Engineering Escherichia coli via introduction of the isopentenol utilization pathway to effectively produce geranyllinalool.

BACKGROUND: Geranyllinalool, a natural diterpenoid found in plants, has a floral and woody aroma, making it valuable in flavors and fragrances. Currently, its synthesis primarily depends on chemical methods, which are environmentally harmful and economically unsustainable. Microbial synthesis through metabolic engineering has shown potential for producing geranyllinalool. However, achieving efficient synthesis remains challenging owing to the limited availability of terpenoid precursors in microorganisms. Thus, an artificial isopentenol utilization pathway (IUP) was constructed and introduced in Escherichia coli to enhance precursor availability and further improve terpenoid synthesis. RESULTS: We first constructed an artificial IUP in E. coli to enhance the supply of precursor geranylgeranyl diphosphate (GGPP) and then screened geranyllinalool synthases from plants to achieve efficient synthesis of geranyllinalool (274.78 ± 2.48 mg/L). To further improve geranyllinalool synthesis, we optimized various cultivation factors, including carbon source, IPTG concentration, and prenol addition and obtained 447.51 ± 6.92 mg/L of geranyllinalool after 72 h of shaken flask fermentation. Moreover, a scaled-up production in a 5-L fermenter was investigated to give 2.06 g/L of geranyllinalool through fed-batch fermentation. To the best of our knowledge, this is the highest reported titer so far. CONCLUSIONS: Efficient synthesis of geranyllinalool in E. coli can be achieved through a two-step pathway and optimization of culture conditions. The findings of this study provide valuable insights into the production of other terpenoids in E. coli.

[Existence forms of Tiantian Capsules and its raw material Aloe in rats].

This study explored the existence forms(original constituents and metabolites) of Tiantian Capsules, Aloe, and Tiantian Capsules without Aloe in rats for the first time, aiming to clarify the contribution of Aloe to the existence form of Tiantian Capsules. Rats were administrated with corresponding drugs by gavage once a day for seven consecutive days. All urine and feces samples were collected during the seven days of administration, and blood samples were collected 0.5, 1, and 1.5 h after the last administration. UHPLC-Q-TOF-MS was employed to detect and identify the original constituents and metabolites in the samples. A total of 34, 28, and 2 original constituents and 64, 94, and 0 metabolites were identified in the samples of rats administrated with Aloe, Tiantian Capsules, and Tiantian Capsules without Aloe, respectively. The main metabolic reactions were methylation, hydrogenation, hydroxylation, dehydroxylation, glucuronidation, and sulfation. This study clarified for the first time the existence forms and partial metabolic pathways of Aloe, Tiantian Capsules, and Tiantian Capsules without Aloe in rats, laying a foundation for revealing their effective forms. The findings are of great significance to the research on the functioning mechanism and quality control of Aloe and Tiantian Capsules.

Liensinine alleviates mouse intestinal injury induced by sepsis through inhibition of oxidative stress, inflammation, and cell apoptosis.

Sepsis is a clinical syndrome triggered by an imbalanced host response to pathogens that can lead to multiple organ dysfunction. The immune response and barrier function of the gut play an important role in the pathogenesis and progression of sepsis. This study aimed to explore the potential role of natural alkaloid Liensinine in the treatment of intestinal injury caused by sepsis and its possible molecular mechanism. In this study, a mouse model of sepsis was established by injecting LPS to explore the protective effect of Liensinine on intestinal injury in sepsis. The results showed that Liensinine could reduce the intestinal damage caused by LPS and increase the number of goblet cells. Furthermore, it decreased the release of inflammatory cytokines by inhibiting NF-kB phosphorylation and NLRP3 inflammasome synthesis. Liensinine also reduced the oxidative stress and ROS accumulation caused by LPS, and played an anti-oxidative stress role by regulating the Nrf2/keap1 signaling pathway. In addition, Liensinine alleviated the inhibition of intestinal autophagy caused by LPS by inhibiting the PI3K/Akt/mTOR pathway. And then it reduced the excessive apoptosis of intestinal cells. This study provides valuable insights for sepsis prevention and treatment, offering a potential therapeutic candidate to protect against intestinal injury and regulate the inflammatory response in sepsis.

