Microbial life-history strategies mediate microbial carbon pump efficacy in response to N management depending on stoichiometry of microbial demand.

The soil microbial carbon pump (MCP) is increasingly acknowledged as being directly linked to soil organic carbon (SOC) accumulation and stability. Given the close coupling of carbon (C) and nitrogen (N) cycles and the constraints imposed by their stoichiometry on microbial growth, N addition might affect microbial growth strategies with potential consequences for necromass formation and carbon stability. However, this topic remains largely unexplored. Based on two multi-level N fertilizer experiments over 10 years in two soils with contrasting soil fertility located in the North (Cambisol, carbon-poor) and Southwest (Luvisol, carbon-rich), we hypothesized that different resource demands of microorganism elicit a trade-off in microbial growth potential (Y-strategy) and resource-acquisition (A-strategy) in response to N addition, and consequently on necromass formation and soil carbon stability. We combined measurements of necromass metrics (MCP efficacy) and soil carbon stability (chemical composition and mineral associated organic carbon) with potential changes in microbial life history strategies (assessed via soil metagenomes and enzymatic activity analyses). The contribution of microbial necromass to SOC decreased with N addition in the Cambisol, but increased in the Luvisol. Soil microbial life strategies displayed two distinct responses in two soils after N amendment: shift toward A-strategy (Cambisol) or Y-strategy (Luvisol). These divergent responses are owing to the stoichiometric imbalance between microbial demands and resource availability for C and N, which presented very distinct patterns in the two soils. The partial correlation analysis further confirmed that high N addition aggravated stoichiometric carbon demand, shifting the microbial community strategy toward resource-acquisition which reduced carbon stability in Cambisol. In contrast, the microbial Y-strategy had the positive direct effect on MCP efficacy in Luvisol, which greatly enhanced carbon stability. Such findings provide mechanistic insights into the stoichiometric regulation of MCP efficacy, and how this is mediated by site-specific trade-offs in microbial life strategies, which contribute to improving our comprehension of soil microbial C sequestration and potential optimization of agricultural N management.

Pursuing sustainable productivity with millions of smallholder farmers.

Sustainably feeding a growing population is a grand challenge, and one that is particularly difficult in regions that are dominated by smallholder farming. Despite local successes, mobilizing vast smallholder communities with science- and evidence-based management practices to simultaneously address production and pollution problems has been infeasible. Here we report the outcome of concerted efforts in engaging millions of Chinese smallholder farmers to adopt enhanced management practices for greater yield and environmental performance. First, we conducted field trials across China's major agroecological zones to develop locally applicable recommendations using a comprehensive decision-support program. Engaging farmers to adopt those recommendations involved the collaboration of a core network of 1,152 researchers with numerous extension agents and agribusiness personnel. From 2005 to 2015, about 20.9 million farmers in 452 counties adopted enhanced management practices in fields with a total of 37.7 million cumulative hectares over the years. Average yields (maize, rice and wheat) increased by 10.8-11.5%, generating a net grain output of 33 million tonnes (Mt). At the same time, application of nitrogen decreased by 14.7-18.1%, saving 1.2 Mt of nitrogen fertilizers. The increased grain output and decreased nitrogen fertilizer use were equivalent to US$12.2 billion. Estimated reactive nitrogen losses averaged 4.5-4.7 kg nitrogen per Megagram (Mg) with the intervention compared to 6.0-6.4 kg nitrogen per Mg without. Greenhouse gas emissions were 328 kg, 812 kg and 434 kg CO2 equivalent per Mg of maize, rice and wheat produced, respectively, compared to 422 kg, 941 kg and 549 kg CO2 equivalent per Mg without the intervention. On the basis of a large-scale survey (8.6 million farmer participants) and scenario analyses, we further demonstrate the potential impacts of implementing the enhanced management practices on China's food security and sustainability outlook.

A steady-state N balance approach for sustainable smallholder farming.

