Microbial genomes encode numerous biosynthetic gene clusters (BGCs) that may produce natural products with diverse applications in medicine, agriculture, the environment, and materials science. With the advent of genome sequencing and bioinformatics, heterologous expression of BGCs is of increasing interest in bioactive natural product (NP) discovery. However, this approach has had limited success because expression of BGCs relies heavily on the physiology of just a few commonly available host chassis. Expanding and diversifying the chassis portfolio for heterologous BGC expression may greatly increase the chances for successful NP production. In this review, we first discuss genetic and genome engineering technologies used to clone, modify, and transform BGCs into multiple strains and to engineer chassis strains. We then highlight studies that employed the multi-chassis approach successfully to optimize NP production, discover previously uncharacterized NPs, and better understand BGC function.