Diverse N-doped carbon-supported non-noble metal nanostructures (Ni, Co, Fe, Cu) are designed, and explored in selective butadiene hydrogenation. Focusing on particle size and composition, optimal catalytic performance is observed with Ni catalysts, where smaller metallic Ni particles of ca. 6.2 nm exhibit superior activity and larger ones (14-47 nm) display much higher total butene selectivity. An integral approach combining detailed kinetics, chemisorption, and dual-beam Fourier transform infrared spectroscopic study is performed to rationalize the Ni particle size effect. The findings reveal that smaller Ni particles offer improved activation of butadiene and hydrogen due to advantageous adsorption dynamics. Spectroscopic examinations further suggest different adsorption configurations existing on Ni particles, with larger particles displaying strong it-adsorption, which impedes hydrogen replacement. Additionally, the stability of the catalysts is scrutinized under various reaction conditions, revealing that deactivation occurs more rapidly at lower temperatures, primarily due to mild coke deposition.

Alternaria panax, the primary pathogen that causes ginseng Alternaria leaf blight disease, can lead to a 20–30% reduction in ginseng yield. WD40 repeat-containing proteins are evolutionarily conserved proteins with diverse functions between different organisms. In this study, we characterized the roles of a WD40 repeat-containing protein in A. panax. The deletion of ApWD40a impaired the mycelial growth, reduced the sporulation, and significantly decreased the efficiency in utilizing various carbon sources. The ΔApwd40a mutant showed increased sensitivity to osmotic stress and metal ion stress induced by sorbitol, NaCl, and KCl, but decreased the sensitivity to a cell wall stress factor (SDS) and oxidative stress factors (paraquat and H2O2). Pathogenicity assays performed on detached ginseng leaves and roots revealed that the disruption of ApWD40a significantly decreased the fungal virulence through attenuating melanin and mycotoxin production by A. panax. A comparative transcriptome analysis revealed that ApWD40a was involved in many metabolic and biosynthetic processes, including amino acid metabolism, carbon metabolism, sulfate metabolic pathways, and secondary metabolite pathways. In particular, a significantly upregulated gene that encoded a sulfate permease 2 protein in ΔApwd40a, named ApSulP2, was deleted in the wild-type strain of A. panax. The deletion of ApSulP2 resulted in reduced biomass under sulfate-free conditions, demonstrating that the sulfate transport was impaired. Taken together, our findings highlight that ApWD40a played crucial roles in different biological processes and the pathogenicity of A. panax through modulating the expressions of genes involved in various primary and secondary metabolic processes.

Nitrogen and carbon are the two most essential nutrient elements, and their metabolism is tightly coupled in single carbon metabolic microorganisms. However, the nitrogen metabolism and the nitrogen/carbon (N/C) metabolic balance in single-carbon metabolism is poorly studied. In this study, the nitrogen metabolism pattern of the fast growing methanotrophs Methylomonas sp. ZR1 grown in methane and methanol was studied. Effect study of different nitrogen sources on the cell growth of ZR1 indicates that nitrate salts are the best nitrogen source supporting the growth of ZR1 using methane and methanol as carbon source. However, its metabolic intermediate ammonium was found to accumulate with high N/C ratio in the medium and consequently inhibit the growth of ZR1. Studies of carbon and nitrogen metabolic kinetic under different N/C ratio conditions indicate that the accumulation of NH4+ is caused by the imbalanced nitrogen and carbon metabolism in ZR1. Feeding carbon skeleton α-ketoglutaric acid could effectively relieve the inhibition effect of NH4+ on the growth of ZR1, which further confirms this assumption. qPCR analysis of the expression level of the central metabolic key enzyme gene indicates that the nitrogen metabolic intermediate ammonium has strong regulation effect on the central nitrogen and carbon metabolism in ZR1. qPCR-combined genomic analysis confirms that a third ammonium assimilation pathway glycine synthesis system is operated in ZR1 to balance the nitrogen and carbon metabolism. Based on the qPCR result, it was also found that ZR1 employs two strategies to relieve ammonium stress in the presence of ammonium: assimilating excess ammonium or cutting off the nitrogen reduction reactions according to the available C1 substrate. Validating the connections between single-carbon and nitrogen metabolism and studying the accumulation and assimilation mechanism of ammonium will contribute to understand how nitrogen regulates cellular growth in single-carbon metabolic microorganisms.