Hundreds of millions of smallholders in emerging countries substantially overuse nitrogen (N) fertilizers, driving local environmental pollution and global climate change. Despite local demonstration-scale successes, widespread mobilization of smallholders to adopt precise N management practices remains a challenge, largely due to associated high costs and complicated sampling and calculations. Here, we propose a long-term steady-state N balance (SSNB) approach without these complications that is suitable for sustainable smallholder farming. The hypothesis underpinning the concept of SSNB is that an intensively cultivated soil-crop system with excessive N inputs and high N losses can be transformed into a steady-state system with minimal losses while maintaining high yields. Based on SSNB, we estimate the optimized N application range across 3,824 crop counties for the three staple crops in China. We evaluated SSNB first in ca. 18,000 researcher-managed on-farm trials followed by testing in on-farm trials with 13,760 smallholders who applied SSNB-optimized N rates under the guidance of local extension staff. Results showed that SSNB could significantly reduce N fertilizer use by 21 to 28% while maintaining or increasing yields by 6 to 7%, compared to current smallholder practices. The SSNB approach could become an effective tool contributing to the global N sustainability of smallholder agriculture.

Updated loss factors and high-resolution spatial variations for reactive nitrogen losses from Chinese rice paddies.

Anthropogenic reactive nitrogen (Nr) loss has been a critical environmental issue. However, due to the limitations of data availability and appropriate methods, the estimation of Nr loss from rice paddies and associated spatial patterns at a fine scale remain unclear. Here, we estimated the background Nr loss (BNL, i.e., Nr loss from soils without fertilization) and the loss factors (the percentage of Nr loss from synthetic fertilizer, LFs) for five loss pathways in rice paddies and identified the national 1 × 1 km spatial variations using data-driven models combined with multi-source data. Based on established machine learning models, an average of 23.4% (15.3-34.6%, 95% confidence interval) of the synthetic N fertilizer was lost to the environment, in the forms of NH3 (17.4%, 10.9-26.7%), N2O (0.5%, 0.3-0.8%), NO (0.2%, 0.1-0.4%), N leaching (3.1%, 0.8-5.7%), and runoff (2.3%, 0.6-4.5%). The total Nr loss from Chinese rice paddies was estimated to be 1.92 ± 0.52 Tg N yr-1 in 2021, in which synthetic fertilizer-induced Nr loss accounted for 69% and BNL accounted for the other 31%. The hotspots of Nr loss were concentrated in the middle and lower regions of the Yangtze River, an area with extensive rice cultivation. This study improved the estimation accuracy of Nr losses and identified the hotspots, which could provide updated insights for policymakers to set the priorities and strategies for Nr loss mitigation.

Data-driven estimation of nitric oxide emissions from global soils based on dominant vegetation covers.

Soils are a major source of global nitric oxide (NO) emissions. However, estimates of soil NO emissions have large uncertainties due to limited observations and multifactorial impacts. Here, we mapped global soil NO emissions, integrating 1356 in-situ NO observations from globally distributed sites with high-resolution climate, soil, and management practice data. We then calculated global and national total NO budgets and revealed the contributions of cropland, grassland, and forest to global soil NO emissions at the national level. The results showed that soil NO emissions were explained mainly by N input, water input and soil pH. Total above-soil NO emissions of the three vegetation cover types were 9.4 Tg N year-1 in 2014, including 5.9 Tg N year-1 (1.04, 95% confidence interval [95% CI]: 0.09-1.99 kg N ha-1 year-1 ) emitted from forest, 1.7 Tg N year-1 (0.68, 95% CI: 0.10-1.26 kg N ha-1 year-1 ) from grassland, and 1.8 Tg N year-1 (0.98, 95% CI: 0.42-1.53 kg N ha-1 year-1 ) from cropland. Soil NO emissions in approximately 57% of 213 countries surveyed were dominated by forests. Our results provide updated inventories of global and national soil NO emissions based on robust data-driven models. These estimates are critical to guiding the mitigation of soil NO emissions and can be used in combination with biogeochemical models.

An enhanced electron transport chain improved astaxanthin production in Phaffia rhodozyma.

