JP2008086238A - Method for producing polyhydroxyalkanoate - Google Patents
Method for producing polyhydroxyalkanoate Download PDFInfo
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- JP2008086238A JP2008086238A JP2006269730A JP2006269730A JP2008086238A JP 2008086238 A JP2008086238 A JP 2008086238A JP 2006269730 A JP2006269730 A JP 2006269730A JP 2006269730 A JP2006269730 A JP 2006269730A JP 2008086238 A JP2008086238 A JP 2008086238A
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 36
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 title claims abstract description 16
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- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical compound CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 claims abstract description 66
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- HPMGFDVTYHWBAG-UHFFFAOYSA-N 3-hydroxyhexanoic acid Chemical compound CCCC(O)CC(O)=O HPMGFDVTYHWBAG-UHFFFAOYSA-N 0.000 claims abstract description 13
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Images
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、微生物を用いて製造される３−ヒドロキシ酪酸（以下、３ＨＢとも記す）及び３−ヒドロキシヘキサン酸（以下、３ＨＨとも記す）のモノマーユニットを含有するポリヒドロキシアルカノエート共重合体（以下、ＰＨＡとも記す）並びにその製造方法に関し、さらに詳しくは、３−ヒドロキシブチレート単位と３−ヒロドキシヘキサノエート単位との共重合比率をコントロールする方法に関する。 The present invention relates to a polyhydroxyalkanoate copolymer (hereinafter referred to as 3-hydroxybutanoic acid (hereinafter also referred to as 3HB) and 3-hydroxyhexanoic acid (hereinafter also referred to as 3HH) monomer units produced using a microorganism. In particular, the present invention relates to a method for controlling the copolymerization ratio of 3-hydroxybutyrate units and 3-hydroxyhexanoate units.
ＰＨＡは、広範な微生物によって生成されるポリエステルの１種である。ＰＨＡは生分解性を有すること、熱可塑性高分子であること、及び、再生可能資源から産生されることから、環境調和型素材或い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。
現在までに数多くの微生物において、エネルギー貯蔵物質としてポリエステルを菌体内に蓄積することが知られている。その代表例が３ＨＢのホモポリマーであるポリ−３−ヒドロキシ酪酸（以下、Ｐ（３ＨＢ）とも記す）であり、１９２５年にＢａｃｉｌｌｕｓ ｍｅｇａｔｅｒｉｕｍ内で最初に発見された。Ｐ（３ＨＢ）は熱可塑性高分子であり、自然環境中で生物的に分解されることから、環境にやさしいプラスチックとして注目されている。しかし、Ｐ（３ＨＢ）は結晶性が高いため、硬くて脆い性質を持っていることから実用的には応用範囲が限られる。この為、この性質の改良を目的とした研究がなされてきた。
PHA is a kind of polyester produced by a wide range of microorganisms. Since PHA is biodegradable, is a thermoplastic polymer, and is produced from renewable resources, it is industrially produced as an environmentally conscious material or a biocompatible material and used in various industries. Attempts have been made.
To date, it has been known that polyesters accumulate in cells as an energy storage substance in many microorganisms. A typical example is poly-3-hydroxybutyric acid (hereinafter also referred to as P (3HB)), which is a homopolymer of 3HB, and was first discovered in 1925 in Bacillus megaterium. P (3HB) is a thermoplastic polymer and is biologically decomposed in the natural environment, and thus has attracted attention as an environmentally friendly plastic. However, since P (3HB) has high crystallinity, it has a hard and brittle nature, so its practical application range is limited. For this reason, research aimed at improving this property has been conducted.
その中で、３−ヒドロキシ酪酸（３ＨＢ）と３−ヒドロキシ吉草酸（以下、３ＨＶとも記す）とからなる共重合体（以下、Ｐ（３ＨＢ−ｃｏ−３ＨＶ）とも記す）の製造方法が開示されている（例えば特許文献１、特許文献２）。このＰ（３ＨＢ−ｃｏ−３ＨＶ）は、Ｐ（３ＨＢ）に比べると柔軟性に富むため、幅広い用途に応用できると考えられた。しかしながら、実際にはＰ（３ＨＢ−ｃｏ−３ＨＶ）の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、特にフィルム等へ加工するために要求される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、硬質成型体の限られた分野にしか利用されなかった。 Among them, a method for producing a copolymer (hereinafter also referred to as P (3HB-co-3HV)) comprising 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (hereinafter also referred to as 3HV) is disclosed. (For example, Patent Document 1 and Patent Document 2). Since P (3HB-co-3HV) is more flexible than P (3HB), it was considered that it could be applied to a wide range of applications. However, actually, even if the 3HV mole fraction of P (3HB-co-3HV) is increased, the change in physical properties associated therewith is poor, and in particular, the flexibility required for processing into a film or the like does not improve. It was used only in limited fields such as shampoo bottles and disposable razor handles.
近年、３ＨＢと３ＨＨからなる共重合体（以下、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）とも記す）及びその製造方法について研究がなされた（特許文献３、特許文献４）。これら報告におけるＰ（３ＨＢ−ｃｏ−３ＨＨ）の製造方法は、土壌より単離されたＡｅｒｏｍｏｎａｓ ｃａｖｉａｅを用い、オレイン酸等の脂肪酸やオリーブオイル等の油脂を炭素源として発酵生産するものであった。また、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）の物性に関する研究もなされている（非特許文献１）。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Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅを培養し、３ＨＨ組成比が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、硬質ポリマーから軟質ポリマー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（低３ＨＨ組成）から、フィルム等のように柔軟性を要求されるもの（高３ＨＨ組成）まで、幅広い分野への応用が期待できる。しかしながら、前記製造方法では菌体生産量４ｇ／Ｌ、ポリマー含量が３０重量％とポリマー生産性は低いものであり、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）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ポリマー含量が得られる方法が探索されている。 In recent years, research has been conducted on a copolymer comprising 3HB and 3HH (hereinafter also referred to as P (3HB-co-3HH)) and a method for producing the same (Patent Document 3 and Patent Document 4). The production method of P (3HB-co-3HH) in these reports was to fermentate and produce Aeromonas caviae isolated from soil using fatty acids such as oleic acid and fats and oils such as olive oil as a carbon source. Studies on the physical properties of P (3HB-co-3HH) have also been made (Non-Patent Document 1). In this report, Aeromonas caviae is cultured using a fatty acid having 12 or more carbon atoms as a sole carbon source, and P (3HB-co-3HH) having a 3HH composition ratio of 11 to 19 mol% is produced by fermentation. This P (3HB-co-3HH) gradually becomes more flexible from the hard and brittle nature of P (3HB) as the 3HH composition increases, and the flexibility exceeds P (3HB-co-3HV). It was made clear to show. In other words, P (3HB-co-3HH) has a wide range of physical properties that can be applied from hard polymers to soft polymers by changing the 3HH composition. The composition can be expected to be applied in a wide range of fields, from films to films that require flexibility (high 3HH composition). However, in the above production method, the yield of bacterial cells is 4 g / L, the polymer content is 30% by weight, and the polymer productivity is low. Since it must be said that it is sufficient, a method for obtaining a higher polymer content for practical use is being sought.
