な性質(物性、機能、生理活性又は免疫学的活性等)を変化させないという観点からは、例えば、同族アミノ酸(極性アミノ酸、非極性アミノ酸、疎水性アミノ酸、親水性アミノ酸、陽性荷電アミノ酸、陰性荷電アミノ酸及び芳香族アミノ酸等)の間での相互の置換は容易に想定される。
(mutant squalene-hopene cyclase)
The mutant squalene-hopene cyclase of the present invention comprises (1) the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4; (2) the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4; 1 to 20, preferably 1 to 15, more preferably 1 to 10, most preferably 1 to 5 amino acids have been substituted, deleted, inserted and/or added, and SEQ ID NOs: 2 to 4 An amino acid sequence having the activity of ring-closing squalene to produce pentacyclic hopene, which is possessed by a polypeptide consisting of the amino acid sequence set forth in any one of (3) the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4. an amino acid sequence having an activity of ring-closing squalene to produce pentacyclic hopene, which is possessed by a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 4. including.
In addition, when introducing mutations into a peptide, from the viewpoint of not changing the basic properties of the peptide (physical properties, functions, physiological activities, immunological activities, etc.), for example, homologous amino acids (polar amino acids, non-polar amino acids, Mutual substitutions between hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) are easily envisaged.
(本発明のスクリーニング方法)
本発明のスクアレン消費酵素のスクリーニング方法は、スクアレン消費酵素の探索を行うため、基質から色素を合成することができる細胞にスクアレン消費酵素遺伝子を導入し発現させて、基質の消費をスクアレン消費酵素と競合する酵素と競合させ、色素合成量の増減を検出することによる、スクアレン消費酵素遺伝子のスクリーニング方法に関する。
さらに、本発明は、スクアレン消費酵素遺伝子ライブラリ(特に、ランダム変異を導入したスクアレン消費酵素遺伝子ライブラリ)を色素合成可能な細胞に導入し、色素合成量の増減を検出することによる、野生型スクアレン消費酵素と比較して活性の高い変異型スクアレン消費酵素遺伝子のスクリーニング方法に関する。
(Screening method of the present invention)
In order to search for squalene consuming enzymes, the squalene consuming enzyme screening method of the present invention introduces and expresses a squalene consuming enzyme gene into cells capable of synthesizing pigments from substrates, thereby controlling the consumption of substrates from squalene consuming enzymes. This invention relates to a method for screening squalene-consuming enzyme genes by detecting an increase or decrease in the amount of pigment synthesis in competition with a competing enzyme.
Furthermore, the present invention is capable of consuming wild type squalene by introducing a squalene consuming enzyme gene library (in particular, a squalene consuming enzyme gene library into which random mutations have been introduced) into cells capable of pigment synthesis, and detecting an increase or decrease in the amount of pigment synthesis. This invention relates to a method for screening mutant squalene-consuming enzyme genes that have higher activity than other enzymes.
(スクアレン消費酵素)
本発明での「スクアレン消費酵素」とは、スクアレンの消費に関与する酵素(複数の酵素群も含む)である。特に、スクアレン消費酵素は、スクアレンを直接又は間接的に色素に転換する作用を有すれば特に限定されない。
例えば、スクアレン-ホペン環化酵素、スクアレン不飽和化酵素、CrtNb(4, 4'-diapolycopene oxidase)、スクアレンエポキシダーゼ、アンブレイン合成酵素、スクアレン水素化酵素及びスクアレン飽和化酵素等を例示することができる。
また、本発明での「スクアレン消費酵素遺伝子」とは、スクアレン消費酵素をコードす
る遺伝子である。「その変異体」とは、スクアレン消費酵素遺伝子のDNAがその一部のヌクレオチドの置換、欠失、付加などにより変異した遺伝子の概念である。自然界で生じた変異でもよいし、人工的になされた変異でもよい。
変異は、発現酵素の酵素活性、発現量又は安定性が高くなる変異が好ましい。また基質特異性を高めるような変異も好ましい。特に、酵素活性を高める変異が好ましい。
変異体のライブラリを作成し、そのライブラリから、その宿主において酵素活性、発現量又は安定性が高い変異体を本発明の方法により、スクリーニングできる。
本発明における「スクアレンを経由して合成される色素合成可能な細胞へ導入されるスクアレン消費酵素遺伝子」とは、スクアレン消費酵素遺伝子若しくはその変異体を含むと考えられる遺伝子又は遺伝子群である。天然の細胞から抽出した遺伝子、作製したライブラリの遺伝子、化学合成遺伝子、PCR増幅遺伝子など由来は問わない。本発明のスクリーニング方法は、発現する酵素の基質消費能を指標とするため、未同定の遺伝子をスクリーニングすることができる。
スクアレン消費酵素遺伝子又はその変異体の、スクアレンを経由して合成される色素合成可能な細胞への導入は、遺伝子組み換え技術で用いられている常法によることができる。例えば、ベクターにスクリーニング対象の遺伝子を組み込み、細胞を形質転換する等である。
(squalene consuming enzyme)
The "squalene consuming enzyme" in the present invention is an enzyme (including a plurality of enzyme groups) involved in the consumption of squalene. In particular, the squalene consuming enzyme is not particularly limited as long as it has the function of directly or indirectly converting squalene into a pigment.
Examples include squalene-hopene cyclase, squalene desaturase, CrtNb (4, 4'-diapolycopene oxidase), squalene epoxidase, unbrain synthase, squalene hydrogenase, and squalene saturase. can.
Moreover, the "squalene consuming enzyme gene" in the present invention is a gene encoding a squalene consuming enzyme. The term "variant" refers to a gene in which the DNA of a squalene consuming enzyme gene has been mutated due to partial nucleotide substitution, deletion, addition, or the like. It may be a mutation that occurs in nature, or it may be an artificial mutation.
Preferably, the mutation is a mutation that increases the enzyme activity, expression level, or stability of the expressed enzyme. Mutations that increase substrate specificity are also preferred. In particular, mutations that increase enzyme activity are preferred.
