An endogenous promoter LpSUT2 discovered in duckweed: a promising transgenic tool for plants.

Promoters are one of the most critical elements in regulating gene expression. They are considered essential biotechnological tools for heterologous protein production. The one most widely used in plants is the 35S promoter from cauliflower mosaic virus. However, our study for the first time discovered the 35S promoter reduced the expression of exogenous proteins under increased antibiotic stress. We discovered an endogenous strong promoter from duckweed named LpSUT2 that keeps higher initiation activity under antibiotic stress. Stable transformation in duckweed showed that the gene expression of eGFP in the LpSUT2:eGFP was 1.76 times that of the 35S:eGFP at 100 mg.L-1 G418 and 6.18 times at 500 mg.L-1 G418. Notably, with the increase of G418 concentration, the gene expression and the fluorescence signal of eGFP in the 35S:eGFP were weakened, while the LpSUT2:eGFP only changed slightly. This is because, under high antibiotic stress, the 35S promoter was methylated, leading to the gene silencing of the eGFP gene. Meanwhile, the LpSUT2 promoter was not methylated and maintained high activity. This is a previously unknown mechanism that provides us with new insights into screening more stable promoters that are less affected by environmental stress. These outcomes suggest that the LpSUT2 promoter has a high capacity to initiate the expression of exogenous proteins. In conclusion, our study provides a promoter tool with potential application for plant genetic engineering and also provides new insights into screening promoters.

Interpretation of the adsorption process of toxic Cd2+ removal by modified sweet potato residue.

Cadmium (Cd) is a common and toxic non-essential heavy metal that must be effectively treated to reduce its threat to the environment and public health. Adsorption with an adsorbent, such as agricultural waste, is widely used to remove heavy metals from wastewater. Sweet potato, the sixth most abundant food crop worldwide, produces a large amount of waste during postharvest processing that could be used as an economic adsorbent. In this study, the feasibility of using sweet potato residue (SPR) as an adsorbent for Cd2+ adsorption was assessed. To enhance the removal rate, SPR was modified with NaOH, and the effects of the modification and adsorption conditions on the removal of Cd2+ from wastewater were investigated. The results showed that modified sweet potato residue (MSPR) could be adapted to various pH and temperatures of simulated wastewater, implying its potential for multi-faceted application. Under optimized conditions, the removal of Cd2+ by MSPR was up to 98.94% with a maximum adsorption capacity of 19.81 mg g-1. Further investigation showed that the MSPR exhibited rich functional groups, a loose surface, and a mesoporous structure, resulting in advantageous characteristics for the adsorption of Cd2+. In addition, the MSPR adsorbed Cd2+ by complexation, ion exchange, and precipitation during a monolayer chemisorption adsorption process. This work demonstrates a sustainable and environment friendly strategy for Cd2+ removal from wastewater and a simple approach for the preparation of MSPR and also revealed the adsorption mechanism of Cd2+ by MSPR, thus providing a suitable adsorbent and strategy for the removal of other heavy metals.

Insight into broad substrate specificity and synergistic contribution of a fungal α-glucosidase in Chinese Nong-flavor daqu.