Ｐ（３ＨＢ−ｃｏ−３ＨＨ）の工業生産を目指した取組みもなされている。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、菌体量９５．７ｇ／Ｌ、ポリマー含量４５．２重量％、３ＨＨ組成１７ｍｏｌ％のＰ（３ＨＢ−ｃｏ−３ＨＨ）が生産された（非特許文献２）。また、炭素源としてグルコース及びラウリン酸を用いてＡｅｒｏｍｏｎａｓ ｈｙｄｒｏｐｈｉｌａの培養をし、菌体量５０ｇ／Ｌ、ポリマー含量５０重量％を達成した（非特許文献３）。しかしながら、Ａｅｒｏｍｏｎａｓ ｈｙｄｒｏｐｈｉｌａはヒトに対して病原性を有する（非特許文献４）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。さらには、ポリマー含量も５０重量％以下と低い。
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。Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅよりクローニングされたポリヒドロキシアルカン酸（ＰＨＡ）合成酵素遺伝子をＣｕｐｒｉａｖｉｄｕｓ ｎａｃａｔｏｒ（旧分類：Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ，Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｈｕｓ）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、オクタン酸を炭素源として菌体生産性は菌体量４ｇ／Ｌ、ポリマー含量は３０重量％、３ＨＨ組成は２２ｍｏｌ％であった（特許文献５、非特許文献５）。更に前記形質転換体を、炭素源として植物油脂を用いて培養した結果、菌体量４ｇ／Ｌ、ポリマー含量８０重量％が達成された（非特許文献６）。しかしながら、前記製造方法は安価な植物油脂を炭素源とするものの、ポリマーの生産性は低く、該ポリマーの３ＨＨ組成は５ｍｏｌ％であった。また、フラクトースを炭素源としてＰ（３ＨＢ−ｃｏ−３ＨＨ）生産できるＣｕｐｒｉａｖｉｄｕｓ ｎａｃａｔｏｒも構築されたが、本菌株のＰ（３ＨＢ−ｃｏ−３ＨＨ）生産におけるポリマー含量は５０重量％、３ＨＨ組成は１．５ｍｏｌ％と低く、実生産に適しているとはいえなかった（非特許文献７）。
Efforts aimed at industrial production of P (3HB-co-3HH) have also been made. In the culture using Aeromonas hydrophila, P (3HB-co) with a cell amount of 95.7 g / L, a polymer content of 45.2% by weight, a 3HH composition of 17 mol% in fed-batch culture for 43 hours using oleic acid as a carbon source. -3HH) was produced (Non-Patent Document 2). In addition, Aeromonas hydrophila was cultured using glucose and lauric acid as a carbon source to achieve a cell amount of 50 g / L and a polymer content of 50% by weight (Non-patent Document 3). However, Aeromonas hydrophila has pathogenicity to humans (Non-Patent Document 4), and thus cannot be said to be a suitable species 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. Furthermore, the polymer content is as low as 50% by weight or less.
For this reason, efforts were made to improve production and productivity in a safe host. Production of P (3HB-co-3HH) using a transformant in which a polyhydroxyalkanoic acid (PHA) synthase gene cloned from Aeromonas caviae was introduced into Cupriavidus nacator (formerly Ralstonia eutropha, Alcaligenes eutrophus) As a result, the cell productivity was 4 g / L, the polymer content was 30% by weight, and the 3HH composition was 22 mol% using octanoic acid as a carbon source (Patent Document 5, Non-Patent Document 5). Furthermore, as a result of culturing the transformant using vegetable oil as a carbon source, a bacterial cell amount of 4 g / L and a polymer content of 80% by weight were achieved (Non-patent Document 6). However, although the said manufacturing method uses cheap vegetable fats and oils as a carbon source, the productivity of a polymer is low and the 3HH composition of this polymer was 5 mol%. In addition, a Cupriavidus nacator capable of producing P (3HB-co-3HH) using fructose as a carbon source was also constructed. The polymer content of this strain in the production of P (3HB-co-3HH) was 50% by weight, and the composition of 3HH was 1. It was as low as 5 mol% and could not be said to be suitable for actual production (Non-patent Document 7).
大腸菌を宿主としたＰ（３ＨＢ−ｃｏ−３ＨＨ）生産株も構築された。Ａｅｒｏｍｏｎａｓ属のＰＨＡ合成酵素遺伝子、Ｃｕｐｒｉａｖｉｄｕｓ ｎａｃａｔｏｒ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４０．８時間培養した結果、菌体量７９ｇ／Ｌ、ポリマー含量２７．２重量％、３ＨＨ組成比１０．８ｍｏｌ％であった（非特許文献８）。Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ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。ラウリン酸を含む培地で該大腸菌を培養すると、菌体生産性は菌体量約１ｇ／Ｌ、ポリマー含量約１６重量％、３ＨＨ組成約１６ｍｏｌ％であった（非特許文献９）。しかしながら、これらの大腸菌ではポリマー含量が低く、工業的生産への適用は困難であった。 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 of the genus Aeromonas, the NADP-acetoacetyl Co-A reductase gene of Cupriavidus nucleator and the like were introduced into Escherichia coli. As a result of culturing the Escherichia coli for 40.8 hours using dodecane as a carbon source, the amount of cells was 79 g / L, the polymer content was 27.2% by weight, and the 3HH composition ratio was 10.8 mol% (Non-patent Document 8). An Escherichia coli into which the PHA synthase gene, enoyl CoA hydratase gene and acyl CoA dehydrogenase gene of Aeromonas caviae were introduced was also constructed. When the Escherichia coli was cultured in a medium containing lauric acid, the cell productivity was about 1 g / L, the polymer content was about 16% by weight, and the 3HH composition was about 16 mol% (Non-patent Document 9). However, these Escherichia coli has a low polymer content and is 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（非特許文献１０）。Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ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報告されている。しかし、これらは未だポリマー含量が約１３重量％と低いことから、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。
Ｔ．Ｆｕｋｕｉ等が作製した、Ｃｕｐｒｉａｖｉｄｕｓ ｎａｃａｔｏｒを宿主としてＰ（３ＨＢ−ｃｏ−３ＨＨ）を生産した菌株に導入されていたＰＨＡ合成酵素発現プラスミドは、ｐＪＲＤ２１５（ＡＴＣＣ ３７５３３）にポリエステル合成酵素遺伝子やＤ−エノイルＣｏ−Ａヒドラターゼ遺伝子等を導入したｐＪＲＤＥＥ３２やｐＪＲＤＥＥ３２ｄ１３等（特許文献５参照）であった。本菌株の菌体量は４ｇ／Ｌと低かったが、植物油脂を炭素源とした前記菌株の培養条件改善により、菌体量４５ｇ／Ｌ、ポリマー含量６２．５重量％、３ＨＨ組成８．１ｍｏｌ％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）。
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）。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％のポリエステルを生産することが可能となり、種々の物性を有する（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。
In order to improve the productivity of P (3HB-co-3HH) and control the 3HH composition, artificial modification of PHA synthase was performed (Non-patent Document 10). Among the mutants of PHA synthase derived from Aeromonas caviae, a mutant enzyme in which the 149th amino acid asparagine is replaced with serine, and a mutant enzyme in which the 171st aspartic acid is replaced with glycine are PHA in E. coli. It has been reported that synthase activity and 3HH composition are improved. However, since these polymers still have a low polymer content of about 13% by weight, further improvements for industrial production utilizing the characteristics of these mutant enzymes were necessary.