A library of mutants can be created, and from the library, mutants with high enzyme activity, high expression level, or high stability in the host can be screened by the method of the present invention.
In the present invention, "a squalene consuming enzyme gene introduced into a cell capable of synthesizing a pigment synthesized via squalene" is a gene or gene group that is considered to include a squalene consuming enzyme gene or a variant thereof. The origin does not matter, such as a gene extracted from natural cells, a gene from a prepared library, a chemically synthesized gene, or a PCR amplified gene. Since the screening method of the present invention uses the substrate consuming ability of the expressed enzyme as an index, it is possible to screen unidentified genes.
The squalene-consuming enzyme gene or its mutant can be introduced into cells capable of synthesizing a pigment that is synthesized via squalene by a conventional method used in gene recombination technology. For example, a gene to be screened is inserted into a vector and cells are transformed.
(スクアレン消費酵素と競合する酵素)
本発明での「スクアレン消費酵素と競合する酵素」とは、基質の消費をスクアレン消費酵素と競合する特性を有する。
例えば、下記の表1に記載の酵素を例示することができる。
(enzyme that competes with squalene consuming enzyme)
In the present invention, an "enzyme that competes with a squalene consuming enzyme" has the property of competing with a squalene consuming enzyme for substrate consumption.
For example, the enzymes listed in Table 1 below can be exemplified.
(色素合成に関与又は競合する酵素の基質)
本発明での「色素合成に関与又は競合する酵素の基質」とは、スクアレンだけでなく、スクアレンの前駆物質や、スクアレン派生物質であるジアポニューロスポレン、ジアポリコペン等も対象とする。
(Substrate for enzymes involved in or competing with pigment synthesis)
In the present invention, "a substrate for an enzyme that participates in or competes with pigment synthesis" refers not only to squalene but also to squalene precursors and squalene derivatives such as diaponeurosporene and diapolicopene.
(本発明のスクリーニング方法での組合せ)
本発明のスクリーニング方法では、下記の表1に記載の組合せを例示することができるが、特に限定されない。
(Combination in the screening method of the present invention)
In the screening method of the present invention, the combinations listed in Table 1 below can be exemplified, but the combinations are not particularly limited.
例えば、色素合成に関与又は競合する酵素の基質がスクアレンであり、スクアレン消費酵素がスクアレン-ホペン環化酵素であり、スクアレン消費酵素と競合する酵素がスクアレン不飽和化酵素の時、スクアレン消費酵素の活性が高い場合にはジアポニューロスポレン(黄色)の合成量が低下し(ホペン(無色)の合成量が増加し)、スクアレン消費酵素
の活性が低い場合にはジアポニューロスポレン(黄色)の合成量が上昇する(ホペン(無色)の合成量が低下する)。
例えば、色素合成に関与又は競合する酵素の基質がスクアレンであり、スクアレン消費酵素がスクアレン不飽和化酵素であり、スクアレン消費酵素と競合する酵素がスクアレン-ホペン環化酵素の時、スクアレン消費酵素の活性が高い場合にはジアポニューロスポレン(黄色)の合成量が上昇し(ホペン(無色)の合成量が低下する)、スクアレン消費酵素の活性が低い場合にはジアポニューロスポレン(黄色)の合成量が減少する(ホペン(無色)の合成量が増加する)。
例えば、色素合成に関与又は競合する酵素の基質がスクアレンであり、スクアレン消費酵素がスクアレン-ホペン環化酵素であり、スクアレン消費酵素と競合する酵素がスクアレン不飽和化酵素CrtN及びCrtNb(4, 4'-diapolycopene oxidase)のオペロンの時、スクアレン消費酵素の活性が高い場合には赤色色素であるジアポリコペンジアールの合成量が低下し(ホペンの合成量が増加し)、スクアレン消費酵素の活性が低い場合には赤色色素であるジアポリコペンジアールの合成量が増加する(ホペンの合成量が低下する)。
例えば、色素合成に関与又は競合する酵素の基質がスクアレンであり、スクアレン消費酵素がスクアレンエポキシダーゼであり、スクアレン消費酵素と競合する酵素がスクアレン不飽和化酵素の時、スクアレン消費酵素の活性が高い場合にはジアポニューロスポレン(黄色)の合成量が低下する。
例えば、色素合成に関与又は競合する酵素の基質がスクアレンであり、スクアレン消費酵素がアンブレイン合成酵素であり、スクアレン消費酵素と競合する酵素がスクアレン不飽和化酵素の時、スクアレン消費酵素の活性が高い場合にはジアポニューロスポレン(黄色)の合成量が低下する。
例えば、色素合成に関与又は競合する酵素の基質がスクアレンであり、スクアレン消費酵素がスクアレン水素化酵素(スクアレン飽和化酵素)であり、スクアレン消費酵素と競合する酵素がスクアレン不飽和化酵素の時、スクアレン消費酵素の活性が高い場合にはジアポニューロスポレン(黄色)の合成量が低下する。
For example, when the substrate of an enzyme that participates in or competes with pigment synthesis is squalene, the squalene consuming enzyme is squalene-hopene cyclase, and the enzyme that competes with the squalene consuming enzyme is squalene desaturase, the squalene consuming enzyme When the activity is high, the amount of diaponeurosporene (yellow) synthesized decreases (the amount of hopene (colorless) synthesized increases), and when the activity of the squalene consuming enzyme is low, the amount of diaponeurosporene (yellow) synthesized decreases (the amount of hopene (colorless) synthesized increases). ) increases (the amount of hopene (colorless) synthesized decreases).
For example, when the substrate of an enzyme that participates in or competes with pigment synthesis is squalene, the squalene consuming enzyme is squalene desaturase, and the enzyme that competes with the squalene consuming enzyme is squalene-hopene cyclase, the squalene consuming enzyme When the activity is high, the amount of diaponeurosporene (yellow) synthesized increases (the amount of hopene (colorless) synthesized decreases), and when the activity of the squalene consuming enzyme is low, the amount of diaponeurosporene (yellow) synthesized increases (the amount of hopene (colorless) synthesized decreases). ) decreases (the amount of hopene (colorless) synthesized increases).
