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        絲狀真菌纖維素酶合成誘導(dǎo)及轉(zhuǎn)錄調(diào)控

        2016-12-12 05:01:42張飛白鳳武趙心清
        生物工程學(xué)報(bào) 2016年11期
        關(guān)鍵詞:絲狀里氏木霉

        張飛,白鳳武,,趙心清

        1 大連理工大學(xué) 生命科學(xué)與技術(shù)學(xué)院,遼寧 大連 116024 2 微生物代謝國(guó)家重點(diǎn)實(shí)驗(yàn)室 上海交通大學(xué) 生命科學(xué)技術(shù)學(xué)院,上海 200240

        絲狀真菌纖維素酶合成誘導(dǎo)及轉(zhuǎn)錄調(diào)控

        張飛1,白鳳武1,2,趙心清2

        1 大連理工大學(xué) 生命科學(xué)與技術(shù)學(xué)院,遼寧 大連 116024 2 微生物代謝國(guó)家重點(diǎn)實(shí)驗(yàn)室 上海交通大學(xué) 生命科學(xué)技術(shù)學(xué)院,上海 200240

        張飛, 白鳳武, 趙心清. 絲狀真菌纖維素酶合成誘導(dǎo)及轉(zhuǎn)錄調(diào)控. 生物工程學(xué)報(bào), 2016, 32(11): 1481-1495.

        Zhang F, Bai FW, Zhao XQ. Induction and regulation of cellulase expression in filamentous fungi: a review. Chin J Biotech, 2016, 32(11): 1481-1495.

        利用廉價(jià)可再生木質(zhì)纖維素資源水解產(chǎn)生的可發(fā)酵糖生產(chǎn)生物能源和生物基化學(xué)品是近年來國(guó)內(nèi)外研究的熱點(diǎn)。纖維素酶酶解是木質(zhì)纖維素原料生物降解的重要手段,但目前纖維素酶生產(chǎn)成本過高,限制了纖維素生物轉(zhuǎn)化和生物煉制的工業(yè)化應(yīng)用。對(duì)絲狀真菌纖維素酶基因表達(dá)和調(diào)控進(jìn)行研究,有利于進(jìn)一步選育纖維素酶高產(chǎn)菌株,降低纖維素酶生產(chǎn)成本。隨著高通量測(cè)序及絲狀真菌遺傳操作等技術(shù)的進(jìn)步,對(duì)絲狀真菌纖維素酶誘導(dǎo)和基因表達(dá)調(diào)控機(jī)理有了更深入的認(rèn)識(shí)。本文綜述了近年來絲狀真菌纖維素酶誘導(dǎo)和纖維素酶基因表達(dá)調(diào)控的最新進(jìn)展,重點(diǎn)論述糖轉(zhuǎn)運(yùn)蛋白、轉(zhuǎn)錄因子和染色質(zhì)重塑對(duì)纖維素酶表達(dá)調(diào)控的影響,并對(duì)利用人工鋅指蛋白進(jìn)行絲狀真菌纖維素酶誘導(dǎo)調(diào)控研究進(jìn)行了展望。

        纖維素酶,基因表達(dá)調(diào)控,轉(zhuǎn)錄因子,里氏木霉

        利用可再生生物質(zhì)資源生產(chǎn)生物能源和生物基化學(xué)品對(duì)實(shí)現(xiàn)經(jīng)濟(jì)可持續(xù)發(fā)展具有重要意義。由于化石能源的不可再生,以及其燃燒利用后產(chǎn)生的全球變暖等環(huán)境效應(yīng),發(fā)展生物能源作為化石能源的替代品受到普遍關(guān)注。木質(zhì)纖維素生物質(zhì)是地球上儲(chǔ)量豐富的廉價(jià)可再生生物質(zhì)資源,通過其降解及發(fā)酵產(chǎn)生燃料乙醇,是國(guó)內(nèi)外學(xué)者關(guān)注的熱點(diǎn)[1]。木質(zhì)纖維素主要由纖維素、半纖維素和多酚類的木質(zhì)素組成,這些成分構(gòu)成緊密交聯(lián)的天然屏障,這種緊密的結(jié)構(gòu)使生物質(zhì)具有極強(qiáng)的抗逆性,因此需要利用木質(zhì)纖維素降解酶進(jìn)行水解,釋放可發(fā)酵糖。目前限制木質(zhì)纖維素原料高效生物轉(zhuǎn)化的瓶頸之一是纖維素降解酶的生產(chǎn)成本較高,因此,研究如何提高纖維素酶的高效生產(chǎn)成為當(dāng)前生物技術(shù)研究的重要內(nèi)容。

        對(duì)纖維素的高效分解和利用的微生物幾乎分布在整個(gè)真菌界中,因此真菌被認(rèn)為是自然界中纖維素物質(zhì)的主要降解微生物。絲狀真菌分泌的纖維素酶是一類聚合酶,可以通過酶催化模式與結(jié)構(gòu)特征將其酶系分為3類,分別是內(nèi)切葡聚糖酶,或稱1,4-β-D-葡聚糖-葡聚糖水解酶 (EC3.2.1.4),簡(jiǎn)稱EG (Endo-glucanase);外切葡聚糖酶,或稱1,4-β-D-葡聚糖-纖維二糖水解酶 (EC3.2.1.91),簡(jiǎn)稱CBH (Cellobiohydrolase)和β-葡萄糖苷酶 (EC3.2.1.21),簡(jiǎn)稱BGL (β-glucosidase),上述主要3類纖維素酶通過協(xié)同作用來對(duì)纖維素進(jìn)行降解[2]。纖維素降解除了纖維素酶起主要作用,一些輔助蛋白對(duì)纖維素的輔助作用近年來也受到普遍關(guān)注,這些輔助蛋白包括膨脹素 (Swollenin)[3]和溶解性多糖單加氧酶 (Lytic polysaccharide monooxygenases, LPMOs)[4]。當(dāng)前研究較深入的產(chǎn)纖維素酶真菌主要是里氏木霉Trichoderma reesei、草酸青霉Penicillium oxalicum、黑曲霉Aspergillus niger和粗糙脈孢菌Neurospora crassa等[5],其中里氏木霉是工業(yè)上的主要纖維素酶產(chǎn)生菌[6]。過去幾十年,對(duì)絲狀真菌纖維素酶的誘導(dǎo)及調(diào)控有了初步的了解,國(guó)內(nèi)學(xué)者近年來也對(duì)絲狀真菌纖維素酶誘導(dǎo)物及調(diào)控機(jī)理進(jìn)行了綜述[7-9],但目前對(duì)2013年以來報(bào)道的結(jié)果涉及很少,而近兩年來,大量最新研究結(jié)果進(jìn)一步揭示了絲狀真菌誘導(dǎo)產(chǎn)生纖維素酶的新機(jī)理。本文對(duì)纖維素酶的誘導(dǎo)及表達(dá)調(diào)控的最新進(jìn)展進(jìn)行了全面綜述,將碳信號(hào)與纖維素酶誘導(dǎo)和轉(zhuǎn)錄調(diào)控等過程整合,系統(tǒng)整理了纖維素酶誘導(dǎo)表達(dá)調(diào)控的分子機(jī)理。

