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Construction of a controllable β‐carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae
Author(s) -
Xie Wenping,
Liu Min,
Lv Xiaomei,
Lu Wenqiang,
Gu Jiali,
Yu Hongwei
Publication year - 2014
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25002
Subject(s) - saccharomyces cerevisiae , yeast , biosynthesis , plasmid , metabolic pathway , metabolic engineering , synthetic biology , gene , biochemistry , biology , chemistry , computational biology
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 β‐carotene biosynthesis pathway (∼16 kb) 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 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 S. cerevisisae . Biotechnol. Bioeng. 2014;111: 125–133. © 2013 Wiley Periodicals, Inc.