Transcriptomic analysis of tea plant (Camellia sinensis) revealed the co-expression network of 4111 paralogous genes and biosynthesis of quality-related key metabolites under multiple stresses.

The tea plant is an essential economic plant in many countries. However, its growing season renders them vulnerable to stresses. To understand the transcriptomic influences of these stresses on tea plants, we sequenced and analyzed the transcriptomes under drought, high-temperature, and pest. Paralogs were identified by comparing 14 evolutionarily close genomes. The differentially expressed paralog (DEPs) genes were analyzed regarding single or multiple stresses, and 1075 of the 4111 DEPs were commonly found in all the stresses. The co-expression network of the DEPs and TFs indicated that genes of catechin biosynthesis were associated with most transcription factors specific to each stress. The genes playing a significant role in the late response to drought and pest stress mainly functioned in the early response to high-temperature. This study revealed the relationship between stress and regulation of QRM synthesis and the role of QRMs in response to these (a)biotic stresses.

Mutations in the regulatory regions result in increased streptomycin resistance and keratinase synthesis in Bacillus thuringiensis.

Keratinases are a group of proteases of great industrial significance. To take full advantage of Bacillus species as an inherent superior microbial producer of proteases, we performed the ribosome engineering to improve the keratinase synthesis capacity of the wild-type Bacillus thuringiensis by inducing streptomycin resistance. Mutant Bt(Str-O) was identified as a stable keratinase overproducer. Comparative characterization of the two strains revealed that, although the resistance to Streptomycin increased by eight-fold in MIC, the mutant's resistance to other commonly used antibiotics was not affected. Furthermore, the mutant exhibited an enhanced keratinase synthesis (1.5-fold) when cultured in a liquid LB medium. In the whole feather degradation experiment, the mutant could secret twofold keratinase into the medium, reaching 640 U/mL per 107 CFU. By contrast, no significant differences were found in the scanning electron microscopic analysis and spore formation experiment. To understand the genetic factors causing these phenotypic changes, we cloned and analyzed the rpsL gene. No mutation was observed. We subsequently determined the genome sequences of the two strains. Comparing the rpsL gene revealed that the emergence of streptomycin resistance was not necessarily dependent on the mutation(s) in the generally recognized "hotspot." Genome-wide analysis showed that the phenotypic changes of the mutant were the collective consequence of the genetic variations occurring in the regulatory regions and the non-coding RNA genes. This study demonstrated the importance of genetic changes in regulatory regions and the effectiveness of irrational ribosome engineering in creating prokaryotic microbial mutants without sufficient genetic information.

The differential expression patterns and co-expression networks of paralogs as an indicator of the TNM stages of lung adenocarcinoma and squamous cell carcinoma.

Cancers constitute a severe threat to human health. Elucidating the association between the expression patterns of the paralogous genes and transcription factors (TF) and the progression of cancers by comprehensively investigating the expression patterns and co-expression networks will contribute to the in-depth understanding of the pathogenesis of cancers. Here, we identified the paralogous gene pairs and systematically analyzed the expression patterns of these paralogs and the known TFs to elucidate the associations with Tumor, Node, Metastasis (TNM) staging information across ten cancers. We found that the expression of ~60% paralogs was cancer-dependent, and more than 50% of the differentially expressed TFs pairs showed positive expression correlations. The down-regulation patterns of paralogs and TFs were closely associated with the M and N developmental stages of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Our results will help to understand the roles of paralogs and TFs in cancer progression and to screen prognostic biomarkers for early cancer diagnosis.

Small design from big alignment: engineering proteins with multiple sequence alignment as the starting point.

Multiple sequence alignment (MSA) is a fundamental way to gain information that cannot be obtained from the analysis of any individual sequence included in the alignment. It provides ways to investigate the relationship between sequence and function from a perspective of evolution. Thus, the MSA of proteins can be employed as a reference for protein engineering. In this paper, we reviewed the recent advances to highlight how protein engineering was benefited from the MSA of proteins. These methods include (1) engineering the thermostability or solubility of proteins by making it closer to the consensus sequence of the alignment through introducing site mutations; (2) structure-based engineering proteins with comparative modeling; (3) creating paleoenzymes featured with high thermostability and promiscuity by constructing the ancestral sequences derived from multiple sequence alignment; and (4) incorporating site-mutations targeting the evolutionarily coupled sites identified from multiple sequence alignment.

