文章信息
- 查代明, 张炳火, 李汉全, 闫云君
- ZHA Dai-ming, ZHANG Bing-huo, LI Han-quan, YAN Yun-jun
- 假单胞菌属脂肪酶的分子生物学研究进展
- Research Advances in Molecular Biology of Pseudomonas Lipases
- 中国生物工程杂志, 2015, 35(9): 114-121
- China Biotechnology, 2015, 35(9): 114-121
- http://dx.doi.org/10.13523/j.cb.20150916
-
文章历史
- 收稿日期: 2015-04-22
- 修回日期: 2015-04-30
2 华中科技大学生命科学与技术学院 武汉 430074
2 College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
脂肪酶(triacylglycerol acylhydrolase,EC 3.1.1 .3 )属于α/β型水解酶超家族,广泛存在于各种动物、植物和微生物中[1]。它们在油水界面上能催化长链酰基甘油水解为脂肪酸和二酰基甘油、一酰基甘油或甘油,而在微水相或非水相中能催化多种化学反应,如酯化反应、转酯反应、醇解反应、氨解反应、酸解反应等,催化的反应具有高的化学选择性、区域选择性和/或立体选择性[2, 3, 4]。由于微生物脂肪酶具有资源丰富、催化活性多样、遗传操作简单、产量较高、不随季节波动的稳定供应、生产周期短、生产成本低、稳定性好等优点,因此已成为商品化脂肪酶的主要来源,并被广泛应用于食品、饮料、油脂、洗涤剂、饲料、纺织、皮革、新型材料、精细化工、医药、化妆品、造纸、环境治理、生物能源等诸多工业领域[5, 6, 7, 8]。而在各种来源的微生物脂肪酶中,以细菌脂肪酶催化反应的类型最多、活性最高、稳定性最好,其中又以假单胞菌属(Pseudomonas)脂肪酶的性能最为优越[4, 9, 10]。
假单胞菌属细菌是一类革兰氏阴性菌,属于γ-变形杆菌纲(Proteobacteria)。由于该属细菌广泛存在于自然界中,在微生物学发展早期就能观察到它们的存在,并一直成为科研人员的热门研究对象。该属细菌种类繁多、功能类群多样,有些是动物或植物病原体,如铜绿假单胞菌(P. aeruginosa)、丁香假单胞菌(P. syringae);有些对某些植物病害具有防治作用,如荧光假单胞菌(P. fluorescens)、防御假单胞菌(P. protegens)、绿针假单胞菌(P. chlororaphis)、橘黄假单胞菌(P. aurantiaca);有些具有生物降解能力,如产碱假单胞菌(P. alcaligenes)、门多萨假单胞菌(P. mendocina)、恶臭假单胞菌(P. putida);有些能引起食物腐败,如荧光假单胞菌、莓实假单胞菌(P. fragi)、隆德假单胞菌(P. lundensis)[11, 12, 13]。尽管假单胞菌属细菌分布广泛、功能类群多样,但大多能分泌脂肪酶以分解脂类物质供自身新陈代谢所需。作为性能最为优越、应用最为广泛的一类脂肪酶,对假单胞菌属脂肪酶在基因资源挖掘及克隆、基因表达调控及分泌机制、活性过表达策略、蛋白质结晶及3D结构解析、蛋白质工程等分子生物学研究方向上做了大量的研究工作,本文就这些研究方向进行归纳和述评,并对其未来研究方向进行展望。
1 基因资源挖掘及克隆假单胞菌属脂肪酶基因的挖掘及克隆主要通过以下两种方法。
一种是基于基因组文库构建、活性筛选的方法,包括两种技术,即可培养微生物的基因组文库和不可培养微生物的宏基因组文库。采用基因组文库克隆微生物基因起始较早,已有多个研究小组克隆到假单胞菌属脂肪酶基因[11, 14, 15, 16, 17, 18, 19, 20]。宏基因组文库是一种能从不可培养微生物中克隆新基因的有效手段,据报道不可培养微生物超过原核生物总量的99%[21]。为从这一巨大资源中克隆新的假单胞菌属脂肪酶基因,Hardeman和Sjoling[21]、Khan和Jithesh[22]分别对海底沉积物和土壤开展了相关研究工作并取得了预期结果。
