A general scheme for the directed evolution of multiple proteins within a metabolic pathway. (1) Diversity is introduced to the genes through error-prone PCR, DNA shuffling, or other mutagenesis techniques. (2) The genes are then assembled into the pathway through homologous recombination. Each fragment is designed with flanking homologous DNA regions to allow for the recombination. P1 and T1 are the promoter/terminator regions of the first gene, while P2 and T2 are the promoter/terminator for the second gene. No promoter or terminator region was mutagenized. (3, 4) The library is screened based on a high-throughput method to interrogate the phenotype of interest. In this study, screening was accomplished via colony size on cellobiose agar plates. Large colonies are associated with faster growth and chosen for further analysis in flask fermentation. (5) To confirm that improved phenotype is a result of the mutated genes of the pathway, the plasmids containing the mutated pathways were isolated and retransformed. (6) Improved mutants are chosen for a second round of diversification and screening.