芽孢桿菌表達系統的特點及研究進展

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(中國農業大學食品科學與營養工程學院,北京 100083)

芽孢桿菌表達系統的特點及研究進展

苗苗,黃昆侖,梁志宏*

(中國農業大學食品科學與營養工程學院,北京100083)

芽孢桿菌表達系統作為基因工程表達系統,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、無致病性等多種優勢而被廣泛的研究和應用,很多種工業酶都在芽孢桿菌表達系統中獲得成功表達。本文主要介紹了芽孢桿菌表達系統的研究進展,特點以及其在工業上的應用。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,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,廣泛存在于環境中[1]。如今,芽孢桿菌作為基因工程表達系統得到了越來越廣泛的應用,該系統可表達耐熱性酶制劑、多肽類藥物和殺蟲蛋白等外源蛋白,有的已經投入大規模生產,具有良好的應用前景[2]。特別是枯草芽孢桿菌,因其無內毒素產生,無致病性的優點,現已被開發成為食品級表達系統,在食物加工、醫學藥品、生物農藥、生物飼料、生物降污等工業應用上都具有特殊的重要性。雖然芽孢桿菌表達系統有很多優勢,但該系統存在著分泌蛋白酶和質粒不穩定的缺點,國內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、巨大芽孢桿菌表達系統、短小芽孢桿菌表達系統、短芽孢桿菌表達系統、蘇云金芽孢桿菌表達系統和地衣芽孢桿菌表達系統的研究進展、應用和特點,主要介紹了枯草芽孢桿菌表達系統。

1 構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,表達的外源蛋白分離純化。可通過提高表達載體的穩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[3-5]。

1.1 提高質粒的穩定性

自主復制質粒、整合質粒和噬菌體都可用作表達載體,自主復制的質粒應用較多。第一代主要來自金黃色葡萄球菌、革蘭氏陰性菌,通過重組構成帶有雙抗性標志的嵌合質粒[6]。第二代質粒載體是穿梭質粒,可以在大腸桿菌表達系統和枯草芽孢桿菌表達系統中表達外源蛋白[7]。第三代質粒表達載體是由枯草芽孢桿菌的隱形質粒和大腸桿菌的質粒共同構成嵌合質粒[8]。除了質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粒載體和整合載體見表1。

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

表2 啟動子的選擇與優化Table 2 The selection and optimization of promoter

1.2 表達調控元件的選擇

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,常用的有葡萄糖誘導系統和蔗糖誘導系統。使用較多的誘導系統有葡萄糖、木糖、IPTG誘導系統。Pxyl使用木糖誘導,但木糖較貴,還沒有在工業上取得廣泛應用,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 芽孢桿菌表達系統的構建Table 3 The construction of the expression system of Bacillus

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

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

2 芽孢桿菌表達系統的應用現狀

2.1 B.subtilis芽孢桿菌表達系統的應用

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),并在培養上清液中將其提取[27]。各種工業酶和飼用酶[28]都可在芽孢桿菌表達系統獲得高效表達,如中性蛋白酶和淀粉酶等,結合國內外對芽孢桿菌表達系統的研究,將枯草芽孢桿菌表達系統在工業上的應用總結如下,見表4。

2.2 其他芽孢桿菌表達系統的應用

巨大芽孢桿菌具有誘導劑廉價、胞外蛋白酶活性低、質粒穩定性好的優點。是比大腸桿菌更具有優勢的表達宿主,該表達系統作為外源蛋白的表達在國內外研究逐漸深入,并已經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。該菌具有結晶內含物,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,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,該表達系統還可用于真核基因的表達,表達產物直接分泌到胞外,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,見表5。

3 芽孢桿菌表達系統的特點及發展前景

隨著分子生物學技術的發展和芽孢桿菌研究的深入,使用芽孢桿菌表達系統已經克隆和表達了大量基因,有些已進行大規模工業生產,多種酶和臨床需要的化學藥品或者工業產品都通過芽孢桿菌來表達生產。例如維生素B6、內切酶葡聚糖酶、堿性蛋白酶和脂肽[36-38],結合發酵技術,該系統還可用于工業上生產飼用酶和益生菌[41-42]。芽孢桿菌表達系統具有很強的蛋白質分泌功能,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粒都可以用作克隆載體,這是其他原核菌表達系統都不具備的優勢。通過對表達調控元件的優化[54]、強啟動子的使用[45-46]、發酵工藝調整[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的工業化應用具有重大意義。

[1]閆子祥,楊然 李秀婷.微生物表達系統研究進展[J]. 中國食品學報,2013,37(15):126-135.

