Biodegradation of Polyethylene Terephthalate

Polyethylene terephthalate
Biodegradation
Hydrolases
Chassis

一、介绍1. Introduction

聚对苯二甲酸乙二醇酯(PET)是人们生活中使用最广泛的合成塑料之一[ 1 ]。它由对苯二甲酸 (TPA) 和乙二醇 (EG) 通过酯键聚合而成 [ 2 ]。自20世纪PET首次用于生产一次性塑料瓶以来，受到世界范围的欢迎，成为人们生活中不可或缺的一部分[ 3 ]。由于 PET 对自然降解具有很强的抵抗力，因此鼓励对 PET 进行回收利用 [ 4 ]。目前，处理 PET 废弃物的主要方法包括填埋、焚烧以及物理和化学回收 [ 5 , 6]]。这些方法通常会对环境造成二次污染，消耗大量能源，既不经济也不环保。由于回收利用策略不当，塑料制品的机械性能强，造成土壤污染、海洋生态系统扰乱等严重的环境问题[ 7 ]。因此，PET 生物降解作为一种环境友好的替代方法受到了关注，与其他回收方法相比，它需要更温和的温度和更低的能耗 [ 8 , 9 ]。此外，降解单体可以很容易地回收利用，希望将 PET 转化为高价值的化学品。Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics in people’s lives [1]. It is polymerized by terephthalic acid (TPA) and ethylene glycol (EG) through ester bonds [2]. Since PET was first used to produce disposable plastic bottles in the 20th century, it has been welcomed worldwide and has become an indispensable part of people’s lives [3]. As PET is highly resistant to natural degradation, the recycling of PET has been encouraged [4]. At present, the main methods for managing PET waste include landfilling, incineration, as well as physical and chemical recycling [5,6]. These methods usually cause secondary pollution to the environment and consume huge amounts of energy, which is not economical or environmentally friendly. Due to the improper recycling strategies and the strong mechanical properties of plastic products, serious environmental problems, such as soil pollution and the disturbance of marine ecosystems, have occurred [7]. Therefore, PET biodegradation has attracted attention as an environmentally friendly alternative, requiring milder temperatures and lower energy consumption than other recycling methods [8,9]. Additionally, the degradation monomers can easily be recycled, with the hope of converting PET into high value chemicals.

1977 年，报道了几种商业脂肪酶和酯酶可以水解各种聚酯 [ 10 ]。从那时起，许多 PET 水解酶，例如脂肪酶、角质酶和酯酶，已被各种微生物发现并表征 [ 1 , 11 ]。2016 年，从废物回收站分离到了Ideonella sakaiensis 201-F6 [ 12 ]。发现它可以产生 PET 水解酶 (PETase) 和对苯二甲酸单羟乙酯 (MHET) 水解酶 (MHETase)，它们可以在 30°C 下将 PET 降解为中间产品。然后，对两种酶的结构进行了分析，并进行了一系列有效的酶修饰 [ 13 , 14 , 15 ], 16 , 17 ]，有效提高了两种酶的活性和稳定性。PETase和MHETase的发现和修饰为常温降解PET废物提供了重要依据。In 1977, several commercial lipases and an esterase were reported to hydrolyze various kinds of polyesters [10]. Since then, many PET hydrolases, such as lipases, cutinases and esterases, have been discovered and characterized by various microorganisms [1,11]. In 2016, Ideonella sakaiensis 201-F6 was isolated from a waste recycling station [12]. It was found to produce PET hydrolase (PETase) and monohydroxyethyl terephthalate (MHET) hydrolase (MHETase), which can degrade PET into intermediate products at 30 °C. Then, the structures of the two enzymes were analyzed and a series of effective enzyme modifications were carried out [13,14,15,16,17], efficiently improving the activity and stability of the two enzymes. The discovery and modification of PETase and MHETase has provided an important basis for the degradation of PET waste under ambient temperatures.