Effective isolation of antioxidant Phelligridin LA from the fermentation broth of Inonotus baumii by macroporous resin.

Phelligridin LA (PLA) is a natural product with vigorous free radical scavenging activities accumulated in the liquid fermentation of herbal medicinal fungus Inonotus baumii. Aiming to establish an efficient isolation method of PLA from the fermentation broth, we evaluated the adsorption of PLA by macroporous resins. The best resin ADS-17 was screened for six candidates with various physical properties and adsorption behaviors. Studies on the thermodynamics and kinetics of the process revealed that the adsorption reaction could take place spontaneously, which implied that the heat generated in adsorption might compensate for the decrease in entropy. The Freundlich theory could be utilized to fit the experimental data. The pseudo-second-order equation could describe the process, and the adsorption rate was primarily controlled by liquid film diffusion and pore diffusion. The influencing operation factors (temperature, pH, and the ratio of fermentation broth to resin) of the adsorption process were optimized with response surface methodology. The optimized condition (temperature 22.81 °C, pH 5.19, and the ratio of fermentation broth to resin or RLS 5.11) supported an adsorption rate of 97.03%. These findings would be indispensable for further optimization of the efficient separation of PLA from the fermentation broth, and the fermentation production of PLA in which separation would be included.

The high-efficient production of phelligridin LA by Inonotus baumii with an integrated fermentation-separation process.

The phelligridin LA was one of the valuable metabolites synthesized by the medicinal fungus Sanghuang in liquid fermentation. In the improvement of PLA productivity by fermentation, we investigated the optimal conditions for the efficient separation of PLA from the fermentation broth with a chromatographic column packed with the macroporous resin ADS-17. Based on the findings, we further developed an integrated bioreactor system that coupled the fermentation and separation of PLA. Fermentation experiments with the bioreactor system testified the performance of our design in fortification of the PLA production: an improvement of PLA production by 2.14 folds was successfully achieved due to the prompt removal of the PLA, while the formation of hyphae biomass was not affected. Also, the integrated system could afford a simultaneous purification of PLA to a purity of 92.95% with a recovery of 84.3%, which was comparable to that of the PLA purified with an additional process (97.53%), at a reasonable recovery. This study provided a feasible approach for the improved production of PLA by fermentation. Besides, the design of the integrated bioreactor system offered a useful reference for the fermentation process development of fungi for the production of diverse valuable metabolites.

Incorporation of nonstandard amino acids into proteins: principles and applications.

The cellular ribosome shows a naturally evolved strong preference for the synthesis of proteins with standard amino acids. An in-depth understanding of the translation process enables scientists to go beyond this natural limitation and engineer translating systems capable of synthesizing proteins with artificially designed and synthesized non-standard amino acids (nsAA) featuring more bulky sidechains. The sidechains can be functional groups, with chosen biophysical or chemical activities, that enable the direct application of these proteins. Alternatively, the sidechains can be designed to contain highly reactive groups: enabling the ready formation of conjugates via a covalent bond between the sidechain and other chemicals or biomolecules. This co-translational incorporation of nsAAs into proteins allows for a vast number of possible applications. In this paper, we first systematically summarized the advances in the engineering of the translation system. Subsequently, we reviewed the extensive applications of these nsAA-containing proteins (after chemical modification) by discussing representative reports on how they can be utilized for different purposes. Finally, we discussed the direction of further studies which could be undertaken to improve the current technology utilized in incorporating nsAAs in order to use them to their full potential and improve accessibility across disciplines.

Strategical isolation of efficient chicken feather-degrading bacterial strains from tea plantation soil sample.

