JP7425783B2 - Transformed microorganism and method for producing polyhydroxyalkanoic acid - Google Patents
Transformed microorganism and method for producing polyhydroxyalkanoic acid Download PDFInfo
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- JP7425783B2 JP7425783B2 JP2021501777A JP2021501777A JP7425783B2 JP 7425783 B2 JP7425783 B2 JP 7425783B2 JP 2021501777 A JP2021501777 A JP 2021501777A JP 2021501777 A JP2021501777 A JP 2021501777A JP 7425783 B2 JP7425783 B2 JP 7425783B2
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- 235000013930 proline Nutrition 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 101150025220 sacB gene Proteins 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 235000004400 serine Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229920001169 thermoplastic Polymers 0.000 description 1
- 239000004416 thermosoftening plastic Substances 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 235000008521 threonine Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000001132 ultrasonic dispersion Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
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Description
本発明は、ポリヒドロキシアルカン酸を生産可能な形質転換微生物、及び、当該形質転換微生物を用いたポリヒドロキシアルカン酸の製造方法に関する。 The present invention relates to a transformed microorganism capable of producing polyhydroxyalkanoic acid, and a method for producing polyhydroxyalkanoic acid using the transformed microorganism.
環境問題、食糧問題、健康及び安全に対する意識の高まり、天然又は自然志向の高まりなどを背景に、微生物を利用した物質製造（発酵生産、バイオ変換など）の意義及び重要性が益々高まっており、タンパク質医薬品や遺伝子治療用の核酸などの製造にも、微生物による物質生産が応用されている。例えば、酵母やバクテリアなどの微生物を利用したエタノール、酢酸、医療用タンパク質の生産などが活発に産業応用されている。 Against the background of environmental issues, food issues, increasing awareness of health and safety, and growing interest in natural products, the significance and importance of substance production using microorganisms (fermentation production, bioconversion, etc.) is increasing. Substance production using microorganisms is also applied to the production of protein drugs and nucleic acids for gene therapy. For example, the production of ethanol, acetic acid, and medical proteins using microorganisms such as yeast and bacteria is actively being applied industrially.
その一例として、生分解性プラスチックとしての産業利用が期待されているポリヒドロキシアルカン酸（以下、ＰＨＡともいう）の微生物による生産が挙げられる（非特許文献１を参照）。ＰＨＡは、多くの微生物種の細胞にエネルギー蓄積物質として産生、蓄積される熱可塑性ポリエステルであり、生分解性を有している。現在、環境への意識の高まりから非石油由来のプラスチックが注目されるなか、特に、微生物が菌体内に産生、蓄積するＰＨＡは、自然界の炭素循環プロセスに取り込まれることから生態系への悪影響が小さいと予想されており、その実用化が切望されている。微生物を利用したＰＨＡ生産では、例えば、カプリアビダス属細菌に炭素源として糖、植物油脂や脂肪酸を与え、細胞内にＰＨＡを蓄積させることでＰＨＡを生産することが知られている（非特許文献２及び３を参照）。 One example is the production by microorganisms of polyhydroxyalkanoic acid (hereinafter also referred to as PHA), which is expected to be used industrially as a biodegradable plastic (see Non-Patent Document 1). PHA is a thermoplastic polyester that is produced and accumulated as an energy storage substance in the cells of many microbial species, and is biodegradable. Currently, non-petroleum-based plastics are attracting attention due to growing environmental awareness. In particular, PHA, which is produced and accumulated within the cells of microorganisms, is incorporated into the carbon cycle process in the natural world and has no negative impact on the ecosystem. It is expected to be small, and its practical application is eagerly awaited. In PHA production using microorganisms, it is known that, for example, PHA is produced by feeding Capriavidus bacteria with sugar, vegetable oil, or fatty acid as a carbon source and accumulating PHA within the cells (Non-patent Document 2). and 3).
しかしながら、微生物を利用した物質生産においては、目的生産物の分離回収工程が煩雑となり、生産コストが高くなることが問題になるケースがある。従って、目的生産物の分離回収効率を向上させることは、生産コストの低減のための大きな課題である。 However, in the production of substances using microorganisms, there are cases in which the separation and recovery process of the target product becomes complicated and the production cost becomes high. Therefore, improving the separation and recovery efficiency of target products is a major challenge for reducing production costs.
非特許文献４には、カプリアビダス属細菌においてフェイシンタンパク質をコードする遺伝子ｐｈａＰ１を破壊することで、非破壊の場合より大粒子径のＰＨＡを蓄積したことが報告されており、これを目的生産物の分離回収効率を改善する方法として採用することが考えられる。しかし、該ｐｈａＰ１破壊株はＰＨＡ蓄積量が著しく減少することが示されており、工業生産に適したものではなかった。 Non-Patent Document 4 reports that by destroying the gene phaP1 encoding the phasin protein in Capriavidus bacteria, PHA with a larger particle size than in the case of non-destruction was accumulated, and this was used to produce the target product. It is considered that this method can be adopted as a method to improve the separation and recovery efficiency of However, the phaP1-disrupted strain was shown to have a markedly reduced amount of accumulated PHA, and was not suitable for industrial production.
ＰＨＡは微生物細胞内において粒子状に蓄積される。微生物細胞内に蓄積されたＰＨＡを生分解性プラスチックとして利用するためには、細胞を破砕してＰＨＡ粒子を取り出し、他の細胞成分から分離し、回収する。分離回収の手法は、大きくは有機溶媒系による方法と水系による方法に分けられるが、有機溶媒の使用は高環境負荷、高コストとなるため、工業的には水系による方法が好ましい。水系による方法では、例えば、ＰＨＡ粒子を含む細胞破砕液から、遠心分離機や分離膜等によってＰＨＡ粒子を分離することができる。このような場合、分離回収の効率はＰＨＡ粒子の大きさに依存することになる。即ち、微生物細胞内に蓄積されたＰＨＡ粒子が大きいほど、遠心分離機や分離膜等を用いた分離回収を容易に実施でき、生産コストの低減につながる。 PHA accumulates in particulate form within microbial cells. In order to utilize PHA accumulated in microbial cells as a biodegradable plastic, the cells are crushed to extract PHA particles, separated from other cell components, and recovered. Separation and recovery methods can be broadly divided into organic solvent-based methods and aqueous-based methods, but since the use of organic solvents results in high environmental impact and high cost, the aqueous-based methods are preferred from an industrial perspective. In the aqueous method, for example, PHA particles can be separated from a cell disruption solution containing PHA particles using a centrifuge, a separation membrane, or the like. In such a case, the efficiency of separation and recovery will depend on the size of the PHA particles. That is, the larger the PHA particles accumulated in microbial cells, the easier it is to separate and recover them using a centrifuge, separation membrane, etc., leading to a reduction in production costs.
