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(中國(guó)農(nóng)業(yè)大學(xué)食品科學(xué)與營(yíng)養(yǎng)工程學(xué)院,北京 100083)

芽孢桿菌表達(dá)系統(tǒng)的特點(diǎn)及研究進(jìn)展

苗苗,黃昆侖,梁志宏*

(中國(guó)農(nóng)業(yè)大學(xué)食品科學(xué)與營(yíng)養(yǎng)工程學(xué)院,北京100083)

芽孢桿菌表達(dá)系統(tǒng)作為基因工程表達(dá)系統(tǒng),因具有外源蛋白易分離純化、表達(dá)的外源蛋白不會(huì)形成包涵體、無(wú)致病性等多種優(yōu)勢(shì)而被廣泛的研究和應(yīng)用,很多種工業(yè)酶都在芽孢桿菌表達(dá)系統(tǒng)中獲得成功表達(dá)。本文主要介紹了芽孢桿菌表達(dá)系統(tǒng)的研究進(jìn)展,特點(diǎn)以及其在工業(yè)上的應(yīng)用。對(duì)芽孢桿菌表達(dá)體系存在的缺陷提出了解決方法,并說(shuō)明了芽孢桿菌今后發(fā)展的主要方向,為芽孢桿菌表達(dá)體系的研究提供參考。

芽孢桿菌,表達(dá)系統(tǒng),枯草芽孢桿菌,研究進(jìn)展

Abstract:Bacillusexpression system as gene engineering expression system has many advantages such as excellent secretion ability,successful heterologous protein expression system,not forming inclusion bodies,non-endotoxin,so it is widely studied and applied,many kinds of industrial enzymes were expressed inBacillussubtilisexpression system. In this review,it mainly talks about the advances,characters and application ofBacillusexpression system,and presents a solution to the defects in the expression system ofBacillus,offering the main research directions forBacillusexpression system.

Keywords:Bacillus;expression system;Bacillussubtilis;advances

芽孢桿菌是一種可以厭氧繁殖、能生成抗逆性孢子的菌,廣泛存在于環(huán)境中[1]。如今,芽孢桿菌作為基因工程表達(dá)系統(tǒng)得到了越來(lái)越廣泛的應(yīng)用,該系統(tǒng)可表達(dá)耐熱性酶制劑、多肽類藥物和殺蟲蛋白等外源蛋白,有的已經(jīng)投入大規(guī)模生產(chǎn),具有良好的應(yīng)用前景[2]。特別是枯草芽孢桿菌,因其無(wú)內(nèi)毒素產(chǎn)生,無(wú)致病性的優(yōu)點(diǎn),現(xiàn)已被開發(fā)成為食品級(jí)表達(dá)系統(tǒng),在食物加工、醫(yī)學(xué)藥品、生物農(nóng)藥、生物飼料、生物降污等工業(yè)應(yīng)用上都具有特殊的重要性。雖然芽孢桿菌表達(dá)系統(tǒng)有很多優(yōu)勢(shì),但該系統(tǒng)存在著分泌蛋白酶和質(zhì)粒不穩(wěn)定的缺點(diǎn),國(guó)內(nèi)外研究的解決方法主要是使用蛋白酶缺陷菌株或添加強(qiáng)啟動(dòng)子來(lái)保證遺傳的穩(wěn)定性。芽孢桿菌中巨大芽孢桿菌(B.megaterium)、短芽孢桿菌(B.brevis)、解淀粉芽孢桿菌(B.amyloliquefaciens)、短小芽孢桿菌(B.pumilus)、地衣芽孢桿菌(B.licheniformis)、球形芽孢桿菌(B.sphaericus)、蘇云金芽孢桿菌(B.thuringiensis)和嗜堿芽孢桿菌(B.alcalophilus)等都可用作表達(dá)宿主菌。本文介紹了枯草芽孢桿菌(B.subtilis)表達(dá)系統(tǒng)、巨大芽孢桿菌表達(dá)系統(tǒng)、短小芽孢桿菌表達(dá)系統(tǒng)、短芽孢桿菌表達(dá)系統(tǒng)、蘇云金芽孢桿菌表達(dá)系統(tǒng)和地衣芽孢桿菌表達(dá)系統(tǒng)的研究進(jìn)展、應(yīng)用和特點(diǎn),主要介紹了枯草芽孢桿菌表達(dá)系統(tǒng)。

1 構(gòu)建高效表達(dá)的芽孢桿菌表達(dá)體系

芽孢桿菌表達(dá)體系是食用級(jí)安全的表達(dá)系統(tǒng),表達(dá)的外源蛋白分離純化?？赏ㄟ^(guò)提高表達(dá)載體的穩(wěn)定性和提高芽孢桿菌表達(dá)體系的分泌表達(dá)效率,獲得更加高效的芽孢桿菌表達(dá)系統(tǒng)[3-5]。

