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This study describes the biosynthesis of novel sulfur-containing polyhydroxyalkanoates (PHAs), which consist exclusively of hydroxypropylthioalkanoic acid containing thioether groups in the side chains. In addition, the utilization of alkylthioalkanoic acids (lthia fatty acids) by various bacteria was investigated. Based on feedings with propylthiooctanoic acid (PTO) or propylthiohexanoic acid, the metabolically engineered PHAnegative mutant PHB N 4 of Ralstonia eutropha, which harbours plasmid pBBR1 ::phaC1 expressing the PHA synthase of Pseudomonas mendocina, synthesized two novel poly(3-hydroxy-S-propyl-ω-thioalkanoic) acids [poly(3HPTA)s]. A terpolyester consisting of 3-hydroxypropylthiobutyric acid (3HPTB), 3-hydroxypropylthiohexanoic acid (3HPTHx) and 3-hydroxypropylthiooctanoic acid (3HPTO) was synthesized from PTO, whereas a co-polyester of 3HPTB and 3HPTHx was synthesized from propylthiohexanoic acid. Fed-batch fermentation of R. eutropha PHB N 4(pBBR1 ::phaC1) on PTO was done on a 26-litre scale, providing a cell density of 73 g l N1 , from which 45 g of the novel poly(3HPTB-co-3HPTHx-co-3HPTO) were isolated. The chemical structures of the poly(3HPTA)s were identified by gas chromatography/mass spectrometry, elemental sulfur analysis, partial pyrolysis and detailed mass spectrometric analysis, exhibiting 3HPTB, 3HPTHx and 3HPTO as constituents. These novel, hitherto undescribed, constituents of PHAs were randomly distributed in the co-polyesters.

2-Methylcitrate synthase (2-MCS1) and citrate synthase (CS) of Ralstonia eutropha strain H16 were separated by affinity chromatography and analyzed for their substrate specificities. 2-MCS1 used not only the primary substrate propionyl-CoA but also acetyl-CoA and, at a low rate, even butyryl-CoA and valeryl-CoA for condensation with oxaloacetate. The KM values for propionyl-CoA and acetyl-CoA were 0.061 or 0.35 mM, respectively. This enzyme is therefore a competitor for acetyl-CoA during biosynthesis of poly(3-hydroxybutyrate) (PHB) and has to be taken into account if metabolic fluxes are calculated for PHB biosynthesis. In contrast, CS could not use propionyl-CoA as a substrate. The gene-encoding CS (cisY) of R. eutropha was cloned and encodes for a protein consisting of 433 amino acids with a calculated molecular weight of 48,600 Da; it is not truncated in the N-terminal region. Furthermore, a gene encoding a second functionally active 2-methylcitrate synthase (2-MCS2, prpC2) was identified in the genome of R. eutropha. The latter was localized in a gene cluster with genes for an NAD(H)-dependent malate dehydrogenase and a putative citrate lyase. RT-PCR analysis of R. eutropha growing on different carbon sources revealed the transcription of prpC2. In addition, cells of recombinant Escherichia coli strains harboring prpC2 of R. eutropha exhibited high 2-MCS activity of 0.544 U mg-1. A prpC2 knockout mutant of R. eutropha exhibited an identical phenotype as the wild type if grown on different media. 2-MCS2 seems to be dispensable, and a function could not be revealed for this enzyme.