基于增强青霉淀粉诱导信号的新型表达系统构建及优化

Filamentous fungi, which have excellent capability in protein expression, is not only the resource library of glycoside hydrolases and fungal metabolites, but also an important expression host of high-added value proteins. In this study, we remodeling the amylases expression pathway by dominantly activating G protein-cAMP pathway, overexpressing amylase transcriptional activator AmyR and deleting the protease activator of PrtT and cellodextrin transporters CdtD and CdtG in Penicillium oxalicum. As a result, the efficiency of amylase Amy15A promoter could be highly enhanced. Moreover, a “concentrated” promoter would be obtained by deleting the redundancy part of amy15A promoter. Then, an efficient protein expression vector would be constructed with hybrid-synthetic promoter, which formed by series-connecting multiple copies of the “concentrated” amy15A promoters or inserting multiple copies of the AmyR binding sequence into the upstream of “concentrated” promoter. Finally, a host strain will be achieved with cellulase-free background by using the economic carbon source starch, which could not induce cellulases expression. In addition, a host with lowest background would be obtained by identifying and deleting those few proteins that secreted by Penicillium oxalicum on starch. The new style expression system not only helps efficient expression and rapid identification of those “silencing proteins” in filamentous fungi on a large scale, but also give a better choice for expression of those high value-added pharmaceutical proteins.

丝状真菌不仅是糖苷水解酶及真菌代谢产物的资源库，同时还是高附加值蛋白的重要表达宿主。以草酸青霉为研究对象，通过激活G蛋白-cAMP途径、过表达淀粉酶转录激活因子AmyR、敲除蛋白酶转录激活因子PrtT及纤维寡糖转运蛋白CdtD和CdtC等近年新发现与淀粉酶Amy15A特异相关的调控因子来增强淀粉诱导信号，使Amy15A启动子效率得到大幅提升。然后，再对Pamy15A启动子进行去冗余分析，通过串联“浓缩”的启动子或插入上游激活序列构建杂合启动子，再进一步提高Pamy15A的启动效率。最后，通过以淀粉为碳源，在无诱导纤维素酶分泌的同时敲除该条件下胞外分泌的少数蛋白从而获得极低分泌背景的宿主菌，并且将该宿主和包含改造后启动子的载体组成能以淀粉为唯一碳源的低背景高效表达系统。该新型表达系统的建成不仅有利于丝状真菌中重要酶性质研究和蛋白资源的规模化挖掘，同时也将为高附加值药用蛋白的表达提供更好选择。

丝状真菌不仅是糖苷水解酶及真菌代谢产物的资源库，同时还是高附加值蛋白的重要表达宿主。项目以草酸青霉为研究对象，通过优化碳源，激活G蛋白-cAMP途径、过表达淀粉酶转录激活因子AmyR、敲除碳代谢阻遏因子CreA等转录调控因子使得淀粉酶Amy15A启动子效率得到大幅提升。通过以淀粉为碳源，在无诱导纤维素酶分泌的同时敲除该条件下胞外分泌的主要蛋白Amy15A以及Amy13A， 从而获得极低分泌背景的宿主菌。同时，构建了含Amy15A启动子（P15A）和终止子（T15A）的通用蛋白表达载体，将宿主和通用表达载体组成能以淀粉为唯一碳源的低背景高效表达系统。最后，以该表达系统为基础，对本源“沉默”的蛋白以及多种真核宿主来源的酶类蛋白及药物多肽进行表达，其中酶类蛋白能获得了较好的分泌。该丝状真菌表达系统的成功构建，为丝状真菌中重要酶性质研究和蛋白资源的规模化挖掘提供更好选择。此外，在进行人源干扰素（HuIFN-β）及虎纹捕鸟蛛来源的药用多肽克胰素（HWTX-XI）的表达时，结果并未检测到靶标蛋白的胞外分泌，推测可能被青霉自身蛋白酶降解所致。研究中还发现过表达蛋白酶转录激活因子PrtT时，SDS-PAGE上显示一个大小为20 kDa的蛋白显著增强，可能有助于进一步揭示PrtT的调控机制。