For example, the substrate of an enzyme that participates in or competes with pigment synthesis is squalene, the squalene consuming enzyme is squalene-hopene cyclase, and the enzyme that competes with the squalene consuming enzyme is squalene desaturase CrtN and CrtNb (4, 4 '-diapolycopene oxidase) operon, if the activity of the squalene consuming enzyme is high, the amount of synthesis of diapolycopene dial, a red pigment, decreases (the amount of hopene synthesis increases), and the activity of the squalene consuming enzyme decreases. When this is low, the amount of diapolicopendial, which is a red pigment, synthesized increases (the amount of hopene synthesized decreases).
For example, when the substrate of an enzyme that participates in or competes with pigment synthesis is squalene, the squalene consuming enzyme is squalene epoxidase, and the enzyme that competes with the squalene consuming enzyme is squalene desaturase, the activity of the squalene consuming enzyme is high. In some cases, the amount of diaponeurosporene (yellow) synthesized decreases.
For example, when the substrate of an enzyme that participates in or competes with pigment synthesis is squalene, the squalene consuming enzyme is unbrain synthase, and the enzyme that competes with the squalene consuming enzyme is squalene desaturase, the activity of the squalene consuming enzyme is When it is high, the amount of diaponeurosporene (yellow) synthesized decreases.
For example, when the substrate of an enzyme that participates in or competes with pigment synthesis is squalene, the squalene consuming enzyme is squalene hydrogenase (squalene saturase), and the enzyme that competes with the squalene consuming enzyme is squalene desaturase, When squalene-consuming enzyme activity is high, the amount of diaponeurosporene (yellow) synthesized decreases.
(スクアレンを経由して合成される色素合成可能な細胞)
本発明での「スクアレンを経由して合成される色素合成可能な細胞」は、上記の酵素により色素を生合成することができる細胞であれば制限されない。天然の細胞でも、遺伝子組み換えにより形質転換されている細胞でもよい。遺伝子組み換え細胞は、遺伝子組み換えの操作に簡便な細胞である、大腸菌、枯草菌、酵母などが挙げられる。
本発明で用いる好ましい色素合成可能な細胞は、色素合成に関与する酵素遺伝子若しくはその変異体、及び/又は、色素合成に競合する酵素遺伝子若しくはその変異体により形質転換されている細胞である。また、さらにイソペンテニル二リン酸イソメラーゼ(idi)遺伝子により、Idiを細胞に導入し、イソプレノイド経路を強化した細胞が好ましい。
本発明で用いる色素合成可能な細胞は、必要に応じて、ジアポフィトエン合成酵素(CrtM)遺伝子若しくはその変異体、及び/又はデヒドロスクアレン不飽和化酵素(CrtN)遺伝子若しくはその変異体を形質転換された細胞でも良い。その色素合成可能細胞は、C15PPを基質として、ジアポニューロスポレン(黄色)ジアポリコペン(赤色)等を生成し、細胞が黄色や赤色等になる。CrtM遺伝子とCrtN遺伝子又はそれらの変異体は、同一のベクターに組み込まれていてもよいし、別のベクターに組み込まれていてもよい。
また、本発明で用いる色素合成可能な細胞は、必要に応じて、CrtM遺伝子若しくはその変異体、及び/又はCrtN遺伝子若しくはその変異体に加えさらにゲラニル二リン酸合成酵素(GPS)遺伝子又はその変異体により形質転換された細胞でも良い。その色素合成可能な細胞は、C10PPを基質として、ジアポニューロスポレン(黄色)ジアポリコペン(赤色)等を生成し、細胞が黄色や赤色等になる。CrtM遺伝子、CrtN遺伝子及びGPS遺伝子又はそれらの変異体は、同一のベクターに組み込まれていてもよいし、別のベクターに組み込まれていてもよい。
また、本発明で用いる色素合成可能な細胞は、必要に応じて、フィトエン合成酵素(Cr
tB)遺伝子若しくはその変異体、及び/又はフィトエン脱水素酵素(CrtI)遺伝子若しくはその変異体により形質転換された細胞でも良い。その色素合成可能細胞は、C20PPを基質として、リコペン(赤色)等を生成し、細胞がピンク色等になる。CrtB遺伝子及びCrtI遺伝子又はそれらの変異体は、同一のベクターに組み込まれていてもよいし、別のベクターに組み込まれていてもよい。
また、本発明で用いる色素合成可能な細胞は、必要に応じて、CrtB遺伝子若しくはその変異体、及び/又はCrtI遺伝子若しくはその変異体に加えさらにゲラニルゲラニル二リン酸合成酵素(CrtE)遺伝子又はその変異体により形質転換された細胞でも良い。その色素合成可能細胞は、C20PPを基質として、リコペン(赤色)等を生成し、細胞がピンク色等になる。特にC20PPを合成しない宿主(例えば大腸菌)にはCrtE遺伝子の導入が必要になる。CrtB遺伝子、CrtI遺伝子及びCrtE遺伝子又はそれらの変異体は、同一のベクターに組み込まれていてもよいし、別のベクターに組み込まれていてもよい。
(Cells capable of synthesizing pigments synthesized via squalene)
In the present invention, "cells capable of synthesizing pigments synthesized via squalene" are not limited as long as they are cells capable of biosynthesizing pigments using the enzymes described above. They may be natural cells or cells that have been transformed by genetic recombination. Examples of genetically modified cells include E. coli, Bacillus subtilis, and yeast, which are cells that are easy to perform genetic recombination operations.
Preferred cells capable of pigment synthesis used in the present invention are cells transformed with an enzyme gene involved in pigment synthesis or a mutant thereof, and/or an enzyme gene or mutant thereof that competes with pigment synthesis. Furthermore, cells in which Idi is introduced into the cells using the isopentenyl diphosphate isomerase (idi) gene to strengthen the isoprenoid pathway are preferable.
