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標題: 以合成生物學技術研發新世代類胡蘿蔔素生產平台
Development of synthetic biology platform for biosynthesis of carotenoid
作者: Caroline Thia
關鍵字: 合成生物學;PGASO;蝦青素;細胞工廠;synthetic biology;PGASO;Astaxanthin;cell factory
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In the post-genome era, synthetic biology is a new approach to design a new biological system or to re-design natural biological systems for new function. Carotenoids, including β-carotene, canthaxanthin and zeaxanthin, are antioxidants and the downstream product, astaxanthin, is the stronger one but the lower contestant. We propose to develop a 'cell factory' for producing carotenoids, via a synthetic biology tool, PGASO, that can simultaneously transform and regulatory express multiple gene in the yeast. In this proposal, engineering of the carotenogenic pathway will be achieved in a thermo- and toxin-tolerant yeast, Kluyveromyces marxianus KY3. The suitable Astaxanthin synthesis genes, including crtE, crtYB, crtI, tHMG1, crtS, crtR, bkt, and chyb, from suitable host, such as Xanthophyllomyces dendrorhous and green algae (Chlamydomonas reinhardtii, Chlorella zofingiensis, and Haematococcus pluvialis) were selected. We check the conformation of the engineered strain Cz30 via high performance liquid chromatography (HPLC) and it contains β-carotene and canthaxanthin in the red color cell. Compared to the wild type strain, the engineered strain showed higher ethanol tolerance and higher bio-ethanol productivity, in addition to producing carotenoids. The results suggested that the production of antioxidant carotenoids can improve toxin tolerance, damage recovering, and cell membrane maintenance via free radical removal. Furthermore, the strain could utilize glycerol, which is a common waste product from biodiesel industry, to improve the cost-effectiveness of carotenoids production. In conclusion, this system can serve as a biorefinery for both biofuel and value-added metabolites production

合成生物學是後基因體時代的新策略,可以重新設計自然界的生物系統來產生新的功能,透過合成生物學的概念建構一個細胞工廠,用以生產天然抗氧化劑類胡蘿蔔素。類胡蘿蔔素是提供生物抗氧化要素之一,番茄紅素、β-胡蘿蔔素、玉米黃質、角黃素等均為類胡蘿蔔素生物合成的中間產物,而蝦青素為其下游最終產物,其抗氧化能力最強但自然界含量卻很少。本實驗利用多基因單一步驟轉殖技術「PGASO」,將分別來自不同物種例如紅酵母「Xanthophyllomyces dendrorhous」和綠藻「Chlamydomonas reinhardtii, Chlorella zofingiensis 與 Haematococcus pluvialis」,挑選一些適合的基因包括crtE (GGPP synthase)、 crtYB (phytoene synthase/lycopene cyclase)、crtI (phytoene desaturase)、tHMG1 (修飾後 3-hydroxy-3-methylglutaryl–coenzyme A reductase)、bkt (β-carotene ketolase) 與chyb (β-carotene hydroxylase) 進行基因轉殖,以進入一株耐熱的酵母菌「Kluyveromyces marxianus KY3」之基因體中,且每個基因透過獨立的啟動子進行調控。研究初步結果,轉殖株 Cz30 (其 chyb 來自 C. zofingiensis) 細胞有明顯的呈橘紅色變化,進一步利用高壓液相層析儀 (HPLC) 分析探討內容物成分,確實有生產數種類胡蘿蔔素包括 β-胡蘿蔔素與角黃素等。實驗發現相關酵素作用溫度與關鍵基因之表現量會影響類胡蘿蔔素的含量及成份,並且可因而提升轉殖株之抗氧化能力以抵抗 UV、酒精等環境傷害。此外,轉殖株 Cz30 可以運用甘油當原料,來生產高價值的天然類胡蘿素,為具有應用價值的綠色生物製程之成功例子。
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