山东大学 微生物技术国家重点实验室，山东 济南 250100
收稿日期：2016-09-21;接收日期：2017-01-09; 网络出版时间：2017-01-16 基金项目：国家重点基础研究发展计划 (973计划) (No. 2011CB707402)，国家自然科学基金 (Nos. 31272370, 30870085) 资助
摘要： 漆酶属于多铜氧化酶家族中的一种，广泛存在于昆虫、植物、真菌和细菌中。由于其作用的底物范围较广，因此在纺织、制浆、食品以及木质素的降解等方面有广阔的应用前景。但是自然界中的漆酶存在表达量和酶活低、高温易失活等问题，限制了它的应用。对漆酶进行大量高效的异源表达，是解决这一问题的有效途径。近年来，越来越多不同来源的漆酶基因被克隆，并在不同宿主中异源表达。但这些大多局限于实验室研究，还未达到工业化生产的水平。笔者对真核生物来源漆酶的异源表达研究进展进行综述，重点介绍了真核生物来源的漆酶在不同表达系统中的异源表达情况以及在酵母细胞中表达漆酶时提高表达量和酶活性能的方法，以期为研究者们提供参考。
关键词： 真核漆酶 异源表达 酶活力 稳定性
Advance of heterologous expression study of eukaryote-origin laccases
Na Ning, Huijun Tan, Xinxin Sun, Jinfeng Ni
State Key Laboratory of Microbial Technology, Shandong University, Ji'nan 250100, Shandong, China
Received: September 21, 2016; Accepted: January 9, 2017; Published: January 16, 2017
Supported by: National Basic Research and Development Program of China (973 Program) (No. 2011CB707402), National Natural Science Foundation of China (Nos. 31272370, 30870085)
Corresponding author:Jinfeng Ni. Tel: +86-531-88363323; E-mail: jinfgni@sdu.edu.cn
Abstract: Laccases are enzymes belonging to the group of multi-copper oxidases. These enzymes are widely distributed in insects, plants, fungi and bacteria. In general, laccases can oxidize an exceptionally high number of substrates, so they have broad applications in textile, pulp, food and the degradation of lignin. However, low yield, low activity and thermo-instability of laccase in nature limit the application of laccase. High efficient heterologous expression of the protein is an effective way for solving this problem. Here, we summarize the research advances of heterologous expression of eukaryote-origin laccases. We focus on the overexpression of eukaryote-origin laccases using different expression system and the method for improving the production yield and enzyme activity in yeast cells. Information provided in this review would be helpful for researchers in the field.
