Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
Interfaculty Institute for Microbiology and Infection Medicine Tübingen, University of Tübingen, Tübingen, Germany.
Nat Commun. 2021 Aug 13;12(1):4929. doi: 10.1038/s41467-021-25142-0.
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.
合成代谢途径对工程菌来说是一种负担，但潜在的机制往往难以捉摸。在这里，我们表明，转录因子 Cra 的失调活性是导致甘油过量生产的大肠杆菌生长负担的原因。甘油生产降低了 1,6-二磷酸果糖 (FBP) 的浓度，然后激活 Cra，导致糖酵解酶下调和糖异生酶上调。由于细胞以葡萄糖为食，糖异生的不当激活和伴随的糖酵解抑制可能会在甘油途径的更高诱导下损害生长。我们通过在控制甘油途径限速酶表达的启动子中设计一个 Cra 结合位点来解决这种失调问题，以保持 FBP 水平足够高。我们通过将 Cra 依赖性调节设计到一组组成型和诱导型启动子中，展示了这种方法的广泛适用性，并使用其中一个启动子在大肠杆菌中过量生产类胡萝卜素。