T.A. A PHA synthase expression plasmid introduced into a strain that produced P (3HB-co-3HH) using Cupriavidus nacator as a host produced by Fukui et al. Was found in pJRD215 (ATCC 37533) as a polyester synthase gene or D-enoyl Co -PJRDEE32, pJRDEE32d13, etc. into which A hydratase gene or the like was introduced (see Patent Document 5). Although the bacterial mass of this strain was as low as 4 g / L, by improving the culture conditions of the strain using vegetable oil as a carbon source, the bacterial mass was 45 g / L, the polymer content was 62.5% by weight, and the 3HH composition was 8.1 mol. As a result, a method for producing P (3HB-co-3HH) having a high 3HH composition by a culture method was also studied (Patent Document 6).
A method for controlling the physical properties of P (3HB-co-3HH) is also disclosed (Patent Document 6). By using at least two types of fats and oils and / or fatty acids having different numbers of carbons as a carbon source, it becomes possible to produce a polyester having a 3HH composition of 1 to 40 mol% and have various physical properties (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.
さらに、酪酸やブタノールの代謝に関与する遺伝子を組み込んだ大腸菌にて、グルコースと酪酸またはブタノールからＰＨＢＨを生産させた方法も開示されているが、３ＨＨ組成は１〜３ｍｏｌ％程度であった。（特許文献７）。
上記の様に、様々なＰＨＡの生産検討が行なわれてきたが、ＰＨＡの３ＨＨ組成を高くする様にコントロールするには高価なヘキサン酸やオクタン酸などの脂肪酸を炭素源にしなければならなかった。また、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）に限らず、他のＰＨＡの生産菌においても、生産菌の安全性や、ＰＨＡの生産性など、柔軟性に富むＰＨＡの工業生産には多くの課題が残されている。
何れにしても、これまでのＰ（３ＨＢ−ｃｏ−３ＨＨ）の発酵生産では、ポリマー含量が低い、３ＨＨ組成が５ｍｏｌ％以下と低い、高価な炭素源を必要とする、宿主の人への安全性が低いなどの問題があった。
As described above, various PHA production studies have been conducted, but expensive fatty acids such as hexanoic acid and octanoic acid had to be used as a carbon source in order to control the PHA to have a high 3HH composition. . Further, not only P (3HB-co-3HH) but also other PHA producing bacteria, there are many problems in the industrial production of PHA with high flexibility such as the safety of producing bacteria and the productivity of PHA. It is left.
In any case, the conventional fermentative production of P (3HB-co-3HH) requires an expensive carbon source with a low polymer content and a low 3HH composition of 5 mol% or less. There were problems such as low nature.
本発明の目的は、３ＨＢ及び３ＨＨを含有するＰＨＡにおいて、菌体中のポリマー含量を低下させずに、３ＨＢ組成に対する３ＨＨ組成が向上したＰＨＡの発酵生産方法を提供することである。好ましくは３ＨＨ組成が７ｍｏｌ％以上のＰ（３ＨＢ−ｃｏ−３ＨＨ）の発酵生産において、安全な生産菌であるＣｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒを用い、高価な脂肪酸を使用せず、ポリマー含量を６５重量％以上と高水準に維持し且つ３ＨＨ組成が上昇する様にコントロールできる発酵生産方法を提供することである。 An object of the present invention is to provide a method for fermentative production of PHA having an improved 3HH composition relative to the 3HB composition without reducing the polymer content in the cells in the PHA containing 3HB and 3HH. Preferably, in the fermentative production of P (3HB-co-3HH) having a 3HH composition of 7 mol% or more, a safe producing bacterium, Cupriavidus necator, is used, an expensive fatty acid is not used, and the polymer content is as high as 65 wt% or more. It is to provide a fermentation production method that can be controlled to maintain the level and increase the 3HH composition.
本発明者らは、上記課題を解決するために鋭意研究を重ねた結果、Ｃｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒが保有するβケトチオラーゼ遺伝子（ｐｈｂＡ遺伝子）の不活性化を行い、炭素源として酪酸及び／又はブタノールを必ず使用し、培養する事によって、効果的に３ＨＢ組成に対する３ＨＨ組成比率の向上したＰＨＡが生成し、蓄積する事を見出し、また、炭素源中の酪酸及び／又はブタノールの含有量をコントロールする事で３ＨＨ組成をコントロール可能である事を見出し、本発明を完成するに至った。
即ち、本発明の第一は、３−ヒドロキシ酪酸（以下、３ＨＢとも記す）及び３−ヒドロキシヘキサン酸（以下、３ＨＨとも記す）のモノマーユニットを含有したポリヒドロキシアルカノエート共重合体（以下ＰＨＡとも記す）を生産可能であり、且つｐｈｂＡ遺伝子が不活性化されていることを特徴とするＣｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒを用い、さらに使用する炭素源が酪酸及び／又はブタノールを含むことを特徴とするＰＨＡの発酵生産方法に関する。好ましい実施態様は、ＰＨＡが３ＨＢと３ＨＨのモノマーユニットのみからなるポリヒドロキシアルカノエート共重合体（以下、Ｐ（３ＨＢ−ｃｏ−３ＨＨ）とも記す）であることを特徴とする上記記載の発酵生産方法に関する。より好ましくは、炭素源中の酪酸及び／又はブタノールの含有量を調整することにより、ＰＨＡの３ＨＨ組成比を上昇させることを特徴とする上記記載の発酵生産方法、更に好ましくは、炭素源全体中、酪酸及び／又はブタノールの含有量が、酪酸及び／又はブタノール以外の炭素源１００重量部に対して５〜４０重量部であることを特徴とする上記記載の発酵生産方法、最も好ましくは、酪酸及び／又はブタノール以外の炭素源が、炭素数６以上の脂肪酸又は植物油脂であることを特徴とする上記記載の発酵生産方法、に関する。
As a result of intensive studies to solve the above-mentioned problems, the present inventors inactivate the β-ketothiolase gene (phbA gene) possessed by Cupriavidus necator and always use butyric acid and / or butanol as a carbon source. By culturing, it was found that PHA having an improved 3HH composition ratio relative to 3HB composition was generated and accumulated, and 3HH was controlled by controlling the content of butyric acid and / or butanol in the carbon source. The present inventors have found that the composition can be controlled and have completed the present invention.
