聚對苯二甲酸乙二酯（PET）在過去半個世紀以來的需求急遽上升，達到每年3000 萬噸，也因此產生了大量PET廢棄物，以傳統方法處理PET廢棄物會對環境造成破壞。生物法可在溫和的溫度環境下將PET聚合物降解為單體對苯二甲酸單（2-羥乙基）酯（MHET）、對苯二甲酸（TPA）和乙二醇（EG），所需的耗能低，對環境相當友善。日前許多研究已使用細菌系統生產PETase來降解PET，但因細菌具有內毒素或需要豐富的碳源生長，成為對環境有害的汙染物，因此選擇對環境友善的宿主至關重要。萊茵衣藻因其生長成本低、易於培養以及具有高等生物相同的轉譯後和轉錄後修飾，被認為是一個理想的表達宿主，更重要的是，萊茵衣藻為一光合自營生物，提供了一種有效將溫室氣體二氧化碳及太陽能轉化為目標蛋白的途徑，在生產和代謝不產生有毒廢物。因此本研究以萊茵衣藻作為宿主，開發表面展示及分泌系統，生產、分泌PETase，並固定PETase於萊茵衣藻表面，以快速且便利地回收PETase，同時提高PETase的穩定性、催化功能。
本研究在細胞壁缺陷的CC-400萊茵衣藻表面成功展示了Spytag。至於分泌系統部分，CC-124、CC-125及CC-400均能夠分泌PETase並且水解PET，在30天於30°C恆溫光照培養下能水解PET粉末並分別產生最多16.13 μM、25.51 μM、6.63 μM濃度的TPA，其中以CC-125水解PET效率最高。最後於共培養階段，雖然並非所有組別顯示出更高的PET水解效率，但由HPLC分析結果得知，共培養對PET水解效率不會產生負面影響。未來可嘗試將分泌MHETase之萊茵衣藻一同培養，以增加副產物MHET的分解。另外，後續將持續調整表達系統，例如更換啟動子，或於基因中插入內含子，以增強萊茵衣藻轉基因表達。

The demand for PET has surged, resulting in 30 million tons of annual production and significant PET waste. Conventional waste processing methods pose environmental threats. To address this, the biological approach degrades PET into mono（2-Hydroxyethyl） terephthalate （MHET）, terephthalic acid （TPA）and ethylene glycol（EG） under mild conditions, ensuring low energy consumption and environmental friendliness. However, bacterial systems used for PETase production often release endotoxins or require rich carbon sources, making them environmentally harmful. Chlamydomonas reinhardtii, with low growth cost and ease of cultivation, is an ideal expression host. It possesses post-translational and post-transcriptional modifications and efficiently converts carbon dioxide and solar energy into target proteins without toxic waste. This study employs Chlamydomonas reinhardtii as a host for surface display and secretion of PETase, enabling convenient recovery, stability, and catalytic function enhancement.
The study confirms Spytag display on the surface of cell wall-deficient CC-400 Chlamydomonas reinhardtii. CC-124, CC-125, and CC-400 exhibit PETase secretion and PET hydrolysis. After a 30-day culture, they produce TPA from PET powder at concentrations of 16.13 μM, 25.51 μM, and 6.63 μM, respectively. CC-125 displays the highest PET hydrolysis efficiency. Co-cultivation does not adversely affect PET hydrolysis efficiency according to HPLC analysis. Future research may focus on cultivating Chlamydomonas reinhardtii capable of secreting MHETase to enhance MHET decomposition. Additionally, the expression system will be refined to enhance transgene expression in Chlamydomonas reinhardtii, such as through promoter replacement or gene intron insertion.

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