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Oxidative stress causes chronic inflammation, and mediates various diseases.

Abnormal vasoconstriction, inflammation, and vascular remodeling can be promoted by angiotensin II (Ang II) in the renin-angiotensin system (RAS), leading to vascular dysfunction diseases such as hypertension and atherosclerosis. Researchers have recently focused on angiotensin I-converting enzyme inhibitory peptides (ACEIPs), that have desirable efficacy in vascular dysfunction therapy due to Ang II reduction by inhibiting ACE activity. Promising methods for the large-scale preparation of ACEIPs include selective enzymatic hydrolysis and microbial fermentation. Thus far, ACEIPs have been widely reported to be hydrolyzed from protein-rich sources, including animals, plants, and marine organisms, while many emerging microorganism-derived ACEIPs are theoretically biosynthesized through the nonribosomal peptide synthase (NRPS) pathway. Notably, vasodilatation, anti-inflammation, and vascular reconstruction reversal of ACEIPs are strongly correlated. However, the related molecular mechanisms underlying signal transduction regulation in vivo remain unclear. We provide a comprehensive update of the ACE-Ang II-G protein-coupled type 1 angiotensin receptor (AT1R) axis signaling and its functional significance for potential translation into therapeutic strategies, particularly targeting AT1R by ACEIPs, as well as specific related signaling pathways. Future studies are expected to verify the biosynthetic regulatory mechanism of ACEIPs via the NRPS pathway, the effect of gut microbiota metabolism on vascular dysfunction and rigorous studies of ACE-Ang II-AT1R signaling pathways mediated by ACEIPs in large animals and humans.

This protocol describes a method for verifying the specific transcription factor regulating glycerol dehydratase (GDH) expression in Klebsiella. DNA pull-down accompanied with mass spectrometry is used to screen and identify the transcription factor interacting with the promoter region of the key gene in Klebsiella. EMSA method is used to validate the specific binding of the transcription factor to the promoter region in vitro. In addition, the target DNA fragments are constructed by fusion PCR to prepare competent cells from Klebsiella for electrical transformation and further transformed to obtain key gene deletion strains to verify the transcription factor responsible for the target gene expression in Klebsiella.

Ganoderic acids (GAs) are well recognized as important pharmacological components of the medicinal species belonging to the basidiomycete genus Ganoderma. However, transcription factors directly regulating the expression of GA biosynthesis genes remain poorly understood. Here, the genome of Ganoderma lingzhi was de novo sequenced. Using DAP-seq, we identified putative targets of the transcription factor SREBP, including the genes of triterpenoid synthesis and lipid metabolism. Interactions between SREBP and the targets were verified by EMSA. RNA-seq showed that SREBP targets, mevalonate kinase and 3-hydroxy-3-methylglutaryl CoA synthetase in mevalonate pathway, sterol isomerase and lanosterol 14-demethylase in ergosterol biosynthesis, were significantly upregulated in the SREBP overexpression strain. In addition, 3 targets involved in glycerophospholipid/glycerolipid metabolism were upregulated. Then, the contents of mevalonic acid, lanosterol, ergosterol and 13 different GAs as well as a variety of lipids were significantly increased in this strain. Furthermore, the effects of SREBP overexpression on triterpenoid and lipid metabolisms were recovered when OE::SREBP strain were treated with exogenous fatostatin, a specific inhibitor of SREBP. Taken together, our genome-wide study clarified the role of SREBP in triterpenoid and lipid metabolisms of G. lingzhi.

Ganoderic acids (GAs) are well recognized as important pharmacological components of the medicinal species belonging to the basidiomycete genus Ganoderma. However, transcription factors directly regulating the expression of GA biosynthesis genes remain poorly understood. Here, the genome of Ganoderma lingzhi is de novo sequenced. Using DNA affinity purification sequencing, we identify putative targets of the transcription factor sterol regulatory element-binding protein (SREBP), including the genes of triterpenoid synthesis and lipid metabolism. Interactions between SREBP and the targets are verified by electrophoretic mobility gel shift assay. RNA-seq shows that SREBP targets, mevalonate kinase and 3-hydroxy-3-methylglutaryl coenzyme A synthetase in mevalonate pathway, sterol isomerase and lanosterol 14-demethylase in ergosterol biosynthesis, are significantly upregulated in the SREBP overexpression (OE::SREBP) strain. In addition, 3 targets involved in glycerophospholipid/glycerolipid metabolism are upregulated. Then, the contents of mevalonic acid, lanosterol, ergosterol and 13 different GAs as well as a variety of lipids are significantly increased in this strain. Furthermore, the effects of SREBP overexpression on triterpenoid and lipid metabolisms are recovered when OE::SREBP strain are treated with exogenous fatostatin, a specific inhibitor of SREBP. Taken together, our genome-wide study clarify the role of SREBP in triterpenoid and lipid metabolisms of G. lingzhi.

