Deciphering the biodesulfurization pathway employing marine mangrove Bacillus aryabhattai strain NM1-A2 according to whole genome sequencing and transcriptome analyses.

In the biogeochemical cycle, sulfur oxidation plays a vital role and is typically referred to as the elemental sulfur or reductive sulfide oxidation process. This study aimed to characterize a subtropical mangrove-isolated bacterial strain using biochemical, whole-genome, and transcriptome sequencing analyses to enhance our understanding of sulfur metabolism and biodegradation from a molecular genetic perspective. Strain NM1-A2 was characterized as Gram-positive and found to have a close molecular phylogenetic relationship with Bacillus aryabhattai. NM1-A2 efficiently converted dibenzothiophene (DBT) into 2-hydroxybiphenyl (2-HBP) via a 4S pathway with 95% efficiency, using enzymes encoded by the dsz operon (dszA, dszB, and dszC), which determine monooxygenases (DszA & DszC) and desulfinase (DszB). The whole-genome sequence of NM1-A2 had a length of approximately 5,257,678 bp and included 16 sulfur metabolism-related genes, featuring the ABC transport system, small subunit (ssu) and cysteine (cys) gene families, and adenosine 5'-phosphosulfate (APS) and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis-related genes. Transcriptomic analysis of NM1-A2 using three sulfur groups-magnesium sulfate (MS), sulfur powder (SP), and sodium thiosulfate (ST) resulted in a significant number of differentially expressed genes (1200, 2304, and 2001, respectively). This analysis revealed that intracellular cysteine concentration directly regulated the expression of cys and ssu genes. Sulfate did not directly affect cys gene expression but repressed ssu gene expression. The cys gene expression levels decreased during the conversion of sulfate to sulfide and cysteine. The transcriptomic data was validated by analyzing the expression patterns of NM1-A2 using real-time quantitative PCR validation analysis. The expression levels of cysl, mccB, and nrnA were significantly upregulated, while cysH, metB, and sat were downregulated in the SP, ST, and MS groups, respectively. This research contributes to our understanding of marine mangrove microorganisms' bacterial efficiency through characterization, whole-genome, and transcriptome sequencing-based molecular degradation of organic compounds in the mangrove ecosystem, which may enhance nutrient availability.

Effect of ammonium stress on phosphorus solubilization of a novel marine mangrove microorganism Bacillus aryabhattai NM1-A2 as revealed by integrated omics analysis.

BACKGROUND: Phosphorus is one of the essential nutrients for plant growth. Phosphate-solubilizing microorganisms (PSMs) can alleviate available P deficiency and enhance plant growth in an eco-friendly way. Although ammonium toxicity is widespread, there is little understanding about the effect of ammonium stress on phosphorus solubilization (PS) of PSMs. RESULTS: In this study, seven PSMs were isolated from mangrove sediments. The soluble phosphate concentration in culture supernatant of Bacillus aryabhattai NM1-A2 reached a maximum of 196.96 mg/L at 250 mM (NH4)2SO4. Whole-genome analysis showed that B. aryabhattai NM1-A2 contained various genes related to ammonium transporter (amt), ammonium assimilation (i.e., gdhA, gltB, and gltD), organic acid synthesis (i.e., ackA, fdhD, and idh), and phosphate transport (i.e., pstB and pstS). Transcriptome data showed that the expression levels of amt, gltB, gltD, ackA and idh were downregulated, while gdhA and fdhD were upregulated. The inhibition of ammonium transporter and glutamine synthetase/glutamate synthase (GS/GOGAT) pathway contributed to reducing energy loss. For ammonium assimilation under ammonium stress, accompanied by protons efflux, the glutamate dehydrogenase pathway was the main approach. More 2-oxoglutarate (2-OG) was induced to provide abundant carbon skeletons. The downregulation of formate dehydrogenase and high glycolytic rate resulted in the accumulation of formic acid and acetic acid, which played key roles in PS under ammonium stress. CONCLUSIONS: The accumulation of 2-OG and the inhibition of GS/GOGAT pathway played a key role in ammonium detoxification. The secretion of protons, formic acid and acetic acid was related to PS. Our work provides new insights into the PS mechanism, which will provide theoretical guidance for the application of PSMs.

Integration of Microbial Transformation Mechanism of Polyphosphate Accumulation and Sulfur Cycle in Subtropical Marine Mangrove Ecosystems with Spartina alterniflora Invasion.