另一种是基于PCR的方法,包括两种技术,即基于基因组步移(genome walking)的二步法克隆基因全长和基于已报道假单胞菌属脂肪酶基因侧翼保守序列设计引物的一步法克隆基因全长。二步法首先也需要根据现有假单胞菌属脂肪酶基因的保守序列设计引物克隆到部分基因序列,然后根据这一序列采用基因组步移PCR即可获得基因全长[23, 24, 25]。随着数据库中假单胞菌属基因组序列和脂肪酶基因序列的日益增多,基于一步法克隆基因全长的研究越来越多[4, 10, 26, 27, 28, 29, 30, 31],并表现出简单、快捷、成本低等优势。
2 基因表达调控及分泌机制截止目前,已克隆了大量假单胞菌属脂肪酶基因,但对其表达调控机制的研究仍处于探索性阶段,主要集中在基于NtrB/C蛋白超家族的双组分系统(two-component system)[32, 33, 34, 35]、基于酰基高丝氨酸内酯(acyl-homoserine lactones,acyl-HSL)的群体感应系统(quorum sensing system)[32, 36]、Gac/Rsm信号转导系统(Gac/Rsm signal transduction system)[36, 37, 38, 39]、渗透压感应双组分系统EnvZ/OmpR[40]和精氨酸代谢调控因子ArgR[41]对脂肪酶基因的表达调控(图 1)。从图 1可知,只有Gac/Rsm信号转导系统的调控机制被阐明,通过途径①“Gac/Rsm信号转导系统→QS系统→NtrB/C蛋白超家族→脂肪酶”间接激活脂肪酶基因转录(发现于铜绿假单胞菌中[31, 32, 33, 34, 35, 36, 37],图 2)或通过途径②“Gac/Rsm信号转导系统→脂肪酶”直接激活脂肪酶基因翻译(本课题组发现于防御假单胞菌中[39],图 3)。然而,Gac/Rsm信号转导系统还通过其他未知途径对脂肪酶基因的表达进行微调,如抑制铜绿假单胞菌脂肪酶基因lipA的转录(图 2)、激活防御假单胞菌脂肪酶基因lipA的转录(图 3),虽然它们不占主导作用,但是调控的分子机制值得进一步研究。基于上述分析并结合相关文献[39, 42],我们对Gac/Rsm信号转导系统激活假单胞菌属脂肪酶基因表达的调控机制提出以下假设:当假单胞菌属细菌拥有基于acyl-HSL的QS系统时,主要通过途径①激活脂肪酶基因转录;当假单胞菌属细菌缺乏基于acyl-HSL的QS系统时,主要通过途径②激活脂肪酶基因翻译。
假单胞菌属细菌是一类革兰氏阴性菌,主要通过两种途径分泌胞外脂肪酶(表 1)[6, 32]。Type I型分泌途径:脂肪酶C端信号序列首先被ABC输出子特异性识别,然后通过ABC蛋白复合体形成的通道直接分泌到胞外。Type II型分泌途径:脂肪酶前体N端信号肽首先被内膜(质膜)上的多亚基蛋白复合体Sec装置识别并进入周质,此过程伴随信号肽的切除;然后在同源折叠酶Lif和二硫键形成蛋白Dsb的作用下折叠成正确构象并形成二硫键,此过程中错误折叠的脂肪酶将被周质蛋白酶所降解;成熟脂肪酶最后在Xcp分泌子的介导下通过外膜分泌到胞外。
Secretion pathways | Secretion systems | Secretion signals | Accessory factors | Representative lipases |
Type I | ABC输出子:由内膜ATP酶、膜融合蛋白 MFP和外膜蛋白OMP组成 | C端信号序列,不被切除 | 无 | 荧光假单胞菌 SIK W1脂肪酶 |
Type II | 内膜Sec装置:由多个亚基组成;内外膜 Xcp分泌子:由≤ 14个蛋白质组成 | N端信号肽,被切除 | Lif、Dsb | 铜绿假单胞菌 PAO1脂肪酶 |
脂肪酶的大量活性表达一直是困扰科研人员的难题,其原因主要有两:其一,宿主细胞常常没有与异源脂肪酶相对应的分泌系统,大量表达的异源脂肪酶容易对宿主细胞产生较大毒性而过早死亡,或者为避免毒害宿主细胞而形成无活性包涵体;其二,多数脂肪酶需要在同源折叠酶的作用下才能形成正确构象的活性蛋白质,而异源宿主细胞常常没有对应的折叠酶[43]。由于假单胞菌属脂肪酶的优越性,人们对其活性过表达开展了一系列研究,归纳起来主要有以下几种策略。