The cells capable of pigment synthesis used in the present invention are transformed with the diapophytoene synthase (CrtM) gene or its mutant, and/or the dehydrosqualene desaturase (CrtN) gene or its mutant, as necessary. Cells that have been treated may also be used. These pigment-synthesizing cells use C15PP as a substrate to produce diaponurosporene (yellow), diapolicopene (red), etc., making the cells yellow or red. The CrtM gene and CrtN gene or variants thereof may be integrated into the same vector or into separate vectors.
In addition, the cells capable of pigment synthesis used in the present invention may contain the CrtM gene or a variant thereof, and/or the CrtN gene or a variant thereof, as well as the geranyl diphosphate synthase (GPS) gene or a variant thereof. Cells transformed by the body may also be used. Cells capable of pigment synthesis use C10PP as a substrate to produce diaponeurosporene (yellow), diapolicopene (red), etc., making the cells yellow or red. The CrtM gene, CrtN gene, and GPS gene or variants thereof may be integrated into the same vector or into separate vectors.
In addition, the cells capable of pigment synthesis used in the present invention may contain phytoene synthase (Cr
tB) gene or a mutant thereof, and/or a cell transformed with the phytoene dehydrogenase (CrtI) gene or a mutant thereof. These pigment-synthesizing cells use C20PP as a substrate to produce lycopene (red), making the cells pink. The CrtB gene and CrtI gene or variants thereof may be integrated into the same vector or into separate vectors.
In addition, the cells capable of pigment synthesis used in the present invention may contain the CrtB gene or a mutant thereof, and/or the CrtI gene or a mutant thereof, as well as the geranylgeranyl diphosphate synthase (CrtE) gene or a mutant thereof, as necessary. Cells transformed by the body may also be used. These pigment-synthesizing cells use C20PP as a substrate to produce lycopene (red), making the cells pink. In particular, it is necessary to introduce the CrtE gene into hosts that do not synthesize C20PP (eg, E. coli). The CrtB gene, CrtI gene, and CrtE gene or variants thereof may be integrated into the same vector or into separate vectors.
(色素合成量の増加又は低下)
本発明での「色素合成量の増加又は低下」は、細胞における色素生合成の量が増加又は低下し、細胞中の色素量が増加又は減少することである。スクアレン消費酵素とスクアレン消費酵素と競合する酵素が基質の消費を競合することにより、色素合成量が増加又は低下する。すなわち、本発明のスクリーニング方法では、スクアレン消費酵素遺伝子を導入した色素合成可能な細胞における色素合成量を、スクアレン消費酵素遺伝子を導入していない色素合成可能な細胞における色素合成量と比較し、色素合成量が増加又は低下した細胞を選択し、その細胞に導入された該遺伝子をスクアレン消費酵素遺伝子であると決定する。
(Increase or decrease in pigment synthesis amount)
In the present invention, "increase or decrease in the amount of pigment synthesis" means that the amount of pigment biosynthesis in cells increases or decreases, and the amount of pigments in cells increases or decreases. The amount of pigment synthesis increases or decreases as a squalene consuming enzyme and an enzyme that competes with the squalene consuming enzyme compete for substrate consumption. That is, in the screening method of the present invention, the amount of pigment synthesis in cells capable of pigment synthesis into which a squalene consuming enzyme gene has been introduced is compared with the amount of pigment synthesis in cells capable of pigment synthesis without introducing a squalene consuming enzyme gene. Cells in which the amount of synthesis increases or decreases are selected, and the gene introduced into the cells is determined to be a squalene consuming enzyme gene.
色素合成量の増加若しくは低下を検出する方法、又は色素合成量の増加若しくは低下を測定する方法は、視認により実施できる。また、各種測定機器、マイクロプレートリーダー、分光光度計、測色光度計などの各種測定機器により検出できる。
本発明のスクリーニング方法を用いれば、細胞を破壊することなく、スクアレン消費酵素の活性を検出又は測定することができる。具体的には、視認によるときは、細胞のコロニーをプレート上に作成し、コロニー色を観察する。視認によるものであれば、0.3-1.5 mm、特に0.5-1.0 mm直径のコロニーを形成させ、目視により探索する。
スループットをあげる必要があるときは、低倍率拡大鏡などを用い、より小さなコロニーを密集させる手法を用いることができる。また、画像解析ソフトウエアを用いることによって、コロニー色の強弱を定量化し、コロニーピッカーを用いてスクリーニング全般を半自動化することもできる。
A method for detecting an increase or decrease in the amount of pigment synthesis or a method for measuring an increase or decrease in the amount of pigment synthesis can be carried out by visual confirmation. In addition, it can be detected by various measuring instruments such as a microplate reader, a spectrophotometer, and a colorimeter.
Using the screening method of the present invention, the activity of squalene-consuming enzymes can be detected or measured without destroying cells. Specifically, when visual recognition is used, a colony of cells is created on a plate and the color of the colony is observed. If by visual inspection, form a colony with a diameter of 0.3-1.5 mm, especially 0.5-1.0 mm, and search visually.
When it is necessary to increase the throughput, a method can be used in which smaller colonies are concentrated using a low-power magnifying glass. Furthermore, by using image analysis software, the intensity of colony color can be quantified, and the overall screening can be semi-automated using a colony picker.
