Flipphi M J, Visser J, van der Veen P, de Graaff L H
Department of Genetics, Wageningen Agricultural University, The Netherlands.
Appl Microbiol Biotechnol. 1993 Jun;39(3):335-40. doi: 10.1007/BF00192088.
Using L-arabitol as an inducer, simple induction conditions were established that resulted in high-level expression of alpha-L-arabinofuranosidase A by an Aspergillus niger D-xylulose kinase mutant strain. These conditions were adapted to construct a cDNA expression library from which an alpha-L-arabinofuranosidase A cDNA clone was isolated using specific antiserum. The corresponding gene encoding alpha-L-arabinofuranosidase A (abfA) was isolated from a genomic library and cloned into a high copy plasmid vector. By co-transformation of uridine auxotrophic mutants lacking orotidine-5-phosphate decarboxylase activity, the afbA gene was introduced both in A. niger and A. nidulans, using the A. niger pyrA gene as selection marker. The identity of the abfA gene was confirmed by overexpression of the gene product by A. niger and A. nidulans transformants, upon growth using sugar beet pulp as the carbon source.
以L-阿拉伯糖醇作为诱导剂，建立了简单的诱导条件，可使黑曲霉D-木酮糖激酶突变株高水平表达α-L-阿拉伯呋喃糖苷酶A。这些条件被用于构建一个cDNA表达文库，利用特异性抗血清从中分离出α-L-阿拉伯呋喃糖苷酶A的cDNA克隆。从基因组文库中分离出编码α-L-阿拉伯呋喃糖苷酶A（abfA）的相应基因，并将其克隆到高拷贝质粒载体中。通过共转化缺乏乳清苷-5'-磷酸脱羧酶活性的尿苷营养缺陷型突变体，以黑曲霉pyrA基因作为选择标记，将afbA基因导入黑曲霉和构巢曲霉中。在以甜菜粕作为碳源生长时，通过黑曲霉和构巢曲霉转化体对基因产物的过表达，证实了abfA基因的身份。
