Microbial biocatalysis has emerged to a standard technology in the food, feed, pharmaceutical, and fine chemical industries. Since microorganisms are optimized by nature to maximize survival and typically not for the high-level accumulation of any sort of product, effective engineering strategies are required to satisfy the growing demand of new, economically competitive, and environmentally friendly products and processes. Random mutagenesis and subsequent selection used to be successful strateges, despite the fact that genetic changes were often not clear and include potentially unwanted detrimental alterations. The recent advances in systems biology research allow a genome-scale characterization of the complex composition and function of cells. We consider it important to recognize that the comprehensive understanding of a bioprocess is not restricted to the quantitative description of cellular functions but, in addition, includes analyses and evaluations on the reaction and process level. Using today's unprecedented toolbox, new whole-cell catalyst design targets can be systematically identified and the properties of microbial hosts can be specifically fine-tuned according to the requirements of a given bioprocess. This review proposes an integrated strategy as a holistic knowledge-driven approach for systems biotechnology and summarizes recent advances on the basis of selected examples.