ISOLATION AND CHARACTERIZATION OF KLUYVEROMYCES-MARXIANUS MUTANTS DEFICIENT IN MALATE TRANSPORT

In malic acid-grown cells of the strains ATCC 10022 and KMS3 of Kluyve romyces, marxianus the transport of malic acid occurred by a malate-pr oton symport, which accepted L-malic, D-malic, succinic and fumaric ac ids, but not tartaric, malonic or maleic acids. The system was inducib le and subjected to glucose repression. Mutants of the strain KMS3, un able to grow in a medium with malic acid, were isolated and checked fo r their capacity to utilize several carbon sources and to transport di carboxylic acids by the malate-proton symport. Two distinct clones aff ected on malate transport were obtained. Both were able to grow on a m edium with glycerol or ethanol but not with DL-malic, succinic, oxoglu taric and oxaloacetic acids as the sole carbon and energy sources. How ever, while one of the mutants (Mal7) displayed activity levels for th e enzymes malate dehydrogenase, isocitrate lyase, and phosphoenolpyruv ate carboxykinase similar to those of the wild strain: in the other mu tant type (Mal6) the activities for the same enzymes were significantl y reduced. Plasma membranes from derepressed cells of the wild strain and of the mutants Mal6 and Mal7 were isolated and the protein analyse d by SDS-PAGE. The electrophoretic patterns of these preparations diff ered in a polypeptide with an apparent molecular mass of about 28 kDa, which was absent only in the mutant Mal7. The results indicated that Mal7 can be affected in a gene that encodes a malate carrier in K. mar xianus. (C) 1998 John Wiley & Sons, Ltd.