Key words: eukaryote-origin laccases heterologous expression enzyme activity stability
漆酶 (Laccase，苯二酚：双氧氧化还原酶；EC 1.10.3.2) 与抗坏血酸氧化酶和各种铁氧化酶同属于多铜氧化酶家族[1]。漆酶最早被发现于植物中[2]，随着分子生物学和生物信息学技术的发展，人们陆续在细菌[3]、真菌以及昆虫[4]中发现了漆酶。不同来源的漆酶具有不同的体内功能。细菌漆酶主要与细菌色素的形成和金属离子抵抗有关[5-6]。也有报道称芽孢杆菌中的漆酶，在抗紫外线孢子的发育中起作用[7]。目前研究最多的是真菌漆酶，主要集中于担子菌和子囊菌中的漆酶，这类菌株多为白腐真菌[8]。白腐真菌是目前所知道的唯一能够利用自身氧化酶系统将木质素降解为二氧化碳的微生物[9]，漆酶在此过程中起了重要作用。另外，真菌漆酶还是一些致病真菌的毒性成分，在真菌的分化和色素形成中起重要作用[10]。植物漆酶虽然发现较早，但研究却相对较少。植物中的漆酶主要与木质素的合成以及损伤部位的修复有关[2]。也有少量的报道证明植物中的漆酶在植物种间竞争[11]以及植物抵抗微生物的侵染[12]中起作用。昆虫漆酶根据生理作用的不同可以分为漆酶1和漆酶2。目前昆虫漆酶的研究多集中于漆酶2在昆虫外骨骼的硬化、表皮色素沉积以及角质层鞣化方面的作用，而对可能在木质素降解或食物脱毒中起作用的漆酶1的报道较少[13]。
漆酶底物范围广泛，主要包括酚类化合物、芳香族化合物、脂肪胺和无机阳离子等[14]。漆酶可以利用铜离子特有的氧化还原能力，对还原性底物进行单电子氧化，同时传递4个电子，将作为第二底物的氧气还原成水[15]。基于漆酶底物范围的广泛性以及副产物只有水的环境友善性，使得漆酶在木质素降解、造纸工业、染料脱色、食品和饮料业等方面都具有潜在的应用价值[16]。然而，自然界中分离到的野生菌株的漆酶有产量和活性较低、纯化困难、高温易失活、不易操作等缺点，很大程度上阻碍了对漆酶生理生化性质的基础研究及其工业化应用[10, 17-18]。近年来，越来越多不同来源的漆酶基因被克隆，并在不同宿主中异源表达。但这些大都局限于实验室研究，还未达到工业化生产的水平。提高漆酶的表达量、重组蛋白的耐热性和pH稳定性是目前漆酶工业应用需要面对的问题。对于细菌漆酶的研究进展已经有****对其进行了总结[19-20]，因此文中将结合笔者的研究工作，对近年来真核来源的漆酶 (植物、真菌和昆虫来源的漆酶) 的异源表达情况进行概述，以期为相关研究者们提供参考。
1 真核生物来源的漆酶在不同表达系统中的表达目前为止，异源表达真核生物来源的漆酶 (以下简称真核漆酶) 所用表达宿主主要有细菌、酵母、丝状真菌和昆虫杆状细胞，其中使用最为广泛的宿主是毕赤酵母Pichia pastoris。下面根据所使用表达系统的不同，对真核漆酶的异源表达情况进行介绍。
1.1 真核漆酶在细菌中的表达由于细菌具有好操作、培养周期短、经济实惠等特点，经常被用来表达漆酶。其中大肠杆菌表达系统是应用最广泛的原核表达系统。表 1显示了真核漆酶在细菌中的表达情况。杨建强等[21]将野生革耳Panus rudis来源的漆酶基因在大肠杆菌中表达，得到可溶性漆酶蛋白，在可溶漆酶蛋白中添加CuSO4并在室温下孵育复性，获得有活性的漆酶，这是首次报道的真菌来源的漆酶基因在大肠杆菌中实现表达。另外来自黑蛋巢菌Cyathus bulleri [22]和白腐担子菌类木硬孔菌Rigidoporus lignosus [23]的真菌漆酶也在大肠杆菌中成功异源表达。
表 1 漆酶在细菌中的表达Table 1 The expression of laccase genes in bacteria
| Laccase origin | Expression hosts | Substrates | Active | References |
| Panus rudis | Escherichia coli | ABTS | Yes | [21] |
| Cyathus bulleri | Escherichia coli | ABTS | Yes | [22] |
| Rigidoporus lignosus | Escherichia coli | Syringaldazine | Yes | [23] |
| Termitomyce albuminosus | Escherichia coli | ABTS | No | [24] |
| Pleurotus eryngii | Lactobacillus buchneri | NR | NR | [25] |
