Fig. 5: Implementation of module 3 of THETA in vivo.

a, A simplified scheme of the THETA cycle, consisting of three modules. b, The scheme for implementing module 3 in vivo. The conversion of (2S)-methylsuccinyl-CoA to mesaconyl-C₄-CoA, which was initially planned to take place through a CoA-based route (involving Mco or Mcd), was replaced with an acid bypass, in which native thioesterases (TesB and YciA) hydrolyse (2S)-methylsuccinyl-CoA into methylsuccinate, which is further converted to mesaconate and mesaconyl-C₄-CoA using Sdh and Ict, respectively. c, Expression of Ccr and DmdB1 enabled HH61 to produce methylsuccinate (left) and mesaconate (right) in M9 minimal medium with 1% glucose, 10 mM acetate, 0.5 μM coenzyme B12 and 10 mM crotonate, indicating that the upper part of module 3 (Ccr, Epi and Ecm) worked in vivo so that E. coli converted methylsuccinate to mesaconate (through Sdh). Production of methylsuccinate was substantially decreased in a thioesterase double knockout (ΔtesB ΔyciA), indicating that these native enzymes are responsible for methylsuccinyl-CoA hydrolysis. d, Expression of Ict (Y. pestis) and the lower part of module 3 (Meh and Ccl steps) enabled HH61 to produce non-labelled pyruvate in M9 minimal medium containing 1% [U-¹³C]glucose, 10 mM [U-¹³C]acetate and 10 mM mesaconate, indicating that the lower part from mesaconate to pyruvate and acetyl-CoA was functional in vivo. e, Overexpression of E. coli native Sdh resulted in a notable increase in mesaconate production in M9 minimal medium with 1% glucose, 10 mM acetate and 10 mM methylsuccinate. f, Expression of module 3 (integrated epi and ecm, pTE4501 and pTE4502) enabled HH422 to produce non-labelled pyruvate in M9 minimal medium containing 1% [U-¹³C]glucose, 2% LB, 0.5 μM coenzyme B12 and 10 mM crotonate, indicating that module 3 worked in vivo. To confirm that the non-labelled pyruvate originated from crotonate and not from LB, two negative controls were used: HH422 with module 3 in the same medium in the absence of crotonate, and HH422 without module 3 in the same medium containing crotonate. All media contained 0.5 mM IPTG and appropriate antibiotics. Metabolites measured in c–f are colour-coded according to b. The data represent the mean ± s.d determined from n = 3 independent experiments. Yp, Yersinia pestis.