Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration
- 9 May 2016
- journal article
- research article
- Published by Wiley in Biotechnology & Bioengineering
- Vol. 113 (11), 2462-2473
- https://doi.org/10.1002/bit.26004
Abstract
The CEN/ARS-based low-copy plasmids and 2 μ-based high-copy plasmids have been broadly used for both fundamental studies and practical applications in Saccharomyces cerevisiae. However, the relative low copy numbers and narrow dynamic range limit their applications in many cases. In this study, the expression level of the selection marker proteins was engineered to increase the plasmid copy numbers. A series of plasmids with step-wise increased copy numbers were constructed. The copy number of the plasmids with engineered dominant markers (5–100 copies per cell) showed a positive correlation with the concentration of antibiotics supplemented to the growth media. Based on this finding, we developed a simple yet highly efficient strategy, named Pathway Optimization by Tuning Antibiotic Concentrations (POTAC) to rapidly balance the flux of multi-gene pathways at the DNA level in S. cerevisiae. As proof of concept, POTAC was used to optimize the lycopene and n-butanol biosynthetic pathways, increasing the production of lycopene and n-butanol by 10- and 100-fold, respectively. Additionally, multiplex genome integration with controllable copy numbers was attempted by combining the engineered dominant markers with the CRISPR/Cas9 system. Biotechnol. Bioeng. 2016;113: 2462–2473.Keywords
Funding Information
- Energy Biosciences Institute (OO7G21)
This publication has 52 references indexed in Scilit:
- Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systemsNucleic Acids Research, 2013
- Customized optimization of metabolic pathways by combinatorial transcriptional engineeringNucleic Acids Research, 2012
- Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acidsNature Biotechnology, 2012
- Engineering microbial factories for synthesis of value-added productsJournal of Industrial Microbiology & Biotechnology, 2011
- Isoprenoid Pathway Optimization for Taxol Precursor Overproduction in Escherichia coliScience, 2010
- Automated design of synthetic ribosome binding sites to control protein expressionNature Biotechnology, 2009
- DNA assembler, an in vivo genetic method for rapid construction of biochemical pathwaysNucleic Acids Research, 2008
- Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acidBMC Biotechnology, 2008
- Production of the antimalarial drug precursor artemisinic acid in engineered yeastNature, 2006
- A URA3‐Promoter Deletion in a pYES Vector Increases the Expression Level of a Fungal Lipase in Saccharomyces cerevisiaeAnnals of the New York Academy of Sciences, 1996