Chicken feather waste is generally insufficiently utilized despite its high content of protein, constituting an environmental issue. Biodegradation of the waste with enabling microbes provides an advantageous option among the available solutions. In this study, an efficient whole feather-degrading strain was strategically isolated from a soil sample taken from a local tea plantation that has little or nothing to do with feathers. The strain was identified as Bacillus thuringiensis (designated as FDB-10) according to the cloned complete 16S rRNA sequence. The FDB-10 could efficiently degrade briefly heat-treated whole feather (102 °C, 5 min; up to 90% of a maximum concentration of 30 g/L) in a salt medium supplemented with 0.1 g/L yeast extract within 24 h (37 °C, 150 rpm). Addition of carbon sources (glycerol, glucose, starch, Tween 20, Tween 80, 1.25 g/L as glycerol) to the fermentation medium could improve the degradation. However, significant inhibition could be observed when the added carbon source reached the amount usually adopted in the investigation of carbon source preference (1%). Nitrogen source (NH4Cl, (NH4)2SO4, peptone) adversely influenced the performance of the strain. When the molar concentrations of NH4+ were equal for the two salt, the inhibitory effect on degradation of whole feathers was similar. Entirely different from other reported feather-degrading strains showing a preference to melanin-free feather substrates, the strain isolated in this study could degrade melanin-containing feather equally efficiently, and higher protease activity could be detected in the digest mix. As a plus, the strain could degrade feathers in rice wash produced in daily cooking, indicating its potential use in the simultaneous treatment of rice cooker wastewater produced by a rice processing plant. All these results imply that the FDB-10 is a strain with great potential in the biodegradation of feather waste.

Design and screening of a chimeric survivin-specific nanobody and its anticancer activities in vitro.

Survivin is a strong inhibitor of apoptosis protein and a promising target for cancer prevention and treatment. Here, we report the design and preparation of novel chimeric nanobodies (Nbs) that could specifically bind to survivin. We screened the peptides from phage-displayed libraries (7-mer, 12-mer) for nonconserved sequences of complementarity-determining regions (CDRs) in the scaffold of the Nb. By a combination of the nonconserved sequences for CDRs, the corresponding chimeric Nbs (10 Nbs) were prepared with genetic operations. The antisurvivin Nb TAT-Nb4A (a fusion with cellular transduction peptide TAT) was found to be the most efficient antibody on the basis of the results from enzyme-linked immunosorbent assay, MTT, and flow cytometry when these nanobodies were tested with hepatoma carcinoma cell HepG2. TAT-Nb4A could inhibit the growth of HepG2 and promote cancer cell apoptosis significantly in a dose-dependent and time-dependent manner: the apoptosis rate reached 52.5% when the concentration of TAT-Nb4A was 120 µg/ml. Western blotting with cells expressing survivin showed that the prepared nanobody could efficiently bind to expressed survivin and blocked the signaling pathway in which survivin played a role. This study provided a convenient and feasible method of obtaining a novel specific Nb with the case of survivin as a good example.

Discovery of the cell-penetrating function of A2 domain derived from LTA subunit of Escherichia coli heat-labile enterotoxin.

Heat-labile enterotoxin (LT) is a protein toxin produced by enterotoxigenic Escherichia coli (ETEC). As a bacterial toxin, LT holotoxin can enter intestinal epithelial cells and cause diarrhea. In addition, LT is also a powerful mucosal adjuvant capable of enhancing the strong immune responses to co-administered antigens. However, the LT immunological mechanism is still not clear in some aspects, especially with the respect to how the LTA subunit functions alone. Here, we discovered that the A2 domain of LTA could carry a fluorescent protein into cells, whose function is similar to a cell-penetrating peptide. The transmembrane-transporting ability of the A2 domain is non-specific in its cell-penetrating function, which was shown through testing with different cell types. Moreover, the LTA2 fusion protein penetrated a fluorescently labeled cell membrane that identified LTA2 internalization through membrane transport pathways, and showed it finally localized in the endoplasmic reticulum. Furthermore, low-temperature stress and pharmacological agent treatments showed that the LTA2 internalization route is a temperature-dependent process involving the clathrin-mediated endocytosis and the macropinocytosis pathways. These results could explain the internalization of the LTA subunit alone without the LTB pentamer, contributing to a better understanding of LTA working as a mucosal adjuvant; they also suggest that the A2 domain could be used as a novel transport vehicle for research and treatment of disease.

Investigating the expression of F10 and G11 xylanases in Aspergillus niger A09 with qPCR.