Fungi uniquely synthesize lysine through the α-aminoadipate pathway. The saccharopine reductase ScLys9 catalyzes the formation of saccharopine from ɑ-aminoadipate 6-semialdehyde, the seventh step in the lysine biosynthesis pathway in Saccharomyces cerevisiae. Here, we characterized the functions of TrLys9, an ortholog of S. cerevisiae ScLys9 in the industrial filamentous fungus Trichoderma reesei. Transcription-level analysis indicated that TrLYS9 expression was higer in the conidia stage than in other stages. Disruption of TrLYS9 led to lysine auxotrophy. Phenotype analysis of the ΔTrlys9 mutant showed that TrLYS9 was involved in fungal development including vegetative growth, conidiation, and conidial germination and lysine biosynthesis. Cellulase production was also impaired in the ΔTrlys9 mutant due to the failure of conidial germination in liquid cellulase-inducing liquid medium. Defects in radial growth and asexual development of the ΔTrlys9 mutant were fully recovered when exogenous lysine was added to the medium. These results imply that TrLys9 is involved in fungal development and lysine biosynthesis in T. reesei.

Supported metal clusters comprising of well-tailored low-nuclearity heteroatoms have great potentials in catalysis owing to the maximized exposure of active sites and metal synergy. However, atomically precise design of these architectures is still challenging for the lack of practical approaches. Here, we report a defect-driven nanostructuring strategy through combining defect engineering of nitrogen-doped carbons and sequential metal depositions to prepare a series of Pt and Mo ensembles ranging from single atoms to sub-nanoclusters. When applied in continuous gas-phase decomposition of formic acid, the low-nuclearity ensembles with unique Pt3Mo1N3 configuration deliver high-purity hydrogen at full conversion with unexpected high activity of 0.62 molHCOOH molPt-1 s-1 and remarkable stability, significantly outperforming the previously reported catalysts. The remarkable performance is rationalized by a joint operando dual-beam Fourier transformed infrared spectroscopy and density functional theory modeling study, pointing to the Pt-Mo synergy in creating a new reaction path for consecutive HCOOH dissociations.

Cordyceps militaris is a high-value medicinal and edible fungus that produces many bioactive compounds, including carotenoid, and thus, improving the carotenoid productivity of C. militaris will increase its commercial value. However, little is known about the genetic regulatory mechanism of carotenoid biosynthesis in C. militaris. To further understanding the regulatory mechanism of carotenoid biosynthesis, we performed a large-scale screen of T-DNA insertional mutant library and identified a defective mutant, denoted T111, whose colonies did not change color from white to yellow upon exposure to light. Mutation analysis confirmed that a single T-DNA insertion occurred in the gene encoding a 695-amino-acid putative fungal-specific transcription factor with a predicted Zn2Cys6 binuclear cluster DNA-binding domain found uniquely in fungi. Targeted deletion of this gene, denoted C. militaris carotenogenesis regulatory factor 1 (Cmcrf1), generated the ΔCmcrf1 mutant that exhibited drastically reduced carotenoid biosynthesis and failed to generate fruiting bodies. In addition, the ΔCmcrf1 mutant showed significantly increased conidiation and increased hypersensitivity to cell-wall-perturbing agents compared with the wild-type strain. However, the Cmcrf1 gene did not have an impact on the mycelia growth of C. militaris. These results show that Cmcrf1 is involved in carotenoid biosynthesis and is required for conidiation and fruiting body formation in C. militaris.

Acetylene coupling with ethylene dichloride,which uses both coal and oil resources,is attractive for sustainable PVC manufacturing.Herein,highly active and stable carbon nitride-based catalysts were developed by a novel pre-oxidation-pyrolysis process,affording unprecedented dehydrochlorina-tion activity with good durability.The best-performing system was further modified with different precious metals (Au,Pt,and Ru) to promote the hydrochlorination chemistry between the in-situ formed hydrogen chloride and acetylene co-feed.The presence of metal centers intensifies the hy-drochlorination activity but weakens the dehydrochlorination ability due to competitive adsorption between the two reactants at the metal sites.Superior coupling performance was achieved over C3N4/AC and single-atom Au/C3N4/AC catalysts in cascade reactors.Our results strongly suggest that dehydrochlorination is an essential step in the coupling reaction,and the activation of acetylene and ethylene dichloride molecules requires different active sites that should be engineered in future work.

