JP2008029218A - Method for producing copolymerized polyester by using microorganism having lowered enzymatic activity - Google Patents
Method for producing copolymerized polyester by using microorganism having lowered enzymatic activity Download PDFInfo
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- JP2008029218A JP2008029218A JP2006203401A JP2006203401A JP2008029218A JP 2008029218 A JP2008029218 A JP 2008029218A JP 2006203401 A JP2006203401 A JP 2006203401A JP 2006203401 A JP2006203401 A JP 2006203401A JP 2008029218 A JP2008029218 A JP 2008029218A
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Abstract
Description
本発明は、柔軟な性質を示す３−ヒドロキシヘキサン酸のモノマーユニットの比率が高い共重合ポリエステルを生産できるよう改良された微生物を用いる、生分解性ポリエステルの製造方法に関する。 The present invention relates to a method for producing a biodegradable polyester using microorganisms improved so as to produce a copolyester having a high ratio of 3-hydroxyhexanoic acid monomer units exhibiting flexible properties.
ポリヒドロキシアルカン酸は、広範な微生物によって生成されるポリエステル型有機分子ポリマーである。これらのポリマーは生分解性を有し、熱可塑性高分子であること、また、再生可能資源から産生されることから、環境調和型素材または生体適合型素材として工業的に生産し、多様な産業へ利用する試みが行われている。
このポリエステルを構成するモノマー単位は一般名３−ヒドロキシアルカン酸であって、具体的には３−ヒドロキシ酪酸、３−ヒドロキシ吉草酸、３−ヒドロキシヘキサン酸、３−ヒドロキシオクタン酸、あるいはよりアルキル鎖の長い３−ヒドロキシアルカン酸が単重合もしくは共重合することにより、ポリマー分子が形成されている。３−ヒドロキシ酪酸（以下３ＨＢと略す）のホモポリマーであるポリ−３−ヒドロキシ酪酸（以下、Ｐ（３ＨＢ）と略す）は、１９２５年にバチルス・メガテリウム（Ｂａｃｉｌｌｕｓ ｍｅｇａｔｅｒｉｕｍ）で最初に発見されたが、このＰ（３ＨＢ）は結晶性が高いため、硬くて脆い性質を持っており、実用的には応用範囲が限られている。
Polyhydroxyalkanoic acid is a polyester-type organic molecular polymer produced by a wide range of microorganisms. Since these polymers are biodegradable, are thermoplastic polymers, and are produced from renewable resources, they are industrially produced as environmentally friendly materials or biocompatible materials, and are used in various industries. Attempts have been made to use.
The monomer unit constituting this polyester is a general name 3-hydroxyalkanoic acid, specifically 3-hydroxybutyric acid, 3-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, or a more alkyl chain. Polymer molecules are formed by homopolymerization or copolymerization of a long 3-hydroxyalkanoic acid. Poly-3-hydroxybutyric acid (hereinafter abbreviated as P (3HB)), a homopolymer of 3-hydroxybutyric acid (hereinafter abbreviated as 3HB), was first discovered in 1925 in Bacillus megaterium. Since P (3HB) has high crystallinity, it has a hard and brittle nature, and its practical application range is limited.
また、３−ヒドロキシ酪酸（３ＨＢ）と３−ヒドロキシ吉草酸（３ＨＶ）とからなる共重合体（以下Ｐ（３ＨＢ−ｃｏ−３ＨＶ）と略す）はＰ（３ＨＢ）に比べると柔軟性に富むことが知られ、製造方法も開示されている（例えば特許文献１、特許文献２参照）。しかしながら、実際にはＰ（３ＨＢ−ｃｏ−３ＨＶ）は３ＨＶモル分率を増加させても、それに伴う物性の変化が乏しく、特に柔軟性が向上しないため、シャンプーボトルや使い捨て剃刀の取っ手等、硬質成型体の分野にしか利用されなかった。
また、アルキル鎖の炭素数が６から１６の３−ヒドロキシアルカン酸で構成される中鎖ＰＨＡはＰ（３ＨＢ）やＰ（３ＨＢ−ｃｏ−３ＨＶ）よりも結晶性が低く、弾力に富んでいるので（非特許文献１参照）、異なる分野への用途が期待されている。中鎖ＰＨＡの製造研究は、シュードモナス属のＰＨＡ合成酵素遺伝子を、シュードモナス属、ラルストニア属、大腸菌に導入することによって行われてきたが、いずれも生産性が低く工業生産に適したものではなかった（非特許文献２、非特許文献３、非特許文献４参照）。
A copolymer composed of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) (hereinafter abbreviated as P (3HB-co-3HV)) is more flexible than P (3HB). And a manufacturing method is also disclosed (see, for example, Patent Document 1 and Patent Document 2). In practice, however, P (3HB-co-3HV) increases the 3HV mole fraction, so that the change in physical properties associated therewith is poor, and the flexibility is not particularly improved. Therefore, it is difficult to use a hard shampoo bottle or disposable razor handle. It was used only in the field of molded products.
Further, medium chain PHA composed of 3-hydroxyalkanoic acid having 6 to 16 carbon atoms in the alkyl chain has lower crystallinity and rich elasticity than P (3HB) and P (3HB-co-3HV). Therefore (see Non-Patent Document 1), it is expected to be used in different fields. Research on production of medium chain PHA has been carried out by introducing a PHA synthase gene of Pseudomonas genus into Pseudomonas genus, Ralstonia genus, E. coli, but none of them is low in productivity and suitable for industrial production (See Non-Patent Document 2, Non-Patent
また、ポリヒドロキシアルカン酸の一種として、３ＨＢと３−ヒドロキシヘキサン酸（以下、３ＨＨと略す）との２成分共重合ポリエステル（以下Ｐ（３ＨＢ−ｃｏ−３ＨＨ）と略す）が知られており、その製造方法が開示されている（特許文献３、特許文献４参照）。これら報告のＰ（３ＨＢ−ｃｏ−３ＨＨ）の製造方法は、土壌より単離されたアエロモナス・キャビエ（Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ）を用いてオレイン酸等の脂肪酸やオリーブオイル等の油脂から発酵生産するものであった。また、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）の性質に関する研究もなされている（非特許文献５参照）。この報告では炭素数が１２個以上の脂肪酸を唯一の炭素源としてアエロモナス・キャビエを培養し、３ＨＨ組成が１１〜１９ｍｏｌ％のＰ（３ＨＢ−ｃｏ−３ＨＨ）を発酵生産している。このＰ（３ＨＢ−ｃｏ−３ＨＨ）は３ＨＨ組成の増加にしたがって、Ｐ（３ＨＢ）の硬くて脆い性質から次第に柔軟な性質を示すようになり、Ｐ（３ＨＢ−ｃｏ−３ＨＶ）を上回る柔軟性を示すことが明らかにされた。すなわちＰ（３ＨＢ−ｃｏ−３ＨＨ）は３ＨＨ組成を変えることで、硬質ポリエステルから軟質ポリエステルまで応用可能な幅広い物性を持つため、テレビの筐体等のように硬さを要求されるものからフィルム等のように柔軟性を要求されるものまで、幅広い分野への応用が期待できる。しかしながら、本製造方法ではポリエステル生産性は１．２ｇ／Ｌと低いものであり、本ポリエステルの実用化に向けた生産方法としては未だ不十分と言わざるを得なかったため、実用化に向けて更に高い生産性が得られる方法が探索された。
As one kind of polyhydroxyalkanoic acid, a two-component copolymerized polyester of 3HB and 3-hydroxyhexanoic acid (hereinafter abbreviated as 3HH) (hereinafter abbreviated as P (3HB-co-3HH)) is known. The manufacturing method is disclosed (see
Ｐ（３ＨＢ−ｃｏ−３ＨＨ）の工業生産を目指した取組みもなされている。アエロモナス・ハイドロフィラ（Ａｅｒｏｍｏｎａｓ ｈｙｄｒｏｐｈｉｌａ）を用いた培養では、オレイン酸を炭素源とした４３時間の流加培養において、ポリエステル生産性４３ｇ／Ｌ、３ＨＨ組成１７％のＰ（３ＨＢ−ｃｏ−３ＨＨ）が生産された（非特許文献６参照）。また、アエロモナス・ハイドロフィラを炭素源としてグルコース及びラウリン酸を用いて培養し、菌体生産量５０ｇ／Ｌ、ポリエステル含量５０％を達成した（非特許文献７参照）。しかしながら、アエロモナス・ハイドロフィラはヒトに対して病原性を有する（非特許文献８参照）ことから、工業生産に適した種とはいえない。また、これらの培養生産では高価な炭素源を使用するため、製造コストの観点より安価な炭素源の利用も求められている。
このため、安全な宿主での生産及び生産性の向上を目指した取組みが行なわれた。アエロモナス・キャビエよりポリヒドロキシアルカン酸（ＰＨＡ）合成酵素遺伝子がクローニングされた（特許文献５参照）。本遺伝子をワーテルシア・ユートロファ（Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ、別名カプリアビダス・ニケーター、旧名ラルストニア・ユートロファ、アルカリゲネス・ユートロファス）ＰＨＢ−４株に導入した形質転換体を用いてＰ（３ＨＢ−ｃｏ−３ＨＨ）の生産を行った結果、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）生産性は２．９ｇ／Ｌ、３ＨＨ組成４％であった。さらに、植物油脂を炭素源とした同菌株の培養条件改善によりＰ（３ＨＢ−ｃｏ−３ＨＨ）生産性２８ｇ／Ｌ、３ＨＨ組成８．１％にまで向上したように、培養方法によって高３ＨＨ組成のＰ（３ＨＢ−ｃｏ−３ＨＨ）を生産する方法も研究された（特許文献６参照）。しかしながら、工業的生産に応用するには、未だ不十分な生産性であった。
Efforts aimed at industrial production of P (3HB-co-3HH) have also been made. In the culture using Aeromonas hydrophila, in a fed-batch culture for 43 hours using oleic acid as a carbon source, P (3HB-co-3HH) having a polyester productivity of 43 g / L and a 3HH composition of 17% is obtained. Produced (see Non-Patent Document 6). In addition, Aeromonas hydrophila was cultured using glucose and lauric acid as a carbon source to achieve a cell production of 50 g / L and a polyester content of 50% (see Non-Patent Document 7). However, since Aeromonas hydrophila is pathogenic to humans (see Non-Patent Document 8), it cannot be said to be a species suitable for industrial production. In addition, since an expensive carbon source is used in these culture productions, utilization of an inexpensive carbon source is also required from the viewpoint of manufacturing cost.
For this reason, efforts were made to improve production and productivity in a safe host. A polyhydroxyalkanoic acid (PHA) synthase gene was cloned from Aeromonas caviae (see Patent Document 5). P (3HB-co-3HH) was produced using a transformant in which this gene was introduced into a Waterscia eutropha (Watersia eutropha, also known as Capriavidas nickater, formerly Ralstonia eutropha, Alkalinenes eutrophas) PHB-4 strain. As a result, P (3HB-co-3HH) productivity was 2.9 g / L, and 3HH composition was 4%. Furthermore, P (3HB-co-3HH) productivity was improved to 28 g / L and 3HH composition to 8.1% by improving the culture conditions of the same strain using vegetable oil as a carbon source. A method for producing P (3HB-co-3HH) was also studied (see Patent Document 6). However, the productivity is still insufficient for application to industrial production.
また、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）の物性を制御する方法も開示されている（特許文献６参照）。少なくとも２種類の炭素数の異なる油脂および／または脂肪酸を炭素源として用いることによって、３ＨＨ組成が１〜４０ｍｏｌ％のＰ（３ＨＢ−ｃｏ−３ＨＨ）を生産することが可能となり、種々の物性を有するＰ（３ＨＢ−ｃｏ−３ＨＨ）が生産できるようになった。しかしながら、本製造方法では、３ＨＨ組成制御のために比較的高価なヘキサン酸、オクタン酸等を添加する必要があり、また高濃度のヘキサン酸は細胞毒性を示すことから菌体生産性が低下する結果となっている。また、多成分の炭素源を添加するため、生産設備が複雑・高価なものになりかねない。 Further, a method for controlling physical properties of P (3HB-co-3HH) is also disclosed (see Patent Document 6). By using at least two types of fats and oils and / or fatty acids having different carbon numbers as a carbon source, it becomes possible to produce P (3HB-co-3HH) having a 3HH composition of 1 to 40 mol%, and various physical properties. P (3HB-co-3HH) can be produced. However, in this production method, it is necessary to add relatively expensive hexanoic acid, octanoic acid or the like for 3HH composition control, and high concentration hexanoic acid exhibits cytotoxicity, so that the cell productivity decreases. It is the result. In addition, since a multi-component carbon source is added, production facilities can be complicated and expensive.
また、フラクトースを炭素源としてＰ（３ＨＢ−ｃｏ−３ＨＨ）を生産できるワーテルシア・ユートロファも構築されたが、本菌株のポリエステル生産性は低く、実生産に適しているとはいえなかった（非特許文献９参照）。
大腸菌を宿主としたＰ（３ＨＢ−ｃｏ−３ＨＨ）生産株も構築された。アエロモナス属のＰＨＡ合成酵素遺伝子及びワーテルシア・ユートロファのＮＡＤＰ−アセトアセチルＣｏ−Ａ還元酵素遺伝子等を大腸菌に導入した株が構築された。ドデカンを炭素源として同大腸菌を培養した結果、４０．８時間培養でＰ（３ＨＢ−ｃｏ−３ＨＨ）生産性２１ｇ／Ｌ、３ＨＨ組成１０．８％であった（非特許文献１０参照）。
アエロモナス・キャビエのＰＨＡ合成酵素遺伝子、エノイルＣｏＡヒドラターゼ遺伝子及びアシルＣｏＡデヒドロゲナーゼ遺伝子を導入した大腸菌も構築された。同大腸菌をラウリン酸を含む培地で培養すると、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）含量約１６％、３ＨＨ組成約１６％であった（非特許文献１１参照）。これらの大腸菌では生産性は低く、工業的生産への適用は困難であった。
In addition, Watersia eutropha, which can produce P (3HB-co-3HH) using fructose as a carbon source, was constructed, but the polyester productivity of this strain was low and could not be said to be suitable for actual production (non-patented) Reference 9).
