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グラム陰性細菌Burkholderia stabilis由来コレステロールエステラーゼの基質結合メカニズムと高生産化に関する研究

小西, 健司 北海道大学

2023.03.23

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グラム陰性細菌Burkholderia stabilis由来コレステロールエステラーゼの基質結合メカニズムと高生産化に関
する研究

小西, 健司

北海道大学. 博士(農学) 甲第15294号

2023-03-23

10.14943/doctoral.k15294

http://hdl.handle.net/2115/89486

theses (doctoral)

Konishi_Kenji.pdf

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Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

博士学位論文

グラム陰性細菌 Burkholderia stabilis 由来
コレステロールエステラーゼの基質結合メカニズムと
高生産化に関する研究

小西

健司

北海道大学大学院農学院
生命フロンティアコース

応用生物化学ユニット

基礎環境微生物学
2023 年 3 月

略語表
略称

正式名称

Che

コレステロールエステラーゼ

Lip

リパーゼ

Lif

Lipase-specific foldase

POD

ペルオキシダーゼ

LB 培地

Luria-Bertani 培地

YS 培地

Yeast sorbitol 培地

YSO 培地

Yeast sorbitol oleic acid 培地

NB 培地

Nutrient broth 培地

TYSS 培地

Tryptone yeast sodium chloride sucrose 培地

RNA-seq

RNA sequence

DNA-seq

DNA sequence

BLAST

Basic Local Alignment Search Tool

Chr

Chromosome

CLL

コレステロールリノレート

4-AA

4-アミノアンチピリン

NTG

N-メチル-N'-ニトロ-N-ニトロソグアニジン

SDS

ドデシル硫酸ナトリウム

SDS-PAGE

SDS ポリアクリルアミド電気泳動

RMSD

平均二乗偏差

FPKM

Fragments per kilobase of exon per million reads mapped

CFU

Colony forming unit

DUF

Domain of unknown function

SMC

Structural maintenance of chromosome

NCBI

National Center for Biotechnology Information

PDB

Protein Data Bank

目次
序論 .......................................................................................................................................... 1
(1)研究の背景................................................................................................................ 1
コレステロールエステラーゼについて.................................................................................................... 1
リパーゼについて ........................................................................................................................................... 5

Burkholderia stabilis 由来 Che と Lip について ....................................................................................... 9
BsChe と Lip は大腸菌での組換え発現が困難 .................................................................................... 15

Burkholderia stabilis を用いた発現系の構築 ........................................................................................ 18

(2)研究の目的..............................................................................................................22

本論 ........................................................................................................................................23
第1章 コレステロールエステラーゼの基質結合メカニズムの解析 ................23
第1節

緒言 ................................................................................................................................................. 23

第2節

材料および方法 ........................................................................................................................... 24

1-2-1. 菌株、プラスミド、培地 ........................................................................................................ 24
1-2-2. 遺伝子クローニング ................................................................................................................. 27
1-2-3. BsChe の精製 ............................................................................................................................... 29
1-2-4. 結晶化条件の最適化 ................................................................................................................. 29
1-2-5. 構造決定と精密化...................................................................................................................... 30
1-2-6. ドッキングシミュレーション解析 ....................................................................................... 34
1-2-7. His タグ付き組換え酵素の作製 .............................................................................................. 34

1-2-8. B. stabilis のコンピテントセル作製と形質転換法 ............................................................ 36
1-2-9. 形質転換体の培養および Che 精製 ...................................................................................... 36
1-2-10. Che および Lip 活性の測定.................................................................................................... 36
第3節

結果および考察 ........................................................................................................................... 40

1-3-1. BsChe の結晶構造解析.............................................................................................................. 40
1-3-2. コレステロールリノレートを用いたドッキングシミュレーション解析 ................. 47
1-3-3. BsChe のコレステロールエステル特異性に関するアミノ酸 ...................................... 50
1-3-4. L266/I287 の導入による Burkholderia Lip への Che 活性付与...................................... 52

第2章

コレステロールエステラーゼの高生産化.................................................58

第1節

緒言 ................................................................................................................................................. 58

第2節

材料および方法 ........................................................................................................................... 59

2-2-1. 菌株、プラスミド、培地 ........................................................................................................ 59
2-2-2. 遺伝子クローニング ................................................................................................................. 62
2-2-3. B. stabilis のコンピテントセル作製と形質転換法 ............................................................ 62
2-2-4. NTG 変異導入 ............................................................................................................................. 66
2-2-5. トランスポゾン変異導入 ........................................................................................................ 66
2-2-6. リコンビナントタンパク質(BsChe、BcLip、BpLip)の発現検討 .......................... 67
2-2-7. BsChe、BcLip、BpLip 活性測定 ............................................................................................ 68
2-2-8. SDS-PAGE とウェスタンブロッティングによる BsChe の検出.................................. 68
2-2-9. DNA-seq 解析 .............................................................................................................................. 69
2-2-10. 遺伝子破壊 ................................................................................................................................ 69
2-2-11. 遺伝子相補実験 ........................................................................................................................ 73
2-2-12. RNA-seq 解析 ............................................................................................................................ 73
2-2-13. プラスミドのコピー数測定 .................................................................................................. 74
2-2-14. プラスミド保持アッセイ ...................................................................................................... 76
2-2-15. 染色体保持アッセイ ............................................................................................................... 76
2-2-16. 形質転換効率の測定 ............................................................................................................... 76