        1 纖維素酶初始誘導(dǎo)

        木質(zhì)纖維素底物常以不可溶形式存在,影響了其直接被吸收利用。因此真菌需要在纖維素底物存在時(shí)形成基本的纖維素酶來完成底物的初步降解和胞內(nèi)纖維素酶基因的大量誘導(dǎo),這個(gè)過程是真菌在自然界中生存的基礎(chǔ)。目前實(shí)現(xiàn)初始誘導(dǎo)有幾種不同假說。第一種假說是少量的特定纖維素酶本底組成型表達(dá),并初步對(duì)纖維素進(jìn)行降解,釋放少量的寡糖,這些寡糖進(jìn)入細(xì)胞內(nèi)誘導(dǎo)后續(xù)的纖維素酶合成。該假說的依據(jù)是當(dāng)有抗纖維素酶抗體存在時(shí),可以阻止細(xì)胞內(nèi)纖維素酶誘導(dǎo)形成[10];另外,里氏木霉在非誘導(dǎo)條件下,可以檢測(cè)到少量纖維二糖水解酶Cel7A和Cel7B[11]。第二種假說是分子孢子表面存在一系列不同的纖維素降解酶可參與纖維素底物的最初降解,這些酶的去除能嚴(yán)重影響孢子在纖維素條件下的萌發(fā)過程[12],在沒有外界誘導(dǎo)物存在下,里氏木霉孢子初始萌發(fā)階段也檢測(cè)到了大量糖苷水解酶基因的轉(zhuǎn)錄[13]。其次,在碳源耗盡20?30 h后,從菌絲體中可以檢測(cè)到纖維素酶的轉(zhuǎn)錄[14],但這些轉(zhuǎn)錄不是簡(jiǎn)單因?yàn)樘即x阻遏效應(yīng)去除的結(jié)果,可能是由于真菌細(xì)胞壁中釋放的多糖誘導(dǎo)纖維素酶產(chǎn)生。

        通過初始纖維素降解會(huì)產(chǎn)生寡糖片段、纖維二糖和葡萄糖,其中寡糖和纖維二糖可作為特異誘導(dǎo)物來激活后續(xù)大量纖維素酶的表達(dá)分泌。葡萄糖會(huì)產(chǎn)生較強(qiáng)的碳代謝阻遏效應(yīng),無法有效激活纖維素酶表達(dá)。纖維二糖可誘導(dǎo)里氏木霉纖維素酶的產(chǎn)生,在纖維素培養(yǎng)基中釋放的纖維二糖被胞外β葡萄糖苷酶降解的效率不如進(jìn)入細(xì)胞內(nèi)轉(zhuǎn)化為誘導(dǎo)物效率高,更容易作為誘導(dǎo)物被利用[15]。里氏木霉中主要纖維素酶可以通過纖維素和一系列二糖衍生物,包括乳糖、纖維二糖和槐糖進(jìn)行誘導(dǎo),尤其槐糖對(duì)里氏木霉纖維素酶的誘導(dǎo)能力最顯著,相反果糖、葡萄糖和甘油卻沒有誘導(dǎo)效果[14]。里氏木霉基因組中至少有11個(gè)β葡萄糖苷酶,主要分布在糖苷水解酶1和3家族[16],其中胞內(nèi)的β葡萄糖苷酶轉(zhuǎn)糖苷作用對(duì)誘導(dǎo)物的形成很關(guān)鍵[17],最新研究結(jié)果顯示,β葡萄糖苷酶BGLII的V409F突變體β葡萄糖苷酶活性降低,反之轉(zhuǎn)糖苷活性明顯提高,將該突變體轉(zhuǎn)入里氏木霉后,纖維素酶活性也相應(yīng)提高[18]。當(dāng)培養(yǎng)基中加入β葡萄苷酶抑制劑野艽霉素 (Nojirimycin)后,槐糖的誘導(dǎo)能力消失,推測(cè)最終的誘導(dǎo)過程可能還有其他轉(zhuǎn)糖苷產(chǎn)物參與[19]。因此,絲狀真菌產(chǎn)纖維素酶的真正誘導(dǎo)物,以及如何誘導(dǎo)還需要進(jìn)一步深入研究。

        2 糖轉(zhuǎn)運(yùn)蛋白

        纖維二糖和其轉(zhuǎn)糖苷產(chǎn)物寡糖都可以作為里氏木霉的纖維素酶誘導(dǎo)物,而寡糖和纖維二糖進(jìn)入細(xì)胞時(shí),糖轉(zhuǎn)運(yùn)蛋白對(duì)這些誘導(dǎo)物的運(yùn)輸和功能行使起到關(guān)鍵的作用。糖轉(zhuǎn)運(yùn)蛋白在纖維素酶誘導(dǎo)過程中的作用早有報(bào)道[20]。在草酸青霉中分別過表達(dá)CdtC、CdtD和CdtG,可以明顯提高纖維素酶產(chǎn)量[21]。通過對(duì)粗糙脈孢菌在纖維素條件下的轉(zhuǎn)錄組數(shù)據(jù)進(jìn)行分析,發(fā)現(xiàn)了2個(gè)重要的糖轉(zhuǎn)運(yùn)蛋白CDT-1和CDT-2,其缺失突變體在微晶纖維素上無法誘導(dǎo)產(chǎn)生纖維素酶。除了功能性轉(zhuǎn)運(yùn)蛋白之外,還有一類膜蛋白具有與糖轉(zhuǎn)運(yùn)蛋白相同的蛋白結(jié)構(gòu),但是這類蛋白不具有糖轉(zhuǎn)運(yùn)的功能,機(jī)能上傾向于感受纖維素誘導(dǎo)信號(hào),為了驗(yàn)證這2個(gè)糖轉(zhuǎn)運(yùn)蛋白的碳信號(hào)感應(yīng)功能,研究人員突變了CDT-1和CDT-2中糖轉(zhuǎn)運(yùn)功能關(guān)鍵氨基酸殘基,發(fā)現(xiàn)二者的突變體對(duì)纖維二糖下的纖維素酶誘導(dǎo)能力沒有影響,說明這2種糖轉(zhuǎn)運(yùn)蛋白不但具有轉(zhuǎn)運(yùn)寡糖功能,對(duì)纖維素酶誘導(dǎo)信號(hào)的介導(dǎo)也具有重要作用[22]。最近在粗糙脈孢菌中發(fā)現(xiàn)的CLP1蛋白,其與CDT-2蛋白序列高度同源,結(jié)構(gòu)預(yù)測(cè)也是一種糖轉(zhuǎn)運(yùn)蛋白。通過實(shí)驗(yàn)分析發(fā)現(xiàn)CLP1沒有糖轉(zhuǎn)運(yùn)蛋白功能,其更多參與到了纖維素酶誘導(dǎo)信號(hào)轉(zhuǎn)導(dǎo)過程中。同時(shí)敲除CDT-2、CLP1和胞內(nèi)三類β葡萄糖苷酶基因的突變體菌株,可使纖維二糖誘導(dǎo)能力提高到出發(fā)菌株的6.9倍,CLP1和CDT-2共同作用來識(shí)別和傳遞纖維二糖信號(hào),并控制纖維素酶誘導(dǎo)途徑下游基因的表達(dá)[23]。