本発明は、上記現状に鑑み、大粒子径のＰＨＡを蓄積可能な形質転換微生物、及び、当該形質転換微生物を用いたＰＨＡの製造方法を提供することを課題とする。 In view of the above-mentioned current situation, it is an object of the present invention to provide a transformed microorganism capable of accumulating PHA with a large particle size, and a method for producing PHA using the transformed microorganism.
本発明者らは鋭意検討した結果、カプリアビダス属細菌において機能未知とされているＡ２３６５遺伝子（例えば配列番号１に記載のアミノ酸配列をコードする遺伝子）の発現を強化することで、工業的に望ましいＰＨＡ蓄積量を維持しながら、微生物細胞内に蓄積されるＰＨＡの粒子径を拡大できることを見出し、本発明に至った。 As a result of extensive studies, the present inventors have found that by enhancing the expression of the A2365 gene (for example, the gene encoding the amino acid sequence shown in SEQ ID NO: 1), which is said to have an unknown function in bacteria of the genus Capriavidus, an industrially desirable PHA can be produced. It was discovered that the particle size of PHA accumulated in microbial cells can be expanded while maintaining the accumulated amount, leading to the present invention.
すなわち本発明は、ポリヒドロキシアルカン酸合成酵素遺伝子を有し、Ａ２３６５遺伝子の発現が強化された、形質転換微生物に関する。前記形質転換微生物は、カプリアビダス属に属することが好ましく、カプリアビダス・ネカトールの形質転換微生物であることがより好ましい。
また本発明は、前記形質転換微生物を、炭素源の存在下で培養する工程を含む、ポリヒドロキシアルカン酸の製造方法にも関する。炭素源は、油脂あるいは脂肪酸を含有してもよく、また、糖を含有してもよく、二酸化炭素を含有してもよい。ポリヒドロキシアルカン酸は、２種以上のヒドロキシアルカン酸の共重合体であることが好ましく、３－ヒドロキシヘキサン酸をモノマーユニットとして含有する共重合体であることがより好ましく、３－ヒドロキシ酪酸と３－ヒドロキシヘキサン酸との共重合体であることがさらに好ましい。
That is, the present invention relates to a transformed microorganism that has a polyhydroxyalkanoic acid synthase gene and has enhanced expression of the A2365 gene. The transformed microorganism preferably belongs to the genus Capriavidus, and is more preferably a transformed microorganism of Capriavidus necator.
The present invention also relates to a method for producing polyhydroxyalkanoic acid, which includes the step of culturing the transformed microorganism in the presence of a carbon source. The carbon source may contain fats and oils or fatty acids, sugar, or carbon dioxide. The polyhydroxyalkanoic acid is preferably a copolymer of two or more hydroxyalkanoic acids, more preferably a copolymer containing 3-hydroxyhexanoic acid as a monomer unit, and 3-hydroxybutyric acid and 3-hydroxybutyric acid. - A copolymer with hydroxyhexanoic acid is more preferable.
本発明によれば、大粒子径のＰＨＡを蓄積可能な形質転換微生物、及び、当該形質転換微生物を用いたＰＨＡの製造方法を提供することができる。本発明によると、微生物細胞内に大粒子径のＰＨＡ粒子が蓄積されるため、細胞破砕後に細胞成分からのＰＨＡの分離回収が容易となり、生産コストの低減を実現することができる。 According to the present invention, it is possible to provide a transformed microorganism capable of accumulating PHA with a large particle size, and a method for producing PHA using the transformed microorganism. According to the present invention, since large-diameter PHA particles are accumulated within microbial cells, PHA can be easily separated and recovered from cell components after cell disruption, and production costs can be reduced.
以下、本発明の実施形態を詳細に説明する。
本発明に係る形質転換微生物は、ＰＨＡ合成酵素遺伝子を有し、Ａ２３６５遺伝子の発現が強化された、形質転換微生物である。
Embodiments of the present invention will be described in detail below.
The transformed microorganism according to the present invention has a PHA synthase gene and has enhanced expression of the A2365 gene.
（微生物）
本発明に係る形質転換微生物は、ＰＨＡ合成酵素遺伝子を有し、かつ、Ａ２３６５遺伝子の発現が強化されるように形質転換された微生物である（以下、Ａ２３６５遺伝子発現強化株ともいう）。本発明に係る形質転換微生物の宿主は、ＰＨＡ合成酵素遺伝子を有する微生物であれば特に限定されない。当該細菌としては、例えば、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、アエロモナス（Ａｅｒｏｍｏｎａｓ）属、エシェリキア（Ｅｓｃｈｅｒｉｃｈｉａ）属、アルカリゲネス（Ａｌｃａｌｉｇｅｎｅｓ）属、シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属等に属する細菌類が好ましい例として挙げられる。安全性及びＰＨＡ生産性の観点から、より好ましくはラルストニア属、カプリアビダス属、アエロモナス属、ワウテルシア属に属する細菌であり、さらに好ましくはカプリアビダス属又はアエロモナス属に属する細菌であり、さらにより好ましくはカプリアビダス属に属する微生物であり、特に好ましくはカプリアビダス・ネカトール（Ｃｕｐｒｉａｖｉｄｕｓ ｎｅｃａｔｏｒ）である。
(microorganisms)
The transformed microorganism according to the present invention is a microorganism that has a PHA synthase gene and has been transformed so that the expression of the A2365 gene is enhanced (hereinafter also referred to as an A2365 gene expression-enhanced strain). The host of the transformed microorganism according to the present invention is not particularly limited as long as it has a PHA synthase gene. Examples of the bacteria include the genus Ralstonia, the genus Cupriavidus, the genus Wautersia, the genus Aeromonas, the genus Escherichia, the genus Alcaligenes, and the genus Pseudomonas. omonas) etc. Preferred examples include bacteria belonging to the same group. From the viewpoints of safety and PHA productivity, bacteria belonging to the genus Ralstonia, Capriavidus, Aeromonas, and Wautersia are more preferred, bacteria belonging to the genus Capriavidus or Aeromonas are even more preferred, and even more preferred are bacteria belonging to the genus Capriavidus. A particularly preferred microorganism is Cupriavidus necator.
