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Metabolome and proteome analyses of glycerol overproducing E. coli unravel transcriptional misregulation of glycolysis and guide optimization strategies

ABSTRACT: Feedback-regulation of gene expression in synthetic metabolic pathways can improve production titers and yields. However, the dynamic nature of metabolism makes it difficult to engineer such regulation on a rational basis. Here, we expressed enzymes in a synthetic glycerol production pathway with static or feedback-regulated promoters, and explored the consequences for E. coli metabolism. Expressing the synthetic glycerol pathway with static promoters caused a strong growth burden, resulting in low biomass-levels and low glycerol titers. We show that the growth burden is due to a regulatory interaction between fructose-1,6-bisphoshate (FBP) and the transcription factor Cra, which switches between glycolysis and gluconeogenesis in E. coli. This regulation activated gluconeogenesis at higher induction of the glycerol pathway, because FBP levels were low. Feedback-regulated promoters, in contrast, stabilized FBP levels and enabled robust expression of the synthetic glycerol pathway. We show the broad utility of this approach by engineering different feedback-regulated promoters (pBAD, pTet, pConstitutive) and by applying one of them to carotenoid production in E. coli.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Escherichia Coli

TISSUE(S): Cell Line Cell

SUBMITTER: Timo Glatter

LAB HEAD: Timo Glatter

PROVIDER: PXD022965 | Pride | 2021-07-21

REPOSITORIES: Pride

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