Improving CoQ10 productivity by strengthening glucose transmembrane of Rhodobacter sphaeroides.

BACKGROUND: Several Rhodobacter sphaeroides have been widely applied in commercial CoQ10 production, but they have poor glucose use. Strategies for enhancing glucose use have been widely exploited in R. sphaeroides. Nevertheless, little research has focused on the role of glucose transmembrane in the improvement of production. RESULTS: There are two potential glucose transmembrane pathways in R. sphaeroides ATCC 17023: the fructose specific-phosphotransferase system (PTSFru, fruAB) and non-PTS that relied on glucokinase (glk). fruAB mutation revealed two effects on bacterial growth: inhibition at the early cultivation phase (12-24 h) and promotion since 36 h. Glucose metabolism showed a corresponding change in characteristic vs. the growth. For ΔfruAΔfruB, maximum biomass (Biomax) was increased by 44.39% and the CoQ10 content was 27.08% more than that of the WT. glk mutation caused a significant decrease in growth and glucose metabolism. Over-expressing a galactose:H+ symporter (galP) in the ΔfruAΔfruB relieved the inhibition and enhanced the growth further. Finally, a mutant with rapid growth and high CoQ10 titer was constructed (ΔfruAΔfruB/tac::galPOP) using several glucose metabolism modifications and was verified by fermentation in 1 L fermenters. CONCLUSIONS: The PTSFru mutation revealed two effects on bacterial growth: inhibition at the early cultivation phase and promotion later. Additionally, biomass yield to glucose (Yb/glc) and CoQ10 synthesis can be promoted using fruAB mutation, and glk plays a key role in glucose metabolism. Strengthening glucose transmembrane via non-PTS improves the productivity of CoQ10 fermentation.

Biopotentiality of High Efficient Aerobic Denitrifier Bacillus megaterium S379 for Intensive Aquaculture Water Quality Management.

Excessive nitrite accumulation is a very tough issue for intensive aquaculture. A high efficient aerobic denitrifier Bacillus megaterium S379 with 91.71±0.17% of NO2--N (65 mg L-1) removal was successfully isolated for solving the problem. Denitrification of S379 showed excellent environment adaptation that it kept high nitrite removal ratio (more than 85%) when temperature ranged from 25°C to 40°C and pH varied between 7.0 and 9.0, and could endure as high as 560 mg L-1 of NO2--N. Immobilization of S379 could enhance denitrification even when NO2--N adding amount got to 340 mg L-1. Immobilized cells also showed well pollutants removal performance in aquaculture wastewater treatment. Moreover, S379 possessed positive hydrolase activities for starch, casein, cellulose and fat and bore more than 60 ppt of salinity. Totally, all the results revealed significant potentiality of immobilized S379 applied in aquaculture water quality management.

Biochemical analysis and the preliminary crystallographic characterization of D-tagatose 3-epimerase from Rhodobacter sphaeroides.

BACKGROUND: D-Tagatose 3-epimerase epimerizes D-fructose to yield D-psicose, which is a rare sugar that exists in small quantities in nature and is difficult to synthesize chemically. We aim to explore potential industrial biocatalysts for commercial-scale manufacture of this rare sugar. A D-tagatose 3-epimerase from Rhodobacter sphaeroides (RsDTE) has recently been identified as a D-tagatose 3-epimerase that can epimerize D-fructose to yield D-psicose with a high conversion rate. RESULTS: The purified RsDTE by Ni-affinity chromatography, ionic exchange chromatography and gel filtration forms a tetramer in solution. The maximal activity was in Tris-HCl buffer pH 8.5, and the optimal temperature was at 35 °C. The product, D-psicose, was confirmed using HPLC and NMR. Crystals of RsDTE were obtained using crystal kits and further refined under crystallization conditions such as 10% PEG 8000,0.1 M HEPES pH 7.5, and 8% ethylene glycol at 20 °C using the sitting-drop vapor diffusion method. The RsDTE homology model showed that it possessed the characteristic TIM-barrel fold. Four residues, Glu156, Asp189, Gln215 and Glu250, forms a hydrogen bond network with the active Mn(II) for the hydride transfer reaction. These residues may constitute the catalytic tetrad of RsDTE. The residues around O1, O2 and O3 of the substrates were conserved. However, the binding-site residues are different at O4, O5 and O6. Arg118 formed the unique hydrogen bond with O4 of D-fructose which indicates RsDTE's preference of D-fructose more than any other family enzymes. CONCLUSIONS: RsDTE possesses a different metal-binding site. Arg118, forming unique hydrogen bond with O4 of D-fructose, regulates the substrate recognition. The research on D-tagatose 3-epimerase or D-psicose 3-epimerase enzymes attracts enormous commercial interest and would be widely used for rare sugar production in the future.

