Extended Data Fig. 7: Reverse engineering of P. putida ALE-mutant Ahx2-322.
From: Upcycling of polyamides through chemical hydrolysis and engineered Pseudomonas putida
a, Genetic context of the identified mutation in ALE mutant Ahx2-322. The mutation (C→T) (red flash) was located in close proximity to the predicted promoter regions of PP_2176 (green) and PP_2177-80 (blue). Downstream of PP_2177-80, its repressor PP_2181 is encoded that represses expression of the operon in the presence of HMDA and other amines. The mutation might enable constitutive expression of PP_2177-80 or prevent binding of PP_2181 thereby activating expression of PP_2177-80 in the absence of inducers. Promoter regions were predicted using SAPPHIRE. b-d, Combinations of the identified mutations were implemented in P. putida NYL P14f –nylB and screened individually or in combination yielding the final reverse engineered strain P. putida NYLON-B. Strains were cultivated in MSM supplemented with 15 mM of Ahx2 (b) and Ahx3 (c) or the PA6 soluble fraction (d). The mean values and standard deviations of three replicates are shown (n = 3).
