Xuepeng Yang

Pyrroloquinoline quinone biosynthesis in Escherichia coli through expression of the Gluconobacter oxydans pqqABCDE gene cluster.

We have expressed the pqqABCDE gene cluster from Gluconobacter oxydans, which is involved in pyrroloquinoline quinone (PQQ) biosynthesis, in Escherichia coli, resulting in PQQ accumulation in the medium. Since the gene cluster does not include the tldD gene needed for PQQ production, this result suggests that the E. coli tldD gene, which shows high homology to the G. oxydanstldD gene, carries out that function. The synthesis of PQQ activated d-glucose dehydrogenase in E. coli and the growth of the recombinant was improved. In an attempt to increase the production of PQQ, which acts as a vitamin or growth factor, we transformed E. coli with various recombinant plasmids, resulting in the overproduction of the PQQ synthesis enzymes and, consequently, PQQ accumulation—up to 6 mM—in the medium. This yield is 21.5-fold higher than that obtained in previous studies.

Membrane-Bound Pyrroloquinoline Quinone-Dependent Dehydrogenase in Gluconobacter oxydans M5, Responsible for Production of 6-(2-Hydroxyethyl) Amino-6-Deoxy-l-Sorbose

ABSTRACT A membrane-bound protein purified from Gluconobacter oxydans M5 was confirmed to be a pyrroloquinoline quinone-dependent d-sorbitol dehydrogenase. Gene disruption and complementation experiments demonstrated that this enzyme is responsible for the oxidation of 1-(2-hydroxyethyl) amino-1-deoxy-d-sorbitol (1NSL) to 6-(2-hydroxyethyl) amino-6-deoxy-l-sorbose (6NSE), which is the precursor of an antidiabetic drug, miglitol.

Characterization of Enzymes in the Oxidation of 1,2-Propanediol to d-(−)-Lactic Acid by Gluconobacter oxydans DSM 2003

Although Gluconobacter oxydans can convert 1,2-propanediol to d-(−)-lactic acid, the enzyme(s) responsible for the conversion has remain unknown. In this study, the membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter oxydans DSM 2003 was purified and confirmed to be essential for the process of d-(−)-lactic acid production by gene knockout and complementation studies. A 25 percent decrease in d-(−)-lactic acid production was found for the aldehyde dehydrogenase (ALDH) deficient strain of G. oxydans DSM 2003, indicating that this enzyme is involved in the reaction but not necessary. It is the first report that reveals the function of ADH and ALDH in the biooxidation of 1,2-propanediol to d-(−)-lactic acid by G. oxydans DSM 2003.

Pyrroloquinoline quinone from Gluconobacter oxydans fermentation broth enhances superoxide anion-scavenging capacity of Cu/Zn-SOD.

A bioassay-guided fractionation of extract from Gluconobacter oxydans fermentation broth afforded Compound 1, which was identified as pyrroloquinoline quinone (PQQ) by spectroscopic methods. PQQ has been shown to enhance the superoxide anion-scavenging capacity significantly for Cu/Zn-SOD. To illustrate the mechanism, the interaction between PQQ and Cu/Zn-SOD was investigated. The multiple binding sites involving hydrogen bonds and van der Waals force between PQQ and Cu/Zn-SOD were revealed by isothermal titration calorimetry. The α-helix content was increased in the Cu/Zn-SOD structure with the addition of PQQ into the solution through ultraviolet (UV) spectroscopy. These results indicated that PQQ could change the conformation of Cu/Zn-SOD through interaction, which could enhance its superoxide anion-scavenging capacity. Therefore, PQQ is a potential natural antioxidant.

Degradation of phytosterols in tobacco waste extract by a novel Paenibacillus sp.

Phytosterols have been demonstrated to be precursors of polycyclic aromatic hydrocarbons (PAHs) formed during biomass pyrolysis. Here, a novel Paenibacillus sp. was evaluated for its ability to degrade phytosterols in tobacco waste extract (TWE). The optimal conditions for cell growth and stigmasterol (a representative of phytosterols) degradation were 37 °C, pH 7.0, 1.0 g/L yeast extract, and 6.0 g/L glucose. Paenibacillus sp. could degrade stigmasterol under high concentrations of glucose (up to 130 g/L) and tolerate wide pH (5.0–9.0) and temperature (25–42 °C) ranges. The new strain could degrade stigmasterol completely into CO2 and H2O, and no intermediate steroids were detected during the degradation process. Phytosterol degradation in TWE was demonstrated by high‐performance liquid chromatography–tandem mass spectrometry. Under optimal conditions (37 °C, pH 7.0, with the exponential‐phase cells), the total degradation ratio of phytosterols reached 38.5% in TWE, including 45.2% of stigmasterol, 37.4% of β‐sitosterol, 27.3% of campesterol, and 28.7% of cholesterol. These results showed that Paenibacillus sp. is a candidate for phytosterol degradation in TWE and other biomass and is potentially useful in reducing the PAHs generated from biomass pyrolysis.

An Efficient Separation of Pyrroloquinoline Quinone Using Chemical Complexation Extraction

Pyrroloquinoline quinone (PQQ), an important natural product, is biosynthesized by Gluconobacter oxydans. In order to prepare the product in large scale, a rapid and selective chemical complexation extraction (CCE) is proposed to separate and purify PQQ from fermentation broth. In this work, the effect of extraction solvent system, stripping agent, and inorganic ion on the extraction were investigated, respectively. As a result, trioctylamine-octanol system showed the most extraction constants (log Kex = 5.07). Ammonia water was the most stripping agent (log Kex = 4.85). Several inorganic salts were determined to have negative effect on the extraction, which suggest that inorganic salts should be removed from broth before extraction. Finally, the optimal extraction condition is Trioctylamine-octanol system (1:1, v/v), 28 °C, and pH=6.5, which leads to more than 90% of extraction rate. In conclusion, trioctylamine-octanol system and ammonia water showed excellent extraction efficiency, which can be used to prepare PQQ.