There exist significant differences between the 2 main types of xylanases, family F10 and G11. A clear understanding of the expression pattern of microbial F10 and G11 under different culture conditions would facilitate better production and industrial application of xylanase. In this study, the fungal xylanase producer Aspergillus niger A09 was systematically investigated in terms of induced expression of xylanase F10 and G11. Results showed that carbon and nitrogen sources could influence xylanase F10 and G11 transcript abundance, with G11 more susceptible to changes in culture media composition. The most favorable carbon and nitrogen sources for high G11 and low F10 production by A. niger A09 were xylan (2%) and (NH4)2C2O4 (0.3%), respectively. Following cultivation at 33 °C for 60 h, the highest xylanase activity (1132 IU per gram of wet mycelia) was observed. On the basis of differential gene expression of F10 and G11, as well as their different properties, we deduced that the F10 protein initially targeted xylan and hydrolyzed it into fragments including xylose, after which xylose acted as the inducer of F10 and G11 gene expression. These speculations also accounted for our failure to identify conditions favoring the high production of F10 but a low production of G11.

Spatially programmed assembling of oxidoreductases with single-stranded DNA for cofactor-required reactions.

Cofactor is especially important for biotransformation catalyzed by oxidoreductases. Many attempts in enhancing performance of the reactions by improving cofactor utilization have been reported. In this study, efficiency of cofactor-requiring biocatalysis was enhanced by improving cofactor recycling via spatially programmed assembling glycerol dehydrogenase (GlyDH, Escherichia coli MG1655) and glutamate dehydrogenase (GluDH, Bacillus subtilis str168), with the aid of single-stranded DNA (ssDNA). The two enzymes were first independently expressed as molecules fused with a phage protein A* that could covalently link ssDNA with certain features. After an enzymatic cross-linking reaction taking place under mild conditions, the conjugate of fused enzyme and ssDNA was assembled into desired structures through base pairing enabled by the ssDNA. Results showed that, to some extent, the fusion with protein A* could improve the specific activity of the enzymes (GlyDH-A*/GlyDH = 116.0 %; GluDH-A*/GluDH = 105.2 %). Additionally, in the coupled reaction system with glycerol and α-ketoglutaric acid as substrates, regarding the production of glutamic acid based on HPLC analysis, the efficiency of cofactor utilization was significantly enhanced (by 23.8- to 41.9-folds), indicating the existence of a substrate-channeling mechanism for cofactor utilization in the assembled reaction system due to the proximity effects. The degree of substrate channeling was calculated as from 1.65 to 1.73. Furthermore, the efficiency of cofactor utilization was influenced in an architecture-dependent manner when complexes with different stoichiometry of GlyDH and GluDH were utilized in biotransformation. This study demonstrated a novel strategy of cofactor recycling for enhanced performance of coupled oxidoreductive reactions.

Dynamics design of a non-natural transcription factor responding to androst-4-ene-3,17-dione.

The production of androst-4-ene-3,17-dione (AD) by the steroidal microbial cell factory requires transcription factors (TFs) to participate in metabolic regulation. However, microbial cell factory lacks effective TFs that can respond to AD in its metabolic pathway. Additionally, finding and obtaining natural TFs that specifically respond to AD is a complex and onerous task. In this study, we devised an artificial TF that responds to AD, termed AdT, based on structure-guided molecular dynamics (MD) simulation. According to MD analysis of the conformational changes of AdT after binding to AD, an LBD in which the N- and C-termini exhibited convergence tendencies was used as a microswitch to guide the assembly of a DNA-binding domain lexA, a linker (GGGGS)2, and a transcription activation domain B42 into an artificial TF. As a proof of design, a AD biosensor was designed and constructed in yeast on the basis of the ligand-binding domain (LBD) of hormone receptor. In addition, the transcription factor activity of AdT was increased by 1.44-fold for its variant F320Y. Overall, we created non-natural TF elements for AD microbial cell factory, and expected that the design TF strategy will be applied to running in parallel to the signaling machinery of the host cell.

Enhanced production of L-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH.