A P (3HB-co-3HH) producing strain using E. coli as a host was also constructed. A strain was constructed in which the PHA synthase gene belonging to the genus Aeromonas and the NADP-acetoacetyl Co-A reductase gene from Watersia eutropha were introduced into E. coli. As a result of culturing the same Escherichia coli using dodecane as a carbon source, the P (3HB-co-3HH) productivity was 21 g / L and the 3HH composition was 10.8% after 40.8 hours of cultivation (see Non-Patent Document 10).
Escherichia coli into which the PHA synthase gene of Aeromonas caviae, the enoyl CoA hydratase gene and the acyl CoA dehydrogenase gene were introduced was also constructed. When the E. coli was cultured in a medium containing lauric acid, the P (3HB-co-3HH) content was about 16% and the 3HH composition was about 16% (see Non-Patent Document 11). These E. coli have low productivity and are difficult to apply to industrial production.
Ｐ（３ＨＢ−ｃｏ−３ＨＨ）の生産性向上並びに３ＨＨ組成制御を目指して、ＰＨＡ合成酵素の人為的な改変が行なわれた。アエロモナス・キャビエ由来のＰＨＡ合成酵素変異体のなかで、１４９番目のアミノ酸アスパラギンがセリンに置換された変異体酵素や、１７１番目のアスパラギン酸がグリシンに置換された変異体酵素は、大腸菌内でのＰＨＡ合成酵素活性や３ＨＨ組成が向上していることが示された（非特許文献１２参照）。また、同酵素の５１８番目のフェニルアラニンがイソロイシンに置換された変異体酵素や２１４番目のバリンがグリシンに置換された変異体酵素は大腸菌でのＰＨＡ合成酵素活性やＰ（３ＨＢ−ｃｏ−３ＨＨ）含量が向上したことが報告されている（非特許文献１３参照）。しかし、これらは宿主として特殊な大腸菌を用いており未だＰ（３ＨＢ−ｃｏ−３ＨＨ）含量は低いことから、これらの変異体酵素の特徴を活かした工業的生産に向けた更なる改良が必要であった。 In order to improve the productivity of P (3HB-co-3HH) and control the 3HH composition, artificial modification of PHA synthase was performed. Among the mutants of PHA synthase derived from Aeromonas caviae, the mutant enzyme in which the 149th amino acid asparagine is replaced with serine, and the mutant enzyme in which the 171st aspartic acid is replaced with glycine are It was shown that PHA synthase activity and 3HH composition are improved (see Non-Patent Document 12). The mutant enzyme in which the 518th phenylalanine of the enzyme is replaced with isoleucine and the mutant enzyme in which the 214th valine is replaced with glycine are PHA synthase activity and P (3HB-co-3HH) content in Escherichia coli. Has been reported to improve (see Non-Patent Document 13). However, since these use special E. coli as the host and the P (3HB-co-3HH) content is still low, further improvements for industrial production utilizing the characteristics of these mutant enzymes are necessary. there were.
ワーテルシア・ユートロファをＰ（３ＨＢ−ｃｏ−３ＨＨ）生産用の宿主として用いる場合、合成経路で重要な酵素遺伝子はβ−ケトチオラーゼ遺伝子（ｐｈｂＡ）およびアセトアセチル−ＣｏＡ還元酵素遺伝子（ｐｈｂＢ）である（図１参照）。この両酵素は基質特異性の特徴から３−ヒドロキシ酪酸のモノマーユニットを優先的に供給していると考えられている。従って、両酵素の活性を低下させることによって相対的に３ＨＨ組成の高いＰ（３ＨＢ−ｃｏ−３ＨＨ）が得られると期待できる。しかしながら宿主として頻用されているワーテルシア・ユートロファＰＨＢ−４株は、β−ケトチオラーゼ活性およびアセトアセチル−ＣｏＡ還元酵素活性が親株であるＨ１６株に比べてそれぞれ２０分の１程度に減少している（非特許文献１４参照）にもかかわらず、この株を宿主とした３ＨＨ組成が高くかつ生産性が高いＰ（３ＨＢ−ｃｏ−３ＨＨ）生産株の報告例はなく、ＰＨＢ−４株以外のβ−ケトチオラーゼ活性あるいはアセトアセチル−ＣｏＡ還元酵素活性が低下した株を用いた３ＨＨ組成が高くかつ生産性が高いＰ（３ＨＢ−ｃｏ−３ＨＨ）生産株の報告例もない。
上述したようにＰ（３ＨＢ−ｃｏ−３ＨＨ）の工業的生産のために多くの取り組みがなされているが、低３ＨＨ組成Ｐ（３ＨＢ−ｃｏ−３ＨＨ）生産系に比べ、柔軟な性質を活かしたフィルム等への加工に適した高３ＨＨ組成Ｐ（３ＨＢ−ｃｏ−３ＨＨ）の安全且つ安価な生産系が確立されていないため、その高生産系開発が望まれていた。
本発明の目的は、高３ＨＨ組成のＰ（３ＨＢ−ｃｏ−３ＨＨ）を従来よりも効率よく生産し、その生産収率を増大させるのに有効な方法を提供することにある。
As described above, many efforts have been made for the industrial production of P (3HB-co-3HH), but it made use of its flexible properties compared to the production system of low 3HH composition P (3HB-co-3HH). Since a safe and inexpensive production system of a high 3HH composition P (3HB-co-3HH) suitable for processing into a film or the like has not been established, development of the high production system has been desired.
An object of the present invention is to provide an effective method for producing P (3HB-co-3HH) having a high 3HH composition more efficiently than before and increasing its production yield.
本発明者は、前記課題を解決するために鋭意研究した結果、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）生産能を有する微生物において、そのＰ（３ＨＢ−ｃｏ−３ＨＨ）の基質モノマーの一つである３ヒドロキシ酪酸ＣｏＡの生合成系酵素の活性、特にアセトアセチル−ＣｏＡ還元酵素及びβ−ケトチオラーゼ活性が欠損若しくは減少した変異株を作製し、これを培養して生産されたＰ（３ＨＢ−ｃｏ−３ＨＨ）の３ＨＨ組成を比較したところ、親株よりも高い３ＨＨ組成を示すことを見いだし、これらの知見に基づいて本発明を完成するに至った。
即ち本発明は以下の通りである。
［１］以下に示す（ａ）（ｂ）のいずれか一方もしくは両方の特徴を有し、かつ、３−ヒドロキシ酪酸と３−ヒドロキシヘキサン酸のモノマーユニットで構成される共重合ポリエステルの生産能を有するワーテルシア属、カプリアビダス属、ラルストニア属、アルカリゲネス属及びシュードモナス属からなる群より選択される微生物。
（ａ）アセトアセチル−ＣｏＡ還元酵素遺伝子の全部もしくは一部が欠失されたことにより、またはアセトアセチル−ＣｏＡ還元酵素遺伝子の開始コドンより上流１０塩基以内に存在するリボソーム結合部位の塩基配列に少なくとも１塩基以上の置換、欠失もしくは挿入がなされたことにより、アセトアセチル−ＣｏＡ還元酵素活性が０．３Ｕ／ｍｇ蛋白質以下である。
（ｂ）β−ケトチオラーゼ遺伝子のＤＮＡ配列に少なくとも１塩基以上の置換、欠失もしくは挿入がなされたことにより、β−ケトチオラーゼ活性が欠損又は減少されている。
［２］ワーテルシア・ユートロファであることを特徴とする、［１］に記載の微生物。
［３］３−ヒドロキシ酪酸と３−ヒドロキシヘキサン酸のモノマーユニットで構成される共重合ポリエステルの重合酵素遺伝子を染色体上に保持していることを特徴とする、［１］又は［２］に記載の微生物。
［４］重合酵素遺伝子がアエロモナス・キャビエ由来の酵素又は変異体をコードするものである、［３］に記載の微生物。
［５］重合酵素遺伝子が、配列番号１のアミノ酸配列で示されるアエロモナス・キャビエ由来の変異体酵素をコードするものである、［３］に記載の微生物。
［６］配列番号２に示す遺伝子配列の１２９６番目から２０３６番目までを欠失していることを特徴とする、［２］〜［５］のいずれか１項に記載の微生物。
［７］配列番号２に示す遺伝子配列の１２８７番目の塩基グアニンがシトシンに置換され、かつ１２８９番目の塩基アデニンがシトシンに置換されていることを特徴とする、［２］〜［５］のいずれか１項に記載の微生物。
［８］配列番号２に示す遺伝子配列の１２８８番目の塩基グアニンがチミンに置換され、かつ１２９０番目の塩基グアニンがシトシンに置換されていることを特徴とする、［２］〜［５］のいずれか１項に記載の微生物。
［９］配列番号３に示すβ−ケトチオラーゼ遺伝子の１６番目のアミノ酸であるリジンが終止コドンに置換していることを特徴とする、［２］〜［８］のいずれか１項に記載の微生物。
［１０］ワーテルシア・ユートロファＨ１６株の染色体上にある、配列番号２４に示す遺伝子配列の８４２番目から２６１１番目までが、配列番号２５に示す遺伝子配列に置換され、かつ配列番号２に示す遺伝子配列の１２９６番目から２０３６番目までを欠失している微生物。
［１１］ワーテルシア・ユートロファＨ１６株の染色体上にある、配列番号２４に示す遺伝子配列の８４２番目から２６１１番目までが、配列番号２５に示す遺伝子配列に置換され、配列番号２に示す遺伝子配列の１２８７番目の塩基グアニンがシトシンに置換され、かつ１２８９番目の塩基アデニンがシトシンに置換されている微生物。
［１２］ワーテルシア・ユートロファＨ１６株の染色体上にある、配列番号２４に示す遺伝子配列の８４２番目から２６１１番目までが、配列番号２５に示す遺伝子配列に置換され、配列番号２に示す遺伝子配列の１２８８番目の塩基グアニンがチミンに置換され、かつ１２９０番目の塩基グアニンがシトシンに置換されている微生物。
［１３］ワーテルシア・ユートロファＨ１６株の染色体上にある、配列番号２４に示す遺伝子配列の８４２番目から２６１１番目までが、配列番号２５に示す遺伝子配列に置換され、配列番号２に示す遺伝子配列の８５番目の塩基アデニンがチミンに置換され、かつ８８番目の塩基チミンがシトシンに置換されている微生物。
［１４］ワーテルシア・ユートロファＨ１６株の染色体上にある、配列番号２４に示す遺伝子配列の８４２番目から２６１１番目までが、配列番号２５に示す遺伝子配列に置換され、配列番号２に示す遺伝子配列の８５番目の塩基アデニンがチミンに置換され、８８番目の塩基チミンがシトシンに置換され、かつ１２９６番目から２０３６番目までを欠失している微生物。
［１５］ワーテルシア・ユートロファＨ１６株の染色体上にある、配列番号２４に示す遺伝子配列の８４２番目から２６１１番目までが、配列番号２５に示す遺伝子配列に置換され、配列番号２に示す遺伝子配列の８５番目の塩基アデニンがチミンに置換され、８８番目の塩基チミンがシトシンに置換され、１２８７番目の塩基グアニンがシトシンに置換され、かつ１２８９番目の塩基アデニンがシトシンに置換されている微生物。
［１６］ワーテルシア・ユートロファＨ１６株の染色体上にある、配列番号２４に示す遺伝子配列の８４２番目から２６１１番目までが、配列番号２５に示す遺伝子配列に置換され、配列番号２に示す遺伝子配列の８５番目の塩基アデニンがチミンに置換され、８８番目の塩基チミンがシトシンに置換され、１２８８番目の塩基グアニンがチミンに置換され、かつ１２９０番目の塩基グアニンがシトシンに置換されている微生物。
［１７］［１］〜［１６］のいずれか１項に記載の微生物を用いた、３−ヒドロキシ酪酸と３−ヒドロキシヘキサン酸のモノマーユニットで構成される共重合ポリエステルの製造方法。
As a result of intensive studies to solve the above problems, the inventor is one of the substrate monomers of P (3HB-co-3HH) in microorganisms having P (3HB-co-3HH) production ability. P (3HB-co-3HH) produced by producing a mutant strain in which the activity of the biosynthetic enzyme of hydroxybutyric acid CoA, in particular, acetoacetyl-CoA reductase and β-ketothiolase activity is deficient or reduced When the 3HH composition was compared, it was found that the 3HH composition was higher than that of the parent strain, and the present invention was completed based on these findings.
That is, the present invention is as follows.
[1] The ability to produce a copolyester having either one or both of the following (a) and (b) characteristics and comprising monomer units of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid A microorganism selected from the group consisting of the genus Watersia, Capriavidas, Ralstonia, Alkagenes and Pseudomonas.
(A) at least in the base sequence of the ribosome binding site present by deletion of all or part of the acetoacetyl-CoA reductase gene or within 10 bases upstream from the start codon of the acetoacetyl-CoA reductase gene As a result of substitution, deletion or insertion of one or more bases, the acetoacetyl-CoA reductase activity is 0.3 U / mg protein or less.
(B) β-ketothiolase activity is deficient or reduced by substitution, deletion or insertion of at least one base in the DNA sequence of the β-ketothiolase gene.
[2] The microorganism according to [1], which is Watercia utropha.
[3] The polymerization enzyme gene of a copolyester composed of monomer units of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid is retained on the chromosome, and described in [1] or [2] Microorganisms.
[4] The microorganism according to [3], wherein the polymerase enzyme gene encodes an enzyme or mutant derived from Aeromonas caviae.
[5] The microorganism according to [3], wherein the polymerizing enzyme gene encodes a mutant enzyme derived from Aeromonas caviae represented by the amino acid sequence of SEQ ID NO: 1.
[6] The microorganism according to any one of [2] to [5], wherein the gene sequence represented by SEQ ID NO: 2 is deleted from position 1296 to position 2036.
[7] Any one of [2] to [5], wherein the 1287th base guanine in the gene sequence shown in SEQ ID NO: 2 is substituted with cytosine and the 1289th base adenine is substituted with cytosine 2. The microorganism according to item 1.
[8] Any of [2] to [5], wherein the 1288th base guanine in the gene sequence shown in SEQ ID NO: 2 is substituted with thymine and the 1290th base guanine is substituted with cytosine 2. The microorganism according to item 1.
[9] The microorganism according to any one of [2] to [8], wherein lysine, which is the 16th amino acid of the β-ketothiolase gene shown in SEQ ID NO: 3, is substituted with a stop codon. .
