van Gemeren I A, Beijersbergen A, Musters W, Gouka R J, van den Hondel C A, Verrips C T
Department of Molecular and Cellular Biology, University of Utrecht, The Netherlands.
Appl Microbiol Biotechnol. 1996 Jul;45(6):755-63. doi: 10.1007/s002530050759.
A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A. awamori endoxylanase II (exlA) promoter and terminator. The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated at the pyrG locus. Transformants containing a construct encoding a direct, inframe fusion of the xylanase pre-peptide to the mature cutinase showed a 2-fold higher cutinase production level compared to strains containing constructs with an additional cutinase pro-peptide. The effect of multicopy integration of the expression cassette on cutinase production was analysed in strains with different numbers of a cutinase construct containing its own pre-pro-sequence. The multicopy strains showed a 6-to 12-fold increased production of extracellular cutinase relative to the single-copy strains. No linear dose response relation to the number of expression cassettes present in the strains was observed. The amount of active enzyme produced by the strains correlated with the amount of cutinase-specific mRNA, suggesting that cutinase overproduction is not limited at the level of translation or secretion.
使用泡盛曲霉内切木聚糖酶II（exlA）启动子和终止子，在泡盛曲霉中表达了豌豆镰孢菌角质酶cDNA的合成衍生物。在含有整合于pyrG位点的表达盒的单拷贝转化体中，分析了前导序列的来源和前肽的存在对细胞外角质酶产生效率的影响。与含有带有额外角质酶前肽构建体的菌株相比，含有编码木聚糖酶前肽与成熟角质酶直接读码框融合体构建体的转化体，其角质酶产生水平高出2倍。在含有不同数量带有自身前原肽序列的角质酶构建体的菌株中，分析了表达盒多拷贝整合对角质酶产生的影响。多拷贝菌株相对于单拷贝菌株，细胞外角质酶产量增加了6至12倍。未观察到菌株中表达盒数量与线性剂量反应关系。菌株产生的活性酶量与角质酶特异性mRNA量相关，表明角质酶过量产生在翻译或分泌水平不受限制。