(色素合成可能細胞の製造方法)
色素合成可能細胞は、常法により製造することができる。例えば、各遺伝子を、購入又はPCRなどにより常法により調製し、その遺伝子を宿主に適する発現ベクターに常法により組み込み、目的のベクターを選択し、そのベクターにより宿主細胞を常法により形質転換することにより得られる。
用いる宿主細胞は、制限されないが、培養時間の短縮やクローニングの容易さを考えれば、大腸菌、枯草菌、酵母などが好ましい。特に大腸菌、酵母が好ましく、好適な大腸菌としては、Escherichia coli XL1-Blueのようなクローニング株、HB101やBL21などの発現株に加え、テルペン前駆体の合成量が豊富な遺伝子ノックアウト株、たとえばJW1750 ΔgdhA (glutamate dehydrogenase欠損)、JW0110 ΔaceE(pyruvate dehydrogenase欠損)(Baba,T. et al.; Mol Syst Biol 2, 2006 0008 (2006))等が挙げられ、好適な酵母としては、標準的な発芽酵母、INVSc1(invitrogen)、YPH499(stratagene)等が挙げられる。
好適なベクターは、特に制限はなく、一般に用いられているベクターでよい。例えば、宿主が大腸菌であるときは、pUC18、pACYC184、などに由来するものが挙げられ、宿主が枯草菌であるときはpUB110、pE194、pC194、pHY300PLK DNAなどが、宿主が酵母であると
きは、pRS303、YEp213、TOp2609等が挙げられる。
(Method for producing cells capable of pigment synthesis)
Cells capable of pigment synthesis can be produced by conventional methods. For example, each gene is prepared by a conventional method by purchasing or by PCR, the gene is inserted into an expression vector suitable for the host by a conventional method, a target vector is selected, and a host cell is transformed by the vector by a conventional method. It can be obtained by
The host cell to be used is not limited, but Escherichia coli, Bacillus subtilis, yeast, and the like are preferred in view of shortening culture time and ease of cloning. Escherichia coli and yeast are particularly preferred, and suitable examples of Escherichia coli include cloning strains such as Escherichia coli XL1-Blue, expression strains such as HB101 and BL21, and gene knockout strains that synthesize abundant amounts of terpene precursors, such as JW1750 ΔgdhA. (glutamate dehydrogenase deficient), JW0110 ΔaceE (pyruvate dehydrogenase deficient) (Baba, T. et al.; Mol Syst Biol 2, 2006 0008 (2006)), etc. Suitable yeasts include standard germinating yeast, Examples include INVSc1 (invitrogen) and YPH499 (stratagene).
Suitable vectors are not particularly limited and may be commonly used vectors. For example, when the host is E. coli, examples include those derived from pUC18, pACYC184, etc. When the host is Bacillus subtilis, examples include pUB110, pE194, pC194, pHY300PLK DNA, etc. When the host is yeast, examples include DNA derived from pUC18, pACYC184, etc. Examples include pRS303, YEp213, TOp2609, and the like.
(スクアレン消費酵素遺伝子又はその変異体のスクリーニング用キット)
本発明のスクアレン消費酵素遺伝子又はその変異体のスクリーニング用キットは、色素合成可能細胞を含むものであり得る。色素合成可能細胞は、冷凍保存してバイアル内に封入したものが好適である。あるいは、本発明に係るキットは、所望の細胞を色素合成可能細胞に形質転換するための色素合成酵素遺伝子発現ベクターを構成要素として含むものであり得る。
さらに、上記本発明に係るキットは、被検遺伝子用の発現ベクター、クローニング酵素セット、メタゲノムソース、あるいは配列データベースをもとに作製された(候補)遺伝子ライブラリ、マイクロプレート、イソプレノイド経路(テルペン上流経路)の強化された菌株、培地や添加剤などのスクリーニングに有用な物質や器具(画像スキャナや測色光度計、色見本、コロニーピッカー)などをキットの構成要素として加えることができる。
(Kit for screening squalene consuming enzyme gene or its mutant)
The kit for screening squalene consuming enzyme genes or variants thereof of the present invention may contain cells capable of pigment synthesis. It is preferable that cells capable of pigment synthesis be stored frozen and sealed in a vial. Alternatively, the kit according to the present invention may contain as a component a pigment synthase gene expression vector for transforming desired cells into cells capable of pigment synthesis.
Furthermore, the kit according to the present invention includes an expression vector for a test gene, a cloning enzyme set, a metagenomic source, or a (candidate) gene library prepared based on a sequence database, a microplate, an isoprenoid pathway (terpene upstream pathway ), materials and equipment (image scanners, photometers, color samples, colony pickers) useful for screening enhanced bacterial strains, culture media, additives, etc. can be added as components of the kit.
以下、実施例を挙げて本発明を説明するが、本発明はこれら実施例により何ら限定されるものではない。 The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples in any way.
(SHCライブラリの作製)
以下の通り、スクアレン消費酵素であるスクアレン・ホペンサイクラーゼ(スクアレン-ホペン環化酵素)の遺伝子全域にerror-prone PCRを施してランダム変異を導入し、SHCライブラリ(pJ211 -plac-[SHC]lib)を作製した。概要を図1に示す。
(Creation of SHC library)
As shown below, random mutations were introduced into the entire gene of squalene-hopene cyclase, a squalene-consuming enzyme, by error-prone PCR, and the SHC library (pJ211 -plac- [SHC] lib) was created. An overview is shown in Figure 1.
(ランダム変異導入)
(1)テンプレートとしてpJ211-plac-SHCwt(図2)からPCR増幅したSHCwt(5ng)、TaqDNAポリメラーゼ(5-units、NEB)、プライマー(pJ211-SHCF:5'-TTTTGAATTCTCGGGAGGTACATATGGCTGAGCAGTTGGTGGA-3':配列番号8及びpJ211-SHCR:5'-TTTTACTAGTTTACCTGCGCTCGATGGCTT-3':配列番号9)、MnCl2(0、10又は20μM)、dNTP(各2mM)、5%DMSO及び超純水(残部)を混合し総量50μLとした。
(2)94℃5秒、続いて(94℃15秒-57℃15秒-68℃2分)×25サイクル、最後に68℃5分のサイクルでPCRを行い、MnCl2濃度0μM、10μM及び20μMにおいて得られたPCR産物をそれぞれプラスミドライブラリ化に用いるインサートとした。
(3)PCR産物をアガロースゲル電気泳動し、PCR産物のシングルバンドの所見より増幅効率を確認した。
その結果、PCR増幅収量は、MnCl2が0μMの場合5μg、MnCl2が10μMの場合5μg、及びMnCl2が20μMの場合3μgであり、増幅効率はそれぞれ1000倍、1000倍、及び600倍であったことを確認した。
(Random mutation introduction)
(1) SHCwt (5 ng) PCR amplified from pJ211-plac-SHCwt (Figure 2) as a template, Taq DNA polymerase (5-units, NEB), primer (pJ211-SHCF: 5'-TTTTGAATTCTCGGGAGGTACATATGGCTGAGCAGTTGGTGGA-3': SEQ ID NO: 8 and pJ211-SHCR:5'-TTTTACTAGTTTACCTGGCGCTCGATGGCTT-3': SEQ ID NO: 9), MnCl2 (0, 10 or 20 μM), dNTPs (2 mM each), 5% DMSO and ultrapure water (remainder) were mixed to make a total volume of 50 μL. .