Genetic and biochemical characterization of genes involved in hyaluronic acid synthesis in Streptococcus zooepidemicus.

The biosynthetic pathway for hyaluronic acid (HA) has been proposed; however, a thorough genetic and functional analysis is required to further elucidate the roles of genes involved in HA production. Previously, we developed a markerless gene-deletion system for Streptococcus zooepidemicus and confirmed that hasA is essential for HA synthesis. Here, we constructed a comprehensive set of deletion mutants and investigated the roles of ten additional predicted genes in the HA synthetic pathway. Phenotypic assays revealed that all ten genes play a role in cell growth and/or HA synthesis. As expected, the deletion of hasA or hasB abolished HA production with little effect on growth, while the deletion of genes that are also required for peptidoglycan biosynthesis (hasE, glmM, and glmS) significantly reduced cell growth and HA production. Either of the glmU homologues (hasD and gcaD) was sufficient for optimal growth and the mucoid phenotype, while no double mutant could be isolated. Of the two UDP-glucose pyrophosphorylase (UGPase) paralogues, the operon-encoded hasC1 was responsible for 65 % of the activity, while hasC2 was responsible for the remaining 35 %. The deletion of hasC1 had no effect on cell growth and caused only a moderate decrease in the UDP-glucose level and HA production. The deletion of both hasC1 and hasC2 resulted in a severe growth defect and negligible UDP-glucose accumulation, HA production, and pyrophosphorylase activity. Of the two phosphoglucomutase paralogues, pgm1 and pgm2, the former is responsible for around 10 % of activity, while the latter is responsible for 90 %. The deletion of pgm1 showed no apparent effect on HA synthesis and growth, while the deletion of pgm2 resulted in the abolishment of HA synthesis and a significantly slower growth. These results should guide the metabolic engineering of S. zooepidemicus to improve HA productivity and quality.

[Physiological response to acetic acid stress of Acetobacter pasteuranus during vinegar fermentation].

OBJECTIVE: The aim of the study is to propose a dynamic acetic acid resistance mechanism through analysis on response of cellular morphology, physiology and metabolism of A. pasteurianus CICIM B7003 during vinegar fermentation. METHODS: Vinegar fermentation was carried out in a Frings 9 L acetator by strain B7003 and cultures were sampled at different cellular growth phases. Simultaneously, percentage of capsular polysaccharide versus dry cells weight, ratio of unsaturated fatty acids to saturated fatty acids, transcription of acetic acid resistance genes, activity of alcohol respiratory chain enzymes and ATPase were detected for these samples to assay the responses of bacterial morphology, physiology and metabolism. RESULTS: When acetic acid was existed, no obvious capsular polysaccharide was secreted by cells. As vinegar fermentation proceeding, percentage of capsular polysaccharide versus dry cells weight was reduced from 2.5% to 0.89%. Ratio of unsaturated fatty acids to saturated fatty acids was increased obviously which can improve membrane fluidity. Also transcription level of acetic acid resistance genes was promoted. Interestingly, activity of alcohol respiratory chain and ATPase was not inhibited but promoted obviously with acetic acid accumulation which could provide enough energy for acetic acid resistance mechanism. CONCLUSION: On the basis of the results obtained from the experiment, A. pasteurianus CICIM B7003 relies mainly on the cooperation of changes of extracellular capsular polysaccharide and membrane fatty acids, activation of acid resistance genes transcription, enhancement of activity of alcohol respiratory chain and rapid energy production to tolerate acidic environment.

A novel strategy to improving Rhodobacter azotoformans denitrification efficiency: Insight into the role of a two-component system NtrX/Y in denitrification regulation.