BACKGROUND: Chinese Nong-favor daqu, the presentative liquor starter of Baijiu, has been enriched with huge amounts of enzymes in degrading various biological macromolecules by openly man-made process for thousand years. According to previous metatranscriptomics analysis, plenty of α-glucosidases were identified to be active in NF daqu and played the key role in degrading starch under solid-state fermentation. However, none of α-glucosidases was characterized from NF daqu, and their actual functions in NF daqu were still unknown. RESULTS: An α-glucosidase (NFAg31A, GH31-1 subfamily), the second highest expressed α-glucosidases in starch degradation of NF daqu, was directly obtained by heterologous expression in Escherichia coli BL21 (DE3). NFAg31A exhibited the highest sequence identities of 65.8% with α-glucosidase II from Chaetomium thermophilum, indicating its origin of fungal species, and it showed some similar features with homologous α-glucosidase IIs, i.e., optimal activity at pH ~ 7.0 and litter higher temperature of 45 ℃, well stability at 41.3 ℃ and a broad pH range of pH 6.0 to pH 10.0, and preference on hydrolyzing Glc-α1,3-Glc. Besides this preference, NFAg31A showed comparable activities on Glc-α1,2-Glc and Glc-α1,4-Glc, and low activity on Glc-α1,6-Glc, indicating its broad specificities on α-glycosidic substrates. Additionally, its activity was not stimulated by any of those detected metal ions and chemicals, and could be largely inhibited by glucose under solid-state fermentation. Most importantly, it exhibited competent and synergistic effects with two characterized α-amylases of NF daqu on hydrolyzing starch, i.e., all of them could efficiently degrade starch and malto-saccharides, two α-amylases showed advantage in degrading starch and long-chain malto-saccharides, and NFAg31A played the competent role with α-amylases in degrading short-chain malto-saccharides and the irreplaceable contribution in hydrolyzing maltose into glucose, thus alleviating the product inhibitions of α-amylases. CONCLUSIONS: This study provides not only a suitable α-glucosidase in strengthening the quality of daqu, but also an efficient way to reveal roles of the complicated enzyme system in traditional solid-state fermentation. This study would further stimulate more enzyme mining from NF daqu, and promote their actual applications in solid-state fermentation of NF liquor brewing, as well as in other solid-state fermentation of starchy industry in the future.

Improving the quality of barren rocky soil by culturing sweetpotato, with special reference to plant-microbes-soil interactions.

Biological process is an effective strategy to improve soil quality in agroecosystems. Sweetpotato has long been cultivated in barren rocky soil (BRS) to improve soil fertility and obtain considerably high yield. However, how sweetpotato cultivation affects soil quality is still unclear. We cultured sweetpotato in virgin BRS, and investigated its transcriptome, rhizospheric microbial community and soil properties. A high sweetpotato yield (22.69 t.ha-1) was obtained through upregulating the expression of genes associated with stress resistance, nitrogen/phosphorus/potassium (N/P/K) uptake, and root exudates transport. Meanwhile, the rhizospheric microbial diversity in BRS increased, and the rhizospheric microbial community structure became more similar to that of fertile soil, which might benefit from the increased root exudates. Notably, the relative abundances of N-fixing and P/K-solubilizing microbes increased, and the copy number of nifH increased 6.67 times. Moreover, the activities of acid, neutral, and alkaline phosphatases increased strongly from 0.63, 0.02, and 1.15-1.58, 0.31, and 2.11 mg phenol·g-1·d-1, respectively, and total carbon, dissolved organic carbon, available N/P content also increased, while bulk density and pH of BRS decreased, indicating the enhanced soil fertility. Our study found sweetpotato cultivation improved BRS quality through shaping microbial communities, which has important guiding significance for sustainable agriculture.

Rapid and Highly Efficient Genetic Transformation and Application of Interleukin-17B Expressed in Duckweed as Mucosal Vaccine Adjuvant.

Molecular farming utilizes plants as a platform for producing recombinant biopharmaceuticals. Duckweed, the smallest and fastest growing aquatic plant, is a promising candidate for molecular farming. However, the efficiency of current transformation methods is generally not high in duckweed. Here, we developed a fast and efficient transformation procedure in Lemna minor ZH0403, requiring 7-8 weeks from screening calluses to transgenic plants with a stable transformation efficiency of 88% at the DNA level and 86% at the protein level. We then used this transformation system to produce chicken interleukin-17B (chIL-17B). The plant-produced chIL-17B activated the NF-κB pathway, JAK-STAT pathway, and their downstream cytokines in DF-1 cells. Furthermore, we administrated chIL-17B transgenic duckweed orally as an immunoadjuvant with mucosal vaccine against infectious bronchitis virus (IBV) in chickens. Both IBV-specific antibody titer and the concentration of secretory immunoglobulin A (sIgA) were significantly higher in the group fed with chIL-17B transgenic plant. This indicates that the duckweed-produced chIL-17B enhanced the humoral and mucosal immune responses. Moreover, chickens fed with chIL-17B transgenic plant demonstrated the lowest viral loads in different tissues among all groups. Our work suggests that cytokines are a promising adjuvant for mucosal vaccination through the oral route. Our work also demonstrates the potential of duckweed in molecular farming.