Malvidin alleviates LPS-induced septic intestinal injury through the nuclear factor erythroid 2-related factor 2/reactive oxygen species/NLRP3 inflammasome pathway.

Emerging evidence suggests that the gastrointestinal tract plays a crucial role in the pathophysiology of sepsis, a leading cause of mortality among patients admitted to the intensive care unit (ICU). Malvidin, belonging to the flavonoid family of compounds, exhibits a range of capabilities including anti-inflammatory and antioxidant properties. Studies have demonstrated that Malvidin exhibits a dose-dependent effect in mitigating sepsis-induced intestinal injury. The advantageous impact of Malvidin in safeguarding against sepsis-induced intestinal injury is associated with its capacity to counteract oxidative stress, inhibit cellular apoptosis, diminish the secretion of pro-inflammatory cytokines, and regulate the synthesis of inflammasomes. The findings indicate that Malvidin, a natural compound, exhibits protective effects on the gut by activating the nuclear factor erythroid 2-related factor 2/reactive oxygen species/NLRP3 inflammasome pathway. These results have significant implications for potential clinical applications and offer valuable insights into the treatment of sepsis-induced intestinal injury.

Silybin mitigates chronic Avermectin exposure-induced intestinal damage and growth inhibition in carp.

Avermectins, as a new type of environmental pollutant, have received significant attention in recent years. Previous research has shown that acute exposure to avermectins can induce oxidative stress and inflammation in non-target fish species, such as carp. Flavonoid lignans, particularly Silybin, have demonstrated promising biological activities, including regulation of non-alcoholic fatty liver and cerebral ischemia-reperfusion injury. This study aims to investigate the impact of dietary supplementation with Silybin on the intestinal damage in carp caused by chronic exposure to avermectins and to improve the health status and production of carp in aquaculture. Silybin was used as a dietary supplement by adding it to the experimental feed, and an animal experimental model was utilized to assess its effects on oxidative stress, inflammation, and cell apoptosis in carp intestine. Additionally, intestinal barrier integrity, digestive capacity, and fish growth were evaluated. The results indicated that dietary supplementation with Silybin effectively alleviated the oxidative stress induced by chronic exposure to avermectins in carp intestine. Furthermore, Silybin improved intestinal barrier integrity and digestive capacity by modulating the Nrf2/Keap1 pathway. This study demonstrates that dietary supplementation with Silybin can effectively mitigate the intestinal damage caused by chronic exposure to avermectins in carp, providing a sustainable solution for the aquaculture industry to enhance the overall health and production of cultured fish. The research expands our understanding of avermectin environmental pollution and offers a potential remediation approach.

Ferulic acid alleviates carp brain damage and growth inhibition caused by avermectin by modulating the Nrf2/Keap1 and NF-κB signaling pathways.

The increasing concern over environmental pollution caused by the pesticide avermectin used in aquaculture has attracted significant attention. The use of avermectin, a neurotoxic pesticide, in aquatic environments leads to toxic effects on non-target organisms, particularly causing harm to fish. The phenolic compound ferulic acid possesses excellent anti-inflammatory and antioxidant capabilities. This study was conducted by establishing a chronic exposure experiment to avermectin, proposes the use of ferulic acid as a dietary additive to protect the carp brain from damage caused by exposure to avermectin. Furthermore, it investigates the anti-inflammatory and antioxidant effects of ferulic acid in the carp brain under chronic exposure to avermectin. The experimental results demonstrate that ferulic acid can alleviate brain tissue inflammation and oxidative stress by modulating the Nrf2/Keap1 and NF-κB signaling pathways. It protects the carp brain from chronic avermectin-induced damage, preserves the integrity of the carp blood-brain barrier, enhances the levels of feeding factors, and thereby alleviates carp growth inhibition. These findings provide new therapeutic strategies and a theoretical foundation for the sustainable development of carp aquaculture.

Liensinine, a alkaloid from lotus plumule, mitigates lipopolysaccharide-induced sepsis-associated encephalopathy through modulation of nuclear factor erythroid 2-related factor-mediated inflammatory biomarkers and mitochondria apoptosis.

