Appl Microbiol Biotechnol (1992) 36:535-537 Short contribution Applied Microbiology Biotechnology © Springer-Verlag 1992 Highly efficient bioconversion of glucose into fructose diphosphate with fed-batch-grown Saccharomyces cerevisiae cells Concetta Compagno, Aldo Tura, Bianca Maria Ranzi, and Enzo Martegani Dipartimento di Fisiologia e Biochimica Generali, Universitfi di Milano, Milan, Italy Received 24 May 1991/Accepted 13 September 1991 Summary. One of the methods commonly used for manufacturing fructose 1,6-diphosphate is based on the enzymatic phosphorylation of glucose with inorganic phosphate using permeabilized brewer's yeast cells. Our results demonstrate that a substantial improvement in the yield of bioconversion can be achieved using fed- batch-grown Saccharomyces cerevisiae cells. Under an appropriate glucose and phosphate to cell ratio the effi- ciency of bioconversion reaches 70% of the theoretical value. Introduction Fructose 1,6-diphosphate (FDP), an important glyco- lytic intermediate, has been the subject of several phar- macological investigations and its salts find several clinical and pharmaceutical applications (Markov et al. 1981). At present, one of the methods commonly used for manufacturing FDP is based on the enzymatic phosphorylation of glucose with inorganic phosphate (Pi) using permeabilized brewer's yeast cells. Organic solvents such as diethyl ether or toluene, have been employed in the commercial preparation of FDP from yeast (Leisola and Linko 1974). Recently, other procedures for cell permeabilization have been developed (Bisso and Melelli 1986; Melelli et al. 1989). The mechanism of FDP accumulation during glucose fermentation by permeabilized yeast in the presence of Pi is not completely understood and the relevance of the physiological condition of the yeast cells for the op- timization of the process has not been exploited. There- fore, we undertook an analysis of FDP production us- ing either commercial baker's yeast or laboratory strain $288C of Saccharomyces cerevisiae grown in fed-batch culture. Our results demonstrate that a substantial im- provement in the yield of bioconversion can be achieved using fed-batch-grown S. cerevisiae $288C cells. Under appropriate glucose and phosphate to cell ratios, the efficiency of bioconversion reaches 70% of the theoretical value. Materials and methods Strains and growth conditions. The experiments were performed with commercial baker's yeast (Gist-Brocades, Pavia, Italy) and with the haploid strain $288C (MATa, gal2, SUC2, MAL) of S. cerevisiae. The production of $288C was done in our laboratory in a fed-batch reactor (1.21) by a control process for fed-batch fer- mentation based on measured ethanol in the exit gases, as de- scribed elsewhere (Porro et al. 1991). The fed-batch process was performed at 30° C and the pH was controlled at 4.950 by ammonia addition. The feed medium con- tained 50% glucose as carbon source and concentrated salts as previously described (Porro et al. 199I). Yeast cells were grown at a final density of 100 g dry weight/1. The centrifuged yeast cells were stored at 4°C until used (this was within a week from the end of fed-batch fermentation for $288C). Bioconversion cycles. Yeast cells were resuspended in 40 ml me- dium to give different cell concentrations (87, 175 and 375 g wet weight/l). The medium solution contained different concentra- tions of glucose and Na2HPO4 (5 or 10% glucose and 2.5 or 5% Na2HPO4). Cell permeabilization was obtained by the addition of 10% tol- uene and vigorous shaking of the suspension. The cultures were then stirred in a water bath at 30° C. Analytical methods. FDP was determined on supernatant samples of the suspension taken at different times after deproteinization. This was obtained by adding 1 ml of 1 M perehloric acid to 1-ml samples and allowing to stand for 15 min at 0° C. After neutraliza- tion with 0.5 ml of 2.5 M KHCO3 for 15 rain on ice, the samples were centrifuged and the supernatants used for the determination of metabolites. Glucose assay was performed by an enzymatic method (Peri- dochrom, Boehringer Mannheim, FRG). Ethanol was determined according to Bergmeyer (1984). The FDP yield was calculated as moles of FDP formed from moles of glucose consumed during bioconversion. Offprint requests to: C. Compagno