JPS6221510B2 - - Google Patents
Info
- Publication number
- JPS6221510B2 JPS6221510B2 JP4336184A JP4336184A JPS6221510B2 JP S6221510 B2 JPS6221510 B2 JP S6221510B2 JP 4336184 A JP4336184 A JP 4336184A JP 4336184 A JP4336184 A JP 4336184A JP S6221510 B2 JPS6221510 B2 JP S6221510B2
- Authority
- JP
- Japan
- Prior art keywords
- plasmid
- anaerobic
- psh62
- thermoanaerobacter
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 239000013612 plasmid Substances 0.000 claims description 25
- 241000186339 Thermoanaerobacter Species 0.000 claims description 11
- 108091008146 restriction endonucleases Proteins 0.000 claims description 11
- 238000003776 cleavage reaction Methods 0.000 claims description 7
- 230000007017 scission Effects 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 description 14
- 102000004190 Enzymes Human genes 0.000 description 14
- 229940088598 enzyme Drugs 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000007221 ypg medium Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 229930193140 Neomycin Natural products 0.000 description 3
- 201000004283 Shwachman-Diamond syndrome Diseases 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 229960004927 neomycin Drugs 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 241000588624 Acinetobacter calcoaceticus Species 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 241000606768 Haemophilus influenzae Species 0.000 description 2
- 241000606766 Haemophilus parainfluenzae Species 0.000 description 2
- 235000013878 L-cysteine Nutrition 0.000 description 2
- 239000004201 L-cysteine Substances 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 229940047650 haemophilus influenzae Drugs 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- QHYHLOUVASDBGV-UHFFFAOYSA-N CCO.CCOC(=O)OC(=O)OCC Chemical compound CCO.CCOC(=O)OC(=O)OCC QHYHLOUVASDBGV-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 102000002265 Human Growth Hormone Human genes 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- VHWAMQKROBKDQY-UHFFFAOYSA-N sporogen Natural products O=C1C2(C(C)=O)OC2C2(C)C(C)C(O)CCC2=C1 VHWAMQKROBKDQY-UHFFFAOYSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【発明の詳細な説明】
本発明は嫌気性好熱菌を宿主とする組換え
DNA実験のベクターとして有用な新規なプラス
ミドを保有する新規な微生物に関するものであ
り、より詳しくはその分子量が約3.3メガダルト
ンであり、図に示される制限酵素開裂地図により
特徴づけられる新規なプラスミドを保有する新規
なサーモアナエロバクターに関するものである。[Detailed Description of the Invention] The present invention relates to a recombinant method using an anaerobic thermophilic bacterium as a host.
This article concerns a novel microorganism that possesses a novel plasmid that is useful as a vector for DNA experiments, and more specifically, a novel plasmid that has a molecular weight of approximately 3.3 megadaltons and is characterized by the restriction enzyme cleavage map shown in the figure. This relates to the new Thermoanaerobacter.
従来、組換えDNA実験は主として大腸菌を宿
主とする系で広く研究がおこなわれインシユリ
ン、インターフエロン、ヒト成長ホルモン等が大
腸菌で量産されるなど大きな成果を挙げている。
大腸菌の宿主・ベクター系はほぼ完成されてお
り、また大腸菌以外にも酵母、枯草菌などで宿
主・ベクター系が開発され応用への道が検討され
つつある。 Up until now, recombinant DNA experiments have been widely conducted mainly using E. coli as a host system, and great results have been achieved, such as the mass production of insulin, interferon, human growth hormone, etc. using E. coli.
The host-vector system for E. coli has almost been completed, and other host-vector systems have been developed for yeast, Bacillus subtilis, etc., and ways to apply them are being considered.