That is, the first of the present invention is a polyhydroxyalkanoate copolymer (hereinafter also referred to as PHA) containing monomer units of 3-hydroxybutyric acid (hereinafter also referred to as 3HB) and 3-hydroxyhexanoic acid (hereinafter also referred to as 3HH). And the fermentative production of PHA, wherein the carbon source to be used contains butyric acid and / or butanol, and is characterized in that the phbA gene is inactivated. Regarding the method. In a preferred embodiment, the PHA is a polyhydroxyalkanoate copolymer (hereinafter also referred to as P (3HB-co-3HH)) comprising only 3HB and 3HH monomer units. About. More preferably, the 3HH composition ratio of PHA is increased by adjusting the content of butyric acid and / or butanol in the carbon source, and more preferably in the whole carbon source. The fermentation production method as described above, wherein the content of butyric acid and / or butanol is 5 to 40 parts by weight with respect to 100 parts by weight of a carbon source other than butyric acid and / or butanol, most preferably butyric acid And / or the carbon source other than butanol is a fatty acid having 6 or more carbon atoms or a vegetable oil, wherein the fermentation production method is described above.
以下、本発明につき、さらに詳細に説明する。
本発明における３ＨＢと３ＨＨのモノマーユニットを含有するＰＨＡとは、化学式（１）（式中のｌ、ｍ及びｎはポリマーのモノマー単位を表し、１以上の整数。Ｒ１は炭素数２又は４以上１３以下であるアルキル基を表し、Ｒ１が複数ある場合、ポリマー中同一でもよいし、異なっていても良い。）に示されるような３ＨＢと３ＨＨのモノマーユニットを含有するポリヒドロキシアルカノエート共重合体である。本発明において、柔軟性に富むＰＨＡの生産という観点及び３ＨＨ組成比をコントロールするという観点からは、３ＨＨ組成比が７．５〜１２ｍｏｌ％であることが好ましい。
より好ましくは、以下の化学式（２）（式中のｍ及びｎはポリマーのモノマー単位を表し、１以上の整数）に示すような３−ヒロドキシ酪酸（３ＨＢ）と３−ヒロドキシヘキサン酸（３ＨＨ）のみをモノマー単位とする共重合体であるＰ（３ＨＢ−ｃｏ−３ＨＨ）である。
Hereinafter, the present invention will be described in more detail.
PHA containing 3HB and 3HH monomer units in the present invention is represented by chemical formula (1) (wherein l, m and n represent monomer units of the polymer and are integers of 1 or more. R 1 has 2 or 4 carbon atoms. A polyhydroxyalkanoate containing a monomer unit of 3HB and 3HH as shown in the above formula, wherein the alkyl group is 13 or less, and when there are a plurality of R 1 s , they may be the same or different in the polymer. It is a polymer. In the present invention, the 3HH composition ratio is preferably 7.5 to 12 mol% from the viewpoint of production of PHA having high flexibility and from the viewpoint of controlling the 3HH composition ratio.
More preferably, 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HB) as shown in the following chemical formula (2) (wherein m and n represent monomer units of the polymer and are integers of 1 or more) P (3HB-co-3HH) which is a copolymer having only 3HH) as a monomer unit.
本発明において、前記ＰＨＡを合成可能な宿主とは、ポリメラーゼ遺伝子やβ−ケトチオラーゼ遺伝子などのＰＨＡ合成関連遺伝子が元来発現し、酪酸及び／又はブタノールを炭素源に生育でき、その結果ＰＨＡを産生し得る微生物であれば特に限定はないが、天然から単離された微生物や、遺伝子操作された微生物等を使用できる。具体的にはラルストニア（Ｒａｌｓｔｏｎｉａ）属、カプリアビダス（Ｃｕｐｒｉａｖｉｄｕｓ）属、ワウテルシア（Ｗａｕｔｅｒｓｉａ）属、アルカリゲネス（Ａｌｃａｌｉｇｅｎｅｓ）属、シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属等の細菌類を使用することができる。安全性及び生産性の観点から好ましくはラルストニア属、カプリアビダス属、ワウテルシア属であり、本発明においてはＣｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒ（分類学上、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ、Ｗａｕｔｅｒｓｉａ ｅｕｔｒｏｐｈａと同一）を用いる。勿論、前記微生物を人工的突然変異処理して得られる変異株、遺伝子工学的手法により変異処理された類縁菌株も使用できる。 In the present invention, the host capable of synthesizing PHA originally expresses PHA synthesis-related genes such as a polymerase gene and β-ketothiolase gene, and can grow butyric acid and / or butanol on a carbon source, resulting in production of PHA. The microorganism is not particularly limited as long as it can be used, but a microorganism isolated from nature, a genetically engineered microorganism, and the like can be used. Specifically, bacteria such as Ralstonia genus, Capriavidus genus, Wautersia genus, Alcaligenes genus, Pseudomonas genus and the like can be used. From the viewpoint of safety and productivity, the genus Ralstonia, Capriavidas, and Wautercia are preferable, and in the present invention, Cupriavidus nector (taxonically identical to Ralstonia eutropha and Watersia eutropha) is used. Of course, mutant strains obtained by artificial mutation treatment of the above-mentioned microorganisms and related strains mutant-treated by genetic engineering techniques can also be used.