Excessive phosphorus can lead to eutrophication in marine and coastal ecosystems. Sulfur metabolism-associated microorganisms stimulate biological phosphorous removal. However, the integrating co-biotransformation mechanism of phosphorus and sulfur in subtropical marine mangrove ecosystems with Spartina alterniflora invasion is poorly understood. In this study, an ecological model of the coupling biotransformation of sulfur and phosphorus is constructed using metagenomic analysis and quantitative polymerase chain reaction strategies. Phylogenetic analysis profiling, a distinctive microbiome with high frequencies of Gammaproteobacteria and Deltaproteobacteria, appears to be an adaptive characteristic of microbial structures in subtropical mangrove ecosystems. Functional analysis reveals that the levels of sulfate reduction, sulfur oxidation, and poly-phosphate (Poly-P) aggregation decrease with increasing depth. However, at depths of 25-50 cm in the mangrove ecosystems with S. alterniflora invasion, the abundance of sulfate reduction genes, sulfur oxidation genes, and polyphosphate kinase (ppk) significantly increased. A strong positive correlation was found among ppk, sulfate reduction, sulfur oxidation, and sulfur metabolizing microorganisms, and the content of sulfide was significantly and positively correlated with the abundance of ppk. Further microbial identification suggested that Desulfobacterales, Anaerolineales, and Chromatiales potentially drove the coupling biotransformation of phosphorus and sulfur cycling. In particular, Desulfobacterales exhibited dominance in the microbial community structure. Our findings provided insights into the simultaneous co-biotransformation of phosphorus and sulfur bioconversions in subtropical marine mangrove ecosystems with S. alterniflora invasion.

Characterization of NMCR-2, a new non-mobile colistin resistance enzyme: implications for an MCR-8 ancestor.

MCR-4 and MCR-8 are two recently identified members of an ongoing MCR family of colistin resistance. Although that aquatic reservoir for MCR-4 is proposed, the origin and mechanism of MCR-8 is poorly understood. Here we report a previously unrecognized non-mobile colistin resistance enzyme, termed NMCR-2, originating from the plant pathogen Kosakonia pseudosacchari. NMCR-2 (551aa) gives 67.3% identity to MCR-8 (565aa). NMCR-2 is placed as a progenitor/ancestor for MCR-8 in phylogeny of MCR members. Genetic study reveals that nmcr-2 is comparable to mcr-8 in the ability of producing phenotypic colistin resistance. Biochemical analyses determine that these two enzymes catalyse the transfer of PEA from the donor PE lipid substrate to the recipient lipid A molecule by a putative 'ping-pong' trade-off. Further experiment of protein engineering demonstrates that the two motifs (TM region and catalytic domain) of NMCR-2 are functionally exchangeable with that of MCR-8, rather than MCR-1. Physiological impacts of nmcr-2 and/or mcr-8 are detected in Escherichia coli, featuring with fitness cost. Evidently, the action and mechanism of NMCR-2 is analogous to that of MCR-8. Therefore, our finding underlines that NMCR-2 might be a possible progenitor of MCR-8.

Whole genome sequencing and metabolomics analyses reveal the biosynthesis of nerol in a multi-stress-tolerant Meyerozyma guilliermondii GXDK6.

BACKGROUND: Nerol (C10H18O), an acyclic monoterpene, naturally presents in plant essential oils, and is used widely in food, cosmetics and pharmaceuticals as the valuable fragrance. Meanwhile, chemical synthesis is the only strategy for large-scale production of nerol, and the disadvantages of chemical synthesis greatly limit the production and its application. These defects drive the interests of researchers shift to the production of nerol by eco-friendly methods known as biosynthesis methods. However, the main technical bottleneck restricting the biosynthesis of nerol is the lacking of corresponding natural aroma-producing microorganisms. RESULTS: In this study, a novel multi-stress-tolerant probiotics Meyerozyma guilliermondii GXDK6 with aroma-producing properties was identified by whole genome sequencing and metabolomics technology. GXDK6 showed a broad pH tolerance in the range of 2.5-10.0. The species also showed salt tolerance with up to 12% NaCl and up to 18% of KCl or MgCl2. GXDK6 exhibited heavy-metal Mn2+ tolerance of up to 5494 ppm. GXDK6 could also ferment with a total of 21 kinds of single organic matter as the carbon source, and produce abundant aromatic metabolites. Results from the gas chromatography-mass spectrometry indicated the production of 8-14 types of aromatic metabolites (isopentanol, nerol, geraniol, phenylethanol, isobutanol, etc.) when GXDK6 was fermented up to 72 h with glucose, sucrose, fructose, or xylose as the single carbon source. Among them, nerol was found to be a novel aromatic metabolite from GXDK6 fermentation, and its biosynthesis mechanism had also been further revealed. CONCLUSION: A novel aroma-producing M. guilliermondii GXDK6 was identified successfully by whole genome sequencing and metabolomics technology. GXDK6 showed high multi-stress-tolerant properties with acid-base, salty, and heavy-metal environments. The aroma-producing mechanism of nerol in GXDK6 had also been revealed. These findings indicated the aroma-producing M. guilliermondii GXDK6 with multi-stress-tolerant properties has great potential value in the fermentation industry.