Astaxanthin (AX) is a carotenoid pigment with antioxidant properties widely used as a feed supplement. Wild-type strains of Phaffia rhodozyma naturally produce low AX yields, but we increased AX yields 50-fold in previous research using random mutagenesis of P. rhodozyma CBS6938 and fermentation optimization. On that study, genome changes were linked with phenotype, but relevant metabolic changes were not resolved. In this study, the wild-type and the superior P. rhodozyma mutant strains were grown in chemically defined media and instrumented fermenters. Differential kinetic, metabolomics, and transcriptomics data were collected. Our results suggest that carotenoid production was mainly associated with cell growth and had a positive regulation of central carbon metabolism metabolites, amino acids, and fatty acids. In the stationary phase, amino acids associated with the TCA cycle increased, but most of the fatty acids and central carbon metabolism metabolites decreased. TCA cycle metabolites were in abundance and media supplementation of citrate, malate, α-ketoglutarate, succinate, or fumarate increased AX production in the mutant strain. Transcriptomic data correlated with the metabolic and genomic data and found a positive regulation of genes associated with the electron transport chain suggesting this to be the main driver for improved AX production in the mutant strain.

Fertilization as the most critical factor affecting yield response and agronomic efficiency of phosphorus in Chinese rice production: evidence from multi-location field trials.

BACKGROUND: Efficient utilization of phosphorus (P) has been a major challenge for sustainable agriculture. However, the responses of fertilizer rate, region, soil properties, cropping systems and genotypes to P have not been investigated comprehensively and systematically. RESULTS: A comprehensive analysis of 9863 fertilizer-P experiments on rice cultivation in China showed that rice yield  increased first and then fell down with the addition of P fertilizer, and the highest yield of 7963 kg ha-1 was observed under 100% P treatment. Under 100% P treatment, the yield response of applied P (YRP ) and agronomic efficiency of applied P (AEP ) were 12.8% and 30.1 kg ha-1 , respectively. Lower soil pH (< 5.5) and organic matter (< 30.0 g kg-1 ) were associated with lower YRP and AEP . By contrast, soil available P < 25.0 mg kg-1 resulted in decreased YRP (15.3 to 11.4%) and AEP (32.3 kg kg-1 to 26.2 kg kg-1 ), whereas soil available P > 25.0 mg kg-1 maintained the relatively stable YRP and AEP . Also, the YRP and AEP were significantly higher for single-cropping rice compared to other cropping systems. Moreover, the rice genotypes such as 'Longdun', 'Kendao' and 'Jigeng' had higher YRP and AEP than the average value. Overall, the fertilizer-P rate was the primary factor affecting YRP and AEP , and the recommended P fertilizer rate can be reduced by 9-21 kg P ha-1 compared to existing expert recommendations. CONCLUSION: The present study highlights the role of fertilizer-P rate in maximizing the YRP and AEP , thereby providing a strong basis for future fertilizer management in rice cultivation systems. © 2023 Society of Chemical Industry.

Evaluation of variation in background nitrous oxide emissions: A new global synthesis integrating the impacts of climate, soil, and management conditions.

Robust global simulation of soil background N2 O emissions (BNEs) is a challenge due to the lack of a comprehensive system for quantification of the variations in their magnitude and location. We mapped global BNEs based on 1353 field observations from globally distributed sites and high-resolution climate and soil data. We then calculated global and national total BNE budgets and compared them to the IPCC-estimated values. The average BNE was 1.10, 0.92, and 0.84 kg N ha-1  year-1 with variations from 0.18 to 3.47 (5th-95th percentile, hereafter), 0.20 to 3.44, and -1.16 to 3.70 kg N ha-1  year-1 for cropland, forestland, and grassland, respectively. Soil pH, soil N mineralization, atmospheric N deposition, soil volumetric water content, and soil temperature were the principle significant drivers of BNEs. The total BNEs of three land use types was lower than IPCC-estimated total BNEs by 0.83 Tg (1012  g) N year-1 , ranging from -47% to 94% across countries. The estimated BNE with cropland values were slightly higher than the IPCC estimates by 0.11 Tg N year-1 , and forestland and grassland lower than the IPCC estimates by 0.4 and 0.54 Tg N year-1 , respectively. Our study underlined the necessity for detailed estimation of the spatial distribution of BNEs to improve the estimates of global N2 O emissions and enable the establishment of more realistic and effective mitigation measures.