The present study aims to investigate the role of liensinine in life-threatened sepsis-associated encephalopathy (SAE) mice and the underlying mechanism. Here, seventy-two mice were divided into six groups, including the control group, SAE group, liensinine-treated group, and three doses of liensinine-treated SAE groups. Lipopolysaccharide triggered cerebrum necrosis and disrupted the integrity and permeability of blood-brain barrier (BBB). While liensinine restored cerebrum structure and improved BBB integrity with upregulated tight junction proteins, decreased evans blue leakage and fibrinogen expression with decreased matrix metalloproteinases 2/9 in serum, thereby reducing BBB permeability. Moreover, lipopolysaccharide triggered cerebrum oxidative stress and inflammation, whereas liensinine enhanced antioxidant enzymes activities and weakened malondialdehyde through nuclear factor erythroid 2-related factor. Meanwhile, liensinine inhibited inflammation by activating inducible nitric oxide synthase. Tunel staining combined with transmission electron microscope indicated that lipopolysaccharide induced cerebrum apoptosis, whereas liensinine blocked apoptosis through decreasing B-cell lymphoma-2 associated X (Bax) expression and cytochrome C (Cyto-c) release, increasing B-cell lymphoma-2 (Bcl-2) expression, blocking apoptosome assembly, inhibiting caspase-3 activation, thereby suppressing intrinsic mitochondria apoptosis. Recovering of inflammatory homeostasis and inhibition of mitochondria apoptosis by liensinine ultimately restored cognitive function in SAE mice. Altogether, liensinine attenuated lipopolysaccharide-induced SAE via modulation of Nrf2-mediated inflammatory biomarkers and mitochondria apoptosis.

Engineering Corynebacterium glutamicum for de novo production of 2-phenylethanol from lignocellulosic biomass hydrolysate.

BACKGROUND: 2-Phenylethanol is a specific aromatic alcohol with a rose-like smell, which has been widely used in the cosmetic and food industries. At present, 2-phenylethanol is mainly produced by chemical synthesis. The preference of consumers for "natural" products and the demand for environmental-friendly processes have promoted biotechnological processes for 2-phenylethanol production. Yet, high 2-phenylethanol cytotoxicity remains an issue during the bioproduction process. RESULTS: Corynebacterium glutamicum with inherent tolerance to aromatic compounds was modified for the production of 2-phenylethanol from glucose and xylose. The sensitivity of C. glutamicum to 2-phenylethanol toxicity revealed that this host was more tolerant than Escherichia coli. Introduction of a heterologous Ehrlich pathway into the evolved phenylalanine-producing C. glutamicum CALE1 achieved 2-phenylethanol production, while combined expression of the aro10. Encoding 2-ketoisovalerate decarboxylase originating from Saccharomyces cerevisiae and the yahK encoding alcohol dehydrogenase originating from E. coli was shown to be the most efficient. Furthermore, overexpression of key genes (aroGfbr, pheAfbr, aroA, ppsA and tkt) involved in the phenylpyruvate pathway increased 2-phenylethanol titer to 3.23 g/L with a yield of 0.05 g/g glucose. After introducing a xylose assimilation pathway from Xanthomonas campestris and a xylose transporter from E. coli, 3.55 g/L 2-phenylethanol was produced by the engineered strain CGPE15 with a yield of 0.06 g/g xylose, which was 10% higher than that with glucose. This engineered strain CGPE15 also accumulated 3.28 g/L 2-phenylethanol from stalk hydrolysate. CONCLUSIONS: In this study, we established and validated an efficient C. glutamicum strain for the de novo production of 2-phenylethanol from corn stalk hydrolysate. This work supplied a promising route for commodity 2-phenylethanol bioproduction from nonfood lignocellulosic feedstock.

Recent progress in adaptive laboratory evolution of industrial microorganisms.

Adaptive laboratory evolution (ALE) is a technique for the selection of strains with better phenotypes by long-term culture under a specific selection pressure or growth environment. Because ALE does not require detailed knowledge of a variety of complex and interactive metabolic networks, and only needs to simulate natural environmental conditions in the laboratory to design a selection pressure, it has the advantages of broad adaptability, strong practicability, and more convenient transformation of strains. In addition, ALE provides a powerful method for studying the evolutionary forces that change the phenotype, performance, and stability of strains, resulting in more productive industrial strains with beneficial mutations. In recent years, ALE has been widely used in the activation of specific microbial metabolic pathways and phenotypic optimization, the efficient utilization of specific substrates, the optimization of tolerance to toxic substance, and the biosynthesis of target products, which is more conducive to the production of industrial strains with excellent phenotypic characteristics. In this paper, typical examples of ALE applications in the development of industrial strains and the research progress of this technology are reviewed, followed by a discussion of its development prospects.