Nitrogen-doped carbons (NC) and carbon nitride (C3N4) belong to the two fundamental pillars of functionalized carbon materials that have broad applications in different fields. The synthesis of NC and C3N4 often starts from different precursors, and a general approach that can offer both architectures is still highly sought. Herein, a facile Triton X-100-assisted copolymerization-carbonization strategy is reported to fulfill this task. By moderating the weight ratios of Triton X-100 and several conventional precursors of C3N4 (e.g., dicyandiamide, urea, and cyanamide), a series of both types of N-functionalized carbons with controllable N dopants is successfully afforded. The yielded solids transform from graphitic C3N4 with low surface areas to porous NC when the weight ratio reaches the critical values. This phenomenon might be attributed to the intensified copolymerization between the intermediates of both starting precursors during the carbonization, resulting in increased oxygen content and decreased N:C ratio in the products. Both the bulk and activated carbon-supported materials are then designed and used as metal-free catalysts in the dehydrochlorination of ethylene dichloride, a key reaction in polyvinyl chloride manufacture. It is found that NC is significantly more active than C3N4, but the corresponding supported catalysts exhibit an opposing trend. The structure-activity correlations suggest both the number of different N defects and the surface areas might play key roles in determining the dehydrochlorination chemistry.

Low enzymatic hydrolysis efficiency prevents the economic biotechnological conversion of lignocellulose to valuable products. To improve the lignocellulose degradation, artificial fusion enzymes of swollenin (SWOI) and xylanase (XYNII) from Trichoderma reesei were constructed and utilized in lignocellulose hydrolysis. Different connection modes between swo1 and xyn2 were shown to have a significant effect on the activity of the fusion enzyme. Xylanase activity of fusion enzyme SWOI-XYNII (S-X) obtained by connecting C-terminal of SWOI and N-terminal of XYNII increased, while that of XYNII-SWOI (X-S) decreased. Xylanase activity of SWOI-(GGGGS)2XYNII (S-2X) with a connecting peptide (GlyGlyGlyGlySer)2 between SWOI and XYNII was further improved comparing to that of S-X. Compared with XYNII, S-2X improved the production of reducing sugar from alkalitreated corn (Zea mays L.) cob by 42 %. When acting on alkali-treated corn cob together with endoglucanase EGII, S-2X increased the yield of reducing sugar by 58 % comparing with XYNII. The swollenin-xylanase fusion enzyme was proved to be conducive to plant cell wall decomposition and could be a promising candidate to enhance the efficiency of enzyme cocktails for lignocellulose degradation.

Methylomonas sp. ZR1 was an isolated new methanotrophs that could utilize methane and methanol growing fast and synthesizing value added compounds such as lycopene. In this study, the genomic study integrated with the comparative transcriptome analysis were taken to understanding the metabolic characteristic of ZR1 grown on methane and methanol at normal and high temperature regime. Complete Embden-Meyerhof-Parnas pathway (EMP), Entner–Doudoroff pathway (ED), Pentose Phosphate Pathway (PP) and Tricarboxy Acid Cycle (TCA) were found to be operated in ZR1. In addition, the energy saving ppi-dependent EMP enzyme, coupled with the complete and efficient central carbon metabolic network might be responsible for its fast growing nature. Transcript level analysis of the central carbon metabolism indicated that formaldehyde metabolism was a key nod that may be in charge of the carbon conversion efficiency (CCE) divergent of ZR1 grown on methanol and methane. Flexible nitrogen and carotene metabolism pattern were also investigated in ZR1. Nitrogenase genes in ZR1 were found to be highly expressed with methane even in the presence of sufficient nitrate. It appears that, higher lycopene production in ZR1 grown on methane might be attributed to the higher proportion of transcript level of C40 to C30 metabolic gene. Higher transcript level of exopolysaccharides metabolic gene and stress responding proteins indicated that ZR1 was confronted with severer growth stress with methanol than with methane. Additionally, lower transcript level of the TCA cycle, the dramatic high expression level of the nitric oxide reductase and stress responding protein, revealed the imbalance of the central carbon and nitrogen metabolic status, which would result in the worse growth of ZR1 with methanol at 30 °C.

为了选育高产二十碳五烯酸(eicosapentaenoic acid,EPA)和二十二碳六烯酸(docosahexaenoic acid,DHA)的裂殖壶菌,本研究采用了紫外诱变的方法对原始菌株B4D1进行诱变,将诱变的突变菌株进行培养和油脂提取,然后用气相色谱仪(GC)进行脂肪酸各组分含量的分析,最终获得一株高产EPA和DHA的菌株11-9E,它的EPA和DHA含量分别达到总油脂的0.47％和41.24％,而原始菌株的EPA和DHA仅占总油脂含量的0.32％和36.92％.相对于原始菌株,突变菌株11-9E的EPA和DHA含量分别提高了 46.88％和11.70％.本研究为裂殖壶菌的发酵产业提供了优良菌株,对EPA和DHA的产业化生产具有重大意义.

