Glutamic acid + Ethylamine → L-Theanine
Catalytic enzyme: gamma-glutamyl transpeptidase (GGT)
The intracellular glutamate concentration in E. coli is very low, so an additional pathway is introduced to enhance glutamate transport in E. coli.
Ethylamine is toxic, pungent, and expensive
Exogenous ethylamine = unnatural, unsafe, and unattractive
High concentrations can kill E. coli
Allow bacteria to gently produce ethylamine within the cells. Achieve: fully endogenous, no exogenous addition, closed-loop synthesis of L-theanine.
The use of B0032 and B0032, which are moderately weak RBSs, aims to regulate ethylamine levels and prevent excessive ethylamine from harming cells.
Theanine accumulates more and more in cells:
Will inhibit PsGGT enzyme activity (feedback inhibition)
Will slow down cell growth
So add a line specifically to release theanine outside the cell.
Therefore, the synthesis of theanine efflux transporters reduces intracellular product accumulation, relieves enzyme feedback inhibition, and significantly increases yield.