| ABTS: 2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate); NR: not reported. | ||||
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然而，潘程远等[24]在大肠杆菌中表达来源于鸡枞菌Termitomyce albuminosus的漆酶cDNA (talcc) 时，得到重组漆酶蛋白，但未检测到漆酶活性。大肠杆菌虽然操作简便，但一般不具备对重组蛋白进行二级结构修饰的能力，而漆酶是一种高糖基化修饰的酶，因此在大肠杆菌中表达真核来源的漆酶时往往会得到没有酶活性的重组蛋白。
1.2 真核漆酶在酵母细胞中的表达酵母是最低等的真核生物。具有操作简单、生长速率快、可进行高密度发酵；培养基廉价，适用于工业生产；不含有病原体、热原体和病毒包涵体；可对重组蛋白进行蛋白酶解、形成二硫键和糖基化等翻译后修饰的优点。但是对在酵母中异源表达的漆酶进行糖基化分析，发现酵母对漆酶的糖基化有时会影响甚至破坏漆酶的酶活性质和酶活力[26-27]。
尽管有一些酵母菌会产生自己的漆酶[28-29]，但是很多不同的酵母，例如酿酒酵母Saccharomyces cerevisiae、毕赤酵母Pichia pastoris、乳酸克鲁维酵母Kluyveromyces lactis、解脂耶氏酵母Yarrowia lipolytica、隐球酵母Cryptococcus sp.作为宿主，都成功表达了其他真菌 (担子菌门和子囊菌门) 来源的漆酶。表 2总结了2007年以来截止到目前在酵母中成功表达的部分真核漆酶。非常有趣的是，漆酶的表达水平因酵母宿主和漆酶亚型的不同而不同。例如，在乳酸克鲁维酵母K. lactis和酿酒酵母S. cerevisiae中表达来自于糙皮侧耳Pleurotus ostreatus中的漆酶POX3时，酿酒酵母中表达的漆酶酶活性比在乳酸克鲁维酵母中的高[30]。Gu等[31]在毕赤酵母P. pastoris中表达来自于毛头鬼伞Coprinus comatus的漆酶Lac3和Lac4时，Lac4的酶活可达到1 465 U/L，而Lac3的酶活为689 U/L。表达漆酶时在培养基中加入不同浓度的硫酸铜 (0.1-1.0 mmol/L)，可以促进漆酶的表达并提高酶活力。Pezzella等[30]在酿酒酵母中表达来自于糙皮侧耳P. ostreatus中的漆酶，不加硫酸铜时酶活为30 U/L，加入0.6 mmol/L硫酸铜时酶活提高至75 U/L。另外在培养过程中，达到最大酶活的时间差异较大，从48-504 h不等，这种差异主要与漆酶亚型、表达载体以及培养规模等相关。酵母菌产生的重组漆酶在应用方面的研究主要集中于染料脱色方面 (表 2)，也有关于漆酶对酚类化合物的降解作用[32]以及对植物生长的促进作用[33]的相关研究。
表 2 漆酶在酵母细胞中的表达Table 2 Expression of laccase genes in yeast cells
| Origin | Laccases | Expression hosts | Expression plasmids | CuSO4 concentration (mmol/L) | Culture time (h) | Maximum activity (U/L) | Applications of the recombinant laccases | References |
| Colletotrichum lagenarium | CILACII | Pichia pastoris GS115 | pPIC9K | NR | NR | NR | Decolorization of dyes | [34] |
| Moniliophthora perniciosa FA553 | LacMP | Pichia pastoris X33 | pPICZaA-6AA-LacMP | 0.4 | 96 | 232 | NR | [35] |
| Volvariella volvacea | Vvlcc3 | Pichia pastoris GS115 | pPIC9K-Vvlcc3 | 0.3 | 504 | 296.83 | NR | [36] |
| Basidiomycete cerrena sp. | Lac1 | Pichia pastoris X33 | pPICZα-Lac1 | 1.0 | 216 | 6 300 | Decolorization of dyes | [37] |