前記ポリメラーゼ遺伝子とは、Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ由来のｐｈａＣ遺伝子などが挙げられ、３−ヒドロキシヘキサノイルＣｏＡを基質とし３ＨＨ組成比の高いＰＨＡを産生させる観点からは、Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ由来のｐｈａＣ遺伝子が好ましい。さらにポリメラーゼ遺伝子としては、上記以外にＡｅｒｏｍｏｎａｓ ｐｕｎｃｔａｔａ由来ｐｈａＣ遺伝子、Ａｅｒｏｍｏｎａｓ ｈｙｄｒｏｐｈｉｌａ由来ｐｈａＣ遺伝子などが挙げられる。
本発明においてＣｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒを宿主として使用する場合、前記野生株は３−ヒロドキシヘキサノイルＣｏＡをＰＨＡへと変換させる事が殆どできない性質である為、Ｃｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒが持つＰＨＡ合成酵素遺伝子（ｐｈｂＣ遺伝子）よりも、３−ヒドロキシヘキサノイルＣｏＡをＰＨＡへと変換する能力の高い酵素を発現する遺伝子を、染色体への挿入したり、プラスミドベクターなどで導入する事で、細胞内にて合成された３−ヒドロキシヘキサノイルＣｏＡをポリ−３−ヒドロキシヘキサン酸（以下、Ｐ（３ＨＨ）とも記す）へと変換する効率を向上する様に操作している。Ｐ（３ＨＨ）へと変換する能力の高い酵素を発現する遺伝子としては、Ａｅｒｏｍｏｎａｓ ｃａｖｉａｅ由来のｐｈａＣ遺伝子が好ましい。
Examples of the polymerase gene include a phaC gene derived from Aeromonas caviae. From the viewpoint of producing PHA having a high 3HH composition ratio using 3-hydroxyhexanoyl CoA as a substrate, a phaC gene derived from Aeromonas caviae is preferable. Furthermore, examples of the polymerase gene include Aeromonas punctata-derived phaC gene, Aeromonas hydrophila-derived phaC gene and the like.
In the present invention, when Cupriavidus necator is used as a host, the wild strain has a property that 3-hydroxyhexanoyl CoA can hardly be converted into PHA. Therefore, the PHA synthase gene (phbC gene) possessed by Cupriavidus necator 3) synthesized in a cell by inserting a gene expressing an enzyme having a higher ability to convert 3-hydroxyhexanoyl CoA into PHA into a chromosome or introducing it with a plasmid vector or the like 3 -It operates to improve the efficiency of converting hydroxyhexanoyl CoA into poly-3-hydroxyhexanoic acid (hereinafter also referred to as P (3HH)). As a gene expressing an enzyme having a high ability to convert to P (3HH), the phaC gene derived from Aeromonas caviae is preferable.
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、Ｋｉｃｈｉｓｅ等、Ａｐｐｌ．Ｅｎｖｉｒｏｎ．Ｍｉｃｒｏｂｉｏｌ．，６８：２４１１−２４１９（２００２）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メラーゼ遺伝子としては、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）がより好ましい。 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, Kichise et al., Appl. Environ. Microbiol. 68: 2411-2419 (2002), 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 asparagine of the 171st amino acid A PHA synthase gene (D171G mutant gene) derived from Aeromonas caviae in which the acid is replaced with glycine, or a PHA synthase gene derived from Aeromonas caviae in which the above two amino acid substitutions are combined is preferably used. be able to. The polymerase gene used in the present invention is preferably one that encodes an enzyme or mutant derived from Aeromonas caviae, wherein Aeromonas 149 is substituted with serine for the 149th amino acid asparagine and glycine for the 171st amino acid aspartic acid. Those encoding a mutant enzyme derived from Cavier (N149S / D171G mutant) are more preferred.
また前記β−ケトチオラーゼ遺伝子とは、βケトチオラーゼ活性を持つタンパク質をコードする遺伝子であれば特に限定はないが、例えばｐｈｂＡ遺伝子、ｂｋｔＢ遺伝子などが挙げられる（Ｓ．ＳＬＡＴＥＲ等，Ｊ． ｏｆ Ｂａｃｔｅｒｉｏｌｏｇｙ，Ａｐｒ，ｐ．１９７９−１９８７，ｖｏｌ．１８０，Ｎｏ．８，１９９８）。
本発明においてＣｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒを使用する場合は、Ｃｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒが有する数々のβ−ケトチオラーゼ遺伝子の中で特にｐｈｂＡ遺伝子を不活性化する事によって、３ＨＨ比率をさらに高くすることができる。ｐｈｂＡ遺伝子を不活性化すると、ｐｈｂＡ遺伝子から発現されるアセトアセチルＣｏＡチオラーゼによって触媒されるアセチルＣｏＡの二量化によるアセトアセチルＣｏＡの生産を行う代謝経路を遮断する事ができるためと考える。アセトアセチルＣｏＡは３ＨＢのモノマー合成経路の中間体である事から、アセトアセチルＣｏＡの生産経路の１つを遮断する事で３ＨＢの比率が低下し、３ＨＨ組成比率が向上する。
The β-ketothiolase gene is not particularly limited as long as it is a gene encoding a protein having β-ketothiolase activity, and examples thereof include phbA gene and bktB gene (S. SLATER et al., J. of Bacteriology, Apr. , P. 1979-1987, vol. 180, No. 8, 1998).
In the case of using Cupriavidus necator in the present invention, the 3HH ratio can be further increased by inactivating the phbA gene among a number of β-ketothiolase genes possessed by Cupriavidus necator. It is thought that inactivation of the phbA gene can block a metabolic pathway that produces acetoacetyl CoA by dimerization of acetyl CoA catalyzed by acetoacetyl CoA thiolase expressed from the phbA gene. Since acetoacetyl CoA is an intermediate in the 3HB monomer synthesis pathway, blocking one of the acetoacetyl CoA production pathways reduces the 3HB ratio and improves the 3HH composition ratio.
β−ケトチオラーゼ遺伝子を不活性化するには、通常の変異処理方法が採用できる。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。 In order to inactivate the β-ketothiolase gene, a usual mutation treatment method can be 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).
β−ケトチオラーゼ遺伝子を特異的に不活性化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、目的遺伝子のみを不活性化させることができ、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 inactivating the β-ketothiolase gene, there is a chromosomal gene replacement method by genetic manipulation. According to this method, it is possible to inactivate only the target gene without damaging the gene other than the target gene, and this method is particularly preferable because the synthetic pathway other than the target gene is not changed. . 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.
β−ケトチオラーゼ遺伝子を特異的に不活性化させるには、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、ｐｈｂＡ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。
Ｃｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒの場合、配列番号２に示すｐｈｂＡ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１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１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。本発明においては、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性化するのがより好ましい。
Specific inactivation of the β-ketothiolase gene includes substitution, deletion or insertion of at least one base in the DNA sequence of the β-ketothiolase gene.
