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(中國農(nóng)業(yè)大學食品科學與營養(yǎng)工程學院,北京 100083)

芽孢桿菌表達系統(tǒng)的特點及研究進展

苗苗,黃昆侖,梁志宏*

(中國農(nóng)業(yè)大學食品科學與營養(yǎng)工程學院,北京100083)

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

芽孢桿菌,表達系統(tǒng),枯草芽孢桿菌,研究進展

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系統(tǒng)得到了越來越廣泛的應用,該系統(tǒng)可表達耐熱性酶制劑、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),具有良好的應用前景[2]。特別是枯草芽孢桿菌,因其無內(nèi)毒素產(chǎn)生,無致病性的優(yōu)點,現(xiàn)已被開發(fā)成為食品級表達系統(tǒng),在食物加工、醫(yī)學藥品、生物農(nóng)藥、生物飼料、生物降污等工業(yè)應用上都具有特殊的重要性。雖然芽孢桿菌表達系統(tǒng)有很多優(yōu)勢,但該系統(tǒng)存在著分泌蛋白酶和質(zhì)粒不穩(wěn)定的缺點,國內(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啟動子來保證遺傳的穩(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)等都可用作表達宿主菌。本文介紹了枯草芽孢桿菌(B.subtilis)表達系統(tǒng)、巨大芽孢桿菌表達系統(tǒng)、短小芽孢桿菌表達系統(tǒng)、短芽孢桿菌表達系統(tǒng)、蘇云金芽孢桿菌表達系統(tǒng)和地衣芽孢桿菌表達系統(tǒng)的研究進展、應用和特點,主要介紹了枯草芽孢桿菌表達系統(tǒng)。

1 構(gòu)建高效表達的芽孢桿菌表達體系

芽孢桿菌表達體系是食用級安全的表達系統(tǒng),表達的外源蛋白分離純化。可通過提高表達載體的穩(wěn)定性和提高芽孢桿菌表達體系的分泌表達效率,獲得更加高效的芽孢桿菌表達系統(tǒng)[3-5]。

1.1 提高質(zhì)粒的穩(wěn)定性

自主復制質(zhì)粒、整合質(zhì)粒和噬菌體都可用作表達載體,自主復制的質(zhì)粒應用較多。第一代主要來自金黃色葡萄球菌、革蘭氏陰性菌,通過重組構(gòu)成帶有雙抗性標志的嵌合質(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系統(tǒng)和枯草芽孢桿菌表達系統(tǒng)中表達外源蛋白[7]。第三代質(zhì)粒表達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-大腸桿菌穿梭表達載體,成功表達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。常見的大腸桿菌-枯草芽孢桿菌穿梭質(zhì)粒載體和整合載體見表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 啟動子的選擇與優(yōu)化Table 2 The selection and optimization of promoter

1.2 表達調(diào)控元件的選擇

1.3 提高表達效率

芽孢桿菌自身會分泌多種蛋白酶,如枯草芽孢桿菌自身會分泌300多種蛋白酶[23],這些蛋白酶的分泌常常會導致外源蛋白被降解,對于這一問題可使用誘導系統(tǒng),常用的有葡萄糖誘導系統(tǒng)和蔗糖誘導系統(tǒng)。使用較多的誘導系統(tǒng)有葡萄糖、木糖、IPTG誘導系統(tǒng)。Pxyl使用木糖誘導,但木糖較貴,還沒有在工業(yè)上取得廣泛應用,而IPTG本身對微生物有毒害作用,且本身成本也較高,也因此在工業(yè)上沒有得到廣泛使用[20-21]。另外提高表達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,其蛋白酶突變菌株有DB104、DB105、DB431、WB600、WB700、LB700。對于表達載體、宿主菌及誘導因素和啟動子等方面選擇[24-25],整理了一些常見的表達體系見表3。

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

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

表5 芽孢桿菌表達系統(tǒng)的應用Table 5 The application of Bacillus expression system

2 芽孢桿菌表達系統(tǒng)的應用現(xiàn)狀

2.1 B.subtilis芽孢桿菌表達系統(tǒng)的應用

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植物的體表中,其細胞外膜很薄,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等人利用其簡單的細胞壁,在一種工程枯草芽孢桿菌中表達了堿性成纖維細胞生長因子(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系統(tǒng)獲得高效表達,如中性蛋白酶和淀粉酶等,結(jié)合國內(nèi)外對芽孢桿菌表達系統(tǒng)的研究,將枯草芽孢桿菌表達系統(tǒng)在工業(yè)上的應用總結(jié)如下,見表4。

2.2 其他芽孢桿菌表達系統(tǒng)的應用

巨大芽孢桿菌具有誘導劑廉價、胞外蛋白酶活性低、質(zhì)粒穩(wěn)定性好的優(yōu)點。是比大腸桿菌更具有優(yōu)勢的表達宿主,該表達系統(tǒng)作為外源蛋白的表達在國內(nèi)外研究逐漸深入,并已經(jīng)成功應用于多個基因和外源蛋白的分泌表達如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子,這種雙啟動子有加強基因表達的作用;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越多的重視,因其還具有細胞壁蛋白會脫落的獨特性質(zhì),使用攜帶細胞壁蛋白基因的啟動子等表達元件,可使目的基因通過分泌方式得以高效表達;地衣芽孢桿菌細胞壁表面的蛋白基因也具有雙啟動子,該表達系統(tǒng)還可用于真核基因的表達,表達產(chǎn)物直接分泌到胞外,容易分離純化。對于除枯草芽孢桿菌表達體系的其他芽孢桿菌表達體系的應用進行總結(jié),見表5。

3 芽孢桿菌表達系統(tǒng)的特點及發(fā)展前景

隨著分子生物學技術(shù)的發(fā)展和芽孢桿菌研究的深入,使用芽孢桿菌表達系統(tǒng)已經(jīng)克隆和表達了大量基因,有些已進行大規(guī)模工業(yè)生產(chǎn),多種酶和臨床需要的化學藥品或者工業(yè)產(chǎn)品都通過芽孢桿菌來表達生產(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]。芽孢桿菌表達系統(tǒng)具有很強的蛋白質(zhì)分泌功能,分泌的外源蛋白不易形成包涵體,無顯著的密碼子偏愛性,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系統(tǒng)都不具備的優(yōu)勢。通過對表達調(diào)控元件的優(yōu)化[54]、強啟動子的使用[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]等方法提高表達外源蛋白的表達效率,這對于芽孢桿菌表達系統(tǒng)的工業(yè)化應用具有重大意義。

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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。

國家自然科學基金(31671947)。

10.13386/j.issn1002-0306.2017.18.059