Soils and sediments host Thermoplasmata archaea encoding novel copper membrane monooxygenases (CuMMOs)

Copper membrane monooxygenases (CuMMOs) play critical roles in the global carbon and nitrogen cycles. Organisms harboring these enzymes perform the first, and rate limiting, step in aerobic oxidation of ammonia, methane, or other simple hydrocarbons. Within archaea, only organisms in the order Nitrososphaerales (Thaumarchaeota) encode CuMMOs, which function exclusively as ammonia monooxygenases. From grassland and hillslope soils and aquifer sediments, we identified 20 genomes from distinct archaeal species encoding divergent CuMMO sequences. These archaea are phylogenetically clustered in a previously unnamed Thermoplasmatota order, herein named the Ca. Angelarchaeales. The CuMMO proteins in Ca. Angelarchaeales are more similar in structure to those in Nitrososphaerales than those of bacteria, and contain all functional residues required for general monooxygenase activity. Ca. Angelarchaeales genomes are significantly enriched in blue copper proteins (BCPs) relative to sibling lineages, including plastocyanin-like electron carriers and divergent nitrite reductase-like (nirK) 2-domain cupredoxin proteins co-located with electron transport machinery. Ca. Angelarchaeales also encode significant capacity for peptide/amino acid uptake and degradation and share numerous electron transport mechanisms with the Nitrososphaerales. Ca. Angelarchaeales are detected at high relative abundance in some of the environments where their genomes originated from. While the exact substrate specificities of the novel CuMMOs identified here have yet to be determined, activity on ammonia is possible given their metabolic and ecological context. The identification of an archaeal CuMMO outside of the Nitrososphaerales significantly expands the known diversity of CuMMO enzymes in archaea and suggests previously unaccounted organisms contribute to critical global nitrogen and/or carbon cycling functions.

Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells

Cytochromes P450 catalyze oxidation of chemically diverse compounds and thus offer great potential for biocatalysis. Due to the complexity of these enzymes, their dependency of nicotinamide cofactors and redox partner proteins, recombinant microbial whole cells appear most appropriate for effective P450-mediated biocatalysis. However, some drawbacks exist that require individual solutions also when P450 whole-cell catalysts are used. Herein, we compared wet resting cells and lyophilized cells of recombinant E. coli regarding P450-catalyzed oxidation and found out that lyophilized cells are well-appropriate as P450-biocatalysts. E. coli harboring CYP105D from Streptomyces platensis DSM 40041 was used as model enzyme and testosterone as model substrate. Conversion was first enhanced by optimized handling of resting cells. Co-expression of the alcohol dehydrogenase from Rhodococcus erythropolis for cofactor regeneration did not affect P450 activity of wet resting cells (46% conversion) but was crucial to obtain sufficient P450 activity with lyophilized cells reaching a conversion of 72% under the same conditions. The use of recombinant lyophilized E. coli cells for P450 mediated oxidations is a promising starting point towards broader application of these enzymes.

A sensitive, accurate, and high-throughput gluco-oligosaccharide oxidase based HRP colorimetric method for lytic polysaccharide monooxygenase activity assay

Background The AA9 (auxiliary activities) family of lytic polysaccharide monooxygenases (AA9 LPMOs) are ubiquitous and diverse group of enzymes amongst the fungal kingdom. They catalyze the oxidative cleavage of glycosidic bonds in lignocellulose and exhibit great potential for secondary biorefinery applications. Screening of AA9 LPMOs for desirable properties is crucial for biorefinery industrial applications. However, robust, high-throughput and direct method for AA9 LPMO activity assay, which is prerequisite for screening of LPMOs with excellent properties, is still lacking. Here, we have described a gluco-oligosaccharide oxidase (GOOX) based horseradish peroxidase (HRP) colorimetric method for AA9 LPMO activity assay. Results We cloned and expressed a GOOX gene from Sarocladium strictum in Trichoderma reesei, purified the recombinant SsGOOX, validated its properties, and set up a SsGOOX based HRP colorimetric method for cellobiose concentration assay. Then we expressed two AA9 LPMOs from Thielavia terrestris, TtAA9F and TtAA9G in T. reesei, purified the recombinant proteins, and analyzed their product profiles and regioselectivity towards phosphoric acid swollen cellulose (PASC). TtAA9F was characterized as a C1 type (class 1) LPMO, while TtAA9G was characterized as a C4 type (class 2) LPMO. Finally, the SsGOOX based HRP colorimetric method was used to quantify the total concentration of reducing lytic products from LPMO reaction, and consequently, the activities of both C1 and C4 types of LPMOs were analyzed. These LPMOs could be effectively analyzed with limits of detection (LoDs) lower than 30 nmol/L, and standard curves between A515 and LPMO concentrations with determination coefficients greater than 0.994 were obtained. Conclusions A novel, sensitive and accurate assay method that directly targets the main activity of both C1 and C4 type of AA9 LPMOs was established. This method is easy to use and could be performed on a microtiter plate ready for high-throughput screening of AA9 LPMOs with high properties.