Examples of modifications that lack the activity expression of the β-ketothiolase gene include modifications that insert a heterologous gene, such as a kanamycin resistance gene fragment, into the β-ketothiolase gene, and a stop codon that is generated in the β-ketothiolase gene. Examples of such modifications include insertion, deletion or substitution of simple nucleotide sequences, and deletion of part or all of the β-ketothiolase gene.
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 modifications that insert, delete or substitute nucleotide sequences into the promoter and / or ribosome binding site of the phbA gene.
In the case of Cupriavidus necator, it is preferable to replace the 85th base adenine of the gene sequence shown in SEQ ID NO: 1 with thymine so that the lysine, which is the 16th amino acid of the phbA gene shown in SEQ ID NO: 2, is replaced with a stop codon. . 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: 1 You can also. In the present invention, it is more preferable to inactivate by substitution of a base sequence that causes a stop codon in the structural gene.
β−ケトチオラーゼ活性が欠損または減少するようなｐｈｂＡ遺伝子の改変によって、染色体上でｐｈｂＡ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。 Modification of the phbA gene such that the β-ketothiolase activity is deficient or decreased affects the expression of the acetoacetyl-CoA reductase gene existing downstream of the phbA gene on the chromosome and decreases the acetoacetyl-CoA reductase activity. In some cases, a microorganism obtained in this manner can also be suitably used as the microorganism of the present invention.
本発明に用い得る炭素源としては、酪酸及び／又はブタノールを必須とし、酪酸及び／又はブタノール以外の炭素源を含む方が好ましい。酪酸及び／又はブタノール以外の炭素源を全く含まないと、生産菌の増殖が阻害される場合がある。酪酸及び／又はブタノール以外に用いる炭素源としては、例えばグルコース、フラクトース等の糖類やメタノール、エタノールなどのアルコール類；酢酸、プロピオン酸、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、ミリスチン酸等の炭素数が６以上の脂肪酸類；或いはこれら脂肪酸のエステルや塩等の脂肪酸誘導体、乳酸、ヤシ油、パーム油、パーム核油（以下ＰＫＯＯ）等の植物油脂が挙げられ、それらの群より選ばれる少なくとも１種を酪酸及び／又はブタノールと共に用いることができる。酪酸及び／又はブタノールと、酪酸及び／又はブタノール以外の炭素源との併用が好ましく、油脂と酪酸の組合せ、又は油脂とブタノールの組合せがより好ましい。 As a carbon source that can be used in the present invention, it is preferable that butyric acid and / or butanol is essential and a carbon source other than butyric acid and / or butanol is included. If no carbon source other than butyric acid and / or butanol is contained, the growth of the producing bacteria may be inhibited. Examples of carbon sources used in addition to butyric acid and / or butanol include sugars such as glucose and fructose, alcohols such as methanol and ethanol; acetic acid, propionic acid, hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, and palmitic acid. Fatty acids having 6 or more carbon atoms such as acid, linoleic acid, linolenic acid, myristic acid; or fatty acid derivatives such as esters and salts of these fatty acids, lactic acid, coconut oil, palm oil, palm kernel oil (hereinafter PKOO), etc. A vegetable oil is mentioned, At least 1 sort (s) chosen from those groups can be used with a butyric acid and / or butanol. A combination of butyric acid and / or butanol and a carbon source other than butyric acid and / or butanol is preferable, and a combination of fat and butyric acid or a combination of fat and butanol is more preferable.
酪酸及び／又はブタノールと、酪酸及び／又はブタノール以外の炭素源との併用により培養する際の酪酸及び／又はブタノールの使用量としては、菌株が増殖し、ＰＨＡを合成できる範囲であれば特に限定は無いが、好ましくは酪酸及び／又はブタノール以外の炭素源１００重量部に対して５重量部〜１００重量部である。より好ましくは酪酸及び／又はブタノール以外の炭素源１００重量部に対して５重量部〜７０重量部である。さらに好ましくは酪酸及び／又はブタノール以外の炭素源１００重量部に対して５重量部〜４０重量部である。酪酸及び／又はブタノールの添加量が５重量部より少ないと、ＰＨＡ中の３ＨＨ組成比率が上がらない場合があり、１００重量部よりも多いと菌株が増殖しなくなる場合がある。そして少なくとも菌株の増殖に問題のない、酪酸及び／又はブタノール以外の炭素源１００重量部に対する酪酸及び／又はブタノールの使用量が５重量部〜４０重量部の範囲では、その使用量が多いほど３ＨＨ組成比は高くすることができる。
本発明においては、微生物細胞内の脂肪酸代謝経路であるβ酸化反応によって酪酸及び／又はブタノールから生産されたアセチルＣｏＡと、同じく酪酸及び／又はブタノールが代謝されて生産されたブチリルＣｏＡから、より多量の３−ヒロドキシヘキサノイルＣｏＡを生産し、それをＰ（３ＨＨ）へと変換する事によって、３ＨＨ組成比のより高いＰＨＡが産生されると考えられる。
The amount of butyric acid and / or butanol used when cultivated in combination with butyric acid and / or butanol and a carbon source other than butyric acid and / or butanol is particularly limited as long as the strain can grow and synthesize PHA. However, it is preferably 5 to 100 parts by weight with respect to 100 parts by weight of a carbon source other than butyric acid and / or butanol. More preferably, it is 5 to 70 parts by weight with respect to 100 parts by weight of a carbon source other than butyric acid and / or butanol. More preferably, it is 5 to 40 parts by weight with respect to 100 parts by weight of a carbon source other than butyric acid and / or butanol. If the amount of butyric acid and / or butanol added is less than 5 parts by weight, the 3HH composition ratio in PHA may not increase, and if it exceeds 100 parts by weight, the strain may not grow. And at least when the amount of butyric acid and / or butanol used is 5 to 40 parts by weight with respect to 100 parts by weight of a carbon source other than butyric acid and / or butanol, which does not cause any problem of growth of the strain, the more the amount used, 3HH The composition ratio can be increased.
In the present invention, a larger amount of acetyl CoA produced from butyric acid and / or butanol by β-oxidation reaction, which is a fatty acid metabolic pathway in microbial cells, and butyryl CoA produced by metabolizing butyric acid and / or butanol as well. It is considered that PHA having a higher composition ratio of 3HH is produced by producing 3-hydroxyhexanoyl CoA and converting it to P (3HH).