Local endocytosis of sucrose transporter 2 in duckweed reveals the role of sucrose transporter 2 in guard cells.

The local endocytosis of membrane proteins is critical for many physiological processes in plants, including the regulation of growth, development, nutrient absorption, and osmotic stress response. Much of our knowledge on the local endocytosis of plasma membrane (PM) protein only focuses on the polar growth of pollen tubes in plants and neuronal axon in animals. However, the role of local endocytosis of PM proteins in guard cells has not yet been researched. Here, we first cloned duckweed SUT2 (sucrose transporter 2) protein and then conducted subcellular and histological localization of the protein. Our results indicated that LpSUT2 (Landoltia punctata 0202 SUT2) is a PM protein highly expressed on guard cells. In vitro experiments on WT (wild type) lines treated with high sucrose concentration showed that the content of ROS (reactive oxygen species) in guard cells increased and stomatal conductance decreased. We observed the same results in the lines after overexpression of the LpSUT2 gene with newfound local endocytosis of LpSUT2. The local endocytosis mainly showed that LpSUT2 was uniformly distributed on the PM of guard cells in the early stage of development, and was only distributed in the endomembrane of guard cells in the mature stage. Therefore, we found the phenomenon of guard cell LpSUT2 local endocytosis through the changes of duckweed stomata and concluded that LpSUT2 local endocytosis might be dependent on ROS accumulation in the development of duckweed guard cells. This paper might provide future references for the genetic improvement and water-use efficiency in other crops.

Effects of various spectral compositions on micro-polluted water purification and biofuel feedstock production using duckweed.

The purification of micro-polluted water for drinking water can play an important role in solving water crisis. To investigate the effects of spectral composition on nutrient removal and biofuel feedstock production using duckweed, Landoltia punctata was cultivated in different spectral compositions in micro-polluted water. Results showed that the nitrogen and phosphorus removal efficiency were 99.4% and 93.5% at an recommended red and blue light photon intensity mixture ratio of 2:1. Meanwhile, maximum growth rate of duckweed (11.37 g/m2/day) was observed at red/blue = 2:1. In addition, maximum starch accumulation rate of duckweed was found to be 6.12 g/m2/day, with starch content of 36.63% at red/blue = 4:1, which was three times higher when compared to that of white light. Moreover, the recommended ratio of red and blue light was validated by economic efficiency analysis of energy consumptions. These findings provide a sustainable environmental restoration method to transform water micro-pollutants to available substances.

Multi-Omics Reveal the Efficient Phosphate-Solubilizing Mechanism of Bacteria on Rocky Soil.

Phosphate-solubilizing bacteria (PSB) can alleviate available phosphorus (AP)-deficiency without causing environmental pollution like chemical phosphate fertilizers. However, the research and application of PSB on the barren rocky soil is very rare. We screened six PSB from sweetpotato rhizosphere rocky soil. Among them, Ochrobactrum haematophilum FP12 showed the highest P-solubilizing ability of 1,085.00 mg/L at 7 days, which was higher than that of the most reported PSB. The assembled genome of PSB FP12 was 4.92 Mb with P-solubilizing and plant growth-promoting genes. In an AP-deficient environment, according to transcriptome and metabolomics analysis, PSB FP12 upregulated genes involved in gluconic acid synthesis and the tricarboxylic acid cycle, and increased the concentration of gluconic acid and malic acid, which would result in the enhanced P-solubilizing ability. Moreover, a series of experiments in the laboratory and field confirmed the efficient role of the screened PSB on significantly increasing AP in the barren rocky soil and promoting sweetpotato yield. So, in this study, we screened highly efficient PSB, especially suitable for the barren rocky soil, and explored the P-solubilizing mechanism. The research will reduce the demand for chemical phosphate fertilizers and promote the environment-friendly agricultural development.

