Nuclear Magnetic Resonance Studies of Poly(3-Hydroxybutyrate) and Polyphosphate Metabolism in Alcaligenes eutrophus

Abstract

The metabolic pathways of poly(3-hydroxybutyrate) (PHB) and polyphosphate in the microorganism Alcaligenes eutrophus H16 were studied by 1 H, 13 C, and 31 P nuclear magnetic resonance (NMR) spectroscopy and by conventional analytical techniques. A. eutrophus cells accumulated two storage polymers of PHB and polyphosphate in the presence of carbon and phosphate sources under aerobic conditions after exhaustion of nitrogen sources. The solid-state cross-polarization/magic-angle spinning 13 C NMR spectroscopy was used to study the biosynthetic pathways of PHB and other cellular biomass components from 13 C-labeled acetate. The solid-state 13 C NMR analysis of lyophilized intact cells grown on [1- 13 C]acetate indicated that the carbonyl carbon of acetate was selectively incorporated both into the carbonyl and methine carbons of PHB and into the carbonyl carbons of proteins. The 31 P NMR analysis of A. eutrophus cells in suspension showed that the synthesis of intracellular polyphosphate was closely related to the synthesis of PHB. The roles of PHB and polyphosphate in the cells were studied under conditions of carbon, phosphorus, and nitrogen source starvation. Under both aerobic and anaerobic conditions PHB was degraded, whereas little polyphosphate was degraded. The rate of PHB degradation under anaerobic conditions was faster than that under aerobic conditions. Under anaerobic conditions, acetate and 3-hydroxybutyrate were produced as the major extracellular metabolites. The implications of this observation are discussed in connection with the regulation of PHB and polyphosphate metabolism in A. eutrophus.