STUDIES ON COPOLYESTER SYNTHESIS BY RHODOCOCCUS-RUBER AND FACTORS INFLUENCING THE MOLECULAR MASS OF POLYHYDROXYBUTYRATE ACCUMULATED BY METHYLOBACTERIUM-EXTORQUENS AND ALCALIGENES-EUTROPHUS

Rhodococcus ruber NCIMB 40126 synthesizes copolyesters (polyhydroxyalkanoates) of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), containing primarily 3HV monomer units, from a variety of single, chemically unrelated substrates. The 3HV content of the polymer is dependent on the choice of substrate and succinic acid yields high values, e.g., 92-95 mol%. The C-13-NMR spectrum of copolymer produced from [2,3-C-13]succinate displayed similar enrichment of signals representing C-1,C-2, C-4 and C-5 of the 3HV component, whereas the C-3 signal showed negligible enrichment. Label from [1,4-C-13]succinate was incorporated into the C-3 position of the 3HV monomer unit. The key enzymes of polyhydroxybutyrate (PHB) synthesis (3-ketothiolase, acetoacetyl-CoA reductase and PHB synthase) are present, have been studied and their specificities shown to permit the synthesis of poly(3HB-co-3HV). Attempts to detect putative enzymes of succinate metabolism leading to propionyl-CoA formation have so far proved inconclusive. The organism can readily utilise as nitrogen source the amino acids valine and isoleucine, which yield propionyl-CoA in their metabolism. Under sulphate-limited conditions the copolymer accumulated from valine possesses an enhanced 3HV content relative to glucose as substrate. The weight-average molecular mass (M(r)) of PHB accumulated by Methylobacterium extorquens can be considerably higher (1.7 x 10(6)) than previously reported and is dependent on the choice of carbon source, methanol yielding lower values (up to 6 x 10(5)) than succinate (up to 1.7 x 10(6)). The M(r) of the PHB accumulated by both Alcaligenes eutrophus and M. extorquens is affected significantly by the concentration of the carbon source supplied, having lower values and higher polydispersities at higher substrate concentrations. During batch fermentation the M(r) of PHB in A. eutrophus decreases gradually with time whereas the converse is true for M. extorquens.