Native broken-rice starch was used to create starch nanoparticles (StNPs) with particle sizes ranging from 100 nm to 800 nm. The fluorescent isothiocyanate poly-l-lysine StNPs (FITC-PLL-StNPs) were created in two steps. First, the StNPs were electrostatically modified by poly-l-lysine (PLL) molecules rich in amino acids. Second, fluorescein isothiocyanate reacted with some amino groups on PLL molecules (FITC). Fluorescence spectrophotometry was used to determine the degree of substitution (DS) and fluorescent properties of fluorescent starches. The study found that FITC-PLL-StNP-200 has higher fluorescence stability, more phagocytic cells, and a better and clearer fluorescence detecting effect than FITC-PLL-St, FITC-PLL-StNP-100, FITC-PLL-StNP-400, and FITC-PLL-StNP-800. The biological evaluation results showed that FITC-PLL-StNP-200 did not affect the viability of HeLa cells at the lysosome labeling concentration. These findings suggest that FITC-PLL-StNP-200 has strong and stable fluorescence, indicating that FITC-PLL-StNP-200 can be used as a fluorescent probe and lysosome marker in a variety of applications, particularly in biomedicine.

3-Hydroxypropionic acid (3-HP) is an emerging platform chemical, which can be used for the production of various value-added chemicals. The traditional chemical synthesis of 3-HP has some difficulties in meeting the new sustainable development goals. Accordingly, microbial cell factories offer a green and sustainable method for the biosynthesis of 3-HP. Herein, a novel oxaloacetate pathway was successfully established for 3-HP biosynthesis. Firstly, a 3-HP production module was designed and constructed in Saccharomyces cerevisiae by overexpressing pyruvate carboxylase, benzoylformate decarboxylase, and 3-hydroxyisobutyrate dehydrogenase. Secondly, promoter engineering was used to optimize the glc7 expression, which could regulate the dephosphorylation of hexokinase to enhance the cytoplasmic energy metabolism, resulting in a 74.4% increase in 3-HP titer. Furthermore, a quorum-sensing system was applied to regulate the ptc7 expression, which could control the dephosphorylation of citrate synthase and mitochondrial hydroxylase to enhance the mitochondrial energy metabolism, resulting in a 98.0% increase in 3-HP titer. Finally, to further improve the 3-HP production, dynamic control of dephosphorylation was achieved to optimize the cytoplasmic and mitochondrial energy metabolism, resulting in a 4.2-fold increase in 3-HP titer in a 5 L bioreactor. The final engineered strain, thTAM-47, produced 18.1 g L−1 3-HP, which is the highest level of 3-HP production in S. cerevisiae to date. This work showed the shortest pathway for 3-HP production using glucose as a substrate in S. cerevisiae at present. Also, this is the first report on improving the pathway efficiency of 3-HP biosynthesis using a dephosphorylation regulation strategy to increase the intracellular ATP level.

Broken-rice starch nanoparticles with different mean particle diameters for 100, 200, 400 and 800 nm were prepared by nanoprecipitation, alkali freezing, cross-linking and H2SO4 hydrolysis methods respectively, and their structural, morphological and physicochemical properties were systematically characterized. The results showed that broken-rice starch nanoparticles had higher water absorption rate, and the maximum water absorption rate was obtained from the 100 nm starch granules being 91.53%, which means an increase about 2.07-fold in water absorption rate as compared with native rice starch. The stability of native rice starch is the worst, but the viscosity characteristic value is always higher than that of starch nanoparticles in the whole gelatinization process. The FT-IR spectrum showed that only starch nanoparticles prepared by cross-linking method showed the characteristic peak of secondary amide structure at 1714 cm−1, but the structure of was basically the same as native starch. The X-ray diffraction pattern revealed that there were obvious characteristic diffraction peaks near 2θ for 15°, 17°, 19° and 23° for the 800 nm starch nanoparticles and native rice starch, while the characteristic diffraction peaks of other starch nanoparticles disappeared in varying degrees due to the changed crystal structure.

