JP7464921B2 - ヘパロサンの製造方法及びヘパロサン生産能を有するエシェリヒア属細菌 - Google Patents
ヘパロサンの製造方法及びヘパロサン生産能を有するエシェリヒア属細菌 Download PDFInfo
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- JP7464921B2 JP7464921B2 JP2022560347A JP2022560347A JP7464921B2 JP 7464921 B2 JP7464921 B2 JP 7464921B2 JP 2022560347 A JP2022560347 A JP 2022560347A JP 2022560347 A JP2022560347 A JP 2022560347A JP 7464921 B2 JP7464921 B2 JP 7464921B2
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- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 101150019416 trpA gene Proteins 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229940088594 vitamin Drugs 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
- 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
- 235000019164 vitamin B2 Nutrition 0.000 description 1
- 239000011716 vitamin B2 Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
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Description
1.以下の(1)の遺伝子改変を有し、かつヘパロサン生産能を有するエシェリヒア属細菌を培地で培養し、ヘパロサンを培地中に生成させることを含む、ヘパロサンの製造方法。
(1)kpsS遺伝子の発現を増大させる遺伝子改変
2.前記エシェリヒア属細菌が、さらに以下の(2)及び(3)の少なくとも一方の遺伝子改変を有する前記1に記載のヘパロサンの製造方法。
(2)kfiA、kfiB、kfiC及びkfiD遺伝子から選択される少なくとも1の遺伝子の発現を増大させる遺伝子改変
(3)yhbJ遺伝子の機能を欠損させる遺伝子改変
3.前記(1)の前記遺伝子改変が、前記kpsS遺伝子の発現調節領域の改変及びコピー数を高めることの少なくとも一方である前記1または2に記載のヘパロサンの製造方法。
4.前記(2)の前記遺伝子改変が、前記kfiA、前記kfiB、前記kfiC及び前記kfiD遺伝子から選択される少なくとも1の遺伝子の発現調節領域の改変及び前記遺伝子のコピー数を高めることの少なくとも一方である、前記2または3に記載のヘパロサンの製造方法。
5.前記(3)の前記遺伝子改変が、前記yhbJ遺伝子の欠損である、前記2~4のいずれか1に記載のヘパロサンの製造方法。
6.前記エシェリヒア属細菌がエシェリヒア・コリである、前記1~5のいずれか1に記載のヘパロサンの製造方法。
7.前記kpsS遺伝子が、配列番号33に示す塩基配列を含むDNAまたは、配列番号33に示す塩基配列と90%以上の同一性を有する塩基配列を含みかつヘパロサン生産能を有するエシェリヒア属細菌において発現量を増大させた際に同細菌のヘパロサン生産能を増大させる性質を有するDNAである、前記1~6のいずれか1に記載のヘパロサンの製造方法。
8.前記kfiA遺伝子が、配列番号34に示す塩基配列を含むDNAまたは、配列番号34に示す塩基配列と90%以上の同一性を有する塩基配列を含みかつヘパロサン生産能を有するエシェリヒア属細菌において発現量を増大させた際に同細菌のヘパロサン生産能を増大させる性質を有するDNAであり、
前記kfiB遺伝子が、配列番号35に示す塩基配列を含むDNAまたは、配列番号35に示す塩基配列と90%以上の同一性を有する塩基配列を含みかつヘパロサン生産能を有するエシェリヒア属細菌において発現量を増大させた際に同細菌のヘパロサン生産能を増大させる性質を有するDNAであり、