Closing yield gaps in China by empowering smallholder farmers.

Sustainably feeding the world's growing population is a challenge, and closing yield gaps (that is, differences between farmers' yields and what are attainable for a given region) is a vital strategy to address this challenge. The magnitude of yield gaps is particularly large in developing countries where smallholder farming dominates the agricultural landscape. Many factors and constraints interact to limit yields, and progress in problem-solving to bring about changes at the ground level is rare. Here we present an innovative approach for enabling smallholders to achieve yield and economic gains sustainably via the Science and Technology Backyard (STB) platform. STB involves agricultural scientists living in villages among farmers, advancing participatory innovation and technology transfer, and garnering public and private support. We identified multifaceted yield-limiting factors involving agronomic, infrastructural, and socioeconomic conditions. When these limitations and farmers' concerns were addressed, the farmers adopted recommended management practices, thereby improving production outcomes. In one region in China, the five-year average yield increased from 67.9% of the attainable level to 97.0% among 71 leading farmers, and from 62.8% to 79.6% countywide (93,074 households); this was accompanied by resource and economic benefits.

Mitigation of Multiple Environmental Footprints for China's Pig Production Using Different Land Use Strategies.

Pig production contributes considerably to land use and greenhouse gas (GHG) and reactive nitrogen (Nr) emissions. Land use strategies were widely proposed, but the spillover effects on biological flow are rarely explored. Here, we simultaneously assessed the carbon (C), nitrogen (N), and cropland footprints of China's pig production at the provincial scale in 2017. The environmental impacts of land use strategies were further evaluated. Results show that one kg live-weight pig production generated an average of 1.9 kg CO2-equiv and 59 g Nr emissions, occupying 3.5 m2 cropland, with large regional variations. A large reduction in GHG (58-64%) and Nr (12-14%) losses and occupied cropland (10-11%) could be achieved simultaneously if combined strategies of intensive crop production, improved feed-protein utilization efficiency, and feeding co-products were implemented. However, adopting a single strategy may have environmental side-effects. Reallocating cropland that pigs used for feed to plant food alternatives would enhance human-edible energy (3-20 times) and protein delivery (1-5 times) and reduce C and N footprints, except for rice and vegetables. Reallocating cropland to beef and milk production would decrease energy and protein supply. Therefore, a proper combination of land use strategies is essential to alleviate land use changes and nutrient emissions without sacrificing food supply.

Safeguarding Food Supply and Groundwater Safety for Maize Production in China.

Quantifying sustainable nitrogen (N) management at the national scale is critical for developing targeted policies and strategies to simultaneously achieve food security and groundwater protection. In this study, we report county-scale optimization scenarios for Chinese maize production and evaluate their outcomes for safeguarding food supply and groundwater safety. First, we performed random forest regression modeling to simulate in situ NO3- leaching based on a meta-analysis that integrates climate, soil, water, and N balance parameters. The NO3- leaching was then mapped for 1406 counties based on data compiled from 2.89 million farmer surveys. Average NO3- leaching during the maize growth season was estimated to be 27.6 kg N ha-1, and 56% of counties had groundwater whose nitrate concentrations exceeded drinking water safety levels during 2005-2014. The top 5% farmers in each county produced not only more grain but also greater NO3- leaching. Scenario analysis of potential management changes found that when these top producers combined optimal N management practices, national N use in Chinese maize system was reduced by 25%, from 9.1 to 6.9 Mt, while maize production increased by 6.1%. Modeled NO3- leaching was 0.58 Mt, which was 31% lower than groundwater safety levels and 53% lower than the current leaching amount. This study provides evidence that integrated crop and N management practices implemented at the county level safeguard both maize crop food security and enhance environment sustainability.

