龐慶霄 梁泉峰 祁慶生(山東大學(xué)生命科學(xué)學(xué)院 微生物技術(shù)國(guó)家重點(diǎn)實(shí)驗(yàn)室,濟(jì)南 250100)
合成生物學(xué)開(kāi)關(guān)在代謝工程中的應(yīng)用
龐慶霄 梁泉峰 祁慶生
(山東大學(xué)生命科學(xué)學(xué)院 微生物技術(shù)國(guó)家重點(diǎn)實(shí)驗(yàn)室,濟(jì)南 250100)
在代謝工程研究領(lǐng)域中合成生物學(xué)開(kāi)關(guān)主要用于調(diào)控基因的表達(dá)。傳統(tǒng)的代謝工程改造主要通過(guò)敲除和過(guò)表達(dá)來(lái)改變特定基因的表達(dá)量。但基因敲除通常會(huì)導(dǎo)致菌體生長(zhǎng)的下降。因此,我們需要適時(shí)的關(guān)閉和激活特定基因的表達(dá)。合成生物學(xué)開(kāi)關(guān)就是解決這一問(wèn)題的關(guān)鍵工具。目前,在代謝工程中常用的合成生物學(xué)開(kāi)關(guān)有光控開(kāi)關(guān)、溫度誘導(dǎo)開(kāi)關(guān)、撥動(dòng)開(kāi)關(guān)和核糖開(kāi)關(guān)。其中,撥動(dòng)開(kāi)關(guān)和核糖開(kāi)關(guān)在動(dòng)態(tài)調(diào)節(jié)基因表達(dá)上擁有更大優(yōu)勢(shì)。介紹了代謝工程中常用的幾種合成生物學(xué)開(kāi)關(guān),以及它們?cè)诖x工程的應(yīng)用。
合成生物學(xué);基因表達(dá)調(diào)控;代謝工程;合成生物學(xué)開(kāi)關(guān);動(dòng)態(tài)調(diào)控
代謝工程的主要目標(biāo)之一就是通過(guò)基因調(diào)控增加化合物產(chǎn)量。隨著DNA分子技術(shù)的進(jìn)步,我們現(xiàn)在能夠在基因水平對(duì)代謝網(wǎng)絡(luò)進(jìn)行操作。通過(guò)針對(duì)途徑中的關(guān)鍵酶的編碼基因,進(jìn)行敲除、擴(kuò)增和下調(diào)[1,2]?;蚯贸椒ㄖ饕獌?yōu)勢(shì)是增加底物向目標(biāo)化合物的轉(zhuǎn)化率。然而基因敲除常常導(dǎo)致生長(zhǎng)率的下降,從而致使產(chǎn)量的減少。權(quán)衡產(chǎn)量和產(chǎn)率使沖突最小化,這樣的設(shè)計(jì)和操作的優(yōu)化是至關(guān)重要的[3]。在抵消基因敲除對(duì)產(chǎn)量的影響上[4],基因表達(dá)動(dòng)態(tài)調(diào)節(jié)比靜態(tài)的方法在最大化產(chǎn)物濃度上更有利。對(duì)于基因敲除影響產(chǎn)量這一問(wèn)題,動(dòng)態(tài)調(diào)控的解決方法就是保持動(dòng)態(tài)的高度表達(dá)直到獲得足夠的生物量,然后關(guān)掉這些基因。合成生物學(xué)開(kāi)關(guān)就是實(shí)現(xiàn)動(dòng)態(tài)基因表達(dá)控制系統(tǒng)的重要元件,它能感應(yīng)相關(guān)條件的變化,在適當(dāng)?shù)臅r(shí)間控制基因表達(dá)的“開(kāi)”或“關(guān)”。目前,在代謝工程中常用的合成生物學(xué)開(kāi)關(guān)有光控開(kāi)關(guān)、溫度誘導(dǎo)開(kāi)關(guān)、撥動(dòng)開(kāi)關(guān)和核糖開(kāi)關(guān)。
光是一個(gè)高度靈活的外界刺激,它在不改變代謝條件的前提下,實(shí)現(xiàn)時(shí)間空間上控制基因的精確表達(dá)。因此,在功能基因組學(xué)、系統(tǒng)生物學(xué)和生物技術(shù)領(lǐng)域光介導(dǎo)的基因表達(dá)調(diào)控研究進(jìn)展迅速。光誘導(dǎo)的生物反應(yīng)主要通過(guò)使用光封閉分子,或者使用帶有光敏感結(jié)構(gòu)域(發(fā)色團(tuán))的光感應(yīng)器來(lái)實(shí)現(xiàn)光控開(kāi)關(guān)的觸發(fā)。光封閉化合物和有發(fā)色團(tuán)分子吸收一定波長(zhǎng)的光,然后起始一個(gè)光觸發(fā)的反應(yīng)。光誘導(dǎo)反應(yīng)導(dǎo)致封閉基團(tuán)不可逆釋放或者使光感受器處于可逆的激活態(tài)。因此這兩種機(jī)制都用于光依賴(lài)的基因表達(dá)控制[5]。