50〜80℃の高温条件下でセルロース、ヘミセル
ロース、デンプン等を分解し、アルコール類、有
機酸、メタン等を生産する嫌気性好熱菌は、バイ
オマス変換の効率的生体触媒として、あるいは耐
熱性、安定性に優れる有用酵素の供給源として注
目を集めている。従つて、嫌気性好熱性細菌の育
種が重要と考えられるが、その為の一つの、しか
も有力な手段と考えられる嫌気性好熱性細菌の宿
主・ベクター系の開発研究は、これまで全く行な
われていない。しかも、ベクターの開発研究の基
礎となるべきプラスミドDNAの検索という点に
ついても、嫌気性好熱菌を材料とした研究は全く
知られていない。そこで、本発明者らは、嫌気性
好熱菌より、選択マーカー(そのプラスミドが宿
主内に存在していることを示すマーカー)を有
し、しかも分子量の小さいプラスミドの検索を行
つた。その結果ネオマイシン耐性を示したサーモ
アナエロバクターから分子量約3.3メガダルトン
のプラスミドを単離する事に成功した。 Anaerobic thermophilic bacteria that decompose cellulose, hemicellulose, starch, etc. under high-temperature conditions of 50 to 80°C to produce alcohols, organic acids, methane, etc. can be used as efficient biocatalysts for biomass conversion, or as heat-resistant, It is attracting attention as a source of useful enzymes with excellent stability. Therefore, breeding of anaerobic thermophilic bacteria is considered to be important, but research to develop a host-vector system for anaerobic thermophilic bacteria, which is considered to be one of the effective means for this purpose, has not been conducted to date. Not yet. Moreover, there is no known research using anaerobic thermophilic bacteria as a material for searching for plasmid DNA, which should form the basis of vector development research. Therefore, the present inventors searched for a plasmid from anaerobic thermophilic bacteria that has a selection marker (a marker indicating that the plasmid is present in the host) and has a small molecular weight. As a result, we successfully isolated a plasmid with a molecular weight of approximately 3.3 megadaltons from Thermoanaerobacter that showed neomycin resistance.
このプラスミドは、前記の制限酵素開裂地図に
示される如く、分子量が小さくしかも数種の制限
酵素による切断点を特異的に有している(以下、
本プラスミドをpSH62と略称する)。 As shown in the above-mentioned restriction enzyme cleavage map, this plasmid has a small molecular weight and has specific cleavage points for several types of restriction enzymes (hereinafter referred to as
This plasmid is abbreviated as pSH62).
なお、図に示されている制限酵素の略称は次の
とおりである。 The abbreviations of the restriction enzymes shown in the figure are as follows.
EcoRはエシヤリシア・コリ由来の酵素、
Hpaはハエモフイルス・パラインフルエンザエ
由来の酵素、Claはカリオフアノン・ラツム由
来の酵素、Accはアシネトバクター・カルコア
セテイカス由来の酵素、Bglはバチルス・グロ
ビギイ由来の酵素、Hind はハエモフイル
ス・インフルエンザ由来の酵素をそれぞれ示して
いる。 EcoR is an enzyme derived from Eschialsia coli,
Hpa is an enzyme derived from Haemophilus parainfluenzae, Cla is an enzyme derived from Karyophanon ratum, Acc is an enzyme derived from Acinetobacter calcoaceticus, Bgl is an enzyme derived from Bacillus globigii, and Hind is an enzyme derived from Haemophilus influenzae. It shows.
