Degradation of (S)-1,3-butanediol in Pseudomonas putida KT2440 reveals a CoA-activation route and enantiomeric crosstalk between (R)- and (S)-3-hydroxybutyrate catabolism.

1,3-Butanediol (1,3-BDO) exists as (R)- and (S)-enantiomers, yet how aerobic bacteria catabolize the (S)-form has been unknown. Here, we define (S)-1,3-BDO degradation pathway in Pseudomonas putida KT2440 and uncover an enantioselective enzyme that both controls the pathway and enables a chiral resolution. Transcriptomics, quantitative RT-PCR, and systematic gene deletions show that the ped cluster oxidizes (S)-1,3-BDO to (S)-3-hydroxybutyrate [(S)-3-HB] using the same upstream machinery as (R)-1,3-BDO and 1,4-BDO, but that the downstream route then diverges sharply. Rather than the free-acid dehydrogenase route used for (R)-3-HB, (S)-3-HB is catabolized through an acyl-CoA synthetase, Acs (PP_3458): a Δacs mutant cannot grow on (S)-3-HB, and complementation restores growth, establishing that PP_3458 activates (S)-3-HB to (S)-3-hydroxybutyryl-CoA for β-oxidation. This ATP-dependent CoA-activation route is mechanistically distinct from the ATP-independent (S)-3-hydroxybutyrate dehydrogenase (3SHBDH) pathway of anaerobic Clostridia, identifying a different enzymatic solution to (S)-3-HB assimilation in aerobes. We further show that PP_3458 strongly prefers (S)- over (R)-3-HB and is inhibited by (R)-3-HB, a crosstalk that explains why a (R)-3-HB-accumulating mutant arrests on racemic 3-HB. Two applications follow directly: whole-cell kinetic resolution that converts racemic 3-HB to (S)-3-HB at high enantiomeric excess (ee > 0.99), and, in a strain unable to degrade (S)-3-HB, near-quantitative recovery-providing a chiral pharmaceutical building block; and ped-based biosensors that report 1,3-BDO across both enantiomers and function in P. putida and Escherichia coli. Some mechanistic details-the inhibition mode and the individual downstream β-oxidation steps-remain to be fully resolved.IMPORTANCE1,3-Butanediol is a commercially important C4 diol, but the microbial fate of its (S)-enantiomer in aerobic bacteria has been a gap in our understanding of diol metabolism. We show that Pseudomonas putida KT2440 degrades (S)-1,3-BDO through a route that, after a shared ped-dependent oxidation to (S)-3-hydroxybutyrate, depends on an acyl-CoA synthetase (PP_3458) to activate the (S)-acid as its CoA thioester. This ATP-dependent CoA-activation step is a distinct enzymatic strategy from the ATP-independent dehydrogenase route that anaerobic bacteria use for the same substrate, showing that aerobes and anaerobes solve (S)-3-HB assimilation differently. The work also turns this enzymology into utility: because PP_3458 is enantioselective and is inhibited by (R)-3-hydroxybutyrate, the strain can be used for whole-cell kinetic resolution that yields enantiopure (S)-3-hydroxybutyrate-a chiral pharmaceutical building block-from racemic 3-hydroxybutyrate, and the ped-based biosensors reported here detect 1,3-BDO in both P. putida and Escherichia coli, providing a portable tool for strain screening.