Journal of Biotechnology 2012-12-31

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

Eline H Huisjes, Marijke A H Luttik, Marinka J H Almering, Markus M M Bisschops, Dieu H N Dang, Michiel Kleerebezem, Roland Siezen, Antonius J A van Maris, Jack T Pronk

Index: J. Biotechnol. 162(2-3) , 303-10, (2012)

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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 gpd1Δ gpd2Δ 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.Copyright © 2012 Elsevier B.V. All rights reserved.