defragrans strains growing with different monoterpenes   α-Phellandrene Limonene β-Myrcene 65Phen ΔgeoA ΔgeoAcomp 65Phen ΔgeoA ΔgeoAcomp 65Phen ΔgeoA ΔgeoAcomp MaxOD660 find more 0.321 0.217 0.342 0.318 0.174 0.347 0.155 0.066 0.149 Generation time [h] 9.8 34.9 13.5 25.4 50.8 44.9 46.9 57.1 45.8 NO3 – consumed [mM] 10 10 10 10 10 10 7.3 5.8 8.1 NO2 – formed [mM] 0 0 0 0 0 0.01 0.22 0 0.009 Biomass formed [g/L] 0.34 0.23 0.32 0.35 0.22 0.35 0.14 0.08 0.17 C. defragrans

strains 65Phen (wild type), Δgeo A and Δgeo Acomp were grown under standard conditions at 28°C for 280 h (α-phellandrene, limonene) or for 304 h (β-myrcene) with 4 mM monoterpene (in HMN) and 10 mM nitrate. As negative control served a culture without inoculum. The growth phenotype of the wild type was recovered in the mutant strain by complementation with the geoA gene located on a broad-host range plasmid. The in trans complemented mutant C. defragrans ΔgeoAcomp revealed

physiological characteristics similar to C. defragrans 65Phen: growth rate and yield, monoterpene consumption and nitrate reduction were almost identical suggesting that the wild type phenotype was restored by GeDH constitutively expressed from the plasmid pBBR1MCS-2geoA (Table  2, Figure  3). The absence of GeDH was expected to reduce the rate of geranic acid formation. In this study, geranic acid was detected in cultures grown on selleck 6 mM monoterpene in the presence of HMN and 10 mM nitrate (Table  1). Cultures were sampled after nitrate depletion. Geranic acid RGFP966 price concentrations of acidified and lysed cultures were 9 ± 1 μM in the medium of the wild type and 12 ± 1 μM in the medium of the complemented mutant, but only 5 ± 2 μM in the medium of C. defragrans ΔgeoA, thus revealing a limited capacity to form geranic acid in the absence of GeDH. The ΔgeoA phenotype has still the capacity to degrade monoterpenes, an indication for the presence of another alcohol dehydrogenase that catalyzes the geraniol oxidation. Thus, we tested the GeDH activity

spectrophotometrically in cell-free, cytosolic extracts of C. defragrans strains 65Phen, ΔgeoA and ΔgeoAcomp. Under standard conditions, with 0.8 mM geraniol as substrate and identical DOK2 protein concentrations in the assay, the geraniol oxidation rates were 5.8 nkat mg-1 protein for C. defragrans 65Phen and 1.05 nkat mg-1 protein for C. defragrans ΔgeoA. Complementation restored the activity to 9.4 nkat mg-1 protein in C. defragrans ΔgeoAcomp. The in vivo concentration of geraniol inside the cell is expected to be in the micromolar range [47]. The GeDH activity in the extracts of C. defragrans ΔgeoA catalyzed the reaction with a high affinity, the apparent concentration for half-maximal rate was below 10 μM geraniol (Figure  4). This indicated an activity of the second alcohol dehydrogenase at physiological conditions. Figure 4 Initial specific GeDH activity of C. defragrans strains 65Phen, Δ geoA and Δ geoA comp.