These data confirmed that the identified pqqABCDEF operon was essential, at minimum, for several steps of the PQQ biosynthetic pathway in P. ananatis. However, it cannot be excluded that some additional genes from other loci of the P. ananatis SC17(0) chromosome participated in PQQ synthesis as well. To test this possibility, the cloned pqq operon was transferred from P. ananatis SC17(0) into E. coli. www.selleckchem.com/products/GDC-0941.html In E. coli, the primary pathway for glucose consumption is the phosphoenolpyruvate/carbohydrate phosphotransferase system (PTS) (for a review, see Deutscher et al., 2006). The GDH-mediated pathway

in this organism does not work because of the absence of the PQQ biosynthesis route. In E. coli, mGDH is synthesized only in apoenzyme form; however, the holoenzyme can be formed in the presence of exogenous PQQ. We expected to observe a similar effect after integration of the P. ananatis putative pqq operon into the E. coli chromosome. To support this hypothesis,

one copy of the pqq operon was introduced into the double mutant strain, with inactivated PTS and the mannose permease Alectinib mouse system. The strain used as a recipient, named MG1655-2Δ, is unable to grow on the glucose minimal medium because of the absence of effective glucose uptake. Synthesis of PQQ in the MG1655-2Δ-pqq strain, which has pqq operon integrated at the φ80attB site, could lead to direct oxidation of glucose to gluconic acid by PQQ-mGDH. The growth properties of MG1655-2Δ-pqq were compared with those

of the wild-type strain and to MG1655-2Δ, grown with the addition of exogenous PQQ, on the minimal medium with glucose as the sole carbon source. As shown in Fig. 1, integration of the pqq operon resulted in the restoration of MG1655-2Δ-pqq growth on glucose minimal medium. However, MG1655-2Δ-pqq showed a prolonged lag time unlike the wild-type strain or MG1655-2Δ growing in the Lonafarnib in vitro presence of PQQ in the medium (+PQQ). In addition, MG1655-2Δ-pqq grew at a slower rate than MG1655-2Δ under +PQQ conditions; however, it had a higher final OD. Comparison of the growth properties of MG1655-2Δ and MG1655-2Δ-pqq suggests that the introduction of the pqq operon allowed the formation of an active GDH, resulting in the production of gluconic acid from glucose and its further utilization. We attempted to determine whether E. coli strains containing the pqq operon are able to accumulate PQQ in the culture medium. In our experiments, we could detect about 0.25 μg L−1 of PQQ in the assay system. However, no PQQ was observed during MG1655-2Δ-pqq growth on the minimal medium with gluconate as the sole carbon source. It is possible that pqq genes cloned with their native regulatory regions from P. ananatis are poorly expressed in E. coli. Conversely, the P. ananatis SC17(0) strain with the native pqq operon accumulates up to 9 mg L−1 of PQQ.