| Phytophthora capsici | PCLAC2 | Pichia pastoris GS115 | pPIC9K/ Pclac2 | NR | 168 | 84 000 | NR | [38] |
| Coprinopsis cinerea | CcLcc2 | Pichia pastoris GS115 | pYM7898 | NR | 72 | NR | Dyes decolorization | [39] |
| Okayama-7#130 | ||||||||
| Coprinus comatus | Lac3 | Pichia pastoris KM71H | pPICZαB-10AALac3 | 0.5 | 336 | 689 | Dyes decolorization | [31] |
| Lac4 | pPICZαB-10AALac4 | 1 465 | ||||||
| Phomopsis liquidambari | LACB3 | Schizosacchar-omyces pombe | pESP-3-lacB3 | NR | NR | NR | Growth promotion of plants | [33] |
| Volvaria volvacea | vv-lac1 | Pichia pastoris GS115 | pPIC9K-vv-lac1 | NR | NR | 333.17 | NR | [40] |
| vv-1ac6 | pPIC9K-vv-lac6 | 227.63 | ||||||
| Canoderma lucidum TR6 | glacTR6 | Pichia pastoris GS115 | pPIC9K-glacTR6 | 0.3 | 432 | 685.8 | NR | [41] |
| Coprinus comatus | Lac1 | Pichia pastoris KM71H | pPICZαB-lac1 | 0.5 | 144 | 550 | Dyes decolorization | [42] |
| Cyathus bulleri | Lcc | Pichia pastoris X33 | pPICZαBlcc-5 | 0.4 | 72 | 7 200 | NR | [43] |
| Monilinia fructigena | MfLcc | Pichia pastoris GS115 | pYM8025 | NR | 48 | NR | Degradation of phenolic compounds | [32] |
| Botrytis aclada | BaLac | Pichia pastoris X33 | pGAPZA-BaLac | 0.1 | 76 | 53 300 | NR | [44] |
| Ganoderma lucidum | GlLCCI | Pichia pastoris GS115 | pYM 7909 | 1.0 | 72 | NR | Decolorization of methylorange | [45] |
| Polyporus grammocephalus | Lac-T16 | Pichia pastoris GS115 | pPIC3.5K-TR16lac | 0.3 | 264 | 320·8 | NR | [46] |
| TR16 | pPIC9K-TR16lac | NR | NR | NR | ||||
| Pleurotus ostreatus | rPOX3 | Saccharomyces cerevisiae W30 | pSAL4-POX3 | NA | 48 | 30 | NR | [30] |
| 3-1A | 0.6 | 48 | 75 | |||||
| Kluyveromyces lactis | pYG132-POX3 | NA | 72 | 12 | ||||
| Pleurotus eryngii | Ery3 | Free Saccharomyces cerevisiae cells | pY-Ery3 | 0.5 | 72 | 88 | NR | [47] |
| Immobilized Saccharomyces cerevisiae cells | 0.5 | 120 | 139 | |||||
| Pleurotus ostreatus | POXA3 | Kluyveromyces lactis | A3L | 1.0 | 336-408 | 20 | NR | [48] |
