Systematic analysis of the effects of different nitrogen source and ICDH knockout on glycolate synthesis in...

Glycolate is an important α-hydroxy carboxylic acid widely used in industrial and consumer applications. The production of glycolate from glucose in Escherichia coli is generally carried out by glycolysis and glyoxylate shunt pathways, followed by reduction to glycolate. Glycolate accumulation was significantly affected by nitrogen sources and isocitrate dehydrogenase (ICDH), which influenced carbon flux distribution between the tricarboxylic acid (TCA) cycle and the glyoxylate shunt, however, the mechanism was unclear.

Herein, we used RNA-Seq to explore the effects of nitrogen sources and ICDH knockout on glycolate production. The Mgly534 strain and the Mgly624 strain (with the ICDH deletion in Mgly534), displaying different phenotypes on organic nitrogen sources, were also adopted for the exploration. Though the growth of Mgly534 was improved on organic nitrogen sources, glycolate production decreased and acetate accumulated, while Mgly624 achieved a balance between cell growth and glycolate production, reaching 0.81 g glycolate/OD (2.6-fold higher than Mgly534). To further study Mgly624, the significant changed genes related to N-regulation, oxidative stress response and iron transport were analyzed. Glutamate and serine were found to increase the biomass and productivity respectively. Meanwhile, overexpressing the arginine transport gene argT accelerated the cell growth rate and increased the biomass. Further, the presence of Fe²⁺ also speeded up the cells growth and compensated for the lack of reducing equivalents.

Our studies identified that ICDH knockout strain was more suitable for glycolate production. RNA-Seq provided a better understanding of the ICDH knockout on cellular physiology and glycolate production.