[10] The gene sequence shown in SEQ ID NO: 24 from the 842th to 2611th of the gene sequence shown in SEQ ID NO: 24 on the chromosome of Watersia Eutropha H16 strain is replaced with the gene sequence shown in SEQ ID NO: 2 Microorganism lacking from 1296th to 2036th.
[11] The gene sequence shown in SEQ ID NO: 24 from the 842th to 2611th on the chromosome of Watersia Eutropha H16 strain is replaced with the gene sequence shown in SEQ ID NO: 25, and 1287 of the gene sequence shown in SEQ ID NO: 2 A microorganism in which the 2nd base guanine is substituted with cytosine and the 1289th base adenine is substituted with cytosine.
[12] The gene sequence shown in SEQ ID NO: 24 from the 842th to 2611th positions on the chromosome of Watersia Eutropha H16 strain is replaced with the gene sequence shown in SEQ ID NO: 25, and 1288 of the gene sequence shown in SEQ ID NO: 2 A microorganism in which the 2nd base guanine is substituted with thymine and the 1290th base guanine is substituted with cytosine.
[13] The gene sequence shown in SEQ ID NO: 24 from the 842th to 2611th positions on the chromosome of Watersia Eutropha H16 strain is replaced with the gene sequence shown in SEQ ID NO: 25, and 85 of the gene sequence shown in SEQ ID NO: 2 A microorganism in which the base adenine is substituted with thymine and the 88th base thymine is substituted with cytosine.
[14] The 842th to 2611th positions of the gene sequence shown in SEQ ID NO: 24 on the chromosome of Watersia Eutropha strain H16 are replaced with the gene sequence shown in SEQ ID NO: 25, and 85 of the gene sequence shown in SEQ ID NO: 2 A microorganism in which the first base adenine is replaced with thymine, the 88th base thymine is replaced with cytosine, and the 1296th to 2036th positions are deleted.
[15] The gene sequence shown in SEQ ID NO: 24 from the 842th to 2611th positions on the chromosome of Watersia Eutropha H16 strain is replaced with the gene sequence shown in SEQ ID NO: 25, and 85 of the gene sequence shown in SEQ ID NO: 2 A microorganism in which the first base adenine is replaced with thymine, the 88th base thymine is replaced with cytosine, the 1287th base guanine is replaced with cytosine, and the 1289th base adenine is replaced with cytosine.
[16] The gene sequence shown in SEQ ID NO: 24 from the 842th to 2611th positions on the chromosome of Watersia Eutropha strain H16 is replaced with the gene sequence shown in SEQ ID NO: 25, and 85 of the gene sequence shown in SEQ ID NO: 2 A microorganism in which the base adenine is substituted with thymine, the 88th base thymine is substituted with cytosine, the 1288th base guanine is substituted with thymine, and the 1290th base guanine is substituted with cytosine.
[17] A method for producing a copolyester comprising a monomer unit of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, using the microorganism according to any one of [1] to [16].
以下に本発明を詳細に説明する。
本発明における３−ヒドロキシ酪酸と３−ヒドロキシヘキサン酸のモノマーユニットで構成される共重合ポリエステル（以下、ポリヒドロキシアルカン酸ともいう）とは、以下の一般式：
The present invention is described in detail below.
In the present invention, the copolymerized polyester (hereinafter, also referred to as polyhydroxyalkanoic acid) composed of monomer units of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid is represented by the following general formula:
（式中、ｍおよびｎは該ポリマーのモノマー単位数を表し、１以上である。）で表される共重合ポリマーである。
本発明の方法によって製造されるＰ（３ＨＢ−ｃｏ−３ＨＨ）は、３ＨＨ組成が７．５ｍｏｌ％〜４０ｍｏｌ％のものが好ましく、より好ましくは１０ｍｏｌ％から２５ｍｏｌ％である。
(Wherein m and n represent the number of monomer units of the polymer and are 1 or more).
P (3HB-co-3HH) produced by the method of the present invention preferably has a 3HH composition of 7.5 mol% to 40 mol%, more preferably 10 mol% to 25 mol%.
本発明に使用する微生物としては、ワーテルシア属、カプリアビダス属、ラルストニア属、アルカリゲネス属及びシュードモナス属のいずれかに属する微生物であればいかなるものでもよく、例えばＷａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ（分類学上、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ、Ｃｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒと同一）、Ａｌｃａｌｉｇｅｎｅｓ ｌａｔｕｓ、Ｐｓｅｕｄｏｍｏｎａｓ ａｅｒｕｇｉｎｏｓａ、Ｐｓｅｕｄｏｍｏｎａｓ ｐｕｔｉｄａ等の細菌類が含まれる。安全性及び生産性の観点から好ましくはワーテルシア属であり、より好ましくはＷａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａであり、更に好ましくはＷａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株である。 The microorganism used in the present invention may be any microorganism belonging to any of the genus Watersia, Capriavidas, Ralstonia, Alkaligenes, and Pseudomonas, such as Watersia eutropha (taxonically, Ralstonia eutropha, Cupriavidus necator). And Bacteria such as Alcaligenes latus, Pseudomonas aeruginosa and Pseudomonas putida. From the viewpoint of safety and productivity, the genus is preferably Watersia, more preferably Watersia eutropha, and still more preferably Watersia eutropha H16 strain.
本発明に使用する微生物は、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）の生産能を有するように、少なくともポリヒドロキシアルカン酸合成酵素遺伝子を細胞内に保持し、発現することが必要であるが、そのポリヒドロキシアルカン酸合成酵素遺伝子は細胞内のプラスミド上で存在しても、染色体上に存在してもよい。生産能の安定性の観点から染色体上に存在する方がより好ましい。
本発明に使用する微生物に存在するポリヒドロキシアルカン酸合成酵素遺伝子は各種のＰＨＡ蓄積生物に由来する遺伝子のうち、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）を蓄積させるものであればどのようなものでもよい。そのような遺伝子としては例えば、アエロモナス・キャビエ（特許文献５）およびアエロモナス・ハイドロフィラ（ＧｅｎＢａｎｋアクセッション番号ＡＹ０９３６８５）などから単離されているポリヒドロキシアルカン酸合成酵素遺伝子がある。
The microorganism used in the present invention is required to retain and express at least the polyhydroxyalkanoate synthase gene in the cell so as to have the ability to produce P (3HB-co-3HH). The hydroxyalkanoic acid synthase gene may be present on an intracellular plasmid or on a chromosome. It is more preferable that it exists on the chromosome from the viewpoint of the stability of productivity.
The polyhydroxyalkanoate synthase gene present in the microorganism used in the present invention may be any gene that accumulates P (3HB-co-3HH) among genes derived from various PHA accumulating organisms. . Examples of such genes include polyhydroxyalkanoate synthase genes isolated from Aeromonas cabie (Patent Document 5) and Aeromonas hydrophila (GenBank accession number AY093685).
また、目的とする酵素活性が失われない範囲内でアミノ酸配列が改変するようにそれらの遺伝子の塩基配列の一部を改変したものも使用することができる。例えば、非特許文献１２に記載されている、１４９番目のアミノ酸のアスパラギンがセリンに置換されたアエロモナス・キャビエ由来であるＰＨＡ合成酵素遺伝子（Ｎ１４９Ｓ変異体遺伝子）、１７１番目のアミノ酸のアスパラギン酸がグリシンに置換されたアエロモナス・キャビエ由来であるＰＨＡ合成酵素遺伝子（Ｄ１７１Ｇ変異体遺伝子）、または、上記の２つのアミノ酸置換が組み合わされたアエロモナス・キャビエ由来であるＰＨＡ合成酵素遺伝子等を好ましく用いることができる。本発明で用いるポリヒドロキシアルカン酸合成酵素遺伝子としては、アエロモナス・キャビエ由来の酵素又は変異体をコードするものが好ましく、１４９番目のアミノ酸のアスパラギンをセリンに、かつ１７１番目のアミノ酸のアスパラギン酸をグリシンに置換したアエロモナス・キャビエ由来の変異体酵素をコードするもの（Ｎ１４９Ｓ／Ｄ１７１Ｇ変異体、配列番号１）がより好ましい。ワーテルシア・ユートロファＨ１６株場合、染色体上にある配列番号２４に示す遺伝子配列の８４２番目から２６１１番目までを、配列番号２５に示す遺伝子配列に置換することが好ましい。 Moreover, what modified a part of the base sequence of those genes so that an amino acid sequence may modify | change within the range in which the target enzyme activity is not lost can also be used. For example, as described in Non-Patent Document 12, the PHA synthase gene (N149S mutant gene) derived from Aeromonas caviae in which the asparagine of the 149th amino acid is substituted with serine, the aspartic acid of the 171st amino acid is glycine. PHA synthase gene (D171G mutant gene) derived from Aeromonas caviae substituted with the above, or the PHA synthase gene derived from Aeromonas caviae combined with the above two amino acid substitutions can be preferably used. . The polyhydroxyalkanoic acid synthase gene used in the present invention preferably encodes an enzyme or a mutant derived from Aeromonas caviae, and the 149th amino acid asparagine is used as serine and the 171st amino acid aspartic acid is used as glycine. Those encoding a mutant enzyme derived from Aeromonas caviae substituted with (N149S / D171G mutant, SEQ ID NO: 1) are more preferred. In the case of Watersia Eutropha H16 strain, it is preferable to substitute the gene sequence shown in SEQ ID NO: 25 from the 842nd to 2611th of the gene sequence shown in SEQ ID NO: 24 on the chromosome.
本発明の微生物は、（ａ）アセトアセチル−ＣｏＡ還元酵素活性が０．３Ｕ／ｍｇ蛋白質以下となっているか、及び／又は、（ｂ）β−ケトチオラーゼ遺伝子が不活化されている。
前記した微生物を親株として用いて、アセトアセチル−ＣｏＡ還元酵素活性が欠損もしくは減少した微生物とするには、通常に行われる変異処理方法が採用される。その変異処理方法としては、公知の方法、例えば遺伝子操作による方法、細胞に変異源性のある薬剤を接触させる方法、またＸ線、紫外線、放射線、光などを照射する方法などを挙げることができる。前記の薬剤を接触させる方法に用いられる薬剤としては、例えばＮ−メチル−Ｎ’−ニトロソグアニジン（ＮＴＧ）、メタンスルホン酸エチル（ＥＭＳ）などを挙げることができる。
The microorganism of the present invention has (a) an acetoacetyl-CoA reductase activity of 0.3 U / mg protein or less and / or (b) a β-ketothiolase gene is inactivated.
In order to use the above-mentioned microorganism as a parent strain to obtain a microorganism having a deficient or reduced acetoacetyl-CoA reductase activity, a usual mutation treatment method is employed. Examples of the mutation treatment method include a known method, for example, a method by genetic manipulation, a method of contacting a cell with a mutagenic agent, a method of irradiating X-rays, ultraviolet rays, radiation, light, etc. . Examples of the drug used in the method for contacting the drug include N-methyl-N′-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS).
アセトアセチル−ＣｏＡ還元酵素の活性を特異的に欠損又は減少させる方法としては、遺伝子操作による染色体遺伝子置換法がある。この方法によれば、目的以外の遺伝子に損傷を与えることなく、目的遺伝子の活性発現のみを欠損又は減少させることができ、目的遺伝子以外の合成経路に変動を与えることがないことから、この方法が特に好ましい。具体的には、アセトアセチル−ＣｏＡ還元酵素遺伝子の発現領域を含む遺伝子断片をクローニングし、アセトアセチル−ＣｏＡ還元酵素活性が欠損又は減少するようにＤＮＡ配列の変更を施して改変遺伝子を構築する。この改変遺伝子を親株に形質転換すると、親株の正常な同領域との間で相同組換えの機構によって遺伝子の組換えが起こり、改変遺伝子に置換された変異株を得ることができる。 As a method for specifically deleting or reducing the activity of acetoacetyl-CoA reductase, there is a chromosomal gene replacement method by genetic manipulation. According to this method, only the active expression of the target gene can be deleted or reduced without damaging the non-target gene, and the synthetic pathway other than the target gene is not changed. Is particularly preferred. Specifically, a gene fragment containing the expression region of the acetoacetyl-CoA reductase gene is cloned, and a modified gene is constructed by changing the DNA sequence so that the acetoacetyl-CoA reductase activity is lost or reduced. When this modified gene is transformed into a parent strain, recombination of the gene occurs by the homologous recombination mechanism with the normal region of the parent strain, and a mutant strain substituted with the modified gene can be obtained.
改変の対象となるアセトアセチル−ＣｏＡ還元酵素遺伝子の例としてはワーテルシア・ユートロファのｐｈｂＢ遺伝子（ＧｅｎＢａｎｋアクセッション番号Ｊ０４９８７）、シュードモナス プチダのｐｈｂＢ遺伝子（ＧｅｎＢａｎｋアクセッション番号ＡＢ０８５８１６）、シュードモナス ｓｐ．６１−３のｐｈｂＢ遺伝子（ＧｅｎＢａｎｋアクセッション番号ＡＢ０１４７５７）が挙げられる。 Examples of acetoacetyl-CoA reductase genes to be modified include Waterscia eutropha phbB gene (GenBank Accession No. J04987), Pseudomonas putida phbB gene (GenBank Accession No. AB085816), Pseudomonas sp. 61-3 phbB gene (GenBank accession number AB014757).
アセトアセチル−ＣｏＡ還元酵素活性が欠損するような改変の例としては、アセトアセチル−ＣｏＡ還元酵素の構造遺伝子の内部に異種遺伝子、例えばカナマイシン耐性遺伝子断片を挿入する改変、アセトアセチル−ＣｏＡ還元酵素の構造遺伝子内に終止コドンが生成するような塩基配列の挿入、欠失又は置換をする改変、アセトアセチル−ＣｏＡ還元酵素の構造遺伝子の一部又は全部を欠失する改変などが挙げられる。アセトアセチル−ＣｏＡ還元酵素遺伝子の活性発現を完全に欠損させるには、アセトアセチル−ＣｏＡ還元酵素の構造遺伝子の全部を欠失（ワーテルシア・ユートロファの場合、配列番号２に示す遺伝子配列の１２９６番目から２０３６番目までを欠失）する改変が好ましい。
配列番号２は、ＤＮＡデータベースＤＤＢＪのＡｃｃｅｓｓｉｏｎ Ｎｏ．Ｊ０４９８７の全配列であり、アセトアセチル−ＣｏＡ還元酵素遺伝子及びβ−ケトチオラーゼ遺伝子を含む。
Examples of modifications that lack acetoacetyl-CoA reductase activity include modifications that insert a heterologous gene such as a kanamycin resistance gene fragment into the structural gene of acetoacetyl-CoA reductase, acetoacetyl-CoA reductase Examples include modification that inserts, deletes, or replaces a base sequence that generates a stop codon in the structural gene, modification that deletes part or all of the structural gene of acetoacetyl-CoA reductase, and the like. In order to completely delete the active expression of the acetoacetyl-CoA reductase gene, all of the structural gene of acetoacetyl-CoA reductase is deleted (in the case of Watersia Eutropha, from position 1296 of the gene sequence shown in SEQ ID NO: 2). Modifications that delete up to 2036) are preferred.