3.1 脂肪酶基因与同源分泌系统基因簇共表达Ahn等[44]将荧光假单胞菌SIK W1的脂肪酶基因tliA及同源ABC输出子基因簇tliDEF在假单胞菌属菌株中共表达,研究了同源分泌系统对假单胞菌属细菌产脂肪酶的影响。结果表明,共表达tliDEFA显著性优于单独表达tliA,其中同源表达tliDEFA效果最好,是同源表达tliA的69倍;同源共表达显著性优于异源共表达,但同源表达tliA不一定优于异源表达tliA;TliDEF介导的TliA分泌具有温度依赖性,TliDEF在培养温度高于30℃时丧失分泌功能。然而,并不是所有同源共表达都优于异源共表达。Song等[45]的研究表明,tliDEFA在黏质沙雷氏菌(Serratia marcescens)中共表达显著性优于同源共表达,可能是因为TliDEF介导的TliA分泌在黏质沙雷氏菌中没有生长阶段依赖性,能持续表达并分泌TliA直至稳定生长期。对于Type II型分泌途径分泌的脂肪酶,xcp基因簇的过表达能增加脂肪酶的产量[46, 47]。
3.2 脂肪酶基因与同源折叠酶基因共表达多数假单胞菌属脂肪酶表达后,需要在同源折叠酶的作用下才能形成活性蛋白质,它们的异源表达经常形成无活性包涵体。大量研究表明,假单胞菌属脂肪酶基因与同源折叠酶基因的共表达能很好地克服这一瓶颈[15, 29, 48, 49, 50]。Omori等[48]以假单胞菌属菌株109的脂肪酶操纵子lipL/limL为研究对象,系统研究了表达宿主、启动子、基因拷贝数及其组合对lipL/limL共表达的影响。结果显示:在弱启动子tac-lac UV5的控制下,同源表达产脂肪酶的效果是原始菌株的7.1倍;在强启动子T7的控制下,铜绿假单胞菌中异源表达产脂肪酶的效果是原始菌株的13倍;lipL和/或limL基因拷贝数的组合也会影响脂肪酶产量,其中(lipL)3-limL组合最优,其产脂肪酶的效果是原始菌株的67倍。因此,表达宿主适配性、启动子强弱、共表达基因的拷贝数及组合都是影响脂肪酶产量的主要因素。
3.3 脂肪酶表面展示Lee等[51, 52]利用铜绿假单胞菌外膜蛋白OprF成功将TliA展示在大肠杆菌(Escherichia coli)XL10-Gold和恶臭假单胞菌KT2442表面,与大肠杆菌表面展示系统相比,恶臭假单胞菌表面展示系统在酶活力、有机溶剂耐受性、温度稳定性、手性拆分能力等方面表现出更大优势。Jung等[53, 54]利用丁香假单胞菌冰核蛋白INP将TliA成功展示在大肠杆菌JM109和恶臭假单胞菌GM730表面,恶臭假单胞菌表面展示系统也全面优于大肠杆菌表面展示系统。
3.4 脂肪酶基因在酵母中表达与大肠杆菌、酿酒酵母(Saccharomyces cerevisiae)表达系统相比,巴斯德毕赤酵母(Pichia pastoris)表达系统具有无可比拟的优势[55],作为异源宿主也常常被应用于表达原核基因。Jiang等[23]、王海燕[55]、Yang等[27]利用巴斯德毕赤酵母作为异源宿主成功实现了荧光假单胞菌脂肪酶基因的表达和分泌,这是因为该类脂肪酶属于I.3亚家族,无需同源折叠酶等辅助因子的帮助就能折叠成活性构象。
4 蛋白质结晶及3D结构解析脂肪酶3D结构在理论方面有助于解释催化机制和研究构效关系,在工程技术方面可以辅助蛋白质工程。到目前为止,有三个假单胞菌属脂肪酶的3D结构被解析。
Boston等[56]解析了门多萨假单胞菌脂肪酶的3D结构(PDB ID:2FX5),并基于结构分析研究了点突变对脂肪酶活力的影响,认为增加甘油三酯底物的酰基链结合是提高脂肪酶活力的关键。Sibille等[57]在此基础上,通过NMR分析和分子动力学模拟提出了降低脂肪酶的分子柔性是提高其稳定性的基础。
Nardini等[58]解析了铜绿假单胞菌PAO1脂肪酶(PAL)开放构象的3D结构(PDB ID:1EX9),根据结构分析得出酰基口袋大小、sn-2结合口袋大小及相互作用是PAL具有区域和立体选择性的决定性因素。
Angkawidjaja等[59]解析了假单胞菌属菌株MIS38脂肪酶(PML)及其铂结合S445C突变体封闭构象的3D结构(PDB ID:2Z8X、2Z8Z),发现PML有两个盖子结构(Lid1和Lid2)。基于结构分析得出了盖子结构的开放机制,即Lid1在开放构象时,Ca2+结合到Ca1有助于Lid1定位于开放构象;Lid1在封闭构象时,Ca1消失、Lid1的结构和位置发生巨大改变;Lid2在开放构象时形成一个螺旋状发夹结构,而在封闭构象时不形成该发夹结构并覆盖活性位点。为研究Ca2+结合位点对PML活力和稳定性的重要性,该研究小组解析了两个突变体D157A和D337A的3D结构(PDB ID:2ZJ6、2ZJ7),结果表明活性位点的完全开放需要Ca1、Ca2位点,Ca2、Ca3位点有助于PML的稳定[60]。该研究小组又根据PML不同构象的3D结构(PDB ID:2ZVD、3A6Z、3A70)结合分子动力学模拟研究了PML的界面激活机制,结果表明Ca1的功能类似于钩子,有助于稳定Lid1的完全开放构象和引发Lid2的随后开放[61]。