(2) PCR was performed at 94°C for 5 seconds, followed by (94°C for 15 seconds - 57°C for 15 seconds - 68°C for 2 minutes) x 25 cycles, and a final cycle of 68°C for 5 minutes, with MnCl2 concentrations of 0 μM, 10 μM and 20 μM. The PCR products obtained in the above were used as inserts for constructing a plasmid library.
(3) The PCR product was subjected to agarose gel electrophoresis, and the amplification efficiency was confirmed from the observation of a single band of the PCR product.
As a result, the PCR amplification yield was 5 μg when MnCl2 was 0 μM, 5 μg when MnCl2 was 10 μM, and 3 μg when MnCl2 was 20 μM, and the amplification efficiency was 1000 times, 1000 times, and 600 times, respectively. confirmed.
(プラスミドライブラリ化)
(1)pJ211-plac-SHCwtをテンプレートとしてQ5ポリメラーゼ(NEB)でベクター部分を増幅した。
(2)前記ベクター部分及び前記インサートをEcoRI/Spelで切断し、ライゲーションした。
(3)BW25113エレクトロポレーション用コンピテントセルに形質転換し、ライブラリを回収し、MnCl2濃度0μM、10μM及び20μM(0μMライブラリ、10μMライブラリ及び20μMライブラリ)におけるライブラリサイズを確認した。
結果を表2に示す。
(Plasmid library)
(1) Using pJ211-plac-SHCwt as a template, the vector portion was amplified with Q5 polymerase (NEB).
(2) The vector portion and the insert were cut with EcoRI/Spel and ligated.
(3) BW25113 competent cells for electroporation were transformed, the library was collected, and the library size was confirmed at MnCl2 concentrations of 0 μM, 10 μM, and 20 μM (0 μM library, 10 μM library, and 20 μM library).
The results are shown in Table 2.
(SHCライブラリのスクアレン消費酵素スクリーニングのためのCrtNの選択)
実施例3のSHCライブラリを使用したスクアレン消費酵素スクリーニングで使用するスクアレンを経由して合成される色素合成に関与する酵素であるCrtNを選択するため、pUC-hSQSCrtNopt、大腸菌に最適化していないpUC-hSQS-CrtN及びCrtNを合成しないpUC-hSQSを用いて評価した。
(CrtN selection for squalene consuming enzyme screening of SHC library)
In order to select CrtN, an enzyme involved in pigment synthesis synthesized via squalene, used in squalene-consuming enzyme screening using the SHC library in Example 3, pUC-hSQSCrtNopt and pUC-, which has not been optimized for E. coli, were used. Evaluation was performed using hSQS-CrtN and pUC-hSQS that does not synthesize CrtN.
(手順)
(1)大腸菌コドンに最適化されたCrtN(CrtNopt)について、翻訳開始配列を3通りに変え、それぞれ開始効率を高(CrtNopt100,000)・中(CrtNopt10,000)・低(CrtNopt500)に設計し、3種類のpUC-hSQS-CrtNoptを作製した。
(2)作製したpUC-hSQSCrtNopt、大腸菌に最適化していないpUC-hSQS-CrtN及びCrtNを合成しないpUC-hSQSをpJ211-Φ、pJ211-SHCwt、pJ211-SHCdead、pJ211-SHCE45D、pJ211-SHCF437Lと共にXL1Bに導入し、ニトロセルロース膜上に植菌した。37℃で48時間静置培養した後、コロニーの色を観察した。
(procedure)
(1) Regarding CrtN (CrtNopt), which is optimized for E. coli codons, the translation initiation sequence was changed in three ways, and the initiation efficiency was designed to be high (CrtNopt100,000), medium (CrtNopt10,000), and low (CrtNopt500), respectively. , three types of pUC-hSQS-CrtNopt were created.
(2) The prepared pUC-hSQSCrtNopt, pUC-hSQS-CrtN not optimized for E. coli, and pUC-hSQS that does not synthesize CrtN together with pJ211-Φ, pJ211-SHCwt, pJ211-SHCdead, pJ211-SHCE45D, and pJ211-SHCF437L. XL1B and inoculated onto a nitrocellulose membrane. After statically culturing at 37°C for 48 hours, the color of the colonies was observed.
(結果)
結果を図3に示す。
植菌後37℃で44時間静置培養したプレートを観察した結果、CrtNの種類によらず、色の濃さはpJ211-Φ≒pJ211-SHCdead>pJ211-SHCwt>pJ211-SHCE45D>pJ211-SHCF437Lとなった。
なお、SHCE45Dは野生型よりも低温活性型、ΦはSHCを発現しない型、SHCdeadはスクアレン?ホペン環化酵素の活性がない型(D376R)、SHCF437LはSHCE45Dよりもさらに低温活性型を意味する。
よって、pUC-hSQS-CrtNopt10,000(図4)、pUC-hSQS-CrtNopt1,000、pUC-hSQS-CrtNopt500及びpUC-hSQS-CrtNの全てについて、SHC活性(スクアレン消費活性)のスクリーニングに適していた。
次に、植菌後37℃で48時間静置培養したプレートを観察した結果、CrtNopt500では、pJ211-SHCwtの方が低温適応型(大腸菌内活性が高いと思われる)として知られるpJ211-SHCE45Dよりも色素蓄積量の違いが明確であった。すなわち、CrtNopt500株は野生型よりも高い活性を見分けるのに適していることが確認できた。
また、CrtNopt10,000では、pJ211-SHCwt及びpJ211-SHCE45Dの色素蓄積量は同程度であった。すなわち、pJ211-SHCE45Dよ
りもさらに強い活性の探索に適していることが確認できた。
(result)
The results are shown in Figure 3.