合成生物学和代谢工程策略已应用于 PET 废物的生物降解和生物转化，特别是在 PET 水解酶的修饰、微生物底盘的优化和降解途径的重建方面。目前，一些细菌、真菌和海洋微藻已被报道为PET生物降解的良好微生物基质。全细胞生物催化剂已经能够实现PET的初始降解。TPA 和 EG 的生物转化途径已经确定。一些微生物已被设计为从 PET 单体生产高价值化学品，这是 PET 升级回收的一个重要发展方向。基于这些目前的进展，Synthetic biology and metabolic engineering strategies have been applied to the biodegradation and bioconversion of PET waste, especially in the modification of PET hydrolases, optimization of microbial chassis, and reconstruction of degradation pathways. At present, some bacteria, fungi, and marine microalgae have been reported as being good microbial chassis for PET biodegradation. The whole-cell biocatalysts have been able to achieve the initial degradation of PET. The bioconversion pathways of TPA and EG have been identified. Some microorganisms have been engineered to produce high value chemicals from PET monomers, which is an important development direction for PET upcycling. Based on these current advances, developing enhanced microbial chassis and constructing artificial microbial consortia to couple the biodegradation of PET by secreted PET hydrolases with the bioconversion of high value chemicals from monomers is a promising method to realize the circular economy of PET waste.

2. PET 生物降解2. PET Biodegradation

在 PET 生物降解过程中，微生物首先附着在 PET 薄膜表面，然后分泌细胞外 PET 水解酶，与 PET 薄膜结合并启动生物降解过程 [ 18 , 19 ]。PET水解酶作用于PET的酯键，将其水解为TPA和EG，生成不完全水解产物，如MHET和对苯二甲酸双(2-羟乙基)酯(BHET)。在I. sakaiensis 201-F6 中，MHET在MHETase的作用下可进一步水解为TPA 和EG [ 12 ]。据报道，MHETase 对末端生成的 PET 膜具有水解活性，证明了该酶的外切 PETase 功能 [ 20]。PET 水解酶可以进一步水解 BHET，产生 MHET、TPA 和 EG [ 12 ]。TPA和EG可以用不同的微生物可以使用，并可以进一步代谢成三羧酸循环（TCA循环）中的产品[ 21，22，23，24，25，26，27，28，29 ]。此外，这些 PET 生物降解的中间产品和最终产品已被确定为 PET 水解酶的竞争性抑制剂 [ 30 , 31 ]。During PET biodegradation, the microorganisms first adhere onto the surface of PET films and then secrete extracellular PET hydrolases, which bind to the PET films and initiate the biodegradation process [18,19]. PET hydrolases act on the ester bond of PET, hydrolyzing it into TPA and EG and generating incomplete hydrolysis products, such as MHET and Bis-(2-hydroxyethyl) terephthalate (BHET). In I. sakaiensis 201-F6, MHET can be further hydrolyzed into TPA and EG under the action of MHETase [12]. It was reported that MHETase has a hydrolysis activity against the termini-generated PET film, demonstrating the exo-PETase function of the enzyme [20]. PET hydrolases can further hydrolyze BHET to produce MHET, TPA, and EG [12]. The products TPA and EG can be used by different microorganisms and be further metabolized into the tricarboxylic acid cycle (TCA cycle) [21,22,23,24,25,26,27,28,29]. Additionally, these intermediate and final products of PET biodegradation have been identified as competitive inhibitors of PET hydrolases [30,31].

