Toward pectin fermentation by Saccharomyces cerevisiae: expression of the first two steps of a bacterial pathway for D-galacturonate metabolism.
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.
Department of Biotechnology, Delft University of Technology and Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands.
Eline H
EH
Huisjes
Marijke A H
MA
Luttik
Marinka J H
MJ
Almering
Markus M M
MM
Bisschops
Dieu H N
DH
Dang
Michiel
M
Kleerebezem
Roland
R
Siezen
Antonius J A
AJ
van Maris
Jack T
JT
Pronk
D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D056804:Biofuels; D019149:Bioreactors / Q000382:microbiology; D002237:Carbohydrate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D003062:Codon; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D006603:Hexuronic Acids / Q000378:metabolism*; D007535:Isomerases / Q000235:genetics / Q000378:metabolism; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods; D053858:Metabolic Networks and Pathways; D010368:Pectins / Q000378:metabolism*; D010802:Phylogeny; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
D056804:Biofuels; D003062:Codon; D006603:Hexuronic Acids; D000431:Ethanol; C007819:galacturonic acid; D010368:Pectins; D002237:Carbohydrate Dehydrogenases; C100035:tagaturonate reductase; D007535:Isomerases