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PROCESS FOR PRODUCTION OF POLYHYDROXYALKANOIC ACID USING GENETICALLY MODIFIED MICROORGANISM HAVING ENOYL-CoA HYDRATASE GENE INTRODUCED THEREIN

Foreign code F120006526
File No. S2010-0631-C0
Posted date May 8, 2012
Country WIPO
International application number 2011JP053861
International publication number WO 2011105379
Date of international filing Feb 22, 2011
Date of international publication Sep 1, 2011
Priority data
  • P2010-043017 (Feb 26, 2010) JP
Title PROCESS FOR PRODUCTION OF POLYHYDROXYALKANOIC ACID USING GENETICALLY MODIFIED MICROORGANISM HAVING ENOYL-CoA HYDRATASE GENE INTRODUCED THEREIN
Abstract Poly(3-hydroxybutanoic acid-co-3-hydroxyhexnoic acid) [P(3HB-co-3HHx)] having a high 3-hydroxyhexnoic acid fraction can be produced using a plant oil as a base raw material. Disclosed is a process for producing a microorganism capable of producing poly(3-hydroxybutanoic acid-co-3-hydroxyhexnoic acid) having a high 3-hydroxyhexnoic acid fraction using a plant oil as a base raw material, which comprises introducing a gene that encodes an R-hydratase capable of converting a β-oxidation intermediate for a fatty acid to an (R)-3-hydroxyacyl-CoA [R-3HA-CoA] (which is a monomer) into a genetically modified Cupriavidus necator strain imparted with a P(3HB-co-3HHx) production capability.
Outline of related art and contending technology BACKGROUND ART
Which is an essential material in the modern society petroleum synthetic plastic is inexpensive and easily processable on the other hand, when the non-degradable discarded because it is a question of processing. Therefore, many microbes polyhydroxyalkanes accumulates intracellularly as an energy source is acid (PHA), rather than petroleum biomass as a raw material, and in an environment with a low load type biodegradable plastic material as expected. Poly (3 - hydroxybutanoic acid) (herein 'P (3HB) ' abbreviated as.) Is the biosynthesis of many microbes in the exemplary PHA. However, the physical property (3HB) to be hard and brittle P for practical use is difficult.
On the other hand, Aeromonas, synthesized from vegetable oils such as by caviae (Aeromonas caviae) poly ((R) -3 - -3 - (R) - co - hydroxybutanoic acid hexanoic acid) copolymer P (3HB-co-3HHx) superior properties in flexibility. Further, the copolymer is, depending on the composition 3HHx from a rigid polymer properties in a wide range applicable up to soft polymer for, application of various applications can be expected (non-patent document 1).
Heretofore, Aeromonas, caviae is, vegetable oils as a raw material, the fraction of 3HHx (hexanoic acid) 9-13mol % P (3HB-co-3HHx) in may not be known. However, about 6-12 weight % fungus body accumulation rate low, they can be applied to the real production difficult (patent document 1, patent document 2, non-patent document 1). Therefore, Aeromonas, caviae-derived PHA synthase (polymerase) gene phaCAc P efficiently kupuriavidasu hydrogen producing bacteria (3HB) bacteria, of accumulation capacity nekatoru PHA (Cupriavidus necator) deficient strain PHB- 4can be introduced into the strain, from vegetable oils P (3HB-co-3HHx) (70-80 weight %) high accumulation rate in the past in recombinant strains produced by the present inventors. However, in this case is as low as 3HHx 4-5mol % fraction, also PHB- 4strain in cell lines and H16 wild type strain and a decrease in the capacity to proliferate is was a problem (patent document 3, non-patent document 2). A polymer having a practically useful flexibility in order to make the 3HHx fraction is preferably from 7-15mol %, high 3HHx fraction P (3HB-co-3HHx) accumulated efficiently, growth and the method for preparing a recombinant strain having a high ability have been searched. The inventors have found that phaCAc kupuriavidasu to, nekatoru H16 high by introducing into the chromosome of the proliferative capacity and high PHA productivity strains which were fabricated, the reduction of the 3HHx fraction was observed (non-patent document 3). Also Aeromonas, caviae-derived PHA synthase gene of the mutant enzymes phaCNSDG introduced in the chromosome, β - ketothiolase enzyme feeding in a monomer 3HB acetoacetyl - CoA reductase gene and inactivated disclosed method (patent document 4).
On the other hand, by the present inventors, Aeromonas, caviae in PHA from an analysis of the biosynthesis, the gene located immediately downstream of the PHA synthase phaJAc is, as an intermediate of fatty acid β - enoyl - CoA oxidation system to convert R-3HA-CoA enoyl CoA hydratase R-specific (or less, simply 'R - hydratase' is sometimes to be abbreviated as.) Encode (Fig. 1) revealed (patent document 3, non-patent document 4). This phaJAc and phaCAc E. coli by introducing both, does not have the ability to synthesize PHA original E. coli P fatty acid as a carbon source (3HB-co-3HHx) confer the ability to biosynthesis can be shown by the present inventors (non-patent document 5). In addition, Aeromonas, caviae in subsequent R- hydratase discovery, various microorganism and similar R- hydratase and its gene has been found, does not have the ability to synthesize PHA PHA polymerase suitable E. coli along with the gene can be introduced into the alkanoic acids having a carbon number of 4-6 or a copolymer comprising PHA, 3 - hydroxy alkanoic acid having a carbon number of 6-12 in long chain PHA copolymer can be used in the synthesis of the microorganisms shown in (non-patent document 6, 7 and 8, Patent Document 5). However when E. coli a fatty acid carbon source due to the low proliferative capacity of, in the example of these PHA lower productivity, the cost of the carbon source is also E. coli cannot be used vegetable oils was a problem.
On the other hand, the above-described kupuriavidasu, nekatoru PHB- 4in the creation of recombinant strains derived from the, phaCAc and phaJAc introduced to produce the various recombinant strains, octanoic acid and the carbon source has been studied biosynthesis PHA is, or 3HHx of these strains in the PHA accumulation rate and the fraction phaJAc and the introduction of a clear correlation was confirmed (patent document 3, non-patent document 9, 10). Therefore, kupuriavidasu, P nekatoru line was used in the biosynthesis (3HB-co-3HHx) is known about the effect of introducing R- hydratase gene is not. In addition, kupuriavidasu, is considered to have a hydratase nekatoru R- has not been reported.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 ポリ(3-ヒドロキシブタン酸-co-3-ヒドロキシヘキサン酸)生産能を付与した組換えクプリアヴィダス・ネカトール(Cupriavidus necator)株の染色体にR体特異的エノイルCoAヒドラターゼ遺伝子を相同性組換えによって形質転換し、又は前記株に該遺伝子が組み込まれた自律複製ベクターを導入することによって形質転換し、炭素源として植物油を含有する培地で形質転換体を増殖させることを含む、3-ヒドロキシヘキサン酸の分率が5~20モル%であり、及び形質転換体内蓄積率が50~90重量%であるポリ(3-ヒドロキシブタン酸-co-3-ヒドロキシヘキサン酸)を製造する方法。