De novo Synthesis of 2-phenylethanol from Glucose by Metabolically Engineered Escherichia coli.

2-Phenylethanol (2- PE) is an aromatic alcohol with wide applications, but there is still no efficient microbial cell factory for 2-PE based on Escherichia coli. In this study, we constructed a metabolically engineered E. coli capable of de novo synthesis of 2-PE from glucose. Firstly, the heterologous styrene-derived and Ehrlich pathways were individually constructed in an L-Phe producer. The results showed that the Ehrlich pathway was better suited to the host than the styrene-derived pathway, resulting in a higher 2-PE titer of ∼0.76 ± 0.02 g/L after 72 h of shake flask fermentation. Furthermore, the phenylacetic acid synthase encoded by feaB was deleted to decrease the consumption of 2-phenylacetaldehyde, and the 2-PE titer increased to 1.75 ± 0.08 g/L. As phosphoenolpyruvate (PEP) is an important precursor for L-Phe synthesis, both the crr and pykF genes were knocked out, leading to ∼35% increase of the 2-PE titer, which reached 2.36 ± 0.06 g/L. Finally, a plasmid-free engineered strain was constructed based on the Ehrlich pathway by integrating multiple ARO10 cassettes (encoding phenylpyruvate decarboxylases) and overexpressing the yjgB gene. The engineered strain produced 2.28 ± 0.20 g/L of 2-PE with a yield of 0.076 g/g glucose and productivity of 0.048 g/L/h. To our best knowledge, this is the highest titer and productivity ever reported for the de novo synthesis of 2-PE in E. coli. In a 5-L fermenter, the 2-PE titer reached 2.15 g/L after 32 h of fermentation, suggesting that the strain has the potential to efficiently produce higher 2-PE titers following further fermentation optimization.

Avermectin induces carp neurotoxicity by mediating blood-brain barrier dysfunction, oxidative stress, inflammation, and apoptosis through PI3K/Akt and NF-κB pathways.

Avermectin, a "low toxicity insecticide", has been widely used in recent years, but its non-target toxicity, especially to aquatic organisms, has been neglected. In this study, we evaluated the neurotoxic effects of avermectin on carp by establishing a 96 h avermectin acute toxicity test, and its possible mechanism was discussed. The 96 h LC50 of avermectin in carp was found to be 24.04 µg/L. Therefore, 3.005 µg/L and 12.02 µg/L were used as the low-dose and high-dose groups, respectively, to investigate the neurotoxic effects of avermectin on carp. The results of high-performance liquid chromatography (HPLC) analysis showed that avermectin accumulated in the carp brain. Histopathological observation and immunohistochemical analysis (IHC) of TNF-α and Bax showed that avermectin exposure led to inflammatory cell infiltration and neuronal necrosis. The mRNA levels of tight junction genes and the IHC results of ZO-1 and Occludin showed that the structure of the blood-brain barrier (BBB) was destroyed. Biochemical analysis showed that avermectin induced the accumulation of MDA in the brain and decreased the activity of antioxidant enzymes CAT and SOD, leading to oxidative stress. In addition, avermectin induces brain inflammation by activating NF-κB pathway and releasing inflammatory factors IL-1ß, IL-6, TNF-α and iNOS. TEM and TUNEL assays showed that exposure to avermectin induced apoptosis in brain. what is more, the expression of apoptosis-related genes and proteins suggested that avermectin-induced apoptosis may be associated with inhibition of the PI3K/Akt signaling pathway. This study also showed that avermectin-induced NF-κB signaling activation was partially dependent on its upstream PI3K/Akt signaling pathway. Therefore, this study concludes that avermectin can induce neurotoxicity in carp by disrupting the blood-brain barrier structure and generating oxidative stress, inflammation, and apoptosis and that NF-κB and PI3K/Akt signaling pathways are involved in this process.

Improvement of the catalytic performance of glycerol kinase from Bacillus subtilis by chromosomal site-directed mutagenesis.

