Construction of a native promoter-containing transposon vector for the stable monitoring of the denitrifying bacterium Pseudomonas stutzeri LYS-86 by chromosomal-integrated gfp.

Green fluorescent protein (GFP) is the most potential useful marker for the in situ monitoring of biofilm microbes. The objective of this study was to construct and compare the efficacy of transposon vectors containing native and foreign promoters in monitoring the denitrifying bacterium Pseudomonas stutzeri LYS-86 by chromosomal-integrated gfp. The promoter of nitrite reductase (Pnir) was cloned from LYS-86 and utilized to construct the transposon vector pUT/mini-Tn5-km2-Pnir-gfp. Another transposon vector, pUT/mini-Tn5-km2-Plac-gfp, containing the lactose promoter Plac was also constructed. These two transposon vectors and pUT-luxAB-gfp containing the promoter PpsbA were individually inserted into the chromosome of P. stutzeri LYS-86 by conjugation. Three GFP-tagged recombinant strains, LYS-Plac-gfp, LYS-Pnir-gfp, and LYS-PpsbA-gfp, were selected from the conjugants. Green fluorescence was observed only in LYS-Pnir-gfp, suggesting that the native promoter Pnir may be more suitable for GFP expression in P. stutzeri than the foreign promoters Plac and PpsbA. Indeed, LYS-Pnir-gfp maintained stable GFP fluorescence over 16 subcultures without significant changes in the denitrifying capacity.

Coenzyme Q(10) production by immobilized Sphingomonas sp. ZUTE03 via a conversion-extraction coupled process in a three-phase fluidized bed reactor.

A three-phase fluidized bed reactor (TPFBR) was designed to evaluate the potential of CoQ(10) production by gel-entrapped Sphingomonas sp. ZUTE03 via a conversion-extract coupled process. In the reactor, the CoQ(10) yield reached 46.99 mg/L after 8 h of conversion; a high-level yield of about 45 mg/L was maintained even after 15 repetitions (8 h/batch). To fully utilize the residual precursor (para-hydroxybenzoic acid, PHB) in the aqueous phase, the organic phase was replaced with new solution containing 70 mg/L solanesol for each 8 h batch. The CoQ(10) yield of each batch was maintained at a level of about 43 mg/L until the PHB ran out. When solid solanesol was fed to the organic phase for every 8 h batch, CoQ(10) could accumulate and reach a yield of 171.52 mg/L. When solid solanesol and PHB were fed to the conversion system after every 8 h batch, the CoQ(10) yield reached 441.65 mg/L in the organic phase after 20 repetitions, suggesting that the conversion-extract coupled process could enhance CoQ(10) production in the TPFBR.

Coenzyme Q(10) production directly from precursors by free and gel-entrapped Sphingomonas sp. ZUTE03 in a water-organic solvent, two-phase conversion system.

In a water-organic solvent, two-phase conversion system, CoQ(10) could be produced directly from solanesol and para-hydroxybenzoic acid (PHB) by free cells of Sphingomonas sp. ZUTE03 and CoQ(10) concentration in the organic solvent phase was significantly higher than that in the cell. CoQ(10) yield reached a maximal value of 60.8 mg l(-1) in the organic phase and 40.6 mg g(-1)-DCW after 8 h. CoQ(10) also could be produced by gel-entrapped cells in the two-phase conversion system. Soybean oil and hexane were found to be key substances for CoQ(10) production by gel-entrapped cells of Sphingomonas sp. ZUTE03. Soybean oil might improve the release of CoQ10 from the gel-entrapped cells while hexane was the suitable solvent to extract CoQ(10) from the mixed phase of aqueous and organic. The gel-entrapped cells could be re-used to produce CoQ(10) by a repeated-batch culture. After 15 repeats, the yield of CoQ(10) kept at a high level of more than 40 mg l(-1). After 8 h conversion under optimized precursor's concentration, CoQ(10) yield of gel-trapped cells reached 52.2 mg l(-1) with a molar conversion rate of 91% and 89.6% (on PHB and solanesol, respectively). This is the first report on enhanced production of CoQ(10) in a two-phase conversion system by gel-entrapped cells of Sphingomonas sp. ZUTE03.