Transcription factors (TFs) can manage the coordinated expression of genes clusters or multiple genes. TF was used to improve bacterial denitrification ability in this study. During denitrification, the ntrY of R. azotoformans, which encodes the sensor of NtrX/Y system, was significantly upregulated in transcription. Denitrification of the mutant △ntrY was significantly inhibited, and it was recovered after replenishing this gene to the mutant, which indicates the NtrX/Y system plays an important role in regulating bacterial denitrification. According to additional research, the NtrX/Y system regulates bacterial denitrification by directly promoting the expression of the nitrite reductase. ntrY overexpression appears to accelerate bacterial denitrification, and the introduction of a strong promoter tac in conjunction with iron supply optimization increases the rate by 72% further. This study realizes bacterial denitrification enhancement from the perspective of global transcription regulation, which provides a novel strategy for improving microbial ability to degrade pollutants.

Ammonia assimilation: A double-edged sword influencing denitrification of Rhodobacter azotoformans and for nitrogen removal of aquaculture wastewater.

NO3--N and NH4+-N are two prevalent nitrogenous pollutants in aquaculture wastewater posing a significant health risk to aquatic animals. R. azotoformans ATCC17025 can rapidly denitrify to remove NO3--N, assimilating NH4+-N. The study investigated the influence of ammonia assimilation on bacterial denitrification. Results revealed that low concentration of NH4+-N (≤0.3 mM) accelerated denitrification, whereas high concentration inhibited it. RT-qPCR indicated that the inhibition of NO reduction under high concentration of NH4+-N was the primary cause of denitrification depression, whereas low concentration of NH4+-N enhanced the synthesis of practically all enzymes involved in denitrification. Finally, nitrogen-rich aquaculture effluent was effectively treated in lab-scale using a semi-continuous operation that provided an appropriate NH4+-N concentration for denitrification. This semi-continuous operation treated wastewater 2 times faster than the batch operation and the content of nitrogen decreased to effluent standard. The study can provide guidance for nitrogen removal of aquaculture wastewater with bioaugmentation.

Improving microbial bioremediation efficiency of intensive aquacultural wastewater based on bacterial pollutant metabolism kinetics analysis.

How to effectively bioremediate aquacultural wastewater using microbes is an urgent issue for the application of aquaculture beneficial microorganisms. Purple non-sulfur bacteria (PNSB) are beneficial in preventing related pollution in aquaculture applications. An autochthonous PNSB Rhodobacter sphaeroides was employed in this study to explore an effective bioremediation strategy of aquacultural wastewater. The test bacterium showed high performance in the removal of ammonium (97.50% ± 0.78% of 42 mg L-1 NH4+-N) and phosphate (93.24% ± 0.71% of 50 mg L-1 PO43--P) in the synthetic wastewater, which are the two crucial indicators of the aquacultural wastewater bioremediation. The study also unveiled that the imbalanced ratio of nutrients in water was the principal reason for limiting the efficient bioremediation of shrimp-culture wastewater. Therefore, an effective microbial bioremediation strategy was proposed by comprehensively considering bacterial pollutant metabolism kinetics constants such as specific consumption yields of chemical oxygen demand (COD)/phosphorous and nitrogen/phosphorous. Finally, COD, total nitrogen (TN), total phosphorus (TP), and ammonium (NH4+-N) in the wastewater were examined, and the results showed that they all decreased to the acceptable values. In conclusion, this study suggested a novel method for improved bioremediation efficiency of aquacultural wastewater, and the findings revealed that this strategy is promising due to its characteristics to be used in various aquaculture wastewater types.

Improving fermented quality of cider vinegar via rational nutrient feeding strategy.

This work aimed to find a rational nutrient feeding strategy for cider vinegar fermentation based on adequate information on the nutritional requirement of acetic acid bacteria. Through single nutrient lack experiment assay, necessary nutrient recipe for Acetobacter pasteurianus CICIM B7003 in acetous fermentation was confirmed. Compounds from the essential nutrient recipe were tested further to find out the key substrates significantly influencing cider vinegar fermentation. The findings showed that aspartate, glutamate, proline and tryptophan should be considered in detail for optimizing nutritional composition of cider. Finally, a nutrient feeding strategy that simultaneously adds proline, glutamate, aspartate and tryptophan to form final concentrations of 0.02g/L, 0.03g/L, 0.01g/L and 0.005g/L in cider was achieved by orthogonal experiment design. Comparing to the original fermentation, the yield of acetic acid from alcohol reached 93.3% and the concentration of most volatile flavor compounds increased with the rational nutrient feeding strategy.