然而封閉化合物有一些限制,光信號(hào)到達(dá)特定細(xì)胞的過(guò)程和這種機(jī)制在大范圍不同的生物體的應(yīng)用都有一定障礙,而且封閉化合物光分解后不可逆轉(zhuǎn)。因此,基因編碼的人工光受體蛋白的發(fā)展有助于規(guī)避這些系統(tǒng)固有的限制。相比較而言光敏感調(diào)控系統(tǒng)被用于代謝工程中進(jìn)行重組開(kāi)關(guān)的構(gòu)建[6]。人工光受體蛋白在光照射下激活其激酶活性,從而磷酸化某些啟動(dòng)子轉(zhuǎn)錄所需的關(guān)鍵轉(zhuǎn)錄因子,轉(zhuǎn)錄因子被激活起始轉(zhuǎn)錄(圖1)。Binder等[7]將光控開(kāi)關(guān)用于谷氨酸棒狀桿菌中朱欒倍半萜的生產(chǎn),獲得了目前為止朱欒倍半萜最高產(chǎn)量和滴定量。朱欒倍半萜對(duì)菌體生長(zhǎng)有嚴(yán)重影響,而且Binder等使用的谷氨酸棒狀桿菌細(xì)胞膜對(duì)IPTG透性較差。他們將帶有封閉分子的IPTG加LAC操縱子的光控開(kāi)關(guān)用于調(diào)節(jié)朱欒倍半萜合酶的表達(dá),在生長(zhǎng)期抑制朱欒倍半萜的合成,當(dāng)菌體達(dá)到一定密度后,用光誘導(dǎo)朱欒倍半萜合酶基因表達(dá)。從而在不影響菌體生長(zhǎng)情況下,獲得了高產(chǎn)量的朱欒倍半萜。
圖1 光介導(dǎo)的基因表達(dá)的控制
溫度作為代謝過(guò)程的重要環(huán)境條件很早就已經(jīng)應(yīng)用在代謝調(diào)控中。在大腸桿菌中含有λ噬菌體的PL啟動(dòng)子的質(zhì)粒常用來(lái)表達(dá)外源基因。通過(guò)引入λ噬菌體的CI抑制子,PL啟動(dòng)子下的基因表達(dá)可以被溫度控制[8]。這種溫度誘導(dǎo)需要使用一個(gè)溫度敏感的抑制子,在32℃時(shí)抑制子抑制基因的表達(dá),在42℃時(shí)誘導(dǎo)基因表達(dá)。Gu等[9]發(fā)現(xiàn)了另一種溫度控制基因表達(dá)機(jī)制。這種機(jī)制通過(guò)溫度控制σE因子是否與LuxR啟動(dòng)子(Plux)結(jié)合來(lái)控制基因表達(dá)。LuxR是群體感應(yīng)的重要因子,將群體感應(yīng)原理與溫度控制開(kāi)關(guān)結(jié)合可以構(gòu)建出“與”門(mén)邏輯通路。當(dāng)溫度達(dá)到37℃并且Plux啟動(dòng)子被群體信號(hào)分子AHL所誘導(dǎo)時(shí),Plux啟動(dòng)子下游的基因才能表達(dá)。溫度誘導(dǎo)在大規(guī)模應(yīng)用上來(lái)說(shuō)可能是最方便的方法,溫度可以通過(guò)加熱和冷卻來(lái)迅速切換。1990年,Shi等[10]將溫度誘導(dǎo)開(kāi)關(guān)應(yīng)用于聚-3-羥基丁酸(PHB)的生產(chǎn),在34℃下培養(yǎng)菌體10 h促進(jìn)菌體生長(zhǎng),然后在40℃誘導(dǎo)PHB合酶表達(dá),5 h后溫度降至37℃緩解熱休克,他們使用這種方法大大提高了PHB的產(chǎn)量和產(chǎn)率。2016年,Zhang等[11]將溫度誘導(dǎo)開(kāi)關(guān)用于α-酮丁酸的生產(chǎn),在35℃保證細(xì)胞生長(zhǎng),然后切換到40℃誘導(dǎo)蘇氨酸脫氨酶表達(dá),使α-酮丁酸的產(chǎn)量在發(fā)酵26 h后達(dá)40.8 g/L,達(dá)到了工業(yè)生產(chǎn)要求的產(chǎn)量。