Producing more grain with lower environmental costs.

Agriculture faces great challenges to ensure global food security by increasing yields while reducing environmental costs. Here we address this challenge by conducting a total of 153 site-year field experiments covering the main agro-ecological areas for rice, wheat and maize production in China. A set of integrated soil-crop system management practices based on a modern understanding of crop ecophysiology and soil biogeochemistry increases average yields for rice, wheat and maize from 7.2 million grams per hectare (Mg ha(-1)), 7.2 Mg ha(-1) and 10.5 Mg ha(-1) to 8.5 Mg ha(-1), 8.9 Mg ha(-1) and 14.2 Mg ha(-1), respectively, without any increase in nitrogen fertilizer. Model simulation and life-cycle assessment show that reactive nitrogen losses and greenhouse gas emissions are reduced substantially by integrated soil-crop system management. If farmers in China could achieve average grain yields equivalent to 80% of this treatment by 2030, over the same planting area as in 2012, total production of rice, wheat and maize in China would be more than enough to meet the demand for direct human consumption and a substantially increased demand for animal feed, while decreasing the environmental costs of intensive agriculture.

Phosphorus flow analysis in the maize based food-feed-energy systems in China.

Phosphorus (P) is an essential and limiting nutrient for agricultural systems, where the demand for agricultural products such as food, feed, and bio-fuel are the major drivers of the intensification of agricultural production systems. Globally, maize is one of three main cereal crops, a main feedstock for animal production and a substrate for the production of bio-ethanol. This study investigated P flows through the multiple utilization systems of maize (as represented by the subsystems of food, feed and energy production) at a crop level of 2016 as reference year and made future predictions of P flows for the year 2030 based on different scenarios for food-feed-energy systems in China. For 2016, the subsystem of animal production resulted in the highest waste of P due to inappropriate manure management, but the subsystem of value-added products (Bio-fuel production, distillers dried grains with solubles (DDGS), maize-oil) showed the lowest P use efficiency (39%). From the value-added subsystem, 17% of P from the process flow to the subsystem of animal production as DDGS, and 61% of P is wasted associated with wastewater and sludge. Future scenarios of structural adjustments in the maize consumption system predict that the supply of maize for animal feed will be threatened if the policy of the Biofuel National Promotion before 2020 is fully implemented in China, as current maize production will not meet the future demand of food, feed and energy simultaneously. The results emphasized the use of P waste resources and better sludge management from a systems perspective. This also implied the importance of exploring coordinated development and integrated strategies for sustainable P flow management in multiple utilization systems.

Oligonucleotide-mediated tRNA sequestration enables one-pot sense codon reassignment in vitro.

Sense codon reassignment to unnatural amino acids (uAAs) represents a powerful approach for introducing novel properties into polypeptides. The main obstacle to this approach is competition between the native isoacceptor tRNA(s) and orthogonal tRNA(s) for the reassigned codon. While several chromatographic and enzymatic procedures for selective deactivation of tRNA isoacceptors in cell-free translation systems exist, they are complex and not scalable. We designed a set of tRNA antisense oligonucleotides composed of either deoxy-, ribo- or 2'-O-methyl ribonucleotides and tested their ability to efficiently complex tRNAs of choice. Methylated oligonucleotides targeting sequence between the anticodon and variable loop of tRNASerGCU displayed subnanomolar binding affinity with slow dissociation kinetics. Such oligonucleotides efficiently and selectively sequestered native tRNASerGCU directly in translation-competent Escherichia coli S30 lysate, thereby, abrogating its translational activity and liberating the AGU/AGC codons. Expression of eGFP protein from the template harboring a single reassignable AGU codon in tRNASerGCU-depleted E. coli lysate allowed its homogeneous modification with n-propargyl-l-lysine or p-azido-l-phenylalanine. The strategy developed here is generic, as demonstrated by sequestration of tRNAArgCCU isoacceptor in E. coli translation system. Furthermore, this method is likely to be species-independent and was successfully applied to the eukaryotic Leishmania tarentolae in vitro translation system. This approach represents a new direction in genetic code reassignment with numerous practical applications.