本発明に係る形質転換微生物の宿主は、ＰＨＡ合成酵素遺伝子を本来的に有する野生株であってもよいし、そのような野生株を人工的に突然変異処理して得られる変異株や、あるいは、遺伝子工学的手法により外来のＰＨＡ合成酵素遺伝子が導入された菌株であってもよい。外来のＰＨＡ合成酵素遺伝子を導入する方法は特に限定されず、宿主の染色体上に遺伝子を直接挿入または置換する方法、宿主が保有するメガプラスミド上に遺伝子を直接挿入または置換する方法、あるいはプラスミド、ファージ、ファージミドなどのベクター上に遺伝子を配置して導入する方法などが選択でき、これらの方法のうち２つ以上を併用しても良い。導入遺伝子の安定性を考慮すると、好ましくは、宿主の染色体上または宿主が保有するメガプラスミド上に遺伝子を直接挿入または置換する方法であり、より好ましくは、宿主の染色体上に遺伝子を直接挿入または置換する方法である。 The host of the transformed microorganism according to the present invention may be a wild strain that inherently has the PHA synthase gene, a mutant strain obtained by artificially mutating such a wild strain, or It may also be a strain into which a foreign PHA synthase gene has been introduced by genetic engineering techniques. The method of introducing the foreign PHA synthase gene is not particularly limited, and may include a method of directly inserting or replacing the gene onto the chromosome of the host, a method of directly inserting or replacing the gene onto a megaplasmid carried by the host, or a plasmid. Methods such as placing genes on vectors such as phages and phagemids for introduction can be selected, and two or more of these methods may be used in combination. Considering the stability of the introduced gene, the preferred method is to insert or replace the gene directly onto the chromosome of the host or onto the megaplasmid possessed by the host. More preferably, the method involves directly inserting or substituting the gene onto the chromosome of the host. This is a method of replacement.
（ＰＨＡ合成酵素遺伝子）
ＰＨＡ合成酵素遺伝子としては特に限定されないが、ラルストニア属、カプリアビダス属、ワウテルシア属、アルカリゲネス属、アエロモナス属、シュードモナス属、ノルカディア属、クロモバクテリウム属に類する生物に由来するＰＨＡ合成酵素遺伝子や、それらの改変体などが挙げられる。前記改変体としては、１以上のアミノ酸残基が欠失、付加、挿入、又は置換されたＰＨＡ合成酵素をコードする塩基配列などを用いることができる。例えば、配列番号２～６のいずれかに記載のアミノ酸配列で示されるポリペプチドをコードする塩基配列を有する遺伝子、及び、該アミノ酸配列に対して８５％以上の配列相同性を有するアミノ酸配列で示され、かつＰＨＡ合成酵素活性を有するポリペプチドをコードする塩基配列を有する遺伝子などが挙げられる。上記配列相同性としては好ましくは９０％以上、より好ましくは９５％以上、さらに好ましくは９７％以上、特に好ましくは９９％以上である。
(PHA synthase gene)
PHA synthase genes are not particularly limited, but include PHA synthase genes derived from organisms similar to the genus Ralstonia, Capriavidus, Wautersia, Alcaligenes, Aeromonas, Pseudomonas, Norcadia, and Chromobacterium; Examples include variants. As the variant, a base sequence encoding a PHA synthase in which one or more amino acid residues are deleted, added, inserted, or substituted can be used. For example, a gene having a nucleotide sequence encoding a polypeptide represented by the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 6, and an amino acid sequence having 85% or more sequence homology to the amino acid sequence. and a gene having a base sequence encoding a polypeptide having PHA synthase activity. The above sequence homology is preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more.
（ＰＨＡ）
本発明のＡ２３６５遺伝子発現強化株が生産するＰＨＡの種類としては、微生物が生産し得るＰＨＡである限り特に限定されないが、炭素数４～１６の３－ヒドロキシアルカン酸から選択される１種のモノマーの単独重合体、炭素数４～１６の３－ヒドロキシアルカン酸から選択される１種のモノマーとその他のヒドロキシアルカン酸（例えば、炭素数４～１６の２－ヒドロキシアルカン酸、４－ヒドロキシアルカン酸、５－ヒドロキシアルカン酸、６－ヒドロキシアルカン酸など）の共重合体、及び、炭素数４～１６の３－ヒドロキシアルカン酸から選択される２種以上のモノマーの共重合体が好ましい。例えば、３－ヒドロキシ酪酸（略称：３ＨＢ）のホモポリマーであるＰ（３ＨＢ）、３ＨＢと３－ヒドロキシ吉草酸（略称：３ＨＶ）の共重合体Ｐ（３ＨＢ－ｃｏ－３ＨＶ）、３ＨＢと３－ヒドロキシヘキサン酸（略称：３ＨＨ）の共重合体Ｐ（３ＨＢ－ｃｏ－３ＨＨ）（略称：ＰＨＢＨ）、３ＨＢと４－ヒドロキシ酪酸（略称：４ＨＢ）の共重合体Ｐ（３ＨＢ－ｃｏ－４ＨＢ）、乳酸（略称：ＬＡ）を構成成分として含むＰＨＡ、例えば３ＨＢとＬＡの共重合体Ｐ（ＬＡ－ｃｏ－３ＨＢ）などが挙げられるが、これらに限定されない。この中でも、ポリマーとしての応用範囲が広いという観点から、ＰＨＢＨが好ましい。なお、生産されるＰＨＡの種類は、目的に応じて、使用する微生物の保有するあるいは別途導入されたＰＨＡ合成酵素遺伝子の種類や、その合成に関与する代謝系の遺伝子の種類、培養条件などによって適宜選択しうる。
(PHA)
The type of PHA produced by the A2365 gene expression-enhanced strain of the present invention is not particularly limited as long as it is a PHA that can be produced by microorganisms, but one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms is used. A homopolymer of 3-hydroxyalkanoic acid having 4 to 16 carbon atoms and other hydroxyalkanoic acids (for example, 2-hydroxyalkanoic acid and 4-hydroxyalkanoic acid having 4 to 16 carbon atoms) , 5-hydroxyalkanoic acid, 6-hydroxyalkanoic acid, etc.), and a copolymer of two or more monomers selected from 3-hydroxyalkanoic acid having 4 to 16 carbon atoms. For example, P(3HB) is a homopolymer of 3-hydroxybutyric acid (abbreviation: 3HB), P(3HB-co-3HV) is a copolymer of 3HB and 3-hydroxyvaleric acid (abbreviation: 3HV), Copolymer P (3HB-co-3HH) of hydroxyhexanoic acid (abbreviation: 3HH) (abbreviation: PHBH), copolymer P (3HB-co-4HB) of 3HB and 4-hydroxybutyric acid (abbreviation: 4HB), Examples include, but are not limited to, PHA containing lactic acid (abbreviation: LA) as a constituent, such as a copolymer P of 3HB and LA (LA-co-3HB). Among these, PHBH is preferred from the viewpoint of a wide range of applications as a polymer. The type of PHA produced depends on the purpose, the type of PHA synthase gene possessed by the microorganism used or separately introduced, the type of metabolic system gene involved in its synthesis, culture conditions, etc. Can be selected as appropriate.
