外源添加代谢中间体对产琥珀酸放线杆菌厌氧发酵制备丁二酸的影响

Effect of adding intermediate metabolites on succinate production by Actinobacillus succinogenes

考察了外源添加中间代谢产物对菌体生长及发酵产酸的影响,结果表明添加0.5g/L磷酸烯醇式丙酮酸(PEP)时丁二酸产量最高。围绕产琥珀酸放线杆菌NJ113厌氧发酵产丁二酸的代谢网络进行代谢通量分析,发现添加PEP后己糖磷酸途径(HMP)与糖酵解途径(EMP)的通量比由39.4∶60.3提高至76.8∶22.6,解决了丁二酸合成过程中还原力不足的矛盾,导致PEP生成草酰乙酸的通量提高了23.8%,丁二酸代谢通量从99.8mmol/(gDCW·h)增至124.4mmol/(gDCW·h),而副产物乙酸及甲酸的代谢通量分别降低了22.9%、15.4%;关键酶活分析结果表明,添加0.5g/LPEP后PEP羧化激酶比酶活达到1910U/mg,与对照相比提高了74.7%,而丙酮酸激酶的比酶活降低了67.5%。最终丁二酸浓度为29.1g/L,收率达到76.2%,比未添加PEP时提高了11.0%。

We investigated the effect of adding intermediate metabolites on cell growth and succinate production. The yield of succinic acid achieved to the highest when 0.5 g/L phosphoenolpyruvic acid （PEP） was added. According to the metabolic network of Actinobaccilus succinogenes NJ113,the metabolic flux was calculated by metabolic flux analysis. The ratio of hexose monophosphate pathway to glycolytic pathway increased from 39.4：60.3 to 76.8：22.6 after adding 0.5 g/L PEP,thus the reducing power was better balanced. The flux of PEP to oxaloacetate was 23.8% higher,which made the succinic acid flux improve from 99.8 mmol/（g DCW·h） to 124.4 mmol/（g DCW·h） and the flux of acetic acid and formic acid decreased by 22.9% and 15.4%,respectively. The key enzyme activity analysis showed that the specific activity of PEP carboxykinase reached to 1910 U/mg with 0.5 g/L PEP addition,which was 74.7% higher than the control; and the specific activity of pyruvate kinase decreased by 67.5%. Finally,the concentration of succinic acid was 29.1 g/L with the yield of 76.2%.