| Ganoderma lucidum | GLlac1 | Pichia pastoris | pPIC-RCL1 | NR | 168 | NR | Antioxidative properties | [49] |
| Trametes sp. 420 | rLacD | Pichia pastoris GS115 | pPIC9K-LacD | 0.3 | NR | 83 000 | Decolorization of dyes | [50] |
| Trametes sp. 420 | rLacC | Pichia pastoris GS115 | pPIC9K-LacC | 0.3 | 216 | 16 200 | Dye decolorization | [51] |
| ABTS: 2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate); NR: not reported; NA: not added. | ||||||||
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目前为止，还没有昆虫来源的漆酶在酵母中成功异源表达的相关报道。笔者尝试在毕赤酵母中表达来自黄翅大白蚁Macrotermes barneyi唾液腺-前肠的漆酶基因，尽管使用了不同菌株作为宿主并优化了培养条件，但并未得到重组蛋白。可能是由于密码子的偏好性、糖基化位点以及二硫键数目较多等原因而使昆虫来源的漆酶难以在毕赤酵母中正常表达。
1.3 真核漆酶在丝状真菌中的表达目前为止，漆酶已经在曲霉属Aspergillus sp.[52-57]、里氏木霉Trichoderma reesei [58-60]和灰白青霉Penicillium canescens [61]中表达 (表 3)。大多数情况下丝状真菌用来表达来源于白腐真菌的漆酶。例如栓菌属Trametes sp.的漆酶在里氏木霉[59]和曲霉[57-62]中成功表达。除了真菌漆酶，也有细菌来源的 (天蓝色链霉菌 Streptomyces coelicolor ) 漆酶在米曲霉中成功表达，并解析了它的三维结构[63]。由于在丝状真菌中表达重组漆酶时有一个相对较高的蛋白产量，因此有利于研究漆酶的分子量[52, 64-65]、反应机制[66]和三维结构[63-67]。丝状真菌产生的重组漆酶在纸浆脱色[68-69]、合成染料的生物转化[54]以及植物修复[62]中广泛应用。另外丝状真菌表达的重组漆酶还用于食品工业黄曲霉毒素的消除、环境中酚类化合物的检测中。
表 3 部分丝状真菌中表达的重组漆酶的特性Table 3 Properties of some recombinant laccases expressed in filamentous fungi
| Laccase origin | Expression hosts | Substrates | Maximum enzyme activity (U/L) | Optimum temperature (℃) | Optimum pH | References |
| Trametes villosa | Aspergillus oryzae | Syringaldazine | NR | NR | 5.0-5.5 | [52] |
| ABTS | 8 250 | NR | 2.7 | |||
| Ceriporiopsis subvermispora | Aspergillus nidulans | ABTS | 230 | NR | NR | [53] |
| Aspergillus niger | ||||||
| Phanerochaete flavido-alba | Aspergillus niger | ABTS | 2 500 | NR | 3.0 | [54] |
| Trichoderma reesei i | Trichoderma reesei | ABTS | 46 800 | NR | NR | [58] |
| Guaiacol | NR | 5.0-7.0 | ||||
| Trametes sp. AH28-2 | Trichoderma reesei | ABTS | 3 620 | 50 | 3.0 | [59] |
| Guaiacol | NR | 50 | 4.2 | |||
| Pleurotus ostreatus | Trichoderma reesei | ABTS | 237 134 | 50 | 3.0 | [60] |