Sequence number 2 is Accession No. of DNA database DDBJ. This is the entire sequence of J04987, including the acetoacetyl-CoA reductase gene and the β-ketothiolase gene.
アセトアセチル−ＣｏＡ還元酵素活性が減少するような改変の例としては、アセトアセチル−ＣｏＡ還元酵素のアミノ酸配列中の一箇所又は二箇所以上のアミノ酸が他のアミノ酸に変わるような、塩基配列の挿入、欠失又は置換をする改変、アセトアセチル−ＣｏＡ還元酵素遺伝子のプロモーターおよび／又はリボソーム結合部位への塩基配列の挿入、欠失又は置換をする改変などが挙げられる。
本発明においてはアセトアセチル−ＣｏＡ還元酵素遺伝子の開始コドンより上流１０塩基以内のＤＮＡ配列に少なくとも１塩基以上の置換、欠失もしくは挿入を行う。アセトアセチル−ＣｏＡ還元酵素遺伝子の開始コドンから上流１０塩基内に存在するリボソーム結合部位の塩基配列を置換する改変が、改変の容易さから好ましい。ワーテルシア・ユートロファの場合、配列番号２に示す遺伝子配列の１２８７番目の塩基グアニンをシトシンに置換し、かつ１２８９番目の塩基アデニンをシトシンに置換するか、又は、配列番号２に示す遺伝子配列の１２８８番目の塩基グアニンをチミンに置換し、かつ１２９０番目の塩基グアニンをシトシンに置換することが好ましい。
Examples of modifications that reduce acetoacetyl-CoA reductase activity include insertion of a base sequence such that one or more amino acids in the amino acid sequence of acetoacetyl-CoA reductase are changed to other amino acids , Alterations for deletion or substitution, and alterations for insertion, deletion or substitution of the base sequence into the promoter and / or ribosome binding site of the acetoacetyl-CoA reductase gene.
In the present invention, at least one base substitution, deletion, or insertion is performed in the DNA sequence within 10 bases upstream from the start codon of the acetoacetyl-CoA reductase gene. Modification that replaces the base sequence of the ribosome binding site present within 10 bases upstream from the start codon of the acetoacetyl-CoA reductase gene is preferable from the viewpoint of ease of modification. In the case of Watersia Eutropha, the 1287th base guanine of the gene sequence shown in SEQ ID NO: 2 is replaced with cytosine and the 1289th base adenine is replaced with cytosine, or the 1288th gene sequence shown in SEQ ID NO: 2 It is preferable to substitute the base guanine with thymine and the 1290th base guanine with cytosine.
β−ケトチオラーゼ活性が欠損若しくは減少した微生物とするには、前記したアセトアセチル−ＣｏＡ還元酵素活性が欠損若しくは減少した微生物とする方法と同様の変異処理方法が採用できる。すなわちその変異処理方法としては、公知の方法、例えば遺伝子操作による方法、細胞に変異源性のある薬剤を接触させる方法、またＸ線、紫外線、放射線、光などを照射する方法などを挙げることができる。前記の薬剤を接触させる方法に用いられる薬剤としては、例えばＮ−メチル−Ｎ’−ニトロソグアニジン（ＮＴＧ）、メタンスルホン酸エチル（ＥＭＳ）などを挙げることができる。 In order to obtain a microorganism having a deficient or reduced β-ketothiolase activity, a mutation treatment method similar to the method for obtaining a microorganism having a deficient or reduced acetoacetyl-CoA reductase activity can be employed. That is, examples of the mutation treatment method include known methods such as a method by genetic manipulation, a method in which a cell is brought into contact with a mutagenic agent, a method in which X-rays, ultraviolet rays, radiation, light, etc. are irradiated. it can. Examples of the drug used in the method for contacting the drug include N-methyl-N′-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS).
β−ケトチオラーゼ活性を特異的に欠損又は減少させる方法としては、遺伝子操作による染色体遺伝子置換法がある。この方法によれば、目的以外の遺伝子に損傷を与えることなく、目的遺伝子の活性発現のみを欠損または減少させることができ、目的遺伝子以外の合成経路に変動を与えることがないことから、この方法が特に好ましい。具体的には、β−ケトチオラーゼ遺伝子の発現領域を含む遺伝子断片をクローニングし、β−ケトチオラーゼ活性が欠損又は減少するようにＤＮＡ配列の変更を施して改変遺伝子を構築する。この改変遺伝子を親株に形質転換すると、親株の正常な同領域との間で相同組換えの機構によって遺伝子の組換えが起こり、改変遺伝子に置換された変異株を得ることができる。 As a method for specifically deleting or reducing β-ketothiolase activity, there is a chromosomal gene replacement method by genetic manipulation. According to this method, only the active expression of the target gene can be deleted or decreased without damaging the non-target gene, and the synthetic pathway other than the target gene is not changed. Is particularly preferred. Specifically, a gene fragment containing the expression region of the β-ketothiolase gene is cloned, and a modified gene is constructed by changing the DNA sequence so that the β-ketothiolase activity is deleted or reduced. When this modified gene is transformed into a parent strain, recombination of the gene occurs by the homologous recombination mechanism with the normal region of the parent strain, and a mutant strain substituted with the modified gene can be obtained.
改変の対象となるβ−ケトチオラーゼ遺伝子の例としてはワーテルシア・ユートロファのｐｈｂＡ遺伝子（ＧｅｎＢａｎｋアクセッション番号Ｊ０４９８７）およびｂｋｔＢ遺伝子（ＧｅｎＢａｎｋアクセッション番号ＡＦ０２６５４４）、シュードモナス プチダのｐｈｂＡ遺伝子（ＧｅｎＢａｎｋアクセッション番号ＡＢ０８５８１６）、シュードモナス ｓｐ．６１−３のｐｈｂＡ遺伝子（ＧｅｎＢａｎｋアクセッション番号ＡＢ０１４７５７）が挙げられる。 Examples of the β-ketothiolase gene to be modified include Waterscia Eutropha phbA gene (GenBank accession number J04987) and bktB gene (GenBank accession number AF026544), Pseudomonas putida phbA gene (GenBank accession number AB085816), Pseudomonas sp. 61-3 phbA gene (GenBank accession number AB014757).
β−ケトチオラーゼ活性を特異的に欠損又は減少させるには、β−ケトチオラーゼ遺伝子のＤＮＡ配列に少なくとも１塩基以上の置換、欠失もしくは挿入することが挙げられる。
β−ケトチオラーゼ遺伝子の活性発現が欠損するような改変の例としては、β−ケトチオラーゼの構造遺伝子の内部に異種遺伝子、例えばカナマイシン耐性遺伝子断片を挿入する改変、β−ケトチオラーゼの構造遺伝子内に終止コドンが生成するような塩基配列の挿入、欠失又は置換をする改変、β−ケトチオラーゼの構造遺伝子の一部又は全部を欠失する改変などが挙げられる。
β−ケトチオラーゼ活性が減少するような改変の例としては、β−ケトチオラーゼのアミノ酸配列中の一箇所又は二箇所以上のアミノ酸が他のアミノ酸に変わるような、塩基配列の挿入、欠失又は置換をする改変、β−ケトチオラーゼ遺伝子のプロモーターおよび／又はリボソーム結合部位への塩基配列の挿入、欠失又は置換をする改変などが挙げられる。ワーテルシア・ユートロファの場合、配列番号３に示すβ−ケトチオラーゼ遺伝子の１６番目のアミノ酸であるリジンが終止コドンに置換するように、配列番号２に示す遺伝子配列の８５番目の塩基アデニンをチミンに置換することが好ましい。この際、操作を容易にするために、上記置換と同時に、配列番号２に示す遺伝子配列の８８番目の塩基チミンをシトシンに置換することにより、制限酵素ＮｈｅＩ切断部位が生じるような塩基置換を行うこともできる。
In order to specifically delete or reduce the β-ketothiolase activity, substitution, deletion or insertion of at least one base or more is included in the DNA sequence of the β-ketothiolase gene.
Examples of modifications that lack active expression of the β-ketothiolase gene include modifications that insert a heterologous gene such as a kanamycin resistance gene fragment into the structural gene of β-ketothiolase, and a stop codon within the structural gene of β-ketothiolase. Modification for deleting, inserting, deleting or substituting a base sequence such that a part of or the entire structural gene of β-ketothiolase is deleted.
Examples of modifications that reduce β-ketothiolase activity include insertion, deletion, or substitution of nucleotide sequences such that one or more amino acids in the amino acid sequence of β-ketothiolase are changed to other amino acids. And the modification which inserts, deletes or substitutes the base sequence into the promoter and / or ribosome binding site of the β-ketothiolase gene. In the case of Watersia eutropha, the 85th base adenine of the gene sequence shown in SEQ ID NO: 2 is replaced with thymine so that the lysine, which is the 16th amino acid of the β-ketothiolase gene shown in SEQ ID NO: 3, is replaced with a stop codon. It is preferable. At this time, in order to facilitate the operation, at the same time as the above substitution, base substitution is performed such that a restriction enzyme NheI cleavage site is generated by substituting cytosine for the 88th base thymine of the gene sequence shown in SEQ ID NO: 2. You can also.
ワーテルシア・ユートロファを親株とする場合は、酵素の基質特異性の観点からｐｈｂＡ遺伝子を不活化するのが好ましく、構造遺伝子内に終止コドンが生ずるような塩基配列の置換によって不活化するのがより好ましい。
β−ケトチオラーゼ活性が欠損または減少するようなβ−ケトチオラーゼ遺伝子の改変によって、染色体上でβ−ケトチオラーゼ遺伝子の下流に存在するアセトアセチル−ＣｏＡ還元酵素遺伝子の発現に影響を与え、アセトアセチル−ＣｏＡ還元酵素活性が減少した菌株が得られる場合があるが、そのようにして得られた微生物も本発明の微生物として好適に用いることができる。
In the case of using Watersia eutropha as a parent strain, it is preferable to inactivate the phbA gene from the viewpoint of the substrate specificity of the enzyme, and it is more preferable to inactivate it by substitution of a base sequence that causes a stop codon in the structural gene. .
Modification of the β-ketothiolase gene so that the β-ketothiolase activity is deficient or decreased affects the expression of the acetoacetyl-CoA reductase gene existing downstream of the β-ketothiolase gene on the chromosome and reduces acetoacetyl-CoA reductase Although a strain with reduced enzyme activity may be obtained, the microorganism thus obtained can also be suitably used as the microorganism of the present invention.
本発明の微生物の例として、Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株のポリヒドロキシアルカン酸合成酵素遺伝子をアエロモナス・キャビエ由来であるポリヒドロキシアルカン酸合成酵素変異体遺伝子に置換した株ＫＮＫ−００５株を親株として用い、前記した遺伝子操作による方法でアセトアセチル−ＣｏＡ還元酵素活性が欠損若しくは減少した微生物の取得例を後述の実施例に示す。
取得した株は、β−ケトチオラーゼ遺伝子（ｐｈｂＡ）の構造遺伝子内に終止コドンが生成するように塩基配列を置換した株（ＫＮＫ−００５ＡＳ）、アセトアセチル−ＣｏＡ還元酵素遺伝子（ｐｈｂＢ）のリボソーム結合部位の塩基配列を置換した株（ＫＮＫ−００５ＳＤＭ１およびＫＮＫ−００５ＳＤＭ２）、ｐｈｂＢ遺伝子の構造遺伝子の全部を欠失した株（ＫＮＫ−００５ΔｐｈｂＢ）、ｐｈｂＡ遺伝子の構造遺伝子内に終止コドンが生成するように塩基配列を置換し、かつｐｈｂＢ遺伝子のリボソーム結合部位の塩基配列を置換した株（ＫＮＫ−００５ＡＳＳＤＭ１およびＫＮＫ−００５ＡＳＳＤＭ２）、ｐｈｂＡ遺伝子の構造遺伝子内に終止コドンが生成するように塩基配列を置換し、かつｐｈｂＢ遺伝子の構造遺伝子の全部を欠失した株（ＫＮＫ−００５ＡＳΔｐｈｂＢ）である。親株であるＫＮＫ−００５と比べて、これらの株のアセトアセチル−ＣｏＡ還元酵素活性はいずれも低いことが確認された。そしてこれらの株を培養して得られるＰ（３ＨＢ−ｃｏ−３ＨＨ）の３ＨＨ組成はいずれも、ＫＮＫ−００５を培養して得られるＰ（３ＨＢ−ｃｏ−３ＨＨ）の３ＨＨ組成よりも高いことが確認された。
As an example of the microorganism of the present invention, the strain KNK-005 strain in which the polyhydroxyalkanoate synthase gene of Watersia eurotropa H16 strain is replaced with the polyhydroxyalkanoate synthase mutant gene derived from Aeromonas caviae is used as the parent strain, Examples of obtaining microorganisms in which the acetoacetyl-CoA reductase activity is deficient or reduced by the genetic manipulation method described below are shown in the Examples below.
The obtained strain is a ribosome binding site of a strain (KNK-005AS), a acetoacetyl-CoA reductase gene (phbB) in which the base sequence is substituted so that a stop codon is generated in the structural gene of the β-ketothiolase gene (phbA). (KNK-005SDM1 and KNK-005SDM2), strains in which all of the structural gene of the phbB gene has been deleted (KNK-005ΔphbB), and bases so that a stop codon is generated in the structural gene of the phbA gene. Strains substituted for the sequence and the base sequence of the ribosome binding site of the phbB gene (KNK-005ASSDM1 and KNK-005ASSDM2), the base sequence is replaced so that a stop codon is generated in the structural gene of the phbA gene, and Missing all structural genes of phbB gene Lost strain (KNK-005ASΔphbB). These strains were confirmed to have low acetoacetyl-CoA reductase activity compared to the parent strain KNK-005. The 3HH composition of P (3HB-co-3HH) obtained by culturing these strains should be higher than the 3HH composition of P (3HB-co-3HH) obtained by culturing KNK-005. confirmed.