前記kfiC遺伝子が、配列番号36に示す塩基配列を含むDNAまたは、配列番号36に示す塩基配列と90%以上の同一性を有する塩基配列を含みかつヘパロサン生産能を有するエシェリヒア属細菌において発現量を増大させた際に同細菌のヘパロサン生産能を増大させる性質を有するDNAであり、
前記kfiD遺伝子が、配列番号37に示す塩基配列を含むDNAまたは、配列番号37に示す塩基配列と90%以上の同一性を有する塩基配列を含みかつヘパロサン生産能を有するエシェリヒア属細菌において発現量を増大させた際に同細菌のヘパロサン生産能を増大させる性質を有するDNAである、前記2~7のいずれか1に記載のヘパロサンの製造方法。
9.前記yhbJ遺伝子が、配列番号38に示す塩基配列を含むDNAまたは、配列番号38に示す塩基配列と90%以上の同一性を有する塩基配列を含みかつヘパロサン生産能を有するエシェリヒア属細菌において発現量を低下させた際に同細菌のヘパロサン生産能を増大させる性質を有するDNAである、前記2~8のいずれか1に記載のヘパロサンの製造方法。
10.エシェリヒア属細菌が以下の(4)の遺伝子改変を有していない、前記1~9のいずれか1に記載のヘパロサンの製造方法。
(4)kpsC遺伝子の発現を増大させる遺伝子改変
11.ヘパロサン生産能を有するエシェリヒア属細菌であって、以下の(1)の遺伝子改変を有する、エシェリヒア属細菌。
(1)kpsS遺伝子の発現を増大させる遺伝子改変
12.さらに以下の(2)及び(3)の少なくとも一方の遺伝子改変を有する、前記11に記載のエシェリヒア属細菌。
(2)kfiA、kfiB、kfiC及びkfiD遺伝子から選択される少なくとも1の遺伝子の発現を増大させる遺伝子改変
(3)yhbJ遺伝子の機能を欠損させる遺伝子改変
13.エシェリヒア属細菌が以下の(4)の遺伝子改変を有していない、前記11または12に記載のエシェリヒア属細菌。
(4)kpsC遺伝子の発現を増大させる遺伝子改変
本発明のヘパロサンの製造方法は、以下の(1)の遺伝子改変を有し、かつヘパロサン生産能を有するエシェリヒア属細菌(以下、本発明の細菌とも略す。)を用いる。
(1)kpsS遺伝子の発現を増大させる遺伝子改変
(2)kfiA、kfiB、kfiC及びkfiD遺伝子から選択される少なくとも1の遺伝子の発現を増大させる遺伝子改変
(3)yhbJ遺伝子の機能を欠損させる遺伝子改変
したがって、前記エシェリヒア属細菌における遺伝子改変としては、前記(1)及び(2)、前記(1)及び(3)、前記(1)~(3)が挙げられる。
(4)kpsC遺伝子の発現を増大させる遺伝子改変
「遺伝子の発現の増大」とは、非改変株と比較して該遺伝子の発現が上昇することをいう。遺伝子の発現の増大の一態様としては、例えば、非改変株と比較して、該遺伝子の発現が好ましくは1.5倍以上、より好ましくは2倍以上、さらに好ましくは3倍以上に上昇していることが挙げられる。
上記した(3)のyhbJ遺伝子の機能を欠損させる遺伝子改変としては、宿主であるエシェリヒア属細菌のゲノムDNAのうちyhbJに相当する部分をコードするDNAに改変を加えることにより、yhbJ遺伝子の機能を欠損させるyhbJに相当する部分がコードするタンパク質の機能を低減若しくは完全に停止させることが挙げられる。
(a)yhbJに相当する部分をコードするDNAの全部または一部を除去する。
(b)yhbJに相当する部分をコードするDNAに、1または数個の置換、欠失もしくは付加する。
(c)yhbJに相当する部分をコードするDNAを、改変前のDNA配列との同一性が80%未満であるDNA配列と置き換える。
エシェリヒア属細菌としては、特に制限されないが、微生物学の専門家に知られている分類によりエシェリヒア属に分類されている細菌が挙げられる。エシェリヒア属細菌としては、例えば、文献[Backmann, B. J. 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In F. D. Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology/Second Edition, American Society for Microbiology Press, Washington, D.C.]に記載されたものが挙げられる。
本発明のヘパロサンの製造法は、本発明の細菌を培地で培養してヘパロサンを該培地中に生成蓄積することを含む。本発明のヘパロサンの製造法は、必要であれば、該培地よりヘパロサンを採取することを含んでもよい。
(a)遺伝子欠損用マーカー遺伝子断片の構築