| Trametes hirsute | Penicillium canescens | NR | 3 000 | NR | NR | [61] |
| ABTS: 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate); NR: not reported. | ||||||
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1.4 真核漆酶在植物中的表达植物主要用来表达来自不同植物或真菌的漆酶。主要的植物表达宿主有拟南芥Arabidopsis thaliana、番茄Lycopersicon esculentum、水稻Oryza sativa、烟草Nicotiana tabacum、甘蔗Saccharum officinarum、玉米Zea mays等。在番茄中过量表达来自马铃薯的多酚氧化酶，表达植株对丁香假单胞菌Pseudomonas syringae的抵抗能力明显增强[70]。有些情况下，构建表达漆酶的转基因植物是为了建立植物修复系统，来降解双酚、三氯苯酚、五氯苯酚及其他酚类化合物[72]。在甘蔗中表达漆酶，有助于阐明漆酶在木质化过程中的作用[73]。
1.5 真核漆酶在昆虫细胞中的表达目前发现的动物来源的漆酶主要集中于昆虫漆酶，在甲壳类以及棘皮动物中也有发现。根据昆虫漆酶的功能和表达部位的不同，将分布于昆虫唾液腺、马氏管和中肠组织中的漆酶称为漆酶1 (laccase 1)，此类漆酶可能参与食物的脱毒作用[4]；而将表达于表皮、卵壳等组织的漆酶称为漆酶2 (laccase 2)，此类漆酶与昆虫角质层的黑化作用有关[73]。
Dittmer等[74]利用昆虫杆状病毒表达系统，成功表达了烟草天蛾Manduca sexta来源的多铜氧化酶漆酶2。该研究表明在昆虫杆状病毒表达系统中表达的重组昆虫漆酶可以代替内源的酶来测定生化性质，帮助我们更广泛地了解烟草天蛾来源的漆酶以及其他昆虫来源漆酶的酶学性质和功能。Coy等[75]通过RACE的方法获得了美洲散白蚁Reticulitermes flavipes唾液腺-前肠中的两个漆酶基因 (RfLacA和RfLacB)，因为该酶在微生物缺乏的唾液腺和前肠中活性最高，并且进化上比较特别，推测该酶由白蚁自身产生。将这两个基因在昆虫杆状病毒表达系统中表达，纯化后测定酶活，发现这两个酶对木质素的单体芥子酸及其他几种酚氧化物具有较强的活力，而对黑色素前体没有或有很低的酶活力，这就为白蚁唾液腺和前肠产生的漆酶在木质纤维素降解中起作用提供了重要的证据。然而，我们前期实验尝试将黄翅大白蚁M. barneyi中肠来源的漆酶基因在昆虫杆状病毒系统中表达，但是并没有成功表达。昆虫杆状病毒表达系统的使用，使得对昆虫漆酶的研究成为可能。但是该表达系统与细菌和真菌表达系统相比，操作较为繁琐，价格和成本也比较高。
2 漆酶表达量及酶活性能的提高如前所述，漆酶应用方面存在表达困难、热稳定性和pH耐受性较差和酶活力较低等问题。目前提高漆酶表达量的方法主要有两个方面：构建重组基因和控制培养条件及产酶条件。在提高酶活力和热稳定性方面，主要的方法包括定点突变、随机突变和DNA Shuffling等基因工程的方法。由于酵母是目前表达漆酶应用最广泛的宿主，因此更多的研究集中于漆酶在酵母细胞中的表达优化，下面对此进行介绍。
2.1 启动子的选择在表达漆酶时，首先要选择一个合适的强启动子。在酿酒酵母中表达漆酶时，使用强启动子半乳糖激酶 (GAL1或GAL10) 启动子，利用半乳糖进行诱导，可以使漆酶有一个较高水平的表达[64]。选择铜结合蛋白 (CUP1) 启动子，在培养基中加入CuSO4至终浓度0.4-0.5 mmol/L，不仅可以诱导重组蛋白大量表达，也为漆酶提供了适量的铜离子。尽管这些诱导型启动子的使用，使得漆酶的表达量较高，但是由于诱导物的成本较高，并不适用于工业生产。从生产成本方面考虑，强的组成型启动子更具优势。在酿酒酵母中应用于表达漆酶的组成型启动子主要有乙醇脱氢酶 (ADH1) 启动子、磷酸甘油酸激酶 (PGK1) 启动子、磷酸丙糖异构酶 (TPI1) 启动子、翻译延伸因子-1 (TEF1) 启动子以及甘油醛-3-磷酸脱氢酶 (GPD1) 启动子。在毕赤酵母中表达漆酶时，最常用的是甲醇诱导型的乙醇氧化酶 (AOX1) 启动子[43]。有研究显示当用0.5%的甲醇诱导时，比用2.0%甲醇诱导时表达水平要高出5倍，从2.0 U/mL提高到11.5 U/mL[76]。类似的，在Pichia methalonica中使用甲醇诱导型启动子 (AUG1) 来表达漆酶时，最适合的产漆酶的甲醇浓度为0.8%，同样低于1%[77]。由于甲醇有毒并且成本较高，因此人们也在努力寻求不需甲醇诱导的启动子。在毕赤酵母中利用组成型的GAP启动子，成功表达了来自于木质层孔菌Fomes lignosus [78]、灵芝Ganoderma lucidum [79]、担子菌类[80]、糙皮侧耳Pleurotus ostreatus [79]以及云芝Trametes versicolor [81]来源的漆酶基因。