[2]葉小蘭,楊倩. 枯草芽孢桿菌在防御動物疾病中的研究進展[J]. 中國獸醫科學,2011,41(9):958-988.

[3]楊明明.枯草芽孢桿菌關鍵遺傳調控元件及表達系統的研究[D]. 咸陽:西北農林科技大學,2013:1-17.

[4]郭菁,田寶玉,蔡婉玲,等.側孢短芽孢桿菌蛋白酶基因 BLG4 在枯草芽孢桿菌 WB600中的高效表達[J]. 福建農林大學學報:自然科學版,2011,40(2):165-171.

[5]Ameny Farhat-Khemakhem,Mounira Ben Farhat,Ines Boukhris,et al. Heterologous expression and optimization using experimental designs allowed highly efficient production of the PHY US417 phytase inBacillussubtilis168[J]. AMB Express,2012,2:2-11.

[6]Gryzczan T J.Molecular cloning inBacillussubtilis[J].The Molecular Biology of the Bacilli,1982:21-28.

[7]梁曉梅,黎明,成堏,等.大腸桿菌-枯草芽孢桿菌穿梭質粒的構建及堿性蛋白酶的的表達[J].生物技術通報,2011,4(6):165-169.

[8]夏雨.枯草芽孢桿菌食品級表達系統的構建和分泌表達研究[D].無錫:江南大學,2007:1-10.

[9]屈伸,劉志.分子生物學實驗技術[M]. 北京:化學工業出版社,2008:201-204.

[10]徐子勤.功能基因組學[M]. 北京:科學出版社,2007:213-215.

[11]劉偉,林志偉,陳美霞,等. 枯草芽孢桿菌綠色熒光蛋白高效表達載體的構建[J]. 熱帶作物學報,2012,33(3):467-471.

[12]尚田田.枯草芽孢桿菌表達體系高效表達抗菌肽PNK-19方法的建立[D].新鄉:河南科技學院,2014:8-10.

[13]Kaltwasser M,Wiegert T,Schumann W. Construction and application of epitope-and green fluorescent protein-tagging integration vect ors forBacillussubtilis[J].Applied and Environmental Microbiology,2002,68(5):2624-2628.

[14]Feucht A,Lewis PJ. Improved plasmid vectors for the production of multiple fluorescent protein fu sions inBacillussubtilis[J]. Gene,2001,264(2):289-297.

[15]Guérout-Fleury AM,Frandsen N,Stragier P. Plasmids for ectopic integration inBacillussubtilis[J].Gene,1996,180(1):57-61.

[16]Nagarajan V,Albertson H,Chen M,et al. Modular expression and secretion vectors for Bacillus subtili[J]. Gene,1992,114(1):121-126.

[17]Wu SC,Wong SL. Development of improved pUB110-based vect ors for expression and secretion studies inBacillussubtilis[J].Journal of Biotechnology,1999,72(3):185-195.

[18]Wang LF,Wong SL,Lee SG,et al. Expres sion and secretion of human atrial natriureticα-factor inBacillussubtilisusing the subtilisin signal peptide[J]. Gene,1988,69(1):39-47.

[19]董晨,曹娟,張跡. 耐高溫α-淀粉酶基因在枯草芽孢桿菌中的高效表達[J]. 應用與環境生物學報,2008,14(4):534-538.

[20]Long Liu,Yanfeng Liu,Hyun-dong Shin,et al. DevelopingBacillusspp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology[J]. Appl Microbiol Biotechnol,2013,97:6113-6127.

[21]Michel S,Dominique L C. The DNA sequence of the gene for the secretedBacillussubtilisenzyme levansucrase and its genetic control sites[J]. Molecular and General Genetics,1985,200:220-228.

[22]周勇,徐剛,楊立榮,等.信號肽優化在枯草芽孢桿菌體系中對脂肪酶分泌表達的影響[J].中國生物工程雜志,2015,35(9):42-49.

[23]Tjalsma H,Bolhuis A,Jongbloed JDH,et al. Signal peptide-dependent protein transport inBacillussubtilis:agenome-based survey of the secretome[J]. Microbiology and Molecular Biology Reviews,2000,64:515-547.

[24]李靜靜.高產α-乙酰乳酸脫羧酶重組枯草芽孢桿菌的構建及其發酵優化[D].無錫:江南大學,2013:25-44.

[25]丁偉,張明俐,史吉平,等.表達谷氨酸脫羧酶重組枯草芽孢桿菌的構建及其發酵條件的優化[J].生物工程,2015,36(23):194-198.

[26]郭蘇. 芽胞桿菌屬來源的普魯蘭酶基因的克隆表達及枯草芽孢桿菌表達體系的構建[D]. 上海:華東理工,2012:35-48.

