A vector set for systematic metabolic engineering in Saccharomyces cerevisiae

Authors

  • Fang Fang,

    1. Department of Biological Chemistry, School of Medicine, University of California, Irvine, CA, USA
    Search for more papers by this author
  • Kirsty Salmon,

    1. The Computational Biology Research Laboratory, University of California, Irvine, CA, USA
    Current affiliation:
    1. Verdezyne Inc., 2715 Loker Street, Carlsbad, CA 92010, USA.
    Search for more papers by this author
  • Michael W. Y. Shen,

    1. Department of Chemical Engineering and Materials Science, The Henry Samueli School of Engineering, University of California, Irvine, CA, USA
    Search for more papers by this author
  • Kimberly A. Aeling,

    1. The Computational Biology Research Laboratory, University of California, Irvine, CA, USA
    Current affiliation:
    1. Verdezyne Inc., 2715 Loker Street, Carlsbad, CA 92010, USA.
    Search for more papers by this author
  • Elaine Ito,

    1. The Computational Biology Research Laboratory, University of California, Irvine, CA, USA
    Current affiliation:
    1. Verdezyne Inc., 2715 Loker Street, Carlsbad, CA 92010, USA.
    Search for more papers by this author
  • Becky Irwin,

    1. Department of Biological Chemistry, School of Medicine, University of California, Irvine, CA, USA
    Search for more papers by this author
  • Uyen Phuong C. Tran,

    1. Department of Biological Chemistry, School of Medicine, University of California, Irvine, CA, USA
    Search for more papers by this author
  • G. Wesley Hatfield,

    1. The Computational Biology Research Laboratory, University of California, Irvine, CA, USA
    2. Institute for Genomics and Bioinformatics, University of California, Irvine, CA, USA
    Search for more papers by this author
  • Nancy A. Da Silva,

    Corresponding author
    1. Institute for Genomics and Bioinformatics, University of California, Irvine, CA, USA
    2. Department of Chemical Engineering and Materials Science, The Henry Samueli School of Engineering, University of California, Irvine, CA, USA
    • Department of Chemical Engineering and Materials Science University of California, Irvine, CA 92697 USA.
    Search for more papers by this author
  • Suzanne Sandmeyer

    Corresponding author
    1. Department of Biological Chemistry, School of Medicine, University of California, Irvine, CA, USA
    2. Institute for Genomics and Bioinformatics, University of California, Irvine, CA, USA
    • Department of Biological Chemistry, School of Medicine, University of California, Irvine, CA 92697, USA.
    Search for more papers by this author

Abstract

A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 µ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high- and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering. Copyright © 2010 John Wiley & Sons, Ltd.

Ancillary