2.1. 工程 PET 水解酶2.1. Engineered PET Hydrolases

水解酶，包括脂肪酶[ 31，32，33，34 ]，角质酶[ 35，36，37，38，39，40，41，42 ]，酯酶[ 43，44，45，46 ]，PETase [ 12 ]和MHETase [ 12]，已经确定可以降解 PET。其中，脂肪酶对PET的水解活性最低，主要是因为它们的催化中心被盖结构覆盖，限制了水解酶与底物PET的接触和催化作用。角质酶总是具有很强的PET水解能力，因为它们具有大的底物结合口袋，没有盖子结构，有利于PET与其活性中心的结合。然而，角质酶通常在高温（50-70°C）下降解 PET，而 PETase 和 MHETase 可以在 30°C 下高效且特异性地水解 PET [ 12]]。PETase和MHETase的发现有助于实现PET在常温下的高效生物降解。目前，对这两种酶的结构进行了广泛的研究，并出现了更多的高活性水解酶变体。The hydrolases, including lipases [31,32,33,34], cutinases [35,36,37,38,39,40,41,42], esterases [43,44,45,46], PETase [12] and MHETase [12], that can degrade PET have been identified. Among them, lipases have the lowest hydrolysis activity of PET mainly because their catalytic centers are covered by lid structures, which limits the hydrolases’ contact and catalysis with the substrate PET. Cutinases always have a strong PET hydrolysis ability due to their large substrate binding pockets without lid structures, which is conducive to the combination of PET with their active centers. However, cutinases usually degrade PET at high temperatures (50–70 °C), while PETase and MHETase can efficiently and specifically hydrolyze PET at 30 °C [12]. The discovery of PETase and MHETase is helpful in achieving the high efficiency biodegradation of PET at ambient temperatures. At present, the structures of these two enzymes have been studied extensively, and more high activity hydrolases variants have appeared.

自然界中鉴定出的PET水解酶稳定性差、活性低、表达水平低，限制了其大规模工业化应用。已经提出了一系列可以提高 PET 水解酶催化活性的策略 [ 13 ]。The PET hydrolases identified in nature always have poor stability, low activity, and low expression levels, which limit their large-scale industrial application. A series of strategies that could enhance the catalytic activity of PET hydrolases have been proposed [13].

一种策略是设计结合口袋，这可以提高 PET 水解酶的特异性并增加酶和底物的有效吸附 [ 15 , 47 , 48 , 49 ]。我们的实验室之前专注于 PETase 与底物结合附近的六个关键氨基酸，并进行了定点突变。成功筛选了 R61A、L88F 和 I179F 突变体，与野生型 PETase 相比，酶活性分别增加了 1.4 倍、2.1 倍和 2.5 倍 [ 50 ]。席尔瓦等人。[ 51 ] 对来自Thermobifida fusca的角质酶进行修饰_0883通过定点诱变构建了单突变Ile218Ala和双突变Q132A/T101A，扩大了催化空间，提高了PET生物降解效率。陈等人。[ 52 ] 通过 PETase 的结构分析确定了独特的氨基酸 S214 和 I218，并指出它们与 W185 摆动和 β6-β7 环灵活性有关。这项研究有助于设计增加底物结合口袋灵活性的 PETase 突变体。One strategy is to engineer the binding pocket, which can improve the specificity of the PET hydrolases and increase the effective adsorption of enzymes and substrates [15,47,48,49]. Our laboratory previously focused on six key amino acids near the binding of PETase to the substrate and conducted site-directed mutations. The R61A, L88F, and I179F mutants were successfully screened, and the enzyme activity increased 1.4-fold, 2.1-fold, and 2.5-fold, respectively, in comparison to wild-type PETase [50]. Silva et al. [51] modified the cutinase from Thermobifida fusca_0883 by site-directed mutagenesis and constructed a single mutation Ile218Ala and a double mutation Q132A/T101A, which expanded the catalytic space and improved the efficiency of the PET biodegradation. Chen et al. [52] identified the unique amino acids S214 and I218 through the structural analysis of PETase and noted that they are associated with W185 wobbling and β6-β7 loop flexibility. This research is helpful in designing PETase mutants that increase the flexibility of the substrate binding pocket.

