产丁二酸工程菌的构建及其厌氧发酵  

Construction and Anaerobic Fermentation of Metabolically Engineered Escherichia coli Producing Succinate

作　　者：谢鑫[1] 陈可泉[1] 刘忠敏[1] 姜岷[1] 韦萍[1] 

机构地区：[1]南京工业大学制药与生命科学学院,材料化学工程国家重点实验室,南京210009

出　　处：《生物工程学报》2008年第1期101-105,共5页Chinese Journal of Biotechnology

基　　金：国家高技术发展计划“863”(No．2006AA022235);国家自然科学基金资助(No．20606017);国家自然科学基金重点项目(No．20336010);江苏省高校自然科学研究计划(No．05KJB180043)资助~~

摘　　要：为了考察过量表达苹果酸酶对于E.coli NZN111(IdhA::Kan pfl::Cam)厌氧发酵产丁二酸的影响,将连接有苹果酸酶基因sfcA的表达载体pTrc99a-sfcA转化进NZN111中,构建了重组NZN111(pTrc99a-sfcA)。0.5 mmol/LIPTG诱导8 h后,测定的苹果酸酶比酶活为30.67 u/mg,比受体菌提高了140倍。采用两阶段发酵模式,结果表明:过量表达的苹果酸酶在NZN111体内催化了从丙酮酸到苹果酸的逆向反应,丁二酸是发酵过程中积累的主要有机酸,且当加入0.7 mmol/L IPTG诱导,初始葡萄糖糖浓度为18.5 g/L时,选择对数生长期后期的菌种以10%的接种量转入厌氧发酵,发酵结束时发酵液中丁二酸的浓度为12.84 g/L,对葡萄糖的收率为69.43%,乙酸为0.58 g/L,二者浓度比为22:1,没有检测到甲酸和乳酸。构建的菌种具有高产丁二酸和副产物极少的优点,在同类菌种中处于先进水平。To study the effect of malic enzyme overexpression on succinate production in the pfl ldh double mutant Escherichia coli NZNlll （ldhA：：Kan pfl：：Cam） , we transformed the expression vector pTrc99a-sfcA into it and constructed the recombinant NZNlll（pTrc99a-sfcA）. The specific malic enzyme activity of the recombinant was 30.67 u/mg after 8-hour inducement by 0.5 mmol/L Isopropyl β-D- 1-Thiogalactopyranoside, 140 times higher than that of NZN 111. The two-step fermentation was used and the results showed that the overexpression of malic enzyme catalyzed the reverse reaction from pyruvate to malate, which was impossi- ble under general conditions. Succinate accumulated as the major product. Cells at the late exponential phase were inoculated to an anaerobic fermentation with 0.7 mmol/L IPTG in medium containing 18.5 g/L glucose. The final concentration of succinate and acetate was 12.84 g/L and 0.58 g/L, respectively. Formate and lactate were not detected. The constructed metabolically engineered strain had the feature of higher succinate yield and less by-products.

关 键 词：苹果酸酶 NZN111 两阶段发酵 逆向催化 丁二酸 

分 类 号：Q78[生物学—分子生物学]