Metabolic engineering of microorganisms promises to enable the large-scale, renewable production of many valuable molecules that are difficult to synthesize using existing methods. To achieve commercial viability for these processes, the most difficult chal-lenge typically is not identifying a pathway to produce a desired molecule; rather, it is optimizing the production host to maximize titer, yield, and productivity. The process of strain optimization mirrors other engineering disciplines, consisting of three critical phases—rational design, prototyping, and testing—which are performed iteratively un-til the desired specifications have been met. Here, we present new technologies for more efficiently navigating each stage of the engineering cycle in the widely used pro-duction host, Saccharomyces cerevisiae (baker’s yeast), which benefits from a long history in industrial fermentations and a plethora of genetic tools. First, we describe a standard-ized framework for yeast strain prototyping that enables rapid and high-throughput experimentation. We then turn to strain testing, where we focus on optimizing the pro-duction of benzylisoquinoline alkaloids (BIAs), a large family of plant secondary me-tabolites with a variety of therapeutic uses. Using a novel biosensing strategy that links production of a key BIA intermediate to the generation of a highly fluorescent plant pigment, we perform library screening to identify yeast mutants capable of producing BIAs directly from glucose. Finally, we attempt to improve upon our ability to rational-ly design efficient production strains by taking the initial steps to repurpose the peroxi-some as a synthetic organelle. We propose organelle compartmentalization as a general-izable strategy for reducing undesirable and difficult-to-predict interactions between an engineered metabolic pathway and the cellular context in which it is expressed. Taken together, these tools improve our ability to quickly iterate through the engineering cycle to develop yeast strains for the efficient production of valuable molecules.