Generalizable Protein Biosensors Based on Synthetic Switch Modules.

Allosteric protein switches are key controllers of information and energy processing in living organisms and are desirable engineered control tools in synthetic systems. Here we present a generally applicable strategy for construction of allosteric signaling systems with inputs and outputs of choice. We demonstrate conversion of constitutively active enzymes into peptide-operated synthetic allosteric ON switches by insertion of a calmodulin domain into rationally selected sites. Switches based on EGFP, glucose dehydrogenase, NanoLuciferase, and dehydrofolate reductase required minimal optimization and demonstrated a dynamic response ranging from 1.8-fold in the former case to over 200-fold in the latter case. The peptidic nature of the calmodulin ligand enables incorporation of such synthetic switch modules into higher order sensory architectures. Here, a ligand-mediated increase in proximity of the allosteric switch and the engineered activator peptide modulates biosensor's activity. Created biosensors were used to measure concentrations of clinically relevant drugs and biomarkers in plasma, saliva, and urine with accuracy comparable to that of the currently used clinical diagnostic assays. The approach presented is generalizable as it allows rapid construction of efficient protein switches that convert binding of a broad range of analytes into a biochemical activity of choice enabling construction of artificial signaling and metabolic circuits of potentially unlimited complexity.

Newer and select maize, wheat, and rice varieties can help mitigate N footprint while producing more grain.

Sustainably feeding the growing population amid a changing climate and dwindling resources is a grand challenge facing mankind. Decades-long advancement in crop breeding has progressively elevated yield potential, markedly enhancing global food production capacity. However, relevant impact on reactive N (Nr) emissions associated with crop variety improvement has not been explicitly described. Here, we report multitiered evidence that newer and select maize, wheat, and rice varieties developed in China have the capacity to substantially lower Nr losses while producing more grain. First, we pooled studies featuring side-by-side comparison of different varieties, totaling 269 paired observations, to demonstrate that collectively, relatively newer varieties of maize, wheat, and rice had less Nr emissions (9.6%-23.5%) while yielding more grains (7.3%-11.2%) compared to older varieties under wide-ranging conditions. Next, we built an extended database (142 field studies with 833 observations) and comprehensively evaluated the Nr-loss reduction potential of newer varieties (2000 and after) versus older ones (1985-1999). We found that newer varieties had Nr emission factors (N loss as a percentage of N applied after correcting for background emissions) 18.2%-75.7% less for N2 O, 18.3%-75.7% less for NO 3 - , and -8.5% to 22.8% less for NH3 , while producing more grains (16.0%-24.4%). Individual varieties differed markedly in yield-emission scores. A nationwide farmer survey (2.47 million responses) indicated tremendous opportunities for a new way of management intervention. Coupling variety selection with sound N and other agronomic management can help lower N footprint while producing more grain.

Mycobacterium tuberculosis Lipoprotein MPT83 Induces Apoptosis of Infected Macrophages by Activating the TLR2/p38/COX-2 Signaling Pathway.

Tuberculosis caused by Mycobacterium tuberculosis continues to pose a serious global health threat. The attenuated Mycobacterium bovis bacillus Calmette-Guérin, as the only licensed vaccine, has limited protective efficacy against TB. The development of more effective antituberculosis vaccines is urgent and demands for further identification and understanding of M. tuberculosis Ags. MPT83 (Rv2873), a secreted mycobacterial lipoprotein, has been applied into subunit vaccine development and shown protective effects against M. tuberculosis infection in animals; however, the understanding of the underlying mechanism is limited. In present study, we systematically studied the effect of MPT83 on macrophage apoptosis by constructing Mycobacterium smegmatis strain overexpressing MPT83 (MS_MPT83) and purifying rMPT83 protein. We found that MPT83 induced apoptosis in both human and mouse macrophages. MPT83 induced cyclooxygenase-2 (COX-2) expression at both the transcriptional and protein levels in macrophages, whereas silencing or inhibiting COX-2 blocked rMPT83-induced apoptosis or the enhanced apoptotic response to MS_MPT83 in comparison with M. smegmatis transfected with pMV261 vector (MS_Vec), indicating that COX-2 is required for MPT83-induced apoptosis. Additionally, tlr2 deficiency led to significant reduction of COX-2 expression, accompanied by less apoptosis in macrophages stimulated with rMPT83 or infected with MS_MPT83. Moreover, the activation of p38 accounted for MPT83-induced COX-2 expression. Finally, lower bacteria burdens in the lungs and spleens and enhanced survival were observed in mice i.v. infected with MS_MPT83 compared with MS_Vec. Taken together, our results established a proapoptotic effect of MPT83 and identified the TLR2/p38/COX-2 axis in MPT83-induced macrophage apoptosis.

