Engineering of the metabolism of Saccharomyces cerevisiae for anaerobic production of mannitol |
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| Authors: | Costenoble Roeland Adler Lennart Niklasson Claes Lidén Gunnar |
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| Institution: | 1. University of Sfax, ENIS — Laboratory of Biochemistry and Enzymatic Engineering of Lipases, 3038 Sfax, Tunisia;2. Proteabio Europe S.A.S., 290 Chemin de Saint-Dionysy, Bât A, 30980 Langlade, France;3. CNRS, Aix-Marseille Université, UMR 7282 Enzymologie Interfaciale et Physiologie de la Lipolyse, 31 Chemin Joseph Aiguier, 13009 Marseille, France;4. CNRS — Aix-Marseille Université, Plate-forme Protéomique FR3479, IBiSA Marseille-Protéomique, 31 Chemin Joseph Aiguier, 13009 Marseille, France;1. Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, IN 46202, USA;2. Department of Life Sciences, College of Natural Sciences, Sungkyunkwan University, Suwon-Si, Gyeonggi-Do 440-746, Republic of Korea;3. Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea;4. Department of Biophysics and Chemical Biology, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea;1. Faculty of BioSciences and Technology for Food, Agriculture and Environment, University of Teramo, Via C.R. Lerici 1, 64023 Mosciano Sant'' Angelo, Italy;2. Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Ave. Agustín Escardino 7, 46980 Paterna, Valencia, Spain;3. Department of Genetics, Universitat de València, C/Dr. Moliner, 56100 Burjassot, Valencia, Spain |
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| Abstract: | Under anaerobic conditions, Saccharomyces cerevisiae uses NADH-dependent glycerol-3-phosphate dehydrogenase (Gpd1p and Gpd2p) to re-oxidize excess NADH, yielding substantial amounts of glycerol. In a Deltagpd1 Deltagpd2 double-null mutant, the necessary NAD+ regeneration through glycerol production is no longer possible, and this mutant does not grow under anaerobic conditions. The excess NADH formed can potentially be used to drive other NADH-dependent reactions or pathways. To investigate this possibility, a double-null mutant was transformed with a heterologous gene (mtlD) from Escherichia coli, coding for NADH-dependent mannitol-1-phosphate dehydrogenase. Expression of this gene in S. cerevisiae should result in NADH oxidation by the NADH-requiring formation of mannitol-1-phosphate from fructose-6-phosphate. The strain was characterized using step-change experiments, in which, during the exponential growth phase, the inlet gas was changed from air to nitrogen. It was found that the mutant produced mannitol only under anaerobic conditions. However, anaerobic growth was not regained, which was probably due to the excessive accumulation of mannitol in the cells. |
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| Keywords: | Δgpd1Δgpd2 double-null mutant Redox balance NADH-coupled reduction Mannitol-1-phosphate dehydrogenase Glycerol formation |
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