1.1 提高質(zhì)粒的穩(wěn)定性

自主復(fù)制質(zhì)粒、整合質(zhì)粒和噬菌體都可用作表達(dá)載體,自主復(fù)制的質(zhì)粒應(yīng)用較多。第一代主要來(lái)自金黃色葡萄球菌、革蘭氏陰性菌,通過(guò)重組構(gòu)成帶有雙抗性標(biāo)志的嵌合質(zhì)粒[6]。第二代質(zhì)粒載體是穿梭質(zhì)粒,可以在大腸桿菌表達(dá)系統(tǒng)和枯草芽孢桿菌表達(dá)系統(tǒng)中表達(dá)外源蛋白[7]。第三代質(zhì)粒表達(dá)載體是由枯草芽孢桿菌的隱形質(zhì)粒和大腸桿菌的質(zhì)粒共同構(gòu)成嵌合質(zhì)粒[8]。除了質(zhì)粒載體還可以使用噬菌體如sppl,Фl05噬菌體等噬菌體[9-10]。劉偉等人[11-12]構(gòu)建了枯草芽孢桿菌-大腸桿菌穿梭表達(dá)載體,成功表達(dá)了綠色熒光蛋白和抗菌肽。目前常用的穿梭質(zhì)粒載體有 pEB10、pEB60、pUB18和pWB980等,整合型載體,如pMutin4、pMutin-GFP+、pSG1151、pSG1156、pDG1662等。常見(jiàn)的大腸桿菌-枯草芽孢桿菌穿梭質(zhì)粒載體和整合載體見(jiàn)表1。

表1 芽孢桿菌整合和穿梭質(zhì)粒載體Table 1 Bacillus integration and shuttle plasmid vector

表2 啟動(dòng)子的選擇與優(yōu)化Table 2 The selection and optimization of promoter

1.2 表達(dá)調(diào)控元件的選擇

1.3 提高表達(dá)效率

芽孢桿菌自身會(huì)分泌多種蛋白酶,如枯草芽孢桿菌自身會(huì)分泌300多種蛋白酶[23],這些蛋白酶的分泌常常會(huì)導(dǎo)致外源蛋白被降解,對(duì)于這一問(wèn)題可使用誘導(dǎo)系統(tǒng),常用的有葡萄糖誘導(dǎo)系統(tǒng)和蔗糖誘導(dǎo)系統(tǒng)。使用較多的誘導(dǎo)系統(tǒng)有葡萄糖、木糖、IPTG誘導(dǎo)系統(tǒng)。Pxyl使用木糖誘導(dǎo),但木糖較貴,還沒(méi)有在工業(yè)上取得廣泛應(yīng)用,而IPTG本身對(duì)微生物有毒害作用,且本身成本也較高,也因此在工業(yè)上沒(méi)有得到廣泛使用[20-21]。另外提高表達(dá)效率還可使用蛋白酶突變型菌株作為宿主菌。以B.subtilis168為原始菌株的多種蛋白酶突變型菌株被廣泛用于外源基因的表達(dá),其蛋白酶突變菌株有DB104、DB105、DB431、WB600、WB700、LB700。對(duì)于表達(dá)載體、宿主菌及誘導(dǎo)因素和啟動(dòng)子等方面選擇[24-25],整理了一些常見(jiàn)的表達(dá)體系見(jiàn)表3。

表3 芽孢桿菌表達(dá)系統(tǒng)的構(gòu)建Table 3 The construction of the expression system of Bacillus

表4 枯草芽孢桿菌表達(dá)系統(tǒng)的應(yīng)用 Table 4 The application of B.subtilis expression system

表5 芽孢桿菌表達(dá)系統(tǒng)的應(yīng)用Table 5 The application of Bacillus expression system

2 芽孢桿菌表達(dá)系統(tǒng)的應(yīng)用現(xiàn)狀

2.1 B.subtilis芽孢桿菌表達(dá)系統(tǒng)的應(yīng)用

枯草芽孢桿菌是一類好氧型桿狀菌,廣泛存在于自然界和動(dòng)植物的體表中,其細(xì)胞外膜很薄,便于目的蛋白直接分泌到培養(yǎng)基中,易于分離和純化[26]。Keith WY等人利用其簡(jiǎn)單的細(xì)胞壁,在一種工程枯草芽孢桿菌中表達(dá)了堿性成纖維細(xì)胞生長(zhǎng)因子(bFGF),并在培養(yǎng)上清液中將其提取[27]。各種工業(yè)酶和飼用酶[28]都可在芽孢桿菌表達(dá)系統(tǒng)獲得高效表達(dá),如中性蛋白酶和淀粉酶等,結(jié)合國(guó)內(nèi)外對(duì)芽孢桿菌表達(dá)系統(tǒng)的研究,將枯草芽孢桿菌表達(dá)系統(tǒng)在工業(yè)上的應(yīng)用總結(jié)如下,見(jiàn)表4。