Metabolic engineering is a powerful tool which has been widely used for producing valuable products. For improving L-phenylalanine (L-Phe) accumulation in Corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. The genes involved in the biosynthesis of L-Phe were found to be strictly regulated genes by feedback inhibition. As a result, overexpression of the native wild-type genes aroF, aroG or pheA resulted in a slight increase of L-Phe. In contrast, overexpression of aroF (wt) or pheA (fbr) from E. coli significantly increased L-Phe production. Co-overexpression of aroF (wt) and pheA (fbr) improved the titer of L-Phe to 4.46 ± 0.06 g l⁻¹. To further analyze the target enzymes in the aromatic amino acid synthesis pathway between C. glutamicum and E. coli, the wild-type gene aroH from E. coli was overexpressed and evaluated in C. glutamicum. As predicted, upregulation of the wild-type gene aroH resulted in a remarkable increase of L-Phe production. Co-overexpression of the mutated pheA (fbr) and the wild-type gene aroH resulted in the production of L-Phe up to 4.64 ± 0.09 g l⁻¹. Based on these results we conclude that the wild-type gene aroH from E. coli is an appropriate target gene for pathway engineering in C. glutamicum for the production of aromatic amino acids.

Bioinformatics-aided Protein Sequence Analysis and Engineering.

Most of the currently available knowledge about protein structure and function has been obtained from laboratory experiments. As a complement to this classical knowledge discovery activity, bioinformatics-assisted sequence analysis, which relies primarily on biological data manipulation, is becoming an indispensable option for the modern discovery of new knowledge, especially when large amounts of protein-encoding sequences can be easily identified from the annotation of highthroughput genomic data. Here, we review the advances in bioinformatics-assisted protein sequence analysis to highlight how bioinformatics analysis will aid in understanding protein structure and function. We first discuss the analyses with individual protein sequences as input, from which some basic parameters of proteins (e.g., amino acid composition, MW and PTM) can be predicted. In addition to these basic parameters that can be directly predicted by analyzing a protein sequence alone, many predictions are based on principles drawn from knowledge of many well-studied proteins, with multiple sequence comparisons as input. Identification of conserved sites by comparing multiple homologous sequences, prediction of the folding, structure or function of uncharacterized proteins, construction of phylogenies of related sequences, analysis of the contribution of conserved related sites to protein function by SCA or DCA, elucidation of the significance of codon usage, and extraction of functional units from protein sequences and coding spaces belong to this category. We then discuss the revolutionary invention of the "QTY code" that can be applied to convert membrane proteins into water- soluble proteins but at the cost of marginal introduced structural and functional changes. As machine learning has been done in other scientific fields, machine learning has profoundly impacted protein sequence analysis. In summary, we have highlighted the relevance of the bioinformatics-assisted analysis for protein research as a valuable guide for laboratory experiments.

Understanding Protein Functions in the Biological Context.

Proteins are essential biomacromolecules in all living systems because they are the prominent ultimate executives of the genetic information stored in DNA. Thus, studying protein is one of the central tasks in biological sciences. The complexity, diversity, and dynamics of a protein's structure, function, and structure-function relationship, the inherent structural fragility and thus the requirements on handling proteins to maintain protein's structural and functional orderliness make it a rather tricky task to work with protein. The approach to understanding the functions of a protein has been progressing steadily. In this paper, we reviewed the progress on the approach to the functional study of proteins that tremendously contributed to understanding their biological significance. Emphasis was put on the advances in the age in which high-throughput DNA sequencing and bioinformatics analysis are revolutionizing biological study.

Preparation and performance of semiconductor device bonding joints based on Cu@Sn@Ag preform.

Herein, a 110 A commercial, Si continuous current diode with high heat dissipation power is attached to Cu@Sn@Ag preform, formed by electroplating and physical vapor deposition, and pressed into a preformed sheet under a pressure of 5-10 MPa. The prepared dense three-dimensional network of Cu/Cu3Sn/Ag3Sn joint, based on Cu@Sn@Ag preform obtained using transient liquid-phase diffusion soldering technology, can withstand high temperatures up to 600 °C in power device applications. The mechanical and thermal performance and power cycle reliability of the Cu@Sn@Ag joint are investigated and comparatively analyzed with PbSn5Ag2.5 joints. The results show that the average shear strength of the Cu@Sn@Ag joint is ∼35 MPa, which exceeds that of PbSn5Ag2.5 solder joint, and is similar to that of sintered nano-silver solder joint; the minimum thermal resistance of the corresponding device is ∼0.18 K W-1, near to that of PbSn5Ag2.5 joint. The growth rate of the forward voltage drops below 2% following 150 000 active power cycles, with a junction temperature difference below 60 °C, meeting the requirements of reliability test standards for vehicle specifications. It is concluded that the performance and power cycle reliability of the Cu@Sn@Ag joint are better than those of the PbSn5Ag2.5 joint.