一些研究侧重于使用酶工程策略来提高 PET 水解酶的稳定性，以提高 PET 生物降解效率 [ 53 , 54 ]。添加Ca 2+或Mg 2+ [ 38 , 55 ]、引入二硫键和盐桥[ 56 , 57 ]和糖基化等方法均已被证明可以提高PET水解酶的稳定性。研究人员添加二硫键以提高叶枝堆肥角质酶 (LCC) 的热稳定性，并对底物结合附近的热氨基酸进行定点突变，获得组合突变 F243I/D238C/S283C/Y127G (ICCG) [ 53]。最后，90% 的 PET 塑料瓶碎片在 72°C 下降解 10 小时，这是迄今为止最有效的 PET 水解酶 [ 53 ]。Some studies focused on using enzyme engineering strategies to improve the stability of the PET hydrolases to improve PET biodegradation efficiency [53,54]. Methods such as adding Ca²⁺ or Mg²⁺ [38,55], introducing a disulfide bond and salt bridge [56,57], and glycosylation have all been proven to improve the stability of PET hydrolases. Researchers added disulfide bonds to improve the thermal stability of leaf-branch compost cutinase (LCC) and performed site-directed mutations on hot amino acids near the substrate binding to obtain the combined mutation F243I/D238C/S283C/Y127G (ICCG) [53]. Finally, 90% of shredded PET plastic bottles were degraded at 72 °C for 10 h, which is by far the most efficient PET hydrolase [53].

此外，通过工程化PET的水解酶增加了对PET的水解酶的底物可访问性也得到了广泛的研究[ 58，59，60 ]。据报道，纤维素分解热双歧杆菌的 Thc_Cut1 与里氏木霉的疏水蛋白（HFB4 和 HFB7）的融合表达可使PET 的水解效果提高 16 倍以上，而酶和疏水蛋白的混合物仅导致 4-至多增加倍数 [ 61 ]。Additionally, increasing the substrate accessibility for PET hydrolases by engineering the PET hydrolases has also been widely studied [58,59,60]. It is reported that the fusion expression of Thc_Cut1 from Thermobifida cellulosilytica and hydrophobins (HFB4 and HFB7) from Trichoderma reesei can increase the hydrolysis effect of PET by more than 16 times, while a mixture of the enzyme and the hydrophobins led to only a 4-fold increase at most [61].

PET 生物降解的中间产品和最终产品，如对苯二甲酸双（2-羟乙基）酯 (BHET)、对苯二甲酸单羟乙酯 (MHET)、TPA 和 EG，都是 PET 水解酶的竞争性抑制剂 [ 30 ]。因此，协同作用的水解酶或蛋白质工程化策略的降低的酶和产物之间的相互作用的混合物是用于解决抑制[有效的方法62，63，64 ]。The intermediate and final products of PET biodegradation, such as Bis-(2-hydroxyethyl) terephthalate (BHET), monohydroxyethyl terephthalate (MHET), TPA, and EG, are all competitive inhibitors of the PET hydrolases [30]. Therefore, the mixtures of hydrolases that act synergistically or protein engineering strategies that reduce the interaction between the enzymes and products are effective methods for solving the inhibition [62,63,64].

此外，其他策略已被研究以提高酶的活性，提高PET [生物降解36，38，57，59，65 ]。In addition, other strategies have been studied to increase the activity of the enzymes and enhance the biodegradation of PET [36,38,57,59,65].

2.2. 工程 PET 生物降解底盘2.2. Engineered PET Biodegradation Chassis

大多数鉴定出的能够分泌PET水解酶的微生物是非模型微生物，由于其复杂的遗传背景，它们很难进行基因工程。此外，来自野生菌株的 PET 水解酶的表达水平不足以满足大规模降解的需求。因此，有必要开发利用模型微生物高效表达PET水解酶的重组表达系统。PET是由TPA和EG聚合而成的高分子聚合物，不能进入细胞，因此PET的体外酶解得到了广泛的研究。由于PET水解酶的纯化和制备过程耗时且成本高，因此有必要在细胞外有效表达PET水解酶以用于实际应用。76,77].Most of the microorganisms identified that are capable of secreting PET hydrolases are non-model microorganisms and they are difficult to genetically engineer due to their complex genetic background. In addition, the expression level of the PET hydrolases from wild strains is insufficient to satisfy the demand for large-scale degradation. Therefore, it is necessary to develop recombinant expression systems using model microorganisms to express the PET hydrolases efficiently. PET is a high molecular polymer that is polymerized from TPA and EG and cannot enter cells, so in vitro enzymatic degradation of PET has been studied extensively. Owing to the purification and preparation process of PET hydrolases being time-consuming and cost-intensive, the efficient expression PET hydrolases extracellularly for practical applications is necessary [76,77].

