Engineering NADH metabolism in Saccharomyces cerevisiae: formate as an electron donor for glycerol production by anaerobic, glucose-limited chemostat cultures.
Anaerobic Saccharomyces cerevisiae cultures reoxidize the excess NADH formed in biosynthesis via glycerol production. This study investigates whether cometabolism of formate, a well-known NADH-generating substrate in aerobic cultures, can increase glycerol production in anaerobic S. cerevisiae cultures. In anaerobic, glucose-limited chemostat sultures (D=0.10 h(-1)) with molar formate-to-glucose ratios of 0 to 0.5, only a small fraction of the formate added to the cultures was consumed. To investigate whether incomplete formate consumption was by the unfavourable kinetics of yeast formate dehydrogenase (high k(M) for formate at low intracellular NAD(+) concentrations) strains were constructed in which the FDH1 and/or GPD2 genes, encoding formate dehydrogenase and glycerol-3-phosphate dehydrogenase, respectively, were overexpressed. The engineered strains consumed up to 70% of the formate added to the feed, thereby increasing glycerol yields to 0.3 mol mol(-1) glucose at a formate-to-glucose ratio of 0.34. In all strains tested, the molar ratio between formate consumption and additional glycerol production relative to a reference culture equalled one. While demonstrating that that format can be use to enhance glycerol yields in anaerobic S. cerevisiae cultures, This study also reveals kinetic constraints of yeast formate dehydrogenase as an NADH-generating system in yeast mediated reduction processes.
Geertman
Jan-Maarten A
JM
Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
van Dijken
Johannes P
JP
Pronk
Jack T
JT
D000693:Anaerobiosis; D019149:Bioreactors; D004271:DNA, Fungal; D004583:Electrons; D005561:Formates / Q000378:metabolism*; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D004271:DNA, Fungal; D005561:Formates; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; C030544:formic acid; D005947:Glucose; D005990:Glycerol