代谢工程改造大肠杆菌合成丁二酸及发酵罐放大工艺

Production of succinate by a metabolic engineered Escherichia coli and its scale-up process in fermentor

摘要:

【背景】Escherichia coli AFP111发酵生产丁二酸时大量副产乙酸，丁二酸得率低。【目的】代谢工程改造Escherichia coli AFP111，提高丁二酸得率，降低副产物乙酸的生成，建立100 L规模的丁二酸发酵工艺。【方法】一步同源重组敲除乙酸合成途径关键酶基因，改造丁二酸合成途径关键酶启动子实现过表达；单因素优化5 L发酵罐培养条件。【结果】敲除乙酸产生途径编码乙酸激酶和磷酸转乙酰酶的基因ackA-pta、苏氨酸脱羧酶和2-酮丁酸甲酸裂解酶的基因tdcDE获得SX02菌株，摇瓶发酵条件下其乙酸产量下降了53.42%，丁二酸得率提高9.85%。在SX02菌株基础上，经启动子改造过表达编码葡萄糖激酶的基因glk后获得菌株SX03，其Glk酶活性提高3.66倍，乙酸产量下降了31.62%，丁二酸得率提高8.28%。SX03菌株发酵生产丁二酸在5 L发酵罐进行放大，其乙酸产量为3.97 g/L，丁二酸得率为1.62 mol/mol葡萄糖，相比出发菌株的乙酸产量下降了75.76%，丁二酸得率提高19.12%。在5 L发酵罐上对比研究了中和剂Na2CO3和NaOH混合液替换碱式MgCO3的发酵效果，并优化了发酵pH、搅拌转速和葡萄糖浓度，获得如下最适发酵条件：pH 6.8，搅拌转速250 r/min，葡萄糖100 g/L，发酵结束时乙酸产量为2.24 g/L，丁二酸得率为1.66 mol/mol葡萄糖。中和剂替换优化后乙酸产量下降了20.65%，丁二酸得率提高2.47%。菌株SX03发酵工艺进一步在100 L发酵罐上实现放大，其乙酸产量为1.91 g/L，丁二酸得率为1.30 mol/mol葡萄糖。【结论】通过代谢工程改造的大肠杆菌，其副产物乙酸含量显著下降，丁二酸得率提高，并在5 L和100 L发酵罐上实现了工艺放大，展现出较大的工业化利用潜力。

Abstract:

[Background] Escherichia coli AFP111 produces succinate with low yield from glucose and with high production of acetate. [Objective] To reduce acetate production and improve succinate yield. A 100-L-scale fermentation process was established. [Methods] One-step homologous recombination was used for knocking out the key enzyme gene of acetate synthesis pathway and modifying key enzyme promoter of succinate synthesis pathway. The culture conditions were optimized by using single factor design in a 5-L fermentor. [Results] The resulting strain of SX02, deletion of ackA-pta and tdcDE, reduced acetate production by 53.42%, and increased succinate yield by 9.85% as compared to the original strain. The glucokinase activity of SX03, obtained after over-expressing glk, was 3.66 times high than SX02. Its acetate production reduced by 31.62% and its succinate yield increased by 8.28% as compared to SX02. SX03 produced 3.97 g/L acetate, and 1.62 mol of succinate per mol glucose in a 5-L fermentor with a decrease of acetate production by 75.76% and an increase of succinate yield by 19.12% as compared to the original strain. The fermentation conditions including various neutralizers pH values, agitation speed, glucose concentrations were optimized in a 5-L fermentor. The optimal fermentation conditions were pH 6.8, agitation speed of 250 r/min, glucose concentration of 100 g/L. Under the optimal conditions, SX03 produced 2.24 g/L acetate and 1.66 mol of succinate per mol glucose with a decrease of acetate production by 20.65% and an increase of succinate yield by 2.47%. The fermentation using the strain SX03 was scaled up in a 100-L fermentor, which produced 1.91 g/L acetate and 1.30 mol of succinate per mol glucose. [Conclusion] The metabolic engineered Escherichia coli shows great industrial potential for the commercial production of succinate.