本発明の微生物を炭素源存在下で増殖させることにより、微生物体内に共重合ポリエステルを蓄積させることができる。炭素源としては、糖、油脂または脂肪酸を用いることができる。炭素源以外の栄養源として、窒素源、無機塩類、そのほかの有機栄養源を任意に用いることができる。
糖としては、例えばグルコース、フラクトース等の炭水化物が挙げられる。油脂としては、炭素数が１０以上である飽和・不飽和脂肪酸を多く含む油脂、例えばヤシ油、パーム油、パーム核油等が挙げられる。脂肪酸としては、ヘキサン酸、オクタン酸、デカン酸、ラウリン酸、オレイン酸、パルミチン酸、リノール酸、リノレン酸、ミリスチン酸等の飽和・不飽和脂肪酸、あるいはこれら脂肪酸のエステルや塩等の脂肪酸誘導体が挙げられる。
By growing the microorganism of the present invention in the presence of a carbon source, a copolyester can be accumulated in the microorganism. As the carbon source, sugar, fat or fatty acid can be used. Nitrogen sources, inorganic salts, and other organic nutrient sources can be arbitrarily used as nutrient sources other than the carbon source.
Examples of the sugar include carbohydrates such as glucose and fructose. Examples of the fats and oils include fats and oils containing a large amount of saturated / unsaturated fatty acids having 10 or more carbon atoms, such as coconut oil, palm oil, and palm kernel oil. Examples of fatty acids include saturated and unsaturated fatty acids such as hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linoleic acid, linolenic acid, and myristic acid, and fatty acid derivatives such as esters and salts of these fatty acids. Can be mentioned.
窒素源としては、例えばアンモニア、塩化アンモニウム、硫酸アンモニウム、リン酸アンモニウム等のアンモニウム塩の他、ペプトン、肉エキス、酵母エキス等が挙げられる。
無機塩類としては、例えばリン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム等が挙げられる。
そのほかの有機栄養源としては、例えばグリシン、アラニン、セリン、スレオニン、プロリン等のアミノ酸；ビタミンＢ１、ビタミンＢ１２、ビタミンＣ等のビタミン等が挙げられる。
Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, peptone, meat extract, yeast extract, and the like.
Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.
Examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline; vitamins such as vitamin B1, vitamin B12, and vitamin C.
培養温度は、その菌が生育可能な温度であればよいが、２０℃から４０℃が好ましい。培養時間は、特に制限はないが、１〜１０日間程度で良い。
得られた該培養菌体に蓄積されたポリエステルは公知の方法により回収することができる。
例えば次のような方法により行うことができる。培養終了後、培養液から遠心分離器等で菌体を分離し、その菌体を蒸留水およびメタノール等により洗浄し、乾燥させる。この乾燥菌体から、クロロホルム等の有機溶剤を用いてポリエステルを抽出する。このポリエステルを含んだ有機溶剤溶液から、濾過等によって菌体成分を除去し、そのろ液にメタノールやヘキサン等の貧溶媒を加えてポリエステルを沈殿させる。さらに、濾過や遠心分離によって上澄み液を除去し、乾燥させてポリエステルを回収することができる。
The culture temperature may be any temperature at which the bacteria can grow, but is preferably 20 ° C to 40 ° C. The culture time is not particularly limited, but may be about 1 to 10 days.
The polyester accumulated in the cultured cells thus obtained can be recovered by a known method.
For example, it can be performed by the following method. After completion of the culture, the cells are separated from the culture solution with a centrifuge, and the cells are washed with distilled water, methanol, and the like and dried. Polyester is extracted from the dried cells using an organic solvent such as chloroform. The bacterial cell component is removed from the organic solvent solution containing the polyester by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate the polyester. Furthermore, the supernatant can be removed by filtration or centrifugation and dried to recover the polyester.
本発明の方法によれば、高３ＨＨ組成のＰ（３ＨＢ−ｃｏ−３ＨＨ）を従来の方法に比べて効率よく製造することができる。そして、本発明の方法によって製造された高３ＨＨ組成のＰ（３ＨＢ−ｃｏ−３ＨＨ）は柔軟な性質を活かしたフィルム等への加工に適した生分解性プラスチックとして多くの分野で種々の用途に用いることができるので、産業上極めて有用である。 According to the method of the present invention, P (3HB-co-3HH) having a high 3HH composition can be produced more efficiently than the conventional method. P (3HB-co-3HH) having a high 3HH composition produced by the method of the present invention is a biodegradable plastic suitable for processing into a film and the like utilizing its flexible properties, and is used in various fields in various fields. Since it can be used, it is very useful industrially.
以下に実施例で本発明を説明するが、本発明はこれら実施例によって何ら制限されるものではない。
（実施例１）ＰＨＢＨ生産菌親株ＫＮＫ−００５株の作製
アセトアセチル−ＣｏＡ還元酵素の活性及びβ−ケトチオラーゼ活性を欠損又は減少させるために用いたＰＨＢＨ生産菌親株のＫＮＫ−００５株は以下のようにして作製した。
＜遺伝子置換用プラスミドの作製＞
Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株の菌体を鋳型ＤＮＡの供給源として、配列番号４と配列番号５で示されるプライマーを用いてＰＣＲ反応を行い、ポリヒドロキシアルカン酸合成酵素遺伝子（ｐｈａＣＲｅ）の構造遺伝子を含むＤＮＡ断片を得た。ＰＣＲ条件は（１）９４℃で２分、（２）９４℃で３０秒、（３）４５℃で３０秒、（４）７２℃で３分、（２）から（４）を２５サイクル、（５）７２℃で５分であり、ポリメラーゼとしてはＴａＫａＲａ ＬＡ Ｔａｑ（タカラバイオ製）を用いた。ＰＣＲで得たＤＮＡ断片を制限酵素ＢａｍＨＩで切断し、ベクターｐＢｌｕｅｓｃｒｉｐｔＩＩＫＳ（−）（東洋紡社製）を同酵素で切断した部位にサブクローニングした（ｐＢｌｕｅ−ｐｈａＣＲｅ）。
EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
(Example 1) Production of parent strain KNK-005 producing PHBH producing strain The parent strain KNK-005 used for deleting or reducing the activity of acetoacetyl-CoA reductase and β-ketothiolase activity is as follows. It was made.
<Preparation of gene replacement plasmid>
A PCR reaction is carried out using the primers of SEQ ID NO: 4 and SEQ ID NO: 5 using the bacterial body of Watersia europa strain H16 as a template DNA source, and the polyhydroxyalkanoate synthase gene (phaC Re ) is included. A DNA fragment was obtained. PCR conditions were (1) 94 ° C for 2 minutes, (2) 94 ° C for 30 seconds, (3) 45 ° C for 30 seconds, (4) 72 ° C for 3 minutes, (2) to (4) 25 cycles, (5) 5 minutes at 72 ° C., and TaKaRa LA Taq (manufactured by Takara Bio Inc.) was used as the polymerase. The DNA fragment obtained by PCR was cleaved with the restriction enzyme BamHI, and the vector pBluescriptIIKS (−) (manufactured by Toyobo Co., Ltd.) was subcloned into the site cleaved with the enzyme (pBlue-phaC Re ).
Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ由来のポリエステル合成酵素変異体遺伝子であるＮ１４９Ｓ／Ｄ１７１Ｇ変異体は、次のように作製した。まず、ｐＢｌｕｅｓｃｒｉｐｔＩＩＫＳ（−）（東洋紡社製）をＰｓｔＩ処理し、ＤＮＡ Ｂｌｕｎｔｉｎｇ Ｋｉｔ（タカラバイオ製）を用いて平滑末端化しライゲーションすることによりＰｓｔＩサイトを欠失させた。次いでこのプラスミドをＸｈｏＩ処理し、ＤＮＡ Ｂｌｕｎｔｉｎｇ Ｋｉｔ（タカラバイオ製）を用いて平滑末端化しライゲーションすることによりＸｈｏＩサイトを欠失したプラスミドｐＢｌｕｅ−Ｎｅｗを作製した。このプラスミドのＥｃｏＲＩサイトにｐＪＲＤ２１５−ＥＥ３２ｄ１３（特許文献５）より同酵素で切り出したｄ１３断片をクローニングした（ｐＢｌｕｅ−ｄ１３）。次に、理化学研究所より分与されたクローンＥ２−５０由来のプラスミド（非特許文献１３）を鋳型とし、配列番号６と７に記載のプライマーのセット及び配列番号８と９に記載のプライマーのセットを用いてそれぞれＰＣＲ法により増幅、２断片を得た。その条件は（１）９４℃で２分、（２）９４℃で３０秒、（３）５５℃で３０秒、（４）７２℃で２分、（２）から（４）を２５サイクル、（５）７２℃で５分である。増幅された２断片を等モル混合し再びＰＣＲ反応を行い、２断片を結合させた。その条件は（１）９６℃で５分、（２）９５℃で２分、（３）７２℃で１分、（２）から（３）を１２サイクルであり、ポリメラーゼとしてはＰｙｒｏｂｅｓｔポリメラーゼ（タカラバイオ製）を用いた。目的サイズのＤＮＡ断片をアガロース電気泳動ゲルより切り出しＰｓｔＩとＸｈｏＩで処理し、同酵素で処理したｐＢｌｕｅ−ｄ１３に断片を入れ替える形でクローニングした（ｐＢｌｕｅ−Ｎ１４９Ｓ／Ｄ１７１Ｇ）。塩基配列決定を、ＰＥＲＩＫＩＮ ＥＬＭＥＲ ＡＰＰＬＩＥＤ ＢＩＯＳＹＳＴＥＭＳ社製のＤＮＡシークエンサー３１０ Ｇｅｎｅｔｉｃ Ａｎａｌｙｚｅｒを用いて行い、ＰＨＡ合成酵素の１４９番目のアミノ酸であるアスパラギンがセリンに、１７１番目のアミノ酸であるアスパラギン酸がグリシンに置換された変異遺伝子であることを確認した。 The N149S / D171G mutant, which is a polyester synthase mutant gene derived from Aeromonas caviae, was prepared as follows. First, pBluescript IIKS (-) (manufactured by Toyobo Co., Ltd.) was treated with PstI, blunt-ended using DNA Blunting Kit (manufactured by Takara Bio Inc.), and ligated to delete the PstI site. Next, this plasmid was treated with XhoI, blunt-ended using DNA Blunting Kit (manufactured by Takara Bio Inc.) and ligated to prepare plasmid pBlue-New lacking the XhoI site. The d13 fragment excised from pJRD215-EE32d13 (Patent Document 5) with the same enzyme was cloned into the EcoRI site of this plasmid (pBlue-d13). Next, using a plasmid derived from clone E2-50 (Non-patent Document 13) distributed by RIKEN as a template, a set of primers shown in SEQ ID NOs: 6 and 7 and primers shown in SEQ ID NOs: 8 and 9 were used. Each set was amplified by PCR and 2 fragments were obtained. The conditions are (1) 94 ° C. for 2 minutes, (2) 94 ° C. for 30 seconds, (3) 55 ° C. for 30 seconds, (4) 72 ° C. for 2 minutes, (2) to (4) for 25 cycles, (5) 5 minutes at 72 ° C. The two amplified fragments were mixed in an equimolar amount and subjected to a PCR reaction again to bind the two fragments. The conditions are (1) 96 ° C. for 5 minutes, (2) 95 ° C. for 2 minutes, (3) 72 ° C. for 1 minute, and (2) to (3) for 12 cycles. Pyrobest polymerase (Takara) Bio-made) was used. A DNA fragment of the desired size was excised from an agarose electrophoresis gel, treated with PstI and XhoI, and cloned into pBlue-d13 treated with the same enzyme (pBlue-N149S / D171G). The base sequence was determined using a DNA sequencer 310 Genetic Analyzer manufactured by PERIKIN ELMER APPLIED BIOSSYSTEMS. Asparagine, the 149th amino acid of PHA synthase, was replaced with serine, and aspartic acid, the 171st amino acid, was replaced with glycine. It was confirmed that it was a mutant gene.
ｐＢｌｕｅ−Ｎ１４９Ｓ／Ｄ１７１Ｇを鋳型として配列番号１０と配列番号１１で示されるプライマーを用いてＰＣＲ反応を行い、Ｎ１４９Ｓ／Ｄ１７１Ｇ変異体の構造遺伝子ＤＮＡを増幅させた。ＰＣＲ条件は（１）９４℃で２分、（２）９４℃で３０秒、（３）４５℃で３０秒、（４）７２℃で２分、（２）から（４）を２５サイクル、（５）７２℃で５分であり、ポリメラーゼとしてはＴａＫａＲａ ＬＡ Ｔａｑ（タカラバイオ製）を用いた。次に、ｐＢｌｕｅ−ｐｈａＣＲｅを制限酵素ＳｂｆＩとＣｓｐ４５Ｉで処理し、同酵素で処理した上記増幅ＤＮＡ断片をｐｈａＣＲｅ構造遺伝子と入れ替える形でクローニングした（ｐＢｌｕｅ−ｐｈａＣＲｅ：：Ｎ１４９Ｓ／Ｄ１７１Ｇ）。
次に、プラスミドｐＪＲＤ２１５（ＡＴＣＣ３７５３３）を制限酵素ＸｈｏＩとＤｒａＩで処理してカナマイシン耐性遺伝子を含む約１．３ｋｂのＤＮＡ断片を単離後、ＤＮＡブランティングキット（タカラバイオ製）を用いて末端を平滑化し、ｐＢｌｕｅ−ｐｈａＣＲｅ：：Ｎ１４９Ｓ／Ｄ１７１Ｇを制限酵素ＳａｌＩで切断後同様に平滑末端化した部位に挿入した（ｐＢｌｕｅ−ｐｈａＣＲｅ：：Ｎ１４９Ｓ／Ｄ１７１Ｇ−Ｋｍ）。
続いて、プラスミドｐＭＴ５０７１（Ｔｓｕｄａ、ＧＥＮＥ、２０７：３３−４１（１９９８））を制限酵素ＮｏｔＩで処理してｓａｃＢ遺伝子を含む約８ｋｂのＤＮＡ断片を単離し、ｐＢｌｕｅ−ｐｈａＣＲｅ：：Ｎ１４９Ｓ／Ｄ１７１Ｇ−Ｋｍを同酵素で切断した部位に挿入して遺伝子置換用プラスミドｐＢｌｕｅ−ｐｈａＣＲｅ：：Ｎ１４９Ｓ／Ｄ１７１Ｇ−ＫｍＳＡＣを作製した。
PCR reaction was carried out using pBlue-N149S / D171G as a template and the primers shown in SEQ ID NO: 10 and SEQ ID NO: 11 to amplify the structural gene DNA of the N149S / D171G mutant. PCR conditions were (1) 94 ° C for 2 minutes, (2) 94 ° C for 30 seconds, (3) 45 ° C for 30 seconds, (4) 72 ° C for 2 minutes, (2) to (4) 25 cycles, (5) 5 minutes at 72 ° C., and TaKaRa LA Taq (manufactured by Takara Bio Inc.) was used as the polymerase. Next, pBlue-phaC Re was treated with restriction enzymes SbfI and Csp45I, and the amplified DNA fragment treated with the enzymes was cloned in a form replacing the phaC Re structural gene (pBlue-phaC Re :: N149S / D171G).
