近年來，許多研究開始著重於尋找石化原料的替代品，而其中又以FDCA最具有未來性，FDCA可替代塑膠的原料－聚對苯二甲酸乙二酯 (PET) 製程中所需的對苯二甲酸，對環境較PET更為友善。目前FDCA可由HMF氧化成HMFCA或DFF，再氧化成FFCA，最後氧化成FDCA，本研究以基因轉殖技術將文獻提及可生產FDCA的酵素HmfH、HMFO、AAO、UPO、CPO、PAMO及GOase基因分別接入載體，將其轉型入對有機溶劑具有高耐受性的Pseudomonas. putida S12表現不同的酵素，以全細胞生物轉換法將HMF轉化成FDCA。我們首先將含有酵素基因的質體分別轉型入P. putida中，並以強啟動子PHCE表現酵素與50 mM HMF反應，發現HmfH及HMFO可將HMF轉化成FDCA，其轉化率分別約為78.8 %及77.6 %，其餘酵素僅反應至HMFCA即終止。接著我們測試以ATc作為誘導劑的誘導型啟動子PTc表現前次結果無法生產FDCA的酵素，並以20及100 ng/ml的ATc誘導，但所有酵素反應依然止於HMFCA。由於目前的文獻僅GOase及HRP被提及可用E. coli生產，而P. putida與E. coli同為原核生物，因此接下來我們將GOase與HRP一同使用搭配不同的啟動子與信號肽，期望能解決HMFCA累積的問題，但仍然無法生產FDCA，因此我們推測這些酵素可能不適合於P. putida中表現。而為了將酵素基因嵌入P. putida染色體以降低篩選菌株所使用的抗生素成本，我們將三種CRISPR系統SpCas9、SaCas9及FnCas12a應用於P. putida中，發現其搭配λ-Red系統可成功嵌入外源基因，因此我們分別將可生產FDCA的HmfH及HMFO兩個酵素基因分別嵌入P. putida染色體中並與50 mM HMF反應，結果顯示HMF可成功被轉化成FDCA，其轉化率分別約為86.1 %及84.2 %。未來我們會繼續嘗試其他培養條件或酵素，期望能解決HMFCA累積的問題並提高FDCA產量，並設計去除CRISPR質體的系統，以增加菌株的穩定性。

Recently, FDCA becomes one of the most promising bio-based compound. It can be an alternative to the ingredient of PET and is more eco-friendly. FDCA can be produced by oxidizing HMF to HMFCA or DFF, then to FFCA, finally to FDCA. In this study, we expressed several enzymes such as HmfH, HMFO, AAO, UPO, CPO, PAMO and GOase for FDCA production in bacteria Pseudomonas putida S12 with high tolerance to organic solvent by bio-transformation. We first expressed enzymes by strong promoter P¬HCE and reacted with 50 mM HMF. HmfH and HMFO could successfully convert HMF to FDCA at the yield of ~78.8 % and 77.6 %, but others could only produce HMFCA. Next, we expressed enzymes that couldn’t work in previous result by inducible promoter PTc which could be induced by ATc and induced the expression by 20 and 100 ng/ml ATc, but the reaction still stopped at HMFCA. Because GOase and HRP were the only enzymes that could be expressed in E. coli in previous literatures, we then expressed GOase and HRP together combining different promoter and signal peptide. Unfortunately, HMFCA was still the main product. To integrate enzyme gene into the genome of P. putida, we applied 3 CRISPR system SpCas9, SaCas9 and FnCas12a in P. putida, and found that all of them could integrate foreign genes into the genome with the help of λ-Red system. Thus, we integrated HmfH and HMFO genes into its genome, and 50 mM HMF could be successfully transformed to FDCA at the yield of ~86.1 % and 84.2 %. For future work, we will try to optimize our culture condition or express other enzymes, hoping to solve the problem of HMFCA accumulation. Besides, we will design the plasmid curing system for elimination fo CRISPR plasmid to enhance the stability of strain.

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Lee Y, Oh S, Park W. 2009. Inactivation of the Pseudomonas putida KT2440 dsbA gene promotes extracellular matrix production and biofilm formation. FEMS Microbiol Lett 297(1):38-48.
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Poo H, Song JJ, Hong S-P, Choi Y-H, Yun SW, Kim J-H, Lee SC, Lee S-G, Sung MH. 2002. Novel high-level constitutive expression system, pHCE vector, for a convenient and cost-effective soluble production of human tumor necrosis factor-α. Kluwer Academic Publishers 24:1185-1189.
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