研究以甲醇為碳源生產乳酸

Abstract

近年來，利用再生資源生產生質化學品日益受到重視且被廣泛利用，因為它具有前瞻性以及對環境友善的優點。 乳酸是一個很重要的化學製品，被美國能源局列為從生物質中生產的最高附有價值的化學品之一，乳酸主要為酸味劑以及增味劑添加於食品和飲料中，除此之外，乳酸可以經由聚合反應生產聚乳酸，此聚合物為具有生物降解性和耐熱性的塑膠原料。目前工業上乳酸的合成途徑部分是經由石化燃料轉換而成，然而會造成嚴重的環境問題，溫室氣體的釋放，如:二氧化碳。為了要解決這的問題，利用代謝工程技術藉由再生資源來生產化學品相當受到重視。 和醣類不同，甲醇是一種非糧食競爭的原料，也可以用來生產具高價值的化學品。甲醇主要由甲烷轉換而成，甲烷是頁岩中含量豐富的氣體，又稱為天然氣，由於天然氣的可用性來源增加，在未來甲醇的價格將會降低。 本篇論文的研究目標為在大腸桿菌和嗜甲醇桿菌中利用甲醇當作碳源生產乳酸，大腸桿菌為革蘭氏陰性菌，可以生長於有氧以及無氧環境，因為它的基因工程技術的發展健全，我們可以很簡單的修改細菌的基因系統。然而，天然的大腸桿菌是無法利用甲醇當作碳源生長的，為了要建構出一株可以利用甲醇生產乳酸的大腸桿菌，我們在細菌裡表現人工的甲醇轉換途徑。在這個研究中，首先選擇不同甲醇轉換和乳酸生產的基因。發現到在大腸桿菌中建構甲醇轉換路徑來生產乳酸是可行的但是會不太穩定。此外，我們也嘗試了利用B. methanolicus PB1藉由表現乳酸去氫酶來生產乳酸。有趣的是在野生型PB1除了可以天然分泌出15 mg/L左右的乳酸但同時也會消耗些許乳酸。比較在E. coli和PB1中利用甲醇來生產乳酸的實驗得知，現階段利用甲醇來生產乳酸以天然可以利用甲醇的B. methanolicus較有效。

Recently, bio-based chemical feedstock from renewable resources has widely been used because it has prospects and environmental benefits. Lactate is an important chemical which is listed as one of the top value added chemicals from biomass mandated by the U.S Department of Energy. It was used as acidulant and taste enhancer in beverage and food industry. In addition, lactate can be converted into polylactate by polymerization, which is a biodegradable and thermoplastics. Some of lactate are produced through pretroleum, which can increase greenhouse gas releasing like CO2. In order to solve this problem, using metabolic engineering for production of chemical feedstocks from renewable resource is more popular. Unlike sugar, methanol is non-food feedstock for the production of high value chemical. It can be produced from methane, which is an abundant gas from shale. With the increasing availability of natural gas source, the price of methanol will be low. The goal of this research is using methanol as a carbon source to produce lactate in Escherichi coli and native methylotrophic bacteria. E. coli is a gram negative and facultatively anaerobic bacteria. It is easy to modify because of well-developed genetic tool. However, wild type E. coli cannot use methanol as carbon source. To construct a strain of E. coli can produce lactate by methanol, we will introduce a synthetic methanol conversion pathway into E. coli. In this work, we first choose different genes for methanol conversion and lactate production. Lactate synthesis from recombinant E. coli with RuMP pathway was possible but unstable. Furthermore, we also try native methylotrophy to produce lactate by expressing lactate dehydrogenase and test production.Interesting, we can see that wildtype PB1 can secret about 15 mg/L lactate and also consume a small amout of lactate. At this stage, using methanol to produce lactate by B. methanolicus is more effective.