“…[18][19][20][21][22][23][24][25][26][27] Two-stage dynamic control, which decouples growth from production, offers additional potential benefits in large scale bioprocesses, enabling metabolic states to be pushed beyond the boundaries required for growth. 8,21,22,24,28 We have recently implemented two-stage dynamic control in the commonly used and well characterized microbe, E. coli , by utilizing synthetic metabolic valves, which rely on the combination of proteolysis and gene silencing to dynamically reduce levels of key enzymes in the context of a standardized two-stage phosphate depleted process (as illustrated in Figure 1) 17,21,22,29 Proteolysis is accomplished by appending C-terminal degron (DAS+4) tags to a given gene and silencing via expression of the native E. coli CRISPR CASCADE system as well as silencing gRNAs (from pCASCADE plasmids). 30,31 Importantly, in these studies where we produced the organic acid citramalate 22 and the sugar alcohol xylitol, 21 we identified that dynamic deregulation of metabolism was a fundamental mechanism to improve stationary phase metabolic fluxes.…”