Physiological and genetic engineering of cytosolic redox metabolism in Saccharomyces cerevisiae for improved glycerol production.
Previous metabolic engineering strategies for improving glycerol production by Saccharomyces cerevisiae were constrained to a maximum theoretical glycerol yield of 1 mol.(molglucose)(-1) due to the introduction of rigid carbon, ATP or redox stoichiometries. In the present study, we sought to circumvent these constraints by (i) maintaining flexibility at fructose-1,6-bisphosphatase and triosephosphate isomerase, while (ii) eliminating reactions that compete with glycerol formation for cytosolic NADH and (iii) enabling oxidative catabolism within the mitochondrial matrix. In aerobic, glucose-grown batch cultures a S. cerevisiae strain, in which the pyruvate decarboxylases the external NADH dehydrogenases and the respiratory chain-linked glycerol-3-phosphate dehydrogenase were deleted for this purpose, produced glycerol at a yield of 0.90 mol.(molglucose)(-1). In aerobic glucose-limited chemostat cultures, the glycerol yield was ca. 25% lower, suggesting the involvement of an alternative glucose-sensitive mechanism for oxidation of cytosolic NADH. Nevertheless, in vivo generation of additional cytosolic NADH by co-feeding of formate to aerobic, glucose-limited chemostat cultures increased the glycerol yield on glucose to 1.08 mol mol(-1). To our knowledge, this is the highest glycerol yield reported for S. cerevisiae.
Geertman
Jan-Maarten A
JM
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
van Maris
Antonius J A
AJ
van Dijken
Johannes P
JP
Pronk
Jack T
JT
D019149:Bioreactors; D053898:Biosynthetic Pathways; D001709:Biotechnology / Q000379:methods*; D018929:Cell Culture Techniques; D003600:Cytosol / Q000378:metabolism*; D017931:DNA Primers; D005560:Formate Dehydrogenases / Q000378:metabolism; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D015202:Protein Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014305:Triose-Phosphate Isomerase / Q000378:metabolism
D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; C499416:FDH1 protein, S cerevisiae; D005560:Formate Dehydrogenases; D006597:Fructose-Bisphosphatase; D014305:Triose-Phosphate Isomerase; D005990:Glycerol