Turning microorganisms into highly efficient microbial cell factories (MCFs) is a daunting process due to the enormous cellular complexity, i.e., the vast metabolic network superimposed by a multilevel regulatory layer and the fragmentary knowledge thereof. Typically, to steer the strain engineering process a “Design-Build-Test-Learn” (DBTL) cycle is iteratively run. In this project, to move more successfully through this cycle and, hence, to more successfully engineer biological systems we propose to pursue a strategy that synergistically combines modern tools of Synthetic Biology and System Biology. This will certainly be beneficial for the exploitation of the hexosamine biosynthesis pathway (HBP), which requires precursors from all over the metabolism and, moreover, is tightly regulated on all levels of regulatory control. This will be demonstrated using the development of microbial cell factories for the production of the HBP-derived chitooligosaccharides(COS) as proof-of-concept. COS have unique characteristics resulting in numerous (potential) applications in the fields of cosmetics, feed, food and pharma.