すなわち、本発明は、ＰＨＡ中の３ＨＢ組成比率を低下させ、３ＨＨ組成を向上させる事で、より柔軟性に富むＰＨＡを発酵生産する方法である。
さらにＰＨＡの製造方法について説明する。その方法は特に限定するわけではないが、例えば以下のようにして行う事ができる。本発明のＰＨＡの生産においては、油脂と、酪酸又はブタノールとを混合した混合物、或いは、油脂と、酪酸又はブタノールを別々に炭素源として与え、炭素源以外の栄養源である窒素源、無機塩類、そのほかの有機栄養源を含む培地を用いて、上記菌株を培養することができる。例えば、カプリアビダス（Ｃｕｐｒｉａｖｉｄｕｓ）属の微生物を宿主として得られた形質転換体を培養する培地として微生物が資化し得る炭素源を与え、場合によっては、窒素源、無機塩類および有機栄養源のうちのいずれかを制限した培地、例えば窒素源を０．０１から０．１％に制限した培地等を用いることができる。
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。また、培養液中に、発現プラスミドに存在する薬剤耐性遺伝子に対応する抗生物質（カナマイシン等）を添加しても良い。
本発明において、菌体からのＰＨＡの回収は、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機等で菌体を分離し、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。
得られたポリエステルの３ＨＨ組成（ｍｏｌ％）の分析は、ガスクロマトグラフ法や核磁気共鳴法などにより行うことができる。
That is, the present invention is a method for fermenting and producing more flexible PHA by reducing the 3HB composition ratio in PHA and improving the 3HH composition.
Furthermore, the manufacturing method of PHA is demonstrated. The method is not particularly limited, but can be performed, for example, as follows. In the production of PHA of the present invention, a mixture of oil and fat and butyric acid or butanol, or a fat and oil and butyric acid or butanol are separately provided as a carbon source, and a nitrogen source or inorganic salt that is a nutrient source other than the carbon source. The strain can be cultured using a medium containing other organic nutrient sources. For example, a carbon source that can be assimilated by a microorganism is provided as a medium for culturing a transformant obtained by using a microorganism of the genus Cupriavidus as a host, and in some cases, any of nitrogen sources, inorganic salts, and organic nutrient sources For example, a medium in which the nitrogen source is limited to 0.01 to 0.1% can be used.
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. Moreover, you may add antibiotics (kanamycin etc.) corresponding to the drug resistance gene which exists in an expression plasmid in a culture solution.
In the present invention, the recovery of PHA from the bacterial cells can be performed, for example, 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, or 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. Further, the supernatant is removed by filtration or centrifugation, and dried to recover the polyester.
Analysis of the 3HH composition (mol%) of the obtained polyester can be performed by gas chromatography, nuclear magnetic resonance, or the like.
本発明を用いれば、３ＨＢ及び３ＨＨを含有するＰＨＡにおいて、菌体中のポリマー含量を低下させずに、３ＨＢ組成に対する３ＨＨ組成が向上したＰＨＡの発酵生産方法を提供することができる。特に３ＨＨ組成が７ｍｏｌ％以上のＰ（３ＨＢ−ｃｏ−３ＨＨ）の発酵生産において、安全な生産菌であるＣｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒを用い、高価な脂肪酸を使用せず、ポリマー含量が６５重量％以上と高水準を維持し且つ３ＨＨ組成が上昇する様にコントロールできる。 If the present invention is used, a PHA containing 3HB and 3HH can be provided with a PHA fermentation production method in which the 3HH composition is improved relative to the 3HB composition without reducing the polymer content in the cells. In particular, in the fermentation production of P (3HB-co-3HH) having a 3HH composition of 7 mol% or more, a high-level polymer content of 65 wt% or more is used without using expensive fatty acids, using a safe producer, Cupriavidus necator. And can be controlled to increase the 3HH composition.
以下に実施例を示し、本発明をより具体的に説明するが、本発明はこれらの実施例に何ら限定されるものではない。なお全体的な遺伝子操作は、Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ（Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ、（１９８９））に記載されているように行うことができる。また、遺伝子操作に使用する酵素、クローニング宿主等は、市場の供給者から購入し、その説明に従い使用することができる。なお、酵素としては、遺伝子操作に使用できるものであれば特に限定されない。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. The overall genetic manipulation can be performed as described in Molecular Cloning (Cold Spring Harbor Laboratory Press, (1989)). In addition, enzymes, cloning hosts, etc. used for gene manipulation can be purchased from market suppliers and used according to the explanation. The enzyme is not particularly limited as long as it can be used for gene manipulation.
（実施例１） ＫＮＫ−００５株の作製
先ず、遺伝子置換用プラスミドの作製を行った。作製は以下のように行った。
Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株の菌体を鋳型ＤＮＡの供給源として、配列番号３と配列番号４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）。
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５と６に記載のプライマーのセット及び配列番号７と８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，ＰＥＲＫＩＮ 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。
(Example 1) Preparation of KNK-005 strain First, a plasmid for gene replacement was prepared. The production was performed as follows.
Using the cells of Ralstonia eutropha H16 strain as a template DNA source, PCR reaction was performed using the primers shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the polyhydroxyalkanoate synthase gene (phaC Re ) was 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 ).
The N149S / D171G mutant, which is a polyester synthase mutant gene derived from Aeromonas caviae, was prepared as follows. First, pBluescript IIKS (-) (Toyobo Co., Ltd.) was treated with PstI, blunt-ended using DNA Blunting Kit (Takara Bio), and ligated to prepare plasmid pBlue-New lacking the PstI 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: 5 and 6 and a primer set shown in SEQ ID NOs: 7 and 8 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 PERKIN 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９と配列番号１０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 performed using pBlue-N149S / D171G as a template and the primers represented by SEQ ID NO: 9 and SEQ ID NO: 10 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, the plasmid pMT5071 (Tsuda, GENE, 207: 33-41 (1998)) was treated with the 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.
次に、遺伝子置換株の作製を行った。
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、Ｒａｌｓｔｏｎｉａ 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Ｒａｌｓｔｏｎｉａ 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、ＰＥＲＫＩＮ 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１）。
Next, gene replacement strains were prepared.
Escherichia coli S17-1 strain (ATCC 47005) is transformed with the gene replacement plasmid pBlue-phaC Re :: N149S / D171G-KmSAC, and mixed and cultured on Ralstonia eutropha H16 strain and neutral Agar medium (manufactured by Difco). 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, 15 g / L agar, pH 6.8) was selected, and a strain in which the plasmid was integrated on the chromosome of Ralstonia eutropha H16 strain was obtained. 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 PERKIN ELMER APPLIED BIOSSYSTEMS, from the start codon to the stop codon of the phaC Re gene on the chromosome. It was confirmed that the strain was substituted from the start codon to the stop codon of the / D171G mutant gene (FIG. 1).
