AbstractTranscriptional regulation plays a pivotal role in metabolic flow, which can, in turn, affect L-Trp biosynthesis in Escherichia coli. The application of global transcriptional regulation by Crp, FruR, FlhD, and TyrR for L-Trp biosynthesis during shake-flask fermentation of E. coli FB-04/pSV03, an L-Trp-producing strain, was elucidated. Inactivation of TyrR was phenotypically silent, whereas, absence of the Crp caused a noticeable reduction in L-Trp yield and a pronounced slow-growth phenotype. Lacking FlhD led to a decrease in the acetate level; however, the L-Trp yield also decreased. When FruR was inactivated, the L-Trp biosynthesis and the biotransformation rate were improved by 59.5 and 52.4%, respectively. However, using the same strain with deleted fruR-gene, for fed-batch cultivation in a 3-L reactor leads to a significant decrease in L-Trp biosynthesis and the conversion rate.