プラスミドDNAがベクターたり得る為には、
そのプラスミドが宿主内での自律的増殖能、及び
選択マーカー(そのプラスミドが宿主内に存在し
ていることを示すマーカー)を有していることが
必須である。しかし、嫌気性好熱菌の様に、その
生育環境が栄養源に乏しくしかも抗生物質が存在
しない温泉等である菌について考えた場合、薬剤
耐性遺伝子等を有するプラスミドを得る事は容易
ではない。従つて、性質が不明のいわゆるクリプ
テイツク・プラスミドに宿主染色体由来のマーカ
ーを賦与するという方式でベクター開発を行わな
ければならないであろう。その際にpSH62を利用
すれば、極めて便利であるものと考えられる。何
故ならば、第1にpSH62は嫌気性好熱菌で複製が
可能なプラスミドであるからであり、第2には、
小さい分子量を有するという点から、本プラスミ
ドの必須領域、例えば複製開始点領域、複製に関
与する遺伝子等の解析が、容易に行えるという利
点を有しているからである。 In order for plasmid DNA to be used as a vector,
It is essential that the plasmid has the ability to autonomously reproduce within the host and a selection marker (a marker indicating that the plasmid is present within the host). However, when considering bacteria such as anaerobic thermophiles, whose growth environment is hot springs and the like with poor nutritional sources and no antibiotics, it is not easy to obtain plasmids containing drug-resistant genes. Therefore, vector development will have to be carried out by providing a marker derived from the host chromosome to a so-called cryptographic plasmid whose properties are unknown. It would be extremely convenient to use pSH62 in this case. This is because, firstly, pSH62 is a plasmid that can replicate in anaerobic thermophiles, and secondly,
This is because, since it has a small molecular weight, it has the advantage that essential regions of the plasmid, such as the replication origin region, genes involved in replication, etc., can be easily analyzed.
更にpSH62は図からも明らかなように、EcoR
,Bglなどの制限酵素による開裂部位を特定
のしかも限られた位置に有している。このことは
pSH62をベクターとして利用する際に、挿入すべ
き異種遺伝子の導入部位を有意に保持できるとい
う点で有利である。 Furthermore, as is clear from the figure, pSH62 is an EcoR
It has cleavage sites for restriction enzymes such as , Bgl, etc. at specific and limited positions. This thing is
When pSH62 is used as a vector, it is advantageous in that it can significantly retain the introduction site for the heterologous gene to be inserted.
本プラスミドをベクターとして用いることによ
り、バイオマス変換効率が高く、有用化成品を生
産する嫌気性好熱菌の育種が可能となろう。 By using this plasmid as a vector, it will become possible to breed anaerobic thermophiles that have high biomass conversion efficiency and produce useful chemical products.
pSH62の入手は、本発明者らが土壌中から新た
に分離した嫌気性好熱菌、サーモアナエロバクタ
ーNo.62をYPG培地(デイフコ・イーストエキス
トラクト0.2%、ポリペプトン(大五栄養)0.4
%、NaCl0.2%、グルコース1%、Na2CO30.3
%、L−システイン0.2%、PH7.5)により対数増
殖後期迄増殖させて得た菌体を、リゾチーム、
SDS処理によつて溶菌させる事によつて達せられ
るが、本プラスミドを保有する点で本菌株は新規
である。 pSH62 was obtained by growing Thermoanaerobacter No. 62, an anaerobic thermophile newly isolated from soil, in YPG medium (Difco Yeast Extract 0.2%, Polypeptone (Daigo Nutrients) 0.4%).
%, NaCl 0.2%, Glucose 1%, Na 2 CO 3 0.3
%, L-cysteine 0.2%, PH7.5) until the late logarithmic growth stage.
This can be achieved by lysing the bacteria with SDS treatment, but this strain is novel in that it possesses this plasmid.
また、サーモアナエロバクターNo.62の菌学的性
質を表に示すがpSH62を保有する点では従来には
認められない新規な微生物である。本菌株はネオ
マイシン耐性株として土壌中より分離されたもの
である。 In addition, the mycological properties of Thermoanaerobacter No. 62 are shown in the table, and it is a novel microorganism that has not been previously recognized in that it possesses pSH62. This bacterial strain was isolated from soil as a neomycin-resistant strain.
