Klabunde J, Diesel A, Waschk D, Gellissen G, Hollenberg C P, Suckow M
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany.
Appl Microbiol Biotechnol. 2002 May;58(6):797-805. doi: 10.1007/s00253-002-0957-0. Epub 2002 Mar 19.
We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios.
我们研究了多形汉逊酵母这种甲基营养型酵母，将其作为宿主，采用核糖体DNA（rDNA）整合方法来共整合和表达多个异源基因。发现多形汉逊酵母的核糖体DNA（rDNA）由位于II号染色体上的一个约50 - 60个8 kb单位重复序列的单一rDNA簇组成。分离出多形汉逊酵母rDNA的一个2.4 kb片段，该片段包含25S的部分、完整的5S以及25S和18S rDNA之间的非转录间隔区，并将其插入到含有源自酿酒酵母的URA3基因用于筛选的传统整合型多形汉逊酵母质粒中。这些rDNA质粒作为几个独立的簇同源整合到多形汉逊酵母（odc1）宿主的rDNA重复序列中。预计这种多簇整合模式可用于同时整合几种不同的rDNA质粒，于是将宿主菌株与多达三种不同质粒的混合物共转化，所有质粒都带有相同的URA3选择标记。转化确实产生了有丝分裂稳定的菌株，这些菌株含有整合到rDNA中的一个、两个或所有三个质粒。无论涉及的不同质粒数量如何，整合的质粒的总体拷贝数都不超过未转化宿主菌株中存在的rDNA重复序列的数量。含有不同质粒的菌株共表达导入的基因，产生功能蛋白。因此，这种方法为快速产生以所需化学计量比同时共产生几种蛋白质的重组菌株提供了一种新的且有吸引力的工具。
