At an amazing pace, synthetic biology, bio-informatics and systems biology are developing from metabolic engineering tools into major drivers of industrial biotechnology and thus, crucial factors in the transition to a knowledge-based bio-economy. Jointly, they allow the rapid development of bio-processes for products which are new-to-nature and which offer novel opportunities for solving problems not resolvable hitherto. Such a class of molecules for which industry is searching for alternative production technologies are chito-oligosaccharides (COS), as the state-of-the-art processes are seriously flawed. Here, we will tackle the main drawbacks of our previously developed COS biotechnological production platform, i.e., the limited set of COS produced in vivo and the sub-optimal process performance. The product portfolio will be expanded specifically towards a higher degree of polymerization (DP7 or DP8) by employing engineered NodC enzymes. In addition, the process performance will be optimized, by developing a novel tool based on colocalization to i) align all steps in complex (artificial) pathways to each other and to the host’s metabolism, and ii) efficiently channel pathway intermediates between the consecutive enzymatic reactions of the pathway. These technological advances will allow the production of pure COS with high DP, which will subsequently be tested for various applications in the domains of plant, animal and human health