Natural products (NPs) and their derivatives play an important role in modern healthcare as frontline therapeutics for many diseases and as commodity chemicals. Despite their ubiquity, NPs are historically challenging to produce. Many high-value metabolites are produced in nature by organisms that are not ideal for large-scale production. Therefore, interest exists in expressing the biosynthetic pathways that create these compounds in organisms that are more suitable for industrial production, a key example being the yeast Saccharomyces cerevisiae. The explosion of genomic sequence data and the significant advancements in synthetic biology have led to developments in both the discovery of new natural products as well as their corresponding biosynthetic pathways. However, challenges remain in the development of metabolic engineering tools that allow for the reconstruction of complex pathways in new host microorganisms. Every natural product is synthesized from the core metabolism by a large network of enzymes, whose coding genes must be integrated and stably expressed within the new host organism. The integration of these complex genetic pathways has traditionally formed a major bottleneck in the creation of NP-producing microbes. We report a biosynthetic pathway reconstruction platform based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae to allow for the homology-based recombination of exogenous gene fragments into the yeast chromosome. The results presented here demonstrate a one-step system that can insert specific genes into targeted sites of the yeast genome, allowing for the expression of large biosynthetic pathways.