撥動(dòng)開(kāi)關(guān)是一大類(lèi)代謝調(diào)控開(kāi)關(guān),它們的共同特點(diǎn)是能夠隨環(huán)境(誘導(dǎo)劑或其他感應(yīng)底物的有無(wú))的改變來(lái)控制基因表達(dá)的開(kāi)關(guān)。這里簡(jiǎn)單介紹幾種代謝工程中使用的撥動(dòng)開(kāi)關(guān)。2000年,Gardner等[12]描述了一種構(gòu)建在質(zhì)粒上的撥動(dòng)開(kāi)關(guān),它可以在兩個(gè)啟動(dòng)子表達(dá)之間來(lái)回?fù)軇?dòng)來(lái)應(yīng)答外部信號(hào)。開(kāi)關(guān)由兩個(gè)抑制子和兩個(gè)保守的啟動(dòng)子組成(圖2)。每個(gè)啟動(dòng)子都會(huì)被另一個(gè)啟動(dòng)子表達(dá)的抑制子所抑制[13]。這樣通過(guò)控制誘導(dǎo)劑的有無(wú)來(lái)控制開(kāi)關(guān)在“開(kāi)”和“關(guān)”之間切換,從而控制下游基因的表達(dá)。
隨著對(duì)微生物群體感應(yīng)機(jī)制研究的深入,將群體感應(yīng)機(jī)制與撥動(dòng)開(kāi)關(guān)結(jié)合形成了能夠感應(yīng)菌體密度的動(dòng)態(tài)調(diào)節(jié)元件。2004年,Kobayashi等[14]設(shè)計(jì)了一個(gè)由PL*啟動(dòng)子、Ptrc啟動(dòng)子、lacI基因和λcl基因組成的撥動(dòng)開(kāi)關(guān)。2008年,Anesiadis等[15]將群體感應(yīng)應(yīng)用到代謝工程中,在Kobayashi等的開(kāi)關(guān)的基礎(chǔ)上設(shè)計(jì)出了感應(yīng)群體密度的撥動(dòng)開(kāi)關(guān)。細(xì)胞間的交流是通過(guò)一群信號(hào)分子完成的,它們是自誘導(dǎo)物。高絲氨酸內(nèi)酯(AHL)就是一個(gè)這樣的自誘導(dǎo)物,它的濃度跟環(huán)境中的細(xì)胞數(shù)量成一定比例。將AHL的表達(dá)基因設(shè)計(jì)到撥動(dòng)開(kāi)關(guān)中,就構(gòu)建成了感應(yīng)群體密度的撥動(dòng)開(kāi)關(guān)(圖3)。菌體密度決定AHl濃度,AHL濃度決定LacI表達(dá),進(jìn)而決定目的基因表達(dá)[16]。這實(shí)現(xiàn)了基因表達(dá)的動(dòng)態(tài)調(diào)節(jié),解決了因?yàn)榛蚯贸绊懠?xì)胞生長(zhǎng),進(jìn)而影響產(chǎn)量的問(wèn)題。
圖2 雙穩(wěn)態(tài)撥動(dòng)開(kāi)關(guān)
在代謝工程生產(chǎn)目標(biāo)產(chǎn)物時(shí)往往使用阻斷競(jìng)爭(zhēng)途徑和過(guò)表達(dá)瓶頸酶等手段來(lái)增加產(chǎn)量,這種方法往往使中間體和酶積累較高水平,高水平的中間體和酶對(duì)菌體產(chǎn)生毒性,抑制細(xì)胞生長(zhǎng)和降低產(chǎn)物產(chǎn)量。Liu 等在對(duì)大腸桿菌進(jìn)行改造生產(chǎn)脂肪酸時(shí)發(fā)現(xiàn)改造后的菌株中乙酰輔酶A羧化酶積累很高。乙酰輔酶A羧化酶積累導(dǎo)致菌體生長(zhǎng)的下降。Liu等[17]設(shè)計(jì)了一種可以感應(yīng)乙酰輔酶A羧化酶的產(chǎn)物丙二酸單酰輔酶A濃度的撥動(dòng)開(kāi)關(guān)。該開(kāi)關(guān)可以在丙二酸單酰輔酶A濃度低時(shí)提高乙酰輔酶A羧化酶的表達(dá),在丙二酸單酰輔酶A濃度較高時(shí)降低乙酰輔酶A羧化酶的表達(dá)。這樣既可以增加產(chǎn)物積累,又能降低乙酰輔酶A羧化酶積累對(duì)細(xì)胞的毒性。
Tsuruno等[18]將撥動(dòng)開(kāi)關(guān)應(yīng)用于生產(chǎn)3-羥基丙酸,獲得了比傳統(tǒng)敲除方法更高的產(chǎn)量和產(chǎn)率。本實(shí)驗(yàn)室在對(duì)莽草酸生產(chǎn)的研究中,為了解決敲除莽草酸下游代謝基因莽草酸激酶而使菌株變成芳香族化合物營(yíng)養(yǎng)缺陷型的問(wèn)題,設(shè)計(jì)了一種能夠調(diào)節(jié)莽草酸激酶基因表達(dá)量的開(kāi)關(guān),增加了莽草酸的產(chǎn)量。這種方法與直接敲除莽草酸激酶相比避免了外源添加芳香族化合物[19],從而降低了成本。
