里氏木霉是一種具有巨大蛋白分泌潛力的微生物,并且一直以來(lái)被認(rèn)為是安全的工業(yè)生產(chǎn)菌株,在同源蛋白表達(dá)方面,該表達(dá)系統(tǒng)已滿足工業(yè)生產(chǎn)的要求,且質(zhì)量?jī)?yōu)異,產(chǎn)量巨大。但在異源蛋白表達(dá)方面,除僅有的數(shù)種異源蛋白表達(dá)量滿足工業(yè)化生產(chǎn)外,該表達(dá)系統(tǒng)尚不足以進(jìn)行工業(yè)化生產(chǎn)。隨著近年來(lái)里氏木霉基因組學(xué)和蛋白組學(xué)的相關(guān)研究取得突飛猛進(jìn)的發(fā)展,限制異源表達(dá)的瓶頸可能隨時(shí)被打破,另一個(gè)類似于酵母表達(dá)系統(tǒng)穩(wěn)定的異源蛋白表達(dá)系統(tǒng)即將產(chǎn)生。
本研究構(gòu)建的里氏木霉外源基因表達(dá)載體 Ppth15,使用了里氏木霉zui強(qiáng)效的啟動(dòng)子 Pcbh1,并引入 CBH1 自身信號(hào)肽來(lái)介導(dǎo)目的蛋白的分泌性表達(dá)。通過(guò)引物設(shè)計(jì),在 Pcbh1 上游引入 Sal I-Not I 2 個(gè)酶切位點(diǎn),其目的是為后續(xù)實(shí)驗(yàn),如啟動(dòng)子的替換、基因改造等預(yù)留空間;在啟動(dòng)子下游使用 Spe I-EcoR Ⅴ-Afl II 3 個(gè)常用酶切位點(diǎn)構(gòu)成了多克隆位點(diǎn),后續(xù)工作還可以在這個(gè)位點(diǎn)插入多種不同的酶切位點(diǎn)。根據(jù)坎貝爾機(jī)制,本實(shí)驗(yàn)使用里氏木霉 CBH1 的啟動(dòng)子 Pcbh1 與終止子 Tcbh1 形成兩段同源性序列,以達(dá)到將目的基因表達(dá)框與里氏木霉 CBH1 表達(dá)框進(jìn)行定向整合及替換的目的。
表達(dá)載體 Ppth15 上搭載了潮霉素磷酸轉(zhuǎn)移酶基因 (HygB),啟動(dòng) HygB 表達(dá)的是三磷酸甘油醛脫氫酶基因啟動(dòng)子(PgpdA),PgpdA 是組成型啟動(dòng)子,故當(dāng) HygB 整合入里氏木霉基因組后,它的翻譯和表達(dá)不受培養(yǎng)基營(yíng)養(yǎng)成分的影響,由此可使陽(yáng)性轉(zhuǎn)化子的篩選工作簡(jiǎn)單化。本實(shí)驗(yàn)成功地在里氏木霉中表達(dá)了綠色熒光蛋白,通過(guò)熒光顯微鏡可以清楚地觀察到菌絲的頂端、隔膜及培養(yǎng)基中有大量熒光,這與絲狀真菌胞外蛋白主要通過(guò)菌絲頂端分泌的論斷一致。
但我們還發(fā)現(xiàn)一個(gè)有趣的現(xiàn)象,在橫隔附近的細(xì)胞壁上,也可以看到有 eGFP 的堆積,分析原因可能是當(dāng)橫隔小孔轉(zhuǎn)運(yùn) eGFP 的能力飽和后,造成 eGFP 的堆積,誘導(dǎo)宿主激活另一種蛋白分泌機(jī)制,同樣的現(xiàn)象在黑曲霉表達(dá)中也有報(bào)道。通過(guò)對(duì)陽(yáng)性菌株的發(fā)酵上清濃縮后進(jìn)行 SDS-PAGE 分析,可以在預(yù)計(jì)大小位置觀察到條帶,這證明了綠色熒光蛋白被成功表達(dá)并分泌到了胞外。不可否認(rèn)的是,在發(fā)酵液未經(jīng)濃縮處理前,eGFP 的 SDS-PAGE 條帶是不清晰的,說(shuō)明表達(dá)量尚未達(dá)到zui大化、發(fā)酵條件也并非zui優(yōu)。
后續(xù)實(shí)驗(yàn)可以在發(fā)酵條件、密碼子優(yōu)化,啟動(dòng)子修飾及選用蛋白酶缺陷菌株上進(jìn)行綜合優(yōu)化、由此來(lái)提高外源蛋白的表達(dá)量。里氏木霉外源基因表達(dá)載體 Ppth15 的成功構(gòu)建為進(jìn)一步研究里氏木霉表達(dá)其他外源蛋白提供了有效的載體工具,綠色熒光蛋白的表達(dá)揭示了外源蛋白在里氏木霉中分泌的過(guò)程和特點(diǎn),為里氏木霉高產(chǎn)工程菌株的培育提供了研究方向。
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