2.2 其他芽孢桿菌表達(dá)系統(tǒng)的應(yīng)用

巨大芽孢桿菌具有誘導(dǎo)劑廉價(jià)、胞外蛋白酶活性低、質(zhì)粒穩(wěn)定性好的優(yōu)點(diǎn)。是比大腸桿菌更具有優(yōu)勢(shì)的表達(dá)宿主,該表達(dá)系統(tǒng)作為外源蛋白的表達(dá)在國(guó)內(nèi)外研究逐漸深入,并已經(jīng)成功應(yīng)用于多個(gè)基因和外源蛋白的分泌表達(dá)如β-半乳糖苷酶、水解酶、木糖等[29-30];蘇云金芽孢桿菌(Bt)是來(lái)源較為廣泛的土壤菌。該菌具有結(jié)晶內(nèi)含物,具有殺蟲的特性[33]。Bt殺蟲晶體蛋白基因還含有雙啟動(dòng)子,這種雙啟動(dòng)子有加強(qiáng)基因表達(dá)的作用;短小芽孢桿菌開發(fā)成為重組體DNA分子的受體的研究受到越來(lái)越多的重視,因其還具有細(xì)胞壁蛋白會(huì)脫落的獨(dú)特性質(zhì),使用攜帶細(xì)胞壁蛋白基因的啟動(dòng)子等表達(dá)元件,可使目的基因通過(guò)分泌方式得以高效表達(dá);地衣芽孢桿菌細(xì)胞壁表面的蛋白基因也具有雙啟動(dòng)子,該表達(dá)系統(tǒng)還可用于真核基因的表達(dá),表達(dá)產(chǎn)物直接分泌到胞外,容易分離純化。對(duì)于除枯草芽孢桿菌表達(dá)體系的其他芽孢桿菌表達(dá)體系的應(yīng)用進(jìn)行總結(jié),見(jiàn)表5。

3 芽孢桿菌表達(dá)系統(tǒng)的特點(diǎn)及發(fā)展前景

隨著分子生物學(xué)技術(shù)的發(fā)展和芽孢桿菌研究的深入,使用芽孢桿菌表達(dá)系統(tǒng)已經(jīng)克隆和表達(dá)了大量基因,有些已進(jìn)行大規(guī)模工業(yè)生產(chǎn),多種酶和臨床需要的化學(xué)藥品或者工業(yè)產(chǎn)品都通過(guò)芽孢桿菌來(lái)表達(dá)生產(chǎn)。例如維生素B6、內(nèi)切酶葡聚糖酶、堿性蛋白酶和脂肽[36-38],結(jié)合發(fā)酵技術(shù),該系統(tǒng)還可用于工業(yè)上生產(chǎn)飼用酶和益生菌[41-42]。芽孢桿菌表達(dá)系統(tǒng)具有很強(qiáng)的蛋白質(zhì)分泌功能,分泌的外源蛋白不易形成包涵體,無(wú)顯著的密碼子偏愛(ài)性,外源蛋白可直接分泌,易于分離純化[39-40],另外枯草芽孢桿菌噬菌體和質(zhì)粒都可以用作克隆載體,這是其他原核菌表達(dá)系統(tǒng)都不具備的優(yōu)勢(shì)。通過(guò)對(duì)表達(dá)調(diào)控元件的優(yōu)化[54]、強(qiáng)啟動(dòng)子的使用[45-46]、發(fā)酵工藝調(diào)整[24]、篩選適合的蛋白酶缺陷菌株[49-50]等方法提高表達(dá)外源蛋白的表達(dá)效率,這對(duì)于芽孢桿菌表達(dá)系統(tǒng)的工業(yè)化應(yīng)用具有重大意義。

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ThecharactersandadvancesofBacillusexpressionsystem

MIAOMiao,HUANGKun-lun,LIANGZhi-hong*

(College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China)

TS201

A

1002-0306(2017)18-0312-06

2017-02-13

苗苗(1995-)，女，碩士研究生，研究方向:食品工程,E-mail:17801136541@163.com。

*通訊作者:梁志宏(1969-)，女，博士，副教授，研究方向:微生物與食品安全,E-mail:lzh105@cau.edu.cn。

國(guó)家自然科學(xué)基金(31671947)。

10.13386/j.issn1002-0306.2017.18.059