Biochemical and synergistic properties of a novel alpha-amylase from Chinese nong-flavor Daqu.

BACKGROUND: Daqu is the most important fermentation starter for Chinese liquor, with large number of microbes and enzymes being openly enriched in the Daqu system over thousands of years. However, only a few enzymes have been analyzed with crude protein for total liquefying power and saccharifying power of Daqu. Therefore, the complex enzymatic system present in Daqu has not been completely characterized. Moreover, their pivotal and complicated functions in Daqu are completely unknown. RESULTS: In this study, a novel α-amylase NFAmy13B, from GH13_5 subfamily (according to the Carbohydrate-Active enZYmes Database, CAZy) was successfully heterologous expressed by Escherichia coli from Chinese Nong-flavor (NF) Daqu. It exhibited high stability ranging from pH 5.5 to 12.5, and higher specific activity, compared to other GH13_5 fungal α-amylases. Moreover, NFAmy13B did not show activity loss and retained 96% residual activity after pre-incubation at pH 11 for 21 h and pH 12 for 10 h, respectively. Additionally, 1.25 mM Ca2+ significantly improved its thermostability. NFAmy13B showed a synergistic effect on degrading wheat starch with NFAmy13A (GH13_1), another α-amylase from Daqu. Both enzymes could cleave maltotetraose and maltopentaose in same degradation pattern, and only NFAmy13A could efficiently degrade maltotriose. Moreover, NFAmy13B showed higher catalytic efficiency on long-chain starch, while NFAmy13A had higher catalytic efficiency on short-chain maltooligosaccharides. Their different catalytic efficiencies on starch and maltooligosaccharides may be caused by their discrepant substrate-binding region. CONCLUSIONS: This study mined a novel GH13_5 fungal α-amylase (NFAmy13B) with outstanding alkali resistance from Nong-flavor (NF) Daqu. Furthermore, its synergistic effect with NFAmy13A (GH13_1) on hydrolyzing wheat starch was confirmed, and their possible contribution in NF Daqu was also speculated. Thus, we not only provide a candidate α-amylase for industry, but also a useful strategy for further studying the interactions in the complex enzyme system of Daqu.

Ammonium detoxification mechanism of ammonium-tolerant duckweed (Landoltia punctata) revealed by carbon and nitrogen metabolism under ammonium stress.

In this work, the ammonium-tolerant duckweed Landoltia punctata 0202 was used to study the effect of ammonium stress on carbon and nitrogen metabolism and elucidate the detoxification mechanism. The growth status, protein and starch content, and activity of nitrogen assimilation enzymes were determined, and the transcriptional levels of genes involved in ion transport and carbon and nitrogen metabolism were investigated. Under high ammonium stress, the duckweed growth was inhibited, especially when ammonium was the sole nitrogen source. Ammonium might mainly enter cells via low-affinity transporters. The stimulation of potassium transport genes suggested sufficient potassium acquisition, precluding cation deficiency. In addition, the up-regulation of ammonium assimilation and transamination indicated that excess ammonium could be incorporated into organic nitrogen. Furthermore, the starch content increased from 3.97% to 16.43% and 26.02% in the mixed-nitrogen and ammonium-nitrogen groups, respectively. And the up-regulated starch synthesis, degradation, and glycolysis processes indicated that the accumulated starch could provide sufficient carbon skeletons for excess ammonium assimilation. The findings of this study illustrated that the coordination of carbon and nitrogen metabolism played a vital role in the ammonium detoxification mechanism of duckweeds.

