||Nonribosomal peptide synthetases (NRPSs) are large, modular proteins responsible for biosynthesis of many medicinally important peptide natural products. Although detailed genetic and biochemical experiments have yielded deep insights into the principles of nonribosomal peptide biosynthesis in recent years, little is known about the in vitro properties of these synthetases that are generally unstable and difficult to reconstitute. In this thesis, three overlooked cellular factors, macromolecular crowding, protein organization, and proofreading enzyme, that influence the activities of these multifunctional enzymes were investigated using enterobactin synthetase as a model system. First, we investigated the macromolecular crowding effect on the activities of the enterobactin NRPS. It was found that macromolecular crowding significantly increase product specificity of the synthetase. Crowding most likely exerts this effect through inducing conformational changes in the multifunctional EntF of the synthetase. Moreover, we investigated protein organization in the enterobactin pathway in vivo and in vitro. All the enzymes involved in this biosynthetic pathway were found to be co-immunoprecipitated from E. coli cells cross-linked by formaldehyde despite the fact that these biosynthetic enzymes were not found to form stable protein complexes in vitro. Furthermore, the influence of type II thioesterase EntH on enterobactin biosynthesis was investigated. We found that EntH evolves its distinctive ability to recognize the aberrant intermediates from the versatile catalytic platform of hotdog proteins and suggests that type II thioesterases are able to actively search for the wrongly incorporated intermediates in nonribosomal peptide synthesis. In summary, the results described in this thesis show that these three factors are critical to correct understanding of biosynthesis of NRPSs and indispensable in their biotechnological utilizations.