        以上這種碳信號(hào)識(shí)別效應(yīng)在里氏木霉中也有存在,在里氏木霉中發(fā)現(xiàn)的糖轉(zhuǎn)運(yùn)蛋白Stp1和Crt1在誘導(dǎo)物進(jìn)入細(xì)胞和纖維素酶誘導(dǎo)過程中發(fā)揮不同的作用。stp1基因的敲除會(huì)大量激活在纖維素培養(yǎng)基中的纖維素酶誘導(dǎo)產(chǎn)生,在纖維二糖培養(yǎng)基中表現(xiàn)出了相反的效果,但在槐糖條件下卻沒有影響,說明Stp1的轉(zhuǎn)運(yùn)在纖維素酶誘導(dǎo)信號(hào)的傳遞過程中沒有起到積極的作用[24]。而另外一個(gè)糖轉(zhuǎn)運(yùn)蛋白Crt1盡管也定位在細(xì)胞膜上,但其表現(xiàn)出了不同的趨勢(shì),Crt1對(duì)纖維二糖與槐糖的轉(zhuǎn)運(yùn)與消耗并沒有影響,但對(duì)誘導(dǎo)物乳糖的轉(zhuǎn)運(yùn)起到了關(guān)鍵的作用,Crt1的缺失使細(xì)胞無法正常利用乳糖來生長(zhǎng),而且同時(shí)也會(huì)使乳糖和槐糖的纖維素酶誘導(dǎo)效果消失[25],說明糖轉(zhuǎn)運(yùn)蛋白在纖維素酶誘導(dǎo)過程中不僅僅起到了轉(zhuǎn)運(yùn)寡糖的作用,而且在碳信號(hào)識(shí)別、傳遞功能上也會(huì)發(fā)揮很關(guān)鍵的作用。

        3 轉(zhuǎn)錄調(diào)控因子

        絲狀真菌內(nèi)纖維素酶基因的表達(dá)多數(shù)受轉(zhuǎn)錄因子調(diào)控,這些轉(zhuǎn)錄因子響應(yīng)來自外界的碳源誘導(dǎo)物傳遞的信號(hào),從而對(duì)纖維素酶產(chǎn)生激活或者抑制的效應(yīng)。當(dāng)前研究較深入的里氏木霉中至少存在3個(gè)正調(diào)控轉(zhuǎn)錄因子Xyr1、Ace2、Ace3和2個(gè)負(fù)調(diào)控轉(zhuǎn)錄因子Cre1和Ace1參與纖維素酶的調(diào)控。

        里氏木霉中Xyr1是纖維素和半纖維素酶最主要的轉(zhuǎn)錄調(diào)控因子,Xyr1過表達(dá)可以提高纖維素酶轉(zhuǎn)錄表達(dá),其缺失導(dǎo)致所有已知誘導(dǎo)物失去誘導(dǎo)效果[26]。通過對(duì)Xyr1敲除轉(zhuǎn)錄組分析發(fā)現(xiàn),在不同碳源誘導(dǎo)條件下,Xyr1轉(zhuǎn)錄因子的變化可以影響部分轉(zhuǎn)錄因子、轉(zhuǎn)運(yùn)蛋白家族蛋白和大部分糖苷水解酶轉(zhuǎn)錄水平,并影響纖維素酶的誘導(dǎo)產(chǎn)生[27],而且Xyr1進(jìn)入細(xì)胞核發(fā)揮作用必須要有誘導(dǎo)物存在[28]。Xyr1是Zn2Cys6型鋅雙核簇轉(zhuǎn)錄因子,可結(jié)合特定核苷酸5′-GGC (A/T)4并行使調(diào)控功能,而在FTFRMH區(qū)域中的點(diǎn)突變 (A824V),不但會(huì)顯著提高菌株纖維素酶的誘導(dǎo)能力,還會(huì)消除木聚糖酶的誘導(dǎo)效應(yīng)[29]。在槐糖誘導(dǎo)物存在時(shí),Xyr1進(jìn)入細(xì)胞核內(nèi),誘導(dǎo)停止后Xyr1蛋白迅速降解[28],但目前還不清楚轉(zhuǎn)錄激活因子Xyr1如何從不同的誘導(dǎo)物中接受誘導(dǎo)信號(hào)。對(duì)黑曲霉、構(gòu)巢曲霉、里氏木霉、禾谷鐮刀菌和稻瘟病菌中的Xyr1基因功能分析發(fā)現(xiàn),在木聚糖誘導(dǎo)物中Xyr1轉(zhuǎn)錄因子對(duì)糖苷水解酶的誘導(dǎo)調(diào)控具有實(shí)質(zhì)性差異,在里氏木霉和黑曲霉中XlnR/Xyr1對(duì)纖維素酶調(diào)控有主要影響,構(gòu)巢曲霉和稻瘟病菌中纖維素酶基因調(diào)控不受XlnR的影響,而禾谷鐮刀菌中XlnR的缺失會(huì)提高其纖維素酶基因的轉(zhuǎn)錄和表達(dá)[30]。研究表明曲霉中的Xln1/R不同程度的磷酸化決定其功能,Noguchi等研究表明米曲霉中Xyr1轉(zhuǎn)錄因子同源基因Xln1,沒有木糖時(shí)Xln1具有不同的磷酸化程度,當(dāng)培養(yǎng)基中木糖增加時(shí),Xln1的磷酸化程度增加[31];黑曲霉中的XlnR也通過木糖誘導(dǎo),被磷酸化后可進(jìn)入細(xì)胞核,發(fā)揮轉(zhuǎn)錄調(diào)控功能[32]。