Multiple Functions of Compatible Solute Ectoine and Strategies for Constructing Overproducers for Biobased Production.

Ectoine and its derivative 5-hydroxyectoine are compatible solutes initially found in the hyperhalophilic bacterium Ectothiorhodospira halochloris, which inhabits the desert in Egypt. The habitat of ectoine producers implies the primary function of ectoine as a cytoprotectant against harsh conditions such as high salinity, drought, and high radiation. More extensive and in-depth studies have revealed the multiple functions of ectoine in its native producer bacterial cells and other types of cells and its biomolecular components (such as proteins and DNA) as a general protective agent. Its chemical properties as a bio-based amino acid derivative make it attractive for basic scientific research and related industries, such as the food/agricultural industry, cosmetic manufacturing, biologics, and therapeutic agent preparation. This article first discusses the functions and applications of ectoine and 5-hydroxyectoine. Subsequently, more emphasis was placed on advances in bio-based ectoine and/or 5-hydroxyectoine production. Strategies for developing more robust cell factories for highly efficient ectoine and/or 5-hydroxyectoine production are further discussed. We hope this review will provide a valuable reference for studies on the bio-based production of ectoine and 5-hydroxyectoine.

Synthesis, physicochemical characteristics, cytocompatibility, and antibacterial properties of iron-doped biphasic calcium phosphate nanoparticles with incorporation of silver.

The application of biphasic calcium phosphate (BCP) in tissue engineering and regenerative medicine has been widely explored due to its extensively documented multi-functionality. The present study attempts to synthesize a new type of BCP nanoparticles, characterised with favourable cytocompatibility and antibacterial properties via modifications in their structure, functionality and assemblage, using dopants. In this regard, this study initially synthesized iron-doped BCP (FB) nanoparticles with silver subsequently incorporated into FB nanoparticles to create a nanostructured composite (FBAg). The FB and FBAgnanoparticles were then characterized using Fourier transform infrared spectroscopy, x-ray diffraction, ultraviolet-visible spectroscopy, and x-ray photoelectron spectroscopy. The results showed that silver was present in the FBAgnanoparticles, with a positive correlation observed between increasing AgNO3concentrations and increasing shape irregularity and reduced particle size distribution. Additionally, cell culture tests revealed that both FB and FBAgnanoparticles were compatible with bone marrow-derived mesenchymal stem cells (hBMSCs). The antibacterial activity of the FBAgnanoparticles was also tested using Gram-negativeE. coliand Gram-positiveS. aureus, and was found to be effective against both bacteria. The inhibition rates of FBAgnanoparticles againstE. coliandS. aureuswere 33.78 ± 1.69-59.03 ± 2.95%, and 68.48 ± 4.11-89.09 ± 5.35%, respectively. These findings suggest that the FBAgnanoparticles have potential use in future biomedical applications.

Available methods for assembling expression cassettes for synthetic biology.

Studies in the structural biology of the multicomponent protein complex, metabolic engineering, and synthetic biology frequently rely on the efficient over-expression of these subunits or enzymes in the same cell. As a first step, constructing the multiple expression cassettes will be a complicated and time-consuming job if the classic and conventional digestion and ligation based cloning method is used. Some more efficient methods have been developed, including (1) the employment of a multiple compatible plasmid expression system, (2) the rare-cutter-based design of vectors, (3) in vitro recombination (sequence and ligation independent cloning, the isothermally enzymatic assembly of DNA molecules in a single reaction), and (4) in vivo recombination using recombination-efficient yeast (in vivo assembly of overlapping fragments, reiterative recombination for the chromosome integration of foreign expression cassettes). In this review, we systematically introduce these available methods.