As a result of observing the plate that had been statically cultured for 44 hours at 37°C after inoculation, the color intensity was pJ211-Φ≒pJ211-SHCdead>pJ211-SHCwt>pJ211-SHCE45D>pJ211-SHCF437L, regardless of the type of CrtN. became.
Note that SHCE45D is a type that is activated at lower temperatures than the wild type, Φ is a type that does not express SHC, SHCdead is a type that does not have squalene-hopene cyclase activity (D376R), and SHCF437L is a type that is activated at a lower temperature than SHCE45D.
Therefore, pUC-hSQS-CrtNopt10,000 (Figure 4), pUC-hSQS-CrtNopt1,000, pUC-hSQS-CrtNopt500, and pUC-hSQS-CrtN were all suitable for screening for SHC activity (squalene consuming activity). .
Next, as a result of observing the plate that had been statically cultured at 37°C for 48 hours after inoculation, it was found that pJ211-SHCwt was better than pJ211-SHCE45D, which is known as a cold-adapted type (which seems to have higher activity in E. coli), for CrtNopt500. There was also a clear difference in the amount of pigment accumulation. In other words, it was confirmed that the CrtNopt500 strain is suitable for identifying higher activity than the wild type.
Furthermore, in CrtNopt10,000, the amount of pigment accumulation of pJ211-SHCwt and pJ211-SHCE45D was comparable. In other words, it was confirmed that it is suitable for searching for even stronger activity than pJ211-SHCE45D.
(SHCライブラリのスクアレン消費スクリーニング)
pUC-hSQS-CrtNopt10,000を用いて、実施例1で作製したSHCライブラリのSHC活性について、大腸菌コロニーの色から判断するスクアレン消費スクリーニングを行い、活性が上がったSHC変異体を探索した。
(Squalene consumption screening of SHC library)
Using pUC-hSQS-CrtNopt10,000, squalene consumption screening was performed to determine the SHC activity of the SHC library prepared in Example 1 based on the color of E. coli colonies, and SHC mutants with increased activity were searched for.
(手順)
(1)pUC-hSQS-CrtNopt10,000を導入したXL1Blueのケミカルコンピテントセルに、実施例1で作製したSHCライブラリ(0μMライブラリ、10μMライブラリ及び20μMライブラリ)、pJ211-SHCwt又はpJ211-SHCdeadを形質転換し、LB固体培地{カルベニシリン及びカナマイシン含有(Carb.Kan.)}に敷いたNC膜上に植菌した。
(2)プレートを37℃で38時間静置培養した後、室温に取り出し、さらに10時間静置培養し、コロニーの様子を観察した。
(3)pJ211-SHCwtよりも特に色が薄いコロニーをピックし、2 mL LB 液体培地(Carb.Kan.)を分注した48穴ディープウェルで一晩培養した後、プラスミドを回収した。
(procedure)
(1) XL1Blue chemically competent cells introduced with pUC-hSQS-CrtNopt10,000 were transformed with the SHC libraries prepared in Example 1 (0 μM library, 10 μM library, and 20 μM library), pJ211-SHCwt, or pJ211-SHCdead. The cells were then inoculated onto an NC membrane spread on LB solid medium {containing carbenicillin and kanamycin (Carb. Kan.)}.
(2) After statically culturing the plate at 37°C for 38 hours, it was taken out to room temperature, and statically cultured for an additional 10 hours, and the appearance of colonies was observed.
(3) Colonies that were particularly lighter in color than pJ211-SHCwt were picked and cultured overnight in a 48-well deep well into which 2 mL LB liquid medium (Carb.Kan.) was dispensed, and then the plasmid was recovered.
(結果)
結果を図5に示す。
各ライブラリ中に、pJ211-SHCwtよりも色が薄いコロニーが多く見つかった(図5)。その中でも特に色が薄いコロニーをピックして回収した。詳細を下記表3に示す。ミニプレップ、SDS-ポリアクリルアミドゲル電気泳動の結果、SHCの正しいサイズにバンドが検出されたSHC変異体は合計28個であった。
(result)
The results are shown in Figure 5.
In each library, many colonies with a lighter color than pJ211-SHCwt were found (FIG. 5). Among them, colonies with particularly light color were picked and collected. Details are shown in Table 3 below. As a result of miniprep and SDS-polyacrylamide gel electrophoresis, a total of 28 SHC variants were detected with bands at the correct size of SHC.
(SHC変異体のアミノ酸変異マッピング)
実施例3で取得した28個のSHC変異体のうち、24個について配列解析し、公知の方法により、アミノ酸一次配列上へのSHC変異体のアミノ酸変異マッピングを行った。
(Amino acid mutation mapping of SHC mutants)
Of the 28 SHC mutants obtained in Example 3, 24 were sequenced, and amino acid mutations of the SHC mutants were mapped onto the primary amino acid sequence by a known method.
