Nonribosomal peptides (NRPs), a large family of natural products, possess numerous pharmaceutically significant bioactivities. However, many native microbial producers of NRPs are not cultivable or have low production yields making mass production infeasible. The recombinant production of natural products in a surrogate host has emerged as a strategy to overcome these limitations. De novo recombinant production of the NRP antibiotic valinomycin in an engineered Escherichia coli host strain was established with the necessary biosynthetic pathway constituents from Streptomyces tsusimaensis. In the present study, the initially modest valinomycin yields could be significantly increased from 0.3 up to 2.4 mg L⁻¹ by switching from a batch to an enzyme-based fed-batch mode in shake flasks. A subsequent design of experiment-driven optimization of parallel fed-batch cultivations in 24-well plates with online monitoring of dissolved oxygen and pH led to valinomycin yields up to 6.4 mg L⁻¹. Finally, repeated glucose polymer feeding to enzyme-based high cell density cultivations in shake flasks resulted in cell densities of OD₆₀₀>50 and a valinomycin titer of appr. 10 mg L⁻¹. This represents a 33-fold improvement compared to the initial batch cultivations and is the highest concentration of a nonribosomal peptide which has been produced in E. coli without feeding of specific precursors so far to our knowledge. Also, such a small-scale optimization under fed-batch conditions may be generally applicable for the development and scale-up of natural product production processes in E. coli.