ORIGINAL PAPER B.-S. Lee á T. Maurer á H. R. Kalbitzer á E. Holler b-Poly(L-malate) production by Physarum polycephalum 13 C Nuclear magnetic resonance studies Received: 16 March 1999 / Received revision: 5 May 1999 / Accepted: 7 May 1999 Abstract b-Poly(L-malate) (PMLA) production in Physarum polycephalum has been followed by using D-[1- 13 C]glucose and Ca 13 CO 3 . Nuclear magnetic reso- nance studies of PMLA showed that the 13 C label from [1- 13 C]glucose was incorporated in the presence of CaCO 3 into positions C-3 (-CH 2 -) and C-4 (-CO-) of the L-malate repeating unit of PMLA. The 13 C label from Ca 13 CO 3 was incorporated into position C-4 and indi- cated that not only the endogenous CO 2 but also the exogenous CO 2 from CaCO 3 served signi®cantly as a carbon source for PMLA production. In the absence of CaCO 3 , the 13 C labeling pattern of PMLA from D-[1- 13 C]glucose was almost indistinguishable from that for the natural abundance 13 C-NMR spectrum of the polymer. These results indicated that L-malate used for PMLA production is synthesized either via carboxyla- tion of pyruvate and reduction of oxaloacetate in the presence of CaCO 3 or via the oxidative tricarboxylic acid (TCA) cycle in the absence of CaCO 3 . Avidin strongly inhibited the formation of L-malate via car- boxylation; the 13 C labeling pattern of PMLA in the presence of CaCO 3 was almost identical with that for the natural abundance spectrum when avidin was added, indicating that L-malate utilized for PMLA production was supplied under this condition by the oxidative TCA cycle. Introduction Large amounts of b-poly(L-malate) (PMLA) are pro- duced from glucose and CaCO 3 by aerobic cultivation of plasmodial Physarum polycephalum (Lee and Holler 1999). The biosynthetic mechanisms by which PMLA is produced and excreted into the cultural medium (Fisc- her et al. 1989) has not yet been elucidated. The pre- cursor of polymerization is L-malate, as shown by the results of feeding experiments with 14 C-labeled D-glu- cose (Holler et al. 1992; Schmidt et al. 1996) or micro- injection of L-[ 14 C]malate (Willibald and Holler, unpublished data). It was concluded that L-malate was generated from glucose via glycolysis and the tricar- boxylic acid (TCA) cycle. Liu and SteinbuÈchel (1997) suggested that PMLA production by Aureobasidium pullulans followed a similar pathway since it was inhib- ited by tri¯uoroacetic acid. The inhibition was relieved when the medium contained, in addition, succinate or malate. They also reported that malonate, an inhibitor of succinate dehydrogenase, stimulated PMLA produc- tion, suggesting the induction of the glyoxylate shunt as an alternative pathway (Liu and SteinbuÈchel 1997). The production of PMLA by P. polycephalum was consid- erably stimulated by carbonate (Lee and Holler 1999). The eect of avidin, an inhibitor of pyruvate carboxyl- ase, suggested that carbonate was an additional carbon source besides D-glucose. Similarly, the production of L- malate by Aspergillus ¯avus (Peleg et al. 1989) and by Saccharomyces cerevisiae (Schwartz and Radler 1988; Pines et al. 1996) was stimulated by the addition of carbonate. Several lines of evidence suggested that L-malate was formed by a cytosolic reductive pathway which converted pyruvate to L-malate via oxalacetate. In the present contribution, we used 13 C-labeled carbon sources to follow the basic metabolic pathways of PMLA production in the plasmodium of P. polyceph- alum. Materials and methods Conditions for growth and PMLA production Microplasmodia of P. polycephalum (strain M 3 CVII, ATCC 204388) were cultivated in ¯asks and preserved in the form of spherules as described by Daniel and Baldwin (1964). A vegetative Appl Microbiol Biotechnol (1999) 52: 415±420 Ó Springer-Verlag 1999 B.-S. Lee á T. Maurer á H. R. Kalbitzer á E. Holler (&) Institut fuÈ r Biophysik und physikalische Biochemie der UniversitaÈt Regensburg, D-93040 Regensburg, Germany http://www.biologie.uni-regensburg.de/Biophysik/Holler/index.html Tel.: +49-941-9433030 Fax: +49-941-9432813