        里氏木霉中除了Xyr1,鋅雙核簇轉(zhuǎn)錄因子Ace2對(duì)纖維素酶起到正調(diào)控作用。體外實(shí)驗(yàn)表明Ace2可以結(jié)合Cel7A啟動(dòng)的GGCTAATAA核酸序列,Xyr1也可結(jié)合這個(gè)靶位點(diǎn),同時(shí)在Xyr1基因的啟動(dòng)子區(qū)域發(fā)現(xiàn)Ace2的潛在結(jié)合位點(diǎn),推測(cè)可能是Ace2可以微調(diào)Xyr1的表達(dá)來響應(yīng)不同的誘導(dǎo)信號(hào)。Ace2基因被敲除后會(huì)降低不同纖維素酶的轉(zhuǎn)錄量,并且在纖維素誘導(dǎo)條件下總酶活會(huì)下降30%?70%,但在槐糖誘導(dǎo)時(shí),Ace2的缺失對(duì)纖維素酶酶活沒有影響[33]。另外Ace2需要經(jīng)過磷酸化并以二聚體形式發(fā)揮調(diào)控功能[34],而在其他絲狀真菌中沒有發(fā)現(xiàn)Ace2的同源基因,說明了里氏木霉調(diào)控纖維素酶表達(dá)的特異性。對(duì)里氏木霉基因組分析發(fā)現(xiàn),其與其他絲狀真菌不同之處是纖維素酶和其他糖苷水解酶定位在一些分散的基因簇中,轉(zhuǎn)錄調(diào)控因子基因經(jīng)常與β葡萄糖苷酶和轉(zhuǎn)運(yùn)蛋白基因聚集在一起[35]。H?kkinen等利用這個(gè)特點(diǎn)鑒定出了幾個(gè)新的纖維素酶轉(zhuǎn)錄激活因子,其中Ace3在乳糖培養(yǎng)基中可顯著誘導(dǎo)纖維素酶基因表達(dá),敲除該基因會(huì)同時(shí)抑制纖維素酶、半纖維素酶的誘導(dǎo)表達(dá)[36]。

        粗糙脈孢菌產(chǎn)纖維素酶誘導(dǎo)機(jī)制和里氏木霉大不相同,盡管XlnR/Xyr1同源基因轉(zhuǎn)錄因子XLR-1對(duì)粗糙脈孢菌的木聚糖酶的調(diào)控很關(guān)鍵,但其敲除對(duì)纖維素酶的誘導(dǎo)調(diào)控影響甚微[37]。通過對(duì)粗糙脈孢菌轉(zhuǎn)錄因子敲除文庫(kù)篩選分析,獲得了粗糙脈孢菌中鋅雙核簇轉(zhuǎn)錄因子Clr-1和Clr-2,在纖維素條件下纖維素酶誘導(dǎo)產(chǎn)生作用非常顯著,盡管在木聚糖條件下對(duì)木聚糖酶誘導(dǎo)表達(dá)影響不大。當(dāng)Clr-1缺失時(shí),菌株無法在纖維素和纖維二糖上生長(zhǎng),而且也無法產(chǎn)生clr-2轉(zhuǎn)錄體[38]。Clr-2在纖維素酶調(diào)控過程中發(fā)揮更重要的作用,其組成型表達(dá)也會(huì)像里氏木霉中Xyr1突變體一樣,使纖維素酶的產(chǎn)生不受誘導(dǎo)物影響[38]。通過纖維素誘導(dǎo)條件下的組學(xué)分析發(fā)現(xiàn),受到Clr-1和Clr-2調(diào)控的基因包括主要的糖苷水解酶基因,蛋白分泌途徑相關(guān)的基因,以及內(nèi)質(zhì)網(wǎng)蛋白加工和修飾的相關(guān)基因,說明Clr-1和Clr-2對(duì)粗糙脈孢菌纖維素酶表達(dá)的重要作用[39]。研究表明,粗糙脈孢菌中Clr-1和Clr-2都分別以同源二聚體形式發(fā)揮功能,通過免疫共沉淀和轉(zhuǎn)錄組結(jié)合分析找出了Clr-1/-2和Xlr-1的直接靶點(diǎn)基因,Clr-1和Clr-2直接作用調(diào)控大量的纖維素酶和糖轉(zhuǎn)運(yùn)蛋白;而Xlr-1雖然也直接調(diào)控大量胞外分泌蛋白,但并不直接作用在纖維素酶啟動(dòng)子區(qū)域,主要結(jié)合基因是木聚糖降解酶基因,并且其缺失只對(duì)纖維素酶的產(chǎn)生有微弱影響[37,40]。絲狀真菌中廣泛存在clr-1和clr-2的同源基因來調(diào)控纖維素酶的轉(zhuǎn)錄和表達(dá),如構(gòu)巢曲霉中的同源基因clrA和clrB。構(gòu)巢曲霉中調(diào)控纖維素酶表達(dá)的是ClrB,其對(duì)纖維素酶基因表達(dá)至關(guān)重要,但ClrA的缺失也同Clr-1缺失一樣會(huì)影響纖維二糖的利用[39]。相同現(xiàn)象也在草酸青霉中發(fā)現(xiàn),在草酸青霉中,ClrB是纖維素酶基因主要調(diào)控因子,XlnR更多的調(diào)節(jié)半纖維素酶基因發(fā)揮重要作用[41]。最近對(duì)黑曲霉研究發(fā)現(xiàn)ClrA和ClrB的作用功能與粗糙脈孢菌中的同源基因有相似趨勢(shì),ClrB對(duì)糖苷水解酶的調(diào)控有一定作用,但黑曲霉胞內(nèi)的轉(zhuǎn)錄因子XlnR在纖維素酶調(diào)控過程中卻十分重要而顯著,同時(shí)XlnR還正調(diào)控著ClrA/ClrB的轉(zhuǎn)錄[42]。這些結(jié)果都說明了XlnR/Xyr1和ClrB兩組轉(zhuǎn)錄調(diào)控因子在絲狀真菌纖維素酶誘導(dǎo)中的關(guān)鍵作用,二者相互關(guān)聯(lián),但在不同的真菌中這兩類轉(zhuǎn)錄因子對(duì)纖維素酶誘導(dǎo)調(diào)控機(jī)制不同。

