过表达羧化途径及失活苹果酸酶基因对大肠杆菌好氧发酵产苹果酸的影响

Effects of overexpression of carboxylation pathway genes and inactivation of malic enzymes on malic acid production in Escherichia coli

摘要:

苹果酸是一种重要的C4二羧酸，在食品、医药、化工等领域有广泛的应用。本文主要研究羧化途径强化及苹果酸酶失活对大肠杆菌好氧发酵生产苹果酸的影响。首先在大肠杆菌E2中过表达了磷酸烯醇式丙酮酸羧化酶基因ppc，得到菌株E21，苹果酸积累量从0.57 g/L提高到3.83 g/L。随后，分别过表达来自谷氨酸棒杆菌的丙酮酸羧化酶基因pyc和来自琥珀酸放线杆菌的磷酸烯醇式丙酮酸激酶pck基因，相应的工程菌株E21 (pTrcpyc) 和E21 (pTrc-A-pck) 分别产6.04 和5.01 g/L苹果酸，得率分别达到0.79和0.65 mol/mol葡萄糖。敲除E21中的苹果酸酶基因maeA和maeB，苹果酸产量也显著提高了36%，达到5.21 g/L，得率为0.62 mol/mol。然而，在过表达pyc的基础上敲除苹果酸酶基因并不能进一步提高苹果酸的产量。经过摇瓶发酵条件的初步优化，菌株E21 (pTrcpyc) 生产12.45 g/L苹果酸，得率为0.84 mol/mol，达到理论得率的63.2%。

Abstract:

Malic acid is a dicarboxylic acid that is widely used in food, pharmaceutical and chemical industries. We studied the effects of overexpression of carboxylation pathway genes and inactivation of malic enzymes on the aerobic production of malic acid. Over expression of phosphoenolpyruvate (PEP) carboxylase (ppc) generated strain E21, which increased malic acid production from 0.57 g/L to 3.83 g/L. Then pyc gene from Coryenbacterium glutamicus and pck gene from Actinobacillus succinogenes were overexpressed in E21 separately. The resulting strains E21 (pTrcpyc) and E21 (pTrc-A-pck) produced 6.04 and 5.01 g/L malate with a yield of 0.79 and 0.65 mol/mol glucose, respectively. Deleting two malic enzymes (encoded by maeA and maeB) also led to an increase of 36% in malic acid production with a production of 5.21 g/L. However, the combination of malic enzymes deletion and pyc overexpression could not further increase the yield of malic acid. After optimization of fermentation conditions, strain E21 (pTrcpyc) produced 12.45 g/L malic acid with a yield of 0.84 mol/mol which is 63.2% of the theoretical yield.