Thermostable β-xylosidase produced by Aspergillus fumigatus (A. fumigatus) XC6 can be efficiently extracted and purified via an aqueous two-phase system consisting of 20% (m/v) PEG 2000 and 25% (m/v) NaH2PO4 with a purification factor of approximately 16. Maximum activity of the purified β-xylosidase was observed at 75 °C and pH 4.5 using p-nitrophenyl-β-Dxylopyranoside (pNPXyl) as a substrate. The thermal stability of purified β-xylosidase is higher than that of compared fungal strains. Thermotolerant kluyveromyces marxianus was directly added to a fermentation system composed of 50 g L−1 rice straw and 10 g L−1 (NH4)2SO4, but prehydrolysis with A. fumigatus XC6 pellets for 12 h yielded 11.2 g L−1 ethanol within 36 h, an amount 80% of the theoretical value. The results indicated that A. fumigatus XC6 β-xylosidase is a thermostable enzyme with high xylose production activity, and may have promising industrial application for cost-effective lignocellulosic bioethanol production.

Based on a β-cyclodextrin (β-CD) subject-object competition model, a simple and sensitive electrochemical aptamer sensor for the determination of tetracycline (TET) was fabricated. First, the TET aptamer modified with ferrocene (Fc) as a signal molecule was captured by β-CD loaded onto the gold electrode. Subsequently, TET was added to the detection system, causing a binding event between the target and aptamer with strong affinity during which process the aptamer configuration was changed from the original upright linear state to an agglomeration structure, resulting in its departure from the electrode surface. Consequently, an “off-signal” was turned along with the presence of the target. The results indicated that the TET concentration had a linear response to the signal ranging from 0.01 to 100 nM and the accurate detection limit could reach as low as 0.008 nM (3δ). The fabricated TET biosensor also showed outstanding detection specificity. Moreover, the suitability of the developed method was demonstrated in the determination of TET concentrations in different samples comprising water, milk, and bacteria culture medium, achieving acceptable recoveries for spiked samples ranging from 96.0 to 104.4%. This detection system was simple, economical, time-saving but remarkably sensitive, selective, and efficient, potentially rendering services in food safety screening and medical testing.

Enzyme production as well as rice straw saccharification and fermentation were integrated to produce bioethanol in this study. Submerged fermentation of Na2CO3 pretreated rice straw to produce a complex enzyme for saccharification by Aspergillus fumigatus was performed. The major component of rice straw, that is cellulose, hemicellulose and lignin was almost completely degraded in 24 h. Using whole pretreated rice straw slurry as the substrate, the maximum concentrations of reducing sugar and ethanol were obtained with values of 63.6 g/L and 30.9 g/L, respectively, in a total hydrolysis and fermentation time at a substrate concentration of 8% (based on the original amount of rice straw). Fed-batch fermentation was employed for enzymolysis and fermentation of rice straw residue. Under a 32% total substrate concentration, 108.6 g/L ethanol was obtained in a total enzymolysis and fermentation time of 40 h. The results from different fermentation methods showed that the laccase produced by A. fumigatus in situ could effectively promote the enzymatic hydrolysis and fermentation through detoxifying the phenols produced during pretreatment and by the enzymatic hydrolysis processes.

Tung meal is the main byproduct generated during tung oil extraction from tung tree (Vernicia fordii) seeds and contains approximately 36–45 wt% crude protein. The disposal of large amounts of this resource has attracted increasing attention in relation to its value-added utilization. In this study, a new strain Pseudomonas aeruginosa LYT-4, was isolated for the bioconversion of tung meal into free amino acid fertilizer. Solid-state fermentation analysis showed that the protease activity of P. aeruginosa LYT-4 treatment was significantly higher than that under natural treatment throughout the fermentation period. The total percentage of free amino acids increased from 46.5% under natural fermentation to 55.5% under P. aeruginosa LYT-4 treatment. Pot experiments revealed that P. aeruginosa LYT-4 fermented-tung meal efficiently promoted spinach growth by increasing biomass and was more effective than both naturally fermented tung meal and chemical fertilizer. Furthermore, P. aeruginosa LYT-4 exhibited efficient in vitro antagonistic activity against three plant pathogens, Colletotrichum lini, Rhizoctonia solani and Fusarium oxysporum, possibly due to the abundant secondary metabolite clusters in its genome based on genomic analysis. In conclusion, P. aeruginosa LYT-4 exhibited a promising prospect for the industrial production of free amino acid fertilizer from tung meal with simultaneous potential biocontrol capacity.