（Ａ２３６５遺伝子）
本発明で発現が強化されるＡ２３６５遺伝子は、配列番号１に記載のアミノ酸配列で示されるポリペプチド（ＵｎｉＰｒｏｔＫＢ ＩＤ Ｑ０Ｋ９６１）、及び、該アミノ酸配列に対して８５％以上の配列相同性を有するアミノ酸配列で示されるポリペプチドをコードする塩基配列を有する遺伝子である。上記配列相同性としては好ましくは９０％以上、より好ましくは９５％以上、さらに好ましくは９７％以上、特に好ましくは９９％以上である。なお、Ａ２３６５遺伝子が有する機能は未だ報告されていない。
(A2365 gene)
The A2365 gene whose expression is enhanced in the present invention is a polypeptide represented by the amino acid sequence set forth in SEQ ID NO: 1 (UniProtKB ID Q0K961), and an amino acid sequence having 85% or more sequence homology to the amino acid sequence. This is a gene having a base sequence encoding a polypeptide shown in The above sequence homology is preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more. Note that the function of the A2365 gene has not yet been reported.
（遺伝子発現強化）
本発明における遺伝子発現の強化とは、対象遺伝子の発現が強化されていない菌株と比較して、対象遺伝子の転写量または対象遺伝子のコードするポリペプチドの発現量が増加している状態を指す。その増加量は特に限定されないが、対象遺伝子の発現が強化されていない菌株と比較して１倍超であればよく、好ましくは１．１倍以上、より好ましくは１．２倍以上、さらに好ましくは１．５倍以上、さらにより好ましくは２倍以上の増加である。
(enhanced gene expression)
Enhanced gene expression in the present invention refers to a state in which the amount of transcription of the target gene or the amount of expression of the polypeptide encoded by the target gene is increased compared to a strain in which the expression of the target gene is not enhanced. The amount of increase is not particularly limited, but it may be more than 1 times, preferably 1.1 times or more, more preferably 1.2 times or more, even more preferably 1.2 times or more, compared to the strain in which the expression of the target gene is not enhanced. is an increase of 1.5 times or more, even more preferably 2 times or more.
本発明において、Ａ２３６５遺伝子の発現を強化する方法は特に限定されないが、対象遺伝子を宿主に導入する方法、宿主がゲノムＤＮＡ上に元来有する対象遺伝子の発現量を増強する方法、またはその両方を選択することができる。 In the present invention, the method for enhancing the expression of the A2365 gene is not particularly limited, but may include a method of introducing the target gene into the host, a method of enhancing the expression level of the target gene that the host originally has on the genomic DNA, or both. You can choose.
対象遺伝子を宿主に導入する方法としては特に限定されないが、宿主の染色体上に対象遺伝子を直接挿入または置換する方法、宿主が保有するメガプラスミド上に対象遺伝子を直接挿入または置換する方法、あるいはプラスミド、ファージ、ファージミドなどのベクター上に対象遺伝子を配置して導入する方法などが選択でき、これらの方法のうち２つ以上を併用しても良い。 Methods for introducing the target gene into the host include, but are not limited to, direct insertion or replacement of the target gene onto the chromosome of the host, direct insertion or replacement of the target gene onto a megaplasmid possessed by the host, or a plasmid. Methods of introducing the target gene by placing it on a vector such as phage, phagemid, etc. can be selected, and two or more of these methods may be used in combination.
導入遺伝子の安定性を考慮すると、好ましくは、宿主の染色体上または宿主が保有するメガプラスミド上に対象遺伝子を直接挿入または置換する方法であり、より好ましくは、宿主の染色体上に対象遺伝子を直接挿入または置換する方法である。導入する遺伝子を確実に発現させるために、対象遺伝子が、宿主が元来有する「遺伝子発現調節配列」の下流に位置するように導入するか、または、対象遺伝子が、外来の「遺伝子発現調節配列」の下流に位置する形で導入することが好ましい。本発明における「遺伝子発現調節配列」とは、その遺伝子の転写量を制御する塩基配列（例えばプロモーター配列）、及び／または、その遺伝子から転写されたメッセンジャーＲＮＡの翻訳量を調節する塩基配列（例えばシャイン・ダルガノ配列）を含むＤＮＡ配列である。「遺伝子発現調節配列」としては、自然界に存在する任意の塩基配列を利用することもできるし、人工的に構築または改変された塩基配列を利用しても良い。 Considering the stability of the introduced gene, the preferred method is to insert or replace the target gene directly onto the chromosome of the host or onto the megaplasmid possessed by the host, and more preferably, to insert or replace the target gene directly onto the chromosome of the host. This is a method of inserting or replacing. In order to ensure the expression of the introduced gene, the target gene is introduced so that it is located downstream of the "gene expression regulatory sequence" that the host originally has, or the target gene is introduced so that it is located downstream of the "gene expression regulatory sequence" that the host originally has. It is preferable to introduce it in a downstream position. In the present invention, a "gene expression regulatory sequence" refers to a base sequence that controls the amount of transcription of the gene (e.g. promoter sequence) and/or a base sequence that controls the amount of translation of messenger RNA transcribed from the gene (e.g. This is a DNA sequence containing the Shine-Dalgarno sequence. As the "gene expression regulatory sequence", any naturally occurring base sequence may be used, or an artificially constructed or modified base sequence may be used.