        除了以上所論述的轉(zhuǎn)錄激活因子,在里氏木霉中也發(fā)現(xiàn)纖維素酶誘導(dǎo)相關(guān)的轉(zhuǎn)錄抑制因子。Ace1是一個(gè)C2H2型鋅指蛋白抑制因子,其特異結(jié)合序列是5′-AGGGA。敲除ace1的突變體,可以顯著增加在纖維素和槐糖誘導(dǎo)條件下的主要纖維素酶和半纖維素酶[43]。研究表明,ace1基因敲除突變株在木糖條件下xyr1基因的表達(dá)顯著提高,促進(jìn)纖維素酶合成。在構(gòu)巢曲霉中,ace1基因的同源基因stzA編碼對(duì)氨基酸代謝相關(guān)基因的壓力響應(yīng)因子,其可潛在結(jié)合在cpcA基因的啟動(dòng)子區(qū)域,相應(yīng)地在stzA/ace1的啟動(dòng)子區(qū)域也具有CpcA的潛在結(jié)合位點(diǎn)[44];同時(shí)在里氏木霉培養(yǎng)基中加入蛋氨酸后可增強(qiáng)纖維素酶的表達(dá)[45]。以上結(jié)果也說明了培養(yǎng)基中氨基酸種類和纖維素酶基因表達(dá)有著密切的聯(lián)系。

        里氏木霉纖維素酶誘導(dǎo)培養(yǎng)基中含有高濃度葡萄糖時(shí) (葡萄糖濃度大于0.5%),葡萄糖會(huì)激活碳代謝阻遏 (CCR),不會(huì)有纖維素酶產(chǎn)生,當(dāng)培養(yǎng)基中過量葡萄糖耗盡時(shí),誘導(dǎo)物會(huì)繼續(xù)發(fā)揮作用使得纖維素酶產(chǎn)生[14]。葡萄糖的碳代謝阻遏效應(yīng)表現(xiàn)在兩個(gè)方面,一方面,葡萄糖可抑制糖轉(zhuǎn)運(yùn)蛋白對(duì)二糖誘導(dǎo)物的轉(zhuǎn)運(yùn)能力,降低細(xì)胞對(duì)誘導(dǎo)物的攝入[15];另一方面,葡萄糖可抑制里氏木霉中主要的纖維素酶基因的轉(zhuǎn)錄[14]。葡萄糖碳代謝阻遏和纖維素酶誘導(dǎo)條件下比較發(fā)現(xiàn),至少在糖轉(zhuǎn)運(yùn)蛋白和轉(zhuǎn)錄因子在轉(zhuǎn)錄水平上存在顯著差距[46]。

        研究表明里氏木霉C2H2型轉(zhuǎn)錄因子Cre1在CCR過程中起重要調(diào)控作用[47]。在里氏木霉、粗糙脈孢菌中大量的纖維素酶、半纖維素酶轉(zhuǎn)錄都受到CreA/Cre1因子的抑制作用,其也會(huì)對(duì)轉(zhuǎn)錄激活因子Xyr1產(chǎn)生負(fù)效應(yīng),最終會(huì)抑制大量纖維素酶的轉(zhuǎn)錄表達(dá)[48-49]。Cre1結(jié)合位點(diǎn)是兩個(gè)臨近的5'-SYGGRG核酸序列,通過這個(gè)核酸序列可以直接激活Cre1的抑制效應(yīng)。Cre1轉(zhuǎn)錄因子以雙重調(diào)節(jié)形式存在,Cre1除了可以直接作用在相應(yīng)糖苷水解酶基因的啟動(dòng)子區(qū)域來進(jìn)行調(diào)控,其還間接作用于其他功能基因來抑制纖維素酶的產(chǎn)生。如里氏木霉中cre1基因的缺失或者在其結(jié)構(gòu)基因結(jié)合位點(diǎn) (5′-SYGGRG)的突變會(huì)導(dǎo)致主要纖維素酶基因cbh1和半纖維素酶基因xyn1的去阻遏效應(yīng)[50];同時(shí)通過cre1基因缺失的轉(zhuǎn)錄組結(jié)果表明,Cre1轉(zhuǎn)錄因子可以間接調(diào)控纖維素酶轉(zhuǎn)錄激活因子、轉(zhuǎn)運(yùn)蛋白家族蛋白和部分糖苷水解酶,從而抑制纖維素酶的誘導(dǎo)和產(chǎn)生[51-52]。在構(gòu)巢曲霉中也存在上述雙重調(diào)節(jié)現(xiàn)象,CreA不但抑制正調(diào)控轉(zhuǎn)錄因子XlnR的轉(zhuǎn)錄,而且CreA還直接結(jié)合抑制木質(zhì)纖維素降解酶xlnA基因的表達(dá)[53-54]。工業(yè)菌株T. reesei Rut-C30中的cre1基因缺失了一個(gè)鋅指蛋白結(jié)構(gòu),因此其體內(nèi)的纖維素酶轉(zhuǎn)錄表達(dá)具有一定能力的葡萄糖耐受性[55],不過單獨(dú)的由于cre1缺失產(chǎn)生的碳代謝阻遏消除并不能有效的產(chǎn)纖維素酶,細(xì)胞生長(zhǎng)環(huán)境中誘導(dǎo)物的存在是產(chǎn)纖維素酶所必需的[56]。