Two new meroterpenoid compounds (1 and 2) together with five known meroterpenoid derivatives (3-7) were isolated from solid culture of mushroom Panus lecomtei. The structures of new compounds were confirmed by the analysis of NMR and HR-ESI-MS spectroscopic data. The biosynthetic pathway of 1-7 was postulated. All isolated compounds were evaluated for antibacterial activities against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa and Bacillus Calmette-Guerin. Compound 3 exhibited weak antibacterial activity against Bacillus Calmette-Guerin with the inhibition rate of 83.6% at 100 μmol·L-1. Other compounds showed no antibacterial activities against all tested pathogens at 100 μmol·L-1.

Background Lignin is a complex aromatic heteropolymer comprising 15–30% dry weight of the lignocellulose. The complex structural characteristic of lignin renders it difficult for value-added utilization. Exploring efficient lignin-degrading microorganisms and investigating their lignin-degradation mechanisms would be beneficial for promoting lignin valorization. In this study, a newly isolated white-rot basidiomycete, Trametes hirsuta X-13, with capacity to utilize alkaline lignin as the sole substrate was investigated. Results The analysis of the fermentation properties of T. hirsuta X-13 using alkaline lignin as the sole substrate, including the mycelial growth, activities of ligninolytic enzymes and the rates of lignin degradation and decolorization confirmed its great ligninolysis capacity. The maximum lignin degradation rate reached 39.8% after 11 days of T. hirsuta X-13 treatment, which was higher than that of reported fungi under the same condition. Fourier transform infrared spectrometry (FTIR), gas chromatography–mass spectrometry (GC–MS) scanning electron micrographs (SEM), two-dimensional heteronuclear single quantum coherence NMR analysis (2D-HSQC NMR) collaborated with pyrolysis gas chromatography–mass spectrometry (py-GC/MS) analyses proved that lignin structure was severely deconstructed along with amounts of monomer aromatics generated. Furthermore, according to those chemical analysis, in addition to canonical Cα–Cβ breakage, the cleavage of lignin interunit linkages of β–β might also occur by T. hirsuta X-13. Conclusions This study characterized a newly isolated white-rot basidiomycete T. hirsuta X-13 with impressive alkaline lignin degradation ability and provided mechanistic insight into its ligninolysis mechanism, which will be valuable for the development of lignin valorization strategies.

Background Pretreatment is a critical step required for efficient conversion of woody biomass into biofuels and platform chemicals. Fungal pretreatment is regarded as one of the most promising technology for woody biomass conversion but remains challenging for industrial application. The exploration of potential fungus strain with high efficient delignification and less processing time for woody biomass pretreatment will be valuable for development of biorefinery industry. Here, a newly isolated white-rot basidiomycete Peniophora incarnate T-7 was employed for poplar wood pretreatment. Results The chemical component analysis showed that cellulose, hemicellulose and lignin from poplar wood declined by 16%, 48% and 70%, respectively, after 7 days submerged fermentation by P. incarnate T-7. Enzymatic saccharification analysis revealed that the maximum yields of glucose and xylose from 7 days of P. incarnate T-7 treated poplar wood reached 33.4% and 27.6%, respectively, both of which were enhanced by sevenfold relative to the untreated group. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), X-ray diffraction (XRD) and pyrolysis gas chromatography–mass spectrometry (Py-GC/MS) characterization confirmed that lignocellulosic structure of poplar wood was largely broken by P. incarnate T-7, including delignification and de-crystalline of cellulose. Meanwhile, lignin component of poplar wood was selectively degraded by P. incarnate T-7, and G-type unit of lignin was preferentially attacked by the strain. Furthermore, quantitative proteomic analysis revealed that a considerable amount of lignocellulolytic enzymes were detected in the secretory proteins of P. incarnate T-7, especially with high abundance of lignin-degrading enzymes and hemicellulases. Combination of quantitative proteomic with transcriptomic analysis results showed that most of those lignocellulolytic enzymes were highly upregulated on poplar wood substrate compared to glucose substrate. Conclusions This study showed that P. incarnate T-7 could selectively delignify poplar wood by submerged fermentation with short time of 7 days, which greatly improved its enzymatic saccharification efficiency. Our results suggested that P. incarnate T-7 might be a promising candidate for industrial woody biomass pretreatment.