表
(分類学的性質)
生育至適温度;66℃
生育温度範囲;50〜74℃
形 態;周鞭毛を有する桿菌0.3〜0.4×1〜
4μm
胞子形成体;な し
グラム染色;不 定
GC含量;34%
主要生産物;エタノール、酢酸、乳酸
糖の利用性;グルコース、ラクトース、マルトー
ス、マンノース、キシロース、セロ
ビオース、デンプン
以上の菌学的性質から「アーチブ オブ マイ
クロバイオロジー(Archives of
Microbiology),128−129,Bd.,1980−81」によ
り検索したところ、サーモアナエロバクター
(Thermoanaerobacter)と認められた。 Table (Taxonomic properties) Optimum growth temperature: 66℃ Growth temperature range: 50-74℃ Morphology: 0.3-0.4 x 1 ~ rods with periflagella
4μm Sporogen; None Gram staining; Undefined GC content; 34% Main products; Availability of ethanol, acetic acid, lactic sugar; Glucose, lactose, maltose, mannose, xylose, cellobiose, starch More mycological properties ``Archives of Microbiology''
Microbiology, 128-129, Bd., 1980-81'', it was recognized as Thermoanaerobacter.
しかし、種については前記の新規なプラスミド
を保有することから従来のどの種とも云えない点
を考慮し、新規なサーモアナエロバクターNo.62と
命名した。 However, considering the fact that it cannot be said to be any conventional species since it possesses the above-mentioned novel plasmid, the species was named the new Thermoanaerobacter No. 62.
なお、本菌株は微工研菌寄第7493号として寄託
されている。 This strain has been deposited as Microtechnical Research Institute No. 7493.
以下、実施例により本発明をより具体的に詳述
する。 Hereinafter, the present invention will be explained in more detail with reference to Examples.
実施例1 (菌株のスクリーニング)
茨城県地方の土壌約0.5gをYPG培地(デイフ
コ・イーストエキストラクト0.2%、大五栄養ポ
リペプトン0.4%、グルコース1%、NaCl0.2%、
Na2CO30.3%、L−システイン0.2%、PH7.5)10
mlに加え、炭酸ガス嫌気条件下、70℃で約18時間
培養後、ネオマイシン50μg/mlを含むYPGロ
ールチユーブで生育したコロニーの一つからサー
モアナエロバクターNo.62(微工研菌寄第7493号)
が得られた。Example 1 (Screening of bacterial strains) Approximately 0.5 g of soil from the Ibaraki Prefecture region was mixed with YPG medium (Difco Yeast Extract 0.2%, Daigo Nutrient Polypeptone 0.4%, Glucose 1%, NaCl 0.2%,
Na 2 CO 3 0.3%, L-cysteine 0.2%, PH7.5) 10
After culturing for about 18 hours at 70℃ under anaerobic carbon dioxide conditions, one of the colonies grown in a YPG roll tube containing 50 μg/ml of neomycin was infected with Thermoanaerobacter No. issue)
was gotten.
実施例 2
プラスミドpSH62のサーモアナエロバクターNo.
62からの分離
サーモアナエロバクターNo.62(微工研菌寄第
7493号)の生物学的に純粋な培養基から20mlの
YPG培地に接種し炭酸ガス嫌気条件下70℃で12
時間培養する。この培養液を1のYPG培地に
接種し、70℃嫌気条件下12時間培養する。菌体を
遠心によつて集め、アジ化ナトリウム10mMを添
加したTES(20mM Tris−HCl、5mM EDTA、
100mMNaCl、PH7.5)で洗浄後、菌体湿重量4g
当りアジ化ナトリウム10mMを添加した25%シヨ
糖含有TES10mlに懸濁する。リゾチーム(10
mg/ml)を3ml、0.25M EDTA2mlを加え、0℃
20分間放置する。この細胞混合液に300μの10
%ジエチルピロカーボネイトエタノール溶液、16
mlのアルカリSDS溶液(1%SDS、0.2N
NaOH)、12mlの酢酸ナトリウム(3M、PH4.8)を
加え、0℃に2時間放置する。これを7000rpm、
1時間の遠心をおこない上清を得る。この上清に
100mlの95%エタノールを加え、2時間−20℃に
静置し、7000rpm、10分の遠心で沈澱を得る。こ
の沈澱を50mlの95%エタノールで洗浄し減圧乾燥
後、14mlのTESに溶解し、CsCl及びエチジウム
ブロマイドを加えて密度を1.58に調整する。この
試料を38000rpmで30〜40時間、平衝密度勾配遠
心する。生じたプラスミドDNAのバンドを集
め、イソアルミアルコールでエチジウムブロマイ
ドを除去した後、TEN(20mMTris−HCl,1mM
EDTA,20mM NaCl)に透析する事によつてプ
ラスミド溶液が得られる。Example 2 Plasmid pSH62 of Thermoanaerobacter No.