目前，一些微生物底盘如细菌、真菌、海洋微藻等已应用于PET水解酶的分泌和表达，已被研究证明是降解PET的有前景的底盘。几个全细胞生物催化剂已被设计成降低PET，其能够不仅避免酶纯化的复杂的步骤，而且在多步反应中重复使用，相比于自由基于酶的方法[ 78，79 ]。此外，还解决了环境因素影响酶活性降低，甚至酶失活的困难。下面总结了几种适用于PET生物降解的微生物底盘。At present, some microbial chassis such as bacteria, fungi, and marine microalgae have been applied to the secretion and expression of PET hydrolases, which have been studied and proven to be promising chassis to degrade PET. Several whole-cell biocatalysts have been designed to degrade PET, which are able to not only avoid the complicated steps of enzyme purification but also be reused in multi-step reactions, in comparison to the free enzyme-based approach [78,79]. Additionally, the difficulty of the reduced activity of the enzymes, or even enzymes being inactivated, under the influence of environmental factors has been solved. The following is a summary of several microbial chassis that are suitable for PET biodegradation.

2.2.1. 细菌2.2.1. Bacteria

大肠杆菌Escherichia coli

大肠杆菌由于其遗传背景清晰、生长条件简单、高密度培养等优点，是生产重组蛋白的重要模式微生物[ 89 ]。在近年来，随着PET水解酶的连续发现，越来越多的酶已在所取得的异源表达大肠杆菌[ 12，14，16，50，53，69 ]。已经总结了在大肠杆菌中异源表达的 PET 水解酶[ 76 ]，这有助于进一步分析这些酶的晶体结构并探索 PET 的降解机制。E. coli is an important model microorganism for the production of recombinant proteins due to its clear genetic background, simple growth conditions, and its advantages in high density cultivation [89]. In recent years, with the continuous discovery of PET hydrolases, more and more enzymes have achieved the heterologous expression in E. coli [12,14,16,50,53,69]. PET hydrolases heterologously expressed in E. coli have been summarized [76] and it is helpful in further analyzing the crystal structures of these enzymes and explore the degradation mechanism for PET.

最近的研究表明，工程大肠杆菌可用作 PET 生物降解的全细胞生物催化剂。选择最佳信号肽是用于改进异源 PET 水解酶切片的常用策略。一项研究测试了来自大肠杆菌的 Sec 依赖和 SRP 依赖信号肽对分泌 PETase 的影响，并通过融合 SP LamB和 PETase成功生产了 6.2 mg/L PETase [ 80 ]。其他一些研究通过修饰信号肽来提高表达滴度和酶活性。通过随机诱变获得的进化信号肽 PelB (G58A) 已成功用于在大肠杆菌中表达异源 PETase，并使 PETase 分泌量提高 1.7 倍。81 ]。研究了信号肽 B1 的增强剂 (MERACVAV) 来介导 PETase 的排泄，最后，B1PelB 介导的 PETase 的排泄效率比 PelB 的排泄效率提高了 62 倍 [ 82 ]。Recent studies have shown that engineered E. coli can be used as a whole-cell biocatalyst for PET biodegradation. Selecting the optimal signal peptide is a common strategy used to improve the section of heterologous PET hydrolases. A study tested the effects of Sec-dependent and SRP-dependent signal peptides from E. coli in secreting PETase, and successfully produced 6.2 mg/L PETase by fusing SP_LamB and PETase [80]. Some other research improved the expression titer and enzymatic activity by modifying the signal peptide. An evolved signal peptide PelB (G58A) obtained by random mutagenesis was successfully used to express heterologous PETase in E. coli and enabled up to 1.7-fold higher PETase secretion [81]. An enhancer of signal peptides B1 (MERACVAV) was studied to mediate the excretion of PETase, and finally, the excretion efficiency of PETase mediated by B1PelB demonstrated a 62-fold increase over that of PelB [82].