VV Reduction by Polaromonas spp. in Vanadium Mine Tailings.

Vanadium (V) is an important metal with critical industrial and medical applications. Elevated V contamination, however, can be a threat to the environment and human health. Microorganisms can reduce the more toxic and mobile VV to the less toxic and immobile VIV, which could be a detoxification and energy metabolism strategy adopted by V-reducing bacteria (VRB). The limited understanding of microbial responses to V contamination and the mechanisms for VV reduction, however, hamper our capability to attenuate V contamination. This study focused on determining the microbial responses to elevated V concentration and the mechanisms of VV reduction in V tailings. The bacterial communities were characterized and compared between the V tailings and the less contaminated adjacent mineral soils. Further, VV-reducing enrichments indicated that bacteria associated with Polaromonas, a genus belonging to the family Burkholderiaceae, were potentially responsible for VV reduction. Retrieved metagenome-assembled genomes (MAGs) suggested that the Polaromonas spp. encoded genes (cymA, omcA, and narG) were responsible for VV reduction. Additionally, Polaromonas spp. was metabolically versatile and could use both organic and inorganic electron donors. The metabolic versatility of Polaromonas spp. may be important for its ability to flourish in the V tailings.

The Role of Tauroursodeoxycholic Acid on Dedifferentiation of Vascular Smooth Muscle Cells by Modulation of Endoplasmic Reticulum Stress and as an Oral Drug Inhibiting In-Stent Restenosis.

PURPOSE: The role of endoplasmic reticulum (ER) stress in cardiovascular disease is now recognized. Tauroursodeoxycholic acid (TUDCA) is known to have cardiovascular protective effects by decreasing ER stress. This study aimed to assess the ability of TUDCA to decrease ER stress, inhibit dedifferentiation of vascular smooth muscle cells (VSMCs), and reduce in-stent restenosis. METHODS: The effect of TUDCA on dedifferentiation of VSMCs and ER stress was investigated in vitro using wound-healing assays, MTT assays, and western blotting. For in vivo studies, 18 rabbits were fed an atherogenic diet to induce atheroma formation. Bare metal stents (BMS), BMS+TUDCA or Firebird stents were implanted in the left common carotid artery. Rabbits were euthanized after 28 days and processed for scanning electron microscope (SEM), histological examination (HE), and immunohistochemistry. RESULTS: In vitro TUDCA (10-1000 µmol/L) treatment significantly inhibited platelet-derived growth factor (PDGF)-BB-induced proliferation and migration in VSMCs in a concentration-dependent manner and decreased ER stress markers (IRE1, XBP1, KLF4, and GRP78). In vivo, we confirmed no significant difference in neointimal coverage on three stents surfaces; neointimal was significantly lower with BMS+TUDCA (1.6 ± 0.2 mm2) compared with Firebird (1.90 ± 0.1 mm2) and BMS (2.3 ± 0.1 mm2). Percent stenosis was lowest for BMS+TUDCA, then Firebird, and was significantly higher with BMS (28 ± 4%, 35 ± 7%, 40 ± 1%; respectively; P < 0.001). TUDCA treatment decreased ER stress in the BMS+TUDCA group compared with BMS. CONCLUSIONS: TUDCA inhibited dedifferentiation of VSMCs by decreasing ER stress and reduced in-stent restenosis, possibly through downregulation of the IRE1/XBP1 signaling pathway.

