Construction of a controllable β‐carotene biosynthetic pathway by decentralized assembly strategy in <i>Saccharomyces cerevisiae</i>

<jats:title>ABSTRACT</jats:title><jats:sec><jats:label /><jats:p>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 <jats:italic>S. cerevisiae</jats:italic> still remains a significant challenge. Here, we present a strategy for reconstructing controllable multi‐gene pathways by employing the <jats:italic>GAL</jats:italic> regulatory system. A set of marker recyclable integrative plasmids (pMRI) was designed for decentralized assembly of pathways. As proof‐of‐principle, a controllable β‐carotene biosynthesis pathway (∼16 kb) was reconstructed and optimized by repeatedly using <jats:italic>GAL10</jats:italic>–<jats:italic>GAL1</jats:italic> bidirectional promoters with high efficiency (80–100%). By controling the switch time of the pathway, production of 11 mg/g DCW of total carotenoids (72.57 mg/L) and 7.41 mg/g DCW of β‐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 <jats:italic>S. cerevisisae</jats:italic>. Biotechnol. Bioeng. 2014;111: 125–133. © 2013 Wiley Periodicals, Inc.</jats:p></jats:sec>