Fatty acid feedstocks enable a highly efficient glyoxylate-TCA cycle for high-yield production of β-alanine

Metabolic engineering to produce tricarboxylic acid (TCA) cycle-derived chemicals is usually associated with problems of low production yield and impaired cellular metabolism. In this work, we found that fatty acid (FA) feedstocks could enable high-yield production of TCA cycle-derived chemicals, while maintaining an efficient and balanced metabolic flux of the glyoxylate-TCA cycle, which is favorable for both product synthesis and cell growth. Here, we designed a novel synthetic pathway for production of β-alanine, an important TCA cycle-derived product, from FAs with a high theortecial yield of 1.391 g/g. By introducing panD, improving aspA, and knocking out iclR, glyoxylate shunt was highly activated in FAs and the yield of β-alanine reached 0.71 g/g from FAs, much higher than from glucose. Blocking the TCA cycle at icd/sucA/fumAC nodes could increase β-alanine yield in a flask cultivation, but severely reduced cell growth and FA utilization during fed-batch processes. Replenishing oxaloacetate by knocking out aspC and recovering fumAC could restore the growth and lead to a titer of 35.57 g/l. After relieving the oxidative stress caused by FA metabolism, β-alanine production could reach 72.05 g/l with a maximum yield of 1.24 g/g, about 86% of the theoretical yield. Our study thus provides a promising strategy for the production of TCA cycle-derived chemicals.