纖維生質精煉之催化轉換技術開發及應用研究

Abstract

在全球人口的增加下，傳統的糖類生物資源將會逐漸不適合用於發酵生產工業用化學品與生質燃油。因此非糧食類的生物資源將成為合成生質化學的重要一項技術。 近年來，利用甲醇合成其他長碳鏈化合物得到相當的重視，主要因為甲醇是非糧食類資源並可同時由較低成本的石化資源與生質物取得。 另外，因應未來頁岩天然氣的量產，單一碳(C1)的利用也成了一項重要的研發方向。本計畫將利用天然甲基營養生物Bacillus methanolicus進行代謝工程改造使其轉換甲醇成琥珀酸。 琥珀酸(丁二酸)是眾多附加價值化學品(value added chemicals)中極重要的一個，目前廣泛用於食品工業、藥物賦形劑、以及塑膠合成，但更重要的是琥珀酸能進一步衍生成其它重要工業用Ｃ4化學品例如：1,4-丁二醇、丁二烯、順丁烯二酸酐、四氫呋喃等。 本計畫將研究和建構琥珀酸合成代謝途徑並將它們表達於B. methanolicus。 首先我們先將建立一個使用α-酮戊二酸合成琥珀酸的代謝途徑。此合成代謝途徑利用B. methanolicus天然產出穀胺酸的特性可望達到較有效的合成。因為B. methanolicus為嗜熱菌，最佳生長溫度為50°C，我們將找出耐熱的α-酮戊二酸脫羧酶(α-ketoglutarate decarboxylase)與琥珀酸半醛脱氢酶(succinate semialdehyde dehydrogenase)。 另外由於B. methanolicus的基改工具較少，我們將測試其原生啟動子的活性來找出並設計合適表達琥珀酸合成基因的操縱子。最後，由於琥珀酸是中央代謝三羧酸循環(TCA cycle)中的其中一個代謝物， B.methanolicus有可能將其代謝並 消耗，我們將設計基因剔除的方法將會消耗琥珀酸的反應抑制。我們預期這項研究將可達到利用甲醇合成琥珀酸

As world population increases, traditional bioresources such as sugars are less suitable for use in the production of chemicals and fuels. As an alternative, methanol is an attractive non-food feedstock for the production of value added chemicals. Methanol is available at low price from petrochemical resources and can be derived from biomass. Furthermore, with the increase availability of natural gas resources, one carbon (C1) utilization becomes an important direction for technological development. Naturally, methylotrophic microorganisms grow utilizing methanol. It is therefore an appealing direction to metabolically engineer methylotrophic microorganisms for converting methanol to value added products. In this study, we propose to engineer a model methylotrophic gram positive bacteria Bacillus methanolicus for the production of succinate from methanol. Succinate is one of the top most value added chemicals currently used as a food additive, pharmaceutical stabilizer, and the synthesis of synthetic rubber. Derivatives of succinate including 1,4-butanediol, 1,3-butadiene, maleic anhydride, and tetrahydrofuran are common industrial chemical feedstocks with high volume demands. To achieve succinate production from methanol using engineered Bacillus methanolicus, we will first construct a pathway for succinate production from α-ketoglutarate, a pathway potentially suitable for expression in B. methanolicus due to its natural ability for glutamate production. Succinate may be synthesized through decarboxylation of α-ketoglutarate to succinate semialdehyde and followed by an oxidation. First we will implement this pathway in E. coli for validation of its function. Then we will characterize the enzymes’ thermostability as B. methanolicus grows optimally at 50°C. Unlike traditional microbial hosts, B. methanolicus is less well characterized for its genetic tools. Here we will search and characterize several promoters in search of proper strength for expressing the genes necessary for succinate production. Upon identification of proper promoter and suitable enzymes, we will introduce synthetic succinate producing operons into B. methanolicus for succinate production. Lastly, we expect that succinate may be consumed by B. methanolicus as it is a key intermediate of TCA cycle. Therefore we will also investigate and design knockout or knock down tools for regulating the activities of the enzymes consuming succinate. Through these efforts, we expect that we will achieve the first demonstration of succinate production from methanol.