Verdoes J C, van Diepeningen A D, Punt P J, Debets A J, Stouthamer A H, van den Hondel C A
TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene-technology, Rijswijk, The Netherlands.
J Biotechnol. 1994 Aug 15;36(2):165-75. doi: 10.1016/0168-1656(94)90052-3.
To evaluate the possibility of improving glucoamylase (GLA) production in Aspergillus niger strains carrying multiple copies of the GLA encoding gene (glaA), additional glaA copies were introduced either by genetic recombination or retransformation. For strains to be used in such experiments a genetic analysis was first carried out. The results of this analysis clearly revealed that in each transformant integration had occurred at a chromosome corresponding to a single linkage group (LG). The GLA production per gene copy showed considerable variation in these strains, indicating a clear effect of the site of integration on gene expression. Introduction of additional gene copies by genetic recombination experiments was carried out for different combinations of strains, carrying glaA copies in different chromosomes. The introduction of additional glaA gene copies by genetic recombination did not result in a considerable increase in GLA production compared to the parental strains. In some strains recombination resulted in genetic instability, observed by the frequent loss of glaA copies. Also, retransformation of multi-copy glaA strains did not result in an increase in GLA production. In several strains even a decrease in GLA production was found after retransformation. Southern analysis of these transformants suggested that newly introduced gene copies were heavily rearranged, which partly explains why GLA production was not increased. Further analysis of one such transformant provided evidence that the overexpression of the glaA gene is limited by the amount of trans-acting regulatory protein(s) available.
为了评估在携带多拷贝葡糖淀粉酶（GLA）编码基因（glaA）的黑曲霉菌株中提高GLA产量的可能性，通过基因重组或再转化引入了额外的glaA拷贝。对于将用于此类实验的菌株，首先进行了遗传分析。该分析结果清楚地表明，在每个转化体中，整合发生在与单个连锁群（LG）相对应的染色体上。每个基因拷贝的GLA产量在这些菌株中表现出相当大的差异，表明整合位点对基因表达有明显影响。通过基因重组实验，针对在不同染色体上携带glaA拷贝的不同菌株组合引入了额外的基因拷贝。与亲本菌株相比，通过基因重组引入额外的glaA基因拷贝并未导致GLA产量大幅增加。在一些菌株中，重组导致遗传不稳定，表现为glaA拷贝频繁丢失。此外，多拷贝glaA菌株的再转化也未导致GLA产量增加。在几个菌株中，再转化后甚至发现GLA产量下降。对这些转化体的Southern分析表明，新引入的基因拷贝发生了大量重排，这部分解释了GLA产量未增加的原因。对其中一个这样的转化体的进一步分析提供了证据，表明glaA基因的过表达受到可用反式作用调节蛋白数量的限制。