Identification of 5 Hub Genes Related to the Early Diagnosis, Tumour Stage, and Poor Outcomes of Hepatitis B Virus-Related Hepatocellular Carcinoma by Bioinformatics Analysis

Background. The majority of primary liver cancers in adults worldwide are hepatocellular carcinomas (HCCs, or hepatomas). Thus, a deep understanding of the underlying mechanisms for the pathogenesis and carcinogenesis of HCC at the molecular level could facilitate the development of novel early diagnostic and therapeutic treatments to improve the approaches and prognosis for HCC patients. Our study elucidates the underlying molecular mechanisms of HBV-HCC development and progression and identifies important genes related to the early diagnosis, tumour stage, and poor outcomes of HCC. Methods. GSE55092 and GSE121248 gene expression profiling data were downloaded from the Gene Expression Omnibus (GEO) database. There were 119 HCC samples and 128 nontumour tissue samples. GEO2R was used to screen for differentially expressed genes (DEGs). Volcano plots and Venn diagrams were drawn by using the ggplot2 package in R. A heat map was generated by using Heatmapper. By using the clusterProfiler R package, KEGG and GO enrichment analyses of DEGs were conducted. Through PPI network construction using the STRING database, key hub genes were identified by cytoHubba. Finally, KM survival curves and ROC curves were generated to validate hub gene expression. Results. By GO enrichment analysis, 694 DEGs were enriched in the following GO terms: organic acid catabolic process, carboxylic acid catabolic process, carboxylic acid biosynthetic process, collagen-containing extracellular matrix, blood microparticle, condensed chromosome kinetochore, arachidonic acid epoxygenase activity, arachidonic acid monooxygenase activity, and monooxygenase activity. In the KEGG pathway enrichment analysis, DEGs were enriched in arachidonic acid epoxygenase activity, arachidonic acid monooxygenase activity, and monooxygenase activity. By PPI network construction and analysis of hub genes, we selected the top 10 genes, including CDK1, CCNB2, CDC20, BUB1, BUB1B, CCNB1, NDC80, CENPF, MAD2L1, and NUF2. By using TCGA and THPA databases, we found five genes, CDK1, CDC20, CCNB1, CENPF, and MAD2L1, that were related to the early diagnosis, tumour stage, and poor outcomes of HBV-HCC. Conclusions. Five abnormally expressed hub genes of HBV-HCC are informative for early diagnosis, tumour stage determination, and poor outcome prediction.

Effects of Elevated Atmospheric CO2 Concentration on Phragmites australis and Wastewater Treatment Efficiency in Constructed Wetlands

Elevated atmospheric CO2 concentration (e[CO2]) has been predicted to rise to more than 400 ppm by the end of this century. It has received extensive attention with regard to the pros and cons of e[CO2] effects in terrestrial and marine ecosystems, while the effects of e[CO2] on wastewater treatment efficiency in constructed wetlands (CWs) are rarely known. In this study, the atmospheric CO2 concentration was set as 400 ppm (that is, ambient [CO2]) and 800 ppm (that is, e[CO2]). The physiological performance of Phragmites australis and microbial enzyme activities in constructed wetlands in response to e[CO2] were tested. Significantly higher net photosynthetic rate and plant growth were found under e[CO2]. The concentrations of nitrate, total anions, and total ions in the xylem sap of Phragmites australis were reduced, while the uptake of N and P in plants were not affected under e[CO2] condition. In addition, the ammonia monooxygenase activity was reduced, while the phosphatase activity was enhanced by e[CO2]. The increased removal efficiency of chemical oxygen demand and total nitrogen in CWs could be ascribed to the changes in physiological performance of Phragmites australis and activities of microbial enzymes under e[CO2]. These results suggested that the future atmospheric CO2 concentration could affect the wastewater treatment efficiency in CWs, due to the direct effects on plants and microorganisms.

In Vitro Metabolic Fate of the Synthetic Cannabinoid Receptor Agonists QMPSB and QMPCB (SGT-11) Including Isozyme Mapping and Esterase Activity

Quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) and quinolin-8-yl 4-methyl-3-(piperidine-1-carbonyl)benzoate (QMPCB, SGT-11) are synthetic cannabinoid receptor agonists (SCRAs). Knowing their metabolic fate is crucial for the identification of toxicological screening targets and to predict possible drug interactions. The presented study aimed to identify the in vitro phase I/II metabolites of QMPSB and QMPCB and to study the contribution of different monooxygenases and human carboxylesterases by using pooled human liver S9 fraction (pHLS9), recombinant human monooxygenases, three recombinant human carboxylesterases, and pooled human liver microsomes. Analyses were carried out by liquid chromatography high-resolution tandem mass spectrometry. QMPSB and QMPCB showed ester hydrolysis, and hydroxy and carboxylic acid products were detected in both cases. Mono/dihydroxy metabolites were formed, as were corresponding glucuronides and sulfates. Most of the metabolites could be detected in positive ionization mode with the exception of some QMPSB metabolites, which could only be found in negative mode. Monooxygenase activity screening revealed that CYP2B6/CYP2C8/CYP2C9/CYP2C19/CYP3A4/CYP3A5 were involved in hydroxylations. Esterase screening showed the involvement of all investigated isoforms. Additionally, extensive non-enzymatic ester hydrolysis was observed. Considering the results of the in vitro experiments, inclusion of the ester hydrolysis products and their glucuronides and monohydroxy metabolites into toxicological screening procedures is recommended.