        纖維二糖普遍被認(rèn)為作為一個(gè)誘導(dǎo)物前體存在,里氏木霉在纖維二糖培養(yǎng)基中,誘導(dǎo)初期轉(zhuǎn)錄因子BglR可以特異上調(diào)胞內(nèi)β葡萄糖苷酶基因bgl1轉(zhuǎn)錄來誘導(dǎo)纖維素酶的初步產(chǎn)生,但隨著降解的葡萄糖量增多會(huì)產(chǎn)生碳代謝阻遏效應(yīng),因此在纖維二糖培養(yǎng)基中,轉(zhuǎn)錄因子BglR的缺失會(huì)提高纖維素酶產(chǎn)量[57]。里氏木霉中葡萄糖木糖-脫氫酶蛋白Grd1對(duì)纖維素酶表達(dá)具有正向的效應(yīng),同時(shí)其也具有降解胞內(nèi)纖維二糖的作用,但效率很低,不足以影響胞內(nèi)纖維二糖作為基礎(chǔ)產(chǎn)生下一步的纖維素酶誘導(dǎo)信號(hào)[58]。最新研究結(jié)果表明,鋅指蛋白轉(zhuǎn)錄因子Crz1可特異地響應(yīng)環(huán)境中的Ca2+信號(hào),對(duì)纖維素酶基因轉(zhuǎn)錄有激活作用,體外實(shí)驗(yàn)證明Crz1可直接作用在xyr1和cbh1基因的啟動(dòng)子區(qū)域,并且與纖維素酶轉(zhuǎn)錄抑制因子Cre1競(jìng)爭(zhēng)相同的作用靶點(diǎn)[59]。在棘孢曲霉中轉(zhuǎn)錄因子ClbR可以誘導(dǎo)一些不受XlnR調(diào)控的基因來響應(yīng)纖維二糖和纖維素信號(hào)[60]。粗糙脈孢菌中轉(zhuǎn)錄因子Vib-1介導(dǎo)細(xì)胞程序性死亡,在碳饑餓條件下其參與到胞外蛋白水解酶分泌。vib-1基因缺失的突變菌株會(huì)增強(qiáng)細(xì)胞內(nèi)的CCR效應(yīng),從而抑制粗糙脈孢菌中主要轉(zhuǎn)錄因子Clr-2的轉(zhuǎn)錄表達(dá),后者影響胞外主要纖維素酶的轉(zhuǎn)錄和表達(dá)[61]。

        糖轉(zhuǎn)運(yùn)蛋白及轉(zhuǎn)錄因子對(duì)絲狀真菌纖維素酶的誘導(dǎo)見圖1。

        圖1 里氏木霉纖維素酶調(diào)控示意圖[62]Fig. 1 Schematic diagram for the regulation of cellulase genes in Trichoderma reesei (Modified from the reference[62]).

        除了以上纖維素酶轉(zhuǎn)錄調(diào)控因子之外,近年來還發(fā)現(xiàn)了其他轉(zhuǎn)錄調(diào)控因子對(duì)纖維素酶的產(chǎn)生起到了重要的作用。米曲霉中轉(zhuǎn)錄因子ManR最初被認(rèn)為是甘露聚糖基因相關(guān)的調(diào)控因子,研究表明至少參與5種主要纖維素酶的調(diào)控,和XlnR轉(zhuǎn)錄因子共同對(duì)主要纖維素酶的調(diào)控表達(dá)[63]。草酸青霉中AmyR是受到Clr-2和CreA調(diào)控的纖維素酶調(diào)控抑制因子,研究表明其主要功能是控制著淀粉和纖維素碳源利用的平衡。草酸青霉中AmyR的缺失會(huì)顯著提高纖維素酶的產(chǎn)生但同時(shí)會(huì)抑制淀粉酶基因的轉(zhuǎn)錄表達(dá)[41]。構(gòu)巢曲霉中的McmA是SRF型MADS box 調(diào)控蛋白,這一類調(diào)控蛋白時(shí)常與胞內(nèi)其他調(diào)控因子共同作用來發(fā)揮功能,McmA主要參與介導(dǎo)纖維二糖條件下的2個(gè)內(nèi)切酶和1個(gè)外切酶基因的轉(zhuǎn)錄調(diào)控[64]。

        很多纖維素酶轉(zhuǎn)錄調(diào)控蛋白,包括Xyr1、Cre1、Ace1、Ace2、PacC、Clr-1及Clr-2等都是鋅指蛋白。鋅指蛋白廣泛存在于多種微生物,對(duì)微生物的生長(zhǎng)和代謝具有重要調(diào)控作用[65]。C2H2型鋅指蛋白是最簡(jiǎn)單的轉(zhuǎn)錄因子調(diào)控模塊,可特異識(shí)別核酸序列中3個(gè)相鄰的核苷酸序列,根據(jù)鋅指蛋白獨(dú)特的模塊結(jié)構(gòu),將來源和結(jié)合位點(diǎn)不同的C2H2型鋅指模塊通過基因合成,得到三/四鋅指混合的鋅指文庫(kù),鋅指蛋白后加上激活/抑制功能域,便可得到人工鋅指蛋白轉(zhuǎn)錄因子文庫(kù)[65]。將此文庫(kù)轉(zhuǎn)化微生物后,人工轉(zhuǎn)錄因子可以隨機(jī)對(duì)細(xì)胞體內(nèi)不同基因的轉(zhuǎn)錄進(jìn)行調(diào)節(jié),因此可獲得特異表型提高的突變體。韓國(guó)學(xué)者使用人工鋅指蛋白文庫(kù)轉(zhuǎn)化大腸桿菌和酵母,獲得了耐溫性及耐藥性提高的突變體,證明人工轉(zhuǎn)錄因子對(duì)細(xì)胞代謝存在有效控制[66]。本課題組利用人工鋅指蛋白文庫(kù)轉(zhuǎn)化釀酒酵母,獲得了乙醇和乙酸耐性顯著提高的釀酒酵母突變體,并發(fā)現(xiàn)了人工鋅指蛋白可能調(diào)控的基因QDR3與釀酒酵母乙酸耐性相關(guān)[67]。近期本課題組也成功構(gòu)建了可在絲狀真菌中表達(dá)的人工鋅指蛋白文庫(kù),并轉(zhuǎn)化里氏木霉Rut-C30,獲得了多個(gè)纖維素酶產(chǎn)量提高的突變體[68]。通過纖維素酶活性顯著提高轉(zhuǎn)化子的轉(zhuǎn)錄組分析,進(jìn)一步確定了和纖維素酶轉(zhuǎn)錄調(diào)控相關(guān)的調(diào)控通路 (未發(fā)表資料)。人工轉(zhuǎn)錄因子作為新的工具可用于絲狀真菌纖維素酶的轉(zhuǎn)錄調(diào)控,并有希望挖掘新的纖維素酶的調(diào)控機(jī)制。

        除了轉(zhuǎn)錄因子之外,泛素化的相關(guān)蛋白也存在于構(gòu)巢曲霉和里氏木霉的碳代謝抑制途徑中[69]。里氏木霉中泛素羧基末端水解酶Cre2的敲除使不同誘導(dǎo)碳源條件下纖維素酶表達(dá)都有提高,盡管其誘導(dǎo)效果沒有Cre1敲除顯著[70]。而在構(gòu)巢曲霉中的F-box泛素化蛋白FbxA參與半纖維素酶的轉(zhuǎn)錄調(diào)控[71]。表1為近年來發(fā)現(xiàn)的不同絲狀真菌中纖維素酶和半纖維素酶轉(zhuǎn)錄調(diào)控因子的總結(jié)。

