|
|
Assembly of biosynthetic pathways in Saccharomyces cerevisiae using a marker recyclable integrative plasmid toolbox |
Lidan Ye1,2,Xiaomei Lv1,Hongwei Yu1() |
1. Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China 2. Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University, Hangzhou 310027, China |
|
|
Abstract 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.
|
Keywords
pathway assembly
toolbox
reiterative recombination
S. cerevisiae
biosynthesis
|
Corresponding Author(s):
Hongwei Yu
|
Online First Date: 23 November 2016
Issue Date: 17 March 2017
|
|
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): 70–74
https://doi.org/10.1126/science.1191652
|
2 |
Alper H, Miyaoku K, Stephanopoulos G. Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nature Biotechnology, 2005, 23(5): 612–616
https://doi.org/10.1038/nbt1083
|
3 |
Chang M C, Keasling J D. Production of isoprenoid pharmaceuticals by engineered microbes. Nature Chemical Biology, 2006, 2(2): 674–681
https://doi.org/10.1038/nchembio836
|
4 |
Dugar D, Stephanopoulos G. Relative potential of biosynthetic pathways for biofuels and bio-based products. Nature Biotechnology, 2011, 29(12): 1074–1078
https://doi.org/10.1038/nbt.2055
|
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): 16944–16949
https://doi.org/10.1073/pnas.1111558108
|
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): 1–9
https://doi.org/10.1016/j.ymben.2012.08.007
|
7 |
Xie W, Liu M, Lv X, Lu W, Gu J, Yu H. Construction of a controllable b-carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae. Biotechnology and Bioengineering, 2014, 111(1): 125–133
https://doi.org/10.1002/bit.25002
|
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): 8419–8428
https://doi.org/10.1007/s00253-015-6791-y
|
9 |
Xie W, Lv X, Ye L, Zhou P, Yu H. Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering. Metabolic Engineering, 2015, 30: 69–78
https://doi.org/10.1016/j.ymben.2015.04.009
|
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: 12851
https://doi.org/10.1038/ncomms12851
|
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): 1112–1114
https://doi.org/10.1271/bbb.58.1112
|
12 |
Goldstein J L, Brown M S. Regulation of the mevalonate pathway. Nature, 1990, 343(6257): 425–430
https://doi.org/10.1038/343425a0
|
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 push-pull-restrain strategy. Journal of Biotechnology, 2014, 186: 128–136
https://doi.org/10.1016/j.jbiotec.2014.06.024
|
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): 2519–2524
https://doi.org/10.1093/nar/24.13.2519
|
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): 399–405
https://doi.org/10.1002/yea.1365
|
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): 12
https://doi.org/10.1186/1754-1611-5-12
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
|
Shared |
|
|
|
|
|
Discussed |
|
|
|
|