[27]Keith WY Kwong,K L Ng,C C Lam Yule Y Wang,et al. Authentic human basic fibroblast growth factor produced by secretion inBacillussubtilis[J]. Appl Microbiol Biotechnol,2013,97:6803-6811.

[28]賈敏,萬孟,張濤,等. D-阿洛酮糖 3-差向異構酶基因在枯草芽孢桿菌中的表達[J]. 食品與生物技術學報,2014,33(11):1129-1135.

[29]郭德軍,李巖松,王欣,等.巨大芽孢桿菌表達系統的特點及其研究進展[J].生物技術,2010,20(6):92-95.

[30]牟琳,王紅寧,鄒立扣.巨大芽孢桿菌表達外源蛋白的特點及其研究進展[J].中國生物工程雜志,2008,28(4):93-97.

[31]金輝,廖思明,王青艷.堿性α-淀粉酶基因在巨大芽孢桿菌中的表達及酶學性質研究[J]. 生物技術通報,2011(11):154-158.

[32]周海燕,饒力群,吳永堯.甘露聚糖酶man23基因的重組及其在短短芽孢桿菌中的表達[J].浙江大學學報,2008,34(4):389-394.

[33]張杰,宋福平,左雅慧,等.31株蘇云金芽孢桿菌殺蟲晶體蛋白基因型鑒定及表達產物研究[J]. 微生物學報，2000,40(7):372-377.

[34]陳啟民,耿運琪,倪津.短小芽抱桿菌作為芽抱桿菌屬基因工程受體菌的研究[J].遺傳學報,1989,16(3):206-212.

[35]包怡紅,劉偉豐,董志揚.耐堿性木聚糖酶在短小芽孢桿菌中高效分泌表達的研究[J].中國食品學報,2008,8(5):37-43.

[36]Commichau FM,Alzinger A,Sande R,et al. Overexpression of a non-native deoxyxylulose-dependent vitamin B6 pathway inBacillussubtilisfor the production of pyridoxine[J]. Metab Eng,2014,25:38-49.

[37]Zafar M,Ahmed S,Khan MI,et al. Recombinant expression and characterization of a novel endoglucanase fromBacillussubtilisinEscherichiacoli[J]. Molecular Biology Reports,2014,41:3295-302.

[38]Ju Jung,Kyung Ok Yu,Ahmad Bazli Ramzi,et al. Improvement of surfactin production inBacillussubtilisusing synthetic wastewater by overexpression of specific extracellular signaling peptides,comX and phrC[J]. Biotechnol Bioeng,2012,109:2349-2356.

[39]王金斌,陳大超,李文,等.食品級枯草芽孢桿菌表達系統的最新研究進展[J].上海農業學報,2014,30(1):115-120.

[40]Phan TTP,Nguyen HD,Schumann W. Development of a strong intracellular expression system forBacillussubtilisby optimizing promoter elements[J]. Biotechnol,2012,157:167-172.

[41]Zhang Xiao Zhou,Yan Xin,Cui Zhong Li,et al. Recombinant expression and secretion of mpd gene using the promoters of ytkA and ywoF gene fromBacillussubtilis[J]. Chinese Journal of Biotechnology,2006,22(2):249-256.

[42]Shanshan Li,Xiaoqiang Jia,Jianping Wen. Improved 2-methyl-1-propanol production in an engineeredBacillussubtilisby constructing inducible pathways[J].Biotechnol Lett,2012,34:2253-2258.

[43]J Olmos-Soto,R Contreras-Flores.Genetic system constructed toover produce and secrete proinsulin inBacillussubtilis[J]. Appl Microbiol Biotech,2003,62:369-373.

[44]Mauriello,Ducb,Rachele Isticatoa,et al. Display of heterologous antigens on theBacillussubtilisspore coat using CotC as a fusion partner[J].Vaccine,2004,22:1177-1187.

[45]Chengran Guan,Wenjing Cui,Jintao Cheng,et al. Development of an efficient autoinducible expression system by promoter engineering inBacillussubtilis[J].Microbial Cell Factories,2016,15(66):2-12.

[46]Reza Panahi,Ebrahim Vasheghani-Farahani,Seyed Abbas Shojaosadati,et al. Induction ofBacillussubtilisexpression system using environmental stresses and glucose starvation[J]. Ann Microbiol,2014,64:879-882.

[47]畢臺飛,胡雄斌,宋巍,等.枯草芽孢桿菌高效表達系統的構建[J].西北農林科技大學學報,2011,39(11):71-79.

[48]Prax M,Lee CY,Bertram R. An update on the molecular genetics toolbox for staphylococci[J]. Microbiol,2013,159:421-435.