Next, plasmid pJRD215 (ATCC 37533) is treated with restriction enzymes XhoI and DraI to isolate a DNA fragment of about 1.3 kb containing a kanamycin resistance gene, and then blunted using a DNA branding kit (Takara Bio). PBlue-phaC Re :: N149S / D171G was cleaved with the restriction enzyme SalI and then inserted into the same blunt-ended site (pBlue-phaC Re :: N149S / D171G-Km).
Subsequently, plasmid pMT5071 (Tsuda, GENE, 207: 33-41 (1998)) was treated with restriction enzyme NotI to isolate an approximately 8 kb DNA fragment containing the sacB gene, and pBlue-phaC Re :: N149S / D171G- the Km was prepared insert and plasmid for gene substitution pBlue-phaC Re :: N149S / D171G -KmSAC the sites cut with the same enzymes.
＜遺伝子置換株の作製＞
遺伝子置換用プラスミドｐＢｌｕｅ−ｐｈａＣＲｅ：：Ｎ１４９Ｓ／Ｄ１７１Ｇ−ＫｍＳＡＣで大腸菌Ｓ１７−１株（ＡＴＣＣ４７００５）を形質転換し、Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株とＮｕｔｒｉｅｎｔ Ａｇａｒ培地（Ｄｉｆｃｏ社製）上で混合培養して接合伝達を行った。２５０ｍｇ／Ｌのカナマイシンを含むシモンズ寒天培地（クエン酸ナトリウム２ｇ／Ｌ、塩化ナトリウム５ｇ／Ｌ、硫酸マグネシウム・７水塩０．２ｇ／Ｌ、リン酸二水素アンモニウム１ｇ／Ｌ、リン酸水素二カリウム１ｇ／Ｌ、寒天１５ｇ／Ｌ、ｐＨ６．８）上で生育してきた菌株を選択して、プラスミドがＷａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａ Ｈ１６株の染色体上に組み込まれた株を取得した。この株をＮｕｔｒｉｅｎｔ Ｂｒｏｔｈ培地（Ｄｉｆｃｏ社製）で２世代培養した後、１５％のシュークロースを含むＮｕｔｒｉｅｎｔ Ａｇａｒ培地上に希釈して塗布し、生育してきた菌株を選択して、２段階の相同組換えによりプラスミドが脱落した株を取得した。さらにＰＣＲによる解析によりｐｈａＣＲｅ遺伝子がＮ１４９Ｓ／Ｄ１７１Ｇ変異体遺伝子に置換された菌株を単離した。この遺伝子置換株をＫＮＫ−００５株と命名し、塩基配列決定を、ＰＥＲＩＫＩＮ ＥＬＭＥＲ ＡＰＰＬＩＥＤ ＢＩＯＳＹＳＴＥＭＳ社製のＤＮＡシークエンサー３１０ Ｇｅｎｅｔｉｃ Ａｎａｌｙｚｅｒを用いて行い、染色体上のｐｈａＣＲｅ遺伝子の開始コドンから終止コドンまでがＮ１４９Ｓ／Ｄ１７１Ｇ変異体遺伝子の開始コドンから終止コドンまでに置換された株であることを確認した（図２参照）。
<Production of gene replacement strain>
Coli S17-1 strain (ATCC47005) was transformed with the plasmid for gene substitution pBlue-phaC Re :: N149S / D171G -KmSAC, conjugal transfer were mixed cultured on Wautersia eutropha H16 strain and Nutrient Agar medium (Difco Co.) Went. Simmons agar medium containing 250 mg / L kanamycin (sodium citrate 2 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 0.2 g / L, ammonium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, agar 15 g / L, pH 6.8) was selected to obtain a strain in which the plasmid was integrated on the chromosome of the Watersia eutropha H16 strain. This strain is cultured for 2 generations in Nutrient Broth medium (Difco), diluted on a Nutrient Agar medium containing 15% sucrose, applied, and the grown strain is selected to produce a two-stage homologous group. A strain from which the plasmid was removed by replacement was obtained. Furthermore, a strain in which the phaC Re gene was replaced with the N149S / D171G mutant gene was isolated by PCR analysis. This gene replacement strain was named KNK-005 strain, and the nucleotide sequence was determined using the DNA sequencer 310 Genetic Analyzer manufactured by PERIKIN ELMER APPLIED BIOSSYSTEMS, and the phaC Re gene on the chromosome from the start codon to the stop codon was N149S. It was confirmed that the strain was substituted from the start codon to the stop codon of the / D171G mutant gene (see FIG. 2).
（実施例２）β−ケトチオラーゼ遺伝子破壊株の作製
＜β−ケトチオラーゼ遺伝子の破壊用プラスミドの作製＞
プラスミドｐＪＲＤ２１５を鋳型とし、配列番号１２と配列番号１３で示されるプライマーを用いてＰＣＲ反応を行い、約１．２ｋｂｐのカナマイシン耐性遺伝子を含むＤＮＡ断片を調製した。次にｐＭＴ５０７１を制限酵素ＢａｍＨＩで処理し、同酵素で処理した上記ＤＮＡ断片をクロラムフェニコール耐性遺伝子と入れ替える形でクローニングした（ｐＳＡＣＫｍ）。
ＫＮＫ−００５株の菌体を鋳型ＤＮＡの供給源として、配列番号１４と配列番号５で示されるプライマーを用いてＰＣＲ反応を行い、約１．１ｋｂｐのβ−ケトチオラーゼ遺伝子（ｐｈｂＡ遺伝子）を含むＤＮＡ断片を調製した。このＤＮＡ断片を制限酵素ＢａｍＨＩで切断し、ベクターｐＢｌｕｅｓｃｒｉｐｔＩＩＫＳ（−）（東洋紡社製）を同酵素で切断した部位にサブクローニングした。配列番号１５で示される変異プライマーを用い、ＴａＫａＲａ ＬＡ ＰＣＲ ｉｎ ｖｉｔｒｏ Ｍｕｔａｇｅｎｅｓｉｓ Ｓｙｓｔｅｍ（タカラバイオ製）を利用して、配列番号２に示す遺伝子配列の８５番目の塩基アデニンがチミンに置換され、かつ８８番目の塩基チミンがシトシンに置換されることにより、開始コドンから１６残基目のアミノ酸が終止コドンとなり、同時に制限酵素ＮｈｅＩ切断部位が生じるような塩基置換を行った。このプラスミドを制限酵素ＮｏｔＩで切断し、ｐＳＡＣＫｍを同酵素で切断して調製した約５．７ｋｂのＤＮＡ断片を（ｏｒｉＴ＋ＫｍＲ＋ｓａｃＢＲ）を挿入して染色体置換用ベクター（ｐＢｌｕｅＡＳＲＵ）とした。
(Example 2) Preparation of β-ketothiolase gene disruption strain <Preparation of β-ketothiolase gene disruption plasmid>
A PCR reaction was performed using plasmid pJRD215 as a template and the primers shown in SEQ ID NO: 12 and SEQ ID NO: 13 to prepare a DNA fragment containing about 1.2 kbp kanamycin resistance gene. Next, pMT5071 was treated with the restriction enzyme BamHI, and the above-mentioned DNA fragment treated with the enzyme was cloned in the form of replacing the chloramphenicol resistance gene (pSACKm).
DNA containing about 1.1 kbp of β-ketothiolase gene (phbA gene) by performing PCR reaction using primers of SEQ ID NO: 14 and SEQ ID NO: 5 using the bacterial body of KNK-005 strain as a template DNA source Fragments were prepared. This DNA fragment was cleaved with the restriction enzyme BamHI, and the vector pBluescriptIIKS (−) (manufactured by Toyobo) was subcloned into the site cleaved with the enzyme. Using the mutation primer represented by SEQ ID NO: 15 and using TaKaRa LA PCR in vitro Mutagenesis System (manufactured by Takara Bio Inc.), the 85th base adenine of the gene sequence shown in SEQ ID NO: 2 is substituted with thymine, and the 88th position When the base thymine was replaced with cytosine, the amino acid at the 16th residue from the start codon became a stop codon, and at the same time, a base substitution was performed in which a restriction enzyme NheI cleavage site was generated. This plasmid was cleaved with the restriction enzyme NotI and pSACKm was cleaved with the same enzyme, and a DNA fragment of about 5.7 kb was prepared by inserting (oriT + Km R + sacBR) into a chromosome replacement vector (pBlueASRU).
＜染色体置換＞
実施例１の遺伝子置換株の作製方法と同様にして、ＫＮＫ−００５株を親株としてｐＢｌｕｅＡＳＲＵを用いて染色体置換株ＫＮＫ−００５ＡＳ株を作製した。ＫＮＫ−００５ＡＳ株のＤＮＡ塩基配列を解析して、β−ケトチオラーゼ遺伝子がｐＢｌｕｅＡＳＲＵの相同配列部分と置き換わって終止コドンと制限酵素ＮｈｅＩ切断部位が生成していることを確認した（図３参照）。なお、図３中のアミノ酸配列は配列番号３のアミノ酸配列の一部であり、図３中の塩基配列は配列番号２の塩基配列の一部である。
<Chromosome replacement>
In the same manner as the method for preparing the gene replacement strain of Example 1, the chromosome replacement strain KNK-005AS was prepared using pBlueASRU with the KNK-005 strain as the parent strain. The DNA base sequence of the KNK-005AS strain was analyzed, and it was confirmed that the β-ketothiolase gene was replaced with the homologous sequence portion of pBlueASRU to generate a stop codon and a restriction enzyme NheI cleavage site (see FIG. 3). The amino acid sequence in FIG. 3 is a part of the amino acid sequence of SEQ ID NO: 3, and the base sequence in FIG. 3 is a part of the base sequence of SEQ ID NO: 2.
（実施例３）アセトアセチル−ＣｏＡ還元酵素遺伝子リボソーム結合部位変異株の作製
＜リボソーム結合部位変異置換用プラスミドの作製＞
ＫＮＫ−００５株の菌体を鋳型ＤＮＡの供給源として、配列番号１６と配列番号１７で示されるプライマーを用いてＰＣＲ反応を行い、約１ｋｂｐのアセトアセチル−ＣｏＡ還元酵素遺伝子（ｐｈｂＢ）のリボソーム結合部位を含むＤＮＡ断片を調製した。このＤＮＡ断片を制限酵素ＢａｍＨＩで切断し、ベクターｐＢｌｕｅｓｃｒｉｐｔＩＩＫＳ（−）（東洋紡社製）を同酵素で切断した部位にサブクローニングした。配列番号１８および配列番号１９で示される変異プライマーを用い、ＴａＫａＲａ ＬＡ ＰＣＲ ｉｎ ｖｉｔｒｏ Ｍｕｔａｇｅｎｅｓｉｓ Ｓｙｓｔｅｍ（タカラバイオ製）を利用して、それぞれ、配列番号１８を用いた場合は制限酵素ＭｌｕＩ切断部位が生じるように、配列番号１９を用いた場合はＳｃａＩ切断部位が生じるようなリボソーム結合部位の塩基置換を行った。
配列番号１８を用いた場合は、配列番号２に示す遺伝子配列の１２８７番目の塩基グアニンがシトシンに置換され、かつ１２８９番目の塩基アデニンがシトシンに置換されて制限酵素ＭｌｕＩ切断部位が生じ、配列番号１９を用いた場合は、配列番号２に示す遺伝子配列の１２８８番目の塩基グアニンがチミンに置換され、かつ１２９０番目の塩基グアニンがシトシンに置換されてＳｃａＩ切断部位が生じる。
このプラスミドを制限酵素ＮｏｔＩで切断し、ｐＳＡＣＫｍを同酵素で切断して調製した約５．７ｋｂのＤＮＡ断片を（ｏｒｉＴ＋ＫｍＲ＋ｓａｃＢＲ）を挿入して染色体置換用ベクター（ｐＢｌｕｅＳＤＭ１ＲＵ及びｐＢｌｕｅＳＤＭ２ＲＵ）とした。
(Example 3) Preparation of acetoacetyl-CoA reductase gene ribosome binding site mutant <Preparation of ribosome binding site mutation replacement plasmid>
Using a bacterial body of the KNK-005 strain as a template DNA source, PCR reaction was performed using the primers shown in SEQ ID NO: 16 and SEQ ID NO: 17, and ribosome binding of about 1 kbp of acetoacetyl-CoA reductase gene (phbB) A DNA fragment containing the site was prepared. This DNA fragment was cleaved with the restriction enzyme BamHI, and the vector pBluescriptIIKS (−) (manufactured by Toyobo) was subcloned into the site cleaved with the enzyme. Using the mutation primers shown in SEQ ID NO: 18 and SEQ ID NO: 19 and using TaKaRa LA PCR in vitro Mutagenesis System (manufactured by Takara Bio), a restriction enzyme MluI cleavage site is generated when SEQ ID NO: 18 is used, respectively. In addition, when SEQ ID NO: 19 was used, base substitution of the ribosome binding site was performed such that a ScaI cleavage site was generated.
When SEQ ID NO: 18 is used, the 1287th base guanine of the gene sequence shown in SEQ ID NO: 2 is replaced with cytosine, and the 1289th base adenine is replaced with cytosine to generate a restriction enzyme MluI cleavage site. When 19 is used, the 1288th base guanine of the gene sequence shown in SEQ ID NO: 2 is substituted with thymine, and the 1290th base guanine is substituted with cytosine, resulting in a ScaI cleavage site.