Uncovering key residues responsible for the thermostability of a thermophilic 1,3(4)-ß-d-glucanase from Nong flavor Daqu by rational design.

Fungal 1,3(4)-ß-D-glucanases were usually applied in brewing and feedstuff industries, however, the thermostability limits the most their application. The characterized 1,3(4)-ß-D-glucanase (NFEg16A) from Chinese Nong-flavor (NF) Daqu showed the highest thermostability among GH16 fungal 1,3(4)-ß-D-glucanases, with half-lives of thermal inactivation (t1/2) of 44.9 min at 90 °C, so multiple rational designs were used to identify the key residues for its thermostability. Based on protein sequence and 3D structure analyses around the catalytic regions. Nine site-mutants were constructed, among which N173Y and S187A were identified as the most thermotolerant and thermolabile ones, with t1/2 values of 61 min and 14.0 min at 90 °C, respectively. Therefore, N173 and S187 were then selected as "hotspots" for site-saturation mutagenesis. Interestingly, most of the N173 and S187 variants exhibited a similar thermostability to that of N173Y and S187A, respectively, confirming their different roles in the thermostability of NFEg16A. In addition, each S187A and its surrounding substitutions (D144 N and T164 N) was independently detrimental to the thermostability of NFEg16A, since the t1/2 (90 °C) of S187A, D144 N and T164 N were 14.0 min, 20.6 min and 27.2 min, respectively. Surprisingly, combinatorial substitution of S187A with D144 N or T164 N showed positive effects on the thermostability, with the increase of t1/2 (90 °C) to 30.9 min and 63.5 min for S187A-D144 N and S187A-T164 N, respectively. More importantly, S187A-T164 N showed higher thermostability than that of wild type. In short, we successfully identified two key sites and their surrounding residues in response to the thermostability of NFEg16A and further improved its thermostability by several rational designs. These findings could be used for the protein engineering of homologous 1,3(4)-ß-D-glucanases, as well as other enzyme family members with high similarities.

Low Nitrogen Fertilization Alter Rhizosphere Microorganism Community and Improve Sweetpotato Yield in a Nitrogen-Deficient Rocky Soil.

Sweetpotato can be cultivated in the reclaimed rocky soil in Sichuan Basin, China, which benefits from the release of mineral nutrients in the rocky soil by microorganisms. Shortage of nitrogen (N) in the rocky soil limits sweetpotato yield, which can be compensated through N fertilization. Whereas high N fertilization inhibits biological N fixation and induces unintended environmental consequences. However, the effect of low N fertilization on microorganism community and sweetpotato yield in the N-deficient rocky soil is still unclear. We added a low level of 1.5 g urea/m2 to a rocky soil cultivated with sweetpotato, and measured rocky soil physiological and biochemical properties, rhizosphere microbial diversity, sweetpotato physiological properties and transcriptome. When cultivating sweetpotato in the rocky soil, low N fertilization (1.5 g urea/m2) not only improved total N (TN) and available N (AN) in the rocky soil, but also increased available phosphorus (AP), available potassium (AK), and nitrogenase and urease activity. Interestingly, although low N fertilization could reduce bacterial diversity through affecting sweetpotato root exudates and rocky soil properties, the relative abundance of P and K-solubilizing bacteria, N-fixing and urease-producing bacteria increased under low N fertilization, and the relative abundance of plant pathogens decreased. Furthermore, low N fertilization increased the phytohormones, such as zeatin riboside, abscisic acid, and methyl jasmonate contents in sweetpotato root. Those increases were consistent with our transcriptome findings: the inhibition of the lignin synthesis, the promotion of the starch synthesis, and the upregulated expression of Expansin, thus resulting in promoting the formation of tuberous roots and further increasing the sweetpotato yield by half, up to 3.3 kg/m2. This study indicated that low N fertilization in the N-deficient rocky soil improved this soil quality through affecting microorganism community, and further increased sweetpotato yield under regulation of phytohormones pathway.