[49]Sen-Lin Liu,Kun Du. Enhanced expression of an endoglucanase inBacillussubtilisby using the sucrose-inducible sacB promoter and improved properties of the recombinant enzyme[J]. Protein Expression and Purification,2012,83:164-168.

[50]Westers H,Dorenbos R,Dijl J M. Genome engineering reveals large dispensable regions inBacillussubtilis[J].Mol Biol Evol,2003,20(12):2076-2090.

[51]Cui W,Han L,Cheng J,et al. Engineering an inducible gene expression system forBacillussubtilisfrom a strong constitutive promoter and a theophylline-activated synthetic riboswitch[J]. Microbial Cell Factories,2016,15(1):199.

[52]Bambai RPEV-FSASB. Auto-inducible expression system based on the SigB-dependent ohrB promoter inBacillussubtilis[J]. Ann Microbiol,2014(64):879-82.

[53]Phan TT,Tran LT,Schumann W,et al. Development of Pgrac100-based expression vectors allowing high protein production levels inBacillussubtilisand relatively low basal expression inEscherichiacoli[J]. Microbial Cell Factories,2015:14-72.

[54]Toshitaka Minetoki HT,Akio Koda,Kenji Ozeki,et al. Development of high expression system with the improved promoter using the cis-acting element inAspergillusspecies[J]. J Biol Macromol,2003,3(3):89-96.

[55]Hernandez-Garcia CM,Finer JJ. A novel cis-acting element in the GmERF3 promoter contributes to inducible gene expression in soybean and tobacco after wounding[J]. Plant Cell Reports,2016,35(2):303-316.

[56]Guan C,Cui W,Cheng J,et al. Development of an efficient autoinducible expression system by promoter engineering inBacillussubtilis[J]. Microbial Cell Factories,2016,15:60-66.

[57]Liu B,Zhang J,Li B,et al. Expression and characterization of extreme alkaline,oxidation-resistant keratinase fromBacilluslicheniformisin recombinantBacillussubtilisWB600 expression system and its application in wool fiber processing[J]. World Journal of Microbiology & Biotechnology,2013,29(5):825-832.

[58]Cheng J,Guan C,Cui W,et al. Enhancement of a high efficient autoinducible expression system inBacillussubtilisby promoter engineering[J]. Protein Expression and Purification,2016,127:81-87.

[59]Bongers RS,Veening JW,Van Wieringen M,et al. Development and characterization of a subtilin-regulated expression system inBacillussubtilis:strict control of gene expression by addition of subtilin[J]. Applied and Environmental Microbiology,2005,71(12):8813-8826.

[60]Guan C,Cui W,Cheng J,et al. Construction and development of an auto-regulatory gene expression system in Bacillus subtilis[J]. Microbial Cell Factories,2015,14:8818-8824.

[61]Tobias Küppers1 VS,Hendrik Hellmuth1,Timothy O’Connell1,et al. Developing a new production host from a blueprint:Bacilluspumilusas an industrial enzyme producer[J]. Microbial Cell Factories,2014;44(13):2-15.

[62]Chen J,Zhu Y,Fu G,et al. High-level intra-and extra-cellular production of D-psicose 3-epimerase via a modified xylose-inducible expression system inBacillussubtilis[J]. Journal of Industrial Microbiology & Biotechnology,2016,43(11):1577-1591.

[63]Guan C,Cui W,Cheng J,et al. Construction and development of an auto-regulatory gene expression system inBacillussubtilis[J]. Microbial Cell Factories,2015,14:150.

[64]Tobias Küppers,Victoria Steffen,Hendrik Hellmuth. Developing a new production host from a blueprint:Bacilluspumilusas an industrial enzyme producer[J]. Microbial Cell Factories,2014,44(13).

[65]Chen J,Jin Z,Gai Y,et al. A food-grade expression system for d-psicose 3-epimerase production inBacillussubtilisusing an alanine racemase-encoding selection marker[J]. Bioresources and Bioprocessing,2017,4(1):9.

[66]Thomas Rygus WH. Inducible high-level expression of heterologous genes inBacillusmegateriumusing the regulatory elements of the xylose-utilization operon[J]. Appl Microbiol Biotechnol,1991(35):594-599.

[67]T Rygus AS,R Allmansberger, W Hillen. Molecular cloning,structure,promoters and regulatory elements for transcription of theBacillusmegateriumencoded regulon for xylose utilization[J].Arch Microbiol,1991(155):535-542.

[68]Peng DH,Pang CY,Wu H. The expression and crystallization of Cry65Aa require two C-termini,revealing a novel evolutionary strategy ofBacillusthuringiensisCry proteins[J]. Scientific Reports,2015,5:8291.

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