The aroma characteristics of Pu-erh tea were studied in this work. A total of 117 volatile compounds were identified by HS-SPME combined with GC–TOF/MS, of which 29 active aroma compounds were identified by olfactometry. The active aroma compounds of Pu-erh tea were analyzed by intensity aroma (IA), aroma extraction dilution (AEDA), sensory quantification value (MF), detection frequency (DFA) and odor activity value (OAV). 24, 21 and 23 active aroma components were identified by DFA, AEDA and OAV, while 20 by IA and MF. Recombination of the obtained active aroma components indicated that OAV method was best for determining the active aroma compounds as the aroma profile of OAV recombination model was the most similar to that of Pu-erh tea sample. Omission test of the OAV recombination model furtherly identified 19 characteristic active aroma compounds of Pu-erh tea, and linalool, 1,2,3-trimenthoxybenzebe, 1,2,4-trimethoxybenzene and ɑ-ionone were the key characteristic active aromatic compounds.

The hybridization of abundant transition metals with facile nitrogen-doped carbons is an effective strategy to fabricate advanced functional materials for targeted applications. Herein, we demonstrate a novel and straightforward synthetic approach of preparing a series of different transition metal-modified nitrogen-doped carbons (M/NC, M=Fe, Co, Ni) through the addition of corresponding metal sulfates during the polymerization of aniline, followed by high-temperature (873-1273 K) pyrolysis treatment. While Fe/NC features predominantly single Fe atoms, nanoparticles of mixed metal oxides and or carbonates are formed on Co/NC and Ni/NC. When employed in the selective oxidation of ethyl benzene by using tert-butyl hydroperoxide both as the solvent and the oxidant, these materials showed promoted catalytic response with the order of Fe>Co>Ni as compared with the metal-free analogue. The performance can be further tuned by controlling the pyrolysis temperature, and the best performance can be achieved over Fe/NC-1273 (0.153 wt.% Fe), affording an unprecedented acetophenone yield of 98 % and excellent cycling performance.

Genetically encoded biosensors are powerful tools used to screen metabolite-producing microbial strains. Traditionally, biosensor-based screening approaches also use fluorescence-activated cell sorting (FACS). However, these approaches are limited by the measurement of intracellular fluorescence signals in single cells, rather than the signals associated with populations comprising multiple cells. This characteristic reduces the accuracy of screening because of the variability in signal levels among individual cells. To overcome this limitation, we introduced an approach that combined biosensors with droplet microfluidics (i.e., fluorescence-activated droplet sorting, FADS) to detect labeled cells at a multi-copy level and in an independent droplet microenvironment. We used our previously reported genetically encoded biosensor, 3-dehydroshikimic acid (3-DHS), as a model with which to establish the biosensor-based FADS screening method. We then characterized and compared the effects of the sorting method on the biosensor-based screening system by subjecting the same mutant library to FACS and FADS. Notably, our developed biosensor-enabled, droplet microfluidics-based FADS screening system yielded an improved positive mutant enrichment rate and increased productivity by the best mutant, compared with the single-cell FACS system. In conclusion, the combination of a biosensor and droplet microfluidics yielded a more efficient screening method that could be applied to the biosensor-based high-throughput screening of other metabolites.

Decorating small copper nanoparticles with isolated and/or clustered oxophilic rhenium species enables the first low-copper catalytic technology for intensified and stable ethanol production under mild conditions in dimethyl oxalate hydrogenation. Addition of rhenium induces hydrogenolysis functionality and strong electronic interactions with copper, accounting for the remarkable performance.