Ganoderma lucidum (G. lucidum) has been widely used in Asia to treat hypertension, but the active substances responsible for its antihypertensive effects remain unclear. Using the well-established angiotensin I-converting enzyme (ACE) as a target, we identified three ACE inhibitory peptides (ACEIPs), Gln-Leu-Val-Pro (QLVP), Gln-Asp-Val-Leu (QDVL), and Gln-Leu-Asp-Leu (QLDL), which account for the antihypertensive activity of G. lucidum. Notably, QLVP worked in a mixed-type manner against ACE with an IC50 value of 127.9 μmol/L. Molecular dynamics simulation suggested that the potent charge energy of QLVP, which interacted with Gln242 and Lys472 of ACE via a hydrogen bond and a salt bridge, potentially contributed to ACE inhibitory activity. Moreover, QLVP markedly activated angiotensin I-mediated phosphorylation of endothelial nitric oxide synthase in human umbilical vein endothelial cells and partly reduced mRNA and protein expression of the vasoconstrictor factor endothelin-1. This is the first report of the antihypertensive activity of small ACEIPs originating from G. lucidum mycelia, paving the way for the possible application of these peptides as potent drug candidates for treating hypertension.

Recent progress has been made in adding exogenous vegetable oils in culture media to promote bioactive metabolite production in several medicinal mushrooms, but the mechanism is still unclear. In this study, we found that the vegetable oil coix seed oil (CSO) could induce the biosynthesis of triterpene acids (TAs) and also significantly increase cytoplasmic nitric oxide (NO) and hydrogen peroxide (H2O2) concentrations in the mycelium of Ganoderma lingzhi. The change in TA biosynthesis caused by CSO could be reversed by adding NO scavenger or H2O2 scavenger, and adding NO scavenger or H2O2 scavenger resulted in the reduction of the cytoplasmic H2O2 or NO concentration under CSO treatment, respectively. Moreover, adding NO scavenger or H2O2 scavenger reversed TA biosynthesis, which could be rescued by H2O2 or NO donor, respectively. Taken together, our study indicated that both NO and H2O2 were involved in the regulation of TA biosynthesis, and CSO-activated NO and H2O2 were interdependent but independently regulated the TA biosynthesis under CSO treatment in G. lingzhi.

Smartly coupling exonuclease-induced target recycling signal amplifications with β-cyclodextrin host-guest recognition, a novel “signal-on” aptamer sensor for sensitive determination of ochratoxin A (OTA) was proposed for the first time. Firstly, the formation of double-strand DNA (dsDNA) was occurred by hybridizing OTA aptamer with its complementary DNA (cDNA) and as the probe DNA the cDNA at its 3′ terminal was labeled with methylene blue (MB). Next, when OTA was present, the aptamer tended to form aptamer-OTA complex with conformation of G-quadruplex instead of aptamer-cDNA duplex, leading to thus the probe DNA separating from dsDNA complex. Then the RecJf exonuclease was added, demolishing partially G-quadruplex structure and releasing a certain number of OTA. Sequentially, those released OTA would continue to react with the rest of aptamer in dsDNA, drawn into development of a new round of G-quadruplex complex, where the target cycling was realized. Meanwhile, as a signal molecule, MB modified on cDNA was liberated along with the cDNA being digested into monoucleotides by RecJf exonuclease, capable of diffusing onto the electrode surface due to host-guest recognition with β-cyclodextrin, whereupon the signal was enriched and yielded. In this way, cycles of target with continuous output of signal indicators were undergone, in which the detection of target was in return fulfilled with signal amplification owing to the joint endeavor of exonuclease and β-cyclodextrin. Under the optimal conditions, the raising signal maintained a linear relation with the logarithm of the target concentrations ranging from 10 pg/mL to 10.0 ng/mL and the detection limit reached as low as 3 pg/mL. This brand-new strategy was simple and low-cost but satisfactory in terms of detection limit, range and sensitivity, in all possibility to be applied extensively for diverse targets detection by easily alternating the corresponding aptamers.