相同組換えを用いたEscherichia coliの遺伝子欠損のためのマーカー遺伝子として用いるクロラムフェニコール耐性遺伝子(cat)およびレバンシュークラーゼ遺伝子(sacB)を含むDNA断片を以下のようにして調製した。
配列番号7および8で表される塩基配列からなる合成DNA、並びに配列番号9および10で表される塩基配列からなる合成DNAをそれぞれプライマーセットとして用い、常法により調製したEscherichia coli Nissle 1917株[DSM 6601、Mutaflor(Pharma-Zentrale GmbH)、以下Nissle株と略す]のゲノムDNAを鋳型として一回目のPCRを行い、それぞれyhbJ遺伝子の開始コドン付近からその上流1000bp、およびyhbJ遺伝子の終始コドン付近からその下流約1000bpを含むDNA断片を取得した。PCRはprimeSTAR Max DNA Polymerase(タカラバイオ社製)を用いて、説明書に記載の通りに行った。
以下に示す方法でkfiA遺伝子のプロモーター領域置換株を造成した。配列番号14および15で表される塩基配列からなる合成DNA、並びに配列番号16および17で表される塩基配列からなる合成DNAをそれぞれプライマーセットとして用い、常法により調製したEscherichia coli Nissle株のゲノムDNAを鋳型として一回目のPCRを行い、それぞれkfiA遺伝子の開始コドンより上流100bp付近からその上流1000bp、およびkfiA遺伝子の開始コドン付近からその下流約1000bpを含むDNA断片を取得した。
また、配列番号20および21で表される塩基配列からなる合成DNAをプライマーセットとして用い、常法により調製したEscherichia coli W株(ATCC 9637)のゲノムDNAを鋳型としてPCRを行い、約300bpのuspAプロモーターを含むDNA断片を得た。
(a)ヘパロサン生産株の培養
実施例1で得られたyhbJ欠損変異株NY株、および実施例2で得られたkfiAプロモーター置換株NA株、kfiAプロモーター置換yhbJ欠損株NYA株、および親株であるNissle株をLB寒天培地で30℃、24時間培養した後、それぞれを前培養培地[10 g/L大豆ペプチド(ハイニュートAM;不二製油社製)、5 g/L 塩化ナトリウム、5 g/L 酵母エキスパウダー(AY-80;アサヒフードアンドヘルスケア社製)をpH7.2となるよう水酸化ナトリウムで調整] 330mLの入った2Lバッフルに植菌し、30℃で18時間培養した。
蒸留水で10倍に希釈した培養液を100℃で30分間インキュベートした後、遠心分離により培養液から菌体を除去し、得られた上清200μLを1.5mLエッペンドルフチューブに移した。0.5mol/L 硫酸ナトリウム水溶液40μLを加えて混合した後、10g/L 塩化ヘキサデシルピリジニウム水溶液400μLを加えて転倒混合し、37℃で1時間静置した。
粗ヘパロサン溶液 20μLを氷冷しながら、硫酸液 [9.5g/L四ホウ酸ナトリウム十水和物を濃硫酸に溶解させた液] 100μLを加えて混合した後、100℃で10分間インキュベートした。該溶液を再度氷冷しながら、カルバゾール液 [1.25g/L カルバゾールを100%エタノールに溶解させた液] 4μLを加えて混合し、100℃で15分間インキュベートした。
上記の方法で測定した場合のヘパロサンの蓄積量を表1に示す。
Escherichia coli Nissle株の染色体DNAを鋳型とし、配列番号23および24で表される塩基配列からなる合成DNAをプライマーセットとして用いてPCR反応を行い、kpsC、kpsS遺伝子領域を含む約3.3kbpのDNA断片(以下kpsCS遺伝子増幅断片という)を得た。
実施例1で得られたNY株に、pMW118プラスミドおよび、実施例4で得られたpMW118-PuspA-kpsS、pMW118-PuspA-kpsCS、pMW118-PuspA-kpsFEDUCSをそれぞれ形質転換した。得られた形質転換体をそれぞれNY/pMW118株、NY/pMW118-PuspA-kpsS株、NY/pMW118-PuspA-kpsCS株、NY/pMW118-PuspA-kpsFEDUCS株と命名した。
Escherichia coli Nissleの野生株に、pMW118プラスミドおよび、実施例4で得られたpMW118-PuspA-kpsS、pMW118-PuspA-kpsCS、pMW118-PuspA-kpsFEDUCSをそれぞれ形質転換した。得られた形質転換体をそれぞれN/pMW118株、N/pMW118-PuspA-kpsS株、N/pMW118-PuspA-kpsCS株、N/pMW118-PuspA-kpsFEDUCS株と命名した。