        4 染色質(zhì)調(diào)控及其他相關(guān)調(diào)控方式

        近期絲狀真菌纖維素酶誘導(dǎo)過程中染色質(zhì)重塑作用也受到了關(guān)注。研究表明,在cbh1基因啟動(dòng)子和編碼區(qū)區(qū)域內(nèi),誘導(dǎo)和阻遏條件下該基因的核小體定位不同。里氏木霉在槐糖誘導(dǎo)物存在時(shí)核小體只出現(xiàn)在啟動(dòng)子區(qū)域,此時(shí)核小體重新定位的區(qū)域使轉(zhuǎn)錄激活因子有效作用,同時(shí)抑制因子作用的區(qū)域被封閉;野生型里氏木霉在葡萄糖條件下,核小體定位在cbh1啟動(dòng)子和編碼區(qū)域,核小體在cbh1基因啟動(dòng)子上結(jié)合區(qū)域有兩段,一段是在-840 bp到-1 140 bp,其包括Ace1結(jié)合位點(diǎn),另一段是在-340 bp到-500 bp,與激活轉(zhuǎn)錄因子Xyr1和Ace2結(jié)合位點(diǎn)相鄰,核小體的存在阻礙了其靶結(jié)合序列。值得注意的是,此時(shí)截短或缺失的Cre1轉(zhuǎn)錄因子起不到與完整Cre1轉(zhuǎn)錄因子相同的效果,導(dǎo)致在Cbh1編碼區(qū)的核小體消失,說明Cre1不但可以直接作用在被抑制基因的啟動(dòng)子區(qū)域,也可以調(diào)節(jié)核小體重塑來調(diào)控轉(zhuǎn)錄[73]。整個(gè)過程中是否存在Cre1調(diào)控的染色質(zhì)重塑因子來參與有待于進(jìn)一步揭示。另外,里氏木霉中纖維素酶基因cel6A啟動(dòng)子區(qū)域的CCAAT盒與HAP2/3/5復(fù)合物和轉(zhuǎn)錄抑制因子Cre1結(jié)合,繼而會(huì)影響核小體的定位,從而對(duì)cel6A的轉(zhuǎn)錄起到了嚴(yán)格而特異的調(diào)節(jié)作用[74]。最近研究表明Xyr1對(duì)纖維素酶基因具有直接調(diào)控之外,還通過影響纖維素酶基因的染色質(zhì)狀態(tài)來影響基因的轉(zhuǎn)錄和表達(dá)。研究表明,即使在有槐糖誘導(dǎo)物條件下,xyr1基因的缺失也會(huì)使主要纖維素酶基因cbh1、cbh2的染色質(zhì)壓縮,以至于阻遏了有效地展開和轉(zhuǎn)錄,通過分析找出了10個(gè)受Xyr1調(diào)控并與染色質(zhì)重塑相關(guān)的基因,Xyr1直接作用還是受其調(diào)控的下游基因發(fā)揮作用有待于進(jìn)一步揭示[75]。

        在構(gòu)巢曲霉中,CreA的功能依賴于組蛋白乙?;癄顟B(tài),組蛋白酰基轉(zhuǎn)移酶GceE可以看作是CreA的共調(diào)控因子,激活或者抑制靶基因的轉(zhuǎn)錄[76]。研究表明,里氏木霉體內(nèi)gcn5基因編碼組氨酸乙?;D(zhuǎn)移酶,其對(duì)纖維素酶表達(dá)是必需的,該基因的缺失使在cbh1啟動(dòng)子區(qū)域核小體的H3K9和H3K14乙?;虲BH1的表達(dá)大大降低[77]。過表達(dá)另外一個(gè)CGN5乙?;D(zhuǎn)移酶,可以使纖維素酶表達(dá)增加一倍[36]。同時(shí)發(fā)現(xiàn)這個(gè)CGN5?;D(zhuǎn)移酶受到Lae1的調(diào)控,Lae1并不直接作用在纖維素酶的組蛋白上,而是間接調(diào)控纖維素酶的轉(zhuǎn)錄和表達(dá)[78]。

        表1 不同真菌中新發(fā)現(xiàn)的纖維素酶和半纖維素酶調(diào)控子Table 1 Novel candidate regulators for cellulase and hemicellulase genes of different fungi

        甲基轉(zhuǎn)移酶是具有對(duì)核蛋白甲基化的蛋白,在許多絲狀真菌中甲基轉(zhuǎn)移酶作為全局調(diào)控子影響次級(jí)代謝產(chǎn)物的表達(dá)。里氏木霉中的甲基轉(zhuǎn)移酶Lae1可調(diào)節(jié)細(xì)胞內(nèi)大量基因轉(zhuǎn)錄的變化,其中包括調(diào)控主要纖維素酶基因表達(dá),在缺失Lae1的突變體中,無法表達(dá)纖維素酶和其糖苷水解酶;Lae1過表達(dá)時(shí),這些糖苷水解酶會(huì)急劇增加,說明Lae1在纖維素酶表達(dá)中起重要作用,并且其功能和轉(zhuǎn)錄因子Xyr1相互依賴。研究表明,盡管Lae1在曲霉中的等位基因LaeA參與組蛋白甲基化修飾,在染色質(zhì)水平調(diào)控次級(jí)代謝產(chǎn)物的轉(zhuǎn)錄水平[79],但是里氏木霉中Lae1不直接對(duì)主要纖維素酶基因的組蛋白進(jìn)行甲基化修飾,其主要和vel1等基因產(chǎn)物形成VELVET蛋白復(fù)合物[80],這個(gè)復(fù)合物調(diào)節(jié)絲狀真菌中的次級(jí)代謝和糖苷水解酶基因[81]。在里氏木霉中VELVET復(fù)合物中的Lae1和Vel1缺失不誘導(dǎo)纖維素酶產(chǎn)生,并且VELVET的激活方式不受光影響,而其他真菌中光可抑制VELVET的形成和進(jìn)入細(xì)胞核[72]。

