Antonie van Leeuwenhoek 80: 287–295, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 287 Enzymes of the Entner–Doudoroff and pyruvate decarboxylation pathways in Zymomonas mobilis wild-type CP4 and mutant strains grown in continuous culture Alexander L. Savvides 1 , Kalliopi I. Chalkou 1 , Milton A. Typas 2 & Amalia D. Karagouni 1,∗ 1 Department of Botany, Faculty of Biology, University of Athens, 15781 Athens, Greece; 2 Department of Genetics and Biotechnology, Faculty of Biology, University of Athens, 15781 Athens, Greece; ( ∗ Author for correspondence; Tel: +30-1-7274526; Fax: +30-1-7274702; E-mail: akar@biol.uoa.gr) Received 29 August 2000; accepted 22 May 2001 Key words: beet molasses, continuous culture, enzyme activity, Zymomonas mobilis Abstract The osmotolerant Zymomonas mobilis strain suc 40, (containing plasmid pDS3154-inaZ), which is capable of pro- ducing simultaneously ethanol and ice nuclei protein, was cultivated in a chemically defined complete sucrose medium, as well as in a sugar beet molasses medium in continuous culture. The strain exhibited the normal Monod’s relationship between biomass and dilution rate, and between growth substrate concentration and dilution rate. Specific activities of a number of enzymes that appear to control important steps in the metabolic flux of the Entner–Doudoroff and pyruvate decarboxylation pathways were investigated over a range of growth rates in steady state cultures. With the exception of glucose-6-phosphate dehydrogenase and gluconate kinase, all of the enzymes exhibited a very similar pattern for the wild type Z. mobilis CP4 and for the osmotolerant mutants, independent of the media used; the enzyme patterns remained relatively constant over the studied growth range. The specific activity of glucose-6-phosphate dehydrogenase was increased 2-fold by decreasing dilution rate in sugar beet molasses. The specific activity of gluconate kinase was 2-fold lower at medium growth rates compared with that at either low or high growth rates. Pyruvate kinase, pyruvate decarboxylase and alcohol dehydrogenase activities were significantly higher compared with those of the enzymes governing the early steps of the Entner– Doudoroff pathway. The present study, which was designed to determine the behaviour of important enzymes in sucrose metabolism of Z. mobilis suc 40 /pDS3154-inaZ grown in continuous culture showed that the microorganism required regulation of specific enzyme activities at the transcriptional level when sugar beet molasses were used as the growth medium. Introduction Zymomonas mobilis ferments glucose, fructose and sucrose exclusively via the Entner–Doudoroff path- way, yielding one mole of ATP per mole of sugar with 96% efficiency followed by pyruvate decarboxylation (Gibbs & DeMoss 1954; Buchholz et al. 1987). In this way, it competes favourably with yeasts for ethanol production, as it displays significantly higher specific rates of sugar uptake, higher ethanol productivities in continuous cell recycle systems and lower bio- mass production (Rogers et al. 1980; Barrow et al. 1984). The interest in enzyme synthesis and activity has been focussed for the past 15 years on the gly- colytic flux and on enzymes in the lower part of the fermentative pathway (Osman et al. 1987). During this period all of the enzymes involved have been purified and characterised (for review see Dawes et al. 1966; Bringer-Meyer & Sahm 1988; Doelle et al. 1993). The ethanol yield obtained from sucrose in batch and continuous cultures is generally lower than that from glucose. This is due to the production of levan and other byproducts, such as sorbitol and fructo- oligomers at higher levels during sucrose metabolism. In contrast, the optimization of fermentation condi- tions led to an inhibition in the formation of these