Deletion of gene gnd encoding 6-phosphogluconate dehydrogenase promotes L-serine biosynthesis in a genetically engineered strain of Methylobacterium sp. MB200.

OBJECTIVE: To identify potential target genes involved in L-serine biosynthesis in Methylobacterium sp. MB200 and to evaluate the gnd genetically-engineered strains for L-serine production. RESULTS: Five genes that are not associated with the central metabolic pathway but with L-serine biosynthesis were identified from Methylobacterium sp. MB200 mutants. Gene gnd, encoding 6-phosphogluconate dehydrogenase (PGDH), was selected for further evaluation. The gnd deletion mutant showed a 600% increase in D-serine tolerance and an 80% decrease in PGDH activity compared to Methylobacterium sp. MB200. gnd over-expression did not affect D-serine tolerance, whereas it did increase enzyme-activity up to 136%. Additionally, analysis revealed that in Methylobacterium sp. MB200, L-serine inhibited PGDH activity. The deletion of gnd did not affect growth, whereas it did enhance the biosynthesis of L-serine, resulting in a 225% increase in production of L-serine compared to the wild-type. CONCLUSION: gnd, one of the five genes identified here that is associated with L-serine synthesis, can be developed as a potential candidate for metabolic engineering to promote L-serine synthesis in Methylobacterium sp. MB200.

Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived ß-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside.

Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived ß-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (kcat/Km of 561.72 mM-1 s-1) toward ρ-nitrophenyl-ß-d-glucopyranoside (ρNPG) that increased by 23-fold, half-life of inactivation by 10-fold, and further retained transglycosidation activity at 50 °C as compared with the wild-type Bgl1D protein. Site-directed mutagenesis also revealed that Asp44 residue was essential to ß-glucosidase activity of Bgl1D. This study improved our understanding of the key amino acids of the novel ß-glucosidases and presented a raw material with enhanced catalytic activity and thermostability for the synthesis of butyl glucosides.

Folic acid delays development of atherosclerosis in low-density lipoprotein receptor-deficient mice.

Many studies support the cardioprotective effects of folic acid (FA). We aimed to evaluate the utility of FA supplementation in preventing the development of atherosclerotic in low-density lipoprotein receptor-deficient (LDLR-/-) mice and to elucidate the molecular processes underlying this effect. LDLR-/- mice were randomly distributed into four groups: control group, HF group, HF + FA group and the HF + RAPA group. vascular smooth muscle cells (VSMCs) were divided into the following four groups: control group, PDGF group, PDGF + FA group and PDGF + FA + RAPA group. Blood lipid levels, oxidative stress and inflammatory cytokines were measured. Atherosclerosis severity was evaluated with oil red O staining. Haematoxylin and eosin (H&E) staining was used to assess atherosclerosis progression. Immunohistochemical staining was performed with antismooth muscle α-actin (α-SMA) antibodies and anti-osteopontin (OPN) antibodies that demonstrate VSMC dedifferentiation. The protein expression of α-SMA, OPN and mechanistic target of rapamycin (mTOR)/p70S6K signalling was detected by Western blot analysis. FA and rapamycin reduced serum levels of total cholesterol, triacylglycerol, LDL, inhibiting oxidative stress and the inflammatory response. Oil red O and H&E staining demonstrated that FA and rapamycin inhibited atherosclerosis. FA and rapamycin treatment inhibited VSMC dedifferentiation in vitro and in vivo, and FA and rapamycin attenuated the mTOR/p70S6K signalling pathway. Our findings suggest that FA attenuates atherosclerosis development and inhibits VSMC dedifferentiation in high-fat-fed LDLR-/- mice by reduced lipid levels and inhibiting oxidative stress and the inflammatory response through mTOR/p70S6K signalling pathway.

Molecular Characterization and Directed Evolution of a Metagenome-Derived l-Cysteine Sulfinate Decarboxylase.

l-Cysteine sulfinate decarboxylase (CSD, EC 4.1.1.29), the rate-limiting enzyme in taurine synthesis pathway, catalyzes l-cysteine sulfinic acid to form hypotaurine. Identification of the novel CSD that could improve the biosynthetic efficiency of taurine is important. An unexplored decarboxylase gene named undec1A was identified in a previous work through sequence-based screening of uncultured soil microorganisms. Random mutagenesis through sequential error-prone polymerase chain reaction was used in Undec1A. A mutant Undec1A-1180, which was obtained from mutagenesis library, had 5.62-fold higher specific activity than Undec1A at 35 °C and pH=7.0. Molecular docking results indicated that amino acid residues Ala235, Val237, Asp239, Ile267, Ala268, and Lys298 in the Undec1A-1180 protein helped recognize and catalyze the substrate molecules of l-cysteine sulfinic acid. These results could serve as a basis for elucidating the characteristics of the Undec1A-1180. Directed evolution technology is a convenient way to improve the biotechnological applications of metagenome-derived genes.

