Heparosan, the capsular polysaccharide of Escherichia coli K5 having a carbohydrate backbone similar to that of heparin, has become a potential precursor for bioengineering heparin. In the heparosan biosynthesis pathway, the gene waaR encoding α-1-, 2-glycosyltransferasecatalyzes the third glucosyl residues linking to the oligosaccharide chain. In the present study, a waaR deletion mutant of E. coli K5 was constructed. The mutant showed improvement of capsule polysaccharide yield. It is interesting that the heparosan molecular weight of the mutant is reduced and may become more suitable as a precursor for the production of low molecular weight heparin derived from the wild-type K5 capsular polysaccharide.

•Δ4,5 unsaturated uronate residue is heparin-lyases-depolymerized products of GAGs.•Recovery of unsaturated uronate is necessary to prepare low molecular weight heparin.•Enhanced soluble express of Δ4,5Δ20 glycuronidase was achieved using pMAL-c2x.•Resultant Δ4,5Δ20 glycuronidase can recover unsaturated uronate residues of GAGs.The Δ4,5 unsaturated uronate (4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid) residue is produced through the depolymerization of heparin, heparosan, and heparan sulfate with heparin lyases. The recovery of unsaturated uronate containing products is necessary to prepare low molecular weight heparin (LMWH) from heparin or heparosan. In this study, the gene of Δ4,5 and Δ4,5Δ20 unsaturated glycuronidase (EC# 3.2.1.56) from Pedobacter heparinus (formerly Flavobacterium heparinum) was cloned into pMAL-c2x plasmid. Its fusion protein with MBP was expressed in Escherichia coli TB1. After purification, Δ4,5 unsaturated glycuronidase was evaluated. The Δ4,5Δ20 glycuronidase showed excellent activity on the unsaturated bonds of the different depolymerized products from Hep I, Hep II, and Hep III on heparin, heparosan, and heparan sulfate.

In the present study, the ability of a newly isolated strain, Methylobacterium sp. XJLW to degrade formaldehyde was investigated in shake flasks and in a bioreactor. The resting cells of Methylobacterium sp. XJLW showed high formaldehyde tolerance (60 g L−1) and high degradation rate (1,687.5 mg L−1 h−1) in shake flasks. This biodegradation was initiated by a dismutation reaction since formic acid was formed and caused significant dropping of pH in the media. The addition of CaCO3 to the media was found as an effective strategy to control the pH and keep the cells in high degradation bioactivity. A three-phase fluidized bed reactor (TPFBR) was designed to test the formaldehyde-biodegrading ability of immobilized Methylobacterium sp. XJLW. Using a repeated-batch degradation mode, the immobilized cells were able to degrade 5 g L−1 formaldehyde (with a maximal degradation rate of 464.5 mg L−1 h−1 under the optimum conditions) and showed stable bioactivity after 20 batches of reuse in the TPFBR.

In a water-organic solvent, two-phase conversion system, CoQ10 could be produced directly from solanesol and para-hydroxybenzoic acid (PHB) by free cells of Sphingomonas sp. ZUTE03 and CoQ10 concentration in the organic solvent phase was significantly higher than that in the cell. CoQ10 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. CoQ10 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 CoQ10 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 CoQ10 from the mixed phase of aqueous and organic. The gel-entrapped cells could be re-used to produce CoQ10 by a repeated-batch culture. After 15 repeats, the yield of CoQ10 kept at a high level of more than 40 mg l−1. After 8 h conversion under optimized precursor’s concentration, CoQ10 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 CoQ10 in a two-phase conversion system by gel-entrapped cells of Sphingomonas sp. ZUTE03.

The use of coenzyme Q10 (CoQ10) as a complementary therapy in heart failure will increase in proportion to the growth of the ageing population and the expansion of statins consumption. Economical production of CoQ10 by microbes will become more important due to the growing demands of the pharmaceutical industry. Process simplification and integration might be one desirable pathway for economic production of CoQ10 by microbial fermentation. In this report, the effect of a coupled fermentation–extraction process on CoQ10 production by newly isolated Sphingomonas sp. ZUTEO3 was evaluated. It was found that the CoQ10 yield of the coupled process was significantly higher than that of the traditional process. As optimal conditions in our experiment, 2% soybean oil was added to the original culture to enhance cell membrane permeability, and 50 mL hexane was added to the 30 h culture as an extracting solvent for the subsequent coupled fermentation–extraction process. The maximal yield of CoQ10 reached 43.2 mg/L and 32.5 mg/g dry cell weight after 38 h of total fermentation period. The coupled process represents one potential pathway for CoQ10 production with even higher yield and lower cost. This is the first report of CoQ10 production by Sphingomonas sp. using a coupled fermentation–extraction process.

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.Highlights► The promoter of nitrite reductase (Pnir) was cloned from Pseudomonas stutzeri LYS-86. ► Two transposons, pUT/mTn5-km2-Pnir-gfp and pUT/mTn5-km2-Plac-gfp were constructed. ► Transposons were inserted into the chromosome of LYS-86. ► Only pUT/mTn5-km2-Pnir-gfp expressed significantly green fluorescence in LYS-86. ► The native promoter might be more suitable for the gfp expression in LYS-86.

A three-phase fluidized bed reactor (TPFBR) was designed to evaluate the potential of CoQ10 production by gel-entrapped Sphingomonas sp. ZUTE03 via a conversion–extract coupled process. In the reactor, the CoQ10 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 CoQ10 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, CoQ10 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 CoQ10 yield reached 441.65 mg/L in the organic phase after 20 repetitions, suggesting that the conversion–extract coupled process could enhance CoQ10 production in the TPFBR.Highlights► We design a three-phase fluidized bed reactor (TPFBR) for CoQ10 production by Sphingomonas sp. ► CoQ10 production via a conversion–extract coupled process was accomplished in the TPFBR. ► CoQ10 yield reached 46.99 mg/L after 8 h of conversion. ► 441.65 mg/L CoQ10 accumulated after 20 repetitions when solid precursors were fed in every batch.