Novel biosynthesis of L-malate based on overflow metabolism in Escherichia coli

Overflow metabolism has been extensively studied over the past few decades, which is characterized by excretion of large amount of by-products. A systematic investigation of L-malate overflow metabolism was carried out with Escherichia coli AFP111 in this study. The results showed that nitrogen-limitation was one critical factor provoking the overflow metabolism of L-malate when AFP111 used succinate as carbon source under aerobic condition. The RT-qPCR analysis showed that the transcription levels of mdh, sfcA, maeB, ndh and nuo were significantly down-regulated while sdhA, sdhB and fumC were dramatically up-regulated under nitrogen-limited conditions compared with those under nitrogen sufficiency. It indicated that efficiency of L-malate catabolism and electron transport chain decreased. Meanwhile, lower NAD⁺ and NAD⁺/NADH ratio were detected which might be due to down-regulated ndh and nuo. Furthermore, nuo and/or ndh of AFP111 were inactivated by CRISPR-Cas9 system. Either ndh or nuo knockout strain exhibited significantly increased L-malate accumulation accompanying with decreased NAD⁺ and NAD(H/+) pool. The fermentation results showed that the △NDH-2 strain could accumulate up to 20.37 g/L L-malate with yield of 0.65 g/g while the △NDH-1 strain could accumulate 17.27 g/L L-malate with yield of 0.82 g/g, respectively. This study extends our understanding of overflow metabolism and to date it is the first time to report L-malate production based on overflow metabolism by E. coli.