MicroRNA-384-mediated Herpud1 upregulation promotes angiotensin II-induced endothelial cell apoptosis.

BACKGROUND: Angiotensin II (Ang II)-induced damage to endothelial cells (ECs) plays a crucial role in the pathogenesis of atherosclerosis. This study aimed to investigate the role of microRNA-384 (miR-384) in endothelial cell apoptosis. METHODS: The expression of five various miRNAs in Ang II-treated human umbilical vein endothelial cells (HUVECs) were detected by qPCR. The Ang II-induced apoptosis of HUVECs was determined by flow cytometry, TUNEL staining and western blot. Endoplasmic reticulum (ER) stress markers were detected by western blot analysis. The target gene of miR-384 was determined by bioinformatics analyses. qPCR, western blotting and immunofluorescence were performed to determine the expression level of homocysteine inducible ER protein with ubiquitin like domain 1 (Herpud1). RESULTS: miR-384 expression level was significantly decreased in Ang II-treated HUVECs. Ang II-induced HUVEC apoptosis was accompanied by the occurrence of ER stress. A decreased rate of HUVEC apoptosis and a decreased rate of ER stress were observed following restoration of miR-384 expression. Herpud1 expression level was increased in HUVECs treated with Ang II, and miR-384 mimics effectively inhibited Herpud1 expression. Mechanistically, miR-384 directly targets the 3'-untranslated region of Herpud1. Furthermore, effects of miR-384 on HUVECs apoptosis and ER stress were at least partly reversed by knockdown of Herpud1 expression. CONCLUSION: The results of the present study collectively indicated that miR-384 expression level was downregulated in Ang II-treated HUVECs and miR-384 overexpression protected HUVECs against Ang II-induced apoptosis by negatively regulating Herpud1. These findings point towards new strategies by which apoptosis of ECs can be suppressed.

Primary Culture of Rat Aortic Vascular Smooth Muscle Cells: A New Method.

BACKGROUND Developing a simple and efficient method of obtaining primary cultured VSMCs is necessary for basic cardiovascular research. MATERIAL AND METHODS The procedure of our new method mainly includes 6 steps: isolation of the aortic artery, removal of the fat tissue around the artery, separation of the media, cutting the media into small tissue blocks, transferring the tissue blocks to cell culture plates, and incubation until the cells reach confluence. The cells were identified as VSMCs by morphology and immunofluorescence. Then, VSMCs obtained by this new tissue explants method, the traditional tissue explants method, the enzyme digestion method, and A7r5 cell line were divided into 4 groups. The purity of cells was test by multiple fluorescent staining. Western blotting was used to investigate the phenotype of VSMCs obtained by different methods. RESULTS Cells began to grow out at about 8 days and became relatively confluent within 16 days. Compared with VSMCs from the traditional tissue explants method and enzyme digestion method or A7r5 cell line, VSMCs obtained by our method showed higher purity and manifested a more "contractile" phenotype characteristic. CONCLUSIONS We have conquered the disadvantages in the previous primary culture methods and established a simple and reliable way to isolate and culture rat aortic VSMCs with high purity and stability.

Polyphenols and Polypeptides in Chinese Rice Wine Inhibit Homocysteine-induced Proliferation and Migration of Vascular Smooth Muscle Cells.

