Itoh N, Kawanami T, Nitta C, Iwata N, Usami S, Abe Y, Koide Y
Biotechnology Research Center, Toyama Prefectural University, Kurokawa 5180, 939-0398, Kosugi, Toyama, Japan.
Appl Microbiol Biotechnol. 2003 May;61(3):240-6. doi: 10.1007/s00253-002-1195-1. Epub 2003 Jan 9.
The complete nucleotide sequence of pNI10 (3.75 kb), from which pNI105 and pNI107 were constructed as medium-host-range vectors for Gram-negative bacteria, was determined. A fragment of about 2.1 kb of pNI10 was essential for replication in Escherichia coli and Pseudomonas fluorescens. This fragment encodes a putative origin of replication ( ori) and one putative replication-controlling protein (Rep). An improved version of the medium-host-range plasmid vector pNUK73 (5.13 kb) was constructed with the basic-replicon of pNI10 and pHSG298 (2.68 kb). We show that expression in pseudomonads of the bromoperoxidase gene ( bpo) of Pseudomonas putida, inserted downstream of the lac promoter in pNUK73, resulted in about 30% (13.6 U/l culture) of the enzyme level obtained in E. coli.
测定了pNI10（3.75 kb）的完整核苷酸序列，pNI105和pNI107就是基于该序列构建的革兰氏阴性菌中宿主范围载体。pNI10约2.1 kb的片段对于在大肠杆菌和荧光假单胞菌中复制至关重要。该片段编码一个假定的复制起点（ori）和一个假定的复制控制蛋白（Rep）。用pNI10的基本复制子和pHSG298（2.68 kb）构建了中宿主范围质粒载体pNUK73（5.13 kb）的改进版本。我们发现，插入到pNUK73中lac启动子下游的恶臭假单胞菌溴过氧化物酶基因（bpo）在假单胞菌中的表达量，约为在大肠杆菌中所获酶水平的30%（13.6 U/升培养物）。
