Salt stress perception and metabolic regulation network analysis of a marine probiotic Meyerozyma guilliermondii GXDK6

IntroductionExtremely salt-tolerant microorganisms play an important role in the development of functional metabolites or drug molecules.MethodsIn this work, the salt stress perception and metabolic regulation network of a marine probiotic Meyerozyma guilliermondii GXDK6 were investigated using integrative omics technology.ResultsResults indicated that GXDK6 could accept the salt stress signals from signal transduction proteins (e.g., phosphorelay intermediate protein YPD1), thereby contributing to regulating the differential expression of its relevant genes (e.g., CTT1, SOD) and proteins (e.g., catalase, superoxide dismutase) in response to salt stress, and increasing the salt-tolerant viability of GXDK6. Omics data also suggested that the transcription (e.g., SMD2), translation (e.g., MRPL1), and protein synthesis and processing (e.g., inner membrane protein OXA1) of upregulated RNAs may contribute to increasing the salt-tolerant survivability of GXDK6 by improving protein transport activity (e.g., Small nuclear ribonucleoprotein Sm D2), anti-apoptotic ability (e.g., 54S ribosomal protein L1), and antioxidant activity (e.g., superoxide dismutase). Moreover, up to 65.9% of the differentially expressed genes/proteins could stimulate GXDK6 to biosynthesize many salt tolerant-related metabolites (e.g., β-alanine, D-mannose) and drug molecules (e.g., deoxyspergualin, calcitriol), and were involved in the metabolic regulation of GXDK6 under high NaCl stress.DiscussionThis study provided new insights into the exploration of novel functional products and/or drugs from extremely salt-tolerant microorganisms.Graphical Abstrac

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

L-cistein sulfinat dekarboksilaza (EC 4.1.1.29) je enzim koji određuje brzinu biosinteze taurina, te katalizira dekarboksilaciju L-cistein sulfinskekiseline u hipotaurin. Identifikacija nove L-cistein sulfinat dekarboksilaze koja bi mogla poboljšati biosintetičku učinkovitost taurina vrlo je bitna. Još neistraženi gen za dekarboksilazu, undec1A, identificiran je u prethodnom radu probirom sekvencija DNA iz neuzgojenih mikroorganizama iz tla. Uproteinu Undec1A primjenjena je nasumična mutageneza pomoću sekvencijalne lančane reakcije polimeraze s pogreškama. Mutirani protein Undec1A-1180, izdvojen iz zbirke tako dobivenih različitih mutiranih proteina, imao je 5,62 puta veću specifičnu aktivnost od ishodnogproteina Undec1A pri temperaturi od 35 °C i pH=7,0. Rezultati molekulskog uklapanja potvrđuju da aminokiselinski ostaci Ala235, Val237, Asp239, Ile267, Ala268 i Lys298 u proteinu Undec1A-1180 pomažu pri prepoznavanju i katalizi molekula L-cistein sulfinske kiseline. Ovi bi rezultati mogli poslužiti kao osnova za definiranje svojstava proteina Undec1A-1180.Tehnologija usmjerene evolucije prikladna je za unapređenje biotehnološke primjene gena dobivenih iz metagenoma.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

Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

<p>Abstract</p> <p>Background</p> <p>Fumarase catalyzes the reversible hydration of fumarate to <smcaps>L</smcaps>-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of <smcaps>L</smcaps>-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications.</p> <p>Results</p> <p>A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF) was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from <it>Bacteroides </it>sp. 2_1_33B and <it>Parabacteroides distasonis </it>ATCC 8503 (26% identical and 43% similar). The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in <it>E. coli </it>BL21(DE3)pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form <smcaps>L</smcaps>-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg²⁺. The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: <it>K</it>_m= 0.48 mM, <it>V</it>_max= 827 μM/min/mg, and <it>k</it>_cat/<it>K</it>_m= 1900 mM/s.</p> <p>Conclusions</p> <p>We isolated a novel fumarase gene, <it>fumF</it>, from a sequence-based screen of a plasmid metagenomic library from uncultivated marine microorganisms. The properties of FumF protein may be ideal for the industrial production of <smcaps>L</smcaps>-malate under higher temperature conditions. The identification of FumF underscores the potential of marine metagenome screening for novel biomolecules.</p

Aspect of Clusters Correlation at Light Nuclei Excited State

The correlation of $\alpha\alpha$ was probed via measuring the transverse momentum $p_{T}$ and width $\delta p_{T}$ of one $\alpha$, for the first time, which represents the spatial and dynamical essentialities of the initial coupling state in $^{8}$Be nucleus. The weighted interaction vertex of 3$\alpha$ reflected by the magnitudes of their relative momentums and relative emission angles proves the isosceles triangle configuration for 3$\alpha$ at the high excited energy analogous Hoyle states.Comment: 8 pages, 9 figure

