Abstract
Synthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Here we show that the misregulated activity of the transcription factor Cra is responsible for the growth burden of glycerol overproducing E. coli. Glycerol production decreases the concentration of fructose-1,6-bisphoshate (FBP), which then activates Cra resulting in the downregulation of glycolytic enzymes and upregulation of gluconeogenesis enzymes. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway. We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. We show the broad applicability of this approach by engineering Cra-dependent regulation into a set of constitutive and inducible promoters, and use one of them to overproduce carotenoids in E. coli.
Highlights
Synthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive
To investigate how induction of a synthetic metabolic pathway impacts the metabolism of the host, we expressed the glycerol biosynthesis pathway from yeast in E. coli (Fig. 1a)
The glycerol pathway is a two-step pathway that starts from the glycolytic metabolite dihydroxyacetone phosphate (DHAP)
Summary
Synthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. Optimal control of enzyme expression has been achieved at various levels of transcription and translation, for example by engineering promoters[8] or ribosome-binding sites[9]. Metabolomics and proteomics data indicated that the growth burden was caused by a transcriptional response in glycolysis, notably the activation of gluconeogenesis by the transcription factor Cra. we combined theoretical and experimental analysis to show that insertion of a Cra-binding site into the pBAD promoter enables higher growth rates at higher glycerol production rates. We show that this approach is generally applicable to synthetic pathways that utilize glycolytic metabolites such as carotenoid production
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