枯草芽孢杆菌Bacillus subtilis

革兰氏阳性枯草芽孢杆菌具有分泌量高、生长快、无外膜等优点，与通常形成包涵体的大肠杆菌相比，被认为是分泌异源蛋白的优良微生物底盘[ 90 , 91 ]。此外，枯草芽孢杆菌对恶劣环境具有很强的抵抗力，它已被用来分泌可以降解许多污染物的蛋白质，这就是为什么它被认为是一种有前途的生物降解微生物底盘 [ 92 , 93 ]。Gram-positive B. subtilis has the advantages of high secretion capacity, fast growth, and the lack of an outer membrane, and it is regarded as an excellent microbial chassis for secreting heterologous proteins compared to E. coli, which usually forms an inclusion body [90,91]. Additionally, B. subtilis has a strong resistance to harsh environments and it has been used to secrete proteins that can degrade many pollutants, which is why it is considered to be a promising microbial chassis for biodegradation [92,93].

在 PET 生物降解方面，枯草芽孢杆菌已被设计为分泌 PET 水解酶。据报道，枯草芽孢杆菌168在其天然信号肽（SP PETase）的指导下，成功地将PETase分泌到培养基中。SP PETase被预测为双精氨酸信号肽，双精氨酸易位 (Tat) 复合物的失活使 PETase 的分泌量提高了 3.8 倍 [ 83 ]。另外两个PET水解酶（BhrPETase和LCC）也表示在枯草芽孢杆菌，和BhrPETase的表达滴度和LCC中的工程化的分子伴侣表达达到0.66克/ L和0.89克/升的枯草芽孢杆菌分别[42 ]。此外，优化了信号肽和启动子的组合以促进枯草芽孢杆菌WB600中PETase的表达，并且信号肽SP amy和弱启动子P43的组合被证明是最好的[ 84 ]。In terms of PET biodegradation, B. subtilis has been engineered to secrete PET hydrolases. It is reported that PETase was successfully secreted into the medium by B. subtilis 168 under the direction of its native signal peptide (SP_PETase). SP_PETase is predicted to be a twin-arginine signal peptide, and the inactivation of twin-arginine translocation (Tat) complexes improved the secretion amount of PETase 3.8-fold [83]. Another two PET hydrolases (BhrPETase and LCC) were also expressed in B. subtilis, and the expression titer of BhrPETase and LCC reached 0.66 g/L and 0.89 g/L in an engineered chaperone-overexpression of B. subtilis, respectively [42]. Additionally, the combinations of signal peptides and promoters were optimized to promote the expression of PETase in B. subtilis WB600, and the combination of the signal peptide SP_amy and the weak promoter P43 was proved to be best [84].

嗜热细菌Thermophilic Bacteria

大多数能够降解PET的水解酶，包括脂肪酶、角质酶和酯酶，在较高温度下具有更高的酶活性，而大多数能够产生异源PET水解酶的模型微生物的最佳生长温度通常为30-40°C。全细胞生物催化剂与某些仅在高温下起作用的 PET 水解酶不兼容 [ 94 ]。因此，需要一个嗜热表达系统来提高 PET 生物降解的效率 [ 36 , 95 ]。除了具有成熟基因操作平台的热纤梭菌（Clostridium thermocellum）外，大多数嗜热微生物通常难以进行基因改造[ 94 ]。C. 热纤已被设计用于木质纤维素生物转化 [ 96 ] 和生物燃料生产 [ 97 ]，这就是为什么它被认为是 PET 生物降解的潜在微生物底盘。Most of the hydrolases capable of degrading PET, including lipases, cutinases and esterases, have higher enzymatic activity at higher temperatures, while the optimal growth temperature of most model microorganisms that can produce heterologous PET hydrolases is usually 30–40 °C. Whole-cell biocatalyst is not compatible with some PET hydrolases that are only functional at high temperatures [94]. Therefore, a thermophilic expression system is necessary to improve the efficiency of PET biodegradation [36,95]. Most thermophilic microorganisms are usually difficult to genetically engineer except for Clostridium thermocellum, which has a mature genetic manipulation platform [94]. C. thermocellum has been engineered for lignocellulose bioconversion [96] and biofuel production [97], which is why it is regarded as a potential microbial chassis for the biodegradation of PET.