The beneficial effect of Chinese rice wine on atherosclerosis has been proved, but the exact components that have the cardiovascular protective effect are still unknown. This study aimed to explore the exact ingredients in Chinese rice wine that could inhibit homocysteine (Hcy)-induced vascular smooth muscle cell (VSMC) proliferation and migration. VSMCs were divided into 7 groups: control, Hcy (1 mmol/L), Hcy + oligosaccharide, Hcy + polypeptides, Hcy + polyphenols, Hcy + alcohol, and Hcy + Chinese rice wine. methyl thiazolyl tetrazolium (MTT) assay, Transwell chambers, and wound-healing assay were used to test the proliferation and migratory ability of the VSMCs. Western blot and gelatin zymography were used to investigate the expressions and activities of metal matrix proteinase 2/9 (MMP-2/9) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in VSMCs. Polypeptides and polyphenols in the Chinese rice wine reduced the proliferation and migration ability of the VSMCs. Furthermore, they also decreased the expression and activity of MMP-2/9 but had no obvious impact on the expression of TIMP-2 in each group. This study further confirms that polypeptides and polyphenols in the Chinese rice wine could inhibit Hcy-induced proliferation and migration of VSMCs and maintain the balance between matrix metalloproteinases (MMPs) and TIMPs.

A novel D-amino acid oxidase from a contaminated agricultural soil metagenome and its characterization.

A novel D-amino acid oxidase (DAAO) gene designated as daoE was cloned by the sequence-based screening of a plasmid metagenomic library of uncultured microorganisms from contaminated agricultural soil. The deduced amino acid sequence comparison and phylogenetic analysis indicated that daoE and other putative DAAOs are closely related. The putative DAAO gene was subcloned into a pETBlue-2 vector and overexpressed in Escherichia coli Tunner(DE3)pLacI. The recombinant protein was purified to homogeneity. The maximum activity of DaoE protein occurred at pH 8.0 and 37 °C. DaoE recombinant protein had an apparent K m of 2.96 mM, V max of 0.018 mM/min, k cat of 10.9/min, and k cat/K m of 1.16 × 10(4)/mol/min. The identification of this novel DAAO gene demonstrated the importance of metagenomic libraries in exploring new D-amino acid oxidases from environmental microorganisms to optimize their applications.

Yellow wine polyphenolic compounds inhibit matrix metalloproteinase-2, -9 expression and improve atherosclerotic plaque in LDL-receptor-knockout mice.

Many epidemiological studies have strongly suggested an inverse correlation between dietary polyphenol consumption and reduced risks of cardiovascular diseases. Yellow rice wine is a Chinese specialty and one of the three most ancient wines in the world (Shaoxing rice wine, beer, and grape wine). There is a large amount of polyphenol substances in yellow rice wine. This experiment was designed to study the potential beneficial effects of yellow wine polyphenolic compounds (YWPC) from yellow rice wine on progression of atherosclerosis in vivo and to further explore its underlying mechanisms. Six-week-old male LDL-receptor-knockout mice were treated with high-fat diet to establish the mouse model with atherosclerosis. Animals received 10, 30, or 50 mg/kg per day of YWPC or 10 mg/kg per day rosuvastatin or water (vehicle) for 14 weeks. The results indicated that YWPC and rosuvastatin significantly decreased circulating total cholesterol and low-density lipoprotein cholesterol. Compared to the control group, the atherosclerosis lesion area in the rosuvastatin-intervention group and YWPC at doses of 10, 30, and 50 mg/kg per day intervention groups decreased by 74.14%, 18.51%, 40.09%, and 38.42%, respectively. YWPC and rosuvastatin decreased the expression and activity of matrix metalloproteinases (MMP)-2, 9, whereas the expression of the endogenous inhibitors of these proteins, namely, tissue inhibitors of matrix metalloproteinases (TIMP)-1, 2, increased when compared to the control group. It can be concluded that the YWPC is similar to the benefic effects of rosuvastatin on cardiovascular system. These effects may be attributed to their anti-atherosclerotic actions by lowering lipid and modulating the activity and expression of MMP-2, 9 and TIMP-1, 2.

Screening of Burkholderia sp. WGB31 producing anisic acid from anethole and optimization of fermentation conditions.

Anisic acid, the precursor of a variety of food flavors and industrial raw materials, can be bioconversed from anethole which extracted from star anise fruits. WGB31 strain with anisic acid molar production rate of 10.25% was isolated and identified as Burkholderia sp. Three significant influential factors, namely, glucose concentration, initial pH value, and medium volume were selected and their effects were evaluated by Box-Behnken Design (BBD). Regression analysis was performed to determine response surface methodology and the significance was tested to obtain the process model of optimal conditions for producing anisic acid. The fermentation conditions at the stable point of the model were obtained: glucose 6 g L(-1) , pH 6.2, culture medium volume 61 mL in a triangular flask with 250 ml volume. Verification test indicated that the production rate of anisic acid was 30.7%, which was three times of that before optimizing. The results provide a basis and reference for producing anisic acid by microbial transformation.

