Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Canada.
Department of Biochemistry, University of Toronto, Toronto, Canada.
Microb Cell Fact. 2022 Dec 31;21(1):280. doi: 10.1186/s12934-022-02007-9.
Over the 70 years since the introduction of plastic into everyday items, plastic waste has become an increasing problem. With over 360 million tonnes of plastics produced every year, solutions for plastic recycling and plastic waste reduction are sorely needed. Recently, multiple enzymes capable of degrading PET (polyethylene terephthalate) plastic have been identified and engineered. In particular, the enzymes PETase and MHETase from Ideonella sakaiensis depolymerize PET into the two building blocks used for its synthesis, ethylene glycol (EG) and terephthalic acid (TPA). Importantly, EG and TPA can be re-used for PET synthesis allowing complete and sustainable PET recycling.
In this study we used Saccharomyces cerevisiae, a species utilized widely in bioindustrial fermentation processes, as a platform to develop a whole-cell catalyst expressing the MHETase enzyme, which converts monohydroxyethyl terephthalate (MHET) into TPA and EG. We assessed six expression architectures and identified those resulting in efficient MHETase expression on the yeast cell surface. We show that the MHETase whole-cell catalyst has activity comparable to recombinant MHETase purified from Escherichia coli. Finally, we demonstrate that surface displayed MHETase is active across a range of pHs, temperatures, and for at least 12 days at room temperature.
We demonstrate the feasibility of using S. cerevisiae as a platform for the expression and surface display of PET degrading enzymes and predict that the whole-cell catalyst will be a viable alternative to protein purification-based approaches for plastic degradation.
自塑料进入日常用品以来的 70 多年里，塑料废物已成为一个日益严重的问题。每年生产超过 3.6 亿吨塑料，因此迫切需要解决塑料回收和减少塑料废物的问题。最近，已经鉴定出并工程改造了多种能够降解 PET（聚对苯二甲酸乙二醇酯）塑料的酶。特别是，来自 Ideonella sakaiensis 的酶 PETase 和 MHETase 将 PET 解聚成用于其合成的两种构建块，即乙二醇（EG）和对苯二甲酸（TPA）。重要的是，EG 和 TPA 可重新用于 PET 合成，从而实现完全和可持续的 PET 回收。
在这项研究中，我们使用酿酒酵母作为平台，该酵母是生物工业发酵过程中广泛使用的物种，来开发表达 MHETase 酶的全细胞催化剂，该酶将单羟乙基对苯二甲酸酯（MHET）转化为 TPA 和 EG。我们评估了六种表达架构，并确定了那些在酵母细胞表面有效表达 MHETase 的架构。我们表明，MHETase 全细胞催化剂的活性可与从大肠杆菌中纯化的重组 MHETase 相媲美。最后，我们证明了表面展示的 MHETase 在广泛的 pH 值、温度范围内以及在室温下至少 12 天内都具有活性。
我们证明了使用酿酒酵母作为表达和表面展示 PET 降解酶的平台是可行的，并预测全细胞催化剂将是基于蛋白质纯化的塑料降解方法的可行替代方法。