Claims (11)
- 以下の(1)の遺伝子改変を有し、以下の(4)の遺伝子改変を有しておらず、かつヘパロサン生産能を有するエシェリヒア属細菌を培地で培養し、ヘパロサンを培地中に生成させることを含む、ヘパロサンの製造方法。
(1)kpsS遺伝子の発現を非改変株と比較して上昇させる遺伝子改変
(4)kpsC遺伝子の発現を非改変株と比較して上昇させる遺伝子改変 - 前記エシェリヒア属細菌が、さらに以下の(2)及び(3)の少なくとも一方の遺伝子改変を有する請求項1に記載のヘパロサンの製造方法。
(2)kfiA、kfiB、kfiC及びkfiD遺伝子から選択される少なくとも1の遺伝子の発現を増大させる遺伝子改変
(3)yhbJ遺伝子の機能を欠損させる遺伝子改変 - 前記(1)の前記遺伝子改変が、前記kpsS遺伝子の発現調節領域の改変及びコピー数を高めることの少なくとも一方である請求項1または2に記載のヘパロサンの製造方法。
- 前記(2)の前記遺伝子改変が、前記kfiA、前記kfiB、前記kfiC及び前記kfiD遺伝子から選択される少なくとも1の遺伝子の発現調節領域の改変及び前記遺伝子のコピー数を高めることの少なくとも一方である、請求項2または3に記載のヘパロサンの製造方法。
- 前記(3)の前記遺伝子改変が、前記yhbJ遺伝子の欠損である、請求項2~4のいずれか1項に記載のヘパロサンの製造方法。
- 前記エシェリヒア属細菌がエシェリヒア・コリである、請求項1~5のいずれか1項に記載のヘパロサンの製造方法。
- 前記kpsS遺伝子が、配列番号33に示す塩基配列を含むDNAまたは、配列番号33に示す塩基配列と90%以上の同一性を有する塩基配列を含みかつヘパロサン生産能を有するエシェリヒア属細菌において発現量を増大させた際に同細菌のヘパロサン生産能を増大させる性質を有するDNAである、請求項1~6のいずれか1項に記載のヘパロサンの製造方法。
- 前記kfiA遺伝子が、配列番号34に示す塩基配列を含むDNAまたは、配列番号34に示す塩基配列と90%以上の同一性を有する塩基配列を含みかつヘパロサン生産能を有するエシェリヒア属細菌において発現量を増大させた際に同細菌のヘパロサン生産能を増大させる性質を有するDNAであり、
前記kfiB遺伝子が、配列番号35に示す塩基配列を含むDNAまたは、配列番号35に示す塩基配列と90%以上の同一性を有する塩基配列を含みかつヘパロサン生産能を有するエシェリヒア属細菌において発現量を増大させた際に同細菌のヘパロサン生産能を増大させる性質を有するDNAであり、
前記kfiC遺伝子が、配列番号36に示す塩基配列を含むDNAまたは、配列番号36に示す塩基配列と90%以上の同一性を有する塩基配列を含みかつヘパロサン生産能を有するエシェリヒア属細菌において発現量を増大させた際に同細菌のヘパロサン生産能を増大させる性質を有するDNAであり、
前記kfiD遺伝子が、配列番号37に示す塩基配列を含むDNAまたは、配列番号37に示す塩基配列と90%以上の同一性を有する塩基配列を含みかつヘパロサン生産能を有するエシェリヒア属細菌において発現量を増大させた際に同細菌のヘパロサン生産能を増大させる性質を有するDNAである、請求項2~7のいずれか1項に記載のヘパロサンの製造方法。 - 前記yhbJ遺伝子が、配列番号38に示す塩基配列を含むDNAまたは、配列番号38位に示す塩基配列と90%以上の同一性を有する塩基配列を含みかつヘパロサン生産能を有するエシェリヒア属細菌において発現量を低下させた際に同細菌のヘパロサン生産能を増大させる性質を有するDNAである、請求項2~8のいずれか1項に記載のヘパロサンの製造方法。
- ヘパロサン生産能を有するエシェリヒア属細菌であって、以下の(1)の遺伝子改変を有し、かつ以下の(4)の遺伝子改変を有していない、エシェリヒア属細菌。
(1)kpsS遺伝子の発現を非改変株と比較して上昇させる遺伝子改変
(4)kpsC遺伝子の発現を非改変株と比較して上昇させる遺伝子改変 - さらに以下の(2)及び(3)の少なくとも一方の遺伝子改変を有する、請求項10に記載のエシェリヒア属細菌。
(2)kfiA、kfiB、kfiC及びkfiD遺伝子から選択される少なくとも1の遺伝子の発現を増大させる遺伝子改変
(3)yhbJ遺伝子の機能を欠損させる遺伝子改変
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