(SHC変異体の生産物解析)
配列解析したSHC変異体についてhSQSと共発現して生産物解析を行った。方法は以下の通りである。
(手順)
(1)XL1BlueをpUC-hSQS並びにpJ211-SHCwt若しくは上記で配列解析したSHC変異体(pJ211-SHCmutants)で共形質転換しコロニーを形成させた。
(2)コロニーをピックしてLB液体培地(Carb.Kan.)培地で37°C、1
6時間培養した。
(3)培養液を50mLフラスコに分注したTB(Carb.Kan.)培地10mLに1/100量(100μL)植菌し37°Cで48時間培養した。
(4)各サンプル培養液10mLを遠心(3750rpm、4℃、15分)により集菌し、生理食塩水で洗浄し、ペレットにアセトン10mLを加えて脂溶性画分を抽出した。
(5)脂溶性画分をlmLヘキサンで抽出し、MgSO4を加え水分を除去した。このうちヘキサン700μLを分取し、TFH:MeOH=6:4の溶媒70μLに濃縮した。
(6)得られたサンプルのうち5μLを用いてHPLC-MSによる解析を行った。固定相にはShimpackカラム(150×2mm)、移動相にはメタノールを用い、流速は0.4mL/minとした。検出にはフォトダイオードアレイ検出器及び質量分析器(イオン化:APCI)を用いた。生産物量はMSのSIMモードの411.4mZzのイオンで得られたクロマトグラムのピーク面積から、スクアレン標品を用いて作製した検量線を用いて算出した。
(Product analysis of SHC mutants)
The sequence-analyzed SHC mutants were co-expressed with hSQS and product analysis was performed. The method is as follows.
(procedure)
(1) XL1Blue was co-transformed with pUC-hSQS and pJ211-SHCwt or the SHC mutants sequenced above (pJ211-SHCmutants) to form colonies.
(2) Pick a colony and place it in LB liquid medium (Carb.Kan.) at 37°C for 1
Cultured for 6 hours.
(3) A 1/100 amount (100 μL) of the culture solution was inoculated into 10 mL of TB (Carb. Kan.) medium, which was dispensed into a 50 mL flask, and cultured at 37° C. for 48 hours.
(4) 10 mL of each sample culture solution was collected by centrifugation (3750 rpm, 4° C., 15 minutes), washed with physiological saline, and 10 mL of acetone was added to the pellet to extract the fat-soluble fraction.
(5) The fat-soluble fraction was extracted with 1 mL hexane, and MgSO4 was added to remove water. Of this, 700 μL of hexane was collected and concentrated to 70 μL of a solvent of TFH:MeOH=6:4.
(6) HPLC-MS analysis was performed using 5 μL of the obtained sample. A Shimpack column (150 x 2 mm) was used as the stationary phase, methanol was used as the mobile phase, and the flow rate was 0.4 mL/min. A photodiode array detector and a mass spectrometer (ionization: APCI) were used for detection. The amount of product was calculated from the peak area of the chromatogram obtained with the 411.4 mZz ion in the SIM mode of MS using a calibration curve prepared using a squalene standard.
(結果)
アミノ酸変異マッピングと生産物解析の結果を併せた結果を図7及び表4に示す。
生産物解析の結果を図6に示す。
今回解析したSHC変異体は、全て細胞あたりのホペン合成量が野生型よりも増えていた。最も高い変異体は29倍も多くなっていた(図6のサンプルNo.10-2)。
単独で活性を上げる変異が5個あり、複数の変異体に見られた変異箇所が6個あった(表4のV128及びM181はどちらにも当てはまる)。これらを組み合わせることによって、さらに活性の高い変異体が得られる。
(result)
The combined results of amino acid mutation mapping and product analysis are shown in FIG. 7 and Table 4.
The results of product analysis are shown in Figure 6.
All of the SHC mutants analyzed this time synthesized more hopene per cell than the wild type. The highest variant had a 29-fold increase (sample No. 10-2 in Figure 6).
There were 5 mutations that individually increased the activity, and 6 mutations that were found in multiple mutants (V128 and M181 in Table 4 apply to both). By combining these, even more active variants can be obtained.
本発明は、野生型スクアレン-ホペン環化酵素と比較してホペン合成量が高い変異型スクアレン-ホペン環化酵素並びに該酵素のスクリーニング方法を提供することができる。 The present invention can provide a mutant squalene-hopene cyclase that synthesizes a higher amount of hopene than the wild-type squalene-hopene cyclase, and a method for screening the enzyme.
Claims (6)
ここで、アミノ酸置換を導入する酵素の野生型のアミノ酸配列が配列番号1であり、配列番号2又は4に記載のアミノ酸配列と90%以上の同一性を有し、かつ、配列番号1に記載の野生型スクアレン-ホペン環化酵素と比較してホペン合成量が高い、変異型スクアレン-ホペン環化酵素。
(1)F437L、R131Q、M159I
(2)F437L、G259S、K335R、Q525R、A574T
A mutant squalene-hopene cyclase having an amino acid substitution selected from the following (1) or (2),
Here, the wild-type amino acid sequence of the enzyme introducing the amino acid substitution is SEQ ID NO: 1 , has 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 2 or 4, and is set as SEQ ID NO: 1. A mutant squalene-hopene cyclase that synthesizes a higher amount of hopene than the wild-type squalene-hopene cyclase .
(1) F437L, R131Q, M159I
(2) F437L, G259S, K335R, Q525R, A574T
The mutant squalene-hopene cyclase according to claim 1, further comprising an amino acid substitution of V440L.
The mutant squalenehopene cyclase according to claim 1, further comprising an amino acid substitution of V128L.
The mutant squalene-hopene cyclase according to claim 1, further comprising an amino acid substitution of L136P.
(1)配列番号2又は配列番号4に記載のアミノ酸配列からなるポリペプチドをコードする遺伝子
(2)配列番号5又は配列番号7に記載の塩基配列からなるDNAからなる遺伝子
A mutant squalene-hopene cyclase gene consisting of the following gene (1) or (2).
(1) Gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. (2) Gene consisting of DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7.
(1)配列番号2又は配列番号4に記載のアミノ酸配列。 A mutant squalene-hopene cyclase consisting of the following amino acid sequence (1).
(1) Amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4.
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WO2016170099A1 (en) | 2015-04-24 | 2016-10-27 | Givaudan Sa | Enzymes and applications thereof |
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SATO,Tsutomu, et al.,Bioscience. Biotechnology. and Biochemistry,2004年,Vol. 68, No. 3,pp. 728-738 |
SIEDENBURG,Gabriele, et al.,Applied and Environmental Microbiology,2011年,Vol. 77, No. 12,pp. 3905-3915 |
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