2.2 信号肽的选择通常真核生物中的漆酶均分泌到胞外，这是由于在N-末端带有信号肽，促使蛋白分泌，分泌到胞外的蛋白利于纯化和应用。在酵母细胞中异源表达漆酶时，除了利用漆酶自身的信号肽之外，也可使用酵母的信号肽。通常应用比较多的是来源于酿酒酵母的信号肽序列α-factor以及它的修改序列[80]。另外，蔗糖酶基因 (SUC2)、胞外碱性蛋白酶基因 (XPR2)、木聚糖酶 (Xylanase) 基因的信号肽也可以用于漆酶基因在酵母中的高效表达[16]。值得注意的是，有时使用漆酶自身的信号肽比使用α-factor信号肽序列得到的重组蛋白活力要高，而有时情况则相反。例如，在毕赤酵母中表达来自于云芝T. versicolor的漆酶基因laccase Ⅳ时，使用α-factor信号肽，致使分泌的重组蛋白N-端残留了一个四肽，使酶比活相对于使用自身信号肽时下降了25%，从0.88 U/mg下降为0.68 U/mg[82]。而在P. methalonica中表达来自云芝的漆酶基因Lcc1，使用α-factor信号肽时的活力 (3.17 U/mL) 是自身信号肽 (1.87 U/mL) 的1.7倍[83]。类似的，在毕赤酵母中表达来自Physisporinus rivulosus的漆酶时，使用α-factor信号肽时的酶活是使用自身信号肽的1.6倍，分别为4.9 μkat/μg和3.14 μkat/μg [84]。以上这些结果的不同说明对于不同的漆酶和不同的宿主，需要对信号肽进行特定的优化来增加表达量和酶活力。
2.3 漆酶基因改造很多研究报道，合成酵母密码子偏好的漆酶基因，比用原始的漆酶序列获得的重组蛋白更多。通过密码子优化的方式成功表达的漆酶有来源于灰盖鬼伞Coprinus cinereus的漆酶基因LCC5I [85]、灵芝G. lucidum的漆酶基因LCCI [45-79]、桃褐腐病菌M. fructigena的漆酶基因LCC2 [32]和糙皮侧耳P. ostreatus的漆酶基因POXA1b [79]。
改造漆酶编码基因，除了可以提高表达量，还可以提高漆酶的稳定性、改变最适pH、改变K m和K cat以及提高底物亲和力。改造基因使用的主要技术包括随机突变、定向突变以及DNA Shuffling等。Bulter等[86]通过10轮的分子进化使得来自于嗜热毁丝霉Myceliophthora thermophila的漆酶在酿酒酵母中的异源表达量提高了8倍，达到18 mg/L，总酶活提高了170倍，K cat值提高22倍 (从 (80±2.5) min-1提高到 (1 740±34) min-1)。Festa等[87]将糙皮侧耳P. ostreatus来源的漆酶POXA1b在毕赤酵母中表达，通过易错PCR和DNA Shuffling的方法使重组漆酶活性从 (183±1) U/mg提高到 (454±2) U/mg，在60 ℃、pH 7.0条件下的半衰期t 1/2从2.2 h提高至3.1 h。
另一种使得漆酶成功表达的方法是在漆酶基因N-端或C-端加入氨基酸标签来融合表达。例如Gu等[31]通过在来源于毛头鬼伞C. comatus的两个新型漆酶同工酶的N-末端加入10个氨基酸组成的标签，使得这两个漆酶同工酶成功地在毕赤酵母中异源表达，可能是增加这10个氨基酸标签后，有利于蛋白的分泌表达。
3 展望毫无疑问，漆酶在工业和生物技术中都有着广泛的应用前景，是一种环境友好型的木质素降解酶。在自然界中，白腐真菌是漆酶主要的生产者，但野生型真菌漆酶的产量低、培养周期长，限制了漆酶的大规模应用。随着分子生物学技术和基因工程技术的发展，已经有一些不同来源的漆酶在真核和原核表达系统中实现异源表达。但是，目前真核漆酶的异源表达情况还不理想，还未达到工业化水平。酵母细胞具有真核中的翻译后修饰能力、培养基廉价、遗传操作简单和可以分泌表达等优势，因此用作漆酶异源表达的宿主是非常有前景的。未来将加强分子生物学和分子遗传学方面的研究，更加全面详细地了解漆酶的结构和功能，进而在漆酶的基因水平上加以改造，逐步实现真正的高效异源表达，达到工业化水平。
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