Isolation and characterization of a gene associated with sulfate assimilation in Sinorhizobium fredii WGF03.

Sulfur is an essential element for rhizobia, such as sulfated modified Nod factors and nitrogenase. To investigate the role of sulfur metabolism in Rhizobium-Soybean symbiosis, a transponson random insertional mutants' library was constructed and a sulfur assimilation-related gene was isolated and characterized. A mutant strain unable to utilized sulfate was screened from 11,000 random insertional mutants of Sinorhizobium fredii WGF03. Sequencing analysis showed that a sulfate assimilation-related gene (cysDN) was inserted by the Tn transponson. Mutants inactivated in cysD and cysN (SMcysDF and SMcysNF) were constructed by homologous recombination using the suicide plasmid pK18mob. The mutants SMcysDF and SMcysNF could no longer utilize sulfate as sulfur source. Phenotype analysis revealed that mutation of cysDN had multiple effects on S. fredii WGF03. Root hair deformation assay showed that the activity of Nod factors secreted by mutants SMcysDR and SMcysNR elicited minimal hair initiation only. Soybean plant tests indicated that the mutant strains delayed 1-2 days to nodulate and exhibited lower nodulation efficiency and symbiotic efficiency than the wild-type strain. The complementary strain of cysD and cysN (HcysDF and HcysNF) could restore the nodulation efficiency.

Regulatory mechanisms of dopamine metabolism in a marine Meyerozyma guilliermondii GXDK6 under NaCl stress as revealed by integrative multi-omics analysis.

Dopamine can be used to treat depression, myocardial infarction, and other diseases. However, few reports are available on the de novo microbial synthesis of dopamine from low-cost substrate. In this study, integrated omics technology was used to explore the dopamine metabolism of a novel marine multi-stress-tolerant aromatic yeast Meyerozyma guilliermondii GXDK6. GXDK6 was found to have the ability to biosynthesize dopamine when using glucose as the substrate. 14 key genes for the biosynthesis of dopamine were identified by whole genome-wide analysis. Transcriptomic and proteomic data showed that the expression levels of gene AAT2 encoding aspartate aminotransferase (regulating dopamine anabolism) were upregulated, while gene AO-I encoding copper amine oxidase (involved in dopamine catabolism) were downregulated under 10 % NaCl stress compared with non-NaCl stress, thereby contributing to biosynthesis of dopamine. Further, the amount of dopamine under 10 % NaCl stress was 2.51-fold higher than that of zero NaCl, which was consistent with the multi-omics results. Real-time fluorescence quantitative PCR (RT-qPCR) and high-performance liquid chromatography (HPLC) results confirmed the metabolic model of dopamine. Furthermore, by overexpressing AAT2, AST enzyme activity was increased by 24.89 %, the expression of genes related to dopamine metabolism was enhanced, and dopamine production was increased by 56.36 % in recombinant GXDK6AAT2. In conclusion, Meyerozyma guilliermondii GXDK6 could utilize low-cost carbon source to synthesize dopamine, and NaCl stress promoted the biosynthesis of dopamine.

Identification of a metagenome-derived prephenate dehydrogenase gene from an alkaline-polluted soil microorganism.

A novel prephenate dehydrogenase gene designated pdhE-1 was cloned by sequence-based screening of a plasmid metagenomic library from uncultured alkaline-polluted microorganisms. The deduced amino acid sequence comparison and phylogenetic analysis indicated that PdhE-1 and other putative prephenate dehydrogenases were closely related. The putative prephenate dehydrogenase gene was subcloned into pETBlue-2 vector and overexpressed in Escherichia coli BL21(DE3) pLacI. The recombinant protein was purified to homogeneity. The maximum activity of the PdhE-1 protein occurred at pH 8.0 and 45 °C using prephenic acid as the substrate. The prephenate dehydrogenase had an apparent K m value of 0.87 mM, a V max value of 41.5 U/mg, a k cat value of 604.8/min and a k cat/K m value of 1.16 × 10(4)/mol/s. L-Tyrosine did not obviously inhibit the recombinant PdhE-1 protein. The identification of a metagnome-derived prephenate dehydrogenase provides novel material for studies and application of proteins involved in tyrosine biosynthesis.