[請求項2]
 前記組換えクプリアヴィダス・ネカトール株が、NSDG株、NSDGΔA株、又はMF01株であることを特徴とする、請求項1に記載の方法。

[請求項3]
 R体特異的エノイルCoAヒドラターゼ遺伝子が、アエロモナス・キャビエ(Aeromonas caviae)株由来であり、
(a)配列番号1で表される塩基配列を含む核酸;又は
(b)配列番号1で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつ脂肪酸β-酸化系中間体を(R)-3-ヒドロキシアシル-CoAに変換する活性を有するタンパク質をコードする核酸
からなる、請求項1又は2に記載の方法。

[請求項4]
 R体特異的エノイルCoAヒドラターゼ遺伝子が、クプリアヴィダス・ネカトール株由来であり、
(a)配列番号2で表される塩基配列を含む核酸;又は
(b)配列番号2で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつ脂肪酸β-酸化系中間体を(R)-3-ヒドロキシアシル-CoAに変換する活性を有するタンパク質をコードする核酸
からなる、請求項1又は2に記載の方法。

[請求項5]
 R体特異的エノイルCoAヒドラターゼ遺伝子が、クプリアヴィダス・ネカトール株由来であり、
(a)配列番号3で表される塩基配列を含む核酸;又は
(b)配列番号3で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつ脂肪酸β-酸化系中間体を(R)-3-ヒドロキシアシル-CoAに変換する活性を有するタンパク質をコードする核酸
からなる、請求項1又は2に記載の方法。

[請求項6]
 R体特異的エノイルCoAヒドラターゼ遺伝子が、
 - アエロモナス・キャビエ株由来であり、
 (a)配列番号1で表される塩基配列を含む核酸;又は
 (b)配列番号1で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつ脂肪酸β-酸化系中間体を(R)-3-ヒドロキシアシル-CoAに変換する活性を有するタンパク質をコードする核酸;及び
 - クプリアヴィダス・ネカトール株由来であり、
 (c)配列番号2で表される塩基配列を含む核酸;又は
 (d)配列番号2で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつ脂肪酸β-酸化系中間体を(R)-3-ヒドロキシアシル-CoAに変換する活性を有するタンパク質をコードする核酸
からなる、請求項1又は2に記載の方法。

[請求項7]
 R体特異的エノイルCoAヒドラターゼ遺伝子が、
 - アエロモナス・キャビエ株由来であり、
 (a)配列番号1で表される塩基配列を含む核酸;又は
 (b)配列番号1で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつ脂肪酸β-酸化系中間体を(R)-3-ヒドロキシアシル-CoAに変換する活性を有するタンパク質をコードする核酸;及び
 - クプリアヴィダス・ネカトール株由来であり、
 (c)配列番号3で表される塩基配列を含む核酸;又は
 (d)配列番号3で表される塩基配列を含む核酸とストリンジェントな条件下でハイブリダイズし、かつ脂肪酸β-酸化系中間体を(R)-3-ヒドロキシアシル-CoAに変換する活性を有するタンパク質をコードする核酸
からなる、請求項1又は2に記載の方法。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • Tokyo Institute of Technology
  • Inventor
  • FUKUI, Toshiaki
  • ORITA, Izumi
  • MIFUNE, Jun
  • KAWASHIMA, Yui
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PE PG PH PL PT RO RS RU SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ MD RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG
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