LCC 已成功地从工程热纤梭菌中获得。这种工程化的全细胞生物催化剂实现了 LCC 的高水平表达，14 天后，超过 60% 的商用 PET 膜在 60°C 下转化为可溶性单体 [ 85 ]。此嗜热全细胞降解系统具有同时酶的生产和PET降解的优点相比，只有使用游离酶，这就是为什么它是有希望的策略使用其它高温水解酶[降解PET 98，99 ]。除了嗜热全细胞降解系统外，还报道了耐碱全细胞催化系统 [ 100 , 101 , 102 ]].LCC has been successfully obtained from an engineered C. thermocellum. This engineered whole-cell biocatalyst realized a high level expression of LCC and more than 60% of a commercial PET film was converted into soluble monomers at 60 °C after 14 days [85]. This thermophilic whole-cell degradation system has the advantage of simultaneous enzyme production and PET degradation compared to only using free enzymes, which is why it is a promising strategy to degrade PET using other high temperature hydrolases [98,99]. In addition to the thermophilic whole-cell degradation system, an alkali-tolerant whole-cell catalytic system has also been reported [100,101,102].

2.2.2. 菌类2.2.2. Fungi

除细菌外，一些酵母，包括潜在的巴斯德毕赤酵母和脂耶氏酵母，在PET的生物降解中使用进行了研究。P. pastoris具有强大的分泌表达系统和可扩展的发酵能力，已成为工业应用中蛋白质生产的常见菌株。研究人员已经表示BurPL（H344S / F348I）和PETase在巴斯德毕赤酵母和大肠杆菌，并指出，从生产的两种酶巴斯德毕赤酵母表现出更高的活性比在表达大肠杆菌因为蛋白质半衰期保护机制的P . 帕斯托里斯[ 52 , 103]。通过在巴斯德毕赤酵母表面展示 PETase 开发了一种全细胞生物催化剂，与纯化的 PETase 相比，PETase 的酶活性增加了 36 倍，形成高度结晶的 PET [ 86 ]。此外，这种全细胞生物催化剂可以重复使用七次而没有明显的活性损失，这有助于开发其他用于PET生物降解的全细胞生物催化剂[ 86 ]。考虑到P. pastoris进行N-连接糖基化的能力，有研究人员研究了糖基化对P. pastoris中表达的LCC的影响，发现LCC的动力学稳定性和活性都得到了提高[ 35 ]。解脂耶氏酵母也是用于生物修复的绝佳微生物底盘 [ 104 ]。研究人员分离出能够将 PET 转化为 MHET 的Y. lipolytica IMUFRJ 50682，并验证了 PET 单体可能在脂肪酶生产过程中充当诱导剂 [ 105 ]，这表明Y. lipolytica是一种潜在的 PET 生物降解微生物底盘。其他研究在Y. lipolytica Po1f 中表达了 PETase和来自脂肪酶的信号肽，并证实工程菌株可以将 BHET 和 PET 粉末水解成单体 [ 106]。表面展示系统和全细胞生物催化剂为实现 PET 水解酶的高效表达和促进 PET 生物降解提供了新的思路和策略 [ 77 , 107 , 108 ]。酵母与有效的遗传工具一起，已被用作生物降解和生物转化的重要微生物基质 [ 109 ]。In addition to bacteria, the potential of some yeasts, including Pichia pastoris and Yarrowia lipolytica, being used in PET biodegradation has been studied. P. pastoris, with a great secretion expression system and scalable fermentation capability, has become a common strain for protein production in industrial applications. Researchers have expressed BurPL (H344S/F348I) and PETase in P. pastoris and E. coli and noted that both enzymes produced from P. pastoris showed higher activity than that expressed in E. coli because of the protein half-life protection mechanism of P. pastoris [52,103]. A whole-cell biocatalyst was developed by displaying PETase on the surface of P. pastoris and the enzymatic activity of PETase increased 36-fold towards a highly crystalline PET in comparison to that of purified PETase [86]. Additionally, this whole-cell biocatalyst can be reused seven times without obvious activity loss, which is helpful in developing other whole-cell biocatalysts for PET biodegradation [86]. Considering the ability of P. pastoris to perform N-linked glycosylation, some researchers studied the effects of glycosylation on the LCC expressed in P. pastoris and found that the kinetic stability and activity of LCC were both improved [35]. Y. lipolytica is also a great microbial chassis for bioremediation [104]. Researchers isolated Y. lipolytica IMUFRJ 50682 with the ability to convert PET into MHET and verified that the PET monomers may act as inducers in the process of lipase production [105], which showed that Y. lipolytica is a potential microbial chassis for PET biodegradation. Other research expressed PETase in Y. lipolytica Po1f with a signal peptide from lipase and confirmed that the engineered strain could hydrolyze BHET and PET powder into the monomers [106]. Surface display systems and whole-cell biocatalysts provide novel ideas and strategies for achieving the high efficiency expression of PET hydrolases and promoting PET biodegradation [77,107,108]. Yeasts, together with efficient genetic tools, have been used as great microbial chassis for biodegradation and bioconversion [109].

