An important group of bioactive natural products produced by bacteria and fungi are the nonribosomal peptides. The biosynthesis of these structurally diverse metabolites is derived by the modular enzymology of nonribosomal peptide synthetases (NRPSs). The modularity of NRPSs reside in a set of repeating catalytic domains that work together to recognize and incorporate aryl or amino acid precursors into the final product. The repetitive nature of NRPSs makes them perfect candidates for the rational design of these enzymes by the rearrangement of modules or domains to modify the nonribosomal peptide chemical scaffolds. Successes of NRPS combinatorial biosynthesis approaches have been scarce. Recent findings by the Thomas laboratory and others revealed that many NRPSs in bacteria require members of the MbtH-like proteins (MLP) superfamily for their solubility or function. MLP/NRPS interactions and their complexity are an important factor that the field has not addressed during combinatorial biosynthesis efforts. Proper pairing of MLP/NRPS systems might be the key to successful combinatorial biosynthesis efforts. In this thesis I propose two approaches to overcome the MLP challenge in combinatorial biosynthesis. First, the identification or evolution of a “universal” MLP that can act as a molecular tool and interact with multiple NRPSs. In Chapter 2, I characterized an orphan MLP, MXAN_3118, from M. xanthus DK1622 and identify it can naturally interact with at least seven cognate NRPS systems and the enterobactin system from E. coli. The ability of MXAN_3118 to naturally interact with multiple NRPSs suggests this enzyme is a candidate universal MLP that can potentially interact with MLP-dependent NRPSs from different systems. My second approach is to understand how we can change an MLP-dependent NRPS to be independent of the MLP. In Chapter 3, I studied the recently discovered enterobactin (ENT) system from yeast and identified that after acquisition of the biosynthetic gene cluster and further evolution the yeast system is MLP-independent. I hypothesize that the yeast ENT system lost the requirement for MLP from a combination of factors; including changes in the protein structure and the yeast cell physiology. The work in this thesis to overcome the MLP-dependence of NRPS systems, or MLP challenge, provided groundwork that can contribute to the future success of natural product engineering.