Gardner W L, Whitman W B
Department of Microbiology, University of Georgia, Athens, Georgia 30602-2605, USA.
Genetics. 1999 Aug;152(4):1439-47. doi: 10.1093/genetics/152.4.1439.
A series of integrative and shuttle expression vectors was developed for use in Methanococcus maripaludis. The integrative expression vectors contained the Methanococcus voltae histone promoter and multiple cloning sites designed for efficient cloning of DNA. Upon transformation, they can be used to overexpress specific homologous genes in M. maripaludis. When tested with ilvBN, which encodes the large and small subunits of acetohydroxyacid synthase, transformants possessed specific activity 13-fold higher than that of the wild type. An expression shuttle vector, based on the cryptic plasmid pURB500 and the components of the integrative vector, was also developed for the expression of heterologous genes in M. maripaludis. The beta-galactosidase gene from Escherichia coli was expressed to approximately 1% of the total cellular protein using this vector. During this work, the genes for the acetohydroxyacid synthase (ilvBN) and phosphoenolpyruvate synthase (ppsA) were sequenced from a M. maripaludis genomic library.
构建了一系列用于马氏甲烷球菌的整合型和穿梭表达载体。整合型表达载体包含沃氏甲烷球菌组蛋白启动子和为高效克隆DNA设计的多克隆位点。转化后，它们可用于在马氏甲烷球菌中过表达特定的同源基因。用编码乙酰羟酸合酶大亚基和小亚基的ilvBN进行测试时，转化体的比活性比野生型高13倍。还构建了一种基于隐蔽质粒pURB500和整合型载体元件的表达穿梭载体，用于在马氏甲烷球菌中表达异源基因。使用该载体，大肠杆菌的β-半乳糖苷酶基因表达量约占细胞总蛋白的1%。在这项工作中，从马氏甲烷球菌基因组文库中对乙酰羟酸合酶（ilvBN）和磷酸烯醇丙酮酸合酶（ppsA）的基因进行了测序。