Variation of Tensor Force due to Nuclear Medium Effect

The enhancement of $J^{\pi}(T)$=3$^{+}$(0) state with isospin $T=0$ excited by the tensor force in the free $^{6}$Li nucleus has been observed, for the first time, relative to a shrinkable excitation in the $^{6}$Li cluster component inside its host nucleus. Comparatively, the excitation of $J^{\pi}(T)$=0$^{+}$(1) state with isospin $T=1$ for these two $^{6}$Li formations take on an approximately equal excitation strength. The mechanism of such tensor force effect was proposed due to the intensive nuclear medium role on isospin $T$=0 state.Comment: 6 pages, 4 figure

Multi-alpha Boson Gas state in Fusion Evaporation Reaction and Three-body Force

The experimental evidence for the $\alpha$ Boson gas state in the $^{11}$C+$^{12}$C$\rightarrow$$^{23}$Mg$^{\ast}$ fusion evaporation reaction is presented. By measuring the $\alpha$ emission spectrum with multiplicity 2 and 3, we provide insight into the existence of a three-body force among $\alpha$ particles. The observed spectrum exhibited distinct tails corresponding to $\alpha$ particles emitted in pairs and triplets consistent well with the model-calculations of AV18-UX and chiral effective field theory of NV2-3-la*, indicating the formation of $\alpha$ clusters with three-body force in the Boson gas state.Comment: 7 pages, 6 figure

Assessment of Multiple Anaerobic Co-Digestions and Related Microbial Community of Molasses with Rice-Alcohol Wastewater

Molasses is a highly dense and refined byproduct produced in the sugarcane industry, and it contains high amounts of degradable compounds. Through bioconversion, these compounds can be transformed into renewable products. However, the involved biological process is negatively influenced by the high chemical oxygen demand (COD) of molasses and ion concentration. The co-digestion of molasses with rice-alcohol wastewater (RAW) was compared with its mono-digestion at an increasing organic loading rate (OLR). Both processes were assessed by detecting the COD removal rate, the methane contents of biogas, and the structure and composition of microbial communities at different stages. Results showed that the co-digestion is stable up to a maximum OLR of 16 g COD L&minus;1 d&minus;1, whereas after the acclimatization phase, the mono-digestion process was disturbed two times, which occurred at a maximum OLR of 9 and 10 g COD L&minus;1 d&minus;1. The volatile fatty acids (VFAs) observed were 2059.66 mg/L and 1896.9 mg/L, which in mono-digestion causes the inhibition at maximum OLRs. In the co-digestion process, the concomitant COD removal rates and methane content recorded was 90.72 &plusmn; 0.63% 64.47% &plusmn; 0.59% correspondingly. While in the mono-digestion process, high COD removal rate and methane contents observed were 89.29 &plusmn; 0.094% and 61.37 &plusmn; 1.06% respectively. From the analysis of microbial communities, it has been observed that both the bacterial and archaeal communities respond differently at unlike stages. However, in both processes, Propionibacteriaceae was the most abundant family in the bacterial communities, whereas Methanosaetaceae was abundant in the archaeal communities. From the current study, it has been concluded that that rice-alcohol wastewater could be a good co-substrate for the anaerobic digestion of molasses in terms of COD removal rate and methane contents production, that could integrate molasses into progressive biogas production with high OLR

L-Cysteine Synthase Enhanced Sulfide Biotransformation in Subtropical Marine Mangrove Sediments as Revealed by Metagenomics Analysis

High sulfides concentrations can be poisonous to environment because of anthropogenic waste production or natural occurrences. How to elucidate the biological transformation mechanisms of sulfide pollutants in the subtropical marine mangrove ecosystem has gained increased interest. Thus, in the present study, the sulfide biotransformation in subtropical mangroves ecosystem was accurately evaluated using metagenomic sequencing and quantitative polymerase chain reaction analysis. Most abundant genes were related to the organic sulfur transformation. Furthermore, an ecological model of sulfide conversion was constructed. Total phosphorus was the dominant environmental factor that drove the sulfur cycle and microbial communities. We compared mangrove and non-mangrove soils and found that the former enhanced metabolism that was related to sulfate reduction when compared to the latter. Total organic carbon, total organic nitrogen, iron, and available sulfur were the key environmental factors that effectively influenced the dissimilatory sulfate reduction. The taxonomic assignment of dissimilatory sulfate-reducing genes revealed that Desulfobacterales and Chromatiales were mainly responsible for sulfate reduction. Chromatiales were most sensitive to environmental factors. The high abundance of cysE and cysK could contribute to the coping of the microbial community with the toxic sulfide produced by Desulfobacterales. Collectively, these findings provided a theoretical basis for the mechanism of the sulfur cycle in subtropical mangrove ecosystems

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

Abstract 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