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部位特異的遺伝子増幅機能を応用した放線菌二次代謝産物生産能力増強システムの構築

利用位点特异性基因扩增功能增强放线菌次生代谢产物生产能力的系统构建

基本信息

批准号：
19K05779
负责人：
小松護
金额：
$ 2.33万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2019
资助国家：
日本
起止时间：
2019-04-01 至 2022-03-31
项目状态：
已结题

项目摘要

放線菌Streptomyces avermitilisのゲノム縮小株(SUKA)における、S. kanamyceticusならびにStreptomyces sp. RM04株由来のZouAの部位特異的遺伝子増幅機能を応用した二次代謝産物生産能力増強システムを構築した。zouA_kanをRsB-attBs-RsA-zouA_kanの並びでカセット化し、線状プラスミドSAP1に導入した。chloramphenicol(Cml)生合成遺伝子を含むpCML2をSAP1上のattBサイトに導入した。pCML2の選択マーカーであるapramycin耐性遺伝子を利用し、培地中のapramycin濃度を段階的に低濃度から高濃度に添加量を増やし、高濃度のapramycinに対して耐性を示す株を液体培養によって集積した結果、Cmlクラスターが増幅して巨大化したと推測されるSAP1が観察された。増幅されたpCML2を含むSAP1の存在比は極めて低かったが、Cmlの生産量はzouA非導入株の2倍程度(0.4g/L)まで増加した。一方で、zouA_RM04の場合では、pCML2をSAP1ベクター上への導入のみで、効率良くcmlが増幅された。更に効率の良い遺伝子増幅効果を期待して、Cumateを誘導基質とした誘導型プロモーター(Pcmt：P21-cmt-CymRシステム)を用いた結果、cumate添加の有無にかかわらず、効率の良いpCML2の増幅が確認された。RM04株由来の野生型プロモーターを利用した当初の結果と比較しても顕著な増幅効果が見られたことから、今後はPcmtを利用することによって、効率の良い遺伝子増幅が期待できる。また、パルスフィールド電気泳動の結果、増幅された遺伝子領域は欠失することなく安定的に保持されており、観察できる限り、11コピーまで増幅されていた。

Streptomyces avermitilis isolates were isolated from small plants (SUKA), and S. kanamyceticus strains were isolated from Streptomyces sp. The RM04 strain was derived from the special part of the ZouA, and the frame machine was able to use the second generation to strengthen the production ability of the product. ZouA_ kanthan RsB-attBs-RsA-zouA_kan, and make sure that you don't know what to do, and that you don't know what to do, and that you don't know what to do. You need to know that SAP1 is imported. Chloramphenicol (Cml) produces synthetic razor cups that contain "attB" on the pCML2 SAP1. In pCML2, you can select "low", "high", "low", "high" apramycin tolerance, "low", "high" apramycin "tolerance", "liquid culture", "Cml tolerance", "maximization", "SAP1 tolerance", "low temperature", "high temperature", "low temperature", "high temperature" and "high temperature". The content of SAP1 in pCML2 was lower than that in 0.4g/L, and the production of zouA was twice as high as that of 0.4g/L. One side, zouA_RM04, pCML2, SAP1, and so on, and the rate is good. Cml is good. To improve the performance, the results are expected, the Cumate is used to guide the Pcmt:P21-cmt-CymR response, the cumate is added, and the pCML2 is confirmed. The origin of the RM04 strain is wild type. The results of the previous results were compared and the results were compared. In the future, the Pcmt strain was used to compare the results of the previous results, and now the results of the wild-type strains were used. The results of the electrophoretic exercise, the results of the electrophoretic exercise, the failure of the field, the maintenance of the stability, the monitoring of the limit, the limit of the limit, the limit of the limit, the results of the swimming exercise, the loss of stability in the field, the limit of the limit, the results of the swimming exercise, the loss of stability in the field, the limit of the limit, the result of the swimming exercise, the results of the swimming exercise,