2023-01-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/659590全球暖化與糧食短缺威脅人類和生態圈的永續共存，凸顯發展綠色科技來降低碳排放及增產糧食的迫切與必要性。在眾多自營生物中，藍綠菌因能快速生長、用太陽能進行固碳反應、和可基因改造的優勢，近年來被選為發展碳中和及負碳技術的模式物種，學界與業界都冀望將藍綠菌改造成製作營養品、生質能源、及高價化學物質的綠色工廠。維生素 B12 是人類和許多生物生長必須的養分，本團隊期望用藍綠菌 (Synechococcus elongatus PCC 7942) 來生產這結構複雜且昂貴的有機化合物 (每克約60美元，與黃金同價)。前人推測外源表達三種酵素即可令此菌株合成維生素B12，主要的挑戰在提升其產量。為達成此目標，本團隊擬定三階段的遺傳工程：(一) 透過外源基因表達，在藍綠菌體內構築完整的維生素B12 生合成途徑；(二) 建構可偵測並獎勵藍綠菌產生維生素B12的基因迴路；(三) 以基改藍綠菌進行實驗演化，篩選可促進維生素B12 生合成的遺傳變異。自然界的維生素B12 主要產自異營微生物，高耗能的維生素B12 生合成不但造成碳排放且限制了水生生態系的生育力，本計畫提出的創新策略將自營生物的固碳作用轉變成碳捕捉的維生素B12 製程，可行但也極具挑戰性，將是合成生物學、綠色科技、與再生農業的具體實踐與重要貢獻。 The threat of global warming and food shortage urges development of green technology to reduce greenhouse gas emissions and expand nutrition sources. Among autotrophic organisms, cyanobacteria are promising hosts for carbon-neutral or even carbon-negative production of nutrients, biofuels, and valuable chemicals because they grow fast, utilize solar energy for carbon dioxide assimilation, and are amenable to genetic manipulation. My research team intends to engineer cyanobacterium Synechococcus elongatus PCC 7942 for efficient production of vitamin B12, a complex and pricy compound (~60 USD/g, similar to the gold price) essential to humans and many life forms. Prior work suggests expression of three exogenous enzymes could enable this microbe to generate vitamin B12. The major challenge is to enhance its vitamin B12 productivity. To achieve this goal, I propose a three-stage engineering plan: (1) assembly of a de novo vitamin B12 biosynthetic pathway in Synechococcus 7942 through expression of exogenous enzymes; (2) construction of a reward genetic circuit for vitamin B12 production; (3) laboratory evolution of the engineered cyanobacterium to select for mutations that increase vitamin B12 productivity. This proposal presents an innovative and feasible strategy to tailor autotrophic microbes for clean vitamin B12 production. The energy-costly vitamin B12 biosynthesis, performed mainly by heterotrophic prokaryotes in Nature, emits carbon dioxide and limits the fertility of aquatic ecosystems. Our engineering attempt, if successful, will make significant contributions to synthetic biology, green technology, and regenerative agriculture.碳捕捉;綠色科技;合成生物學;代謝工程;維生素B12;carbon capture; green technology; synthetic biology; metabolic engineering; vitamin B12