Full Genome Sequencing of Pseudomonas putida Isolates Following Growth on Cyclopentanone as Sole C-Source or Exposure to 2-cyclopenten-1-one Stress

A Pseudomonas putida strain engineered for the consumption of cyclopentanone as a sole carbon source (RH300-P. putida EM42 PP_5042:Ptac:mekAB:cpnDE) was only able to grow in M9 minimal medium supplemented with 10 mM cyclopentanone as a sole carbon source following a 120 h lag phase. Individual isolates from a stationary phase culture of RH300 were obtained following growth in M9 minimal medium containing 10 mM CP. When these isolates were again grown using 10 mM CP as a sole carbon source, the lag phase was reduced by about 50 h, as compared to RH300. Five isolates (AJB087-AJB091) were sequenced, and all contained mutations predicted to disrupt paaX expression.Additionally, a P. putida strain able to produce glutaric acid from cyclopentanone and 2-cyclopenten-1-one (AJB137-P. putida EM42 PP_5042:Ptac:mekAB:cpnDE gcdH:gcoT- csiD-) showed a significant sensitivity to 2CP at concentrations above 2.5 mM. AJB137 was subjected to Tolerance Adaptive Laboratory Evolution (TALE), by way of sequential passaging in M9 +20 mM D-glucose medium supplemented with 2CP increasing from 2.5 mM to 5.5 mM. Six of the resultant isolates (AJB171-AJB172, AJB175-AJB176, and AJB179-AJB180) were sequenced to determine the genetic determinants for improved 2CP tolerance. Comparison of these isolates against AJB137 revealed that 3/6 isolates contained mutations in their chromosome upstream of the nemA locus and 3/6 harbored mutations in the CDS of nemA, where two of the CDS mutations led to a predicted frame shift in the nemA gene.