Unraveling the Contribution of High Temperature Stage to Jiang-Flavor Daqu, a Liquor Starter for Production of Chinese Jiang-Flavor Baijiu, With Special Reference to Metatranscriptomics.

Jiang-flavor (JF) daqu is a liquor starter used for production of JF baijiu, a well-known distilled liquor in China. Although a high temperature stage (70°C) is necessary for qualifying JF daqu, little is known regarding its active microbial community and functional enzymes, along with its role in generating flavor precursors for JF baijiu aroma. In this investigation, based on metatranscriptomics, fungi, such as Aspergillus and Penicillium, were identified as the most active microbial members and 230 carbohydrate-active enzymes were identified as potential saccharifying enzymes at 70°C of JF daqu. Notably, most of enzymes in identified carbohydrate and energy pathways showed lower expression levels at 70°C of JF daqu than those at the high temperature stage (62°C) of Nong-flavor (NF) daqu, indicating lowering capacities of saccharification and fermentation by high temperature stage. Moreover, many enzymes, especially those related to the degradation of aromatic compounds, were only detected with low expression levels at 70°C of JF daqu albeit not at 62°C of NF daqu, indicating enhancing capacities of generating special trace aroma compounds in JF daqu by high temperature stage. Additionally, most of enzymes related to those capacities were highly expressed at 70°C by fungal genus of Aspergillus, Coccidioides, Paracoccidioides, Penicillium, and Rasamsonia. Therefore, this study not only sheds light on the crucial functions of high temperature stage but also paves the way to improve the quality of JF baijiu and provide active community and functional enzymes for other fermentation industries.

Characterization of a fungal thermostable endoglucanase from Chinese Nong-flavor daqu by metatranscriptomic method.

Chinese Nong-flavor (NF) daqu has been enriched with plenty of active enzymes by man-made environment for thousand years. Based on our previous metatranscriptomics, an endo-ß-glucanase gene (NFEg16A), which showed high expression level in NF daqu, was directly obtained and expressed in Escherichia coli BL21 (DE3). NFEg16A shared the highest sequence identity of 87% with endo-1,3-1,4-ß-glucanase from Paecilomyces thermophile. It was optimally active at pH 6.5 and 60 °C and highly stable (>75% residual activity) at pH 3-8 and temperature 30-90 °C. The activity of NFEg16A was strongly inhibited by 10 mM Fe3+ and Hg2+. Compared with endoglucanases with high similarities, NFEg16A was more stable at 70 °C and had higher half-lives of 3.4 h and 1.4 h at 80 °C and 90 °C, respectively. Its specific activity was 85.3 U/mg on barley ß-glucan. Moreover, NFEg16A could efficiently hydrolyze substrate at high concentration of 15 mg/mL, and released glucose and cellobiose as its main end-products. Therefore, this work to some extent verified the important role of NFEg16A in NF daqu, and it would stimulate the acquisition of more enzymes from NF daqu to improve the baijiu quality in future. High thermostability of NFEg16A could also strengthen its potential applications in feed industry.

Directly mining a fungal thermostable α-amylase from Chinese Nong-flavor liquor starter.

