Saccharomyces cerevisiae is an important platform organism for the synthesis of a great number of natural products. However, the assembly of controllable and genetically stable heterogeneous biosynthetic pathways in S. cerevisiae still remains a significant challenge. Here, we present a strategy for reconstructing controllable multi-gene pathways by employing the GAL regulatory system. A set of marker recyclable integrative plasmids (pMRI) was designed for decentralized assembly of pathways. As proof-of-principle, a controllable -carotene biosynthesis pathway (approximate to 16kb) was reconstructed and optimized by repeatedly using GAL10-GAL1 bidirectional promoters with high efficiency (80-100%). By controling the switch time of the pathway, production of 11mg/g DCW of total carotenoids (72.57mg/L) and 7.41mg/g DCW of -carotene was achieved in shake-flask culture. In addition, the engineered yeast strain exhibited high genetic stability after 20 generations of subculture. The results demonstrated a controllable and genetically stable biosynthetic pathway capable of increasing the yield of target products. Furthermore, the strategy presented in this study could be extended to construct other pathways in S. cerevisisae. Biotechnol. Bioeng. 2014;111: 125-133. (c) 2013 Wiley Periodicals, Inc.