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

摘要:

为了考察过量表达苹果酸酶对于E.coli NZN111(ldhA::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/L IPTG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。

Abstract:

To study the effect of malic enzyme overexpression on succinate production in the pfl ldh double mutant Escherichia coli NZN111 (ldhA::Kan pfl::Cam) , we transformed the expression vector pTrc99a-sfcA into it and constructed the recombinant NZN111(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 b-D-1-Thiogalactopyranoside, 140 times higher than that of NZN111. 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 impossible 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.