Glycerol kinase is the key enzyme in glycerol metabolism, and its catalytic efficiency has an important effect on glycerol utilization. Based on an analysis of the glycerol utilization pathway and regulation mechanism in B. subtilis, we conducted site-directed mutagenesis of the key glycerol kinase gene (glpK) on the chromosome to improve the glycerol utilization efficiency of Bacillus subtilis. Recombinant wild-type Bacillus subtilis glycerol kinase (BsuGlpKWT) and two mutants (BsuGlpKM270I and BsuGlpKS71V) were successfully overexpressed in Escherichia coli BL21(DE3) and purified by Ni-IDA metal chelate chromatography. The specific activity of the BsuGlpKM270I mutant (62.6 U/mg) was significantly higher (296.2%) than that of wild-type BsuGlpKWT (15.8 U/mg). By contrast, the mutant BsuGlpKS71V (4.89 U/mg) exhibited lower (69.1%) activity than BsuGlpKWT, which suggested that variant S71V exhibited reduced catalytic efficiency for the substrate. Furthermore, the mutant strain B. subtilis M270I was constructed using a markerless delivery system, and exhibited a higher specific growth rate (improved by 11.3%, from 0.453 ± 0.012 to 0.511 ± 0.017 h-1) and higher maximal biomass (cell dry weight increased by 16%, from 0.577 ± 0.033 to 0.721 ± 0.015 g/L) than the parental strain with a shortened lag phase (2 ~ 4 h shorter) in M9 minimal medium with glycerol. These results indicate that the mutated glpK resulted in improved glycerol utilization, which has broad application prospects.

Graph Clustering With Graph Capsule Network.

Graph clustering, which aims to partition a set of graphs into groups with similar structures, is a fundamental task in data analysis. With the great advances made by deep learning, deep graph clustering methods have achieved success. However, these methods have two limitations: (1) they learn graph embeddings by a neural language model that fails to effectively express graph properties, and (2) they treat embedding learning and clustering as two isolated processes, so the learned embeddings are unsuitable for the subsequent clustering. To overcome these limitations, we propose a novel capsule-based graph clustering (CGC) algorithm to cluster graphs. First, we construct a graph clustering capsule network (GCCN) that introduces capsules to capture graph properties. Second, we design an iterative optimization strategy to alternately update the GCCN parameters and clustering assignment parameters. This strategy leads GCCN to learn cluster-oriented graph embeddings. Experimental results show that our algorithm achieves performance superior to that of existing graph clustering algorithms in terms of three standard evaluation metrics: ACC, NMI, and ARI. Moreover, we use visualization results to analyze the effectiveness of the capsules and demonstrate that GCCN can learn cluster-oriented embeddings.

Malvidin alleviates mitochondrial dysfunction and ROS accumulation through activating AMPK-α/UCP2 axis, thereby resisting inflammation and apoptosis in SAE mice.

This study aimed to explore the protective roles of malvidin in life-threatened sepsis-associated encephalopathy (SAE) and illustrate the underlying mechanism. SAE mice models were developed and treated with malvidin for subsequently protective effects evaluation. Malvidin restored neurobehavioral retardation, declined serum S100ß and NSE levels, sustained cerebrum morphological structure, improved blood-brain barrier integrity with elevated tight junction proteins, and decreased evans blue leakage, and finally protect SAE mice from brain injury. Mechanistically, malvidin prevented cerebrum from mitochondrial dysfunction with enhanced JC-1 aggregates and ATP levels, and ROS accumulation with decreased lipid peroxidation and increased antioxidant enzymes. UCP2 protein levels were found to be decreased after LPS stimulation in the cerebrum and BV-2 cells, and malvidin recovered its levels in a ROS dependent manner. In vivo inhibition of UCP2 with genipin or in vitro interference with siRNA UCP2 both disrupted the mitochondrial membrane potential, decreased ATP levels and intensified DCF signals, being a key target for malvidin. Moreover, dorsomorphin block assays verified that malvidin upregulated UCP2 expression through phosphorylating AMPK in SAE models. Also, malvidin alleviated SAE progression through inhibition of ROS-dependent NLRP3 inflammasome activation mediated serum pro-inflammatory cytokines secretion and mitochondrial pathway mediated apoptosis with weakened apoptosis body formation and tunel positive signals, and decreased Bax, cytochrome C, caspase-3 and increased Bcl-2 protein levels. Overall, this study illustrated that malvidin targeted AMPK-α/UCP2 axis to restore LPS-induced mitochondrial dysfunction and alleviate ROS accumulation, which further inhibits NLRP3 inflammasome activation and mitochondrial apoptosis in a ROS dependent way, and ultimately protected SAE mice, providing a reference for the targeted development of SAE prophylactic approach.

