title: Construction of a controllable β-carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Institute of Bioengineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, PR China.
authors.forename: Wenping
authors.initials: W
authors.lastname: Xie
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Lv
authors.forename: Wenqiang
authors.initials: W
authors.lastname: Lu
authors.forename: Jiali
authors.initials: J
authors.lastname: Gu
authors.forename: Hongwei
authors.initials: H
authors.lastname: Yu
mesh_terms: D005819:Genetic Markers / Q000235:genetics*; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000187:drug effects / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D019207:beta Carotene / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D005819:Genetic Markers; D019207:beta Carotene; D005947:Glucose
keywords: biosynthetic pathway; decentralized assembly; functional module; pMRI; β-carotene