Isolation from Thermoanaerobacter No. 62
7493) from biologically pure culture medium.
Inoculate YPG medium and store at 70℃ under carbon dioxide gas anaerobic conditions for 12 days.
Incubate for hours. This culture solution is inoculated into YPG medium No. 1, and cultured for 12 hours under anaerobic conditions at 70°C. Bacterial cells were collected by centrifugation and mixed with TES (20mM Tris-HCl, 5mM EDTA,
After washing with 100mMNaCl, PH7.5), bacterial cell wet weight 4g
Suspend in 10 ml of TES containing 25% sucrose to which 10 mM of sodium azide has been added. Lysozyme (10
Add 3 ml of 0.25 M EDTA and 2 ml of 0.25 M EDTA to 0°C.
Leave for 20 minutes. 10 to 300μ of this cell mixture
% diethylpyrocarbonate ethanol solution, 16%
ml of alkaline SDS solution (1% SDS, 0.2N
Add 12 ml of sodium acetate (3M, PH4.8) and leave at 0°C for 2 hours. This at 7000rpm,
Centrifuge for 1 hour to obtain supernatant. In this supernatant
Add 100 ml of 95% ethanol, let stand at -20°C for 2 hours, and centrifuge at 7000 rpm for 10 minutes to obtain a precipitate. This precipitate is washed with 50 ml of 95% ethanol and dried under reduced pressure, then dissolved in 14 ml of TES, and the density is adjusted to 1.58 by adding CsCl and ethidium bromide. This sample is subjected to density gradient centrifugation at 38000 rpm for 30-40 hours. The resulting plasmid DNA bands were collected, ethidium bromide was removed with isoaluminium alcohol, and then TEN (20mM Tris-HCl, 1mM
A plasmid solution is obtained by dialysis against EDTA, 20mM NaCl).
pSH62の特性決定の手順
pSH62の分子量は、その超らせん構造
(supercoiled structure)のDNA及び制限酵素に
よつて切断された断片のアガロースゲル電気泳動
及びポリアクリルアミド・ゲル電気泳動より得ら
れた。この際の分子量マーカーはpBR322DNA
(2.67md)、ColEIDNA(4.2md)及びラムダDNA
のHind分解断片(14.6、5.84、4.05、2.67、
1.30、1.17、0.34md)、ラムダDNAのEcoR分解
断片(13.7、4.74、3.73、3.48、3.02、2.13md)、
φ×174DNAのHae分解断片(0.836、0.666、
0.539、0.373、0.192、0.174、0.167、0.145、
0.120、0.073、0.044md)を用いた。制限酵素に
よる切断は、プラスミドDNA溶液からエタノー
ル沈澱によつてDNAを沈澱させ、適当な緩衝液
に溶解して行なつた。制限酵素は宝酒造及び、ベ
ーリンガー・マンハイム社よりの市販品を用い
た。アガロースゲル電気泳動はシーケム社のアガ
ロースを0.5%又は0.7%の濃度で用い、水平ゲル
電気泳動槽によつてゲル長さ1cm当り1.5Vの定
電圧で15〜17時間行なつた。 Procedure for characterizing pSH62 The molecular weight of pSH62 was obtained from agarose gel electrophoresis and polyacrylamide gel electrophoresis of its supercoiled DNA and fragments cleaved with restriction enzymes. The molecular weight marker in this case is pBR322DNA.