2.2.3. 海洋微藻2.2.3. Marine Microalgae

目前，现有的能够生产PET水解酶的天然和工程微生物底盘通常难以适应复杂的海洋环境，产生大量的PET废物。最近，一些海洋微藻已被用作 PET 生物降解的底盘 [ 110 ]。据报道，一种光合微藻三角褐指藻被设计为能够将 PETase 突变体分泌到培养基中的底盘，并且重组 PETase 能够有效降解不同的底物，包括 PET 薄膜、聚对苯二甲酸乙二醇酯-1,4-环己基二甲基对苯二甲酸酯) (PETG) 薄膜和 PET 碎纸，在 30 °C 或什至在中温温度 (21 °C) [ 87 ]。此外，Chlamydomonas reinhardtii这种绿藻也被成功改造为产生具有降解活性的 PETase，在培养 4 周后，PET 膜上出现了化学和形态变化 [ 88 ]。作为在盐水环境中生物降解 PET 废物的环保底盘，海洋微藻在未来生物技术应用中具有潜在的 PET 污染海水降解潜力 [ 87 ]。At present, the existing native and engineered microbial chassis that are capable of producing PET hydrolases are usually difficult to adapt to the complexity of the marine environment and produce much PET waste. Recently, some marine microalgae have been used as chassis for PET biodegradation [110]. A photosynthetic microalga Phaeodactylum tricornutum has been reported as being engineered as a chassis capable of secreting a PETase mutant into the culture medium, and the recombinant PETase was able to efficiently degrade different substrates, including PET films, poly (ethylene terephthalateco-1,4-cylclohexylenedimethylene terephthalate) (PETG) film, and shredded PET, at 30 °C or even at mesophilic temperatures (21 °C) [87]. Additionally, Chlamydomonas reinhardtii, the green algae, was also successfully engineered to produce PETase with degrading activity, and the chemical and morphological changes appeared on the PET films after 4 weeks of culture [88]. As environmentally friendly chassis for the biodegradation of PET waste in a saltwater-based environment, marine microalgae have the potential for future biotechnological applications in the degradation of PET polluted seawater [87].