Metabolic energy (ME) homeostasis is essential for the survival and proper functioning of microbial cell factories. However, it is often disrupted during bioproduction because of inefficient ME supply and excessive ME consumption. In this review, we propose strategies, including reinforcement of the capacity of ME-harvesting systems in autotrophic microorganisms; enhancement of the efficiency of ME-supplying pathways in heterotrophic microorganisms; and reduction of unessential ME consumption by microbial cells, to address these issues. This review highlights the potential of biotechnology in the engineering of microbial ME homeostasis and provides guidance for the higher efficient bioproduction of microbial cell factories.

The development of fungal fruiting bodies from a hyphal thallus is inducible under low temperature (cold stress). The molecular mechanism has been subject to surprisingly few studies. Analysis of gene expression level has become an important means to study gene function and its regulation mechanism. But identification of reference genes (RGs) stability under cold stress have not been reported in famous medicinal mushroom-forming fungi Cordyceps militaris. Herein, 12 candidate RGs had been systematically validated under cold stress in C. militaris. Three different algorithms, geNorm, NormFinder and BestKeeper were applied to evaluate the expression stability of the RGs. Our results showed that UBC and UBQ were the most stable RGs for cold treatments in short and long periods, respectively. 2 RGs (UBC and PP2A) and 3 RGs (UBQ, TUB and CYP) were the suitable RGs for cold treatments in short and long periods, respectively. Moreover, target genes, two-component-system histidine kinase genes, were selected to validate the most and least stable RGs under cold treatment, which indicated that use of unstable expressed genes as RGs leads to biased results. Our results provide a good starting point for accurate reverse transcriptase quantitative polymerase chain reaction normalization by using UBC and UBQ in C. militaris under cold stress and better support for understanding the mechanism of response to cold stress and fruiting body formation in C. militaris and other mushroom-forming fungi in future research.

Cell division can perturb the metabolic performance of industrial microbes. The C period of cell division starts from the initiation to the termination of DNA replication, whereas the D period is the bacterial division process. Here, we first shorten the C and D periods of E. coli by controlling the expression of the ribonucleotide reductase NrdAB and division proteins FtsZA through blue light and near-infrared light activation, respectively. It increases the specific surface area to 3.7 μm−1 and acetoin titer to 67.2 g·L−1. Next, we prolong the C and D periods of E. coli by regulating the expression of the ribonucleotide reductase NrdA and division protein inhibitor SulA through blue light activation-repression and near-infrared (NIR) light activation, respectively. It improves the cell volume to 52.6 μm3 and poly(lactate-co-3-hydroxybutyrate) titer to 14.31 g·L−1. Thus, the optogenetic-based cell division regulation strategy can improve the efficiency of microbial cell factories., Manipulation of genes controlling microbial shapes can affect bio-production. Here, the authors employ an optogenetic method to realize dynamic morphological engineering of E. coli replication and division and show the increased production of acetoin and poly(lactate-co-3-hydroxybutyrate).

Ganoderma lingzhi is a famous medicinal mushroom used as Chinese medicine or functional food and has been accepted across the globe. It is important to enhance the contents of bioactive compounds, which in turn improves the quality and biological activity of G. lingzhi fruiting body. In this work, freshly harvested G. lingzhi fruiting bodies were treated continuously with air or with 60 and 80% oxygen for 6 days. Samples were collected and determined initially and at 1 day interval during treatment. A high total ganoderic acid content of 29.44 g kg–1 was obtained in samples treated with 60% oxygen at day 3. Quantitative reverse transcriptase (qRT)-PCR and high-performance liquid chromatography (HPLC) analysis showed that the expression levels of hydroxymethylglutaryl-CoA synthase, squalene synthase, and oxidosqualene cyclase genes were substantially increased, resulting in the increase of ganoderic acids A, B, and C2 and ganoderenic acid B. The scavenging activities with 1,1-diphenyl-2-picrylhydrazyl radical, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical, hydroxyl radical, and superoxide radical and the DNA damage protective activity were also enhanced by high oxygen treatment. The results of this work provided a potential method to enhance the active metabolite synthesis in the fruiting body of G. lingzhi.