圖3 群體感應(yīng)撥動(dòng)開(kāi)關(guān)
近些年來(lái),一種新的不需要蛋白轉(zhuǎn)錄因子參與而直接由核酸感受胞內(nèi)信號(hào)分子或胞外環(huán)境變化的調(diào)控模式得到越來(lái)越多關(guān)注并被應(yīng)用于一些研究領(lǐng)域,這種調(diào)控模式被稱(chēng)為核糖開(kāi)關(guān)(riboswitch)。核糖開(kāi)關(guān)結(jié)構(gòu)首先由Winkler等[20]于2002年在大腸桿菌中發(fā)現(xiàn)并命名。迄今為止發(fā)現(xiàn)的核糖開(kāi)關(guān)有20多種,包括Ado-cbl核糖開(kāi)關(guān)、SAM核糖開(kāi)關(guān)、TPP核糖開(kāi)關(guān)、FMN核糖開(kāi)關(guān)、賴(lài)氨酸核糖開(kāi)關(guān)及glmS核糖開(kāi)關(guān)等廣泛應(yīng)用的核糖開(kāi)關(guān)(表1)。因?yàn)楹颂情_(kāi)關(guān)是由適體結(jié)構(gòu)域和表達(dá)結(jié)構(gòu)域的組合,一個(gè)結(jié)構(gòu)域更改并不影響另一個(gè)區(qū)域功能的發(fā)揮。人們可以在不改變調(diào)控法則的情況下更改核糖開(kāi)關(guān)配體結(jié)合的特異性。于是通過(guò)盡可能的綁定新的配體進(jìn)而篩選出高能力的基因調(diào)控適體。核糖開(kāi)關(guān)可實(shí)時(shí)感應(yīng)配體的濃度,并在配體濃度低時(shí)表現(xiàn)出“開(kāi)”的狀態(tài),基因可以正常表達(dá);在配體濃度高時(shí)表現(xiàn)出“關(guān)”的狀態(tài),抑制基因表達(dá)。核糖開(kāi)關(guān)通過(guò)感應(yīng)底物濃度高低來(lái)實(shí)現(xiàn)對(duì)基因表達(dá)的動(dòng)態(tài)調(diào)節(jié)。
核糖開(kāi)關(guān)這種調(diào)節(jié)元件已經(jīng)被用于代謝工程生產(chǎn)。Zhou和Zeng[21]將賴(lài)氨酸核糖開(kāi)關(guān)應(yīng)用于賴(lài)氨酸的生產(chǎn)。三羧酸循環(huán)(TCA)是氨基酸合成前體的重要來(lái)源,檸檬酸合酶是TCA循環(huán)的第一個(gè)酶(圖4)。有實(shí)驗(yàn)證明檸檬酸合酶活性降低有助于賴(lài)氨酸產(chǎn)量增加。但是檸檬酸合酶是細(xì)菌能量代謝的重要酶,直接敲除將導(dǎo)致菌體生長(zhǎng)嚴(yán)重下降。Wang等[22]用賴(lài)氨酸核糖開(kāi)關(guān)替換掉檸檬酸合酶編碼基因gltA前端啟動(dòng)子和起始密碼子之間的序列。核糖開(kāi)關(guān)跟隨gltA一起轉(zhuǎn)錄,在生產(chǎn)菌株產(chǎn)生一定的賴(lài)氨酸時(shí),賴(lài)氨酸與核糖開(kāi)關(guān)結(jié)合,下調(diào)下游基因gltA的表達(dá)。這樣既避免了基因敲除導(dǎo)致的菌體生長(zhǎng)下降又提高了賴(lài)氨酸的產(chǎn)量。本實(shí)驗(yàn)室在對(duì)大腸桿菌生產(chǎn)賴(lài)氨酸的研究中,將雙重篩選基因tetA與核糖開(kāi)關(guān)相結(jié)合組成核糖開(kāi)關(guān)篩選系統(tǒng),實(shí)現(xiàn)了賴(lài)氨酸生產(chǎn)菌株的高通量篩選,提高了賴(lài)氨酸的產(chǎn)量。
圖4 賴(lài)氨酸合成代謝途徑