BACKGROUND: Chinese Nong-flavor (NF) liquor is continuously and stably produced by solid-state fermentation technology for 1000 years, resulting in enrichment of special microbial community and enzymes system in its starter. Based on traditional culture-dependent methods, these functional enzymes are hardly obtained. According to our previous metatranscriptomic analysis, which identifies plenty of thermostable carbohydrate-active enzymes in NF liquor starter, the aim of this study is to provide a direct and efficient way to mine these thermostable enzymes. RESULTS: In present study, an alpha-amylase (NFAmy13A) gene, which showed the highest expression level of enzymes in starch degradation at high temperature stage (62 °C), was directly obtained by functional metatranscriptomics from Chinese Nong-flavor liquor starter and expressed in Pichia pastoris. NFAmy13A had a typical signal peptide and shared the highest sequence identity of 64% with α-amylase from Aspergillus niger. The recombinant enzyme of NFAmy13A showed an optimal pH at 5.0-5.5 and optimal temperature at 60 °C. NFAmy13A was activated and stabilized by Ca2+, and its half-lives at 60 and 70 °C were improved significantly from 1.5 and 0.4 h to 16 and 0.7 h, respectively, in the presence of 10 mM CaCl2. Meanwhile, Hg2+, Co2+ and SDS largely inhibited its activity. NFAmy13A showed the maximum activity on amylopectin, followed by various starches, amylose, glycogen, and pullulan, and its specificity activity on amylopectin was 200.4 U/mg. Moreover, this α-amylase efficiently hydrolyzed starches (from corn, wheat, and potato) at high concentrations up to 15 mg/ml. CONCLUSIONS: This study provides a direct way to mine active enzymes from man-made environment of NF liquor starter, by which a fungal thermostable α-amylase (NFAmy13A) is successfully obtained. The good characteristics of NFAmy13A in degrading starch at high temperature are consistent with its pivotal role in solid-state fermentation of NF liquor brewing. This work would stimulate mining more enzymes from NF liquor starter and studying their potentially synergistic roles in NF liquor brewing, thus paving the way toward the optimization of liquor production and improvement of liquor quality in future.

New microbial resource: microbial diversity, function and dynamics in Chinese liquor starter.

Traditional Chinese liquor (Baijiu) solid state fermentation technology has lasted for several thousand years. The microbial communities that enrich in liquor starter are important for fermentation. However, the microbial communities are still under-characterized. In this study, 454 pyrosequencing technology was applied to comprehensively analyze the microbial diversity, function and dynamics of two most-consumed liquor starters (Jiang- and Nong-flavor) during production. In total, 315 and 83 bacterial genera and 72 and 47 fungal genera were identified in Jiang- and Nong-flavor liquor starter, respectively. The relatively high diversity was observed when the temperature increased to 70 and 62 °C for Jiang- and Nong-flavor liquor starter, respectively. Some thermophilic fungi have already been isolated. Microbial communities that might contribute to ethanol fermentation, saccharification and flavor development were identified and shown to be core communities in correlation-based network analysis. The predictively functional profile of bacterial communities showed significant difference in energy, carbohydrate and amino acid metabolism and the degradation of aromatic compounds between the two kinds of liquor starters. Here we report these liquor starters as a new functionally microbial resource, which can be used for discovering thermophilic and aerobic enzymes and for food and feed preservation.

Insights into the roles of non-catalytic residues in the active site of a GH10 xylanase with activity on cellulose.

Bifunctional glycoside hydrolases have potential for cost-savings in enzymatic decomposition of plant cell wall polysaccharides for biofuels and bio-based chemicals. The N-terminal GH10 domain of a bifunctional multimodular enzyme CbXyn10C/Cel48B from Caldicellulosiruptor bescii is an enzyme able to degrade xylan and cellulose simultaneously. However, the molecular mechanism underlying its substrate promiscuity has not been elucidated. Herein, we discovered that the binding cleft of CbXyn10C would have at least six sugar-binding subsites by using isothermal titration calorimetry analysis of the inactive E140Q/E248Q mutant with xylo- and cello-oligosaccharides. This was confirmed by determining the catalytic efficiency of the wild-type enzyme on these oligosaccharides. The free form and complex structures of CbXyn10C with xylose- or glucose-configured oligosaccharide ligands were further obtained by crystallographic analysis and molecular modeling and docking. CbXyn10C was found to have a typical (ß/α)8-TIM barrel fold and "salad-bowl" shape of GH10 enzymes. In complex structures with xylo-oligosaccharides, seven sugar-binding subsites were found, and many residues responsible for substrate interactions were identified. Site-directed mutagenesis indicated that 6 and 10 amino acid residues were key residues for xylan and cellulose hydrolysis, respectively. The most important residues are centered on subsites -2 and -1 near the cleavage site, whereas residues playing moderate roles could be located at more distal regions of the binding cleft. Manipulating the residues interacting with substrates in the distal regions directly or indirectly improved the activity of CbXyn10C on xylan and cellulose. Most of the key residues for cellulase activity are conserved across GH10 xylanases. Revisiting randomly selected GH10 enzymes revealed unreported cellulase activity, indicating that the dual function may be a more common phenomenon than has been expected.