A new simple and highly sensitive electrochemical method for pyrophosphatase (PPase) activity detection was developed based on the peroxidase-like activity of G-quadruplex-Cu2+ DNAzyme. In the absence of PPase, Cu2+ could coordinate with pyrophosphate (PPi) to form Cu2+-PPi compound. While in the presence of PPase, it could destroy the coordinate compound because PPase catalyzed the hydrolysis of PPi into inorganic phosphate and produced free Cu2+, which then could be coupled with G-rich DNA to form G-quadruplex-Cu2+ DNAzyme. The formation of a mimic enzyme (G-quadruplex-Cu2+ DNAzyme) was immobilized on the surface of screen-printed gold electrode (SPGE). Using 3, 3', 5, 5'-tetramethylbenzidine (TMB) as a redox mediator and H2O2 as an enzyme substrate, the DNAzyme catalyzed the reduction of H2O2 to generate quantitative chronoamperometric signal. The catalytic activity of G-quadruplex-Cu2+ DNAzyme for TMB-H2O2 reaction was proportional to the activity of PPase, based on which, a simple and sensitive turn-on electrochemical method for PPase activity was thus developed for the first time. The chronoamperometric intensity of the system had a linear relationship with the PPase activities in the range of 1.0-50.0mU/mL and the detection limit could be down to 0.6mU/mL (S/N = 3). This proposed method was selective, cost-effective and convenient without any labels or complicated operations, which was furthermore applied to screen the inhibitor for PPase with high efficiency.

A study on the fed-batch semi-simultaneous saccharification and fermentation (FB-S-SSF) of sodium carbonate-pretreated rice straw at biomass loads between 10.0 and 30.0% (m/v) was conducted. Three different feeding modes, i.e., enzyme feeding (E feeding), substrate feeding (SR feeding) and the combination of both E feeding and SR feeding as well as batch mode were conducted for comparison under identical biomass loads. High ethanol concentrations above 90 g/L were obtained in all modes at a biomass load ≥25.0%. The SR feeding modes achieved higher final ethanol concentrations than those of the E feeding mode and batch mode. At biomass loads of 25.0%, 116.8 ± 3.3 and 118.9 ± 3.6 g/L ethanol was obtained for the SR feeding mode and the combination of E feeding and SR feeding modes, respectively. For batch mode and E feeding mode, the ethanol concentrations were 112.3 ± 3.6 and 108.6 ± 3.0 g/L, respectively. The present study shows that the production cost of bioethanol from lignocellulose can be reduced by adopting in situ-produced crude enzymes produced by Aspergillus fumigatus combined with FB-S-SSF of sodium carbonate-pretreated rice straw at a high biomass load, which provides a new idea for effectively reducing the production cost of bioethanol from lignocellulose.

Triterpene acids are among the major bioactive constituents of lucidum. However, submerged fermentation techniques for isolating triterpene acids from G. lucidum have not been optimized for commercial use, and the antitumor activity of the mycelial triterpene acids needs to be further proven. The aim of this work was to optimize the conditions for G. lucidum culture with respect to triterpene acid production, scaling up the process, and examining the in vitro antitumor activity of mycelial triterpene acids. The key conditions (i.e., initial pH, fermentation temperature, and rotation speed) were optimized using response surface methodology, and the in vitro antitumor activity was evaluated using the MTT method. The optimum key fermentation conditions for triterpene acid production were pH 6.0; rotation speed, 161.9 rpm; and temperature, 30.1°C, resulting in a triterpene acid yield of 291.0 mg/L in the validation experiment in a 5-L stirred bioreactor; this yield represented a 70.8% increase in titer compared with the nonoptimized conditions. Furthermore, the optimized conditions were then successfully scaled up to a production scale of 200 L, and a triterpene productivity of 47.9 mg/L/day was achieved, which is, to our knowledge, the highest reported in the large-scale fermentation of G. lucidum. In addition, the mycelial triterpene acids were found to be cytotoxic to the SMMC-7721 and SW620 cell lines in vitro. Chemical analysis showed that the key active triterpene acid compounds, ganoderic acids T and Me, predominated in the extract, at 69.2 and 41.6 mg/g, respectively. Thus, this work develops a simple and feasible batch fermentation technique for the large-scale production of antitumor triterpene acids from G. lucidum.