隨著微生物代謝工程產(chǎn)物不斷的研發(fā)并應(yīng)用于生產(chǎn)中,對(duì)于代謝工程的各種工具的研究也不斷深入。由于基因敲除導(dǎo)致生產(chǎn)菌株生長(zhǎng)率下降進(jìn)而影響產(chǎn)物產(chǎn)量,研究的方向也開(kāi)始轉(zhuǎn)移到了動(dòng)態(tài)調(diào)控相關(guān)基因的表達(dá)。近年來(lái)合成生物學(xué)開(kāi)關(guān)的研究已成為熱點(diǎn),特別是代謝工程中開(kāi)關(guān)的應(yīng)用相關(guān)研究具有獨(dú)特的吸引力,逐步深入并取得了很大進(jìn)展。預(yù)計(jì)合成生物學(xué)的開(kāi)關(guān)將應(yīng)用于代謝工程各種產(chǎn)品的生產(chǎn),將極大推動(dòng)相關(guān)領(lǐng)域的發(fā)展。
表1 常見(jiàn)的核糖開(kāi)關(guān)
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(責(zé)任編輯 馬鑫)
Application of Switch for Synthetic Biology in Metabolic Engineering
PANG Qing-xiao LIANG Quan-feng QI Qing-sheng
(State Key Laboratory of Microbiology,College of Life Sciences,Shandong University,Ji’nan 250100)
Translational switches for synthetic biology are mainly used to regulate gene expression in the field of metabolic engineering research. Traditional metabolic engineering regulates the expression of specific genes by using knockout and overexpression. However,the knockout of genes usually leads to the decrease in growth,therefore,we need to close and activate the expression of specific gene at proper time,and translational switche for synthetic biology is the key tool to solve such issue. At present,light-controlled switch,temperatureinduced switch,toggle switch,and riboswitch are commonly used in metabolic engineering,of which the toggle switch and riboswitch present a great advantage in the dynamic regulation of gene expression. In this paper,several kinds of translational switches for synthetic biology and their applications in metabolic engineering are reviewed.
synthetic biology;gene expression regulation;metabolic engineering;switch for synthesis biology;dynamic regulation
10.13560/j.cnki.biotech.bull.1985.2017.01.006
2016-11-02
國(guó)家“973”項(xiàng)目子課題(2012CB725202),山東省科技發(fā)展計(jì)劃項(xiàng)目(2015GSF121042)
龐慶霄,男,碩士,研究方向:代謝工程及合成生物學(xué);E-mail:lnupqx@163.com
梁泉峰,男,博士,副教授,研究方向:代謝工程及合成生物學(xué);E-mail:liangquanfeng@sdu.edu.cn