また、宿主がゲノムＤＮＡ上に元来有する対象遺伝子の発現量を増強する方法としては特に限定されないが、対象遺伝子の上流に位置する「遺伝子発現調節配列」を改変する方法、対象遺伝子の上流に外来の「遺伝子発現調節配列」を導入する方法、あるいは、対象遺伝子及び／またはその周辺の塩基配列を改変することにより、転写されたメッセンジャーＲＮＡの安定性を向上させる方法などが挙げられる。 In addition, methods for enhancing the expression level of the target gene that the host originally has on the genomic DNA are not particularly limited, but methods that include modifying the "gene expression regulatory sequence" located upstream of the target gene, Examples include a method of introducing a foreign "gene expression regulatory sequence" and a method of improving the stability of transcribed messenger RNA by modifying the target gene and/or its surrounding base sequence.
「遺伝子発現調節配列」に含まれるプロモーター配列やシャイン・ダルガノ配列としては、例えば、配列番号７～１３のいずれかに示される塩基配列、または、これら塩基配列の一部を含む塩基配列などが挙げられるが、特に限定されない。 Examples of promoter sequences and Shine-Dalgarno sequences included in the "gene expression regulatory sequence" include base sequences shown in any of SEQ ID NOs: 7 to 13, or base sequences containing part of these base sequences. However, there are no particular limitations.
ゲノムＤＮＡの少なくとも一部の置換、欠失、挿入及び／又は付加は、当業者に周知の方法により行うことができる。代表的な方法としてはトランスポゾンと相同組換えの機構を利用した方法（Ohman et al., J. Bacteriol., 162:1068-1074 (1985)）や、相同組換えの機構によって起こる部位特異的な組み込みと第二段階の相同組換えによる脱落を原理とした方法（Noti et al., Methods Enzymol., 154:197-217 (1987)）などがある。また、Bacillus subtilis由来のsacB遺伝子を共存させて、第二段階の相同組換えによって遺伝子が脱落した微生物株をスクロース耐性株として容易に単離する方法（Schweizer, Mol. Microbiol., 6:1195-1204 (1992)、Lenz et al., J. Bacteriol., 176:4385-4393 (1994)）も利用することができる。さらに別の方法として、標的ＤＮＡを改変するためのＣＲＩＳＰＲ／Ｃａｓ９システムによるゲノム編集技術（Y. Wang et al., ACS Synth Biol. 2016, 5(7):721-732）も利用することができる。ＣＲＩＳＰＲ／Ｃａｓ９システムでは、ガイドＲＮＡ（ｇＲＮＡ）は改変すべきゲノムＤＮＡの塩基配列の一部に結合しうる配列を有しており、Ｃａｓ９を標的に運ぶ役割をもつ。 Substitution, deletion, insertion, and/or addition of at least a portion of genomic DNA can be performed by methods well known to those skilled in the art. Typical methods include methods that utilize transposons and the homologous recombination mechanism (Ohman et al., J. Bacteriol., 162:1068-1074 (1985)), and site-specific methods that occur using the homologous recombination mechanism. There are methods based on the principle of integration and elimination through homologous recombination in the second step (Noti et al., Methods Enzymol., 154:197-217 (1987)). In addition, a method of coexisting the sacB gene derived from Bacillus subtilis and easily isolating a microbial strain in which the gene has been dropped by the second step of homologous recombination as a sucrose-resistant strain (Schweizer, Mol. Microbiol., 6:1195- 1204 (1992), Lenz et al., J. Bacteriol., 176:4385-4393 (1994)). As another method, genome editing technology using the CRISPR/Cas9 system (Y. Wang et al., ACS Synth Biol. 2016, 5(7):721-732) can also be used to modify the target DNA. . In the CRISPR/Cas9 system, guide RNA (gRNA) has a sequence that can bind to a part of the base sequence of the genomic DNA to be modified, and has the role of transporting Cas9 to the target.
細胞へのベクターの導入方法としても特に限定されないが、例えば、塩化カルシウム法、エレクトロポレーション法、ポリエチレングリコール法、スフェロプラスト法等が挙げられる。
Methods for introducing vectors into cells are not particularly limited, but include, for example, the calcium chloride method, electroporation method, polyethylene glycol method, and spheroplast method.
本発明のＡ２３６５遺伝子発現強化株を培養することで、菌体内にＰＨＡを蓄積させることができる。本発明のＡ２３６５遺伝子発現強化株を培養する方法としては、常法の微生物培養法に従うことができ、適切な炭素源が存在する培地中で培養を行なえばよい。培地組成、炭素源の添加方法、培養スケール、通気攪拌条件や、培養温度、培養時間などは特に限定されない。炭素源は、連続的に、または間欠的に培地に添加することが好ましい。 By culturing the A2365 gene expression-enhanced strain of the present invention, PHA can be accumulated within the bacterial cells. The A2365 gene expression-enhanced strain of the present invention can be cultured by a conventional microbial culture method, and may be cultured in a medium containing an appropriate carbon source. There are no particular limitations on the medium composition, carbon source addition method, culture scale, aeration/agitation conditions, culture temperature, culture time, etc. Preferably, the carbon source is added to the medium continuously or intermittently.
培養時の炭素源としては、本発明のＡ２３６５遺伝子発現強化株が資化可能であればどのような炭素源でも使用可能である。特に限定されないが、例えば、グルコース、フルクトース、シュークロースなどの糖類；パーム油やパーム核油（これらを分別した低融点分画であるパームオレイン、パームダブルオレイン、パーム核油オレインなども含む）、コーン油、やし油、オリーブ油、大豆油、菜種油、ヤトロファ油などの油脂やその分画油類、あるいはその精製副産物；ラウリン酸、オレイン酸、ステアリン酸、パルミチン酸、ミリンスチン酸などの脂肪酸やそれらの誘導体、あるいはグリセロール等が挙げられる。また、本発明のＡ２３６５遺伝子発現強化株が二酸化炭素、一酸化炭素、メタン、メタノール、エタノールなどのガスやアルコール類を利用可能である場合、これらを炭素源として使用することもできる。 As a carbon source during culture, any carbon source can be used as long as it can be assimilated by the A2365 gene expression-enhanced strain of the present invention. Examples include, but are not limited to, sugars such as glucose, fructose, and sucrose; palm oil and palm kernel oil (including low-melting fractions such as palm olein, palm double olein, and palm kernel oil olein); Fats and oils such as corn oil, coconut oil, olive oil, soybean oil, rapeseed oil, and jatropha oil, their fractionated oils, or their refining byproducts; fatty acids such as lauric acid, oleic acid, stearic acid, palmitic acid, myrinstic acid, etc. or glycerol. Furthermore, if the A2365 gene expression-enhanced strain of the present invention can utilize gases and alcohols such as carbon dioxide, carbon monoxide, methane, methanol, and ethanol, these can also be used as carbon sources.