(2.67md), ColEIDNA (4.2md) and Lambda DNA
Hind decomposition fragment (14.6, 5.84, 4.05, 2.67,
1.30, 1.17, 0.34md), EcoR degradation fragment of lambda DNA (13.7, 4.74, 3.73, 3.48, 3.02, 2.13md),
Hae-digested fragments of φ×174 DNA (0.836, 0.666,
0.539, 0.373, 0.192, 0.174, 0.167, 0.145,
0.120, 0.073, 0.044md) were used. Cleavage with restriction enzymes was performed by precipitating DNA from a plasmid DNA solution by ethanol precipitation and dissolving it in an appropriate buffer. Restriction enzymes used were commercially available products from Takara Shuzo and Boehringer Mannheim. Agarose gel electrophoresis was carried out using SeaChem agarose at a concentration of 0.5% or 0.7% in a horizontal gel electrophoresis chamber at a constant voltage of 1.5 V per cm of gel length for 15 to 17 hours.
ポリアクリルアミド・ゲル電気泳動は、生化学
工業社製のポリアクリルアミド・ビスアクリルア
ミドを用い、5%濃度30:1の架橋度のゲルによ
つて垂直型スラブゲル電気泳動槽により、ゲル長
さ1cmあたり10Vの定電圧によつて2〜3時間行
つた。 Polyacrylamide gel electrophoresis was performed using polyacrylamide/bisacrylamide manufactured by Seikagaku Kogyo Co., Ltd., using a 5% concentration gel with a degree of crosslinking of 30:1 in a vertical slab gel electrophoresis chamber at 10 V per 1 cm of gel length. This was carried out for 2 to 3 hours using a constant voltage of .
従来、嫌気性好熱菌においてプラスミドが検出
された例は知られておらず、嫌気性好熱菌サーモ
アナエロバクターNo.62の有するpSH62は全く新規
なプラスミドである。 Until now, there has been no known example of a plasmid being detected in an anaerobic thermophile, and pSH62 possessed by the anaerobic thermophile Thermoanaerobacter No. 62 is a completely new plasmid.
図−1はpSH62の制限酵素開裂地図を示し、図
中のEcoRはエシヤリシア・コリ由来の酵素、
Hpaはハエモフイルス・パラインフルエンザエ
由来の酵素、Claはカリオフアノン・ラツム由
来の酵素、Accはアシネトバクター・カルコア
セテイカス由来の酵素、Bglはバチルス・グロ
ビギイ由来の酵素、Hind はハエモフイル
ス・インフルエンザエ由来の酵素をそれぞれ示し
ている。
Figure 1 shows the restriction enzyme cleavage map of pSH62. EcoR in the figure is an enzyme derived from Eschialsia coli.
Hpa is an enzyme derived from Haemophilus parainfluenzae, Cla is an enzyme derived from Karyophanon ratum, Acc is an enzyme derived from Acinetobacter calcoaceticus, Bgl is an enzyme derived from Bacillus globigii, and Hind is an enzyme derived from Haemophilus influenzae. are shown respectively.
Claims (1)
素BamH及びPstにより開裂されず、図に示
される制限酵素開裂地図で特徴づけられるプラス
ミドを保有する新規なサーモアナエロバクターNo.
62。 [Claims] 1. A novel Thermoanaerobacter No. 1 having a molecular weight of approximately 3.3 megadaltons, which possesses a plasmid that is not cleaved by the restriction enzymes BamH and Pst and is characterized by the restriction enzyme cleavage map shown in the figure.
62.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4336184A JPS60186277A (en) | 1984-03-07 | 1984-03-07 | Novel microorganism having novel plasmid |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP4336184A JPS60186277A (en) | 1984-03-07 | 1984-03-07 | Novel microorganism having novel plasmid |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS60186277A JPS60186277A (en) | 1985-09-21 |
JPS6221510B2 true JPS6221510B2 (en) | 1987-05-13 |
Family
ID=12661713
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP4336184A Granted JPS60186277A (en) | 1984-03-07 | 1984-03-07 | Novel microorganism having novel plasmid |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS60186277A (en) |
-
1984
- 1984-03-07 JP JP4336184A patent/JPS60186277A/en active Granted
Also Published As
Publication number | Publication date |
---|---|
JPS60186277A (en) | 1985-09-21 |
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