利用新型可移动单复制子载体pLF在枯草芽孢杆菌中表达芽孢杆菌谷氨酰内肽酶基因。

Expression of bacillar glutamyl endopeptidase genes in Bacillus subtilis by a new mobilizable single-replicon vector pLF.

作者信息

Shevelev A B, Aleoshin V V, Trachuk L A, Granovsky A E, Kogan Y N, Rumer L M, Serkina A V, Semenova E V, Bushueva A M, Livshits V A, Kostrov S V, Shcheglov A S, Novikova S I, Chestukhina G G

机构信息

Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia.

出版信息

Plasmid. 2000 May;43(3):190-9. doi: 10.1006/plas.1999.1455.

DOI:10.1006/plas.1999.1455

PMID:10783297

Abstract

The pLF1311 natural plasmid from Lactobacillus fermentum 1311 was used to construct a single-replicon vector suitable for rapid cloning in a wide range of gram-positive hosts and Escherichia coli. The new vector is capable of conjugative mobilization from E. coli to various hosts by conjugal transfer. The final vector (3.4 kb) showed a high segregational and structural stability and a high copy number. Glutamyl endopeptidase genes from Bacillus licheniformis (gseBL) and B. intermedius (gseBI) were cloned in both pLF9 and pLF14 vectors and introduced to B. subtilis. The yield of enzymes in the pLF-derived producers was 6- to 30-fold more than in the natural producers and reached 100-150 mg/L of mature protease.

摘要

来自发酵乳杆菌1311的pLF1311天然质粒被用于构建一种单复制子载体，该载体适用于在多种革兰氏阳性宿主和大肠杆菌中进行快速克隆。这种新载体能够通过接合转移从大肠杆菌接合转移至各种宿主。最终的载体（3.4 kb）表现出高分离稳定性和结构稳定性以及高拷贝数。地衣芽孢杆菌（gseBL）和中间芽孢杆菌（gseBI）的谷氨酰胺内肽酶基因被克隆到pLF9和pLF14载体中，并导入枯草芽孢杆菌。源自pLF的生产菌株中酶的产量比天然生产菌株高6至30倍，成熟蛋白酶产量达到100 - 150 mg/L。