Enhanced nitrogen deposition over China.

China is experiencing intense air pollution caused in large part by anthropogenic emissions of reactive nitrogen. These emissions result in the deposition of atmospheric nitrogen (N) in terrestrial and aquatic ecosystems, with implications for human and ecosystem health, greenhouse gas balances and biological diversity. However, information on the magnitude and environmental impact of N deposition in China is limited. Here we use nationwide data sets on bulk N deposition, plant foliar N and crop N uptake (from long-term unfertilized soils) to evaluate N deposition dynamics and their effect on ecosystems across China between 1980 and 2010. We find that the average annual bulk deposition of N increased by approximately 8 kilograms of nitrogen per hectare (P < 0.001) between the 1980s (13.2 kilograms of nitrogen per hectare) and the 2000s (21.1 kilograms of nitrogen per hectare). Nitrogen deposition rates in the industrialized and agriculturally intensified regions of China are as high as the peak levels of deposition in northwestern Europe in the 1980s, before the introduction of mitigation measures. Nitrogen from ammonium (NH4(+)) is the dominant form of N in bulk deposition, but the rate of increase is largest for deposition of N from nitrate (NO3(-)), in agreement with decreased ratios of NH3 to NOx emissions since 1980. We also find that the impact of N deposition on Chinese ecosystems includes significantly increased plant foliar N concentrations in natural and semi-natural (that is, non-agricultural) ecosystems and increased crop N uptake from long-term-unfertilized croplands. China and other economies are facing a continuing challenge to reduce emissions of reactive nitrogen, N deposition and their negative effects on human health and the environment.

Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation.

Cellulosomes are multi-enzyme complexes assembled by cellulases and hemicellulases through dockerin-cohesin interactions, which are the most efficient system for the degradation of lignocellulosic resources in nature. Recent genomic analysis of a cellulosome-producing anaerobe Clostridium clariflavum DSM 19732 revealed that two expansin-like proteins, Clocl_1298 and Clocl_1862, contain a dockerin module, which suggests that they are components of the cellulosome. Bacterial expansin-like proteins do not have hydrolytic activities, but can facilitate the degradation of cellulosic biomass via synergistic effects with cellulases. In this study, the synergistic effect of the expansin-like proteins with both native and designer cellulosomes was investigated. The free expansin-like proteins, including expansin-like domains of Clocl_1298 and Clocl_1862, as well as a well-studied bacterial expansin-like protein BsEXLX1 from Bacillus subtilis, promoted the cellulose degradation by native cellulosomes, indicating the cellulosomal expansin-like proteins have the synergistic function. When they were integrated into a trivalent designer cellulosome, the synergistic effect was further amplified. The sequence and structure analyses indicated that these cellulosomal expansin-like proteins share the conserved functional mechanism with other bacterial expansin-like proteins. These results indicated that non-catalytic expansin-like proteins in the cellulosome can enhance the activity of the cellulosome in lignocellulose degradation. The involvement of functional expansin-like proteins in the cellulosome also implies new physiological functions of bacterial expansin-like proteins and cellulosomes.

Semisynthetic tRNA complement mediates in vitro protein synthesis.

Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.