TY - JOUR
T1 - Toward pectin fermentation by Saccharomyces cerevisiae: Expression of the first two steps of a bacterial pathway for d-galacturonate metabolism.
AU - Huisjes, E.H.
AU - Luttik, M.A.
AU - Almering, M.J.
AU - Bisschops, M.M.
AU - Dang, D.H.
AU - Kleerebezem, M.
AU - Siezen, R.J.
AU - van Maris, A.J.
AU - Pronk, J.T.
PY - 2012
Y1 - 2012
N2 - 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.
AB - 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.
KW - uronic acid metabolism
KW - limited chemostat cultures
KW - neighbor-joining method
KW - mold hypocrea-jecorina
KW - xylose isomerase gene
KW - d-altronic acid
KW - escherichia-coli
KW - l-arabinose
KW - alcoholic fermentation
KW - shuttle vectors
U2 - 10.1016/j.jbiotec.2012.10.003
DO - 10.1016/j.jbiotec.2012.10.003
M3 - Article
VL - 162
SP - 303
EP - 310
JO - Journal of Biotechnology
JF - Journal of Biotechnology
SN - 0168-1656
IS - 2-3
ER -