本発明におけるＰＨＡの製造では、上記炭素源、炭素源以外の栄養源である窒素源、無機塩類、その他の有機栄養源を含む培地を用いて、前記微生物を培養することが好ましい。下記に限定されないが、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；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２水素カリウム、リン酸水素２ナトリウム、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。その他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。 In the production of PHA in the present invention, it is preferable to culture the microorganism using a medium containing the carbon source, a nitrogen source that is a nutrient source other than the carbon source, inorganic salts, and other organic nutrient sources. Examples of the nitrogen source include, but are not limited to, ammonia; ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate; peptone, meat extract, yeast extract, and the like. Examples of the inorganic salts include potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride. Examples of other organic nutritional sources include amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C.
培養を適切な時間行なって菌体内にＰＨＡを蓄積させた後、周知の方法を用いて菌体からＰＨＡを回収する。回収方法については特に限定されないが、例えば、培養終了後、培養液から遠心分離機等で菌体を分離し、乾燥させた後、乾燥菌体から、クロロホルム等の有機溶剤を用いてＰＨＡを抽出し、このＰＨＡを含んだ有機溶剤溶液から濾過等によって細胞成分を除去し、その濾液にメタノールやヘキサン等の貧溶媒を加えてＰＨＡを沈殿させ、濾過や遠心分離によって上澄み液を除去し、乾燥させてＰＨＡを回収することができる。また、界面活性剤やアルカリ、酵素などを用いてＰＨＡ以外の細胞成分を水に溶解させた後、濾過や遠心分離によってＰＨＡ粒子を水相から分離し乾燥させて回収することもできる。本発明により製造され得る大粒径のＰＨＡは、このような水系による分離回収が容易に実施できるため好ましい。 After culturing is carried out for an appropriate period of time to accumulate PHA within the bacterial cells, PHA is recovered from the bacterial cells using a well-known method. The recovery method is not particularly limited, but for example, after the culture is completed, the bacterial cells are separated from the culture solution using a centrifuge, dried, and then PHA is extracted from the dried bacterial cells using an organic solvent such as chloroform. Then, cell components are removed from this PHA-containing organic solvent solution by filtration, etc., a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA, the supernatant is removed by filtration or centrifugation, and then dried. PHA can be recovered by Alternatively, after dissolving cellular components other than PHA in water using a surfactant, alkali, enzyme, etc., PHA particles can be separated from the aqueous phase by filtration or centrifugation, dried, and recovered. PHA with a large particle size that can be produced according to the present invention is preferable because it can be easily separated and recovered using an aqueous system.
以下、実施例により本発明をさらに具体的に説明する。ただし、本発明は、これら実施例に限定されるものではない。なお全体的な遺伝子操作は、例えば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。 Hereinafter, the present invention will be explained in more detail with reference to Examples. However, the present invention is not limited to these Examples. The overall genetic manipulation can be performed, for example, as described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)). Furthermore, enzymes, cloning hosts, etc. used for genetic manipulation can be purchased from commercial suppliers and used according to their instructions. Note that the enzyme is not particularly limited as long as it can be used for genetic manipulation.
（製造例）Ａ２３６５遺伝子発現強化株の作製
まず、Ａ２３６５遺伝子発現用プラスミドｐＣＵＰ２－ｔｒｃ－Ａ２３６５の作製を行った。作製は以下のように行った。
(Production Example) Preparation of A2365 Gene Expression Enhanced Strain First, a plasmid pCUP2-trc-A2365 for A2365 gene expression was prepared. The preparation was performed as follows.
合成オリゴＤＮＡを用いたＰＣＲにより、カプリアビダス・ネカトールＨ１６株のゲノムＤＮＡを鋳型として、Ａ２３６５遺伝子配列を有するＤＮＡ断片（配列番号１４）を得た。このＤＮＡ断片を制限酵素ＭｕｎＩおよびＳｐｅＩで消化し、得られたＤＮＡ断片を、国際公開２００７／０４９７１６号に記載のプラスミドベクターｐＣＵＰ２をＭｕｎＩおよびＳｐｅＩで切断したものと連結して、プラスミドベクターｐＣＵＰ２－Ａ２３６５を得た。 By PCR using synthetic oligo DNA, a DNA fragment (SEQ ID NO: 14) having the A2365 gene sequence was obtained using the genomic DNA of Capriavidus necator H16 strain as a template. This DNA fragment was digested with restriction enzymes MunI and SpeI, and the resulting DNA fragment was ligated with the plasmid vector pCUP2 described in WO 2007/049716 cut with MunI and SpeI to create plasmid vector pCUP2-A2365. I got it.
さらに、合成オリゴＤＮＡを用いたＰＣＲにより、大腸菌に由来するプロモーター配列を有するＤＮＡ断片（配列番号１５）を得た。このＤＮＡ断片を制限酵素ＥｃｏＲＩおよびＭｕｎＩで消化し、得られたＤＮＡ断片を、プラスミドベクターｐＣＵＰ２－Ａ２３６５をＭｕｎＩで切断したものと連結した。得られたプラスミドベクターから、Ａ２３６５遺伝子配列がプロモーター配列の下流側に位置する向きで連結されたプラスミドベクターを選別し、Ａ２３６５遺伝子発現用プラスミドベクターｐＣＵＰ２－ｔｒｃ－Ａ２３６５とした。 Furthermore, a DNA fragment (SEQ ID NO: 15) having a promoter sequence derived from E. coli was obtained by PCR using synthetic oligo DNA. This DNA fragment was digested with restriction enzymes EcoRI and MunI, and the resulting DNA fragment was ligated to plasmid vector pCUP2-A2365 cut with MunI. From the obtained plasmid vectors, a plasmid vector in which the A2365 gene sequence was ligated in an orientation located downstream of the promoter sequence was selected and designated as a plasmid vector for A2365 gene expression pCUP2-trc-A2365.