        蛋白磷酸化的轉(zhuǎn)錄后修飾可以調(diào)節(jié)蛋白功能、蛋白質(zhì)轉(zhuǎn)換、蛋白質(zhì)之間相互作用和胞內(nèi)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)等作用。通過對(duì)槐糖誘導(dǎo)條件下對(duì)里氏木霉蛋白磷酸化分析,發(fā)現(xiàn)共1 721個(gè)磷酸化位點(diǎn)。被磷酸化的蛋白主要參與信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄調(diào)控[82]。誘導(dǎo)條件下糖轉(zhuǎn)運(yùn)蛋白的磷酸化使細(xì)胞獲取胞外纖維素酶碳信號(hào)分子,同時(shí)也發(fā)現(xiàn)中心碳代謝的一些蛋白有磷酸化現(xiàn)象。另外轉(zhuǎn)錄因子Ace2、Cre1等的磷酸化對(duì)其在調(diào)控纖維素酶基因轉(zhuǎn)錄過程功能有重要影響[34,83]。最近研究表明,Hap2在誘導(dǎo)條件下磷酸化程度很高,但磷酸化對(duì)其功能的影響還需要進(jìn)一步研究[82]。除此之外,一些信號(hào)途徑中的蛋白酶具有明顯的磷酸化現(xiàn)象,其中包括腺苷酸環(huán)化酶(Acy1) 和光信號(hào)感應(yīng)蛋白 (VeA),RAS信號(hào)途徑組件和MAPK信號(hào)途徑組件中的蛋白。這些蛋白在胞外信號(hào)到胞內(nèi)傳遞過程中起到了關(guān)鍵的作用。在纖維素培養(yǎng)基中RAS信號(hào)組件對(duì)纖維素酶的誘導(dǎo)存在重要的調(diào)節(jié)作用,里氏木霉的Ras2蛋白可感應(yīng)和調(diào)節(jié)來自胞外的信號(hào),通過下游途徑介導(dǎo)纖維素酶基因的主要調(diào)控因子的表達(dá)和調(diào)控,繼而對(duì)纖維素酶產(chǎn)生進(jìn)行調(diào)節(jié)[84]。最近發(fā)現(xiàn)里氏木霉中促分裂原激活蛋白激酶 (MAPK) 途徑也參與纖維素酶的調(diào)控和表達(dá)。MAP激酶TMK3對(duì)纖維素酶產(chǎn)生有正調(diào)節(jié)效應(yīng)[85],而TMK2則對(duì)纖維素酶生產(chǎn)起抑制效果[86]。里氏木霉中另外一個(gè)來自MAPK家族的Ime2蛋白激酶的敲除顯著提高纖維素酶產(chǎn)生,并發(fā)現(xiàn)cre1和xyr1的轉(zhuǎn)錄量同時(shí)降低,推測(cè)可能受Cre1的磷酸化缺失影響[87]。

        5 展望

        近年來,越來越廣泛的系統(tǒng)生物學(xué)方法 (基因、轉(zhuǎn)錄、蛋白、代謝工具) 對(duì)揭示絲狀真菌感應(yīng)纖維素信號(hào)和纖維素酶誘導(dǎo)產(chǎn)生起到巨大推進(jìn)作用,在纖維素酶產(chǎn)生的轉(zhuǎn)錄和響應(yīng)調(diào)控方面的研究有了更多進(jìn)展,然而纖維素酶誘導(dǎo)機(jī)制、信號(hào)傳導(dǎo)途徑等過程的分子基礎(chǔ)還有許多不明之處有待揭示。同時(shí)基因組、轉(zhuǎn)錄組、代謝組和蛋白組等海量數(shù)據(jù)將會(huì)提供更詳盡的關(guān)于纖維素酶誘導(dǎo)調(diào)控的生化途徑和調(diào)控信息。這種整合不但有助于進(jìn)一步揭示纖維素酶產(chǎn)生的分子基礎(chǔ),也為科研工作者提供更多的改造靶點(diǎn)和通路,從而促進(jìn)纖維素酶高效生產(chǎn),并推進(jìn)生物質(zhì)的有效降解。本課題組的最新研究工作證明了人工轉(zhuǎn)錄因子可調(diào)控絲狀真菌產(chǎn)纖維素酶,通過人工轉(zhuǎn)錄因子技術(shù)的應(yīng)用,可以定向設(shè)計(jì)纖維素酶的全局調(diào)控方式,有助于更加深入理解各種環(huán)境信號(hào)和信號(hào)傳遞通路在纖維素酶誘導(dǎo)調(diào)控過程中起到的作用。此外,利用人工鋅指蛋白有希望發(fā)現(xiàn)新的纖維素酶基因表達(dá)調(diào)控的位點(diǎn),深入對(duì)絲狀真菌纖維素酶基因表達(dá)調(diào)控的機(jī)理認(rèn)識(shí),并在此基礎(chǔ)上實(shí)現(xiàn)對(duì)纖維素酶表達(dá)調(diào)控的定向控制。未來可以結(jié)合絲狀真菌內(nèi)源的調(diào)控元件及人工調(diào)控元件,設(shè)計(jì)出更高效的纖維素酶調(diào)控系統(tǒng),提高纖維素酶合成和分泌的效率,并結(jié)合發(fā)酵控制技術(shù),提高纖維素酶的產(chǎn)量,降低生產(chǎn)成本,促進(jìn)纖維素酶在木質(zhì)纖維素原料生物煉制中的應(yīng)用。

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        (本文責(zé)編 陳宏宇)

        Induction and regulation of cellulase expression in filamentous fungi: a review

        Fei Zhang1, Fengwu Bai1,2, and Xinqing Zhao2

        1 School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, Liaoning, China 2 State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

        Production of bioenergy and bio-based chemicals by using fermentable sugars released from low-costrenewable lignocellulosic biomass has

        great attention. Efficient cellulolytic enzymes are crucial for lignocellulose bioconversion, but high cellulase production cost is limiting the bioconversion efficiency of cellulosic biomass and industrial applications of lignocellulose biorefinery. Studies on induction and regulation of cellulase in filamentous fungi will help to further develop superior fungal strains for efficient cellulase production and reduce cellulase production cost. With the advances in high-throughput sequencing and gene manipulation technology using fungal strains, an in-depth understanding of cellulase induction and regulation mechanisms of enzyme expression has been achieved. We reviewed recent progresses in the induction and regulation of cellulase expression in several model filamentous fungi, emphasizing sugar transporters, transcription factors and chromatin remodeling. Future prospects in application of artificial zinc finger proteins for cellulase induction and regulation in filamentous fungi were discussed.

        March 15, 2016; Accepted: April 20, 2016

        Xinqing Zhao. Tel: +86-21-34206673; Fax: +86-21-34208028; E-mail: xqzhao@sjtu.edu.cn

        cellulase, gene expression and regulation, transcription factor, Trichoderma reesei

        Supported by: National Natural Science Foundation of China (Nos. 31461143029, 51561145014), National High Technology Research and Development Program of China (863 Program) (No. 2012AA021205).

        國(guó)家自然科學(xué)基金 (Nos. 31461143029, 51561145014),國(guó)家高技術(shù)研究發(fā)展計(jì)劃 (863計(jì)劃) (No. 2012AA021205) 資助。

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