Polythioester synthesis in Ralstonia eutropha H16: Novel insights into 3,3′-thiodipropionic acid and 3,3′-dithiodipropionic acid catabolism

• Catabolism of polythioester precursors in Ralstonia eutropha H16.
• Deletion mutant study to elucidate uptake and metabolic reactions.
• New insights into the poly(3HB-co-3MP) biosynthesis pathway.

Ralstonia eutropha H16 is capable of utilizing 3,3′-thiodipropionic acid (TDP) and 3,3’-dithiodipropionic acid (DTDP) as precursor substrates for biosynthesis of a polythioester (PTE) heteropolymer consisting of 3-hydroxybutyric acid (3HB) and 3-mercaptopropionic acid (3MP). To elucidate the hitherto unknown catabolic pathways of TDP and DTDP in R. eutropha H16, 19 defined deletion mutants were generated based on extensive functional genome analyses. Deletions of two ABC-type transporter clusters (H16_A0357-0359, H16_A3658-3660) resulted in an alteration of poly(3HB-co-3MP) composition with TDP as precursor to only 10.2 ± 1.9 mol% 3MP in comparison to 15.1 ± 5.5 mol% in the wild type. A mutant strain of H16 lacking Bordetella uptake gene-like substrate binding proteins (H16_A2779, H16_A0337) incorporated only 7.4 ± 3.8 mol% 3MP into PTE heteropolymers with DTDP as precursor in comparison to 24.5 ± 14.5 mol% in the wild type. Therefore, both gene products are probably involved in transport processes of this compound into the cells. However, the most significant reduction in 3MP contents of the heteropolymers with DTDP as precursor occurred upon the deletion of a gene encoding the putative thiol-disulfide interchange protein DsbD (H16_A3455, 3.9 ± 2.6 mol% 3MP). DsbD is proposed to be involved in the reduction of DTDP into two molecules of 3MP, the common cleavage product of TDP and DTDP.