次に、Ａ２３６５遺伝子発現用プラスミドベクターｐＣＵＰ２－ｔｒｃ－Ａ２３６５をＫＮＫ－００５株に導入して、Ａ２３６５遺伝子発現強化株を得た。ＫＮＫ００５株は、カプリアビダス・ネカトールＨ１６株の染色体上にアエロモナス・キャビエ由来のＰＨＡ合成酵素遺伝子（配列番号４に記載のアミノ酸配列を有するＰＨＡ合成酵素をコードする遺伝子）が導入された形質転換体であり、米国特許第７３８４７６６号明細書に記載の方法に準じて作成することができる。 Next, the plasmid vector pCUP2-trc-A2365 for A2365 gene expression was introduced into the KNK-005 strain to obtain a strain with enhanced A2365 gene expression. The KNK005 strain is a transformant in which a PHA synthase gene derived from Aeromonas caviae (a gene encoding a PHA synthase having the amino acid sequence set forth in SEQ ID NO: 4) has been introduced onto the chromosome of the Capriavidus necator H16 strain. , according to the method described in US Pat. No. 7,384,766.
プラスミドベクターの細胞への導入は以下のように電気導入によって行った。遺伝子導入装置はＢｉｏｒａｄ社製のジーンパルサーを用い、キュベットは同じくＢｉｏｒａｄ社製のｇａｐ０．２ｃｍを用いた。キュベットに、コンピテント細胞４００μｌと発現ベクター２０μｌを注入してパルス装置にセットし、静電容量２５μＦ、電圧１．５ｋＶ、抵抗値８００Ωの条件で電気パルスをかけた。パルス後、キュベット内の菌液をＮｕｔｒｉｅｎｔＢｒｏｔｈ培地（ＤＩＦＣＯ社製）で３０℃、３時間振とう培養し、選択プレート（ＮｕｔｒｉｅｎｔＡｇａｒ培地（ＤＩＦＣＯ社製）、カナマイシン１００ｍｇ／Ｌ）で、３０℃にて２日間培養して、生育してきたＡ２３６５遺伝子発現強化株を取得した。 The plasmid vector was introduced into cells by electrotransfer as described below. Gene Pulser manufactured by Biorad was used as the gene transfer device, and a gap 0.2 cm cuvette also manufactured by Biorad was used as the cuvette. 400 μl of competent cells and 20 μl of expression vector were injected into a cuvette, set in a pulse device, and an electric pulse was applied under the conditions of a capacitance of 25 μF, a voltage of 1.5 kV, and a resistance value of 800 Ω. After the pulse, the bacterial solution in the cuvette was cultured with shaking at 30°C for 3 hours in Nutrient Broth medium (manufactured by DIFCO), and incubated at 30°C for 2 hours on a selection plate (Nutrient Agar medium (manufactured by DIFCO), kanamycin 100 mg/L). After culturing for days, a growing A2365 gene expression-enhanced strain was obtained.
（比較例）ＫＮＫ－００５株によるＰＨＡ生産
下記の条件でＫＮＫ－００５株を用いた培養検討を行なった。
(Comparative Example) PHA production using KNK-005 strain A culture study using KNK-005 strain was conducted under the following conditions.
（培地）
種母培地の組成は１ｗ／ｖ％ Ｍｅａｔ－ｅｘｔｒａｃｔ、１ｗ／ｖ％ Ｂａｃｔｏ－Ｔｒｙｐｔｏｎｅ、０．２ｗ／ｖ％ Ｙｅａｓｔ－ｅｘｔｒａｃｔ、０．９ｗ／ｖ％ Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ 、０．１５ｗ／ｖ％ ＫＨ２ＰＯ４、（ｐＨ６．８）とした。
(Culture medium)
The composition of the seed medium is 1w/v% Meat-extract, 1w/v% Bacto-Tryptone, 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).
前培養培地の組成は１．１ｗ／ｖ％ Ｎａ２ＨＰＯ４・１２Ｈ２Ｏ、０．１９ｗ／ｖ％ＫＨ２ＰＯ４、１．２９ ｗ／ｖ％（ＮＨ４）２ＳＯ４ 、０．１ｗ／ｖ％ ＭｇＳＯ４・７Ｈ２Ｏ、２．５ｗ／ｖ％ パームオレインオイル、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。炭素源としてパームオレインオイルを１０ｇ／Ｌの濃度で一括添加した。 The composition of the preculture medium is 1.1w/v% Na 2 HPO 4 .12H 2 O, 0.19w/v% KH 2 PO 4 , 1.29 w/v% (NH 4 ) 2 SO 4 , 0.1w /v% MgSO 4.7H 2 O, 2.5w/v% Palm olein oil, 0.5v/v% Trace metal salt solution (1.6w/v% FeCl 3.6H 2 O, 1w in 0.1N hydrochloric acid) Dissolve /v% CaCl 2.2H 2 O, 0.02w/v% CoCl 2.6H 2 O, 0.016w/v% CuSO 4.5H 2 O, 0.012w/v% NiCl 2.6H 2 O It was made into Palm olein oil was added at once at a concentration of 10 g/L as a carbon source.
ＰＨＡ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）とした。 The composition of the PHA production medium is 0.385w/v% Na2HPO4.12H2O , 0.067w/v% KH2PO4 , 0.291w/v% ( NH4 ) 2SO4 , 0.1w /v%. v% MgSO 4 ·7H 2 O, 0.5 v/v% trace metal salt solution (1.6 w/v% FeCl 3 ·6H 2 O, 1 w/v% CaCl 2 ·2H 2 O, 0 in 0.1 N hydrochloric acid) .02w/v% CoCl2.6H2O , 0.016w/v % CuSO4.5H2O , and 0.012w/v % NiCl2.6H2O ) .
