Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae
Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae
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DOI：
10.1007/s00253-007-1088-4
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发表时间：
2007-10-01
影响因子：
5
通讯作者：
Kitamoto, Katsuhiko
中科院分区：
文献类型：
--
作者：
Jin, Feng Jie;Watanabe, Taisuke;Kitamoto, Katsuhiko
研究了蛋白酶基因破坏对黑曲霉生产异源蛋白的影响。选择人溶菌酶（HLY）作为异源蛋白的模型用于重组生产。将与α-淀粉酶（AmyB）融合的串联HLY构建体通过A.其中Kex 2切割位点插入在HLY的上游。成功地从AmyB加工出HLY并在培养基中产生。我们用adeA选择标记对HLY生产菌株中的5个蛋白酶基因（pepA、pepE、alpA、tppA和palB）进行了系统的破坏分析。比较分析表明，破坏编码三肽基肽酶的tppA基因导致HLY产量的最高增加（36%）。我们进一步用另一个选择标记argB缺失pepE或palB破坏物中的tppA基因。因此，与对照菌株相比，tppA和pepE基因的双重破坏导致HLY产量增加63%。这是第一个研究报告的tppA和pepE基因的双重破坏提高了丝状真菌的异源蛋白质的生产水平。
In this study, we investigated the effects of proteinase gene disruption on heterologous protein production by Aspergillus oryzae. The human lysozyme (HLY) was selected for recombinant production as a model for the heterologous protein. A tandem HLY construct fused with alpha-amylase (AmyB) was expressed by A. oryzae in which the Kex2 cleavage site was inserted at the upstream of HLY. HLY was successfully processed from AmyB and produced in the medium. We performed a systematic disruption analysis of five proteinase genes (pepA, pepE, alpA, tppA, and palB) in the HLY-producing strain with the adeA selectable marker. Comparative analysis indicated that disruption of the tppA gene encoding a tripeptidyl peptidase resulted in the highest increase (36%) in the HLY production. We further deleted the tppA gene in the pepE or palB disruptant with another selectable marker, argB. Consequently, a double disruption of the tppA and pepE genes led to a 63% increase in the HLY production compared to the control strain. This is the first study to report that the double disruption of the tppA and pepE genes improved the production level of a heterologous protein by filamentous fungi.