2-2-17. 遺伝子機能の予測 ................................................................................................................... 77
2-2-18. 統計解析と再現性 ................................................................................................................... 77
第3節

結果 ................................................................................................................................................. 78

2-3-1. BsChe 高生産化に Chr3 が関与している ............................................................................ 78
2-3-2. Chr3 上の BSFP_068720、BSFP_068730 は BsChe の生産を抑制する .................... 83
2-3-3. BSFP_068720/30/40 の機能予測 .......................................................................................... 99
2-3-4. BSFP_068720/30/40 はプラスミドのコピー数抑制と安定性低下に関与する ..... 105
2-3-5. BSFP_068720/30/40 の欠失は、Chr3 の安定性に影響しない .................................. 115
2-3-6. BSFP_068720/30/40 の破壊により、類縁酵素の生産性も向上する ...................... 117
第4節

考察 ............................................................................................................................................... 119

2-4-1. Ch3 はリコンビナント BsChe の生産量抑制に寄与する ............................................. 119
2-4-2. BSFP_068720/30/40 はプラスミドのコピー数低下と安定性低下に寄与する ..... 120
2-4-3. BSFP_068720/30/40 は従来の SMC タンパク質とは異なるメカニズムで働く .. 123
2-4-4. まとめ.......................................................................................................................................... 130

総括 ..................................................................................................................................... 131

参考文献 ............................................................................................................................ 132

謝辞 ..................................................................................................................................... 142

序論
(1)研究の背景
コレステロールエステラーゼについて
コレステロールエステラーゼ(Che、EC 番号:3.1.1.13)は、コレステロールエステルの
加水分解を触媒し、コレステロールと脂肪酸を生成する(図 1)
。Che は原核生物から哺乳
類に至るまで様々な生物に普遍的に存在している。特に哺乳類の Che は、生化学分野にお
いて最も研究が進んでいる酵素の一つであり、例えばヒトの Che として知られる胆汁酸塩
刺激リパーゼは、脂質代謝や腸管内腔からの遊離コレステロールの取り込みを促進する
(Mukherjee 2003; Shamir et al. 1996)。胆汁酸塩刺激リパーゼは分子量約 110 kDa の単量体
であり、活性型となるためには胆汁酸塩が必要である(Chen et al. 1998; Comte et al. 2006;
Hyun et al. 1972)。真核生物では例えば Aspergillus 属や Saccharomyces 属において Che が
確認されており(Köffel et al. 2005; Tőke et al. 2007)、様々な生物がコレステロールエステ
ルを炭素源として利用する能力を有していると考えられる(表 1)
。特に、微生物の Che は、
異なる環境下で多種多様な脂肪酸エステルの分解に関与することから、様々な基質特異性
や、有機溶媒耐性、耐熱性などを示すことが知られている。アシルグリセロールやアリール
エステルなどのエステル結合を含む幅広い化合物の加水分解や合成を触媒するもの
(Vaquero et al. 2016)や、パルプ中のピッチと呼ばれる粘着性材料の構成成分であるステロ
ールを分解することにより、紙の品質を向上させるもの(Calero-Rueda et al. 2002)、また食
品や化粧品の添加物として有用なステロールエステルを生産するための触媒として利用さ
れているもの(Vaquero et al. 2016)などが知られており、商業利用のために特許出願されて
いるものも多数存在する(表 2)
。また、血清中の総コレステロール値を測定する体外診断
用医薬品にも使用されている(Allain et al. 1974)。このように、Che は産業上非常に重要な
酵素の一つである。

1

図 1.コレステロールエステラーゼの触媒反応
コレステロールエステラーゼはコレステロールエステルをコレステロールと脂肪酸に加水分解する。図中の R は炭化水素を表す。

2

表 1. Che を持つことが報告されている微生物・真菌
(Vaquero et al. ...

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謝辞

本研究の進行にあたり、終始多大なご指導を賜った指導教官の田村具博先生に深く感謝

申し上げます。

共同研究者である産業技術総合研究所の安武義晃さんには結晶構造解析に加え論文投稿

へのご助言など多岐にわたってご指導いただきました。ここに感謝申し上げます。また、同

じく共同研究者であった元産業技術総合研究所、現札幌医科大学 吉田圭太朗さんには特に

宿主ベクターの開発にて多大なご助言をいただきました。ここに感謝申し上げます。産業技

術総合研究所の油谷幸代さん、石谷孔司さんにおかれましても多大なご助言を賜りました。

ここに感謝申し上げます。

旭化成ファーマ株式会社 診断薬製品部の酒瀬川信一さん、村松周治さんにおかれまして

は、本研究の遂行にあたり、多大なご指導を賜りました。ここに感謝申し上げます。また、

多数の実験を実施いただいた同所属の村田里美さんに感謝申し上げます。

結晶 X 線回折データは、高エネルギー加速器研究機構(KEK;茨城県つくば市)のシン

クロトロン放射光科学研究施設、Photon Factory (PF)の共同利用実験により取得しました。

放射光実験において多くのサポートをいただいた PF ビームラインスタッフの皆様にお礼

申し上げます。

なお、本研究は国立研究開発法人新エネルギー・産業技術総合開発機構(NEDO)の委託

業務および助成業務(プロジェクトコード:P16009)、さらに日本学術振興会 (JSPS) の科

学研究費助成事業(助成番号: 21H05230)により得られたものです。

142

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