title: Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration.
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. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana 61801, Illinois.
authors.forename: Jiazhang
authors.initials: J
authors.lastname: Lian
authors.affiliation: School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois.
authors.forename: Run
authors.initials: R
authors.lastname: Jin
authors.affiliation: Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana 61801, Illinois. zhao5@illinois.edu.|Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois. zhao5@illinois.edu.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D018628:Gene Dosage / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods*; D016681:Genome, Fungal / Q000235:genetics*; D060847:Metabolic Engineering / Q000379:methods; D010957:Plasmids / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics
chemical_list: D029701:Saccharomyces cerevisiae Proteins
keywords: CRISPR/Cas9; multiplex genome integration; pathway optimization; plasmid copy number; synthetic biology