This plasmid was cleaved with the restriction enzyme NotI, and a DNA fragment of about 5.7 kb prepared by cleaving pSACKm with the same enzyme was inserted into (oriT + Km R + sacBR) to obtain chromosome replacement vectors (pBlueSDM1RU and pBlueSDM2RU).
＜染色体置換＞
実施例１の遺伝子置換株の作製方法と同様にして、ＫＮＫ−００５株を親株としてｐＢｌｕｅＳＤＭ１ＲＵ及びｐＢｌｕｅＳＤＭ２ＲＵを用いてそれぞれ染色体置換株ＫＮＫ−００５ＳＤＭ１株及びＫＮＫ−００５ＳＤＭ２株を作製した。また、ＫＮＫ−００５ＡＳ株を親株として染色体置換株ＫＮＫ−００５ＡＳＳＤＭ１株およびＫＮＫ−００５ＡＳＳＤＭ２株を作製した。これら４株の染色体置換株のＤＮＡ塩基配列を解析して、アセトアセチル−ＣｏＡ還元酵素遺伝子のリボソーム結合部位に所定の変異が入っていることを確認した（図４参照）。
<Chromosome replacement>
In the same manner as the method for producing the gene replacement strain of Example 1, the chromosome substitution strains KNK-005SDM1 and KNK-005SDM2 were prepared using pBlueSDM1RU and pBlueSDM2RU, respectively, with the KNK-005 strain as the parent strain. In addition, chromosome substitution strains KNK-005ASSDM1 and KNK-005ASSDM2 were prepared using the KNK-005AS strain as a parent strain. The DNA base sequences of these four chromosome replacement strains were analyzed, and it was confirmed that a predetermined mutation was contained in the ribosome binding site of the acetoacetyl-CoA reductase gene (see FIG. 4).
（実施例４）アセトアセチル−ＣｏＡ還元酵素遺伝子破壊株の作製
＜アセトアセチル−ＣｏＡ還元酵素遺伝子の破壊用プラスミドの作製＞
ＫＮＫ−００５株の菌体を鋳型ＤＮＡの供給源として、ポリメラーゼとしてＰｙｒｏｂｅｓｔポリメラーゼ（タカラバイオ製）を用いてＰＣＲ反応を行い、アセトアセチル−ＣｏＡ還元酵素遺伝子の開始コドンより上流約４００ｂａｓｅのＤＮＡ断片と終止コドンより下流約５６０ｂａｓｅのＤＮＡ断片とを調製した。上流側の断片の調製には配列番号２０と配列番号２１で示されるプライマーを用い、下流側の断片の調製には配列番号２２と配列番号２３で示されるプライマーを用いた。両ＤＮＡ断片をそれぞれ末端リン酸化処理後、制限酵素ＢａｍＨＩで消化し、ベクターｐＢｌｕｅｓｃｒｉｐｔＩＩＫＳ（−）のＢａｍＨＩ部位に連結挿入した。このプラスミドを制限酵素ＮｏｔＩで切断し、ｐＳＡＣＫｍを同酵素で切断して調製した約５．７ｋｂのＤＮＡ断片を（ｏｒｉＴ＋ＫｍＲ＋ｓａｃＢＲ）を挿入して染色体置換用ベクター（ｐＢｌｕｅΔｐｈｂＢＲＵ）とした。
(Example 4) Production of acetoacetyl-CoA reductase gene disruption strain <Production of acetoacetyl-CoA reductase gene disruption plasmid>
Using the KNK-005 strain as a template DNA source, PCR reaction was performed using Pyrobest polymerase (manufactured by Takara Bio Inc.) as a polymerase, and a DNA fragment of about 400 bases upstream from the start codon of the acetoacetyl-CoA reductase gene A DNA fragment of about 560 bases downstream from the stop codon was prepared. Primers represented by SEQ ID NO: 20 and SEQ ID NO: 21 were used for the preparation of the upstream fragment, and primers represented by SEQ ID NO: 22 and SEQ ID NO: 23 were used for the preparation of the downstream fragment. Both DNA fragments were each phosphorylated at the end, digested with the restriction enzyme BamHI, and ligated into the BamHI site of the vector pBluescriptIIKS (−). This plasmid was digested with the restriction enzyme NotI and pSACKm was digested with the same enzyme, and a DNA fragment of about 5.7 kb was prepared by inserting (oriT + Km R + sacBR) into a chromosome replacement vector (pBlueΔphbBRU).
＜染色体置換＞
実施例１の遺伝子置換株の作製方法と同様にして、ＫＮＫ−００５株を親株としてｐＢｌｕｅΔｐｈｂＢＲＵを用いて染色体置換株ＫＮＫ−００５ΔｐｈｂＢ株を作製した。また、ＫＮＫ−００５ＡＳ株を親株として染色体置換株ＫＮＫ−００５ＡＳΔｐｈｂＢ株を作製した。これら２株の染色体置換株のＤＮＡ塩基配列を解析して、アセトアセチル−ＣｏＡ還元酵素遺伝子の開始コドンから終止コドンまでの塩基配列が脱落（配列番号２に示す遺伝子配列の１２９６番目から２０３６番目までが欠失）していることを確認した（図５参照）。
<Chromosome replacement>
In the same manner as the method for producing a gene replacement strain of Example 1, a chromosome substitution strain KNK-005ΔphbB strain was prepared using pBlueΔphbBRU with the KNK-005 strain as a parent strain. In addition, a chromosome substitution strain KNK-005ASΔphbB strain was prepared using the KNK-005AS strain as a parent strain. By analyzing the DNA base sequences of these two chromosomal replacement strains, the base sequence from the start codon to the stop codon of the acetoacetyl-CoA reductase gene was lost (from the 1296th to 2036th positions of the gene sequence shown in SEQ ID NO: 2). (See Fig. 5).
（実施例５）アセトアセチル−ＣｏＡ還元酵素活性およびβ−ケトチオラーゼ活性の測定
まず、前培養培地（１ｗ／ｖ％ Ｍｅａｔ−ｅｘｔｒａｃｔ、１ｗ／ｖ％ Ｂａｃｔｏ−Ｔｒｙｐｔｏｎ、０．２ｗ／ｖ％ Ｙｅａｓｔ−ｅｘｔｒａｃｔ、０．９ｗ／ｖ％ Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、０．１５ｗ／ｖ％ ＫＨ２ＰＯ４、（ｐＨ６．８））５ｍｌに実施例２〜４で得られた菌株を植菌して３０℃で１晩培養したものを、５ｍｌの酵素活性測定用培地（１．１ｗ／ｖ％ Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、０．１９ｗ／ｖ％ ＫＨ２ＰＯ４、０．２９ｗ／ｖ％（ＮＨ４）２ＳＯ４、０．１ｗ／ｖ％ ＭｇＳＯ４・７Ｈ２Ｏ、０．５ｗ／ｖ％フラクトース、０．５ｖ／ｖ％ 微量金属塩溶液（０．１Ｎ塩酸に１．６ｗ／ｖ％ ＦｅＣｌ３・６Ｈ２Ｏ、１ｗ／ｖ％ ＣａＣｌ２・２Ｈ２Ｏ、０．０２ｗ／ｖ％ ＣｏＣｌ２・６Ｈ２Ｏ、０．０１６ｗ／ｖ％ ＣｕＳＯ４・５Ｈ２Ｏ、０．０１２ｗ／ｖ％ ＮｉＣｌ２・６Ｈ２Ｏを溶かしたもの））に対して０．０５ｍｌ接種して、３０℃で２４時間培養した。この培養液２ｍｌを４℃で１００００×ｇ、１分間の遠心分離を行い菌体を集めた。この菌体を緩衝液（１００ｍＭ Ｔｒｉｓ−塩酸緩衝液、１ｍＭ ＥＤＴＡ、ｐＨ７．５）で２回洗浄し、１ｍｌの同緩衝液に懸濁した。これを超音波処理して菌体を破砕した後、１５０００×ｇ、４℃、５分間の遠心分離した上清を粗酵素液として用いた。
(Example 5) Measurement of acetoacetyl-CoA reductase activity and β-ketothiolase activity First, pre-culture medium (1 w / v% Meat-extract, 1 w / v% Bacto-Trypton, 0.2 w / v% Yeast-extract 0.9 w / v% Na 2 HPO 4 · 12H 2 O, 0.15 w / v% KH 2 PO 4 , (pH 6.8)) 5 ml was inoculated with the strain obtained in Examples 2-4. 5 ml of enzyme activity measurement medium (1.1 w / v% Na 2 HPO 4 · 12H 2 O, 0.19 w / v% KH 2 PO 4 , 0.29 w / v%) was cultured overnight at 30 ° C. (NH 4 ) 2 SO 4 , 0.1 w / v% MgSO 4 .7H 2 O, 0.5 w / v% fructose, 0.5 v / v% Trace metal salt solution (1.6 w / v in 0.1N hydrochloric acid) % FeCl 3 6H 2 O, 1 w / v% CaCl 2 .2H 2 O, 0.02 w / v% CoCl 2 .6H 2 O, 0.016 w / v% CuSO 4 .5H 2 O, 0.012 w / v% NiCl 2 -0.05 ml inoculated with 6H 2 O dissolved))) and cultured at 30 ° C for 24 hours. Centrifugation of 2 ml of this culture solution at 10000 × g for 1 minute at 4 ° C. collected the cells. The cells were washed twice with a buffer (100 mM Tris-HCl buffer, 1 mM EDTA, pH 7.5) and suspended in 1 ml of the same buffer. This was sonicated to disrupt the cells, and the supernatant obtained by centrifugation at 15000 × g, 4 ° C. for 5 minutes was used as the crude enzyme solution.
アセトアセチル−ＣｏＡ還元酵素活性は反応液（１００ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）、０．１ｍＭ アセトアセチル−ＣｏＡ、０．１ｍＭ ＮＡＤＰＨ）に前記粗酵素液０．０５ｍｌを添加して、全量を０．５ｍｌとし、２５℃で反応させてＮＡＤＰＨの酸化を３４０ｎｍの吸光度で測定した。アセトアセチル−ＣｏＡ還元酵素活性は、１分間に１μｍｏｌのＮＡＤＰＨを酸化する酵素量を１ユニットとした。比活性はタンパク質１ｍｇあたりのユニットとした。なお、タンパク質の定量はウシ血清アルブミンをスタンダードとして、Ｂｉｏ−Ｒａｄプロテインアッセイ試薬（バイオラッド社製）を用いたブラッドフォード法で測定した。
β−ケトチオラーゼ活性は反応液（１００ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）、０．０６ｍＭ アセトアセチル−ＣｏＡ、０．１ｍＭ ＣｏＡ−ＳＨ、２０ｍＭ ＭｇＣｌ２）に前記粗酵素液０．０１ｍｌを添加して、全量を０．５ｍｌとし、２５℃で反応させてアセトアセチル−ＣｏＡの分解を３０３ｎｍの吸光度で測定した。β−ケトチオラーゼ活性は、１分間に１μｍｏｌのアセトアセチル−ＣｏＡを分解する酵素量を１ユニットとした。比活性はタンパク質１ｍｇあたりのユニットとした。なお、タンパク質の定量はウシ血清アルブミンをスタンダードとして、Ｂｉｏ−Ｒａｄプロテインアッセイ試薬（バイオラッド社製）を用いたブラッドフォード法で測定した。
親株と実施例２、３、および４で作製した変異株のアセトアセチル−ＣｏＡ還元酵素活性とβ−ケトチオラーゼ活性を表１に示す。
The acetoacetyl-CoA reductase activity was added to the reaction solution (100 mM Tris-HCl (pH 8.0), 0.1 mM acetoacetyl-CoA, 0.1 mM NADPH) by adding 0.05 ml of the crude enzyme solution to bring the total amount to 0. 0.5 ml, reacted at 25 ° C., and the oxidation of NADPH was measured at an absorbance of 340 nm. In the acetoacetyl-CoA reductase activity, the amount of enzyme that oxidizes 1 μmol of NADPH per minute was defined as 1 unit. Specific activity was in units per mg of protein. Protein quantification was measured by the Bradford method using Bio-Rad protein assay reagent (manufactured by Bio-Rad) with bovine serum albumin as a standard.
β-ketothiolase activity was obtained by adding 0.01 ml of the crude enzyme solution to a reaction solution (100 mM Tris-HCl (pH 8.0), 0.06 mM acetoacetyl-CoA, 0.1 mM CoA-SH, 20 mM MgCl 2 ), The total amount was 0.5 ml, reacted at 25 ° C., and the decomposition of acetoacetyl-CoA was measured at an absorbance of 303 nm. The β-ketothiolase activity was defined as 1 unit of the amount of enzyme that decomposes 1 μmol of acetoacetyl-CoA per minute. Specific activity was in units per mg of protein. Protein quantification was measured by the Bradford method using Bio-Rad protein assay reagent (manufactured by Bio-Rad) with bovine serum albumin as a standard.
Table 1 shows the acetoacetyl-CoA reductase activity and β-ketothiolase activity of the parent strain and the mutant strains prepared in Examples 2, 3, and 4.
表１に示すとおり、β−ケトチオラーゼ遺伝子に改変を加えたＫＮＫ−００５ＡＳ株、ＫＮＫ−００５ＡＳＳＤＭ１株、ＫＮＫ−００５ＡＳＳＤＭ２株、ＫＮＫ−００５ＡＳΔｐｈｂＢ株はいずれもβ−ケトチオラーゼ活性が親株であるＫＮＫ−００５株の１０分の１程度であった。アセトアセチル−ＣｏＡ還元酵素活性は作製したすべての変異株でＫＮＫ−００５株より低く、０．３Ｕ／ｍｇ蛋白質以下であった。中でも、アセトアセチル−ＣｏＡ還元酵素遺伝子を完全に欠失させたＫＮＫ−００５ΔｐｈｂＢ株およびＫＮＫ−００５ＡＳΔｐｈｂＢ株はアセトアセチル−ＣｏＡ還元酵素活性が実質的に失われていた。 As shown in Table 1, all of the KNK-005AS strain, KNK-005ASSDM1, KNK-005ASSDM2, and KNK-005ASΔphbB strains, which are modified from the β-ketothiolase gene, are all of the KNK-005 strains whose β-ketothiolase activity is the parent strain. It was about 1/10. The acetoacetyl-CoA reductase activity was lower than that of the KNK-005 strain in all of the prepared mutants, and was 0.3 U / mg protein or less. Among them, the KNK-005ΔphbB strain and the KNK-005ASΔphbB strain in which the acetoacetyl-CoA reductase gene was completely deleted had substantially lost acetoacetyl-CoA reductase activity.