Metatranscriptomics Reveals the Functions and Enzyme Profiles of the Microbial Community in Chinese Nong-Flavor Liquor Starter.

Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products.

Removal of the free cysteine residue reduces irreversible thermal inactivation of feruloyl esterase: evidence from circular dichroism and fluorescence spectra.

Feruloyl esterase A from Aspergillus niger (AnFaeA) contains three intramolecular disulfide bonds and one free cysteine at position 235. Saturated mutagenesis at Cys235 was carried out to produce five active mutants, all of which displayed unusual thermal inactivation patterns with the most residual activity achieved at 75°C, much higher than the parental AnFaeA. But their optimal reaction temperatures were lower than the parental AnFaeA. Extensive investigation into their free thiol and disulfide bond, circular dichroism spectra and fluorescence spectra revealed that the unfolding of the parental enzyme was irreversible on all the tested conditions, while that of the Cys235 mutants was reversible, and their ability to refold was highly dependent on the denaturing temperature. Mutants denatured at 75°C were able to efficiently reverse the unfolding to regain native structure during the cooling process. This study provided valid evidence that free cysteine substitutions can reduce irreversible thermal inactivation of proteins.

Molecular and biochemical analyses of CbCel9A/Cel48A, a highly secreted multi-modular cellulase by Caldicellulosiruptor bescii during growth on crystalline cellulose.

During growth on crystalline cellulose, the thermophilic bacterium Caldicellulosiruptor bescii secretes several cellulose-degrading enzymes. Among these enzymes is CelA (CbCel9A/Cel48A), which is reported as the most highly secreted cellulolytic enzyme in this bacterium. CbCel9A/Cel48A is a large multi-modular polypeptide, composed of an N-terminal catalytic glycoside hydrolase family 9 (GH9) module and a C-terminal GH48 catalytic module that are separated by a family 3c carbohydrate-binding module (CBM3c) and two identical CBM3bs. The wild-type CbCel9A/Cel48A and its truncational mutants were expressed in Bacillus megaterium and Escherichia coli, respectively. The wild-type polypeptide released twice the amount of glucose equivalents from Avicel than its truncational mutant that lacks the GH48 catalytic module. The truncational mutant harboring the GH9 module and the CBM3c was more thermostable than the wild-type protein, likely due to its compact structure. The main hydrolytic activity was present in the GH9 catalytic module, while the truncational mutant containing the GH48 module and the three CBMs was ineffective in degradation of either crystalline or amorphous cellulose. Interestingly, the GH9 and/or GH48 catalytic modules containing the CBM3bs form low-density particles during hydrolysis of crystalline cellulose. Moreover, TM3 (GH9/CBM3c) and TM2 (GH48 with three CBM3 modules) synergistically hydrolyze crystalline cellulose. Deletion of the CBM3bs or mutations that compromised their binding activity suggested that these CBMs are important during hydrolysis of crystalline cellulose. In agreement with this observation, seven of nine genes in a C. bescii gene cluster predicted to encode cellulose-degrading enzymes harbor CBM3bs. Based on our results, we hypothesize that C. bescii uses the GH48 module and the CBM3bs in CbCel9A/Cel48A to destabilize certain regions of crystalline cellulose for attack by the highly active GH9 module and other endoglucanases produced by this hyperthermophilic bacterium.