Toward pectin fermentation by Saccharomyces cerevisiae: Expression of the first two steps of a bacterial pathway for d-galacturonate metabolism.

E.H. Huisjes, M.A. Luttik, M.J. Almering, M.M. Bisschops, D.H. Dang, M. Kleerebezem, R.J. Siezen, A.J. van Maris, J.T. Pronk

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Abstract

Saccharomyces cerevisiae cannot metabolize d-galacturonate, an important monomer of pectin. Use of S. cerevisiae for production of ethanol or other compounds of interest from pectin-rich feedstocks therefore requires introduction of a heterologous pathway for d-galacturonate metabolism. Bacterial d-galacturonate pathways involve d-galacturonate isomerase, d-tagaturonate reductase and three additional enzymes. This study focuses on functional expression of bacterial d-galacturonate isomerases in S. cerevisiae. After demonstrating high-level functional expression of a d-tagaturonate reductase gene (uxaB from Lactococcus lactis), the resulting yeast strain was used to screen for functional expression of six codon-optimized bacterial d-galacturonate isomerase (uxaC) genes. The L. lactis uxaC gene stood out, yielding a tenfold higher enzyme activity than the other uxaC genes. Efficient expression of d-galacturonate isomerase and d-tagaturonate reductase represents an important step toward metabolic engineering of S. cerevisiae for bioethanol production from d-galacturonate. To investigate in vivo activity of the first steps of the d-galacturonate pathway, the L. lactis uxaB and uxaC genes were expressed in a gpd1¿ gpd2¿ S. cerevisiae strain. Although d-tagaturonate reductase could, in principle, provide an alternative means for re-oxidizing cytosolic NADH, addition of d-galacturonate did not restore anaerobic growth, possibly due to absence of a functional d-altronate exporter in S. cerevisiae.
Original languageEnglish
Pages (from-to)303-310
JournalJournal of Biotechnology
Volume162
Issue number2-3
DOIs
Publication statusPublished - 2012

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Keywords

  • uronic acid metabolism
  • limited chemostat cultures
  • neighbor-joining method
  • mold hypocrea-jecorina
  • xylose isomerase gene
  • d-altronic acid
  • escherichia-coli
  • l-arabinose
  • alcoholic fermentation
  • shuttle vectors

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