5 蛋白质工程蛋白质工程以基因工程为基础,根据蛋白质的结构和功能,通过基因的人工定向改造获得性质改变的蛋白质以满足人类需求。蛋白质工程主要有三种技术,分别为理性设计(rational design)、非理性设计(irrational design)和半理性设计(semi-rational design)[62]。
5.1 理性设计理性设计需要蛋白质的3D结构和功能信息,通过分析结构与功能的关系确定需要突变的氨基酸残基,最后利用定点突变技术达到突变氨基酸残基的目的[63]。Santarossa等[64]通过理性设计定点突变了莓实假单胞菌脂肪酶(PFL)盖子区的3个极性氨基酸残基,T137和T138的突变增加了PFL对C8底物的相对活力和PFL的温度稳定性,而S141的突变破坏PFL的稳定。
5.2 非理性设计非理性设计又称为定向进化或体外分子进化,不需要蛋白质的3D结构和功能信息,通过模拟自然进化随机突变目的基因,定向筛选后获得预期的改良蛋白质[65]。Jung等[53]利用非理性设计随机突变tliA,通过三丁酸甘油酯平板筛选得到4个水解活力增高的TliA突变体(T48、T54、T61和T68)。Fujii等[66]随机突变铜绿假单胞菌脂肪酶基因,通过油酰基-2-萘酰胺(oleoyl 2-naphthylamide)水解活力筛选得到5个酰胺水解酶活力增高的突变体,其中3个突变位点(F207S、A213D和F265L)影响酰胺水解酶/酯酶活力比;这些突变位点的组合进一步提高酰胺水解酶活力,其中F207S/A213D组合的活力最高;3D结构模拟显示这些突变位点远离活性中心而靠近蛋白质表面的Ca2+结合位点。
5.3 半理性设计半理性设计是理性设计与非理性设计的结合,融合了理性设计和非理性设计的优点,需要蛋白质的3D结构或功能信息。一般来说,半理性设计包括4种方案:对特定氨基酸残基进行随机突变、随机突变与定点饱和突变同时进行、随机突变筛选突变位点后进行定点饱和突变、利用计算方法进行半理性设计和组合设计[62]。Carballeira等[67]采用组合活性中心饱和试验(combinatorial active site saturation test,CAST)的策略定向筛选到一个具有高度对应选择性的铜绿假单胞菌脂肪酶突变体(Leu162Phe)。Nakagawa等[68]利用随机突变和饱和突变相结合的策略定点突变P. aeruginosa脂肪酶底物结合位点的所有氨基酸残基,鉴定出残基Leu252是提高酰胺水解酶活力的热点,Met或Phe替换Leu252显著性增加该脂肪酶的酰胺水解酶活力而酯酶活力不受影响,其中三联突变体(F207S/A213D/M252F)的酰胺水解酶活力是原始酶的28倍。
可以预期,随着脂肪酶3D结构解析的日益增多,理性设计和半理性设计将会成为脂肪酶蛋白质工程的首选技术。
6 展望假单胞菌属脂肪酶性能优越、应用范围广泛,在生物转化领域具有非常重要的作用,一直是脂肪酶研究领域的热点对象。虽然已在前文所述分子生物学研究方向上开展了大量研究,并取得了丰硕成果,但是在工业化生产、基因资源挖掘及克隆、蛋白质结晶及3D结构解析、生产系统构建等方向仍需大量而深入的研究,具体研究内容见表 2。
Research directions | Research contents |
工业化生产 | 基因表达调控机制、基因工程菌构建、发酵条件优化 |
基因资源挖掘及克隆 | 极端环境微生物脂肪酶基因、宏基因组筛选不可培养微生物脂肪酶基因、基因组数据库中的脂肪 酶基因、蛋白质工程改造现有脂肪酶基因 |
蛋白质结晶及3D结构解析 | 解释催化机制、研究构效关系、辅助蛋白质工程 |
生产系统构建 | 用于不同来源脂肪酶的表达与分泌 |
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