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

Abstract

Abstract 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\u202fg/L L-malate with yield of 0.65\u202fg/g while the △NDH-1 strain could accumulate 17.27\u202fg/L L-malate with yield of 0.82\u202fg/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.