（実施例６）ポリエステルの生産、精製および３ＨＨ組成（ｍｏｌ％）分析
種母培地の組成は１ｗ／ｖ％ Ｍｅａｔ−ｅｘｔｒａｃｔ、１ｗ／ｖ％ Ｂａｃｔｏ−Ｔｒｙｐｔｏｎ、０．２ｗ／ｖ％Ｙｅａｓｔ−ｅｘｔｒａｃｔ、０．９ｗ／ｖ％Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、０．１５ｗ／ｖ％ＫＨ２ＰＯ４、（ｐＨ６．８）とした。
前培養培地の組成は１．１ｗ／ｖ％ Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、０．１９ｗ／ｖ％ ＫＨ２ＰＯ４、０．２９ｗ／ｖ％（ＮＨ４）２ＳＯ４、０．１ｗ／ｖ％ ＭｇＳＯ４・７Ｈ２Ｏ、２．５ｗ／ｖ％パームＷオレイン油、０．５ｖ／ｖ％ 微量金属塩溶液（０．１Ｎ塩酸に１．６ｗ／ｖ％ ＦｅＣｌ３・６Ｈ２Ｏ、１ｗ／ｖ％ ＣａＣｌ２・２Ｈ２Ｏ、０．０２ｗ／ｖ％ ＣｏＣｌ２・６Ｈ２Ｏ、０．０１６ｗ／ｖ％ ＣｕＳＯ４・５Ｈ２Ｏ、０．０１２ｗ／ｖ％ ＮｉＣｌ２・６Ｈ２Ｏを溶かしたもの。）とした。
Example 6 Polyester Production, Purification and 3HH Composition (mol%) Analysis The composition of the seed mother medium was 1 w / v% Meat-extract, 1 w / v% Bacto-Trypton, 0.2 w / v% Yeast-extract, 0.9 w / v% Na 2 HPO 4 · 12H 2 O, 0.15 w / v% KH 2 PO 4 , (pH 6.8).
The composition of the preculture medium is 1.1 w / v% Na 2 HPO 4 · 12H 2 O, 0.19 w / v% KH 2 PO 4 , 0.29 w / v% (NH 4 ) 2 SO 4 , 0.1 w / v% MgSO 4 · 7H 2 O , 2.5w / v% palm W olein oil, 0.5 v / v% trace metal salt solution (1.6 w in 0.1N HCl / v% FeCl 3 · 6H 2 O, 1w / V% CaCl 2 · 2H 2 O, 0.02 w / v% CoCl 2 · 6H 2 O, 0.016 w / v% CuSO 4 · 5H 2 O, 0.012 w / v% NiCl 2 · 6H 2 O ).
ポリエステル生産培地の組成は０．３８５ｗ／ｖ％ Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、０．０６７ｗ／ｖ％ ＫＨ２ＰＯ４、０・２９１ｗ／ｖ％（ＮＨ４）２ＳＯ４、０．１ｗ／ｖ％ ＭｇＳＯ４・７Ｈ２Ｏ、０．５ｖ／ｖ％ 微量金属塩溶液（０．１Ｎ塩酸に１．６ｗ／ｖ％ ＦｅＣｌ３・６Ｈ２Ｏ、１ｗ／ｖ％ ＣａＣｌ２・２Ｈ２Ｏ、０．０２ｗ／ｖ％ ＣｏＣｌ２・６Ｈ２Ｏ、０．０１６ｗ／ｖ％ ＣｕＳＯ４・５Ｈ２Ｏ、０．０１２ｗ／ｖ％ ＮｉＣｌ２・６Ｈ２Ｏを溶かしたもの。）、０．０５ｗ／ｖ％ＢＩＯＳＰＵＭＥＸ２００Ｋ（消泡剤：コグニスジャパン製）とした。炭素源はパーム核油を分別した低融点画分であるパーム核油オレインを単一炭素源として用い、培養全般を通じ、比基質供給速度が０．０８〜０．１（ｇ油脂）×（ｇ正味乾燥菌体重量）−１×（ｈ）−１となるように流加した。
試験菌株のグリセロールストック（５０μｌ）を種母培地（１０ｍｌ）に接種して２４時間培養し、１．８Ｌの前培養培地を入れた３Ｌジャーファーメンター（丸菱バイオエンジ製ＭＤＬ−３００型）に１．０ｖ／ｖ％接種した。運転条件は、培養温度３０℃、攪拌速度５００ｒｐｍ、通気量１．８Ｌ／ｍｉｎとし、ｐＨは６．７〜６．８の間でコントロールしながら２８時間培養した。ｐＨコントロールには７％水酸化アンモニウム水溶液を使用した。
The composition of the polyester production medium is 0.385 w / v% Na 2 HPO 4 · 12H 2 O, 0.067 w / v% KH 2 PO 4 , 0 · 291 w / v% (NH 4 ) 2 SO 4 , 0.1 w / v% MgSO 4 · 7H 2 O , 0.5v / v% trace metal salt solution (1.6 w in 0.1N HCl / v% FeCl 3 · 6H 2 O, 1w / v% CaCl 2 · 2H 2 O, 0 0.02 w / v% CoCl 2 · 6H 2 O, 0.016 w / v% CuSO 4 · 5H 2 O, 0.012 w / v% NiCl 2 · 6H 2 O)), 0.05 w / v% BIOSPUMEX 200K (antifoaming agent: manufactured by Cognis Japan) was used. As a carbon source, palm kernel oil olein, which is a low melting point fraction obtained by separating palm kernel oil, is used as a single carbon source. Throughout the entire culture, the specific substrate supply rate is 0.08 to 0.1 (g oil) x (g Net dry cell weight) -1 x (h) -1 was fed.
Glycerol stock (50 μl) of the test strain was inoculated into seed medium (10 ml), cultured for 24 hours, and transferred to a 3 L jar fermenter (MDL-300 model manufactured by Maruhishi Bioengineer) containing 1.8 L of preculture medium. 1.0 v / v% was inoculated. The operating conditions were a culture temperature of 30 ° C., a stirring speed of 500 rpm, an aeration rate of 1.8 L / min, and a culture for 28 hours while controlling the pH between 6.7 and 6.8. A 7% aqueous ammonium hydroxide solution was used for pH control.
ポリエステル生産培養は６Ｌの生産培地を入れた１０Ｌジャーファーメンター（丸菱バイオエンジ製ＭＤＬ−１０００型）に前培養種母を５．０ｖ／ｖ％接種した。運転条件は、培養温度２８℃、攪拌速度４００ｒｐｍ、通気量３．６Ｌ／ｍｉｎとし、ｐＨは６．７から６．８の間でコントロールした。ｐＨコントロールには７％水酸化アンモニウム水溶液を使用した。培養は約４８時間行い、培養終了後、遠心分離によって菌体を回収、メタノールで洗浄、凍結乾燥し、乾燥菌体重量を測定した。
得られた乾燥菌体約１ｇに１００ｍｌのクロロホルムを加え、室温で一昼夜攪拌して、菌体内のポリエステルを抽出した。菌体残渣をろ別後、エバポレーターで総容量が約３０ｍｌになるまで濃縮後、約９０ｍｌのヘキサンを徐々に加え、ゆっくり攪拌しながら、１時間放置した。析出したポリエステルをろ別後、５０℃で３時間真空乾燥した。乾燥ポリエステルの重量を測定し、菌体内のポリエステル含量を算出した。そして、培養液１Ｌあたりの乾燥菌体重量にポリエステル含量を掛けることによってポリエステル生産性を算出した。
In the polyester production culture, a 10 L jar fermenter (MDL-1000, manufactured by Maruhishi Bioengine) containing 6 L of production medium was inoculated with 5.0 v / v% of the preculture seed. The operating conditions were a culture temperature of 28 ° C., a stirring speed of 400 rpm, an aeration rate of 3.6 L / min, and a pH controlled between 6.7 and 6.8. A 7% aqueous ammonium hydroxide solution was used for pH control. Cultivation was carried out for about 48 hours. After completion of the cultivation, the cells were collected by centrifugation, washed with methanol, lyophilized, and the dry cell weight was measured.
100 ml of chloroform was added to about 1 g of the obtained dried microbial cells, and stirred overnight at room temperature to extract the polyester in the microbial cells. The bacterial cell residue was filtered off and concentrated with an evaporator until the total volume reached about 30 ml. Then, about 90 ml of hexane was gradually added, and the mixture was allowed to stand for 1 hour with slow stirring. The precipitated polyester was filtered off and then vacuum dried at 50 ° C. for 3 hours. The weight of the dried polyester was measured, and the polyester content in the cells was calculated. Then, the polyester productivity was calculated by multiplying the dry cell weight per liter of the culture solution by the polyester content.
生産されたポリエステルの３ＨＨ組成分析は、以下のようにガスクロマトグラフィーによって行った。
乾燥ポリエステルの約２０ｍｇに２ｍｌの硫酸−メタノール混液（１５：８５）と２ｍｌのクロロホルムを添加して密栓し、１００℃で１４０分間加熱することでポリエステル分解物のメチルエステルを得た。冷却後、これに１．５ｇの炭酸水素ナトリウムを少しずつ加えて中和し、炭酸ガスの発生がとまるまで放置した。４ｍｌのジイソプロピルエーテルを添加してよく混合した後、遠心して、上清中のポリエステル分解物のモノマーユニット組成をキャピラリーガスクロマトグラフィーにより分析した。ガスクロマトグラフは島津製作所ＧＣ−１７Ａ、キャピラリーカラムはＧＬサイエンス社製ＮＥＵＴＲＡ ＢＯＮＤ−１（カラム長２５ｍ、カラム内径０．２５ｍｍ、液膜厚０．４μｍ）を用いた。キャリアガスとしてＨｅを用い、カラム入口圧１００ｋＰａとし、サンプルは１μｌを注入した。温度条件は、初発温度１００〜２００℃まで８℃／分の速度で昇温、さらに２００〜２９０℃まで３０℃／分の速度で昇温した。
以上のようにして親株と実施例２、３、および４で作製した変異株を培養してポリエステルを生産した成績及び３ＨＨ組成を表２に示す。
The 3HH composition analysis of the produced polyester was performed by gas chromatography as follows.
To 20 mg of the dried polyester, 2 ml of a sulfuric acid-methanol mixture (15:85) and 2 ml of chloroform were added and sealed, and heated at 100 ° C. for 140 minutes to obtain a methyl ester of a polyester degradation product. After cooling, 1.5 g of sodium bicarbonate was added little by little to neutralize it, and the mixture was allowed to stand until the generation of carbon dioxide gas stopped. After adding 4 ml of diisopropyl ether and mixing well, the mixture was centrifuged and the monomer unit composition of the polyester degradation product in the supernatant was analyzed by capillary gas chromatography. The gas chromatograph used was Shimadzu Corporation GC-17A, and the capillary column used was GL Science's NEUTRA BOND-1 (column length 25 m, column inner diameter 0.25 mm, liquid film thickness 0.4 μm). He was used as the carrier gas, the column inlet pressure was set to 100 kPa, and 1 μl of the sample was injected. As temperature conditions, the temperature was raised from the initial temperature of 100 to 200 ° C. at a rate of 8 ° C./min, and further from 200 to 290 ° C. at the rate of 30 ° C./min.
Table 2 shows the results and 3HH composition of culturing the parent strain and the mutant strains prepared in Examples 2, 3, and 4 to produce polyester.
表２に示すとおり作製した変異株のポリエステル生産性は４８〜７９ｇ／Ｌと高く、３ＨＨ組成は７．５〜１６．８ｍｏｌ％の高い比率を示した。 Polyester productivity of the mutants produced as shown in Table 2 was as high as 48 to 79 g / L, and the 3HH composition showed a high ratio of 7.5 to 16.8 mol%.
本発明の方法によれば、高３ＨＨ組成のＰ（３ＨＢ−ｃｏ−３ＨＨ）を従来の方法に比べて効率よく製造することができる。そして、本発明の方法によって製造された高３ＨＨ組成のＰ（３ＨＢ−ｃｏ−３ＨＨ）は柔軟な性質を活かしたフィルム等への加工に適した生分解性プラスチックとして多くの分野で種々の用途に用いることができるので、産業上極めて有用である。 According to the method of the present invention, P (3HB-co-3HH) having a high 3HH composition can be produced more efficiently than the conventional method. P (3HB-co-3HH) having a high 3HH composition produced by the method of the present invention is a biodegradable plastic suitable for processing into a film and the like utilizing its flexible properties, and is used in various fields in various fields. Since it can be used, it is very useful industrially.
Claims (17)
（ａ）アセトアセチル−ＣｏＡ還元酵素遺伝子の全部もしくは一部が欠失されたことにより、またはアセトアセチル−ＣｏＡ還元酵素遺伝子の開始コドンより上流１０塩基以内に存在するリボソーム結合部位の塩基配列に少なくとも１塩基以上の置換、欠失もしくは挿入がなされたことにより、アセトアセチル−ＣｏＡ還元酵素活性が０．３Ｕ／ｍｇ蛋白質以下である。
（ｂ）β−ケトチオラーゼ遺伝子のＤＮＡ配列に少なくとも１塩基以上の置換、欠失もしくは挿入がなされたことにより、β−ケトチオラーゼ活性が欠損又は減少されている。 Waterthesia genus having the characteristics of any one or both of the following (a) and (b), and having the ability to produce a copolyester composed of monomer units of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid A microorganism selected from the group consisting of the genus Capriavidas, Ralstonia, Alkagenes and Pseudomonas.
(A) at least in the base sequence of the ribosome binding site present by deletion of all or part of the acetoacetyl-CoA reductase gene or within 10 bases upstream from the start codon of the acetoacetyl-CoA reductase gene As a result of substitution, deletion or insertion of one or more bases, the acetoacetyl-CoA reductase activity is 0.3 U / mg protein or less.
(B) β-ketothiolase activity is deficient or reduced by substitution, deletion or insertion of at least one base in the DNA sequence of the β-ketothiolase gene.
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