Frontiers of Chemical Science and Engineering

, Volume 11, Issue 1, pp 126–132 | Cite as

Assembly of biosynthetic pathways in Saccharomyces cerevisiae using a marker recyclable integrative plasmid toolbox



A robust and versatile tool for multigene pathway assembly is a key to the biosynthesis of high-value chemicals. Here we report the rapid construction of biosynthetic pathways in Saccharomyces cerevisiae using a marker recyclable integrative toolbox (pUMRI) developed in our research group, which has features of ready-to-use, convenient marker recycling, arbitrary element replacement, shuttle plasmid, auxotrophic marker independence, GAL regulation, and decentralized assembly. Functional isoprenoid biosynthesis pathways containing 4–11 genes with lengths ranging from ~10 to ~22 kb were assembled using this toolbox within 1–5 rounds of reiterative recombination. In combination with GAL-regulated metabolic engineering, high production of isoprenoids (e.g., 16.3 mg∙g–1 dcw carotenoids) was achieved. These results demonstrate the wide range of application and the efficiency of the pUMRI toolbox in multigene pathway construction of S. cerevisiae.


pathway assembly toolbox reiterative recombination S. cerevisiae biosynthesis 


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This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 21406196 and 21576234), Zhejiang Provincial Natural Science Foundation of China (Grant No. LQ14B060005), and Qianjiang Talents Project.


  1. 1.
    Ajikumar P K, Xiao W H, Tyo K E J, Wang Y, Simeon F, Leonard E, Mucha O, Phon T H, Pfeifer B, Stephanopoulos G. Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli. Science, 2010, 330(6000): 70–74CrossRefGoogle Scholar
  2. 2.
    Alper H, Miyaoku K, Stephanopoulos G. Construction of lycopeneoverproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nature Biotechnology, 2005, 23(5): 612–616CrossRefGoogle Scholar
  3. 3.
    Chang MC, Keasling J D. Production of isoprenoid pharmaceuticals by engineered microbes. Nature Chemical Biology, 2006, 2(2): 674–681CrossRefGoogle Scholar
  4. 4.
    Dugar D, Stephanopoulos G. Relative potential of biosynthetic pathways for biofuels and bio-based products. Nature Biotechnology, 2011, 29(12): 1074–1078CrossRefGoogle Scholar
  5. 5.
    Lange B M, Croteau R B. Improving peppermint essential oil yield and composition by metabolic engineering. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(41): 16944–16949CrossRefGoogle Scholar
  6. 6.
    Tai M, Stephanopoulos G. Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metabolic Engineering, 2013, 15(1): 1–9CrossRefGoogle Scholar
  7. 7.
    Xie W, Liu M, Lv X, Lu W, Gu J, Yu H. Construction of a controllable β-carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae. Biotechnology and Bioengineering, 2014, 111(1): 125–133CrossRefGoogle Scholar
  8. 8.
    Zhou P, Ye L, Xie W, Lv X, Yu H. Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt. Applied Microbiology and Biotechnology, 2015, 99(20): 8419–8428CrossRefGoogle Scholar
  9. 9.
    Xie W, Lv X, Ye L, Zhou P, Yu H. Construction of lycopeneoverproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering. Metabolic Engineering, 2015, 30: 69–78CrossRefGoogle Scholar
  10. 10.
    Lv X, Wang F, Zhou P, Ye L, Xie W, Xu H, Yu H. Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae. Nature Communications, 2016, 7: 12851CrossRefGoogle Scholar
  11. 11.
    Yamano S, Ishii T, Nakagawa M, Ikenaga H, Misawa N. Metabolic engineering for production of beta-carotene and lycopene in Saccharomyces cerevisiae. Bioscience, Biotechnology, and Biochemistry, 1994, 58(6): 1112–1114CrossRefGoogle Scholar
  12. 12.
    Goldstein J L, Brown M S. Regulation of the mevalonate pathway. Nature, 1990, 343(6257): 425–430CrossRefGoogle Scholar
  13. 13.
    Lv X, Xie W, Lu W, Fei G, Gu J, Yu H, Ye L. Enhanced isoprene biosynthesis in Saccharomyces cerevisiae by engineering of the native acetyl-CoA and mevalonic acid pathways with a pushpull-restrain strategy. Journal of Biotechnology, 2014, 186: 128–136CrossRefGoogle Scholar
  14. 14.
    Güldener U, Heck S, Fielder T, Beinhauer J, Hegemann J H. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research, 1996, 24(13): 2519–2524CrossRefGoogle Scholar
  15. 15.
    Akada R, Kitagawa T, Kaneko S, Toyonaga D, Ito S, Kakihara Y, Hoshida H, Morimura S, Kondo A, Kida K. PCR-mediated seamless gene deletion and marker recycling in Saccharomyces cerevisiae. Yeast (Chichester, England), 2006, 23(5): 399–405CrossRefGoogle Scholar
  16. 16.
    Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D. BglBrick vectors and datasheets: A synthetic biology platform for gene expression. Journal of Biological Engineering, 2011, 5(1): 12CrossRefGoogle Scholar

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© Higher Education Press and Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Institute of Bioengineering, College of Chemical and Biological EngineeringZhejiang UniversityHangzhouChina
  2. 2.Key Laboratory of Biomass Chemical Engineering of Ministry of EducationZhejiang UniversityHangzhouChina

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