（実施例２）ＫＮＫ−００５ＡＳ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１１と配列番号１２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１３と配列番号４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）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１４で示される変異プライマーＰＨＢＡＳＴＯＰ２を用い、ＴａＫａＲａ ＬＡ ＰＣＲ ｉｎ ｖｉｔｒｏ Ｍｕｔａｇｅｎｅｓｉｓ Ｓｙｓｔｅｍ（タカラバイオ製）を利用して開始コドンから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 KNK-005AS strain <Preparation of plasmid for disruption of β-ketothiolase gene>
A PCR reaction was performed using the plasmid pJRD215 as a template and the primers represented by SEQ ID NO: 11 and SEQ ID NO: 12 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).
A DNA fragment containing a β-ketothiolase gene (phbA) of about 1.1 kbp by performing a PCR reaction using the KNK-005 strain cells as a template DNA source and using the primers shown in SEQ ID NO: 13 and SEQ ID NO: 4. 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 primer PHBASTOP2 shown in SEQ ID NO: 14, using the TaKaRa LA PCR in vitro Mutagenesis System (manufactured by Takara Bio), the 16th amino acid from the start codon becomes a stop codon, and at the same time a restriction enzyme NheI cleavage site is generated. Such base substitution was performed. 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２）。
<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 (FIG. 2).
＜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１、２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。
<Measurement of β-ketothiolase activity>
First, pre-culture medium (1w / v% Meat-extract , 1w / v% Bacto-Trypton, 0.2w / v% Yeast-extract, 0.9w / v% Na 2 HPO 4 · 12H 2 O, 0.15w / V% KH 2 PO 4 , (pH 6.8)) 5 ml of the enzyme activity measurement medium (1) after inoculating the strains obtained in Examples 1 and 2 in 5 ml and culturing at 30 ° C. overnight. 0.1 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, 0.5w / v% fructose, 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, 0.02w / v% CoCl 2 · 6 H 2 O, 0.016 w / v% CuSO 4 .5H 2 O, 0.012 w / v% NiCl 2 .6H 2 O dissolved in 0.05 ml)) and inoculated at 30 ° C. for 24 hours Cultured. 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.
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。
実施例１、２で作製した株のβ−ケトチオラーゼ活性を表１に示す。
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 β-ketothiolase activity of the strains prepared in Examples 1 and 2.
（実施例３） 酪酸添加培養
実施例１、２にて作製したＫＮＫ−００５株及び、ＫＮＫ−００５ＡＳ株の培養を行った。
培養は次の様に行った。前培地の組成は１ｗ／ｖ％Ｍｅａｔ−ｅｘｔｒａｃｔ、１ｗ／ｖ％Ｂａｃｔｏ−Ｔｒｙｐｔｏｎ、０．２ｗ／ｖ％Ｙｅａｓｔ−ｅｘｔｒａｃｔ、０．９ｗ／ｖ％Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、０．１５ｗ／ｖ％ＫＨ２ＰＯ４、ｐＨ６．７とした。ポリエステル生産培地の組成は１．１ｗ／ｖ％Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、０．１９ｗ／ｖ％ＫＨ２ＰＯ４、０．６ｗ／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％ＮｉＣｌ３・６Ｈ２Ｏ、０．０１ｗ／ｖ％ＣｒＣｌ３・６Ｈ２Ｏを溶かしたもの。）、炭素源はＰＫＯＯとＰＫＯＯ１００重量部に対して酪酸を２０から３０重量部にて混合して使用し、培養は炭素源を流加する流加培養にて行った。
ＫＮＫ−００５株及び、ＫＮＫ−００５ＡＳ株のグリセロールストックを前培地に接種して２０時間培養し、２．５Ｌの生産培地を入れた５Ｌジャーファーメンター（丸菱バイオエンジ製ＭＤＳ−Ｕ５０型）に１０ｖ／ｖ％接種した。運転条件は、培養温度２８℃、攪拌速度４２０ｒｐｍ、通気量０．６ｖｖｍとし、ｐＨは６．６から６．８の間でコントロールした。コントロールには１４％のアンモニア水を使用した。培養は５０時間まで行った。培養後遠心分離によって菌体を回収し、メタノールで洗浄後、凍結乾燥し、３ＨＨ組成比率を分析した。
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測定した。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。上記条件にて分析した結果、化学式（２）に示すような共重合体ポリエステル（３ＨＢ−Ｃｏ−３ＨＨ）であり、３ＨＨ比率は表２に示す。
(Example 3) Culture with addition of butyric acid The KNK-005 strain and the KNK-005AS strain prepared in Examples 1 and 2 were cultured.
The culture was performed as follows. The composition of the pre-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 / It was set to v% KH 2 PO 4 , pH 6.7. The composition of the polyester production medium is 1.1 w / v% Na 2 HPO 4 · 12H 2 O, 0.19 w / v% KH 2 PO 4 , 0.6 w / v% (NH 4 ) 2 SO 4 , 0.1 w / v% MgSO 4 .7H 2 O, 0.5 v / v% trace metal salt solution (1.6 W / v% FeCl 3 .6H 2 O in 0.1N hydrochloric acid, 1 w / 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 3 .6H 2 O, 0.01 w / v% CrCl 3 .6H 2 O The carbon source was used by mixing 20 to 30 parts by weight of butyric acid with 100 parts by weight of PKOO and PKOO, and the culture was performed by fed-batch culture in which the carbon source was fed.
KNK-005 strain and glycerol stock of KNK-005AS strain were inoculated into the previous medium, cultured for 20 hours, and placed in a 5L jar fermenter (MDS-U50 type, manufactured by Maruhishi Bioengineer) containing 2.5L of production medium. 10% v / v was inoculated. The operating conditions were a culture temperature of 28 ° C., a stirring speed of 420 rpm, an aeration rate of 0.6 vvm, and a pH controlled between 6.6 and 6.8. For control, 14% aqueous ammonia was used. Incubation was performed for up to 50 hours. After incubation, the cells were collected by centrifugation, washed with methanol, lyophilized, and analyzed for the 3HH composition ratio.
The 3HH composition analysis of the produced polyester was measured 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. As a result of analysis under the above conditions, it is a copolymer polyester (3HB-Co-3HH) as shown in chemical formula (2), and the 3HH ratio is shown in Table 2.
（比較例１） 酪酸を添加しない培養
実施例３と同じ培養条件にて、炭素源に酪酸を添加しない培養をＫＮＫ−００５株及びＫＮＫ−００５ＡＳ株について行った。３ＨＨ組成は表２に示す。
(Comparative example 1) Culture without adding butyric acid Under the same culture conditions as in Example 3, the culture without adding butyric acid to the carbon source was performed on the KNK-005 strain and the KNK-005AS strain. The 3HH composition is shown in Table 2.
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