酪丁酸梭菌代謝工程菌的構建及其發酵特性

楊貴清，劉剛，楊長得

1 深圳大學生命科學學院 深圳市微生物基因工程重點實驗室，深圳 518060 2 深圳市羅湖疾病預防控制中心，深圳 518020

酪丁酸梭菌代謝工程菌的構建及其發酵特性

楊貴清1,2，劉剛1，楊長得1

1 深圳大學生命科學學院 深圳市微生物基因工程重點實驗室，深圳 518060 2 深圳市羅湖疾病預防控制中心，深圳 518020

酪丁酸梭菌Clostridium tyrobutyricum可以利用葡萄糖、木糖、纖維二糖、阿拉伯糖等多種底物進行產酸發酵，主要發酵產物為丁酸和乙酸，是一種適合于木質纖維素同步糖化發酵生產丁酸的菌種。將酪丁酸梭菌乙酸發酵關鍵基因取代為丁酸發酵關鍵基因來構建突變株，可使突變株丁酸發酵量增多，乙酸發酵量減少。分別獲得來源于丙酮丁醇梭菌的丁酸代謝關鍵酶基因——乙酰乙酰輔酶A轉移酶基因(thl)、來源于酪丁酸梭菌本身的乙酸代謝關鍵酶基因片段——磷酸轉乙酰基酶基因片段(pta)和來源于質粒pIMP1的紅霉素抗性基因(em)。將它們與質粒pUC19相連構建為非復制性質粒pUC19-EPT。通過電轉化將其導入酪丁酸梭菌中。利用紅霉素抗性平板篩選獲得轉化子，通過PCR驗證發現，獲得的突變株染色體上pta被thl替換。在以葡萄糖為底物的發酵中，突變株丁酸得率為0.47，較野生型增大了34%，乙酸得率為0.05，較野生型下降了29%。

酪丁酸梭菌，丁酸，乙酰乙酰輔酶A轉移酶，磷酸轉乙酰基酶，代謝工程

Abstract:Clostridium tyrobutyricum is suitable for simultaneous saccharification and fermentation of lignocellulosic.It can produce butyric acid, acetic acid as its main fermentation products from a wide variety of carbohydrates such as glucose, xylose,cellobiose and arabinose.In order to decrease acetic acid content and increase butyric acid content in C.tyrobutyricum, we replaced genes on the acetic acid fermentation pathway with genes on the butyric acid fermentation pathway.Three genes were selected.They were acetyl-CoA acetylrtansfers gene(thl)which is the key enzyme gene associated with acetic acid fermentation pathway from Clostridium acetobutylicum, erythromycin gene(em)from plasmid pIMP1 and phosphotransacetylase gene(pta)which is the key enzyme gene associated with butyric acid fermentation pathway from C.tyrobutyricum.We fused these genes with pUC19 to construct nonreplicative integrated plasmids pUC19-EPT.Then we transformed pUC19-EPT into C.tyrobutyricum through electroporation.The recombinant transformants grown on plates containing erythromycin were validated by PCR.A mutant whose pta gene was displaced by thl gene on the chromosome was selected.In the fermentation from glucose, the mutant’s yield of butyric acid is 0.47, increased by 34% compared with wild type; and the yield of acetic acid is 0.05, decreased by 29%compared with wild type.

Keywords:Clostridium tyrobutyricum, butyric acid, acetyl coenzyme A acetyltransferase, phosphotransacetylase, metabolic engineering

正丁酸(分子式CH3(CH2)2COOH，以下簡稱為丁酸)是一種用途廣泛的化工原料和醫藥原料，可以用于各類丁酸酯類、丁酸纖維、清漆、香料等的生產制造，也可以用作乳化劑、殺菌劑和萃取劑[1]，還可作為組蛋白去乙酰化酶的抑制劑抑制多種腫瘤細胞的增殖，誘導腫瘤細胞分化、衰老和調亡[2-4]。……