The metabolic intermediates of nitrogen source have been proved to have multiple functions on the metabolism of mehthanotrophs. In this study, accumulation and assimilation mechanism of the nitrate metabolic intermediate ammonium in the fast growing Methylomonas sp. ZR1 was analyzed. Although, nitrate salt was the best nitrogen source supporting the growth of ZR1, its metabolic intermediate ammonium would accumulate and inhibit ZR1. Kinetic studies indicated that accumulation of NH4+ was deduced from the imbalance of nitrogen and carbon metabolism. Compensation of carbon skeleton α-keto-glutaramate could effectively relieve the inhibition of NH4+ to ZR1, which further approved the assumption. qPCR analysis indicated a third ammonium assimilation pathway Glycine synthesis system may function in ZR1 under high ammonium tension. In the presence of ammonium, ZR1 might employ two strategies to relieve the ammonium stress, one was assimilating the excess ammonium, and another one was cutting off the nitrogen reduction reactions. Investigation of the nitrogen metabolism and its influence to the carbon metabolism is meaningful to systematically understand and control the C1 feedstock bioconversion process in methanotrophs.Importance The nitrogen metabolism in methanotrophs has long been concerned. However, there are lots of research problems yet to be solved. In this study, the accumulation and assimilation mechanism of the nitrogen metabolic intermediate ammonium in the fast growing Methylomonas sp. ZR1 was analyzed. Owing to the imbalance metabolism of carbon and nitrogen source, ammonium would accumulate to high concentrations to inhibit cell growth. Compensation of carbon skeleton was an effective strategy to relieve the inhibition of NH4+. A third ammonium assimilation pathway related genes were proved actively expressing in ZR1 when it confronted with high ammonium tension. When confronted with ammonium tension, ZR1 might employ different strategies to relieve the ammonium stress according to the edible carbon source. Revealing the endogenous ammonium accumulation mechanism and its metabolic adjustment effect on the central metabolism of methanotrophs, was meaningful to reveal the complex coordination metabolic mechanism of nitrogen and carbon in methanotrophs.

An inducer is crucial for cellulase production. In this study, duckweed was used as an inducer of cellulase production by Trichoderma reesei RUT C30. In a reaction induced by 50 g l-1 duckweed in shake flasks, the filter-paper activity (FPA) reached 6.5 FPU ml-1, a value comparable to that induced by avicel. The enzyme-hydrolysis rate induced by steam-exploded corn stalks was 54.2%, representing a 28% improvement over that induced by avicel. The duckweed starch was hydrolyzed to glucose, which was subsequently used for biomass accumulation during the fermentation process. Furthermore, to optimize control of the fermentation process, a combined substrate of avicel and duckweed was used to induce cellulase production by T. reesei RUT C30. The cellulase production and hydrolysis rates for the combined substrate, compared with avicel alone, were 39.6% and 36.7% higher, respectively. The results of this study suggest that duckweed is a good inducer of cellulase production in T. reesei, and it might aid in decreasing the cost of lignocellulosic-material hydrolysis.

The selection of improved producers among the huge number of variants in mutant libraries is a key issue in filamentous fungi of industrial biotechnology. Here, we developed a droplet-based microfluidic high-throughput screening platform for selection of high-cellulase producers from filamentous fungus Trichoderma reesei. The screening system used a fluorogenic assay to measure amount of cellulase and its activity. The key effectors such as cellulase-inducing medium, spore germination, droplet cultivation time, droplet fluorescence signal detection, and droplet cell sorting were studied. An artificial pre-mixed library of high- and low-cellulase-producing T. reesei strains was screened successfully to verify the feasibility of our method. Finally, two cellulase hyperproducers exhibiting improvements in cellulase activity of 27% and 46% were isolated from an atmospheric and room-temperature plasma (ARTP)-mutated library. This high-throughput screening system could be applied to the engineering of T. reesei strains and other industrially valuable protein-producing filamentous fungi.

Nitrogen-doped carbons are promising materials for a broad range of applications. However, their rational design is greatly hampered by the lack of efficient methods to control the nitrogen speciation, which not only causes controversy about the roles of specific nitrogen functionalities but also hinders investigations into other physicochemical characteristics of these materials. We herein present a cutting-edge strategy that allows a systematic tuning of the electrical conductivity of polyaniline-derived N-doped carbons at a defined nitrogen speciation and content, and similar porous properties. By application of these model systems in acetylene hydrochlorination, a major industrial technology for the production of polyvinyl chloride, we provide insights into the active sites and the reaction mechanism and disclose two key catalytic descriptors for N-doped carbons in this reaction: (i) a high content of pyrrolic-N functionalities, promoting the adsorption of the reactants, and (ii) good electrical conductivity, likely influencing the surface diffusion of adsorbed species. Since increasing the electrical conductivity leads to a reduced nitrogen content, the interplay between these two properties must be carefully controlled to maximize catalytic performance. This understanding enabled the design of the first N-doped carbon catalyst that rivals the unprecedented activity of benchmark gold-based systems in acetylene hydrochlorination.

The filamentous fungus Trichoderma reesei, the most widely used cellulase producer, also has promising applications in lignocellulose-based biorefinery: consolidated bioprocessing for the production of high value-added products. However, such applications are thwarted by the time-consuming metabolic engineering processes (design–build–test–learn cycle) for T. reesei, resulted from (i) the spore separation-mediated purification as the multinucleate hyphae, (ii) transformant screening for high expression levels since unavailable of episomal expression system, and (iii) cases of inexpressible heterologous proteins.