（ＰＨＡ蓄積量割合の測定方法）
ＰＨＡ蓄積量の割合は次のように測定した。遠心分離によって培養液から菌体を回収、エタノールで洗浄、凍結乾燥し、乾燥菌体を取得し、重量を測定した。得られた乾燥菌体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ＰＨＡ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３０ｍｌになるまで濃縮後、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ＰＨＡをろ別後、５０℃で３時間真空乾燥した。乾燥ＰＨＡの重量を測定し、乾燥菌体量に対してＰＨＡ蓄積量が占める割合を算出した。
(Method for measuring PHA accumulation ratio)
The rate of PHA accumulation was measured as follows. Bacterial cells were collected from the culture medium by centrifugation, washed with ethanol, freeze-dried, and dried to obtain dry cells, and their weight was measured. 100 ml of chloroform was added to 1 g of the obtained dried bacterial cells, and the mixture was stirred at room temperature all day and night to extract PHA within the bacterial cells. After removing the bacterial cell residue by filtration, the mixture was concentrated using an evaporator to a total volume of 30 ml, and 90 ml of hexane was gradually added thereto, and the mixture was allowed to stand for 1 hour while being slowly stirred. After filtering off the precipitated PHA, it was vacuum dried at 50° C. for 3 hours. The weight of the dried PHA was measured, and the ratio of the accumulated amount of PHA to the amount of dried bacterial cells was calculated.
（ＰＨＡ粒子径の測定方法）
ＰＨＡ粒子径は次のように測定した。培養後の培養液を６５℃で６０分間処理し、菌体細胞不活化を行った後、３．３ｗ／ｖ％ ドデシル硫酸ナトリウム水溶液により１５０倍に希釈し、超音波破砕によりＰＨＡ抽出液を得た。超音波破砕にはＳＭＴ社製超音波分散機ＵＨ?６００を用い、最大出力で４０秒、４回の処理を行った。得られたＰＨＡ抽出液をレーザー回折・散乱式粒子径分布測定装置（Ｍｉｃｒｏｔｒａｃ ＭＴ３３００ＥＸＩＩ）により解析し、ＰＨＡ粒子の体積平均径（ＭＶ）を測定した。測定は標準的な設定（粒子透過性：透過、粒子屈折率：１．８１、粒子形状：非球形、溶媒屈折率：１．３３３）で行った。
(Method for measuring PHA particle size)
The PHA particle size was measured as follows. After culturing, the culture solution was treated at 65°C for 60 minutes to inactivate the bacterial cells, and then diluted 150 times with a 3.3 w/v% sodium dodecyl sulfate aqueous solution, and a PHA extract was obtained by ultrasonic disruption. Ta. For ultrasonic crushing, an ultrasonic dispersion machine UH-600 manufactured by SMT was used, and the treatment was performed four times for 40 seconds at maximum output. The obtained PHA extract was analyzed using a laser diffraction/scattering particle size distribution analyzer (Microtrac MT3300EXII) to measure the volume average diameter (MV) of the PHA particles. Measurements were performed with standard settings (particle permeability: transmission, particle refractive index: 1.81, particle shape: non-spherical, solvent refractive index: 1.333).
（ＰＨＡ生産培養）
ＰＨＡ生産培養は次のように行った。まず、ＫＮＫ－００５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時間培養し種母培養を行なった。次に種母培養液を、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３３℃、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、前培養を行なった。ｐＨコントロールには１４％水酸化アンモニウム水溶液を使用した。
(PHA production culture)
PHA production culture was performed as follows. First, a glycerol stock (50 μl) of the KNK-005 strain was inoculated into a seed medium (10 ml) and cultured for 24 hours to perform seed culture. Next, the seed mother culture solution was inoculated at 1.0 v/v% into a 3 L jar fermenter (model MDL-300 manufactured by Marubishi Bioengineering) containing 1.8 L of preculture medium. The operating conditions were a culture temperature of 33° C., a stirring speed of 500 rpm, and an aeration rate of 1.8 L/min. Preculture was performed by culturing for 28 hours while controlling the pH between 6.7 and 6.8. A 14% ammonium hydroxide aqueous solution was used for pH control.
次に、前培養液を、２．５ＬのＰＨＡ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５．０ｖ／ｖ％接種した。運転条件は、培養温度３３℃、攪拌速度４２０ｒｐｍ、通気量２．１Ｌ／ｍｉｎとし、ｐＨは６．７～６．８の間でコントロールした。ｐＨコントロールには２５％水酸化アンモニウム水溶液を使用した。炭素源は断続的に添加した。炭素源としてはパームオレインオイルを使用した。培養は、乾燥菌体量に対するＰＨＡ蓄積量の割合が９０％程度に達するまで行った。ＰＨＡ蓄積量の割合およびＰＨＡ粒子径は前述のように測定した。結果を表１に示す。 Next, the preculture solution was inoculated at 5.0 v/v% into a 5 L jar fermenter (model MDS-U50 manufactured by Marubishi Bioengineering) containing 2.5 L of PHA production medium. The operating conditions were a culture temperature of 33° C., a stirring speed of 420 rpm, and an aeration rate of 2.1 L/min, and the pH was controlled between 6.7 and 6.8. A 25% ammonium hydroxide aqueous solution was used for pH control. The carbon source was added intermittently. Palm olein oil was used as a carbon source. Cultivation was continued until the ratio of PHA accumulation amount to dry bacterial cell amount reached approximately 90%. The percentage of PHA accumulation and PHA particle size were measured as described above. The results are shown in Table 1.
（実施例）Ａ２３６５遺伝子発現強化株によるＰＨＡ生産
比較例と同様の条件でＡ２３６５遺伝子発現強化株を用いた培養検討を行なった。ＰＨＡ蓄積量の割合およびＰＨＡ粒子径の測定結果を表１に示す。
(Example) PHA production using the A2365 gene expression-enhanced strain A culture study using the A2365 gene expression-enhanced strain was conducted under the same conditions as in the comparative example. Table 1 shows the measurement results of the percentage of PHA accumulation and the PHA particle size.
培養検討の結果、Ａ２３６５遺伝子発現強化株によって生産されたＰＨＡの粒子径は、Ａ２３６５遺伝子の発現を強化していない株（ＫＮＫ－００５株）によって生産されたＰＨＡの粒子径と比較して、増大が認められた。 As a result of culture studies, the particle size of PHA produced by the A2365 gene expression-enhanced strain was increased compared to the PHA particle size produced by the strain (KNK-005 strain) in which A2365 gene expression was not enhanced. was recognized.
なお、比較例および実施例の培養検討によって生産されたＰＨＡはＰＨＢＨであることをＨＰＬＣ分析にて確認した。 In addition, it was confirmed by HPLC analysis that the PHA produced by the culture studies in Comparative Examples and Examples was PHBH.
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| 佐藤 俊輔ほか，微生物による生分解性ポリマーPHBH製造法の開発，生物工学会誌，2019年02月25日，Vol. 97，No. 2，pp. 66-74 |
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