Aeromonas sobria regulates proinflammatory immune response in mouse macrophages via activating the MAPK, AKT, and NF-κB pathways.

Aeromonas sobria, a Gram-negative bacterium that can colonize both humans and animals, is found in a variety of environments, including water, seafood, meat, and vegetables (Cahill, 1990; Galindo et al., 2004; Song et al., 2019). Aeromonas spp. are conditionally pathogenic bacteria in aquaculture, which can rapidly proliferate, causing disease and even death in fish, especially when the environment is degraded (Neamat-Allah et al., 2020, 2021a, 2021b). In developing countries, Aeromonas spp. have been associated with a wide spectrum of infections in humans, including gastroenteritis, wound infections, septicemia, and lung infections (San Joaquin and Pickett, 1988; Wang et al., 2009; Su et al., 2013). Infections caused by Aeromonas spp. are usually more severe in immunocompromised individuals (Miyamoto et al., 2017). The presence of a plasmid encoding a ß|-lactamase in A. sobria that confers resistance to ß-lactam antibiotics poses a huge challenge to the treatment of diseases caused by this microorganism (Lim and Hong, 2020). Consequently, an in-depth understanding of the interaction between A. sobria and its hosts is urgently required to enable the development of effective strategies for the treatment of A. sobria infections.

Aeromonas sobria Induces Proinflammatory Cytokines Production in Mouse Macrophages via Activating NLRP3 Inflammasome Signaling Pathways.

Aeromonas sobria, a common conditional pathogenic bacteria, is widely distributed in the environment and causes gastroenteritis in humans or septicemia in fish. Of all Aeromonas species, A. sobria is the most frequently isolated from human infections especially in immunocompromised subjects. Innate immunity is the first protection system of organism to resist non-specific pathogens invasion; however, the immune response process of hosts against A. sobria infection re\mains unexplored. The present study established an A. sobria infection model using primary mouse peritoneal macrophages (PMφs). The adherence and cytotoxicity of A. sobria on PMφs were determined by May-Grünwald Giemsa staining and LDH release measurement. Pro-inflammatory cytokine expression levels were measured using qPCR, western blotting, and ELISA methods. We also investigated the levels of ASC oligomerization and determined the roles of active caspase-1 in IL-1ß secretion through inhibition assays and explored the activated pattern recognition receptors through immunofluorescence. We further elucidated the roles of activated inflammasome in regulating the host's inflammatory response through inhibition combined with ELISA assays. Our results showed that A. sobria induced lytic cell death and LDH release, whereas it had no adhesive properties on PMφs. A. sobria triggered various proinflammatory cytokine transcription level upregulation, and IL-1ß occupied the highest levels. The pro-IL-1ß protein expression levels increased in a dose-dependent manner with MOI ranging from 1 to 100. This process was regulated by ASC-dependent inflammasome, which cleavage pro-IL-1ß into active IL-1ß p17 with activated caspase-1 p20. Meanwhile, the expression levels of NLRP3 receptor significantly increased, location analysis revealed puncta-like surrounding nuclear, and inhibition of NLRP3 inflammasome downregulated caspase-1 activation and IL-1ß secretion. Blocking of NLRP3 inflammasome activation through K+ efflux and cathepsin B or caspase approaches downregulated A. sobria-induced proinflammatory cytokine production. Overall, these data indicated that A. sobria induced proinflammatory cytokine production in PMφs through activating NLRP3 inflammasome signaling pathways.

Separation and purification of pyrroloquinoline quinone from Gluconobacter oxydans fermentation broth using supramolecular solvent complex extraction.

