Takala T M, Saris P E J
Department of Applied Chemistry and Microbiology, Division of Microbiology, Viikinkaari 9, P.O. Box 56, 00014 University of Helsinki, Finland.
Appl Microbiol Biotechnol. 2002 Aug;59(4-5):467-71. doi: 10.1007/s00253-002-1034-4. Epub 2002 Jun 4.
A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB 590, was constructed entirely of lactococcal DNA: the pSH 71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG 1614 with 60 international units (IU) nisin/ml selection yielded approximately 10(5) transformants/ micro g DNA. MG 1614 carrying pLEB 590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB 590 was successfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications.
利用乳链菌肽免疫基因nisI作为选择标记，构建了一种新的用于乳酸菌的食品级克隆载体。食品级质粒pLEB 590完全由乳球菌DNA构建而成：pSH 71复制子、nisI基因以及用于nisI表达的组成型启动子P45。用60国际单位（IU）/毫升的乳链菌肽进行选择，将其电穿孔导入乳酸乳球菌MG 1614，每微克DNA可产生约10⁵个转化子。携带pLEB 590的MG 1614在含有最高250 IU/毫升乳链菌肽的培养基中能够生长。质粒pLEB 590成功转化到携带多个隐蔽质粒的工业用乳酸乳球菌奶酪发酵剂中。通过在乳酸乳球菌和植物乳杆菌中克隆并表达瑞士乳杆菌的脯氨酸亚氨基肽酶基因pepI，证实了其适用于分子克隆。这些结果表明，本文报道的食品级表达系统在乳酸菌中表达外源基因以构建用于食品应用的改良发酵剂方面具有潜力。
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