Biological network construction for Saccharomyces cerevisiae is a widely used approach for simulating phenotypes and designing cell factories. However, due to a complicated regulatory mechanism governing the translation of genotype to phenotype, precise prediction of phenotypes remains challenging. Here, we present WM_S288C, a computational whole-cell model that includes 15 cellular states and 26 cellular processes and which enables integrated analyses of physiological functions of Saccharomyces cerevisiae. Using WM_S288C to predict phenotypes of S. cerevisiae, the functions of 1140 essential genes were characterized and linked to phenotypes at five levels. During the cell cycle, the dynamic allocation of intracellular molecules could be tracked in real-time to simulate cell activities. Additionally, one-third of non-essential genes were identified to affect cell growth via regulating nucleotide concentrations. These results demonstrated the value of WM_S288C as a tool for understanding and investigating the phenotypes of S. cerevisiae.

Recent progress has been made in adding exogenous vegetable oils in culture media to promote bioactive metabolite production in several medicinal mushrooms, but the mechanism is still unclear. In this study, we found that the vegetable oil coix seed oil (CSO) could induce the biosynthesis of triterpene acids (TAs) and also significantly increase cytoplasmic nitric oxide (NO) and hydrogen peroxide (H

Crude glycerol is largely generated as the main by-product of the biodiesel industry and is unprofitable for industrial application without costly purification. The direct bioconversion of crude glycerol into 1,3-propanediol (1,3-PDO) by microorganisms is a promising alternative for effective and economic utilization. In this study, Klebsiella pneumoniae 2e was newly isolated for the conversion of crude glycerol into 1,3-PDO. Batch fermentation analysis confirmed that crude glycerol and its main impurities had slight impacts on the growth, key enzyme activity, and 1,3-PDO production of K. pneumoniae 2e. The 1,3-PDO yield from crude glycerol by K. pneumoniae 2e reached 0.64 mol 1,3-PDO/mol glycerol, which was higher than that by most reported 1,3-PDO-producing Klebsiella strains. Genomic profiling revealed that K. pneumoniae 2e possesses 30 genes involved in glycerol anaerobic metabolism and 1,3-PDO biosynthesis. Quantitative real-time PCR analysis of these genes showed that the majority of the genes encoding the key enzymes for glycerol metabolism and 1,3-PDO biosynthesis were significantly upregulated during culture in crude glycerol relative to that in pure glycerol. Further comparative genomic analysis revealed a novel glycerol uptake facilitator protein in K. pneumoniae 2e and a higher number of stress response proteins than in other Klebsiella strains. This work confirms the adaptability of a newly isolated 1,3-PDO-producing strain, K. pneumoniae 2e, to crude glycerol and provides insights into the molecular mechanisms involved in its crude glycerol tolerance, which is valuable for industrial 1,3-PDO production from crude glycerol. IMPORTANCE The rapid development of the biodiesel industry has led to tremendous crude glycerol generation. Due to the presence of complex impurities, crude glycerol has low value for industry without costly purification. Obtaining novel microorganisms capable of direct and efficient bioconversion of crude glycerol to value-added products has great economic potential for industrial application. In this work, we characterized a newly isolated strain, Klebsiella pneumoniae 2e, with the capacity to efficiently produce 1,3-propanediol (1,3-PDO) from crude glycerol and demonstrated its adaptation to crude glycerol. Our work provides insights into the molecular mechanisms of K. pneumoniae 2e adaptation to crude glycerol and the expression patterns of its genes involved in 1,3-PDO biosynthesis, which will contribute to the development of industrial 1,3-PDO production from crude glycerol.

Native and acetylated broken-rice starches (nanocrystals) with different degrees of substitution (DS) and their corresponding films were individually prepared, and the drug release profiles, weight loss, solubility and dispersion and surface morphology were comparatively studied. Bovine serum albumin (BSA) was used as a model drug. Acetylated native starch (ANS) DS 2.58, acetylated starch nanocrystals (ASN) DS 0.98, ASN DS 1.86, and ASN DS 2.72 were observed to be very soluble in chloroform. BSA was released rapidly from the native rice starch (NS) and ANS DS 2.58 films. ASN with high DS significantly slowed down the release of BSA from films, the percentages of BSA released from film ASN DS 2.72 only reached to 13% after 3.5 weeks release, and the release data followed Korsmeyer-Peppas equation. Further studies reveal that the particle size of ASN DS 2.72 was smallest, and the weight loss of ASN DS 2.72 film was lowest. The results demonstrate that acetylation and nanometer particle form of rice starch film can effectively retard protein drug release, and the prepared films based on ASN with high DS from broken rice may be suitable for the controlled protein delivery.