In this paper, new supramolecular extractants, which contained surfactant, alkane and alkanol, were designed and used to separate PQQ. After a series of tests, the optimal extractant composition was determined as benzalkalonium (C8-C16) chloride (BC): n-hexane:n-pentanol, and the highest extraction rate could reach 98%. The extraction equilibrium could be reached in five minutes. The mechanism of the extraction selectivity was inferred as an ion-pair and π-π complexation interaction between PQQ and BC, which was indicated by UV and fluorescence quenching experiments. To recycle the organic extractant, the extract was back-extracted with sodium chloride solution. After extraction, back extraction and crystallization, an isolated product with a purity of 97.5% was obtained from G. oxydans fermentation broth. The product was identified as PQQ by HPLC analysis and MS. Above all, the present research developed a simple and efficient method for the separation of PQQ from fermentation broth.

Bioconversion of recombinantly produced precursor peptide pqqA into pyrroloquinoline quinone (PQQ) using a cell-free in vitro system.

Pyrroloquinoline quinone (PQQ) has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P)+) and flavins (FAD, FMN). It plays important physiological roles in various organisms and has strong antioxidant properties. The biosynthetic pathway of PQQ involves a gene cluster composed of 4-7 genes, named pqqA-G, among which pqqA is a key gene for PQQ synthesis, encoding the precursor peptide PqqA. To produce recombinant PqqA in E. coli, fusion tags were used to increase the stability and solubility of the peptide, as well simplify the scale-up of the fermentation process. In this paper, pqqA from Gluconobacter oxydans 621H was expressed in E. coli BL21 (DE3) as a fusion protein with SUMO and purified using a hexahistidine (His6) tag. The SUMO fusion protein and His6 tag were specifically recognized and cleaved by the SUMO specific ULP protease, and immobilized-metal affinity chromatography was used to obtain high-purity precursor peptide PqqA. Expression and purification of target proteins was confirmed by Tricine-SDS-PAGE. Finally, the synthesis of PQQ in a cell-free enzymatic reaction in vitro was confirmed by LC-MS.

Improved Carotenoid Productivity and COD Removal Efficiency by Co-culture of Rhodotorula glutinis and Chlorella vulgaris Using Starch Wastewaters as Raw Material.

Utilization of low-cost raw materials for the bio-based chemical production, such as carotenoids, by the co-culture of Rhodotorula glutinis and Chlorella vulgaris has recently become an attractive option. In this study, the primary nutrients of starch wastewater were analyzed, which were used for carotenoid production by the co-culture strategy in a 5-L fermenter around 4000 Lux light intensity. Synergistic effect of gas utilization revealed that the two species could build up the beneficial balance on mutualism. The maximum carotenoid productivity and COD removal efficiency were 12.34 mg/L and 79.6%, respectively, which were higher than those of monoculture yeast (8.31 mg/L and 54.1%). The organic acids, amino acids, and sugar removal efficiencies were increased by 85%, 31%, and 44%, respectively, and more than three kinds of carotenoids were identified compared with those of monoculture yeast. The results demonstrated that the co-culture strategy of two different nutritional microorganisms could significantly improve carotenoid productivity and COD removal efficiency.

Cellular Response of Escherichia coli to Photocatalysis: Flagellar Assembly Variation and Beyond.

Bacterial cells can be inactivated by external reactive oxygen species (ROS) produced by semiconductor photocatalysis. However, little is known about cellular responses to photocatalysis. For a better understanding of this issue, one strain of Escherichia coli ( E. coli, hereafter named as MT), which has an increased ability to metabolize carbon sources, was screened out from the wild-type (WT) E. coli K12 by repeated exposure to photocatalysis with palladium oxide modified nitrogen-doped titanium dioxide. In this study, transcriptome sequencing of the WT and MT strains that were exposed or unexposed to photocatalysis were carried out. Cellular responses to photocatalysis were inferred from the functions of genes whose transcripts were either increased or decreased. Upregulation of expression of bacterial flagellar assembly genes used for chemotaxis was detected in cells exposed to semilethal photocatalytic conditions of the WT E. coli. Increased capability to degrade superoxide radicals and decreased bacterial flagellar assembly and chemotaxis were observed in MT E. coli compared to WT cells. We conclude that the differences in motility and intracellular ROS between MT and WT are directly related to survivability of E. coli during exposure to photodisinfection.